Biology subjects

Choudhary, J. S.

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

26 records · Page 2Linked to original sources

bioRxiv2019

Decoding the genomic basis of osteoarthritis

Osteoarthritis causes pain and functional disability for a quarter of a billion people worldwide, with no disease-stratifying tools nor modifying therapy. Here, we use primary cartilage and synovium from osteoarthritis patients to construct a molecular quantitative trait locus map of gene expression and protein abundance. By integrating data across omics levels, we identify likely effector genes for osteoarthritis-associated genetic signals. We detect pronounced molecular differences between macroscopically intact and highly degenerated cartilage. We identify molecularly-defined patient subgroups that correlate with clinical characteristics, stratifying patients on the basis of their molecular profile. We construct and validate a 7-gene classifier that reproducibly distinguishes between these disease subtypes, and identify potentially actionable compounds for disease modification and drug repurposing.

genomics↗

bioRxiv2019

Single-cell transcriptomics identifies an effectorness gradient shaping the response of CD4+ T cells to cytokines

Naive CD4+ T cells coordinate the immune response by acquiring an effector phenotype in response to cytokines. However, the cytokine responses in memory T cells remain largely understudied. We used quantitative proteomics, bulk RNA-seq and single-cell RNA-seq of over 40,000 human naive and memory CD4+ T cells to generate a detailed map of cytokine-regulated gene expression programs. We demonstrated that cytokine response differs substantially between naive and memory T cells and showed that memory cells are unable to differentiate into the Th2 phenotype. Moreover, memory T cells acquire a Th17-like phenotype in response to iTreg polarization. At the single-cell level, we demonstrated that T cells form a continuum which progresses from naive to effector memory T cells. This continuum is accompanied by a gradual increase in the expression levels of chemokines and cytokines and thus represents an effectorness gradient. Finally, we found that T cell cytokine responses are determined by where the cells lie in the effectorness gradient and identified genes whose expression is controlled by cytokines in an effectorness-dependent manner. Our results shed light on the heterogeneity of T cells and their responses to cytokines, provide insight into immune disease inflammation and could inform drug development.

immunology↗

bioRxiv2019

Cyclin B1-Cdk1 binding to MAD1 links nuclear pore disassembly to chromosomal stability

How the cell completely reorganises its architecture when it divides is a problem that has fascinated researchers for almost 150 years. We now know that the core regulatory machinery is highly conserved in eukaryotes but how these multiple protein kinases, protein phosphatases, and ubiquitin ligases are coordinated to remodel the cell in a matter of minutes remains a major question. Cyclin B-CDK is the primary kinase that drives mitotic remodelling and here we show that it is targeted to the nuclear pore complex (NPC) by binding an acidic face of the spindle assembly checkpoint protein, MAD1. This localised Cyclin B1-CDK1 activity coordinates NPC disassembly with kinetochore assembly: it is needed for the proper release of MAD1 from the embrace of TPR at the nuclear pore, which enables MAD1 to be recruited to kinetochores before nuclear envelope breakdown, thereby strengthening the spindle assembly checkpoint to maintain genomic stability.

cell biology↗

bioRxiv2018

An E2-ubiquitin thioester-driven approach to identify substrates modified with ubiquitin and ubiquitin-like molecules

Covalent modifications of proteins with ubiquitin and ubiquitin-like molecules are instrumental to many biological processes. However, identifying the E3 ligase responsible for these modifications remains a major bottleneck in ubiquitin research. Here, we present an E2-thioester-driven identification (E2~dID) method for the targeted identification of substrates of specific E2 and E3 enzyme pairs. E2~dID exploits the central position of E2-conjugating enzymes in the ubiquitination cascade and provides in vitro generated biotinylated E2~ubiquitin thioester conjugates as the sole source for ubiquitination in extracto. This enables purification and mass spectrometry-based identification of modified proteins under stringent conditions independently of the biological source of the extract. We demonstrate the sensitivity and specificity of E2-dID by identifying and validating substrates of APC/C in human cells. Finally, we perform E2~dID with SUMO in S. cerevisiae, showing that this approach can be easily adapted to other ubiquitin-like modifiers and experimental models.

biochemistry↗

bioRxiv2018

Evaluation of a Dual Isolation Width Acquisition (DIWA) method for isobaric labelling ratio decompression

ABSRACTIsobaric labelling is a highly precise approach for protein quantification. However, due to the isolation interference problem, isobaric tagging suffers from ratio underestimation at the MS2 level. The use of narrow isolation widths is a rational approach to alleviate the interference problem; however, this approach compromises proteome coverage. We reasoned that although a very narrow isolation window will result in loss of peptide fragment ions, the reporter ion signals will be retained for a significant portion of the spectra. Based on this assumption we have designed a Dual Isolation Width Acquisition (DIWA) method, in which each precursor is first fragmented with HCD using a standard isolation width for peptide identification and preliminary quantification, followed by a second MS2 HCD scan using a much narrower isolation width for the acquisition of quantitative spectra with reduced interference. We leverage the quantification obtained by the \"narrow\" scans to build linear regression models and apply these to decompress the fold-changes measured at the \"standard\" scans. We evaluate the DIWA approach using a nested two species/gene knockout TMT-6plex experimental design and discuss the perspectives of this approach.

systems biology↗

bioRxiv2018

Cell-type specific visualization and biochemical isolation of endogenous synaptic proteins in mice

In recent years, the remarkable molecular complexity of synapses has been revealed, with over 1000 proteins identified in the synapse proteome. Although it is known that different receptors and other synaptic proteins are present in different types of neurons and synapses, the extent of synapse diversity across the brain is largely unknown, mainly owing to technical limitations. Combining mouse genetics and proteomics we have previously reported highly efficient methods for purification of synaptic protein complexes under native conditions. In that approach, tandem affinity purification (TAP) tags were fused to the carboxyl terminus of PSD95 using gene targeting in mice. Here we report an approach that restricts tagging of endogenous PSD95 to cells expressing Cre recombinase. In addition, we developed a labelling strategy enabling visualization of endogenous PSD95 tagged by fluorescent proteins in Cre-expressing cells. We demonstrate the feasibility of proteomic characterisation of synapse proteomes and visualization of synapse proteins in specific cell types. We find that composition of PSD95 complexes purified from specific cell types differs from those extracted from tissues with diverse cellular composition. Therefore, these novel conditional PSD95 tagging lines will not only serve as powerful tools for precisely dissecting synapse diversity in specific subsets of regions/neuronal cells, but also provide an opportunity to better understand brain region-specific alterations associated with various psychiatric/neurological diseases. The newly developed conditional gene tagging methods can be applied to many different synaptic proteins and will thus facilitate research on the molecular complexity of synapses.

neuroscience↗

bioRxiv2017

A screen for combination therapies in BRAF/NRAS wild type melanoma identifies nilotinib plus MEK inhibitor as a synergistic combination

Despite recent therapeutic advances in the management of BRAFV600-mutant melanoma, there is still a compelling need for more effective treatments for patients who developed BRAF/NRAS wild type disease. Since the activity of single targeted agents is limited by innate and acquired resistance, we performed a high-throughput drug screen using 180 drug combinations to generate over 18,000 viability curves, with the aim of identifying agents that synergise to kill BRAF/NRAS wild type melanoma cells. From this screen we observed strong synergy between the tyrosine kinase inhibitor nilotinib and MEK inhibitors and validated this combination in an independent cell line collection. We found that AXL expression was associated with synergy to the nilotinib/MEK inhibitor combination, and that both drugs work in concert to suppress pERK. This finding was supported by genome-wide CRISPR screening which revealed that resistance mechanisms converge on regulators of the MAPK pathway. Finally, we validated the synergy of nilotinib/trametinib combination in vivo using patient-derived xenografts. Our results indicate that a nilotinib/MEK inhibitor combination may represent an effective therapy in BRAF/NRAS wild type melanoma patients.

cancer biology↗

bioRxiv2016

Genomic determinants of protein abundance variation in colorectal cancer cells

Assessing the extent to which genomic alterations compromise the integrity of the proteome is fundamental in identifying the mechanisms that shape cancer heterogeneity. We have used isobaric labelling and tribrid mass spectrometry to characterize the proteomic landscapes of 50 colorectal cancer cell lines and to decipher the relationships between genomic and proteomic variation. The robust quantification of 12,000 proteins and 27,000 phosphopeptides revealed how protein symbiosis translates to a co-variome which is subjected to a hierarchical order and exposes the collateral effects of somatic mutations on protein complexes. Targeted depletion of key chromatin modifiers confirmed the transmission of variation and the directionality as characteristics of protein interactions. Protein level variation was leveraged to build drug response predictive models towards a better understanding of pharmacoproteomic interactions in colorectal cancer. Overall, we provide a deep integrative view of the molecular structure underlying the variation of colorectal cancer cells.\n\nHighlightsO_LIThe cancer cell functional \"co-variome\" is a strong attribute of the proteome.\nC_LIO_LIMutations can have a direct impact on protein levels of chromatin modifiers.\nC_LIO_LITransmission of genomic variation is a characteristic of protein interactions.\nC_LIO_LIPharmacoproteomic models are strong predictors of response to DNA damaging agents.\nC_LI\n\nAbbreviations

systems biology↗