Cardiac Pathophysiology of Proteasome Phosphoregulation

蛋白酶体磷酸调节的心脏病理生理学

• 批准号: 10033517
• 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/10033517
• 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.

心脏病是人类死亡率和发病率的主要原因。泛素 - 蛋白酶体系统（UPS） 对于细胞中的蛋白质数量和质量控制是关键的。 UPS失调，尤其是蛋白酶体功能 功能不全，在从大部分心脏病的发展到心力衰竭的过程中起着重要作用， 因此，蛋白酶体的增强被视为一种新策略，以增加心脏病 蛋白毒性应激（IPTS）。但是，要开发药理手段以增强蛋白酶体，需要 了解蛋白酶体活动如何受到调节机制的调节 利用以增强蛋白酶体。细胞生物学的最新进展表明， 蛋白酶体通常会增加蛋白酶体的活性，但体内生理意义是蛋白酶体的 尚未建立磷酸化。因此，该项目的目的是提高我们对 特定的蛋白酶体磷酸化如何调节心脏生理和病理生理学。我们的试点研究 在遗传学上证实了小鼠，Ser14处的RPN6/PSMD11的磷酸化负责 蛋白酶体依赖性蛋白激酶（PKA）激活。我们的初步数据进一步表明（1） 在遗传的小鼠中，心肌Ser14-PhopShoryTated RPN6（称为P-RPN6）显着改变 患有心肌缺血或反式运动收缩（TAC）和（2）遗传阻滞的IPT和小鼠 P-RPN6的模仿大大减轻了对各种应激源的心脏反应。因此，我们建议 测试中心假设，即P-RPN6对于26S PSM激活至关重要，以满足对增加的需求 通过追求这些特定目的，在压力心脏肌肉中的选择性蛋白水解：（1）确定 在基线时心脏蛋白质和心脏功能中P-RPN6的必要性，（2）确定 与IPTS的遗传性心脏病中P-RPN6增加，（3）确定P-RPN6在 带有IPT的心脏病。使用基因编辑创建的新鼠标模型以阻止或模拟P-RPN6， 以及P-RPN6特异性抗体以及建立良好的UPS性能记者。 基于串联的质量标签（TMT）多路复用与串联质谱法相结合将用于配置 由P-RPN6在压力心脏中形成的泛素组。这项研究将提供最终的体内 证明了PKA引诱的蛋白酶体激活的分子基础，明确确定 第一次在完整动物中，该关键蛋白酶体磷酸化的（病情）生理意义，以及 说明该法规是否可以用于治疗目的。