Mechanism of Heat Shock Protein Induction by Glutamine

谷氨酰胺诱导热激蛋白的机制

• 批准号: 7617867
• 负责人: PAUL E WISCHMEYER
• 金额: $ 27.14万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-05-01 至 2013-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7617867
• 关键词: Accident and Emergency department; Address; Animal Model; Animals; Binding; Biological Assay; Cell Line; Cell Survival; Cell Volumes; Cell model; Cells; Chemicals; Clinical; Confidential Information; Critical Illness; Data; Enhancers; Enzymes; Epithelial Cells; Evaluation; Experimental Models; Family; Fibroblasts; Funding; Gene Activation; Gene Knock-Out Model; Gene Silencing; Genes; Genetic Transcription; Genus Cola; Glutamine; Goals; Health; Heat Stress Disorders; Heat shock proteins; Heat-Shock Proteins 70; Heating; Human; Inflammatory; Injury; Instruction; Intestines; Laboratories; Language; Link; Measures; Mediating; Metabolism; Methods; Mission; Modeling; Nuclear Translocation; Operative Surgical Procedures; Outcome; Pathway interactions; Patients; Plasmids; Promoter Regions; Proteins; Public Health; Reporter; Research; Research Design; Role; Sepsis; Small Interfering RNA; Specificity; Stress; Techniques; Tissues; Transactivation; Transcription factor genes; Transcriptional Activation; cell injury; chemical genetics; enzyme pathway; glycosylation; heat-shock factor 1; ileum; improved; in vivo; inhibitor/antagonist; injured; promoter; protein expression; research study; tool; transcription factor

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.

描述(由申请人提供)：增强的热休克蛋白(HSP)表达保护细胞和组织免受伤害。此外，在危重疾病的实验模型中，增强的HSP表达提高了存活率。然而，这些发现还没有应用于临床环境，因为实验室中热休克蛋白的诱导剂对人类给药是不安全的。因此，这一可能显著改善临床结果的强大工具尚未得到利用。我们的实验室已经证明，谷氨酰胺(Gln)可以安全地增强危重和损伤动物组织中HSP的表达，并且确定HSP的诱导对于Gln在实验性疾病后的有益作用是必要的。在一项对危重患者的试验中，我们证明了谷氨酰胺可提高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)来诱导HSP的表达，O-GlcNAc是已知的依赖于Gln作为限速底物的途径。该途径可以激活热休克蛋白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途径的活性，并增加了HSP途径的关键诱导物如Sp1和热休克因子-1(HSF-1)的核转位和激活。此外，抑制O-GlcNAc途径的关键酶之一的siRNA显著钝化Gln介导的HSP表达的增加。这一建议的主要焦点是确定Gln诱导HSP表达的机制。我们的假设是，Gln通过转运到细胞内并通过O-GlcNAc途径进行代谢，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HSPs的表达。为了解决这一假设，我们提出了三个特定的目标：本项目将利用疾病/损伤的细胞和动物模型，并使用化学/遗传抑制这些途径中的关键酶来解决以下特定目标：1)评估通过O-GlcNAc途径的Gln运输和代谢在应激/损伤中的作用。2)通过截短启动子来评估哪些启动子区域是谷氨酰胺介导的热休克蛋白表达的关键。然后，确定谷氨酰胺对导致HSP表达的关键转录因子的核转位和反式激活的影响。3)评估谷氨酰胺转运、代谢和转录激活对下列细胞和体内终点的影响：a)热休克蛋白表达(多个热休克蛋白家族)，b)组织/细胞损伤，c)细胞体积。本项目将阐明谷氨酰胺如何在临床疾病和损伤中诱导热休克蛋白的表达。我们相信，谷氨酰胺将能够在手术前或在危重疾病/组织损伤开始时(进入ICU/急诊室)作为一种药理学药物给予，以增强HSP的表达并改善存活率。本项目将阐明谷氨酰胺如何在临床疾病和损伤中诱导保护性热休克蛋白的表达。我们相信，谷氨酰胺将能够在手术前或在危重疾病/组织损伤开始时(进入ICU/急诊室)作为一种药理学药物给予，以增强热休克蛋白的表达，提高存活率。