CYTOPROTECTIVE ROLE OF HEAT SHOCK PROTEINS IN IBD

热休克蛋白在 IBD 中的细胞保护作用

• 批准号: 2016790
• 负责人: EUGENE B CHANG
• 金额: $ 23.3万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-09-30 至 2002-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2016790
• 关键词: cell growth regulation; cell line; cytoskeleton; dihydrofolate reductase; enzyme activity; gastrointestinal epithelium; genetic transcription; hyperthermia; immunocytochemistry; inflammatory bowel diseases; oxidative stress; oxidizing agents; protein biosynthesis; sodium potassium exchanging ATPase; stress proteins; tight junctions; transfection

DESCRIPTION: This is an application for continuation of studies to focus on the cytoprotective role of heat shock protein in gastrointestinal epithelial cells. The PI proposes the following specific aims: 1. The effects of IBD-relevant agents and conditions that induce a unique 72 kD heat shock protein (hsp 72) response in intestinal epithelial IEC18 cells will be investigated. In all instances, comparative studies of the expression of hsp 72 and its constitutively-expressed counterpart, hsc 73 will be measured in IEC18 and HeLa cells to determine the specificity of hsp 72 induction. To examine the basis of IEC-specific differences in the hsp 72 response, potential involvement of transcriptional and post-transcriptional processes will be investigated. The functional significance to cell protection of induced hsp 72 will be assessed by anti-sense inhibition of hsp 72 expression. 2. Next, the interaction and protection by hsp 72 of vital IEC targets adversely affected by oxidant and/or thermal stress will be investigated. As part of these studies, the investigator will test the hypothesis that the "activation" of hsp 72 involves its mobilization and binding to vital cellular targets central for survival. The mechanism of hsp 72 protection of the cytosolic enzyme dehyrdofolate reductase (DHFR), with respect to activity and protein structure under conditions of oxidant and thermal stress will be investigated. The nature and potential sites of interaction of the hsp 72-DHFR complex will be examined by competitive binding assays using synthetic peptides of putative binding domains of each molecule. The relative roles of ATP/ADP and DHFR unfolding in promoting this interaction will be studied. 3. Next, hsp interaction and protection of the components of the cytoskeleton during stress will be studied. Both in vitro and intact cell experiments will be performed, the later in Caco-2/C2BBE (c2) cells stably transfected with hsp 72 anti-sense to inhibit their constitutive expression of hsp 72. These studies will provide important insights into how each heat shock protein protects the intestinal epithelium during inflammation-induced stress. This is a relatively unexplored area of investigation and has relevance to the understanding of the mechanisms of intestinal mucosal protection in IBD.

描述：这是一个继续研究的申请，重点是 热休克蛋白对胃肠道上皮细胞保护作用 细胞 PI提出了以下具体目标：1。 的影响 诱导独特72 kD热休克的IBD相关药物和条件 肠上皮IEC 18细胞中的蛋白（hsp 72）应答将被 研究了 在所有情况下，比较研究的表达， 将测量HSP 72及其组成型表达对应物HSC 73 在IEC 18和HeLa细胞中测定hsp 72诱导的特异性。 为了研究hsp 72反应中IEC特异性差异的基础， 转录和转录后过程的潜在参与 将进行调查。 对细胞保护的功能意义 诱导的HSP 72将通过HSP 72的反义抑制来评估 表情 2. 其次，通过热休克蛋白72的相互作用和保护的重要IEC 受到氧化剂和/或热应力不利影响的目标将 研究了 作为这些研究的一部分，研究者将测试 假设HSP 72的“活化”涉及其动员， 与生存的重要细胞靶点结合 的机理 hsp 72保护胞质酶脱氢叶酸还原酶（DHFR）， 在氧化条件下的活性和蛋白质结构方面 和热应力进行研究。 的性质和潜在地点 hsp 72-DHFR复合物的相互作用将通过竞争性的 使用每种蛋白的推定结合结构域的合成肽的结合测定 分子。 ATP/ADP和DHFR去折叠在促进 将研究这种相互作用。 3. 接下来，HSP的相互作用和保护 将研究细胞骨架的组成部分在压力下的变化。 两 将进行体外和完整细胞实验，后者在 用hsp 72反义稳定转染的Caco-2/C2BBE（c2）细胞抑制HSP 72的表达。 它们的热休克蛋白72的组成型表达。 这些研究将提供 每种热休克蛋白如何保护肠道的重要见解 在炎症诱导的应激期间的上皮。 这是一个相对 未探索的调查领域，并与理解有关 IBD肠粘膜保护机制的研究进展。