Novel Signaling Mechanisms and Molecular Targets in the Stressed Myocardium

描述(由申请人提供)： 氧化应激已日益被认为是不同形式的心脏病的共同特征。活性氧簇(ROS)的升高被认为是导致缺血性或非缺血性心力衰竭的汇聚性信号信使。尽管在许多与ROS相关的调查领域取得了重大进展，但仍有两个基本问题尚未解决，并将构成这项拟议调查的中心。第一个问题是，谁是病变心肌中ROS的调节者？第二个问题是ROS的分子靶点是什么，ROS诱导的分子修饰是如何导致心功能障碍的？这一应用的灵感来自于我们将PP2Cm确定为一种新的ROS调节因子的令人兴奋的数据；以及有趣的初步证据，即心脏蛋白酶体复合体是ROS的一类新的分子靶标。因此，拟议的研究将解决ROS信号的一个新方面：PP2Cm对其的调节，并将开始一个基本上未被探索的研究领域：ROS升高的功能后果--它对蛋白酶体系统及其底物的影响。这项应用将确定PP2Cm在ROS生物学中的新角色；它将建立蛋白酶体亚单位作为一组新的ROS升高的分子靶点；它将系统地表征正常和应激心肌中受扰动的蛋白质降解途径。此外，该应用程序将确定潜在的治疗窗口，从而可以挽救中断的蛋白质质量控制。为了实现我们的目标，我们采用了两种相关的心脏应激模型--横主动脉缩窄引起的压力超负荷(TAC)和心肌缺血损伤(I/R)。提出了三个具体的目标：目标1将阐明PP2Cm介导的心脏保护的潜在机制；它将确定其在调控ROS调节中的作用，并使用新建立的PP2Cm内部遗传模型(Null/LacZ敲入KO和心脏条件性诱导Tet-off)来研究PP2Cm基因扰动在TAC和I/R中的影响。目的2将确定20S和26S蛋白酶体在两种应激模型中与蛋白酶体复合体的组装、功能和降解能力有关的作用；它将破译导致ROS损伤的20S和26S蛋白酶体的分子事件。它将应用有针对性的蛋白质组学方法来描述蛋白酶体亚单位的分子修饰；它将定义这种修饰的功能意义。目的3将确定ROS损伤的20S和26S蛋白酶体在两种病理模型中的功能后果；它将描述正常和应激心肌中20S和26S蛋白酶体底物的特征。我们的研究计划得到了一个技术工具箱的支持，该工具箱结合了研究团队收集的既定方法和创新方法。它包括遗传模型、蛋白酶体生物学、ROS生物学、疾病模型、定量蛋白质组学和高分辨率成像。总之，建议的研究将最终确定PP2Cm对应激心肌中ROS的调节；它将建立蛋白酶体亚单位作为ROS的新靶点；它将为两种应激模型中的蛋白质动态平衡提供机械性的见解。

DESCRIPTION (provided by applicant): Oxidative stress has been increasingly recognized as a common feature among different forms of heart disease. Elevated reactive oxygen species (ROS) has been shown as a convergent signaling messenger leading to failing heart either of ischemic or non-ischemic origin. Despite significant progress in many areas of ROS related investigations, two fundamental issues remain unresolved and will constitute the center of this proposed investigation. The first question is who are the regulators of ROS in the diseased myocardium? The second question is what are the molecular targets of ROS and how do ROS-induced molecular modifications result in cardiac dysfunction? This application is inspired by our exciting data identifying PP2Cm as a novel regulator of ROS; and by the intriguing preliminary evidence that the cardiac proteasome complexes are a new class of molecular targets for ROS. Accordingly, the proposed investigation will address a novel aspect of ROS signaling: its modulation by PP2Cm, and it will embark on a largely unexplored area of research: the functional consequences of ROS elevation--its impact on the proteasome systems and their substrates. The application will determine the emerging role of PP2Cm in ROS biology; it will establish proteasome subunits as a new set of molecular targets for the elevated ROS; and it will systematically characterize perturbed protein degradation pathways in the normal and stressed myocardium. Furthermore, the application will identify potential therapeutic windows whereby disrupted protein quality control may be rescued. To accomplish our goals, two related models of cardiac stress--pressure overload by transverse-aortic constriction (TAC) and myocardial ischemic injury (I/R)-are employed. Three specific aims are proposed: Aim 1 will elucidate mechanisms underlying PP2Cm mediated protection of the heart; it will determine its role in governing ROS regulation and examine the impact of genetic perturbations of PP2Cm in TAC and I/R using the newly established PP2Cm genetic models in-house (the null/LacZ knock-in KO and the cardiac conditional inducible Tet-Off). Aim 2 will establish roles of the 20S and 26S proteasomes in the two stress models with respect to proteasome complex assembly, function, and degradation capacity; it will decipher molecular events underlying ROS damaged 20S and 26S proteasomes. It will apply a targeted proteomic approach to delineate the molecular modification of proteasome subunits; and it will define the functional significance of such modifications. Aim 3 will define functional consequences of ROS-injured 20S and 26S proteasomes in the two pathological models; it will characterize the substrate repertoire of 20S and 26S proteasomes in the normal and stressed myocardium. Our research plan is supported by "a technology tool box" combining established methods and innovative approaches assembled by the investigator team. It encompasses genetic models, proteasome biology, ROS biology, disease models, quantitative proteomics, and high-resolution imaging. Collectively, the proposed studies will conclusively characterize PP2Cm regulation of ROS in the stress myocardium; it will establish proteasome subunits as novel targets of ROS; and it will provide mechanistic insights into protein homeostasis in the two stress models.