Biology subjects

Clarke, J. J.

Publications and source records attributed to Clarke, J. J..

3 recordsLinked to original sources

bioRxiv2025

T-cell signaling relies on partial CD45-exclusion at sub-micron sized cellular contacts

How cell contact initiates T-cell activation is uncertain. The local exclusion of the receptor-type protein tyrosine phosphatase CD45 at cell contacts is believed to trigger immune receptor signaling but this is yet to be observed for T cells interacting with authentic cellular targets. Here, quantitative imaging of T cells interacting with tumor cells presenting either native or clinically relevant bi-specific TCR ligands, revealed that they form multiple sub-micron sized close contacts with their targets. The contacts were stabilised by the adhesion protein CD2, but efficient ligand detection required both CD2 and integrin ligation. CD45 was excluded from close contacts at the time of ZAP70 recruitment and signaling, but only partially (30- 40%). A single-cell, mass cytometric analysis showed that this change in kinase/phosphatase activity provoked strong T-cell activation and potent cytotoxicity via very small changes in signaling fluxes. Spatial stochastic simulations suggested that the proximal T-cell signaling network is optimised for efficient antigen discrimination in the setting of partial CD45 exclusion. Our work re-frames early T-cell activation as a process initiated by relatively subtle changes in kinase/phosphatase activity acting on small numbers of signaling effectors at minute cellular contacts.

immunology↗

bioRxiv2025

AlphaFold 3-enabled in silico exploration of PGAM1 interactions in cancer

Cancer cell metabolism is commonly reprogrammed to favour glycolysis over oxidative phosphorylation, even under aerobic conditions, a phenomenon known as the Warburg effect. A key enzyme implicated in this shift is phosphoglycerate mutase 1 (PGAM1), which catalyses the conversion of 3-phosphoglycerate to 2-phosphoglycerate. The human enzyme is dependent on cofactor 2,3-bisphosphoglycerate which can phosphorylate and thereby activate the enzyme at histidine 11 (H11). A recently characterised moonlighting activity of pyruvate kinase M2 (PKM2), in its monomeric or dimeric forms, leads to phosphoenolpyruvate (PEP)-dependent phosphorylation of PGAM1 at the same position. Crucially, this phosphorylation is dependent on prior tyrosine 119 (Y119) phosphorylation of PGAM1 by Src kinase, itself activated by oncogenic growth factors. Using AlphaFold 3 (AF3), this study models the conformational changes induced by PGAM1 Y119 phosphorylation and investigates the molecular basis for its role in facilitating PEP-dependent phosphorylation at H11. Structural comparisons suggest that Y119 phosphorylation induces rearrangement of the C-terminal tail of PGAM1, opening the catalytic site around H11 to enable binding of phosphoenolpyruvate (PEP). Use of molecular docking (Webina, SwissDock, and DiffDock) found that AF3 generated models of PGAM1 with Y119 phosphorylation showed enhanced binding of PEP in comparison to non-phosphorylated PGAM1. However, extensive protein-protein docking (ClusPro, AF3 multimer generation) failed to identify configurations of PGAM1 and PKM2 where catalytic sites were proximal. Overall, this study supports a model in which phosphorylation of PGAM1 at Y119 enables access to the active site for PEP, thus enhancing its enzymatic activation. These findings underscore the critical role of post-translational modifications in modulating protein function and exemplify the utility of AF3 in predicting PTM-induced structural changes relevant to cancer metabolism.

bioinformatics↗

bioRxiv2024

Remote force modulation of the T-cell receptor reveals an NFAT-threshold for CD4+ T cell activation

Abstract/SummaryMechano-modulation of cell surface proteins to influence cell activation has been shown as a promising new advanced therapy for regenerative medicine applications. These strategies rely on the manipulation of mechanosensitive cell surface receptors to initiate intracellular signal transduction. The cell surface receptor of T lymphocytes (TCR), which recognises peptide-MHC molecules central to driving the adaptive immune response, has recently been suggested to be mechano-responsive. Despite this advance, little is known as to whether the TCR can be mechanically modulated to achieve TCR signalling and subsequent T cell activation, and whether these characteristics can be exploited for immunotherapies. Here, we describe a magnetic particle-based platform for mechanical modulation of the TCR and outline how this platform can be utilised to achieve CD4+ T cell activation. We demonstrate that mechanical manipulation of the TCR induces cell surface clustering of the TCR and downstream TCR signalling, leading to eventual TCR downregulation and T cell activation. We investigate the temporal relationship between mechanical modulation of the TCR and subsequent T cell activation, hereby identifying that accumulation of signalling events within the NFAT-pathway is required to reach the threshold required for CD4+ T cell activation, outlining an axis which controls the CD4+ T cell response to external mechanical cues. These findings identify how CD4+ T cells can modulate their function in response to such cues, whilst also outlining a remote-magnetic particle-based platform that may be used for the control of T cell responses.

immunology↗