Prevalence and functional roles of intrinsic disorder in the [F-actin]-monooxygenase MICAL family members and their interactors

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Uversky This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7782044/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 23 Dec, 2025 Read the published version in Cell Biochemistry and Biophysics → Version 1 posted 9 You are reading this latest preprint version Abstract Members of the family of molecule interacting with CASL protein (MICAL) proteins play crucial roles in the cytoskeleton rearrangements by promoting depolymerization of F-actin via oxidation of the specific methionine residues to methionine-sulfoxides. These proteins also have multiple other functions and are related to the pathogenesis of various maladies. However, not much is known about the prevalence and functionality of intrinsically disordered regions in MICALs. To fill this gap, we conducted a comprehensive bioinformatics analysis of five human MICAL protein family members, MICAL1, MICAL2, MICAL3, MICAL-L1, and MICAL-L2, to assess their intrinsic disorder characteristics. We also focused on the top 10 most disordered interactors of these proteins. We utilized a set of bioinformatics tools, FuzDrop, RIDAO, InterPro, IuPred, STRING, AlphaFold, and UniProt, for the extensive analysis of these proteins. This analysis revealed that all five MICALs are highly disordered; they take part in myriad of cellular functions, and are also involved in pathogenesis of various diseases, such as neurodegeneration, cancer, and muscle dystrophy. Our study focused on the intrinsic disorder of these proteins to highlight important implications of disorder in protein functionality. These insights may help to find out potential therapeutic targets in biological processes controlled by MICAL proteins. intrinsically disordered proteins intrinsically disordered regions protein-protein interactions posttranslational modifications liquid-liquid phase separation alternative splicing MICAL Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 Figure 15 1. Introduction 1.1. Brief introduction of MICALs MICAL (molecule interacting with CasL (CRK-associated substrate-related protein)) proteins represent a family of actin regulatory enzymes (methionine monooxygenases) involved in the cytoskeleton rearrangements by promoting depolymerization of F-actin [1]. Here, MICALs mediate oxidation of specific methionine residues on F-actin leading to the formation of methionine-sulfoxide, thereby resulting in actin filament disassembly and preventing repolymerization [1]. In the absence of actin, MICALs can act as NADPH oxidases producing H 2 O 2 [1–3]. In vertebrates, there are three MICAL genes ( MICAL-1 , MICAL-2 , and MICAL-3 ), whereas Drosophila has one Mical gene [1]. The proteins of this family have a characteristic N-terminal redox-domain followed by other important functional regions [4]. In addition to the MICAL proteins, vertebrates also have two MICAL-like proteins, MICAL-L1 (also known as molecule interacting with Rab13, MIRab13) and MICAL-L2 (also known as junctional Rab13-binding protein, JRAB) with the domain organization similar to that of MICALs but lacking the redox domain [4]. MICALs can oxidize their substrate using two approaches, one is the direct approach, in which MICALs enzyme directly interact with the substrate like F-actin to oxidize them, in the indirect mechanism, MICALs produce ROS to do the job. Whether MICALs utilize both mechanisms or only use one at a time is not fully understood [5]. Clearly, the direct mechanism represents a controllable way of modification of specific methionines in target proteins, whereas the ROS generated via the indirect mechanism would hit all accessible sites sensitive to oxidative modification. The major substrate of MICALs is actin, an integral part of cytoskeleton. However, being capable of the structural remodeling of actin, these enzymes are involved in multiple key biological functions, particularly playing a number of significant roles in controlling the shape of the cells, as well as cellular movement, cytokinesis, and navigation [5]. Because of the importance of actin in multiple cellular processes, the significance of actin binding proteins involved in the regulation of its action cannot be overlooked [4]. Among such actin binding proteins are the members of the MICAL family, which are involved in mediating the redox reaction of actin protein [4] and which represent the focus of our article. Being more than 1000 amino acids long, MICALs are characterized by complex domain organization, where along with the conserved N-terminal domain known as hydroxylase or flavoprotein monooxygenase domain, there are several other notable regions, such as proline rich region, a calponin homology (CH) domain, Lin11, Isl-1, Mec-3 (LIM) zinc-biding domain that does not bind DNA but act as interface for protein-protein interaction, bMERB (bivalent Mical/EHBP Rab binding) domain, coiled-coil regions, and other functional motifs, through which these regulatory proteins interact with other proteins to carry out their various roles in cells. In humans, each MICAL family protein is directly involved in destabilization of F-actin, being capable of selective association with actin filaments and reshaping cells [1]. MICALs play very important role in numerous cellular functions, such as T cell maturation, intracellular vesicular trafficking, and axon growth [3]. However, the most notable function of the MICAL proteins is their involvement in a very important regulatory mechanism, oxidation of F-actin during cytokinesis [6]. One of the specific aspects of the flavoprotein monooxygenase domain mechanism that is unique to the MICALs is that these proteins interact physically with the substrate [6]. In the scenario related to F-actin oxidation, destabilization, and disassembly, crucial for the dynamics and remodeling of the cellular cytoskeleton, MICALs have regions capable of direct interaction with F-actin. This interaction places the catalytic domain of MICALs in the proximity of F-actin to oxidize it primarily at two methionine residues Met44 and Met47 within the actin molecule [7]. The N-terminal redox domain binds to the co-factor flavin adenine dinucleotide (FAD) and transfers the electrons from NADPH to oxygen to generate ROS (reactive oxygen species), H 2 O 2 is a byproduct of this reaction [6,8]. The reaction renders the actin molecules interactions weak and leads to the disassembly of F-actin molecules, which is very important for cytoskeleton remodeling allowing cells to modify their shape and assist in directional growth. Generations of ROS further facilitate the oxidative modifications in the local environment of the cell [6]. By modifying actin assembly and disassembly MICAL proteins are involved in several biological processes and are closely linked to cellular signaling [6,9]. MICALs are involved in controlling morphology of neuronal dendrites and are essential for the proper development and regeneration of neurons. This is because actin dynamics are very important for several neuronal functions, such as synapse formation, and axonal repulsion, which is a directional growth of the neurons away from some areas and towards other areas to develop new connections [6]. MICALs also play crucial roles in protein trafficking into axons, which is essential for the proper growth and functions of axons, as well as in docking of the vesicles that are fused at the plasma membrane, all of these are vital for the normal signal transmission in nervous systems [6]. MICALs also have important roles in the pathogenesis of various maladies, e.g. cancer progression. For example, MICAL1 was knocked out in MDA MB 231 human breast cancer cell line to identify the implication of MICAL1 on cancer progression [10]. The changes were observed in cell size and motility, implying that MICAL1 does play an important role in the normal progression of malignant cells [10]. The knockout cells in the mouse models show reduced tumor growth, and analysis of human cancer data highlights the upregulation of MICAL1 expression in the invasive breast cancer cells compared to the normal cells [10]. All of these are pointing out in the direction of MICAL1 influence in the cancer progression [10]. Because of their high interactability, misbehavior of MICALs is related to the progression of neurodegenerative diseases [4]. For example, MICAL has been identified as an important interactor of human tau protein, which is one of the major proteins involved in the pathogenesis of Alzheimer disease (AD) in the brain of drosophila AD model. Furthermore, MICAL was shown to increase the propensity of tau aggregation in tauopathies [11]. Normally, Tau protein stabilizes the microtubules but the effect of the MICAL can change this function, as the interaction of MICAL increases the toxicity and aggregation of Tau proteins [11]. MICAL did this by using its redox domain to oxidize tau at specific cysteine residues, thereby modifying the normal functioning of tau protein [11]. Furthermore, this was validated in human tauopathy samples, as the human MICAL protein was upregulated in the brains of the patients with taupathies [11]. MICALs are important in muscle movement and morphology [12]. If MICAL is mutated, the synaptic boutons fails to spread normally across the surface of the muscles and tend to clusters at nerve entry points, suggesting that MICAL play very important role in neuromuscular junction structure [12]. Mutation in MICAL affect the normal distribution of actin and myosin filaments beneath the cell membrane, which are normally distributed in the sarcomeric manner, but the MICAL mutant might cause a notable disorganization of muscle filaments [12]. 1.2. Intrinsic disorder in proteins Since forever the classic “lock-and-key” concept coined in 1893 by Hermann Emil Louis Fischer (1852–1919) [13,14] converted to the “one gene – one enzyme – one biochemical reaction” notion (proposed for the first time in 1941 by George Wells Beadle (1903–1989) and Edward Lawrie Tatum (1909–1975) [15]) and later transformed into the classic model of a protein science, where a unique amino acid sequence of a protein encodes its unique structure required for unique function (derived from the classic experiments of Christian Boehmer Anfinsen Jr. (1916–1995) [16–18]) was a dominant belief in the structural biology community. It took a while for the scientists to start opening to the fluidity/flexibility of protein structure related to its function. The concept of proteins not always following the conventional “structure-function” paradigm was somewhat there in the 20th century literature, being dispersed in the scientific publications, where such “non-canonical” proteins were given different names, such as chameleon, dancing, flexible, floppy, malleable, mobile, partially folded, pliable, rheomorphic, vulnerable, or 4D proteins, protein clouds or proteins waiting for partners, as well as different combinations of “inherently-intrinsically-natively-naturally” with “denatured-disordered-unfolded-unstructured” (for the historic overview, see [19,20]). Despite all these observations, it took long time to debunk the one structure – one function model. One reason for this challenge was the unshaken belief of protein community in the validity of the lock-and-key model supported by the development of specific and more and more advanced experimental approaches for finding and characterizing structure in proteins. However, the accumulated factual scientific discoveries eventually helped the acceptance of the protein intrinsic disorder concept, and advancement of experimental techniques provided a way for researchers to look deeper into the behavior of these uniquely interesting proteins. Intrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs) are unstructured under physiological conditions. Because they typically have a high number of charged residues and low content of hydrophobic residues, many of them have poorly defined hydrophobic core (if any) and typically exist in somewhat extended form. Since they lack defined 3D shapes, IDPs/IDRs are very adaptable towards their environment [21–24]. Looking at the intrinsic disorder in members of MICAL family is expected to provide some important insights. Since MICALs are involved in the actin dynamic, having intrinsic disorder regions in these enzymes may allow them to switch between their active and inactive state more effectively, and give them more structural flexibility, which might be crucial for the MICALs to interact with actin and multiple binding partners. In the context of binding promiscuity, looking at the IDRs of MICALs can enhance understanding of the roles of these proteins in various signaling pathways, as IDRs allow interactions with multiple different partners. This might help MICAL proteins to interact with actin and different other proteins. IDRs are targets for various post-translational modifications (PTMs) [25]. Modifications in these regions like acylation, palmitoylation, and phosphorylation can alter their activity, stability, and localization, which might be responsible for regulation of the enzymatic activity of MICAL proteins [26]. For example, phosphorylation was shown to affect interaction of these proteins with their partners, such as RAB GTPases and their enzymatic activity. This is illustrated by the facts that phosphorylation of MICAL1 by PAK1 regulates MICAL binding to GTPase [27], whereas ARG-induced phosphorylation of MICAL2 promotes MICAL2-mediated F-actin disassembly [28]. The importance of IDPs and IDRs has been well established across various biological functions, whereas alterations in the IDPs/IDRs could potentially contribute towards the propagation of diseases, such as cancers and neurological disorders [29–31]. Changes in MICAL proteins can accelerate or slow down the propagation of cancer, and since these proteins are involved in the directional growth of neurons, unwanted mutations in them are detrimental, especially those located in the IDRs, as they could possibly aggravate the damage. These IDRs can serve as potential targets for drug development, especially if these regions are in functionally important areas of protein length. We can also get evolutionary insights through investigating the conserved IDR regions in MICAL proteins across various species, highlighting their evolutionary importance and their fundamental role in the protein function. 2. Materials and Methods 2.1. Protein datasets We have gathered five MICAL proteins of MICAL family for the comprehensive analysis of their intrinsic disorder predispositions through comprehensive literature review. We first retrieved the FASTA sequences of these proteins using UniPROT, and then ran these sequences though platforms discussed below. The global STRING analysis of all five proteins revealed 67 interactors within PPI network. We further analyzed these interactors by obtaining their FASTA sequences and submitting these sequences in RIDAO for disorder prediction analysis. Then the top 10 most disordered proteins were selected for the in-depth analysis. 2.2. Bioinformatics tools utilized in this study 2.2.1. STRING Protein-protein interactions research was carried out through STRING database. In the first stage of our study, we used the STRING database to conduct a global PPI network analysis of all five MICALs, identifying 174 interactors within the protein-protein interaction (PPI) network. In the second stage of Study, we used the platform to generate the PPI network for individual proteins using medium confidence and maximum number of interactor 500 in the 1st shell of the network [157]. 2.2.2. RIDAO Six well known predictors PONDR ® VLXT, PONDR ® VSL2, PONDR ® VL3, IUPred Short, IUPred long, and PONDR ® FIT are combined in one platform, Rapid Intrinsic Disorder Analysis Online (RIDAO), which is a web-based predictor, efficiently generating per-residue prediction of intrinsic disorder predisposition in individual query proteins or in protein datasets [158]. RIDAO also provides mean disorder prediction (MDP) by averaging outputs of individual predictors. The residues with the disorder score from 0.5 to 1.0 are considered as disordered, residues with disorder score between 0.15 and 0.5 are considered flexible, whereas residues with disorder score below 0.15 are classified as ordered. Base on their percent of predicted intrinsically disordered residues (PPIDR) values, proteins are traditionally classified as ordered, moderately disordered and highly disordered if their PPIDR < 10%, 10% ≤ PPIDR < 30%, and PPIDR ≥ 30%, respectively [90,91]. RIDAO uses the outputs from two binary predictors, the charge-hydropathy (CH) plot [159] and the cumulative distribution function (CDF) plot [95,160], to generate a CH-CDF phase diagram [96,161]. This allowed for the classification of proteins based on their location within the CH-CDF phase space. Proteins in Quadrant I can be categorized as ordered (predicted to be ordered by both models), Quadrant III can be categorized as native coils or pre-molten globules (disordered by both methods), Quadrant II can be categorized as hybrid proteins or putative native molten globules (ordered by CH but disordered by CDF), and Quadrant IV can be categorized as proteins that are disordered according to the CH plot but ordered by the CDF plot [95]. 2.2.3. IUPred IUPred2A [162,163] was used to predict intrinsic disorder tendencies, focusing on predictions of long disordered regions along with ANCHOR2-based prediction [164,165] of molecular recognition features (MoRFs); i.e., disorder-based binding site that represent short intrinsically disordered regions undergoing disorder to order transition upon interaction with their binding partner [166–169]. These features are present in many IDPs, with some of them being even capable to fold differently at interaction with different partners [170–172]. 2.2.4. InterPro InterPro, a resource for the classification of protein sequences into families, was utilized to analyze the functional domains and motifs within the MICAL proteins and to understand how structural features correlate with protein function [173,174]. 2.2.5. FuzDrop An internet platform FuzDrop that predicts the probability of proteins to undergo liquid-liquid phase separation (LLPS) was used to study regions of proteins contributing to 'fuzziness’, and to identify droplet drivers, enhancing our understanding of how the disorder impacts protein interaction capabilities [175]. The ability of a protein to have fuzziness can lead to the LLPS, which in turn gives rise to the membrane-less organelles (MLOs) or biomolecular condensates (BMCs), performing crucial cellular functions in body like stress granules and p-bodies [176]. 2.2.6. AlphaFold 3D structures of query proteins were modeled using AlphaFold, which is an AI-based computational tool that predicts the 3D structure of a protein given its amino acid sequence [177]. Corresponding structural models were retrieved from the AlphaFold Protein Structure Database ( https://alphafold.ebi.ac.uk/ ), which provides structure coverage for over 214 million protein sequences [178]. These models were used to correlate structural predictions with other results. 2.2.7. D 2 P 2 Focused analysis of human MICALs was accomplished through D 2 P 2 website ( http://d2p2.pro ), which seeks to present standardized and comprehensive disorder predictions for each of the currently mapped genes with molecular inscriptions [179]. To predict location of disordered regions that undergo binding-induced folding during protein-protein interactions, D 2 P 2 presently combines ANCHOR outputs [164,165] with the outputs of PONDR ® VLXT [180], PONDR ® VSL2B [181], IUPred (both short and long forms) [162,163], PrDOS [182], PV2, and three variants of ESpritz (NMR, DisProt, and X-ray) [183]. Given the FASTA sequence or a protein/gene name, position of consensus intrinsic disordered regions in addition to range and length of Molecular Recognition Features (MoRFs) and positions of various posttranslational modifications (PTMs) is given by the D 2 P 2 platform for each of the proteins analyzed in this study [179]. 3. Results and Discussion 3.1. Functional disorder analysis of human MICALs 3.1.1. MICAL1 (UniProt ID: Q8TDZ2) [F-actin]-monooxygenase MICAL1 also known as molecule interacting with CasL protein 1 (MICAL1) or neural precursor cell expressed developmentally down-regulated protein 9 (NEDD9)-interacting protein with calponin homology and LIM domains is a cytoplasmic 1067-residue-long monooxygenase that mediates the F-actin disassembly via oxidation of specific methionine residues along the length of actin filaments to methionine sulfoxides [1]. If the F-actin is absent, the MICAL1 act as a NADPH oxidase producing reactive oxygen species H 2 O 2 [3]. It can also act as a Rab (Ras-like GTPase) effector to play a role in the vesicular trafficking [32]. Human MICAL1 contains a signature catalytic monooxygenase (MO) domain (residues 1-489), a calponin homology (CH) domain (residues 506–612) that is often involved in the actin binding, a LIM zinc-binding domain (residues 695–757), and a coiled-coil (CC) domain (residues 919–1027), which is also known as ERM α-like domain or Rab-binding domain (RBD) that participates in interaction with Rab GTPases via two coiled-coil regions (residues 919–962 and 999–1027) acting as two distinct Rab-binding sites with different affinities [6,9]. In addition to GTP-bound Rab proteins CC/RBD also binds plexins, which are the receptors for one of the largest protein families of extracellular guidance cues, semaphorins, that includes over twenty family members conserved from invertebrates to humans [33]. C-terminal half of MICAL1 includes a region important for interaction with RAB8A (residues 918–1067), and a bMERB domain (residues 918–1067) that mediates binding to predominantly RAB8, RAB10, RAB13, and RAB15 (in their GTP-bound forms) [34]. The four MICAL1 domains are linked via three linker regions, with the shortest linker, L1 (residues 490–507), bridging the MO and CH domains, the intermediate L2 linker (residues 613–694) connecting the CH and LIM domains, and the longest linker, L3 (residues 758 to 907), serving as a bridge between the LIM and the CC/RBD domain [35]. It was shown that the C-terminal coiled-coil domain of MICAL1 interacts with the N-terminal catalytic domain leading to the autoinhibition of this protein [35]. Furthermore, CC/RBD domain can also bind to the assembly of CH-LIM domains, with the L2α1 helix from the L2 linker region that play an important role in the stability of the CH-LIM-CC assembly being wedged between the CC/RBD, CH, and LIM domains [35]. Formation of the CH- L2α1-LIM-CC assembly is crucial for the MICAL activation and autoinhibition as well [35]. Our bioinformatics analysis revealed that human MICAL1 belongs to the category of highly disordered proteins, as it is characterized by the PONDR ® VSL2-based content of disordered residues, PPIDR PONDR ® VSL2 = 45.83% and PONDR ® VSL2-based average disorder score, ADS PONDR ® VSL2 = 0.48 ± 0.28. In line with these observations, PPIDR values generated by other predictors included into RIDAO platform were 22.31%, 24.93%, 31.3%, 33.93%, 40.67%, and 41.7%, for IUPred Short, IUPred long, PONDR ® FIT, MDP (mead disorder prediction), PONDR ® VL3, and PONDR ® VLXT, respectively. Figure 1 A shows that although the N-terminal half of human MICAL1 is predicted as mostly ordered, the C-terminal half of this protein contains high levels of predicted intrinsic disorder, with regions 610–694, 740–990, and 100–1067 being predicted as highly disordered. Figure 1 A also indicates that MICAL1 is predicted by ANCHOR2 to contain 4 MoRFs (residues 474–489, 634–682, 749–872, and 879–906) that cover 44.0% of its disordered segments, which corresponds to 20.3% of the entire sequence. This observation indicates that a very significant part of MICAL1 can be involved in disorder-based interactions with various partners. Curiously, Fig. 1 B shows that human MICAL1 is characterized by high probability of spontaneous liquid-liquid phase separation (LLPS), p LLPS = 0.80, and contains 8 droplet-promoting regions (DPRs (residues 1–11, 182–192, 475–513, 613–632, 647–696, 733–843, 864–889, and 955–967), indicating that this protein is capable of spontaneous LLPS and can potentially serve as LLPS driver, and thereby can control the formation of membrane-less organelles [36]. Furthermore, one can fine several aggregation hot spots in this protein (residues 182–189, 488–501, 613–618, 625–631, 652–666, 683–696, 733–751, 832,-838, 866–872, and 955–967), which are can drive aggregation within the condensates generated by LLPS [37]. In line with the ANCHOR-2 outputs predicting the presence of four disorder-based interactions sites, MoRFs, capable of disorder-to-order transition at interaction with binding partners, Fig. 1 C shows that MICAL1 contains multiple regions with context-dependent interactions that change protein interaction behavior and binding modes under different cellular conditions (residues 7–35, 84–101, 174–189, 426–433, 437–451, 463–478, 488–501, 512–522, 592–608, 611–618, 634–649, 652–666, 683–699, 701–707, 729–738, 740–751, 832–838, 866–872, 885–896, 932–941, 952–970, 978–983, 990–997, 1000–1005, 1019–1037, 1040–1046, and 1048–1061) [38,39]. Figure 1 D provides further support for the highly disordered status of human MICAL1 and shows that the C-terminal half of this protein is predicted as disordered by most of the tools included in the D 2 P 2 platform. It also shows that IDRs have functional roles, containing multiple MoRFs and numerous PTMs (mostly phosphorylation sites). In line with these observations, Fig. 1 E represents the AlphaFold-modeled 3D structure, which indicates that the structure within the N-terminal part of the protein is predicted with higher confidence, whereas the C-terminal region has lower per-residue confidence score (p LDDT ), with several regions being characterized by low (70 > p LDDT >50) and very low (p LDDT < 50) scores, indicating that this regions can be disordered in isolation. Human MICAL1 is known to exist in 4 isoforms generated by alternative splicing. The MICAL-1b contains 981 residues, being different from the canonical MICAL-1a by missing the 312–397 region [40]. As a result, intrinsic disorder propensity of this isoform is slightly increased, to PPIDR PONDR ® VSL2 = 46.99% and ADS PONDR ® VSL2 = 0.50 ± 0.29. In the isoform 3 (332 residues), the long N-terminal region that includes the MO and CH domains and half of LIM domain (residues 1-735) is missing, whereas the 736–768 region is changed from YEQHPGDGHFYCLQHLPQTD HKAEGSDRGPESP to MPRLTFAPKGWPHPPTSLHPGQVTDQTTWWLFQ [41]. With PPIDR PONDR ® VSL2 = 92.47% and ADS PONDR ® VSL2 = 0.75 ± 0.16, this shortest isoform is characterized by the most disordered nature among the other MICAL1 variants. Finally, in isoform 4 (1086 residues), the N-terminal methionine residue is changed to MSCLSHSSLPSCCPPQ EASM [42], and the global disorder level is slightly increased relative to the canonical isoform, to PPIDR PONDR ® VSL2 = 47.24% and ADS PONDR ® VSL2 = 0.50 ± 0.29. The observation that disorder status of MICAL1 is affected by alternative splicing is in line with the known fact that polypeptide segments affected by alternative splicing are most often intrinsically disordered, supporting the idea that alternative splicing represents an important means for regulation of the IDP and IDR binding functions [43]. LLPS potential of MICAL1 is also regulated by alternative splicing, as MICAL1-1a, MICAL1-1b, MICAL1-3, and MICAL1-4 are characterized by the p LLPS of 0.8000, 0.8392, 0.9639, and 0.8457, respectively. It is tempting to hypothesize that one of the potential functions of the shortest MICAL1-3 isoform, which is the most disordered MICAL1 isoform missing the MO and CH domains and half of LIM domain, is related to the control of the biogenesis of some cellular MLOs due to its very high LLPS potential. Clearly, this hypothesis requires experimental validation. To evaluate the interactivity of human MICAL1, we used STRING platform, where we used the threshold of 0.400 (medium confidence) for minimum required interaction score with the maximum interactors 500 in the 1st shell. One should keep in mind that the use of STRING in functional analysis has some logical restrictions. For example, splice isoforms and/or post-translational modifications are collapsed, i.e. each node represents all the proteoforms produced from a single, protein-coding gene locus. Furthermore, although associations identified by STRING are meant to be specific and meaningful, where the assembled proteins jointly contribute to a shared function, this does not necessarily mean that they physically interact each other. In fact, the edges in the STRING-generated PPI networks indicate both functional and physical protein associations derived using three groups of evidence: known interactions derived from curated databases or experimentally determined, predicted interactions derived from gene neighborhood, gene fusions, and gene co-occurrence, as well as other evidence, such as text mining, co-expression, and protein homology. Obviously, confidence of a given protein-protein interaction (or functional association) is determined by the number of active interaction sources used for its determination. Therefore, we used here an option to build the full STRING network containing functional and physical associations. Figure 1 F represents the resulting PPI network centered at human MICAL1. This network is characterized by PPI enrichment value of < 1.0×10 –16 , the number of nodes of 35 and the number of edges of 136, which significantly exceeds the 39 interactions expected for a random set of proteins of the same size and degree distribution drawn from the genome. The average local coefficient of this network is 0.676, indicating that the network has densely connected clusters. In fact, k-means clustering revealed that the MICAL1-centered network contains two clusters, where the first cluster includes 20 proteins related to the Sema3A PAK dependent axon repulsion and semaphorin-plexin signaling pathway involved in neuron projection guidance, and MASA syndrome, and the second cluster contains 15 proteins which are Rab subfamily of small GTPases, and proteins involved in RAB GEFs exchange GTP for GDP on RABs and endocytic recycling. Members of this network have 81, 22, and 39 Gene Ontology (GO) terms significantly enriched in Biological Process, Molecular Function, and Cellular Component categories, respectively. The most significantly enriched biological processes (i.e., the processes with the lowest false discovery rates, FRDs, defined as p-values corrected for multiple testing within each category using the Benjamini–Hochberg procedure) are actin polymerization or depolymerization (GO:0008154; p = 9.01×10 − 9 ), neuron projection development (GO:0031175; p = 3.18×10 − 8 ), plasma membrane bounded cell projection organization (GO:0120036; p = 8.85×10 − 8 ), endocytic recycling (GO:0032456; p = 1.36×10 − 7 ), and actin filament organization (GO:0007015; p = 1.36×10 − 7 ). Among the most enriched molecular functions associated with the members of this network are cytoskeletal protein binding (GO:0008092; p = 2.63×10 − 7 ), actin binding (GO:0003779; p = 5.95×10 − 7 ), GTP binding (GO:0005525; p = 1.61×10 − 6 ), GTPase activity (GO:0003924; p = 3.26×10 − 6 ), and semaphorin receptor activity (GO:0017154; p = 1.05×10 − 5 ). The most enriched cellular components are recycling endosome (GO:0055037; p = 1.08×10 − 7 ), insulin-responsive compartment (GO:0032593; p = 6.00×10 − 6 ), semaphorin receptor complex (GO:0002116; p = 7.61×10 − 6 ), recycling endosome membrane (GO:0055038; p = 1.05×10 − 5 ), and extracellular exosome (GO:0070062; p = 1.05×10 − 5 ). The most enriched KEGG (Kyoto Encyclopedia of Genes and Genomes) [44] pathways are axon guidance (hsa04360; p = 3.13×10 − 7 ), endocytosis (hsa04144; p = 0.0116), and tight junction (hsa04530; p = 0.0205). And among the 13 enriched pathways from the REACTOME pathway database [45,46] the most enriched are RAB geranylgeranylation (HSA-8873719; p = 1.32×10 –11 ), RAB GEFs exchange GTP for GDP on RABs (HSA-8876198; p = 9.14×10 –11 ), Sema3A PAK dependent axon repulsion (HSA-399954; p = 8.26×10 –10 ), CRMPs in Sema3A signaling (HSA-399956; p = 8.26×10 –10 ), and semaphorin interactions (HSA-373755 p = 1.36×10 − 8 ). 3.1.2. [F-actin]-monooxygenase MICAL2 (UniProt ID: O94851) MICAL2 is a methionine monooxygenase that oxidizes MET 44 and MET 47 on actin filaments to facilitate the actin disassembly. It regulates the dynamics of actin protein within the nucleus, by facilitating the breakdown of nuclear actin to the monomeric form G-actin [47]. This depolymerization of nuclear actin through redox reaction decreases the amount of nuclear G-actin, which allows the myocardin-related transcription factor (MRTF-A) to accumulate in the nucleus [47]. G-actin normally interacts with MRTF-A to assist its transportation out of the nucleus [47]. Lowering the G-actin levels in the nucleus facilitates the accumulation of MRTF-A within the nucleus, which is mediated through MICAL-2. The accumulation of MRTF-A promotes the formation of transcriptionally active complex with SRF (serum transcription factor). These complexes are important for mediating gene transcription in response to signals, such as signals generated by the G-protein coupled receptors and nerve growth factor [47]. These complexes are important for several cellular functions, such as neurite formation and cellular migration. In short, MICAL2 is responsible for the redox dependent regulation of nuclear actin, which in turn is regulating the SRF/MRTF-A signaling [47]. MICAL-2 is a 1957-residue-long protein encoded by the MICAL2 gene. It contains MO domain (residues 2-494), CH domain (residues 516–619), LIM-type zinc domain (residues 1000–1062), ProQ/FinO domain (residues 1627–1721), and bMERB domain (residues 1796–1945). There are also nuclear localization signals (residues 660–681) and region for interaction with MAPK1 (residues 1324–1363). LIM domains consist of two zinc binding motifs; the domain is very important in proteins involved in multiple interaction. Proteins with LIM domain are involved in developmental and cellular functions [48]. Proteins containing ProQ/FinO domain are capable of RNA binding and considered as RNA chaperones [49]. The bMERB function is to mediate interaction between MICAL proteins and the RAB protein, which are GTPases that play important roles in vesicular trafficking in the cell [34]. Since MICAL2 does not have C-terminal CC/RBD domain, it cannot be autoinhibited via the mechanism described for MICAL1. As per UniProt and InterPro annotations, there are also eight IDRs in human MICAL2 (residues 660–714, 886–942, 1070–1143, 1168–1243, 1258–1345, 1361–1431, 1467–1626, and 1675–1779). In line with these observations, Fig. 2 A represents the RIDAO-generated disorder profile that clearly shows a very high level of intrinsic disorder in most of this protein sequence. Here, most of the long segment spanning residues 670–1957 have disorder score above 0.5. In fact, based on the outputs of PONDR ® VLXT, PONDR ® VSL2, PONDR ® VL3, IUPred Short, IUPred long, PONDR ® FIT, and MDP, the corresponding PPIDR values are 50.23%, 63.67%, 57.95%, 38.99%, 51.51%, 50.74% and 52.68%. There are also multiple MoRF regions at residue position 678–700, 714–752, 778–798, 834–845, 858–963, 1093–1114, 1122–1217, 1221–1239, 1242–1279, and 1680–1776 respectively (see Fig. 2 A). Also, human MICAL2 contains 7 phosphorylated serine residues at positions 515, 631, 696, 991, 1623, 1660, and 1688. Figure 2 B depicts results of the FuzDrop analysis of human MICAL2 and shows that this protein has a very high p LLPS value of 0.9946, indicating high LLPS potential of this protein and ability to serve as a droplet driver. Along its length, MICAL2 has a set of 10 DPRs, residues 1–13, 655–783, 875–950, 1072–1248, 1252–1438, 1474–1632, 1641–1654, 1685–1779, 1813–1844, and 1872–1922. Furthermore, Fig. 2 C shows that this protein is predicted to have a very large number of regions with cellular context dependent interactions (residues 10–26, 66–80, 84–95, 345–360, 496–504, 611–622, 653–665, 736–742, 776–781, 790–798, 811–817, 828–839, 848–856, 861–874, 883–903, 942–956, 986–999, 1071–1080, 1098–1104, 1142–1160, 1162–1167, 1189–1197, 1204–1210, 1242–1255, 1326–1331, 1341–1348, 1372–1383, 1473–1485, 1535–1541, 1551–1561, 1618–1657, 1664–1707, 1767–1792, 1800–1821, 1838–1858, and 1917–1941) indicating high binding potential of this protein. Alternative splicing of MICAL2 mRNA is responsible for generation of 7 isoforms. The canonical form of the protein is isoform 7 consisting of 1957 residues and showing PPIDR PONDR ® VSL2 = 63.67% and ADS PONDR ® VSL2 = 0.63 ± 0.30. Isoform 1 (1124 residues) differs from the canonical isoform by DEPTSPKRPKSIS → VHFSLPVLHPLLG change in the 1112–1124 region and by missing residues 1125–1957. It is characterized by PPIDR PONDR ® VSL2 = 38.52% and ADS PONDR®VSL2 = 0.46 ± 0.28. With PPIDR PONDR ® VSL2 = 29.21% and ADS PONDR ® VSL2 = 0.40 ± 0.26, isoform 2 (784 residues) differs from the canonical form by missing regions 1-171, 740–986, and 1203–1957 and changes in the 1112–1202 region from DEPTSPKRPKSISEPQHSDAEGDAASPLPSEWTSVRISPGEEAAGQDVLAVRVLVTSEDSSSDTESDYG GSEGSHTEPCEEKPWRPGSPHL to GISTSFFRKVLGWPLRLPRDLCNWMQGLLQAAGLHIRDNAYNYCYMYELLSLGLPLLWAFSEVLAAMYRESEGSLE SICNWVLRCFPVK LR. Residues 929–949 and 1125–1957 are missing in the isoform 3, which also has DEPTSPKRPKSIS → VHFSLPVLHPLLG replacement in region 1112–1124. This isoform contains 1103 residues and is characterized by PPIDR PONDR ® VSL2 = 38.53% and ADS PONDR ® VSL2 = 0.45 ± 0.27. In isoform 4 (979 residues), regions 740–929, 950–985, and 1203–1957 are missing, whereas the 1112–1202 region is changed similarly to change found in isoform 2. This isoform has PPIDR PONDR ® VSL2 = 29.77% and ADS PONDR ® VSL2 = 0.38 ± 0.26. With the length of 934 residues, PPIDR PONDR ® VSL2 = 29.23%, and ADS PONDR ® VSL2 = 0.40 ± 0.25, isoform 5 is different from the canonical form by missing regions 740–929 and 112–1957 and having changes in the 1112–1124 region identical to those found in isoform 3. Finally, isoform 6 (955 residues, PPIDR PONDR ® VSL2 = 26.39%, and ADS PONDR ® VSL2 = 0.37 ± 0.26) has changed 1112–1202 region, which is identical to those of isoforms 2 and 4 and is missing regions 740–986 and 1203–1957. Similar to MICAL1, alternative splicing affects the LLPS potential of human MICAL2, as based on their p LLPS values the seven isoforms can be arranged as MICAL2-4 (0.2433) < MICAL2-2 (0.2540) < MICAL2-6 (0.2352) < MICAL2-5 (0.3923) < MICAL2-1 (0.5744) < MICAL2-3 (0.6380) < MICAL2-7/canonical (0.9946). Therefore, only two alternatively spliced MICAL2 isoforms, 2 and 7, are capable of spontaneous LLPS and can act droplet drivers. Although the probabilities of spontaneous LLPS of the five remaining isoforms are below the 0.6 threshold, all of them contain DPRs and therefore can serve as droplet clients. Although D 2 P 2 does not have information about the canonical form of human MICAL2, it contains the functional disorder profile of the alternatively spiced isoform 1, which is missing a very significant portion of the highly disordered C-terminal half of the canonical form. Figure 2 D shows functional disorder profile for this truncated form. Despite missing most of the C-terminal IDR, this isoform still shows a high level of intrinsic disorder, where almost half of the protein is predicted to be intrinsically disordered. This long IDR contains multiple disorder-based ninding sites, MoRFs, and various PTM sites. In line with high levels of predicted disorder Fig. 2 D represents the 3D structural model of MICAL2 and shows the presence of a substantial number of regions with low and very low per-residue confidence score values, which are mostly concentrated toward the C-terminus of the protein, whereas structure of the N-terminal region is predicted with very high confidence. STRING-based analysis revealed that human MICAL2 forms a PPI network containing 17 nodes linked by 35 edges, which is larger than the 19 expected edges (see Fig. 2 E). Since the enrichment p-value of this network is 0.000439, it has more interactions than expected. Although the average node degree is 4.12, the network is characterized by the average local coefficient of 0.88. Analysis of functional enrichment of the members of the MICAL2-centered network revealed that among the 24 significantly enriched biological processes are vesicle docking involved in exocytosis (GO:0006904; p = 7.29×10 − 6 ), Golgi vesicle fusion to target membrane (GO:0048210; p = 5.07×10 − 5 ), regulation of exocytosis (GO:0017157; p = 5.07×10 − 5 ), protein secretion (GO:0009306; p = 0.00012), and actin cytoskeleton organization (GO:0030036; p = 0.00029). The most enriched molecular functions are GTPase activity (GO:0003924; p = 0.00097), GTP binding (GO:0005525; p = 0.0014), GDP binding (GO:0019003; p = 0.0156), nucleotide binding (GO:0000166; p = 0.0156), and NAD(P)H oxidase H 2 O 2 -forming activity (GO:0016174; p = 0.0157). The members of this network are most abundantly found in insulin-responsive compartment (GO:0032593; p = 0.00024), recycling endosome (GO:0055037; p = 0.00053), recycling endosome membrane (GO:0055038; p = 0.00058), synaptic vesicle (GO:0008021; p = 0.00058), and endosome (GO:0005768; p = 0.00058). Finally, the most enriched pathways from the REACTOME database are RAB geranylgeranylation (HSA-8873719; p = 6.67×10 − 6 ), RAB GEFs exchange GTP for GDP on RABs (HSA-8876198; p = 0.0012), translocation of SLC2A4 (GLUT4) to the plasma membrane (HSA-1445148; p = 0.0190), Spry regulation of FGF signaling (HSA-1295596; p = 0.0403), and post-translational protein modification (HSA-597592; p = 0.0403). 3.1.3. [F-actin]-monooxygenase MICAL3 (UniProt ID: Q7RTP6) Similar to other members of the MICAL family of proteins, MICAL3 is a flavoprotein monooxygenase involved in the redox-mediated F-actin disassembly, and, in the absence of actin, it also produces H 2 O 2 , functioning as a NADPH oxidase. MICAL3 operates as RAB effector protein and is crucial for the regulation of complexes involved in vesicular trafficking. This protein is a central player in the pathways regulating the transport of vehicles from Golgi apparatus to plasma membrane, serving as a linker between RAB8A and ESKL [50]. The monooxygenase enzymatic activity of protein is crucial for vesicular docking and fusion, as the mutated form of protein with an inactive monooxygenase domain leads to the accumulation of unfused vesicles [50]. It was emphasized that MICAL3 (and MICAL-L1, see below) are involved in cell division, but this action does not involve oxidoreduction, as the role of MICAL3 during cytokinesis does not require redox activity, as MICAL3 does not regulate actin levels during cytokinesis [51]. Instead, the Rab8A-positive vesicles are tethered to the midbody via direct interaction of MICAL3 with the centralspindlin component mitotic kinesin-like protein 1 (MKLP1 (also known as kinesin-like protein 5 or kinesin-like protein KIF23) and recruiting the adaptor protein ELKS/Rab6-interacting/CAST family member 1 (ERC1) [51]. Human MICAL3 is a 2002-residue-long protein encoded by MICAL3 gene. It includes the monooxygenase domain (residues 1-494), CH domain (residues 518–621), LIM domain (residues 762–824), a ProQ domain (residue 1627–1721), and CC/RBD (residues 1841–2002), which is also classifies as bMERB domain acting as a Rab effector domain. Furthermore, MICAL3 contains a nuclear localization signal (residues 663–684) and multiple PTM sites that are spread throughout the protein, such as serine phosphorylation at 509, 649, 687, 977, 1134, 1143, 1160, 1192, 1274, 1278, 1310, 1337, 1371, 1384, 1433, 1649, 1701, 1704, and 1912, as well as threonine phosphorylation at positions 887, 1276, 1341, 1454, and 1651. As per UniProt annotations, there are several long IDRs in this protein (residues 658–706, 835–883, 907–1313, 1335–1776, and 1791–1821), and these IDRs are characterized by excessive compositional biases containing polar, basic, and acidic residues or enriched in proline residues (e.g., 1239–1258 and 1456–1467 segments). Figure 3 shows that human MICAL3 is expected to contain high levels of disorder. For example, Fig. 3 A shows the RIDAO plot illustrating the disorder profile of this protein, where the structure from the middle of protein towards the C terminus (residues 829–2002) is highly disordered. PPIDR values calculated from the outputs the predictors included to RIDAO, PONDR ® VLXT, PONDR ® VSL2, PONDR ® VL3, IUPred Short, IUPred long, PONDR ® FIT, and MDP, are 60.59%, 67.83%, 63.34%, 47.85%, 54.8%, 59.44%, and 60.28%, respectively, clearly classifying human MICAL3 as a highly disordered protein. Furthermore, ANCHOR-2 analysis showed that MoRF regions (i.e., regions with the ability to undergo disorder to order transition upon binding with their partner [52]) are abundant within the disordered segments of this protein and can be found at the following positions: 650–662, 697–726, 858–1344, 1354–1502, 1508–1553, and 1558–1707, 1725–1751, 1771–1805, and 1848–1853. The results of the FuzDrop-based analysis of human MICAL3 are shown in Fig. 3 B that clearly indicates that this protein has a very high ability to LLPS. In fact, its p LLPS value of 0.9993 suggests that human MICAL3 can serve as a droplet driver, cotrolling the process of MLO formation. FuzDrop analysis predicts the existence of 12 DPR (residues 1–12, 70–81, 587–612, 645–706, 822–891, 897–1322, 1329–1586, 1598–1775, 1788–1819, 1834–1844, 1875–1890, and 1982–2002). There are 34 short aggregation spots that are scattered through the 2/3 of the C-terminal part of protein sequence. The protein is also predicted to contain almost 50 regions with context-dependent interactions (i.e., regions that change interaction behavior and binding modes under different cellular conditions) (see Fig. 3 C), supporting the ANCHOR-2-based analysis that revealed the MoRFs cover 47.1% of the MICAL3 sequence. This is further supported by Fig. 3 D presenting the D 2 P 2 -genearted functional disorder profile of this protein, where a very significant portion of a protein (~ 60%) is shown to be disordered and highly decorated with multiple PTMs, that are likely to modulate binding activities of numerous MoRFs found in this protein. Additional support for the highly disordered nature of human MICAL3 is given by Fig. 3 E representing the 3D stricture of this protein modeled by AlphaFold. This predicted structure has the p LDDT confidence score of 58.13, which is further reflected in the fact that most of the protein is shown in form of “orange noodles” which represent regions with very low p LDDT values (below 50) that are expected to be unstructured in isolation. Five MICAL3 isoforms are generated by alternative splicing. Canonical isoform is 2002-residue long and is characterized by PPIDR PONDR ® VSL2 = 67.83%, ADS PONDR ® VSL2 = 0.69 ± 0.32, and p LLPS = 0.9993. MICAL3-2 (948 residues) is missing C-terminal half (residues 949–2002) and has 934–948 region changed from SSSESEMEEEGEEEE to RSARRAAGRPPATRP. This isoform is characterized by PPIDR PONDR ® VSL2 = 32.59%, ADS PONDR ® VSL2 = 0.42 ± 0.26, and p LLPS = 0.5798. The MICAL3-3 isoform (976 residues), which in addition to the sequence changes found in MICAL2-2 has R → RQLTQERGASQPSCCLPGQVRPAPTPRWK change at position 746, shows PPIDR PONDR ® VSL2 = 35.45%, ADS PONDR ® VSL2 = 0.43 ± 0.26, and p LLPS = 0.6162. In the MICAL2-4 isoform (966 residues), region 967–2002 is missing, whereas region 934–966 is changed from SSSESEMEEEGEEEEEEPRLPPSDLGGVPWKEA to RDWVSPWLPRMVSNS WAQMIHPPQPPTVLGSQM, resulting in PPIDR PONDR ® VSL2 = 32.40%, ADS PONDR ® VSL2 = 0.41 ± 0.24, and p LLPS = 0.5957. Finally, MICAL3-5 isoform (1073 residues) has most alterations in the sequence, as it is missing the C-terminal region (residues 950–2002), has changed 971 − 949 region from GVNGLEEPSIAKRLRGTPERIELENYRLSLRQAEALQEVPEETQAEHNLSSVLDTGAEEDVASSSSESEMEEEG EEEE to LTSLFGWVARHSLGLCDKAKGMSQHLQSNISSFGQQVAQNPLDSFFMCQLLAFGVPFLYGLSEVLVQIRG EFHWQAVAQ, and Q 747 changed to QQREKECSRTCPKKVITLSPPPTPPPCRAHGGQQTYRDLDADNRGKQS PHHERPEPEPPRRFFVDQWELSLSLRSSARPASPSSDSLRQKYIKMYTGGVSSLAEQIANQLQRKEQPKALLDKKEL. This isoform is characterized by PPIDR PONDR ® VSL2 = 36.16%, ADS PONDR ® VSL2 = 0.43 ± 0.26, and p LLPS = 0.5957. Figure 3 F shows that MICAL3 interacts with 31 partners and forms a relatively dense PPI network characterized by the average local clustering coefficient of 0.853 and containing 32 nodes connected by 89 edges (average node degree is 5.56). Since the expected number of edges for the random network of this size is 36, this MICAL3-centerd network has significantly more interactions than expected (PPI enrichment p-value is 3.79×10 –14 ). Proteins in this PPI network are involved in the following biological processes: Golgi vesicle transport (GO:0048193; p = 4.57×10 − 7 ), vesicle-mediated transport (GO:0016192; p = 4.57×10 − 7 ), cellular component organization (GO:0016043; p = 4.57×10 − 7 ), endosomal transport (GO:0016197; p = 1.02×10 − 5 ), and neuron projection development (GO:0031175; p = 1.41×10 − 5 ). Among the most significantly enriched molecular functions of these proteins are GTPase activity (GO:0003924; p = 1.06×10 − 8 ), nucleoside-triphosphatase activity (GO:0017111; p = 2.95×10 − 8 ), GTP binding (GO:0005525; p = 2.99×10 − 7 ), semaphorin receptor activity (GO:0017154; p = 5.60×10 − 6 ), and anion binding (GO:0043168; p = 0.00011). These proteins are most significantly enriched in the following cellular components GO:0005794 (Golgi apparatus; p = 1.78×10 − 7 ), transport vesicle (GO:0030133; p = 1.09×10 − 6 ), exocytic vesicle (GO:0070382; p = 2.30×10 − 6 ), semaphorin receptor complex (GO:0002116; p = 3.93×10 − 6 ), and recycling endosome membrane (GO:0055038; p = 4.86×10 − 6 ). Finally, the most enriched pathways from the REACTOME database associated with the members of the MICAL3-centered PPI are RAB geranylgeranylation (HSA-8873719; p = 4.65×10 –14 ), RAB GEFs exchange GTP for GDP on RABs (HSA-8876198; p = 3.68×10 –11 ), membrane trafficking (HSA-199991; p = 2.59×10 –10 ), SEMA3A-Plexin repulsion signaling by inhibiting Integrin adhesion (HSA-399955; p = 6.53×10 − 6 ), and Sema3A PAK dependent Axon repulsion (HSA-399954; p = 8.84×10 − 6 ). 3.1.4. Human MICAL-like protein 1 (MICAL-L1; UniProt ID: Q8N3F8) By modulating the actin cytoskeleton, human molecule interacting with CasL-like1 (MICAL-L1 protein, which is also known as MIRab13 for molecule interacting with Rab13) is involved in the extension and guidance of neurites, which are the extensions of neurons that form synapses and facilitate communication between nerve cells [9,53,54]. Furthermore, being a lipid binding protein, MICAL-L1 shows higher affinity for phosphatidic acid, which is enriched in recycling endosome membranes. C-terminus of the protein contains two hydrophobic residues through which the protein binds to the phosphatidic acid, and this interaction is vital for the anchoring of MICAL-L1 to the membrane structure and can also influence its role in membrane cargo trafficking [55]. As a result, MICAL-L1 is recruited to the endosome membranes, where it acts as a downstream effector of Rab proteins engaging cytosolic proteins to regulate membrane tubulation [55–58]. MICAL-L1 acts as one of the Rab8 effectors, regulates receptor recycling and recruits Rab8 and EH-domain containing protein 1 (EHD1) to tubular RE membranes that emanate from the perinuclear region [58]. MICAL-L1 also plays a role in endosomal functions, being involved in the late stages of the endocytosis. MICAL-L1 is localized to the tubular membranes and the C-terminal of the protein has coiled-coil region, necessary for this localization that plays an important role in the recycling of receptors and lipids [58]. The protein interacts with EDH1 that acts as a key regulator of endocytosis. Both proteins colocalize to the tubular membrane, indicating functional role in endocytic recycling. Depleting MICAL-L1 has consequences on the endocytic recycling process, it leads to the disruption of normal processing of these functions, suggesting the protein is integral to maintain the function of protein complexes in tubular membranes [58]. Along with this, the depletion also results in the loss of the interaction of Rab8a and EDH1 protein and their absence in tubular membrane [58]. By recruiting EHD1 to primary cilium where it is anchored to the centriole through interaction with tubulins, MICAL-L1 plays a role in ciliogenesis coordination [59]. MICAL-L1 is also crucial for the remodeling of the elongated tubular endosomal network (ETEN) that originates from endosomal recycling compartment in human dendritic cells in response to specific stimuli [59]. ETEN remodeling is very important for the stable delivery of membrane proteins to their designated locations, and is crucial for the antigen presentation and activation of T-cells pointing out the role of MICAL-L1 in immune response [60]. Furthermore, MICAL-L1 regulates endocytosed-EGF receptor trafficking [61], slow endocytic recycling of endocytosed proteins back to the plasma membrane [58], and in cargo protein delivery to the plasma membrane [55]. MICAL-L1 protein is 863-residue-long, which is encoded by the MICALL1 gene. Despite its multifunctionality, MICAL-L1 is a mostly uncharacterized member of the MICAL family containing an N-terminal CH domain (residues 2-108), a LIM domain (residues 162–225, two NPF motifs possessing asparagine-proline-phenylalanine sequences (residues 425–427 and 633–635), bMERB domain (residues 671–818), and C-terminal CC domain (residues 785–830) [58]. The bMERB domain is present in the MICAL family and EH domain-binding protein (EHBP) family, both playing a role in the endosomal trafficking [62]. Region 652–863 is known to mediates the interaction of MICAL-L1 with RAB13 and RAB35 and intramolecular interaction with the CH domain with two coiled-coil regions (residues 682–711, and 785–830) [61,63], whereas 700–863 region, which is necessary and sufficient to associate with tubular recycling endosome membranes, is known to mediate phosphatidic acid-binding and membrane tubulation [56]. There is also a long IDR (residues 224–670) containing multiple low complexity regions and regions with composition bias in human MICAL-L1. The protein contains serine phosphorylation sites at positions 295, 309, 318, 391, 471, 484, 578, 621, and 740. Figure 4 A represents the RIDAO-generated disorder profile of human MICAL-L1 and shows that this protein is predicted to have a long IDR located in the middle (residues 201–695). Therefore, it is not surprising that PPIDR values evaluated from the outputs of PONDR ® VLXT. PONDR ® VSL2, PONDR ® VL3, IUPred Short, IUPred long, PONDR ® FIT, and MDP mounts to 75.9%, 78.1%, 77.64%, 63.27%, 66.16%, 65.7%, and 70.57%, respectively. Figure 4 A also shows that MICAL-L1 contains four MoRF regions (residues 136–149, 227–473, 478–686, and 837–855) predicted by ANCHOR-2, indicating that almost entire central IDR can serve as a protein binding platform. In line with these consideration, it was shown that MICAL-L1 acts as a hub connecting Rab8, Rab10, Rab35, Arf6, Eps15 homology domain protein 1 (EHD-1), and syndapin2 (an F-BAR protein) required for the formation and maintenance of endosomal tubules [56,63–66]. As per FuzDrop analysis, MICAL-L1 is characterized by a very high P LLPS value of 0.9990, suggesting that this protein has a strong tendency to undergo spontaneous LLPS and to form droplets (see Fig. 4 B). MICAL-L1 is predicted to contain 4 DPRs (residues 109–166, 219–669, 673–689, and 825–863) and 10 aggregation hot spots (residues 117–122, 159–166, 238–245, 316–337, 372–378, 429–435, 661–669, 673–682, 711–716, and 825–836) located within the DPR regions. Figure 4 C indicates that MICAL-L1 has 15 regions with context-dependent interactions, with longest spanning regions at 159–179, 316–337, 661–682, 700–730, and 819–836. Figure 4 D shows that the central intrinsically disordered region of this protein is predicted to have multiple PTMs and MoRFs. The AlphaFold predicted structure of human MICAL-L1 has p LLDT value of around 52% and contains a very significant number of “orange noodles”, reflecting high disorder content of this protein (see Fig. 4 E). STRING analysis predicted 119 nodes and 3891 interactions for human MICAL-L1 with p-value of < 1.0×10 –16 , indicating that this network is statistically significant. High degree of connectivity is indicated by an average node degree of 65.4 (see Fig. 4 F). The numbers of edges and nodes in MICAL-L1-centered network are higher than those in MICAL1, MICAL2, and MICAL3 networks. Curiously, it seems that the number of predicted nodes and interaction in MICAL PPI networks are proportional to the disorder status of the protein, suggesting that the interactivity of MICALs correlates with their flexibility or intrinsic disorder content. Proteins in MICAL-L1-centered PPI network are involved in the following biological processes: translation (GO:0006412; p = 2.75×10 –85 ), cytoplasmic translation (GO:0002181; p = 1.20×10 –71 ), organonitrogen compound biosynthetic process (GO:1901566; p = 2.55×10 –51 ), cellular nitrogen compound biosynthetic process (GO:0044271; p = 3.97×10 –48 ), and gene expression (GO:0010467; p = 2.28×10 –40 ). The most enriched molecular functions include structural constituent of ribosome (GO:0003735; p = 8.50×10 –55 ), RNA binding (GO:0003723; p = 8.73×10 –40 ), translation initiation factor activity (GO:0003743; p = 7.21×10 –30 ), structural molecule activity (GO:0005198; p = 1.22×10 –29 ), and translation regulator activity (GO:0045182; p = 5.09×10 –29 ). Cellular components associated with members of this network are small ribosomal subunit (GO:0015935; p = 6.09×10 –64 ), cytosolic small ribosomal subunit (GO:0022627; p = 1.82×10 –57 ), ribosomal subunit (GO:0044391; p = 1.21×10 –55 ), ribosome (GO:0005840; p = 1.33×10 –55 ), and ribonucleoprotein complex (GO:1990904; p = 4.51×10 –55 ). The most enriched KEGG pathways are ribosome (hsa03010; p = 2.06×10 –58 ), RNA transport (hsa03013; p = 4.73×10 –15 ), and endocytosis (hsa04144; p = 3.67×10 –10 ). Among the 46 most enriched pathways from the REACTOME database are formation of the ternary complex, and subsequently, the 43S complex (HSA-72695; p = 2.20×10 –80 ), ribosomal scanning and start codon recognition (HSA-72702; p = 2.20×10 –80 ), translation initiation complex formation (HSA-72649; p = 9.31×10 –79 ), GTP hydrolysis and joining of the 60S ribosomal subunit (HSA-72706; p = 2.39×10 –74 ), and translation (HSA-72766; p = 3.42×10 –73 ). Using more stringent criteria (the threshold of 0.400 (medium confidence) for minimum required interaction score and “text mining” as active interaction source) STRING generated a MICAL-L1-centered network containing 24 nodes linked by 78 edges and characterized by the average node degree of 6.5, average local clustering coefficient of 0.750 and a PPI-enrichment p-value below 10 –16 (data not shown). This was done to avoid uncertainties associated with inclusion of EIF3L and its interactors to the MICAL network (see below). 3.1.5. MICAL-L2 (MICAL-LIKE 2 or JRAB; UniProt ID: Q8IY33) Molecule interacting with CasL-like 2 (MICAL-L2), which is also known as junctional Rab13-binding protein (JRAB), plays an important role in cell adhesion and cell spreading and contributes towards the neurite growth [67]. The role of MICAL-L2 in neurite growth is context-dependent, as this protein can both inhibit and promote neurite outgrowth. For example, although the neurite outgrowth is inhibited by expression of MICAL-L2/JRAB alone, this inhibitory effect is reversed by coexpression of MICAL-L2/JRAB with the dominant active form of Rab13 [67]. The process of neurite outgrowth regulation involves interaction with a small G protein Rab13, which stimulates the interaction of MICAL-L2/JRAB with actinin-4, an actin-binding protein localizes to the cell body and the tips of the neurites, suggesting that Rab13 and MICAL-L2/JRAB may act to transfer actinin-4 from the cell body to the tips of neurites, where it is involved in the reorganization of the actin cytoskeleton which results in neurite outgrowth [67]. When bound to Rab13, MICAL-L2/JRAB induces membrane ruffles [68]. Being an effector protein for Rab13, MICAL-L2/JRAB regulates the endocytic recycling of tight junctional cell adhesion molecule occludin and controls the adhesion and repulsion of epithelial cells [69]. The spatiotemporal regulation of actin cytoskeleton during collective cell migration is linked to the ability of MICAL-L2/JRAB to undergo a conformational change between its closed and open forms depending on the association with Rab13 [70]. MICAL-L2/JRAB protein is involved in tubulation of recycling endosomes directly and is activated by Rab8A [71]. MICAL-L2/JRAB protein induce liquid-liquid phase separation to drive the segregation of cytoplasmic contents to form compartment of distinct phases, a process critical for the regulation of tubular recycling endosomes [71]. The intrinsically disordered region located between the globular N- and C-terminal domains is important for the formation of condensates as it promotes the LLPS process [71]. The interaction of MICAL-L2/JRAB with Rab8A induces formation of a closed MICAL-L2/JRAB form (in which the N-terminal CH and LIM domains interact with the C-terminal CC domain) functioning in the tubulation of recycling endosomes. These data indicate that the biogenesis of tubular recycling endosomes involves two sequential MICAL-L2/JRAB-dependent steps, where the LLPS is first driven by MICAL-L2/JRAB and then the endosomal tubulation is promoted by the closed form of MICAL-L2/JRAB formed by interaction with Rab8A [71]. By forming the distinct Rab8-MICAL-L2/ JRAB and Rab13-MICAL-L2/JRAB complexes, MICAL-L2/JRAB coordinates the assembly of adherens junctions (AJs) and tight junctions (TJs), respectively [72]. Furthermore, MICAL-L2/JRAB was shown to control the transport of claudins, occludin, and E-cadherin to the plasma membrane (PM), as well as Rab8 regulated the Rab13-independent transport of E-cadherin to the PM [72]. MICAL-L2 is highly expressed and promotes cell migration and invasion in several cancer types, such as gastric cancer [73], ovarian cancer [74], breast cancer [75], lung cancer [76], and colon cancer [77]. The molecular mechanisms of MICAL-L2/JRAB involvement in different cancers is very diverse and depend on the cancer type. For example, MICAL-L2/JRAB is upregulated in ovarian cancer, which is a cancer with the highest mortality rate among the gynecologic malignancies [74]. Silencing the expression of MICAL-L2/JRAB prevents the proliferation and invasion of the cancerous cells, suggesting the role of the MICAL-L2/JRAB in the aggressive behavior of malignant cells [74]. The mutation of MICAL-L2/JRAB hindered the translocation of β-catenin to the nucleus, which stimulates the β-catenin/Wnt signaling pathway involved in the cell proliferation and migration, suggesting the possible involvement of MICAL-L2/JRAB protein in the ovarian cancer progression through the modulation of this pathway [75]. In breast cancer, MICAL-L2/JRAB interacts with DENN domain-containing protein 2B (DENND2B) leading to the localization of this protein to the cell surface, where it selectively activates Rab13 on membrane ruffles, at the dynamic leading edge of migrating cells required for cell migration and invasion [75]. Proliferation of lung cancer cells is facilitated by MICAL-L2/JRAB via the de-ubiquitination of c-Myc thereby blocking its degradation [76]. MICAL-L2/JRAB is involved in directing the cells to move together in groups by changing its conformation [70]. Therefore, the impaired conformational plasticity of this protein can disturb the group migration of cells and result in the loss of directionality [70]. MICAL-L2/JRAB is highly disordered as in the AlphaFold structure, most of the protein is highly unstructured. This high level of disorder may allow the conformationally flexible protein to govern the cells to migrate as single units [70]. MICAL-L2/JRAB is a 904-residue-long protein encoded by the MICALL2 gene. Similar to other MICAL proteins MICAL-L2/JRAB contains CH domain (residues 1-107, which is typically found in protein families involved in the actin binding, suggesting the role of protein in actin dynamic [78]), LIM domain (residues 186–248), bMERB domain (residues 723–874), CC domain (residues 735–771), and a regions mediating interaction with RAB13 required for transition from the closed to the opened conformation (residues 807–903). This protein also has a long IDR (residues 251–722) containing a multitude of segments with compositional bias, such as low complexity segments and segments enriched in proline residues, or segments with polar or basic and acidic residues, indicative of intrinsic disorder. Peculiarities of disorder distribution in human MACAL-L2 are further illustrated by Fig. 5 . Figure 5 A shows that with PPIDR values of 72.01%, 79.98%, 78.1%, 61.39%, 65.71%, 71.9%, and 74.78% derived from the outputs of PONDR ® VLXT, PONDR ® VSL2, PONDR ® VL3, IUPred Short, IUPred long, PONDR ® FIT, and MDP, respectively, human MICAL-L2 is predicted as a highly disordered protein. In fact, this is the most disordered member of the human MICAL family. This protein has four MoRF regions (residues 132–168, 174–180, 249–270, and 276–740), that cover 58.7% of its sequence, suggesting that the human MICAL-L2 interactivity relies on intrinsic disorder and disorder-to-order transitions. In line with the established capability of human MICAL-L2/JRAB to undergo LLPS and drive the formation of cellular biomolecular condensates [71], Fig. 5 B represents the results of the FuzDrop analysis revealing that most of the MICAL-L2/JRAB residues have a p DP value above 0.6, so it is indeed a droplet driver with the p LLPS value of 0.9997. This protein is predicted to contain the following droplet-promoting regions along its length: 107–185, 222–233, 245–674, 679–737, and 893–904. It also has 13 aggregation hot spots, with the most noticeable being 172–185, 265–281, 444–469, 578–597, and 679–700 (see Fig. 5 B). Furthermore, Fig. 5 C indicates that the interactivity of human MICAL-L2/JRAB is expected to be dependent on cellular context, as it has a plethora of regions with the context dependent interactions that are scattered along the length of the protein. These regions are expected to behave differently based on the cellular environment and their binding partners, adding a layer of flexibility towards the adaptability of protein. Prevalence of functional intrinsic disorder in this protein is further illustrated by Fig. 5 D showing D 2 P 2 disorder profile, where one can find multiple MoRFs and PTM sites within the central disordered region. Figure 5 E provides another view of high intrinsic disorder content of human MICAL-L2/JRAB showing its AlphaFold-generated 3D structural model, which clearly contains a multitude of regions with very low p LDDT . Human MICAL-L2 exists in five alternatively spliced isoforms. Canonical isoform is 904-residue long and is characterized by PPIDR PONDR ® VSL2 = 79.98%, ADS PONDR ® VSL2 = 0.78 ± 0.28, and p LLPS = 0.9997. Isoform 2, which is 680-residue-long, has altered 1–48 and 656–904 regions, which are changed to MFLSSR and GPPPHPAAGDWPSLPASTFVTTGFGRSPLARKPECRAGRRRRRNLTFRANQGDPCPRPMSLLCLARR. As a result, this isoform has PPIDR PONDR ® VSL2 = 84.41%, ADS PONDR ® VSL2 = 0.82 ± 0.25, and p LLPS = 0.9998. In isoform 3, the 1-570 region is changed to MALSSWAQGTSWAAKGFSRSFSLAEFSLLKPRAGSCRTQEPRKPADGQPWLRCSPCTGGQRIWVHGAHPATSPPIR QKGKLRPRGRESFPQGHTAQESQLGAPPLTPCPVLLMPPGRLAVGVSEGGVAMGRWQGEAQPPLQTPHSQHSFLT PRPLASHP, leading to PPIDR PONDR ® VSL2 = 95.33%, ADS PONDR ® VSL2 = 0.82 ± 0.15, and p LLPS = 0.9974. Isoform 4 (572 residues) is generated by replacing the 571–904 region to GE. It is characterized by PPIDR PONDR ® VSL2 = 72.38%, ADS PONDR ® VSL2 = 0.74 ± 0.32, and p LLPS = 0.9996. Finally, region 290240 is missing in isoform 5 (692 residues), which has PPIDR PONDR ® VSL2 = 92.49%, ADS PONDR ® VSL2 = 0.86 ± 0.20, and p LLPS = 0.9996. Therefore, all isoforms of human MICAL-L2 are highly disordered and all are characterized by very high LLPS potential, suggesting that all of them can be related to the regulation of the biogenesis of cellular MLOs. Figure 5 F represents the STRING-generated PPI network centered at human MICAL-L2. The network includes 17 proteins involved in the 35 interactions. Although this network is not large (the average node degree is 4.12), it is characterized by the high average local clustering coefficient of 0.824. Since the expected number of edges for the random network of similar size is 18, the MICAL-L2 has significantly more interactions than expected (PPI enrichment p-value is 0.000175). The interaction of MICAL-L2 with actinins ACTN1, ACTN2, and ACTN4 (which are the F-actin cross-linking proteins anchoring actin to a variety of intracellular structures) highlights the involvement of this protein in the cytoskeleton organization, and actin remodeling. Proteins in this MICAL-L2-centered PPI network are involved in the following biological processes: vesicle docking involved in exocytosis (GO:0006904; p = 6.36×10 − 6 ), vesicle-mediated transport to the plasma membrane (GO:0098876; p = 6.36×10 − 6 ), regulation of exocytosis (GO:0017157; p = 6.36×10 − 6 ), endosomal transport (GO:0016197; p = 6.36×10 − 6 ), and vesicle-mediated transport (GO:0016192; p = 6.36×10 − 6 ). The most enriched molecular functions include GDP binding (GO:0019003; p = 1.86×10 − 7 ), GTPase activity (GO:0003924; p = 2.64×10 − 7 ), GTP binding (GO:0005525; p = 7.31×10 − 7 ), myosin V binding (GO:0031489; p = 0.00025), and cytoskeletal protein binding (GO:0008092; p = 0.00033). Cellular components where these proteins are most significantly localized include cell junction (GO:0030054; p = 2.86×10 − 8 ), recycling endosome membrane (GO:0055038; p = 1.69×10 − 7 ), recycling endosome (GO:0055037; p = 1.69×10 − 7 ), synaptic vesicle (GO:0008021; p = 1.72×10 − 7 ), and secretory vesicle (GO:0099503; p = 8.84×10 − 7 ). The most enriched KEGG pathway is tight junction (hsa04530; p = 1.12×10 − 6 ). Among the most enriched pathways from the REACTOME database are RAB geranylgeranylation (HSA-8873719; p = 1.28×10 –12 ), RAB GEFs exchange GTP for GDP on RABs (HSA-8876198; p = 6.84×10 –12 ), membrane trafficking (HSA-199991; p = 1.03×10 − 8 ), nephrin family interactions (HSA-373753; p = 0.00052), and platelet activation, signaling and aggregation (HSA-76002; p = 0.00075). 3.2. Global analysis of the interactome of human MICAL proteins Next, we checked the interactability of human MICAL proteins among themselves. This analysis revealed that at the used STRING setting, MICAL1 interacted with MICAL2, MICAL3, and MICAL-L2, whereas MICAL-L1 was not included in the intra-set interactions. Then, we analyzed the global interactability of human MICALs by building their common interactome. To this end, we used STRING platform utilizing somewhat greedy approach with the threshold of 0.400 (medium confidence) for minimum required interaction score and full STRING network option, where the edges indicate both functional and physical protein associations, such as known interactions from curated databases and experimentally determined, predicted interactions from gene neighborhood, gene fusions, and gene co-occurrence, and others associations from text mining and co-expression. This analysis revealed that the joint network of human MICALs includes 174 nodes connected by 4262 edges. The average node degree is 49, indicating that each individual protein in this PPI network is interacting with 49 other proteins. Since the expected number of edges for the random network of similar size is 1095, the PPI network centered at five human MICALs has significantly more interactions than expected (PPI enrichment p-value is < 10 –16 ). The average local clustering coefficient is 0.738, which means that the network tends to make clusters, as shown in Fig. 6 A. In fact, applying the STRING-embedded k-means clustering protocol that finds a defined number of clusters based on their centroids found three distinct clusters in this network. Here, cluster 1 includes 127 members involved in the formation of the ternary complex, and subsequently, the 43S complex as well as in the cytoplasmic translation. The 31 members of the cluster 2 are involved in RAB GEFs exchange GTP for GDP on RABs and in Rab subfamily of small GTPases, whereas 16 members of cluster 3 are related to the semaphorin-plexin signaling pathway involved in neuron projection guidance, SEMA3A-plexin repulsion signaling by inhibiting integrin adhesion, and semaphorin receptor complex. Functional enrichment of the members of this network in term of GO terms associated with biological processes, molecular functions, and cellular components are shown Figs. 6 B, 6 C, and 6 D, respectively, whereas Fig. 6 D represents REACTOME pathways enrichment. Although MICAL-L1 does not directly interact with other members of MICAL family, it is well-integrated into the joint MICAL network via its interactors. Since the number of nodes in this joint MICAL network (174) is smaller than the sum of the nodes in the PPI networks centered at the individual MICALs (35 + 17 + 32 + 119 + 17 = 220), the joint network contains 46 shared partners. Among partners shared by all five MICALs are NEDD9, RAB10, RAB13, RAB8A, and RAB8B. RAB15 is shared by MICAL1, MICAL2, MICAL3, and MICAL-L1. RAB35 is shared by MICAL1, MICAL3, MICAL-L1, and MICAL-L2. MICAL1, MICAL2, and MICAL3 share PLXN4. MICAL1 and MICAL3 share PLXNA1, PLXNA2, PLXNA3, RAB1A, RAB1B, and RAB6A, whereas MSRB1 is shared by MICAL1 and MICAL2, whereas EHD1 interacts with MICAL1 and MICAL-L1. Analysis of the STRING-identified MICAL interactors revealed some apparent inconsistencies. For example, ribosomal proteins constitute a large fraction of a “red” cluster shown in Fig. 6 A, which therefore is annotated as related to the cytoplasmic translation. It seems that this association is questionable as MICALs, which are involved in the redox signaling and actin cytoskeleton regulation, were not shown to be directly linked to translation. However, one cannot exclude a possibility that this protein family still can affect translation via modulation of the actin cytoskeleton. In fact, among the multitude of the crucial roles of the actin cytoskeleton in various cellular processes, is its relation to the protein biosynthesis [79]. It was shown that the densely branched cortical actin patches (CAPs) found in yeast or the focal adhesions (FAs) in mammals might provide a platform for mRNA translation [80–85]. Furthermore, polyribosomes were shown to be associated with cytoskeletal filaments, indicating that ribosomes can interact with actin filaments [86,87]. Therefore, MICAL-mediated regulation of the actin cytoskeleton can affect the efficiency and accuracy of translation. Furthermore, since the actin cytoskeleton is involved in the organization of the endoplasmic reticulum (ER), where protein synthesis (translation) takes place and controls is dynamics, including ER movement, positioning, and remodeling [88], MICALs can indirectly affect translation by inducing changes in the ER organization. Although the aforementioned facts suggest that MICALs can be involved in protein biosynthesis via their effects on actin cytoskeleton, these considerations do not explain the observed association of MICAL-L1 with multiple ribosomal proteins (see Fig. 6 A and 4 F). To understand what can cause these otherwise unexpected associations, we looked at the evidence provided by STRING. This analysis revealed that STRING database identified MICAL-L1 as a distant homolog of the EIF3L (eukaryotic translation initiation factor 3 subunit L) protein (UniProt ID: Q9Y262). Based on the outputs of the EMBOSS Stretcher platform [89] used for the pairwise sequence alignment, MICAL-L1 and EIF3L are characterized by low sequence identity (14.3%) and weak sequence similarity (24.7%), placing these proteins within the "twilight zone" of protein sequence alignment and indicating that the relationship between them is not clearly defined in terms of structure and homology. However, since the actual EIF3L interacts with other translation initiation factors, multiple ribosomal proteins, and other specific proteins, this weak sequence similarity between MICAL-L1 and EIF3L brought these EIF3L interactors to the MICAL-L1 interactome. To avoid the uncertainties associated with inclusion of EIF3L interactors to the MICAL network, we repeated our analysis using more stringent criteria. Figure 7 represents result of this analysis, where MICAL-centered PPI network was generated by STRING using the threshold of 0.400 (medium confidence) for minimum required interaction score and “text mining” as active interaction source. These setting allowed us avoiding inclusion of translation initiation factors and ribosomal proteins to the network. Figure 7 A shows that the size of this network noticeably decreased from 174 to 67. This network has 233 edges and is characterized by the average node degree of 6.96 and an average local clustering coefficient of 0.507. Since the expected number of edges for the random set of proteins of the same size and degree distribution drawn from the genome is 77, the proteins in this MICAL-centered PPI network have more interactions among themselves than what would be expected (PPI enrichment p-value is below 10 –16 ). Application of the STRING-embedded k-means clustering protocol revealed that this network contains three clusters, where 26 proteins in the “red” cluster are related to RAB geranylgeranylation, Rab subfamily of small GTPases, and recycling endosome membrane, 24 proteins in the “green” cluster are related to the Sema3A PAK dependent axon repulsion, and 17 proteins from the “blue” cluster are related to endocytic recycling, recycling endosome membrane, and Eps15 homology domain (see Fig. 7 A). “Red” cluster contains proteins interacting with MICAL3 and MICALL2, “green” cluster includes interactors of MICAL1 and MICAL2, whereas proteins interacting with MICAL-L1 are included to the ‘”blue” cluster. Analysis of the functional enrichment of the members of this network in term of GO terms associated with biological processes, molecular functions, and cellular components are shown Figs. 7 B, 7 C, and 7 D, respectively, with Fig. 7 D representing the REACTOME pathways enrichment. Note that members of this “reduced” PPI network does not contain terms associated with protein translation. Despite stringent criteria used in creation of this network it still contains multiple partners shared by different MICALs. For example, NEDD9, shared by all five MICALs, RAB8A, RAB10, and RAB35 are shared by MICAL1, MICAL3, MICAL-L1, and MICAL-L2, whereas RAB13 is shared by MICAL1, MICAL2, MICAL-L1, and MICAL-L2. PLXNA4 is shared by MICAL1, MICAL2, and MICAL3, and RAB36 is shared by MICAL3, MICAL-L1, and MICAL-L2. MSRB1 is shared by MICAL1 and MICAL2, ACAP2 interacts with MICAL1 an MICAL-L1, and RAB6A and RAB1B are shared by MICAL1 and MICAL3. To avoid potential erroneous conclusions associated with the inclusion of proteins interacting with EIF3L to the MICAL PPI network, at the subsequent stages we exclusively analyzed the “reduced” STRING-generated network. 3.3. Global analysis of the prevalence of intrinsic disorder in MICAL interactome At the next stage, we evaluated the global prevalence of intrinsic disorder in proteins interacting with human MICALs. Figure 8 represents the results of this analysis and shows that these proteins are characterized by various levels of intrinsic disorder, being, in general, not very different from those observed in the entire human proteome. In fact, based on the seven per-residue disorder predictors used in this study (PONDR ® VLXT, PONDR ® VSL2, PONDR ® VL3, PONDR ® FIT, IUPRed_Short, IUPRed_Long, and MDP), the MICAL-interacting proteins are characterized by the mean PPIDR values of 32.0 ± 16.1%, 39.0 ± 21.9%, 30.6 ± 24.7%, 24.9 ± 20.4%, 17.4 ± 14.1%, 19.0 ± 19.7%, and 23.6 ± 23.2%, respectively, in comparison with 34.1 ± 20.3%, 43.6 ± 26.2%, 36.6 ± 29.0%, 30.4 ± 24.0%, 21.2 ± 19.3%, 22.8 ± 24.8%, and 28.3 ± 26.3% evaluated for the entire proteome. The mean ADS values of MICAL interactors are 0.35 ± 0.12, 0.46 ± 0.14, 0.38 ± 0.16, 0.33 ± 0.13, 0.28 ± 0.09, 0.31 ± 0.12, and 0.33 ± 0.13, whereas the members of the human proteome are characterized by the corresponding mean ADS values of 0.36 ± 0.17, 0.48 ± 0.19, 0.42 ± 0.19, 0.37 ± 0.17, 0.28 ± 0.14, 0.31 ± 0.18, and 0.36 ± 0.17. Based on the Mann-Whitney rank sum test, the observed difference in the median PPIDR MDP values between the MICAL-interacting proteins and members of the human proteome is not a statistically significant (P = 0.267); i.e., the difference is not great enough to exclude the possibility that the difference is due to random sampling variability. Similarly, difference between the ADS MDP values between these two groups are not statistically significant as well (P = 0.145). Figure 9A provides another way to look at the global disorder predisposition of the MICAL-interacting by showing the PONDR ® VSL2 score (ADS) vs . PONDR ® VSL2 (%) (PPIDR) plot. Typically, PPIDR values are used to classify proteins as highly ordered, moderately disordered, and highly disordered, if their corresponding PPIDR values are below 10%, between 10% and 30%, and above 30%, respectively [90,91]. An additional angle is provided by the analysis of the protein ADS values that provides another means for protein classification as highly ordered, moderately disordered/flexible, or highly disordered if their ADS < 0.15, 0.15 ≤ ADS < 0.5, and ADS ≥ 0.5. Based on these criteria, all MICAL interactors are clearly classified as moderately or highly disordered by PONDR ® VSL2, and none of these proteins are predicted as highly ordered by ADS and/or PPIDR (as none of the proteins is located in dark blue or cyan areas). This analysis also shows that half of these proteins are predicted as highly disordered. For comparison, the analogous analysis of the entire human proteome (20,317 proteins) revealed that 0.4%, 5.1%, 33.7%, 21.0%, and 39.8% proteins were located within dark blue, cyan, dark pink, light pink, and red areas, respectively [92]. Therefore, with their 0.0% – 0.0% – 50.0% – 24.2% – 25.8% distribution, MICAL interactors are rather close to the whole proteome. FIGURE 9 . Global disorder analysis of human MICAL-interacting proteins. A . The PONDR ® VSL2 score (ADS) vs . PONDR ® VSL2 (%) (PPIDR) plot. Here, each point corresponds to a query protein, coordinates of which are evaluated from the corresponding PONDR ® VSL2 data as its ADS and PPIDR values. Color blocks are used to visualize proteins based on the accepted classification, with red, pink/light pink, and blue/light blue regions containing highly disordered, moderately disordered, and ordered proteins, respectively (see the text). Dark blue or pink areas correspond to the regions where PPIDR agrees with ADS, whereas areas in which only one of these criteria applies are shown by light blue or light pink. Data for the MICAL interactome is shown by gray circles, whereas the positions of individual MICALs are shown by differently colored symbols. B . Charge-Hydropathy and Cumulative Distribution Function (CH-CDF) analysis of query proteins. The ΔCH-ΔCDF plot is a two-dimensional representation that integrates the CH plot, which correlates a protein's net charge with its hydrophobicity, and the CDF, which correlates the cumulative frequency of disorder scores with the disorder scores. In the resulting ΔCH-ΔCDF plot, the Y-axis (ΔCH) represents the protein's distance from the CH boundary, while the X-axis (ΔCDF) represents the deviation of a protein's disorder frequency from the CDF boundary. Proteins are then stratified into four quadrants: Quadrant 1 (bottom right) indicates proteins likely to be structured; Quadrant 2 (bottom left) includes proteins that may be in a molten globule state or lack a unique 3D structure; Quadrant 3 (top left) consists of proteins predicted to be highly disordered; Quadrant 4 (top right) captures proteins that present a mixed prediction of being disordered according to CH but ordered according to CDF. C . Analysis of the interactability of the members of MICAL interactome in a form of the dependence of the node degree on disorder content evaluated as PONDR ® VSL2-based PPIDR. Vertical lines show 10% and 30% PPIDR thresholds, whereas the horizontal line corresponds to the average node degree of the analyzed joint MICAL interactome (7). D . Analysis of the LLPS predisposition of the human MICAL interactors with their intrinsic disorder propensity. Vertical lines show 10% and 30% PPIDR thresholds, whereas the horizontal line corresponds to the 0.6 p LLPS threshold. In plots C and D, circles are colored based on the relation of the corresponding proteins to LLPS, with droplet drivers and droplet clients shown by red and pink circles, and with proteins not related to LLPS (showing p LLPS below 0.6 and not containing DPRs) are shown by blue circles. Figure 9B represents the results of global disorder analysis of human proteins interacting with MICALs in the form of the ΔCH-ΔCDF plot that can be used for further classification of proteins as mostly ordered, molten globule-like or hybrid, or highly disordered based on their positions within the resulting CH-CDF phase space [93–96]. This analysis provided further support to the idea that the MICAL-interacting proteins include noticeable levels of disorder, which are a bit lower than those found in human proteome, which contains 59.1%, 25/5%, 12.3%, and 3.1% proteins in quadrants Q1, Q2, Q3, and Q4, respectively [92]. Figure 9C shows correlation between interactability of the members of the MICAL interactome (in terms of their node degree) with their intrinsic disorder status and demonstrates that there is a weak positive correlation between these two parameters. This is an expected behavior, as disordered proteins are typically prone more to be promiscuous binders. Interestingly, this dataset contains noticeably more proteins with the node degrees below the average node degree of the entire joint MICAL interactome (7) (38 vs . 29). The most connected proteins in this PPI network are MICAL1, MICAL3, MICALL1, RAB8A, RAB6A, RAB11A, ARF6, MICAL2, and MICALL2 interacting with 34, 31, 24, 19, 17, 17, 16, 16, and 16 partners, respectively. Next, we analyzed the LLPS predispositions of the members of the MICAL interactome using FuzDrop and looked at the correlation between the propensity of all the query proteins to undergo LLPS and their intrinsic disorder status. Figure 9D represents the results of this analysis as the p LLPS vs . PPIDR plot and clearly shows that all the proteins capable of spontaneous LLPS are predicted as highly disordered. These observations support the known notion that high levels of intrinsic disorder represent one of the important features of proteins undergoing LLPS. On the other hand, Fig. 9D shows that not all highly disordered proteins are expected to act as droplet-drivers. In fact, out of 37 droplet clients (i.e., proteins with the probabilities of spontaneous LLPS below the 0.6 threshold but containing DPRs), 14 are classified as highly disordered, being characterized by the PPIDR PONDR®VSL2 exceeding 30%. The remaining proteins in this category are moderately disordered. Figure 9D also shows that even among the 10 proteins not associated with LLPS in any way (i.e., showing p LLPS below 0.6 and not containing any DPRs) there were 2 highly disordered proteins: CFL1 (36.75%) and CFL2 (34.94%). Therefore, although all analyzed in this study proteins capable of spontaneous LLPS (droplet drivers) and many droplet clients are highly disordered, not all highly disordered proteins can promote phase separation or become engaged in biomolecular condensate as clients. In other words, the correlation between disorder and LLPS propensity is not symmetric, as “high LLPS potential means high disorder propensity” is not equivalent to “high disorder propensity means high LLPS potential”. Furthermore, there is also a possibility that even mostly ordered proteins can find their way into some biomolecular condensates or MLS being bound to the droplet-clients or droplet-drivers. Finally, Fig. 9C illustrates that there is no obvious correlation between the LLPS potential of a protein and its interactivity, as some LLPS drivers do not have many partners, and many proteins unrelated to LLPS are highly connected. 3.4. Functional disorder analysis of 5 most disordered MICAL interactors 3.4.1. ELKS/Rab6-interacting/CAST family member 1 (ERC1; UniProt ID: Q8IUD2; PPIDR PONDR ® VSL2 = 94.44%) ELKS/Rab6-interacting/CAST family member 1 (ERC1, also known as Rab6-interacting protein 2 (RAB6IP2); the name ELKS is derived from protein rich in the amino acids E, L, K and S [97]) is a regulatory subunit of the IKK complex (IκB kinase or IkappaB kinase) [98], which is an enzyme complex that is involved in propagating the cellular response to inflammation acting as a central regulator of NF-κB activation [99]. ERC1 can also play roles in the organization of the cytomatrix at the nerve terminals active zone (CAZ) as well as vesicle trafficking at CAZ, and in the regulation of endosomes to Goldi apparatus by RAB6 [98]. Complex formed by MICAL3, ELKS and Rab8A was shown to be involved in exocytotic vesicle trafficking controlling the fusion of exocytotic carriers with the plasma membrane [50,100]. It was also shown that by interaction with the centralspindlin complex via the kinesin-6 family protein mitotic kinesin-like protein 1 (MKLP1), which recruits MICAL3 to the central spindle, MICAL3 participates in targeting its binding partners ELKS and Rab8A to the midbody (a transient structure formed during cell division that plays a crucial role in separating two daughter cells), being therefore required for cytokinesis [51]. ERC1 is a member of a family of RIM-binding proteins, with RIMs being regulators of neurotransmitter release at active zone. The protein is known to have multiple isoforms generated by alternative splicing. The canonical isoform is ELKS epsilon, which is a 1,116-residue-long protein characterized by PPIDR PONDR ® VSL2 = 94.44%, ADS PONDR ® VSL2 = 0.82 ± 0.17, and p LLPS of 0.9912. The isoform 2 (ELKS beta, 992 residues) is different from the canonical form by missing regions 440–467 and 1017 − 116 and by having glutamate at position 783 changed to ESLTS and region 1009–1016 changed to DEEEGIWA [101]. As a result, this isoform is characterized by PPIDR PONDR ® VSL2 = 98.89%, ADS PONDR ® VSL2 = 0.85 ± 0.12, and p LLPS of 0.9912. The isoform 3 (ELKS delta, 1088 residues) is missing 440–467 region and has PPIDR PONDR ® VSL2 = 94.30%, ADS PONDR ® VSL2 = 0.82 ± 0.17, and p LLPS of 0.9962. In the isoform 4 (ELKS alpha, 948 residues), regions 440–467, 830–873, and 1017–1116 are missing, whereas glutamate at position 783 is changed to ESLTS and region 1009–1016 is changed to DEEEGIWA. This isoform has PPIDR PONDR ® VSL2 = 98.84%, ADS PONDR ® VSL2 = 0.85 ± 0.12, and p LLPS of 0.9955. The isoform 5 (ELKS gamma) is the shortest version of ERC1 (720 residues). In addition to missing regions 224–523 and 1017–1116, it has changes at position 783 (E → ESLTS) and 1009 − 116 (IIQPLLEL → DEEEGIWA). It is characterized by PPIDR PONDR ® VSL2 = 99.17%, ADS PONDR ® VSL2 = 0.86 ± 0.13, and p LLPS of 0.9986 [101]. Due to a premature stop codon in the mRNA leading to nonsense-mediated mRNA decay, ELKSs beta, alpha, and gamma may be produced at very low levels. Furthermore, in a papillary thyroid carcinoma, translocation t(10;12)(q11;p13) fuses ELKS to receptor-type tyrosine kinase RET [101]. Human ERC1 contains two coiled coil domains (residues 144–988 and 1060–1100) and a FIP-RBD domain (residues 1046–1108), which is a C-terminally located Rab-binding domain (RBD) found in a family of Rab11 interacting proteins (FIPs) that selectively recognize the active form of Rab11. Figure 10 A shows that human ERC1/RAB6IP2 is predicted to have a high level of intrinsic disorder. This is further supported by the RIDAO outputs, which showed that the PONDR ® VLXT, PONDR ® VSL2, PONDR ® VL3, IUPred Short, IUPred long, PONDR ® FIT, and MDP PPIDR values evaluated for this protein were 68.28%, 94.44%, 91.22%, 36.11%, 65.23%, 79.93%, and 84.32%, respectively. There are 24 MoRFs and multiple phosphorylation and acetylation sites in this protein, with the N-terminal 200 residues containing 16 phosphorylation sites. Figure 10 B represents the AlphaFold-generated 3D structural model of this protein, which is dominated by the extremely long α-helices (residues 141–187, 194–250, 264–318, 324–369, 418–701, and 801–943), which do not form a compact core. These α-helices, especially the longest ones, cannot physically exist in unbound form and therefore represent “hallucinations”; i.e., plausible-looking structures where the real protein is actually unstructured. Note that these long α-helices are located within the very long N-terminal coiled coil domain. FuzDrop analysis showed that human ERC1 can serve as droplet driver, since it has a high pLLPS of 0.9912 and contains 8 DPRs (residues 1-118, 172–193, 243–255, 625–704, 713–755, 855–873, 894–919, 945–969, and 973–1017). There are also numerous aggregation hot spots and regions with context-dependent interactions. These observations indicate that ERC1 can act as a scaffold protein generating a disorder-based platform for its binding partners. In line with the FuzDrop predictions, it was shown that ERC1 induces formation of cytoplasmic condensates with liquid-like properties that specifically host partners of a network relevant to cell motility, including liprin-α1 [102]. Importantly, in migrating cells, ERC1, liprin-α, and LL5 proteins (which are the pleckstrin homology-like domain family members) form polarized plasma membrane-associated platforms (PMAPs), which are liquid condensates associated with the regulation of cell motility [102]. Figure 10 C represents the STRING-generated PPI network centered at human ERC1 and shows that this protein is engaged in interaction with 101 partners forming a network with 102 nodes and 673 edges. Among the 198 significantly enriched GO-terms related to biological processes that are associated with this PPI network are neurotransmitter secretion (GO:0007269; signal = 4.57), vesicle-mediated transport in synapse (GO:0099003; signal = 4.3), synaptic vesicle cycle (GO:0099504; signal = 4.23), neurotransmitter transport (GO:0006836; signal = 3.78), and synaptic vesicle exocytosis (GO:0016079; signal = 3.63). Among the 27 significantly enriched molecular function GO terms are syntaxin-1 binding (GO:0017075; signal = 1.43), syntaxin binding (GO:0019905; signal = 1.27), GTPase binding (GO:0051020; signal = 0.91), transmembrane transporter binding (GO:0044325; signal = 0.84), and myosin V binding (GO:0031489; signal = 0.82). Among the 92 significantly enriched cellular component GO terms are presynaptic active zone (GO:0048786; = 4.55), presynaptic active zone cytoplasmic component (GO:0098831; signal = 3.82), cell cortex region (GO:0099738; signal = 3.22), IkappaB kinase complex (GO:0008385; signal = 2.95), and presynapse (GO:0098793; signal = 2.82). Among the 53 significantly enriched KEGG pathways are NF-kappa B signaling pathway (hsa04064; signal = 3.44), NOD-like receptor signaling pathway (hsa04621; signal = 2.06), IL-17 signaling pathway (hsa04657; signal = 1.91), shigellosis (hsa05131; signal = 1.77), and RIG-I-like receptor signaling pathway (hsa04622; signal = 1.63). And among the 78 REACTOME pathways the most enriched are serotonin neurotransmitter release cycle (HSA-181429; signal = 5.59), dopamine neurotransmitter release cycle (HSA-212676; signal = 5.27), acetylcholine neurotransmitter release cycle (HSA-264642; signal = 4.24), norepinephrine neurotransmitter release cycle (HSA-181430; signal = 4.19), and glutamate neurotransmitter release cycle (HSA-210500; signal = 3.84). 3.4.2. Myocardin-related transcription factor A (MRTFA; UniProt ID: Q969V6; PPIDR PONDR ® VSL2 = 93.69%) Myocardin-related transcription factor A (MRTFA, also known as MKL/myocardin-like protein 1, megakaryoblastic leukemia 1 protein (MKL1), and megakaryocytic acute leukemia protein (MAL)), is a 931-residue-long transcription coactivator involved in the control of the expression of the genes to regulate the cytoskeleton during cell migration, development, and morphogenesis [103]. This activity relies on the interaction of MKL1/MAL/MRTFA with the serum response factor (SRF) transcription factor, with the activity of the resulting SRF-MRTFA complex being regulated by MRTFA binding to cellular globular actin (G-actin) via its RPEL repeats (core sequence RPxxxEL) as well as by filamentous actin (F-actin) in the nucleus. SRF-MRTFA complex couples cytoskeletal gene expression to cytoskeletal dynamics being responsive to the Rho GTPase-induced changes in the G-actin concentration [103]. MRTFA is found in cytoplasm [47] and nucleus [47,104], and its subcellular localization is tightly regulated by actin both in cytoplasm and nucleus, where MRTFA binding to G-actin in cytoplasm masks the nuclear localization signal (NLS) and thereby inhibits nuclear import, whereas binding to the nuclear G-actin stimulates nuclear export of MRTFA to the cytoplasm. MICAL2 regulates nuclear localization of MRTFA by mediating nuclear actin depolymerization that leads to the decrease in the nuclear G-actin pool and consequent retention of MRTFA in the nucleus and formation of an active SRF-MRTFA complex [47]. It was also shown that MICAL2 facilitates gastric cancer cell migration via positive regulation of nuclear translocation of MRTFA and subsequent CDC42 activation and MMP9 expression [105]. By utilizing SRF/MRTFA signaling pathway, MICAL2 was shown to regulate myofibroblasts differentiation in epidural fibrosis [106]. A loss-of-function homozygous mutation of MRTFA is associated with immunodeficiency and severe susceptibility to bacterial infection [103]. Deregulated activity of MRTFA in transgenic mice with selective endothelial Progerin expression resulted in the development of the myocardial and perivascular fibrosis, left ventricular hypertrophy, and premature death [107]. MRTFA belongs to the MRTF family of transcription factors sharing evolutionary conserved domains essential for actin-binding, high-order chromatin organization, homo- and heterodimerization, and transcriptional activation [108]. Human MRTFA has a complex multidomain organization and includes an N-terminal region mediating interaction with suppressor of cancer cell invasion (SCAI) and beta-actin (ACTB) (residues 1-256) that contains intervening spacer sequences (residues 6–23 and 60–67) RPEL repeats (residues 24–29 and 50–67), a bipartite Nuclear localization signal (residues 27–65), a SAP domain (residues 347–381, named after three proteins known to contain it, SAF-A/B, Acinus, and PIAS), a coiled coil domain (residue 515–563), a transactivation domain (residues 564–931) and a multitude of low complexity regions and regions with other compositional biases (e.g., enriched in polar residues, proline residues, and basic and acidic residues). Each of the RPEL and intervening spacer motifs can bind autonomously to G-actin [109]. Mouse MRTFA that contains three RPEL motifs forms a pentavalent complex with G-actin [109]. Proteins with SAP domain are nuclear proteins involved in transcription, DNA repair, RNA processing, or apoptotic chromatin degradation. SAP motif also serves as a putative DNA-binding motif involved in chromosomal organization [110]. Figure 11 shows that human MRTFA is a highly disordered protein containing multiple MoRFs and different PTMs, which are capable of interacting with multiple partners. As per RIDAO analysis, is PONDR ® VLXT, PONDR ® VSL2, PONDR ® VL3, IUPred Short, IUPred long, PONDR ® FIT, and MDP PPIDR values are 80.23%, 93.69%, 92.24%, 59.42%, 77.85%, 83.64%, and 87.47%, respectively. FuzDrop-based analysis revealed that this protein has a very high p LLPS value of 0.9999 and contains 6 DPRs (residues 98–263, 272–343, 385–396, 432–485, 544–664, and 672–866). It also has 17 aggregation hot spots and 27 regions with the context-dependent interactions The presence of multiple disorder-based interactions sites (MoRFs) (Fig. 11 A) and the high overall level of intrinsic disorder (Figs. 11 A and 11 B) define the high binding promiscuity of human MRTFA, which is shown by STRING to form a rich PPI network with 147 nodes and 1398 edges, average node degree of 19, average local clustering coefficient of 0.63, and a PPI enrichment value of < 10 –16 . Among the 185 biological processes GO-terms significantly enriched in this network are ethanol metabolic process (GO:0006067; signal = 4.13), monocarboxylic acid metabolic process (GO:0032787; signal = 4.05), cellular aldehyde metabolic process (GO:0006081; signal = 3.81), small molecule catabolic process (GO:0044282; signal = 3.58), and carboxylic acid metabolic process (GO:0019752; signal = 3.38). Among the 105 significantly enriched molecular function GO-terms are aldehyde dehydrogenase [NAD(P)+] activity (GO:0004030; signal = 6.18), oxidoreductase activity, acting on the aldehyde or oxo group of donors, NAD or NADP as acceptor (GO:0016620; signal = 6.17), oxidoreductase activity, acting on the aldehyde or oxo group of donors (GO:0016903 ; signal = 5.98), aldehyde dehydrogenase (NAD+) activity (GO:0004029; signal = 5.93), and C-acyltransferase activity (GO:0016408; signal = 3.79). Among the 18 significantly enriched cellular component GO-terms are mitochondrial matrix (GO:0005759; signal = 1.66), mitochondrion (GO:0005739; signal = 0.88), phosphopyruvate hydratase complex (GO:0000015; signal = 0.76), peroxisome (GO:0005777; signal = 0.63), and serine C-palmitoyltransferase complex (GO:0017059; signal = 0.6). 3.4.3. Ninein-like protein (NINL; UniProt ID: Q9Y2I6; PPIDR PONDR ® VSL2 = 86.69%) Human centrosomal ninein-like protein (NINL or NLP) is a member of the γ-tubulin complexes binding proteins (GTBPs) that is essential for proper execution of various mitotic events [111]. NINL is involved in the microtubule organization in interphase cells, promoting microtubule nucleation that contributes to centrosome maturation, spindle formation and chromosome segregation [111]. Within a cell, NINL can be found in cytoplasm, cytoskeleton, microtubule organizing center, and centrosome [112,113]. Although inn interphase cells, NINL is transported to the centrosomes by the dynein-dynactin motor complex, direct phosphorylation of NINL by PLK1 during centrosome maturation results in releasing NINL to the cytoplasm [112]. Since the overexpression of NINL promoted anchorage-independent growth and induces cell malignant transformation, this protein was classified as an oncogenic protein [114–116]. It was shown that NINL/NLP promotes autophagy by facilitating the interaction of Rab7 and FYCO1 (which is FYVE (zinc finger domain targeting signal-transducing proteins to cell membranes through binding to the membrane lipid phosphatidylinositol-3-phosphate (PtdIns(3)P) with high specificity) and coiled-coil domain-containing protein 1) [117]. Furthermore, in the process of ciliopathies, NINL/NLP associates with the coiled-coil and C2 domain-containing protein 2A (CC2D2A, a component of the tectonic-like complex localized at the transition zone of primary cilia that acts as a barrier preventing diffusion of transmembrane proteins between the cilia and plasma membranes [118]) and functions in Rab8-MICAL3-regulated vesicle trafficking [119]. Human NINL is a 1,382-residue long calcium-binding protein containing four EF-can domains (residues 7–42, 41–76, 196–231, and 233–268), three coiled coil domains (residues 384–424, 484–579, and 1046–1375), and the KEN (residues 495-497and D (destructive, residues 633–641) boxes required for the cell cycle-controlled NINL degradation by the APC/C pathway [120]. A very significant part of this protein is predicted as intrinsically disordered (see Fig. 12 A). NINL has multiple MoRFs and different PTMs, which are capable of interacting with multiple partners. In line with these considerations, RIDAO analysis indicated that the PONDR ® VLXT, PONDR ® VSL2, PONDR ® VL3, IUPred Short, IUPred long, PONDR ® FIT, and MDP PPIDR values of human NINL were 62.66%, 86.69%, 87.05%, 30.75%, 44.14%, 58.47%, and 66.64%, respectively, whereas the corresponding ADS values were 0.56 ± 0.26, 0.74 ± 0.26, 0.68 ± 0.19, 0.40 ± 0.20, 0.49 ± 0.20, 0.54 ± 0.23, and 0.57 ± 0.19. In addition to the canonical isoform, NINL has an alternative splicing-generated isoform missing residues 735–1083, which represents a molecular link between Usher syndrome and Leber congenital amaurosis [121]. This 1003-residue-long alternatively spiced isoform is characterized by the PPIDR PONDR®VSL2 of 80.54% and ADS PONDR®VSL2 of 0.67. FuzDrop-based analysis revealed that this protein has a high p LLPS value of 0.9821 and contains 10 DPRs (residues 70–104, 120–203, 207–219, 574–611, 653–667, 686–718, 741–782, 810–1012, 1158–1172, and 1271–1287). It also has 18 aggregation hot spots and 43 regions with the context-dependent interactions. Alternative splicing results in an isoform with decreased p LLPS value of 0.8397, reduced number of DPRs (9), DPRs (13), and regions with the context-dependent interactions (31), suggesting that both functionality and LLPS potential of this protein are affected by alternative splicing. Together with the presence of multiple disorder-based interactions sites (MoRFs) (Fig. 12 A) and the high overall level of intrinsic disorder (Figs. 12 A and 12 B) the multitude of the regions with the context-dependent interactions found in human NINL/NLP suggests that this protein can be characterized by high binding promiscuity. Validity of this hypothesis is illustrated by Fig. 12 C representing a STRING-generated PPI network centered at NINL and containing 93 nodes linked by 1046 edges. This network is characterized by the average node degree of 22.5, average local clustering coefficient of 0.745, and a PPI enrichment value of < 10 –16 . Among the 98 biological processes GO-terms significantly enriched in this network are centrosome cycle (GO:0007098; signal = 6.54), microtubule organizing center organization (GO:0031023; signal = 6.38), microtubule cytoskeleton organization (GO:0000226; signal = 5.09), microtubule-based process (GO:0007017; signal = 4.23), and spindle organization (GO:0007051; signal = 4.19). Among the 17 significantly enriched molecular function GO-terms are tubulin binding (GO:0015631; signal = 1.95), microtubule binding (GO:0008017; signal = 1.82), cytoskeletal protein binding (GO:0008092; signal = 1.59), dynein light intermediate chain binding (GO:0051959; signal = 1.48), and dynactin binding (GO:0034452; signal = 1.47). Among the 68 significantly enriched cellular component GO-terms are centrosome (GO:0005813; signal = 5.94), microtubule organizing center (GO:0005815; signal = 5.07), microtubule associated complex (GO:0005875; signal = 4.42), centriole (GO:0005814; signal = 4.18), and mitotic spindle microtubule (GO:1990498; signal = 3.75). 3.4.4. AP-4 complex accessory subunit RUSC2 (UniProt ID: Q8N2Y8; PPIDR PONDR ® VSL2 = 83.05%) AP-4 complex accessory subunit RUSC2 (RUN and SH3 domain containing 2, also known as Iporin for interacting protein of Rab1 [122]) is involved in vesicular trafficking of proteins at the trans-Golgi network being capable of interaction with the adapter-like complex 4 (AP-4), which is an ancient membrane trafficking complex [123]. Here, RUSC2 serves as an AP-4 accessory protein that facilitates the transport of AP-4-derived, ATG9A-positive vesicles from the trans-Golgi network to the cell periphery [123]. This protein can be found in cytoplasm/cytosole showing punctate distribution [122], on cell membrane [124], or in the perinuclear region [122]. RUSC2 can also bind to golgin subfamily A member 2 (GOLGA2, a peripheral membrane component of the cis-Golgi stack that acts as a membrane skeleton maintaining the structure of the Golgi apparatus, and as a vesicle tether facilitating vesicle fusion to the Golgi membrane [125]) and active forms of RAB1A and RAB1B [122]. It also can use its RUN domain to interact with the GTP-bound form of RAB35, thereby being recruited to the plasma membrane [122–124]. Furthermore, RUSK2/Iporin might serve as a link between the targeting of ER derived vesicles, triggered by the Rab1 GTPase and a signaling pathway regulated by molecules containing SH3 and/or poly-proline regions [122]. MICAL1 serves one of the Rab1 interactors that defines a link between the Golgi apparatus associated rab1 and the intermediate filament cytoskeleton [126]. Together with p115 (the vesicle tethering-protein), GM130 (a cis-Golgi-localized coiled-coil protein), golgin-84 (a type II C-terminally anchored cis-Golgi protein with an extensive cytoplasmic coiled-coil domain), RUSC2/Iporin, and MICAL1, an activated form of Rab1 forms a protein complex mediating and regulating intracellular vesicle transport [122]. Although RUSC2/Iporin does not physically interact with MICAL1, these proteins act together within the aforementioned complex, where they both bind to Rab1 [122]. Variants of RUSC2 with missing C-terminal regions (866–1516 or 1318–1516) are linked to intellectual developmental disorder, autosomal recessive 61 (MRT61) characterized by central hypotonia, microcephaly, and moderate to severe intellectual disability [127]. RUSC2/Iporin is a 1,516-residue-long protein that contains a RUN domain (residues 1,031 − 1,175), which being a major part of the Rab1-binding domain (R1BD) is required for the interaction with RAB1A, RAB1B and RAB35 [122,124], an SH3 domain (residues 1,447-1,506) that potentially mediates assembly of specific protein complexes via binding to proline-rich peptides, and multiple regions with composition biases, such as regions containing polar residues, acidic residues, basic and acidic residues, glycine residues, as well as low complexity regions all indicative of intrinsic disorder. In line with this hypothesis Figs. 13 A and 13 B show that a very significant fraction of this protein is predicted as intrinsically disordered. Further support to this idea is coming from RIDAO-based analysis intrinsic disorder predisposition, according to which human RUSC2 is characterized by the PONDR ® VLXT, PONDR ® VSL2, PONDR ® VL3, IUPred Short, IUPred long, PONDR ® FIT, and MDP PPIDR values of 58.91%, 83.05%, 78.96%, 55.21%, 66.36%, 69.92%, and 69.39%, respectively, with the corresponding ADS values being 0.55 ± 0.36, 0.75 ± 0.36, 0.69 ± 0.27, 0.48 ± 0.24, 0.59 ± 0.26, 0.62 ± 0.25, and 0.61 ± 0.24. Figure 13 A also shows that this protein contains a large set of MoRFs and numerous phosphorylation sites. According to AlphaFold analysis (Fig. 13 B), 3D structure of human RUSC2 is dominated by orange “noodles” indicative of very low per-residue confidence score (p LDDT < 50), suggesting that many of these regions are expected to be unstructured in isolation. FuzDrop showed that human RUSC2 is characterized by the p LLPS of 1.0 and contains 17 DPRs (residues 24–57, 67–101, 148–164, 178–281, 292–303, 325–366, 401–446, 450–693, 726–839, 848–893, 900–920, 1012–1027, 1175–1186, 1202–1270, 1277–1406, 1415–1452, and 1505–1516). It possesses 31 aggregation hot spots and 40 regions with the context-dependent interactions. Figure 13 C represents a PPI network generated by STRING for this protein. This network containing 58 nodes and 256 is characterized by the average node degree of 9, average local clustering coefficient of 837, and a PPI enrichment value of < 10 –16 . Among the 33 significantly enriched GO-terms describing biological processes associated with this network are isoprenoid biosynthetic process (GO:0008299: signal = 3.9), small molecule biosynthetic process (GO:0044283; signal = 1.83), isoprenoid metabolic process (GO:0006720; signal = 2.38), cellular lipid metabolic process (GO:0044255; signal = 1.05), and alcohol biosynthetic process (GO:0046165; signal = 1.6). Among the 17 significantly enriched molecular function GO-terms are prenyltransferase activity (GO:0004659; signal = 3.38), transferase activity, transferring alkyl or aryl (other than methyl) groups (GO:0016765; signal = 2.6), small GTPase binding (GO:0031267; signal = 0.44), trans-hexaprenyltranstransferase activity (GO:0000010: signal = 0.47), and isopentenyl-diphosphate delta-isomerase activity (GO:0004452; signal = 0.47). Among the 68 significantly enriched cellular component GO-terms are organelle membrane (GO:0031090; signal = 0.54), AP-4 adaptor complex (GO:0030124; signal = 1.05), organelle subcompartment (GO:0031984; signal = 0.56), cytoplasm (GO:0005737; signal = 0.31), and intracellular anatomical structure (GO:0005622; signal = 0.29). 3.4.5. EH domain-binding protein 1-like protein 1 (EHBP1L1; UniProt ID: Q8N3D4; PPIDR PONDR ® VSL2 = 80.30%) EH domain-binding protein 1-like protein 1 (EHBP1L1) is a human 1,523-residue-long protein acting as RAB effector and playing a role in vesicle trafficking [34]. Together with adaptor proteins of actin regulators CD2-associated protein (CD2AP) and CBL-interacting protein of 85 kDa (CIN85), EHBP1L1 is involved in the length control of cilia, which are organelles that function in signal sensing and transduction and consist of axonemal microtubules and plasma membranes protruding from the cell surface to the extracellular region [128]. This protein is also involved in polarized exocytic trafficking in small intestinal epithelial cells [129]. During terminal erythropoiesis, promotes nuclear polarization and subsequent enucleation of erythroblasts is promoted by an apicobasal polarity regulator EHBP1L1 in coordination with Rab10, Bin1, and dynamin [130]. The formation of apically-destined vesicles at the recycling endosomes is dependent on EHBP1L1, Bin1, and dynamin 1 [131]. In renal cell carcinoma (RCC), interaction of EHBP1L1 with Janus kinase 1 (JAK1) stabilizes JAK1, leading to the formation of an immunosuppressive tumor microenvironment (TME) and thereby driving immune evasion [132]. Similar to MICALs, EHBP1L1 contains a bivalent Mical/EHBP Rab binding (bMERB) domain that can bind two Rabs simultaneously [34]. Therefore, EHBP1L1 and MICALs are functionally related, although no direct physical interaction between these proteins was demonstrated as of yet. EHBP1L1 possesses a Ca 2+ /phospholipid-binding (C2) domain (residues 8-157), a calponin homology (CH) domain (residues 1037–1142), a bivalent Mical/EHBP Rab binding (bMERB) domain (residues 1349–1501) mediating binding to the GTP-bound forms of RAB8, RAB10, RAB10, RAB13, and RAB15 [34], a coiled-coil domain (residues 1363–1515), and CAAX motif (residues 1520–1523) acting as a signal for prenylation and required for endosomal colocalization with RAB8 and RAB10 [34]. Because of the presence of the bMERB domain, EHBP1L1 has two Rab-binding sites and therefore, together with MICAL-1, MICAL-3, and MICAL-L2, belongs to the bivalent RAB8-family effectors [34]. In its farnesylated form, EHBP1L1 is preferentially localized to the intracellular structures resembling endosomes, where it colocalizes with constitutively active RAB8 and RAB10 [34]. Like the other proteins discussed in this section, human EHBP1L1 is predicted to have high level of disorder. For example, based on the RIDAO analysis, this protein is characterized by the PONDR ® VLXT, PONDR ® VSL2, PONDR ® VL3, IUPred Short, IUPred long, PONDR ® FIT, and MDP PPIDR values of 79.71%, 80.3%, 78.79%, 56.8%, 68.29%, 71.11%, and 74.06%, respectively. Figure 14 A represents the functional disorder profile generated for EHBP1L1 by D 2 P 2 and shows that it has multiple MoRFs that almost completely cover its long IDRs. The protein is also densely decorated by PTMs, indicating that the intrinsic disorder-based interactivity of EHBP1L1 can be regulated by PTMs. Figure 14 B provides further support to the highly disordered nature of this protein by showing 3D structure model generated by AlphaFold. Here, with the exception to Ca 2+ /phospholipid-binding (C2) domain (residues 2-172), CH-domain (residues 1030–1143), and portion of CC domain (residues 1352–1445 and 1470–1508), for which the average per-residue model confidence score p LDDT values are 87.92, 87.33, and 83.49, very significant fraction of a protein is given by disordered loops with very low p LDDT < 50. Based on the outputs of FuzDrop analysis, human EHBP1L1 is expected to serve as droplet driver, as it shows a very high p LLPS score of 0.9998 indicative of high potential of this protein to undergo spontaneous LLPS. Protein also includes 8 DPRs (residues 175–595, 648–665, 693–704, 817–864, 870–915, 919–929, 933–1041, and 1151–1369. Although currently, there is no experimental evidence that EHBP1L1 can have a role in the formation or regulation of biomolecular condensates, some of its functions are indicative of potential EHBP1L1-LLPS connections. Because of the aforementioned involvement of this protein in membrane trafficking and interactions with RAB8, BIN1, and dynamin, EHBP1L1 could be implicated in the formation of the biomolecular condensates at the endocytic recycling compartment (ERC, which is a cellular structure responsible for sorting and recycling proteins and lipids back to the cell membrane [129]) or in other membrane-associated processes. In addition to 8 DPRs and 24 MoRFs, human EHBP1L1 contains 21 aggregation hot spots and more than 35 regions with context-dependent interactions. All this suggests that this protein might have high interaction potential. The capability of EHBP1L1 to interact with several proteins was already discussed. Figure 14 C represents further support to the high interactivity of this protein by showing the STRING-generated PPI network centered at EHBP1L1. This network includes 49 nodes connected by 463 edges. The network has an average local clustering coefficient of 0.886 and average node degree of 18.9. Members of this network are taking part in various biological processes such as protein transport from ciliary membrane to plasma membrane (GO:1903445; FDR = 0.0147), Golgi vesicle fusion to target membrane (GO:0048210; FDR = 0.000060), vesicle transport along actin filament (GO:0030050; FDR = 0.0102), vesicle docking involved in exocytosis (GO:0006904; FDR = 0.0036), and establishment of protein localization to plasma membrane (GO:0061951; FDR = 0.0050). The molecular functions represented are muscle alpha-actinin binding (GO:0051371; FDR = 9.26e-13), cadherin binding involved in cell-cell adhesion (GO:0098641; FDR = 0.0070), microfilament motor activity (GO:0000146; FDR = 0.0022), cell-cell adhesion mediator activity (GO:0098632; FDR = 0.0063), and GDP binding (GO:0019003; FDR = 0.0011). Cellular components, where these proteins are localized include filamentous actin (GO:0031941; FDR = 4.11e-61), unconventional myosin complex (GO:0016461; FDR = 0.00013), insulin-responsive compartment (GO:0032593; FDR = 0.0122), stress fiber (GO:0001725; FDR = 1.13e-09), and stereocilium tip (GO:0032426; FDR = 0.0432). 3.5. Functional disorder analysis of the neural precursor cell expressed developmentally down-regulated protein 9 (NEDD9; UniProt ID: Q14511; PPIDR PONDR ® VSL2 = 59.71%), a shared interactor of five MICALs Human enhancer of filamentation 1 (HEF1, also known as neural precursor cell expressed developmentally down-regulated protein 9 (NEDD9), CRK-associated substrate-related protein (CASL) [133], Cas scaffolding protein family member 2 (CASS2), renal carcinoma antigen NY-REN-12 [134], and p105 [135]) is a 834-residue-long scaffolding protein with central coordinating role in tyrosine-kinase-based signaling related to cell adhesion [136]. The protein is a member of the Crk-associated substrate (CAS) protein family and is broadly spread throughout the cell, being found in basolateral cell membrane, cytoplasm [137,138], cell cortex [137], nucleus [138–140], Golgi apparatus [140], cell projections, such as lamellipodium [136,140] and cilium [141], cilium basal body [141], cell junction (focal adhesion) [140,142–144], cytoskeleton [137], and spindle pole [138,145]. Broad distribution of NEDD9 within cell and a multitude of names given to this protein by different researchers suggest its multifunctionality. Being a non-catalytic scaffolding protein [135,140,146], NEDD9 forms complexes involving oncogenic kinases, such as focal adhesion kinase (FAK), ABL, SRC, Aurora-A (AURKA), and others. By acting as a docking protein NEDD9/CASL is involved in signaling pathways related to T-cell differentiation, cell-cell junctions formation, cell migration, in part, in response to integrin activation [147,148]. This defines the capability of this protein to regulate the magnitude and duration of cell signaling cascades controlling multiple cellular processes, which that are crucial for tumorigenesis and metastases, such as apoptosis and cell cycle, migration, adhesion, invasion, and chemotaxis [147–151]. Furthermore, pleiotropic functions of NEDD9 link this protein to various maladies, where poor outcome, metastatic potential, and resistance to chemotherapy in many human cancers are linked to altered expression of NEDD9, and where non-malignant pathological conditions, such as stroke and polycystic kidney disease are linked to altered NEDD9 function [148,152]. In databanks, MICALs are also classified as NEDD9/CASL-interacting proteins [153]. This interaction depends on binding of the proline-rich motif PPKPP (Pro-Pro-Lys-Pro-Pro) of MICALs to the SH3 domain of the Cas family members Cas and CasL [137]. Binding of NEDD9 to MICAL1 was shown to facilitate hypoxia-induced gastric cancer cell migration via Rac1-dependent manner [154]. It was shown that MICAL connects NEDD9/CASL to the intermediate filament [137]. Human NEDD9 has a complex domain organization with multiple functional domains and regions, such as SH3 domain (residues 1–66), regions required for interaction with E3 ubiquitin-protein ligase Itchy homolog (ITCH, residues 1-505) [155], SH2 binding sites (residues 67–351 and 352–626), spindle-regulatory protein D1M1 (residues 102–229), Src substrate cortactin (CTTN, residues 351–834) [136], and serine/threonine-protein kinase PLK1 (residues 710–834) [142]. Conserved C-terminal domain (residues 626–834) includes divergent helix-loop-helix motif (residues 710–760) involved in homodimerization and heterodimerization with a corresponding region of p130 Cas and other HLH motif-containing proteins [156]. NEDD9 also contains multiple regions with low complexity and compositional bias. In additional to the canonical isoform, NEDD9 is known to have two alternatively spiced isoforms, with the isoform 2 missing most of the sequence (residues 175–834) and possessing changes in the remaining C-terminal region (residues 166–174) ITPVRTGHGYVYEYPSRYQK → FQRDGQVSYFLVRASKQTSL [40] and with the isoform 3 containing MKYK → MWTR changes in the N-terminal region (residues 1–4). Figure 15 A shows that as expected from its multifunctionality, human NEDD9 contains high levels of intrinsic disorder and contains 12 MoRFs and multiple PTM sites. In fact, according to the outputs of RIDAO, this protein is characterized by the PONDR ® VLXT, PONDR ® VSL2, PONDR ® VL3, IUPred Short, IUPred long, PONDR ® FIT, and MDP PPIDR values of 36.33%, 59.71%, 54.68%, 39.45%, 47.12%, 39.93%, and 47.00%, respectively, with the corresponding ADS values being 0.40 ± 0.36, 0.55 ± 0.26, 0.48 ± 0.26, 0.40 ± 0.23, 0.46 ± 0.23, 0.40 ± 0.27, and 0.45 ± 0.24. High level of intrinsic disorder in human NEDD9 is further supported by Fig. 15 B showing 3D structure model of this protein generated by AlphaFold. There are three regions with high per-residue model confidence scores p LDDT values, residues 1–61 corresponding to the N-terminal SH3 domain, serine-rich domain (residues 408–559) and conserved C-terminal domain (residues 701–834), whereas the remaining regions (residues 62–407 and 560–700) represent disordered loops with very low p LDDT < 50, which contain 58% of protein sequence. FuzDrop analysis revealed that NEDD9 is characterized by the pLLPS score of 0.9690 and contains 10 DPRs (residues 68–87, 108–129, 136–152, 179–202, 234–262, 282–410, 496–506, 560–628, 654–668, and 671–704) indicating that this protein can undergo spontaneous LLPS and can serve as a droplet driver. The presence of 19 aggregation hot spots and 23 regions with context-dependent interactions in combination with the presence of 12 MoRFs suggests that NEDD9 can be characterized by high interaction potential. Figure 15 C supports this notion and shows that this protein forms a PPI network with 96 nodes and 801 edges. The network has an average local clustering coefficient of 0.671 and average node degree of 16.7. Members of the NEDD9-centered PPI network are taking part in various biological processes, with actin filament organization (GO:0007015; strength = 2.49), supramolecular fiber organization (GO:0097435; strength = 2.34), actin polymerization or depolymerization (GO:0008154; strength = 2.29), integrin-mediated signaling pathway (GO:0007229; strength = 2.11), and enzyme-linked receptor protein signaling pathway (GO:0007167; strength = 2.04) being the most enriched representatives of the 595 corresponding significantly enriched GO-terms. The most highly enriched molecular functions represented in this network (from 59 corresponding significantly enriched GO-terms) are kinase binding (GO:0019900; strength = 2.24), protein kinase binding (GO:0019901; strength = 2.19), cytoskeletal protein binding (GO:0008092; strength = 1.74), enzyme binding (GO:0019899; strength = 1.68), and actin binding GO:0003779; strength = 1.58). The most enriched cellular components, where these proteins are located (from 80 corresponding significantly enriched GO-terms) include cell leading edge (GO:0031252; strength = 2.24), actin cytoskeleton (GO:0015629; strength = 2.01), lamellipodium (GO:0030027; strength = 1.91), focal adhesion (GO:0005925; strength = 1.86), and cytoskeleton (GO:0005856; strength = 1.48). 4. Conclusions Our bioinformatics analysis of MICAL family protein demonstrated that all five key proteins are highly disordered with mean disorder prediction above 30%. Out of 62 interactors of MICALs, approximately 50% are also highly disordered. Furthermore, in-depth analysis of these proteins revealed that disorder indeed has functional implications. Notably, MICALs and their interactors were shown to be involved in multiple interactions. MICALs and many disordered MICAL interactors are predicted to have a high tendency to undergo LLPS, forming biomolecular condensates and membrane-less organelles. These characteristics support and highlight why these proteins play important roles in cellular functions. Diving deep into the disorder profiles of these proteins can help us to identify potential targets for diseases, such as neurodegeneration and cancer. Declarations Author Contributions: Conceptualization, V.N.U.; methodology, V.N.U.; validation, H.N.A. and V.N.U.; formal analysis, H.N.A. and V.N.U.; investigation, H.N.A. and V.N.U.; data curation, H.N.A. and V.N.U.; writing—original draft preparation, H.N.A. and V.N.U.; writing—review and editing, H.N.A. and V.N.U.; visualization, V.N.U.; supervision, V.N.U. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data are contained within the article. Conflicts of Interest: The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. References Wu, H.; Yesilyurt, H.G.; Yoon, J.; Terman, J.R. 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Cite Share Download PDF Status: Published Journal Publication published 23 Dec, 2025 Read the published version in Cell Biochemistry and Biophysics → Version 1 posted Editorial decision: Revision requested 05 Nov, 2025 Reviews received at journal 31 Oct, 2025 Reviewers agreed at journal 16 Oct, 2025 Reviewers agreed at journal 09 Oct, 2025 Reviewers agreed at journal 08 Oct, 2025 Reviewers invited by journal 08 Oct, 2025 Editor assigned by journal 06 Oct, 2025 Submission checks completed at journal 06 Oct, 2025 First submitted to journal 04 Oct, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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09:22:27","extension":"xml","order_by":33,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":330785,"visible":true,"origin":"","legend":"","description":"","filename":"319b4b036bae4880b7ad427b016bfbe61structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7782044/v1/5ec484f465b7a318210fcc14.xml"},{"id":94006401,"identity":"9763cc45-152c-45f6-ab2f-b17374b3716a","added_by":"auto","created_at":"2025-10-21 09:22:27","extension":"html","order_by":34,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":344040,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7782044/v1/6fa7a61ebbfb6b90e27a1cd6.html"},{"id":94005993,"identity":"9b5e895e-efa3-4922-b00e-a680f6310e31","added_by":"auto","created_at":"2025-10-21 09:14:26","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":988001,"visible":true,"origin":"","legend":"\u003cp\u003eThe outputs of PONDR\u003csup\u003e®\u003c/sup\u003e VLXT, PONDR\u003csup\u003e®\u003c/sup\u003e VSL2, PONDR\u003csup\u003e®\u003c/sup\u003e VL3, PONDR\u003csup\u003e®\u003c/sup\u003e FIT, IUPred long, and IUPred short are shown by black, red, green, pink, blue, and yellow lines, respectively. The output of the ANCHOR-2 algorithm is shown by dark yellow circles connected by dark yellow lines. Mean disorder profile (or mean disorder prediction, MDP) calculated as an average of outputs of these six predictors is shown by dashed dark pink line, whereas error distribution is shown as light pink shadow. In this per-residue disorder analysis, a disorder score was assigned to each residue. A residue with disorder scores equal to or above 0.5 is considered as disordered and a residue with disorder score below 0.5 is predicted as ordered. Residues/regions with disorder scores between 0.15 and 0.5 were considered as ordered but flexible. The corresponding thresholds are shown by solid (0.5) and long-dashed lines (0.15). Positions of the predicted disorder-based binding sites (MoRF regions) are defined as regions with the scores exceeding the 0.5 threshold. \u003cstrong\u003eB\u003c/strong\u003e. Evaluation of LLPS potential of human MICAL1 using FuzDrop in a form of the residue-based droplet-promoting probability (p\u003csub\u003eDP\u003c/sub\u003e) distribution. Residues with p\u003csub\u003eDP\u003c/sub\u003e ≥ 0.60 is capable to promote LLPS. Droplet-promoting regions (DPRs) are defined by at least five consecutive residues with p\u003csub\u003eDP\u003c/sub\u003e ≥ 0.60. Aggregation hot-spots are DPRs (p\u003csub\u003eDP\u003c/sub\u003e ≥ 0.60) that exhibit high interaction mode divergence (S\u003csub\u003eBIND\u003c/sub\u003e ≥ 2.2). Positions of DPRs and aggregation hot spots are shown by blue and orange bars, respectively. \u003cstrong\u003eC\u003c/strong\u003e. Sequence distribution of cellular context dependent interactability. Regions with context-dependent interactions change protein interaction behavior and binding modes under different cellular conditions. These residues can be ordered or disordered with S\u003csub\u003eBIND\u003c/sub\u003e ≥ 2.25. \u003cstrong\u003eD\u003c/strong\u003e. Functional disorder profile generated by D\u003csup\u003e2\u003c/sup\u003eP\u003csup\u003e2\u003c/sup\u003e shows the outputs of several disorder predictors such as PONDR\u003csup\u003e®\u003c/sup\u003e VLXT, PONDR\u003csup\u003e®\u003c/sup\u003e VSL2b, PrDOS, IU-Pred and Espritz. Consensus disorder predictions are shown by blue-green-white bar, where blue indicates regions are where the disorder predictions intersect the SCOP domain prediction and green indicates regions represent disorder that is not found within a predicted SCOP domain. The colored bar highlighted by blue and green shade represents the consensus disorder prediction. Above this consensus bar, line with numbered, colored bars show the predicted locations of SCOP (Structural Classifications of Proteins) domains. Yellow zigzagged bars show positions of MoRFs, whereas colored circles in the bottom of the plot show the positions of predicted PTMs. \u003cstrong\u003eE\u003c/strong\u003e. 3D structure of MICAL1 modeled by AlphaFold. Structure is colored based on the AlphaFold-generated per-residue confidence score (p\u003csub\u003eLDDT\u003c/sub\u003e) that ranges between 0 and 100, where orange, yellow, cyan, and blue colors correspond to the segments predicted by AlphaFold with very high very low (p\u003csub\u003eLDDT\u003c/sub\u003e \u0026lt; 50), low (70 \u0026gt; p\u003csub\u003eLDDT\u003c/sub\u003e \u0026gt; 50), high (90 \u0026gt; p\u003csub\u003eLDDT\u003c/sub\u003e \u0026gt; 70), and (p\u003csub\u003eLDDT\u003c/sub\u003e \u0026gt; 90) confidence. \u003cstrong\u003eF\u003c/strong\u003e. Protein-protein interaction (PPI) network generated by STRING using seven types of evidence shown by differently colored lines: a black line represents co-expression evidence; a blue line – co-occurrence evidence; a green line - neighborhood evidence; a light blue line – database evidence; a purple line – experimental evidence; a red line – the presence of fusion evidence; and a yellow line – text mining evidence\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-7782044/v1/aea198dbc57c59e93458eea6.png"},{"id":94005996,"identity":"12aa07f4-f84a-4af7-a9ae-8d687b91b96f","added_by":"auto","created_at":"2025-10-21 09:14:26","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":957880,"visible":true,"origin":"","legend":"\u003cp\u003eFunctional disorder analysis of human MICAL2 (UniProt ID: O94851). A. RIDAO-generated per-residue intrinsic disorder propensity profile. B. Evaluation of LLPS potential of human MICAL2 using FuzDrop. C. Sequence distribution of cellular context dependent interactability. D. Functional disorder profile generated by D\u003csup\u003e2\u003c/sup\u003eP\u003csup\u003e2\u003c/sup\u003e for the alternatively spliced isoform 1 of human MICAL2. E. 3D structure of MICAL2 modeled by AlphaFold. F. MICAL2-centered PPI network generated by STRING.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-7782044/v1/99687896954f232a9249ec9d.png"},{"id":94005995,"identity":"5f6f266a-4c97-4c60-b021-a9b950e7c569","added_by":"auto","created_at":"2025-10-21 09:14:26","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1312126,"visible":true,"origin":"","legend":"\u003cp\u003eFunctional disorder Functional disorder analysis of human MICAL3 (UniProt ID: Q7RTP6). A. RIDAO-generated per-residue intrinsic disorder propensity profile. B. Evaluation of LLPS potential of human MICAL3 using FuzDrop. C. Sequence distribution of the MICAL3 cellular context dependent interactability. D. Functional disorder profile generated by D\u003csup\u003e2\u003c/sup\u003eP\u003csup\u003e2\u003c/sup\u003e. E. 3D structure of MICAL3 modeled by AlphaFold. F. MICAL3-centered PPI network generated by STRING.\u0026nbsp;\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-7782044/v1/9477937c54f1abea708993ab.png"},{"id":94006380,"identity":"7ea305c7-87a2-4fed-a56b-e501c3572de5","added_by":"auto","created_at":"2025-10-21 09:22:26","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1443805,"visible":true,"origin":"","legend":"\u003cp\u003eFunctional disorder analysis of human MICAL-L1 (UniProt ID: Q8N3F8). A. RIDAO-generated per-residue intrinsic disorder propensity profile. B. Evaluation of LLPS potential of human MICAL-L1 using FuzDrop. C. Sequence distribution of the MICAL-L1 cellular context dependent interactability. D. Functional disorder profile generated by D\u003csup\u003e2\u003c/sup\u003eP\u003csup\u003e2\u003c/sup\u003e platform. E. 3D structure of MICAL-L1 modeled by AlphaFold. F. STRING-generated PPI network centered at MICAL-L1.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-7782044/v1/8379108c3302b51ff9a963f7.png"},{"id":94006002,"identity":"645aa758-aa64-4338-90a0-4f26672eaf56","added_by":"auto","created_at":"2025-10-21 09:14:26","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":988130,"visible":true,"origin":"","legend":"\u003cp\u003eFunctional disorder analysis of human MICAL-L2 (UniProt ID: Q8IY33). \u003cstrong\u003eA\u003c/strong\u003e. RIDAO-generated per-residue intrinsic disorder propensity profile. \u003cstrong\u003eB\u003c/strong\u003e. Evaluation of LLPS potential of human MICAL-L2 using FuzDrop. \u003cstrong\u003eC\u003c/strong\u003e. Sequence distribution of the MICAL-L2 cellular context dependent interactability. \u003cstrong\u003eD\u003c/strong\u003e. D\u003csup\u003e2\u003c/sup\u003eP\u003csup\u003e2\u003c/sup\u003e-generated functional disorder profile. \u003cstrong\u003eE\u003c/strong\u003e. 3D structure of MICAL-L2 modeled by AlphaFold. \u003cstrong\u003eF\u003c/strong\u003e. STRING-generated PPI network centered at MICAL-L2.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-7782044/v1/4855f82808775f451ae57fc7.png"},{"id":94006381,"identity":"648d25b7-f0e7-459b-89f1-ee4bf60345f1","added_by":"auto","created_at":"2025-10-21 09:22:26","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":716998,"visible":true,"origin":"","legend":"\u003cp\u003eSTRING-based analysis of the global interactivity of human MICAL proteins. \u003cstrong\u003eA\u003c/strong\u003e. STRING-generated PPI network centered at five human MICALs. Network is colored according to three clusters identified by STRING-embedded k-means clustering protocol. Functional enrichment of the joint MICAL nework: \u003cstrong\u003eB\u003c/strong\u003e. 10 most enriched Biological Process (BP) Gene Ontology (GO) terms (of the 121 significantly enriched BP terms). \u003cstrong\u003eC\u003c/strong\u003e. 10 most enriched Molecular Function (MF) GO terms (of 47 significantly enriched MF terms). \u003cstrong\u003eD\u003c/strong\u003e. 10 most enriched Cellular Component (CC) GO terms (of 89 significantly enriched CC terms). E. Enrichment analysis in the REACTOME pathways (of 78 significantly enriched pathways).\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-7782044/v1/39cb4702496b925f09159609.png"},{"id":94006000,"identity":"7b47f322-4e36-43e2-b658-bf194fd338ad","added_by":"auto","created_at":"2025-10-21 09:14:26","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1131078,"visible":true,"origin":"","legend":"\u003cp\u003e“Reduced” STRING-based analysis of the global interactivity of human MICAL proteins. \u003cstrong\u003eA\u003c/strong\u003e. STRING-generated PPI network centered at five human MICALs using the threshold of 0.400 (medium confidence) for minimum required interaction score and “text mining” as active interaction source. Network is colored according to three clusters identified by STRING-embedded k-means clustering protocol (see the text). Functional enrichment of the joint MICAL nework: B. 10 most enriched Biological Process (BP) Gene Ontology (GO) terms (of the 128 significantly enriched BP terms). C. 10 most enriched Molecular Function (MF) GO terms (of 29 significantly enriched MF terms). D. 10 most enriched Cellular Component (CC) GO terms (of 62 significantly enriched CC terms). E. Enrichment analysis in the REACTOME pathways (of 30 significantly enriched pathways).\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-7782044/v1/32c26c5a4fa8c2d14b89d394.png"},{"id":94006010,"identity":"f303aa0b-04cd-4349-b0a8-8a0d28c25110","added_by":"auto","created_at":"2025-10-21 09:14:26","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1516069,"visible":true,"origin":"","legend":"\u003cp\u003eGlobal disorder analysis of the intrinsic disorder predispositions of MICAL interactors (green boxes) and members of human proteome (gray) human proteins by seven per-residue disorder predictors (PONDR\u003csup\u003e®\u003c/sup\u003e VLXT, PONDR\u003csup\u003e®\u003c/sup\u003e VSL2, PONDR\u003csup\u003e®\u003c/sup\u003e VL3, IUPRed_Short, IUPRed_Long, PONDR\u003csup\u003e®\u003c/sup\u003e FIT, and MDP). A. Distributions of proteins based on their percentage of predicted intrinsically disordered residues (PPIDR) values. Horizontal lines in these plots show 10% (dotted lines) and 30% PPIDR thresholds. B. Distributions of proteins based on their average disorder score (ADS) values, which are calculated for each query protein as a protein length-normalized sum of all the per-residue disorder scores. In these plots, the horizontal lines represent 0.15 (dotted lines) and 0.5 ADS thresholds, respectively.\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-7782044/v1/76539a6a94510366b3529b95.png"},{"id":94006382,"identity":"a7858a35-c53d-4671-ac08-145284b90668","added_by":"auto","created_at":"2025-10-21 09:22:26","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":441268,"visible":true,"origin":"","legend":"\u003cp\u003eGlobal disorder analysis of human MICAL-interacting proteins. \u003cstrong\u003eA\u003c/strong\u003e. The PONDR\u003csup\u003e®\u003c/sup\u003e VSL2 score (ADS) \u003cem\u003evs\u003c/em\u003e. PONDR\u003csup\u003e®\u003c/sup\u003e VSL2 (%) (PPIDR) plot. Here, each point corresponds to a query protein, coordinates of which are evaluated from the corresponding PONDR\u003csup\u003e®\u003c/sup\u003e VSL2 data as its ADS and PPIDR values. Color blocks are used to visualize proteins based on the accepted classification, with red, pink/light pink, and blue/light blue regions containing highly disordered, moderately disordered, and ordered proteins, respectively (see the text). Dark blue or pink areas correspond to the regions where PPIDR agrees with ADS, whereas areas in which only one of these criteria applies are shown by light blue or light pink. Data for the MICAL interactome is shown by gray circles, whereas the positions of individual MICALs are shown by differently colored symbols. \u003cstrong\u003eB\u003c/strong\u003e. Charge-Hydropathy and Cumulative Distribution Function (CH-CDF) analysis of query proteins. The ΔCH-ΔCDF plot is a two-dimensional representation that integrates the CH plot, which correlates a protein's net charge with its hydrophobicity, and the CDF, which correlates the cumulative frequency of disorder scores with the disorder scores. In the resulting ΔCH-ΔCDF plot, the Y-axis (ΔCH) represents the protein's distance from the CH boundary, while the X-axis (ΔCDF) represents the deviation of a protein's disorder frequency from the CDF boundary. Proteins are then stratified into four quadrants: Quadrant 1 (bottom right) indicates proteins likely to be structured; Quadrant 2 (bottom left) includes proteins that may be in a molten globule state or lack a unique 3D structure; Quadrant 3 (top left) consists of proteins predicted to be highly disordered; Quadrant 4 (top right) captures proteins that present a mixed prediction of being disordered according to CH but ordered according to CDF. \u003cstrong\u003eC\u003c/strong\u003e. Analysis of the interactability of the members of MICAL interactome in a form of the dependence of the node degree on disorder content evaluated as PONDR\u003csup\u003e®\u003c/sup\u003e VSL2-based PPIDR. Vertical lines show 10% and 30% PPIDR thresholds, whereas the horizontal line corresponds to the average node degree of the analyzed joint MICAL interactome (7). \u003cstrong\u003eD\u003c/strong\u003e. Analysis of the LLPS predisposition of the human MICAL interactors with their intrinsic disorder propensity. Vertical lines show 10% and 30% PPIDR thresholds, whereas the horizontal line corresponds to the 0.6 p\u003csub\u003eLLPS\u003c/sub\u003e threshold. In plots C and D, circles are colored based on the relation of the corresponding proteins to LLPS, with droplet drivers and droplet clients shown by red and pink circles, and with proteins not related to LLPS (showing p\u003csub\u003eLLPS\u003c/sub\u003e below 0.6 and not containing DPRs) are shown by blue circles.\u003c/p\u003e","description":"","filename":"image9.png","url":"https://assets-eu.researchsquare.com/files/rs-7782044/v1/a63f70c5c2b84d15322c70f8.png"},{"id":94006006,"identity":"b85a6991-331e-45e7-8785-b44368b6c783","added_by":"auto","created_at":"2025-10-21 09:14:26","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":973293,"visible":true,"origin":"","legend":"\u003cp\u003eFunctional Disorder analysis of human ERC1 (UniProt ID: Q8IUD2). A. Functional disorder profile generated by D\u003csup\u003e2\u003c/sup\u003eP\u003csup\u003e2\u003c/sup\u003e.\u0026nbsp; B.\u0026nbsp; AlphaFold predicted 3D structure of the colored protein based on the p\u003csub\u003eLDDT\u003c/sub\u003e values. C. STRING-based analysis of the ERC1 interactivity. The interactive version of this network and can be found at the following permalink: \u003ca href=\"https://version-12-0.string-db.org/cgi/network?networkId=bxenJL1ppLUh\"\u003ehttps://version-12-0.string-db.org/cgi/network?networkId=bxenJL1ppLUh\u003c/a\u003e.\u003c/p\u003e","description":"","filename":"image10.png","url":"https://assets-eu.researchsquare.com/files/rs-7782044/v1/fd624e92001bc0a1c24d0ac4.png"},{"id":94006004,"identity":"f60355eb-2ffc-4f24-8e25-b1698c16485b","added_by":"auto","created_at":"2025-10-21 09:14:26","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":1270174,"visible":true,"origin":"","legend":"\u003cp\u003eFunctional Disorder analysis of human MRTFA (UniProt ID: Q969V6). \u003cstrong\u003eA\u003c/strong\u003e. Functional disorder profile generated by D\u003csup\u003e2\u003c/sup\u003eP\u003csup\u003e2\u003c/sup\u003e.\u0026nbsp; \u003cstrong\u003eB\u003c/strong\u003e.\u0026nbsp; AlphaFold predicted 3D structure of the colored protein based on the p\u003csub\u003eLDDT\u003c/sub\u003e values. \u003cstrong\u003eC\u003c/strong\u003e. STRING-based analysis of the MRTFA interactivity. The interactive version of this network and can be found at the following permalink: \u003ca href=\"https://version-12-0.string-db.org/cgi/network?networkId=bsOLdAARTyO5\"\u003ehttps://version-12-0.string-db.org/cgi/network?networkId=bsOLdAARTyO5\u003c/a\u003e.\u003c/p\u003e","description":"","filename":"image11.png","url":"https://assets-eu.researchsquare.com/files/rs-7782044/v1/101bc3ea2c69d384859131b3.png"},{"id":94006384,"identity":"06bea488-c8eb-40f7-aa73-cafbdc0c1b27","added_by":"auto","created_at":"2025-10-21 09:22:26","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":1023062,"visible":true,"origin":"","legend":"\u003cp\u003eFunctional Disorder analysis of human NINL (UniProt ID: Q9Y2I6). A. Functional disorder profile generated by D\u003csup\u003e2\u003c/sup\u003eP\u003csup\u003e2\u003c/sup\u003e.\u0026nbsp; B.\u0026nbsp; AlphaFold predicted 3D structure of the protein colored based on the p\u003csub\u003eLDDT\u003c/sub\u003e values. C. STRING-based analysis of the NINL interactivity. The interactive version of this network and can be found at the following permalink: \u003ca href=\"https://version-12-0.string-db.org/cgi/network?networkId=byzsG3QX4CvT\"\u003ehttps://version-12-0.string-db.org/cgi/network?networkId=byzsG3QX4CvT\u003c/a\u003e.\u003c/p\u003e","description":"","filename":"image12.png","url":"https://assets-eu.researchsquare.com/files/rs-7782044/v1/47847af1e5b4f68671254742.png"},{"id":94007414,"identity":"73eaad92-9662-4c6b-b4be-c1f8dba025c4","added_by":"auto","created_at":"2025-10-21 09:30:26","extension":"png","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":942322,"visible":true,"origin":"","legend":"\u003cp\u003eFunctional Disorder analysis of human RUSC2 (UniProt ID: Q8N2Y8). \u003cstrong\u003eA\u003c/strong\u003e. Functional disorder profile generated by D\u003csup\u003e2\u003c/sup\u003eP\u003csup\u003e2\u003c/sup\u003e.\u0026nbsp; \u003cstrong\u003eB\u003c/strong\u003e.\u0026nbsp; AlphaFold predicted 3D structure of the protein colored based on the p\u003csub\u003eLDDT\u003c/sub\u003e values. \u003cstrong\u003eC\u003c/strong\u003e. STRING-based analysis of the RUSC2 interactivity. The interactive version of this network and can be found at the following permalink: \u003ca href=\"https://version-12-0.string-db.org/cgi/network?networkId=baPbv1Z2HJkg\"\u003ehttps://version-12-0.string-db.org/cgi/network?networkId=baPbv1Z2HJkg\u003c/a\u003e.\u003c/p\u003e","description":"","filename":"image13.png","url":"https://assets-eu.researchsquare.com/files/rs-7782044/v1/272096de64026305bf51dcda.png"},{"id":94006013,"identity":"d1cf5a89-0b1f-45bd-98d3-d00af3bfb3ae","added_by":"auto","created_at":"2025-10-21 09:14:26","extension":"png","order_by":14,"title":"Figure 14","display":"","copyAsset":false,"role":"figure","size":920330,"visible":true,"origin":"","legend":"\u003cp\u003eFunctional Disorder analysis of human EHBP1L1 (UniProt ID: Q8N3D4). \u003cstrong\u003eA\u003c/strong\u003e. Functional disorder profile generated by D\u003csup\u003e2\u003c/sup\u003eP\u003csup\u003e2\u003c/sup\u003e. \u003cstrong\u003eB\u003c/strong\u003e. AlphaFold predicted 3D structure of the protein colored based on the p\u003csub\u003eLDDT\u003c/sub\u003e values. \u003cstrong\u003eC\u003c/strong\u003e. STRING-based analysis of the EHBP1L1 interactivity. The interactive version of this network and can be found at the following permalink: \u003ca href=\"https://version-12-0.string-db.org/cgi/network?networkId=benAs3m86coZ\"\u003ehttps://version-12-0.string-db.org/cgi/network?networkId=benAs3m86coZ\u003c/a\u003e.\u003c/p\u003e","description":"","filename":"image14.png","url":"https://assets-eu.researchsquare.com/files/rs-7782044/v1/19635d0adb7302a7dde5d03f.png"},{"id":94006388,"identity":"64d802e5-c023-4c56-96c5-73b33953cd54","added_by":"auto","created_at":"2025-10-21 09:22:26","extension":"png","order_by":15,"title":"Figure 15","display":"","copyAsset":false,"role":"figure","size":1152490,"visible":true,"origin":"","legend":"\u003cp\u003eFunctional Disorder analysis of human NEDD9 (UniProt ID: Q14511). \u003cstrong\u003eA\u003c/strong\u003e. Functional disorder profile generated by D\u003csup\u003e2\u003c/sup\u003eP\u003csup\u003e2\u003c/sup\u003e. \u003cstrong\u003eB\u003c/strong\u003e. AlphaFold predicted 3D structure of the protein colored based on the p\u003csub\u003eLDDT\u003c/sub\u003e values. \u003cstrong\u003eC\u003c/strong\u003e. STRING-based analysis of the NEDD9 interactivity. The interactive version of this network and can be found at the following permalink: \u003ca href=\"https://version-12-0.string-db.org/cgi/network?networkId=bAXJYRFk9mjS\"\u003ehttps://version-12-0.string-db.org/cgi/network?networkId=bAXJYRFk9mjS\u003c/a\u003e.\u003c/p\u003e","description":"","filename":"image15.png","url":"https://assets-eu.researchsquare.com/files/rs-7782044/v1/eaeada32aaf4cca3e5516300.png"},{"id":99172482,"identity":"8359a8be-78ff-41ae-a447-26df48f24af0","added_by":"auto","created_at":"2025-12-29 16:10:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":18010987,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7782044/v1/8b03f74b-67b8-4919-9d45-047b831eb729.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Prevalence and functional roles of intrinsic disorder in the [F-actin]-monooxygenase MICAL family members and their interactors","fulltext":[{"header":"1. Introduction","content":"\u003cdiv id=\"Sec2\" class=\"Section2\"\u003e\u003ch2\u003e1.1. Brief introduction of MICALs\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eMICAL (molecule interacting with CasL (CRK-associated substrate-related protein)) proteins represent a family of actin regulatory enzymes (methionine monooxygenases) involved in the cytoskeleton rearrangements by promoting depolymerization of F-actin [1]. Here, MICALs mediate oxidation of specific methionine residues on F-actin leading to the formation of methionine-sulfoxide, thereby resulting in actin filament disassembly and preventing repolymerization [1]. In the absence of actin, MICALs can act as NADPH oxidases producing H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e [1\u0026ndash;3]. In vertebrates, there are three \u003cem\u003eMICAL\u003c/em\u003e genes (\u003cem\u003eMICAL-1\u003c/em\u003e, \u003cem\u003eMICAL-2\u003c/em\u003e, and \u003cem\u003eMICAL-3\u003c/em\u003e), whereas Drosophila has one \u003cem\u003eMical\u003c/em\u003e gene [1]. The proteins of this family have a characteristic N-terminal redox-domain followed by other important functional regions [4]. In addition to the MICAL proteins, vertebrates also have two MICAL-like proteins, MICAL-L1 (also known as molecule interacting with Rab13, MIRab13) and MICAL-L2 (also known as junctional Rab13-binding protein, JRAB) with the domain organization similar to that of MICALs but lacking the redox domain [4].\u003c/p\u003e\u003cp\u003eMICALs can oxidize their substrate using two approaches, one is the direct approach, in which MICALs enzyme directly interact with the substrate like F-actin to oxidize them, in the indirect mechanism, MICALs produce ROS to do the job. Whether MICALs utilize both mechanisms or only use one at a time is not fully understood [5]. Clearly, the direct mechanism represents a controllable way of modification of specific methionines in target proteins, whereas the ROS generated via the indirect mechanism would hit all accessible sites sensitive to oxidative modification.\u003c/p\u003e\u003cp\u003eThe major substrate of MICALs is actin, an integral part of cytoskeleton. However, being capable of the structural remodeling of actin, these enzymes are involved in multiple key biological functions, particularly playing a number of significant roles in controlling the shape of the cells, as well as cellular movement, cytokinesis, and navigation [5]. Because of the importance of actin in multiple cellular processes, the significance of actin binding proteins involved in the regulation of its action cannot be overlooked [4]. Among such actin binding proteins are the members of the MICAL family, which are involved in mediating the redox reaction of actin protein [4] and which represent the focus of our article.\u003c/p\u003e\u003cp\u003eBeing more than 1000 amino acids long, MICALs are characterized by complex domain organization, where along with the conserved N-terminal domain known as hydroxylase or flavoprotein monooxygenase domain, there are several other notable regions, such as proline rich region, a calponin homology (CH) domain, Lin11, Isl-1, Mec-3 (LIM) zinc-biding domain that does not bind DNA but act as interface for protein-protein interaction, bMERB (bivalent Mical/EHBP Rab binding) domain, coiled-coil regions, and other functional motifs, through which these regulatory proteins interact with other proteins to carry out their various roles in cells. In humans, each MICAL family protein is directly involved in destabilization of F-actin, being capable of selective association with actin filaments and reshaping cells [1]. MICALs play very important role in numerous cellular functions, such as T cell maturation, intracellular vesicular trafficking, and axon growth [3]. However, the most notable function of the MICAL proteins is their involvement in a very important regulatory mechanism, oxidation of F-actin during cytokinesis [6]. One of the specific aspects of the flavoprotein monooxygenase domain mechanism that is unique to the MICALs is that these proteins interact physically with the substrate [6]. In the scenario related to F-actin oxidation, destabilization, and disassembly, crucial for the dynamics and remodeling of the cellular cytoskeleton, MICALs have regions capable of direct interaction with F-actin. This interaction places the catalytic domain of MICALs in the proximity of F-actin to oxidize it primarily at two methionine residues Met44 and Met47 within the actin molecule [7]. The N-terminal redox domain binds to the co-factor flavin adenine dinucleotide (FAD) and transfers the electrons from NADPH to oxygen to generate ROS (reactive oxygen species), H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e is a byproduct of this reaction [6,8]. The reaction renders the actin molecules interactions weak and leads to the disassembly of F-actin molecules, which is very important for cytoskeleton remodeling allowing cells to modify their shape and assist in directional growth. Generations of ROS further facilitate the oxidative modifications in the local environment of the cell [6].\u003c/p\u003e\u003cp\u003eBy modifying actin assembly and disassembly MICAL proteins are involved in several biological processes and are closely linked to cellular signaling [6,9]. MICALs are involved in controlling morphology of neuronal dendrites and are essential for the proper development and regeneration of neurons. This is because actin dynamics are very important for several neuronal functions, such as synapse formation, and axonal repulsion, which is a directional growth of the neurons away from some areas and towards other areas to develop new connections [6]. MICALs also play crucial roles in protein trafficking into axons, which is essential for the proper growth and functions of axons, as well as in docking of the vesicles that are fused at the plasma membrane, all of these are vital for the normal signal transmission in nervous systems [6].\u003c/p\u003e\u003cp\u003eMICALs also have important roles in the pathogenesis of various maladies, e.g. cancer progression. For example, MICAL1 was knocked out in MDA MB 231 human breast cancer cell line to identify the implication of MICAL1 on cancer progression [10]. The changes were observed in cell size and motility, implying that MICAL1 does play an important role in the normal progression of malignant cells [10]. The knockout cells in the mouse models show reduced tumor growth, and analysis of human cancer data highlights the upregulation of MICAL1 expression in the invasive breast cancer cells compared to the normal cells [10]. All of these are pointing out in the direction of MICAL1 influence in the cancer progression [10].\u003c/p\u003e\u003cp\u003eBecause of their high interactability, misbehavior of MICALs is related to the progression of neurodegenerative diseases [4]. For example, MICAL has been identified as an important interactor of human tau protein, which is one of the major proteins involved in the pathogenesis of Alzheimer disease (AD) in the brain of drosophila AD model. Furthermore, MICAL was shown to increase the propensity of tau aggregation in tauopathies [11]. Normally, Tau protein stabilizes the microtubules but the effect of the MICAL can change this function, as the interaction of MICAL increases the toxicity and aggregation of Tau proteins [11]. MICAL did this by using its redox domain to oxidize tau at specific cysteine residues, thereby modifying the normal functioning of tau protein [11]. Furthermore, this was validated in human tauopathy samples, as the human MICAL protein was upregulated in the brains of the patients with taupathies [11].\u003c/p\u003e\u003cp\u003eMICALs are important in muscle movement and morphology [12]. If MICAL is mutated, the synaptic boutons fails to spread normally across the surface of the muscles and tend to clusters at nerve entry points, suggesting that MICAL play very important role in neuromuscular junction structure [12]. Mutation in MICAL affect the normal distribution of actin and myosin filaments beneath the cell membrane, which are normally distributed in the sarcomeric manner, but the MICAL mutant might cause a notable disorganization of muscle filaments [12].\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e1.2. Intrinsic disorder in proteins\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eSince forever the classic \u0026ldquo;lock-and-key\u0026rdquo; concept coined in 1893 by Hermann Emil Louis Fischer (1852\u0026ndash;1919) [13,14] converted to the \u0026ldquo;one gene \u0026ndash; one enzyme \u0026ndash; one biochemical reaction\u0026rdquo; notion (proposed for the first time in 1941 by George Wells Beadle (1903\u0026ndash;1989) and Edward Lawrie Tatum (1909\u0026ndash;1975) [15]) and later transformed into the classic model of a protein science, where a unique amino acid sequence of a protein encodes its unique structure required for unique function (derived from the classic experiments of Christian Boehmer Anfinsen Jr. (1916\u0026ndash;1995) [16\u0026ndash;18]) was a dominant belief in the structural biology community. It took a while for the scientists to start opening to the fluidity/flexibility of protein structure related to its function. The concept of proteins not always following the conventional \u0026ldquo;structure-function\u0026rdquo; paradigm was somewhat there in the 20th century literature, being dispersed in the scientific publications, where such \u0026ldquo;non-canonical\u0026rdquo; proteins were given different names, such as chameleon, dancing, flexible, floppy, malleable, mobile, partially folded, pliable, rheomorphic, vulnerable, or 4D proteins, protein clouds or proteins waiting for partners, as well as different combinations of \u0026ldquo;inherently-intrinsically-natively-naturally\u0026rdquo; with \u0026ldquo;denatured-disordered-unfolded-unstructured\u0026rdquo; (for the historic overview, see [19,20]).\u003c/p\u003e\u003cp\u003eDespite all these observations, it took long time to debunk the one structure \u0026ndash; one function model. One reason for this challenge was the unshaken belief of protein community in the validity of the lock-and-key model supported by the development of specific and more and more advanced experimental approaches for finding and characterizing structure in proteins. However, the accumulated factual scientific discoveries eventually helped the acceptance of the protein intrinsic disorder concept, and advancement of experimental techniques provided a way for researchers to look deeper into the behavior of these uniquely interesting proteins. Intrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs) are unstructured under physiological conditions. Because they typically have a high number of charged residues and low content of hydrophobic residues, many of them have poorly defined hydrophobic core (if any) and typically exist in somewhat extended form. Since they lack defined 3D shapes, IDPs/IDRs are very adaptable towards their environment [21\u0026ndash;24].\u003c/p\u003e\u003cp\u003eLooking at the intrinsic disorder in members of MICAL family is expected to provide some important insights. Since MICALs are involved in the actin dynamic, having intrinsic disorder regions in these enzymes may allow them to switch between their active and inactive state more effectively, and give them more structural flexibility, which might be crucial for the MICALs to interact with actin and multiple binding partners. In the context of binding promiscuity, looking at the IDRs of MICALs can enhance understanding of the roles of these proteins in various signaling pathways, as IDRs allow interactions with multiple different partners. This might help MICAL proteins to interact with actin and different other proteins. IDRs are targets for various post-translational modifications (PTMs) [25]. Modifications in these regions like acylation, palmitoylation, and phosphorylation can alter their activity, stability, and localization, which might be responsible for regulation of the enzymatic activity of MICAL proteins [26]. For example, phosphorylation was shown to affect interaction of these proteins with their partners, such as RAB GTPases and their enzymatic activity. This is illustrated by the facts that phosphorylation of MICAL1 by PAK1 regulates MICAL binding to GTPase [27], whereas ARG-induced phosphorylation of MICAL2 promotes MICAL2-mediated F-actin disassembly [28].\u003c/p\u003e\u003cp\u003eThe importance of IDPs and IDRs has been well established across various biological functions, whereas alterations in the IDPs/IDRs could potentially contribute towards the propagation of diseases, such as cancers and neurological disorders [29\u0026ndash;31]. Changes in MICAL proteins can accelerate or slow down the propagation of cancer, and since these proteins are involved in the directional growth of neurons, unwanted mutations in them are detrimental, especially those located in the IDRs, as they could possibly aggravate the damage. These IDRs can serve as potential targets for drug development, especially if these regions are in functionally important areas of protein length. We can also get evolutionary insights through investigating the conserved IDR regions in MICAL proteins across various species, highlighting their evolutionary importance and their fundamental role in the protein function.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Protein datasets\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eWe have gathered five MICAL proteins of MICAL family for the comprehensive analysis of their intrinsic disorder predispositions through comprehensive literature review. We first retrieved the FASTA sequences of these proteins using UniPROT, and then ran these sequences though platforms discussed below. The global STRING analysis of all five proteins revealed 67 interactors within PPI network. We further analyzed these interactors by obtaining their FASTA sequences and submitting these sequences in RIDAO for disorder prediction analysis. Then the top 10 most disordered proteins were selected for the in-depth analysis.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Bioinformatics tools utilized in this study\u003c/h2\u003e\u003cdiv id=\"Sec7\" class=\"Section3\"\u003e\u003ch2\u003e2.2.1. STRING\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eProtein-protein interactions research was carried out through STRING database. In the first stage of our study, we used the STRING database to conduct a global PPI network analysis of all five MICALs, identifying 174 interactors within the protein-protein interaction (PPI) network. In the second stage of Study, we used the platform to generate the PPI network for individual proteins using medium confidence and maximum number of interactor 500 in the 1st shell of the network [157].\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section3\"\u003e\u003ch2\u003e2.2.2. RIDAO\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eSix well known predictors PONDR\u003csup\u003e\u0026reg;\u003c/sup\u003e VLXT, PONDR\u003csup\u003e\u0026reg;\u003c/sup\u003e VSL2, PONDR\u003csup\u003e\u0026reg;\u003c/sup\u003e VL3, IUPred Short, IUPred long, and PONDR\u003csup\u003e\u0026reg;\u003c/sup\u003e FIT are combined in one platform, Rapid Intrinsic Disorder Analysis Online (RIDAO), which is a web-based predictor, efficiently generating per-residue prediction of intrinsic disorder predisposition in individual query proteins or in protein datasets [158]. RIDAO also provides mean disorder prediction (MDP) by averaging outputs of individual predictors. The residues with the disorder score from 0.5 to 1.0 are considered as disordered, residues with disorder score between 0.15 and 0.5 are considered flexible, whereas residues with disorder score below 0.15 are classified as ordered. Base on their percent of predicted intrinsically disordered residues (PPIDR) values, proteins are traditionally classified as ordered, moderately disordered and highly disordered if their PPIDR\u0026thinsp;\u0026lt;\u0026thinsp;10%, 10% \u0026le; PPIDR\u0026thinsp;\u0026lt;\u0026thinsp;30%, and PPIDR\u0026thinsp;\u0026ge;\u0026thinsp;30%, respectively [90,91].\u003c/p\u003e\u003cp\u003eRIDAO uses the outputs from two binary predictors, the charge-hydropathy (CH) plot [159] and the cumulative distribution function (CDF) plot [95,160], to generate a CH-CDF phase diagram [96,161]. This allowed for the classification of proteins based on their location within the CH-CDF phase space. Proteins in Quadrant I can be categorized as ordered (predicted to be ordered by both models), Quadrant III can be categorized as native coils or pre-molten globules (disordered by both methods), Quadrant II can be categorized as hybrid proteins or putative native molten globules (ordered by CH but disordered by CDF), and Quadrant IV can be categorized as proteins that are disordered according to the CH plot but ordered by the CDF plot [95].\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\u003ch2\u003e2.2.3. IUPred\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eIUPred2A [162,163] was used to predict intrinsic disorder tendencies, focusing on predictions of long disordered regions along with ANCHOR2-based prediction [164,165] of molecular recognition features (MoRFs); i.e., disorder-based binding site that represent short intrinsically disordered regions undergoing disorder to order transition upon interaction with their binding partner [166\u0026ndash;169]. These features are present in many IDPs, with some of them being even capable to fold differently at interaction with different partners [170\u0026ndash;172].\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section3\"\u003e\u003ch2\u003e2.2.4. InterPro\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eInterPro, a resource for the classification of protein sequences into families, was utilized to analyze the functional domains and motifs within the MICAL proteins and to understand how structural features correlate with protein function [173,174].\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section3\"\u003e\u003ch2\u003e2.2.5. FuzDrop\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eAn internet platform FuzDrop that predicts the probability of proteins to undergo liquid-liquid phase separation (LLPS) was used to study regions of proteins contributing to 'fuzziness\u0026rsquo;, and to identify droplet drivers, enhancing our understanding of how the disorder impacts protein interaction capabilities [175]. The ability of a protein to have fuzziness can lead to the LLPS, which in turn gives rise to the membrane-less organelles (MLOs) or biomolecular condensates (BMCs), performing crucial cellular functions in body like stress granules and p-bodies [176].\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section3\"\u003e\u003ch2\u003e2.2.6. AlphaFold\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e3D structures of query proteins were modeled using AlphaFold, which is an AI-based computational tool that predicts the 3D structure of a protein given its amino acid sequence [177]. Corresponding structural models were retrieved from the AlphaFold Protein Structure Database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://alphafold.ebi.ac.uk/\u003c/span\u003e\u003cspan address=\"https://alphafold.ebi.ac.uk/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), which provides structure coverage for over 214\u0026nbsp;million protein sequences [178]. These models were used to correlate structural predictions with other results.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section3\"\u003e\u003ch2\u003e2.2.7. D\u003csup\u003e2\u003c/sup\u003eP\u003csup\u003e2\u003c/sup\u003e\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eFocused analysis of human MICALs was accomplished through D\u003csup\u003e2\u003c/sup\u003eP\u003csup\u003e2\u003c/sup\u003e website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://d2p2.pro\u003c/span\u003e\u003cspan address=\"http://d2p2.pro\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), which seeks to present standardized and comprehensive disorder predictions for each of the currently mapped genes with molecular inscriptions [179]. To predict location of disordered regions that undergo binding-induced folding during protein-protein interactions, D\u003csup\u003e2\u003c/sup\u003eP\u003csup\u003e2\u003c/sup\u003e presently combines ANCHOR outputs [164,165] with the outputs of PONDR\u003csup\u003e\u0026reg;\u003c/sup\u003e VLXT [180], PONDR\u003csup\u003e\u0026reg;\u003c/sup\u003e VSL2B [181], IUPred (both short and long forms) [162,163], PrDOS [182], PV2, and three variants of ESpritz (NMR, DisProt, and X-ray) [183]. Given the FASTA sequence or a protein/gene name, position of consensus intrinsic disordered regions in addition to range and length of Molecular Recognition Features (MoRFs) and positions of various posttranslational modifications (PTMs) is given by the D\u003csup\u003e2\u003c/sup\u003eP\u003csup\u003e2\u003c/sup\u003e platform for each of the proteins analyzed in this study [179].\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"3. Results and Discussion","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.1. Functional disorder analysis of human MICALs\u003c/h2\u003e\u003cdiv id=\"Sec16\" class=\"Section3\"\u003e\u003ch2\u003e3.1.1. MICAL1 (UniProt ID: Q8TDZ2)\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e[F-actin]-monooxygenase MICAL1 also known as molecule interacting with CasL protein 1 (MICAL1) or neural precursor cell expressed developmentally down-regulated protein 9 (NEDD9)-interacting protein with calponin homology and LIM domains is a cytoplasmic 1067-residue-long monooxygenase that mediates the F-actin disassembly via oxidation of specific methionine residues along the length of actin filaments to methionine sulfoxides [1]. If the F-actin is absent, the MICAL1 act as a NADPH oxidase producing reactive oxygen species H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e [3]. It can also act as a Rab (Ras-like GTPase) effector to play a role in the vesicular trafficking [32].\u003c/p\u003e\u003cp\u003eHuman MICAL1 contains a signature catalytic monooxygenase (MO) domain (residues 1-489), a calponin homology (CH) domain (residues 506\u0026ndash;612) that is often involved in the actin binding, a LIM zinc-binding domain (residues 695\u0026ndash;757), and a coiled-coil (CC) domain (residues 919\u0026ndash;1027), which is also known as ERM α-like domain or Rab-binding domain (RBD) that participates in interaction with Rab GTPases via two coiled-coil regions (residues 919\u0026ndash;962 and 999\u0026ndash;1027) acting as two distinct Rab-binding sites with different affinities [6,9]. In addition to GTP-bound Rab proteins CC/RBD also binds plexins, which are the receptors for one of the largest protein families of extracellular guidance cues, semaphorins, that includes over twenty family members conserved from invertebrates to humans [33]. C-terminal half of MICAL1 includes a region important for interaction with RAB8A (residues 918\u0026ndash;1067), and a bMERB domain (residues 918\u0026ndash;1067) that mediates binding to predominantly RAB8, RAB10, RAB13, and RAB15 (in their GTP-bound forms) [34]. The four MICAL1 domains are linked via three linker regions, with the shortest linker, L1 (residues 490\u0026ndash;507), bridging the MO and CH domains, the intermediate L2 linker (residues 613\u0026ndash;694) connecting the CH and LIM domains, and the longest linker, L3 (residues 758 to 907), serving as a bridge between the LIM and the CC/RBD domain [35]. It was shown that the C-terminal coiled-coil domain of MICAL1 interacts with the N-terminal catalytic domain leading to the autoinhibition of this protein [35]. Furthermore, CC/RBD domain can also bind to the assembly of CH-LIM domains, with the L2α1 helix from the L2 linker region that play an important role in the stability of the CH-LIM-CC assembly being wedged between the CC/RBD, CH, and LIM domains [35]. Formation of the CH- L2α1-LIM-CC assembly is crucial for the MICAL activation and autoinhibition as well [35].\u003c/p\u003e\u003cp\u003eOur bioinformatics analysis revealed that human MICAL1 belongs to the category of highly disordered proteins, as it is characterized by the PONDR\u003csup\u003e\u0026reg;\u003c/sup\u003e VSL2-based content of disordered residues, PPIDR\u003csub\u003ePONDR\u003c/sub\u003e\u003csup\u003e\u0026reg;\u003c/sup\u003e\u003csub\u003eVSL2\u003c/sub\u003e = 45.83% and PONDR\u003csup\u003e\u0026reg;\u003c/sup\u003e VSL2-based average disorder score, ADS\u003csub\u003ePONDR\u003c/sub\u003e\u003csup\u003e\u0026reg;\u003c/sup\u003e\u003csub\u003eVSL2\u003c/sub\u003e = 0.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28. In line with these observations, PPIDR values generated by other predictors included into RIDAO platform were 22.31%, 24.93%, 31.3%, 33.93%, 40.67%, and 41.7%, for IUPred Short, IUPred long, PONDR\u003csup\u003e\u0026reg;\u003c/sup\u003e FIT, MDP (mead disorder prediction), PONDR\u003csup\u003e\u0026reg;\u003c/sup\u003e VL3, and PONDR\u003csup\u003e\u0026reg;\u003c/sup\u003e VLXT, respectively. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA shows that although the N-terminal half of human MICAL1 is predicted as mostly ordered, the C-terminal half of this protein contains high levels of predicted intrinsic disorder, with regions 610\u0026ndash;694, 740\u0026ndash;990, and 100\u0026ndash;1067 being predicted as highly disordered. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA also indicates that MICAL1 is predicted by ANCHOR2 to contain 4 MoRFs (residues 474\u0026ndash;489, 634\u0026ndash;682, 749\u0026ndash;872, and 879\u0026ndash;906) that cover 44.0% of its disordered segments, which corresponds to 20.3% of the entire sequence. This observation indicates that a very significant part of MICAL1 can be involved in disorder-based interactions with various partners.\u003c/p\u003e\u003cp\u003eCuriously, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB shows that human MICAL1 is characterized by high probability of spontaneous liquid-liquid phase separation (LLPS), p\u003csub\u003eLLPS\u003c/sub\u003e = 0.80, and contains 8 droplet-promoting regions (DPRs (residues 1\u0026ndash;11, 182\u0026ndash;192, 475\u0026ndash;513, 613\u0026ndash;632, 647\u0026ndash;696, 733\u0026ndash;843, 864\u0026ndash;889, and 955\u0026ndash;967), indicating that this protein is capable of spontaneous LLPS and can potentially serve as LLPS driver, and thereby can control the formation of membrane-less organelles [36]. Furthermore, one can fine several aggregation hot spots in this protein (residues 182\u0026ndash;189, 488\u0026ndash;501, 613\u0026ndash;618, 625\u0026ndash;631, 652\u0026ndash;666, 683\u0026ndash;696, 733\u0026ndash;751, 832,-838, 866\u0026ndash;872, and 955\u0026ndash;967), which are can drive aggregation within the condensates generated by LLPS [37]. In line with the ANCHOR-2 outputs predicting the presence of four disorder-based interactions sites, MoRFs, capable of disorder-to-order transition at interaction with binding partners, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC shows that MICAL1 contains multiple regions with context-dependent interactions that change protein interaction behavior and binding modes under different cellular conditions (residues 7\u0026ndash;35, 84\u0026ndash;101, 174\u0026ndash;189, 426\u0026ndash;433, 437\u0026ndash;451, 463\u0026ndash;478, 488\u0026ndash;501, 512\u0026ndash;522, 592\u0026ndash;608, 611\u0026ndash;618, 634\u0026ndash;649, 652\u0026ndash;666, 683\u0026ndash;699, 701\u0026ndash;707, 729\u0026ndash;738, 740\u0026ndash;751, 832\u0026ndash;838, 866\u0026ndash;872, 885\u0026ndash;896, 932\u0026ndash;941, 952\u0026ndash;970, 978\u0026ndash;983, 990\u0026ndash;997, 1000\u0026ndash;1005, 1019\u0026ndash;1037, 1040\u0026ndash;1046, and 1048\u0026ndash;1061) [38,39].\u003c/p\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD provides further support for the highly disordered status of human MICAL1 and shows that the C-terminal half of this protein is predicted as disordered by most of the tools included in the D\u003csup\u003e2\u003c/sup\u003eP\u003csup\u003e2\u003c/sup\u003e platform. It also shows that IDRs have functional roles, containing multiple MoRFs and numerous PTMs (mostly phosphorylation sites). In line with these observations, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE represents the AlphaFold-modeled 3D structure, which indicates that the structure within the N-terminal part of the protein is predicted with higher confidence, whereas the C-terminal region has lower per-residue confidence score (p\u003csub\u003eLDDT\u003c/sub\u003e), with several regions being characterized by low (70\u0026thinsp;\u0026gt;\u0026thinsp;p\u003csub\u003eLDDT\u003c/sub\u003e \u0026gt;50) and very low (p\u003csub\u003eLDDT\u003c/sub\u003e \u0026lt; 50) scores, indicating that this regions can be disordered in isolation.\u003c/p\u003e\u003cp\u003eHuman MICAL1 is known to exist in 4 isoforms generated by alternative splicing. The MICAL-1b contains 981 residues, being different from the canonical MICAL-1a by missing the 312\u0026ndash;397 region [40]. As a result, intrinsic disorder propensity of this isoform is slightly increased, to PPIDR\u003csub\u003ePONDR\u003c/sub\u003e\u003csup\u003e\u0026reg;\u003c/sup\u003e\u003csub\u003eVSL2\u003c/sub\u003e = 46.99% and ADS\u003csub\u003ePONDR\u003c/sub\u003e\u003csup\u003e\u0026reg;\u003c/sup\u003e\u003csub\u003eVSL2\u003c/sub\u003e = 0.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29. In the isoform 3 (332 residues), the long N-terminal region that includes the MO and CH domains and half of LIM domain (residues 1-735) is missing, whereas the 736\u0026ndash;768 region is changed from YEQHPGDGHFYCLQHLPQTD HKAEGSDRGPESP to MPRLTFAPKGWPHPPTSLHPGQVTDQTTWWLFQ [41]. With PPIDR\u003csub\u003ePONDR\u003c/sub\u003e\u003csup\u003e\u0026reg;\u003c/sup\u003e\u003csub\u003eVSL2\u003c/sub\u003e = 92.47% and ADS\u003csub\u003ePONDR\u003c/sub\u003e\u003csup\u003e\u0026reg;\u003c/sup\u003e\u003csub\u003eVSL2\u003c/sub\u003e = 0.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16, this shortest isoform is characterized by the most disordered nature among the other MICAL1 variants. Finally, in isoform 4 (1086 residues), the N-terminal methionine residue is changed to MSCLSHSSLPSCCPPQ EASM [42], and the global disorder level is slightly increased relative to the canonical isoform, to PPIDR\u003csub\u003ePONDR\u003c/sub\u003e\u003csup\u003e\u0026reg;\u003c/sup\u003e\u003csub\u003eVSL2\u003c/sub\u003e = 47.24% and ADS\u003csub\u003ePONDR\u003c/sub\u003e\u003csup\u003e\u0026reg;\u003c/sup\u003e\u003csub\u003eVSL2\u003c/sub\u003e = 0.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29. The observation that disorder status of MICAL1 is affected by alternative splicing is in line with the known fact that polypeptide segments affected by alternative splicing are most often intrinsically disordered, supporting the idea that alternative splicing represents an important means for regulation of the IDP and IDR binding functions [43]. LLPS potential of MICAL1 is also regulated by alternative splicing, as MICAL1-1a, MICAL1-1b, MICAL1-3, and MICAL1-4 are characterized by the p\u003csub\u003eLLPS\u003c/sub\u003e of 0.8000, 0.8392, 0.9639, and 0.8457, respectively. It is tempting to hypothesize that one of the potential functions of the shortest MICAL1-3 isoform, which is the most disordered MICAL1 isoform missing the MO and CH domains and half of LIM domain, is related to the control of the biogenesis of some cellular MLOs due to its very high LLPS potential. Clearly, this hypothesis requires experimental validation.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eTo evaluate the interactivity of human MICAL1, we used STRING platform, where we used the threshold of 0.400 (medium confidence) for minimum required interaction score with the maximum interactors 500 in the 1st shell. One should keep in mind that the use of STRING in functional analysis has some logical restrictions. For example, splice isoforms and/or post-translational modifications are collapsed, i.e. each node represents all the proteoforms produced from a single, protein-coding gene locus. Furthermore, although associations identified by STRING are meant to be specific and meaningful, where the assembled proteins jointly contribute to a shared function, this does not necessarily mean that they physically interact each other. In fact, the edges in the STRING-generated PPI networks indicate both functional and physical protein associations derived using three groups of evidence: known interactions derived from curated databases or experimentally determined, predicted interactions derived from gene neighborhood, gene fusions, and gene co-occurrence, as well as other evidence, such as text mining, co-expression, and protein homology. Obviously, confidence of a given protein-protein interaction (or functional association) is determined by the number of active interaction sources used for its determination. Therefore, we used here an option to build the full STRING network containing functional and physical associations.\u003c/p\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF represents the resulting PPI network centered at human MICAL1. This network is characterized by PPI enrichment value of \u0026lt;\u0026thinsp;1.0\u0026times;10\u003csup\u003e\u0026ndash;16\u003c/sup\u003e, the number of nodes of 35 and the number of edges of 136, which significantly exceeds the 39 interactions expected for a random set of proteins of the same size and degree distribution drawn from the genome. The average local coefficient of this network is 0.676, indicating that the network has densely connected clusters. In fact, k-means clustering revealed that the MICAL1-centered network contains two clusters, where the first cluster includes 20 proteins related to the Sema3A PAK dependent axon repulsion and semaphorin-plexin signaling pathway involved in neuron projection guidance, and MASA syndrome, and the second cluster contains 15 proteins which are Rab subfamily of small GTPases, and proteins involved in RAB GEFs exchange GTP for GDP on RABs and endocytic recycling. Members of this network have 81, 22, and 39 Gene Ontology (GO) terms significantly enriched in Biological Process, Molecular Function, and Cellular Component categories, respectively. The most significantly enriched biological processes (i.e., the processes with the lowest false discovery rates, FRDs, defined as p-values corrected for multiple testing within each category using the Benjamini\u0026ndash;Hochberg procedure) are actin polymerization or depolymerization (GO:0008154; p\u0026thinsp;=\u0026thinsp;9.01\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;9\u003c/sup\u003e), neuron projection development (GO:0031175; p\u0026thinsp;=\u0026thinsp;3.18\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;8\u003c/sup\u003e), plasma membrane bounded cell projection organization (GO:0120036; p\u0026thinsp;=\u0026thinsp;8.85\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;8\u003c/sup\u003e), endocytic recycling (GO:0032456; p\u0026thinsp;=\u0026thinsp;1.36\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003e), and actin filament organization (GO:0007015; p\u0026thinsp;=\u0026thinsp;1.36\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003e). Among the most enriched molecular functions associated with the members of this network are cytoskeletal protein binding (GO:0008092; p\u0026thinsp;=\u0026thinsp;2.63\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003e), actin binding (GO:0003779; p\u0026thinsp;=\u0026thinsp;5.95\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003e), GTP binding (GO:0005525; p\u0026thinsp;=\u0026thinsp;1.61\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e), GTPase activity (GO:0003924; p\u0026thinsp;=\u0026thinsp;3.26\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e), and semaphorin receptor activity (GO:0017154; p\u0026thinsp;=\u0026thinsp;1.05\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e). The most enriched cellular components are recycling endosome (GO:0055037; p\u0026thinsp;=\u0026thinsp;1.08\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003e), insulin-responsive compartment (GO:0032593; p\u0026thinsp;=\u0026thinsp;6.00\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e), semaphorin receptor complex (GO:0002116; p\u0026thinsp;=\u0026thinsp;7.61\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e), recycling endosome membrane (GO:0055038; p\u0026thinsp;=\u0026thinsp;1.05\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e), and extracellular exosome (GO:0070062; p\u0026thinsp;=\u0026thinsp;1.05\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e). The most enriched KEGG (Kyoto Encyclopedia of Genes and Genomes) [44] pathways are axon guidance (hsa04360; p\u0026thinsp;=\u0026thinsp;3.13\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003e), endocytosis (hsa04144; p\u0026thinsp;=\u0026thinsp;0.0116), and tight junction (hsa04530; p\u0026thinsp;=\u0026thinsp;0.0205). And among the 13 enriched pathways from the REACTOME pathway database [45,46] the most enriched are RAB geranylgeranylation (HSA-8873719; p\u0026thinsp;=\u0026thinsp;1.32\u0026times;10\u003csup\u003e\u0026ndash;11\u003c/sup\u003e), RAB GEFs exchange GTP for GDP on RABs (HSA-8876198; p\u0026thinsp;=\u0026thinsp;9.14\u0026times;10\u003csup\u003e\u0026ndash;11\u003c/sup\u003e), Sema3A PAK dependent axon repulsion (HSA-399954; p\u0026thinsp;=\u0026thinsp;8.26\u0026times;10\u003csup\u003e\u0026ndash;10\u003c/sup\u003e), CRMPs in Sema3A signaling (HSA-399956; p\u0026thinsp;=\u0026thinsp;8.26\u0026times;10\u003csup\u003e\u0026ndash;10\u003c/sup\u003e), and semaphorin interactions (HSA-373755 p\u0026thinsp;=\u0026thinsp;1.36\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;8\u003c/sup\u003e).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section3\"\u003e\u003ch2\u003e3.1.2. [F-actin]-monooxygenase MICAL2 (UniProt ID: O94851)\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eMICAL2 is a methionine monooxygenase that oxidizes MET 44 and MET 47 on actin filaments to facilitate the actin disassembly. It regulates the dynamics of actin protein within the nucleus, by facilitating the breakdown of nuclear actin to the monomeric form G-actin [47]. This depolymerization of nuclear actin through redox reaction decreases the amount of nuclear G-actin, which allows the myocardin-related transcription factor (MRTF-A) to accumulate in the nucleus [47]. G-actin normally interacts with MRTF-A to assist its transportation out of the nucleus [47]. Lowering the G-actin levels in the nucleus facilitates the accumulation of MRTF-A within the nucleus, which is mediated through MICAL-2. The accumulation of MRTF-A promotes the formation of transcriptionally active complex with SRF (serum transcription factor). These complexes are important for mediating gene transcription in response to signals, such as signals generated by the G-protein coupled receptors and nerve growth factor [47]. These complexes are important for several cellular functions, such as neurite formation and cellular migration. In short, MICAL2 is responsible for the redox dependent regulation of nuclear actin, which in turn is regulating the SRF/MRTF-A signaling [47].\u003c/p\u003e\u003cp\u003eMICAL-2 is a 1957-residue-long protein encoded by the \u003cem\u003eMICAL2\u003c/em\u003e gene. It contains MO domain (residues 2-494), CH domain (residues 516\u0026ndash;619), LIM-type zinc domain (residues 1000\u0026ndash;1062), ProQ/FinO domain (residues 1627\u0026ndash;1721), and bMERB domain (residues 1796\u0026ndash;1945). There are also nuclear localization signals (residues 660\u0026ndash;681) and region for interaction with MAPK1 (residues 1324\u0026ndash;1363). LIM domains consist of two zinc binding motifs; the domain is very important in proteins involved in multiple interaction. Proteins with LIM domain are involved in developmental and cellular functions [48]. Proteins containing ProQ/FinO domain are capable of RNA binding and considered as RNA chaperones [49]. The bMERB function is to mediate interaction between MICAL proteins and the RAB protein, which are GTPases that play important roles in vesicular trafficking in the cell [34]. Since MICAL2 does not have C-terminal CC/RBD domain, it cannot be autoinhibited via the mechanism described for MICAL1.\u003c/p\u003e\u003cp\u003eAs per UniProt and InterPro annotations, there are also eight IDRs in human MICAL2 (residues 660\u0026ndash;714, 886\u0026ndash;942, 1070\u0026ndash;1143, 1168\u0026ndash;1243, 1258\u0026ndash;1345, 1361\u0026ndash;1431, 1467\u0026ndash;1626, and 1675\u0026ndash;1779). In line with these observations, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA represents the RIDAO-generated disorder profile that clearly shows a very high level of intrinsic disorder in most of this protein sequence. Here, most of the long segment spanning residues 670\u0026ndash;1957 have disorder score above 0.5. In fact, based on the outputs of PONDR\u003csup\u003e\u0026reg;\u003c/sup\u003e VLXT, PONDR\u003csup\u003e\u0026reg;\u003c/sup\u003e VSL2, PONDR\u003csup\u003e\u0026reg;\u003c/sup\u003e VL3, IUPred Short, IUPred long, PONDR\u003csup\u003e\u0026reg;\u003c/sup\u003e FIT, and MDP, the corresponding PPIDR values are 50.23%, 63.67%, 57.95%, 38.99%, 51.51%, 50.74% and 52.68%. There are also multiple MoRF regions at residue position 678\u0026ndash;700, 714\u0026ndash;752, 778\u0026ndash;798, 834\u0026ndash;845, 858\u0026ndash;963, 1093\u0026ndash;1114, 1122\u0026ndash;1217, 1221\u0026ndash;1239, 1242\u0026ndash;1279, and 1680\u0026ndash;1776 respectively (see Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Also, human MICAL2 contains 7 phosphorylated serine residues at positions 515, 631, 696, 991, 1623, 1660, and 1688.\u003c/p\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB depicts results of the FuzDrop analysis of human MICAL2 and shows that this protein has a very high p\u003csub\u003eLLPS\u003c/sub\u003e value of 0.9946, indicating high LLPS potential of this protein and ability to serve as a droplet driver. Along its length, MICAL2 has a set of 10 DPRs, residues 1\u0026ndash;13, 655\u0026ndash;783, 875\u0026ndash;950, 1072\u0026ndash;1248, 1252\u0026ndash;1438, 1474\u0026ndash;1632, 1641\u0026ndash;1654, 1685\u0026ndash;1779, 1813\u0026ndash;1844, and 1872\u0026ndash;1922. Furthermore, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC shows that this protein is predicted to have a very large number of regions with cellular context dependent interactions (residues 10\u0026ndash;26, 66\u0026ndash;80, 84\u0026ndash;95, 345\u0026ndash;360, 496\u0026ndash;504, 611\u0026ndash;622, 653\u0026ndash;665, 736\u0026ndash;742, 776\u0026ndash;781, 790\u0026ndash;798, 811\u0026ndash;817, 828\u0026ndash;839, 848\u0026ndash;856, 861\u0026ndash;874, 883\u0026ndash;903, 942\u0026ndash;956, 986\u0026ndash;999, 1071\u0026ndash;1080, 1098\u0026ndash;1104, 1142\u0026ndash;1160, 1162\u0026ndash;1167, 1189\u0026ndash;1197, 1204\u0026ndash;1210, 1242\u0026ndash;1255, 1326\u0026ndash;1331, 1341\u0026ndash;1348, 1372\u0026ndash;1383, 1473\u0026ndash;1485, 1535\u0026ndash;1541, 1551\u0026ndash;1561, 1618\u0026ndash;1657, 1664\u0026ndash;1707, 1767\u0026ndash;1792, 1800\u0026ndash;1821, 1838\u0026ndash;1858, and 1917\u0026ndash;1941) indicating high binding potential of this protein.\u003c/p\u003e\u003cp\u003eAlternative splicing of \u003cem\u003eMICAL2\u003c/em\u003e mRNA is responsible for generation of 7 isoforms. The canonical form of the protein is isoform 7 consisting of 1957 residues and showing PPIDR\u003csub\u003ePONDR\u003c/sub\u003e\u003csup\u003e\u0026reg;\u003c/sup\u003e\u003csub\u003eVSL2\u003c/sub\u003e = 63.67% and ADS\u003csub\u003ePONDR\u003c/sub\u003e\u003csup\u003e\u0026reg;\u003c/sup\u003e\u003csub\u003eVSL2\u003c/sub\u003e = 0.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30. Isoform 1 (1124 residues) differs from the canonical isoform by DEPTSPKRPKSIS \u0026rarr; VHFSLPVLHPLLG change in the 1112\u0026ndash;1124 region and by missing residues 1125\u0026ndash;1957. It is characterized by PPIDR\u003csub\u003ePONDR\u003c/sub\u003e\u003csup\u003e\u0026reg;\u003c/sup\u003e\u003csub\u003eVSL2\u003c/sub\u003e = 38.52% and ADS\u003csub\u003ePONDR\u0026reg;VSL2\u003c/sub\u003e = 0.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28. With PPIDR\u003csub\u003ePONDR\u003c/sub\u003e\u003csup\u003e\u0026reg;\u003c/sup\u003e\u003csub\u003eVSL2\u003c/sub\u003e = 29.21% and ADS\u003csub\u003ePONDR\u003c/sub\u003e\u003csup\u003e\u0026reg;\u003c/sup\u003e\u003csub\u003eVSL2\u003c/sub\u003e = 0.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26, isoform 2 (784 residues) differs from the canonical form by missing regions 1-171, 740\u0026ndash;986, and 1203\u0026ndash;1957 and changes in the 1112\u0026ndash;1202 region from DEPTSPKRPKSISEPQHSDAEGDAASPLPSEWTSVRISPGEEAAGQDVLAVRVLVTSEDSSSDTESDYG GSEGSHTEPCEEKPWRPGSPHL to GISTSFFRKVLGWPLRLPRDLCNWMQGLLQAAGLHIRDNAYNYCYMYELLSLGLPLLWAFSEVLAAMYRESEGSLE SICNWVLRCFPVK LR. Residues 929\u0026ndash;949 and 1125\u0026ndash;1957 are missing in the isoform 3, which also has DEPTSPKRPKSIS \u0026rarr; VHFSLPVLHPLLG replacement in region 1112\u0026ndash;1124.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThis isoform contains 1103 residues and is characterized by PPIDR\u003csub\u003ePONDR\u003c/sub\u003e\u003csup\u003e\u0026reg;\u003c/sup\u003e\u003csub\u003eVSL2\u003c/sub\u003e = 38.53% and ADS\u003csub\u003ePONDR\u003c/sub\u003e\u003csup\u003e\u0026reg;\u003c/sup\u003e\u003csub\u003eVSL2\u003c/sub\u003e = 0.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27. In isoform 4 (979 residues), regions 740\u0026ndash;929, 950\u0026ndash;985, and 1203\u0026ndash;1957 are missing, whereas the 1112\u0026ndash;1202 region is changed similarly to change found in isoform 2. This isoform has PPIDR\u003csub\u003ePONDR\u003c/sub\u003e\u003csup\u003e\u0026reg;\u003c/sup\u003e\u003csub\u003eVSL2\u003c/sub\u003e = 29.77% and ADS\u003csub\u003ePONDR\u003c/sub\u003e\u003csup\u003e\u0026reg;\u003c/sup\u003e\u003csub\u003eVSL2\u003c/sub\u003e = 0.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26. With the length of 934 residues, PPIDR\u003csub\u003ePONDR\u003c/sub\u003e\u003csup\u003e\u0026reg;\u003c/sup\u003e\u003csub\u003eVSL2\u003c/sub\u003e = 29.23%, and ADS\u003csub\u003ePONDR\u003c/sub\u003e\u003csup\u003e\u0026reg;\u003c/sup\u003e\u003csub\u003eVSL2\u003c/sub\u003e = 0.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25, isoform 5 is different from the canonical form by missing regions 740\u0026ndash;929 and 112\u0026ndash;1957 and having changes in the 1112\u0026ndash;1124 region identical to those found in isoform 3. Finally, isoform 6 (955 residues, PPIDR\u003csub\u003ePONDR\u003c/sub\u003e\u003csup\u003e\u0026reg;\u003c/sup\u003e\u003csub\u003eVSL2\u003c/sub\u003e = 26.39%, and ADS\u003csub\u003ePONDR\u003c/sub\u003e\u003csup\u003e\u0026reg;\u003c/sup\u003e\u003csub\u003eVSL2\u003c/sub\u003e = 0.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26) has changed 1112\u0026ndash;1202 region, which is identical to those of isoforms 2 and 4 and is missing regions 740\u0026ndash;986 and 1203\u0026ndash;1957. Similar to MICAL1, alternative splicing affects the LLPS potential of human MICAL2, as based on their p\u003csub\u003eLLPS\u003c/sub\u003e values the seven isoforms can be arranged as MICAL2-4 (0.2433)\u0026thinsp;\u0026lt;\u0026thinsp;MICAL2-2 (0.2540)\u0026thinsp;\u0026lt;\u0026thinsp;MICAL2-6 (0.2352)\u0026thinsp;\u0026lt;\u0026thinsp;MICAL2-5 (0.3923)\u0026thinsp;\u0026lt;\u0026thinsp;MICAL2-1 (0.5744)\u0026thinsp;\u0026lt;\u0026thinsp;MICAL2-3 (0.6380)\u0026thinsp;\u0026lt;\u0026thinsp;MICAL2-7/canonical (0.9946). Therefore, only two alternatively spliced MICAL2 isoforms, 2 and 7, are capable of spontaneous LLPS and can act droplet drivers. Although the probabilities of spontaneous LLPS of the five remaining isoforms are below the 0.6 threshold, all of them contain DPRs and therefore can serve as droplet clients.\u003c/p\u003e\u003cp\u003eAlthough D\u003csup\u003e2\u003c/sup\u003eP\u003csup\u003e2\u003c/sup\u003e does not have information about the canonical form of human MICAL2, it contains the functional disorder profile of the alternatively spiced isoform 1, which is missing a very significant portion of the highly disordered C-terminal half of the canonical form. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD shows functional disorder profile for this truncated form. Despite missing most of the C-terminal IDR, this isoform still shows a high level of intrinsic disorder, where almost half of the protein is predicted to be intrinsically disordered. This long IDR contains multiple disorder-based ninding sites, MoRFs, and various PTM sites.\u003c/p\u003e\u003cp\u003eIn line with high levels of predicted disorder Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD represents the 3D structural model of MICAL2 and shows the presence of a substantial number of regions with low and very low per-residue confidence score values, which are mostly concentrated toward the C-terminus of the protein, whereas structure of the N-terminal region is predicted with very high confidence.\u003c/p\u003e\u003cp\u003eSTRING-based analysis revealed that human MICAL2 forms a PPI network containing 17 nodes linked by 35 edges, which is larger than the 19 expected edges (see Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Since the enrichment p-value of this network is 0.000439, it has more interactions than expected. Although the average node degree is 4.12, the network is characterized by the average local coefficient of 0.88. Analysis of functional enrichment of the members of the MICAL2-centered network revealed that among the 24 significantly enriched biological processes are vesicle docking involved in exocytosis (GO:0006904; p\u0026thinsp;=\u0026thinsp;7.29\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e), Golgi vesicle fusion to target membrane (GO:0048210; p\u0026thinsp;=\u0026thinsp;5.07\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e), regulation of exocytosis (GO:0017157; p\u0026thinsp;=\u0026thinsp;5.07\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e), protein secretion (GO:0009306; p\u0026thinsp;=\u0026thinsp;0.00012), and actin cytoskeleton organization (GO:0030036; p\u0026thinsp;=\u0026thinsp;0.00029). The most enriched molecular functions are GTPase activity (GO:0003924; p\u0026thinsp;=\u0026thinsp;0.00097), GTP binding (GO:0005525; p\u0026thinsp;=\u0026thinsp;0.0014), GDP binding (GO:0019003; p\u0026thinsp;=\u0026thinsp;0.0156), nucleotide binding (GO:0000166; p\u0026thinsp;=\u0026thinsp;0.0156), and NAD(P)H oxidase H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-forming activity (GO:0016174; p\u0026thinsp;=\u0026thinsp;0.0157). The members of this network are most abundantly found in insulin-responsive compartment (GO:0032593; p\u0026thinsp;=\u0026thinsp;0.00024), recycling endosome (GO:0055037; p\u0026thinsp;=\u0026thinsp;0.00053), recycling endosome membrane (GO:0055038; p\u0026thinsp;=\u0026thinsp;0.00058), synaptic vesicle (GO:0008021; p\u0026thinsp;=\u0026thinsp;0.00058), and endosome (GO:0005768; p\u0026thinsp;=\u0026thinsp;0.00058). Finally, the most enriched pathways from the REACTOME database are RAB geranylgeranylation (HSA-8873719; p\u0026thinsp;=\u0026thinsp;6.67\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e), RAB GEFs exchange GTP for GDP on RABs (HSA-8876198; p\u0026thinsp;=\u0026thinsp;0.0012), translocation of SLC2A4 (GLUT4) to the plasma membrane (HSA-1445148; p\u0026thinsp;=\u0026thinsp;0.0190), Spry regulation of FGF signaling (HSA-1295596; p\u0026thinsp;=\u0026thinsp;0.0403), and post-translational protein modification (HSA-597592; p\u0026thinsp;=\u0026thinsp;0.0403).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section3\"\u003e\u003ch2\u003e3.1.3. [F-actin]-monooxygenase MICAL3 (UniProt ID: Q7RTP6)\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eSimilar to other members of the MICAL family of proteins, MICAL3 is a flavoprotein monooxygenase involved in the redox-mediated F-actin disassembly, and, in the absence of actin, it also produces H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, functioning as a NADPH oxidase. MICAL3 operates as RAB effector protein and is crucial for the regulation of complexes involved in vesicular trafficking. This protein is a central player in the pathways regulating the transport of vehicles from Golgi apparatus to plasma membrane, serving as a linker between RAB8A and ESKL [50]. The monooxygenase enzymatic activity of protein is crucial for vesicular docking and fusion, as the mutated form of protein with an inactive monooxygenase domain leads to the accumulation of unfused vesicles [50]. It was emphasized that MICAL3 (and MICAL-L1, see below) are involved in cell division, but this action does not involve oxidoreduction, as the role of MICAL3 during cytokinesis does not require redox activity, as MICAL3 does not regulate actin levels during cytokinesis [51]. Instead, the Rab8A-positive vesicles are tethered to the midbody via direct interaction of MICAL3 with the centralspindlin component mitotic kinesin-like protein 1 (MKLP1 (also known as kinesin-like protein 5 or kinesin-like protein KIF23) and recruiting the adaptor protein ELKS/Rab6-interacting/CAST family member 1 (ERC1) [51].\u003c/p\u003e\u003cp\u003eHuman MICAL3 is a 2002-residue-long protein encoded by \u003cem\u003eMICAL3\u003c/em\u003e gene. It includes the monooxygenase domain (residues 1-494), CH domain (residues 518\u0026ndash;621), LIM domain (residues 762\u0026ndash;824), a ProQ domain (residue 1627\u0026ndash;1721), and CC/RBD (residues 1841\u0026ndash;2002), which is also classifies as bMERB domain acting as a Rab effector domain. Furthermore, MICAL3 contains a nuclear localization signal (residues 663\u0026ndash;684) and multiple PTM sites that are spread throughout the protein, such as serine phosphorylation at 509, 649, 687, 977, 1134, 1143, 1160, 1192, 1274, 1278, 1310, 1337, 1371, 1384, 1433, 1649, 1701, 1704, and 1912, as well as threonine phosphorylation at positions 887, 1276, 1341, 1454, and 1651. As per UniProt annotations, there are several long IDRs in this protein (residues 658\u0026ndash;706, 835\u0026ndash;883, 907\u0026ndash;1313, 1335\u0026ndash;1776, and 1791\u0026ndash;1821), and these IDRs are characterized by excessive compositional biases containing polar, basic, and acidic residues or enriched in proline residues (e.g., 1239\u0026ndash;1258 and 1456\u0026ndash;1467 segments).\u003c/p\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e shows that human MICAL3 is expected to contain high levels of disorder. For example, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA shows the RIDAO plot illustrating the disorder profile of this protein, where the structure from the middle of protein towards the C terminus (residues 829\u0026ndash;2002) is highly disordered. PPIDR values calculated from the outputs the predictors included to RIDAO, PONDR\u003csup\u003e\u0026reg;\u003c/sup\u003e VLXT, PONDR\u003csup\u003e\u0026reg;\u003c/sup\u003e VSL2, PONDR\u003csup\u003e\u0026reg;\u003c/sup\u003e VL3, IUPred Short, IUPred long, PONDR\u003csup\u003e\u0026reg;\u003c/sup\u003e FIT, and MDP, are 60.59%, 67.83%, 63.34%, 47.85%, 54.8%, 59.44%, and 60.28%, respectively, clearly classifying human MICAL3 as a highly disordered protein. Furthermore, ANCHOR-2 analysis showed that MoRF regions (i.e., regions with the ability to undergo disorder to order transition upon binding with their partner [52]) are abundant within the disordered segments of this protein and can be found at the following positions: 650\u0026ndash;662, 697\u0026ndash;726, 858\u0026ndash;1344, 1354\u0026ndash;1502, 1508\u0026ndash;1553, and 1558\u0026ndash;1707, 1725\u0026ndash;1751, 1771\u0026ndash;1805, and 1848\u0026ndash;1853.\u003c/p\u003e\u003cp\u003eThe results of the FuzDrop-based analysis of human MICAL3 are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB that clearly indicates that this protein has a very high ability to LLPS. In fact, its p\u003csub\u003eLLPS\u003c/sub\u003e value of 0.9993 suggests that human MICAL3 can serve as a droplet driver, cotrolling the process of MLO formation. FuzDrop analysis predicts the existence of 12 DPR (residues 1\u0026ndash;12, 70\u0026ndash;81, 587\u0026ndash;612, 645\u0026ndash;706, 822\u0026ndash;891, 897\u0026ndash;1322, 1329\u0026ndash;1586, 1598\u0026ndash;1775, 1788\u0026ndash;1819, 1834\u0026ndash;1844, 1875\u0026ndash;1890, and 1982\u0026ndash;2002). There are 34 short aggregation spots that are scattered through the 2/3 of the C-terminal part of protein sequence. The protein is also predicted to contain almost 50 regions with context-dependent interactions (i.e., regions that change interaction behavior and binding modes under different cellular conditions) (see Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), supporting the ANCHOR-2-based analysis that revealed the MoRFs cover 47.1% of the MICAL3 sequence. This is further supported by Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD presenting the D\u003csup\u003e2\u003c/sup\u003eP\u003csup\u003e2\u003c/sup\u003e-genearted functional disorder profile of this protein, where a very significant portion of a protein (~\u0026thinsp;60%) is shown to be disordered and highly decorated with multiple PTMs, that are likely to modulate binding activities of numerous MoRFs found in this protein.\u003c/p\u003e\u003cp\u003eAdditional support for the highly disordered nature of human MICAL3 is given by Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE representing the 3D stricture of this protein modeled by AlphaFold. This predicted structure has the p\u003csub\u003eLDDT\u003c/sub\u003e confidence score of 58.13, which is further reflected in the fact that most of the protein is shown in form of \u0026ldquo;orange noodles\u0026rdquo; which represent regions with very low p\u003csub\u003eLDDT\u003c/sub\u003e values (below 50) that are expected to be unstructured in isolation.\u003c/p\u003e\u003cp\u003eFive MICAL3 isoforms are generated by alternative splicing. Canonical isoform is 2002-residue long and is characterized by PPIDR\u003csub\u003ePONDR\u003c/sub\u003e\u003csup\u003e\u0026reg;\u003c/sup\u003e\u003csub\u003eVSL2\u003c/sub\u003e = 67.83%, ADS\u003csub\u003ePONDR\u003c/sub\u003e\u003csup\u003e\u0026reg;\u003c/sup\u003e\u003csub\u003eVSL2\u003c/sub\u003e = 0.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32, and p\u003csub\u003eLLPS\u003c/sub\u003e = 0.9993. MICAL3-2 (948 residues) is missing C-terminal half (residues 949\u0026ndash;2002) and has 934\u0026ndash;948 region changed from SSSESEMEEEGEEEE to RSARRAAGRPPATRP. This isoform is characterized by PPIDR\u003csub\u003ePONDR\u003c/sub\u003e\u003csup\u003e\u0026reg;\u003c/sup\u003e\u003csub\u003eVSL2\u003c/sub\u003e = 32.59%, ADS\u003csub\u003ePONDR\u003c/sub\u003e\u003csup\u003e\u0026reg;\u003c/sup\u003e\u003csub\u003eVSL2\u003c/sub\u003e = 0.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26, and p\u003csub\u003eLLPS\u003c/sub\u003e = 0.5798. The MICAL3-3 isoform (976 residues), which in addition to the sequence changes found in MICAL2-2 has R \u0026rarr; RQLTQERGASQPSCCLPGQVRPAPTPRWK change at position 746, shows PPIDR\u003csub\u003ePONDR\u003c/sub\u003e\u003csup\u003e\u0026reg;\u003c/sup\u003e\u003csub\u003eVSL2\u003c/sub\u003e = 35.45%, ADS\u003csub\u003ePONDR\u003c/sub\u003e\u003csup\u003e\u0026reg;\u003c/sup\u003e\u003csub\u003eVSL2\u003c/sub\u003e = 0.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26, and p\u003csub\u003eLLPS\u003c/sub\u003e = 0.6162. In the MICAL2-4 isoform (966 residues), region 967\u0026ndash;2002 is missing, whereas region 934\u0026ndash;966 is changed from SSSESEMEEEGEEEEEEPRLPPSDLGGVPWKEA to RDWVSPWLPRMVSNS WAQMIHPPQPPTVLGSQM, resulting in PPIDR\u003csub\u003ePONDR\u003c/sub\u003e\u003csup\u003e\u0026reg;\u003c/sup\u003e\u003csub\u003eVSL2\u003c/sub\u003e = 32.40%, ADS\u003csub\u003ePONDR\u003c/sub\u003e\u003csup\u003e\u0026reg;\u003c/sup\u003e\u003csub\u003eVSL2\u003c/sub\u003e = 0.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24, and p\u003csub\u003eLLPS\u003c/sub\u003e = 0.5957. Finally, MICAL3-5 isoform (1073 residues) has most alterations in the sequence, as it is missing the C-terminal region (residues 950\u0026ndash;2002), has changed 971\u0026thinsp;\u0026minus;\u0026thinsp;949 region from GVNGLEEPSIAKRLRGTPERIELENYRLSLRQAEALQEVPEETQAEHNLSSVLDTGAEEDVASSSSESEMEEEG EEEE to LTSLFGWVARHSLGLCDKAKGMSQHLQSNISSFGQQVAQNPLDSFFMCQLLAFGVPFLYGLSEVLVQIRG EFHWQAVAQ, and Q\u003csub\u003e747\u003c/sub\u003e changed to QQREKECSRTCPKKVITLSPPPTPPPCRAHGGQQTYRDLDADNRGKQS PHHERPEPEPPRRFFVDQWELSLSLRSSARPASPSSDSLRQKYIKMYTGGVSSLAEQIANQLQRKEQPKALLDKKEL. This isoform is characterized by PPIDR\u003csub\u003ePONDR\u003c/sub\u003e\u003csup\u003e\u0026reg;\u003c/sup\u003e\u003csub\u003eVSL2\u003c/sub\u003e = 36.16%, ADS\u003csub\u003ePONDR\u003c/sub\u003e\u003csup\u003e\u0026reg;\u003c/sup\u003e\u003csub\u003eVSL2\u003c/sub\u003e = 0.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26, and p\u003csub\u003eLLPS\u003c/sub\u003e = 0.5957.\u003c/p\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF shows that MICAL3 interacts with 31 partners and forms a relatively dense PPI network characterized by the average local clustering coefficient of 0.853 and containing 32 nodes connected by 89 edges (average node degree is 5.56). Since the expected number of edges for the random network of this size is 36, this MICAL3-centerd network has significantly more interactions than expected (PPI enrichment p-value is 3.79\u0026times;10\u003csup\u003e\u0026ndash;14\u003c/sup\u003e). Proteins in this PPI network are involved in the following biological processes: Golgi vesicle transport (GO:0048193; p\u0026thinsp;=\u0026thinsp;4.57\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003e), vesicle-mediated transport (GO:0016192; p\u0026thinsp;=\u0026thinsp;4.57\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003e), cellular component organization (GO:0016043; p\u0026thinsp;=\u0026thinsp;4.57\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003e), endosomal transport (GO:0016197; p\u0026thinsp;=\u0026thinsp;1.02\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e), and neuron projection development (GO:0031175; p\u0026thinsp;=\u0026thinsp;1.41\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e). Among the most significantly enriched molecular functions of these proteins are GTPase activity (GO:0003924; p\u0026thinsp;=\u0026thinsp;1.06\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;8\u003c/sup\u003e), nucleoside-triphosphatase activity (GO:0017111; p\u0026thinsp;=\u0026thinsp;2.95\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;8\u003c/sup\u003e), GTP binding (GO:0005525; p\u0026thinsp;=\u0026thinsp;2.99\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003e), semaphorin receptor activity (GO:0017154; p\u0026thinsp;=\u0026thinsp;5.60\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e), and anion binding (GO:0043168; p\u0026thinsp;=\u0026thinsp;0.00011). These proteins are most significantly enriched in the following cellular components GO:0005794 (Golgi apparatus; p\u0026thinsp;=\u0026thinsp;1.78\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003e), transport vesicle (GO:0030133; p\u0026thinsp;=\u0026thinsp;1.09\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e), exocytic vesicle (GO:0070382; p\u0026thinsp;=\u0026thinsp;2.30\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e), semaphorin receptor complex (GO:0002116; p\u0026thinsp;=\u0026thinsp;3.93\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e), and recycling endosome membrane (GO:0055038; p\u0026thinsp;=\u0026thinsp;4.86\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e). Finally, the most enriched pathways from the REACTOME database associated with the members of the MICAL3-centered PPI are RAB geranylgeranylation (HSA-8873719; p\u0026thinsp;=\u0026thinsp;4.65\u0026times;10\u003csup\u003e\u0026ndash;14\u003c/sup\u003e), RAB GEFs exchange GTP for GDP on RABs (HSA-8876198; p\u0026thinsp;=\u0026thinsp;3.68\u0026times;10\u003csup\u003e\u0026ndash;11\u003c/sup\u003e), membrane trafficking (HSA-199991; p\u0026thinsp;=\u0026thinsp;2.59\u0026times;10\u003csup\u003e\u0026ndash;10\u003c/sup\u003e), SEMA3A-Plexin repulsion signaling by inhibiting Integrin adhesion (HSA-399955; p\u0026thinsp;=\u0026thinsp;6.53\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e), and Sema3A PAK dependent Axon repulsion (HSA-399954; p\u0026thinsp;=\u0026thinsp;8.84\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section3\"\u003e\u003ch2\u003e3.1.4. Human MICAL-like protein 1 (MICAL-L1; UniProt ID: Q8N3F8)\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eBy modulating the actin cytoskeleton, human molecule interacting with CasL-like1 (MICAL-L1 protein, which is also known as MIRab13 for molecule interacting with Rab13) is involved in the extension and guidance of neurites, which are the extensions of neurons that form synapses and facilitate communication between nerve cells [9,53,54]. Furthermore, being a lipid binding protein, MICAL-L1 shows higher affinity for phosphatidic acid, which is enriched in recycling endosome membranes. C-terminus of the protein contains two hydrophobic residues through which the protein binds to the phosphatidic acid, and this interaction is vital for the anchoring of MICAL-L1 to the membrane structure and can also influence its role in membrane cargo trafficking [55]. As a result, MICAL-L1 is recruited to the endosome membranes, where it acts as a downstream effector of Rab proteins engaging cytosolic proteins to regulate membrane tubulation [55\u0026ndash;58]. MICAL-L1 acts as one of the Rab8 effectors, regulates receptor recycling and recruits Rab8 and EH-domain containing protein 1 (EHD1) to tubular RE membranes that emanate from the perinuclear region [58]. MICAL-L1 also plays a role in endosomal functions, being involved in the late stages of the endocytosis. MICAL-L1 is localized to the tubular membranes and the C-terminal of the protein has coiled-coil region, necessary for this localization that plays an important role in the recycling of receptors and lipids [58]. The protein interacts with EDH1 that acts as a key regulator of endocytosis. Both proteins colocalize to the tubular membrane, indicating functional role in endocytic recycling. Depleting MICAL-L1 has consequences on the endocytic recycling process, it leads to the disruption of normal processing of these functions, suggesting the protein is integral to maintain the function of protein complexes in tubular membranes [58]. Along with this, the depletion also results in the loss of the interaction of Rab8a and EDH1 protein and their absence in tubular membrane [58]. By recruiting EHD1 to primary cilium where it is anchored to the centriole through interaction with tubulins, MICAL-L1 plays a role in ciliogenesis coordination [59].\u003c/p\u003e\u003cp\u003eMICAL-L1 is also crucial for the remodeling of the elongated tubular endosomal network (ETEN) that originates from endosomal recycling compartment in human dendritic cells in response to specific stimuli [59]. ETEN remodeling is very important for the stable delivery of membrane proteins to their designated locations, and is crucial for the antigen presentation and activation of T-cells pointing out the role of MICAL-L1 in immune response [60]. Furthermore, MICAL-L1 regulates endocytosed-EGF receptor trafficking [61], slow endocytic recycling of endocytosed proteins back to the plasma membrane [58], and in cargo protein delivery to the plasma membrane [55].\u003c/p\u003e\u003cp\u003eMICAL-L1 protein is 863-residue-long, which is encoded by the \u003cem\u003eMICALL1\u003c/em\u003e gene. Despite its multifunctionality, MICAL-L1 is a mostly uncharacterized member of the MICAL family containing an N-terminal CH domain (residues 2-108), a LIM domain (residues 162\u0026ndash;225, two NPF motifs possessing asparagine-proline-phenylalanine sequences (residues 425\u0026ndash;427 and 633\u0026ndash;635), bMERB domain (residues 671\u0026ndash;818), and C-terminal CC domain (residues 785\u0026ndash;830) [58]. The bMERB domain is present in the MICAL family and EH domain-binding protein (EHBP) family, both playing a role in the endosomal trafficking [62]. Region 652\u0026ndash;863 is known to mediates the interaction of MICAL-L1 with RAB13 and RAB35 and intramolecular interaction with the CH domain with two coiled-coil regions (residues 682\u0026ndash;711, and 785\u0026ndash;830) [61,63], whereas 700\u0026ndash;863 region, which is necessary and sufficient to associate with tubular recycling endosome membranes, is known to mediate phosphatidic acid-binding and membrane tubulation [56]. There is also a long IDR (residues 224\u0026ndash;670) containing multiple low complexity regions and regions with composition bias in human MICAL-L1. The protein contains serine phosphorylation sites at positions 295, 309, 318, 391, 471, 484, 578, 621, and 740.\u003c/p\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA represents the RIDAO-generated disorder profile of human MICAL-L1 and shows that this protein is predicted to have a long IDR located in the middle (residues 201\u0026ndash;695). Therefore, it is not surprising that PPIDR values evaluated from the outputs of PONDR\u003csup\u003e\u0026reg;\u003c/sup\u003e VLXT. PONDR\u003csup\u003e\u0026reg;\u003c/sup\u003e VSL2, PONDR\u003csup\u003e\u0026reg;\u003c/sup\u003e VL3, IUPred Short, IUPred long, PONDR\u003csup\u003e\u0026reg;\u003c/sup\u003e FIT, and MDP mounts to 75.9%, 78.1%, 77.64%, 63.27%, 66.16%, 65.7%, and 70.57%, respectively. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA also shows that MICAL-L1 contains four MoRF regions (residues 136\u0026ndash;149, 227\u0026ndash;473, 478\u0026ndash;686, and 837\u0026ndash;855) predicted by ANCHOR-2, indicating that almost entire central IDR can serve as a protein binding platform. In line with these consideration, it was shown that MICAL-L1 acts as a hub connecting Rab8, Rab10, Rab35, Arf6, Eps15 homology domain protein 1 (EHD-1), and syndapin2 (an F-BAR protein) required for the formation and maintenance of endosomal tubules [56,63\u0026ndash;66].\u003c/p\u003e\u003cp\u003eAs per FuzDrop analysis, MICAL-L1 is characterized by a very high P\u003csub\u003eLLPS\u003c/sub\u003e value of 0.9990, suggesting that this protein has a strong tendency to undergo spontaneous LLPS and to form droplets (see Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). MICAL-L1 is predicted to contain 4 DPRs (residues 109\u0026ndash;166, 219\u0026ndash;669, 673\u0026ndash;689, and 825\u0026ndash;863) and 10 aggregation hot spots (residues 117\u0026ndash;122, 159\u0026ndash;166, 238\u0026ndash;245, 316\u0026ndash;337, 372\u0026ndash;378, 429\u0026ndash;435, 661\u0026ndash;669, 673\u0026ndash;682, 711\u0026ndash;716, and 825\u0026ndash;836) located within the DPR regions. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC indicates that MICAL-L1 has 15 regions with context-dependent interactions, with longest spanning regions at 159\u0026ndash;179, 316\u0026ndash;337, 661\u0026ndash;682, 700\u0026ndash;730, and 819\u0026ndash;836. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD shows that the central intrinsically disordered region of this protein is predicted to have multiple PTMs and MoRFs. The AlphaFold predicted structure of human MICAL-L1 has p\u003csub\u003eLLDT\u003c/sub\u003e value of around 52% and contains a very significant number of \u0026ldquo;orange noodles\u0026rdquo;, reflecting high disorder content of this protein (see Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eSTRING analysis predicted 119 nodes and 3891 interactions for human MICAL-L1 with p-value of \u0026lt;\u0026thinsp;1.0\u0026times;10\u003csup\u003e\u0026ndash;16\u003c/sup\u003e, indicating that this network is statistically significant. High degree of connectivity is indicated by an average node degree of 65.4 (see Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). The numbers of edges and nodes in MICAL-L1-centered network are higher than those in MICAL1, MICAL2, and MICAL3 networks. Curiously, it seems that the number of predicted nodes and interaction in MICAL PPI networks are proportional to the disorder status of the protein, suggesting that the interactivity of MICALs correlates with their flexibility or intrinsic disorder content. Proteins in MICAL-L1-centered PPI network are involved in the following biological processes: translation (GO:0006412; p\u0026thinsp;=\u0026thinsp;2.75\u0026times;10\u003csup\u003e\u0026ndash;85\u003c/sup\u003e), cytoplasmic translation (GO:0002181; p\u0026thinsp;=\u0026thinsp;1.20\u0026times;10\u003csup\u003e\u0026ndash;71\u003c/sup\u003e), organonitrogen compound biosynthetic process (GO:1901566; p\u0026thinsp;=\u0026thinsp;2.55\u0026times;10\u003csup\u003e\u0026ndash;51\u003c/sup\u003e), cellular nitrogen compound biosynthetic process (GO:0044271; p\u0026thinsp;=\u0026thinsp;3.97\u0026times;10\u003csup\u003e\u0026ndash;48\u003c/sup\u003e), and gene expression (GO:0010467; p\u0026thinsp;=\u0026thinsp;2.28\u0026times;10\u003csup\u003e\u0026ndash;40\u003c/sup\u003e). The most enriched molecular functions include structural constituent of ribosome (GO:0003735; p\u0026thinsp;=\u0026thinsp;8.50\u0026times;10\u003csup\u003e\u0026ndash;55\u003c/sup\u003e), RNA binding (GO:0003723; p\u0026thinsp;=\u0026thinsp;8.73\u0026times;10\u003csup\u003e\u0026ndash;40\u003c/sup\u003e), translation initiation factor activity (GO:0003743; p\u0026thinsp;=\u0026thinsp;7.21\u0026times;10\u003csup\u003e\u0026ndash;30\u003c/sup\u003e), structural molecule activity (GO:0005198; p\u0026thinsp;=\u0026thinsp;1.22\u0026times;10\u003csup\u003e\u0026ndash;29\u003c/sup\u003e), and translation regulator activity (GO:0045182; p\u0026thinsp;=\u0026thinsp;5.09\u0026times;10\u003csup\u003e\u0026ndash;29\u003c/sup\u003e). Cellular components associated with members of this network are small ribosomal subunit (GO:0015935; p\u0026thinsp;=\u0026thinsp;6.09\u0026times;10\u003csup\u003e\u0026ndash;64\u003c/sup\u003e), cytosolic small ribosomal subunit (GO:0022627; p\u0026thinsp;=\u0026thinsp;1.82\u0026times;10\u003csup\u003e\u0026ndash;57\u003c/sup\u003e), ribosomal subunit (GO:0044391; p\u0026thinsp;=\u0026thinsp;1.21\u0026times;10\u003csup\u003e\u0026ndash;55\u003c/sup\u003e), ribosome (GO:0005840; p\u0026thinsp;=\u0026thinsp;1.33\u0026times;10\u003csup\u003e\u0026ndash;55\u003c/sup\u003e), and ribonucleoprotein complex (GO:1990904; p\u0026thinsp;=\u0026thinsp;4.51\u0026times;10\u003csup\u003e\u0026ndash;55\u003c/sup\u003e). The most enriched KEGG pathways are ribosome (hsa03010; p\u0026thinsp;=\u0026thinsp;2.06\u0026times;10\u003csup\u003e\u0026ndash;58\u003c/sup\u003e), RNA transport (hsa03013; p\u0026thinsp;=\u0026thinsp;4.73\u0026times;10\u003csup\u003e\u0026ndash;15\u003c/sup\u003e), and endocytosis (hsa04144; p\u0026thinsp;=\u0026thinsp;3.67\u0026times;10\u003csup\u003e\u0026ndash;10\u003c/sup\u003e). Among the 46 most enriched pathways from the REACTOME database are formation of the ternary complex, and subsequently, the 43S complex (HSA-72695; p\u0026thinsp;=\u0026thinsp;2.20\u0026times;10\u003csup\u003e\u0026ndash;80\u003c/sup\u003e), ribosomal scanning and start codon recognition (HSA-72702; p\u0026thinsp;=\u0026thinsp;2.20\u0026times;10\u003csup\u003e\u0026ndash;80\u003c/sup\u003e), translation initiation complex formation (HSA-72649; p\u0026thinsp;=\u0026thinsp;9.31\u0026times;10\u003csup\u003e\u0026ndash;79\u003c/sup\u003e), GTP hydrolysis and joining of the 60S ribosomal subunit (HSA-72706; p\u0026thinsp;=\u0026thinsp;2.39\u0026times;10\u003csup\u003e\u0026ndash;74\u003c/sup\u003e), and translation (HSA-72766; p\u0026thinsp;=\u0026thinsp;3.42\u0026times;10\u003csup\u003e\u0026ndash;73\u003c/sup\u003e). Using more stringent criteria (the threshold of 0.400 (medium confidence) for minimum required interaction score and \u0026ldquo;text mining\u0026rdquo; as active interaction source) STRING generated a MICAL-L1-centered network containing 24 nodes linked by 78 edges and characterized by the average node degree of 6.5, average local clustering coefficient of 0.750 and a PPI-enrichment p-value below 10\u003csup\u003e\u0026ndash;16\u003c/sup\u003e (data not shown). This was done to avoid uncertainties associated with inclusion of EIF3L and its interactors to the MICAL network (see below).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section3\"\u003e\u003ch2\u003e3.1.5. MICAL-L2 (MICAL-LIKE 2 or JRAB; UniProt ID: Q8IY33)\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eMolecule interacting with CasL-like 2 (MICAL-L2), which is also known as junctional Rab13-binding protein (JRAB), plays an important role in cell adhesion and cell spreading and contributes towards the neurite growth [67]. The role of MICAL-L2 in neurite growth is context-dependent, as this protein can both inhibit and promote neurite outgrowth. For example, although the neurite outgrowth is inhibited by expression of MICAL-L2/JRAB alone, this inhibitory effect is reversed by coexpression of MICAL-L2/JRAB with the dominant active form of Rab13 [67]. The process of neurite outgrowth regulation involves interaction with a small G protein Rab13, which stimulates the interaction of MICAL-L2/JRAB with actinin-4, an actin-binding protein localizes to the cell body and the tips of the neurites, suggesting that Rab13 and MICAL-L2/JRAB may act to transfer actinin-4 from the cell body to the tips of neurites, where it is involved in the reorganization of the actin cytoskeleton which results in neurite outgrowth [67]. When bound to Rab13, MICAL-L2/JRAB induces membrane ruffles [68]. Being an effector protein for Rab13, MICAL-L2/JRAB regulates the endocytic recycling of tight junctional cell adhesion molecule occludin and controls the adhesion and repulsion of epithelial cells [69]. The spatiotemporal regulation of actin cytoskeleton during collective cell migration is linked to the ability of MICAL-L2/JRAB to undergo a conformational change between its closed and open forms depending on the association with Rab13 [70].\u003c/p\u003e\u003cp\u003eMICAL-L2/JRAB protein is involved in tubulation of recycling endosomes directly and is activated by Rab8A [71]. MICAL-L2/JRAB protein induce liquid-liquid phase separation to drive the segregation of cytoplasmic contents to form compartment of distinct phases, a process critical for the regulation of tubular recycling endosomes [71]. The intrinsically disordered region located between the globular N- and C-terminal domains is important for the formation of condensates as it promotes the LLPS process [71]. The interaction of MICAL-L2/JRAB with Rab8A induces formation of a closed MICAL-L2/JRAB form (in which the N-terminal CH and LIM domains interact with the C-terminal CC domain) functioning in the tubulation of recycling endosomes. These data indicate that the biogenesis of tubular recycling endosomes involves two sequential MICAL-L2/JRAB-dependent steps, where the LLPS is first driven by MICAL-L2/JRAB and then the endosomal tubulation is promoted by the closed form of MICAL-L2/JRAB formed by interaction with Rab8A [71]. By forming the distinct Rab8-MICAL-L2/ JRAB and Rab13-MICAL-L2/JRAB complexes, MICAL-L2/JRAB coordinates the assembly of adherens junctions (AJs) and tight junctions (TJs), respectively [72]. Furthermore, MICAL-L2/JRAB was shown to control the transport of claudins, occludin, and E-cadherin to the plasma membrane (PM), as well as Rab8 regulated the Rab13-independent transport of E-cadherin to the PM [72].\u003c/p\u003e\u003cp\u003eMICAL-L2 is highly expressed and promotes cell migration and invasion in several cancer types, such as gastric cancer [73], ovarian cancer [74], breast cancer [75], lung cancer [76], and colon cancer [77]. The molecular mechanisms of MICAL-L2/JRAB involvement in different cancers is very diverse and depend on the cancer type. For example, MICAL-L2/JRAB is upregulated in ovarian cancer, which is a cancer with the highest mortality rate among the gynecologic malignancies [74]. Silencing the expression of MICAL-L2/JRAB prevents the proliferation and invasion of the cancerous cells, suggesting the role of the MICAL-L2/JRAB in the aggressive behavior of malignant cells [74]. The mutation of MICAL-L2/JRAB hindered the translocation of β-catenin to the nucleus, which stimulates the β-catenin/Wnt signaling pathway involved in the cell proliferation and migration, suggesting the possible involvement of MICAL-L2/JRAB protein in the ovarian cancer progression through the modulation of this pathway [75]. In breast cancer, MICAL-L2/JRAB interacts with DENN domain-containing protein 2B (DENND2B) leading to the localization of this protein to the cell surface, where it selectively activates Rab13 on membrane ruffles, at the dynamic leading edge of migrating cells required for cell migration and invasion [75]. Proliferation of lung cancer cells is facilitated by MICAL-L2/JRAB via the de-ubiquitination of c-Myc thereby blocking its degradation [76].\u003c/p\u003e\u003cp\u003eMICAL-L2/JRAB is involved in directing the cells to move together in groups by changing its conformation [70]. Therefore, the impaired conformational plasticity of this protein can disturb the group migration of cells and result in the loss of directionality [70]. MICAL-L2/JRAB is highly disordered as in the AlphaFold structure, most of the protein is highly unstructured. This high level of disorder may allow the conformationally flexible protein to govern the cells to migrate as single units [70].\u003c/p\u003e\u003cp\u003eMICAL-L2/JRAB is a 904-residue-long protein encoded by the \u003cem\u003eMICALL2\u003c/em\u003e gene. Similar to other MICAL proteins MICAL-L2/JRAB contains CH domain (residues 1-107, which is typically found in protein families involved in the actin binding, suggesting the role of protein in actin dynamic [78]), LIM domain (residues 186\u0026ndash;248), bMERB domain (residues 723\u0026ndash;874), CC domain (residues 735\u0026ndash;771), and a regions mediating interaction with RAB13 required for transition from the closed to the opened conformation (residues 807\u0026ndash;903). This protein also has a long IDR (residues 251\u0026ndash;722) containing a multitude of segments with compositional bias, such as low complexity segments and segments enriched in proline residues, or segments with polar or basic and acidic residues, indicative of intrinsic disorder. Peculiarities of disorder distribution in human MACAL-L2 are further illustrated by Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA shows that with PPIDR values of 72.01%, 79.98%, 78.1%, 61.39%, 65.71%, 71.9%, and 74.78% derived from the outputs of PONDR\u003csup\u003e\u0026reg;\u003c/sup\u003e VLXT, PONDR\u003csup\u003e\u0026reg;\u003c/sup\u003e VSL2, PONDR\u003csup\u003e\u0026reg;\u003c/sup\u003e VL3, IUPred Short, IUPred long, PONDR\u003csup\u003e\u0026reg;\u003c/sup\u003e FIT, and MDP, respectively, human MICAL-L2 is predicted as a highly disordered protein. In fact, this is the most disordered member of the human MICAL family. This protein has four MoRF regions (residues 132\u0026ndash;168, 174\u0026ndash;180, 249\u0026ndash;270, and 276\u0026ndash;740), that cover 58.7% of its sequence, suggesting that the human MICAL-L2 interactivity relies on intrinsic disorder and disorder-to-order transitions.\u003c/p\u003e\u003cp\u003eIn line with the established capability of human MICAL-L2/JRAB to undergo LLPS and drive the formation of cellular biomolecular condensates [71], Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB represents the results of the FuzDrop analysis revealing that most of the MICAL-L2/JRAB residues have a p\u003csub\u003eDP\u003c/sub\u003e value above 0.6, so it is indeed a droplet driver with the p\u003csub\u003eLLPS\u003c/sub\u003e value of 0.9997. This protein is predicted to contain the following droplet-promoting regions along its length: 107\u0026ndash;185, 222\u0026ndash;233, 245\u0026ndash;674, 679\u0026ndash;737, and 893\u0026ndash;904. It also has 13 aggregation hot spots, with the most noticeable being 172\u0026ndash;185, 265\u0026ndash;281, 444\u0026ndash;469, 578\u0026ndash;597, and 679\u0026ndash;700 (see Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003eFurthermore, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC indicates that the interactivity of human MICAL-L2/JRAB is expected to be dependent on cellular context, as it has a plethora of regions with the context dependent interactions that are scattered along the length of the protein. These regions are expected to behave differently based on the cellular environment and their binding partners, adding a layer of flexibility towards the adaptability of protein. Prevalence of functional intrinsic disorder in this protein is further illustrated by Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD showing D\u003csup\u003e2\u003c/sup\u003eP\u003csup\u003e2\u003c/sup\u003e disorder profile, where one can find multiple MoRFs and PTM sites within the central disordered region. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE provides another view of high intrinsic disorder content of human MICAL-L2/JRAB showing its AlphaFold-generated 3D structural model, which clearly contains a multitude of regions with very low p\u003csub\u003eLDDT\u003c/sub\u003e.\u003c/p\u003e\u003cp\u003eHuman MICAL-L2 exists in five alternatively spliced isoforms. Canonical isoform is 904-residue long and is characterized by PPIDR\u003csub\u003ePONDR\u003c/sub\u003e\u003csup\u003e\u0026reg;\u003c/sup\u003e\u003csub\u003eVSL2\u003c/sub\u003e = 79.98%, ADS\u003csub\u003ePONDR\u003c/sub\u003e\u003csup\u003e\u0026reg;\u003c/sup\u003e\u003csub\u003eVSL2\u003c/sub\u003e = 0.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28, and p\u003csub\u003eLLPS\u003c/sub\u003e = 0.9997. Isoform 2, which is 680-residue-long, has altered 1\u0026ndash;48 and 656\u0026ndash;904 regions, which are changed to MFLSSR and GPPPHPAAGDWPSLPASTFVTTGFGRSPLARKPECRAGRRRRRNLTFRANQGDPCPRPMSLLCLARR. As a result, this isoform has PPIDR\u003csub\u003ePONDR\u003c/sub\u003e\u003csup\u003e\u0026reg;\u003c/sup\u003e\u003csub\u003eVSL2\u003c/sub\u003e = 84.41%, ADS\u003csub\u003ePONDR\u003c/sub\u003e\u003csup\u003e\u0026reg;\u003c/sup\u003e\u003csub\u003eVSL2\u003c/sub\u003e = 0.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25, and p\u003csub\u003eLLPS\u003c/sub\u003e = 0.9998. In isoform 3, the 1-570 region is changed to MALSSWAQGTSWAAKGFSRSFSLAEFSLLKPRAGSCRTQEPRKPADGQPWLRCSPCTGGQRIWVHGAHPATSPPIR QKGKLRPRGRESFPQGHTAQESQLGAPPLTPCPVLLMPPGRLAVGVSEGGVAMGRWQGEAQPPLQTPHSQHSFLT PRPLASHP, leading to PPIDR\u003csub\u003ePONDR\u003c/sub\u003e\u003csup\u003e\u0026reg;\u003c/sup\u003e\u003csub\u003eVSL2\u003c/sub\u003e = 95.33%, ADS\u003csub\u003ePONDR\u003c/sub\u003e\u003csup\u003e\u0026reg;\u003c/sup\u003e\u003csub\u003eVSL2\u003c/sub\u003e = 0.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15, and p\u003csub\u003eLLPS\u003c/sub\u003e = 0.9974. Isoform 4 (572 residues) is generated by replacing the 571\u0026ndash;904 region to GE. It is characterized by PPIDR\u003csub\u003ePONDR\u003c/sub\u003e\u003csup\u003e\u0026reg;\u003c/sup\u003e\u003csub\u003eVSL2\u003c/sub\u003e = 72.38%, ADS\u003csub\u003ePONDR\u003c/sub\u003e\u003csup\u003e\u0026reg;\u003c/sup\u003e\u003csub\u003eVSL2\u003c/sub\u003e = 0.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32, and p\u003csub\u003eLLPS\u003c/sub\u003e = 0.9996. Finally, region 290240 is missing in isoform 5 (692 residues), which has PPIDR\u003csub\u003ePONDR\u003c/sub\u003e\u003csup\u003e\u0026reg;\u003c/sup\u003e\u003csub\u003eVSL2\u003c/sub\u003e = 92.49%, ADS\u003csub\u003ePONDR\u003c/sub\u003e\u003csup\u003e\u0026reg;\u003c/sup\u003e\u003csub\u003eVSL2\u003c/sub\u003e = 0.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20, and p\u003csub\u003eLLPS\u003c/sub\u003e = 0.9996. Therefore, all isoforms of human MICAL-L2 are highly disordered and all are characterized by very high LLPS potential, suggesting that all of them can be related to the regulation of the biogenesis of cellular MLOs.\u003c/p\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF represents the STRING-generated PPI network centered at human MICAL-L2. The network includes 17 proteins involved in the 35 interactions. Although this network is not large (the average node degree is 4.12), it is characterized by the high average local clustering coefficient of 0.824. Since the expected number of edges for the random network of similar size is 18, the MICAL-L2 has significantly more interactions than expected (PPI enrichment p-value is 0.000175). The interaction of MICAL-L2 with actinins ACTN1, ACTN2, and ACTN4 (which are the F-actin cross-linking proteins anchoring actin to a variety of intracellular structures) highlights the involvement of this protein in the cytoskeleton organization, and actin remodeling.\u003c/p\u003e\u003cp\u003eProteins in this MICAL-L2-centered PPI network are involved in the following biological processes: vesicle docking involved in exocytosis (GO:0006904; p\u0026thinsp;=\u0026thinsp;6.36\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e), vesicle-mediated transport to the plasma membrane (GO:0098876; p\u0026thinsp;=\u0026thinsp;6.36\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e), regulation of exocytosis (GO:0017157; p\u0026thinsp;=\u0026thinsp;6.36\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e), endosomal transport (GO:0016197; p\u0026thinsp;=\u0026thinsp;6.36\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e), and vesicle-mediated transport (GO:0016192; p\u0026thinsp;=\u0026thinsp;6.36\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e). The most enriched molecular functions include GDP binding (GO:0019003; p\u0026thinsp;=\u0026thinsp;1.86\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003e), GTPase activity (GO:0003924; p\u0026thinsp;=\u0026thinsp;2.64\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003e), GTP binding (GO:0005525; p\u0026thinsp;=\u0026thinsp;7.31\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003e), myosin V binding (GO:0031489; p\u0026thinsp;=\u0026thinsp;0.00025), and cytoskeletal protein binding (GO:0008092; p\u0026thinsp;=\u0026thinsp;0.00033). Cellular components where these proteins are most significantly localized include cell junction (GO:0030054; p\u0026thinsp;=\u0026thinsp;2.86\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;8\u003c/sup\u003e), recycling endosome membrane (GO:0055038; p\u0026thinsp;=\u0026thinsp;1.69\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003e), recycling endosome (GO:0055037; p\u0026thinsp;=\u0026thinsp;1.69\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003e), synaptic vesicle (GO:0008021; p\u0026thinsp;=\u0026thinsp;1.72\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003e), and secretory vesicle (GO:0099503; p\u0026thinsp;=\u0026thinsp;8.84\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003e). The most enriched KEGG pathway is tight junction (hsa04530; p\u0026thinsp;=\u0026thinsp;1.12\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e). Among the most enriched pathways from the REACTOME database are RAB geranylgeranylation (HSA-8873719; p\u0026thinsp;=\u0026thinsp;1.28\u0026times;10\u003csup\u003e\u0026ndash;12\u003c/sup\u003e), RAB GEFs exchange GTP for GDP on RABs (HSA-8876198; p\u0026thinsp;=\u0026thinsp;6.84\u0026times;10\u003csup\u003e\u0026ndash;12\u003c/sup\u003e), membrane trafficking (HSA-199991; p\u0026thinsp;=\u0026thinsp;1.03\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;8\u003c/sup\u003e), nephrin family interactions (HSA-373753; p\u0026thinsp;=\u0026thinsp;0.00052), and platelet activation, signaling and aggregation (HSA-76002; p\u0026thinsp;=\u0026thinsp;0.00075).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003e3.2. Global analysis of the interactome of human MICAL proteins\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eNext, we checked the interactability of human MICAL proteins among themselves. This analysis revealed that at the used STRING setting, MICAL1 interacted with MICAL2, MICAL3, and MICAL-L2, whereas MICAL-L1 was not included in the intra-set interactions. Then, we analyzed the global interactability of human MICALs by building their common interactome. To this end, we used STRING platform utilizing somewhat greedy approach with the threshold of 0.400 (medium confidence) for minimum required interaction score and full STRING network option, where the edges indicate both functional and physical protein associations, such as known interactions from curated databases and experimentally determined, predicted interactions from gene neighborhood, gene fusions, and gene co-occurrence, and others associations from text mining and co-expression. This analysis revealed that the joint network of human MICALs includes 174 nodes connected by 4262 edges. The average node degree is 49, indicating that each individual protein in this PPI network is interacting with 49 other proteins. Since the expected number of edges for the random network of similar size is 1095, the PPI network centered at five human MICALs has significantly more interactions than expected (PPI enrichment p-value is \u0026lt;\u0026thinsp;10\u003csup\u003e\u0026ndash;16\u003c/sup\u003e). The average local clustering coefficient is 0.738, which means that the network tends to make clusters, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eIn fact, applying the STRING-embedded k-means clustering protocol that finds a defined number of clusters based on their centroids found three distinct clusters in this network. Here, cluster 1 includes 127 members involved in the formation of the ternary complex, and subsequently, the 43S complex as well as in the cytoplasmic translation. The 31 members of the cluster 2 are involved in RAB GEFs exchange GTP for GDP on RABs and in Rab subfamily of small GTPases, whereas 16 members of cluster 3 are related to the semaphorin-plexin signaling pathway involved in neuron projection guidance, SEMA3A-plexin repulsion signaling by inhibiting integrin adhesion, and semaphorin receptor complex. Functional enrichment of the members of this network in term of GO terms associated with biological processes, molecular functions, and cellular components are shown Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD, respectively, whereas Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD represents REACTOME pathways enrichment.\u003c/p\u003e\u003cp\u003eAlthough MICAL-L1 does not directly interact with other members of MICAL family, it is well-integrated into the joint MICAL network via its interactors. Since the number of nodes in this joint MICAL network (174) is smaller than the sum of the nodes in the PPI networks centered at the individual MICALs (35\u0026thinsp;+\u0026thinsp;17\u0026thinsp;+\u0026thinsp;32\u0026thinsp;+\u0026thinsp;119\u0026thinsp;+\u0026thinsp;17\u0026thinsp;=\u0026thinsp;220), the joint network contains 46 shared partners. Among partners shared by all five MICALs are NEDD9, RAB10, RAB13, RAB8A, and RAB8B. RAB15 is shared by MICAL1, MICAL2, MICAL3, and MICAL-L1. RAB35 is shared by MICAL1, MICAL3, MICAL-L1, and MICAL-L2. MICAL1, MICAL2, and MICAL3 share PLXN4. MICAL1 and MICAL3 share PLXNA1, PLXNA2, PLXNA3, RAB1A, RAB1B, and RAB6A, whereas MSRB1 is shared by MICAL1 and MICAL2, whereas EHD1 interacts with MICAL1 and MICAL-L1.\u003c/p\u003e\u003cp\u003eAnalysis of the STRING-identified MICAL interactors revealed some apparent inconsistencies. For example, ribosomal proteins constitute a large fraction of a \u0026ldquo;red\u0026rdquo; cluster shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, which therefore is annotated as related to the cytoplasmic translation. It seems that this association is questionable as MICALs, which are involved in the redox signaling and actin cytoskeleton regulation, were not shown to be directly linked to translation. However, one cannot exclude a possibility that this protein family still can affect translation via modulation of the actin cytoskeleton. In fact, among the multitude of the crucial roles of the actin cytoskeleton in various cellular processes, is its relation to the protein biosynthesis [79]. It was shown that the densely branched cortical actin patches (CAPs) found in yeast or the focal adhesions (FAs) in mammals might provide a platform for mRNA translation [80\u0026ndash;85]. Furthermore, polyribosomes were shown to be associated with cytoskeletal filaments, indicating that ribosomes can interact with actin filaments [86,87]. Therefore, MICAL-mediated regulation of the actin cytoskeleton can affect the efficiency and accuracy of translation. Furthermore, since the actin cytoskeleton is involved in the organization of the endoplasmic reticulum (ER), where protein synthesis (translation) takes place and controls is dynamics, including ER movement, positioning, and remodeling [88], MICALs can indirectly affect translation by inducing changes in the ER organization.\u003c/p\u003e\u003cp\u003eAlthough the aforementioned facts suggest that MICALs can be involved in protein biosynthesis via their effects on actin cytoskeleton, these considerations do not explain the observed association of MICAL-L1 with multiple ribosomal proteins (see Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). To understand what can cause these otherwise unexpected associations, we looked at the evidence provided by STRING. This analysis revealed that STRING database identified MICAL-L1 as a distant homolog of the EIF3L (eukaryotic translation initiation factor 3 subunit L) protein (UniProt ID: Q9Y262). Based on the outputs of the EMBOSS Stretcher platform [89] used for the pairwise sequence alignment, MICAL-L1 and EIF3L are characterized by low sequence identity (14.3%) and weak sequence similarity (24.7%), placing these proteins within the \"twilight zone\" of protein sequence alignment and indicating that the relationship between them is not clearly defined in terms of structure and homology. However, since the actual EIF3L interacts with other translation initiation factors, multiple ribosomal proteins, and other specific proteins, this weak sequence similarity between MICAL-L1 and EIF3L brought these EIF3L interactors to the MICAL-L1 interactome.\u003c/p\u003e\u003cp\u003eTo avoid the uncertainties associated with inclusion of EIF3L interactors to the MICAL network, we repeated our analysis using more stringent criteria. Figure\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e represents result of this analysis, where MICAL-centered PPI network was generated by STRING using the threshold of 0.400 (medium confidence) for minimum required interaction score and \u0026ldquo;text mining\u0026rdquo; as active interaction source. These setting allowed us avoiding inclusion of translation initiation factors and ribosomal proteins to the network. Figure\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA shows that the size of this network noticeably decreased from 174 to 67. This network has 233 edges and is characterized by the average node degree of 6.96 and an average local clustering coefficient of 0.507. Since the expected number of edges for the random set of proteins of the same size and degree distribution drawn from the genome is 77, the proteins in this MICAL-centered PPI network have more interactions among themselves than what would be expected (PPI enrichment p-value is below 10\u003csup\u003e\u0026ndash;16\u003c/sup\u003e).\u003c/p\u003e\u003cp\u003eApplication of the STRING-embedded k-means clustering protocol revealed that this network contains three clusters, where 26 proteins in the \u0026ldquo;red\u0026rdquo; cluster are related to RAB geranylgeranylation, Rab subfamily of small GTPases, and recycling endosome membrane, 24 proteins in the \u0026ldquo;green\u0026rdquo; cluster are related to the Sema3A PAK dependent axon repulsion, and 17 proteins from the \u0026ldquo;blue\u0026rdquo; cluster are related to endocytic recycling, recycling endosome membrane, and Eps15 homology domain (see Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). \u0026ldquo;Red\u0026rdquo; cluster contains proteins interacting with MICAL3 and MICALL2, \u0026ldquo;green\u0026rdquo; cluster includes interactors of MICAL1 and MICAL2, whereas proteins interacting with MICAL-L1 are included to the \u0026lsquo;\u0026rdquo;blue\u0026rdquo; cluster. Analysis of the functional enrichment of the members of this network in term of GO terms associated with biological processes, molecular functions, and cellular components are shown Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC, and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD, respectively, with Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD representing the REACTOME pathways enrichment. Note that members of this \u0026ldquo;reduced\u0026rdquo; PPI network does not contain terms associated with protein translation. Despite stringent criteria used in creation of this network it still contains multiple partners shared by different MICALs.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eFor example, NEDD9, shared by all five MICALs, RAB8A, RAB10, and RAB35 are shared by MICAL1, MICAL3, MICAL-L1, and MICAL-L2, whereas RAB13 is shared by MICAL1, MICAL2, MICAL-L1, and MICAL-L2. PLXNA4 is shared by MICAL1, MICAL2, and MICAL3, and RAB36 is shared by MICAL3, MICAL-L1, and MICAL-L2. MSRB1 is shared by MICAL1 and MICAL2, ACAP2 interacts with MICAL1 an MICAL-L1, and RAB6A and RAB1B are shared by MICAL1 and MICAL3.\u003c/p\u003e\u003cp\u003eTo avoid potential erroneous conclusions associated with the inclusion of proteins interacting with EIF3L to the MICAL PPI network, at the subsequent stages we exclusively analyzed the \u0026ldquo;reduced\u0026rdquo; STRING-generated network.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003e3.3. Global analysis of the prevalence of intrinsic disorder in MICAL interactome\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eAt the next stage, we evaluated the global prevalence of intrinsic disorder in proteins interacting with human MICALs. Figure\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e represents the results of this analysis and shows that these proteins are characterized by various levels of intrinsic disorder, being, in general, not very different from those observed in the entire human proteome. In fact, based on the seven per-residue disorder predictors used in this study (PONDR\u003csup\u003e\u0026reg;\u003c/sup\u003e VLXT, PONDR\u003csup\u003e\u0026reg;\u003c/sup\u003e VSL2, PONDR\u003csup\u003e\u0026reg;\u003c/sup\u003e VL3, PONDR\u003csup\u003e\u0026reg;\u003c/sup\u003e FIT, IUPRed_Short, IUPRed_Long, and MDP), the MICAL-interacting proteins are characterized by the mean PPIDR values of 32.0\u0026thinsp;\u0026plusmn;\u0026thinsp;16.1%, 39.0\u0026thinsp;\u0026plusmn;\u0026thinsp;21.9%, 30.6\u0026thinsp;\u0026plusmn;\u0026thinsp;24.7%, 24.9\u0026thinsp;\u0026plusmn;\u0026thinsp;20.4%, 17.4\u0026thinsp;\u0026plusmn;\u0026thinsp;14.1%, 19.0\u0026thinsp;\u0026plusmn;\u0026thinsp;19.7%, and 23.6\u0026thinsp;\u0026plusmn;\u0026thinsp;23.2%, respectively, in comparison with 34.1\u0026thinsp;\u0026plusmn;\u0026thinsp;20.3%, 43.6\u0026thinsp;\u0026plusmn;\u0026thinsp;26.2%, 36.6\u0026thinsp;\u0026plusmn;\u0026thinsp;29.0%, 30.4\u0026thinsp;\u0026plusmn;\u0026thinsp;24.0%, 21.2\u0026thinsp;\u0026plusmn;\u0026thinsp;19.3%, 22.8\u0026thinsp;\u0026plusmn;\u0026thinsp;24.8%, and 28.3\u0026thinsp;\u0026plusmn;\u0026thinsp;26.3% evaluated for the entire proteome. The mean ADS values of MICAL interactors are 0.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12, 0.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14, 0.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16, 0.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13, 0.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09, 0.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12, and 0.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13, whereas the members of the human proteome are characterized by the corresponding mean ADS values of 0.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17, 0.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19, 0.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19, 0.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17, 0.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14, 0.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18, and 0.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17. Based on the Mann-Whitney rank sum test, the observed difference in the median PPIDR\u003csub\u003eMDP\u003c/sub\u003e values between the MICAL-interacting proteins and members of the human proteome is not a statistically significant (P\u0026thinsp;=\u0026thinsp;0.267); i.e., the difference is not great enough to exclude the possibility that the difference is due to random sampling variability. Similarly, difference between the ADS\u003csub\u003eMDP\u003c/sub\u003e values between these two groups are not statistically significant as well (P\u0026thinsp;=\u0026thinsp;0.145).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eFigure 9A provides another way to look at the global disorder predisposition of the MICAL-interacting by showing the PONDR\u003csup\u003e\u0026reg;\u003c/sup\u003e VSL2 score (ADS) \u003cem\u003evs\u003c/em\u003e. PONDR\u003csup\u003e\u0026reg;\u003c/sup\u003e VSL2 (%) (PPIDR) plot. Typically, PPIDR values are used to classify proteins as highly ordered, moderately disordered, and highly disordered, if their corresponding PPIDR values are below 10%, between 10% and 30%, and above 30%, respectively [90,91]. An additional angle is provided by the analysis of the protein ADS values that provides another means for protein classification as highly ordered, moderately disordered/flexible, or highly disordered if their ADS\u0026thinsp;\u0026lt;\u0026thinsp;0.15, 0.15\u0026thinsp;\u0026le;\u0026thinsp;ADS\u0026thinsp;\u0026lt;\u0026thinsp;0.5, and ADS\u0026thinsp;\u0026ge;\u0026thinsp;0.5. Based on these criteria, all MICAL interactors are clearly classified as moderately or highly disordered by PONDR\u003csup\u003e\u0026reg;\u003c/sup\u003e VSL2, and none of these proteins are predicted as highly ordered by ADS and/or PPIDR (as none of the proteins is located in dark blue or cyan areas). This analysis also shows that half of these proteins are predicted as highly disordered. For comparison, the analogous analysis of the entire human proteome (20,317 proteins) revealed that 0.4%, 5.1%, 33.7%, 21.0%, and 39.8% proteins were located within dark blue, cyan, dark pink, light pink, and red areas, respectively [92]. Therefore, with their 0.0% \u0026ndash; 0.0% \u0026ndash; 50.0% \u0026ndash; 24.2% \u0026ndash; 25.8% distribution, MICAL interactors are rather close to the whole proteome.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e\u003cb\u003eFIGURE 9\u003c/b\u003e. Global disorder analysis of human MICAL-interacting proteins. \u003cb\u003eA\u003c/b\u003e. The PONDR\u003csup\u003e\u0026reg;\u003c/sup\u003e VSL2 score (ADS) \u003cem\u003evs\u003c/em\u003e. PONDR\u003csup\u003e\u0026reg;\u003c/sup\u003e VSL2 (%) (PPIDR) plot. Here, each point corresponds to a query protein, coordinates of which are evaluated from the corresponding PONDR\u003csup\u003e\u0026reg;\u003c/sup\u003e VSL2 data as its ADS and PPIDR values. Color blocks are used to visualize proteins based on the accepted classification, with red, pink/light pink, and blue/light blue regions containing highly disordered, moderately disordered, and ordered proteins, respectively (see the text). Dark blue or pink areas correspond to the regions where PPIDR agrees with ADS, whereas areas in which only one of these criteria applies are shown by light blue or light pink. Data for the MICAL interactome is shown by gray circles, whereas the positions of individual MICALs are shown by differently colored symbols. \u003cb\u003eB\u003c/b\u003e. Charge-Hydropathy and Cumulative Distribution Function (CH-CDF) analysis of query proteins. The ΔCH-ΔCDF plot is a two-dimensional representation that integrates the CH plot, which correlates a protein's net charge with its hydrophobicity, and the CDF, which correlates the cumulative frequency of disorder scores with the disorder scores. In the resulting ΔCH-ΔCDF plot, the Y-axis (ΔCH) represents the protein's distance from the CH boundary, while the X-axis (ΔCDF) represents the deviation of a protein's disorder frequency from the CDF boundary. Proteins are then stratified into four quadrants: Quadrant 1 (bottom right) indicates proteins likely to be structured; Quadrant 2 (bottom left) includes proteins that may be in a molten globule state or lack a unique 3D structure; Quadrant 3 (top left) consists of proteins predicted to be highly disordered; Quadrant 4 (top right) captures proteins that present a mixed prediction of being disordered according to CH but ordered according to CDF. \u003cb\u003eC\u003c/b\u003e. Analysis of the interactability of the members of MICAL interactome in a form of the dependence of the node degree on disorder content evaluated as PONDR\u003csup\u003e\u0026reg;\u003c/sup\u003e VSL2-based PPIDR. Vertical lines show 10% and 30% PPIDR thresholds, whereas the horizontal line corresponds to the average node degree of the analyzed joint MICAL interactome (7). \u003cb\u003eD\u003c/b\u003e. Analysis of the LLPS predisposition of the human MICAL interactors with their intrinsic disorder propensity. Vertical lines show 10% and 30% PPIDR thresholds, whereas the horizontal line corresponds to the 0.6 p\u003csub\u003eLLPS\u003c/sub\u003e threshold. In plots C and D, circles are colored based on the relation of the corresponding proteins to LLPS, with droplet drivers and droplet clients shown by red and pink circles, and with proteins not related to LLPS (showing p\u003csub\u003eLLPS\u003c/sub\u003e below 0.6 and not containing DPRs) are shown by blue circles.\u003c/p\u003e\u003cp\u003eFigure 9B represents the results of global disorder analysis of human proteins interacting with MICALs in the form of the ΔCH-ΔCDF plot that can be used for further classification of proteins as mostly ordered, molten globule-like or hybrid, or highly disordered based on their positions within the resulting CH-CDF phase space [93\u0026ndash;96]. This analysis provided further support to the idea that the MICAL-interacting proteins include noticeable levels of disorder, which are a bit lower than those found in human proteome, which contains 59.1%, 25/5%, 12.3%, and 3.1% proteins in quadrants Q1, Q2, Q3, and Q4, respectively [92].\u003c/p\u003e\u003cp\u003eFigure 9C shows correlation between interactability of the members of the MICAL interactome (in terms of their node degree) with their intrinsic disorder status and demonstrates that there is a weak positive correlation between these two parameters. This is an expected behavior, as disordered proteins are typically prone more to be promiscuous binders. Interestingly, this dataset contains noticeably more proteins with the node degrees below the average node degree of the entire joint MICAL interactome (7) (38 \u003cem\u003evs\u003c/em\u003e. 29). The most connected proteins in this PPI network are MICAL1, MICAL3, MICALL1, RAB8A, RAB6A, RAB11A, ARF6, MICAL2, and MICALL2 interacting with 34, 31, 24, 19, 17, 17, 16, 16, and 16 partners, respectively.\u003c/p\u003e\u003cp\u003eNext, we analyzed the LLPS predispositions of the members of the MICAL interactome using FuzDrop and looked at the correlation between the propensity of all the query proteins to undergo LLPS and their intrinsic disorder status. Figure\u0026nbsp;9D represents the results of this analysis as the p\u003csub\u003eLLPS\u003c/sub\u003e \u003cem\u003evs\u003c/em\u003e. PPIDR plot and clearly shows that all the proteins capable of spontaneous LLPS are predicted as highly disordered. These observations support the known notion that high levels of intrinsic disorder represent one of the important features of proteins undergoing LLPS.\u003c/p\u003e\u003cp\u003eOn the other hand, Fig.\u0026nbsp;9D shows that not all highly disordered proteins are expected to act as droplet-drivers. In fact, out of 37 droplet clients (i.e., proteins with the probabilities of spontaneous LLPS below the 0.6 threshold but containing DPRs), 14 are classified as highly disordered, being characterized by the PPIDR\u003csub\u003ePONDR\u0026reg;VSL2\u003c/sub\u003e exceeding 30%. The remaining proteins in this category are moderately disordered. Figure\u0026nbsp;9D also shows that even among the 10 proteins not associated with LLPS in any way (i.e., showing p\u003csub\u003eLLPS\u003c/sub\u003e below 0.6 and not containing any DPRs) there were 2 highly disordered proteins: CFL1 (36.75%) and CFL2 (34.94%). Therefore, although all analyzed in this study proteins capable of spontaneous LLPS (droplet drivers) and many droplet clients are highly disordered, not all highly disordered proteins can promote phase separation or become engaged in biomolecular condensate as clients. In other words, the correlation between disorder and LLPS propensity is not symmetric, as \u0026ldquo;high LLPS potential means high disorder propensity\u0026rdquo; is not equivalent to \u0026ldquo;high disorder propensity means high LLPS potential\u0026rdquo;. Furthermore, there is also a possibility that even mostly ordered proteins can find their way into some biomolecular condensates or MLS being bound to the droplet-clients or droplet-drivers. Finally, Fig.\u0026nbsp;9C illustrates that there is no obvious correlation between the LLPS potential of a protein and its interactivity, as some LLPS drivers do not have many partners, and many proteins unrelated to LLPS are highly connected.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e\u003ch2\u003e3.4. Functional disorder analysis of 5 most disordered MICAL interactors\u003c/h2\u003e\u003cdiv id=\"Sec24\" class=\"Section3\"\u003e\u003ch2\u003e3.4.1. ELKS/Rab6-interacting/CAST family member 1 (ERC1; UniProt ID: Q8IUD2; PPIDR\u003csub\u003ePONDR\u003c/sub\u003e\u003csup\u003e\u0026reg;\u003c/sup\u003e\u003csub\u003eVSL2\u003c/sub\u003e = 94.44%)\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eELKS/Rab6-interacting/CAST family member 1 (ERC1, also known as Rab6-interacting protein 2 (RAB6IP2); the name ELKS is derived from protein rich in the amino acids E, L, K and S [97]) is a regulatory subunit of the IKK complex (IκB kinase or IkappaB kinase) [98], which is an enzyme complex that is involved in propagating the cellular response to inflammation acting as a central regulator of NF-κB activation [99]. ERC1 can also play roles in the organization of the cytomatrix at the nerve terminals active zone (CAZ) as well as vesicle trafficking at CAZ, and in the regulation of endosomes to Goldi apparatus by RAB6 [98]. Complex formed by MICAL3, ELKS and Rab8A was shown to be involved in exocytotic vesicle trafficking controlling the fusion of exocytotic carriers with the plasma membrane [50,100]. It was also shown that by interaction with the centralspindlin complex via the kinesin-6 family protein mitotic kinesin-like protein 1 (MKLP1), which recruits MICAL3 to the central spindle, MICAL3 participates in targeting its binding partners ELKS and Rab8A to the midbody (a transient structure formed during cell division that plays a crucial role in separating two daughter cells), being therefore required for cytokinesis [51]. ERC1 is a member of a family of RIM-binding proteins, with RIMs being regulators of neurotransmitter release at active zone.\u003c/p\u003e\u003cp\u003eThe protein is known to have multiple isoforms generated by alternative splicing. The canonical isoform is ELKS epsilon, which is a 1,116-residue-long protein characterized by PPIDR\u003csub\u003ePONDR\u003c/sub\u003e\u003csup\u003e\u0026reg;\u003c/sup\u003e\u003csub\u003eVSL2\u003c/sub\u003e = 94.44%, ADS\u003csub\u003ePONDR\u003c/sub\u003e\u003csup\u003e\u0026reg;\u003c/sup\u003e\u003csub\u003eVSL2\u003c/sub\u003e = 0.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17, and p\u003csub\u003eLLPS\u003c/sub\u003e of 0.9912. The isoform 2 (ELKS beta, 992 residues) is different from the canonical form by missing regions 440\u0026ndash;467 and 1017\u0026thinsp;\u0026minus;\u0026thinsp;116 and by having glutamate at position 783 changed to ESLTS and region 1009\u0026ndash;1016 changed to DEEEGIWA [101]. As a result, this isoform is characterized by PPIDR\u003csub\u003ePONDR\u003c/sub\u003e\u003csup\u003e\u0026reg;\u003c/sup\u003e\u003csub\u003eVSL2\u003c/sub\u003e = 98.89%, ADS\u003csub\u003ePONDR\u003c/sub\u003e\u003csup\u003e\u0026reg;\u003c/sup\u003e\u003csub\u003eVSL2\u003c/sub\u003e = 0.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12, and p\u003csub\u003eLLPS\u003c/sub\u003e of 0.9912. The isoform 3 (ELKS delta, 1088 residues) is missing 440\u0026ndash;467 region and has PPIDR\u003csub\u003ePONDR\u003c/sub\u003e\u003csup\u003e\u0026reg;\u003c/sup\u003e\u003csub\u003eVSL2\u003c/sub\u003e = 94.30%, ADS\u003csub\u003ePONDR\u003c/sub\u003e\u003csup\u003e\u0026reg;\u003c/sup\u003e\u003csub\u003eVSL2\u003c/sub\u003e = 0.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17, and p\u003csub\u003eLLPS\u003c/sub\u003e of 0.9962. In the isoform 4 (ELKS alpha, 948 residues), regions 440\u0026ndash;467, 830\u0026ndash;873, and 1017\u0026ndash;1116 are missing, whereas glutamate at position 783 is changed to ESLTS and region 1009\u0026ndash;1016 is changed to DEEEGIWA. This isoform has PPIDR\u003csub\u003ePONDR\u003c/sub\u003e\u003csup\u003e\u0026reg;\u003c/sup\u003e\u003csub\u003eVSL2\u003c/sub\u003e = 98.84%, ADS\u003csub\u003ePONDR\u003c/sub\u003e\u003csup\u003e\u0026reg;\u003c/sup\u003e\u003csub\u003eVSL2\u003c/sub\u003e = 0.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12, and p\u003csub\u003eLLPS\u003c/sub\u003e of 0.9955. The isoform 5 (ELKS gamma) is the shortest version of ERC1 (720 residues). In addition to missing regions 224\u0026ndash;523 and 1017\u0026ndash;1116, it has changes at position 783 (E \u0026rarr; ESLTS) and 1009\u0026thinsp;\u0026minus;\u0026thinsp;116 (IIQPLLEL \u0026rarr; DEEEGIWA). It is characterized by PPIDR\u003csub\u003ePONDR\u003c/sub\u003e\u003csup\u003e\u0026reg;\u003c/sup\u003e\u003csub\u003eVSL2\u003c/sub\u003e = 99.17%, ADS\u003csub\u003ePONDR\u003c/sub\u003e\u003csup\u003e\u0026reg;\u003c/sup\u003e\u003csub\u003eVSL2\u003c/sub\u003e = 0.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13, and p\u003csub\u003eLLPS\u003c/sub\u003e of 0.9986 [101]. Due to a premature stop codon in the mRNA leading to nonsense-mediated mRNA decay, ELKSs beta, alpha, and gamma may be produced at very low levels. Furthermore, in a papillary thyroid carcinoma, translocation t(10;12)(q11;p13) fuses ELKS to receptor-type tyrosine kinase RET [101].\u003c/p\u003e\u003cp\u003eHuman ERC1 contains two coiled coil domains (residues 144\u0026ndash;988 and 1060\u0026ndash;1100) and a FIP-RBD domain (residues 1046\u0026ndash;1108), which is a C-terminally located Rab-binding domain (RBD) found in a family of Rab11 interacting proteins (FIPs) that selectively recognize the active form of Rab11. Figure\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e10\u003c/span\u003eA shows that human ERC1/RAB6IP2 is predicted to have a high level of intrinsic disorder. This is further supported by the RIDAO outputs, which showed that the PONDR\u003csup\u003e\u0026reg;\u003c/sup\u003e VLXT, PONDR\u003csup\u003e\u0026reg;\u003c/sup\u003e VSL2, PONDR\u003csup\u003e\u0026reg;\u003c/sup\u003e VL3, IUPred Short, IUPred long, PONDR\u003csup\u003e\u0026reg;\u003c/sup\u003e FIT, and MDP PPIDR values evaluated for this protein were 68.28%, 94.44%, 91.22%, 36.11%, 65.23%, 79.93%, and 84.32%, respectively. There are 24 MoRFs and multiple phosphorylation and acetylation sites in this protein, with the N-terminal 200 residues containing 16 phosphorylation sites.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e10\u003c/span\u003eB represents the AlphaFold-generated 3D structural model of this protein, which is dominated by the extremely long α-helices (residues 141\u0026ndash;187, 194\u0026ndash;250, 264\u0026ndash;318, 324\u0026ndash;369, 418\u0026ndash;701, and 801\u0026ndash;943), which do not form a compact core. These α-helices, especially the longest ones, cannot physically exist in unbound form and therefore represent \u0026ldquo;hallucinations\u0026rdquo;; i.e., plausible-looking structures where the real protein is actually unstructured. Note that these long α-helices are located within the very long N-terminal coiled coil domain.\u003c/p\u003e\u003cp\u003eFuzDrop analysis showed that human ERC1 can serve as droplet driver, since it has a high pLLPS of 0.9912 and contains 8 DPRs (residues 1-118, 172\u0026ndash;193, 243\u0026ndash;255, 625\u0026ndash;704, 713\u0026ndash;755, 855\u0026ndash;873, 894\u0026ndash;919, 945\u0026ndash;969, and 973\u0026ndash;1017). There are also numerous aggregation hot spots and regions with context-dependent interactions. These observations indicate that ERC1 can act as a scaffold protein generating a disorder-based platform for its binding partners. In line with the FuzDrop predictions, it was shown that ERC1 induces formation of cytoplasmic condensates with liquid-like properties that specifically host partners of a network relevant to cell motility, including liprin-α1 [102]. Importantly, in migrating cells, ERC1, liprin-α, and LL5 proteins (which are the pleckstrin homology-like domain family members) form polarized plasma membrane-associated platforms (PMAPs), which are liquid condensates associated with the regulation of cell motility [102].\u003c/p\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e10\u003c/span\u003eC represents the STRING-generated PPI network centered at human ERC1 and shows that this protein is engaged in interaction with 101 partners forming a network with 102 nodes and 673 edges. Among the 198 significantly enriched GO-terms related to biological processes that are associated with this PPI network are neurotransmitter secretion (GO:0007269; signal\u0026thinsp;=\u0026thinsp;4.57), vesicle-mediated transport in synapse (GO:0099003; signal\u0026thinsp;=\u0026thinsp;4.3), synaptic vesicle cycle (GO:0099504; signal\u0026thinsp;=\u0026thinsp;4.23), neurotransmitter transport (GO:0006836; signal\u0026thinsp;=\u0026thinsp;3.78), and synaptic vesicle exocytosis (GO:0016079; signal\u0026thinsp;=\u0026thinsp;3.63). Among the 27 significantly enriched molecular function GO terms are syntaxin-1 binding (GO:0017075; signal\u0026thinsp;=\u0026thinsp;1.43), syntaxin binding (GO:0019905; signal\u0026thinsp;=\u0026thinsp;1.27), GTPase binding (GO:0051020; signal\u0026thinsp;=\u0026thinsp;0.91), transmembrane transporter binding (GO:0044325; signal\u0026thinsp;=\u0026thinsp;0.84), and myosin V binding (GO:0031489; signal\u0026thinsp;=\u0026thinsp;0.82). Among the 92 significantly enriched cellular component GO terms are presynaptic active zone (GO:0048786; = 4.55), presynaptic active zone cytoplasmic component (GO:0098831; signal\u0026thinsp;=\u0026thinsp;3.82), cell cortex region (GO:0099738; signal\u0026thinsp;=\u0026thinsp;3.22), IkappaB kinase complex (GO:0008385; signal\u0026thinsp;=\u0026thinsp;2.95), and presynapse (GO:0098793; signal\u0026thinsp;=\u0026thinsp;2.82). Among the 53 significantly enriched KEGG pathways are NF-kappa B signaling pathway (hsa04064; signal\u0026thinsp;=\u0026thinsp;3.44), NOD-like receptor signaling pathway (hsa04621; signal\u0026thinsp;=\u0026thinsp;2.06), IL-17 signaling pathway (hsa04657; signal\u0026thinsp;=\u0026thinsp;1.91), shigellosis (hsa05131; signal\u0026thinsp;=\u0026thinsp;1.77), and RIG-I-like receptor signaling pathway (hsa04622; signal\u0026thinsp;=\u0026thinsp;1.63). And among the 78 REACTOME pathways the most enriched are serotonin neurotransmitter release cycle (HSA-181429; signal\u0026thinsp;=\u0026thinsp;5.59), dopamine neurotransmitter release cycle (HSA-212676; signal\u0026thinsp;=\u0026thinsp;5.27), acetylcholine neurotransmitter release cycle (HSA-264642; signal\u0026thinsp;=\u0026thinsp;4.24), norepinephrine neurotransmitter release cycle (HSA-181430; signal\u0026thinsp;=\u0026thinsp;4.19), and glutamate neurotransmitter release cycle (HSA-210500; signal\u0026thinsp;=\u0026thinsp;3.84).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\u003ch2\u003e3.4.2. Myocardin-related transcription factor A (MRTFA; UniProt ID: Q969V6; PPIDR\u003csub\u003ePONDR\u003c/sub\u003e\u003csup\u003e\u0026reg;\u003c/sup\u003e\u003csub\u003eVSL2\u003c/sub\u003e = 93.69%)\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eMyocardin-related transcription factor A (MRTFA, also known as MKL/myocardin-like protein 1, megakaryoblastic leukemia 1 protein (MKL1), and megakaryocytic acute leukemia protein (MAL)), is a 931-residue-long transcription coactivator involved in the control of the expression of the genes to regulate the cytoskeleton during cell migration, development, and morphogenesis [103]. This activity relies on the interaction of MKL1/MAL/MRTFA with the serum response factor (SRF) transcription factor, with the activity of the resulting SRF-MRTFA complex being regulated by MRTFA binding to cellular globular actin (G-actin) via its RPEL repeats (core sequence RPxxxEL) as well as by filamentous actin (F-actin) in the nucleus. SRF-MRTFA complex couples cytoskeletal gene expression to cytoskeletal dynamics being responsive to the Rho GTPase-induced changes in the G-actin concentration [103]. MRTFA is found in cytoplasm [47] and nucleus [47,104], and its subcellular localization is tightly regulated by actin both in cytoplasm and nucleus, where MRTFA binding to G-actin in cytoplasm masks the nuclear localization signal (NLS) and thereby inhibits nuclear import, whereas binding to the nuclear G-actin stimulates nuclear export of MRTFA to the cytoplasm. MICAL2 regulates nuclear localization of MRTFA by mediating nuclear actin depolymerization that leads to the decrease in the nuclear G-actin pool and consequent retention of MRTFA in the nucleus and formation of an active SRF-MRTFA complex [47]. It was also shown that MICAL2 facilitates gastric cancer cell migration via positive regulation of nuclear translocation of MRTFA and subsequent CDC42 activation and MMP9 expression [105]. By utilizing SRF/MRTFA signaling pathway, MICAL2 was shown to regulate myofibroblasts differentiation in epidural fibrosis [106]. A loss-of-function homozygous mutation of MRTFA is associated with immunodeficiency and severe susceptibility to bacterial infection [103]. Deregulated activity of MRTFA in transgenic mice with selective endothelial Progerin expression resulted in the development of the myocardial and perivascular fibrosis, left ventricular hypertrophy, and premature death [107].\u003c/p\u003e\u003cp\u003eMRTFA belongs to the MRTF family of transcription factors sharing evolutionary conserved domains essential for actin-binding, high-order chromatin organization, homo- and heterodimerization, and transcriptional activation [108]. Human MRTFA has a complex multidomain organization and includes an N-terminal region mediating interaction with suppressor of cancer cell invasion (SCAI) and beta-actin (ACTB) (residues 1-256) that contains intervening spacer sequences (residues 6\u0026ndash;23 and 60\u0026ndash;67) RPEL repeats (residues 24\u0026ndash;29 and 50\u0026ndash;67), a bipartite Nuclear localization signal (residues 27\u0026ndash;65), a SAP domain (residues 347\u0026ndash;381, named after three proteins known to contain it, SAF-A/B, Acinus, and PIAS), a coiled coil domain (residue 515\u0026ndash;563), a transactivation domain (residues 564\u0026ndash;931) and a multitude of low complexity regions and regions with other compositional biases (e.g., enriched in polar residues, proline residues, and basic and acidic residues). Each of the RPEL and intervening spacer motifs can bind autonomously to G-actin [109]. Mouse MRTFA that contains three RPEL motifs forms a pentavalent complex with G-actin [109]. Proteins with SAP domain are nuclear proteins involved in transcription, DNA repair, RNA processing, or apoptotic chromatin degradation. SAP motif also serves as a putative DNA-binding motif involved in chromosomal organization [110].\u003c/p\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e11\u003c/span\u003e shows that human MRTFA is a highly disordered protein containing multiple MoRFs and different PTMs, which are capable of interacting with multiple partners. As per RIDAO analysis, is PONDR\u003csup\u003e\u0026reg;\u003c/sup\u003e VLXT, PONDR\u003csup\u003e\u0026reg;\u003c/sup\u003e VSL2, PONDR\u003csup\u003e\u0026reg;\u003c/sup\u003e VL3, IUPred Short, IUPred long, PONDR\u003csup\u003e\u0026reg;\u003c/sup\u003e FIT, and MDP PPIDR values are 80.23%, 93.69%, 92.24%, 59.42%, 77.85%, 83.64%, and 87.47%, respectively. FuzDrop-based analysis revealed that this protein has a very high p\u003csub\u003eLLPS\u003c/sub\u003e value of 0.9999 and contains 6 DPRs (residues 98\u0026ndash;263, 272\u0026ndash;343, 385\u0026ndash;396, 432\u0026ndash;485, 544\u0026ndash;664, and 672\u0026ndash;866). It also has 17 aggregation hot spots and 27 regions with the context-dependent interactions\u003c/p\u003e\u003cp\u003eThe presence of multiple disorder-based interactions sites (MoRFs) (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e11\u003c/span\u003eA) and the high overall level of intrinsic disorder (Figs.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e11\u003c/span\u003eA and \u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e11\u003c/span\u003eB) define the high binding promiscuity of human MRTFA, which is shown by STRING to form a rich PPI network with 147 nodes and 1398 edges, average node degree of 19, average local clustering coefficient of 0.63, and a PPI enrichment value of \u0026lt;\u0026thinsp;10\u003csup\u003e\u0026ndash;16\u003c/sup\u003e. Among the 185 biological processes GO-terms significantly enriched in this network are ethanol metabolic process (GO:0006067; signal\u0026thinsp;=\u0026thinsp;4.13), monocarboxylic acid metabolic process (GO:0032787; signal\u0026thinsp;=\u0026thinsp;4.05), cellular aldehyde metabolic process (GO:0006081; signal\u0026thinsp;=\u0026thinsp;3.81), small molecule catabolic process (GO:0044282; signal\u0026thinsp;=\u0026thinsp;3.58), and carboxylic acid metabolic process (GO:0019752; signal\u0026thinsp;=\u0026thinsp;3.38). Among the 105 significantly enriched molecular function GO-terms are aldehyde dehydrogenase [NAD(P)+] activity (GO:0004030; signal\u0026thinsp;=\u0026thinsp;6.18), oxidoreductase activity, acting on the aldehyde or oxo group of donors, NAD or NADP as acceptor (GO:0016620; signal\u0026thinsp;=\u0026thinsp;6.17), oxidoreductase activity, acting on the aldehyde or oxo group of donors (GO:0016903 ; signal\u0026thinsp;=\u0026thinsp;5.98), aldehyde dehydrogenase (NAD+) activity (GO:0004029; signal\u0026thinsp;=\u0026thinsp;5.93), and C-acyltransferase activity (GO:0016408; signal\u0026thinsp;=\u0026thinsp;3.79). Among the 18 significantly enriched cellular component GO-terms are mitochondrial matrix (GO:0005759; signal\u0026thinsp;=\u0026thinsp;1.66), mitochondrion (GO:0005739; signal\u0026thinsp;=\u0026thinsp;0.88), phosphopyruvate hydratase complex (GO:0000015; signal\u0026thinsp;=\u0026thinsp;0.76), peroxisome (GO:0005777; signal\u0026thinsp;=\u0026thinsp;0.63), and serine C-palmitoyltransferase complex (GO:0017059; signal\u0026thinsp;=\u0026thinsp;0.6).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec26\" class=\"Section3\"\u003e\u003ch2\u003e3.4.3. Ninein-like protein (NINL; UniProt ID: Q9Y2I6; PPIDR\u003csub\u003ePONDR\u003c/sub\u003e\u003csup\u003e\u0026reg;\u003c/sup\u003e\u003csub\u003eVSL2\u003c/sub\u003e = 86.69%)\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eHuman centrosomal ninein-like protein (NINL or NLP) is a member of the γ-tubulin complexes binding proteins (GTBPs) that is essential for proper execution of various mitotic events [111]. NINL is involved in the microtubule organization in interphase cells, promoting microtubule nucleation that contributes to centrosome maturation, spindle formation and chromosome segregation [111]. Within a cell, NINL can be found in cytoplasm, cytoskeleton, microtubule organizing center, and centrosome [112,113]. Although inn interphase cells, NINL is transported to the centrosomes by the dynein-dynactin motor complex, direct phosphorylation of NINL by PLK1 during centrosome maturation results in releasing NINL to the cytoplasm [112]. Since the overexpression of NINL promoted anchorage-independent growth and induces cell malignant transformation, this protein was classified as an oncogenic protein [114\u0026ndash;116]. It was shown that NINL/NLP promotes autophagy by facilitating the interaction of Rab7 and FYCO1 (which is FYVE (zinc finger domain targeting signal-transducing proteins to cell membranes through binding to the membrane lipid phosphatidylinositol-3-phosphate (PtdIns(3)P) with high specificity) and coiled-coil domain-containing protein 1) [117]. Furthermore, in the process of ciliopathies, NINL/NLP associates with the coiled-coil and C2 domain-containing protein 2A (CC2D2A, a component of the tectonic-like complex localized at the transition zone of primary cilia that acts as a barrier preventing diffusion of transmembrane proteins between the cilia and plasma membranes [118]) and functions in Rab8-MICAL3-regulated vesicle trafficking [119].\u003c/p\u003e\u003cp\u003eHuman NINL is a 1,382-residue long calcium-binding protein containing four EF-can domains (residues 7\u0026ndash;42, 41\u0026ndash;76, 196\u0026ndash;231, and 233\u0026ndash;268), three coiled coil domains (residues 384\u0026ndash;424, 484\u0026ndash;579, and 1046\u0026ndash;1375), and the KEN (residues 495-497and D (destructive, residues 633\u0026ndash;641) boxes required for the cell cycle-controlled NINL degradation by the APC/C pathway [120]. A very significant part of this protein is predicted as intrinsically disordered (see Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e12\u003c/span\u003eA). NINL has multiple MoRFs and different PTMs, which are capable of interacting with multiple partners. In line with these considerations, RIDAO analysis indicated that the PONDR\u003csup\u003e\u0026reg;\u003c/sup\u003e VLXT, PONDR\u003csup\u003e\u0026reg;\u003c/sup\u003e VSL2, PONDR\u003csup\u003e\u0026reg;\u003c/sup\u003e VL3, IUPred Short, IUPred long, PONDR\u003csup\u003e\u0026reg;\u003c/sup\u003e FIT, and MDP PPIDR values of human NINL were 62.66%, 86.69%, 87.05%, 30.75%, 44.14%, 58.47%, and 66.64%, respectively, whereas the corresponding ADS values were 0.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26, 0.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26, 0.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19, 0.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20, 0.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20, 0.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23, and 0.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19. In addition to the canonical isoform, NINL has an alternative splicing-generated isoform missing residues 735\u0026ndash;1083, which represents a molecular link between Usher syndrome and Leber congenital amaurosis [121]. This 1003-residue-long alternatively spiced isoform is characterized by the PPIDR\u003csub\u003ePONDR\u0026reg;VSL2\u003c/sub\u003e of 80.54% and ADS\u003csub\u003ePONDR\u0026reg;VSL2\u003c/sub\u003e of 0.67.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eFuzDrop-based analysis revealed that this protein has a high p\u003csub\u003eLLPS\u003c/sub\u003e value of 0.9821 and contains 10 DPRs (residues 70\u0026ndash;104, 120\u0026ndash;203, 207\u0026ndash;219, 574\u0026ndash;611, 653\u0026ndash;667, 686\u0026ndash;718, 741\u0026ndash;782, 810\u0026ndash;1012, 1158\u0026ndash;1172, and 1271\u0026ndash;1287). It also has 18 aggregation hot spots and 43 regions with the context-dependent interactions. Alternative splicing results in an isoform with decreased p\u003csub\u003eLLPS\u003c/sub\u003e value of 0.8397, reduced number of DPRs (9), DPRs (13), and regions with the context-dependent interactions (31), suggesting that both functionality and LLPS potential of this protein are affected by alternative splicing.\u003c/p\u003e\u003cp\u003eTogether with the presence of multiple disorder-based interactions sites (MoRFs) (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e12\u003c/span\u003eA) and the high overall level of intrinsic disorder (Figs.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e12\u003c/span\u003eA and \u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e12\u003c/span\u003eB) the multitude of the regions with the context-dependent interactions found in human NINL/NLP suggests that this protein can be characterized by high binding promiscuity. Validity of this hypothesis is illustrated by Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e12\u003c/span\u003eC representing a STRING-generated PPI network centered at NINL and containing 93 nodes linked by 1046 edges. This network is characterized by the average node degree of 22.5, average local clustering coefficient of 0.745, and a PPI enrichment value of \u0026lt;\u0026thinsp;10\u003csup\u003e\u0026ndash;16\u003c/sup\u003e. Among the 98 biological processes GO-terms significantly enriched in this network are centrosome cycle (GO:0007098; signal\u0026thinsp;=\u0026thinsp;6.54), microtubule organizing center organization (GO:0031023; signal\u0026thinsp;=\u0026thinsp;6.38), microtubule cytoskeleton organization (GO:0000226; signal\u0026thinsp;=\u0026thinsp;5.09), microtubule-based process (GO:0007017; signal\u0026thinsp;=\u0026thinsp;4.23), and spindle organization (GO:0007051; signal\u0026thinsp;=\u0026thinsp;4.19). Among the 17 significantly enriched molecular function GO-terms are tubulin binding (GO:0015631; signal\u0026thinsp;=\u0026thinsp;1.95), microtubule binding (GO:0008017; signal\u0026thinsp;=\u0026thinsp;1.82), cytoskeletal protein binding (GO:0008092; signal\u0026thinsp;=\u0026thinsp;1.59), dynein light intermediate chain binding (GO:0051959; signal\u0026thinsp;=\u0026thinsp;1.48), and dynactin binding (GO:0034452; signal\u0026thinsp;=\u0026thinsp;1.47). Among the 68 significantly enriched cellular component GO-terms are centrosome (GO:0005813; signal\u0026thinsp;=\u0026thinsp;5.94), microtubule organizing center (GO:0005815; signal\u0026thinsp;=\u0026thinsp;5.07), microtubule associated complex (GO:0005875; signal\u0026thinsp;=\u0026thinsp;4.42), centriole (GO:0005814; signal\u0026thinsp;=\u0026thinsp;4.18), and mitotic spindle microtubule (GO:1990498; signal\u0026thinsp;=\u0026thinsp;3.75).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec27\" class=\"Section3\"\u003e\u003ch2\u003e3.4.4. AP-4 complex accessory subunit RUSC2 (UniProt ID: Q8N2Y8; PPIDR\u003csub\u003ePONDR\u003c/sub\u003e\u003csup\u003e\u0026reg;\u003c/sup\u003e\u003csub\u003eVSL2\u003c/sub\u003e = 83.05%)\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eAP-4 complex accessory subunit RUSC2 (RUN and SH3 domain containing 2, also known as Iporin for interacting protein of Rab1 [122]) is involved in vesicular trafficking of proteins at the trans-Golgi network being capable of interaction with the adapter-like complex 4 (AP-4), which is an ancient membrane trafficking complex [123]. Here, RUSC2 serves as an AP-4 accessory protein that facilitates the transport of AP-4-derived, ATG9A-positive vesicles from the trans-Golgi network to the cell periphery [123]. This protein can be found in cytoplasm/cytosole showing punctate distribution [122], on cell membrane [124], or in the perinuclear region [122].\u003c/p\u003e\u003cp\u003eRUSC2 can also bind to golgin subfamily A member 2 (GOLGA2, a peripheral membrane component of the cis-Golgi stack that acts as a membrane skeleton maintaining the structure of the Golgi apparatus, and as a vesicle tether facilitating vesicle fusion to the Golgi membrane [125]) and active forms of RAB1A and RAB1B [122]. It also can use its RUN domain to interact with the GTP-bound form of RAB35, thereby being recruited to the plasma membrane [122\u0026ndash;124]. Furthermore, RUSK2/Iporin might serve as a link between the targeting of ER derived vesicles, triggered by the Rab1 GTPase and a signaling pathway regulated by molecules containing SH3 and/or poly-proline regions [122]. MICAL1 serves one of the Rab1 interactors that defines a link between the Golgi apparatus associated rab1 and the intermediate filament cytoskeleton [126]. Together with p115 (the vesicle tethering-protein), GM130 (a cis-Golgi-localized coiled-coil protein), golgin-84 (a type II C-terminally anchored cis-Golgi protein with an extensive cytoplasmic coiled-coil domain), RUSC2/Iporin, and MICAL1, an activated form of Rab1 forms a protein complex mediating and regulating intracellular vesicle transport [122]. Although RUSC2/Iporin does not physically interact with MICAL1, these proteins act together within the aforementioned complex, where they both bind to Rab1 [122]. Variants of RUSC2 with missing C-terminal regions (866\u0026ndash;1516 or 1318\u0026ndash;1516) are linked to intellectual developmental disorder, autosomal recessive 61 (MRT61) characterized by central hypotonia, microcephaly, and moderate to severe intellectual disability [127].\u003c/p\u003e\u003cp\u003eRUSC2/Iporin is a 1,516-residue-long protein that contains a RUN domain (residues 1,031\u0026thinsp;\u0026minus;\u0026thinsp;1,175), which being a major part of the Rab1-binding domain (R1BD) is required for the interaction with RAB1A, RAB1B and RAB35 [122,124], an SH3 domain (residues 1,447-1,506) that potentially mediates assembly of specific protein complexes via binding to proline-rich peptides, and multiple regions with composition biases, such as regions containing polar residues, acidic residues, basic and acidic residues, glycine residues, as well as low complexity regions all indicative of intrinsic disorder. In line with this hypothesis Figs.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e13\u003c/span\u003eA and \u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e13\u003c/span\u003eB show that a very significant fraction of this protein is predicted as intrinsically disordered. Further support to this idea is coming from RIDAO-based analysis intrinsic disorder predisposition, according to which human RUSC2 is characterized by the PONDR\u003csup\u003e\u0026reg;\u003c/sup\u003e VLXT, PONDR\u003csup\u003e\u0026reg;\u003c/sup\u003e VSL2, PONDR\u003csup\u003e\u0026reg;\u003c/sup\u003e VL3, IUPred Short, IUPred long, PONDR\u003csup\u003e\u0026reg;\u003c/sup\u003e FIT, and MDP PPIDR values of 58.91%, 83.05%, 78.96%, 55.21%, 66.36%, 69.92%, and 69.39%, respectively, with the corresponding ADS values being 0.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36, 0.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36, 0.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27, 0.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24, 0.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26, 0.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25, and 0.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24. Figure\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e13\u003c/span\u003eA also shows that this protein contains a large set of MoRFs and numerous phosphorylation sites. According to AlphaFold analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e13\u003c/span\u003eB), 3D structure of human RUSC2 is dominated by orange \u0026ldquo;noodles\u0026rdquo; indicative of very low per-residue confidence score (p\u003csub\u003eLDDT\u003c/sub\u003e \u0026lt; 50), suggesting that many of these regions are expected to be unstructured in isolation.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eFuzDrop showed that human RUSC2 is characterized by the p\u003csub\u003eLLPS\u003c/sub\u003e of 1.0 and contains 17 DPRs (residues 24\u0026ndash;57, 67\u0026ndash;101, 148\u0026ndash;164, 178\u0026ndash;281, 292\u0026ndash;303, 325\u0026ndash;366, 401\u0026ndash;446, 450\u0026ndash;693, 726\u0026ndash;839, 848\u0026ndash;893, 900\u0026ndash;920, 1012\u0026ndash;1027, 1175\u0026ndash;1186, 1202\u0026ndash;1270, 1277\u0026ndash;1406, 1415\u0026ndash;1452, and 1505\u0026ndash;1516). It possesses 31 aggregation hot spots and 40 regions with the context-dependent interactions. Figure\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e13\u003c/span\u003eC represents a PPI network generated by STRING for this protein. This network containing 58 nodes and 256 is characterized by the average node degree of 9, average local clustering coefficient of 837, and a PPI enrichment value of \u0026lt;\u0026thinsp;10\u003csup\u003e\u0026ndash;16\u003c/sup\u003e. Among the 33 significantly enriched GO-terms describing biological processes associated with this network are isoprenoid biosynthetic process (GO:0008299: signal\u0026thinsp;=\u0026thinsp;3.9), small molecule biosynthetic process (GO:0044283; signal\u0026thinsp;=\u0026thinsp;1.83), isoprenoid metabolic process (GO:0006720; signal\u0026thinsp;=\u0026thinsp;2.38), cellular lipid metabolic process (GO:0044255; signal\u0026thinsp;=\u0026thinsp;1.05), and alcohol biosynthetic process (GO:0046165; signal\u0026thinsp;=\u0026thinsp;1.6). Among the 17 significantly enriched molecular function GO-terms are prenyltransferase activity (GO:0004659; signal\u0026thinsp;=\u0026thinsp;3.38), transferase activity, transferring alkyl or aryl (other than methyl) groups (GO:0016765; signal\u0026thinsp;=\u0026thinsp;2.6), small GTPase binding (GO:0031267; signal\u0026thinsp;=\u0026thinsp;0.44), trans-hexaprenyltranstransferase activity (GO:0000010: signal\u0026thinsp;=\u0026thinsp;0.47), and isopentenyl-diphosphate delta-isomerase activity (GO:0004452; signal\u0026thinsp;=\u0026thinsp;0.47). Among the 68 significantly enriched cellular component GO-terms are organelle membrane (GO:0031090; signal\u0026thinsp;=\u0026thinsp;0.54), AP-4 adaptor complex (GO:0030124; signal\u0026thinsp;=\u0026thinsp;1.05), organelle subcompartment (GO:0031984; signal\u0026thinsp;=\u0026thinsp;0.56), cytoplasm (GO:0005737; signal\u0026thinsp;=\u0026thinsp;0.31), and intracellular anatomical structure (GO:0005622; signal\u0026thinsp;=\u0026thinsp;0.29).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec28\" class=\"Section3\"\u003e\u003ch2\u003e3.4.5. EH domain-binding protein 1-like protein 1 (EHBP1L1; UniProt ID: Q8N3D4; PPIDR\u003csub\u003ePONDR\u003c/sub\u003e\u003csup\u003e\u0026reg;\u003c/sup\u003e\u003csub\u003eVSL2\u003c/sub\u003e = 80.30%)\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eEH domain-binding protein 1-like protein 1 (EHBP1L1) is a human 1,523-residue-long protein acting as RAB effector and playing a role in vesicle trafficking [34]. Together with adaptor proteins of actin regulators CD2-associated protein (CD2AP) and CBL-interacting protein of 85 kDa (CIN85), EHBP1L1 is involved in the length control of cilia, which are organelles that function in signal sensing and transduction and consist of axonemal microtubules and plasma membranes protruding from the cell surface to the extracellular region [128]. This protein is also involved in polarized exocytic trafficking in small intestinal epithelial cells [129]. During terminal erythropoiesis, promotes nuclear polarization and subsequent enucleation of erythroblasts is promoted by an apicobasal polarity regulator EHBP1L1 in coordination with Rab10, Bin1, and dynamin [130]. The formation of apically-destined vesicles at the recycling endosomes is dependent on EHBP1L1, Bin1, and dynamin 1 [131]. In renal cell carcinoma (RCC), interaction of EHBP1L1 with Janus kinase 1 (JAK1) stabilizes JAK1, leading to the formation of an immunosuppressive tumor microenvironment (TME) and thereby driving immune evasion [132]. Similar to MICALs, EHBP1L1 contains a bivalent Mical/EHBP Rab binding (bMERB) domain that can bind two Rabs simultaneously [34]. Therefore, EHBP1L1 and MICALs are functionally related, although no direct physical interaction between these proteins was demonstrated as of yet.\u003c/p\u003e\u003cp\u003eEHBP1L1 possesses a Ca\u003csup\u003e2+\u003c/sup\u003e/phospholipid-binding (C2) domain (residues 8-157), a calponin homology (CH) domain (residues 1037\u0026ndash;1142), a bivalent Mical/EHBP Rab binding (bMERB) domain (residues 1349\u0026ndash;1501) mediating binding to the GTP-bound forms of RAB8, RAB10, RAB10, RAB13, and RAB15 [34], a coiled-coil domain (residues 1363\u0026ndash;1515), and CAAX motif (residues 1520\u0026ndash;1523) acting as a signal for prenylation and required for endosomal colocalization with RAB8 and RAB10 [34]. Because of the presence of the bMERB domain, EHBP1L1 has two Rab-binding sites and therefore, together with MICAL-1, MICAL-3, and MICAL-L2, belongs to the bivalent RAB8-family effectors [34]. In its farnesylated form, EHBP1L1 is preferentially localized to the intracellular structures resembling endosomes, where it colocalizes with constitutively active RAB8 and RAB10 [34].\u003c/p\u003e\u003cp\u003eLike the other proteins discussed in this section, human EHBP1L1 is predicted to have high level of disorder. For example, based on the RIDAO analysis, this protein is characterized by the PONDR\u003csup\u003e\u0026reg;\u003c/sup\u003e VLXT, PONDR\u003csup\u003e\u0026reg;\u003c/sup\u003e VSL2, PONDR\u003csup\u003e\u0026reg;\u003c/sup\u003e VL3, IUPred Short, IUPred long, PONDR\u003csup\u003e\u0026reg;\u003c/sup\u003e FIT, and MDP PPIDR values of 79.71%, 80.3%, 78.79%, 56.8%, 68.29%, 71.11%, and 74.06%, respectively. Figure\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e14\u003c/span\u003eA represents the functional disorder profile generated for EHBP1L1 by D\u003csup\u003e2\u003c/sup\u003eP\u003csup\u003e2\u003c/sup\u003e and shows that it has multiple MoRFs that almost completely cover its long IDRs. The protein is also densely decorated by PTMs, indicating that the intrinsic disorder-based interactivity of EHBP1L1 can be regulated by PTMs. Figure\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e14\u003c/span\u003eB provides further support to the highly disordered nature of this protein by showing 3D structure model generated by AlphaFold. Here, with the exception to Ca\u003csup\u003e2+\u003c/sup\u003e/phospholipid-binding (C2) domain (residues 2-172), CH-domain (residues 1030\u0026ndash;1143), and portion of CC domain (residues 1352\u0026ndash;1445 and 1470\u0026ndash;1508), for which the average per-residue model confidence score p\u003csub\u003eLDDT\u003c/sub\u003e values are 87.92, 87.33, and 83.49, very significant fraction of a protein is given by disordered loops with very low p\u003csub\u003eLDDT\u003c/sub\u003e \u0026lt; 50.\u003c/p\u003e\u003cp\u003eBased on the outputs of FuzDrop analysis, human EHBP1L1 is expected to serve as droplet driver, as it shows a very high p\u003csub\u003eLLPS\u003c/sub\u003e score of 0.9998 indicative of high potential of this protein to undergo spontaneous LLPS. Protein also includes 8 DPRs (residues 175\u0026ndash;595, 648\u0026ndash;665, 693\u0026ndash;704, 817\u0026ndash;864, 870\u0026ndash;915, 919\u0026ndash;929, 933\u0026ndash;1041, and 1151\u0026ndash;1369. Although currently, there is no experimental evidence that EHBP1L1 can have a role in the formation or regulation of biomolecular condensates, some of its functions are indicative of potential EHBP1L1-LLPS connections. Because of the aforementioned involvement of this protein in membrane trafficking and interactions with RAB8, BIN1, and dynamin, EHBP1L1 could be implicated in the formation of the biomolecular condensates at the endocytic recycling compartment (ERC, which is a cellular structure responsible for sorting and recycling proteins and lipids back to the cell membrane [129]) or in other membrane-associated processes.\u003c/p\u003e\u003cp\u003eIn addition to 8 DPRs and 24 MoRFs, human EHBP1L1 contains 21 aggregation hot spots and more than 35 regions with context-dependent interactions. All this suggests that this protein might have high interaction potential. The capability of EHBP1L1 to interact with several proteins was already discussed. Figure\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e14\u003c/span\u003eC represents further support to the high interactivity of this protein by showing the STRING-generated PPI network centered at EHBP1L1. This network includes 49 nodes connected by 463 edges. The network has an average local clustering coefficient of 0.886 and average node degree of 18.9.\u003c/p\u003e\u003cp\u003eMembers of this network are taking part in various biological processes such as protein transport from ciliary membrane to plasma membrane (GO:1903445; FDR\u0026thinsp;=\u0026thinsp;0.0147), Golgi vesicle fusion to target membrane (GO:0048210; FDR\u0026thinsp;=\u0026thinsp;0.000060), vesicle transport along actin filament (GO:0030050; FDR\u0026thinsp;=\u0026thinsp;0.0102), vesicle docking involved in exocytosis (GO:0006904; FDR\u0026thinsp;=\u0026thinsp;0.0036), and establishment of protein localization to plasma membrane (GO:0061951; FDR\u0026thinsp;=\u0026thinsp;0.0050). The molecular functions represented are muscle alpha-actinin binding (GO:0051371; FDR\u0026thinsp;=\u0026thinsp;9.26e-13), cadherin binding involved in cell-cell adhesion (GO:0098641; FDR\u0026thinsp;=\u0026thinsp;0.0070), microfilament motor activity (GO:0000146; FDR\u0026thinsp;=\u0026thinsp;0.0022), cell-cell adhesion mediator activity (GO:0098632; FDR\u0026thinsp;=\u0026thinsp;0.0063), and GDP binding (GO:0019003; FDR\u0026thinsp;=\u0026thinsp;0.0011). Cellular components, where these proteins are localized include filamentous actin (GO:0031941; FDR\u0026thinsp;=\u0026thinsp;4.11e-61), unconventional myosin complex (GO:0016461; FDR\u0026thinsp;=\u0026thinsp;0.00013), insulin-responsive compartment (GO:0032593; FDR\u0026thinsp;=\u0026thinsp;0.0122), stress fiber (GO:0001725; FDR\u0026thinsp;=\u0026thinsp;1.13e-09), and stereocilium tip (GO:0032426; FDR\u0026thinsp;=\u0026thinsp;0.0432).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e\u003cem\u003e3.5. Functional disorder analysis of the neural precursor cell expressed developmentally down-regulated protein 9 (NEDD9; UniProt ID: Q14511; PPIDR\u003c/em\u003e\u003csub\u003e\u003cem\u003ePONDR\u003c/em\u003e\u003c/sub\u003e\u003csup\u003e\u003cem\u003e\u0026reg;\u003c/em\u003e\u003c/sup\u003e\u003csub\u003e\u003cem\u003eVSL2\u003c/em\u003e\u003c/sub\u003e \u003cem\u003e= 59.71%), a shared interactor of five MICALs\u003c/em\u003e\u003c/p\u003e\u003cp\u003eHuman enhancer of filamentation 1 (HEF1, also known as neural precursor cell expressed developmentally down-regulated protein 9 (NEDD9), CRK-associated substrate-related protein (CASL) [133], Cas scaffolding protein family member 2 (CASS2), renal carcinoma antigen NY-REN-12 [134], and p105 [135]) is a 834-residue-long scaffolding protein with central coordinating role in tyrosine-kinase-based signaling related to cell adhesion [136]. The protein is a member of the Crk-associated substrate (CAS) protein family and is broadly spread throughout the cell, being found in basolateral cell membrane, cytoplasm [137,138], cell cortex [137], nucleus [138\u0026ndash;140], Golgi apparatus [140], cell projections, such as lamellipodium [136,140] and cilium [141], cilium basal body [141], cell junction (focal adhesion) [140,142\u0026ndash;144], cytoskeleton [137], and spindle pole [138,145].\u003c/p\u003e\u003cp\u003eBroad distribution of NEDD9 within cell and a multitude of names given to this protein by different researchers suggest its multifunctionality. Being a non-catalytic scaffolding protein [135,140,146], NEDD9 forms complexes involving oncogenic kinases, such as focal adhesion kinase (FAK), ABL, SRC, Aurora-A (AURKA), and others. By acting as a docking protein NEDD9/CASL is involved in signaling pathways related to T-cell differentiation, cell-cell junctions formation, cell migration, in part, in response to integrin activation [147,148]. This defines the capability of this protein to regulate the magnitude and duration of cell signaling cascades controlling multiple cellular processes, which that are crucial for tumorigenesis and metastases, such as apoptosis and cell cycle, migration, adhesion, invasion, and chemotaxis [147\u0026ndash;151]. Furthermore, pleiotropic functions of NEDD9 link this protein to various maladies, where poor outcome, metastatic potential, and resistance to chemotherapy in many human cancers are linked to altered expression of NEDD9, and where non-malignant pathological conditions, such as stroke and polycystic kidney disease are linked to altered NEDD9 function [148,152]. In databanks, MICALs are also classified as NEDD9/CASL-interacting proteins [153]. This interaction depends on binding of the proline-rich motif PPKPP (Pro-Pro-Lys-Pro-Pro) of MICALs to the SH3 domain of the Cas family members Cas and CasL [137]. Binding of NEDD9 to MICAL1 was shown to facilitate hypoxia-induced gastric cancer cell migration via Rac1-dependent manner [154]. It was shown that MICAL connects NEDD9/CASL to the intermediate filament [137].\u003c/p\u003e\u003cp\u003eHuman NEDD9 has a complex domain organization with multiple functional domains and regions, such as SH3 domain (residues 1\u0026ndash;66), regions required for interaction with E3 ubiquitin-protein ligase Itchy homolog (ITCH, residues 1-505) [155], SH2 binding sites (residues 67\u0026ndash;351 and 352\u0026ndash;626), spindle-regulatory protein D1M1 (residues 102\u0026ndash;229), Src substrate cortactin (CTTN, residues 351\u0026ndash;834) [136], and serine/threonine-protein kinase PLK1 (residues 710\u0026ndash;834) [142]. Conserved C-terminal domain (residues 626\u0026ndash;834) includes divergent helix-loop-helix motif (residues 710\u0026ndash;760) involved in homodimerization and heterodimerization with a corresponding region of p130\u003csup\u003eCas\u003c/sup\u003e and other HLH motif-containing proteins [156]. NEDD9 also contains multiple regions with low complexity and compositional bias. In additional to the canonical isoform, NEDD9 is known to have two alternatively spiced isoforms, with the isoform 2 missing most of the sequence (residues 175\u0026ndash;834) and possessing changes in the remaining C-terminal region (residues 166\u0026ndash;174) ITPVRTGHGYVYEYPSRYQK \u0026rarr; FQRDGQVSYFLVRASKQTSL [40] and with the isoform 3 containing MKYK \u0026rarr; MWTR changes in the N-terminal region (residues 1\u0026ndash;4).\u003c/p\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e15\u003c/span\u003eA shows that as expected from its multifunctionality, human NEDD9 contains high levels of intrinsic disorder and contains 12 MoRFs and multiple PTM sites. In fact, according to the outputs of RIDAO, this protein is characterized by the PONDR\u003csup\u003e\u0026reg;\u003c/sup\u003e VLXT, PONDR\u003csup\u003e\u0026reg;\u003c/sup\u003e VSL2, PONDR\u003csup\u003e\u0026reg;\u003c/sup\u003e VL3, IUPred Short, IUPred long, PONDR\u003csup\u003e\u0026reg;\u003c/sup\u003e FIT, and MDP PPIDR values of 36.33%, 59.71%, 54.68%, 39.45%, 47.12%, 39.93%, and 47.00%, respectively, with the corresponding ADS values being 0.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36, 0.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26, 0.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26, 0.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23, 0.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23, 0.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27, and 0.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24. High level of intrinsic disorder in human NEDD9 is further supported by Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e15\u003c/span\u003eB showing 3D structure model of this protein generated by AlphaFold. There are three regions with high per-residue model confidence scores p\u003csub\u003eLDDT\u003c/sub\u003e values, residues 1\u0026ndash;61 corresponding to the N-terminal SH3 domain, serine-rich domain (residues 408\u0026ndash;559) and conserved C-terminal domain (residues 701\u0026ndash;834), whereas the remaining regions (residues 62\u0026ndash;407 and 560\u0026ndash;700) represent disordered loops with very low p\u003csub\u003eLDDT\u003c/sub\u003e \u0026lt; 50, which contain 58% of protein sequence.\u003c/p\u003e\u003cp\u003eFuzDrop analysis revealed that NEDD9 is characterized by the pLLPS score of 0.9690 and contains 10 DPRs (residues 68\u0026ndash;87, 108\u0026ndash;129, 136\u0026ndash;152, 179\u0026ndash;202, 234\u0026ndash;262, 282\u0026ndash;410, 496\u0026ndash;506, 560\u0026ndash;628, 654\u0026ndash;668, and 671\u0026ndash;704) indicating that this protein can undergo spontaneous LLPS and can serve as a droplet driver. The presence of 19 aggregation hot spots and 23 regions with context-dependent interactions in combination with the presence of 12 MoRFs suggests that NEDD9 can be characterized by high interaction potential. Figure\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e15\u003c/span\u003eC supports this notion and shows that this protein forms a PPI network with 96 nodes and 801 edges. The network has an average local clustering coefficient of 0.671 and average node degree of 16.7.\u003c/p\u003e\u003cp\u003eMembers of the NEDD9-centered PPI network are taking part in various biological processes, with actin filament organization (GO:0007015; strength\u0026thinsp;=\u0026thinsp;2.49), supramolecular fiber organization (GO:0097435; strength\u0026thinsp;=\u0026thinsp;2.34), actin polymerization or depolymerization (GO:0008154; strength\u0026thinsp;=\u0026thinsp;2.29), integrin-mediated signaling pathway (GO:0007229; strength\u0026thinsp;=\u0026thinsp;2.11), and enzyme-linked receptor protein signaling pathway (GO:0007167; strength\u0026thinsp;=\u0026thinsp;2.04) being the most enriched representatives of the 595 corresponding significantly enriched GO-terms. The most highly enriched molecular functions represented in this network (from 59 corresponding significantly enriched GO-terms) are kinase binding (GO:0019900; strength\u0026thinsp;=\u0026thinsp;2.24), protein kinase binding (GO:0019901; strength\u0026thinsp;=\u0026thinsp;2.19), cytoskeletal protein binding (GO:0008092; strength\u0026thinsp;=\u0026thinsp;1.74), enzyme binding (GO:0019899; strength\u0026thinsp;=\u0026thinsp;1.68), and actin binding GO:0003779; strength\u0026thinsp;=\u0026thinsp;1.58). The most enriched cellular components, where these proteins are located (from 80 corresponding significantly enriched GO-terms) include cell leading edge (GO:0031252; strength\u0026thinsp;=\u0026thinsp;2.24), actin cytoskeleton (GO:0015629; strength\u0026thinsp;=\u0026thinsp;2.01), lamellipodium (GO:0030027; strength\u0026thinsp;=\u0026thinsp;1.91), focal adhesion (GO:0005925; strength\u0026thinsp;=\u0026thinsp;1.86), and cytoskeleton (GO:0005856; strength\u0026thinsp;=\u0026thinsp;1.48).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"4. Conclusions","content":"\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eOur bioinformatics analysis of MICAL family protein demonstrated that all five key proteins are highly disordered with mean disorder prediction above 30%. Out of 62 interactors of MICALs, approximately 50% are also highly disordered. Furthermore, in-depth analysis of these proteins revealed that disorder indeed has functional implications. Notably, MICALs and their interactors were shown to be involved in multiple interactions. MICALs and many disordered MICAL interactors are predicted to have a high tendency to undergo LLPS, forming biomolecular condensates and membrane-less organelles. These characteristics support and highlight why these proteins play important roles in cellular functions. Diving deep into the disorder profiles of these proteins can help us to identify potential targets for diseases, such as neurodegeneration and cancer.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u0026nbsp;\u003c/strong\u003eConceptualization, V.N.U.; methodology, V.N.U.; validation, H.N.A. and V.N.U.; formal analysis, H.N.A. and V.N.U.; investigation, H.N.A. and V.N.U.; data curation, H.N.A. and V.N.U.; writing—original draft preparation, H.N.A. and V.N.U.; writing—review and editing, H.N.A. and V.N.U.; visualization, V.N.U.; supervision, V.N.U. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThis research received no external funding.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInstitutional Review Board Statement:\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed Consent Statement:\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement:\u0026nbsp;\u003c/strong\u003eThe data are contained within the article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest:\u0026nbsp;\u003c/strong\u003eThe authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003e Wu, H.; Yesilyurt, H.G.; Yoon, J.; Terman, J.R. The MICALs are a family of F-actin dismantling oxidoreductases conserved from Drosophila to humans. \u003cem\u003eScientific reports\u003c/em\u003e \u003cb\u003e2018\u003c/b\u003e, \u003cem\u003e8\u003c/em\u003e, 937.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e Vitali, T.; Maffioli, E.; Tedeschi, G.; Vanoni, M.A. Properties and catalytic activities of MICAL1, the flavoenzyme involved in cytoskeleton dynamics, and modulation by its CH, LIM and C-terminal domains. \u003cem\u003eArch Biochem Biophys\u003c/em\u003e \u003cb\u003e2016\u003c/b\u003e, \u003cem\u003e593\u003c/em\u003e, 24\u0026ndash;37, doi:10.1016/j.abb.2016.01.016.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e Zucchini, D.; Caprini, G.; Pasterkamp, R.J.; Tedeschi, G.; Vanoni, M.A. Kinetic and spectroscopic characterization of the putative monooxygenase domain of human MICAL-1. \u003cem\u003eArch Biochem Biophys\u003c/em\u003e \u003cb\u003e2011\u003c/b\u003e, \u003cem\u003e515\u003c/em\u003e, 1\u0026ndash;13, doi:10.1016/j.abb.2011.08.004.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e Alto, L.T.; Terman, J.R. MICALs. \u003cem\u003eCurrent Biology\u003c/em\u003e \u003cb\u003e2018\u003c/b\u003e, \u003cem\u003e28\u003c/em\u003e, R538-R541.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e Giridharan, S.S.; Caplan, S. MICAL-family proteins: Complex regulators of the actin cytoskeleton. \u003cem\u003eAntioxid Redox Signal\u003c/em\u003e \u003cb\u003e2014\u003c/b\u003e, \u003cem\u003e20\u003c/em\u003e, 2059\u0026ndash;2073, doi:10.1089/ars.2013.5487.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e Rajan, S.; Terman, J.R.; Reisler, E. MICAL-mediated oxidation of actin and its effects on cytoskeletal and cellular dynamics. \u003cem\u003eFrontiers in Cell and Developmental Biology\u003c/em\u003e \u003cb\u003e2023\u003c/b\u003e, \u003cem\u003e11\u003c/em\u003e, 1124202.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e Grintsevich, E.E.; Ge, P.; Sawaya, M.R.; Yesilyurt, H.G.; Terman, J.R.; Zhou, Z.H.; Reisler, E. 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ESpritz: accurate and fast prediction of protein disorder. \u003cem\u003eBioinformatics\u003c/em\u003e \u003cb\u003e2012\u003c/b\u003e, \u003cem\u003e28\u003c/em\u003e, 503\u0026ndash;509, doi:10.1093/bioinformatics/btr682.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cell-biochemistry-and-biophysics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cbbi","sideBox":"Learn more about [Cell Biochemistry and Biophysics](http://link.springer.com/journal/12013)","snPcode":"12013","submissionUrl":"https://submission.nature.com/new-submission/12013/3","title":"Cell Biochemistry and Biophysics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"intrinsically disordered proteins, intrinsically disordered regions, protein-protein interactions, posttranslational modifications, liquid-liquid phase separation, alternative splicing, MICAL","lastPublishedDoi":"10.21203/rs.3.rs-7782044/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7782044/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMembers of the family of molecule interacting with CASL protein (MICAL) proteins play crucial roles in the cytoskeleton rearrangements by promoting depolymerization of F-actin via oxidation of the specific methionine residues to methionine-sulfoxides. These proteins also have multiple other functions and are related to the pathogenesis of various maladies. However, not much is known about the prevalence and functionality of intrinsically disordered regions in MICALs. To fill this gap, we conducted a comprehensive bioinformatics analysis of five human MICAL protein family members, MICAL1, MICAL2, MICAL3, MICAL-L1, and MICAL-L2, to assess their intrinsic disorder characteristics. We also focused on the top 10 most disordered interactors of these proteins. We utilized a set of bioinformatics tools, FuzDrop, RIDAO, InterPro, IuPred, STRING, AlphaFold, and UniProt, for the extensive analysis of these proteins. This analysis revealed that all five MICALs are highly disordered; they take part in myriad of cellular functions, and are also involved in pathogenesis of various diseases, such as neurodegeneration, cancer, and muscle dystrophy. Our study focused on the intrinsic disorder of these proteins to highlight important implications of disorder in protein functionality. These insights may help to find out potential therapeutic targets in biological processes controlled by MICAL proteins.\u003c/p\u003e","manuscriptTitle":"Prevalence and functional roles of intrinsic disorder in the [F-actin]-monooxygenase MICAL family members and their interactors","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-21 09:14:21","doi":"10.21203/rs.3.rs-7782044/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-11-05T14:45:27+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-31T19:31:10+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"94085673492954913797049498323633663985","date":"2025-10-16T20:00:02+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"318620202448348808407342139095491674018","date":"2025-10-09T08:15:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"28523128238002852876746177302131998848","date":"2025-10-08T18:31:34+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-08T15:05:04+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-06T15:33:13+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-06T15:31:23+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cell Biochemistry and Biophysics","date":"2025-10-04T22:43:45+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-biochemistry-and-biophysics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cbbi","sideBox":"Learn more about [Cell Biochemistry and Biophysics](http://link.springer.com/journal/12013)","snPcode":"12013","submissionUrl":"https://submission.nature.com/new-submission/12013/3","title":"Cell Biochemistry and Biophysics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"0b8e1c51-c269-4758-be6e-c7a6d6cf63ec","owner":[],"postedDate":"October 21st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-29T16:05:56+00:00","versionOfRecord":{"articleIdentity":"rs-7782044","link":"https://doi.org/10.1007/s12013-025-01978-x","journal":{"identity":"cell-biochemistry-and-biophysics","isVorOnly":false,"title":"Cell Biochemistry and Biophysics"},"publishedOn":"2025-12-23 15:58:16","publishedOnDateReadable":"December 23rd, 2025"},"versionCreatedAt":"2025-10-21 09:14:21","video":"","vorDoi":"10.1007/s12013-025-01978-x","vorDoiUrl":"https://doi.org/10.1007/s12013-025-01978-x","workflowStages":[]},"version":"v1","identity":"rs-7782044","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7782044","identity":"rs-7782044","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}