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：
8311645
负责人：
CAROLYN K SUZUKI
金额：
$ 13.36万
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 2013-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8311645
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.

描述（由申请人提供）：蛋白质错误折叠和聚集与衰老以及各种人类疾病有关，包括帕金森氏病，囊性纤维化，肌萎缩性侧面硬化症（ALS），短链酰基酰基-COAA酰基-COA脱氢酶（SCAD）缺乏症（SCAD）缺乏症和过度性心脏瘤性。线粒体蛋白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依赖性蛋白酶组成，ATP依赖性蛋白酶降解异常和受损的蛋白质以及分子伴侣，介导蛋白质折叠并促进蛋白质降解。在线粒体基质中，ATP依赖性蛋白酶和CLPXP在响应代谢需求和不断变化的环境条件下选择性降解正常和异常蛋白质。线粒体基质伴侣mortalin和tid1一起起作用以折叠新生的多肽，并帮助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还介导蛋白质分解。该项目的目的是：（1）阐明玛alin和tid1在蛋白质分解，重新分解和重折叠的功能，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）确定这些蛋白酶和伴侣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 - （a）防止异常蛋白质的毒性积累，（b）促进非本地多肽的降解，并且（c）一旦蛋白质被否决了蛋白质的功能状态。我们的研究策略是优化生物化学和细胞培养测定系统，以研究伴侣辅助的蛋白质分解，重新激活/重新折叠和降解报告基质底物。定量生化测定将用于测量纯化的玛alin和tid1分解和重新激活不溶性和酶无活性的6-磷酸脱氢酶（AGGG6PDH）的能力。同时，将使用用于TID1，LON或CLPP的敲低或过表达细胞系的细胞培养实验，以确定这些质量控制蛋白在鸟氨酸经钙化酶（Aggotc）的聚合易受共同突变体的展开和降解中之间的相互作用。最后，将采取一种蛋白质组学方法来识别TID1，LON和CLPP的内源性线粒体底物。这种方法得到了电子显微镜数据的支持，表明包含物体在lon耗尽细胞的线粒体内的积累。我们预测在clPP或TID1耗尽的线粒体中类似的表型。 LON，CLPP或TID1耗尽后积累或聚集的线粒体蛋白可能是底物。从该项目获得的结果将提供利用这些线粒体伴侣的功能以及蛋白酶在蛋白质不满意和可能其他疾病的管理或治疗中所需的知识和实验测定。