Neddylation and cardiac protein quality control

描述(由申请人提供)：泛素和泛素样蛋白的翻译后修饰对于蛋白质质量控制是必不可少的。泛素介导的蛋白降解缺陷可导致多种心脏疾病，包括结蛋白相关性心肌病(DRC)和心肌缺血再灌注(I/R)损伤。然而，对于NEDD8等泛素样蛋白在心肌细胞(CM)生理学和病理生理学中的重要性知之甚少。这一应用的初步数据揭示了NEDD8在CM中的一个新的、被低估的角色，并形成了我们长期目标的基础：了解NEDD8信号如何被调节以调节CM功能，最终调节心脏疾病。NEDD8与靶蛋白的结合或去结合(去结合或去结合)由一系列酶动态调节。去核酸化调节各种基本的细胞过程，包括蛋白质分解。我们的初步数据显示，在心脏疾病的动物模型中，以及在患有缺血性或扩张性心肌病的人类衰竭心脏中，Nedylated蛋白质显著增加。由于缺乏NEDD8去结合酶的小鼠通过损害蛋白质分解而患上严重的心肌病，因此NEDD8去结合酶的积聚具有重要的功能。通过多种途径减弱胞内代谢，保护CMS免受细胞损伤。这些发现共同表明，过多的去信号蛋白损害蛋白分解，对CMS是有毒的，而预防/正常化去信号蛋白的策略促进了CM的存活和功能。NUB1L是代谢的负性调节因子。NUB1L的表达减少了蛋白质的缺失，增强了蛋白酶体的功能，防止了应激诱导的CM细胞死亡。因此，本项目的目标是确定NUB1L在心脏代谢和蛋白质降解调节中的作用。我们将验证我们的中心假设，即NUB1L减少蛋白质代谢并保护心脏免受蛋白毒性压力的影响。目的1利用一种新的转基因小鼠模型，确定NUB1L是如何调节心脏排泄的，以及排泄减少对心脏结构和功能的影响。目的2将利用转基因报告测试NUB1L的表达是否增强心脏蛋白酶体功能，并确定抑制内切是否调节心脏中错误折叠蛋白的移除并改变DRC的进展。目的3确定药物抑制剂和NUB1L表达对心肌I/R损伤的抑制作用。减少心肌梗死的策略可能是治疗DRC和缺血性心肌病的有效策略。这一建议的翻译结果包括在心肌梗死时保留肌肉和预防心力衰竭，这是美国国立卫生研究院的主要任务。这项拟议的研究是首次针对心脏疾病模型中的蛋白质缺失进行研究，并将采用一种创新的方法，使用转基因小鼠和药物干预。

DESCRIPTION (provided by applicant): Posttranslational modification by ubiquitin and ubiquitin-like protein is essential for protein quality control. Defects in ubiquitin-mediated proteolysis are pathogenic to various cardiac diseases including desmin-related cardiomyopathy (DRC) and myocardial ischemia reperfusion (I/R) injury. However, little is known about the importance of ubiquitin-like proteins such as NEDD8 in cardiac myocytes (CM) physiology and pathophysiology. Preliminary data in this application reveal a novel and underappreciated role of NEDD8 in the CM and form the basis of our long-term goal: to understand how NEDD8 signaling can be modulated to regulate CM function and ultimately cardiac disease. Conjugation or deconjugation of NEDD8 to target proteins (neddylation or deneddylation) is dynamically regulated by a family of enzymes. Neddylation regulates a variety of essential cellular processes including proteolysis. Our preliminary data reveal a striking increase of neddylated proteins in the hearts from animal models of cardiac diseases, as well as in human failing hearts with ischemic or dilated cardiomyopathy. The buildup of neddylated proteins is functionally significant, as the mice deficient of a NEDD8 deconjuation enzyme develop severe cardiomyopathy through impairing proteolysis. Attenuation of neddylation by multiple means protects CMs from cell injury. These findings collectively indicate that the excessive neddylated proteins impairs proteolysis and are toxic to CMs, and that strategies to prevent/normalize neddylated proteins promote CM survival and function. NUB1L is a negative regulator of neddylation. NUB1L expression reduces neddylated proteins, enhances proteasomal function, and prevents stress-induced CM cell death. Therefore, the goal of the current project is to define the role of NUB1L in regulation of neddylation and protein degradation in the heart. We will test our central hypothesis that NUB1L reduces neddylated proteins and protects the heart against proteotoxic stress. Aim 1 will determine how NUB1L regulates neddylation and the impact of diminished neddylation on cardiac structure and function, using a novel transgenic mouse model. Aim 2 will test whether NUB1L expression enhances cardiac proteasomal function using a transgenic reporter, and determine whether suppression of neddylation regulates the removal of misfolded proteins in the heart and alters DRC progression. Aim 3 will determine the efficacy of suppression of neddylation, by both pharmacologic inhibitor and NUB1L expression, on myocardial I/R injury. Strategies to reduce neddylation may be effective therapeutic strategies to treat DRC and ischemic cardiomyopathy. The translational outcomes of this proposal include the preservation of muscle in myocardial infarction and prevention of heart failure, key missions of the NIH. The proposed studies are the first to target protein neddylation in models of cardiac diseases and will employ an innovative approach using genetically modified mice and pharmacologic intervention.