Quantitative Mitochondrial Proteomics of Healthy and Diabetic Mice

描述(由申请人提供)：本申请涉及广泛的挑战领域(03)：生物标记物的发现和验证，以及特定的挑战主题03-DK-103：识别与NIDDK疾病相关的亚细胞器的正常和患病蛋白质组。线粒体功能障碍被认为是2型糖尿病(T2 DM)的统一主题，目前仅在美国就有2000多万人受到影响。线粒体含量和氧化能力降低，脂肪酸氧化改变，线粒体衍生的活性氧增加与胰岛素抵抗和T2 DM的发生有关。然而，这些观察的重要性最近受到了挑战，目前还不清楚线粒体组成和/或功能的改变实际上对T2 DM的病因有何影响。这项建议的长期目标是通过以前所未有的分辨率阐明伴随着这种疾病发生的成分变化，从而更全面地理解线粒体功能障碍在T2 DM中的重要性。尽管有上述观察，但由于至少两个原因，人们对T2 DM线粒体功能障碍的分子基础缺乏了解。首先，直到最近，许多哺乳动物线粒体蛋白质组本身都是未知的，因此无法对这种疾病不同阶段的线粒体组成进行全面比较。其次，用于调查健康和糖尿病患者之间基因表达的全球差异的微阵列分析，由于细胞mRNA和蛋白质水平之间的相关性明显较差，已被削弱。在这里，我们建议使用最先进的定量蛋白质组学(AIM1)来建立肥胖依赖型T2 DM发病过程中发生的蛋白质组和磷蛋白质组变化的综合图谱。为此，我们将利用两个小鼠品系：C57BL/6(B6)Leptinob/ob小鼠，它们对糖尿病具有抵抗力，以及BTBR Leptinob/ob小鼠，它们随着年龄的增长而患上严重的糖尿病。在这个项目的两年时间框架内，我们将专注于骨骼肌中的线粒体，这是体内胰岛素介导的葡萄糖处置的主要部位。这个蛋白质组资源也将提供一个机会来探索这些线粒体改变发生的机制。特别是，我们将使用整合基因组学来阐明在控制线粒体基因表达和蛋白质功能(AIM2)中发挥作用的转录后和翻译后机制。我们的目标的完成将澄清伴随着T2 DM发病的骨骼肌中线粒体的重构，为糖尿病社区提供丰富的定量蛋白质组资源，并为识别蛋白质生物标志物和设计针对该细胞器的肌肉特异性疗法治疗T2 DM奠定基础。 公共卫生相关性：线粒体功能障碍是2型糖尿病(T2 DM)的一个显著特征，但这种功能障碍的潜在基础尚不清楚。我们建议使用最先进的蛋白质组学技术来建立肥胖诱导的糖尿病发作期间骨骼肌中发生的线粒体改变的图谱。这一目标的完成将有助于确定这种疾病的线粒体生物标记物，并为设计针对该细胞器的治疗T2 DM的药物提供一个框架。

DESCRIPTION (provided by applicant): This application addresses broad Challenge Area (03): Biomarker Discovery and Validation, and specific Challenge Topic 03-DK-103: Identify the normal and diseased proteome of subcellular organelles of relevance to NIDDK diseases. Mitochondrial dysfunction has been postulated as a unifying theme of type 2 diabetes mellitus (T2DM), which currently affects more than 20 million individuals in the US alone. Decreased mitochondrial content and oxidative capacity, altered fatty acid oxidation, and a rise in mitochondria-derived reactive oxygen species have been associated with the development of insulin resistance and T2DM. However, the importance of these observations has recently been challenged, and it remains unclear which, if any, changes to mitochondrial composition and/or function actually contribute to T2DM etiology. The long-term objective of this proposal is to arrive at a fuller understanding of the importance of mitochondrial dysfunction in T2DM by elucidating the compositional changes that accompany the onset of this disease at unprecedented resolution. Despite the observations noted above, an understanding of the molecular basis for mitochondrial dysfunction in T2DM has largely been lacking for at least two reasons. First, until recently, much of the mammalian mitochondria proteome itself was undefined, making it impossible to conduct comprehensive comparisons of mitochondrial composition during different stages of this disease. Second, microarray analyses used to investigate global differences in gene expression between healthy and diabetic patients have been crippled by the markedly poor correlation between cellular mRNA and protein levels. Here, we propose to establish a comprehensive map of the proteomic and phosphoproteomic changes that occur during the onset of obesity-dependent T2DM using state-of-the-art quantitative proteomics (AIM1). To do so, we will take advantage of two mouse strains: C57BL/6 (B6) leptinob/ob mice, which are resistant to diabetes, and BTBR leptinob/ob mice, which develop severe diabetes as they age. During the two-year timeframe of this project, we will focus on mitochondria from skeletal muscle, the primary site of insulin- mediated glucose disposal in the body. This proteomic resource will also provide an opportunity to explore the mechanisms by which these mitochondrial alterations occur. In particular, we will use integrative genomics to elucidate the post-transcriptional and post-translational mechanisms at play in the control of mitochondrial gene expression and protein function (AIM2). Completion of our aims will clarify the mitochondrial restructuring in skeletal muscle that accompanies the onset of T2DM, provide a rich quantitative proteomic resource for the diabetes community, and lay the groundwork for identifying protein biomarkers and designing muscle-specific therapies targeted against this organelle to treat T2DM. PUBLIC HEALTH RELEVANCE: Mitochondrial dysfunction is a prominent feature of type 2 diabetes mellitus (T2DM), but the underlying basis for this dysfunction is not well understood. We propose to use state-of-the-art proteomics technologies to establish a map of mitochondrial alterations that occur in skeletal muscle during the onset of obesity-induced diabetes. Completion of this goal will help identify mitochondrial biomarkers for this disease, and provide a framework for designing therapies targeted against this organelle to treat T2DM.