Preservation of organ function during transplantation, routine surgical procedures, and after trauma is an important aspect in critical care medicine which in spite of intensive research has not been satisfactorily resolved. One alternative that may help in preserving organ function is to utilize natural mechanisms of cellular protection. The understanding of such cytoprotective mechanisms should permit the development of strategies that can be applied clinically to improve preservation of organ function after surgical stress. One of the most basic mechanisms of cellular protection is the expression of a set of genes known as heat shock or stress proteins (hsps). The induction of hsps expression has been correlated with the phenomenon of acquired stress tolerance, defined as the capacity of stress primed cells to survive a second, otherwise lethal thermotolerance stress. During the last several years, evidence has accumulated that links the development of thermotolerance with the preservation of the cellular capacity to synthesize proteins during stress and this has been correlated with the cellular levels of the inducible form of hsp-70, hsp-72. More recently hsp-72 has been found interacting with polysomes of thermotolerant cells during stress. The objective of the present proposal is to investigate whether the interaction of hsp-72 with nascent polypeptides within polysomes promotes translational elongation and stabilizes protein synthesis during the stress and thus, contributing to cytoprotection.
The specific aims of the investigation are: I- Mechanism of cytoprotection induced by hsp-72.
This aim i s divided into two parts: 1- To determine the step of translation that is stabilized during stress in thermotolerant cells. 2-Evaluate the contribution of hsp-72 to the recovery of protein synthesis during heat shock. These studies will be directed toward measuring the rates of translation elongation and total protein synthesis in thermotolerant cells, cells transfected with the gene coding for hsp-72, and in an in vitro translation system under conditions that mimic the effect of stress in protein synthesis. II-Molecular basis for the biological activity of hsp-72. The objective os this part of the proposal: 1-Determine the polysome component(s) that is interacting with hsp-72 in thermotolerant cells. 2-To elucidate the region of hsp-72 involved in the interaction with polysomes and denatured proteins. Amino acid substitutions of the proposed peptide binding site of hsp-72 with nascent polypeptides, promoting elongation and stabilizing protein synthesis during stress will provide a better understanding of the process of cytoprotection induced by heat shock proteins and will also provide more information about the process of translation. Ultimately, this information may facilitate the design of strategies which exploit this cytoprotective mechanism for the preservation of organ function following trauma or surgical stress.
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