Bioinformatics analysis of actin interactome: Characterization of the nuclear and cytoplasmic actin-binding proteins

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Mokin, Olga I. Povarova, Iuliia A. Antifeeva, Alexey V. Artemov, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4014138/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract Actin is present in the cytoplasm and nucleus of every eukaryotic cell. In the cytoplasm, framework and motor functions of actin are associated with its ability to polymerize to form F-actin. In the nucleus, globular actin plays a significant functional role. For a globular protein, actin has a uniquely large number of proteins with which it interacts. Bioinformatics analysis of the actin interactome showed that only a part of actin-binding proteins are both cytoplasmic and nuclear. There are proteins that interact only with cytoplasmic, or only with nuclear actin. The first pool includes proteins associated with the formation, regulation, and functioning of the actin cytoskeleton predominate, while nuclear actin-binding proteins are involved in the majority of key nuclear processes, from regulation of transcription to DNA damage response. Bioinformatics analysis of the structure of actin-binding proteins showed that these are mainly intrinsically disordered proteins, many of which are part of membrane-less organelles. Interestingly, although the number of actin-binding proteins in the nucleus is greater than in the cytoplasm, the drivers for the formation of the membrane-less organelles in the cytoplasm are significantly (four times) greater than in the nucleus. actin actin-binding proteins intrinsically disordered proteins liquid-liquid phase separation protein-protein interactions Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Actin was first isolated from the muscle cells, in which this protein is one of the main components of the actomyosin motor [ 1 ]. Subsequently, it turned out that actin is present in every cell of the body. At the end of the twentieth century, modern ideas about the role of actin in the formation of the cytoskeleton, cell motility, interaction of cells with the environment, and cell division were formed [ 2 – 5 ]. The process of actin polymerization - depolymerization was studied in detail, the main actin-bound proteins were identified that are involved in the initiation of the polymerization of actin monomers, elongation and branching of F-actin, the formation of the cytoskeleton in the cytoplasm of cells and contractile fibers in muscle cells [ 6 ]. Almost simultaneously with actin in the cytoplasm of non-muscle cells, actin was found in the cell nucleus [ 7 – 11 ]. However, the existence of nuclear actin has long caused great skepticism among researchers [ 12 ]. This was mainly due to the fact that actin filaments (F-actin) could not be detected in the nucleus. In addition, the abundance of actin in the cytoplasm did not exclude the possibility that the actin detected in the nucleus or nuclear fractions was an admixture of the cytoplasmic actin. Finally, the absence of F-actin in the nucleus has called into question the functional significance of nuclear actin. All this slowed down the development of nuclear actin research for several decades. However, achievements over the past 15 years have convincingly demonstrated not only the existence of nuclear actin, but also its significant functional role in various nuclear processes [ 13 , 14 ]. In recent years, thanks to the development of new technologies, a detailed study of nuclear actin has become possible, and many studies have appeared devoted to the study of its functional role. In particular, it was shown that although actin does not have a nuclear localization signal, monomeric G-actin constantly moves between the nucleus and the cytoplasm. It has been shown that actin moves from the cytoplasm to the nucleus through nuclear pores in a complex with caffeline and importin 9, and from the nucleus into the cytoplasm in a complex with profilin and exportin 6 [ 15 , 16 ]. It has been shown that most nuclear actin exists in monomeric form, with dimers and oligomers also observed [ 17 – 20 ]. If in the cytoplasm, actin monomers are considered as simple building blocks of F-actin filaments, then in the nucleus the functional role of G-actin is much more diverse. Nuclear G-actin is a subunit of several chromatin remodeling and modifying complexes that control chromatin structure and accessibility by regulating nucleosome repositioning and histone modifications [ 21 , 22 ]. It has been shown that there are two components of chromatin mobility: Brownian motion and actin / ATP-dependent motion, which allows reordering of chromatin compaction that is important during transcription or repair [ 23 , 24 ]. Nuclear actin regulates various transcription factors and interacts with all RNA polymerases [ 25 – 28 ]. Since changes in chromatin and genome architecture are known attributes of embryonic and postembryonic cell differentiation and correlate with various diseases, an important role of nuclear actin is that it participates in the differentiation programs during neurogenesis, myogenesis, organ formation, and the development of various diseases [ 29 , 30 ]. The multifunctionality of nuclear actin suggests that nuclear actin is also important for immune cell differentiation and function [ 31 – 33 ]. In addition, nuclear actin is involved in apoptosis [ 34 ], counteracting viral infection [ 35 ], changing the structure of the nuclear membrane [ 36 – 39 ], and DNA repair [ 40 ]. Therefore, if in the cytoplasm actin is mainly present in the F-form and plays a structural-motor role, then in the cell nucleus, most of the actin is present in the G-form and interacts with nuclear proteins, chromatin, and DNA. Furthermore, in the nucleus, there are polymeric forms of actin that differ from F-actin [ 41 – 43 ], called “short oligomers” of nuclear actin or “rods” [ 44 ]. It is known that under normal conditions, these structures are dynamic, but under stress and disease they are persistent and their number increases [ 33 , 44 , 45 ]. We assume that “short oligomers” of nuclear actin may be similar to the so-called inactivated actin, a monodisperse associate consisting of 14–16 monomers that appears in vitro under any denaturing influences [ 46 – 48 ]. We also assume that the formation of inactivated actin is associated with a specific pathway of oligomerization of actin monomers in vitro and that this situation may be associated with the differences in actin-binding proteins in the cytoplasm and in the cell nucleus. Cells are crowded with macromolecules and all proteins are permanently in contact with their neighbor. This became especially evident with the discovery of intrinsically disordered proteins (IDPs). IDPs are always in interaction with other proteins or nucleic acids. The actin interactome is significantly wider than that of other globular proteins [ 49 ]. Actin, which has more than 800 proteins in its interactome, is more similar to an IDP hub than to a globular protein [ 50 ]. Well-studied actin-binding proteins are only a small part of the proteins interacting with cytoplasmic and nuclear actin. In this regard, we decided to conduct a bioinformatics analysis of the actin interactome and compare the interactomes of cytoplasmic and nuclear actin. Materials & Methods A dataset of actin interaction network was assembled using STRING http://string-db.org/ and BIOGRID (v.4.4) https://thebiogrid.org/ databases. 887 proteins that physically interact with actin were selected for analysis. The intrinsic disorder predispositions analysis of the studied proteins was performed by the RIDAO online platform [ 51 ]. The PER(VSL2B) indicator was used as a measure of protein structural disorder. Proteins with disorder score more than 0.5 were marked as “disorder”. The tendency of studied proteins to spontaneous and induced phase separation was elevated using FuzDrop [ 52 ] and PSPredictor [ 53 ]. Proteins with PSPredcitor score > 0.5 and FuzDrop score > 0.6 were marked as prone to LLPS. According to FuzDrop, proteins with pLPS ≥ 0.60 were marked as drivers of liquid-liquid phase separation and proteins with droplet-promoting regions, defined as consecutive residues with pDP ≥ 0.60 were marked as droplet-clients. The charge, hydrophobicity and aggregation propensity were calculated as described in [ 54 ]. Gene Onthology (GO) was performed using the Enrichr resource ( https://maayanlab.cloud/Enrichr ). The p-values were computed from the Fisher exact test Results and Discussion The β-actin interactome was constructed using a comparative analysis of the StringDB and Biogrid databases. The β-actin interactome derived from StringDB showed that actin-binding proteins include proteins that are present only in the cytoplasm, only in the nucleus, and proteins that are present in both the nucleus and the cytoplasm. However, the network of interactions of any protein in STRING analysis cannot exceed 500 partners. Therefore, for further work, we focused on the analysis of the interactome using the BioGRID database. We analyzed 887 proteins shown to physically interact with actin. The results of the initial analysis are presented in Fig. 1 B, Table S1 . It turned out that in this database there are 887 actin-binding proteins, including 293 cytoplasmic, 77 nuclear proteins, 427 proteins found in both the cytoplasm and the nucleus, as well as 90 others (including those localized in membranes and exosomes). Bioinformatics analysis fully confirmed that the main role of actin in the nucleus is associated with interaction with chromatin and DNA. Actin-binding proteins that interact only with nuclear actin are involved in regulation of response to DNA damage stimulus, regulation of nucleobase-containing compound metabolic process, regulation of double-strand break repair, chromosome organization, chromatin organization, etc. (Fig. 2 ). At the same time, the actin-binding proteins found only in the cytoplasm are associated exclusively with the formation, regulation, and functioning of the actin cytoskeleton (Fig. 3 ). Interestingly, the functions of these proteins do not overlap. Actin-binding proteins, which are present both in the cytoplasm and in the nucleus, are associated with various metabolic processes. It is not surprising that actin-binding proteins, found only in the nucleus or only in the cytoplasm so significantly different in their functions, also have large variability in their structural properties. We analyzed these proteins for their structure (dis)orderedness, propensity for liquid–liquid phase separation (LLPS), and ability to initiate the formation of membrane-less organelles (MLOs) or to enter the already formed MLOs (Fig. 4 ). Most actin-binding proteins are intrinsically disordered proteins. Only 2% of cytoplasmic actin-binding proteins are globular proteins, the rest either have disordered regions or a completely disordered structure. The percentage of disordered proteins is significantly higher in the nucleus compared to cytoplasmic proteins (Fig. 4 , panel A). Assessing the propensity of proteins to liquid–liquid phase separation (LLPS) showed that among cytoplasmic actin-binding proteins, only 25% are prone to LLPS, while among nuclear actin-binding proteins, 44% are capable of LLPS (Fig. 4 , panel B ). At the same time, only 3% of nuclear actin-binding proteins can be classified as proteins capable of initiating the formation of MLOs, which is significantly lower than the corresponding number of the cytoplasmic actin-binding proteins (Fig. 4 , panel C). Apparently, intrinsically disordered proteins are extremely important for actin to perform all its functions. At the moment, there is already evidence of how actin polymerization occurs inside MLOs [ 55 , 56 ]. By now it has become obvious that the formation of MLOs is associated with most, if not all intracellular processes (see e.g. [ 57 – 59 ]). However, the details of actin functioning in the cell nucleus remain to be elucidated. Conclusions Summarizing the analysis of the actin interactome in the cell, it can be noted that most of the actin interactors are intrinsically disordered proteins, many of which are prone to LLPS. This is especially important in connection with the currently developing ideas about the role of LLPS and MLO in the organization of all intracellular processes. The significant difference in the properties of nuclear and cytoplasmic actin-binding proteins indicates that their closer study should also shed light on the features of the structural forms and functions of the still poorly studied oligomeric forms of nuclear actin. Declarations Funding The work was supported by the Russian Science Foundation (project No. 23-15-00494, IMK) Competing Interests We confirm that there are no relevant financial or non-financial competing interests to report. Data availability All data supporting the findings of this manuscript are available from the corresponding author upon reasonable request. Author Contributions Yakov I. Mokin: conceptualization; software, investigation; validation; formal analysis; writing – review and editing. Olga I. Povarova: visualization; project administration; writing – original draft; writing – review and editing. Iuliia A. Antifeeva: visualization; project administration; writing – original draft; writing – review and editing. Konstantin K. Turoverov: conceptualization; supervision; writing – original draft; writing – review and editing. Alexey V. Artermov: visualization; writing – original draft; writing – review and editing. Vladimir N. Uversky: conceptualization; supervision, writing – review and editing. Alexander V. Fonin: conceptualization; supervision, writing – original draft; writing – review and editing. Irina M. Kuznetsova: conceptualization; supervision; funding acquisition; project administration; resources; writing – original draft; writing – review and editing. References Halliburton WD (1887) On Muscle-Plasma. 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Supplementary Files FigureS1.svg TableS1.xlsx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 26 Mar, 2024 Reviews received at journal 24 Mar, 2024 Reviewers agreed at journal 12 Mar, 2024 Reviewers invited by journal 12 Mar, 2024 Editor assigned by journal 05 Mar, 2024 Submission checks completed at journal 05 Mar, 2024 First submitted to journal 04 Mar, 2024 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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Mokin","email":"","orcid":"","institution":"Institute of Cytology","correspondingAuthor":false,"prefix":"","firstName":"Yakov","middleName":"I.","lastName":"Mokin","suffix":""},{"id":276488010,"identity":"3c66e7c1-ff9d-4e1b-b4f8-cbb03bf86c48","order_by":1,"name":"Olga I. Povarova","email":"","orcid":"","institution":"Institute of Cytology","correspondingAuthor":false,"prefix":"","firstName":"Olga","middleName":"I.","lastName":"Povarova","suffix":""},{"id":276488011,"identity":"ed5b3de3-e816-4e5d-8742-9523d0d1f5a6","order_by":2,"name":"Iuliia A. Antifeeva","email":"","orcid":"","institution":"Institute of Cytology","correspondingAuthor":false,"prefix":"","firstName":"Iuliia","middleName":"A.","lastName":"Antifeeva","suffix":""},{"id":276488012,"identity":"8ca0d859-7f97-4e00-8bd3-a4146397779c","order_by":3,"name":"Alexey V. Artemov","email":"","orcid":"","institution":"Institute of Cytology","correspondingAuthor":false,"prefix":"","firstName":"Alexey","middleName":"V.","lastName":"Artemov","suffix":""},{"id":276488013,"identity":"87da80aa-0a25-4f74-a7d2-16574c34c7f4","order_by":4,"name":"Vladimir N. Uversky","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAvUlEQVRIiWNgGAWjYPCCBAZ+IPmBgYGZBC2SDQyMM0jTYnCAWC0GN3IMH1f8SUvcfCP9YQNDhXViAxFajA3PtuUkbgPqbWA4k05Yi+SMtDTJxoYKkBb2B4xth4nSkv6z4U9F4uYZQIcx/iNCC79E8jHGBracxA0SCYYNjA3EaOF5fFiysS3NeMaZN4YNCcfSjQlqYWNPbPzY8CdZtr8d6LAPNdayBLXAgCNYZQKxykHAnhTFo2AUjIJRMMIAAF3rQ/6NaLI3AAAAAElFTkSuQmCC","orcid":"","institution":"University of South Florida","correspondingAuthor":true,"prefix":"","firstName":"Vladimir","middleName":"N.","lastName":"Uversky","suffix":""},{"id":276488014,"identity":"bda48002-29d2-4981-b9ee-886c8e65ad07","order_by":5,"name":"Konstantin K. Turoverov","email":"","orcid":"","institution":"Institute of Cytology","correspondingAuthor":false,"prefix":"","firstName":"Konstantin","middleName":"K.","lastName":"Turoverov","suffix":""},{"id":276488015,"identity":"f24ce86c-7869-474c-93d2-2eed3917c5c8","order_by":6,"name":"Irina M. Kuznetsova","email":"","orcid":"","institution":"Institute of Cytology","correspondingAuthor":false,"prefix":"","firstName":"Irina","middleName":"M.","lastName":"Kuznetsova","suffix":""},{"id":276488016,"identity":"e4768df1-6385-47c4-8820-79e35ed44c41","order_by":7,"name":"Alexander V. Fonin","email":"","orcid":"","institution":"Institute of Cytology","correspondingAuthor":false,"prefix":"","firstName":"Alexander","middleName":"V.","lastName":"Fonin","suffix":""}],"badges":[],"createdAt":"2024-03-04 19:15:25","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4014138/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4014138/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":52131117,"identity":"59c65a9e-3436-4007-9644-2fe7d23eadfe","added_by":"auto","created_at":"2024-03-07 08:11:32","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":660912,"visible":true,"origin":"","legend":"\u003cp\u003eβ-Actin interactome. Panel A. Actin interactome constructed from StringDB. Blue represents cytoplasmic proteins, red represents nuclear proteins, blue/red represents proteins found both in the cytoplasm and in the nucleus. Panel B. The actin interactome based on the BioGRID database. The blue sector corresponds to cytoplasmic proteins, red – nuclear, blue-red – proteins found both in the cytoplasm and in the nucleus, gray – to other proteins.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4014138/v1/0e4e0475ee2f60521a680663.png"},{"id":52131116,"identity":"e994d6f7-c161-4d68-8864-6d4c6cb83669","added_by":"auto","created_at":"2024-03-07 08:11:32","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":74740,"visible":true,"origin":"","legend":"\u003cp\u003eDiagram representing biological processes involving actin-binding proteins that interact only with the cytoplasmic actin.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4014138/v1/8109407c9c163a8151c3a0bd.png"},{"id":52131115,"identity":"9888dd50-511f-4546-97c2-0ac0c8e403db","added_by":"auto","created_at":"2024-03-07 08:11:32","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":79935,"visible":true,"origin":"","legend":"\u003cp\u003eDiagram representing biological processes involving actin-binding proteins that interact only with the nuclear actin.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4014138/v1/af7c494c98ae3d1a82f9e1db.png"},{"id":52131119,"identity":"2bf725f0-5341-4696-915a-9954e4b15f6e","added_by":"auto","created_at":"2024-03-07 08:11:32","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":96169,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of the degree of structure (A), propensity to undergo LLPS (B) and relation to the formation of MLO (C) of the actin-binding proteins located just in cytoplasm (inner circle), just in nucleus (outer circle) and both in cytoplasm and nucleus (middle circle).\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4014138/v1/1c932b8eede4694115b69587.png"},{"id":52131670,"identity":"ea72758b-58ca-4db2-9009-fd5f29409ae1","added_by":"auto","created_at":"2024-03-07 08:19:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1015298,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4014138/v1/fb9f57b8-200c-428a-a775-e9c7e00c5628.pdf"},{"id":52131121,"identity":"10cfbe47-a808-47d1-b029-5db18c6e4b5c","added_by":"auto","created_at":"2024-03-07 08:11:33","extension":"svg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":19312182,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS1.svg","url":"https://assets-eu.researchsquare.com/files/rs-4014138/v1/f872904f154dbb7d9611e6bf.svg"},{"id":52131118,"identity":"564ec390-2f52-40ba-b043-f44575b4f187","added_by":"auto","created_at":"2024-03-07 08:11:32","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":10836,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4014138/v1/f1f60d15035eb5c63389cf99.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Bioinformatics analysis of actin interactome: Characterization of the nuclear and cytoplasmic actin-binding proteins","fulltext":[{"header":"Introduction","content":"\u003cp\u003eActin was first isolated from the muscle cells, in which this protein is one of the main components of the actomyosin motor [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Subsequently, it turned out that actin is present in every cell of the body. At the end of the twentieth century, modern ideas about the role of actin in the formation of the cytoskeleton, cell motility, interaction of cells with the environment, and cell division were formed [\u003cspan additionalcitationids=\"CR3 CR4\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The process of actin polymerization - depolymerization was studied in detail, the main actin-bound proteins were identified that are involved in the initiation of the polymerization of actin monomers, elongation and branching of F-actin, the formation of the cytoskeleton in the cytoplasm of cells and contractile fibers in muscle cells [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAlmost simultaneously with actin in the cytoplasm of non-muscle cells, actin was found in the cell nucleus [\u003cspan additionalcitationids=\"CR8 CR9 CR10\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. However, the existence of nuclear actin has long caused great skepticism among researchers [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. This was mainly due to the fact that actin filaments (F-actin) could not be detected in the nucleus. In addition, the abundance of actin in the cytoplasm did not exclude the possibility that the actin detected in the nucleus or nuclear fractions was an admixture of the cytoplasmic actin. Finally, the absence of F-actin in the nucleus has called into question the functional significance of nuclear actin. All this slowed down the development of nuclear actin research for several decades. However, achievements over the past 15 years have convincingly demonstrated not only the existence of nuclear actin, but also its significant functional role in various nuclear processes [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn recent years, thanks to the development of new technologies, a detailed study of nuclear actin has become possible, and many studies have appeared devoted to the study of its functional role. In particular, it was shown that although actin does not have a nuclear localization signal, monomeric G-actin constantly moves between the nucleus and the cytoplasm. It has been shown that actin moves from the cytoplasm to the nucleus through nuclear pores in a complex with caffeline and importin 9, and from the nucleus into the cytoplasm in a complex with profilin and exportin 6 [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. It has been shown that most nuclear actin exists in monomeric form, with dimers and oligomers also observed [\u003cspan additionalcitationids=\"CR18 CR19\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. If in the cytoplasm, actin monomers are considered as simple building blocks of F-actin filaments, then in the nucleus the functional role of G-actin is much more diverse. Nuclear G-actin is a subunit of several chromatin remodeling and modifying complexes that control chromatin structure and accessibility by regulating nucleosome repositioning and histone modifications [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. It has been shown that there are two components of chromatin mobility: Brownian motion and actin / ATP-dependent motion, which allows reordering of chromatin compaction that is important during transcription or repair [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNuclear actin regulates various transcription factors and interacts with all RNA polymerases [\u003cspan additionalcitationids=\"CR26 CR27\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Since changes in chromatin and genome architecture are known attributes of embryonic and postembryonic cell differentiation and correlate with various diseases, an important role of nuclear actin is that it participates in the differentiation programs during neurogenesis, myogenesis, organ formation, and the development of various diseases [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The multifunctionality of nuclear actin suggests that nuclear actin is also important for immune cell differentiation and function [\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. In addition, nuclear actin is involved in apoptosis [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], counteracting viral infection [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], changing the structure of the nuclear membrane [\u003cspan additionalcitationids=\"CR37 CR38\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], and DNA repair [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTherefore, if in the cytoplasm actin is mainly present in the F-form and plays a structural-motor role, then in the cell nucleus, most of the actin is present in the G-form and interacts with nuclear proteins, chromatin, and DNA. Furthermore, in the nucleus, there are polymeric forms of actin that differ from F-actin [\u003cspan additionalcitationids=\"CR42\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], called \u0026ldquo;short oligomers\u0026rdquo; of nuclear actin or \u0026ldquo;rods\u0026rdquo; [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. It is known that under normal conditions, these structures are dynamic, but under stress and disease they are persistent and their number increases [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. We assume that \u0026ldquo;short oligomers\u0026rdquo; of nuclear actin may be similar to the so-called inactivated actin, a monodisperse associate consisting of 14\u0026ndash;16 monomers that appears \u003cem\u003ein vitro\u003c/em\u003e under any denaturing influences [\u003cspan additionalcitationids=\"CR47\" citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. We also assume that the formation of inactivated actin is associated with a specific pathway of oligomerization of actin monomers \u003cem\u003ein vitro\u003c/em\u003e and that this situation may be associated with the differences in actin-binding proteins in the cytoplasm and in the cell nucleus.\u003c/p\u003e \u003cp\u003eCells are crowded with macromolecules and all proteins are permanently in contact with their neighbor. This became especially evident with the discovery of intrinsically disordered proteins (IDPs). IDPs are always in interaction with other proteins or nucleic acids. The actin interactome is significantly wider than that of other globular proteins [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Actin, which has more than 800 proteins in its interactome, is more similar to an IDP hub than to a globular protein [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Well-studied actin-binding proteins are only a small part of the proteins interacting with cytoplasmic and nuclear actin. In this regard, we decided to conduct a bioinformatics analysis of the actin interactome and compare the interactomes of cytoplasmic and nuclear actin.\u003c/p\u003e"},{"header":"Materials \u0026 Methods","content":"\u003cp\u003eA dataset of actin interaction network was assembled using STRING \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://string-db.org/\u003c/span\u003e\u003cspan address=\"http://string-db.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e and BIOGRID (v.4.4) \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://thebiogrid.org/\u003c/span\u003e\u003cspan address=\"https://thebiogrid.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e databases. 887 proteins that physically interact with actin were selected for analysis. The intrinsic disorder predispositions analysis of the studied proteins was performed by the RIDAO online platform [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. The PER(VSL2B) indicator was used as a measure of protein structural disorder. Proteins with disorder score more than 0.5 were marked as \u0026ldquo;disorder\u0026rdquo;. The tendency of studied proteins to spontaneous and induced phase separation was elevated using FuzDrop [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e] and PSPredictor [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Proteins with PSPredcitor score\u0026thinsp;\u0026gt;\u0026thinsp;0.5 and FuzDrop score\u0026thinsp;\u0026gt;\u0026thinsp;0.6 were marked as prone to LLPS. According to FuzDrop, proteins with pLPS\u0026thinsp;\u0026ge;\u0026thinsp;0.60 were marked as drivers of liquid-liquid phase separation and proteins with droplet-promoting regions, defined as consecutive residues with pDP\u0026thinsp;\u0026ge;\u0026thinsp;0.60 were marked as droplet-clients. The charge, hydrophobicity and aggregation propensity were calculated as described in [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Gene Onthology (GO) was performed using the Enrichr resource (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://maayanlab.cloud/Enrichr\u003c/span\u003e\u003cspan address=\"https://maayanlab.cloud/Enrichr\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The p-values were computed from the Fisher exact test\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003eThe β-actin interactome was constructed using a comparative analysis of the StringDB and Biogrid databases. The β-actin interactome derived from StringDB showed that actin-binding proteins include proteins that are present only in the cytoplasm, only in the nucleus, and proteins that are present in both the nucleus and the cytoplasm. However, the network of interactions of any protein in STRING analysis cannot exceed 500 partners. Therefore, for further work, we focused on the analysis of the interactome using the BioGRID database. We analyzed 887 proteins shown to physically interact with actin. The results of the initial analysis are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. It turned out that in this database there are 887 actin-binding proteins, including 293 cytoplasmic, 77 nuclear proteins, 427 proteins found in both the cytoplasm and the nucleus, as well as 90 others (including those localized in membranes and exosomes).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBioinformatics analysis fully confirmed that the main role of actin in the nucleus is associated with interaction with chromatin and DNA. Actin-binding proteins that interact only with nuclear actin are involved in regulation of response to DNA damage stimulus, regulation of nucleobase-containing compound metabolic process, regulation of double-strand break repair, chromosome organization, chromatin organization, etc. (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). At the same time, the actin-binding proteins found only in the cytoplasm are associated exclusively with the formation, regulation, and functioning of the actin cytoskeleton (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Interestingly, the functions of these proteins do not overlap. Actin-binding proteins, which are present both in the cytoplasm and in the nucleus, are associated with various metabolic processes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIt is not surprising that actin-binding proteins, found only in the nucleus or only in the cytoplasm so significantly different in their functions, also have large variability in their structural properties. We analyzed these proteins for their structure (dis)orderedness, propensity for liquid\u0026ndash;liquid phase separation (LLPS), and ability to initiate the formation of membrane-less organelles (MLOs) or to enter the already formed MLOs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMost actin-binding proteins are intrinsically disordered proteins. Only 2% of cytoplasmic actin-binding proteins are globular proteins, the rest either have disordered regions or a completely disordered structure. The percentage of disordered proteins is significantly higher in the nucleus compared to cytoplasmic proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, panel A). Assessing the propensity of proteins to liquid\u0026ndash;liquid phase separation (LLPS) showed that among cytoplasmic actin-binding proteins, only 25% are prone to LLPS, while among nuclear actin-binding proteins, 44% are capable of LLPS (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, panel B ). At the same time, only 3% of nuclear actin-binding proteins can be classified as proteins capable of initiating the formation of MLOs, which is significantly lower than the corresponding number of the cytoplasmic actin-binding proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, panel C).\u003c/p\u003e \u003cp\u003eApparently, intrinsically disordered proteins are extremely important for actin to perform all its functions. At the moment, there is already evidence of how actin polymerization occurs inside MLOs [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. By now it has become obvious that the formation of MLOs is associated with most, if not all intracellular processes (see e.g. [\u003cspan additionalcitationids=\"CR58\" citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]). However, the details of actin functioning in the cell nucleus remain to be elucidated.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eSummarizing the analysis of the actin interactome in the cell, it can be noted that most of the actin interactors are intrinsically disordered proteins, many of which are prone to LLPS. This is especially important in connection with the currently developing ideas about the role of LLPS and MLO in the organization of all intracellular processes. The significant difference in the properties of nuclear and cytoplasmic actin-binding proteins indicates that their closer study should also shed light on the features of the structural forms and functions of the still poorly studied oligomeric forms of nuclear actin.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe work was supported by the Russian Science Foundation (project No. 23-15-00494, IMK)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe confirm that there are no relevant financial or non-financial competing interests to report.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data supporting the findings of this manuscript are available from the corresponding author upon reasonable request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYakov I. Mokin: conceptualization; software, investigation; validation; formal analysis; writing \u0026ndash; review and editing. Olga I. Povarova: visualization; project administration; writing \u0026ndash; original draft; writing \u0026ndash; review and editing. Iuliia A. Antifeeva: visualization; project administration; writing \u0026ndash; original draft; writing \u0026ndash; review and editing. Konstantin K. Turoverov: conceptualization; supervision; writing \u0026ndash; original draft; writing \u0026ndash; review and editing. Alexey V. Artermov: visualization; writing \u0026ndash; original draft; writing \u0026ndash; review and editing. Vladimir N. Uversky: conceptualization; supervision, writing \u0026ndash; review and editing. Alexander V. Fonin: conceptualization; supervision, writing \u0026ndash; original draft; writing \u0026ndash; review and editing. \u0026nbsp;Irina M. Kuznetsova: conceptualization; supervision; funding acquisition; project administration; resources; writing \u0026ndash; original draft; writing \u0026ndash; review and editing.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHalliburton WD (1887) On Muscle-Plasma. J Physiol 8(3\u0026ndash;4):133\u0026ndash;202. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1113/jphysiol.1887.sp000252\u003c/span\u003e\u003cspan address=\"10.1113/jphysiol.1887.sp000252\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePollard TD (1986) Assembly and dynamics of the actin filament system in nonmuscle cells. 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Science 357(6357). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1126/science.aaf4382\u003c/span\u003e\u003cspan address=\"10.1126/science.aaf4382\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\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":"the-protein-journal","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jopc","sideBox":"Learn more about [The Protein Journal](http://link.springer.com/journal/10930)","snPcode":"10930","submissionUrl":"https://submission.nature.com/new-submission/10930/3","title":"The Protein Journal","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"actin, actin-binding proteins, intrinsically disordered proteins, liquid-liquid phase separation, protein-protein interactions","lastPublishedDoi":"10.21203/rs.3.rs-4014138/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4014138/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eActin is present in the cytoplasm and nucleus of every eukaryotic cell. In the cytoplasm, framework and motor functions of actin are associated with its ability to polymerize to form F-actin. In the nucleus, globular actin plays a significant functional role. For a globular protein, actin has a uniquely large number of proteins with which it interacts. Bioinformatics analysis of the actin interactome showed that only a part of actin-binding proteins are both cytoplasmic and nuclear. There are proteins that interact only with cytoplasmic, or only with nuclear actin. The first pool includes proteins associated with the formation, regulation, and functioning of the actin cytoskeleton predominate, while nuclear actin-binding proteins are involved in the majority of key nuclear processes, from regulation of transcription to DNA damage response. Bioinformatics analysis of the structure of actin-binding proteins showed that these are mainly intrinsically disordered proteins, many of which are part of membrane-less organelles. Interestingly, although the number of actin-binding proteins in the nucleus is greater than in the cytoplasm, the drivers for the formation of the membrane-less organelles in the cytoplasm are significantly (four times) greater than in the nucleus.\u003c/p\u003e","manuscriptTitle":"Bioinformatics analysis of actin interactome: Characterization of the nuclear and cytoplasmic actin-binding proteins","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-07 08:11:27","doi":"10.21203/rs.3.rs-4014138/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-03-26T19:52:26+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-03-25T02:42:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"844c4ac2-56e2-4908-bf9d-5f392b1bcb3d","date":"2024-03-12T21:05:29+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-03-12T04:29:57+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-03-05T10:53:36+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-03-05T10:53:24+00:00","index":"","fulltext":""},{"type":"submitted","content":"The Protein Journal","date":"2024-03-04T17:17:55+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"the-protein-journal","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jopc","sideBox":"Learn more about [The Protein Journal](http://link.springer.com/journal/10930)","snPcode":"10930","submissionUrl":"https://submission.nature.com/new-submission/10930/3","title":"The Protein Journal","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"0b8e1c51-c269-4758-be6e-c7a6d6cf63ec","owner":[],"postedDate":"March 7th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-05-09T13:44:44+00:00","versionOfRecord":[],"versionCreatedAt":"2024-03-07 08:11:27","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4014138","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4014138","identity":"rs-4014138","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}