CHARACTERIZING THE HIV MUSCLE MITOCHONDRIAL PROTEOME

HIV 肌肉线粒体蛋白质组的特征

• 批准号: 7721452
• 负责人: KEVIN E YARASHESKI
• 金额: $ 0.61万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-02-01 至 2009-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7721452
• 关键词: Amino Acid Sequence; Complex; Computer Retrieval of Information on Scientific Projects Database; Disease; Fluorescence; Fractionation; Funding; Glucose; Grant; HIV; Human; Institution; Insulin Resistance; Lead; Liquid Chromatography; Mass Spectrum Analysis; Measurement; Metabolic Diseases; Metabolism; Methods; Mitochondria; Muscle; Muscle Proteins; Organism; Pathogenesis; Peptides; Post-Translational Protein Processing; Proteins; Proteome; Proteomics; Research; Research Personnel; Resolution; Resources; Site; Skeletal Muscle; Source; Specimen; Thymidine; Tissues; Treatment Protocols; United States National Institutes of Health; Zidovudine; analog; analytical tool; antiretroviral therapy; base; comparative; fatty acid metabolism; gel electrophoresis; glucose tolerance; human tissue; instrumentation; mass spectrometer; nano; novel; novel strategies; protein expression; tool

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Perturbations in skeletal muscle mitochondrial (mt) protein expression may contribute to the pathogenesis of metabolic disorders in HIV-infected people treated with thymidine-analog-based antiretroviral therapy (AZT- or d4T-ARV). Skeletal muscle is the largest, mt-rich tissue in the body and the primary site for glucose storage. Normal muscle mt protein expression is required for normal glucose and fatty acid metabolism. However, we lack sensitive, specific and comprehensive analytical tools for examining the human muscle mt proteome. We propose to develop sensitive, mt-specific, mass spectrometry-based comparative proteomics tools and approaches that can be used to identify, characterize, and quantify the human muscle mt proteome in specimens previously obtained from 4 groups of well characterized subjects: HIV-seronegative with normal glucose tolerance (NGT); HIV+ naive to ARV with NGT, HIV+ receiving AZT- or d4T-based ARV with NGT; and HIV+ receiving AZT- or d4T-based ARV with insulin resistance. We hypothesize that AZT- or d4T- based regimens impair muscle mt protein expression and induce post-translational modifications to mt proteins that are associated with HIV-related insulin resistance. Specifically, we will separate muscle mt proteins using customized sub-cellular fractionation, protein enrichment and depletion methods, 2D- differential fluorescence gel electrophoresis, and 1D-liquid chromatography. We will identify and characterize muscle protein/peptides using a variety of mass spectrometers (MALDI-TOF, LC-ESI-, nano-LC-FT-tandem MS) for accurate mass measurements and amino acid sequencing. We will discover new and important mt protein forms that will generate novel approaches and new hypotheses about the pathogenesis of muscle mt-based metabolic disorders in HIV-infected people. These analytical approaches and tools have been used to examine proteomes in small organisms, but we need to advance and facilitate their application to complex human tissues (muscle) that are critically involved in disorders of human substrate metabolism, and that might ultimately lead to novel treatment strategies. To accomplish this, we will take advantage of the expertise and the wide variety of high resolution mass spectrometry instrumentation available.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 骨骼肌线粒体 (mt) 蛋白表达的扰动可能导致接受胸苷类似物抗逆转录病毒疗法（AZT 或 d4T-ARV）治疗的 HIV 感染者代谢紊乱的发病机制。骨骼肌是体内最大的、富含 mt 的组织，也是葡萄糖储存的主要场所。正常的肌肉 mt 蛋白表达是正常葡萄糖和脂肪酸代谢所必需的。然而，我们缺乏敏感、特异和全面的分析工具来检查人类肌肉 mt 蛋白质组。我们建议开发敏感的、mt 特异性的、基于质谱的比较蛋白质组学工具和方法，可用于识别、表征和量化先前从 4 组特征明确的受试者中获得的样本中的人类肌肉 mt 蛋白质组：HIV 血清阴性且糖耐量正常 (NGT) 的受试者； HIV+ 未接受过 NGT 抗逆转录病毒疗法，HIV+ 接受基于 AZT 或 d4T 的 ARV 联合 NGT 治疗； HIV+ 接受基于 AZT 或 d4T 的 ARV 治疗且出现胰岛素抵抗。我们假设基于 AZT 或 d4T 的治疗方案会损害肌肉 mt 蛋白表达，并诱导与 HIV 相关胰岛素抵抗相关的 mt 蛋白翻译后修饰。具体来说，我们将使用定制的亚细胞分级分离、蛋白质富集和去除方法、二维差异荧光凝胶电泳和一维液相色谱来分离肌肉 mt 蛋白。我们将使用各种质谱仪（MALDI-TOF、LC-ESI、nano-LC-FT-tandem MS）来识别和表征肌肉蛋白/肽，以进行精确的质量测量和氨基酸测序。我们将发现新的、重要的 mt 蛋白形式，从而产生关于 HIV 感染者肌肉 mt 代谢紊乱发病机制的新方法和新假设。这些分析方法和工具已用于检查小型生物体中的蛋白质组，但我们需要推进和促进它们在复杂的人体组织（肌肉）中的应用，这些组织与人类底物代谢紊乱密切相关，这可能最终导致新的治疗策略。为了实现这一目标，我们将利用专业知识和各种可用的高分辨率质谱仪器。