Regulation of Proteasome Function in Cardiomyopathies

项目摘要蛋白质水解降解是维持蛋白质动态平衡和破坏受损或错误折叠蛋白质的关键过程。作为细胞内蛋白质降解的主要途径，泛素蛋白酶体系统（UPS）需要精确的调控来维持大多数生物过程，其功能的任何扰动都可能产生有害的后果。UPS的过度激活与癌症和骨骼肌萎缩有因果关系，而UPS抑制是神经退行性疾病中蛋白质聚集的原因。先前的研究已经报道了许多心脏病模型中的蛋白酶体功能障碍，但机制在很大程度上是未知的，因果关系尚未确定。该建议独特地关注UPS在心肌病，进行性和经常致命的心肌疾病中的作用。我实验室的初步数据表明，UPS功能在人类肥厚性（HCM）和终末期扩张型（DCM）心肌病中明显受损，但在DCM小鼠模型中激活。与其他疾病相似，我们有理由提出，心脏中UPS活性的激活或抑制都可能是有害的。在本应用中提出的统一假设是，蛋白水解降解的失调对心肌病的病理生理及其进展为心力衰竭有重要作用。准确了解心肌病中蛋白酶体功能障碍的机制对于建立疾病进展的病因学联系以及开发针对缺陷蛋白水解的新特异性治疗方法至关重要。因此，本研究将探索蛋白酶体失调的潜在独立而非互斥机制。目的1将研究人类心肌病中UPS功能障碍的翻译后机制，特别是使用蛋白质组学技术对蛋白酶体进行磷酸化和氧化修饰。还将研究蛋白酶体功能障碍的潜在后果，包括蛋白质聚集和自噬蛋白水解途径的激活。目的2将关注两种小鼠模型——心肌梗死引起的扩张型心肌病和慢性异丙肾上腺素给药——中蛋白酶体磷酸化的变化。Aim 3的目的是确定HCM-linked sarcomere突变基因表达是否足以在体外直接损害成年大鼠心肌细胞的UPS功能，以及突变蛋白稳定性在这种影响中发挥多大作用。这些实验的结果有望为广泛的心肌病中功能失调的蛋白水解降解的潜在机制提供有价值的见解，并确定治疗干预的新靶点。公共卫生相关性：被称为心肌病的心肌疾病是心力衰竭和过早死亡的主要原因，因此是一个需要相当大的科学和临床进步的重大公共卫生问题。然而，我们对最初的压力事件（例如遗传基因改变，心脏病发作）后心肌病如何发展的理解存在很大差距，这阻碍了有效新疗法的发展。这项拨款申请研究了消除心脏受损蛋白质的缺陷机制如何导致心肌病，其长期目标是确定这些毁灭性疾病的新治疗方法的特定靶点。

DESCRIPTION (provided by applicant): Project abstract Proteolytic degradation is a critical process for maintaining a dynamic equilibrium of proteins and destroying damaged or misfolded proteins. As the major pathway for intracellular protein degradation, the ubiquitin proteasome system (UPS) requires precise regulation to sustain most biological processes and any perturbation in its function may have deleterious consequences. Excessive activation of the UPS has been causally linked to cancer and skeletal muscle atrophy, whereas UPS inhibition is responsible for protein aggregation in neurodegenerative diseases. Previous studies have reported proteasome dysfunction in a number of cardiac disease models, but mechanisms are largely unknown, and causality has not yet been established. This proposal uniquely focuses on the role of the UPS in cardiomyopathies, progressive and often fatal heart muscle diseases. Preliminary data from my laboratory indicate that UPS function is markedly impaired in human hypertrophic (HCM) and end-stage, dilated (DCM) cardiomyopathies, but activated in a mouse model of DCM. Drawing parallels to other diseases, it is reasonable to propose that either activation or inhibition of UPS activity in the heart could be detrimental. The unifying hypothesis put forth in this application is that dysregulation of proteolytic degradation contributes significantly to the pathophysiology of cardiomyopathies and their progression to heart failure. A precise understanding of the mechanisms responsible for proteasome dysfunction in cardiomyopathies will be critical for establishing an etiologic link to disease progression and for development of new specific therapies targeting defective proteolysis. This proposal will therefore explore potential independent, but not mutually exclusive, mechanisms of proteasome dysregulation. Aim 1 will examine post-translational mechanisms for UPS dysfunction in human cardiomyopathies, specifically phosphorylation and oxidative modifications to the proteasome using proteomics techniques. Potential consequences of proteasome dysfunction will also be studied, including protein aggregation and activation of autophagic proteolytic pathways. Aim 2 will focus on changes in proteasome phosphorylation in two mouse models - dilated cardiomyopathy induced by myocardial infarction and chronic isoproterenol administration. The goal of Aim 3 is to determine whether HCM-linked sarcomere mutant gene expression is sufficient to directly impair UPS function in adult rat cardiac myocytes in vitro, and to what extent mutant protein stability plays a role in this effect. Results from the proposed experiments are expected to provide valuable insights into potential mechanisms for dysfunctional proteolytic degradation in a broad range of cardiomyopathies and identify new targets for therapeutic intervention. PUBLIC HEALTH RELEVANCE: Cardiac muscle diseases called cardiomyopathies are the principal cause of heart failure and premature death, and are thus a major public health problem in need of considerable scientific and clinical advancement. However, large gaps in our understanding of how cardiomyopathies progress after an initial stressful event (e.g. inherited gene change, heart attack) hinder the development of effective new therapies. This grant application studies how defective mechanisms for elimination of damaged proteins in the heart contribute to cardiomyopathies, with the long term goal of identifying specific targets for new treatments for these devastating diseases.