Direct proteasomal enhancement contributes to PKG-triggered cardioprotection

描述（由申请方提供）：直接蛋白酶体增强有助于PKG触发的心脏保护缺血性心脏病（IHD）是美国最常见的心脏病，是人类发病率和死亡率的主要原因。缺血/再灌注损伤（I/R）是缺血性心脏病（IHD）介入治疗和自然病程中的重要病理过程。除了基因突变，心肌肥大和缺血/再灌注（I/R）也可以迅速增加错误折叠蛋白的产生。在正常情况下，心肌细胞通过泛素蛋白酶体系统去除错误折叠的蛋白质。高度调节的过程需要靶蛋白的泛素化，然后将其转移到蛋白酶体进行降解。心脏蛋白酶体功能不全与泛素化蛋白的积累有关，而泛素化蛋白是大多数心脏疾病的特征。改善蛋白酶体功能将是增强泛素化蛋白质去除的战略方法;然而，这样做的已知方法仍然难以捉摸。蛋白酶体亚基的翻译后修饰引起蛋白酶体活性对受体刺激/应激的即时适应，并且靶向修饰可以证明具有显著的治疗益处。该博士后研究项目的目的是阐明PKG操纵蛋白酶体的新机制，从而增加蛋白酶体活性，并确定蛋白酶体增强是否有助于PKG触发的心脏保护。这项研究的结果将有助于开发一种更巧妙的方法，通过改善蛋白酶体功能来治疗心脏病。 已知对心脏保护具有积极作用的磷酸二酯酶5抑制剂（例如西地那非）可增加PKG活性，但其保护机制尚不清楚。该提案的具体目的是将PKG途径的机制分析与PKG治疗的生理效应结合起来。该项目的第一个目标将采用全面的蛋白质组学方法来确定PKG对蛋白酶体的翻译后修饰位点，并评估PKG诱导的蛋白酶体活性变化。这将使，并且是必不可少的，确定之间的因果关系的特定PKG磷酸化和蛋白酶体功能的变化。第二个目标将测试的假设，蛋白酶体增强PKG操纵有助于心脏保护。一个独特的小鼠模型的心肌细胞限制性中度表达的肽酶失能蛋白酶体亚单位将进行I/R手术与比较西地那非和安慰剂治疗。这项工作将描绘一个新的机制，蛋白酶体功能的调节，并产生重要的新见解PKG途径如何保护心脏。这将最终促进寻找预防和/或更有效地治疗心脏病的新措施。

DESCRIPTION (provided by applicant): Direct proteasomal enhancement contributes to PKG-triggered cardioprotection Ischemic heart disease (IHD) is the most common heart disease in the US, a leading cause of morbidity and mortality in humans. Ischemia/reperfusion (I/R) injury is an important pathological process during not only the intervention but also the natural disease progress of IHD. Besides genetic mutations, cardiac hypertrophy and ischemia/reperfusion (I/R) can both rapidly increase production of misfolded proteins as well. Under normal conditions, cardiomyocytes remove misfolded proteins via the ubiquitin proteasome system. The highly regulated process requires ubiquitination of the targeted protein followed by its transfer to the proteasome for degradation. Cardiac proteasome functional insufficiency has been implicated in the accumulation of ubiquitinated proteins that characterize most heart diseases. Improving proteasome function would be a strategic approach to enhance removal of ubiquitinated proteins; however a known method to do so remains elusive. Posttranslational modifications of proteasome subunits elicit instant adaptation of proteasome activities to receptor stimulation/stress, and targeted modifications could prove of significant therapeutic benefit. The aim of the research project of this postdoctoral fellowship is to elucidate a novel mechanism of PKG manipulation on the proteasome that results in increased proteasome activity and determine if the proteasome enhancement contributes to PKG-triggered cardioprotection. Results of the research would enable development of a more tactful approach to treat heart disease by way of improving proteasome function. PKG activity can be increased by phosphodiesterase 5 inhibitors (e.g. sildenafil) that have known positive effects on cardioprotection, though their mechanism of protection is unknown. The specific aims of this proposal couple mechanistic analysis of the PKG pathway with physiological effect of PKG treatment. The first aim of the project will employ a comprehensive proteomics approach to determine the site(s) of posttranslational modification by PKG on the proteasome and assess PKG-induced changes in proteasome activity. This will enable, and is essential to, determination of the cause-effect relationship between the specific PKG phosphorylation and proteasome functional change. The second aim will test the hypothesis that proteasomal enhancement by PKG manipulation contributes to cardioprotection. A unique mouse model of cardiomyocyte-restricted moderate expression of a peptidase-disabled proteasome subunit will undergo I/R surgery with comparative sildenafil and placebo treatments. The work will delineate a novel mechanism by which proteasome function is regulated and yield significant new insight into how the PKG pathway protects the heart. This will ultimately facilitate the search for new measures to prevent and/or more effectively treat heart disease.