Biology subjects

Mukhopadhyay, R.

Publications and source records attributed to Mukhopadhyay, R..

7 recordsLinked to original sources

bioRxiv2025

Viral Diversity Influences T Cell Responses to Enteric Human Adenoviruses F40 and F41

BackgroundHuman enteric species F adenoviruses are a leading cause of diarrhoea-associated paediatric morbidity and mortality worldwide. The cellular immune response (antigen-specific cytotoxic T cells and secreted cytokines) to human adenovirus (HAdV) infection is known to ameliorate symptoms and is critical for viral clearance. We hypothesised that the capsid proteins (hexon and penton) of HAdV-F40 and 41 (F40, F41) are evolving to escape cellular immune responses. Major histocompatibility complex (MHC) binding of viral peptides is a key step in the presentation of peptide-MHC complexes which activate the T cell receptor and the cytotoxic T cell response. MethodsUsing global HAdV genomic data, we predicted MHC-peptide binding within the hexon and penton proteins of F40 and F41. We focused on MHC class I alleles common in the UK and Kenya and identifying predicted CD8+ T cell epitopes. Eight predicted epitope pairs from the F41 hexon were synthesised as 15mer peptides, comparing the wildtype (1970 F41 reference) to the variant (2019-2022) sequences. Cellular IFN{gamma} responses to these epitopes were measured in healthy donors using FluoroSpot assays. ResultsWe identified multiple predicted CD8+ T cell epitopes shared between HAdV-species C and F, but also unique to species F, and epitopes unique to each genotype. We show that IFN{gamma} and IL2 peripheral blood mononuclear cell (PBMC) responses to HAdV-F are ubiquitous among healthy adult donors from Cambridge, UK. Among predicted CD8+ epitopes within the F41 hexon, 11/16 peptides elicited donor positive IFN{gamma} responses from healthy donor PBMC (at least one epitope from seven out of eight peptide pairs). ConclusionsThe hexon and penton proteins of HAdV-F-40 and F41 are predicted to contain a number of genotype-specific, but conserved, CD8+ T cell epitopes which could be used to inform future vaccine design. Using the hexon of F41 as a case study, we show that predicted T cell epitopes in emergent strains are able to elicit an inflammatory cytokine response from healthy donor PBMC. The role of T cell recognition in driving enteric adenovirus evolution deserves further consideration.

microbiology↗

bioRxiv2025

Employing neutron-encoded monoUbs to study E2/E3 ligase activity and selectivity for assembling Ub chains

While protein ubiquitination is an extensively studied post-translational modification, many aspects of this process remain unclear. Ubiquitin conjugation involves the action of three different types of enzymes working in concert to install ubiquitin onto substrate proteins. Despite efforts, an in vitro mid/high-throughput screen to quickly determine which enzymes work together to build ubiquitin chains and directly analyze the type(s) of chains formed does not exist. In this study, we developed a new multiplexed mass spectrometry-based E1-E2-E3 assay that enables the analysis of whether E2/E3 pairs work together to form ubiquitin chains and concomitantly reports on the nature of the formed ubiquitin chain type(s). The assay employs synthetic modified neutron-encoded monoUb substrates with a distinct molecular weight, enabling the simultaneous analysis of these substrates. Overall, various E2-E3 pairs were screened for their ability to build Ub chains, which furnished a three-dimensional overview of linkage selectivity over time and enzyme concentration. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/655449v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@130203aorg.highwire.dtl.DTLVardef@93c9adorg.highwire.dtl.DTLVardef@9d9bedorg.highwire.dtl.DTLVardef@167ea0c_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

Identification of RNF114 as ADPr-Ub reader through non-hydrolysable ubiquitinated ADP-ribose

Crosstalk between the post-translational modification processes ubiquitination and ADP-ribosylation occurs in DNA-damage and immune-responses, in addition the physical linkage of ADP-ribose and ubiquitin is found during bacterial infection. Here, we study the ubiquitination of ADP-ribose mediated by human Deltex E3 ligases and the subsequent fate of the formed hybrid post-translational modification. We prepare a non-hydrolysable ADPr-Ub probe that we employ in a chemoproteomics approach and identify RNF114 as an interacting protein. Using biophysical and biochemical experiments, we validate that RNF114 preferentially interacts with ubiquitinated ADP-ribose over non-modified ubiquitin. Subsequently, RNF114 can elongate the ubiquitinated ADP-ribose with a K11-linked ubiquitin chain. Using domain deletion analysis, we pinpoint the tandem zinc fingers and ubiquitin interacting motif (ZnF2+ZnF3+UIM) domains of RNF114 to be crucial for recognising ubiquitinated ADP-ribose. Moreover, these domains are essential for the recruitment of RNF114 to the sites of laser-induced DNA damage.

molecular biology↗

bioRxiv2024

USP24 is an ISG15 cross-reactive deubiquitinasethat mediates IFN-I production by de-ISGylatingthe RNA helicase MOV10

The interferon-stimulated gene 15 (ISG15) is a ubiquitin-like modifier induced by type I Interferon (IFN-I) and plays a crucial role in the innate immune response against viral infections. ISG15 is conjugated to target proteins by an enzymatic cascade through a process called ISGylation. While ubiquitin-specific protease 18 (USP18) is a well-defined deISGylase counteracting ISG15 conjugation, ISG15 cross-reactive deubiquitylating enzymes (DUBs) have also been reported. Our study reports USP24 as a novel ISG15 cross-reactive DUB identified through activity-based protein profiling (ABPP). We demonstrate that recombinant USP24 processed pro-ISG15 and ISG15-linked synthetic substrates in vitro. Moreover, the depletion of USP24 significantly increased the accumulation of ISG15 conjugates upon IFN-{beta} stimulation. An extensive proteomic analysis of the USP24-dependent ISGylome, integrating total proteome, GG-peptidome, and ISG15 interactome data, identified the helicase Moloney leukemia virus 10 (MOV10) as a specific target of USP24 for deISGylation. Further validation in cells revealed that ISGylated MOV10 enhances IFN-{beta} production/secretion, whereas USP24 deISGylates MOV10 to negatively regulate the innate immune response. This study showcases USP24s novel roles in modulating ISGylation and modulation of the IFN-I-dependent immune responses, with potential therapeutic implications in infectious diseases, cancer, autoimmunity, and neuroinflammation.

biochemistry↗

bioRxiv2024

Paired transcriptomic analyses of atheromatous and control vessels reveal novel autophagy and immunoregulatory genes in peripheral artery disease

AbstractO_ST_ABSBackgroundC_ST_ABSPeripheral artery disease (PAD), a significant health burden worldwide, affects lower extremities due to atherosclerotic changes in the peripheral vessels. Although mechanisms of PAD have been studied, the molecular milieu of the plaques localized within peripheral arteries in patients are not well understood. Thus, to identify PAD lesion specific gene expression profiles precluding genetic, environmental and dietary biases, we studied the transcriptomic profile of plaque tissues normalized to non-plaque tissues from the same donors. MethodsTranscriptomic analysis of 9 paired samples of PAD patients from south Indian population was performed with institutional ethics approval and written consent. Plaque tissues were histologically confirmed. Expression of a select target gene set was done by qPCR from the same tissues. Bioinformatic and network analyses were performed using various statistical tools. ResultsA total of 296 upregulated, 274 downregulated genes and 186 non-coding RNAs were identified. STAG1, SPCC3, FOXQ1 and E2F3 were key downregulated genes and CD93 was top upregulated gene. Autophagosome assembly, cellular response to UV, cytoskeletal organization, TCR signaling and phosphatase activity were key dysregulated pathways identified in the study. Telomerase regulation and autophagy were identified as novel interacting pathways using network analysis. Plaque tissue was predominantly composed of immune cells and dedifferentiated cell populations indicated by cell specific marker imputed gene expression analysis. ConclusionThe current study identifies novel genes, non-coding RNAs, associated regulatory pathways and cell composition of the plaque tissue in PAD patients. The autophagy and immunoregulatory genes may drive novel mechanisms resulting in atheroma. These novel interacting networks and genes have the potential for PAD specific therapeutic applications.

molecular biology↗

bioRxiv2024

Development of covalent probes to capture Legionella pneumophila effector enzymes

Upon infection of host cells, Legionella pneumophila releases a multitude of effector enzymes into the cells cytoplasm that hijack a plethora of cellular activities, including the hosts ubiquitination pathways. Effectors belonging to the SidE-family are involved in non-canonical serine phosphoribosyl ubiquitination of host substrate proteins contributing to the formation of a Legionella-containing vacuole that is crucial in the onset of Legionnaires disease. This dynamic process is reversed by effectors called Dups that hydrolyse the phosphodiester in the phosphoribosyl ubiquitinated protein. We installed reactive warheads on chemically prepared ribosylated ubiquitin to generate a set of probes targeting these Legionella enzymes. In vitro tests on recombinant DupA revealed that a vinyl sulfonate warhead was most efficient in covalent complex formation. Mutagenesis and x-ray crystallography approaches were used to identify the site of covalent crosslinking to be an allosteric cysteine residue. The subsequent application of this probe highlights the potential to selectively enrich the Dup enzymes from Legionella-infected cell lysates.

cell biology↗

bioRxiv2017

Validation and Implementation of CLIA-Compliant Whole Genome Sequencing (WGS) in Public Health Laboratory

BackgroundPublic health microbiology laboratories (PHL) are at the cusp of unprecedented improvements in pathogen identification, antibiotic resistance detection, and outbreak investigation by using whole genome sequencing (WGS). However, considerable challenges remain due to the lack of common standards.\n\nObjectives1) Establish the performance specifications of WGS applications used in PHL to conform with CLIA (Clinical Laboratory Improvements Act) guidelines for laboratory developed tests (LDT), 2) Develop quality assurance (QA) and quality control (QC) measures, 3) Establish reporting language for end users with or without WGS expertise, 4) Create a validation set of microorganisms to be used for future validations of WGS platforms and multi-laboratory comparisons and, 5) Create modular templates for the validation of different sequencing platforms.\n\nMethodsMiSeq Sequencer and Illumina chemistry (Illumina, Inc.) were used to generate genomes for 34 bacterial isolates with genome sizes from 1.8 to 4.7 Mb and wide range of GC content (32.1%-66.1%). A customized CLCbio Genomics Workbench - shell script bioinformatics pipeline was used for the data analysis.\n\nResultsWe developed a validation panel comprising ten Enterobacteriaceae isolates, five gram-positive cocci, five gram-negative non-fermenting species, nine Mycobacterium tuberculosis, and five miscellaneous bacteria; the set represented typical workflow in the PHL. The accuracy of MiSeq platform for individual base calling was >99.9% with similar results shown for reproducibility/repeatability of genome-wide base calling. The accuracy of phylogenetic analysis was 100%. The specificity and sensitivity inferred from MLST and genotyping tests were 100%. A test report format was developed for the end users with and without WGS knowledge.\n\nConclusionWGS was validated for routine use in PHL according to CLIA guidelines for LDTs. The validation panel, sequencing analytics, and raw sequences will be available for future multi-laboratory comparisons of WGS in PHL. Additionally, the WGS performance specifications and modular validation template are likely to be adaptable for the validation of other platforms and reagents kits.

microbiology↗