Mechanism of Heat Shock Protein Induction by Glutamine

谷氨酰胺诱导热激蛋白的机制

• 批准号: 7777811
• 负责人: PAUL E WISCHMEYER
• 金额: $ 26.79万
• 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/7777811
• 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; Critical Illness; Data; Enhancers; Enzymes; Epithelial Cells; Evaluation; Experimental Models; Family; Fibroblasts; Gene Activation; Gene Knock-Out Model; Gene Silencing; Genes; Genetic Transcription; Genus Cola; Glutamine; Goals; Heat Stress Disorders; Heat shock proteins; Heat-Shock Proteins 70; Heating; Human; Inflammatory; Injury; Intestines; Laboratories; Link; Measures; Mediating; Metabolism; Modeling; Nuclear Translocation; Operative Surgical Procedures; Outcome; Pathway interactions; Patients; Plasmids; Promoter Regions; Proteins; Reporter; 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）表达可保护细胞和组织免受损伤。此外，增强的 HSP 表达可提高危重疾病实验模型的存活率。然而，这些发现尚未应用于临床，因为实验室的 HSP 诱导剂对人体给药并不安全。因此，这种可以显着改善临床结果的强大工具尚未得到利用。我们的实验室已证明谷氨酰胺 (GLN) 可以安全地增强危重和受伤动物组织中的 HSP 表达，并确定 HSP 诱导对于 GLN 在实验疾病后发挥有益作用是必要的。在一项针对危重患者的试验中，我们证明 GLN 可以提高 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 途径的活性以及 HSP 途径关键诱导物的核转位和激活，例如 Sp1 和热休克因子 1 (HSF-1)。此外，siRNA 对关键 O-GlcNAc 途径酶之一的抑制显着减弱了 GLN 介导的 HSP 表达增加。该提案的主要重点是确定 GLN 诱导 HSP 表达的机制。我们的假设是 GLN 通过转运到细胞中并通过 O-GlcNAc 途径代谢来增加 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： 该项目将利用疾病/损伤的细胞和动物模型，并利用这些途径中关键酶的化学/遗传抑制来实现以下具体目标： 1) 评估 GLN 转运的作用应激/损伤时通过 O-GlcNAc 途径进行新陈代谢。 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 对负责 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 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，以及 c) 细胞体积。该项目将阐明 GLN 如何在临床疾病和损伤中诱导 HSP 表达。我们相信 GLN 将能够在手术前或危重疾病/组织损伤发作时（进入 ICU/急诊室）作为药物给药，以增强 HSP 表达并提高生存率。该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表达。我们相信，谷氨酰胺将能够在手术前或危重疾病/组织损伤（进入 ICU/急诊室）发作时作为药物给药，以增强热休克蛋白表达并提高生存率。