Cardiac Pathophysiology of Proteasome Phosphoregulation

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) 是细胞内蛋白质数量和质量控制的关键。UPS调节失调，尤其是蛋白酶体功能 心功能不全在从一大类心脏疾病发展为心力衰竭的过程中起着重要作用， 因此，蛋白酶体增强被认为是治疗心脏病的一种新策略。 蛋白毒性应激(IPTS)。然而，要发展增强蛋白酶体的药理手段，需要 了解蛋白酶体活动是如何被调节的，因为这样的调节机制可能是 被用来增强蛋白酶体。细胞生物学的最新进展表明，细胞周期蛋白的磷酸化 蛋白酶体经常增加蛋白酶体的活性，但蛋白酶体在体内的生理意义 磷调节还没有建立起来。因此，这个项目的目标是增进我们对 特定的蛋白酶体磷酸化如何调节心脏生理学和病理生理学。我们的初步研究 已经在小鼠身上证实了RPN6/PSMD11在Ser14上的磷酸化是导致 CAMP依赖的蛋白激酶(PKA)激活蛋白酶体。我们的初步数据进一步显示(1) 遗传性心脏病小鼠心肌Ser14磷酸化Rpn6(简称p-Rpn6)明显改变 IPTS和小鼠心肌缺血或经主动脉缩窄(TAC)和(2)遗传阻断 P-Rpn6的模拟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激活是必不可少的，以满足日益增长的 应激心肌的选择性蛋白分解，通过追求这些特定的目标：(1)确定 基础状态下p-Rpn6在心脏蛋白平衡和心功能中的必要性，(2)确定p-Rpn6的作用 P-Rpn6在遗传性心脏病合并IPTS中的升高，以及(3)确定p-Rpn6在遗传性心脏病中的作用 一种获得性心脏病合并IPTS。通过基因编辑创建的新小鼠模型可以阻断或模仿p-Rpn6， 以及p-Rpn6特异性抗体将与成熟的UPS性能报告程序一起使用。 基于串联质量标签(TMT)的多路复用与串联质谱学将被用于分析 应激心脏中由p-Rpn6塑造的泛素体。这项研究将提供最终的活体实验 证明了PKA诱导的蛋白酶体激活的分子基础，明确地确定了 首次在完整的动物中发现这一关键的蛋白酶体磷化调节的(病理)生理意义，以及 说明这一规定是否可以用于治疗目的。

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.