Ischemia of whole epithelial tissue, or ATP depletion of epithelial cells in culture, induces a very impressive stress response in the endoplasmic reticulum (ER). This is manifested by substantial induction in mRNA levels of ER stress proteins which act as molecular chaperones, including BiP, glp94, ERp72, and possibly additional ones. Induction of ER molecular chaperones has previously been shown to occur when malfolded proteins accumulate in the ER. This can be due to either: misfolding of proteins, misassembly of multimers or inhibition of the normal degradative mechanism which degrades malfolded proteins. AU of these processes have previously been shown to be critically dependent on the ATP; however, the molecular mechanism of each process remains poorly understood. Nevertheless, because of the tremendous ER stress response observed under ischemic conditions, and because of the critical dependence of ER folding and degradative reactions on ATP, it is hypothesized that one major mechanism underlying the D subcellular pathology in ischemia is the accumulation of malfolded secreted and membrane proteins in the ER Z. This hypothesis is strongly supported by preliminary data (Fig. 15-18); however, it is an area which has yet to be studied in detail. Although other mechanisms are likely to be operative as well (e.g., cytoskeletal dysfunction, effects on: cytosolic Hsp70), this hypothesis provides a potential unifying mechanism for explaining much of the ischemic epithelial phenotype. Under acute, subacute and chronic ischemic conditions, epithelial cells lose cell adhesive mechanisms, develop dysfunctional tight junctions, lose their ability to transport ions vectorially and decrease their cell-substratum interactions. These are mediated, respectively, by cell adhesion molecules, transmembrane tight junctional proteins, transporters appropriately sorted to the apical or basolateral surface of epithelial cells I and integrins: all these proteins are initially folded and assembled in the ER. The ability to recover from ischemic insults would be expected to depend on appropriate """"""""chaperoning"""""""" of these proteins in the ER in order to form new adhesive interactions, new junctions, reestablish vectorial ion transport and develop strong cell-substratum interactions. As already mentioned, ATP is essential for normal ER chaperone function. As is clear from the preliminary data, it has been shown that a set of resident ER proteins, which includes BiP, grp94 (glucose regulatory protein of 94 kD), ERp72 (ER protein of 72 kD), PDI (protein disulfide isomerase), calreticlin, ERp 50, binds to a variety of misfolded proteins and is released from them by ATP hydrolysis in vitro (Fig. 1-3). This same group of proteins also associates with various secretory proteins as they are folded and assembled in the ER in intact cells. Thus, this group of proteins has characteristics of ER molecular chaperones. Furthermore, as indicated by preliminary data, the same group of proteins appears to be involved in the ER degradative pathway thought to be responsible for degradation of malfolded proteins detected by the """"""""quality control"""""""" mechanism in the ER. The goal of this proposal is to investigate, in detail, the mechanisms underlying protein folding and """"""""quality control""""""""/degradation (recognition and degradation of misfolded and misassembled proteins) in the ER and to determine how ischemia affects these ATP-dependent processes, thereby resulting in the accumulation of malfolded proteins in the ER. Three exemplary epithelial secreted and membrane proteins will be studied: thyroglobulin (Tg), E- cadherin and Na/K ATPase. As explained in the body of the application, together, the appropriate folding of these proteins is likely to require a wide variety of folding reactions and their study is likely to elucidate differences in the folding and/or degradation of different classes of proteins transiting the ER Furthermore, the (mis)folding of E-cadherin (the major epithelial cell adhesion molecule) and Na+/K+-ATPaseI (which is critical for vectorial transport of ions and is mispolarized in ischemia) are of particular interest in epithelial ischemia.
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