Kindlin-2泛素化及去泛素化修饰在肾纤维化中的作用机制研究

Kindlin-2 is a novel important protein that promotes renal fibrosis. However, the mechanism of Kindlin-2 degradation in kidney is still unclear. Our previous investigations found that Kindlin-2 interacted with E3 ligase Smurf1 in human renal epithelial cells and Smurf1 induced the degradation of Kindlin-2. Using plasmid- and siRNA based libraries, USP15 was identified as the deubiquitinase of Kindlin-2. In this grant proposal, we will deeply investigate the role of Kindlin-2 ubiquitination and deubiquitination in renal fibrosis. The mechanistic investigations include: confirming the interaction of Kindlin-2 with Smurf1 and USP15; the role of Smurf1 and USP15 in Kindlin-2 ubiquitination, deubiquitination, Kindlin-2 half-life and degradation. Functional investigations include: the role of Kindlin-2 stability regulation by Smurf1 and USP15 in the cellular function of renal epithelial cells including cell spreading , adhesion and migration, etc; the role of Kindlin-2 stability regulation in related signaling activation such as integrin and TGF-β/Smads signalings. In vivo, Smurf1 transgenic mice and USP15 knockout mice will be established by transgenic and CRISPR techniques to uncover the role of Kindlin-2 degradation in the regulation of renal fibrosis. Furthermore, the levels of Smurf1, USP15 and Kindlin-2 will be detected in human fibrotic renal tissues and the correlations among them will be analyzed. We anticipate to identify new evidence for elucidating novel mechanisms underlying renal fibrosis and to design rational strategies for the treatment of chronic kidney diseases.

Kindlin-2是调控肾纤维化的重要蛋白。我们前期研究发现在肾上皮细胞中E3泛素连接酶Smurf1介导Kindlin-2的降解，而USP15是Kindlin-2的去泛素化酶。本申请中，我们将揭示Kindlin-2泛素化及去泛素化修饰在肾纤维化中的作用和机制，包括Smurf1、USP15与Kindlin-2的相互作用及对 Kindlin-2泛素化、去泛素化、半衰期及降解的影响；研究Smurf1和 USP15调控 Kindlin-2稳定性对肾上皮细胞生物学功能及相关通路的调节；我们将建立Smurf1的转基因和USP15基因敲除小鼠，研究体内Kindlin-2降解对肾纤维化进展的影响，并通过肾纤维化病人标本检测Smurf1、USP15与Kindlin-2的表达与纤维化进展的相关性。本研究将揭示Kindlin-2在肾脏中的降解机制，为了解肾纤维化的发病机理提供新信息，为干预肾纤维化提供新靶标。

Kindlin-2是整合素激活调控蛋白，在肾纤维化及多种肿瘤的进展中发挥重要作用。然而Kindlin-2蛋白的降解机制并不清楚。我们旨在探索Kindlin-2蛋白的泛素化及去泛素化修饰机制及其在肾脏纤维化中的作用。我们前期研究发现在肾上皮细胞中E3泛素连接酶S murf1介导Kindlin-2的降解。在随后的研究中我们证实Smurf1确实是Kindlin-2蛋白的E3泛素连接酶，Smurf1与Kindlin-2直接相互作用，通过介导Kindlin-2蛋白的多聚泛素化促进其降解。我们解析出Kindlin-2的泛素化位点及链型。在功能研究中发现Smurf1通过介导Kindlin-2的降解调控整合素的激活和细胞粘附及黏着斑的形成。重要地是，通过Smurf1 KO的小鼠组织和结肠癌病人标本证实了Smurf1与Kindlin-2在体内的负相关。揭示出Kindlin-2的泛素化修饰后，我们进一步展开Kindlin-2蛋白的去泛素化研究。我们结合去泛素化酶（Dub）siRNA文库以及质粒过表达筛选出OTUB2为Kindlin-2蛋白的Dub。我们在肾上皮细胞及几种瘤细胞中证实了OTUB2稳定Kindlin-2的蛋白量，延长其半衰期。OTUB2与Kindlin-2相互作用并介导Kindlin-2的去泛素化。我们将继续研究Smurf1和OTUB2调控 Kindlin-2稳定性对肾上皮细胞生物学功能及相关通路的调节；并建立Smurf1的转基因和 OTUB2基因敲除小鼠，研究体内Kindlin-2降解对肾纤维化进展的影响，并通过肾纤维化病人标 本检测Smurf1、OTUB2与Kindlin-2的表达与纤维化进展的相关性。本研究将揭示Kindlin-2在肾脏中的降解机制，为了解肾纤维化的发病机理提供新信息，为干预肾纤维化提供新靶标。