Targeting ZAKα Interactions and Ribosomes with Novel peptides: The role of ZAKα in Mitochondrial Function and Cancer Cell Proliferation

Targeting ZAKα Interactions and Ribosomes with Novel peptides: The role of ZAKα in Mitochondrial Function and Cancer Cell Proliferation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Targeting ZAKα Interactions and Ribosomes with Novel peptides: The role of ZAKα in Mitochondrial Function and Cancer Cell Proliferation Jaw-Ji Yang, Ying-Chang Hsu, Yang-Shan Yeh This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4003086/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Phage display was used to identify high-affinity and high-specificity peptides that might inhibit ZAKα, a kinase implicated in cellular proliferation, differentiation, and stress response and implicated in both malignant tumor growth and metastasis as well as the pathogenesis of cardiac hypertrophy and fibrotic heart disease. Two peptides were found to specifically bind to ZAKα, PhD30 and PhD35. Their introduction into cancer cells led to the dissociation of ZAKα from RPS20 and the ubiquitination of RPS20 protein resulting in a reduction of essential mitochondria proteins and mitochondria activity impeding the translation of mitochondrial-associated eukaryotic ribosomes, a process we believe to be crucial for tumor growth. This mechanism of action suggests that the primary effect of PhD30 and PhD35 is to lower mitochondrial activity by causing a shortage of essential proteins in mitochondria without changing the number of apoptotic and necrotic cancer cells. Thus, these findings may prove pivotal to the development of a novel approach utilizing these peptides to inhibit cancer cell growth by targeting mitochondrial function, rather than the conventional method of inducing apoptosis. Biological sciences/Cancer/Cancer therapy/Drug development Biological sciences/Biological techniques/Metabolomics ZAKα phage display peptide inhibitors Ribosomal protein S20 Ubiquitylation Translational jams Mitochondria Oxphos tumor growth inhibition Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction ZAK, a member of the MAPKKK (mitogen-activated protein kinase kinase kinase) family, plays a regulatory role in various cellular processes, including proliferation, differentiation, and stress response. ZAK kinase protein is expressed in various tissues and many cancer cells, and it is involved in the regulation of many signal transduction pathways, including the JNK (c-Jun N-terminal kinase) pathways and NFkB 1 . Dysregulation of ZAK kinase activity can lead to the development of cancer or heart disease 2 and ZAK kinase protein has been found to be upregulated in several cancers, including breast, lung, and colorectal cancer, and promote tumor growth and metastasis 3 . In addition, ZAK has been implicated in the pathogenesis of heart disease, particularly cardiac hypertrophy and fibrosis 4 . However, the fundamental molecular mechanisms underlying ZAK-mediated tumorigenesis and cardiac dysfunction are not well understood. ZAK plays an essential role in the regulation of ribosomal protein 5 , 6 . In particular, it has been found to phosphorylate ribosomal protein S6 kinase (S6K), a kinase involved in the regulation of ribosome biogenesis and protein synthesis, processes which require the coordination of numerous proteins and signaling pathways 7 . Ribosomal translation, a process in which ribosomes synthesize proteins using messenger RNA (mRNA) as a template, is crucial for cell growth, development, and survival 8 . The regulation of ribosomal translation is a complex process that enables cells to familiarize themselves with changing environmental conditions and respond to many signals 9 , 10 . This regulation is achieved through a variety of mechanisms that control the initiation, elongation, and termination the synthesis of proteins. The mechanisms involved in these processes include the binding of regulatory proteins to mRNA and ribosomes, the post-translation modification of ribosomal proteins, and the modulation of signaling pathways 11 . One important mechanism underlying this regulation is the binding of regulatory translation factors to mRNA and ribosomes 12 , 13 . These factors can either promote or inhibit translation by controlling the assembly and activity of the ribosome. Additional regulatory proteins, such as RNA-binding proteins, can bind to specific regions of mRNA and affect the stability, localization, or availability of the transcript 14 . Another mechanism underlying the regulation of translation is the modification of ribosomal proteins and RNA 15 . For example, phosphorylation of ribosomal proteins can vary their affinity for mRNA and regulate translation rates 16 . Similarly, modifications to ribosomal RNA, such as those made by methylation or pseudouridylation, can influence ribosome structure and function 17 . Recently, more research attention has been paid to ribosomal proteins (RPs) because they are crucial components of ribosomes, because they are intricately involved in protein synthesis, and because their expressions should be tightly regulated if proper assembly is to be achieved and the ribosomes are to function properly 18 , 19 . Ribosomal protein S20 (RPS20) is a vital component of the ribosome and is located on the small ribosomal subunit, where it plays a crucial role in translation. During the translation process, RPS20 interacts with other ribosomal proteins and rRNA to help position the mRNA and tRNA molecules in the correct orientation for peptide bond formation 20 . It is also required in stabilizing the interactions between the ribosome and mRNA, which is necessary for the precise decoding of the genetic code 21 . Recent studies have shown RPS20 to also be involved in regulating the rate of translation, which is important for the maintenance of cellular homeostasis and reactions to stress 22 . Additionally, mutations in ribosomal RPS20 have been linked to various genetic disorders and cancers, suggesting that this protein contributes substantially to normal cellular functioning 23 – 25 . Ubiquitination, a post-translation modification that involves the covalent linking of ubiquitin molecules to target proteins has been shown to play an important role in the regulating RPs, ribosome biogenesis as well as ribosomal translation rate 26 , 27 . The regulation of RPs by ubiquitination involves a complex interaction between various enzymes, including an activating enzyme (E1), a conjugating enzyme (E2), and a ligase (E3) 28 as well as deubiquitinases and other associated factors 29 . Dysregulation of this process has been associated with many diseases, including cancer and neurodegenerative disorders 30 . Studies have observed that RPS20 ubiquitination can disturb protein translation by regulating the binding of specific mRNA transcripts to the ribosome 31 , 32 . Although there is much evidence that this process regulates protein translation, the fundamental mechanisms underlying RPS20’s ability to do this are not fully understood. Mitochondria are the organelles responsible for energy production in eukaryotic cells. Oxidative phosphorylation, which occurs within the inner mitochondrial membrane, is essential for the generation of ATP in cells. This reaction occurs via protein complexes organized into an electron transport chain (ETC). ETC functioning is strictly regulated, so disturbances in the normal activity of any of the complexes that make up the chain can lead to a variety of pathologies, including metabolic disorders, neurodegenerative diseases, cardiovascular diseases, and cancers 33 , 34 . Therefore, it is very important to understand the mechanisms that govern the effect of the ETC complexes on mitochondrial functioning. This knowledge could lead to the development of therapeutics for diseases involving mitochondrial malfunction. Ribosome co-translation on the mitochondrial membrane requires the synthesis of nascent proteins by the cytoplasmic ribosomes on the mitochondrial surface and the insertion of nascent polypeptides into the mitochondrial membrane. This process enables the newly synthesized proteins to target the mitochondria. Little is known, however, about the mechanisms underlying ribosome co-translation on the mitochondrial membrane and how these mechanisms are regulated. One known regulator of cellular signaling pathways involved in cell proliferation, differentiation, and apoptosis is ZAK. Disruption in ZAK’s ability to regulate such processes can lead to cancer or heart disease 35 , 36 . Its overexpression has also been found in numerous cancer types such as breast, lung, and colorectal cancer and is thought to accelerate tumor development and spread. In addition to its association with cancer, overexpressed ZAK has been linked to the development of heart disease via its regulation of cardiac hypertrophy and fibrosis 4 , 37 . To the best of our knowledge, specific inhibitors of ZAK and their possible effect on cancer and heart disease have not been thoroughly studied. Phage display technology has been used to select peptides, proteins, and antibodies with high specificity and affinity for target molecules. In this study, we used phase display technology to identify peptides that might bind to and inhibit ZAKα, a key member of the mixed lineage kinase family known to play a role in regulating tumor growth, and studied whether they would affect ZAKa-RPS20 binding. In the course of this study, we performed a number of in vitro experiments investigating whether they would hinder ribosomal translation and reduce the mitochondrial proteins involved in ETC complexes. Their impact on mitochondrial activity was assessed by Agilent Seahorse Cell Mito Stress Test to measure basal oxygen consumption in mitochondria of the KB cells. Results Identification and characterization of novel peptide for ZAKα through phage display approach ZAK belongs to the Mixed Lineage Kinase (MLK) family protein, and the ZAK gene can produce two protein isoforms (ZAKα and ZAKβ) through alternative splicing 38 . ZAKα regulates various biological functions. In tumors, for example, the ZAKα protein is upregulated. Using ZAKα specific short hairpin RNA (shRNA) to knockdown ZAKα in oral cancer cells, we previously found that these cells lost their ability to grow under an anchorage-independent condition, suggesting that ZAKα may be required for neoplastic transformation to occur. ZAKα has several domains, including a leucine-zipper (LZ) domain, a sterile-alpha motif (SAM) domain, and an approximately 400-amino-acid non-structural long C-terminal (NSLC) region. The LZ domain is involved in protein-protein interactions and is necessary for the dimerization of ZAKα. The SAM domain in ZAKα also participates in protein-protein interactions and plays an essential role in the formation of protein complexes 39 . The NSLC region is a unique region in ZAKα, and little is known about its functions. In the current study, we subcloned the cDNA corresponding to ZAKa NSLC (amino acid 414–800) into a GST-expressing vector. We purified the GST-ZAK414-800 protein and then added either anti-GST or anti-ZAKα antibody to identify the purified protein by western blot analysis (Fig. 1 A). The Ph.D.-C7C phage display peptide library was used to help identify novel peptides that could bind specifically to this ZAKα NSLC 414–800. We isolated 40 clones. Of these, we found the clones PhD10, PhD30, PhD35, and PhD40 to bind to this protein, repeated 2, 4, 1, and 4 times, respectively (Fig. 1 B). Their corresponding peptides were then tested for their binding ability to ZAKα by in vitro co-precipitation. To do this, we transfected oral cancer cells (KB) with plasmids expressing the four GFP-tagged PhD peptides and immunoprecipitated the protein complexes using an anti-ZAK antibody (0834, generated in the lab). All four GFP-PhD peptides co-precipitated with ZAKα (Fig. 1 C and Supplementary Fig. 1). Identification and characterization of the interaction site within ZAKa for PhD peptides by GST pull-down assay and protein modeling To identify the binding site within ZAKα for these peptides, we fused oligonucleotides corresponding to sequence encoding HIV-TAT (GRKKRRQRRR) and individual PhD peptides with glutathione S-transferase (GST) to generate GST-TAT-PhD fusion proteins. These proteins were then immobilized on glutathione-sepharose resin. Cell lysate obtained from GFP-ZAKα expressed in HEK293 cells was subjected to pull down assay using GST-TAT-PhD fusion proteins. GFP-ZAKα protein was pulled down by all four GST-TAT-PhD proteins (Fig. 2 A), suggesting that ZAKα proteins interacted with the GST-TAT-PhD proteins. To further map the precise binding site within ZAKα protein that interacted with these PhD peptides, we created a series of ZAKα C-terminal deletion mutant clones which we transiently transfected with HEK293 cells (data not shown). Both full-length GFP-ZAK and a C-terminal deletion mutant, GFP-ZAK-380 (amino acids 1-420), were pulled down by GST-TAT-PhD30 and GST-TAT-PhD35 but not GST-TAT-PhD10 (Fig. 2 B). We performed our phage display experiments on ZAKα target region (amino acids 414–800) (Fig. 1 A) and then using deletion experiments we were able to narrow down binding site between ZAKα and the PhD30 or PhD35 peptides to fall within amino acids 414–420. We designated this site as the ZAK phage-binding site (ZAKPBS) with the amino acid sequence, LFHFPPL (Fig. 2 C). Pull down assay using GST-TAT-PhD10, 30, 35, and 40 revealed that only PhD30 and PhD35 might bind to this small region (Fig. 2 D). We confirmed this possibility by performing RoseTTAFold modeling of ZAKa amino acid 321 to amino acid 600 40 and global docking of the PhD30 or PhD35 peptide with ZAKα through PIPER-FlexPepDock 41 – 44 . Figure 2 E shows the predicted docking sites. The PhD30 peptide (red) bound to ZAKPBS next to the End-Helix (EH) site of the ZAKa SAM domain, and the PhD35 peptide (blue) bound to ZAKPBS next to the Middle-Loop (ML) side of the SAM domain. PhD30 and PhD35 peptides modulated mitochondrial protein expression and function in cancer cells Cancer cells alter metabolism more significantly than normal cells, especially glucose metabolism 45 . Most changes are increases glucose consumption and lactate production, a change known as the Warburg effect 46 . These changes may interfere with the energy ATP requirements for cell proliferation and growth. Therefore, we wanted to investigate whether PhD30 or PhD35 peptides in cancer cells would alter the protein levels of oxidative complexes in mitochondria. To find out, we measured the protein levels of oxidative phosphorylation complex III-UQCRC2 (CIII-UQCRC2) and complex V-ATP5A (CV-ATP5A) in KB cells expressing FLAG-PhD30 or FLAG-PhD35. As can be seen in Fig. 3 A, we found that protein levels of complex III and complex V were higher than those cells expressing ZAKα and control cells. We also measured their levels as well as mitochondrial β-actin proteins in isolated mitochondrial fractions and found them to be significantly more decreased in KB cells expressing either PhD30 or PhD35 compared to KB expressing ZAKα and control cells (Fig. 3 A). Together, these findings suggested that PhD30 and PhD35 peptides expressed in KB cells did not suppress the overall protein levels of oxidative phosphorylation complexes in cytosol, but they might have affected mitochondrial proteins levels in this organelle. Similar results were achieved in H9C2 cardiomyoblast cells (Supplementary Fig. 2), indicating that their effect on protein translocation and translation into mitochondria were not cell type dependent. To determine the purity of the isolated mitochondria, we isolated total cellular and isolated mitochondrial RNA and applied them to an agarose gel. Total RNA was found to have clear 18S and 28S rRNA, while isolated mitochondrial RNA only showed 12S and 16S rRNA (Fig. 3 B), indicating the isolated mitochondria were pure and had no contaminated cytosolic fraction. We found lower mitochondrial oxidative phosphorylation complex protein levels in KB expressing PhD30 or PhD35, suggesting that these cells might have lower mitochondrial activity. To test this hypothesis, we used MitoTracker Red CMXROS staining to assess membrane potential of PhD30 or PhD35, either fused with EGFP or FLAG, in KB cells. We found that cells expressing PhD30 or PhD35 had less mitochondrial membrane potential than controls (Fig. 3 C and Supplementary Fig. 3A). We then used the Agilent Seahorse Cell Mito Stress Test to measure the effect of PhD30 or PhD35 on the basal oxygen consumption rate (OCR) in mitochondria of KB cells. We found overexpression of PhD30 or PhD35 with either EGFP or FLAG tags led to a significant reduction in the basal OCR compared to controls (Fig. 3 D, E; Supplementary Fig. 3B, C). These results suggest that PhD30 or PhD35 may bind to ZAKα influencing mitochondrial respiration and energy metabolism in KB cells, which might occur because this binding interferes with the protein translation or translocation of newly synthesized proteins into the mitochondria. Interaction between ZAKα and ribosomal protein We performed a yeast two-hybrid experiment using ZAKa protein as the bait and screened the human heart in the Matchmaker cDNA library to identify proteins that would interact with ZAKa protein. One isolated clone was found to be associated with ZAKa, Ribosomal Protein S20 (RPS20), a protein component located in the small ribosomal subunit thought to play a fundamental role in the translation progression 47 . The association we found between RPS20 and ZAKa suggested that ZAKa might regulate ribosomal translation. To test this hypothesis, we immobilized GST-S20 protein using Glutathione-Sepharose beads and found that it successfully pulled down ZAKa protein. Interestingly, the binding between ZAKa and RPS20 occurred independent of ZAKa intrinsic kinase activity as evidenced by the fact that GST-S20 pulled down both kinase-dead mutant (ZAKdn) and the kinase-constitutively active mutant (ZAKE/E) (Fig. 4 A). We also investigated the potential of ZAKa to phosphorylate RPS20. In vitro kinase assay showed that both ZAKa and ZAKE/E phosphorylated RPS20, whereas ZAKdn did not (Fig. 4 A, lower panel). This finding suggested that ZAKα might regulate RPS20 function through phosphorylation via some unknown mechanism. Regardless, the physical binding of both proteins took place independent of ZAKa kinase activity. To determine the binding sites in ZAKα for RPS20, we constructed serial C-terminal deletion mutants and subjected them to pull down by GST-S20. We found at least three potential binding sites (300–340, 410–460, and 600–700) to respond to RPS20 (Supplementary Fig. 4A). Sites 300–340 and 410–460 were located on the N and C termini of the SAM domain, respectively, possibly implicating SAM domain in ZAKa-RPS20 binding. We constructed a SAM domain deleted ZAKa mutant and found that had a decreased ability to bind with GST-S20 (Supplementary Fig. 4B). We used computer modeling RoseTTAFold 40 to predict ZAKa structures and the HDOCK program 48 to predict ZAKa-RPS20 protein binding sites. We found the predicted binding sites for these two proteins to be located at amino acid residues 323–330, 409–417, and 626–710 in ZAKa (Supplementary Fig. 4C), suggesting that the expression of PhD30 or PhD35 peptides in KB cells might affect the interaction between the two proteins, leading us to want to study the binding ability of ZAKa with RPS20 in the presence of PhD30 or PhD35 peptides. To do this, we transfected HEK293 and KB cells with EGFP-ZAK plasmid with different concentrations FLAG-PhD30 and FLAG-PhD35 plasmid and used GST-S20 pull-down assay to assess ZAK-RPS20 binding ability. In both HEK293 and KB cells, we found the higher the expression FLAG-PhD30 or FLAG-PhD35 plasmid, the greater the decrease in EGFP-ZAKa pulled down by GST-S20 (Fig. 4 B, C). These results suggest that by binding to ZAKa, PhD30 and PhD35 might interfere with the binding of between ZAKa and RPS20. We became interested in exploring the interaction between ZAKa and the mitochondria associated cytoplasm ribosome in the presence of PhD30 and PhD35 because their expression in cells could result in decreased protein levels of oxidative phosphorylation complexes in mitochondria. To do this, we isolated mitochondria from KB cells expressing either FLAG or EGFP fused with PhD30 or PhD35 and found the mitochondrial fractions of the KB cells expressing either PhD30 or PhD35 had decreased protein levels of ZAKa (Fig. 4 D, F), while the protein levels of RPS20 and RPL4 remained unaffected (Fig. 4 E, G). Together, these findings suggest that PhD30 and PhD35 bind to ZAKa, interfering with its ability to bind with RPS20 leading to its separation from the ribosomes. Moreover, we detected both PhD30 and PhD35 in the mitochondrial fraction (Fig. 4 H), also suggesting that they contributed to ZAKα’s ability to affect ribosomal translation on mitochondria. The effects of PhD30 and PhD35 peptides on RPS20 ubiquitination and mitochondrial-associated mRNA levels Numerous studies have found RPS20 to play a crucial role in the regulation of translation, to be involved in the assembly of the ribosome, and to interact with several other ribosomal proteins and rRNA 49 . It has also been shown to interact with specific mRNA sequences and possibly play a role in regulating the initiation of translation 50 . Thus, we wanted to explore how the binding of PhD30 or PhD35 peptide to ZAKa might affect the translation of mitochondria-associated cytoplasm ribosomes. To find out, we isolated the mitochondrial fractions from KB cells expressing either PhD30 or PhD35. In these fractions, we observed the RPS20 protein in multiple ladders with an interval of 8.5 kDa, suggesting that they could be post translational modifications, especially ubiquitination. We immunoprecipitated the mitochondrial fractions using RPS20 antibody and then used ubiquitin antibody to screen for the precipitated RPS20 protein. We found increasing RPS20 ubiquitination levels, which we suspected to be mono-ubiquitinated RPS20, bi-ubiquitinated RPS20, and tri-ubiquitin RPS20 in KB cells expressing PhD30 or PhD35 (Fig. 4 I). The binding of PhD30 or PhD35 to ZAKa protein might result in the dissociation of ZAKa from RPS20, which in turn would increase the ubiquitination of RPS20. This ubiquitination might be implicated in the regulation of translation process 51 . We wanted to investigate whether the expression of PhD30 or PhD35 impedes the ability of mitochondria-associated ribosome to translate nuclear mRNAs. To do this, we first isolated total cellular mRNA and mitochondria-associated mRNA from KB cells expressing PhD30 or PhD35 and then used semi-quantitative RT-PCR to measure the amounts of mRNAs, including CIII-UQCRC2, CV-ATP5A, and β-actin. We found a decrease in CIII-UQCRC2, CV-ATP5A, and β-actin mRNA associated with mitochondria in the KB cells expressing one of the two peptides, compared to control cells, while the mRNA isolated from total cellular fraction remained unchanged (Fig. 4 J, K, L). These findings revealed the reduction of these mitochondria-associated mRNA levels in cells expressing PhD30 or PhD35 correlated with the protein levels in the mitochondria (Fig. 3 A), suggesting that the binding of PhD30 or PhD35 on ZAKa decreased the amount of mRNA associated with mitochondria. This phenomenon was reverse correlated with the amount of ubiquitination on RPS20, suggesting that PhD30 or PhD35 peptide binding to ZAKa might have an impact on mitochondrial function. The above findings suggest that when PhD30 and PhD35 peptide binds to ZAKa, there is an increase in RPS20 ubiquitination impeding the translation of mitochondria-associated ribosomes. We performed a polysome profiling experiment to assess translation efficiency. Total ribosomes were first isolated from the controls, and cells expressing ZAKa, EGFP-PhD30, or EGFP-PhD35 and the fractions were analyzed. We found no significant differences in translation efficiency, although the EGFP-PhD35 cells had a slightly higher polysome fraction compared to the controls (EGFP and EGFP-ZAKa) (Fig. 4 M). We also isolated mitochondria-associated ribosomes from the controls and cells expressing EGFP-PhD30 and EGFP-PhD35 and performed a polysome profiling analysis on them. We found no translation activity in the mitochondria-associated ribosomes isolated from the cells expressing EGFP-PhD35 (Fig. 4 N, left panel) and lower translational activity in the mitochondria-associated ribosomes isolated from cells expressing EGFP-PhD30 (Fig. 4 N, right panel) compared to the controls. Thus, by binding to ZAKa, PhD30 and PhD35 impeded the translation efficiency on mitochondria-associated ribosomes, which means that they could play role in regulating nascent proteins synthesis supplied for mitochondria and energy production. As mentioned above, the binding of PhD30 or PhD35 to ZAKa led to the dissociation of ZAKa from RPS20 in mitochondrial fractions (Fig. 4 D, F). We thought it would be interesting to investigate the association between ZAKα status and elongating polysomes in KB cells expressing EGFP-PhD30 or EGFP-PhD35 in the polysome profile fractions. We found that KB cells expressing PhD30 or PhD35 had decreased the protein levels of ZAKα in the early polysome fractions compared to control cells, suggesting a possible role for ZAKa in regulating the initiation of translation. We also found a correlation between the increased levels of ubiquitinated RPS20 with the dissociation of ZAKα in early fractions. PhD30 and PhD35 were also found in the early fraction, strongly suggesting that they might cause this dissociation by binding to ZAKα. Furthermore, KB cells expressing PhD30 and PhD35 were found to have substantially higher levels of the translation initiation factor, eIF2α, in the early fraction compared to controls (Fig. 4 O), suggesting that the binding of PhD30 or PhD35 to ZAKα led to the dissociation of ZAKa from RPS20, leading to an elevation in RPS20 ubiquitination and inhibiting the dissociation of eIF2α from the ribosomal initiation complexes. Collectively, these findings strongly suggest that PhD30 or PhD35 might play a targeting role in modulating the association of ZAKα with ribosomal initiation complexes leading to a delay or stalling in the initiation of translation. To further examine whether the binding of PhD30 or PhD35 to ZAKa would result in ribosomal translation jams or a complete shutdown in KB cells, we treated KB cells expressing FLAG-PhD30 or FLAG-PhD35 with cycloheximide for eight hours to block ribosomal translation. The cells were then unblocked and mitochondria-associated ribosomes isolated from these cell cultures were subjected to polysome profiling at 24, 48, and 72 hours after the release from blockage. After 24 and 48 hours, the KB cells expressing PhD30 or PhD35 were found to have reduced polysome activity on the mitochondria compared to the controls, suggesting a reduction of ribosome translation efficiency on mitochondria. After 72 hours, the mitochondria-associated ribosomes had accumulated in the monomer stage in KB cells expressing PhD35 and they accumulated in the low-polysome stage in those expressing PhD30, compared to the controls (Fig. 4 P). These findings suggest that the binding of PhD30 or PhD35 to ZAKa could trigger ribosome translation jams or a slow-down on mitochondria, interfering with the initiation of translation. We further found a decrease in mRNA/Ribosome on mitochondria over time in the cells expressing PhD30 or PhD35 (Fig. 4 Q) and that the expression of PhD30 and PhD35 reduced ribosome translation efficiency and slowed the growth of these cells (Fig. 4 R). ZAKa played a critical role in regulating mitochondria-associated ribosomal translation independent of its kinase activity As suggested by the above findings, it is necessary that ZAKα bind to RPS20 for proper ribosomal translation on mitochondria. We wanted to know if there was a specific ZAKα protein required for mitochondria-associated ribosome translation to take place. To find out, we used ZAKα specific shRNA (1712i) to knock down the ZAKα expression in KB cells. The knockdown resulted in the retardation of mitochondria-associated ribosome translation activity as determined by polysome profiling (Fig. 5 A). It also resulted in a reduction in the protein levels of oxidative phosphorylation complexes, such as CV-ATP5A and CIII-UQCRC2 (Fig. 5 B) and an increase in RPS20 ubiquitination in the mitochondria fraction (Fig. 5 C). These results strongly suggest that it was necessary for ZAKα to bind to RPS20 to prevent RPS20 from the further ubiquitination, a process critical to the regulation of mitochondria-associated ribosome translation. The ZAKa protein transduces its signal cascade to downstream effector through its intrinsic kinase activity 52 . Because ZAKa phosphorylated RPS20 (Fig. 4 A bottom panel), we found it essential to our study to investigate whether ZAKa kinase activity played a role in regulating mitochondria-associated ribosomes and to investigate whether this would lead to its phosphorylation of RPS20. Therefore, we studied the possible effect of ZAKdn mutant, one that does not have kinase activity, on KB tumor growth. Interestingly, KB cells expressing ZAKdn were found to retain their ability to grow in the anchorage-independent condition (Fig. 5 D), suggesting that their ability to grow was independent of kinase activity. We performed a polysome profiling assay on cells expressing ZAKdn to further investigate the role of ZAKa kinase activity in regulating ribosome translation efficiency on mitochondria and found that cells expressing ZAKdn exerted ribosome translation activity on mitochondria similar to that of cells expressing ZAKa (Fig. 5 E), indicating that ribosome translation was regulated by some mechanism other than intrinsic kinase activity. Additionally, we found no change in the protein levels of CV-ATP5A and CIII-UQCRC2 or RPS20 in the total cellular fraction or in the mitochondria fraction in KB cells expressing ZAKa or ZAKdn (Fig. 5 F). We also found no change in RPS20 ubiquitination in these cells (Fig. 5 G). MitoTracker Red CMXROS staining also showed no change in mitochondrial activity (Fig. 5 H). Exogenous delivery of GST-TAT-PhD proteins inhibited anchorage-independent growth of KB cells through protein transduction HIV TAT’s (trans-activator of transcription) ability to carry proteins across the cell membrane and deliver proteins across the cell membrane has made TAT an attractive tool for drug delivery and gene therapy 53 . We wanted to determine whether the exogenous supplied proteins might transduce into cells and interfere with anchorage-independent growth. To find out, we purified GST-TAT-PhD30 and GST-TAT-PhD35 proteins and exogenously added them to KB cells cultured in soft agar. Both GST-TAT-PhD30 and GST-TAT-PhD35 were found able to reduce colony formation to 69% or 54%, respectively, at a concentration of 33.3 nM, compared to GST-TAT cells (Fig. 6 A, B). We further examined the uptake capability of the exogenously supplied proteins when different concentrations of GST-TAT-PhD35 were added KB or Rat6 cells for 24 hours. Western blot was used to assess protein penetration. GST-TAT-PhD35 could be detected at a concentration of 10 µg/ml (Fig. 6 C). GST-TAT-PhD30 or GST-TAT-PhD35 proteins were added to separate KB cell culture medium at a concentration of 33.3 nM, and the KB cell growth rates were measured. We found cell growth to be arrested by exogenously supplied GST-TAT-PhD30 or GST-TAT-PhD35 protein to KB cells starting on the fourth day of treatment compared to GST-TAT controls (Fig. 6 D). These findings indicated that continuous treatment of KB cells with exogenously supplied GST-TAT-PhD30 or GST-TAT-PhD35 proteins arrested cancer cell proliferation. Furthermore, the activation of protein levels of caspase 8, caspase 9, and procaspase 3 were not induced in KB cells expressing stable FLAG-PhD30 or FLAG-PhD35 as well as KB cells transiently co-transfected with ZAKa plasmid at different concentrations of FLAG-PhD30 or FLAG-PhD35 plasmids (Supplementary Fig. 5A, B). These findings suggested that the binding of the PhD proteins to ZAKa could potentially be used to develop an apoptosis-free approach to the treatment of cancer cells. PhD30 and PhD35 inhibited oral cancer cell proliferation by targeting ZAKα: evidence from in vitro and in vivo studies To further confirm whether PhD30 or PhD35 peptide expressed in tumor cells affected their proliferation capacity, we measured the growth of KB cells expressing either EGFP or FLAG tagged PhD30 or PhD35. We found growth to be inhibited in EGFP-PhD30 or EGFP-PhD35 KB cells under non-adherent growth conditions (Fig. 7 A, B). These findings suggested that PhD30 or PhD35 peptides expressed in KB cells bound to ZAKa (Fig. 1 C and Supplementary Fig. 1) inhibiting proliferation under non-adherent conditions. Moreover, the growth rates of KB cells expressing EGFP-PhD30 and EGFP-PhD35 were significantly slower than those of the mock control cells (Fig. 7 C). The fact that ZAKa belongs to a MAPKKK family of kinases able to phosphorylate downstream effectors raises an intriguing question. Might these PhD peptides influence ZAKa’s intrinsic kinase activity? To explore ZAKa kinase activities, we performed in vitro kinase assays of KB cells expressing these peptides by using RhoGDIb, an effector known to be phosphorylated by ZAKa 54 as a substrate. When transiently co-transfecting ZAKa plasmid with EGPP-PhD35 or EGFP-PhD30 plasmid, we found that ZAKa kinase activity was inhibited by EGPP-PhD35 but not EGFP-PhD30 (Fig. 7 D), indicating cancer cell growth or mitochondrial activity was inhibited (Fig. 3 C, D, E) by these PhD peptides independent of the intrinsic kinase activity in ZAKa. To prevent the 27kDa GFP protein from influencing these PhD peptides, we constructed FLAG peptide tagged PhD clones. Similar to the results of our study of KB cells expressing EGFP-PhD30 and EGFP-PhD35, those expressing FLAG-PhD30 and FLAG-PhD35 were found to have inhibited colony formation under non-adhesion growth conditions (Fig. 7 E, F). Colony formation was also found to be inhibited in KB cells expressing ZAKb and 1712i, which were our negative growth control cells. KB cells expressing FLAG-PhD30 and FLAG-PhD35 as well as those expressing FLAG-ZAKβ and 1712i had slower growth rates compared to mock or cells expressing FLAG-ZAKα (Fig. 7 G). These findings indicate that these biological effects were indeed affected by these PhD peptides. The ability of cancer cells to migrate and invade plays a critical role in cancer metastasis. Results of Boyden assay revealed that KB cells expressing FLAG-PhD30 and FLAG-PhD35 had significantly impaired ability to migrate and invade (Fig. 7 H, I). Because ZAKPBS is the binding site in ZAK for the PhD30 and PhD35 peptides (Fig. 2 C, D), we wanted to know whether the expression of ZAKPBS in cells could sequester the growth inhibition effect of PhD30 or PhD35 on ZAKα. To find out, we transiently co-transfected FLAG-PhD30 and FLAG-PhD35 plasmid with either a mock vector or GFP-ZAKPBS plasmid for 48 hours and evaluated anchorage-independent growth. The cells transiently expressing PhD30 or PhD35 peptides lost their ability to grow in anchorage-independent conditions, while some anchorage-independent growth ability was restored in the cells co-expressing ZAKPBS with FLAG-PhD30 or FLAG-PhD35, (Fig. 7 J, K). FLAG-ZAKβ or FLAG-CREB1 expressing cells were used as negative growth controls 38 . We used xenografts to study the tumor microenvironment and the growth of these KB cells expressing PhD peptides in a living organism. KB cells expressing four stable FLAG-conjugated PhD peptides (PhD10, PhD30, PhD35, and PhD40) were injected subcutaneously into the backs of nude mice. Four weeks later the mice were killed and tumor sizes were measured. Like the anchorage-independent growth experiment, we found that tumor sizes were smaller in mice injected with KB cells expressing FLAG-PhD30 and FLAG-PhD35 compared to those injected with the mock vector control and FLAG-PhD40 cells (Fig. 7 L, M). The results of our in vitro studies (Fig. 7 A, E) and in vivo studies indicate that the binding of PhD30 or PhD35 to ZAKa resulted in the inhibition of tumor cell growth. The impact of PhD peptides on mitochondrial activity and tumor growth was achieved via modulation of oxidative phosphorylation The binding of PhD30 or PhD35 peptides to ZAKα caused ZAKα to dissociate from RPS20. This dissociation led to the jamming or stalling of translation on mitochondria-associated ribosomes and a significant decrease in protein levels of oxidative phosphorylation complexes, causing an impairment of mitochondrial activity. We wanted to further investigate the relationship between mitochondrial activity and tumor cell growth. To proceed, we challenged KB cells with different concentrations of Antimycin A, an inhibitor of oxidative phosphorylation complex III, and Oligomycin, an inhibitor of oxidative phosphorylation complex V. We found that both inhibitors reduced anchorage-independent growth in a dose-responsive manner. We also found that combination of Antimycin A and Oligomycin had a synergistic effect on the inhibition of colony formation. In particular, we found that this combination treatment at a concentration of 100nM inhibited the colony formation ten times as much as the same concentration of Antimycin A or Oligomycin alone (Fig. 7 N). Furthermore, analyzing MitoTracker Red CMXROS stains, we found that treatment with Antimycin A and Oligomycin alone brought about dose-responsive reductions in mitochondrial membrane potential, while combination treatment at 100nM brought about a much greater reduction in the mitochondrial membrane potential (Fig. 7 O). We concluded that treatment combining Oligomycin and Antimycin at 100nM was sufficient to reduce mitochondrial activities and inhibit tumor cell growth. We wanted to make sure that this combination of inhibitors to treat the cells only reduced the mitochondrial activities leading to the inhibition of tumor growth but not trigger cell death, which would also inhibit growth (Figs. 7 A, E) and mitochondrial activities (Fig. 3 C, D, E) in KB cells expressing PhD30 or PhD35 peptide. To determine whether apoptosis or necrosis was induced, we treated the cells with a combination of the inhibitors (100nM) for 48 hours and used flow cytometry with Annexin V and PI double staining to analyze tumor cell stages. We found no increase in either apoptosis and necrosis compared to controls. However, it should be noted that at a concentration of 500nM, this combined treatment significantly increased both apoptosis and necrosis compared to controls (Supplementary Fig. 6). Together, these findings suggest the reduction of mitochondrial activities by this combination of inhibitors was sufficient to retard tumor growth without killing the tumor cells, highlighting the important relationship between mitochondrial activity and tumor growth and the possible therapeutic potential that Antimycin A and Oligomycin might have on the treatment of tumors. Discussion This study focused on ZAKα, a key member of the mixed lineage kinase family, because it is known to play an important role in regulating tumor growth. Although it is well-known that its increased expression contributes significantly to tumorigenesis, no specific inhibitors targeting ZAKα has been found. Performing phage display assay analysis, we identified four novel peptides (PhD10, PhD30, PhD35, and PhD40) that bound specifically to the non-structural long C-terminal (NSLC) region in ZAKα. PhD30 and PhD35, in particular, were found to have a high affinity for ZAKα, effectively interacting with both exogenously expressed and endogenous forms of the protein. These two peptides were able to bind to a specific region of ZAKα, we designated region as a ZAKPBS (phage-binding site), within the amino acid sequence 414–420. Our results indicated that the binding of these peptides to ZAKα resulted in the dissociation of ZAKα from RPS20, a protein we identified as a ZAKα-associated partner through our studies using yeast two-hybrid screening. This disruption occurred independently of intrinsic kinase activity in ZAKα, suggesting a new mechanism of action for these peptides. Importantly, the binding of PhD30 and PhD35 to ZAKα not only impaired its interaction with RPS20 but it also inhibited tumor growth and affected mitochondrial activity. These findings suggest that these two peptides could potentially be used to develop novel therapeutic agents targeting ZAKα in the treatment of cancer. The peptides PhD30 and PhD35 targeted ZAKα, inhibiting its association with ribosomal initiation complexes, leading to an increase in RPS20 ubiquitination and disrupting the initiation of translation. Other studies have previously suggested that RPS20 ubiquitination plays a role in regulating translational initiation 51 , 55 and studies on ZAKα have indicated that ZAKα plays a novel role in the Ribosome Quality Control (RQC) or Ribotoxic Stress Response (RSR) pathways via its recognition of stalled ribosomes 5 , 6 . Thus, ZAKα may act as a key regulator of protein translation and degradation. When PhD30 or PhD35 peptide binds to ZAKα, ZAKα dissociates from RPS20 allowing RPS20 to be ubiquitinated by E3 ligases. However, the specific E3 ligase for RPS20 ubiquitination has not been identified. ZNF598 could potentially be an E3 ligase for ribosomal subunit protein ubiquitination 56 , and it has been found to orchestrate RQC by ubiquitinating ribosomal proteins and inducing premature translation termination of defective mRNAs 57 . Further studies are needed to determine whether it is responsible for RPS20 ubiquitination. In this study, the expression of PhD30 and PhD35 peptide in cells did not influence their protein levels of RPS20 (Fig. 4 E, G), suggesting that the ubiquitination of RPS20 more likely plays a role in regulation than it does in protein degradation. Studies have indicated that RPS20 ubiquitination is critical to the regulation of the assembly of the 40S ribosomal subunit and the translation of specific mRNAs 58 , 59 and it is also involved in various cellular functions, including ribosome biogenesis, mRNA translation, and protein quality control 51 , 55 , 56 . In this study, the immunoprecipitation of RPS20 from mitochondrial fraction showed that most of the RPS20 proteins were monoubiquitinated in KB cells, suggesting that monoubiquitinated RPS20 is essential for the proper translation on mitochondria associated ribosomes. This finding is in agreement with other studies finding that ubiquitination plays a critical role in regulating protein synthesis, including ribosome-associated quality control (RQC) 27 . These results highlight the complexity of ubiquitin-mediated regulation of translation and the importance of fine control on protein synthesis regular cellular functions. Although mitochondria have their own DNA, which encode a small portion of their proteins, most mitochondrial proteins are encoded by nuclear genes and then translocated into the mitochondria. Studies have found these nuclear encoded mRNAs on the surface of the mitochondrial outer membrane 60 where cytosolic ribosomes associate with them to initiate translation. The binding of ribosomes to the mitochondrial membrane surface ensures the co-translation and translocation of the newly synthesized proteins into mitochondria 61 , 62 . However, in this study, when PhD30 or PhD35 peptide bound to the ZAKα protein, ZAKα dissociated from RPS20 facilitating the ubiquitination of RPS20 (Fig. 4 D, F, I) jamming or stalling this translation. We found that PhD30 and PhD35 peptides interfered with ZAKα’s ability to regulate ribosomal translation (Fig. 4 N, P), resulting in a decrease in crucial oxidative phosphorylation complexes like CV-ATP5A and CIII-UQCRC2 (Fig. 3 A). This decrease in mitochondrial activity highlights the potential of these peptides may hold in modulating mitochondrial activities for cancer therapy. This approach focusing on mitochondrial dysfunction is in alignment with current research exploring the relationship between mitochondria and cancer 63 – 65 . These findings not only elucidate a novel mechanism through which phD30 and PhD35 peptides modulate mitochondrial function through the ZAKα-RPS20 axis but also suggest that the mitochondrial pathway can be targeted as a potential therapeutic strategy in the treatment of cancer. Most kinases exert signaling through the phosphorylation of their specific downstream substrates. Our study discovered that PhD30 and PhD35 peptides reduced mitochondrial activities and inhibited cancer cell growth independent of ZAKα's kinase activity (Fig. 7 D), until now an unknown role for these peptides. We also identified ZAKPBS as the target site for both peptides in ZAKα and demonstrated that exogenous expression of ZAKPBS reversed the ability of these peptides to inhibit growth (Fig. 7 J, K), further confirming it as the binding site. The results of this study suggest an alternative approach to the treatment of cancer, one involving the use of these peptides targeting ZAKα to slow down the proliferation rate without inducing apoptosis. There are various ways to treat cancer, the most common being inducing apoptosis in cancer cells with the use of chemotherapy drugs, radiation therapy, or targeted therapies that specifically target certain proteins in pathways essential for cancer cell survival. There are problems with the use of apoptosis for treatment. One problem is the mutation to drug-resistant cancer cells arising from selection pressure. That is cancer cells can mutate and be selected to resist certain treatment, rendering them harder to kill in the future 66 , 67 . Another problem associated with inducing apoptosis for therapeutic purposes in the collateral damage to normal cells. In contrast, a more sustainable approach to treating cancer might be to slow down or arrest the proliferation of cancer cells, which might help circumvent the development of drug-resistant mutations and collateral damage to normal cells, though this approach would come with its own set of challenges in that it may not be as effective as the killing the cells in the short time and it may not completely eliminate the cancer. This study found that both ZAKα and its kinase-dead mutant, ZAKdn, bound to RPS20 without affecting its ubiquitination status and without influencing anchorage-independent growth activity. This finding suggests that tumor cell growth is not strictly dependent on the kinase activity of ZAKα. This hypothesis was further supported by our finding PhD30 binding to ZAKα did not affect ZAKα’s intrinsic kinase activity but it did interfere with its interaction with RPS20. These findings highlight, the crucial role of ZAKα plays in regulating ribosomal translation on mitochondria and its role in mitochondria’s ability to maintain tumor growth. These findings also indicate that the function of ZAKα may extend beyond its kinase activity, possibly involving physical interactions with RPS20, suggesting a novel mechanism for ZAKα in the regulation of translational control and mitochondrial function. Although both PhD30 and PhD35 peptide bound to the ZAKPBS in ZAKα, only the PhD35 peptide inhibited the kinase activity. The difference might be because PhD30 and PhD35 peptides bind to ZAKPBS at different orientations. PhD30 peptide was predicted to dock at ZAKPBS next to the EH site of ZAKα's SAM domain, while the PhD35 peptide docked at ZAKPBS next to ML side of ZAKα's SAM domain (Fig. 2 E). This difference in binding orientation might lead to differences in protein function, even if peptides bind to the same site. It was interesting to note that although ZAKa bound to PhD30 or PhD35 peptide dissociated from RPS20, interrupting translation of mitochondria associated ribosomes, we found that it did not exert a similar interference in the translation of cytosolic ribosomes (Fig. 4 M, N). This difference raises the question of whether ZAKa might have other associated proteins that are involved in regulating ribosomal translation in cytosol. Thus, further research is needed to identify other ZAKα-associated proteins that might cooperate with ZAKα to regulate the spatial ribosomal translation on mitochondria or in cytosolic compartments. It is also possible that a higher abundance of ribosomes in cytosol and lower expression of the peptides might limit the peptides’ effect on cytosolic translation, whereas in cells, where there are fewer mitochondria-associated ribosomes, the impact of PhD peptides on the translation in the cells might be more significant. This study has some limitations. One limitation is the fact that almost all of experiments we performed in vitro with only one semi in vivo undertaken. Further in vivo studies are needed to study the practicality of this strategy for the treatment of cancer. Another limitation is related to specificity, our ability to target different cancers. In conclusion, this study exploring the effect of two novel ZAKα binding peptides, PhD30 and PhD35, on tumor cell growth, found that by binding to ZAKα, the peptides PhD30 or PhD35 increased ubiquitination of RPS20, significantly impacting translational regulation. It also demonstrated their targeting of ZAKα influenced the translation of nuclear mRNA on mitochondria-associated ribosomes, possibly impacting mitochondrial functions. In particular, PhD30 or PhD35 peptide decreased translation efficiency on mitochondria-associated ribosomes by binding to ZAKα, affecting the building blocks and enzymes required energy production in mitochondria. The results of this study provide new insight into the molecular mechanisms underlying ribosome translation by ZAKα on mitochondria and suggest that the binding of PhD30 or PhD35 peptide on ZAKα might slow down or jam ribosome translation on mitochondria more than likely by interfering with the initiation of translation. We believe that using PhD30 or PhD35 peptide to target ZAKα holds great potential for treatment of cancer. Declarations Acknowledgments This work was supported by a grant from Ington Biotechnologies Co Ltd (105-CSMU82). Authors Contributions J.J.Y. conceived the project, designed, performed all experiments, and wrote the manuscript. I.C.H. and Y.S.Y. performed the molecular modelling and docking confirmations and analyzed the data. All authors discussed the results and commented on the manuscript. Competing Interests The authors declare no competing interests. Methods Cell line and culture conditions KB, HCT116, HEK293, and H9C2 cells were grown in Dulbecco’s modified eagle medium (DMEM) supplemented with 10% fetal bovine serum, 2mM L-glutamine, penicillin and streptomycin. All cells were cultured at 37C in a humidified incubator containing 5% CO2. Mycoplasma testing was performed and found to be a negative. Phage display The cDNA corresponding to ZAKα NSLC 414-800 was subcloned into a GST-expressing vector. The GST-ZAKα NSLC 414-800 protein was expressed in E. coli BL21 (DE3) cells and the protein was purified using Glutathione Sepharose 4B resin. The immobilized Sepharose GST-ZAKα NSLC 414-800 protein was subjected to phage display screening by the Ph.D.-C7C phage display peptide library (New England Biolabs) for 1 hour at room temperature. Unbound phages were washed with PBS containing 0.05% Tween-20 (PBST). The bound phages were eluted using 100 mM triethylamine (TEA) and neutralized with 1 M Tris-HCl, pH 7.5. The eluted phages were further amplified in E. coli and amplified phages were again used to bind Sepharose GST-ZAKα NSLC 414-800 protein, undergoing three rounds of biopanning. We wanted to exclude all phages binding to GST. To do this, we incubated the final panning phage solution with 50 µL of 5 µg/ml Glutathione Sepharose 4B GST for 1 hour at room temperature. The unbound phages were incubated with the host bacteria and poured onto a plate and individual plaques were isolated. Phages were isolated and amplified and genome DNA was extracted for sequencing to identify potential ZAKα NSLC 414-800 binding peptides. Xenografts Six-week-old nude mice were purchased from National Applied Research Laboratories, Taiwan. Stable expressed PhD10, PhD30, PhD35, or PhD40 KB cells were trypsinized and cell concentrations were adjusted to 1x10 7 cells/ml. Subsequently, 200 μL of the cell suspension was injected subcutaneously into the back of nude mice through a 26-gauge needle. Each mouse received one injection on both the left and right sides of the back. The mice were killed four weeks after injection by ether inhalation. Tumors were excised, weights were measured, and photographs were taken. Statistical analysis was conducted comparing tumor weights among the different groups. All animal experiments were performed following universally accepted guidelines for animal care and use, and all necessary precautions were taken to minimize pain and distress to the animals. All animal experimental procedures were performed with the approval of the Institutional Review Board of Taichung Veterans General Hospital (IRBTCVGH No: 950727/C06134). Immunoprecipitation and Western blot analysis Cell lysates were prepared in IP buffer (40 mM Tris-Hcl pH 7.5, 1% NP40, 150 mM NaCl, 5 mM EGTA, 1 mM DTT, 1mM PMSF, 20 mM NaF, proteinase inhibitors, and 1 mM sodium vanadate). Cell extracts (600 mg) were incubated with 5 ml anti-ZAK (0834) polyclonal antibody (generated in the lab) for 6 h at 4 o C. This mixture was then mixed with 20 ml protein-A sepharose suspension and incubated for an additional hour. Immunoprecipitates were collected by centrifugation, washed three times with IP buffer with 0.5% deoxycholate and five times with IP buffer alone, and then subjected to SDS-PAGE. For the western blot analysis, cells were harvested in lysis buffer (50 mM Tris-HCl, pH 8.0/250 mM NaCl/1% NP-40, 2 mM EDTA) containing 1 mM PMSF, 10 ng/ml leupeptin, 50 mM NaF, and 1 mM sodium orthovanadate. Total proteins were then separated on SDS-PAGE and the protein bands were visualized using an ECL chemiluminescent detection system (Amersham). Soft agar assay 2.5 ml of 0.5% Nobel agar was poured into each well of a six-well plate as the bottom agar layer. Cells were suspended in DMEM supplemented with 10% FBS and 0.3% Agar Noble (BD, Difco, MD, USA) and plated onto the bottom layer of Agar Noble. Experiments were performed in six-well plates with 2,500 cells per well in triplicate. Colonies were stained with iodonitro-tetrazolium chloride (INT) solution and counted after two weeks incubation under 37°C and 5% CO2. Colonies were photographed and quantified. The number of colonies was counted under an inverted light microscope at X40 magnification. The data are expressed as mean number of colonies ± standard error (SE) from six fields of three independent wells. Migration and invasion assay A Boyden chamber was coated with Matrigel for invasion assays or without Matrigel for migration assays. The chamber was incubated at 37°C for 30 minutes to allow the matrix to solidify. The Boyden chamber assay was performed by filling the bottom well of the chamber with DMEM medium containing 10% FBS. The wells were covered with polyvinylpyrrolidone-free polycarbonate membranes with 8-μm pores (Neuro Probes, Inc.). We then seeded KB stable expressed FLAG vector control, FLAG-PhD30, FLAG-PhD35, or FLAG-ZAKa at 1500 cells/well in serum-free DMEM to the top chamber. The Boyden chamber was incubated for 24 h at 37°C to allow for the migration or for 48 hours at 37°C to allow for the invasion of cells through the membrane into the bottom chamber. After the incubation, the top chamber was removed the top and the top of the membrane was gently wiped with a cotton swab to remove non-migrated/non-invaded cells. Membranes were stained using Giemsa stain. The membrane was rinsed with distilled water to remove excess stain and air-dried. Migrating and invading cells were imaged under a microscope and counted. In vitro kinase activity assay Protein kinase assays were carried out using glutathione S-transferase (GST)-RhoGDIb or S20 as a substrate. The GST fusion proteins were bound to ZAKa by incubating them with cellular extracts in the kinase buffer (20 mM Hepes, pH 7.6, 1 mM EGTA, 1 mM dithiothreitol, 2 mM MgCl, 2 mM MnCl, 5 mM NaF, 1 mM NaVO, 50 mM NaCl) for 15 min on ice. The beads were pelleted and thoroughly washed with PBST (150 mM NaCl, 16 mM sodium phosphate, pH 7.5, 1% Triton X-100, 2 mM EDTA, 0.1% MeOH, 0.2 mM phenylmethylsulfonyl fluoride, and 5 mM benzamidine) and then incubated with [g- 32 P]ATP (50 cpm/fmol) in the presence of kinase buffer. After washing the phosphorylated GST-RhoGDIb or GST-S20 with PBST seven times, it was boiled in SDS sample buffer. The proteins were run on a 15% SDS-PAGE. The gel was dried, and phosphorylation of the RhoGDIb or S20 substrate was determined by autoradiography. Isolation of mitochondria Cells were cultured to 70-80% confluency. The cells were rinsed twice with ice-cold PBS to remove any remaining media or serum. The cells were then resuspended in hypotonic solution (1.25M sucrose, 10mM MOPS, pH7.2) and allowed to swell for 10 minutes on ice. Swollen cells were suspended in a Dounce and homogenized using six gentle up and down rotating strokes and immediately added to a hypertonic solution (100mM sucrose, 10 mM MOPS, 1 mM EDTA, pH 7.2). The homogenized solution was diluted with two volumes of isolation buffer (75 mM mannitol, 225 mM sucrose, 10mM MOPS, 1 mM EGTA, 0.1% fatty acid free BSA, pH7.2). Sediment nuclei, unbroken cells, and cellular debris were centrifuged at 930 g, 4°C for 5 minutes in a JA-20.1 Beckman rotor. The supernatant was then transferred to Beckman plastic centrifuge tubes. The pellet was added to four ml of isolation buffer, and then homogenization and centrifugation cycles were repeated. All the collected supernatants were combined in Beckman centrifuge tubes and run at 13000 xg under 4°C for 20 minutes. The supernatant was discarded and mitochondrial pellet was resuspend in isolation solution. Polysome profiling Cells at 70%–80% confluency were treated with 100 μg/ml cycloheximide for 5 minutes, washed, and scraped off in ice-cold PBS containing 100 μg/ml cycloheximide. They were then collected by centrifugation at 300 xg for 5 min. The isolated mitochondrial fractions were lysed in 425 ml hypotonic buffer (5 mM Tris-HCl pH 7.5, 1.5 mM KCl, 2.5 mM MgCl 2 ), added with 5ml of 10mg/ml cycloheximide and 1 ml of DTT, and vortexed for 5 seconds. The mixture was then added with 25ml of 10% triton X-100 and 25ml of sodium Deoxycholate. Debris was removed by centrifugation at 21,000 xg for 5 min at 4°C. The lysate concentration was measured by UV spectrophotometer. The same amount of lysate was loaded onto 10%–50% (w/v) sucrose gradient and centrifuged at 35000 xg for 2 hours at 4°C in a Beckman ultracentrifuge with the SW40Ti rotor. After ultracentrifugation, the fractions were collected by pipetting of 0.75 ml for each fraction from the top. Absorbance was recorded using a spectrophotometer set at 254nm. All fractions were immediately frozen on dry ice for future study. Proteins were precipitated with 10% trichloroacetic acid (TCA) at 4°C. The samples were spun down at18000 xg for 15 minutes at 4°C and washed twice in ice-cold acetone. Pellets were resuspended in laemmli buffer containing DTT and boiled at 95°C for 5 minutes. Yeast two hybrid assay The two-hybrid screen assay was performed as described in the user’s manual (Clontech). Briefly, the full-length ZAK was cloned to the pGBKT7 vector as the bait construct. This vector encoding GAL4 DNA binding domain-ZAK fusion (DNA-BD/ZAK) was transformed into AH109 yeast. This pGBKT7-ZAK yeast was mated with Y187 yeast containing a pretransformed MATCHMAKER human heart cDNA library. The yeast was selected following the manufacturer’s directions. The positive clones were isolated and sequenced. Isolate mRNA and RT-PCR Total RNA was isolated from whole cells or purified mitochondrial fractions of KB cells expressing PhD30 or PhD35 using TRIzol (Invitrogen) following the manufacturer's directions. 5 mg of isolated mRNAs was added to the RT-PCR master mix (oligo dT, 10 mM dNTPs, 200 units/μl reverse transcriptase, 5x reaction buffer) at 37°C for 60 minutes. After the reverse transcription reaction, 45 μl of sterile water was added to the mixture. For the PCR reaction, 2 μl of cDNA product from the reverse transcription reaction was used to amplify cDNA with specific primers for CV-ATP5A, CIII-UQCRC2 or actin for 25 cycles. The PCR products were detected by gel electrophoresis. Pull-down assay pGex-S20 plasmids were transferred into BL21 (DE3) and positive clones were selected. The positive clone was induced by IPTG. The bacteria were lysed and GST-S20 protein was immobilized by glutathione-sepharose. 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An ER surface retrieval pathway safeguards the import of mitochondrial membrane proteins in yeast. Science 361 , 1118-1122 (2018). Fulda, S., Galluzzi, L. & Kroemer, G. Targeting mitochondria for cancer therapy. Nature reviews Drug discovery 9 , 447-464 (2010). Wang, L. et al. Selective targeting of gold nanorods at the mitochondria of cancer cells: implications for cancer therapy. Nano letters 11 , 772-780 (2011). Weinberg, S. E. & Chandel, N. S. Targeting mitochondria metabolism for cancer therapy. Nature chemical biology 11 , 9-15 (2015). Housman, G. et al. Drug resistance in cancer: an overview. Cancers 6 , 1769-1792 (2014). Neophytou, C. M., Trougakos, I. P., Erin, N. & Papageorgis, P. Apoptosis deregulation and the development of cancer multi-drug resistance. Cancers 13 , 4363 (2021). Additional Declarations There is NO Competing Interest. Supplementary Files FigureS1.tif Dataset 1 Fig. 1. Specific binding of PhD peptides to MYC-tagged ZAKα protein KB cells were transiently co-transfected with GFP-tagged PhD peptides and MYC-ZAKα plasmids, and the cell lysates were immunoprecipitated with an anti-GFP antibody. Co-precipitated MYC-ZAKα peptides were detected by anti-MYC antibody. All these PhD peptides bound to MYC-tagged ZAKα protein. FigureS2.tif Dataset 2 Fig. 2. The effect of PhD30 and PhD35 peptides on mitochondrial activity in H9C2 cells Western blot of Tet-on inducible ZAK H9C2 Rat cardiomyoblast cells expressing FLAG tagged PhD30 or PhD35 showing their protein levels of CIII-UQCRC2 and CV-ATP5A. The protein of both were found to be decreased. FigureS3ABC.tif Dataset 3ABC Fig. 3. The effect of GFP-PhD30 or GFP-PhD35 peptides on mitochondrial function in KB cells (A) MitoTracker Red CMXROS staining was used to evaluate membrane potential in KB cells expressing GFP-PhD30 or GFP-PhD35 peptide. Cells expressing GFP-PhD30 or GFP-PhD35 peptide had a decrease in membrane potential compared to controls. (B) The basal oxygen consumption rate (OCR) was measured in KB cells expressing EGFP-PhD30 or EGFP-PhD35 peptides. Cells expressing GFP-PhD35 peptides had a reduction in OCR. (C) Quantification of basal OCR in KB cells expressing GFP-PhD30 or GFP-PhD35 peptide. There was a decrease in basal OCR in cells expressing GFP-PhD35 peptide but not GFP-PhD30. FigureS4A.tif Dataset 4 A Fig. 4. Pull-down assay identification of potential binding sites in ZAKα for S20 (A) HEK293 cells expressing ZAKα C-terminal deletion mutants ZAK-100, ZAK-340, and ZAK-460) and cell lysates were subjected to pull down by Sepharose immobilized GST-S20. There was no pull down in ZAKα C-terminal deletion mutants (ZAK-200, ZAK-390, and ZAK-500, suggesting that there are least three regions (300-340, 410-460, and 600-700) within ZAKα that can interact with S20. FigureS4B.tif Dataset 4 B Fig. 4. Pull-down assay identification of potential binding sites in ZAKα for S20 (B) ZAKα with SAM-deleted mutant lost its ability to bind to Sepharose immobilized GST-S20, suggesting that the SAM domain in ZAKα is essential for the maintenance of the correct conformation necessary for the interaction of ZAKα with RPS20. (C) Computer modeling the structures ZAKα (gold) and S20 (green) proteins were predicted by RoseTTAFold, and the HDOCK program was used to predict the potential docking sites for ZAKα and S20. The contact regions between these proteins were found to be located at ZAKα 323-330, 409-417, and 626-710. FigureS4C.tif Dataset 4C Fig. 4. Pull-down assay identification of potential binding sites in ZAKα for S20 (C) Computer modeling the structures ZAKα (gold) and S20 (green) proteins were predicted by RoseTTAFold, and the HDOCK program was used to predict the potential docking sites for ZAKα and S20. The contact regions between these proteins were found to be located at ZAKα 323-330, 409-417, and 626-710. FigureS5AB.tif Fig. 5. PhD30 and PhD35 peptides did not induce caspases in cancer cells (A) The activation of apoptotic caspase was assessed in KB cells stably expressing either FLAG-PhD30 or FLAG-PhD35 to determine whether the two peptides induced apoptosis. Western blot showed no changes in pro-caspase 3, caspase 8 or caspase 9 level in cells expressing PhD30 or PhD35 peptide compared to the control cells. (B) KB cells transiently co-transfected GFP-ZAKα with increasing amounts of FLAG-PhD30 or FLAG-PhD35 plasmids. No changes in the pro-caspase 3, caspase 8 or caspase 9 level in cells expressing PhD30 or PhD35 peptide compared to the vector transfected cells. In addition, the levels of BCL2 protein were also not affected by the expression of PhD30 or PhD35 peptide in cells. These findings suggest that apoptosis was not induced in cancer cells expressing either PhD30 and PhD35 peptide. FigureS6.tif Fig. 6. Low dose combined treatment with Oligomycin and Antimycin A did not induce apoptosis or necrosis in cancer cells Cells were co-treated with both Oligomycin and Antimycin at different concentrations (100nM or 500nM) for 48 hours and flow cytometry was used to study cell population were calculated by Annexin V and PI dual staining to study the effect of combined treatment on apoptosis or necrosis. No change was found in the population of apoptotic or necrotic KB cells treated with this combination of inhibitors at a concentration of 100nM compared to controls, indicating no increase cell death. However, at a concentration of 500nM, there was an increase in apoptosis and necrosis compared to controls. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4003086","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":276990554,"identity":"1ef5ad79-a0b1-4964-aa74-fdcb98475f55","order_by":0,"name":"Jaw-Ji Yang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyUlEQVRIiWNgGAWjYBACxoYDIMoGymUjXksaQgsPkZYdJkELc+MZw88Fv87Ly087Y8Dwoewwg71EAiGHnTGWntl323DD7RwDxhnnDjPwEKHFQJq353aCgXSOATNvG1CLNBG2/ObtOZcgPxuo5S+RWsykeX4cSGAAOoyZkTgtx8qseRuSgX5JKzjYcy6dh+f+A/xaDGcc3nyb54+dvPzs5I0PfpRZy7H3HCCk5YQBA2MbhANSSzgm5fnbge74Q1DdKBgFo2AUjGQAADU/RXnYzqTwAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0001-9385-5275","institution":"Chung-Shan Medical Univeresity","correspondingAuthor":true,"prefix":"","firstName":"Jaw-Ji","middleName":"","lastName":"Yang","suffix":""},{"id":276990555,"identity":"dd8d09b1-96e0-4d9a-9b38-c5c17b7d7753","order_by":1,"name":"Ying-Chang Hsu","email":"","orcid":"","institution":"Chung-Shan Medical University","correspondingAuthor":false,"prefix":"","firstName":"Ying-Chang","middleName":"","lastName":"Hsu","suffix":""},{"id":276990556,"identity":"a310b3a2-99fb-4d26-bebb-35c24f8807ea","order_by":2,"name":"Yang-Shan Yeh","email":"","orcid":"","institution":"Chung-Shan Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yang-Shan","middleName":"","lastName":"Yeh","suffix":""}],"badges":[],"createdAt":"2024-03-01 11:54:58","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4003086/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4003086/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":52755224,"identity":"ede09329-ff83-4646-89e5-4983268a6464","added_by":"auto","created_at":"2024-03-15 11:29:52","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":371158,"visible":true,"origin":"","legend":"\u003cp\u003eIdentification and characterization of novel peptide inhibitors for ZAKα through phage display experiment.\u003c/p\u003e\n\u003cp\u003e(A) Expression and purification of GST-ZAK414-800. GST-tagged ZAKα NSLC 414-800 (GST-ZAK414-800) was expressed in E. coli BL21 (DE3) cells and purified. Purified GST-ZAK414-800 was detected by western blot using anti-GST or anti-ZAKα antibody. (B) Sequences of four ZAKα binding peptides. These four phage clones (PhD10, PhD30, PhD35 and PhD40) were isolated by the binding of Ph.D.-C7C phage display peptide library with GST-ZAK414-800 and sequenced. (C) Binding of PhD peptides to endogenous ZAKα protein. GFP-tagged PhD peptides were transfected into KB cells, and the protein complexes were immunoprecipitated with an anti-ZAKα antibody (0834). Co-precipitated GFP-PhD peptides were detected by using a GFP monoclonal antibody.\u003c/p\u003e","description":"","filename":"Figure1ABC.png","url":"https://assets-eu.researchsquare.com/files/rs-4003086/v1/36e88afa4ec52013a46eef4a.png"},{"id":52755226,"identity":"5e5cf2ec-5781-45a7-8c01-ca7db9e07db5","added_by":"auto","created_at":"2024-03-15 11:29:52","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2938598,"visible":true,"origin":"","legend":"\u003cp\u003eIdentification of the PhD peptides binding site in ZAKα.\u003c/p\u003e\n\u003cp\u003e(A) GFP-ZAKα expressed HEK293 cell lysate was pulled down by Sepharose immobilized GST-TAT-PhD proteins. All four GST-TAT-PhD proteins (PhD10, PhD30, PhD35, and PhD40) were able to pull down GFP-ZAKα. (B) HEK293 cells were transient transfected ZAK C-terminal deleted mutant, GFP-ZAK-380 (amino acids 1-420), or GFP-ZAKα plasmids and cell lysates were subjected to pull down by Sepharose immobilized GST-TAT-PhD proteins. Both full-length GFP-ZAKα and GFP-ZAK-380 were pulled down by GST-TAT-PhD30 and GST-TAT-PhD35 but not GST-TAT-PhD10, suggesting that the binding site in ZAKα for PhD30 or PhD35 peptides could be located at the region of 414-420 in ZAKα. (C) Schema shows the binding site for PhD30 or PhD35 peptide was located next to the SAM domain in ZAKα and designated as ZAKPBS (phage-binding site) with the amino acid sequence LFHFPPL. (D) Upper panel shows HEK293 cell lysate expressed GFP-ZAKPBS was subjected to pull down by GST-TAT-PhD proteins. Both GST-TAT-PhD30 and GST-TAT-35, but not GST-TAT-PhD10 or GST-TAT-PhD40, \u0026nbsp;were pulled down by GFP-ZAKPBS. The lower panel shows that HEK293 cells transiently transfected GFP-ZAKPBS plasmid and that GFP-ZAKPBS protein was detected by GFP antibody. (E) Computer protein modeling and peptide global docking were used to help identify the binding sites of PhD30 and PhD35 peptides on ZAKα. The docking site predictions were made using PIPER-FlexPepDock, which showed the docking sites for PhD30 and PhD35 to be the same as those identified in our experimental results, where PhD30 docked next to the End-Helix (EH) site of ZAKα's SAM domain and PhD35 docked next to the Middle-Loop (ML) side of ZAKα's SAM domain.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4003086/v1/9444feda315da9c77fab8b94.png"},{"id":52755229,"identity":"fd1ce6c8-b565-4678-bbce-4d473a1c5d73","added_by":"auto","created_at":"2024-03-15 11:29:52","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2814912,"visible":true,"origin":"","legend":"\u003cp\u003ePhD30 and PhD35 peptides affected mitochondrial activities in cancer cells.\u003c/p\u003e\n\u003cp\u003e(A) Western blot of the protein levels of CIII-UQCRC2, CV-ATP5A, and β-actin in total cell lysates and isolated mitochondria from stable KB cells expressing FLAG tagged PhD30 or PhD35 KB cells. (B) Total cellular and isolated mitochondrial RNA were isolated and run on a formaldehyde agarose gel. Total RNA had clear 18S and 28S rRNA, while the isolated mitochondrial RNA only showed 12S and 16S rRNA without cellular fraction contamination. (C) MitoTracker Red CMXROS staining shows that KB cells expressing FLAG tagged PhD30 or PhD35 had reduced mitochondrial activity and reduced membrane potential. (D) KB cells expressing FLAG tagged PhD30 or PhD35 had lower Basal oxygen consumption rates (OCR) compared to control cells. (E) Quantification of basal OCR in KB cells expressing FLAG tagged PhD30 or PhD35 compared to control cells.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4003086/v1/3dc6567126832729eeb5021a.png"},{"id":52755899,"identity":"ca6afc06-a49f-467a-8a49-e33d39f43a81","added_by":"auto","created_at":"2024-03-15 11:37:54","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":8904725,"visible":true,"origin":"","legend":"\u003cp\u003eZAKα bound to ribosomal protein S20, while PhD30 and PhD35 influenced the association of ZAKα with mitochondria, S20 ubiquitination, and translation efficiency in KB cells.\u003c/p\u003e\n\u003cp\u003e(A) ZAKα and its kinase-dead mutant (ZAKdn) and kinase-constitutively active mutant (ZAKE/E) was pulled down by Sepharose immobilized GST-S20. \u003cem\u003eIn vitro\u003c/em\u003e kinase assays showed that ZAKα and ZAKE/E, but not ZAKdn, phosphorylated S20. HEK293 (B) or KB (C) cells were transiently co-transfected with EGFP-ZAKα and increasing amounts of FLAG-PhD30 or FLAG-PhD35 plasmids. GST-S20 pull-down assay showed the higher the amount of FLAG-PhD30 or FLAG-PhD35, the greater the decrease in ZAKα protein binding to GST-S20, suggesting that PhD30 and PhD35 may interfere the binding of ZAKα with S20. (D, E, F and G) Mitochondrial fractions were isolated from KB cells stable expressing FLAG or EGFP tagged PhD30 or PhD35 peptides. Western blot of the protein levels of ZAKα in the mitochondrial fractions showed that both PhD30 or PhD35 peptide decreased the association of ZAKα with mitochondria where the protein levels of ribosomal proteins S20 and L4 in the mitochondrial fractions were not changed. (H) PhD30 or PhD35 was also detected in mitochondrial fractions isolated from KB cells expressing either EGFP tagged PhD30 or PhD35 peptide. (I) The mitochondria fraction was immunoprecipitated by anti-S20 antibody. Western blot showed that S20 enhanced ubiquitination in KB cells expressing PhD30 or PhD35 peptide with the formation of mono-, bi-, and tri-ubiquitinated S20 detected by either anti-ubiquitin antibody (upper panel) or anti-S20 antibody (lower panel). (J, K, and L) Total cellular RNA or mitochondria associated nuclear RNA was isolated and the semi-quantitative RT-PCR was used to determine the amount of mRNA, including CIII-UQCRC2, CV-ATP5A, or β-actin. There was a decrease in the three mRNA in the mitochondrial fraction in KB cells expressing PhD30 or PhD35 peptide compared the control cells, while the total amount of these mRNAs was not changed. (M) Total ribosomes were isolated from KB cells expressing ZAKα, PhD30, or PhD35 peptide. No significant changes were found in polysome profiling in these cells. (N) Mitochondria-associated ribosomes were further isolated from mitochondria fraction in KB cells expressing PhD30 or PhD35 peptide and the ribosomal translation activities were analyzed by polysome profiling. Less ribosomal translational activity was observed in EGFP tagged PhD30 (right panel) or PhD35 (left panel) cells. (O) Fractions from the polysome profiling experiment were analyzed to determine the distribution of the regulation proteins , including ZAKα, S20, PhD peptide, or eIF2α, in KB cells expressing EGFP-PhD30 or EGFP-PhD35. Protein levels of ZAKα were lower in the early polysome profiling fractions of KB cells expressing PhD30 or PhD35 compared the control cells, on the contrast, while the protein levels of ubiquitinated S20 and eIF2α were increased in these early fractions. The PhD30 or PhD35 peptide was detected in the early fraction, suggesting that the binding of these peptides ZAKα caused the dissociation of ZAKα from S20. (P) KB cells expressing FLAG-PhD30 or FLAG-PhD35 were blocked with cycloheximide for 8 hours and then unblocked. The mitochondria-associated ribosomes were isolated from these cells at 24, 48, and 72 after unblocking. These ribosomes were subjected to polysome profiling assay. After 24 or 48 hours release from the cycloheximide blockage, KB cells expressing PhD30 or PhD35 were found to have reduced polysome activity on mitochondria compared to control cells, suggesting a reduction in ribosome translation efficiency on mitochondria. After 72 hours of release from the blockage, the mitochondria-associated ribosomes were found to have accumulated in the monomer stage in KB cells expressing PhD35, whereas the mitochondria-associated ribosomes had accumulated in the low-polysome stage on mitochondria in the KB cells expressing PhD30 compared to the control cells. PhD30 or PhD35 cells exhibited fewer polysomes on mitochondria than control cells. (Q) Calculations of the mRNA/Ribosome in mitochondrial fractions revealed a decrease in mRNA/Ribosome contents in mitochondria over time in KB cells expressing PhD30 or PhD35. (R) The effects of PhD30 or PhD35 peptide on the growth rate of KB cells.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4003086/v1/c6e445ad53886d4946f29ea0.png"},{"id":52755227,"identity":"6883d449-5fb1-4f05-bf50-6ba9dd0946d3","added_by":"auto","created_at":"2024-03-15 11:29:52","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1607903,"visible":true,"origin":"","legend":"\u003cp\u003eZAK ZAKα played a crucial role in regulating ribosome translation efficiency on mitochondria and mitochondrial activity.\u003c/p\u003e\n\u003cp\u003e(A) Polysome profiling showed less mitochondria-associated ribosome translation activity in KB cells the experiment knocking down of ZAKα with shRNA 1712i. (B) ZAKα knockdown KB cells had decreased protein levels of the oxidative phosphorylation complexes CV-ATP5A and CIII-UQCRC2. (C) ZAKα knockdown KB cells had increased S20 ubiquitination. (D) KB cells expressing ZAKdn retained their anchorage-independent growth ability. (E) KB cells expressing ZAKdn \u0026nbsp;had no change in the ribosomal translation activity on mitochondria compared to those expressing ZAKα. (F) The protein levels of the oxidative phosphorylation complexes CV-ATP5A and CIII-UQCRC2, and ribosomal proteins S20 and L4 were not altered in the mitochondrial fraction of KB cells expressing ZAKα or ZAKdn. (G) There was no difference in S20 ubiquitination between the KB cells expressing ZAKα and ZAKdn. (H) MitoTracker Red CMXROS staining revealed no change in mitochondrial activity in KB cells expressing either ZAKα or ZAKdn.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4003086/v1/fc7835b34726c30499d6f426.png"},{"id":52755225,"identity":"949bfddd-8378-4fd1-a71a-e5b3a469f5be","added_by":"auto","created_at":"2024-03-15 11:29:52","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":981138,"visible":true,"origin":"","legend":"\u003cp\u003eHIV TAT-fusion PhD peptides inhibited anchorage-independent growth and proliferation of KB cancer cells.\u003c/p\u003e\n\u003cp\u003e(A, B) The colony formation ability of KB cells treated with purified GST-TAT-PhD30 or GST-TAT-PhD35 proteins (33.3nM) was reduced to 69% or 54%, respectively, compared to those treated with GST-TAT. (C) Western blot analysis was used to determine the potential entry of GST-TAT-PhD35 protein into KB and Rat6 cells. The uptake efficiency of GST-TAT-PhD35 was higher in Rat6 cells than in KB cells. (D) The proliferation rate of KB cells treated GST-TAT-PhD30 or GST-TAT-PhD35 proteins at a concentration of 33.3 nM. KB cell growth was arrested by exogenously supplied GST-TAT-PhD30 or HST-TAT-PhD35 starting on the fourth day of treatment.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-4003086/v1/33e5d2b593bf586a962d095d.png"},{"id":52755236,"identity":"6e6f1ebb-9030-436e-8662-61d7f1c0414e","added_by":"auto","created_at":"2024-03-15 11:29:54","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":6176276,"visible":true,"origin":"","legend":"\u003cp\u003ePhD30 and PhD35 inhibited oral cancer cell proliferation and migration by targeting ZAKa.\u003c/p\u003e\n\u003cp\u003e(A and B) Cultures of KB cells stably expressing EGFP-PhD 30 or 35 were grown under non-adherent growth conditions showed that both PhD30 and 35 peptides inhibited KB cell growth. (C) KB cells expressing EGFP-PhD30 and EGFP-PhD35 were found to have lower proliferation rates compared to the control KB cells. (D) \u003cem\u003eIn vitro\u003c/em\u003e kinase revealed an inhibition of ZAKa kinase activity KB cells expressing EGFP-PhD35 but not EGFP-PhD30. (E and F) KB cells stably expressing FLAG-ZAK, FLAG-PhD30, FLAG-PhD35, or FLAG-ZAKβ and ZAK knockdown (1712i) cells grown under anchorage-independent conditions. Cell growth was inhibited in KB cells expressing either FLAG-PhD30 or PhD35 peptide. (G) The proliferation rates KB cells stably expressing FLAG-ZAK, FLAG-PhD30, FLAG-PhD35, or FLAG-ZAKβ and ZAK knockdown (1712i) cells. The proliferation rates were lower in those expressing either FLAG-PhD30 or PhD35 peptide. (H) Loss of migration ability (H) and invasion ability (I) in KB cells expressing FLAG-PhD30 and PhD35 peptide. (J and K) KB cells were transiently co-transfected with GFP-ZAKPBS and either FLAG-PhD30 or FLAG-PhD35 and grown under non-adherent conditions. ZAKPBS restored the growth inhibited by either PhD30 or PhD35. (L and M) KB cells expressing one of four stable FLAG-conjugated PhD peptides (PhD10, PhD30, PhD35, and PhD40) were injected subcutaneously into the backs of nude mice. Four weeks after injection, the mice were killed and their tumor sizes were measured. (N) Antimycin A, an inhibitor of oxidative phosphorylation complex III, and Oligomycin, an inhibitor of oxidative phosphorylation complex V, were used to treat KB cells to study the relationship between mitochondrial activity and tumor growth. KB cells treated with either Antimycin A or Oligomycin were found to have a dose-dependent inhibition of colony formation. Combined treatment of Antimycin A and Oligomycin had a synergistic effect, leading to a more pronounced reduction in colony formation. (O). MitoTracker Red CMXROS staining showed a dose-dependent inhibition of mitochondrial membrane potential in cells treated with either Antimycin A or Oligomycin. Combined treatment at concentration of 100nM resulted in a ten-fold inhibition in that potential, compared to a single using either of these inhibitors alone. (P) PhD30 and PhD35 peptides bound to ZAKα leading to a dissociation of ZAKα from S20 and potentiating S20 ubiquitylation. This ubiquitination may have triggered ribosome translational jams on mitochondria, resulting in a shortage of mitochondrial proteins, particularly oxidative phosphorylation complexes, and a decrease in mitochondrial activity and ATP production. This shortage of energy supply may have caused tumor cells to lose their ability to grow, migrate, and invade. Figure was created using SMART–Servier Medical Art \u003ca href=\"https://smart.servier.com/\"\u003e\u003cstrong\u003ehttps://smart.servier.com\u003c/strong\u003e\u003c/a\u003e\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-4003086/v1/3cb0d15617a486721b65e964.png"},{"id":52757107,"identity":"927b02ff-dd23-4397-b83c-d9a0ed66f6ad","added_by":"auto","created_at":"2024-03-15 11:53:58","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":12003820,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4003086/v1/bc83d263-da08-4d03-b423-6e24003991e7.pdf"},{"id":52755898,"identity":"80f55490-7bc7-4444-b289-0cd3c013f9cb","added_by":"auto","created_at":"2024-03-15 11:37:53","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":14001054,"visible":true,"origin":"","legend":"\u003cp\u003eDataset 1\u003c/p\u003e\n\u003cp\u003eFig. 1. Specific binding of PhD peptides to MYC-tagged ZAKα protein\u003c/p\u003e\n\u003cp\u003eKB cells were transiently co-transfected with GFP-tagged PhD peptides and MYC-ZAKα plasmids, and the cell lysates were immunoprecipitated with an anti-GFP antibody. Co-precipitated MYC-ZAKα peptides were detected by anti-MYC antibody. All these PhD peptides bound to MYC-tagged ZAKα protein.\u003c/p\u003e","description":"","filename":"FigureS1.tif","url":"https://assets-eu.researchsquare.com/files/rs-4003086/v1/5f951bf5e396ddf42cfd21e6.tif"},{"id":52755234,"identity":"e9433cae-21e5-44e1-ab31-836786dc51b0","added_by":"auto","created_at":"2024-03-15 11:29:53","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":14001054,"visible":true,"origin":"","legend":"\u003cp\u003eDataset 2\u003c/p\u003e\n\u003cp\u003eFig. 2. \u0026nbsp;The effect of PhD30 and PhD35 peptides on mitochondrial activity in H9C2 cells\u003c/p\u003e\n\u003cp\u003eWestern blot of Tet-on inducible ZAK H9C2 Rat cardiomyoblast cells expressing FLAG tagged PhD30 or PhD35 showing their protein levels of CIII-UQCRC2 and CV-ATP5A. The protein of both were found to be decreased.\u003c/p\u003e","description":"","filename":"FigureS2.tif","url":"https://assets-eu.researchsquare.com/files/rs-4003086/v1/f9e67616def6cdc005fc08e7.tif"},{"id":52755895,"identity":"439b992b-997f-4076-bffd-b5abbbb2048d","added_by":"auto","created_at":"2024-03-15 11:37:52","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":14001054,"visible":true,"origin":"","legend":"\u003cp\u003eDataset 3ABC\u003c/p\u003e\n\u003cp\u003eFig. 3. The effect of GFP-PhD30 or GFP-PhD35 peptides on mitochondrial function in KB cells\u003c/p\u003e\n\u003cp\u003e(A) MitoTracker Red CMXROS staining was used to evaluate membrane potential in KB cells expressing GFP-PhD30 or GFP-PhD35 peptide. Cells expressing GFP-PhD30 or GFP-PhD35 peptide had a decrease in membrane potential compared to controls. (B) The basal oxygen consumption rate (OCR) was measured in KB cells expressing EGFP-PhD30 or EGFP-PhD35 peptides. Cells expressing GFP-PhD35 peptides had a reduction in OCR. (C) Quantification of basal OCR in KB cells expressing GFP-PhD30 or GFP-PhD35 peptide. There was a decrease in basal OCR in cells expressing GFP-PhD35 peptide but not GFP-PhD30.\u003c/p\u003e","description":"","filename":"FigureS3ABC.tif","url":"https://assets-eu.researchsquare.com/files/rs-4003086/v1/7ea28c753f30a1be645fbc91.tif"},{"id":52755896,"identity":"0202fd9b-e60e-400c-97ea-543d50af0ad0","added_by":"auto","created_at":"2024-03-15 11:37:52","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":14001054,"visible":true,"origin":"","legend":"\u003cp\u003eDataset 4 A\u003c/p\u003e\n\u003cp\u003eFig. 4. Pull-down assay identification of potential binding sites in ZAKα for S20\u003c/p\u003e\n\u003cp\u003e(A) HEK293 cells expressing ZAKα C-terminal deletion mutants ZAK-100, ZAK-340, and ZAK-460) and cell lysates were subjected to pull down by Sepharose immobilized GST-S20. There was no pull down in ZAKα C-terminal deletion mutants (ZAK-200, ZAK-390, and ZAK-500, suggesting that there are least three regions (300-340, 410-460, and 600-700) within ZAKα that can interact with S20.\u003c/p\u003e","description":"","filename":"FigureS4A.tif","url":"https://assets-eu.researchsquare.com/files/rs-4003086/v1/dbbc78097bd30b91ff1fbdd3.tif"},{"id":52755235,"identity":"0b8891df-ebe1-4a44-9db0-c79ae9f1a25f","added_by":"auto","created_at":"2024-03-15 11:29:54","extension":"tif","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":14001054,"visible":true,"origin":"","legend":"\u003cp\u003eDataset 4 B\u003c/p\u003e\n\u003cp\u003eFig. 4. Pull-down assay identification of potential binding sites in ZAKα for S20\u003c/p\u003e\n\u003cp\u003e(B) ZAKα with SAM-deleted mutant lost its ability to bind to Sepharose immobilized GST-S20, suggesting that the SAM domain in ZAKα is essential for the maintenance of the correct conformation necessary for the interaction of ZAKα with RPS20. (C) Computer modeling the structures ZAKα (gold) and S20 (green) proteins were predicted by RoseTTAFold, and the HDOCK program was used to predict the potential docking sites for ZAKα and S20. The contact regions between these proteins were found to be located at ZAKα 323-330, 409-417, and 626-710.\u003c/p\u003e","description":"","filename":"FigureS4B.tif","url":"https://assets-eu.researchsquare.com/files/rs-4003086/v1/0c3c65d4efeef510be4969a5.tif"},{"id":52756715,"identity":"82d08e79-c693-4f32-9bc6-27ee8d2b9c43","added_by":"auto","created_at":"2024-03-15 11:45:52","extension":"tif","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":14001054,"visible":true,"origin":"","legend":"\u003cp\u003eDataset 4C\u003c/p\u003e\n\u003cp\u003eFig. 4. Pull-down assay identification of potential binding sites in ZAKα for S20\u003c/p\u003e\n\u003cp\u003e(C) Computer modeling the structures ZAKα (gold) and S20 (green) proteins were predicted by RoseTTAFold, and the HDOCK program was used to predict the potential docking sites for ZAKα and S20. The contact regions between these proteins were found to be located at ZAKα 323-330, 409-417, and 626-710.\u003c/p\u003e","description":"","filename":"FigureS4C.tif","url":"https://assets-eu.researchsquare.com/files/rs-4003086/v1/17a7f5b9685e2c118cc6f8c2.tif"},{"id":52755233,"identity":"5f858870-6d8a-472a-9e77-abcbd11a83b3","added_by":"auto","created_at":"2024-03-15 11:29:53","extension":"tif","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":14001054,"visible":true,"origin":"","legend":"\u003cp\u003eFig. 5. PhD30 and PhD35 peptides did not induce caspases in cancer cells\u003c/p\u003e\n\u003cp\u003e(A) The activation of apoptotic caspase was assessed in KB cells stably expressing either FLAG-PhD30 or FLAG-PhD35 to determine whether the two peptides induced apoptosis. Western blot showed no changes in pro-caspase 3, caspase 8 or caspase 9 level in cells expressing PhD30 or PhD35 peptide compared to the control cells. (B) KB cells transiently co-transfected GFP-ZAKα with increasing amounts of FLAG-PhD30 or FLAG-PhD35 plasmids. No changes in the pro-caspase 3, caspase 8 or caspase 9 level in cells expressing PhD30 or PhD35 peptide compared to the vector transfected cells. In addition, the levels of BCL2 protein were also not affected by the expression of PhD30 or PhD35 peptide in cells. These findings suggest that apoptosis was not induced in cancer cells expressing either PhD30 and PhD35 peptide.\u003c/p\u003e","description":"","filename":"FigureS5AB.tif","url":"https://assets-eu.researchsquare.com/files/rs-4003086/v1/0c11a42241bd0f869f1b03f3.tif"},{"id":52755240,"identity":"9f168293-cbd0-4e5b-925d-d57f2bd16308","added_by":"auto","created_at":"2024-03-15 11:29:56","extension":"tif","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":14001054,"visible":true,"origin":"","legend":"\u003cp\u003eFig. 6. Low dose combined treatment with Oligomycin and Antimycin A did not induce apoptosis or necrosis in cancer cells\u003c/p\u003e\n\u003cp\u003eCells were co-treated with both Oligomycin and Antimycin at different concentrations (100nM or 500nM) for 48 hours and flow cytometry was used to study cell population were calculated by Annexin V and PI dual staining to study the effect of combined treatment on apoptosis or necrosis. No change was found in the population of apoptotic or necrotic KB cells treated with this combination of inhibitors at a concentration of 100nM compared to controls, indicating no increase cell death. However, at a concentration of 500nM, there was an increase in apoptosis and necrosis compared to controls.\u003c/p\u003e","description":"","filename":"FigureS6.tif","url":"https://assets-eu.researchsquare.com/files/rs-4003086/v1/2f1f8cff9e55ada690b4fee2.tif"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Targeting ZAKα Interactions and Ribosomes with Novel peptides: The role of ZAKα in Mitochondrial Function and Cancer Cell Proliferation","fulltext":[{"header":"Introduction","content":"\u003cp\u003eZAK, a member of the MAPKKK (mitogen-activated protein kinase kinase kinase) family, plays a regulatory role in various cellular processes, including proliferation, differentiation, and stress response. ZAK kinase protein is expressed in various tissues and many cancer cells, and it is involved in the regulation of many signal transduction pathways, including the JNK (c-Jun N-terminal kinase) pathways and NFkB \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Dysregulation of ZAK kinase activity can lead to the development of cancer or heart disease \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e and ZAK kinase protein has been found to be upregulated in several cancers, including breast, lung, and colorectal cancer, and promote tumor growth and metastasis \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. In addition, ZAK has been implicated in the pathogenesis of heart disease, particularly cardiac hypertrophy and fibrosis \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. However, the fundamental molecular mechanisms underlying ZAK-mediated tumorigenesis and cardiac dysfunction are not well understood. ZAK plays an essential role in the regulation of ribosomal protein \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. In particular, it has been found to phosphorylate ribosomal protein S6 kinase (S6K), a kinase involved in the regulation of ribosome biogenesis and protein synthesis, processes which require the coordination of numerous proteins and signaling pathways \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eRibosomal translation, a process in which ribosomes synthesize proteins using messenger RNA (mRNA) as a template, is crucial for cell growth, development, and survival \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. The regulation of ribosomal translation is a complex process that enables cells to familiarize themselves with changing environmental conditions and respond to many signals \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. This regulation is achieved through a variety of mechanisms that control the initiation, elongation, and termination the synthesis of proteins. The mechanisms involved in these processes include the binding of regulatory proteins to mRNA and ribosomes, the post-translation modification of ribosomal proteins, and the modulation of signaling pathways \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. One important mechanism underlying this regulation is the binding of regulatory translation factors to mRNA and ribosomes \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. These factors can either promote or inhibit translation by controlling the assembly and activity of the ribosome. Additional regulatory proteins, such as RNA-binding proteins, can bind to specific regions of mRNA and affect the stability, localization, or availability of the transcript \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Another mechanism underlying the regulation of translation is the modification of ribosomal proteins and RNA \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. For example, phosphorylation of ribosomal proteins can vary their affinity for mRNA and regulate translation rates \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Similarly, modifications to ribosomal RNA, such as those made by methylation or pseudouridylation, can influence ribosome structure and function \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Recently, more research attention has been paid to ribosomal proteins (RPs) because they are crucial components of ribosomes, because they are intricately involved in protein synthesis, and because their expressions should be tightly regulated if proper assembly is to be achieved and the ribosomes are to function properly \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eRibosomal protein S20 (RPS20) is a vital component of the ribosome and is located on the small ribosomal subunit, where it plays a crucial role in translation. During the translation process, RPS20 interacts with other ribosomal proteins and rRNA to help position the mRNA and tRNA molecules in the correct orientation for peptide bond formation \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. It is also required in stabilizing the interactions between the ribosome and mRNA, which is necessary for the precise decoding of the genetic code \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Recent studies have shown RPS20 to also be involved in regulating the rate of translation, which is important for the maintenance of cellular homeostasis and reactions to stress \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Additionally, mutations in ribosomal RPS20 have been linked to various genetic disorders and cancers, suggesting that this protein contributes substantially to normal cellular functioning \u003csup\u003e\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Ubiquitination, a post-translation modification that involves the covalent linking of ubiquitin molecules to target proteins has been shown to play an important role in the regulating RPs, ribosome biogenesis as well as ribosomal translation rate \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. The regulation of RPs by ubiquitination involves a complex interaction between various enzymes, including an activating enzyme (E1), a conjugating enzyme (E2), and a ligase (E3) \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e as well as deubiquitinases and other associated factors \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Dysregulation of this process has been associated with many diseases, including cancer and neurodegenerative disorders \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Studies have observed that RPS20 ubiquitination can disturb protein translation by regulating the binding of specific mRNA transcripts to the ribosome \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Although there is much evidence that this process regulates protein translation, the fundamental mechanisms underlying RPS20\u0026rsquo;s ability to do this are not fully understood.\u003c/p\u003e \u003cp\u003eMitochondria are the organelles responsible for energy production in eukaryotic cells. Oxidative phosphorylation, which occurs within the inner mitochondrial membrane, is essential for the generation of ATP in cells. This reaction occurs via protein complexes organized into an electron transport chain (ETC). ETC functioning is strictly regulated, so disturbances in the normal activity of any of the complexes that make up the chain can lead to a variety of pathologies, including metabolic disorders, neurodegenerative diseases, cardiovascular diseases, and cancers \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Therefore, it is very important to understand the mechanisms that govern the effect of the ETC complexes on mitochondrial functioning. This knowledge could lead to the development of therapeutics for diseases involving mitochondrial malfunction. Ribosome co-translation on the mitochondrial membrane requires the synthesis of nascent proteins by the cytoplasmic ribosomes on the mitochondrial surface and the insertion of nascent polypeptides into the mitochondrial membrane. This process enables the newly synthesized proteins to target the mitochondria. Little is known, however, about the mechanisms underlying ribosome co-translation on the mitochondrial membrane and how these mechanisms are regulated.\u003c/p\u003e \u003cp\u003eOne known regulator of cellular signaling pathways involved in cell proliferation, differentiation, and apoptosis is ZAK. Disruption in ZAK\u0026rsquo;s ability to regulate such processes can lead to cancer or heart disease \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Its overexpression has also been found in numerous cancer types such as breast, lung, and colorectal cancer and is thought to accelerate tumor development and spread. In addition to its association with cancer, overexpressed ZAK has been linked to the development of heart disease via its regulation of cardiac hypertrophy and fibrosis \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. To the best of our knowledge, specific inhibitors of ZAK and their possible effect on cancer and heart disease have not been thoroughly studied.\u003c/p\u003e \u003cp\u003ePhage display technology has been used to select peptides, proteins, and antibodies with high specificity and affinity for target molecules. In this study, we used phase display technology to identify peptides that might bind to and inhibit ZAKα, a key member of the mixed lineage kinase family known to play a role in regulating tumor growth, and studied whether they would affect ZAKa-RPS20 binding. In the course of this study, we performed a number of \u003cem\u003ein vitro\u003c/em\u003e experiments investigating whether they would hinder ribosomal translation and reduce the mitochondrial proteins involved in ETC complexes. Their impact on mitochondrial activity was assessed by Agilent Seahorse Cell Mito Stress Test to measure basal oxygen consumption in mitochondria of the KB cells.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eIdentification and characterization of novel peptide for ZAKα through phage display approach\u003c/h2\u003e \u003cp\u003eZAK belongs to the Mixed Lineage Kinase (MLK) family protein, and the ZAK gene can produce two protein isoforms (ZAKα and ZAKβ) through alternative splicing \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. ZAKα regulates various biological functions. In tumors, for example, the ZAKα protein is upregulated. Using ZAKα specific short hairpin RNA (shRNA) to knockdown ZAKα in oral cancer cells, we previously found that these cells lost their ability to grow under an anchorage-independent condition, suggesting that ZAKα may be required for neoplastic transformation to occur. ZAKα has several domains, including a leucine-zipper (LZ) domain, a sterile-alpha motif (SAM) domain, and an approximately 400-amino-acid non-structural long C-terminal (NSLC) region. The LZ domain is involved in protein-protein interactions and is necessary for the dimerization of ZAKα. The SAM domain in ZAKα also participates in protein-protein interactions and plays an essential role in the formation of protein complexes \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. The NSLC region is a unique region in ZAKα, and little is known about its functions. In the current study, we subcloned the cDNA corresponding to ZAKa NSLC (amino acid 414\u0026ndash;800) into a GST-expressing vector. We purified the GST-ZAK414-800 protein and then added either anti-GST or anti-ZAKα antibody to identify the purified protein by western blot analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The Ph.D.-C7C phage display peptide library was used to help identify novel peptides that could bind specifically to this ZAKα NSLC 414\u0026ndash;800. We isolated 40 clones. Of these, we found the clones PhD10, PhD30, PhD35, and PhD40 to bind to this protein, repeated 2, 4, 1, and 4 times, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Their corresponding peptides were then tested for their binding ability to ZAKα by \u003cem\u003ein vitro\u003c/em\u003e co-precipitation. To do this, we transfected oral cancer cells (KB) with plasmids expressing the four GFP-tagged PhD peptides and immunoprecipitated the protein complexes using an anti-ZAK antibody (0834, generated in the lab). All four GFP-PhD peptides co-precipitated with ZAKα (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eC and Supplementary Fig.\u0026nbsp;1).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eIdentification and characterization of the interaction site within ZAKa for PhD peptides by GST pull-down assay and protein modeling\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo identify the binding site within ZAKα for these peptides, we fused oligonucleotides corresponding to sequence encoding HIV-TAT (GRKKRRQRRR) and individual PhD peptides with glutathione S-transferase (GST) to generate GST-TAT-PhD fusion proteins. These proteins were then immobilized on glutathione-sepharose resin. Cell lysate obtained from GFP-ZAKα expressed in HEK293 cells was subjected to pull down assay using GST-TAT-PhD fusion proteins. GFP-ZAKα protein was pulled down by all four GST-TAT-PhD proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), suggesting that ZAKα proteins interacted with the GST-TAT-PhD proteins. To further map the precise binding site within ZAKα protein that interacted with these PhD peptides, we created a series of ZAKα C-terminal deletion mutant clones which we transiently transfected with HEK293 cells (data not shown). Both full-length GFP-ZAK and a C-terminal deletion mutant, GFP-ZAK-380 (amino acids 1-420), were pulled down by GST-TAT-PhD30 and GST-TAT-PhD35 but not GST-TAT-PhD10 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). We performed our phage display experiments on ZAKα target region (amino acids 414\u0026ndash;800) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) and then using deletion experiments we were able to narrow down binding site between ZAKα and the PhD30 or PhD35 peptides to fall within amino acids 414\u0026ndash;420. We designated this site as the ZAK phage-binding site (ZAKPBS) with the amino acid sequence, LFHFPPL (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Pull down assay using GST-TAT-PhD10, 30, 35, and 40 revealed that only PhD30 and PhD35 might bind to this small region (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). We confirmed this possibility by performing RoseTTAFold modeling of ZAKa amino acid 321 to amino acid 600 \u003csup\u003e40\u003c/sup\u003e and global docking of the PhD30 or PhD35 peptide with ZAKα through PIPER-FlexPepDock \u003csup\u003e\u003cspan additionalcitationids=\"CR42 CR43\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eE shows the predicted docking sites. The PhD30 peptide (red) bound to ZAKPBS next to the End-Helix (EH) site of the ZAKa SAM domain, and the PhD35 peptide (blue) bound to ZAKPBS next to the Middle-Loop (ML) side of the SAM domain.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003ePhD30 and PhD35 peptides modulated mitochondrial protein expression and function in cancer cells\u003c/h2\u003e \u003cp\u003eCancer cells alter metabolism more significantly than normal cells, especially glucose metabolism \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. Most changes are increases glucose consumption and lactate production, a change known as the Warburg effect \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. These changes may interfere with the energy ATP requirements for cell proliferation and growth. Therefore, we wanted to investigate whether PhD30 or PhD35 peptides in cancer cells would alter the protein levels of oxidative complexes in mitochondria. To find out, we measured the protein levels of oxidative phosphorylation complex III-UQCRC2 (CIII-UQCRC2) and complex V-ATP5A (CV-ATP5A) in KB cells expressing FLAG-PhD30 or FLAG-PhD35. As can be seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, we found that protein levels of complex III and complex V were higher than those cells expressing ZAKα and control cells. We also measured their levels as well as mitochondrial β-actin proteins in isolated mitochondrial fractions and found them to be significantly more decreased in KB cells expressing either PhD30 or PhD35 compared to KB expressing ZAKα and control cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Together, these findings suggested that PhD30 and PhD35 peptides expressed in KB cells did not suppress the overall protein levels of oxidative phosphorylation complexes in cytosol, but they might have affected mitochondrial proteins levels in this organelle. Similar results were achieved in H9C2 cardiomyoblast cells (Supplementary Fig.\u0026nbsp;2), indicating that their effect on protein translocation and translation into mitochondria were not cell type dependent.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo determine the purity of the isolated mitochondria, we isolated total cellular and isolated mitochondrial RNA and applied them to an agarose gel. Total RNA was found to have clear 18S and 28S rRNA, while isolated mitochondrial RNA only showed 12S and 16S rRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), indicating the isolated mitochondria were pure and had no contaminated cytosolic fraction. We found lower mitochondrial oxidative phosphorylation complex protein levels in KB expressing PhD30 or PhD35, suggesting that these cells might have lower mitochondrial activity. To test this hypothesis, we used MitoTracker Red CMXROS staining to assess membrane potential of PhD30 or PhD35, either fused with EGFP or FLAG, in KB cells. We found that cells expressing PhD30 or PhD35 had less mitochondrial membrane potential than controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eC and Supplementary Fig.\u0026nbsp;3A). We then used the Agilent Seahorse Cell Mito Stress Test to measure the effect of PhD30 or PhD35 on the basal oxygen consumption rate (OCR) in mitochondria of KB cells. We found overexpression of PhD30 or PhD35 with either EGFP or FLAG tags led to a significant reduction in the basal OCR compared to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, E; Supplementary Fig.\u0026nbsp;3B, C). These results suggest that PhD30 or PhD35 may bind to ZAKα influencing mitochondrial respiration and energy metabolism in KB cells, which might occur because this binding interferes with the protein translation or translocation of newly synthesized proteins into the mitochondria.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eInteraction between ZAKα and ribosomal protein\u003c/h2\u003e \u003cp\u003eWe performed a yeast two-hybrid experiment using ZAKa protein as the bait and screened the human heart in the Matchmaker cDNA library to identify proteins that would interact with ZAKa protein. One isolated clone was found to be associated with ZAKa, Ribosomal Protein S20 (RPS20), a protein component located in the small ribosomal subunit thought to play a fundamental role in the translation progression \u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. The association we found between RPS20 and ZAKa suggested that ZAKa might regulate ribosomal translation. To test this hypothesis, we immobilized GST-S20 protein using Glutathione-Sepharose beads and found that it successfully pulled down ZAKa protein. Interestingly, the binding between ZAKa and RPS20 occurred independent of ZAKa intrinsic kinase activity as evidenced by the fact that GST-S20 pulled down both kinase-dead mutant (ZAKdn) and the kinase-constitutively active mutant (ZAKE/E) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). We also investigated the potential of ZAKa to phosphorylate RPS20. \u003cem\u003eIn vitro\u003c/em\u003e kinase assay showed that both ZAKa and ZAKE/E phosphorylated RPS20, whereas ZAKdn did not (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, lower panel). This finding suggested that ZAKα might regulate RPS20 function through phosphorylation via some unknown mechanism. Regardless, the physical binding of both proteins took place independent of ZAKa kinase activity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo determine the binding sites in ZAKα for RPS20, we constructed serial C-terminal deletion mutants and subjected them to pull down by GST-S20. We found at least three potential binding sites (300\u0026ndash;340, 410\u0026ndash;460, and 600\u0026ndash;700) to respond to RPS20 (Supplementary Fig.\u0026nbsp;4A). Sites 300\u0026ndash;340 and 410\u0026ndash;460 were located on the N and C termini of the SAM domain, respectively, possibly implicating SAM domain in ZAKa-RPS20 binding. We constructed a SAM domain deleted ZAKa mutant and found that had a decreased ability to bind with GST-S20 (Supplementary Fig.\u0026nbsp;4B).\u003c/p\u003e \u003cp\u003eWe used computer modeling RoseTTAFold \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e to predict ZAKa structures and the HDOCK program \u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e to predict ZAKa-RPS20 protein binding sites. We found the predicted binding sites for these two proteins to be located at amino acid residues 323\u0026ndash;330, 409\u0026ndash;417, and 626\u0026ndash;710 in ZAKa (Supplementary Fig.\u0026nbsp;4C), suggesting that the expression of PhD30 or PhD35 peptides in KB cells might affect the interaction between the two proteins, leading us to want to study the binding ability of ZAKa with RPS20 in the presence of PhD30 or PhD35 peptides. To do this, we transfected HEK293 and KB cells with EGFP-ZAK plasmid with different concentrations FLAG-PhD30 and FLAG-PhD35 plasmid and used GST-S20 pull-down assay to assess ZAK-RPS20 binding ability. In both HEK293 and KB cells, we found the higher the expression FLAG-PhD30 or FLAG-PhD35 plasmid, the greater the decrease in EGFP-ZAKa pulled down by GST-S20 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, C). These results suggest that by binding to ZAKa, PhD30 and PhD35 might interfere with the binding of between ZAKa and RPS20.\u003c/p\u003e \u003cp\u003eWe became interested in exploring the interaction between ZAKa and the mitochondria associated cytoplasm ribosome in the presence of PhD30 and PhD35 because their expression in cells could result in decreased protein levels of oxidative phosphorylation complexes in mitochondria. To do this, we isolated mitochondria from KB cells expressing either FLAG or EGFP fused with PhD30 or PhD35 and found the mitochondrial fractions of the KB cells expressing either PhD30 or PhD35 had decreased protein levels of ZAKa (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, F), while the protein levels of RPS20 and RPL4 remained unaffected (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eE, G). Together, these findings suggest that PhD30 and PhD35 bind to ZAKa, interfering with its ability to bind with RPS20 leading to its separation from the ribosomes. Moreover, we detected both PhD30 and PhD35 in the mitochondrial fraction (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eH), also suggesting that they contributed to ZAKα\u0026rsquo;s ability to affect ribosomal translation on mitochondria.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eThe effects of PhD30 and PhD35 peptides on RPS20 ubiquitination and mitochondrial-associated mRNA levels\u003c/h2\u003e \u003cp\u003eNumerous studies have found RPS20 to play a crucial role in the regulation of translation, to be involved in the assembly of the ribosome, and to interact with several other ribosomal proteins and rRNA \u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. It has also been shown to interact with specific mRNA sequences and possibly play a role in regulating the initiation of translation \u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. Thus, we wanted to explore how the binding of PhD30 or PhD35 peptide to ZAKa might affect the translation of mitochondria-associated cytoplasm ribosomes. To find out, we isolated the mitochondrial fractions from KB cells expressing either PhD30 or PhD35. In these fractions, we observed the RPS20 protein in multiple ladders with an interval of 8.5 kDa, suggesting that they could be post translational modifications, especially ubiquitination. We immunoprecipitated the mitochondrial fractions using RPS20 antibody and then used ubiquitin antibody to screen for the precipitated RPS20 protein. We found increasing RPS20 ubiquitination levels, which we suspected to be mono-ubiquitinated RPS20, bi-ubiquitinated RPS20, and tri-ubiquitin RPS20 in KB cells expressing PhD30 or PhD35 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eI). The binding of PhD30 or PhD35 to ZAKa protein might result in the dissociation of ZAKa from RPS20, which in turn would increase the ubiquitination of RPS20. This ubiquitination might be implicated in the regulation of translation process \u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. We wanted to investigate whether the expression of PhD30 or PhD35 impedes the ability of mitochondria-associated ribosome to translate nuclear mRNAs. To do this, we first isolated total cellular mRNA and mitochondria-associated mRNA from KB cells expressing PhD30 or PhD35 and then used semi-quantitative RT-PCR to measure the amounts of mRNAs, including CIII-UQCRC2, CV-ATP5A, and β-actin. We found a decrease in CIII-UQCRC2, CV-ATP5A, and β-actin mRNA associated with mitochondria in the KB cells expressing one of the two peptides, compared to control cells, while the mRNA isolated from total cellular fraction remained unchanged (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ, K, L). These findings revealed the reduction of these mitochondria-associated mRNA levels in cells expressing PhD30 or PhD35 correlated with the protein levels in the mitochondria (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), suggesting that the binding of PhD30 or PhD35 on ZAKa decreased the amount of mRNA associated with mitochondria. This phenomenon was reverse correlated with the amount of ubiquitination on RPS20, suggesting that PhD30 or PhD35 peptide binding to ZAKa might have an impact on mitochondrial function.\u003c/p\u003e \u003cp\u003eThe above findings suggest that when PhD30 and PhD35 peptide binds to ZAKa, there is an increase in RPS20 ubiquitination impeding the translation of mitochondria-associated ribosomes. We performed a polysome profiling experiment to assess translation efficiency. Total ribosomes were first isolated from the controls, and cells expressing ZAKa, EGFP-PhD30, or EGFP-PhD35 and the fractions were analyzed. We found no significant differences in translation efficiency, although the EGFP-PhD35 cells had a slightly higher polysome fraction compared to the controls (EGFP and EGFP-ZAKa) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eM). We also isolated mitochondria-associated ribosomes from the controls and cells expressing EGFP-PhD30 and EGFP-PhD35 and performed a polysome profiling analysis on them. We found no translation activity in the mitochondria-associated ribosomes isolated from the cells expressing EGFP-PhD35 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eN, left panel) and lower translational activity in the mitochondria-associated ribosomes isolated from cells expressing EGFP-PhD30 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eN, right panel) compared to the controls. Thus, by binding to ZAKa, PhD30 and PhD35 impeded the translation efficiency on mitochondria-associated ribosomes, which means that they could play role in regulating nascent proteins synthesis supplied for mitochondria and energy production.\u003c/p\u003e \u003cp\u003eAs mentioned above, the binding of PhD30 or PhD35 to ZAKa led to the dissociation of ZAKa from RPS20 in mitochondrial fractions (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, F). We thought it would be interesting to investigate the association between ZAKα status and elongating polysomes in KB cells expressing EGFP-PhD30 or EGFP-PhD35 in the polysome profile fractions. We found that KB cells expressing PhD30 or PhD35 had decreased the protein levels of ZAKα in the early polysome fractions compared to control cells, suggesting a possible role for ZAKa in regulating the initiation of translation. We also found a correlation between the increased levels of ubiquitinated RPS20 with the dissociation of ZAKα in early fractions. PhD30 and PhD35 were also found in the early fraction, strongly suggesting that they might cause this dissociation by binding to ZAKα. Furthermore, KB cells expressing PhD30 and PhD35 were found to have substantially higher levels of the translation initiation factor, eIF2α, in the early fraction compared to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eO), suggesting that the binding of PhD30 or PhD35 to ZAKα led to the dissociation of ZAKa from RPS20, leading to an elevation in RPS20 ubiquitination and inhibiting the dissociation of eIF2α from the ribosomal initiation complexes. Collectively, these findings strongly suggest that PhD30 or PhD35 might play a targeting role in modulating the association of ZAKα with ribosomal initiation complexes leading to a delay or stalling in the initiation of translation.\u003c/p\u003e \u003cp\u003eTo further examine whether the binding of PhD30 or PhD35 to ZAKa would result in ribosomal translation jams or a complete shutdown in KB cells, we treated KB cells expressing FLAG-PhD30 or FLAG-PhD35 with cycloheximide for eight hours to block ribosomal translation. The cells were then unblocked and mitochondria-associated ribosomes isolated from these cell cultures were subjected to polysome profiling at 24, 48, and 72 hours after the release from blockage. After 24 and 48 hours, the KB cells expressing PhD30 or PhD35 were found to have reduced polysome activity on the mitochondria compared to the controls, suggesting a reduction of ribosome translation efficiency on mitochondria. After 72 hours, the mitochondria-associated ribosomes had accumulated in the monomer stage in KB cells expressing PhD35 and they accumulated in the low-polysome stage in those expressing PhD30, compared to the controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eP). These findings suggest that the binding of PhD30 or PhD35 to ZAKa could trigger ribosome translation jams or a slow-down on mitochondria, interfering with the initiation of translation. We further found a decrease in mRNA/Ribosome on mitochondria over time in the cells expressing PhD30 or PhD35 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eQ) and that the expression of PhD30 and PhD35 reduced ribosome translation efficiency and slowed the growth of these cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eR).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eZAKa played a critical role in regulating mitochondria-associated ribosomal translation independent of its kinase activity\u003c/h2\u003e \u003cp\u003eAs suggested by the above findings, it is necessary that ZAKα bind to RPS20 for proper ribosomal translation on mitochondria. We wanted to know if there was a specific ZAKα protein required for mitochondria-associated ribosome translation to take place. To find out, we used ZAKα specific shRNA (1712i) to knock down the ZAKα expression in KB cells. The knockdown resulted in the retardation of mitochondria-associated ribosome translation activity as determined by polysome profiling (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). It also resulted in a reduction in the protein levels of oxidative phosphorylation complexes, such as CV-ATP5A and CIII-UQCRC2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e5\u003c/span\u003eB) and an increase in RPS20 ubiquitination in the mitochondria fraction (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). These results strongly suggest that it was necessary for ZAKα to bind to RPS20 to prevent RPS20 from the further ubiquitination, a process critical to the regulation of mitochondria-associated ribosome translation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe ZAKa protein transduces its signal cascade to downstream effector through its intrinsic kinase activity \u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. Because ZAKa phosphorylated RPS20 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eA bottom panel), we found it essential to our study to investigate whether ZAKa kinase activity played a role in regulating mitochondria-associated ribosomes and to investigate whether this would lead to its phosphorylation of RPS20. Therefore, we studied the possible effect of ZAKdn mutant, one that does not have kinase activity, on KB tumor growth. Interestingly, KB cells expressing ZAKdn were found to retain their ability to grow in the anchorage-independent condition (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e5\u003c/span\u003eD), suggesting that their ability to grow was independent of kinase activity. We performed a polysome profiling assay on cells expressing ZAKdn to further investigate the role of ZAKa kinase activity in regulating ribosome translation efficiency on mitochondria and found that cells expressing ZAKdn exerted ribosome translation activity on mitochondria similar to that of cells expressing ZAKa (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e5\u003c/span\u003eE), indicating that ribosome translation was regulated by some mechanism other than intrinsic kinase activity. Additionally, we found no change in the protein levels of CV-ATP5A and CIII-UQCRC2 or RPS20 in the total cellular fraction or in the mitochondria fraction in KB cells expressing ZAKa or ZAKdn (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). We also found no change in RPS20 ubiquitination in these cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e5\u003c/span\u003eG). MitoTracker Red CMXROS staining also showed no change in mitochondrial activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e5\u003c/span\u003eH).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eExogenous delivery of GST-TAT-PhD proteins inhibited anchorage-independent growth of KB cells through protein transduction\u003c/h2\u003e \u003cp\u003eHIV TAT\u0026rsquo;s (trans-activator of transcription) ability to carry proteins across the cell membrane and deliver proteins across the cell membrane has made TAT an attractive tool for drug delivery and gene therapy \u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. We wanted to determine whether the exogenous supplied proteins might transduce into cells and interfere with anchorage-independent growth. To find out, we purified GST-TAT-PhD30 and GST-TAT-PhD35 proteins and exogenously added them to KB cells cultured in soft agar. Both GST-TAT-PhD30 and GST-TAT-PhD35 were found able to reduce colony formation to 69% or 54%, respectively, at a concentration of 33.3 nM, compared to GST-TAT cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, B). We further examined the uptake capability of the exogenously supplied proteins when different concentrations of GST-TAT-PhD35 were added KB or Rat6 cells for 24 hours. Western blot was used to assess protein penetration. GST-TAT-PhD35 could be detected at a concentration of 10 \u0026micro;g/ml (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). GST-TAT-PhD30 or GST-TAT-PhD35 proteins were added to separate KB cell culture medium at a concentration of 33.3 nM, and the KB cell growth rates were measured. We found cell growth to be arrested by exogenously supplied GST-TAT-PhD30 or GST-TAT-PhD35 protein to KB cells starting on the fourth day of treatment compared to GST-TAT controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). These findings indicated that continuous treatment of KB cells with exogenously supplied GST-TAT-PhD30 or GST-TAT-PhD35 proteins arrested cancer cell proliferation. Furthermore, the activation of protein levels of caspase 8, caspase 9, and procaspase 3 were not induced in KB cells expressing stable FLAG-PhD30 or FLAG-PhD35 as well as KB cells transiently co-transfected with ZAKa plasmid at different concentrations of FLAG-PhD30 or FLAG-PhD35 plasmids (Supplementary Fig.\u0026nbsp;5A, B). These findings suggested that the binding of the PhD proteins to ZAKa could potentially be used to develop an apoptosis-free approach to the treatment of cancer cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003ePhD30 and PhD35 inhibited oral cancer cell proliferation by targeting ZAKα: evidence from\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003ein vivo\u003c/b\u003e \u003cb\u003estudies\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo further confirm whether PhD30 or PhD35 peptide expressed in tumor cells affected their proliferation capacity, we measured the growth of KB cells expressing either EGFP or FLAG tagged PhD30 or PhD35. We found growth to be inhibited in EGFP-PhD30 or EGFP-PhD35 KB cells under non-adherent growth conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, B). These findings suggested that PhD30 or PhD35 peptides expressed in KB cells bound to ZAKa (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eC and Supplementary Fig.\u0026nbsp;1) inhibiting proliferation under non-adherent conditions. Moreover, the growth rates of KB cells expressing EGFP-PhD30 and EGFP-PhD35 were significantly slower than those of the mock control cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). The fact that ZAKa belongs to a MAPKKK family of kinases able to phosphorylate downstream effectors raises an intriguing question. Might these PhD peptides influence ZAKa\u0026rsquo;s intrinsic kinase activity? To explore ZAKa kinase activities, we performed \u003cem\u003ein vitro\u003c/em\u003e kinase assays of KB cells expressing these peptides by using RhoGDIb, an effector known to be phosphorylated by ZAKa \u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e as a substrate. When transiently co-transfecting ZAKa plasmid with EGPP-PhD35 or EGFP-PhD30 plasmid, we found that ZAKa kinase activity was inhibited by EGPP-PhD35 but not EGFP-PhD30 (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e7\u003c/span\u003eD), indicating cancer cell growth or mitochondrial activity was inhibited (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, D, E) by these PhD peptides independent of the intrinsic kinase activity in ZAKa. To prevent the 27kDa GFP protein from influencing these PhD peptides, we constructed FLAG peptide tagged PhD clones. Similar to the results of our study of KB cells expressing EGFP-PhD30 and EGFP-PhD35, those expressing FLAG-PhD30 and FLAG-PhD35 were found to have inhibited colony formation under non-adhesion growth conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e7\u003c/span\u003eE, F). Colony formation was also found to be inhibited in KB cells expressing ZAKb and 1712i, which were our negative growth control cells. KB cells expressing FLAG-PhD30 and FLAG-PhD35 as well as those expressing FLAG-ZAKβ and 1712i had slower growth rates compared to mock or cells expressing FLAG-ZAKα (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e7\u003c/span\u003eG). These findings indicate that these biological effects were indeed affected by these PhD peptides. The ability of cancer cells to migrate and invade plays a critical role in cancer metastasis. Results of Boyden assay revealed that KB cells expressing FLAG-PhD30 and FLAG-PhD35 had significantly impaired ability to migrate and invade (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e7\u003c/span\u003eH, I). Because ZAKPBS is the binding site in ZAK for the PhD30 and PhD35 peptides (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, D), we wanted to know whether the expression of ZAKPBS in cells could sequester the growth inhibition effect of PhD30 or PhD35 on ZAKα. To find out, we transiently co-transfected FLAG-PhD30 and FLAG-PhD35 plasmid with either a mock vector or GFP-ZAKPBS plasmid for 48 hours and evaluated anchorage-independent growth. The cells transiently expressing PhD30 or PhD35 peptides lost their ability to grow in anchorage-independent conditions, while some anchorage-independent growth ability was restored in the cells co-expressing ZAKPBS with FLAG-PhD30 or FLAG-PhD35, (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e7\u003c/span\u003eJ, K). FLAG-ZAKβ or FLAG-CREB1 expressing cells were used as negative growth controls \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. We used xenografts to study the tumor microenvironment and the growth of these KB cells expressing PhD peptides in a living organism. KB cells expressing four stable FLAG-conjugated PhD peptides (PhD10, PhD30, PhD35, and PhD40) were injected subcutaneously into the backs of nude mice. Four weeks later the mice were killed and tumor sizes were measured. Like the anchorage-independent growth experiment, we found that tumor sizes were smaller in mice injected with KB cells expressing FLAG-PhD30 and FLAG-PhD35 compared to those injected with the mock vector control and FLAG-PhD40 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e7\u003c/span\u003eL, M). The results of our \u003cem\u003ein vitro\u003c/em\u003e studies (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, E) and \u003cem\u003ein vivo\u003c/em\u003e studies indicate that the binding of PhD30 or PhD35 to ZAKa resulted in the inhibition of tumor cell growth.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eThe impact of PhD peptides on mitochondrial activity and tumor growth was achieved via modulation of oxidative phosphorylation\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe binding of PhD30 or PhD35 peptides to ZAKα caused ZAKα to dissociate from RPS20. This dissociation led to the jamming or stalling of translation on mitochondria-associated ribosomes and a significant decrease in protein levels of oxidative phosphorylation complexes, causing an impairment of mitochondrial activity. We wanted to further investigate the relationship between mitochondrial activity and tumor cell growth. To proceed, we challenged KB cells with different concentrations of Antimycin A, an inhibitor of oxidative phosphorylation complex III, and Oligomycin, an inhibitor of oxidative phosphorylation complex V. We found that both inhibitors reduced anchorage-independent growth in a dose-responsive manner. We also found that combination of Antimycin A and Oligomycin had a synergistic effect on the inhibition of colony formation. In particular, we found that this combination treatment at a concentration of 100nM inhibited the colony formation ten times as much as the same concentration of Antimycin A or Oligomycin alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e7\u003c/span\u003eN). Furthermore, analyzing MitoTracker Red CMXROS stains, we found that treatment with Antimycin A and Oligomycin alone brought about dose-responsive reductions in mitochondrial membrane potential, while combination treatment at 100nM brought about a much greater reduction in the mitochondrial membrane potential (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e7\u003c/span\u003eO). We concluded that treatment combining Oligomycin and Antimycin at 100nM was sufficient to reduce mitochondrial activities and inhibit tumor cell growth.\u003c/p\u003e \u003cp\u003eWe wanted to make sure that this combination of inhibitors to treat the cells only reduced the mitochondrial activities leading to the inhibition of tumor growth but not trigger cell death, which would also inhibit growth (Figs.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, E) and mitochondrial activities (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, D, E) in KB cells expressing PhD30 or PhD35 peptide. To determine whether apoptosis or necrosis was induced, we treated the cells with a combination of the inhibitors (100nM) for 48 hours and used flow cytometry with Annexin V and PI double staining to analyze tumor cell stages. We found no increase in either apoptosis and necrosis compared to controls. However, it should be noted that at a concentration of 500nM, this combined treatment significantly increased both apoptosis and necrosis compared to controls (Supplementary Fig.\u0026nbsp;6). Together, these findings suggest the reduction of mitochondrial activities by this combination of inhibitors was sufficient to retard tumor growth without killing the tumor cells, highlighting the important relationship between mitochondrial activity and tumor growth and the possible therapeutic potential that Antimycin A and Oligomycin might have on the treatment of tumors.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study focused on ZAKα, a key member of the mixed lineage kinase family, because it is known to play an important role in regulating tumor growth. Although it is well-known that its increased expression contributes significantly to tumorigenesis, no specific inhibitors targeting ZAKα has been found. Performing phage display assay analysis, we identified four novel peptides (PhD10, PhD30, PhD35, and PhD40) that bound specifically to the non-structural long C-terminal (NSLC) region in ZAKα. PhD30 and PhD35, in particular, were found to have a high affinity for ZAKα, effectively interacting with both exogenously expressed and endogenous forms of the protein. These two peptides were able to bind to a specific region of ZAKα, we designated region as a ZAKPBS (phage-binding site), within the amino acid sequence 414\u0026ndash;420. Our results indicated that the binding of these peptides to ZAKα resulted in the dissociation of ZAKα from RPS20, a protein we identified as a ZAKα-associated partner through our studies using yeast two-hybrid screening. This disruption occurred independently of intrinsic kinase activity in ZAKα, suggesting a new mechanism of action for these peptides. Importantly, the binding of PhD30 and PhD35 to ZAKα not only impaired its interaction with RPS20 but it also inhibited tumor growth and affected mitochondrial activity. These findings suggest that these two peptides could potentially be used to develop novel therapeutic agents targeting ZAKα in the treatment of cancer.\u003c/p\u003e \u003cp\u003eThe peptides PhD30 and PhD35 targeted ZAKα, inhibiting its association with ribosomal initiation complexes, leading to an increase in RPS20 ubiquitination and disrupting the initiation of translation. Other studies have previously suggested that RPS20 ubiquitination plays a role in regulating translational initiation \u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e,\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e and studies on ZAKα have indicated that ZAKα plays a novel role in the Ribosome Quality Control (RQC) or Ribotoxic Stress Response (RSR) pathways via its recognition of stalled ribosomes \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Thus, ZAKα may act as a key regulator of protein translation and degradation. When PhD30 or PhD35 peptide binds to ZAKα, ZAKα dissociates from RPS20 allowing RPS20 to be ubiquitinated by E3 ligases. However, the specific E3 ligase for RPS20 ubiquitination has not been identified. ZNF598 could potentially be an E3 ligase for ribosomal subunit protein ubiquitination \u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e, and it has been found to orchestrate RQC by ubiquitinating ribosomal proteins and inducing premature translation termination of defective mRNAs \u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. Further studies are needed to determine whether it is responsible for RPS20 ubiquitination. In this study, the expression of PhD30 and PhD35 peptide in cells did not influence their protein levels of RPS20 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eE, G), suggesting that the ubiquitination of RPS20 more likely plays a role in regulation than it does in protein degradation. Studies have indicated that RPS20 ubiquitination is critical to the regulation of the assembly of the 40S ribosomal subunit and the translation of specific mRNAs \u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e,\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e and it is also involved in various cellular functions, including ribosome biogenesis, mRNA translation, and protein quality control \u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e,\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e,\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. In this study, the immunoprecipitation of RPS20 from mitochondrial fraction showed that most of the RPS20 proteins were monoubiquitinated in KB cells, suggesting that monoubiquitinated RPS20 is essential for the proper translation on mitochondria associated ribosomes. This finding is in agreement with other studies finding that ubiquitination plays a critical role in regulating protein synthesis, including ribosome-associated quality control (RQC) \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. These results highlight the complexity of ubiquitin-mediated regulation of translation and the importance of fine control on protein synthesis regular cellular functions.\u003c/p\u003e \u003cp\u003eAlthough mitochondria have their own DNA, which encode a small portion of their proteins, most mitochondrial proteins are encoded by nuclear genes and then translocated into the mitochondria. Studies have found these nuclear encoded mRNAs on the surface of the mitochondrial outer membrane \u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e where cytosolic ribosomes associate with them to initiate translation. The binding of ribosomes to the mitochondrial membrane surface ensures the co-translation and translocation of the newly synthesized proteins into mitochondria \u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e,\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e. However, in this study, when PhD30 or PhD35 peptide bound to the ZAKα protein, ZAKα dissociated from RPS20 facilitating the ubiquitination of RPS20 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, F, I) jamming or stalling this translation. We found that PhD30 and PhD35 peptides interfered with ZAKα\u0026rsquo;s ability to regulate ribosomal translation (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eN, P), resulting in a decrease in crucial oxidative phosphorylation complexes like CV-ATP5A and CIII-UQCRC2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). This decrease in mitochondrial activity highlights the potential of these peptides may hold in modulating mitochondrial activities for cancer therapy. This approach focusing on mitochondrial dysfunction is in alignment with current research exploring the relationship between mitochondria and cancer \u003csup\u003e\u003cspan additionalcitationids=\"CR64\" citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e. These findings not only elucidate a novel mechanism through which phD30 and PhD35 peptides modulate mitochondrial function through the ZAKα-RPS20 axis but also suggest that the mitochondrial pathway can be targeted as a potential therapeutic strategy in the treatment of cancer.\u003c/p\u003e \u003cp\u003eMost kinases exert signaling through the phosphorylation of their specific downstream substrates. Our study discovered that PhD30 and PhD35 peptides reduced mitochondrial activities and inhibited cancer cell growth independent of ZAKα's kinase activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e7\u003c/span\u003eD), until now an unknown role for these peptides. We also identified ZAKPBS as the target site for both peptides in ZAKα and demonstrated that exogenous expression of ZAKPBS reversed the ability of these peptides to inhibit growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e7\u003c/span\u003eJ, K), further confirming it as the binding site. The results of this study suggest an alternative approach to the treatment of cancer, one involving the use of these peptides targeting ZAKα to slow down the proliferation rate without inducing apoptosis. There are various ways to treat cancer, the most common being inducing apoptosis in cancer cells with the use of chemotherapy drugs, radiation therapy, or targeted therapies that specifically target certain proteins in pathways essential for cancer cell survival. There are problems with the use of apoptosis for treatment. One problem is the mutation to drug-resistant cancer cells arising from selection pressure. That is cancer cells can mutate and be selected to resist certain treatment, rendering them harder to kill in the future \u003csup\u003e\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e,\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e. Another problem associated with inducing apoptosis for therapeutic purposes in the collateral damage to normal cells. In contrast, a more sustainable approach to treating cancer might be to slow down or arrest the proliferation of cancer cells, which might help circumvent the development of drug-resistant mutations and collateral damage to normal cells, though this approach would come with its own set of challenges in that it may not be as effective as the killing the cells in the short time and it may not completely eliminate the cancer.\u003c/p\u003e \u003cp\u003eThis study found that both ZAKα and its kinase-dead mutant, ZAKdn, bound to RPS20 without affecting its ubiquitination status and without influencing anchorage-independent growth activity. This finding suggests that tumor cell growth is not strictly dependent on the kinase activity of ZAKα. This hypothesis was further supported by our finding PhD30 binding to ZAKα did not affect ZAKα\u0026rsquo;s intrinsic kinase activity but it did interfere with its interaction with RPS20. These findings highlight, the crucial role of ZAKα plays in regulating ribosomal translation on mitochondria and its role in mitochondria\u0026rsquo;s ability to maintain tumor growth. These findings also indicate that the function of ZAKα may extend beyond its kinase activity, possibly involving physical interactions with RPS20, suggesting a novel mechanism for ZAKα in the regulation of translational control and mitochondrial function. Although both PhD30 and PhD35 peptide bound to the ZAKPBS in ZAKα, only the PhD35 peptide inhibited the kinase activity. The difference might be because PhD30 and PhD35 peptides bind to ZAKPBS at different orientations. PhD30 peptide was predicted to dock at ZAKPBS next to the EH site of ZAKα's SAM domain, while the PhD35 peptide docked at ZAKPBS next to ML side of ZAKα's SAM domain (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). This difference in binding orientation might lead to differences in protein function, even if peptides bind to the same site.\u003c/p\u003e \u003cp\u003eIt was interesting to note that although ZAKa bound to PhD30 or PhD35 peptide dissociated from RPS20, interrupting translation of mitochondria associated ribosomes, we found that it did not exert a similar interference in the translation of cytosolic ribosomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eM, N). This difference raises the question of whether ZAKa might have other associated proteins that are involved in regulating ribosomal translation in cytosol. Thus, further research is needed to identify other ZAKα-associated proteins that might cooperate with ZAKα to regulate the spatial ribosomal translation on mitochondria or in cytosolic compartments. It is also possible that a higher abundance of ribosomes in cytosol and lower expression of the peptides might limit the peptides\u0026rsquo; effect on cytosolic translation, whereas in cells, where there are fewer mitochondria-associated ribosomes, the impact of PhD peptides on the translation in the cells might be more significant.\u003c/p\u003e \u003cp\u003eThis study has some limitations. One limitation is the fact that almost all of experiments we performed \u003cem\u003ein vitro\u003c/em\u003e with only one semi \u003cem\u003ein vivo\u003c/em\u003e undertaken. Further in vivo studies are needed to study the practicality of this strategy for the treatment of cancer. Another limitation is related to specificity, our ability to target different cancers.\u003c/p\u003e \u003cp\u003eIn conclusion, this study exploring the effect of two novel ZAKα binding peptides, PhD30 and PhD35, on tumor cell growth, found that by binding to ZAKα, the peptides PhD30 or PhD35 increased ubiquitination of RPS20, significantly impacting translational regulation. It also demonstrated their targeting of ZAKα influenced the translation of nuclear mRNA on mitochondria-associated ribosomes, possibly impacting mitochondrial functions. In particular, PhD30 or PhD35 peptide decreased translation efficiency on mitochondria-associated ribosomes by binding to ZAKα, affecting the building blocks and enzymes required energy production in mitochondria. The results of this study provide new insight into the molecular mechanisms underlying ribosome translation by ZAKα on mitochondria and suggest that the binding of PhD30 or PhD35 peptide on ZAKα might slow down or jam ribosome translation on mitochondria more than likely by interfering with the initiation of translation. We believe that using PhD30 or PhD35 peptide to target ZAKα holds great potential for treatment of cancer.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by a grant from\u0026nbsp;Ington Biotechnologies Co Ltd (105-CSMU82).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJ.J.Y. conceived the project, designed, performed all experiments, and wrote the manuscript. I.C.H. and Y.S.Y. performed the molecular modelling and docking confirmations and analyzed the data. All authors discussed the results and commented on the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eCell line and culture conditions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKB, HCT116, HEK293, and H9C2 cells were grown in Dulbecco’s modified eagle medium (DMEM) supplemented with 10% fetal bovine serum, 2mM L-glutamine, penicillin and streptomycin. All cells were cultured at 37C in a humidified incubator containing 5% CO2. Mycoplasma testing was performed and found to be a negative.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhage display\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe cDNA corresponding to ZAKα NSLC 414-800 was subcloned into a GST-expressing vector. The GST-ZAKα NSLC 414-800 protein was expressed in E. coli BL21 (DE3) cells and the protein was purified using Glutathione Sepharose 4B resin. The immobilized Sepharose GST-ZAKα NSLC 414-800 protein was subjected to phage display screening by the Ph.D.-C7C phage display peptide library (New England Biolabs) for 1 hour at room temperature. Unbound phages were washed with PBS containing 0.05% Tween-20 (PBST). The bound phages were eluted using 100 mM triethylamine (TEA) and neutralized with 1 M Tris-HCl, pH 7.5. The eluted phages were further amplified in E. coli and amplified phages were again used to bind Sepharose GST-ZAKα NSLC 414-800 protein, undergoing three rounds of biopanning. We wanted to exclude all phages binding to GST. To do this, we incubated the final panning phage solution with 50 µL of 5 µg/ml Glutathione Sepharose 4B GST for 1 hour at room temperature. The unbound phages were incubated with the host bacteria and poured onto a plate and individual plaques were isolated. Phages were isolated and amplified and genome DNA was extracted for sequencing to identify potential ZAKα NSLC 414-800 binding peptides.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eXenografts\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSix-week-old nude mice were purchased from National Applied Research Laboratories, Taiwan.\u0026nbsp;Stable expressed PhD10, PhD30, PhD35, or PhD40 KB cells were trypsinized and cell concentrations were adjusted to 1x10\u003csup\u003e7\u003c/sup\u003e cells/ml. Subsequently, 200 μL of the cell suspension was injected subcutaneously into the back of nude mice through a 26-gauge needle. Each mouse received one injection on both the left and right sides of the back. The mice were killed four weeks after injection by ether inhalation. Tumors were excised, weights were measured, and photographs were taken. Statistical analysis was conducted comparing tumor weights among the different groups. All animal experiments were performed following universally accepted guidelines for animal care and use, and all necessary precautions were taken to minimize pain and distress to the animals.\u0026nbsp;All animal experimental procedures were performed with the approval of the Institutional Review Board of Taichung Veterans General Hospital (IRBTCVGH No: 950727/C06134).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunoprecipitation and Western blot analysis\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCell lysates were prepared in IP buffer (40 mM Tris-Hcl pH 7.5, 1% NP40, 150 mM NaCl, 5 mM EGTA, 1 mM DTT, 1mM PMSF, 20 mM NaF, proteinase inhibitors, and 1 mM sodium vanadate). Cell extracts (600 mg) were incubated with 5\u0026nbsp;ml anti-ZAK (0834) polyclonal antibody (generated in the lab) for 6 h at 4\u003csup\u003eo\u003c/sup\u003eC. This mixture was then mixed with 20\u0026nbsp;ml protein-A sepharose suspension and incubated for an additional hour. Immunoprecipitates were collected by centrifugation, washed three times with IP buffer with 0.5% deoxycholate and five times with IP buffer alone, and then subjected to SDS-PAGE.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor the western blot analysis, cells were harvested in lysis buffer (50 mM Tris-HCl, pH 8.0/250 mM NaCl/1% NP-40, 2 mM EDTA) containing 1 mM PMSF, 10 ng/ml leupeptin, 50 mM NaF, and 1 mM sodium orthovanadate. \u0026nbsp;Total proteins were then separated on SDS-PAGE and the protein bands were visualized using an ECL chemiluminescent detection system (Amersham).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSoft agar assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e2.5 ml of 0.5% Nobel agar was poured into each well of a six-well plate as the bottom agar layer.\u0026nbsp;Cells were suspended in DMEM supplemented with 10% FBS and 0.3% Agar Noble (BD, Difco, MD, USA) and plated onto the bottom layer of Agar Noble. Experiments were performed in six-well plates with 2,500 cells per well in triplicate. Colonies were stained with iodonitro-tetrazolium chloride (INT) solution and counted after two weeks incubation under 37°C and 5% CO2. Colonies were photographed and quantified. The number of colonies was counted under an inverted light microscope at X40 magnification. The data are expressed as mean number of colonies ± standard error (SE) from six fields of three independent wells.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMigration and invasion assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA Boyden chamber was coated with Matrigel for invasion assays or without Matrigel for migration assays. The chamber was incubated at 37°C for 30 minutes to allow the matrix to solidify. The Boyden chamber assay was performed by filling the bottom well of the chamber with DMEM medium containing 10% FBS. The wells were covered with polyvinylpyrrolidone-free polycarbonate membranes with 8-μm pores (Neuro Probes, Inc.). We then seeded\u0026nbsp;KB stable expressed FLAG vector control, FLAG-PhD30, FLAG-PhD35, or FLAG-ZAKa\u0026nbsp;at 1500 cells/well in serum-free DMEM to the top chamber. The Boyden chamber was incubated for 24 h at 37°C to allow for the migration or for 48 hours at 37°C to allow for the invasion of cells through the membrane into the bottom chamber.\u0026nbsp;After the incubation, the top chamber was removed the top and the top of the membrane was gently wiped with a cotton swab to remove non-migrated/non-invaded cells. Membranes were stained using Giemsa stain.\u0026nbsp;The membrane was rinsed with distilled water to remove excess stain and air-dried. Migrating and invading cells were imaged under a microscope and counted.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIn vitro\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;kinase activity assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProtein kinase assays were carried out using glutathione S-transferase (GST)-RhoGDIb\u0026nbsp;or S20 as a substrate. The GST fusion proteins were bound to ZAKa\u0026nbsp;by incubating them with cellular extracts in the kinase buffer (20 mM Hepes, pH 7.6, 1 mM EGTA, 1 mM dithiothreitol, 2 mM MgCl, 2 mM MnCl, 5 mM NaF, 1 mM NaVO, 50 mM NaCl) for 15 min on ice. The beads were pelleted and thoroughly washed with PBST (150 mM NaCl, 16 mM sodium phosphate, pH 7.5, 1% Triton X-100, 2 mM EDTA, 0.1% MeOH, 0.2 mM phenylmethylsulfonyl fluoride, and 5 mM benzamidine) and then incubated with [g-\u003csup\u003e32\u003c/sup\u003eP]ATP (50 cpm/fmol) in the presence of kinase buffer. After washing the phosphorylated GST-RhoGDIb\u0026nbsp;or GST-S20 with PBST seven times, it was boiled in SDS sample buffer. The proteins were run on a 15% SDS-PAGE. The gel was dried, and phosphorylation of the RhoGDIb\u0026nbsp;or S20 substrate was determined by autoradiography.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIsolation of mitochondria\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were cultured to 70-80% confluency. The cells were rinsed twice with ice-cold PBS to remove any remaining media or serum. The cells were then resuspended in hypotonic solution (1.25M sucrose, 10mM MOPS, pH7.2) and allowed to swell for 10 minutes on ice. Swollen cells were suspended in a Dounce and homogenized using six gentle up and down rotating strokes and immediately added to a hypertonic solution (100mM sucrose, 10 mM MOPS, 1 mM EDTA, pH 7.2). The homogenized solution was diluted with two volumes of isolation buffer (75 mM mannitol, 225 mM sucrose, 10mM MOPS, 1 mM EGTA, 0.1% fatty acid free BSA, pH7.2). Sediment nuclei, unbroken cells, and cellular debris were centrifuged at 930 g, 4°C for 5 minutes in a JA-20.1 Beckman rotor. The supernatant was then transferred to Beckman plastic centrifuge tubes. The pellet was added to four ml of isolation buffer, and then homogenization and centrifugation cycles were repeated. All the collected supernatants were combined in Beckman centrifuge tubes and run at 13000 xg under 4°C for 20 minutes.\u0026nbsp;The supernatant was discarded and mitochondrial pellet was resuspend in isolation solution.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePolysome profiling\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells at 70%–80% confluency were treated with 100 μg/ml cycloheximide for 5 minutes, washed, and scraped off in ice-cold PBS containing 100 μg/ml cycloheximide. They were then collected by centrifugation at 300 xg for 5 min. The isolated mitochondrial fractions were lysed in 425\u0026nbsp;ml hypotonic buffer (5 mM Tris-HCl pH 7.5, 1.5 mM KCl, 2.5 mM MgCl\u003csub\u003e2\u003c/sub\u003e), added with 5ml of 10mg/ml cycloheximide and 1\u0026nbsp;ml of DTT, and vortexed for 5 seconds. The mixture was then added with 25ml of 10% triton X-100 and 25ml of sodium Deoxycholate. Debris was removed by centrifugation at 21,000 xg for 5 min at 4°C. The lysate concentration was measured by UV spectrophotometer. The same amount of lysate was loaded onto 10%–50% (w/v) sucrose gradient and centrifuged at 35000 xg for 2 hours at 4°C in a Beckman ultracentrifuge with the SW40Ti rotor. After ultracentrifugation, the fractions were collected by pipetting of 0.75 ml for each fraction from the top. Absorbance was recorded using a spectrophotometer set at 254nm. All fractions were immediately frozen on dry ice for future study. Proteins were precipitated with 10% trichloroacetic acid (TCA) at 4°C. The samples were spun down at18000 xg for 15 minutes at 4°C and washed twice in ice-cold acetone. Pellets were resuspended in laemmli buffer containing DTT and boiled at 95°C for 5 minutes.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eYeast two hybrid assay\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe two-hybrid screen assay was performed as described in the user’s manual\u0026nbsp;(Clontech). Briefly, the full-length ZAK was cloned to the pGBKT7 vector as the bait construct. This vector encoding GAL4 DNA binding domain-ZAK fusion (DNA-BD/ZAK) was transformed into AH109 yeast. This pGBKT7-ZAK yeast was mated with Y187 yeast containing a pretransformed MATCHMAKER human heart cDNA library. The yeast was selected following the manufacturer’s directions. The positive clones were isolated and sequenced.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIsolate mRNA and RT-PCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was isolated from whole cells or purified mitochondrial fractions of KB cells expressing PhD30 or PhD35 using TRIzol (Invitrogen) following the manufacturer's directions.\u0026nbsp;\u0026nbsp;5\u0026nbsp;mg of isolated mRNAs was added to the RT-PCR master mix (oligo dT, 10 mM dNTPs, 200 units/μl reverse transcriptase, 5x reaction buffer) at 37°C for 60 minutes. After the reverse transcription reaction, 45 μl of sterile water was added to the mixture. For the PCR reaction, 2 μl of cDNA product from the reverse transcription reaction was used to amplify cDNA with specific primers for CV-ATP5A, CIII-UQCRC2 or actin for 25 cycles. The PCR products were detected by gel electrophoresis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePull-down assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003epGex-S20 plasmids were transferred into BL21 (DE3) and positive clones were selected. The positive clone was induced by IPTG. \u0026nbsp; The bacteria were lysed and GST-S20 protein was immobilized by glutathione-sepharose. HEK293 cells were transfected with 10 μg of pEGFP-ZAKa or serial C-terminal deletion mutants following the calcium phosphate method. \u0026nbsp;Cells were lysed in an immunoprecipitation buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, and a protease inhibitor cocktail). 600 μg of the cell lysate was incubated with 20 ml immobilized GST-S20 at 4°C overnight. The pulled-down complex was washed seven times with the immunoprecipitation buffer and the beads were boiled in Laemmli buffer. The complex was run on SDS-PAGE and the GFP conjugated proteins were detected by GFP antibody.\u003c/p\u003e\n\u003cp\u003e\u003cimg 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\"\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cimg 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\"\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n 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Apoptosis deregulation and the development of cancer multi-drug resistance. \u003cem\u003eCancers\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 4363 (2021).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"ZAKα, phage display, peptide inhibitors, Ribosomal protein S20, Ubiquitylation, Translational jams, Mitochondria, Oxphos, tumor growth inhibition","lastPublishedDoi":"10.21203/rs.3.rs-4003086/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4003086/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePhage display was used to identify high-affinity and high-specificity peptides that might inhibit ZAKα, a kinase implicated in cellular proliferation, differentiation, and stress response and implicated in both malignant tumor growth and metastasis as well as the pathogenesis of cardiac hypertrophy and fibrotic heart disease. Two peptides were found to specifically bind to ZAKα, PhD30 and PhD35. Their introduction into cancer cells led to the dissociation of ZAKα from RPS20 and the ubiquitination of RPS20 protein resulting in a reduction of essential mitochondria proteins and mitochondria activity impeding the translation of mitochondrial-associated eukaryotic ribosomes, a process we believe to be crucial for tumor growth. This mechanism of action suggests that the primary effect of PhD30 and PhD35 is to lower mitochondrial activity by causing a shortage of essential proteins in mitochondria without changing the number of apoptotic and necrotic cancer cells. Thus, these findings may prove pivotal to the development of a novel approach utilizing these peptides to inhibit cancer cell growth by targeting mitochondrial function, rather than the conventional method of inducing apoptosis.\u003c/p\u003e","manuscriptTitle":"Targeting ZAKα Interactions and Ribosomes with Novel peptides: The role of ZAKα in Mitochondrial Function and Cancer Cell Proliferation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-15 11:29:46","doi":"10.21203/rs.3.rs-4003086/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"77452814-2a17-4a62-904b-1819753e695b","owner":[],"postedDate":"March 15th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":29193591,"name":"Biological sciences/Cancer/Cancer therapy/Drug development"},{"id":29193592,"name":"Biological sciences/Biological techniques/Metabolomics"}],"tags":[],"updatedAt":"2024-03-15T11:29:47+00:00","versionOfRecord":[],"versionCreatedAt":"2024-03-15 11:29:46","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4003086","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4003086","identity":"rs-4003086","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}