Mechanism of Heat Shock Protein Induction by Glutamine

说明：请参阅说明。说明应用程序的广泛、长期目标和具体目标，供参考 与该项目的健康有关(即与该机构的任务有关)。简明扼要地描述这项研究 实现这些目标的设计和方法。描述您将用来实现这些目标的基本原理和技术。 此外，用两三句话，简单地描述这项研究与公共卫生的相关性。如果 申请已获资助，此描述将成为公开信息。因此，不包括 专有/机密信息。 增强的热休克蛋白(HSP)表达保护细胞和组织免受损伤。更进一步，增强 在危重疾病的实验模型中，HSP的表达提高了存活率。然而，这些发现已经 没有在临床环境中应用，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 已证明谷氨酰胺(Gln)可以安全地增强危重和受伤动物组织中HSP的表达 GLN在实验性疾病后的有益作用是建立HSP诱导所必需的。在一个 在危重病患者的试验中，我们证明谷氨酰胺提高了HSP-70的水平，这与改善相关 结果。然而，Gln诱导HSP表达的机制尚不清楚。我们假设 谷氨酰胺通过激活O-连接的糖基化途径(O-GlcNAc)诱导HSP的表达，这是 已知依赖谷氨酰胺作为限速底物。这个途径可以激活关键的转录因子。 HSP诱导所必需的。我们的初步数据显示，谷氨酰胺增加了O-GlcNAc的活性 热休克蛋白途径与Sp1、热等关键诱导因子的核转位和激活 休克因子-1(HSF-1)。此外，siRNA显著抑制了O-GlcNAc途径的关键酶之一 钝化Gln介导的HSP表达的增加。这项提案的主要重点是确定 谷氨酰胺诱导热休克蛋白表达的机制我们的假设是谷氨酰胺通过转运进入细胞发挥作用 并通过O-GlcNAc途径代谢，增加关键转录因子的O-连接糖基化 这是热休克蛋白基因激活所必需的，从而增加热休克蛋白的表达。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以及 在这些途径中对关键酶进行化学/遗传抑制，以实现以下具体目标： 1)评估谷氨酰胺通过O-GlcNAc途径的转运和代谢在应激/损伤中的作用。2)评估 通过截短启动子截断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 谷氨酰胺对核转位和转录因子反式激活的影响 HSP表达。3)评估谷氨酰胺转运、代谢和转录激活对 下列细胞和活体终点：a)热休克蛋白表达(多个热休克蛋白家族)，b)组织/细胞 C)细胞体积。该项目将阐明谷氨酰胺是如何在临床疾病中诱导HSP表达的，以及 受伤。我们相信谷氨酰胺将能够在手术前或发病时作为一种药理学药物使用。 危重疾病/组织损伤(进入ICU/急诊室)以增强HSP表达和 提高存活率。该项目将阐明谷氨酰胺如何在临床疾病和疾病中诱导保护性热休克蛋白表达。 受伤。我们相信谷氨酰胺将能够作为一种药理制剂在手术前或在 出现危重疾病/组织损伤(进入ICU/急诊室)以增强热休克蛋白 表达并提高存活率。

DESCRIPTION: See instructions. State the application's broad, long-term objectives and specific aims, making reference to the health relatedness ofthe project (i.e., relevance to the mission of the agency). Describe concisely the research design and methods for achieving these goals. Describethe rationale and techniques you will use to pursue these goals.In addition, in two or three sentences, describe in plain, lay language the relevance of this research to public health. If the application is funded, thisdescription, as is, will become public information. Therefore, do not include proprietary/confidential information. 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.