泛素E3连接酶接头蛋白SPOP控制离子通道KCNQ1蛋白稳定性影响心肌细胞复极化的机制研究

泛素-蛋白酶体系统是细胞内最重要的高选择性蛋白质降解方式，但其在心脏电活动中的角色仍不清楚。泛素E3连接酶接头蛋白SPOP负责识别蛋白底物，在除发育和肿瘤外的病理生理中功能尚不清楚。前期我们通过生物信息学方法在人类全蛋白质组范围内筛选到KCNQ1可能是SPOP的识别底物。鉴于KCNQ1在心肌细胞复极中的重要功能，推测SPOP可能参与心脏电活动。初步实验提示SPOP与KCNQ1存在蛋白互作。本申请计划通过腺病毒介导的心脏特异过表达和干扰SPOP动物模型、心肌细胞电生理分析、心功能检测、体内外蛋白互作、蛋白泛素化降解等实验确定SPOP是否通过泛素化KCNQ1，使其进入泛素-蛋白酶体降解途径，从而调节蛋白稳定性参与心肌细胞复极过程。最后评价小分子药物调节SPOP蛋白水平对细胞电活动的影响。本申请首次研究SPOP在心脏电活动中的功能，为干预SPOP作为抗心律失常潜在治疗手段提供了理论依据。

The ubiquitin-proteasome system (UPS) is a well-controlled, selective mechanism for intracellular protein degradation and turnover. However, the role of UPS played in cardiac electrical activity remain unclear. Speckle-type POZ protein (SPOP), an adaptor for E3 ubiquitin ligase, is a potential regulator of tumorigenesis and organ development. Our preliminary studies showed that, KCNQ1, a key molecule in the depolarization of myocardial cells, was a candidate substrate of SPOP by bioinformatics analysis. We further confirmed the interaction between SPOP and KCNQ1 by CO-IP experiment. Thus, we hypothesized that SPOP might be involved in cardiac electrical activity. In this proposal, we plan to confirm if SPOP could regulate myocardial cell depolarization by ubiquitinating KCNQ1 and further mediate its degradation through UPS. The following experiments: AAV-mediated cardiac-specific SPOP overexpression or knockdown model, electrophysiological analysis of myocardial cells, heart function test, protein interaction in vivo and vitro, ubiquitin-proteasome-triggered degradation, will be performed. Furthermore, small-molecule medication which could regulate SPOP will be used to observe the changes of cardiomyocyte electrical activity. This proposal will for the first time systematically study the function of SPOP in cardiac electrical activity, and the results will provide theoretical basis for novel anti-arrhythmia therapy by targeting SPOP.