Defining Molecular Determinants of Type 2 Diabetes Using Quantitative Proteomics

描述(申请人提供)：拟议研究的目标是确定线粒体蛋白丰度和磷酸化的改变如何导致2型糖尿病(T2 DM)。最近的研究将线粒体功能障碍与胰岛素抵抗联系起来。艾伦·阿蒂博士是这项提案的共同发起人，他利用遗传学来了解患上2型糖尿病的倾向。这项建议的发起人David Pagliarini博士重点介绍了线粒体功能障碍对各种疾病的贡献，并了解了线粒体生物发生的基础科学。Joshua Coon博士是我最初申请的发起人，也是这项提议的密切合作者，他是蛋白质分析用质谱仪(MS)仪器开发和应用的领导者。在从浣熊小组过渡到帕利亚里尼实验室开始第二个博士后工作后，我将继续这个项目，作为我们跨学科协作团队的领导成员。我已经完成了详尽的定量蛋白质组学数据集的收集，这构成了我在最初申请中为这个项目提出的研究的大部分。我在40多只小鼠中观察到肝脏线粒体蛋白丰度和磷酸化的高度重复性变化，这些小鼠在肥胖时要么易患(B6)，要么抵抗(BTBR)。我目前正在使用这个蛋白质组学筛查作为假说驱动的靶向生物学研究的初步数据。这项拟议研究的基本原理是，确定磷酸化如何调节肥胖症中的氧化还原调节蛋白将为开发2型糖尿病的治疗干预措施开辟新的途径。共同赞助的环境将有助于实现我的目标，即建立一个独立的学术实验室，专注于利用蛋白质组学和靶向生物学来研究代谢性疾病中的氧化还原信号。我将使用生化、细胞生物学和靶向蛋白质组学的方法来实现以下目标：目的1.确定肥胖诱导的硒半胱氨酸特异性延伸因子(Eefsec)的磷酸化如何调节氧化还原调节硒蛋白的产生。我将测试一种假设，即肥胖诱导的非线粒体Eefsec亚型的磷酸化增强，在不降低关键线粒体抗氧化剂水平的情况下，减少炎症分泌的硒蛋白的产生，而关键线粒体抗氧化剂受线粒体定位的Eefsec亚型调节。目的2.阐明肥胖诱导的线粒体氧化还原酶磷酸化在调节生物活性亲脂醛丰度中的作用。我将测试肥胖诱导的脱氢酶/还原酶SDR家族成员4(Dhrs4)的磷酸化通过调节该酶的视网膜还原活性来改变糖尿病BTBR小鼠的维甲酸代谢物水平的假设。我将检验的一个平行假设是，诱导乙醛脱氢酶3A2(Aldh3a2)的表达和磷酸化是肥胖患者对解毒活性脂质过氧化产物的一种补偿反应。 公共卫生相关性：2型糖尿病(T2 DM)在美国已达到流行程度。这项拟议的研究依赖于我对40多只小鼠完成的定量蛋白质组图谱作为初步数据，试图确定线粒体蛋白丰度和磷酸化变化是如何导致这种疾病的。这些实验的结果将有助于确定决定T2 DM易感性的机制，为潜在的治疗干预开辟新的途径。

DESCRIPTION (provided by applicant): The objective of the proposed research is to determine how alterations in mitochondrial protein abundance and phosphorylation contribute to Type 2 Diabetes Mellitus (T2DM). Recent studies have linked mitochondrial dysfunction to insulin resistance. Dr. Alan Attie, co-sponsor of this proposal, uses genetics to understand the propensity to develop T2DM. Dr. David Pagliarini, sponsor of this proposal, focuses on the contribution of mitochondrial dysfunction to various diseases and understanding the basic science of mitochondrial biogenesis. Dr. Joshua Coon, the sponsor of my original application and a close collaborator on this proposal, is a leader in the development and application of mass spectrometry (MS) instrumentation for protein analysis. Having transition from the Coon group to start a second post-doc in the Pagliarini lab, I am continuing this project as the leading member of our interdisciplinary collaborative team. I have completed collection of an exhaustive quantitative proteomics dataset that constituted the bulk of my proposed research for this project in my original application. I observed highly reproducible alterations in protein abundance and phosphorylation in liver mitochondria in a cohort of over forty mice, which are either susceptible (B6) or resistant (BTBR) to developing T2DM when made obese. I am currently using this proteomics screen as preliminary data for hypotheses-driven targeted biological investigation. The rationale for the proposed research is that determining how phosphorylation modulates redox-regulatory proteins in obesity will open new avenues for developing therapeutic interventions for type 2 diabetes. The co-sponsoring environment will help facilitate my goal of starting an independent academic laboratory focused on utilizing proteomics and targeted biology to study redox signaling in metabolic disease. I will employ both biochemical, cell biology, and targeted proteomic approaches to carrying out the following Aims: Aim 1. Determine how the obesity-induced phosphorylation of the selenocysteine-specific elongation factor (Eefsec) regulates the production of redox-regulatory selenoproteins. I will test the hypothesis that enhanced obesity-induced phosphorylation of the non-mitochondrial isoform of Eefsec in diabetes- resistant B6 mice attenuates the production of inflammatory secreted selenoproteins without decreasing levels of key mitochondrial antioxidants, which are regulated by a mitochondrial-localized Eefsec isoform. Aim 2. Elucidate the role of obesity-induced phosphorylation of mitochondrial redox enzymes in regulating the abundance of bioactive lipophilic aldehydes. I will test the hypothesis that obesity-induced phosphorylation of dehydrogenase/reductase SDR family member 4 (Dhrs4) alters the levels of retinoid metabolites in diabetic BTBR mice through regulating the enzyme's retinal reducing activity. A parallel hypothesis I will test is that induction of aldehyde dehydrogenase 3A2 (Aldh3a2) expression and phosphorylation is a compensatory response in obesity for detoxifying reactive lipid-peroxidation products. PUBLIC HEALTH RELEVANCE: Type 2 Diabetes Mellitus (T2DM) has reached epidemic proportions in the United States. The proposed research, which relies on my completed quantitative proteomic profiling of over forty mice as preliminary data, seeks to determine how mitochondrial protein abundance and phosphorylation alterations contribute to this disease. The results of these experiments will help define the mechanisms determining T2DM susceptibility, opening new avenues for potential therapeutic interventions.