Dynamic cycling of t-SNARE acylation regulates platelet exocytosis

Jinchao Zhang, Yunjie Huang, Jing Chen, Haining Zhu, Sidney W. Whiteheart

Research output: Contribution to journalArticlepeer-review

6 Scopus citations

Abstract

Platelets regulate vascular integrity by secreting a host of molecules that promote hemostasis and its sequelae. Given the importance of platelet exocytosis, it is critical to understand how it is controlled. The t-SNAREs, SNAP-23 and syntaxin-11, lack classical transmembrane domains (TMDs), yet both are associated with platelet membranes and redistributed into cholesterol-dependent lipid rafts when platelets are activated. Using metabolic labeling and hydroxylamine (HA)/HCl treatment, we showed that both contain thioester-linked acyl groups. Mass spectrometry mapping further showed that syntaxin-11 was modified on cysteine 275, 279, 280, 282, 283, and 285, and SNAP-23 was modified on cysteine 79, 80, 83, 85, and 87. Interestingly, metabolic labeling studies showed incorporation of [3H]palmitate into the t-SNAREs increased although the protein levels were unchanged, suggesting that acylation turns over on the two t-SNAREs in resting platelets. Exogenously added fatty acids did compete with [3H]palmitate for t-SNARE labeling. To determine the effects of acylation, we measured aggregation, ADP/ATP release, as well as P-selectin exposure in platelets treated with the acyltransferase inhibitor cerulenin or the thioesterase inhibitor palmostatin B. We found that cerulenin pretreatment inhibited t-SNARE acylation and platelet function in a dose- and time-dependent manner whereas palmostatin B had no detectable effect. Interestingly, pretreatment with palmostatin B blocked the inhibitory effects of cerulenin, suggesting that maintaining the acylation state is important for platelet function. Thus, our work shows that t-SNARE acylation is actively cycling in platelets and suggests that the enzymes regulating protein acylation could be potential targets to control platelet exocytosis in vivo.

Original languageEnglish
Pages (from-to)3593-3606
Number of pages14
JournalJournal of Biological Chemistry
Volume293
Issue number10
DOIs
StatePublished - Mar 9 2018

Bibliographical note

Funding Information:
This work was supported by NHLBI, National Institutes of Health Grants HL56652 and HL138179; American Heart Association Grant-in-Aid AHA16GRNT27620001; and Veterans Affairs Merit Award (to S. W. W.). This study was also supported in part by NIGMS, National Institutes of Health Grant P30GM110787. The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the respon-sibility of the authors and does not necessarily represent the official views of the National Institutes of Health or reflect the position or policy of the Department of Veterans Affairs or the United States government.

Funding Information:
This work was supported by NHLBI, National Institutes of Health Grants HL56652 and HL138179; American Heart Association Grant-in-Aid AHA16GRNT27620001; and Veterans Affairs Merit Award (to S. W. W.). This study was also supported in part by NIGMS, National Institutes of Health Grant P30GM110787. The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health or reflect the position or policy of the Department of Veterans Affairs or the United States government.

Publisher Copyright:
© 2018 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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