Detection of Protein S-Acylation using Acyl-Resin Assisted Capture

Journal of Biological Chemistry

et al. 2021

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ZAP-70 is a tyrosine kinase essential for T cell immune responses. Upon engagement of the T cell receptor (TCR), ZAP-70 is recruited to the specialized plasma membrane domains, becomes activated, and is released to phosphorylate its laterally segregated targets. A shift in ZAP-70 distribution at the plasma membrane is recognized as a critical step in TCR signal transduction and amplification. However, the molecular mechanism supporting stimulation-dependent plasma membrane compartmentalization of ZAP-70 remains poorly understood. In this study, we identified previously uncharacterized lipidation (S-acylation) of ZAP-70 using Acyl-Biotin Exchange assay, a technique that selectively captures S-acylated proteins. We found that this posttranslational modification of ZAP-70 is dispensable for its enzymatic activity. However, the lipidation-deficient mutant of ZAP-70 failed to propagate the TCR pathway suggesting that S-acylation is essential for ZAP-70 interaction with its protein substrates. The kinetics of ZAP-70 S-acylation were consistent with TCR signaling events indicating that agonist-induced S-acylation is a part of the signaling mechanism controlling T cell activation and function. Taken together, our results suggest that TCR-induced S-acylation of ZAP-70 can serve as a critical regulator of T cell-mediated immunity.

“…Protein S-acylation was determined using the ABE assay modified from previous publications ( 39 , 40 , 41 ). We used 1 × 10 7 cells for each condition.…”

Section: Methodsmentioning

confidence: 99%

T cell receptor–dependent S-acylation of ZAP-70 controls activation of T cells

Tewari¹,

Bieerkehazhi²,

Journal of Biological Chemistry

et al. 2021

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“…Protein S-acylation was determined using ABE assay modified from previous publications (39)(40)(41). We used 1 X 10 7 cells for each condition.…”

Section: Acyl-biotin Exchange (Abe) Assaymentioning

confidence: 99%

“…Next, free thiol groups were blocked by adding 200 µL of 0.2% MMTS (S-Methyl methanethiosulfonate) in 2SHB buffer to each tube to a final concentration of 0.1% MMTS.Blocking was performed for 15 min at 42 ˚C with shaking at 1500 rpm in a thermal shaker. MMTS was removed by three rounds of CM precipitation and protein pellets were dissolved in 2SHB buffer and Buffer A (5 mM EDTA; 100 mM HEPES; pH 7.4) as described(41).Following three rounds of CM precipitation, 10% of the sample was retained as input control and the remaining was split into two equal volumes labeled as +HA and -HA. The +HA group was incubated with 400 mM hydroxylamine (HA, pH 7-7.2, freshly prepared) to promote thioester bond cleavage.…”

mentioning

confidence: 99%

T cell receptor-dependent S-acylation of ZAP-70 controls activation of T cells

Tewari

Bieerkehazhi

et al. 2020

Preprint

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AbstractZAP-70 is a cytoplasmic tyrosine kinase essential for T cell-mediated immune responses. Upon engagement of the T cell receptor, ZAP-70 is quickly recruited to the specialized plasma membrane domains, becomes activated and released to phosphorylate its laterally segregated downstream targets. A shift in ZAP-70 distribution at the plasma membrane is recognized as a critical step in T cell receptor signal transduction and amplification. However, the molecular mechanism supporting stimulation-dependent plasma membrane compartmentalization of ZAP-70 remains poorly understood. In this study, we identified previously uncharacterized reversible lipidation (S-acylation) of ZAP-70. We found that this post-translational modification of ZAP-70 is dispensable for its enzymatic activity. However, the lipidation-deficient mutant of ZAP-70 failed to propagate the T cell receptor signaling cascade suggesting that S-acylation is essential for ZAP-70 interaction with its protein substrates. The kinetics of ZAP-70 S-acylation were consistent with early T cell signaling events indicating that agonist-induced S-acylation is a part of the signaling mechanism controlling T cell activation and function.Significance StatementActivation of T cells is a critical part of the adaptive immune response to pathogen exposure. We found that ZAP-70, a regulatory protein essential for T cell activation, can undergo a post-translational modification with long chain fatty acids, known as S-acylation. In this report, we show that S-acylation of ZAP-70 is T cell receptor-dependent and required for its signaling function. We found that loss of ZAP-70 S-acylation resulted in T cell unresponsiveness to T cell receptor stimulation indicating that abnormalities in protein S-acylation can potentially contribute to the T cell immunodeficiency disorders.

“…To test whether F233 DHHC21 is able to mediate Sacylation of its protein substrates, we isolated CD4 + T cells from spleen and lymph nodes of WT and Zdhhc21 dep mice and subjected them to the acyl-resin assisted capture (Acyl-RAC) assay ( Fig. 2A) (24).…”

Section: Dhhc21 Mediates Tcr-induced S-acylation Of T Cell Signaling mentioning

confidence: 99%

“…Protein S-acylation status was assessed by Acyl-RAC as described (24). Cells were lysed in 1% DDM lysis Phosphatase Inhibitor Cocktail 2 (1:100); Protease Inhibitor Cocktail (1X) and PMSF (10 mM)).…”

Section: Acyl-resin Assisted Capture (Acyl-rac) Assaymentioning

confidence: 99%

Calcium-dependent protein acyltransferase DHHC21 controls activation of CD4⁺T cells

Bieerkehazhi

West

et al. 2020

Preprint

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Despite the recognized significance of reversible protein lipidation (S-acylation) for T cell receptor signal transduction, the enzymatic control of this post-translational modification in T cells remains poorly understood. Here, we demonstrate that DHHC21, a member of the DHHC family of mammalian protein acyltransferases, mediates agonist-induced S-acylation of proximal T cell signaling proteins. Using Zdhhc21dep mice expressing a functionally deficient version of DHHC21, we show that DHHC21 is a calcium/calmodulin-dependent enzyme critical for activation of naïve CD4+ T cells in response to T cell receptor stimulation. We found that disruption of the calcium/calmodulin binding domain of DHHC21 does not affect thymic T cell development but prevents differentiation of peripheral CD4+ T cells into Th1, Th2, and Th17 effector T helper lineages. Our findings identify DHHC21 as an essential component of the T cell receptor signaling machinery and define a new role for protein acyltransferases in regulation of T cell-mediated immunity.SignificanceThis study identifies DHHC21, a member of the DHHC family of mammalian protein acyltransferases, as a novel component of the TCR signaling pathway and demonstrates that this enzyme critically regulates activation and differentiation of CD4+ T cells by mediating rapid TCR-induced S-acylation of signaling proteins. This finding shows that protein acyltransferases can play a vital function in regulation of T cell-mediated immunity and thus serve as potential drug targets in diseases associated with altered immune system homeostasis.