PDF(Pubmed)
Abstract：
S-acylation is an essential post-translational modification, which is mediated by a family of 23 zDHHC enzymes in humans. Several thousand proteins are modified by S-acylation; however, we lack a detailed understanding of how enzyme-substrate recognition and specificity is achieved. Previous work showed that the ankyrin repeat domain of zDHHC17 (ANK17) recognizes a short linear motif, known as the zDHHC ANK binding motif (zDABM) in substrate protein SNAP25, as a mechanism of substrate recruitment prior to S-acylation. Here, we investigated the S-acylation of the Sprouty and SPRED family of proteins by zDHHC17. Interestingly, although Sprouty-2 (Spry2) contains a zDABM that interacts with ANK17, this mode of binding is dispensable for S-acylation, and indeed removal of the zDABM does not completely ablate binding to zDHHC17. Furthermore, the related SPRED3 protein interacts with and is efficiently S-acylated by zDHHC17, despite lacking a zDABM. We undertook mutational analysis of SPRED3 to better understand the basis of its zDABM-independent interaction with zDHHC17. This analysis found that the cysteine-rich SPR domain of SPRED3, which is the defining feature of all Sprouty and SPRED proteins, interacts with zDHHC17. Surprisingly, the interaction with SPRED3 was independent of ANK17. Our mutational analysis of Spry2 was consistent with the SPR domain of this protein containing a zDHHC17-binding site, and Spry2 also showed detectable binding to a zDHHC17 mutant lacking the ANK domain. Thus, zDHHC17 can recognize its substrates through zDABM-dependent and/or zDABM-independent mechanisms, and some substrates display more than one mode of binding to this enzyme.
摘要：
S-酰化是一种重要的翻译后修饰,它是由人类中的23个zDHHC酶家族介导的。通过S-酰化修饰了数千种蛋白质；然而，我们缺乏对如何实现酶-底物识别和特异性的详细了解。先前的工作表明，zDHHC17（ANK17）的锚蛋白重复结构域识别短线性基序，在底物蛋白SNAP25中称为zDHHCANK结合基序（zDABM），作为S酰化之前底物募集的机制。这里,我们通过zDHHC17研究了Sprouty和SPRED蛋白家族的S-酰化。有趣的是,尽管Sprouty-2（Spry2）包含与ANK17相互作用的zDABM，但这种结合方式对于S-酰化作用是不必要的，确实，去除zDABM并不能完全消除与zDHHC17的结合。此外，尽管缺乏zDABM，相关的SPRED3蛋白与zDHHC17相互作用并被zDHHC17有效地S-酰化。我们对SPRED3进行了突变分析，以更好地了解其与zDHHC17的zDABM无关相互作用的基础。该分析发现SPRED3的富含半胱氨酸的SPR结构域，这是所有Sprouty和SPRED蛋白的定义特征，与zDHHC17相互作用。令人惊讶的是,与SPRED3的相互作用独立于ANK17.我们对Spry2的突变分析与含有zDHHC17结合位点的该蛋白的SPR结构域一致，和Spry2还显示可检测的与缺乏ANK结构域的zDHHC17突变体的结合。因此，zDHHC17可以通过zDABM依赖和/或zDABM独立机制识别其底物，和一些底物显示超过一种模式的结合这种酶。
