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）疾病和糖基化先天性疾病 （CDG）是共同常见的代谢疾病，引起了重叠的频谱 大脑发育和多系统疾病的破坏。关于 线粒体糖蛋白酶的性质和调节及其与其潜在相关性 RC功能或糖基化的遗传疾病。我们的初步数据表明：[1] 来自各种物种的线粒体具有独特的N连接甘氨酸修饰。 MALDI-TOF质谱法（MS）； [2]几种RC蛋白是N-糖基化的；和[3] 缺乏N-聚糖酶的患者成纤维细胞系（FCL），秀丽隐杆线虫和小鼠菌株 NGLY1具有线粒体耗竭，RC功能障碍和增加的氧化应激。这 该建议的总体目标是表征线粒体RC糖蛋白酶及其其 调节，并阐明其与RC功能和NGLY1活动的遗传疾病的相关性。 我们假设线粒体糖蛋白的糖基化和脱糖基化， 包括RC复合物的亚基，对于维持正常线粒体至关重要 功能。该提议的具体目的是表征该提案的N连接糖蛋白酶 RC及其被RC疾病改变的方式。在计算机预测中，特定的RC蛋白是 N-糖基化将通过凝集素反应性和PNGASEF，内吞糖苷酶H和F测试 蓝色凝胶的敏感性分离的RC复合物。糖蛋白及其 糖基化位点以及聚糖结构将通过质谱鉴定。 遗传疾病或药理抑制引起的RC抑制对糖蛋白的影响 线粒体RC的修饰将在RC病的患者FCL中进行表征 NGLY1患者以及小鼠NGLY1缺乏MEF模型。这些 研究将鉴定N-糖基化的RC蛋白，并阐明它们如何受RC的影响 疾病。