8-Nitro-cGMP attenuates the interaction between SNARE complex and complexin through S-guanylation of SNAP-25
8-Nitro-cGMP attenuates the interaction between SNARE complex and complexin through S-guanylation of SNAP-25
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8-硝基-cGMP 通过 SNAP-25 的 S-鸟苷酸化减弱 SNARE 复合物和复合蛋白之间的相互作用
DOI：
10.1021/acschemneuro.7b00363
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发表时间：
2018
期刊：
影响因子：
--
通讯作者：
Ihara H.
中科院分区：
文献类型：
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作者：
Kishimoto Y;Kunieda K;Kitamura A;Kakihana Y;Akaike T;Ihara H.
8-硝基鸟苷3′，5 ′-环一磷酸（8-nitro-cGMP）是一氧化氮/活性氧氧化还原信号的第二信使。该分子共价结合蛋白质巯基，称为S-鸟苷化，并发挥各种生物功能。最近，我们已经确定突触体相关蛋白25（SNAP-25）作为S-鸟苷酸化的靶点，并证明SNAP-25的S-鸟苷酸化增强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复合物和复合蛋白（cplx）之间相互作用的影响，复合蛋白（cplx）以高亲和力结合SNARE复合物。下拉测定和免疫共沉淀实验表明，SNAP-25中Cys 90的S-鸟苷酸化减弱了SNARE复合物和cplx之间的相互作用。此外，蓝色native-PAGE后的蛋白质印迹分析显示，在SH-SY 5 Y细胞中，在8-硝基-cGMP处理后，在高分子量下检测到的cplx的量减少。这些结果首次证明SNAP-25的S-鸟苷酸化减弱了SNARE复合物和cplx之间的相互作用。
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.