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.
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