E3泛素化连接酶WWP2调控PARP1在心肌损伤及心衰中的作用及其机制研究

Heart failure is a very important cause of human death and protein homeostasis imbalance plays a key role in the development of heart failure. E3 ubiquitinated ligase is the core of ubiquitin-proteasome system, which regulates protein homeostasis. WWP2 is a HECT E3 ubiquitinated ligase and it is abundantly expressed in myocardial tissue. But the function of WWP2 in cardiac system is still unclear. We were the first to find that expression of WWP2 is decreased in myocardial injury and heart failure. And myocardial cell specific WWP2 knockout significantly aggravated mouse myocardial injury and heart failure. In addition, we identify myocardial injury factor PARP1 as a novel WWP2 binding partner by using mass spectrometry and immunoprecipitation. WWP2 is involved in PARP1 ubiquitin-proteasome system degradation, suggesting that WWP2 takes part in myocardial injury and heart failure by regulating PARP1. This project aims to further elucidate the domain of interaction between WWP2 and PARP1 and the PARP1 ubiquitin-modified site, which would elucidate the mechanism of WWP2 in regulating myocardial injury and heart failure at the molecular, cellular and animal levels. The aim of this study is to reveal the role of a new signaling pathway WWP2-PARP1 in myocardial injury and heart failure and to provide evidences for exploring new drug targets and therapeutic options for myocardial injury and heart failure.

心力衰竭是导致人类死亡的重要原因，蛋白质稳态失衡在心力衰竭的发生发展中扮演重要角色。E3泛素化连接酶是泛素-蛋白酶体系统中控制蛋白质稳态的核心。WWP2是HECT型E3泛素化连接酶，在心肌组织中表达丰富，但其在心脏系统中的功能尚不清楚。我们首次发现WWP2在心肌损伤及心衰发生时表达降低，并且小鼠心肌特异性WWP2敲除显著加重血管紧张素II诱导的心肌损伤及心衰。通过质谱分析及免疫共沉淀，我们发现WWP2与心肌损伤重要因子PARP1相互作用，并且WWP2通过泛素-蛋白酶体系统降解PARP1，提示WWP2可能通过调控PARP1对抗心肌损伤及心衰。本项目拟进一步解析WWP2与PARP1相互作用结构域及泛素化修饰PARP1位点，并在分子、细胞和整体水平阐明WWP2调控心肌损伤及心衰的机制，揭示一条新的WWP2-PARP1信号转导通路在心肌损伤及心衰中的作用，为探索新药物靶点和治疗方案提供依据。

在心脏重构中，多聚ADP-核糖聚合酶-1(PARP1)表达和活性都会升高，即发生多聚ADP核糖基化(PARylation)，从而导致严重的能量消耗和心肌损伤。然而,调节PARP1的机制尚需进一步研究。WWP2是一种HECT型E3泛素连接酶，在心脏中有高表达，但它在心脏中的功能很大程度上是未知的。在本研究中，我们阐明了WWP2在PARP1调控中的作用以及这一调控过程对心脏重构的影响。我们首先明确了在特异性敲除WWP2的心肌中PARP1泛素化水平降低，异丙肾上腺素(ISO)诱导后PARP1和多聚ADP核糖基化的水平增加，进而导致ISO诱导的心肌肥大、心力衰竭和心肌纤维化加重。我们在WWP2基因敲除的H9C2细胞系中通过ISO的诱导，在小鼠心肌组织中得到了一致的结果。而在H9C2细胞系敲减WWP2后再次过表达WWP2后则发现PARP1泛素化水平、PARP1表达水平和多聚ADP核糖基化水平都显著降低。在探究相关机制时，通过免疫共沉淀结果表明WWP2作为PARP1的一种新的相互作用蛋白，主要与其BRCT结构域相互作用，从而通过泛素-蛋白酶体系统介导PARP1的降解。其中，赖氨酸418(K418)和赖氨酸249(K249)位点在WWP2调控的PARP1泛素化降解中具有至关重要的作用。我们的研究首次揭示WWP2-PARP1信号通路参与了心脏重构的调控，为探索与心脏重构相关的心脏疾病的治疗策略提供了理论依据。