Role of Protein Import in the Development of the Diabetic Heart

项目摘要 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 是疾病的核心，它加速收缩障碍，导致死亡。然而，精确 导致糖尿病心脏中线粒体功能障碍的机制仍不清楚。使用2型糖尿病 在人（患者）和小鼠（db/db）模型中，我们观察到线粒体结构的明显破坏， 功能，这与线粒体蛋白质的丢失有关。绝大多数线粒体 蛋白质是核基因组编码的，需要输入到细胞核中。导入通过 一套协调的机器，包含一个主动马达，由线粒体热休克蛋白70驱动 （mtHsp70）。我们观察到2型糖尿病患者心肌线粒体mtHsp 70含量降低， db/db小鼠，和线粒体蛋白质输入减少。输入后，线粒体蛋白质被重新折叠， 使其具有功能。MtHsp 70也参与了重折叠过程， 与Lon肽酶1，线粒体（LonP 1），线粒体基质的AAA+蛋白酶协同作用。我们 还观察到2型糖尿病心脏中LonP 1的减少。当线粒体蛋白质输入不 正常运作时，聚集的核基因组编码蛋白质积累在细胞的外部， 线粒体前体的过度积累应激。目前它 目前还不清楚是什么因素导致蛋白质输入效率和重折叠的降低， 这些过程可以恢复线粒体蛋白质组组成，线粒体功能和心脏收缩功能。 糖尿病心脏的表现。我们提出的研究解决了这一关键的知识差距。的信息 将增强我们对这些过程的理解，并有助于开发治疗策略， 靶向导致线粒体蛋白丢失的特定输入成分。核心假设是 试验表明，2型糖尿病心脏中蛋白质输入和重折叠的减少导致线粒体的损失， 蛋白质和线粒体前体过度积累应激导致线粒体功能障碍， 收缩性损伤本申请的目的是（1）鉴定亚线粒体位置， 2型糖尿病线粒体中蛋白质的输入受损，以及对输入机制的影响;（2）评估 2型糖尿病对线粒体蛋白复性的影响及mtHsp 70的协同作用 和LonP 1;以及（3）确定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，由于线粒体蛋白质输入失败，导致线粒体前体过度积累应激。 这些研究的完成有望为深入了解这些机制提供基本的分子见解。 导致2型糖尿病心脏中核基因组编码的线粒体蛋白的丢失， 细胞后果导致线粒体和心脏功能障碍。

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.