Biology subjects

Choudhuri, A.

Publications and source records attributed to Choudhuri, A..

7 recordsLinked to original sources

bioRxiv2026

Reverse DTNB assay: a novel in vitrobiochemical approach to detect oxidized thiol modifications

The participation of sulfhydryl or thiol functions in a multitude of protein posttranslational modifications, although reflects on the redox versatility of cysteine residues, but their assessment in a dynamic cellular milieu involving the facile inter-conversion of SH to SSG, S-S, SNO, and S-R has been overwhelmingly difficult despite theirimplications in protein folding, enzyme structure and function, signalling and detoxification pathways, and pathophysiological ramifications.The current methodology, in contrast to a wide variety of cumbersome and prolonged techniques,repurposes the conventional DTNB assay for a hassle-free qualitative and quantitative analysisof redox-modified single or multiple susceptible thiol residues of cysteines in pure proteins as well as in a complex mixture of proteins.In this study, we document the thiol content, bearing the susceptibility to undergo reversible, oxidative thiol modifications, utilizing reverse DTNB assay in cell-free lysates and purified proteins that might provide a possible framework for dissecting the physiological phenomena behind the concealment of the susceptible cysteines through their redox-modified forms.

biochemistry↗

bioRxiv2026

Assessing the physiological S nitrosoproteome reveals nitric oxide mediated regulatory networks in rice

Nitric oxide metabolism-based protein post-translational modifications, such as reversible S- nitrosylation, have been at the pinnacle of plant redox research owing to their significant correlation with seed dormancy, interaction with other signaling molecules, plant development and metabolism, biotic and abiotic plant stress responses, immune defense responses against plant pathogens, and senescence. The rapid interconversion of reactive nitrogen species, the abrogation of nitric oxide homeostasis by exogenous supplementation of NO donors and scavengers, the lack of spatio-temporal specificity of NO signaling, and the limited bioavailability or assay sensitivity for detection often limit the effectiveness of identifying and characterizing S-nitrosothiols in plants. Hitherto unknown, we report the first experimental evidence of the total in vivo S-nitrosoproteome in Oryza sativa L. subsp. indica, comprising 134 PSNOs, enriched with 169 putative sites susceptible to S-nitrosylation, without any exogenous supplementation of NO donors. In the present study, mercuric salt- driven facile decomposition of S-nitrosoproteins in the presence of nitrone spin trap 5,5- dimethyl-1-pyrroline N-oxide, resulting in the synthesis of DMPO-nitrone adducts with PSNO-derived protein thiyl radicals in O. sativa, has been demonstrated as an efficient and novel strategy for characterizing the PSNOs using mass spectrometry analysis. The evidence of physiological levels of PSNOs was further re-examined in a bi-directional qualitative and quantitative approach involving the 2,3-diaminonaphthalene assay in tandem with fluorescence-based visualization and fluorometric quantification. In silico analyses, involving both functional enrichment and pathway prediction analyses, have furthermore revealed unique protein-protein interaction networks and signaling pathways among the S- nitrosoproteome candidates and their predictable physiological roles in O. sativa indica, awaiting further in vitro validation for their functional correlation in response to S- nitrosylation. In conclusion, the present study provides novel evidence of nitric oxide signaling in rice cultivars under physiological conditions, bringing new insights into the potential in vivo transnitrosylation of regulatory or active-site cysteine thiols.

biochemistry↗

bioRxiv2024

A PRDM16-CtBP1/2 Complex Interacts with HDAC1/2 to Regulate Transcriptional Programs of Neurogenesis and Guide Cortical Neuron Migration

Chromatin regulation of transcriptional enhancers plays a central role in cell fate specification and differentiation. However, how the coordinated activity of transcription factors and chromatin-modifying enzymes regulates enhancers in neural stem cells (NSCs) and dictates subsequent stages of neuronal differentiation and migration is not well understood. The histone methyltransferase PRDM16 is expressed in NSCs of the developing mouse and human cerebral cortex and is essential for determining the position of upper-layer cortical neurons. Here, we report that PRDM16 interacts with C-terminal binding protein 1 (CtBP1) and CtBP2 to control the transcriptional programs of cortical neurogenesis and regulate upper-layer neuron migration. PRDM16 and CtBP1/2 co-regulate enhancers by interacting with histone deacetylase 1 (HDAC1) and HDAC2, and lysine-specific demethylase 1 (LSD1). In addition, our results suggest that the CCCTC-binding factor CTCF plays a key role in recruiting CtBP1/2 to cortical enhancers. These findings underscore that reduced interactions between PRDM16 and ubiquitous chromatin regulators may contribute to neurodevelopmental deficits in patients with PRDM16 haploinsufficiency.

developmental biology↗

bioRxiv2024

RNA methylation controls stress granule formation and function during erythropoiesis

Stress granules (SGs) are crucial in RNA regulation, affecting cell fate and function. SGs contain RNAs, some of which can be methylated. We studied m6A RNA modifications during the human CD34+ HSPCs (hCD34+) differentiating into erythroid cells and found that mRNAs encoding many erythroid-specific proteins had decreased methylation during differentiation. Increased levels ALKBH5 demethylase during erythropoiesis controls the levels of the 3UTR methylation of these mRNAs. hCD34+ carrying ALKBH5 mutations demonstrated a block in erythropoiesis, and mass-spectrometry studies of the mutant cells showed decreased levels of SG proteins, including the core granule protein ATXN2. ALKBH5 directly regulates the methylation of the mRNA of ATXN2. ATXN2 overexpression accelerated the erythroid differentiation of HSPCs and rescued the erythroid differentiation of ALKBH5 mutant cells. Very few SGs are found in normal human erythroid progenitors. SGs accumulated substantially in ALKBH5 mutant cells, and surprisingly overexpression of ATNX2 reduced SG numbers to normal. Polysome analysis demonstrated m6A-modified RNAs to be enriched in the pre-polysome fractions that were less translated. This work establishes a mechanism by which during stress, ATXN2 facilitates the release of SG-stored m6A-modified RNAs including erythroid-specific and SG-enriched RNAs that are loaded onto functional ribosomes, allowing better translation and accelerated erythroid differentiation during stress.

cell biology↗

bioRxiv2024

Hematopoietic stem cell division is governed by distinct RUNX1 binding partners.

A defined number of hematopoietic stem cell (HSC) clones are born during development and expand to form the pool of adult stem cells. An intricate balance between self-renewal and differentiation of these HSCs supports hematopoiesis for life. HSC fate is determined by complex transcription factor networks that drive cell-type specific gene programs. The transcription factor RUNX1 is required for definitive hematopoiesis, and mutations in Runx1 have been shown to reduce clonal diversity. The RUNX1 cofactor, CBFy, stabilizes RUNX1 binding to DNA, and disruption of their interaction alters downstream gene expression. Chemical screening for modulators of Runx1 and HSC expansion in zebrafish led us to identify a new mechanism for the RUNX1 inhibitor, Ro5-3335. We found that Ro5-3335 increased HSC divisions in zebrafish, and animals transplanted with Ro5-3335 treated cells had enhanced chimerism compared to untreated cells. Using human CD34+ cells, we show that Ro5-3335 remodels the RUNX1 transcription complex by binding to ELF1, independent of CBFy. This allows specific expression of cell cycle and hematopoietic genes that enhance HSC self-renewal and prevent differentiation. Furthermore, we provide the first evidence to show that it is possible to pharmacologically increase the number of stem cell clones in vivo, revealing a previously unknown mechanism for enhancing clonal diversity. Our studies have revealed a mechanism by which binding partners of RUNX1 determine cell fate, with ELF transcription factors guiding cell division. This information could lead to treatments that enhance clonal diversity for blood diseases.

cell biology↗

bioRxiv2024

LoCS-Net: Localizing Convolutional Spiking Neural Network for Fast Visual Place Recognition

Visual place recognition (VPR) is the ability to recognize locations in a physical environment based only on visual inputs. It is a challenging task due to perceptual aliasing, viewpoint and appearance variations and complexity of dynamic scenes. Despite promising demonstrations, many state-of-the-art VPR approaches based on artificial neural networks (ANNs) suffer from computational inefficiency. Spiking neural networks (SNNs), on the other hand, implemented on neuromorphic hardware, are reported to have remarkable potential towards more efficient solutions computationally, compared to ANNs. However, the training of the state-of-the-art (SOTA) SNNs for the VPR task is often intractable on large and diverse datasets. To address this, we develop an end-to-end convolutional SNN model for VPR, that leverages back-propagation for tractable training. Rate-based approximations of leaky integrate-and-fire (LIF) neurons are employed during training to enable back-propagation, and the approximation units are replaced with spiking LIF neurons during inference. The proposed method outperforms the SOTA ANNs and SNNs by achieving 78.2% precision at 100% recall on the challenging Nordland dataset, compared with 53% SOTA performance, and exhibits competitive performance on the Oxford RobotCar dataset while being easier to train and faster in both training and inference when compared to other ANN and SNN-based methods.

neuroscience↗

bioRxiv2022

Stimulation-responsive enhancers regulate inflammatory gene activation through retention and modification of H2A.Z-variant accessible nucleosomes

Prostaglandin E2 (PGE2) and 16,16-dimethyl-PGE2 (dmPGE2) are important regulators of hematopoietic stem and progenitor cell (HSPC) fate and offer potential to enhance stem cell therapies1,2. The mechanism of gene regulation in response to dmPGE2 is poorly understood. Here, we show that dmPGE2 regulates inflammatory gene induction by modulating the chromatin architecture and activity of enhancer elements in human HSPCs. We identified the specific genomic reorganization at stimuli-responsive enhancers that permits rapid transcriptional activation. We found that dmPGE2-inducible enhancers retain MNase-accessible, H2A.Z-variant nucleosomes that are permissive to binding of the transcription factor CREB. CREB binding to enhancer nucleosomes is concomitant with deposition of the histone acetyltransferases p300 and Tip60 on chromatin. Subsequent H2A.Z acetylation improves chromatin accessibility at stimuli-responsive enhancers. Our findings support a model where histone variant nucleosomes retained within inducible enhancers facilitate transcription factor (TF) binding. Acetylation of histone variant nucleosomes by TF-associated nucleosome remodelers creates the accessible nucleosome landscape required for immediate enhancer activation and gene induction. Our work provides a mechanism by which inflammatory mediators such as dmPGE2 lead to acute transcriptional changes and alter HSPC behavior.

developmental biology↗