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: 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
• 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' 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

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多种人类疾病相关，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）缺乏症和肥厚性心肌病。线粒体蛋白质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，线粒体蛋白质对氧化损伤和构象缺陷高度敏感。监督线粒体蛋白质质量控制的监测系统由ATP依赖性蛋白酶和分子伴侣组成，ATP依赖性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变化的环境条件。线粒体基质伴侣Mortalin和Tid 1共同起作用以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Tid 1也介导蛋白质解聚。本研究的目的是：（1）阐明Mortalin和Tid 1在蛋白解聚、再活化和重折叠中的功能，以及它们与线粒体ATP依赖蛋白酶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，和（c）一旦蛋白质分解，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检测系统，研究分子伴侣辅助蛋白解聚，再激活/重折叠和降解的报告底物。将采用定量生物化学测定来测量纯化的Mortalin和Tid 1解聚和再活化不溶性和无酶活性的葡萄糖-6-磷酸脱氢酶（aggG 6PDH）的能力。同时，使用Tid 1、Lon或ClpP的敲低或过表达细胞系的细胞培养实验将用于确定这些质控蛋白在鸟氨酸转氨甲酰酶（aggOTC）的易聚集突变体的解折叠和降解中的相互作用。最后，将进行蛋白质组学方法以鉴定Tid 1、Lon和ClpP的内源性线粒体底物。这种方法是支持电子显微镜数据显示的包涵体内的隆耗尽细胞的线粒体的积累。我们预测在ClpP-或Tid 1-耗尽线粒体类似的表型。Lon、ClpP或Tid 1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。