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

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. 4S. 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 Rabbit Polyclonal to B-RAF potential targets to control platelet exocytosis motifs. Instead, both proteins contain cysteine-rich regions: human syntaxin-11 has eight cysteines with six at its C terminus, and SNAP-23 contains six cysteines with five in a central domain. These cysteine-rich regions have been shown, in some cells, to be important for membrane association and are potential sites for a thioester linkage. Palmitoyl acyl transferases (PATs) are a large family of enzymes, with a characteristic Asp-His-His-Cys (DHHC) motif, that uses fatty acyl-CoAs as a substrate (21,C23). Platelets contain at least 13 PAT isoforms (24). The (30) characterized over 200 proteins in the platelet palmitoylome, many of which appear to be involved in platelet signaling pathways. Consistently, Sim (25) showed that PAT-inhibitor treatment blocked platelet activation in response to several hemostatic agonists. This group also showed that treatment of permeabilized platelets with a recombinant thioesterase released SNAP-23 from platelet membranes. Despite these observations, the dynamics and potential importance of t-SNARE acylation, specifically of syntaxin-11 and SNAP-23, is largely undefined in platelets. In this manuscript, we showed that acylation SNAP-23 and syntaxin-11 are reversibly acylated in platelets and that acyl turnover is important for platelet secretion. We mapped the acylation sites in both t-SNAREs to specific cysteines in their conserved, cysteine-rich regions. Consistent with their acylation, we showed that both t-SNAREs are enriched in detergent-resistant, cholesterol-dependent membrane fractions or rafts, which may be the sites for membrane fusion because the primary v-SNARE, VAMP-8, also localizes to these fractions upon platelet activation. Finally, using PAT (cerulenin) and thioesterase (palmostatin B) inhibitors, we show that the cycling of the DSM265 acyl groups is important for platelet function because palmostatin B pretreatment prevented the inhibitory effect of cerulenin. Given the dynamic nature of t-SNARE acylation in platelets, it may be possible to repurpose the acylation-directed drugs, originally formulated to modulate RAS and RAS-related proteins in tumors, for use as anti-thrombotic therapeutics. Results Syntaxin-11 and SNAP-23 are associated with membranes and lipid rafts Given their lack of identifiable TMDs, we first sought to determine the extent to which syntaxin-11 and SNAP-23 associate with platelet membranes. DSM265 Disrupting the platelets by freeze-thaw cycling, we generated a cytosolic and membrane fraction by centrifugation. In Fig. 1glyceraldehyde 3-phosphate dehydrogenase (GAPDH), were present in the supernatant (S1) and membrane proteins (VAMP-2, -3, -8) in the pelleted fraction (STX + ITX). The two DSM265 syntaxins (syntaxin-2 and -4) with TMDs were pelleted, as were syntaxin-11 and SNAP-23. Two peripheral membrane proteins, Munc18b and Munc13C4, were distributed between the supernatant and pellet. The pelleted membranes were treated with Triton X-100, which solubilized (STX) the TMD-containing syntaxins, but syntaxin-11 and SNAP-23 were found in both Triton X-100 soluble and insoluble fractions (ITX). The Triton X-100 insoluble fraction most likely contains actin cytoskeleton and lipid rafts (31, 32). Because SNAP-23 has been found in detergent-resistant lipid rafts in platelets (33), we fractionated the ITX material on a sucrose density gradient. The sample was loaded into the denser bottom of the gradient and the rafts were floated DSM265 to their lighter density during centrifugation. A significant portion of SNAP-23 and syntaxin-11 from resting ( 0.0001) as determined using an unpaired Student’s test. Acylation of syntaxin-11 and SNAP-23 Given the membrane distribution and raft association of the two TMD-deficient t-SNAREs and their lack of obvious CAA= 455.7301 Da) and Cys-157 (QRDNNEMC157KIR; monoisotopic.