Biochimica et biophysica acta. Proteins and proteomics最新文献

{"title":"Structural insights of the complex formed by KRAS G12V and a novel TIG3 peptide.","authors":"Min Seon Ha, Chang Woo Han, Mi Suk Jeong, Se Bok Jang","doi":"10.1016/j.bbapap.2025.141091","DOIUrl":"https://doi.org/10.1016/j.bbapap.2025.141091","url":null,"abstract":"<p><p>Pancreatic cancer remains a severe malignancy with a dismal 5-year survival rate, and most pancreatic cancer patients harbor KRAS mutations, which are critical targets for anti-cancer drug development. However, the structural characteristics of KRAS present challenges for therapeutic targeting. In this study, we developed a novel peptide derived from TIG3 protein, a type II tumor suppressor, and confirmed its moderate affinity binding to KRAS G12V. X-ray crystallography revealed that this peptide binds near the Switch II domain of KRAS G12V, causing conformational changes likely to affect its activity. Furthermore, the peptide reduced the viability of cancer cell lines harboring the KRAS G12V mutation, thus demonstrating its potential as a KRAS G12V inhibitor. Our results indicate that the developed novel TIG3 peptide is a promising candidate for KRAS-targeted therapy and provide structural insights useful for the development of pancreatic ductal adenocarcinoma therapeutics.</p>","PeriodicalId":8760,"journal":{"name":"Biochimica et biophysica acta. Proteins and proteomics","volume":" ","pages":"141091"},"PeriodicalIF":2.3,"publicationDate":"2025-07-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144768260","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Role of N-terminal of metacaspase of Candida albicans in calcium binding.","authors":"Taiz dos Reis Santos ,&nbsp;Mariana Nascimento Romero Trujilho ,&nbsp;João Pedro Martins Silva Costa ,&nbsp;Laurade Azevedo Maffeis Dalzoto ,&nbsp;Ane Caroline Moreira Duarte ,&nbsp;Karolaine Stella Siqueirade Moraes Valdivia ,&nbsp;Ana Júliade Oliveira Machado ,&nbsp;Vitória Caroline Domingues Rios ,&nbsp;Tatiane Faustino de Moraes ,&nbsp;Wagner Alves de Souza Judice ,&nbsp;Marcelo Ferreira Marcondes ,&nbsp;Maurício Ferreira Marcondes Machado","doi":"10.1016/j.bbapap.2025.141090","DOIUrl":"10.1016/j.bbapap.2025.141090","url":null,"abstract":"<div><div>Metacaspases are members of the CD clan and share structural similarities with mammalian caspases but possess unique features. This study delves into the <em>Candida albicans</em> metacaspase CaMCA-Ia, a type I metacaspase. CaMCA-Ia demonstrates Ca²⁺-dependent autoprocessing and presents a hydrophobic N-terminal, which differs from that of type II metacaspases. Truncated CaMCA-Ia (CaMCA-Ia-ΔN86), lacking 86 N-terminal amino acids, undergoes gradual self-processing and intermolecular processing. Calcium addition induces multistep processing, leading to maturation. Like mammalian caspases, CaMCA-Ia-ΔN86 can activate other molecules, indicate intermolecular activation and accelerating maturation. Distinct binding sites for the full-length and truncated forms of CaMCA-Ia in interaction with Ca²⁺ underscore the N-terminal's role in altering calcium affinity. These findings enhance the understanding of metacaspases' intricate activation and maturation dynamics, offering insights into potential drug targets for pathogenic fungi. The CaMCA mutants D252A, D268A, D299A, and D268/269 A display varied responses to calcium, while the corresponding CaMCA-Ia-ΔN86 mutants exhibit different processing patterns. The D268/299 A mutant showed processing after 48 h of incubation with calcium. Alterations in CaMCA structure and function, such as the deletion of the N-terminus and changes in the aspartates at the calcium-binding site, provide important insights into how CaMCA enzymatic activity and autoprocessing are regulated.</div></div>","PeriodicalId":8760,"journal":{"name":"Biochimica et biophysica acta. Proteins and proteomics","volume":"1873 6","pages":"Article 141090"},"PeriodicalIF":2.3,"publicationDate":"2025-07-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144738199","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Glycoproteomic characterization of Neuropilin-1 reveals critical glycosylation sites for SARS-CoV-2 entry","authors":"Tuhin Das ,&nbsp;Shuhong Luo ,&nbsp;Panning Wang ,&nbsp;Jianmin Fang ,&nbsp;Asif Shajahan ,&nbsp;Lauren Peppi ,&nbsp;Sabyasachi Dash ,&nbsp;Kino Maravillas ,&nbsp;Rochelle N. Wickramasekara ,&nbsp;Parastoo Azadi ,&nbsp;Ruo-Pan Huang","doi":"10.1016/j.bbapap.2025.141089","DOIUrl":"10.1016/j.bbapap.2025.141089","url":null,"abstract":"<div><div>The binding of the SARS-CoV-2 spike (S) glycoprotein to human host receptors, including ACE2, NRP1, and AXL, is essential for viral entry. Glycosylation of both the spike protein and its host receptors can significantly influence these interactions. While NRP1 is recognized as a key host receptor, the role of its glycosylation in spike binding and viral infectivity has not been fully elucidated. In this study, we developed a spike-NRP1 binding assay using recombinant proteins in HEK293 cells and performed LC-MS-based glycoproteomic analysis to characterize NRP1 glycosylation. We identified three N-linked glycosylation sites (N150, N261, N522) and four O-linked glycosylation sites (S612, S637, T638, S641) on NRP1. Mutational analysis revealed that glycosylation at these specific sites is critical for spike binding, as single-site mutants showed significantly reduced interaction. Further characterization of terminal sialic acid linkages, bisecting GlcNAc, and fucosylation patterns provided insight into the complexity of NRP1 glycosylation. Functional assays demonstrated that loss of glycosylation impaired SARS-CoV-2 pseudovirus entry and altered IL-6 secretion, indicating a broader role in host immune modulation. These findings demonstrate that site-specific NRP1 glycosylation modulates spike binding and viral entry in a cell-based model, providing a foundation for future studies to explore the potential of targeting glycosylation-dependent mechanisms in SARS-CoV-2 infection.</div></div>","PeriodicalId":8760,"journal":{"name":"Biochimica et biophysica acta. Proteins and proteomics","volume":"1873 5","pages":"Article 141089"},"PeriodicalIF":2.5,"publicationDate":"2025-07-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144648361","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Modulation of thermomechanical-induced albumin aggregation by extracts of Centella asiatica","authors":"Laipubam Gayatri Sharma,&nbsp;Sanjana SenthilKumar,&nbsp;Latha Rangan,&nbsp;Lalit M. Pandey","doi":"10.1016/j.bbapap.2025.141088","DOIUrl":"10.1016/j.bbapap.2025.141088","url":null,"abstract":"<div><div>In this study, <em>Centella asiatica</em> extracts obtained through sequential extraction in ethyl acetate (CEE), methanol (CME), and water (CWE) were explored for the inhibition of thermal and thermomechanical-induced aggregation of albumin. Th-T fluorescence assay, dynamic light scattering (DLS), circular dichroism (CD) spectroscopy, and TEM analysis were used to study the aggregation behavior. Although CWE contained fewer compounds than CEE, it exhibits comparable inhibition characteristics as determined by High-Resolution Liquid Chromatography-Mass Spectrometry (HR-LCMS) and High-Performance Liquid Chromatography (HPLC) analyses. The rate of aggregation is decreased 1.64 to 0.96 h⁻¹ (i.e. two-fold) in the presence of extracts from under thermomechanical conditions. This effect correlates with the antioxidant capacity of the extract and is further supported by CD analysis, which showed no significant alteration in the native structure of BSA upon interaction with CWE, highlighting its aggregation-inhibiting potential. Isothermal titration calorimetry (ITC) identified the extract fraction with the highest binding affinity to BSA. Molecular docking studies revealed that chlorogenic acid, one of the most abundant compounds in CWE, interacts with albumin with the highest binding free energy (−9.0 kcal/mol), binding predominantly at Sudlow's sites I and III through hydrophobic interactions and hydrogen bonding. This study underscores the potential of natural compounds in preventing protein aggregation and provides a foundation for further investigation of lead bioactive components.</div></div>","PeriodicalId":8760,"journal":{"name":"Biochimica et biophysica acta. Proteins and proteomics","volume":"1873 5","pages":"Article 141088"},"PeriodicalIF":2.5,"publicationDate":"2025-06-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144551835","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Biochemical and kinetic properties of quinone oxidoreductase from Leishmania orientalis, a member of medium-chain dehydrogenase/reductase superfamily","authors":"Panu Pimviriyakul ,&nbsp;Sumita Harungsee ,&nbsp;Theerapat Tangsupatawat ,&nbsp;Yuvarun Kapaothong ,&nbsp;Somchart Maenpuen","doi":"10.1016/j.bbapap.2025.141087","DOIUrl":"10.1016/j.bbapap.2025.141087","url":null,"abstract":"<div><div>Soluble quinone oxidoreductase (QOR) is an attractive target for treating leishmaniasis. In this study, a QOR from <em>Leishmania orientalis</em> PCM2 (<em>Lo</em>QOR) was identified as a member of non<img>Zn²⁺ medium-chain dehydrogenase/reductase (MDR) superfamily. Using steady-state and rapid kinetics approaches, it was found that <em>Lo</em>QOR catalyzes the electron transfer from NADH to menadione <em>via</em> ping-pong kinetics which is unusual for this enzyme. The reaction mechanism was proposed accordingly. The modeled structures created by AlphaFold version 3.0 revealed crucial residues, <em>i.e.,</em> Thr131, Tyr134, and Arg268, which may involve the binding to carboxamide group of the NADH nicotinamide ring. The kinetic analysis of the <em>Lo</em>QOR mutants compared to the wild-type revealed that these residues are not essential for overall catalysis. However, the kinetic mechanism of Tyr134Phe, Tyr134His, and Arg268Gln mutants has shifted to the ternary complex (<em>Lo</em>QOR:NADH:menadione) model, except for Thr131Val and Arg268Lys mutants. It is hypothesized that Tyr134 and Arg268 are likely responsible for determining the mode of the catalytic mechanism. Hence, our findings here provide a fundamental insight into the MDR-type QORs and other related non-metal enzymes in the MDR superfamily.</div></div>","PeriodicalId":8760,"journal":{"name":"Biochimica et biophysica acta. Proteins and proteomics","volume":"1873 5","pages":"Article 141087"},"PeriodicalIF":2.5,"publicationDate":"2025-06-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144367849","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Oxygen-carrying proteins employed in blood substitute candidates: differences in interactions with a model antioxidant molecule","authors":"Maria Lehene ,&nbsp;Jozsef Simon ,&nbsp;Cezara Zagrean-Tuza ,&nbsp;Andreea Aghion ,&nbsp;Bianca Vasile ,&nbsp;Alexandru Sonica ,&nbsp;Stefania Iancu ,&nbsp;Radu Silaghi-Dumitrescu","doi":"10.1016/j.bbapap.2025.141086","DOIUrl":"10.1016/j.bbapap.2025.141086","url":null,"abstract":"<div><div>Glutaraldehyde-polymerized hemoglobin (poly-Hb) has long been explored as a key candidate for blood substitute compositions, to be used in transfusions in order to supplement the oxygen-carrying capacity following severe blood loss. Bovine hemoglobin (bHb) has been the standard choice for such efforts, due to its reasonable availability and to its reduced dependence on organic allosteric effectors. We have recently shown that poly-Hb produced from ovine Hb (poly-oHb) outperforms poly-bHb in in vivo tests employing transfusion after up to 30 % blood loss in animals. This improvement was found to correlate with an increased resistance of ovine hemoglobin (oHb) and of poly-oHb towards oxidative and nitrosative stress agents in vitro. The molecular bases for these differences in reactivity offer an interesting challenge, given the high sequence homology between vertebrate hemoglobins. Reported here is an investigation of these molecular bases using different spectroscopic (fluorescence, resonance Raman, NMR, EPR) and computational (molecular docking) methods to assess the interaction with a convenient probe ligand representative of the class of natural antioxidants, caffeic acid. Fluorescence experiments reveal that ovine Hb fluorescence saturates above 25 μM caffeic acid and indicating full occupancy of fluorescence-responsive binding sites, while resonance Raman and NMR data indicate signals for the heme and indicate the differences between the types of Hbs and the antioxidant binding behavior after polymerization. Computational docking corroborated the spectroscopic data by identifying aromatic residues and distinct affinity patterns for caffeate. The results show that structural differences in oHb may explain a higher redox stability.</div></div>","PeriodicalId":8760,"journal":{"name":"Biochimica et biophysica acta. Proteins and proteomics","volume":"1873 5","pages":"Article 141086"},"PeriodicalIF":2.5,"publicationDate":"2025-06-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144279606","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Human mitochondrial pyrophosphatase hPPA2: In vitro and in silico study of the factors affecting its function","authors":"Svetlana Kurilova ,&nbsp;Nataliya Vorobyeva ,&nbsp;Ekaterina Bezpalaya ,&nbsp;Marina Serebryakova ,&nbsp;Elena Rodina","doi":"10.1016/j.bbapap.2025.141085","DOIUrl":"10.1016/j.bbapap.2025.141085","url":null,"abstract":"<div><div>Biallelic mutations in a nuclear gene PPA2 in human encoding mitochondrial inorganic pyrophosphatase hPPA2 cause mitochondrial disfunctions leading to severe cardiac pathology. This protein catalyzes a Mg²⁺-dependent hydrolysis of pyrophosphate, a by-product of many biosynthetic reactions, thereby providing a thermodynamic pull for these reactions. In order to better understand molecular mechanisms of mitochondrial disfunction caused by mutations in PPA2 gene, detailed characterization of a metabolic role of hPPA2 is required. In this work, we study the <em>in vitro</em> effects of a panel of metabolites, as well as other factors, on the recombinant hPPA2 activity. This study is complemented with the <em>in silico</em> assessment of possible mechanisms of observed patterns. We demonstrate that at Mg²⁺ concentrations typical for the mitochondrial matrix hPPA2 works at under-saturation and therefore the regulatory factors changing Mg²⁺ concentration will have a significant impact on hPPA2 activity. We also demonstrate that hPPA2 activity is regulated by a redox state of cysteine residues. Mass-spectrometry analysis reveals four Cys residues modified by 4-hydroxy-Hg-benzoate, as well as an S<img>S bridge formation within an hPPA2 monomer. Finally, we found that selected metabolites including intermediates of central metabolism affect hPPA2 activity. <em>In silico</em> analysis of hPPA2 allows a structural insight into the observed properties. Of a special interest is an W-loop, unique for PPA2 proteins from animals and shared with acidocalcisomal soluble pyrophosphatases from kinetoplastids, that may be involved in interaction with small-molecule effectors.</div></div>","PeriodicalId":8760,"journal":{"name":"Biochimica et biophysica acta. Proteins and proteomics","volume":"1873 5","pages":"Article 141085"},"PeriodicalIF":2.5,"publicationDate":"2025-06-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144279605","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Native structure of the monoclonal therapeutic CD20 antibody ocrelizumab","authors":"Victor G. Chrone ,&nbsp;Johan C. Jespersen ,&nbsp;Daut C. Asani ,&nbsp;Nicole H. Trier ,&nbsp;Soumik Ray ,&nbsp;Francis Berthias ,&nbsp;Martin Willemoës ,&nbsp;Anja Holm ,&nbsp;Jette L. Frederiksen ,&nbsp;Gunnar Houen ,&nbsp;Peter Højrup","doi":"10.1016/j.bbapap.2025.141084","DOIUrl":"10.1016/j.bbapap.2025.141084","url":null,"abstract":"<div><div>Immunoglobulin G (IgG) is fundamental to adaptive immunity and numerous monoclonal IgGs (monoclonal antibodies (MAbs)) have been developed as therapeutics for various diseases, including ocrelizumab (OMAb), a CD20 MAb used for treating multiple sclerosis, and infliximab (IMAb), a tumor necrosis factor MAb used for treating rheumatoid arthritis and other conditions. Understanding structure-function relationships are essential for understanding the mechanisms of action of IgG MAbs and previous results have shown that IgG has a “closed”, “m”-shaped conformation in native form, which may switch to an “open”, “Y”-shaped conformation upon antigen binding or physico-chemical stress. Supported by immunochemical and biophysical methods and by chemical crosslinking mass spectrometry (XL-MS) we show that both OMAb and IMAb conform to this paradigm. By XL-MS, we identified eighty-five high-confidence cross-links that support the native closed state of OMAb, refining our understanding of IgG architecture. Molecular modeling based on these data further corroborates a compact IgG structure, shielding the Fc domain. This structural insight may increase our understanding of immunoglobulin biology and enhance therapeutic MAb design by optimizing stability and efficacy.</div></div>","PeriodicalId":8760,"journal":{"name":"Biochimica et biophysica acta. Proteins and proteomics","volume":"1873 5","pages":"Article 141084"},"PeriodicalIF":2.5,"publicationDate":"2025-06-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144279607","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"HDAC7 influences ER-⍺ transcription via NCoR-HDAC3 dissociation","authors":"Ishadi K.M. Kodikara,&nbsp;Valentine O. Nwanelo,&nbsp;Angela K. Belanger,&nbsp;Mary Kay H. Pflum","doi":"10.1016/j.bbapap.2025.141083","DOIUrl":"10.1016/j.bbapap.2025.141083","url":null,"abstract":"<div><div>HDAC7 (histone deacetylase 7) is involved in many diseases, including breast cancer. HDAC7 regulates gene expression epigenetically by assisting in the deacetylation of nucleosomal histones to remodel chromatin. However, HDAC7 is a pseudodeacetylase that displays weak enzymatic activity and cannot directly deacetylate histones. Instead, HDAC7 scaffolds histones to active HDAC3 (histone deacetylase 3) via NCoR (nuclear receptor corepressor) to regulate transcription. Recent evidence documented that the inactive pseudo-active site of HDAC7 binds an acetyllysine on the AR (androgen receptor) transcription factor to disrupt HDAC3-NCoR scaffolding and activate transcription. To expand on the acetylation-dependent reversible scaffolding observed with AR, here HDAC7 binding was tested with additional nuclear receptors, including GR (glucocorticoid receptor), PR (progesterone receptor), TR (thyroid receptor), and RXR (retinoid x receptor), with particular focus on ER-⍺ (estrogen receptor alpha). Acetyllysine-dependent HDAC7-NCoR-HDAC3 binding and gene expression was established with ER-⍺ in a physiologically relevant breast cancer cell line, which substantiates acetyllysine-mediated reversible scaffolding by HDAC7 in the epigenetic regulation of nuclear receptor transcriptional activation.</div></div>","PeriodicalId":8760,"journal":{"name":"Biochimica et biophysica acta. Proteins and proteomics","volume":"1873 5","pages":"Article 141083"},"PeriodicalIF":2.5,"publicationDate":"2025-06-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144293184","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Unravelling the phosphoregulatory network of protein kinase C-delta (PKC-δ)","authors":"Darshan Hebbal Raghu ,&nbsp;Leona Dcunha ,&nbsp;Mukhtar Ahmed ,&nbsp;Amal Fahma ,&nbsp;Althaf Mahin ,&nbsp;Athira Perunelly Gopalakrishnan ,&nbsp;Levin John ,&nbsp;Suhail Subair ,&nbsp;Prathik Basthikoppa Shivamurthy ,&nbsp;Susmi Varghese ,&nbsp;Samseera Ummar ,&nbsp;Mahammad Nisar ,&nbsp;Poornima Ramesh ,&nbsp;Inamul Hasan Madar ,&nbsp;Rajesh Raju","doi":"10.1016/j.bbapap.2025.141080","DOIUrl":"10.1016/j.bbapap.2025.141080","url":null,"abstract":"<div><div>Protein kinase C Delta (PRKCD) is a serine/threonine kinase involved in transcription regulation, cytoskeleton organization, DNA damage response, DNA repair and carcinogenesis. Several PRKCD phosphopeptides are frequently detected to be differentially regulated through mass spectrometry-based phosphoproteomics analysis. Here, we utilize publicly available phosphoproteomics data to decipher phosphoregulatory networks associated with PRKCD. Among 315 phosphoproteomics datasets demonstrating the differential regulation of phosphopeptides of PRKCD, the phosphosites S304, Y313, S645, S299, S302, S664, Y334, Y374, T295 and T507 predominantly represent PRKCD hyperphosphorylation in over 83 % of cases. Further, through co-differential regulation analysis of the predominant sites and associated phosphosites on other proteins, we propose that the autophosphorylation sites S302 and S304 are critically associated with the kinase activity of PRKCD and play a crucial role in modulating its downstream signaling pathways. Compared to known activation sites of PRKCD including Y313, Y334 and Y374, the sites S302 and S304 coregulated the most with the experimentally validated and predicted substrates. Besides that, phosphosites Y313, Y334 and Y374 are associated with stress signaling and cancer progression. Current study focuses on critical kinase-activation associated with phosphosites in kinases. This study highlights the sites S302 and S304 as activation sites and as key players in various cellular processes including cell cycle regulation, motility, adhesion and migration. Our approach and the stringent criteria adopted sets a robust platform to analyze differentially regulated phosphoproteome data in diverse biological contexts to interpret the predominant phosphosites, their co-regulation and the overall biological relevance of such regulation in pathophysiological processes.</div></div>","PeriodicalId":8760,"journal":{"name":"Biochimica et biophysica acta. Proteins and proteomics","volume":"1873 5","pages":"Article 141080"},"PeriodicalIF":2.5,"publicationDate":"2025-06-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144212244","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}