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、肌萎缩性侧索硬化症（ALS）、短链酰基辅酶a脱氢酶（SCAD）缺乏症和肥厚性心肌病。线粒体蛋白位于呼吸链附近，是活性氧（ROS）的强大来源，因此蛋白质错误折叠和聚集的风险增加。当暴露于ROS升高时，线粒体蛋白极易受到氧化损伤和构象缺陷的影响。监督线粒体蛋白质质量控制的监视系统由atp依赖的蛋白酶和分子伴侣组成，前者降解错误折叠和受损的蛋白质，后者介导蛋白质折叠并促进蛋白质降解。在线粒体基质中，atp依赖的蛋白酶Lon和ClpXP选择性地降解正常和异常蛋白，以响应代谢需求和不断变化的环境条件。基质伴侣蛋白Mortalin和Tid1协同作用折叠新生多肽，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Tid1也介导蛋白质分解。本项目的目的是：(1)阐明Mortalin和Tid1在蛋白质分解、再激活和再折叠中的功能，以及这些伴侣蛋白与线粒体atp依赖性蛋白酶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)防止异常蛋白的毒性积累，(b)促进非天然多肽的降解，(c)一旦蛋白质被分解，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化和细胞培养实验，以研究伴侣介导的蛋白质分解，再激活/再折叠和报告底物的降解。例如，纯化蛋白系统将用于研究Mortalin和Tid1依赖性的不溶性和酶失活的葡萄糖-6-磷酸脱氢酶（aggG6PDH）的分解和再激活；而变性荧光素酶（unfoldedLuc）将用于检测伴侣辅助的再折叠。此外，Mortalin， Tid1和线粒体蛋白酶在鸟氨酸转氨基酰基酶（aggOTC）聚集易感突变体蛋白质降解中的相互作用将在细胞培养实验中进行，使用Tid1， Lon或ClpP敲除细胞系，以及纯化的伴侣蛋白，蛋白酶和aggOTC。最后，将采用蛋白质组学方法鉴定内源性线粒体底物Tid1， Lon和ClpP。在Lon， ClpP或Tid1耗竭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。电镜数据显示，在线粒体中有包涵体的积累。我们预测在ClpP-或Tid1-耗尽的线粒体中也会出现类似的表型。总之，从这个项目中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分析。

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.