Crosstalk Between 0-GlcNAcylation and Phosphorylation in Diabetic Cardiomyopathy

说明）： 糖尿病是心血管疾病的主要危险因素，最终导致心肌梗死， 失败由于营养过剩和高血糖，延长的高-O-GlcNAc化是一个主要的分子机制。 导致葡萄糖中毒和胰岛素抵抗。0-GlcNAc化增加直接导致糖尿病 心肌病和功能失调的线粒体，可能有助于过度生产的反应性 氧物种（ROS）。尽管0-GlcNAc酰化在糖尿病中明显起着重要作用， 在心血管疾病中，实际上对心肌细胞中的0-GlcNAc化一无所知。这个项目 将阐明0-GlcNAc在糖尿病性心肌病中的作用，并将定义糖尿病性心肌病的“0-GlcNAcome”。 心肌细胞在位点特异性水平。具体目标： 目的1：定量细胞中0-GlcNAc酰化和磷酸化之间的位点特异性串扰。 心肌细胞蛋白质组和纯化的心肌细胞线粒体从正常和糖尿病 大鼠使用化学-酶促光可切割标签富集结合电子转移解离 (ETD)通过串联质谱，我们将定量O-GlcNAc和磷酸盐的位点占用， 心肌细胞收缩蛋白和线粒体蛋白。目标2：确定 正常心肌细胞线粒体中0-GlcNAc化的特异性作用及其作用位点 以及糖尿病诱导的功能障碍的机制，导致ROS的产生。我们将特别修改 0-使用过去20年中开发的方法进行GlcNAc化，并将改变与特定的 线粒体功能目的3：阐明心肌细胞线粒体的特性及其调控 O-GlcNAc转移酶和O-GlcNAc酶的同种型。事实上，我们对线粒体 O-GlcNAc转移酶（OGT）或O-GlcNAc酶（OGA）的同种型。我们将阐明它们的定位， 活动，分子协会和动力学活动的线粒体从正常和糖尿病大鼠。目标4： 评估糖尿病诱导的线粒体功能障碍和O- 心肌细胞收缩机制的GlcNAc化对心脏生理和功能的影响。工作 与核心D密切相关，我们将系统地评估0-GlcNAc酰化之间串扰的重要性， 心肌细胞收缩蛋白和线粒体蛋白的磷酸化对 心肌细胞 这些研究将为理解心脏功能的调节开辟一个新的范例， 糖尿病性心肌病它们将导致完全未探索的可能的治疗干预途径 相关性（参见说明）： 糖尿病是一种主要的流行病，并导致心血管疾病，最终导致 心力衰竭或心肌梗塞。增加的0-GlcNAc化，糖翻译后修饰， 通过影响心肌细胞的收缩功能， 和心肌细胞内的线粒体蛋白质。这些研究将阐明O- GlcNAc在正常和糖尿病心肌细胞生理学中的作用，并可能导致新的治疗方法。

instmctions): Diabetes is a major risk factor for cardiovascular disease, culminating in myocardial infarction, and heart failure. Prolonged hyper-0-GlcNAcylation, due to nutrient excess and hyperglycemia, is a major molecular cause of glucose toxicity and insulin resistance. Increased 0-GlcNAcylation directly contributes to diabetic cardiomyopathy and to dysfunctional mitochondria, perhaps contributing to excessive production of reactive oxygen species (ROS). Even though 0-GlcNAcylation clearly plays an important role in diabetic cardiovascular disease, virtually nothing is known about 0-GlcNAcylation in the cardiomyocyte. This project will elucidate the roles of 0-GlcNAc in diabetic cardiomyopathy and will define the "0-GlcNAcome" of the cardiomyocyte at the site-specific level. Specific Aims: Aim 1: Quantify the Site-Specific Crosstalk Between 0-GlcNAcylation and Phosphorylation in the cardiomyocyte proteome and in purified cardiomyocyte mitochondria from Normal and Diabetic Rats. Using chemico-enzymatic photocleavable tag enrichment combined with electron transfer dissociation (ETD) tandem mass spectrometry, we will quantify site occupancy for both 0-GlcNAc and phosphate in cardiomyocyte contractile and mitochondrial proteins from normal and diabetic rats. Aim 2: Determine the Specific Roles of 0-GlcNAcylation in normal cardiomyocyte mitochondria, and the sites of action and mechanisms of diabetes-induced dysfunction, leading to ROS production. We wilt specifically alter 0-GlcNAcylation using methods developed during the past 20-years, and correlate alterations with specific mitochondrial function. Aim 3: Elucidate the properties and regulation of cardiomyocyte mitochondrial isoforms of O-GlcNAc Transferase and 0-GlcNAcase. Virtually nothing is known about the mitochondrial isoforms of 0-GlcNAc Transferase (OGT) or 0-GlcNAcase (OGA). We will elucidate their localization, activities, molecular associations and kinetic activities in mitochondria from normal and diabetic rats. Aim 4: Evaluate the affects and roles of diabetes-induced mitochondrial dysfunction and increased O- GlcNAcylation of cardiomyocyte contractile machinery on cardiac physiology and function. Working closely with Core D we will systematically evaluate the importance of the crosstalk between 0-GlcNAcylation and phosphorylation of cardiomyocyte contractile and mitochondrial proteins on the physiological functions of cardiomyocytes These studies will open a new paradigm for understanding the regulation of cardiac functions and in diabetic cardiomyopathies. They will lead to totally unexplored avenues of possible therapeutic interventions RELEVANCE (See instructions): Diabetes is a major epidemic and contributes to cardiovascular disease, which ultimately results in heart failure or myocardial infarction. Increased 0-GlcNAcylation, a sugar post-translational modification, underlies molecular events contributing to diabetic cardiomyopathies by affecting the functions of contractile and mitochondrial proteins within the cardiomyocyte. These studies will elucidate the importance of O- GlcNAc in both normal and diabetic cardiomyocyte physiology, and will possibly lead to novel treatments.