Defining Molecular Determinants of Type 2 Diabetes Using Quantitative Proteomics

使用定量蛋白质组学定义 2 型糖尿病的分子决定因素

• 批准号: 8335569
• 负责人: Paul A. Grimsrud
• 金额: $ 5.57万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8335569
• 关键词: 4 hydroxynonenal; Affect; Aldehydes; All-Trans-Retinol; Analytical Chemistry; Antioxidants; Attenuated; BTBR Mouse; Basic Science; Binding; Biochemical; Biochemistry; Biogenesis; Biological; Biological Assay; Biology; Cell Culture Techniques; Cell physiology; Cells; Cellular biology; Collection; Control Animal; Data; Data Set; Development; Diabetes Mellitus; Disease; Doctor of Philosophy; Elongation Factor; Environment; Enzymes; Epidemic; Epitopes; Family member; Functional disorder; Genetic; Goals; Immunoprecipitation; In Vitro; Incidence; Inflammatory; Insulin Resistance; Investigation; Laboratories; Link; Lipid Peroxidation; Liver; Liver Mitochondria; Mass Spectrum Analysis; Metabolic; Metabolic Diseases; Metabolism; Minnesota; Mitochondria; Mitochondrial Proteins; Modeling; Molecular; Monitor; Mus; Mutate; Non-Insulin-Dependent Diabetes Mellitus; Obesity; Oxidation-Reduction; Oxidoreductase; Peripheral; Phosphorylation; Phosphorylation Site; Play; Postdoctoral Fellow; Predisposition; Production; Protein Analysis; Protein Isoforms; Proteins; Proteome; Proteomics; RNA; RNA Interference; Reaction; Recombinant Proteins; Recombinants; Research; Research Project Grants; Resistance; Retinal; Retinoids; Role; Selenocysteine; Signal Transduction; Site-Directed Mutagenesis; Surveys; Testing; Therapeutic Intervention; Tissues; Training; Translations; United States; Universities; Wisconsin; Work; aldehyde dehydrogenases; base; cohort; diabetic; genetic regulatory protein; in vitro activity; insight; instrumentation; member; mitochondrial dysfunction; mutant; novel; overexpression; oxidized lipid; research study; resistant strain; response; selenoprotein; stoichiometry

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.

本研究的目的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 (T2DM)。最近的研究将线粒体功能障碍与胰岛素抵抗联系起来。该提案的共同发起人 Alan Attie 博士利用遗传学来了解罹患 T2DM 的倾向。该提案的发起人 David Pagliarini 博士专注于线粒体功能障碍对各种疾病的影响，并了解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，也是本提案的密切合作者，他是蛋白质分析质谱 (MS) 仪器开发和应用领域的领导者。从 Coon 小组过渡到 Pagliarini 实验室开始第二个博士后工作后，我将作为我们跨学科协作团队的领导成员继续这个项目。 我已经完成了详尽的定量蛋白质组学数据集的收集，该数据集构成了我在最初的申请中为该项目提出的大部分研究。我在 40 多只小鼠的队列中观察到蛋白质丰度和肝线粒体磷酸化的高度可重复的变化，这些小鼠在肥胖时对 T2DM 的发展要么敏感（B6），要么具有抵抗性（BTBR）。我目前正在使用这个蛋白质组学屏幕作为假设驱动的有针对性的生物学研究的初步数据。拟议研究的基本原理是确定磷酸化如何调节肥胖中的氧化还原调节蛋白将为开发 2 型糖尿病治疗干预措施开辟新途径。共同赞助的环境将有助于实现我建立一个独立学术实验室的目标，该实验室专注于利用蛋白质组学和靶向生物学来研究代谢疾病中的氧化还原信号传导。我将采用生物化学、细胞生物学和靶向蛋白质组学方法来实现以下目标： 目标 1. 确定肥胖诱导的硒代半胱氨酸特异性延伸因子 (Eefsec) 磷酸化如何调节氧化还原调节性硒蛋白的产生。我将检验以下假设：在抗糖尿病 B6 小鼠中，肥胖诱导的 Eefsec 非线粒体同工型磷酸化增强会减弱炎症分泌型硒蛋白的产生，而不会降低关键线粒体抗氧化剂的水平，而关键线粒体抗氧化剂的水平受线粒体局部 Eefsec 同工型调节。 目标 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。我将验证以下假设：肥胖诱导的脱氢酶/还原酶 SDR 家族成员 4 (Dhrs4) 的磷酸化通过调节酶的视网膜还原活性来改变糖尿病 BTBR 小鼠中的类视黄醇代谢物水平。我将测试的一个平行假设是，醛脱氢酶 3A2 (Aldh3a2) 表达和磷酸化的诱导是肥胖症中对反应性脂质过氧化产物解毒的一种补偿反应。