Cardiac Pathophysiology of Proteasome Phosphoregulation

蛋白酶体磷酸调节的心脏病理生理学

• 批准号: 10435491
• 负责人: XUEJUN WANG
• 金额: $ 36.75万
• 依托单位: UNIVERSITY OF SOUTH DAKOTA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10435491
• 关键词: 26S proteasome; Adrenergic Agents; Age; Animals; Antibodies; Attenuated; Basic Science; Cardiac; Cardiac Myocytes; Cardiac health; Cause of Death; Cells; Cellular biology; Chronic; Clinical Management; Color; Coupled; Cultured Cells; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Data; Diagnosis; Disease; Functional disorder; Future; Genes; Genetic; Genetic Models; Goals; Healthcare; Heart; Heart Diseases; Heart Hypertrophy; Heart failure; Inherited; Knock-in; Life; Longevity; Molecular; Mus; Myocardial; Myocardial Infarction; Myocardial Ischemia; Myocardium; Nature; Nodal; Pathologic; Pathology; Pathway interactions; Performance; Pharmacology; Phosphorylation; Phosphotransferases; Physiological; Physiology; Pilot Projects; Play; Protein Kinase; Proteins; Proteolysis; Quality Control; Regulation; Reporter; Reporting; Research; Research Project Grants; Role; Severities; Stress; System; Testing; Therapeutic; Time; Touch sensation; Transgenic Mice; Ubiquitin; Virulence Factors; Wild Type Mouse; Work; base; body system; constriction; disability; heart function; human morbidity; human mortality; in vivo; innovation; mimicry; mouse model; multicatalytic endopeptidase complex; novel therapeutic intervention; overexpression; pressure; prevent; protein activation; protein degradation; proteostasis; proteotoxicity; response; stressor; tandem mass spectrometry; tool; virtual

Heart disease is the leading cause of human mortality and morbidity. The ubiquitin-proteasome system (UPS) is pivotal to protein quantity and quality control in the cell. UPS dysregulation, especially proteasome functional insufficiency, plays a major role in the progression from a large subset of heart diseases to heart failure and, accordingly, proteasome enhancement is implicated as a new strategy to treat heart disease with increased proteotoxic stress (IPTS). To develop pharmacological means to enhance the proteasome, however, requires understanding how proteasome activity is regulated as such regulatory mechanisms could potentially be exploited to enhance the proteasome. Recent advances in cell biology show that phosphorylation of the proteasome often increases proteasome activities but the in vivo physiological significance of proteasome phosphoregulation has not been established. Thus, the goal of this project is to advance our understanding on how specific proteasome phosphorylation regulates cardiac physiology and pathophysiology. Our pilot studies have confirmed genetically in mice that phosphorylation of RPN6/PSMD11 at Ser14 is responsible for proteasome activation by cAMP-dependent protein kinase (PKA). Our preliminary data further revealed that (1) myocardial Ser14-phopshorylated Rpn6 (referred to as p-Rpn6) was markedly altered in mice with inherited IPTS and mice subjected to myocardial ischemia or trans-aortic constriction (TAC) and (2) genetic blockade and mimicry of p-Rpn6 substantially mitigated cardiac responses to various stressors. Hence, we propose to test the central hypotheses that p-Rpn6 is essential to 26S Psm activation to meet the increased demand for selective proteolysis in stressed cardiac muscle, via pursuit of these specific aims: (1) to determine the necessity of p-Rpn6 in cardiac proteostasis and cardiac function at baseline, (2) to determine the role of increased p-Rpn6 in the inherited heart disease with IPTS, and (3) to determine the role of increased p-Rpn6 in an acquired heart disease with IPTS. New mouse models created with gene editing to block or mimic p-Rpn6, as well as p-Rpn6 specific antibodies will be used along with a well-established UPS performance reporter. Tandem mass-tags (TMT) based multiplexing coupled with tandem mass spectrometry will be used to profile ubiquitinomes shaped by p-Rpn6 in stressed hearts. This research will provide the ultimate in vivo demonstration for the molecular basis of PKA-elicited proteasome activation, determine unequivocally for the first time the (patho)physiological significance of this key proteasome phosphoregulation in intact animals, and illustrate whether this regulation can be exploited for therapeutic purposes.

心脏病是人类死亡和发病的主要原因。泛素-蛋白酶体系统（UPS） 对细胞中蛋白质的数量和质量控制至关重要。UPS失调，尤其是蛋白酶体功能 心功能不全，在从大部分心脏病发展为心力衰竭中起主要作用， 因此，蛋白酶体增强被认为是治疗心脏病的新策略， 蛋白毒性应激（IPTS）。然而，为了开发增强蛋白酶体的药理学手段，需要 了解蛋白酶体活性是如何调节的，因为这种调节机制可能是 用来增强蛋白酶体细胞生物学的最新进展表明， 蛋白酶体通常增加蛋白酶体活性，但蛋白酶体的体内生理意义 磷酸调节尚未建立。因此，本项目的目标是促进我们对 特异性蛋白酶体磷酸化如何调节心脏生理学和病理生理学。我们的试点研究 已经在小鼠中证实了RPN 6/PSMD 11在Ser 14处的磷酸化是导致 cAMP依赖性蛋白激酶（PKA）激活蛋白酶体。我们的初步数据进一步显示，（1） 心肌Ser 14-磷酸化Rpn 6（简称p-Rpn 6）在遗传性心肌病小鼠中显著改变， IPTS和经受心肌缺血或经主动脉缩窄（TAC）和（2）基因阻断的小鼠 并且p-Rpn 6的模拟物基本上减轻了对各种应激源的心脏反应。因此，我们建议 检验中心假设，即p-Rpn 6对26 S Psm活化是必不可少的，以满足对 应激心肌中的选择性蛋白水解，通过追求这些特定的目标：（1）确定 p-Rpn 6在基线时心脏蛋白质稳态和心脏功能中的必要性，（2）确定 p-Rpn 6在伴有IPTS的遗传性心脏病中的作用，以及（3）确定p-Rpn 6在 患有IPTS的后天性心脏病通过基因编辑来阻断或模拟p-Rpn 6的新小鼠模型， 以及p-Rpn 6特异性抗体将沿着使用完善的UPS性能报告物。 将使用基于串联质量标签（TMT）的多路复用与串联质谱联用来分析 应激心脏中由p-Rpn 6形成的泛素组。这项研究将提供最终的体内 证明PKA引起的蛋白酶体激活的分子基础，明确确定 首次在完整动物中发现了这种关键蛋白酶体磷酸化调节的（病理）生理学意义， 说明这种调节是否可以用于治疗目的。