Mechanism of Heat Shock Protein Induction by Glutamine

描述（由申请人提供）：增强的热休克蛋白（HSP）表达可保护细胞和组织免受损伤。此外，增强的HSP表达提高了危重病实验模型的存活率。然而，这些发现尚未应用于临床环境，因为实验室热休克蛋白诱导剂对人体给药并不安全。因此，这个强大的工具，可以显着改善临床结果，尚未被利用。我们的实验室已经表明谷氨酰胺（GLN）可以安全地增强危重和受伤动物组织中HSP的表达，并且建立HSP诱导对于GLN在实验性疾病后的有益作用是必要的。在一项危重患者的试验中，我们证明了GLN增强HSP-70水平，这与改善预后相关。然而，GLN诱导HSP表达的机制尚不清楚。我们假设GLN通过激活O-连接的糖基化途径（O-GlcNAc）诱导HSP表达，已知该途径依赖于GLN作为限速底物。该途径可以激活HSP诱导所需的关键转录因子。我们的初步数据表明，GLN增加了O-GlcNAc途径和核转位的活性，并激活了HSP途径的关键诱导物，如Sp1和热休克因子-1（HSF-1）。此外，关键O-GlcNAc途径酶之一的siRNA抑制显著减弱了GLN介导的HSP表达增加。该建议的主要重点是确定GLN诱导HSP表达的机制。我们的假设是GLN通过转运进入细胞并通过O-GlcNAc途径代谢，增加HSP基因激活所需的关键转录因子的O-连接糖基化，从而增加HSP的表达。为了解决这一假设，我们提出了三个具体目标：本项目将利用疾病/损伤的细胞和动物模型，并采用这些途径中关键酶的化学/遗传抑制来解决以下具体目标：1）评估GLN通过O-GlcNAc途径的转运和代谢在应激/损伤中的作用。2)通过启动子截短评估哪些启动子区域是GLN介导的HSP表达的关键。然后，确定GLN对核转位和负责HSP表达的关键转录因子的反式激活的影响。3)评价GLN转运、代谢和转录激活对以下细胞和体内终点的影响：a）HSP表达（HSP的多个家族），B）组织/细胞损伤和c）细胞体积。本项目将阐明GLN如何诱导HSP在临床疾病和损伤中的表达。我们相信GLN将能够在手术前或在危重病/组织损伤发作时（进入ICU/急诊室）作为药理学试剂给药，以增强HSP表达并提高生存率。本项目将阐明谷氨酰胺在临床疾病和损伤中如何诱导保护性热休克蛋白表达。我们相信谷氨酰胺将能够在手术前或在危重病/组织损伤发作时（进入ICU/急诊室）作为药理学试剂给药，以增强热休克蛋白表达并提高生存率。

DESCRIPTION (provided by applicant): Enhanced heat shock protein (HSP) expression protects cells and tissues from injury. Further, enhanced HSP expression improves survival in experimental models of critical illness. However, these findings have not been applied in a clinical setting, as laboratory inducers of HSPs are not safe for human administration. Thus, this powerful tool, that may significantly improve clinical outcome, has yet to be utilized. Our laboratory has shown glutamine (GLN) can safely enhance HSP expression in tissues of critically ill and injured animals and established HSP induction is necessary for GLN's beneficial effect following experimental illness. In a trial of critically ill patients we demonstrated GLN enhanced HSP-70 levels, which correlated with improved outcome. However, the mechanism by which GLN induces HSP expression is unknown. We hypothesize GLN induces HSP expression via activation of the O-linked glycosylation pathway (O-GlcNAc), which is known to depend on GLN as a rate limiting substrate. This pathway can activate key transcription factors required for HSP induction. Our preliminary data indicates GLN increases the activity of the O-GlcNAc pathway and nuclear translocation and activation of key inducers of the HSP pathway, such as Sp1 and heat shock factor-1 (HSF-1). Further, siRNA inhibition of one of the key O-GlcNAc pathway enzymes significantly blunts the GLN-mediated increase in HSP expression. The major focus of this proposal is to determine the mechanism by which GLN induces HSP expression. Our hypothesis is GLN acts via transport into the cell and metabolism by the O-GlcNAc pathway to increase O-linked glycosylation of key transcription factors required for HSP gene activation, which then increases the expression of HSPs. To address this hypothesis, we propose three specific aims: This project will utilize cellular and animal models of illness/injury and employ chemical/genetic inhibition of key enzymes in these pathways to address the following specific aims: 1) Evaluate the role of GLN transport and metabolism via the O-GlcNAc pathway in stress/injury. 2) Evaluate via promoter truncation which promoter regions are key for GLN-mediated HSP expression. Then, determine the effect of GLN on nuclear translocation and transactivation of key transcription factors responsible for HSP expression. 3) Evaluate effects of GLN transport, metabolism, and transcriptional activation on the following cellular and in vivo endpoints: a) HSP expression (multiple families of HSPs), b) tissue/cellular injury, and c) cell volume. This project will elucidate how GLN induces HSP expression in clinical illness and injury. We believe GLN will be able to be administered as a pharmacologic agent prior to surgery or at onset of critical illness/tissue injury (with admittance to ICU/emergency room) to enhance HSP expression and improve survival. This project will elucidate how glutamine induces protective heat shock protein expression in clinical illness and injury. We believe glutamine will be able to be administered as a pharmacologic agent prior to surgery or at onset of critical illness/tissue injury (with admittance to ICU/emergency room) to enhance heat shock protein expression and improve survival.