Biology subjects

Chaudhary, J. S.

Publications and source records attributed to Chaudhary, J. S..

10 recordsLinked to original sources

bioRxiv2025

ARID1A stabilises non-homologous end joining factors at DNA breaks induced by the G4 ligand pyridostatin

ARID1A is a subunit of the BAF chromatin remodelling complex that is frequently mutated in cancer. It is challenging to predict how ARID1A loss impacts cancer therapy response because it participates in many different cellular pathways. G quadruplex (G4) binding ligands, such as pyridostatin, have shown anticancer effects, but the pathways and genetic determinants involved in the response to G4 ligands are still not fully understood. Here, we show that ARID1A deficient cells are selectively sensitive to pyridostatin when compared with isogenic controls. Sensitivity to pyridostatin was apparent in ovarian and colorectal cancer cell line models, and in vivo studies suggest that G4 ligands hold promise for treating ARID1A deficient cancers. While we find that ARID1A impacts on pyridostatin-induced transcriptional responses, we find that pyridostatin-mediated toxicity in ARID1A-deficient cells is driven by defective DNA repair of topoisomerase-induced breaks. We show that ARID1A-deficient cells are unable to efficiently accumulate non-homologous end joining proteins on chromatin following pyridostatin exposure. These data uncover a role for ARID1A in the cellular response to G4 ligands, and link remodelling to G4 ligand-induced transcriptional and DNA damage responses.

molecular biology↗

bioRxiv2025

Detection of human unannotated microproteins by mass spectrometry-based proteomics: a community assessment

Thousands of short open reading frames (sORFs) are translated outside of annotated coding sequences. Recent studies have pioneered searching for sORF-encoded microproteins in mass spectrometry (MS)- based proteomics and peptidomics datasets. Here, we assessed literature-reported MS-based identifications of unannotated human proteins. We find that studies vary by three orders of magnitude in the number of unannotated proteins they report. Of nearly 10,000 reported sORF-encoded peptides, 96% were unique to a single study, and 12% mapped to annotated proteins or proteoforms. Manual curation of a benchmark dataset of 406 manually evaluated spectra from 204 sORF-encoded proteins revealed large variation in peptide-spectrum match (PSM) quality between studies, with immunopeptidomics studies generally reporting higher quality PSMs than conventional enzymatic digests of whole cell lysates. We estimate that 65% of predicted sORF-encoded protein detections in immunopeptidomics studies were supported by high-quality PSMs versus 7.8% in non-immunopeptidomics datasets. Our work stresses the need for standardized protocols and analysis workflows to guide future advancements in microprotein detection by MS towards uncovering how many human microproteins exist.

genomics↗

bioRxiv2025

Selection of pre-leukemic hematopoietic stem cells driven by distinct extracellular matrix molecules

Despite rapid advances in mapping genetic drivers and gene expression changes in hematopoietic stem cells (HSCs), there is a relative paucity of studies at the protein level. Here, we perform a deep, multi-omic characterization (epigenome, transcriptome and proteome) of HSCs carrying a loss-of-function mutation in Tet2, a key driver of increased self-renewal in blood cancers. Using state-of-the-art, multiplexed, low-input mass spectrometry (MS)-based proteomics, we profile wildtype (WT) and TET2-deficient (Tet2-/-) HSCs and show that the proteome captures previously unrecognized molecular processes which define the pre-leukemic HSC molecular landscape. Specifically, we obtain more accurate stratification of WT and Tet2-/- HSCs than transcriptomic approaches and identify extracellular matrix (ECM) molecules as novel points of dysregulation upon TET2 loss. HSC expansion assays using ECM-functionalized hydrogels confirm a selective effect on the expansion of Tet2-mutant HSCs. Taken together, our study represents a comprehensive molecular characterization of Tet2-mutant HSCs and identifies a previously unanticipated role of ECM molecules in regulating self-renewal of disease-driving HSCs.

cell biology↗

bioRxiv2024

Integrated multiomic profiling reveals SWI/SNF subunit-specific pathway alterations and targetable vulnerabilities

Mutations in subunits of the SWItch Sucrose Non-Fermentable (SWI/SNF) chromatin remodeling complex occur in {approx}20% of cancers and represent a highly unmet medical need. To identify novel therapeutic approaches, we systematically characterized transcriptomic and proteomic changes caused by the loss of SWI/SNF subunits or other epigenetic enzymes in isogenic cell lines, which we subsequently integrated with high-throughput drug screening and independent genetic screens of the DepMap project. Using an optimized bioinformatics pipeline for pathway enrichment, we identified Metabolism of proteins as the most frequently dysregulated Reactome pathway category in SWI/SNF-defective cell lines. Drug screening and multiomic integration revealed multiple chemicals selectively cytotoxic for SWI/SNF-defective models, including CBP/EP300 or mitochondrial respiration inhibitors. A novel algorithm for the analysis of DepMap CRISPR screens independently identified synthetic lethality between SWI/SNF defects and EP300 or mitochondrial respiration genes, which we further revalidated in disease-relevant models. These results unravel novel genetic dependencies for SWI/SNF-defective cancers.

cancer biology↗

bioRxiv2023

Mechanism of assembly, activation and lysine selection by the SIN3B histone deacetylase complex

Histone deacetylase complexes remove histone lysine acetylation, a key post-translational modification that activates transcription at each gene. Although these complexes are drug targets and crucial regulators of organismal physiology, their structure and mechanisms of action are largely unclear. Here, we present the first structure of a complete human SIN3B histone deacetylase holo-complex with and without a substrate mimic. Remarkably, SIN3B encircles the deacetylase and contacts its allosteric basic patch thereby stimulating catalysis. A SIN3B loop inserts into the catalytic tunnel, rearranges to accommodate the acetyl-lysine moiety and stabilises the substrate for specific deacetylation, which is guided by a substrate receptor subunit. Our findings provide a model of specificity for a main transcriptional regulator conserved from yeast to human and a resource of protein-protein interactions for future drug designs.

molecular biology↗

bioRxiv2022

Characterization of proteome-size scaling by integrative omics reveals mechanisms of proliferation control in cancer

Almost all living cells maintain size uniformity through successive divisions. Proteins that sub- or super-scale with size act as rheostats which regulate cell progression. A comprehensive atlas of these proteins is lacking; particularly in cancer cells where both mitogen and growth signalling are dysregulated. Utilising a multi-omic strategy, that integrates quantitative single cell imaging, phosphoproteomic and transcriptomic datasets, we leverage the inherent size heterogeneity of melanoma cells to investigate how peptides, post-translational modifications, and mRNAs scale with cell size to regulate proliferation. We find melanoma cells have different mean sizes, but all retain uniformity. Across the proteome, we identify proteins and phosphorylation events that sub and super scale with cell size. In particular, G2/M, biosynthetic, and cytoskeletal regulators sub- and super-scale with size. In small cells growth and proliferation processes are tightly coupled by translation which promotes CCND1 accumulation and anabolic increases in mass. Counter intuitively, anabolic growth pathways and translational process are low in large cells, which throttles the expression of factors such as CCND1 and thereby coupling proliferation from anabolic growth. Strikingly, these cells exhibit increased growth and comparable proliferation rates. Mathematical modelling suggests that decoupling growth and proliferative signalling fosters proliferation under mitogenic inhibition. As factors which promote adhesion and actin reorganization super-scale with size or are enriched in large cells, we suggest that growth/proliferation in these cells may be decoupled by cell spreading and mechanics. This study provides one of the first demonstrations of size-scaling phenomena in cancer and how morphology determines the chemistry of the cell.

systems biology↗

bioRxiv2022

HSP90-CDC37-PP5 forms a structural platform for kinase dephosphorylation

Activation of client protein kinases by the HSP90 molecular chaperone system is affected by phosphorylation at multiple sites on HSP90, on the kinase specific co-chaperone CDC37, and the kinase client itself. Removal of regulatory phosphorylation from client kinases and their release from the HSP90-CDC37 system depends on a Ser/Thr phosphatase PP5, which associates with HSP90 via its N-terminal TPR domain. Here we present the cryoEM structure of the oncogenic protein kinase client BRAFV600E bound to HSP90-CDC37, showing how the V600E mutation favours BRAF association with HSP90-CDC37. Structures of HSP90-CDC37-BRAFV600E complexes with PP5, in autoinhibited and activated conformations, together with proteomic analysis of its phosphatase activity, reveal how PP5 is activated by recruitment to HSP90 complexes to comprehensively dephosphorylate client proteins.

biochemistry↗

bioRxiv2021

EROS-mediated control of NOX2 and P2X7 biosynthesis

EROS (Essential for Reactive Oxygen Species) protein is indispensable for expression of gp91phox, the catalytic core of the phagocyte NADPH oxidase. EROS deficiency in humans is a novel cause of the severe immunodeficiency, chronic granulomatous disease (CGD), but its mechanism of action was unknown until now. We elucidate the role of EROS, showing it acts at the earliest stages of gp91phox maturation. It binds the immature 58kDa gp91phox directly, preventing gp91phox degradation and allowing glycosylation via the oligosaccharyltransferase (OST) machinery and the incorporation of the heme prosthetic groups essential for catalysis. EROS also regulates the purine receptors P2X7 and P2X1 through direct interactions and P2X7 is almost absent in EROS deficient mouse and human primary cells. Accordingly, lack of EROS results in markedly abnormal P2X7 signalling, inflammasome activation and T cell responses. The loss of both ROS and P2X7 signalling leads to resistance to influenza infection. Our work identifies EROS as a highly selective chaperone for key proteins in innate and adaptive immunity and a rheostat for immunity to infection. It has profound implications for our understanding of immune physiology, ROS dysregulation and possibly gene therapy.

immunology↗

bioRxiv2020

DOCK5 couples CDC42-GSK3beta activation with focal adhesion morphogenesis to reinforce the establishment of polarity and promote YAP/TAZ activation during migration.

YAP and TAZ are transcriptional co-activators that are often constitutively active in triple negative breast cancer (TNBC) cells driving proliferation, invasion, and drug resistance. Through multiplexed quantitative genetic screens for YAP/TAZ localisation and cell shape, we found that the RhoGEF DOCK5 is essential for YAP/TAZ activation in metastatic cells and is required for the maintenance of polarity during migration. DOCK5 regulates cell shape and thus YAP/TAZ through different genetic interactions with CDC42, RAC, and RHOA GTPases. DOCK5 regulates focal adhesion (FA) morphogenesis in RAC-dependent fashions that promote RHOA mediated actomyosin engagement of FA. Using unbiased systems-level quantification of protein levels by mass spectrometry we show that DOCK5 maintains polarity by stabilising protein levels of the CDC42 effector GSK3{beta}. We conclude DOCK5 acts as a coincidence detector to promote leading edge persistence in subcellular locations where there is both RAC and RHOA dependent FA morphogenesis and active CDC42 mediated cell polarisation.

cell biology↗

bioRxiv2020

Phosphorylation Dependent Assembly of a 14-3-3 Mediated Signaling Complex During Red Blood Cell Invasion by Plasmodium falciparum Merozoites

Red blood cell (RBC) invasion by Plasmodium merozoites requires multiple steps that are regulated by signaling pathways. Exposure of P. falciparum merozoites to the physiological signal of low K+, as found in blood plasma, leads to a rise in cytosolic Ca2+, which mediates microneme secretion, motility, and invasion. We have used global phosphoproteomic analysis of merozoites to identify signaling pathways that are activated during invasion. Using quantitative phosphoproteomics we found 394 protein phosphorylation site changes in merozoites subjected to different ionic environments (high K+/ low K+) out of which 143 were Ca2+-dependent. These included a number of signaling proteins such as catalytic and regulatory subunits of protein kinase A (PfPKAc and PfPKAr) and calcium-dependent protein kinase 1 (PfCDPK1). Proteins of the 14-3-3 family interact with phosphorylated target proteins to assemble signaling complexes. Here, using co-immunoprecipitation and gel filtration chromatography, we demonstrate that Pf14-3-3I binds phosphorylated PfPKAr and PfCDPK1 to mediate the assembly of a multi-protein complex in P. falciparum merozoites. A phospho-peptide, P1, based on the Ca2+ dependent phosphosites of PKAr, binds Pf14-3-3I and disrupts assembly of the Pf14-3-3I-mediated multi-protein complex. Disruption of the multi-protein complex with P1 inhibits microneme secretion and RBC invasion. This study thus identifies a novel signaling complex that plays a key role in merozoite invasion of RBCs. Disruption of this signaling complex could serve as a novel approach to inhibit blood stage growth of malaria parasites. ImportanceInvasion of red blood cells (RBCs) by Plasmodium falciparum merozoites is a complex process that is regulated by intricate signaling pathways. Here, we have used phosphoproteomic profiling to identify the key proteins involved in signaling events during invasion. We found changes in the phosphorylation of various merozoite proteins including multiple kinases previously implicated in the process of invasion. We also found that a phosphorylation dependent multi-protein complex including signaling kinases assembles during the process of invasion. Disruption of this multi-protein complex impairs merozoite invasion of RBCs providing a novel approach for the development of inhibitors to block the growth of blood stage malaria parasites.

microbiology↗