The stress induced or enhanced synthesis of a small number of specific proteins is a fundamental phenomenon. The overall goal of this competing renewal remains the same; that is, to understand the role of heat stress proteins (hsp) in survival and adaptations following hyperthermic stress.
The specific aims of this proposal are: (1) to continue to develop monoclonal antibodies to specific subsets of the hsp. The focus of antibody development will be on lower molecular mass hsp which were synthesized differently in syngeneic rat tumor and endothelial cells, and differently in vitro than in vivo. Focusing studies on these few differences may lead to insights of the hyperthermic biology of tumor and normal cells, of phenotypic differences between these cells, and of the role of some hsp. (2) To ask if changes in the display of cell surface and integral membrane proteins occur with the development of thermotolerance and if any of these are hsp or are associated with hsp. Biotinylation of membrane proteins in situ will be used to probe changes in membrane protein arrangements following slow or rapid temperature transients to hyperthermic temperature. Labeling of membrane proteins and hsp will be used in conjunction with cell-surface-specific biotinylation and isolation techniques. Preliminary studies indicate this approach will detect changes in hsp relationships to membrane proteins after heating. (3) To determine the in situ interactions of hsp with other cellular macromolecules. Photoreactive, cleavable cross-linking reagents will be used to cross-link the hsp to their associated macromolecular structures in thermotolerant cells. The resulting complexes will be analyzed by two-dimensional gel electrophoresis and other standard biochemical and biophysical methods. (4) To use biotinylated macromolecules to probe for the structural characteristics of hsp and for their macromolecular interactions. Biotinylated probes including lectin panels, structural proteins, calmodulin and hsp antibodies will be used to isolate the hsp or macromolecular-hsp complexes. Any interactions of the hsp with these probes will be characterized and will help identify the biochemical and functional roles of the hsp. Preliminary studies indicate this approach will be successful. (5) To determine if any relationship exists between the hsp, a heat effect on the interaction of calmodulin with its binding proteins and thermotolerance to cell killing. The influence of calcium ionophores, organic calcium channel blockers and calmodulin inhibitors on the striking effect of heat on the calmodulin-binding proteins, and on hsp and thermotolerance will be determined. Combined with the results of experiments described in Aims One through Four, these experiments will further clarify the role of hsp in mechanisms of cellular lethality, thermal adaptations and thermotolerance.
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