Mitochondrial chaperones mortalin and Tid1 in protein degradation

蛋白质降解中的线粒体伴侣 mortalin 和 Tid1

• 批准号: 8707617
• 负责人: CAROLYN K SUZUKI
• 金额: $ 4万
• 依托单位: RBHS-NEW JERSEY MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-15 至 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8707617
• 关键词: Mitochondrial; chaperones; mortalin; Tid1; protein

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.

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有关，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）缺乏症和肥厚型心肌病。线粒体蛋白位于呼吸链附近，因此蛋白质错误折叠和聚集的风险增加，而呼吸链是活性氧 (ROS) 的强大来源。当暴露于升高的活性氧时，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 依赖性蛋白酶和分子伴侣组成，前者可降解错误折叠和受损的蛋白质，后者可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 选择性地降解正常和异常蛋白质，以响应代谢需求和不断变化的环境条件。基质伴侣 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 也介导蛋白质解聚。该项目的目的是： (1) 阐明 Mortalin 和 Tid1 在蛋白质解聚、重新激活和重折叠中的功能，以及这些伴侣与线粒体 ATP 依赖性蛋白酶 Lon 和 ClpXP 之间的相互作用； (2)鉴定这些蛋白酶和分子伴侣的内源线粒体底物。我们假设线粒体质量控制蛋白酶和伴侣的功能是：(a) 防止异常蛋白质的毒性积累，(b) 促进非天然多肽的降解，以及 (c) 一旦蛋白质解聚，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测定，以研究分子伴侣介导的蛋白质解聚、重新激活/重折叠和报告底物降解。例如，纯化的蛋白质系统将用于研究 Mortalin 和 Tid1 依赖性解聚和不溶性且酶促无活性的葡萄糖-6-磷酸脱氢酶 (aggG6PDH) 的重新激活；而变性的荧光素酶（unfoldedLuc）将用于检查分子伴侣辅助的重折叠。此外，Mortalin、Tid1 和线粒体蛋白酶在易于聚集的鸟氨酸转氨甲酰酶 (aggOTC) 突变体的蛋白质降解中的相互作用将在细胞培养实验中使用 Tid1、Lon 或 ClpP 的敲低细胞系以及纯化的分子伴侣、蛋白酶和 aggOTC。最后，将采用蛋白质组学方法来鉴定 Tid1、Lon 和 ClpP 的内源线粒体底物。 Lon、ClpP 或 Tid1 耗尽后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 Lon 耗尽细胞线粒体内包涵体的积累。我们预测 ClpP 或 Tid1 耗尽的线粒体中也会出现类似的表型。总而言之，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。