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Results for “Post-translational modifications”

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PubMed2022

Accessing isotopically labeled proteins containing genetically encoded phosphoserine for NMR with optimized expression conditions.

Phosphoserine (pSer) sites are primarily located within disordered protein regions, making it difficult to experimentally ascertain their effects on protein structure and function. Therefore, the production of 15N- (and 13C)-labeled proteins with site-specifically encoded pSer for NMR studies is essential to uncover molecular mechanisms of protein regulation by phosphorylation. While genetic code expansion technologies for the translational installation of pSer in Escherichia coli are well established and offer a powerful strategy to produce site-specifically phosphorylated proteins, methodologies to adapt them to minimal or isotope-enriched media have not been described. This shortcoming exists because pSer genetic code expansion expression hosts require the genomic ΔserB mutation, which increases pSer bioavailability but also imposes serine auxotrophy, preventing growth in minimal media used for isotopic labeling of recombinant proteins. Here, by testing different media supplements, we restored normal BL21(DE3) ΔserB growth in labeling media but subsequently observed an increase of phosphatase activity and mis-incorporation not typically seen in standard rich media. After rounds of optimization and adaption of a high-density culture protocol, we were able to obtain ≥10 mg/L homogenously labeled, phosphorylated superfolder GFP. To demonstrate the utility of this method, we also produced the intrinsically disordered serine/arginine-rich region of the SARS-CoV-2 Nucleocapsid protein labeled with 15N and pSer at the key site S188 and observed the resulting peak shift due to phosphorylation by 2D and 3D heteronuclear single quantum correlation analyses. We propose this cost-effective methodology will pave the way for more routine access to pSer-enriched proteins for 2D and 3D NMR analyses.

Humans↗

PubMed2026

Proteomic and phosphoproteomic profiles of time-dependent dynamic changes in LPS-induced macrophage polarization.

The temporal proteomic and phosphoproteomic reprogramming during early M1 macrophage polarization (0-6 h) remains poorly understood. We performed time-resolved proteomic and phosphoproteomic analyses of LPS-stimulated RAW264.7 macrophages at seven time points within 6 h. Time-clustering of differentially expressed molecules revealed two patterns: initial change with partial recovery, and sustained dysregulation. Upregulated proteins and phosphorylation sites were enriched in the Rho GTPase signaling pathway, T-cell receptor signaling pathway, NF-κB cascade, osteoclast differentiation pathway, and antiviral immune pathway. Downregulated pathways were associated with cell cycle regulation, chromatin remodeling, RNA metabolism, and mRNA processing, indicating resource reallocation to prioritize acute inflammatory responses. Kinase-substrate network analysis confirmed the mitogen-activated protein kinase (MAPK), cyclin-dependent kinase (CDK), protein kinase B (AKT), and ribosomal S6 kinase (RSK) families as core upstream phosphorylation regulators. Integrated analysis revealed synergistic and antagonistic relationships between proteomic and phosphoproteomic changes. This study provides a temporal molecular atlas of M1 polarization, delineating inflammatory signaling dynamics and offering a basis for therapeutic target discovery in inflammatory diseases. SIGNIFICANCE: Macrophage M1 polarization is a central event in innate immune defense against pathogenic invasion, yet its dysregulation is a pivotal driver of the onset and progression of a broad spectrum of inflammation-associated disorders, spanning autoimmune diseases, infectious conditions and inflammatory bone diseases, making the dissection of its molecular regulatory mechanisms an urgent research priority in immunology and translational medicine. Dynamic molecular events within 0-6 h after LPS stimulation are critical for initiating and shaping M1 inflammatory activation, yet systematic time-resolved proteomic and phosphoproteomic profiling remains insufficient.In this study, we comprehensively characterized temporal proteome and phosphoproteome changes at seven consecutive time points during macrophage polarization, clarified two distinct dynamic molecular patterns, identified core signaling pathways and key kinase regulators involved in inflammatory reprogramming, and uncovered the leading role of post-translational phosphorylation modifications in initiating polarization. This work delineates the time-series molecular atlas of early macrophage activation, provides novel insights into the temporal regulatory mechanism of inflammatory signaling networks, and lays a solid experimental foundation for exploring new intervention targets and regulatory nodes in clinical translational research.

Lipopolysaccharides↗

PubMed2026

Tumor-associated macrophages display differential protein cargo sorting in extracellular vesicles associated with poor survival in ovarian cancer.

Ovarian cancer (OC) progression and metastasis are promoted by ascites, which constitutes a central part of the tumor microenvironment (TME). In this fluid, tumor-associated macrophages (TAMs) represent a prominent immune cell type. In addition to tumor and other host cells such as TAMs, ascites is highly enriched in soluble factors as well as extracellular vesicles (EVs). How TAMs contribute to the EV compartment of the OC TME remains, however, underexplored. In this work peripheral blood monocytes from healthy donors were differentiated into monocyte-derived macrophages (MDMs) and polarized into classically activated (M1-like), alternatively activated (M2-like) and TAM-like (by ascites incubation). For all subtypes, serum-free conditioned medium was collected for 24 h and EVs were isolated and characterized by nano-flow cytometry (nFC), label-free mass spectrometry-based proteomics and electron microscopy, among others. Our results demonstrated distinct traits for EV release and cargo across the different macrophage subtypes. Specifically, TAM-like macrophages exhibited impaired release of small EVs and reduced frequency of tetraspanin-positive particles. These EV subpopulations displayed sizing profiles closer to M1-like than to M2-like samples. Also, the low EV release in TAM-like MDMs was accompanied by altered expression of biogenesis-related markers like flotillin-1 (FLOT1) and a decreased N-glycosylation of CD63 protein, which was validated in patient-derived samples. Remarkably, the EV-associated proteome of TAMs displayed significant enrichment in both pro- and anti-inflammatory molecules with clinical value. Markers significantly enriched in the ascites TAM-EV signature were mostly associated with poor prognosis, whereas M1-like EV-related markers (pro-inflammatory) were mostly associated with longer survival. Our results confirmed previous data for proteins like CD163 and MRC1 to be associated to TAM-EVs, while also describing novel candidates with diagnostic (i.e., COLEC12) and/or prognostic (i.e., MSR1) value in plasma. Taken together, our data support a unique secretory profile of TAMs in OC and provide new EV-associated biomarkers with translational impact. Our results pave the way for a better understanding of the mechanisms behind TAM-EV cargo loading and function, and how these cells participate in the TME landscape.

Humans↗

PubMed2025

Identifying key palmitoylation-associated genes in endometriosis through genomic data analysis.

BACKGROUND: Palmitoylation, a post-translational lipid modification, has garnered increasing attention for its role in inflammatory processes and tumorigenesis. Emerging evidence suggests a potential association between palmitoylation and inflammatory responses in the pathogenesis of endometriosis. However, the precise mechanistic interplay remains elusive, necessitating further investigation. METHODS: This study integrated transcriptomic analysis and Mendelian randomization (MR) to identify a causal gene set implicated in endometriosis. Differentially expressed genes (DEGs) were first identified in the training dataset using the limma package in R. Weighted gene co-expression network analysis (WGCNA) was subsequently performed, leveraging Single Sample Gene Set Enrichment Analysis (ssGSEA)-derived scores of palmitoylation-related genes (PRGs) as phenotypic traits to identify key modular genes. The intersection of these key modular genes with DEGs yielded a refined gene set. Machine learning algorithms were then applied to further optimize gene selection, followed by external validation, immune infiltration analysis, RNA network construction, and exploration of potential targeted drug candidates. RESULTS: Through a rigorous screening process, VRK1, GALNT12, and RMI1 emerged as key genes associated with palmitoylation, exhibiting significant downregulation in endometriosis samples (P < 0.05), indicative of a potential protective role. Immune infiltration analysis further revealed strong correlations between these genes and M2 macrophages as well as resting Natural Killer (NK) cells. Additionally, investigations into the targeted RNA network and drug association profiling provided novel insights, laying the groundwork for future high-quality validation studies. CONCLUSIONS: This study employed a comprehensive analytical framework to identify palmitoylation-associated key genes in endometriosis. The integration of immunoinfiltration analysis, RNA network construction, and drug association profiling offers valuable insights for advancing clinical diagnostics, disease monitoring, and therapeutic development in endometriosis.

Humans↗

PubMed2005

SUMO modification of the Ets-related transcription factor ERM inhibits its transcriptional activity.

A variety of transcription factors are post-translationally modified by SUMO, a 97-residue ubiquitin-like protein bound covalently to the targeted lysine. Here we describe SUMO modification of the Ets family member ERM at positions 89, 263, 293, and 350. To investigate how SUMO modification affects the function of ERM, Ets-responsive intercellular adhesion molecule 1 (ICAM-1) and E74 reporter plasmids were employed to demonstrate that SUMO modification causes inhibition of ERM-dependent transcription without affecting the subcellular localization, stability, or DNA-binding capacity of the protein. When the adenoviral protein Gam1 or the SUMO protease SENP1 was used to inhibit the SUMO modification pathway, ERM-dependent transcription was de-repressed. These results demonstrate that ERM is subject to SUMO modification and that this post-translational modification causes inhibition of transcription-enhancing activity.

Adenoviridae↗

PubMed2023

Inferring Metabolic States from Single Cell Transcriptomic Data via Geometric Deep Learning.

The ability to measure gene expression at single-cell resolution has elevated our understanding of how biological features emerge from complex and interdependent networks at molecular, cellular, and tissue scales. As technologies have evolved that complement scRNAseq measurements with things like single-cell proteomic, epigenomic, and genomic information, it becomes increasingly apparent how much biology exists as a product of multimodal regulation. Biological processes such as transcription, translation, and post-translational or epigenetic modification impose both energetic and specific molecular demands on a cell and are therefore implicitly constrained by the metabolic state of the cell. While metabolomics is crucial for defining a holistic model of any biological process, the chemical heterogeneity of the metabolome makes it particularly difficult to measure, and technologies capable of doing this at single-cell resolution are far behind other multiomics modalities. To address these challenges, we present GEFMAP (Gene Expression-based Flux Mapping and Metabolic Pathway Prediction), a method based on geometric deep learning for predicting flux through reactions in a global metabolic network using transcriptomics data, which we ultimately apply to scRNAseq. GEFMAP leverages the natural graph structure of metabolic networks to learn both a biological objective for each cell and estimate a mass-balanced relative flux rate for each reaction in each cell using novel deep learning models.

Preprint↗

PubMed2026

Loss of SUMOylation drives aberrant PRC1 clustering and 3D genome rewiring independent of H3K27me3.

Polycomb repressive complex 1 (PRC1) forms nuclear condensates that organize target chromatin domains. SUMOylation modulates PRC1 clustering, but its impact on condensate properties and 3D genome architecture remains unclear. Here, we show that depletion of small ubiquitin-like modifier (SUMO) in Drosophila wing imaginal discs transforms PRC1 condensates into large structures with reduced molecular dynamics. Biophysical modeling suggests that the changes in PRC1 self-interactions are responsible for the formation of large PRC1 condensates when SUMO is depleted. Interestingly, this biophysical reorganization occurs without global loss of the H3K27me3 mark. Instead, Hi-C reveals widespread rewiring of topologically associating domain (TAD) interactions. PRC1-bound TADs lose specific long-range contacts with each other while gaining ectopic interactions with active chromatin. These topological shifts correlate with gene misregulation independently of changes in Polycomb histone modifications. Our results establish SUMOylation as a critical regulator of PRC1 condensates, demonstrating that post-translational control of biomolecular condensation modulates 3D genome architecture and transcriptional output through mechanisms separable from histone mark deposition.

Animals↗

PubMed2026

Analytical challenges for mapping non-canonical and non-protein ubiquitin/Ubl modifications by mass spectrometry.

INTRODUCTION: Covalent modification by ubiquitin via Lys isopeptide bonds is fundamental for regulating protein turnover and function. Additionally, ubiquitin esterification occurs on Ser/Thr/Tyr residues in proteins and on non-proteinaceous substrates including ribose, saccharides, lipids, and small molecule drugs. Ubiquitin posttranslational modifications may therefore be much more widespread across cell biological pathways. Recent literature (PubMed) reflects the increased interest in analytical methods for mapping of non-canonical substrates modified by ubiquitin and ubiquitin-like (UBL) proteins. AREAS COVERED: Mass spectrometry (MS)-based methodologies involve advanced proteomic techniques to identify ubiquitin modifications on amino acids other than Lys, such as Ser, Thr, Tyr and Cys as well as protein N-termini. After digestion, standard MS workflows identify canonical ubiquitination by detecting a ubiquitin C-terminal tag attached to the amine side chains of Lys residues of substrate-derived peptides suitable for MS/MS sequencing. For non-canonical modifications on proteins and substrates other than proteins, specialized strategies are required, such as using antibodies to enrich N-terminally modified peptides in combination with using high-resolution MS/MS based on softer fragmentation technologies to detect esterification and possibly other types of substrate modifications. EXPERT OPINION: Enabling such technologies will reveal a previously unrecognized angle of the ubiquitin code's complexity in cells.

Humans↗

PubMed2026

Lysine iminylation derived from ω-3 polyunsaturated fatty acids.

Protein posttranslational modifications (PTMs) play a central role for regulating protein function and cellular processes, with many PTMs arising from reactions with electrophilic metabolites. Here we extend the known landscape of PTMs with the identification of "lysine C3-iminylation," the conjugation of protein lysine residues with propionaldehyde. To stabilize iminylation for mass spectrometric analyses and distinguish it from other isomeric PTMs, we developed a fixation and stable-isotope labeling approach based on parallel reduction of proteome with sodium borohydride and borodeuteride. Analyses of protein hydrolysates confirmed the presence of C3-iminylation in Caenorhabditis elegans and mouse. Additionally, proteomics results demonstrated specificity of this PTM in vitro and in vivo and revealed C3-iminylation in proteins related to critical metabolic pathways. Importantly, collective evidence from isotope tracing as well as genetic, dietary, and pharmacological manipulation experiments uncovered that C3-iminylation originates from cytochrome P450 (CYP)-mediated oxidation of omega-3 fatty acids. Correspondingly, C3-iminylation levels were elevated in C. elegans daf-2(e1370) mutants, an aging model, in which CYP activity is generally increased. These findings not only expand our understanding of the biochemical diversity of PTMs but also underscore the complex interplay between lipid metabolism and protein modifications, enabling further exploration of their biological and clinical implications.

Animals↗

PubMed2025

Systematic Identification of Microtubule Posttranslational Modification "Readers" by Quantitative Proteomics.

Microtubules, dynamic polymers assembled from α, β-tubulin dimers, contribute to myriad cellular processes. This is largely attributed to microtubule-associated proteins (MAPs). How MAPs selectively bind microtubules to carry out various functions is not known. The "Tubulin Code" theory proposes that posttranslational modifications (PTMs) of microtubules serve as signs that can be read by specific MAPs, thereby conferring specific functional properties to the microtubules. In support of this hypothesis, "reader" MAPs have been identified for various tubulin PTMs, but, until recently, no systematic screening had been performed to identify readers in an unbiased manner. We addressed this by developing a reader identification pipeline that uses quantitative mass spectrometry to interrogate the microtubule proteome of cells programmed to express specific PTMs. This pipeline can be used to identify readers for any tubulin PTM from various cell types as long as the writer enzymes are known. We also provide an alternative, complementary approach to obtain modified microtubules using a generic writer enzyme in vitro.

Protein Processing, Post-Translational↗

PubMed2025

Genome-Wide Profiling of Histone Modifications in Fission Yeast Using CUT&Tag.

Eukaryotic DNA is organized in the nucleus in the form of chromatin. Nucleosomes, the fundamental unit of chromatin, are subject to many posttranslational modifications (PTMs) as well as compositional variations through incorporation of histone variants. These alterations play important roles in regulation of genome structure and activity. Genome-wide profiling of these regulatory features is essential for understanding of genome function. Chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-Seq) is a widely used method to assay genome-wide localization in fission yeast but suffers from the requirement for a large amount of input chromatin, antibodies, and a cumbersome experimental pipeline. New methods such as Cleavage Under Targets and Tagmentation (CUT&Tag), which combine the specificity of targeted cleavage and adapter insertion with the sensitivity of next-generation sequencing, enable identification and characterization of various epigenetic marks affording low input requirement as well as more streamlined protocols. However, these approaches have not been adapted for use in fission yeast, Schizosaccharomyces pombe. Here, we describe an adapted CUT&Tag protocol for epigenomic profiling in fission yeast using the heterochromatin-associated histone H3K9 methylation PTM for benchmarking.

Schizosaccharomyces↗

PubMed2026

DOT1L-mediated H3K79me3 of ITCH promotes AURKA ubiquitination to suppress ECM degradation in osteoarthritis.

As a prevalent chronic joint disorder, osteoarthritis (OA) is characterized by degenerative changes, primarily driven by the pathological degradation of the chondrocyte extracellular matrix (ECM). Current therapies lack efficacy in halting ECM degradation, making elucidation of its regulatory mechanisms crucial for developing novel OA treatments. This study investigated the role of the DOT1L/ITCH/AURKA axis in ECM degradation during OA development. An in vitro OA model was established by treating rat chondrocytes with 10 ng/mL IL-1β for 24 h. TNF-α and IL-6 secretion was measured by ELISA. ECM content was assessed via alcian blue staining. RT-qPCR, western blot, and immunofluorescence staining analyzed associated molecule expression. Co-IP verified ITCH-AURKA interaction and AURKA ubiquitination. ChIP detected DOT1L and H3K79me3 enrichment at the ITCH promoter. An anterior cruciate ligament transection (ACL-T)-induced OA rat model with intra-articular injection of DOT1L-overexpressing lentivirus was further established, followed by HE staining, safranin O-fast green staining, and IHC analysis. IL-1β stimulation upregulated AURKA but downregulated DOT1L and ITCH expression in rat chondrocytes. ITCH promoted AURKA ubiquitination and degradation, thereby attenuating IL-1β-stimulated degradation of ECM in rat chondrocytes. DOT1L upregulated ITCH expression by mediating H3K79me3 modification at its promoter. DOT1L-dependent H3K79me3 enrichment at the ITCH promoter downregulated AURKA, ultimately inhibiting IL-1β-induced ECM degradation in rat chondrocytes. In vivo, DOT1L overexpression alleviated ACL-T-induced cartilage degeneration and reversed the ACL-T-induced downregulation of ITCH and upregulation of AURKA and ADAMTS5. Collectively, our findings identify the DOT1L/ITCH/AURKA axis as a key epigenetic and post-translational regulatory mechanism that protects against ECM degradation in OA.

Animals↗

PubMed2026

Both L-Lactyl and D-Lactyl Enantiomers Modify Histones in Mouse Testis.

Dynamic histone posttranslational modifications are crucial to precisely orchestrate gene expression programs. The recently discovered histone lysine lactylation has already been explored in various pathological contexts, but less in normal tissues. This modification exists as two enantiomers, L- and D-lactylation; the former may more likely modify histones due to abundant L-lactate produced by glycolysis. Here, we report the identification by proteomics of L- and D-lactylation on lysines of histones H3 and H4 in mouse testis. We developed a targeted proteomic analysis of histone peptides using synthetic sequences modified by L- or D-lactyl, to acquire reliable identification and quantification data. Some histone peptides bearing either enantiomer are separated by reversed-phase chromatography. Interestingly, despite the fact that L-lactate is much more abundant than D-lactate in mouse testis, we estimated abundance ratios of L-over D-lactylation to lie between 0.4 and 1.6 on seven residues of histones H3 and H4. Next, targeted proteomic analyses were performed on histones extracted from meiotic and postmeiotic male germ cells (spermatocytes and round spermatids, respectively), which are known to use L-lactate as a main source of energy. Nonetheless, residues 18 and 23 of histone H3 (H3K18 and H3K23) were reliably quantified and shown to harbor balanced amounts of both enantiomers. The stoichiometry of lactylation is low over the whole sequence of H3 and H4, representing about 0.01 to 0.44%: this contrasts with acetylation which exists at up to 25 to 35% relative abundances on some N-terminal lysines. Yet, lactylation appears to be more abundant than acetylation on the C-terminal half of H3 and H4, where the latter modification is scarce. Collectively, our results suggest a mechanism producing a mixture of the two enantiomers of lactate, or of a more direct substrate for lactylation, that leads to the modification of histones by L- and D-lactylation.

Animals↗

PubMed2025

Histone modifications in the regulation of erythropoiesis.

INTRODUCTION: The pathogenesis of anemia and other erythroid dysphasia are mains poorly understood, primarily due to limited knowledge about the differentiation processes and regulatory mechanisms governing erythropoiesis. Erythropoiesis is a highly complex and precise biological process, that can be categorized into three distinct stages: early erythropoiesis, terminal erythroid differentiation, and reticulocyte maturation, and this complex process is tightly controlled by multiple regulatory factors. Emerging evidence highlights the crucial role of epigenetic modifications, particularly histone modifications, in regulating erythropoiesis. Methylation and acetylation are two common modification forms that affect genome accessibility by altering the state of chromatin, thereby regulating gene expression during erythropoiesis. DISCUSSION: This review systematically examines the roles of histone methylation and acetylation, along with their respective regulatory enzymes, in regulating the development and differentiation of hematopoietic stem/progenitor cells (HSPCs) and erythroid progenitors. Furthermore, we discuss the involvement of these histone modifications in erythroid-specific developmental processes, including hemoglobin switching, chromatin condensation, and enucleation.Conclusions This review summarizes the current understanding of the role of histone modifications in erythropoiesis based on existing research, as a foundation for further research the mechanisms of epigenetic regulatory in erythropoiesis.

Erythropoiesis↗

PubMed2025

ProteoformDB: A Built-In Application to Generate Proteoform Database.

Proteins play essential functions through their complex regulations on cell-type-specific expression, localization, and molecular complexes. Protein complexity is further enhanced by proteoforms, which are the diverse molecular forms that each gene can produce through genomic alterations, transcriptional variations, translational regulations, and protein modifications. Profiling of proteoforms is a promising method for gaining a deeper understanding of the role of proteins in biological pathways and disease mechanisms. Here, we developed ProteoformDB, an application tool for generating proteoform databases, and we cataloged a total of over one million unique single-site human proteoforms. We showed that ProteoformDB can serve as a valuable resource to document the experimentally identified proteoforms in a database, supporting protein characterization in quantitative proteomics for both total protein abundances and modified protein forms.

Humans↗

PubMed2025

Label-Free Quantitative Phosphoproteomics in the Fission Yeast Schizosaccharomyces pombe.

Protein phosphorylation is a dynamic, reversible posttranslational modification that plays an important role in the regulation of cell signaling. Recently, label-free quantitative (LFQ) phosphoproteomics has become a powerful tool to analyze the phosphorylation of proteins within complex samples. In this chapter, we describe how to apply LFQ phosphoproteomics that is based on Fe-IMAC phosphopeptide enrichment followed by strong anion exchange (SAX) and porous graphitic carbon (PGC) fractionation strategies for identification and quantification of changes in the phosphoproteome in the fission yeast Schizosaccharomyces pombe.

Schizosaccharomyces↗

PubMed2025

Malonate promotes CD8+ T cell memory formation via protein malonylation.

Protein malonylation represents a recently identified posttranslational modification whose role in CD8+ T cell differentiation and functionality remains incompletely understood. In this study, we demonstrate that enhancing protein malonylation through sodium malonate (SM) treatment promotes CD8+ T cell memory formation in response to bacterial infection, subsequently potentiating recall responses. Comparative metabolomic analysis between SM-treated and control CD8+ T cells revealed significant metabolic alterations associated with protein malonylation. We present the first comprehensive proteomic analysis of lysine malonylation in murine CD8+ T cells, identifying 77 malonylation sites across 64 proteins involved in diverse cellular processes, particularly metabolic pathways. Malonylation of STAT6 was confirmed via the use of a specific chemical probe. Notably, we established that malonylation at the lysine 374 site of STAT6 results in increased TCF1 expression, due to alleviated transcriptional repression of TCF1 by STAT6. Collectively, our findings provide compelling evidence that protein malonylation plays a significant role in regulating CD8+ T cell memory formation.

Animals↗

PubMed2026

MARK1 suppresses infectious bursal disease virus replication via phosphorylating VP3.

Infectious bursal disease virus (IBDV) of the Birnaviridae family is a non-envelope, double-stranded RNA virus that encodes a VP3 protein with multiple functions, which controls viral genome replication, IFN-β production, and virus traffic in infected cells. Posttranslational modifications (PTMs), such as ubiquitination, of VP3 have been demonstrated for affecting its function and stability. To clarify the mechanism by which VP3 is regulated in IBDV infected cells, we focused on the phosphorylation of VP3. Mass spectrometry analysis identified that microtubule-affinity regulating kinases 1 (MARK1) was a kinase interacting protein of VP3. Inhibitory function of MARK1 in affecting viral replication was validated. We describe the phosphorylation event at the serine 130 (S130) and serine 163 (S163) residues of VP3 mediated by MARK1 via mass spectrometry analysis. Alanine replacement of the phosphorylation sites in VP3 significantly enhanced its RNA-binding activity. Additionally, the mutation of two serine residues led to remarkably improved in its polymerase-enhancing function. We then incorporated the two mutations to rescue recombinant IBDV. Viral growth curve analysis revealed that replication of mutant IBDV was significantly enhanced relative to wild type (WT) virus. In conclusion, we found that VP3 functions are specifically regulated by MARK1 mediated phosphorylation at S130 and S163 and that this regulation suppresses IBDV replication ultimately.

Infectious bursal disease virus↗