Department of Biological Science, Graduate School of Science, Osaka Prefecture University , Sakai, Osaka 599-8531, Japan.
Department of Protein Factory, Translational Research Center, Fukushima Medical University , Fukushima, Fukushima 960-1295, Japan.
ACS Chem Neurosci. 2018 Feb 21;9(2):217-223. doi: 10.1021/acschemneuro.7b00363. Epub 2017 Nov 10.
8-Nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP) is the second messenger in nitric oxide/reactive oxygen species redox signaling. This molecule covalently binds to protein thiol groups, called S-guanylation, and exerts various biological functions. Recently, we have identified synaptosomal-associated protein 25 (SNAP-25) as a target of S-guanylation, and demonstrated that S-guanylation of SNAP25 enhanced SNARE complex formation. In this study, we have examined the effects of S-guanylation of SNAP-25 on the interaction between the SNARE complex and complexin (cplx), which binds to the SNARE complex with a high affinity. Pull-down assays and coimmunoprecipitation experiments have revealed that S-guanylation of Cys90 in SNAP-25 attenuates the interaction between the SNARE complex and cplx. In addition, blue native-PAGE followed by Western blot analysis revealed that the amount of cplx detected at a high molecular weight decreased upon 8-nitro-cGMP treatment in SH-SY5Y cells. These results demonstrated for the first time that S-guanylation of SNAP-25 attenuates the interaction between the SNARE complex and cplx.
8-硝基鸟苷 3',5'-环单磷酸（8-nitro-cGMP）是一氧化氮/活性氧还原信号转导中的第二信使。这种分子与蛋白质巯基共价结合，称为 S-鸟苷化，并发挥各种生物学功能。最近，我们已经确定突触相关蛋白 25（SNAP-25）为 S-鸟苷化的靶标，并证明 SNAP25 的 S-鸟苷化增强了 SNARE 复合物的形成。在这项研究中，我们研究了 SNAP-25 的 S-鸟苷化对 SNARE 复合物与具有高亲和力结合 SNARE 复合物的复合蛋白（cplx）之间相互作用的影响。下拉测定和共免疫沉淀实验表明，SNAP-25 的 Cys90 的 S-鸟苷化减弱了 SNARE 复合物与 cplx 之间的相互作用。此外，蓝色 native-PAGE 随后的 Western blot 分析表明，在 SH-SY5Y 细胞中用 8-nitro-cGMP 处理后，检测到的 cplx 数量在高分子量处减少。这些结果首次证明 SNAP-25 的 S-鸟苷化减弱了 SNARE 复合物与 cplx 之间的相互作用。
