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Dynamic Cycling of t-SNARE Acylation Regulates Platelet Exocytosis.

Dynamic Cycling of t-SNARE Acylation Regulates Platelet Exocytosis.
Author Information (click to view)

Zhang J, Huang Y, Chen J, Zhu H, Whiteheart SW,


Zhang J, Huang Y, Chen J, Zhu H, Whiteheart SW, (click to view)

Zhang J, Huang Y, Chen J, Zhu H, Whiteheart SW,

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The Journal of biological chemistry 2018 01 19() pii jbc.RA117.000140
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 were activated. Using metabolic labeling and hydroxylamine/HCl (HA) 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, while 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 though 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 acyl-transferase 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 while 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.

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