Regulation of O-GlcNAcylation During Injury

摘要 O-连接β-N-乙酰氨基葡萄糖(O-GlcNAc)对细胞内蛋白质的修饰已成为一种 新型细胞保护调节剂。多种形式的细胞损伤，包括心脏缺血预适应 (急性、长期和远程)，在体内和体外模型中都会导致O-GlcNAc水平升高。提升 O-GlcN酰化在诱导细胞损伤之前或之后对缺血模型的保护作用 再灌注(I/R)损伤，以及热应激、氧化应激、内质网应激、低氧和 创伤出血。综上所述，这些数据表明O-GlcNAc是一种新的内源性心脏保护物质 探员。到目前为止，研究I/R损伤模型中O-GlcNAc修饰的工作主要集中在 确定O-GlcNAc介导心脏保护的蛋白质和机制。然而， 了解O-GlcNAc修饰在损伤过程中的调节是至关重要的，目前还没有研究。目标是 这一建议的目的是绘制循环O-GlcNAc修饰的酶的调控网络，即O-GlcNAc修饰 GlcNAc转移酶(OGT)和O-GlcNAcase(OGA)。具体地说，我们将完成以下目标： 目的1.确定蛋白质-蛋白质相互作用对OGT和OGA活性以及底物靶向的影响 在受伤的心里。定量质谱学将用于鉴定OGT的蛋白质相互作用 和OGA。生物化学方法的组合将被用来询问这些效应器的作用- 蛋白质对缺血心脏O-GlcNAc循环的影响 目的2.定位受损心脏中OGT和OGA的共价调节网络。O-GlcNAc 将确定OGT和OGA的修饰位点和其他共价调节剂。O-的影响 在氧化应激和I/R损伤模型中，将评估GlcN酰化对OGT/OGA功能的影响。 总而言之，我们预计这些研究将确定调节OGT、OGA和O- I/R损伤时的GlcN酰化。这一批判性的见解将为研究新的治疗方法提供一个框架 心肌梗死的靶点和对O-GlcNAc介导的应激反应的理解 调控失调导致心血管疾病

Summary The modification of intracellular proteins by O-linked β-N-acetylglucosamine (O-GlcNAc) has emerged as a novel regulator of cytoprotection. Numerous forms of cellular injury, including cardiac ischemic preconditioning (acute, prolonged, and remote), 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 (I/R) 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. To date, the majority of work studying the O-GlcNAc modification in models of I/R injury has focused on identifying the proteins and mechanisms by which O-GlcNAc mediates cardioprotection. However, understanding the regulation of the O-GlcNAc modification during injury is critical and is yet unstudied. The goal of this proposal is to map the regulatory networks of the enzymes that cycle the O-GlcNAc modification, the O- GlcNAc transferase (OGT) and O-GlcNAcase (OGA). Specifically, we will complete the following aims: Aim 1. Define the impact of protein-protein interactions on OGT and OGA activity and substrate targeting in the injured heart. Quantitative mass spectrometry will be used to identify protein-interactors of OGT and OGA. A combination of biochemical approaches will be used to interrogate the role of these effector- proteins on O-GlcNAc cycling in the ischemic heart. Aim 2. Map the OGT and OGA covalent-regulatory networks in the injured heart. The O-GlcNAc modification sites, and other covalent-regulators, of OGT and OGA will be identified. The impact of O- GlcNAcylation on OGT/OGA function will be assessed in models of oxidative stress and I/R injury. Collectively, we anticipate that these studies will define the pathways that regulate OGT, OGA, and O- GlcNAcylation during I/R injury. This critical insight will provide a framework for investigating novel therapeutic targets for myocardial infarction and an understanding about how the O-GlcNAc-mediated stress response is dysregulated contributing to cardiovascular disease