Cyclophilin A associates with and regulates the activity of ZAP70 in TCR/CD3-stimulated T cells.

The ZAP70 protein tyrosine kinase (PTK) couples stimulated T cell antigen receptors (TCRs) to their downstream signal transduction pathways and is sine qua non for T cell activation and differentiation. TCR engagement leads to activation-induced post-translational modifications of ZAP70, predominantly by kinases, which modulate its conformation, leading to activation of its catalytic domain. Here, we demonstrate that ZAP70 in TCR/CD3-activated mouse spleen and thymus cells, as well as human Jurkat T cells, is regulated by the peptidyl-prolyl cis-trans isomerase (PPIase), cyclophilin A (CypA) and that this regulation is abrogated by cyclosporin A (CsA), a CypA inhibitor. We found that TCR crosslinking promoted a rapid and transient, Lck-dependent association of CypA with the interdomain B region, at the ZAP70 regulatory domain. CsA inhibited CypA binding to ZAP70 and prevented the colocalization of CypA and ZAP70 at the cell membrane. In addition, imaging analyses of antigen-specific T cells stimulated by MHC-restricted antigen-fed antigen-presenting cells revealed the recruitment of ZAP70-bound CypA to the immunological synapse. Enzymatically active CypA downregulated the catalytic activity of ZAP70 in vitro, an effect that was reversed by CsA in TCR/CD3-activated normal T cells but not in CypA-deficient T cells, and further confirmed in vivo by FRET-based studies. We suggest that CypA plays a role in determining the activity of ZAP70 in TCR-engaged T cells and impact on T cell activation by intervening with the activity of multiple downstream effector molecules.

Correlation of insulin resistance, beta cell function and insulin sensitivity with serum sFas and sFasL in newly diagnosed type 2 diabetes.

Pancreatic beta cell dysfunction and reduced insulin sensitivity are fundamental factors associated with glucotoxicity, lipotoxicity and oxidative stress in type 2 diabetic patients (T2DM). Diabetic milieu can induce apoptosis in several types of cells. The aim of present study was to compare circulating soluble apoptotic markers (sFas and sFas-L) with HOMA-IR, HOMA-%S, HOMA-%B in the serum of newly diagnosed T2DM and healthy subjects. For this study, 94 T2DM and 60 healthy subjects were enroled and evaluated for various parameters. Biochemical quantifications were performed with Syncron CX5 auto-analyzer. The levels of serum sFas-L, TNF-α and IL-6 were estimated by flowcytometry. The fasting serum insulin and sFas quantified by ELISA. HOMA-IR, HOMA-%S and HOMA-%B were calculated with HOMA calculator v2.2.2. The levels of TC, TG, LDL-C, VLDL-C were augmented and HDL declined significantly (P < 0.001) in diabetics. The levels of serum insulin, TNF-α, IL-6, sFas, HOMA-IR were raised (P < 0.001) and sFas-L, HOMA-%S and HOMA-%B were decreased significantly (P < 0.001) in T2DM subjects than healthy. In diabetics, serum sFas was positively correlated with HOMA-IR (r = 0.720, P < 0.001) and negatively with HOMA-%B (r = -0.642, P < 0.001) significantly while serum sFasL was negatively correlated with HOMA-IR (r = -0.483, P < 0.001) and positively with HOMA-%B (r = 0.466, P < 0.001) significantly. Further, the multivariate stepwise regression analysis shows that HOMA-IR contributes significantly to the variance of sFas and sFasL. Our findings suggest that the pancreatic beta cell dysfunction along with increased insulin resistance appears to be associated with apoptotic markers.