Biology subjects

Gangopadhyay, K.

Publications and source records attributed to Gangopadhyay, K..

5 recordsLinked to original sources

bioRxiv2025

Molecular Basis of Ionic Suppression of ZAP-70 Dependent T Cell Receptor Activation

Ionic imbalance in the tumor microenvironment alters the tumor-infiltrating T lymphocyte function. High extracellular K+ suppresses T cell function by negatively regulating T cell receptor (TCR) signaling. In contrast, elevated extracellular Na+ enhances T cell effector function by boosting the phosphorylation of TCR signaling modules. Here, we presented a mechanism explaining how the two monovalent cations differently regulate TCR function. At rest, high intracellular K+ uncouples allosteric recruitment of ZAP-70, a key signaling module, to the TCR complex. The formation of antigen TCR complex induces K+ efflux, causing spontaneous recruitment of ZAP-70 to the TCR. Increasing extracellular K+ perturbs K+ efflux and slows ZAP-70 recruitment to the TCR complex, even upon antigen binding. This leads to defects in T cell development and arthritis-like symptoms in juvenile mice. We conclude that K+ dynamics is integral to T cell ligand discrimination and fundamental to turning off the signaling during T cell quiescence.

immunology↗

bioRxiv2024

E41K Mutation Activates Brutons Tyrosine Kinase by Stabilizing an Inositol Hexakisphosphate Dependent Invisible Dimer

Brutons tyrosine kinase (BTK) regulates diverse cellular signaling of the innate and adaptive immune system in response to microbial pathogens. Downregulation or constitutive activation of BTK is reported in patients with autoimmune diseases or various B-cell leukemias. BTK is a multidomain protein tyrosine kinase that adopts an Src-like autoinhibited conformation maintained by the interaction between the kinase and PH-TH domains. The PH-TH domain plays a central role in regulating BTK function. The BTK is activated by binding to PIP3 at the plasma membrane upon stimulation by the B-cell receptor (BCR). The PIP3 binding allows dimerization of the PH-TH domain and subsequent transphosphorylation of the activation loop. Alternatively, a recent study shows that the multivalent T-cell-independent (TI) antigen induces BCR response by activating BTK independently of PIP3 binding. It was proposed that a transiently stable IP6-dependent PH-TH dimer may activate BTK during BCR activation by the TI antigens. However, no IP6-dependent PH-TH dimer has been identified yet. Here, we investigated a constitutively active PH-TH mutant (E41K) to determine if the elusive IP6-dependent PH-TH dimer exists. We showed that the constitutively active E41K mutation activates BTK by stabilizing the IP6-dependent PH-TH dimer. We observed that a downregulating mutation in the PH-TH domain (R28H) linked to X-linked agammaglobulinemia impairs BTK activation at the membrane and in the cytosol by preventing PH-TH dimerization. We conclude that the IP6 dynamically remodels the BTK active fraction between the membrane and cytoplasm. Stimulating with IP6 increases the cytosolic fraction of the activated BTK.

biochemistry↗

bioRxiv2023

In Silico Therapeutic Intervention on Cytokine Storm in COVID-19

The recent global COVID-19 outbreak, attributed by the World Health Organization to the rapid spread of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), underscores the need for an extensive exploration of virological intricacies, fundamental pathophysiology, and immune responses. This investigation is vital to unearth potential therapeutic avenues and preventive strategies. Our study delves into the intricate interaction between SARS-CoV-2 and the immune system, coupled with exploring therapeutic interventions to counteract dysfunctional immune responses like the cytokine storm (CS), a driver of disease progression. Understanding these immunological dimensions informs the design of precise multiepitopetargeted peptide vaccines using advanced immunoinformatics and equips us with tools to confront the cytokine storm. Employing a control theory-based approach, we scrutinize the perturbed behavior of key proteins associated with cytokine storm during COVID-19 infection. Our findings support ACE2 activation as a potential drug target for CS control and confirm AT1R inhibition as an alternative strategy. Leveraging deep learning, we identify potential drugs to individually target ACE2 and AT1R, with Lomefloxacin and Fostamatinib emerging as standout options due to their close interaction with ACE2. Their stability within the protein-drug complex suggests superior efficacy among many drugs from our deep-learning analysis. Moreover, there is a significant scope for optimization in fine-tuning protein-drug interactions. Strong binding alone may not be the sole determining factor for potential drugs; precise adjustments are essential. The application of advanced computational power offers novel solutions, circumventing time-consuming lab work. In scenarios necessitating both ACE2 and AT1R targeting, optimal drug combinations can be derived from our analysis of drug-drug interactions, as detailed in the manuscript.

bioinformatics↗

bioRxiv2021

An evolutionary divergent thermodynamic brake in ZAP-70 finetunes the kinetic proofreading in T cell

T cell signaling starts with assembling several tyrosine kinases and adaptor proteins to the T cell receptor (TCR), following the antigen binding. The lifetime of the TCR: antigen complex and the time delay between the recruitment and activation of each kinase determines the T cell response. The mechanism by which the time delays are implemented in TCR signaling is not fully understood. Combining experiments and kinetic modeling, we here report a thermodynamic-brake in the regulatory module of ZAP-70, which determines the ligand selectivity, and may delay the ZAP-70 activation in TCR. Phylogenetic analysis revealed that the evolution of the thermodynamic-brake coincides with the divergence of the adaptive immune system to the cell-mediated and humoral responses. Paralogous kinase Syk expressed in B cells, does not possess such a functional thermodynamic brake, which may explain higher basal activation and lack of ligand selectivity by Syk.

biochemistry↗

bioRxiv2019

An allosteric hot-spot in the tandem-SH2 domain of ZAP-70 regulates T-cell signaling

T-cell receptor (TCR) signaling is initiated by recruiting ZAP-70 to the cytosolic part of TCR. ZAP-70, a non-receptor tyrosine kinase, is composed of an N-terminal tandem SH2 (tSH2) domain connected to the C-terminal kinase domain. The ZAP-70 is recruited to the membrane through binding of tSH2 domain and the doubly-phosphorylated ITAM motifs of CD3 chains in the TCR complex. Our results show that the tSH2 domain undergoes a biphasic structural transition while binding to the doubly-phosphorylated ITAM-{zeta}1 peptide. The C-terminal SH2 domain binds first to the phosphotyrosine residue of ITAM peptide to form an encounter complex leading to subsequent binding of second phosphotyrosine residue to the N-SH2 domain. We decipher a network of non-covalent interactions that allosterically couple the two SH2 domains during binding to doubly-phosphorylated ITAMs. Mutation in the allosteric network residues, for example, W165C, uncouples the formation of encounter complex to the subsequent ITAM binding thus explaining the altered recruitment of ZAP-70 to the plasma membrane causing autoimmune arthritis in mice. The proposed mechanism of allosteric coupling is unique to ZAP-70, which is fundamentally different from Syk, a close homolog of ZAP-70 expressed in B-cells. SignificanceT-cell and B-cell signaling is initiated by the same family of non-receptor tyrosine kinases, ZAP-70 and Syk, respectively. ZAP-70 and Syk share homologous sequence and similar structural architecture, yet the two kinases differ in their mode of ligand recognition. ZAP-70 binds cooperatively to its ligand, whereas Syk binds uncooperatively. Spontaneous mutation (W165C) in the regulatory module of ZAP-70 impairs T-cell signaling causes autoimmune arthritis in SKG mice, the mechanism of which is unknown. We showed that ZAP-70 regulatory module undergoes a biphasic structural transition while binding to its ligand, which is fundamentally different from Syk. We presented a molecular mechanism of cooperativity in ZAP-70 regulatory module that explains altered ligand binding by ZAP-70 mutant found in SKG mice.

biochemistry↗