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，最终导致心肌梗死和心脏病。 失败了。由于营养过剩和高血糖，延长的高-0-GlcN酰化是主要的分子 导致葡萄糖毒性和胰岛素抵抗。0-GlcN酰化增加直接导致糖尿病 心肌病和线粒体功能障碍，可能是过度产生反应性的原因 氧物种(ROS)。尽管0-GlcN酰化显然在糖尿病中起着重要作用 心血管疾病，对心肌细胞中的0-GlcN酰化几乎一无所知。这个项目 将阐明0-GlcNAc在糖尿病心肌病中的作用，并将定义 特定部位水平的心肌细胞。具体目标： 目的1：量化0-GlcN酰化和磷酸化之间的位点特异性串扰 正常和糖尿病心肌细胞蛋白质组和纯化的心肌细胞线粒体 老鼠。化学-酶促光裂解标签富集法与电子转移解离相结合 (ETD)串联质谱仪，我们将量化0-GlcNAc和磷酸盐在 正常和糖尿病大鼠的心肌细胞收缩和线粒体蛋白。目标2：确定 0-GlcN酰化在正常心肌细胞线粒体中的特殊作用及其作用部位 以及糖尿病导致功能障碍的机制，导致ROS的产生。我们会特别修改 使用在过去20年中开发的方法进行的0-GlcN酰化，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0-GlcNAcase的异构体。人们对线粒体几乎一无所知。 0-GlcNAc转移酶(OGT)或0-GlcNAcase(OGA)的异构体。我们将阐明它们的定位， 正常和糖尿病大鼠线粒体的活性、分子结合和动力学活性。目标4： 评估糖尿病引起的线粒体功能障碍和氧自由基增加的影响和作用 心肌细胞收缩机械的GlcN酰化对心脏生理和功能的影响。劳作 与Core D密切合作，我们将系统地评估0-GlcN酰化之间的串扰的重要性 心肌细胞收缩和线粒体蛋白的磷酸化对其生理功能的影响 心肌细胞 这些研究将开启一种新的范式，以了解心脏功能的调节和 糖尿病心肌病。它们将导致可能的治疗干预的完全未知的途径 相关性(请参阅说明)： 糖尿病是一种主要的流行病，会导致心血管疾病，最终导致 心力衰竭或心肌梗死。增加0-GlcN酰化，这是一种糖的翻译后修饰， 通过影响收缩功能而导致糖尿病心肌病的分子事件 和心肌细胞内的线粒体蛋白。这些研究将阐明O-的重要性 GlcNAc在正常和糖尿病心肌细胞生理中的作用，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.