Biology subjects

Ghosh, M.

Publications and source records attributed to Ghosh, M..

At least 37 records · Page 2Linked to original sources

bioRxiv2022

Immunogenicity of non-canonical HLA-tumor ligands identified through proteogenomics

Tumor antigens are central to antitumor immunity. Recent evidence suggests that peptides from non-canonical (nonC) aberrantly translated proteins can be presented on HLA-I by tumor cells. Here, we investigated the immunogenicity of nonC tumor HLA-I ligands (nonC-TL) to better understand their contribution to cancer immunosurveillance and their therapeutic applicability. Using proteogenomics, we identified 517 nonC-TL from 9 patients with melanoma, gynecological, and head and neck cancer. We found no recognition of the 507 nonC-TL tested by autologous ex vivo expanded tumor reactive T-cell cultures while the same cultures demonstrated reactivity to mutated, cancer-germline, or melanocyte differentiation antigens. However, in vitro sensitization of donor peripheral blood lymphocytes against 170 selected nonC-TL, led to the identification of T-cell receptors (TCRs) specific to three nonC-TL, two of which mapped to the 5 UTR regions of HOXC13 and ZKSCAN1, and one mapping to a non-coding spliced variant of C5orf22C. T cells targeting these nonC-TL recognized cancer cell lines naturally presenting their corresponding antigens. Expression of the three immunogenic nonC-TL was shared across tumor types and barely or not detected in normal cells. Our findings predict a limited contribution of nonC-TL to cancer immunosurveillance but demonstrate they may be attractive novel targets for widely applicable immunotherapies.

cancer biology↗

bioRxiv2022

The DEAD-box protein p68 and B-catenin: the crucial regulators of FOXM1 gene expression in arbitrating colorectal cancer

Forkhead box M1 (FOXM1), a vital member of the Forkhead box family of transcription factors, helps in mediating oncogenesis. However, limited knowledge exists regarding the mechanistic insights into the FOXM1 gene regulation. p68, an archetypal member of the DEAD-box family of RNA helicases, shows multifaceted action in cancer progression by arbitrating RNA metabolism and transcriptionally coactivating transcription factors. Here, we report a novel mechanism of alliance between p68 and the Wnt/{beta}-catenin pathway in regulating FOXM1 gene expression and driving colon carcinogenesis. Initial bioinformatic analyses highlighted elevated expression levels of FOXM1 and p68 in colorectal cancer datasets. Immunohistochemical assays confirmed that FOXM1 showed a positive correlation with p68 and {beta}-catenin in both normal and colon carcinoma patient samples. Overexpression of p68 and {beta}-catenin increased the protein and mRNA expression profiles of FOXM1, and the converse correlation occurred during downregulation. Mechanistically, overexpression and knockdown of p68 and {beta}-catenin elevated and diminished FOXM1 promoter activity respectively. Additionally, Chromatin immunoprecipitation assay demonstrated the occupancy of p68 and {beta}-catenin at the TCF4/LEF binding element (TBE) sites on the FOXM1 promoter. Thiostrepton delineated the effect of FOXM1 inhibition on cell proliferation and migration. Colony formation assay, migration assay, and cell cycle data reveal the importance of the p68/{beta}-catenin/FOXM1 axis in oncogenesis. Collectively, our study mechanistically highlights the regulation of FOXM1 gene expression by p68 and {beta}-catenin in colorectal cancer.

cancer biology↗

bioRxiv2022

Recombinant Human Proteoglycan 4 (rhPRG4) Downregulates TNFα-Stimulated NFκB Activity and FAT10 Expression in Human Corneal Epithelial Cells

Dry Eye Disease (DED) is a complex pathology affecting millions of people with significant impact on quality of life. Corneal inflammation, including via the NF{kappa}B pathway, plays a key etiological role in DED. Recombinant human proteoglycan 4 (rhPRG4) has been shown to be a clinically effective treatment for DED that has anti-inflammatory effects in corneal epithelial cells, but the underlying mechanism is still not understood. Our goal was to understand if rhPRG4 affects TNF-stimulated inflammatory activity in corneal epithelial cells. We treated hTERT-immortalized corneal epithelial (hTCEpi) cells {+/-}TNF {+/-}rhPRG4 and performed Western blotting on cell lysate and RNA sequencing. Bioinformatics analysis revealed that rhPRG4 had a significant effect on TNF-mediated inflammation with potential effects on matricellular homeostasis. rhPRG4 reduced activation of key inflammatory pathways and decreased expression of transcripts for key inflammatory cytokines, interferons, interleu-kins, and transcription factors. TNF treatment significantly increased phosphorylation and nuclear translocation of p65, and rhPRG4 significantly reduced both these effects. RNA sequencing identified FAT10, which has not been studied in the context of DED, as a key pro-inflammatory transcript increased by TNF and decreased by rhPRG4. These results were confirmed at the protein level. In summary, rhPRG4 is able to downregulate NF{kappa}B activity in hTCEpi cells, suggesting a potential biological mechanism by which it may act as a therapeutic for DED.

molecular biology↗

bioRxiv2022

Harmonin homology domains of human RTEL1 interact with replication protein A and DNA

The regulator of telomere elongation helicase 1 (RTEL1) is an Fe-S cluster containing helicase that plays important roles in telomere DNA maintenance, DNA repair, and genome stability. It is a modular protein comprising a helicase domain, two tandem harmonin homology domains 1 & 2 (HHD1 and HHD2), and a Zn2+ binding RING domain. In this study, we have unravelled a novel interaction between RTEL1 and replication protein A (RPA) and shown their co-localization upon DNA damage in the cells. Using NMR spectroscopy, we show that 32C domain of RPA and DNA competitively bind with HHD2 of RTEL1. To understand the structural basis of HHD2 - 32C and HHD2 - DNA interactions, we have determined a 1.6 [A] resolution crystal structure of HHD2. NMR chemical shift perturbations-based mapping revealed the 32C and DNA binding surface on HHD2 of RTEL1. Together, these results establish an interplay among RTEL1, RPA, and DNA that provide mechanistic insights into the RTEL1 recruitment at DNA during the processes of replication, repair, and recombination.

biophysics↗

bioRxiv2022

Losartan controls immune checkpoint blocker-induced edema and improves survival in glioblastoma

Immune checkpoint blockers (ICBs) have failed in all Phase III glioblastoma trials. Here, we found that ICBs induce cerebral edema in some patients and mice with glioblastoma. Through single-cell RNA sequencing, intravital imaging, and T cell blocking studies in mice, we demonstrated that this edema results from an inflammatory response following anti-PD1 antibody treatment that disrupts the blood-tumor-barrier. Used in lieu of immunosuppressive corticosteroids, the angiotensin receptor blocker losartan prevented this ICB-induced edema and reprogrammed the tumor microenvironment, curing 20% of mice which increased to 40% in combination with standard of care treatment. Using a bihemispheric tumor model, we identified a "hot" tumor immune signature prior to losartan+anti-PD1 therapy that predicted long-term survival. Our findings provide the rationale and associated biomarkers to test losartan with ICBs in glioblastoma patients. One-Sentence SummaryLosartan prevents immunotherapy-associated edema and enhances the outcome of immunotherapy in glioblastoma.

cancer biology↗

bioRxiv2022

p53 engages the cGAS/STING cytosolic DNA sensing pathway for tumor suppression

Tumor suppression by p53 is known to involve cell autonomous and non-cell autonomous mechanisms. p53 has been shown to suppress tumor growth by modulating immune system functions, however, the mechanistic basis for this activity is not well understood. Here we report that p53 promotes the degradation of the DNA exonuclease TREX1, resulting in cytosolic dsDNA accumulation. We demonstrate that p53 requires the ubiquitin ligase TRIM24 to induce TREX1 ubiquitin-dependent degradation. The accumulation of cytosolic DNA due to p53s suppression of TREX1 activates the cytosolic DNA sensor, cGAS and its downstream effectors STING/TBK1/IRF3 resulting in induction of Type I interferons. TREX1 overexpression sufficed to block WTp53 activation of the cGAS/STING pathway. WTp53 mediated induction of type I interferon (IFNB1) response could be suppressed by cGAS/STING knockout. We find that p53s tumor suppressor activities are compromised by loss of signaling through the cGAS/STING pathway. Thus, our study reveals that p53 utilizes the cGAS/STING innate immune system pathway for both cell intrinsic and cell extrinsic tumor suppressor activities.

cancer biology↗

bioRxiv2021

Regulation of mitochondrial proteostasis by the proton gradient

Mitochondria adapt to different energetic demands reshaping their proteome. Mitochondrial proteases are emerging as key regulators of these adaptive processes. Here, we use a multi-proteomic approach to demonstrate regulation of the m-AAA protease AFG3L2 by the mitochondrial proton gradient, coupling mitochondrial protein turnover to the energetic status of mitochondria. We identify TMBIM5 (previously also known as GHITM or MICS1) as a Ca2+/H+ exchanger in the mitochondrial inner membrane, which binds to and inhibits the m-AAA protease. TMBIM5 ensures cell survival and respiration, allowing Ca2+ efflux from mitochondria and limiting mitochondrial hyperpolarization. Persistent hyperpolarization, however, triggers degradation of TMBIM5 and activation of the m-AAA protease. The m-AAA protease broadly remodels the mitochondrial proteome and mediates the proteolytic breakdown of respiratory complex I to confine ROS production and oxidative damage in hyperpolarized mitochondria. TMBIM5 thus integrates mitochondrial Ca2+ signaling and the energetic status of mitochondria with protein turnover rates to reshape the mitochondrial proteome and adjust the cellular metabolism.

molecular biology↗

bioRxiv2021

The Autophagy Receptor TAX1BP1 (T6BP) is a novel player in antigen presentation by MHC-II molecules.

CD4+ T lymphocytes play a major role in the establishment and maintenance of immunity. They are activated by antigenic peptides derived from extracellular or newly synthesized (endogenous) proteins presented on the surface of antigen presenting cells (APCs) by the MHC-II molecules. The pathways leading to endogenous MHC-II presentation remain poorly characterized. We demonstrate here that the autophagy receptor, T6BP, influences both autophagy-dependent and -independent endogenous presentation of HIV- and HCMV-derived peptides. By studying the immunopeptidome of MHC-II molecules, we show that T6BP affects both the quantity and quality of peptides presented. T6BP silencing induces the mislocalization of the MHC-II-loading compartments and a rapid degradation of the invariant chain (CD74) without altering the expression and internalization kinetics of MHC-II molecules. We determined the interactome of T6BP, identified calnexin as a T6BP partner and show that CANX cytosolic tail is required for this interaction. Remarkably, calnexin silencing replicates the functional consequences of T6BP silencing: decreased CD4+ T cell activation and exacerbated CD74 degradation. Altogether, we unravel T6BP as a key player of the MHC-II-restricted endogenous presentation pathway and we propose one potential mechanism of action.

immunology↗

bioRxiv2021

REPEATED INJURY PROMOTES TRACHEOBRONCHIAL TISSUE STEM CELL ATTRITION

Chronic lung disease has been attributed to stem cell aging and/or exhaustion. To address this issue, we investigated the lifespan of tracheobronchial tissue stem cells (TSC) over time and in response to repeated injury. Chromatin and nucleotide labeling studies in mice indicated that: 1) injury activated a subset of the TSC pool and that this process conserved TSC over time; and 2) activated TSC were predisposed to further proliferation and this activated state lead to terminal differentiation. Analysis of human TSC and clonal isolates indicated that repeated TSC proliferation led to telomere shortening and analysis of TSC from Dyskeratosis Congenita donors indicated that mutations in telomere biology genes accelerated TSC depletion. RNAseq and functional studies indicated that human TSC terminated as a secretory committed cell. These data support a model in which a repeated epithelial injury depletes the TSC pool and initiates the abnormal repair associated with chronic lung disease.

cell biology↗

bioRxiv2020

Differential host response to a herpesvirus: Mareks disease virus peptides on chicken MHC class II molecules are derived from only a few genes and illustrate a new mode of peptide binding

Viral diseases pose major threats to humans and other animals, including the billions of chickens that are an important food source as well as a public health concern due to zoonotic pathogens. Unlike humans and other typical mammals, the major histocompatibility complex (MHC) of chickens can confer decisive resistance or susceptibility to many viral diseases. An iconic example is Mareks disease, caused by an oncogenic herpesvirus with over 100 genes. Classical MHC class I and class II molecules present antigenic peptides to T lymphocytes, and it has been hard to understand how such MHC molecules could be involved in susceptibility to Mareks disease, given the potential number of peptides from over 100 genes. We used a new in vitro infection system and immunopeptidomics to determine peptide motifs for the two class II molecules expressed by the MHC haplotype B2, which is known to confer resistance to Mareks disease. Surprisingly, we found that the vast majority of viral peptide epitopes presented by chicken class II molecules arise from only four viral genes, nearly all having the peptide motif for BL2, the dominantly-expressed class II molecule in chickens. We expressed BL2 linked to several MDV peptides, and determined one X-ray crystal structure, showing how a single small amino acid in the binding site causes a crinkle in the peptide, leading to core binding peptide of ten amino acids, compared to the nine amino acids in all other reported class II molecules. The limited number of potential T cell epitopes from such a complex virus can explain the differential MHC-determined resistance to MDV, but raises questions of mechanism and opportunities for vaccine targets in this important food species, as well as providing a basis for understanding class II molecules in other species including humans.

immunology↗

bioRxiv2020

Proteoglycan 4 (PRG4) expression and functionin dry eye associated inflammation

PurposeDry eye disease (DED) affects hundreds of millions worldwide. Proteoglycan 4 (PRG4) has been shown to improve signs and symptoms of DED in humans. The objectives of this study were to characterize endogenous PRG4 expression by telomerase-immortalized human corneal epithelial (hTCEpi) cells, examine how exogenous recombinant human PRG4 (rhPRG4) modulates cytokine and chemokine secretion in response to TNF and IL-1{beta}, explore rhPRG4 as a potential substrate and/or inhibitor of MMP-9, and to understand how experimental dry (EDE) in mice affects PRG4 expression. MethodsPRG4 secretion was quantified by Western blotting and PRG4 expression by immunocytochemistry. Cytokine/chemokine release was measured by ELISA, and MMP-9 inhibition was quantified using an MMP-9 inhibitor kit. EDE was induced in mice, and PRG4 was visualized by immunohistochemistry in the cornea and Western blotting in lacrimal gland lysate. ResultshTCEpi cells synthesize and secrete PRG4 in vitro, which is inhibited by TNF and IL-1{beta}. TNF and IL-1{beta} significantly increased secretion of cytokine IL-6 and chemokines IL-8, IP-10, RANTES, and ENA-78, and several of these chemokines were downregulated after cotreatment with rhPRG4. Fluorescently-labelled rhPRG4 was internalized by hTCEpi cells. rhPRG4 was not digested by MMP-9 and inhibited in vitro activity of exogenous MMP-9 both in solution and in the presence of human tears. Finally, EDE decreased corneal and lacrimal gland expression of PRG4. ConclusionsThese results demonstrate rhPRG4s anti-inflammatory properties in the corneal epithelium and its contribution to ocular surface homeostasis, furthering our understanding of PRG4s immunomodulatory properties in the context of DED inflammation.

biochemistry↗

bioRxiv2020

Integrated Bioinformatics Analysis Deciphering the microRNA Regulation in Protein-Protein Interaction Network in Lung Adenocarcinoma

BackgroundThe architecture of the protein-protein interaction (PPI) network in any organism relies on their gene expression signature. microRNAs (miRNAs) have recently emerged as major post transcriptional regulators that control PPI by targeting mainly untranslated regions of the gene encoding proteins. Here, we aimed to unveil the role of miRNAs in the PPI network for identifying potential molecular targets for lung adenocarcinoma (LUAD). Materials and methodsThe expression profiles of miRNAs and mRNAs were collected from the NCBI Gene Expression Omnibus (GEO) database (GSE74190 and GSE116959). Abnormally expressed mRNAs from the data were appointed to construct a PPI network and hence incorporated with the miRNA-mRNA regulatory network. The miRNAs and mRNAs in this network were subjected to functional enrichment. Through the network analysis, hubs were identified and their mutation rate and probability of cooccurrence were calculated. ResultsWe identified 17 miRNAs and 429 mRNAs signature for differentially altered transcriptome in LUAD. The combined miRNA-mRNA regulatory network exhibited scale-free characteristics. Network analysis showed 5 miRNA (including hsa-miR-486-5p, hsa-miR-200b-5p, and hsa-miR-130b-5p) and 10 mRNA (including ASPM, CCNB1, TTN, TPX2, and BIRC5) which expressively contribute in the LUAD. We further investigated the hub genes and noticed that ASPM and TTN had the maximum rate of mutation and possessed a high tendency of cooccurrence in LUAD. ConclusionThis study provides a unique network approach to the exploration of the underlying molecular mechanism in LUAD. Identified mRNAs and miRNAs may therefore, serve as significant prognostic predictors and therapeutic targets.

bioinformatics↗

bioRxiv2020

Machine Learning-Based Protein Microarray Digital Assay Analysis

Serial measurement of a large panel of protein biomarkers near the bedside could provide a promising pathway to transform the critical care of acutely ill patients. However, attaining the combination of high sensitivity and multiplexity with a short assay turnaround poses a formidable technological challenge. Here, we developed a rapid, accurate, and highly multiplexed microfluidic digital immunoassay by incorporating machine learning-based autonomous image analysis. The assay achieved 14-plexed biomarker detection at concentrations < 10pg/mL with a sample volume < 10 L, including all processes from sampling to analyzed data delivery within 30 min, while only requiring a 5-min assay incubation. The assay procedure applied both a spatial-spectral microfluidic encoding scheme and an image data analysis algorithm based on machine learning with a convolutional neural network (CNN) for pre-equilibrated single-molecule protein digital counting. This unique approach remarkably reduced errors facing the high-capacity multiplexing of digital immunoassay at low protein concentrations. Longitudinal data obtained for a panel of 14 serum cytokines in human patients receiving chimeric antigen receptor-T (CAR-T) cell therapy manifested the powerful biomarker profiling capability and great potential of the assay for its translation to near-real-time bedside immune status monitoring.

bioengineering↗

bioRxiv2020

Identification and characterisation of the gene encoding an amastine-like surface protein (ALSP) in the Leishmania donovani genome: a putative anti-leishmanial drug target

In the current study, a novel putative protein of Leishmania donovani, amastin-like surface protein (ALSP) has been characterized. The gene was cloned in a bacterial system and the protein was overexpressed. A polyclonal antibody was developed against the protein, which detected a 10 kDa band in the L. donovani amastigote. ALSP mRNA was detected in L. donovani amastigote, which was not expressed in the promastigote. ALSP mRNA was not expressed in either morphological forms of Leishmania major. MALDI-TOF confirmed the molecular weight of ALSP as 10 kDa. I-TASSER predicted the function of ALSP as a lipase, which was confirmed by preliminary in-vitro experiments using amastigotes of L. donovani. ALSP has GAS amino acid sequences, which might act as the active site for its lipase activity. The selective expression of ALSP in amastigotes probably makes it important in virulence mechanisms such as survival in the phagolysosome and modulation of its membrane and other metabolic functions, necessary for parasite survival in the human host. ALSP can act as a peptide vaccine target and maybe detected in the peripheral blood or urine as a molecular biomarker of active disease in visceral leishmaniasis.

molecular biology↗

bioRxiv2020

An innovative approach for HLA typing, molecular tumor testing and the validation of tumor exclusive antigens

DisclaimerThis manuscript has been withdrawn by the corresponding author, as it was submitted and made public in bioRxiv without knowledge and the full consent of all the authors listed. The co-authors are therefore not responsible for the contents of this manuscript. For this reason, the submitting author has chosen to withdraw this preprint as a precautionary measure and would like to state that this work should not be cited as a reference. For any related questions that may arise, please contact the corresponding author.Competing Interest StatementThe authors have declared no competing interest.

immunology↗

bioRxiv2020

A simple and effective F0 knockout method for rapid screening of behaviour and other complex phenotypes

Hundreds of human genes are associated with neurological diseases, but translation into tractable biological mechanisms is lagging. Larval zebrafish are an attractive model to investigate genetic contributions to neurological diseases. However, current CRISPR-Cas9 methods are difficult to apply to large genetic screens studying behavioural phenotypes. To facilitate rapid genetic screening, we developed a simple sequencing-free tool to validate gRNAs and a highly effective CRISPR-Cas9 method capable of converting >90% of injected embryos directly into F0 biallelic knockouts. We demonstrate that F0 knockouts reliably recapitulate complex mutant phenotypes, such as altered molecular rhythms of the circadian clock, escape responses to irritants, and multi-parameter day-night locomotor behaviours. The technique is sufficiently robust to knockout multiple genes in the same animal, for example to create the transparent triple knockout crystal fish for imaging. Our F0 knockout method cuts the experimental time from gene to behavioural phenotype in zebrafish from months to one week.

genetics↗

bioRxiv2020

Instantaneous amplitude and shape of postrhinal theta oscillations differentially encode running speed

Hippocampal theta oscillations have a temporally asymmetric waveform shape, but it is not known if this theta asymmetry extends to all other cortical regions involved in spatial navigation and memory. Here, using both established and improved cycle-by-cycle analysis methods, we show that theta waveforms in the postrhinal cortex are also temporally asymmetric. On average, the falling phase of postrhinal theta cycles lasts longer than the subsequent rising phase. There are, however, rapid changes in both the instantaneous amplitude and instantaneous temporal asymmetry of postrhinal theta cycles. These rapid changes in amplitude and asymmetry are very poorly correlated, indicative of a mechanistic disconnect between these theta cycle features. We show that the instantaneous amplitude and asymmetry of postrhinal theta cycles differentially encode running speed. Although theta amplitude continues to increase at the fastest running speeds, temporal asymmetry of the theta waveform shape plateaus after medium speeds. Our results suggest that the amplitude and waveform shape of individual postrhinal theta cycles may be governed by partially independent mechanisms and emphasize the importance of employing a single cycle approach to understanding the genesis and behavioral correlates of cortical theta rhythms.

neuroscience↗

bioRxiv2020

CD13 is a Critical Regulator of Cell-cell Fusion in Osteoclastogenesis

In vertebrates, bone formation is dynamically controlled by the activity of two specialized cell types: the bone-generating osteoblasts and bone-degrading osteoclasts. Osteoblasts produce the soluble receptor activator of NFκB ligand (RANKL) that binds to its receptor RANK on the surface of osteoclast precursor cells to promote osteoclastogenesis, a process that involves cell-cell fusion and assembly of molecular machinery to ultimately degrade the bone. CD13 is a transmembrane aminopeptidase that is highly expressed in cells of myeloid lineage has been shown to regulate dynamin-dependent receptor endocytosis and recycling and is a necessary component of actin cytoskeletal organization. In the present study, we show that CD13-deficient mice display a normal distribution of osteoclast progenitor populations in the bone marrow, but present a low bone density phenotype. Further, the endosteal bone formation rate is similar between genotypes, indicating a defect in osteoclast-specific function in vivo. Loss of CD13 led to exaggerated in vitro osteoclastogenesis as indicated by significantly enhanced fusion of bone marrow-derived multinucleated osteoclasts in the presence of M-CSF and RANKL, resulting in abnormally large cells with remarkably high numbers of nuclei with a concomitant increase in bone resorption activity. Similarly, we also observed increased formation of multinucleated giant cells (MGC) in CD13KO bone marrow progenitor cells stimulated with IL-4 and IL-13, suggesting that CD13 may regulate cell-cell fusion events via a common pathway, independent of RANKL signaling. Mechanistically, while expression levels of the fusion-regulatory proteins dynamin and DC-STAMP are normally downregulated as fusion progresses in fusion-competent mononucleated progenitor cells, in the absence of CD13 they are uniformly sustained at high levels, even in mature multi-nucleated osteoclasts. Taken together, we conclude that CD13 may regulate cell-cell fusion by controlling expression and localization of key fusion proteins that are critical for both osteoclast and MGC fusion.Competing Interest StatementThe authors have declared no competing interest.View Full Text

immunology↗