Modulation of Cysteine modifications upon short time T-cell activation

Abstract

T cell activation is initiated by rapid and tightly regulated signaling events that rely on reversible post-translational modifications to ensure speed, specificity, and fidelity. While phosphorylation is well established as a central regulator of T cell receptor signaling, the contribution of cysteine-based modifications remains poorly defined at a systems level. Due to their redox-sensitive thiol side chains, cysteine residues can undergo rapid and reversible chemical modifications, enabling them to function as molecular switches that modulate protein structure, activity, and interactions during signal transduction. Engagement of the TCR induces localized redox signaling, primarily through mitochondrial and NADPH oxidase-derived H2O2, leading to selective and reversible oxidation of reactive cysteine residues. These redox events act as spatially and temporally controlled signaling mechanisms rather than global oxidative stress, transiently reshaping phosphorylation-dependent networks through modulation of redox-sensitive kinases, phosphatases, and adaptor proteins. Specificity is conferred by peroxiredoxin-, thioredoxin-, and glutathione-dependent redox control systems that fine-tune cysteine reactivity. In addition, redox-dependent modulation of cysteine residues within zinc-coordinating motifs suggests a coupled redox-zinc signaling axis during T cell activation, where transient redistribution of zinc from cysteine-rich zinc-finger proteins may further regulate kinase activity and transcriptional programs downstream of TCR engagement. Here, we present a quantitative, time-resolved proteomic strategy to systematically interrogate cysteine dynamics during the earliest phases of human T cell activation. By combining cysteine-specific phosphonate adaptable tagging, thiol-disulfide exchange chromatography and TMT-based multiplexed mass spectrometry, we quantified 25,324 free cysteine-containing peptides and 14,853 reversibly modified cysteine peptides across seconds-to-minutes following TCR stimulation. This comprehensive cysteine proteome revealed rapid remodeling of cysteine redox states as early as 30 seconds after activation of T-cells. Functionally, regulated cysteines were enriched in core signaling pathways, including kinases, phosphatases, transcription factors, and nuclear zinc-finger proteins. Notably, key TCR signaling components such as Lck, CD45, ZAP-70, Akt, NFATC3, and RASGRP1 exhibited dynamic cysteine modulation. Together, these findings establish cysteine redox dynamics as a pervasive and functionally relevant regulatory layer in T cell activation, positioning cysteine modification alongside phosphorylation as a central component of immune signal transduction.