Dynamic Palmitoylation Events Following T-Cell Receptor Signaling

Abstract

Palmitoylation is the reversible addition of palmitate to cysteine via a thioester linkage. Following stimulation of the T-cell receptor we find a number of proteins are newly palmitoylated, including those involved in vesiclemediated transport and Ras signal transduction. Among these stimulation-dependent palmitoylation targets are the v-SNARE VAMP7, important for docking of vesicular LAT during TCR signaling, and the largely undescribed palmitoyl acyltransferase DHHC18 that is expressed in two isoforms in T cells. Using our newly developed On-Plate Palmitoylation Assay (OPPA), we show DHHC18 is capable of palmitoylating VAMP7 at Cys183. Cellular imaging shows that the palmitoylation-deficient protein fails to be retained at the Golgi.The initial signaling events following the recognition of a peptide-MHC complex on an antigen-presenting cell (APC) by the T-cell receptor (TCR) involve a number of important molecules that must localize to the plasma membrane (PM) at the immunological synapse (IS). Palmitoylation, the reversible linking of 16-carbon palmitic acid to cysteines via a labile thioester bond, is one strategy used by several important TCR-signaling proteins to drive this localization to and from the membrane (e.g. the soluble Src-family kinases Lck and Fyn) or to and from liquid-ordered microdomains ("lipid rafts") within the membrane (e.g. the transmembrane scaffold LAT) 1 .Unlike other lipid modifications like myristoylation or prenylation, the reversible chemistry of the thioester bond allows palmitoylation to be both added to and removed from specific cysteines; indeed, enzymes have evolved to both add (e.g. the DHHC family of protein acyltransferases [PATs]) and remove (e.g. acyl protein thioesterase 1 [APT1] and ABHD17) palmitate from these sites in eukaryotes 2,3 . This has given rise to palmitoylation cycles, where proteins are palmitoylated in the ER/Golgi, cycled to the PM via vesicle trafficking and localized to the site of activity, then depalmitoylated and recycled back to the ER/Golgi 4 .Such cycles have become well-established in neuronal proteins like PSD-95, as well as the G-protein α subunits Gαi and Gαq and small GTPases H-Ras and N-Ras 4,5 , and pulse-chase experiments have shown that palmitoylation turnover can be accelerated following signaling stimuli, such as glutamate receptor activation with PSD-95, GTP binding of H-Ras, and GPCR signaling for Gα proteins 6-8 . In the context of T-cells, Lck has been shown to undergo relatively rapid palmitate turnover under resting conditions, which is accelerated following Fas receptor stimulation, and this palmitoylation is specifically catalyzed by DHHC21 at the PM 9 . A decrease in the palmitoylation of LAT has been observed in anergic T cells, which affects its ability to localize to lipid rafts and induce an effective stimulatory response 1,10 . Docking of vesicular LAT to the IS and downstream signaling also critically depend on palmitoylated SNARE proteins like SNAP-23 and VAMP7 11,12 . While these targeted approaches suggest the possi...

Metabolic rewiring: a new master of Th17 cell plasticity and heterogeneity

“…Moreover, polyunsaturated fatty acids (PUFAs) inhibit cytoskeleton rearrangements and mitochondrial translocation close to the TCR, events indispensable for effective TCR signaling [77,78]. In contrast, palmitoylation of the linker for activation of T cells (LAT) and the vesicle-associated membrane protein 7 are necessary for their recruitment to the immunological synapse and the prevention of T-cell anergy [79][80][81].…”

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NS1 ‐mediated upregulation of ZDHHC22 acyltransferase in influenza a virus infected cells

“…Using quantitative mass spectrometry, we identified 24 proteins that are acylated in A549 wt cells but not (or at greatly reduced levels) in ZDHHC22 knock‐out cells, suggesting that these proteins are substrates of ZDHHC22. Besides a short isoform of ZDHHC18 (Morrison et al, 2015; Morrison et al, 2020), ZDHHC22 is the shortest member of the ZDHHC family, which is mainly localised in the ER/Golgi. Only two ZDHHC22 substrates have been identified so far, the large conductance calcium‐activated potassium (BK) channel and the nephroblastoma overexpressed protein.…”

Protein cysteine palmitoylation in immunity and inflammation

“…VAMP7 is reported to palmitoylated by ZDHHC18 on Cys183. Interestingly, this study identifies many proteins that are palmitoylated in a TCR signaling‐dependent manner, with VAMP7 being one of them [90]. VAMP7 palmitoylation is important for its trafficking to the Golgi and to localize to the immune synapse upon T‐cell activation.…”