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.

心脏病是人类死亡和发病的主要原因。泛素蛋白酶体系统 (UPS) 对于细胞内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失调 供血不足，在大部分心脏病发展为心力衰竭的过程中发挥着重要作用， 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，要开发增强蛋白酶体的药理学方法，需要 了解蛋白酶体活性如何受到调节，因为这种调节机制可能是 用于增强蛋白酶体。细胞生物学的最新进展表明，磷酸化 蛋白酶体经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，该项目的目标是增进我们对 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 已在小鼠中通过基因证实 RPN6/PSMD11 Ser14 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表明（1） 遗传性小鼠心肌 Ser14 磷酸化 Rpn6（称为 p-Rpn6）显着改变 IPTS 和遭受心肌缺血或经主动脉缩窄 (TAC) 的小鼠和 (2) 基因阻断 p-Rpn6 的模仿大大减轻了心脏对各种应激源的反应。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，以满足日益增长的需求 通过追求以下特定目标，对受压心肌进行选择性蛋白水解：(1) 确定 p-Rpn6 在基线心脏蛋白质稳态和心脏功能中的必要性，(2) 确定 p-Rpn6 在患有 IPTS 的遗传性心脏病中增加，以及 (3) 确定增加的 p-Rpn6 在 患有 IPTS 的后天性心脏病。通过基因编辑创建的新小鼠模型可阻断或模拟 p-Rpn6， 以及 p-Rpn6 特异性抗体将与完善的 UPS 性能报告器一起使用。 基于串联质量标签 (TMT) 的多路复用与串联质谱法相结合将用于分析 在应激心脏中由 p-Rpn6 形成的泛素组。这项研究将提供最终的体内 证明 PKA 引发的蛋白酶体激活的分子基础，明确确定 首次揭示了完整动物中这种关键蛋白酶体磷酸调节的（病理）生理意义，以及 说明该调节是否可用于治疗目的。