去泛素化在Caspase11-Gasdermin-D通路介导的焦亡促进酒精性肝炎进展中的作用机制研究

研究表明Gasdermin D（Gsdmd）介导的焦亡参与酒精性肝炎（alcoholic hepatitis, AH）病程进展，但具体机制不清楚。在前期实验中，我们靶向敲低肝细胞Gsdmd后，AH小鼠肝脏炎症改善，提示Gsdmd介导的焦亡参与AH进展。相较于正常小鼠，AH小鼠肝脏内Gsdmd mRNA表达水平下调，但Gsdmd蛋白表达水平上调，提示肝内存在对Gsdmd蛋白翻译或翻译后水平的调控。鉴于泛素-蛋白酶体途径是细胞内蛋白质降解的主要途径之一，我们推测泛素化修饰在Gsdmd翻译后调控的作用可能与Gsdmd蛋白水平上调有关。本研究将采用酵母双杂交、免疫共沉淀、多维质谱等方法鉴定Gsdmd的去泛素化及多聚泛素侧链修饰，探索Gsdmd的去泛素化参与AH病程的具体机制。通过本研究，我们将首次明确Gsdmd的去泛素化及其在AH病程中的作用，为理解AH发病机制及寻找治疗AH新靶点提供实验室基础。

Hepatic injury and death are the distinguished histological features of alcoholic hepatitis (AH). Pyroptosis mediated by Gasdermin D (Gsdmd) has been proved involving in the pathogenesis of AH. However, roles of Gsdmd in the pathogenesis of AH are not fully understood. In order to identify the roles of hepatocellular pyroptosis mediated by Gsdmd, we knocked down hepatocellular Gsdmd in mice via CRISPR-AAV-SaCas9 system. It was found that hepatocellular Gsdmd knockdown could ameliorate pathological manifestations of AH. It was found that Gsdmd mRNA levels were downregulated in AH mice compared with control mice, whereas Gsdmd protein levels were upregulated. The discrepancy between Gsdmd mRNA levels and Gsdmd protein levels indicated that Gsdmd protein might undergo post-translational modifications. Since the ubiquitin-proteasome pathway is one of the major pathways of protein degradation, we speculated that the regulation of ubiquitination after Gsdmd translation might be related to the up-regulation of Gsdmd protein level..Based on the previous studies, this study intends to further explore the role of deubiquitination of Gsdmd in the progression of AH.The deubiquitination and polyubiquitin side chain modification of Gsdmd were identified by yeast double hybridization, immunopollution, multidimensional mass spectrometry, etc., to determine whether the deubiquitination of Gsdmd was dependent on Caspase11, and to explore the specific mechanism of Gsdmd deubiquitination in the course of AH. Through this study, we will define the deubiquitination of Gsdmd and its role in the course of AH for the first time, so as to provide a laboratory basis for further understanding the pathogenesis of AH and finding new targets for the treatment of AH.