Role of Protein Import in the Development of the Diabetic Heart

项目总结 心血管并发症是糖尿病患者死亡的主要原因。线粒体功能障碍 是这种疾病的核心，它会导致收缩功能受损，导致死亡。然而，准确的 导致糖尿病心脏线粒体功能障碍的机制尚不清楚。使用2型糖尿病 在人类(病人)和小鼠(db/db)模型中，我们观察到线粒体结构和 功能，这与线粒体蛋白的丢失有关。绝大多数线粒体 蛋白质是核基因组编码的，需要输入到线粒体中。导入通过 由线粒体热休克蛋白70驱动的一套包含主动马达的协调机械 (MtHsp70)。我们观察到2型糖尿病患者心肌线粒体mtHsp70含量降低。 Db/db小鼠，线粒体蛋白进口减少。进口后，线粒体蛋白质被重新折叠 变成原生结构才能发挥作用。MtHsp70也参与了重折叠过程，并在 与线粒体基质的AAA+蛋白酶Lon Peptidase 1，线粒体(LonP1)有协同作用。我们 在2型糖尿病心脏中也观察到LonP1的减少。当线粒体蛋白输入不是 功能正常，聚集的核基因组编码蛋白聚集在细胞的外部 线粒体导致一种称为线粒体前体过度积累应激的现象。目前，它 目前还不清楚是什么因素导致蛋白质进口效率和复性的降低，或者是操纵 这些过程可以恢复线粒体蛋白质组组成、线粒体功能和心肌收缩 在糖尿病患者心脏中的表现。我们建议的研究解决了知识中的这一关键差距。这些信息 将增强我们对这些过程的理解，并有助于制定治疗策略 针对导致线粒体蛋白丢失的特定进口成分。核心假设是 试验表明，2型糖尿病心脏中蛋白质输入和复性减少会导致线粒体的丢失 蛋白质和线粒体前体过度堆积应激导致线粒体功能障碍和 收缩功能受损。这项应用的目标是(1)识别亚线粒体位置 2型糖尿病患者线粒体蛋白质输入受损及其对输入机制的影响；(2)评估 2型糖尿病对线粒体蛋白质重折叠的影响及mtHsp70的协同作用 和LonP1；以及(3)确定在2型糖尿病患者中发生的线粒体和蛋白质组应激的程度 心脏，由于线粒体蛋白输入失败，导致线粒体前体过度积聚应激。 这些研究的完成有望提供对这种机制的基本分子洞察。 导致2型糖尿病心脏核基因组编码的线粒体蛋白丢失 导致线粒体和心脏功能障碍的细胞后果。

PROJECT SUMMARY Cardiovascular complications account for the majority of deaths in diabetic patients. Mitochondrial dysfunction is central to the disease and it precipitates contractile impairment, leading to death. However, the precise mechanisms that cause mitochondrial dysfunction in the diabetic heart remain unclear. Using type 2 diabetic human (patient) and mouse (db/db) models, we observed pronounced disruption to mitochondrial structure and function, which were associated with the loss of mitochondrial proteins. The vast majority of mitochondrial proteins are nuclear genome-encoded and require import into the mitochondrion. Import occurs through a coordinated set of machinery containing an active motor, driven by mitochondrial heat shock protein 70 (mtHsp70). We observed decreased mtHsp70 content in cardiac mitochondria from type 2 diabetic patients and db/db mice, and a decrease in mitochondrial protein import. Following import, mitochondrial proteins are refolded into native structures to become functional. MtHsp70 also participates in the refolding process, and works synergistically with Lon Peptidase 1, Mitochondrial (LonP1), an AAA+ protease of the mitochondrial matrix. We have also observed a decrease in LonP1 in the type 2 diabetic heart. When mitochondrial protein import is not functioning properly, aggregated nuclear genome-encoded proteins accumulate on the exterior of the mitochondrion leading to a phenomenon termed mitochondrial precursor over-accumulation stress. Currently, it is unclear what factors contribute to a decrease in protein import efficiency and refolding or whether manipulation of these processes can restore mitochondrial proteomic make-up, mitochondrial function and cardiac contractile performance in the diabetic heart. Our proposed studies address this critical gap in knowledge. The information will enhance our understanding of these processes and aid in the development of therapeutic strategies that target specific import constituents that contribute to loss of mitochondrial proteins. The central hypothesis to be tested is that decreased protein import and refolding in the type 2 diabetic heart causes loss of mitochondrial proteins and mitochondrial precursor over-accumulation stress leading to mitochondrial dysfunction and contractile impairment. The objectives of this application are to (1) identify submitochondrial locations where protein import is compromised in type 2 diabetic mitochondria and the impact on import machinery; (2) evaluate the impact of type 2 diabetes mellitus on mitochondrial protein refolding and the synergistic influence of mtHsp70 and LonP1; and (3) determine the extent of mitochondrial and proteomic stress that occurs in the type 2 diabetic heart, due to failed mitochondrial protein import contributing to mitochondrial precursor over-accumulation stress. Completion of these studies is expected to provide fundamental molecular insight into the mechanisms contributing to the loss of nuclear genome-encoded mitochondrial proteins in the type 2 diabetic heart and the cellular consequences leading to mitochondrial and cardiac dysfunction.