蛋白质巯基亚硝基化修饰在血管内皮功能障碍中的作用研究

Vascular endothelial dysfunction (VED) is a significant initiating factor of vascular disease. The dysregulation of protein s-nitrosylation modification mediated by nitric oxide (NO) relates to the development of various diseases, and the effect of which in VED still needs to be further studied. Our group has been engaged in studying the effect and mechanism of NO in cardiovascular system. Our prior experiments have showed that both the level and distribution of s-nitrosylated proteins have obviously difference in aorta of hypertensive or atherosclerotic rodent models compared with control. We also detected some target proteins, such as CTSB and UCHL1, can be nitrosylated in VED cell models by Biotin switch/tandem mass spectrometry. Therefore, we plan to use specific cysteine mutation, knockout and gene overexpression to not only identify the nitrosylated target proteins, but also discuss the specific effect of s-nitrosylation on protein function and activity in VED. Furthermore, we will confirm the definite mechanism and explore the upstream regulation pathway on which s-nitrosylated target proteins can affect endothelial function in vitro and vivo. This research would hopefully uncover the novel mechanism of endothelial dysfunction and provide novel targets for preventing vascular remodeling-related diseases.

血管内皮功能障碍(vascular endothelial dysfunction, VED)是心血管疾病重要始动因素。一氧化氮（NO）介导的蛋白质巯基亚硝基化修饰调控紊乱涉及多种疾病，其在VED中的作用有待深入研究。本课题组一直从事NO在心血管系统中的作用及机制研究。预实验显示：与正常对照组相比，高血压、动脉粥样硬化动物主动脉内总巯基亚硝基化修饰水平和分布都存在明显差异；在VED细胞模型上，biotin switch/串联质谱技术检测出可发生巯基亚硝基化修饰蛋白（如CTSB/ UCHL1）。我们拟在离体细胞及整体动物上，联合特定半胱氨酸位点突变、基因敲除、过表达等方法，鉴定在VED中发生巯基亚硝基化修饰的蛋白，探讨亚硝基化修饰对靶蛋白的功能、活性及在VED中的作用，明确靶蛋白影响内皮功能的确切作用机制及其上游调控模式。可望揭示VED新机制，为血管重构相关性疾病的防治提供新的干预靶点。

血管内皮功能障碍是心血管疾病重要始动因素。一氧化氮介导的蛋白质巯基亚硝基化(S-nitrosylation, SNO)修饰调控紊乱涉及多种疾病，其在血管内皮功能障碍中的作用有待深入研究。在本项目中，我们通过biotin switch/串联质谱技术检测出可发生SNO修饰蛋白，并在离体细胞及整体动物上，联合特定半胱氨酸位点突变、基因敲除、过表达等方法，鉴定发生SNO修饰的蛋白，探讨SNO修饰对靶蛋白的功能、活性及作用，明确靶蛋白影响内皮功能的确切作用机制及其上游调控模式。本研究结果表明：① OxLDL促进iNOS的表达并诱导eNOS蛋白发生SNO修饰，增加β-catenin信号通路的活化从而导致内皮细胞损伤。② OxLDL促进iNOS的表达并诱导Hsp90蛋白发生SNO修饰，通过影响Hsp90与AHA1及Cdc37的结合上调p65磷酸化水平，从而加重内皮细胞炎症损伤，参与动脉粥样硬化的疾病进程。③ Ang II、OxLDL及HG能够引起CTSB发生SNO修饰，通过MATR3/ADAR1/HuR轴上调CTSB的蛋白水平，参与内皮细胞NLRP3炎症小体活化，导致内皮细胞损伤。④ Ang II上调iNOS的表达并介导PLS3蛋白发生SNO修饰，促进其与cofilin/plectin的结合进而影响内皮细胞间连接完整性，参与主动脉夹层的发生发展。⑤ OxLDL及HG上调iNOS表达并介导GNAI2发生SNO修饰，通过与CXCR5偶联并促进YAP的转录活性，加重内皮炎症并参与糖尿病动脉粥样硬化的疾病进程。⑥ OxLDL诱导ANXA2发生SNO修饰，进而通过PKCα通路导致Pcsk9转录增加，导致内皮炎症损伤并参与动脉粥样硬化的疾病进程。本研究结果揭示了血管内皮功能障碍的新机制，为血管重构相关性疾病的防治提供了新的干预靶点。

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