Mitochondrial chaperones mortalin and Tid1 in protein degradation

蛋白质降解中的线粒体伴侣 mortalin 和 Tid1

基本信息

批准号：
8192595
负责人：
CAROLYN K SUZUKI
金额：
$ 15.32万
依托单位：
UNIV OF MED/DENT OF NJ-NJ MEDICAL SCHOOL
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-08-15 至 2013-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8192595
关键词：
ATP-Dependent Proteases Aging Alzheimer&apos s Disease Amyotrophic Lateral Sclerosis Antineoplastic Agents Apoptosis Archaea Bacteria Biochemical Biological Assay Butyryl-CoA dehydrogenase Cell Culture Techniques Cell Line Cells Chemicals Cystic Fibrosis Data Defect Disease Drug Delivery Systems Electron Microscopy Endoplasmic Reticulum Eukaryotic Cell Exhibits Family member Functional disorder Future Glucose-6-Phosphate Glucosephosphate Dehydrogenase Goals Health Heat shock proteins Homologous Gene Human Hypertrophic Cardiomyopathy Hypoxia Inclusion Bodies Knowledge Link Luciferases Measures Mediating Metabolic Metabolic stress Mitochondria Mitochondrial Matrix Mitochondrial Proteins Molecular Chaperones Ornithine Carbamoyltransferase Outcome Parkinson Disease Pathway interactions Peptide Hydrolases Phenotype Physiological Protein Family Proteins Proteomics Quality Control Reactive Oxygen Species Reporter Research Respiratory Chain Risk Role Source System Testing Time Yeasts age related base endoplasmic reticulum stress environmental change high throughput screening human disease insight mortalin mutant novel overexpression oxidative damage polypeptide prevent protein aggregation protein degradation protein folding protein misfolding research study response small molecule stable cell line tool

项目摘要

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.

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，前者可降解异常和受损的蛋白质，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。线粒体基质伴侣 Mortalin 和 Tid1 共同发挥作用，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，以及这些分子伴侣与线粒体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。我们假设这些质量控制蛋白酶和分子伴侣的功能是：(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。我们的研究策略是优化生化和细胞培养测定系统，以研究分子伴侣辅助的蛋白质解聚、重新激活/重折叠和报告底物的降解。将采用定量生化测定来测量纯化的 Mortalin 和 Tid1 解聚和重新激活不溶性且酶促失活的葡萄糖-6-磷酸脱氢酶 (aggG6PDH) 的能力。同时，使用敲低或过表达 Tid1、Lon 或 ClpP 细胞系的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甲酰酶 (aggOTC) 易聚集突变体的展开和降解中的相互作用。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。这种方法得到了电子显微镜数据的支持，该数据显示 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。 Lon、ClpP 或 Tid1 耗尽后积累或聚集的线粒体蛋白可能是底物。该项目获得的结果将提供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所需的知识和实验测定。公共健康相关性：该 R21 项目的目的是阐明线粒体伴侣 mortalin 和 Tid1 在蛋白质解聚、重折叠和再激活中的功能，以及它们与线粒体 ATP 依赖性蛋白酶 Lon 和 ClpXP 的协作功能。此外，我们的目标是鉴定这些蛋白酶和伴侣的内源线粒体底物。该项目获得的结果将直接有助于扩展现有的知识和实验工具，以阐明和利用这些质量控制蛋白在蛋白质错误折叠和聚集疾病中的功能，例如帕金森病和阿尔茨海默病、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。