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，atp依赖的蛋白酶降解异常和受损的蛋白质，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。线粒体基质伴侣蛋白Mortalin和Tid1一起折叠新生多肽，并协助Lon和ClpXP降解错误折叠的蛋白质。此外，我们的初步结果首次表明，Mortalin和Tid1也介导蛋白质的分解。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，以及它们与线粒体atp依赖性蛋白酶Lon和ClpXP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。我们假设这些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。我们的研究策略是优化生化和细胞培养分析系统，以研究伴侣辅助的蛋白质分解、再激活/再折叠和报告底物的降解。定量生化分析将用于测定纯化的Mortalin和Tid1分解和重新激活不溶性和酶失活的葡萄糖-6-磷酸脱氢酶（aggG6PDH）的能力。同时，使用敲除或过表达Tid1、Lon或ClpP的细胞系进行细胞培养实验，将用于确定这些质量控制蛋白在鸟氨酸转氨基酰基酶（aggOTC）易聚集突变体的展开和降解过程中的相互作用。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。这种方法得到了电镜数据的支持，电镜数据显示，在线粒体中存在包涵体的积累。我们预测在ClpP-或Tid1-耗尽的线粒体中也会出现类似的表型。在Lon、ClpP或Tid1耗竭时积累或聚集的线粒体蛋白可能是底物。从这个项目中获得的结果将提供所需的知识和实验分析，以利用这些线粒体伴侣和蛋白酶在蛋白质错误折叠和可能的其他疾病的管理或治疗中的功能。

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 abnormal 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 both normal and abnormal proteins in response to metabolic demands and changing environmental conditions. The mitochondrial matrix chaperones Mortalin and Tid1, function together to fold nascent polypeptides and assist Lon and ClpXP in the degradation of misfolded proteins. In addition, 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; and (2) to identify endogenous mitochondrial substrates of these proteases and chaperones. We hypothesize that these 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 optimize biochemical and cell culture assay systems to study chaperone-assisted protein disaggregation, reactivation/refolding and degradation of reporter substrates. Quantitative biochemical assays will be employed to measure the ability of purified Mortalin and Tid1 to disaggregate and reactivate insoluble and enzymatically inactive glucose-6- phosphate dehydrogenase (aggG6PDH). In parallel, cell culture experiments using knockdown or overexpressing cell lines for Tid1, Lon or ClpP, will be used to determine the interplay between these quality control proteins in the unfolding and degradation of an aggregation-prone mutant of ornithine transcarbamylase (aggOTC). Lastly, a proteomics approach will be undertaken to identify endogenous mitochondrial substrates of Tid1, Lon and ClpP. This approach is supported by electron microscopy data showing the accumulation of inclusion bodies within mitochondria of Lon-depleted cells. We predict a similar phenotype in ClpP- or Tid1- depleted mitochondria. Mitochondrial proteins that accumulate or aggregate upon depletion of Lon, ClpP or Tid1 are likely to be substrates. The results obtained from this project will provide the knowledge and experimental assays needed to exploit the function of these mitochondrial chaperones and proteases in the management or treatment of protein-misfolding and possibly other diseases. PUBLIC HEALTH RELEVANCE: The aims of this R21 project are to elucidate the functions of the mitochondrial chaperones mortalin and Tid1 in protein disaggregation, refolding and reactivation, as well as their cooperative function with the mitochondrial ATP-dependent proteases Lon and ClpXP. In addition, we aim to identify endogenous mitochondrial substrates of these proteases and chaperones. The results obtained from this project will directly contribute to expanding current knowledge and experimental tools required to elucidate and exploit the function of these quality control proteins in protein misfolding and aggregation diseases such as- Parkinson's and Alzheimer's disease, cystic fibrosis, amyotrophic lateral sclerosis (ALS), short chain acyl-CoA dehydrogenase (SCAD) deficiency and hypertrophic cardiomyopathy.