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.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 骨骼肌线粒体（MT）蛋白表达的扰动可能有助于用基于胸腺苷 - 基于胸苷 - 基于胸腺苷 - 基于基于胸腺苷的抗逆转录病毒疗法（AZT-或D4T-ARV）治疗的HIV感染者的发病机理。骨骼肌是体内最大的，富含MT的组织，是葡萄糖储存的主要部位。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。但是，我们缺乏检查人类肌肉MT蛋白质组的敏感，具体和全面的分析工具。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，这些工具和方法可用于识别，表征和量化的人类肌肉MT蛋白质组先前从4组受试者获得的标本中：HIV-辅助性具有正常的糖糖耐受性（NGT）； HIV+ NGT，HIV+带有NGT的ARV ARV幼稚； HIV+接受具有胰岛素耐药性的基于AZT或D4T的ARV。我们假设基于AZT或D4T的方案会损害与HIV相关胰岛素耐药性相关的MT蛋白的翻译后修饰。具体而言，我们将使用自定义的亚细胞分级，蛋白质富集和耗竭方法，2D-差分荧光凝胶电泳和1D液色谱法分离肌肉MT蛋白。我们将使用多种质谱仪（MALDI-TOF，LC-ESI-，纳米LC-FTANDEM MS）来识别和表征肌肉蛋白/肽，以进行准确的质量测量和氨基酸测序。我们将发现新的重要的MT蛋白质形式，这些形式将产生新的方法和有关HIV感染者基于肌肉MT的代谢性疾病的发病机理的新方法。这些分析方法和工具已用于检查小生物的蛋白质组织，但是我们需要促进并促进它们在复杂的人体组织（肌肉）中的应用，这些人（肌肉）与人类底物代谢的疾病非常重要，这可能最终导致新的治疗策略。为此，我们将利用可用的专业知识和各种高分辨率质谱仪器的优势。