GAPDH硝基化修饰调控SIRT1表达介导DN足细胞损伤相关自噬的机制研究

Protein nitration is an important part in the pathogenesis of diabetic nephropathy (DN). Previous studies have been limited to the use of 3-nitrotyrosine as a biomarker to reflect the degree of nitration, but few studies have been done on the function of nitrated proteins. It was found that tyrosine nitration of 3-phosphate dehydrogenase (GAPDH) was involved in the development of DN. However, the mode of action is not clear. Based on previous reports that GAPDH is involved in the regulation of autophagy mediated by SIRT1, and combining with computer simulation technology, the applicant predicts that tyrosine nitration of GAPDH may inhibit its binding to NAD+, then inhibit its catalytic function. Therefore, the present study intends to identify the functional sites for tyrosine nitration of GAPDH and NAD+. Next, to clarify the mechanism of GAPDH tyrosine nitration on SIRT1, and to confirm the correlation between autophagy mediated by SIRT1 and podocyte injury induced by nitration. Furthermore, to clarify the mechanism that tyrosine nitration of GAPDH inhibits its binding to NAD+, then inhibits the podocyte autophagy mediated by SIRT1, results in kidney damage. Our study in order to reveal a new way to study the pathogenesis of DN, and to provide a theoretical and experimental basis for the discovery of DN treatment.

蛋白质硝基化修饰是糖尿病肾病（DN）发病机制的重要环节。国内外既往研究局限于以硝基化酪氨酸作为标志物来反映机体硝基化程度，针对硝基化蛋白质的功能研究鲜有报道。申请人发现3-磷酸甘油醛脱氢酶（GAPDH）酪氨酸硝基化参与了DN的发生发展，但其作用方式尚不清楚。基于文献报道GAPDH参与调控SIRT1介导的自噬，结合计算机模拟对接技术，申请人理论预测GAPDH酪氨酸硝基化后可能抑制其与NAD+结合，进而抑制其催化功能。本课题拟通过鉴定改变GAPDH与NAD+亲和力的酪氨酸硝基化功能位点，明确GAPDH酪氨酸硝基化对SIRT1的调控机制，确认SIRT1介导的自噬与硝基化作用下足细胞损伤的相关性。进而阐明GAPDH酪氨酸硝基化抑制其与NAD+结合，继而抑制SIRT1介导的足细胞自噬，造成肾脏损伤的作用机制。本研究为揭示DN发病机制提供新思路，为发现治疗DN的新靶点提供理论基础与实验依据。

蛋白质硝基化修饰是糖尿病肾病（DN）发病机制的重要环节。既往研究局限于以硝基化酪氨酸作为标志物来反映机体硝基化程度，但是针对硝基化蛋白的功能研究鲜有报道。申请人前期研究发现3-磷酸甘油醛脱氢酶（GAPDH）酪氨酸硝基化参与了DN的发生发展，基于文献报道推测其机制可能与GAPDH酪氨酸硝基化影响肾脏自噬水平相关。因此，课题组在既往工作基础上，围绕GAPDH酪氨酸硝基化位点检测及自噬表达开展实验，获得以下结果：①通过激光共聚焦观察 db/db小鼠肾皮质中GAPDH及其硝基化表达分布，依据结果确定肾小管上皮细胞（HK-2)为研究靶细胞。②检测高糖培养下HK-2细胞自噬相关蛋白LC3及P62表达水平，结果示高糖刺激下HK-2细胞自噬表达增强。③通过ELISA法测定过氧亚硝基阴离子(ONOO-)浓度，获得了不同高糖刺激条件下细胞的硝基化水平数据，为研究工作提供基础数据和实验依据。同时发现，白藜芦醇可以降低高糖刺激下HK-2细胞的ONOO-浓度，初步证实了白藜芦醇抗硝基化肾脏保护作用，为寻找DN新的治疗方法提供了依据。④通过免疫共沉淀联合串联质谱技术鉴定HK-2细胞GAPDH酪氨酸硝基化位点。结果示，检测到GAPDH蛋白，且无角蛋白等污染蛋白，结果可靠；本次检测暂未获得GAPDH酪氨酸硝基化位点；检测到GAPDH相关蛋白的酪氨酸硝基化位点，证明ONOO-作用浓度及方法可行，为进一步开展后续研究提供了坚实的实验基础，为发现DN发病机制新靶点提供了更多的数据。⑤由于蛋白质酪氨酸硝基化修饰是一个极低表达率和高选择性的过程，所以虽然硝基化修饰在发病机制中意义重大，但研究者鲜有涉及。对于HK-2细胞GAPDH酪氨酸硝基化修饰位点的检测是本课题的难点和关键点。课题组通过质粒转染技术，成功获得GAPDH过表达HK-2细胞，现阶段再次对其酪氨酸硝基化修饰位点进行检测，结果近期回报。综上，课题组以酪氨酸硝基化修饰为切入点，获得高糖培养下的细胞硝基化水平数据，并鉴定出GAPDH相关蛋白硝基化位点，为从全新角度探讨DN发病机制及治疗方法提供了更多的理论依据。