Defining Molecular Determinants of Type 2 Diabetes Using Quantitative Proteomics

描述（由申请人提供）：拟议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发展为2型糖尿病的倾向。David Pagliarini博士是该提案的发起人，他专注于线粒体功能障碍对各种疾病的贡献，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，他是开发和应用质谱（MS）仪器进行蛋白质分析的领导者。从库恩小组转到帕格里亚里尼实验室开始第二个博士后，我作为我们跨学科合作团队的领导成员继续这个项目。我已经完成了一个详尽的定量蛋白质组学数据集的收集，这构成了我在最初的申请中为这个项目提出的大部分研究。我观察了40多只小鼠的肝脏线粒体中蛋白质丰度和磷酸化的高度可重复的变化，这些小鼠在肥胖时对2型糖尿病易感（B6）或抗性（BTBR）。我目前正在使用这种蛋白质组学筛选作为假设驱动的靶向生物学研究的初步数据。这项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型糖尿病的治疗干预开辟新的途径。共同赞助的环境将有助于我建立一个独立的学术实验室的目标，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。我将采用生化、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。我将验证以下假设：在糖尿病抵抗性B6小鼠中，肥胖诱导的Eefsec非线粒体异构体磷酸化增强会减弱炎症分泌的硒蛋白的产生，而不会降低关键线粒体抗氧化剂的水平，这是由线粒体定位的Eefsec异构体调节的。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。我将验证肥胖诱导的脱氢酶/还原酶SDR家族成员4 （Dhrs4）磷酸化通过调节酶的视网膜还原活性改变糖尿病BTBR小鼠类视黄醇代谢物水平的假设。我将验证的一个平行假设是，醛脱氢酶3A2 （Aldh3a2）表达和磷酸化的诱导是肥胖对反应性脂质过氧化产物解毒的代偿反应。

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.