S-nitrosylation of endogenous protein tyrosine phosphatases in endothelial insulin signaling.
S-nitrosylation of endogenous protein tyrosine phosphatases in endothelial insulin signaling.
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DOI：
10.1016/j.freeradbiomed.2016.08.012
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发表时间：
2016-10
影响因子：
7.4
通讯作者：
Meng TC
中科院分区：
文献类型：
--
作者：
Hsu MF;Pan KT;Chang FY;Khoo KH;Urlaub H;Cheng CF;Chang GD;Haj FG;Meng TC
一氧化氮（NO）通过细胞蛋白质的S-亚硝基化发挥其生物学功能。由于这种修饰在生理条件下的不稳定性，在参与信号调节的酶中鉴定S-亚硝基化残基仍然具有技术挑战性。本研究旨在探讨胰岛素刺激下内皮源性MS-1细胞产生的内源性NO是否可能靶向内源性蛋白酪氨酸磷酸酶（PTPs）。为此，我们已经开发了一种方法，使用合成试剂，引入苯乙酰胺基部分的S-亚硝基化半胱氨酸，然后检测与抗苯乙酰胺基半胱氨酸（PAC）抗体。结合用多种基于碘乙酰基的Cys反应性化学物质连续封闭游离巯基，我们采用这种PAC开关方法来显示内源性SHP-2和PTP 1B在暴露于胰岛素的MS-1细胞中被S-亚硝基化。质谱法检测到一个苯乙酰胺基部分，具体存在于SHP-2的活性位点Cys 463上。聚焦于PTP 1B的调节作用，我们发现S-亚硝基化是内皮胰岛素信号传导中主要的Cys可逆氧化还原修饰。PAC开关方法的成像格式说明，一个池的S-亚硝基化的PTP 1B与激活的胰岛素受体共定位到细胞外周，这种事件是内皮NO合酶（eNOS）依赖。此外，异位表达的C215 S突变体的PTP 1B，模拟活性位点Cys 215 S-亚硝基化的形式恢复eNOS消融细胞，这是否则不敏感的胰岛素刺激胰岛素的反应。这项工作不仅介绍了一种新的方法，探讨生理NO在调节信号转导的作用，但也强调了积极的NO通过S-亚硝基化的PTP 1B的活性位点Cys 215促进胰岛素反应性的影响。
Nitric oxide (NO) exerts its biological function through S-nitrosylation of cellular proteins. Due to the labile nature of this modification under physiological condition, identification of S-nitrosylated residue in enzymes involved in signaling regulation remains technically challenging. The present study investigated whether intrinsic NO produced in endothelium-derived MS-1 cells response to insulin stimulation might target endogenous protein tyrosine phosphatases (PTPs). For this, we have developed an approach using a synthetic reagent that introduces a phenylacetamidyl moiety on S-nitrosylated Cys, followed by detection with anti-phenylacetamidyl Cys (PAC) antibody. Coupling with sequential blocking of free thiols with multiple iodoacetyl-based Cys-reactive chemicals, we employed this PAC-switch method to show that endogenous SHP-2 and PTP1B were S-nitrosylated in MS-1 cells exposed to insulin. The mass spectrometry detected a phenylacetamidyl moiety specifically present on the active-site Cys463 of SHP-2. Focusing on the regulatory role of PTP1B, we showed S-nitrosylation to be the principal Cys reversible redox modification in endothelial insulin signaling. The PAC-switch method in an imaging format illustrated that a pool of S-nitrosylated PTP1B was colocalized with activated insulin receptor to the cell periphery, and that such event was endothelial NO synthase (eNOS)-dependent. Moreover, ectopic expression of the C215S mutant of PTP1B that mimics the active-site Cys215 S-nitrosylated form restored insulin responsiveness in eNOS-ablated cells, which was otherwise insensitive to insulin stimulation. This work not only introduces a new method that explores the role of physiological NO in regulating signal transduction, but also highlights a positive NO effect on promoting insulin responsiveness through S-nitrosylation of PTP1B’s active-site Cys215.
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