CHARACTERIZING THE HIV MUSCLE MITOCHONDRIAL PROTEOME

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 在接受基于胸苷类似物的抗逆转录病毒治疗(AZT-或D4T-ARV)的HIV感染者中，骨骼肌线粒体(MT)蛋白表达的紊乱可能与代谢紊乱的发病机制有关。骨骼肌是体内最大的富含mt的组织，也是储存葡萄糖的主要部位。正常的肌肉MT蛋白表达是正常的葡萄糖和脂肪酸代谢所必需的。然而，我们缺乏灵敏、特异和全面的分析工具来研究人类肌肉mt蛋白质组。我们建议开发灵敏的、mt特异的、基于质谱学的比较蛋白质组学工具和方法，可以用来识别、表征和量化先前从4组特征良好的受试者的标本中获得的人类肌肉mt蛋白质组：HIV血清阴性的正常糖耐量(NGT)；HIV+对ARV的幼稚伴NGT；HIV+接受基于AZT或d4T的ARV伴NGT；以及HIV+接受基于AZT或d4T的ARV伴胰岛素抵抗。我们假设，基于AZT或D4T的治疗方案会损害肌肉MT蛋白的表达，并诱导与HIV相关的胰岛素抵抗相关的MT蛋白的翻译后修饰。具体地说，我们将使用定制的亚细胞分离、蛋白质浓缩和去除方法、2D-差示荧光凝胶电泳法和1D-液相色谱分离肌肉mt蛋白。我们将使用各种质谱仪(MALDI-TOF、LC-ESI-、Nano-LC-FT-Tandem MS)对肌肉蛋白质/肽进行鉴定和表征，以实现准确的质量测量和氨基酸排序。我们将发现新的和重要的mt蛋白形式，这些形式将产生关于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被用于研究小生物体中的蛋白质组，但我们需要推动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障碍密切相关，最终可能导致新的治疗策略。为了实现这一目标，我们将利用专业知识和现有的各种高分辨率质谱仪。

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.