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The long range goals of the research are: to determine what role translational regulation plays in the control of cell proliferation, differentiation, and viability; and to determine how teratogenic stress agents (e.g., toxic heavy metal ions) may adversely affect these processes. The rabbit reticulocyte lysate will be utilized initially as a model system. The effects of oxidative stress (heavy metal ions) and heat stress on protein synthesis will be compared. Our principal hypothesis is that the activity of the rabbit reticulocyte heme-regulated eIF-2alpha kinase (HRI) is regulated directly through its interactions with heat shock proteins. We propose that environmental stress agents cause the accumulation of denatured proteins. The interaction of the denatured proteins with hsps, subsequently affects the binding equilibrium of hsps to HRI, leading to the activation of HRI and the inhibition of protein synthesis. The specific goals of the proposal are to: 1) Define the role of hsp binding in the regulation of HRI activity by: (a) characterizing and quantifying the composition and activity of HRI/hsp complexes isolated from reticulocyte lysate in situ; and (b) determining the effect of hsps on the activity of purified HRI in vitro. 2) Define the role played by denatured proteins in the regulation of HRI activity. 3) Determine whether changes in eIF-4E phosphorylation are occurring in reticulocyte lysate in response to heat shock. 4) Characterize changes in hsp levels and translational regulation during reticulocyte maturation. 5) Characterize the effects of heat and oxidative stress on protein synthesis during growth and terminal differentiation of mouse erythroid leukemia (MEL) cells in culture. HRI/hsp interactions will be studied by using monoclonal antibodies to characterize the composition of HRI/hsp complexes during various stages of HRI activation in the reticulocyte lysate. The activation (autophosphorylation) and activity (rate of eIF-2alpha phosphorylation) of HRI in the complexes will be determined. The effects of hsps and chaperonins on the binding of ATP and hemin to HRI, and HRI activity will be quantitated in vitro using purified components. Hsp levels during reticulocyte maturation will be studied by analyzing hsp level in cultured reticulocytes at different times during recovery from anemia. Model substrates and synthetic peptides for the binding of hsp70 will be used to study the relationship between HRI activation and the ability of denatured proteins to block the binding of hsp70 to HRI. The effect of the protein stabilizing agent (D2O) on HRI activity and protein stability during heat shock will be determined. The model substrates for the binding of hsp 70 will be used to quantitate the level of functional hsp70 present in lysate (i.e., the pool of hsp70 available to bind denatured protein). The ability of lysate to restore the activity of marker enzymes (e.g., firefly luciferase) will be used as an assay to purify reticulocyte "chaperonin" activity. The effect of the addition of purified hsps and chaperonins on protein synthesis and HRI activity in reticulocyte lysate will be determined. The effects of stress agents on the levels of hsp70 and chaperonin activity will be determined in situ and in vitro. An in vivo system will be developed to study whether changes in hsp expression occur, which similarly affect the regulation of protein synthesis through eIF-2alpha phosphorylation in cultured MEL cells.
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