Mitochondrial chaperones mortalin and Tid1 in protein degradation

描述（由申请人提供）：蛋白质错误折叠和聚集与衰老以及多种人类疾病相关，包括帕金森病、囊性纤维化、肌萎缩侧索硬化症（ALS）、短链酰基辅酶A脱氢酶（SCAD）缺乏症和肥厚性心肌病。线粒体蛋白质的蛋白质错误折叠和聚集的风险增加，因为它们位于呼吸链附近，这是活性氧（ROS）的强大来源。当暴露于升高的ROS时，线粒体蛋白质对氧化损伤和构象缺陷高度敏感。监督线粒体蛋白质质量控制的监测系统由ATP依赖性蛋白酶和分子伴侣组成，ATP依赖性蛋白酶降解错误折叠和受损的蛋白质，分子伴侣介导蛋白质折叠并促进蛋白质降解。在线粒体基质内，ATP依赖性蛋白酶Lon和ClpXP选择性降解正常和异常蛋白质以响应代谢需求和变化的环境条件。基质分子伴侣Mortalin和Tid 1协同作用以折叠新生多肽并协助Lon和ClpXP介导的蛋白水解。有趣的是，我们的初步结果首次表明，Mortalin和Tid 1也介导蛋白质解聚。本研究的目的是：（1）阐明Mortalin和Tid 1在蛋白质解聚、再活化和重折叠中的功能，以及它们与线粒体ATP依赖蛋白酶Lon和ClpXP之间的相互作用;（2）鉴定这些蛋白酶和伴侣蛋白的内源性线粒体底物。我们假设线粒体质量控制蛋白酶和伴侣蛋白的功能是-（a）防止异常蛋白质的毒性积累，（B）促进非天然多肽的降解，和（c）一旦蛋白质分解，重新激活蛋白质的功能状态。我们的研究策略是开发和优化生物化学和细胞培养试验，以研究分子伴侣介导的蛋白质解聚，再激活/重折叠和降解的报告底物。例如，纯化的蛋白质系统将用于研究Mortalin和Tid 1依赖性解聚和不溶性和无酶活性的葡萄糖-6-磷酸脱氢酶（aggG 6PDH）的再活化;而变性荧光素酶（unfoldedLuc）将用于检查分子伴侣辅助的再折叠。此外，将在细胞培养实验中使用Tid 1、Lon或ClpP的敲低细胞系以及纯化的伴侣蛋白、蛋白酶和aggOTC，进行Mortalin、Tid 1和线粒体蛋白酶在鸟氨酸转氨甲酰酶（aggOTC）的易聚集突变体的蛋白质降解中的相互作用。最后，将进行蛋白质组学方法以鉴定Tid 1、Lon和ClpP的内源性线粒体底物。在Lon、ClpP或Tid 1耗尽时积累或聚集的线粒体蛋白可能是这些质量控制蛋白的底物。电子显微镜数据显示在Lon-depleted细胞的线粒体内包涵体的积累。我们预测在ClpP-或Tid 1-耗尽线粒体类似的表型。总之，从该项目获得的结果将提供进一步阐明和利用这些线粒体伴侣和蛋白酶在蛋白质错误折叠疾病的管理或治疗中的蛋白质质量控制功能所需的知识和实验测定。

DESCRIPTION (provided by applicant): Protein misfolding and aggregation are associated with aging, as well as a variety of human diseases, including Parkinson's disease, cystic fibrosis, amyotrophic lateral sclerosis (ALS), short chain acyl-CoA dehydrogenase (SCAD) deficiency and hypertrophic cardiomyopathy. Mitochondrial proteins are at increased risk of protein misfolding and aggregation as they are located in proximity to the respiratory chain, which is a powerful source of reactive oxygen species (ROS). When exposed to elevated ROS, mitochondrial proteins are highly susceptible to oxidative damage and conformational defects. The surveillance system that oversees mitochondrial protein quality control is composed of ATP-dependent proteases, which degrade misfolded and damaged proteins, as well as molecular chaperones, which mediate protein folding and facilitate protein degradation. Within the mitochondrial matrix, the ATP-dependent proteases Lon and ClpXP selectively degrade normal and abnormal proteins in response to metabolic demands and changing environmental conditions. The matrix chaperones Mortalin and Tid1, function in concert to fold nascent polypeptides and to assist Lon- and ClpXP- mediated proteolysis. Interestingly, our preliminary results show for the first time, that Mortalin and Tid1 also mediate protein disaggregation. The aims of this project are- (1) to elucidate the functions of Mortalin and Tid1 in protein disaggregation, reactivation and refolding, as well as the interplay between these chaperones and the mitochondrial ATP-dependent proteases Lon and ClpXP; (2) to identify endogenous mitochondrial substrates of these proteases and chaperones. We hypothesize that mitochondrial quality control proteases and chaperones function to- (a) prevent the toxic accumulation of abnormal proteins, (b) facilitate the degradation of non-native polypeptides and (c) reactivate the functional state of proteins once they are disaggregated. Our research strategy is to develop and optimize biochemical and cell culture assays to study chaperone-mediated protein disaggregation, reactivation/refolding and degradation of reporter substrates. For example, a purified protein system will be employed to study Mortalin- and Tid1- dependent disaggregation and reactivation of insoluble and enzymatically inactive glucose-6-phosphate dehydrogenase (aggG6PDH); whereas denatured luciferase (unfoldedLuc) will be used to examine chaperone-assisted refolding. In addition, the interplay between Mortalin, Tid1 and mitochondrial proteases in protein degradation of an aggregation-prone mutant of ornithine transcarbamylase (aggOTC) will be carried out in cell culture experiments using knockdown cell lines for Tid1, Lon or ClpP, as well as with purified chaperones, proteases and aggOTC. Lastly, a proteomics approach will be undertaken to identify endogenous mitochondrial substrates of Tid1, Lon and ClpP. Mitochondrial proteins that accumulate or aggregate upon depletion of Lon, ClpP or Tid1 are likely to be substrates of these quality control proteins. Electron microscopy data show the accumulation of inclusion bodies within mitochondria of Lon-depleted cells. We predict a similar phenotype in ClpP- or Tid1- depleted mitochondria. Taken together, the results obtained from this project will provide the knowledge and experimental assays needed to elucidate further and exploit the protein quality control function of these mitochondrial chaperones and proteases in the management or treatment of protein-misfolding diseases.