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Abstract：
S-Acylation of the SNARE protein SNAP25 (synaptosome-associated protein of 25 kDa) is mediated by a subset of Golgi zinc finger DHHC-type palmitoyltransferase (zDHHC) enzymes, particularly zDHHC17. The ankyrin repeat domain of zDHHC17 interacts with a short linear motif known as the zDHHC ankyrin repeat-binding motif (zDABM) in SNAP25 (112VVASQP117), which is downstream of its S-acylated, cysteine-rich domain (85CGLCVCPC92). Here, we investigated the importance of a flexible linker region (amino acids 93-111, referred to hereafter as the \"mini-linker\" region) that separates the zDABM and S-acylated cysteines in SNAP25. Shortening the mini-linker did not affect the SNAP25-zDHHC17 interaction but blocked S-acylation. Insertion of additional flexible glycine-serine repeats had no effect on S-acylation, but extended and rigid alanine-proline repeats perturbed it. A SNAP25 mutant in which the mini-linker region was substituted with a flexible glycine-serine linker of the same length underwent efficient S-acylation. Furthermore, this mutant displayed the same intracellular localization as WT SNAP25, indicating that the amino acid composition of the mini-linker is not important for SNAP25 localization. Using the results of previous peptide array experiments, we generated a SNAP25 mutant predicted to have a higher-affinity zDABM. This mutant interacted with zDHHC17 more strongly but was S-acylated with reduced efficiency in HEK293T cells, implying that a lower-affinity interaction of the SNAP25 zDABM with zDHHC17 is optimal for S-acylation efficiency. These results show that amino acids 93-111 in SNAP25 act as a flexible molecular spacer that ensures efficient coupling of the SNAP25-zDHHC17 interaction and S-acylation of SNAP25.
摘要：
SNARE蛋白的S-酰化SNAP25（25kDa的突触体相关蛋白）由高尔基体锌指DHHC型棕榈酰转移酶（zDHHC）酶的子集介导，特别是zDHHC17。zDHHC17的锚蛋白重复结构域与SNAP25（112VVASQP117）中称为zDHHC锚蛋白重复结合基序（zDABM）的短线性基序相互作用，S-酰化的下游，富含半胱氨酸的结构域(85CGLCVCPC92)。这里,我们研究了将SNAP25中的zDABM和S-酰化半胱氨酸分开的柔性接头区(氨基酸93-111,以下称为“微型接头区”)的重要性.缩短微型接头不影响SNAP25-zDHHC17相互作用,但阻断S-酰化。插入额外的柔性甘氨酸-丝氨酸重复序列对S-酰化没有影响，但是延伸和刚性的丙氨酸-脯氨酸重复干扰了它。其中小接头区被相同长度的柔性甘氨酸-丝氨酸接头取代的SNAP25突变体经历有效的S-酰化。此外，该突变体显示与WTSNAP25相同的细胞内定位,表明小接头的氨基酸组成对于SNAP25定位并不重要。利用先前肽阵列实验的结果，我们产生了一个预测具有更高亲和力zDABM的SNAP25突变体。该突变体与zDHHC17相互作用更强，但在HEK293T细胞中被S-酰化，效率降低，这意味着SNAP25zDABM与zDHHC17的低亲和力相互作用对于S-酰化效率是最佳的。这些结果表明,SNAP25中的氨基酸93-111充当确保SNAP25-zDHHC17相互作用和SNAP25的S-酰化的有效偶联的柔性分子间隔物。
