CHARACTERIZING THE HIV MUSCLE MITOCHONDRIAL PROTEOME

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目及 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 骨骼肌线粒体（mt）蛋白表达的扰动可能有助于代谢紊乱的HIV感染者接受基于胸苷类似物的抗逆转录病毒治疗（AZT或d4 T-ARV）的发病机制。骨骼肌是人体内最大的富含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，我们缺乏灵敏，特异性和全面的分析工具来检查人类肌肉mt蛋白质组。我们建议开发灵敏的、mt特异性的、基于质谱的比较蛋白质组学工具和方法，可用于鉴定、表征和定量先前从4组充分表征的受试者获得的标本中的人类肌肉mt蛋白质组：HIV-血清阴性且葡萄糖耐量正常（NGT）; HIV+初治ARV且NGT; HIV+接受基于AZT或d4 T的ARV且NGT;和HIV+接受AZT或d4 T为基础的ARV与胰岛素抵抗。我们假设，AZT或d4 T为基础的方案损害肌肉mt蛋白的表达，并诱导翻译后修饰mt蛋白，与HIV相关的胰岛素抵抗。具体而言，我们将使用定制的亚细胞分级分离，蛋白质富集和耗尽方法，2D-差分荧光凝胶电泳和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-串联MS）鉴定和表征肌肉蛋白/肽，以进行精确的质量测量和氨基酸测序。我们将发现新的和重要的mt蛋白形式，这将产生新的方法和新的假设，在艾滋病毒感染者的肌肉mt为基础的代谢紊乱的发病机制。这些分析方法和工具已被用于检查小生物体中的蛋白质组，但我们需要推进和促进其应用于复杂的人体组织（肌肉），这些组织与人体底物代谢紊乱密切相关，并可能最终导致新的治疗策略。为了实现这一目标，我们将利用现有的专业知识和各种高分辨率质谱仪器。

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.