Biology subjects

Bhosale, S. D.

Publications and source records attributed to Bhosale, S. D..

5 recordsLinked to original sources

bioRxiv2024

An inflection point in high-throughput proteomics with Orbitrap Astral: analysis of biofluids, cells, and tissues

This technical note presents a comprehensive proteomics workflow for the new combination of Orbitrap and Astral mass analyzers across biofluids, cells, and tissues. Central to our workflow is the integration of Adaptive Focused Acoustics (AFA) technology for cells and tissue lysis, to ensure robust and reproducible sample preparation in a high-throughput manner. Furthermore, we automated the detergent-compatible single-pot, solid-phase-enhanced sample Preparation (SP3) method for protein digestion, a technique that streamlines the process by combining purification and digestion steps, thereby reducing sample loss and improving efficiency. The synergy of these advanced methodologies facilitates a robust and high-throughput approach for cells and tissue analysis, an important consideration in translational research. This work disseminates our platform workflow, analyzes the effectiveness, demonstrates reproducibility of the results, and highlights the potential of these technologies in biomarker discovery and disease pathology. For cells and tissues (heart, liver, lung, and intestine) proteomics analysis by data-independent acquisition mode, identifications exceeding 10,000 proteins can be achieved with a 24-minute active gradient. In 200ng injections of HeLa digest across multiple gradients, an average of more than 80% of proteins have a CV less than 20%, and a 45-minute run covers [~]90% of the expressed proteome. In plasma samples including naive, depleted, perchloric acid precipitated, and Seer nanoparticle captured, all with a 24-minute gradient length, we identified 87, 108, 96 and 137 out of 216 FDA approved circulating protein biomarkers, respectively. This complete workflow allows for large swaths of the proteome to be identified and is compatible across diverse sample types. Graphical abstract created with biorender.com O_FIG O_LINKSMALLFIG WIDTH=152 HEIGHT=200 SRC="FIGDIR/small/591396v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@1cba4c1org.highwire.dtl.DTLVardef@adb231org.highwire.dtl.DTLVardef@18db263org.highwire.dtl.DTLVardef@1babde0_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2023

HIC1 interacts with FOXP3 multi-protein complex: a novel mechanism to regulate human regulatory T cell differentiation and function

Transcriptional repressor, hypermethylated in cancer 1 (HIC1) participates in a range of important biological processes, such as tumor repression, immune suppression, embryonic development and epigenetic gene regulation. Further to these, we previously demonstrated that HIC1 provides a significant contribution to the function and development of regulatory T (Treg) cells. However, the mechanism by which it regulates these processes was not apparent. To address this question, we used affinity-purification mass spectrometry to characterize the HIC1 Interactome in human Treg cells. Altogether 61 high-confidence interactors were identified, including IKZF3, which is a key transcription factor in the development of Treg cells. The biological processes associated with these interacting proteins include protein transport, mRNA processing, non-coding (ncRNA) transcription and RNA metabolism. The results revealed that HIC1 is part of a FOXP3-RUNX1-CBFB protein complex that regulates Treg signature genes thus improving our understanding of HIC1 function during early Treg cell differentiation. HighlightsO_LISystematic characterization of HIC1 interactome in regulatory T cells by Affinity Purification-Mass Spectrometry C_LIO_LIHIC1 binds to the RUNX1 promoter and regulates its expression C_LIO_LIHIC1-a part of FOXP3-RUNX1-CBFB transcriptional complex C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=37 SRC="FIGDIR/small/540505v1_ufig1.gif" ALT="Figure 1"> View larger version (14K): org.highwire.dtl.DTLVardef@1fcb119org.highwire.dtl.DTLVardef@cf1397org.highwire.dtl.DTLVardef@10f512forg.highwire.dtl.DTLVardef@9ae94e_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2021

Mapping Interactome Networks of FOSL1 and FOSL2 in Human Th17 Cells

Dysregulated function of Th17 cells has implications in immunodeficiencies and autoimmune disorders. Th17 cell-differentiation is orchestrated by a complex network of transcription factors, including several members of the activator protein (AP-1) family. Among these, FOSL1 and FOSL2 influence the effector responses of Th17 cells. However, the molecular mechanisms underlying these functions are unclear, owing to the poorly characterized protein interaction networks of these factors. Here, we establish the first interactomes of FOSL1 and FOSL2 in human Th17 cells, using affinity purification-mass spectrometry analysis. In addition to the known JUN proteins, we identified several novel binding partners of FOSL1 and FOSL2. Gene ontology analysis found a major fraction of these interactors to be associated with RNA binding activity, which suggests new mechanistic links. Intriguingly, 29 proteins were found to share interactions with FOSL1 and FOSL2, and these included key regulators of Th17-fate. We further validated the binding partners identified in this study by using parallel reaction monitoring targeted mass-spectrometry and other methods. Our study provides key insights into the interaction-based signaling mechanisms of FOSL factors that potentially govern Th17 cell-differentiation and associated pathologies.

immunology↗

bioRxiv2021

The AP-1 factors FOSL1 and FOSL2 co-regulate human Th17 responses

Th17 cells protect mucosal barriers, but their aberrant activity can cause autoimmunity. Molecular networks dictating human Th17 function are largely unexplored, and this hinders disease-studies. Here, we investigated the roles of the AP-1 factors, FOSL1 and FOSL2, in inducing human Th17 responses. Transient knockdown and over-expression strategies found the two proteins to inhibit Th17-cell identity, while revealing a distinct cooperativity between their functions. Strikingly, FOSL1 plays different roles in human and mouse and FOSL-mediated Th17 regulation is opposed by the AP-1 factor, BATF. Genome-wide occupancy analysis demonstrated the co-localization of FOSL1, FOSL2 and BATF in the vicinity of key Th17 genes. The functional interplay among these factors is possibly governed by sharing interactions with a common set of lineage-associated proteins. We further discovered that the genomic binding sites of these factors harbour a large number of disease-linked SNPs, many of which alter the ability of a given factor to bind DNA. Our findings thus provide crucial insights into the transcriptional regulation of human Th17 function and associated pathologies. ONE SENTENCE SUMMARYFOSL1- and FOSL2-mediated transcription during early human Th17 differentiation

immunology↗

bioRxiv2019

Protein Interactome of the Cancerous Inhibitor of Protein Phosphatase 2A (CIP2A) in Th17 Cells

Cancerous inhibitor of protein phosphatase 2A (CIP2A) is involved in immune response, cancer progression, and in Alzheimers disease. However, an understanding of the mechanistic basis of its function in this wide spectrum of physiological and pathological processes is limited due to its poorly characterized interaction networks. Here we present the first systematic characterization of the CIP2A interactome by affinity-purification mass spectrometry combined with validation by selected reaction monitoring targeted mass spectrometry (SRM-MS) analysis in Th17 cells. In addition to the known regulatory subunit of protein phosphatase PP2A, the catalytic subunit of protein PP2A was found to be interacting with CIP2A. Furthermore, the regulatory (PPP1R18, and PPP1R12A) and catalytic (PPP1CA) subunits of phosphatase PP1 were identified among the top novel CIP2A interactors. Evaluation of the ontologies associated with the proteins in this interactome revealed that they were linked with RNA metabolic processing and splicing, protein traffic, cytoskeleton regulation and ubiquitin-mediated protein degradation processes. Taken together, this network of protein-protein interactions will be important for understanding and further exploring the biological processes and mechanisms regulated by CIP2A both in physiological and pathological conditions.\n\nHighlights{blacksquare} The first characterisation of the CIP2A interactome in Th17 cells.\n{blacksquare}Key interactions were validated by targeted SRM-MS proteomics, western blot and confocal microscopy.\n{blacksquare}Pathway analysis of the interactome revealed interrelationships with proteins across a broad range of processes, in particular associated with mRNA processing.

systems biology↗