Exploring respiratory chain glycoproteomics in mitochondrial disease

探索线粒体疾病中的呼吸链糖蛋白组学

• 批准号: 9333395
• 负责人: YAIR ARGON
• 金额: $ 59.25万
• 依托单位: CHILDREN'S HOSP OF PHILADELPHIA
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-15 至 2019-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9333395
• 关键词: Affinity; Area; Biology; Brain; Caenorhabditis elegans; Cell Line; Cells; Clinical; Complex; Computer Simulation; Congenital Disorders; Data; Development; Disease; Embryo; Endoglycosidases; Fibroblasts; Functional disorder; Gel; Genes; Genetic; Glycoproteins; Glycoside Hydrolases; Goals; Hepatic; Hereditary Disease; Human; Inborn Genetic Diseases; Inherited; Knock-out; Knockout Mice; Knowledge; Lectin; Link; MALDI-TOF Mass Spectrometry; Maintenance; Mass Spectrum Analysis; Meta-Analysis; Metabolic Diseases; Mitochondria; Mitochondrial DNA; Mitochondrial Diseases; Mitochondrial Proteins; Modeling; Modification; Mouse Strains; Mus; Nature; Neurologic; Nuclear; Oxidative Stress; Pathogenesis; Patients; Peptide N-Glycosidase; Peptides; Pharmacology; Polysaccharides; Protein Subunits; Proteins; Proteome; Regulation; Reporting; Respiratory Chain; Role; Rotenone; Severities; Site; Structure; Testing; Time; Transferase; Translations; base; effective therapy; experimental study; glycoproteomics; glycosylation; loss of function mutation; mitochondrial dysfunction; novel; protein folding; therapeutic target; transcriptome

. Primary mitochondrial respiratory chain (RC) diseases and congenital disorders of glycosylation (CDG) are collectively common metabolic diseases that cause an overlapping spectrum of disrupted brain development and multi-systemic disease. Much remains to be discovered about the nature and regulation of the mitochondrial glycoproteome and its potential relevance to inherited disorders of RC function or glycosylation. Our preliminary data suggest that: [1] Mitochondria from diverse species have unique N-linked glycome modifications assessed by MALDI-TOF mass spectrometry (MS); [2] Several RC proteins are N-glycosylated; and [3] Patient fibroblast cell lines (FCL), C. elegans, and mouse strains lacking the N-glycanase NGLY1 have mitochondrial depletion, RC dysfunction, and increased oxidative stress. The overall goal of this proposal is to characterize the mitochondrial RC glycoproteome and its regulation, and clarify its relevance to inherited disorders of RC function and NGLY1 activity. We hypothesize that glycosylation and deglycosylation of mitochondrial glycoproteins, including subunits of RC complexes, are essential for maintenance of normal mitochondrial function. The Specific Aim of this proposal is to characterize the N-linked glycoproteome of the RC and the ways it is altered by RC disease. In silico predictions that specific RC proteins are N-glycosylated will be tested by lectin reactivity and PNGaseF, endoglycosidase H and F sensitivity of blue-native gel separated RC complexes. The glycoproteins and their glycosylation sites as well as the glycan structure will be identified by mass spectrometry. Effects of RC inhibition due to either genetic disease or pharmacologic inhibition on glycoprotein modifications of the mitochondrial RC will be characterized in patient FCLs from RC disease and NGLY1 deficient patients, as well as from mouse NGLY1 deficient MEF models. These studies will identify N-glycosylated RC proteins and elucidate how they are impacted by RC disease.

。 原发性线粒体呼吸链 (RC) 疾病和先天性糖基化障碍 (CDG) 是常见的代谢性疾病，可导致一系列重叠的疾病 大脑发育紊乱和多系统疾病。还有很多事情有待发现 线粒体糖蛋白组的性质和调控及其潜在相关性 RC 功能或糖基化的遗传性疾病。我们的初步数据表明：[1] 来自不同物种的线粒体具有独特的 N 连接糖组修饰，评估方法如下： MALDI-TOF质谱（MS）； [2] 一些 RC 蛋白被 N-糖基化；和[3] 患者成纤维细胞系 (FCL)、线虫和缺乏 N-聚糖酶的小鼠品系 NGLY1 会导致线粒体耗竭、RC 功能障碍和氧化应激增加。这 该提案的总体目标是表征线粒体 RC 糖蛋白组及其 调节，并阐明其与 RC 功能和 NGLY1 活性遗传性疾病的相关性。 我们假设线粒体糖蛋白的糖基化和去糖基化， 包括 RC 复合物的亚基，对于维持正常线粒体至关重要 功能。该提案的具体目标是表征 N-连接糖蛋白组 RC 及其因 RC 疾病而改变的方式。计算机预测特定 RC 蛋白是 N-糖基化将通过凝集素反应性和 PNGaseF、糖苷内切酶 H 和 F 进行测试 蓝色天然凝胶分离的 RC 复合物的敏感性。糖蛋白及其 糖基化位点以及聚糖结构将通过质谱法进行鉴定。 由于遗传疾病或糖蛋白药物抑制而导致的 RC 抑制作用 线粒体 RC 的修饰将在 RC 疾病患者 FCL 中表征 和 NGLY1 缺陷患者，以及小鼠 NGLY1 缺陷 MEF 模型。这些 研究将鉴定 N-糖基化 RC 蛋白并阐明它们如何受到 RC 的影响 疾病。