SenGupta, S. | bioRxiv Science

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↗

bioRxiv2021

Identification of G Protein αi Signaling Partners by Proximity Labeling Reveals a Network of Interactions that Includes PDZ-RhoGEF

G protein-coupled receptors (GPCRs) that couple to the Gi family of G proteins are key regulators of cell and tissue physiology. Our recent work has discovered novel roles for Gi in migration of neutrophils and fibrosarcoma cells downstream of activated chemoattractant receptors, but the molecular target(s) of Gi in these processes remain to be identified. We adopted an intact cell proximity-based labeling approach using BioID2 coupled to tandem mass tag (TMT)-based quantitative proteomics to identify proteins that selectively interact with the GTP-bound form of Gi1. Multiple targets were identified and validated for selective biotinylation by active BioID2-Gi1(Q204L), suggesting a previously unappreciated network of interactions for activated Gi proteins in intact cells. Extensive characterization of one candidate protein, PDZ-RhoGEF (PRG), revealed that active-Gi1 strongly activates PRG. Strikingly, large differences in the ability of Gi1, Gi2, and Gi3 isoforms to activate PRG were observed despite over 85% sequence identity. We also demonstrate the functional relevance of the interaction between active Gi and PRG ex vivo in primary human neutrophils. Identification and characterization of new targets regulated by Gi both individually and in networks provide insights that will aid not only in investigation of diverse functional roles of Gi-coupled GPCRs in biology but also in the development of novel therapeutic approaches. SummaryProximity-based labeling approach was used to identify signaling networks and signaling mechanisms downstream of Gi-coupled receptors.

pharmacology and toxicology↗