Defining the Molecular Mechanisms by Which O-GlcNAc Mediates Cardioprotection

描述(由申请人提供)： 近年来，O-连接的2-N-乙酰氨基葡萄糖(O-GlcNAc)对细胞核、线粒体和细胞质蛋白质的修饰已成为应激反应和细胞存活的一种新的调节因子。多种形式的细胞损伤，包括心脏缺血预适应(急性和长期)，在体内和体外模型中都会导致O-GlcNAc水平升高。在诱导细胞损伤以及热应激、氧化应激、内质网应激、低氧和创伤出血的模型中，在诱导细胞损伤之前或之后升高O-GlcN酰化是有保护作用的。综上所述，这些数据表明O-GlcNAc是一种新的内源性心脏保护剂。然而，O-GlcNAc调节蛋白质功能从而增强细胞存活和心脏保护的分子机制尚未确定。该研究人员的长期目标是在分子水平上确定O-GlcNAc促进细胞存活的机制。本应用的目的是：具体目标1：建立细胞器特异性O-GlcNAc传感器，以评价O-GlcNAc在缺血再灌注损伤中的作用。我们建议开发一套基于FRET的O-GlcNAc传感器，它可以在特定的亚细胞间隔中定位O-GlcN酰化的空间和时间变化，并应用这些传感器来确定O-GlcN酰化是如何在分离的新生心肌细胞中对缺血再灌注损伤做出反应的。具体目的2：明确缺血再灌注损伤心脏动态定位的O-GlcNACome。O-GlcNAc水平升高是对缺血预适应的反应，这是一个已知的增强心脏保护的过程。为了识别这些蛋白质，以及它们被O-GlcNacyl化的位置，我们将开发一种新的工具来丰富O-GlcNAc修饰的多肽。总之，这些研究将描述一种新的心脏内源性防御机制，为提高心脏对缺血再灌注损伤的耐受性的替代策略的开发提供新的靶点。 公共卫生相关性： 糖O-GlcNAc是细胞应激反应的关键成分，可增强细胞和组织在缺血再灌注损伤(如心脏病发作)中存活的能力，但O-GlcNAc保护细胞的机制尚不清楚。我们的目标是在分子水平上了解O-GlcNAc如何在缺血再灌注损伤模型中促进细胞存活，从而为开发提高心脏对缺血再灌注损伤耐受性的替代策略寻找新的靶点。

DESCRIPTION (provided by applicant): Recently, the modification of nuclear, mitochondrial, and cytoplasmic proteins by O-linked 2-N- acetylglucosamine (O-GlcNAc) has emerged as a novel regulator of the stress response and cell survival. Numerous forms of cellular injury, including cardiac ischemic preconditioning (acute and prolonged), lead to elevated levels of O-GlcNAc in both in vivo and in vitro models. Elevating O-GlcNAcylation before, or immediately after, the induction of cellular injury is protective in models of ischemia reperfusion injury, as well as heat stress, oxidative stress, endoplasmic reticulum stress, hypoxia, and trauma hemorrhage. Together, these data suggest that O-GlcNAc is a novel endogenous cardioprotective agent. However, the molecular mechanisms by which O-GlcNAc regulates protein function leading to enhanced cell survival and cardioprotection have not been identified. The long-term goal of this investigator, is to identify at a molecular level the mechanisms by which O-GlcNAc promotes cell survival. The objective of this application is to: Specific Aim 1: Develop and characterize organelle specific O-GlcNAc sensors to assess the role of O-GlcNAc in ischemia reperfusion injury. We propose to develop a suite of FRET-based O-GlcNAc sensors that can map spatial and temporal changes in O-GlcNAcylation in specific subcellular compartments, and apply these sensors to determining how O-GlcNAcylation is regulated in response to ischemia reperfusion injury in isolated neonatal cardiomyocytes. Specific Aim 2: Define the dynamic site-specific O-GlcNAcome of hearts subjected to Ischemia reperfusion injury. O-GlcNAc levels are elevated in response to ischemic preconditioning, a process known to enhance cardioprotection. To identify these proteins, and the sites at which they are dynamically O-GlcNAcylated, we will develop a novel tool to enrich O-GlcNAc-modified peptides. Together, these studies will characterize a novel endogenous defense mechanism of the heart, highlighting new targets for the development of alternative strategies that enhance the hearts tolerance to ischemia reperfusion injury. PUBLIC HEALTH RELEVANCE: The sugar O-GlcNAc is a key component of the cellular stress response that enhances the ability of cells and tissues to survive ischemia reperfusion injury (for example, heart attack), but the mechanisms by which O- GlcNAc protects cells are unknown. Our goal is to understand how O-GlcNAc promotes cell survival in a model of ischemia reperfusion injury at the molecular level, thus identifying new targets for the development of alternative strategies to enhance the heart's tolerance to ischemia reperfusion injury.