Biology subjects

Bhatt, M.

Publications and source records attributed to Bhatt, M..

7 recordsLinked to original sources

bioRxiv2024

Mechanistic Insights into Periplasmic Chaperones as Regulators of Protein Folding and Translocation in Gram-Negative Bacteria

In Gram-negative bacteria, periplasmic chaperones mediate the transport and folding of outer membrane proteins. While their substrate interaction mechanisms are well studied, their functional cycle during periplasmic translocation remains unclear. Using single-molecule force spectroscopy, we demonstrate that SecYEG-associated chaperones PpiD and DsbC act as foldases, delivering up to 45.5 zJ of mechanical work. This activity facilitates substrate extraction from the SecYEG translocation tunnel, reducing ATP consumption during SecA-mediated translocation. In contrast, chaperones such as Spy and Skp function as holdases, stabilizing unfolded substrates to prevent misfolding during translocation. Our findings reveal a unique mechanism by which chaperones adapt to mechanical constraints, optimizing protein folding and translocation in the periplasm.

biophysics↗

bioRxiv2024

Unravelling Antibody-Induced Mechanical Stability of Antigen: Insights from Single-Molecule Studies

The intricate nature of antigen-antibody interactions plays a pivotal role in immunological responses. Despite the multitude of ligand-binding sites on antigens, the influence of antibodies on their mechanical stability remains elusive. This study elucidates the impact of IgM, the largest antibody isotype, on the mechanical stability of protein L, a bacterial superantigen, using single-molecule magnetic tweezers and steered molecular dynamics. Our findings reveal a concentration-dependent elevation in mechanical stability induced by IgM, as demonstrated by prolonged unfolding dwell times. Through steered molecular dynamics simulations, we elucidate the distinct mechanical responses of protein L binding interfaces at various IgM complex states, highlighting their synergistic effect on IgM dimer complex stability. Notably, this enhanced response stems from the altered unfolding pathway of protein L upon IgM interaction, providing significant insights into the generic mechanisms governing antibody-induced mechanical stability of antigenic substrates in physiological conditions, shedding light on the underlying folding dynamics and molecular mechanics of antigen-antibody interaction.

biophysics↗

bioRxiv2024

Unveiling the crucial role of betaine: Modulation of GABA homeostasis via SLC6A1 transporter (GAT1)

Betaine is an endogenous osmolyte that exhibits therapeutic potential by mitigating various neurological disorders. However, the underlying cellular and molecular mechanisms responsible for its neuroprotective effects remain puzzling. In this study, we describe a possible mechanism behind the positive impact of betaine in preserving neurons from excitotoxicity. Using electrophysiology, mass spectroscopy, radiolabelled cellular assay, and molecular dynamics simulation we demonstrate that betaine at mM concentration acts as a slow substrate of GAT1 (slc6a1), the predominant GABA transporter in the central nervous system. Intriguingly, when betaine is present at low concentration (0.01-3 mM) with GABA (at concentration <K0.5), it blocks the GABA reuptake. This GAT1 modulation occurs through the temporal inhibition of the transporter, i.e., the prolonged occupancy by betaine impedes the rapid transition of the transporter to the inward conformation. The temporal inhibition results in a crucial regulatory mechanism contributing to the maintenance of GABA homeostasis, preserving neurons from excitotoxicity.

neuroscience↗

bioRxiv2024

Bioisosteric analogs of MDMA with improved pharmacological profile

3,4-Methylenedioxymethamphetamine (MDMA, ecstasy) is re-emerging in clinical settings as a candidate for the treatment of specific psychiatric disorders (e.g. post-traumatic stress disorder) in combination with psychotherapy. MDMA is a psychoactive drug, typically regarded as an empathogen or entactogen, which leads to transporter-mediated monoamine release. Despite its therapeutic potential, MDMA can induce dose-, individual-, and context-dependent untoward effects outside safe settings. In this study, we investigated whether three new methylenedioxy bioisosteres of MDMA improve its off-target profile. In vitro methods included radiotracer assays, transporter electrophysiology, bioluminescence resonance energy transfer and fluorescence-based assays, pooled human liver microsome/S9 fraction incubation with isozyme mapping, and liquid chromatography coupled to high-resolution mass spectrometry. In silico methods included molecular docking. Compared with MDMA, all three MDMA bioisosteres (ODMA, TDMA, and SeDMA) showed similar pharmacological activity at human serotonin and dopamine transporters (hSERT and hDAT, respectively) but decreased activity at 5-HT2A/2B/2C receptors. Regarding their hepatic metabolism, they differed from MDMA, with N-demethylation being the only metabolic route shared, and without forming phase II metabolites. Additional screening for their interaction with human organic cation transporters (hOCTs) and plasma membrane transporter (hPMAT) revealed a weaker interaction of the MDMA analogs with hOCT1, hOCT2, and hPMAT. Our findings suggest that these new MDMA analogs might constitute appealing therapeutic alternatives to MDMA, sparing the primary pharmacological activity at hSERT and hDAT, but displaying a reduced activity at 5-HT2A/2B/2C receptors and reduced hepatic metabolism. Whether these MDMA bioisosteres may pose lower risk alternatives to the clinically re-emerging MDMA warrants further studies.

pharmacology and toxicology↗

bioRxiv2023

Methotrexate-modulated talin-dynamics drives cellular mechanical phenotypes via YAP signalling

Methotrexate is a well-known antineoplastic drug used to prevent cancer aggravation. Despite being a targeted therapeutic approach, its administration comes with the risk of cancer recurrence, plausibly through its proven off-target effect on focal adhesions. Since FA dynamics is dependent on force transmission through its constituent proteins, including talin, methotrexate might affect the mechanical activity of these proteins. Here we have combined single-molecule studies, computational dynamics, cell-based assays, and genomic analysis to unveil the focal adhesion-regulating role of methotrexate central to its effect on talin dynamics and downstream pathways. Interestingly, our single-molecule force spectroscopic study shows that methotrexate modulates the bimodal force distribution of talin in a concentration-dependent manner. Steered molecular dynamics reveal that methotrexate-talin interactions alter talin mechanical stability exposing their vinculin binding sites. Finally, we found that methotrexate-regulated talin-dynamics remodel cancer cell mechanical phenotypes like cell polarity, adhesion, and migration by regulating talin-vinculin association-mediated YAP signaling. These results further correlate with genomic analysis of methotrexate-treated patients, demonstrating its clinical importance. Taken together, these findings disseminate the effects of methotrexate-modulated mechanosensitivity of adhesion proteins on cellular events.

cell biology↗

bioRxiv2022

Talin-drug interaction reveals a key molecular determinant for biphasic mechanical effect: studied under single-molecule resolution

Talin as an adhesion protein, exhibits a strong force-dependent structure-function dynamics. Being a mechanosensitive focal adhesion (FA) protein, talin might interact to several FA targeting drugs; however, the molecular mechanism of talin-drug interactions remains elusive. Here we combined magnetic tweezers and molecular dynamics (MD) simulation to explore mechanical stability of talin with three drugs based on their talin specificity. Interestingly, our study revealed that talin displays a bimodal force distribution with a low and high unfolding force population. We observed that talin nonspecific drugs (tamoxifen and letrozole) display biphasic effect: increase talin mechanical stability upto optimum concentration, followed by a decrease in stability with further concentration increase. By contrast, talin-specific cyanidin 3-O-glucoside promotes a steady increase to talin mechanical stability with its concentration. We reconciled our observation from the simulation study: tamoxifen enters into talin hydrophobic core, eventually destabilizing the protein; whereas cyanidin 3-O-glucoside stabilizes the protein core by maintaining the inter-helix distance. Finally, we observed a strong correlation among hydrophobicity and cavity analysis, illustrating a detailed mechanistic analysis of drug effect on the mechanosensitive protein. Overall this study presents a novel perspective for drug designing against mechanosensitive proteins and studying off-target effects of already known drugs.

biophysics↗

bioRxiv2020

Unique mutational changes in SARS-CoV2 genome of different state of India

COVID-19 is a global pandemic causing more than 8 million deaths till mid-August, 2020. In India, more than 3 million confirmed cases have been reported although with relatively low death rate of 1.8%. In this study, we sequenced 47 genomes of SARS-CoV-2 from the patients of 13 districts of Uttar Pradesh (UP), the largest state of India using third-generation sequencing technique. The phylogenetic clustering revealed that no UP sample was aligned with the previously defined USA clade, where the mortality was high. We identified 56 distinct SNP variations in the genomes of UP resulting in a unique mutation rate of 1.19% per sequence, which is greater than the value 0.88% obtained for the rest of India. The relatively less death rate in UP indicates that the mutation in the virus is deleterious. Further investigation is required with larger sample size to determine the degree of virulence vis-a-vis SNP variation.

bioinformatics↗