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Patra, S. K.

Publications and source records attributed to Patra, S. K..

8 recordsLinked to original sources
bioRxiv2025

AKNA drives neural stem cell fate transition through differential localization and coordinating the modulation of chromatin from H3K27me3 to H3K27ac

Neuronal stem cells (NSCs) play pivotal role in adult neurogenesis, however, detail mechanisms of obtaining pluripotency or undergoing differentiation remain unknown. Herein, how AT-hook protein AKNA regulates pluripotency and stemness in neuroblastoma cells is demonstrated by gene knockdown, immunofluorescence, chromatin immunoprecipitation (ChIP), localization of AKNA, signaling interactions and transcriptional activity. AKNA was abundant and mostly nuclear during induction of pluripotency and its knockdown reduced stemness, even in presence of other pluripotency factors, including OCT4 and SOX2. During the course of induction of differentiation, AKNA remain localized in the cytosol, essential for regulated differentiation. Cytosolic retention of AKNA is possibly driven by FAK signaling. In the nucleus, AKNA promotes KDM6B demethylase for H3K27me3 demethylation to H3K27, and subsequently promotes CBP/p300 to acetylate H3K27 to H3K27ac deposition on promoter region of respective genes to trigger their transcription. Data obtained from knockdown and overexpression of AKNA and KDM6B further reinforce its importance that, they physically interact to drive pluripotency and stemness. These findings establish AKNA as a critical regulator for NSCs fate determination in association with epigenetic modifiers and signaling pathway, offering potential targets for neuroblastoma therapies and regenerative medicines for neurodegenerative diseases.

molecular biology↗
bioRxiv2024

Screening and identification of gene expression in large cohorts of clinical lung cancer samples unveils the major involvement of EZH2 and SOX2

Lung adenocarcinoma (LUAD), the primary subtype of Non-Small Cell Lung Cancer (NSCLC), accounts for 80% to 85% of cases. Due to suboptimal screening method, LUAD is often detected in late stage, leading to aggressive progression and poor outcomes. Therefore, early disease prognosis for the LUAD is high priority. In order to identify early detection biomarkers, we conducted a meta-analysis of mRNA expression TCGA and GTEx datasets from LUAD patients. A total of 795 differentially expressed genes (DEGs) were identified by exploring the Network-Analyst tool and utilizing combined effect size methods. DEGs refer to genes whose expression levels are significantly different (either higher or lower) compared to their normal baseline expression levels. KEGG pathway enrichment analysis highlighted the TNF signaling pathway as being prominently associated with these DEGs. Subsequently, using the MCODE and CytoHubba plugins in Cytoscape software, we filtered out the top 10 genes. Among these, SOX2 was the only gene exhibiting higher expression, while the others were downregulated. Consequently, our subsequent research focused on SOX2. Further transcription factor-gene network analysis revealed that enhancer of zeste homolog 2 (EZH2) is a significant partner of SOX2, potentially playing a crucial role in euchromatin-heterochromatin dynamics. Structure of SOX2 protein suggest that it is a non-druggable transcription factor, literature survey suggests the same; hence, we drove our focus to investigate on potential drug(s) targeting EZH2. Molecular docking analyses predicted most probable inhibitors of EZH2. We employed several predictive analysis tools and identified GSK343, as a promising inhibitor of EZH2.

genetics↗
bioRxiv2024

Modulation of H3K4 trimethylation by KDM5A and MLLs impacts metabolic adaptability in prostate and cervical cancer cells

AbstractChemical modifications of chromatin modulate gene expression and induce essential metabolic plasticity for tumor growth. Accumulation of H3K4me3 in the promoter of a gene activates transcription by making the promoter accessible to the polymerases. Methylation of H3K4 is catalysed by MLLs and demethylation of H3K4me3 is catalysed by KDM5 family proteins. Herein, we investigated if genes encoding the enzymes involved in glucose metabolism are dependent on KDM5A and MLL1, and if targeting the H3K4me3 would help in modulating the resilience of cancer cells. We present that KDM5A modulates most of the metabolic genes in a demethylase dependent manner as assesses by H3K4me3 occupancy on G6PD and catalase promoters. Targeting its expression would indeed help in sensitizing cancer cells to ROS dependent apoptotic cell death. We elucidated the differences in the epigenetic regulation in cancerous cells originated from cervical and prostate tissues and used a normal skin keratinocyte for comparison. In cervical and prostate cancers - KDM5A activated glycolysis but downregulates other metabolic processes. In cervical cancer, which majorly depends on PPP, changes in KDM5A did not modulate the G6PD expression. Further, we have shown that curcumin treatment enhanced KDM5A expression and downregulated MLL2 in cancer cell lines but not in keratinocyte cells. Curcumin inhibited metabolic pathways and enhanced apoptosis in cancer cells without affecting keratinocyte cells by modulating KDM5A and MLL levels. This work also strengthens the basic concept that, epigenetic modulations of genes in a tissue precisely depends on signal and sites of modification(s).

cancer biology↗
bioRxiv2024

Genomic database furnishes a spontaneous example of a functional Class II glycyl-tRNA synthetase urzyme

The chief barrier to studies of how genetic coding emerged is the lack of experimental models for ancestral aminoacyl-tRNA synthetases (AARS). We hypothesized that conserved core catalytic sites could represent such ancestors. That hypothesis enabled engineering functional "urzymes" from TrpRS, LeuRS, and HisRS. We describe here a fourth urzyme, GlyCA, detected in an open reading frame from the genomic record of the arctic fox, Vulpes lagopus. GlyCA is homologous to a bacterial heterotetrameric Class II GlyRS-B. Alphafold2 predicted that the N-terminal 81 amino acids would adopt a 3D structure nearly identical to the HisRS urzyme (HisCA1). We expressed and purified that N-terminal segment. Enzymatic characterization revealed a robust single-turnover burst size and a catalytic rate for ATP consumption well in excess of that previously published for HisCA1. Time-dependent aminoacylation of tRNAGly proceeds at a rate consistent with that observed for amino acid activation. In fact, GlyCA is actually 35 times more active in glycine activation by ATP than the full-length GlyRS-B -subunit dimer. ATP-dependent activation of the 20 canonical amino acids favors Class II amino acids that complement those favored by HisCA and LeuAC. These properties reinforce the notion that urzymes represent the requisite ancestral catalytic activities to implement a reduced genetic coding alphabet.

evolutionary biology↗
bioRxiv2023

A Zymography technique to study amino acid activation by aminoacyl-tRNA synthetases (aaRS): A broad spectrum, high- throughput tool to screen activities of aaRS and their Urzyme variants.

Amino acyl tRNA synthetases or aaRSs play a key role in assuring the precision of protein translation. They are highly specific for their cognate amino acid and cognate tRNA substrates during protein synthesis, utilizing ATP to ensure that proper assignments are made between amino acid and anticodon. Specific aaRS for each amino acid are present in all cells. We describe a new zymography technique to qualitatively visualize and semi-quantitatively determine the amino acid activation capacity of each type of aaRS molecule by indirect colorimetric detection of released pyrophosphates during the formation of aminoacyl-AMP. Protein samples containing aaRS are subjected to Native PAGE, followed by incubation in buffer containing cognate amino acid and ATP for sufficient time to generate pyrophosphates (PPi) which are then converted to inorganic phosphates by pyrophosphatase treatment. Finally, the generated and localized phosphates around the aaRS protein inside the gel can be visualized after staining by ammonium molybdate and malachite green solution. This technique has been validated by inspecting the substrate specificities of specific aaRSs. This zymography technique is sufficiently sensitive to detect and authenticate activities of much (i.e., ~10-5-fold) less active aaRS "Urzymes", to study alteration of activities of aaRS by various intrinsic or extrinsic factors and to screen aaRS-specific antimicrobial drugs.

biochemistry↗
bioRxiv2021

Physical association of functionally antagonistic enzymes: KDM5A interacts with MLLs to regulate gene expression in a promoter specific manner facilitating EMT and pluripotency.

Differential expression of genes involved in physiological processes are a collaborative outcome of interactions among signalling molecules, downstream effectors and epigenetic modifiers, which together dictate the regulation of genes in response to specific stimuli. MLLs and KDM5A are functionally antagonistic proteins as one acts as writer and the other as eraser of the active chromatin mark, i.e., H3K4me3. KDM5A promotes EMT by occupying promoters of both epithelial and mesenchymal markers. Through this work, it is illustrated that when bound to E-cadherin promoter, KDM5A acts as a classical repressor by demethylating H3K4me3, but on mesenchymal marker promoters, it acts as a transcriptional activator by inhibiting the activity of HDACs and increasing H3K18ac. Further it is demonstrated that KDM5A occupancy enhances either MLL1 or MLL2 by physically interacting with them and that signalling pathways regulate the enzymatic activity of KDM5A probably by phosphorylation. When not active, KDM5A signals for MLL occupancy, a mechanism that can be called epigenetic signalling.

molecular biology↗
bioRxiv2021

In-vivo protein nitration and de-nitration facilitate Vibrio cholerae cell survival under anaerobic condition: Consequences of Nitrite induced protein nitration

Protein tyrosine nitration (PTN), a highly selective post translational modification, occurs in both prokaryotic and eukaryotic cells under nitrosative stress1. It is reported that the activities of many proteins are altered due to PTN2. PTN is found to be associated with many pathophysiological conditions like neurodegenerative and cardiac diseases etc.3. However, its physiological function is not yet clear. Like all other gut pathogens Vibrio cholerae also faces nitrosative stress in the gut environment which makes its proteome more vulnerable to PTN. Here, we report for the first time in-vivo PTN in V. cholerae. We show that in-vivo protein nitration is nitrite dependent and nitration-denitration phenomenon actually facilitates V. cholerae cell survival in anaerobic or hypoxic condition. In our study, we found that the extent of in-vivo nitration is negatively correlated with the intracellular nitrite content and maximum nitration occurs during log phase of V. cholerae. Most interestingly, a significant denitration was associated with increase in intracellular nitrate content during anaerobic incubation of aerobically grown late log phase cultures. In-vivo nitration could provide an avenue for toxic nitrite storage and nitrosative stress tolerance mechanism in many gut pathogens, whereas denitration could supply nitrate for cell survival in anaerobic nitrate deficient environment.

microbiology↗