The long-term objective of this research program is to provide a comprehensive understanding of the biochemical mechanisms and physiological regulation of intracellular protein degradation. This objective has broad significance for understanding numerous cellular processes that utilize protein degradation as a mechanism for their regulation, and for alleviating diseases characterize by tissue atrophy. The general goal of this project is to establish the physiological role of PA28, a protein activation of the proteasome. The proteasome is an intracellular protease required for all ubiquitin-dependent-and an unknown amount of ubiquitin-independent protein degradation in eukaryotic cells. Proteasome function both in vitro and in vivo is mediated by multiple regulatory proteins whose relative cellular roles are poorly defined. PA28 is one of the least understood of these proteins. The cellular role of PA28 will be defined by over-expressing the protein in mammalian cells. These studies will test the hypothesis that PA28 increases overall rates of intracellular protein degradation. The cellular function of PA28 will be examined further by closely related biochemical experiments. These experiments will establish the role of reversible phosphorylation of PA28 as a mechanism for control of its activity by testing the hypothesis that phosphorylation of the PA28 inhibits its proteasome-stimulatory activity. The effect of PA28 on the degradation of model proteins will be studied in vitro by analyzing the kinetics of protein degradation by PA28-proteasome complexes alone and in combination with other proteasome-regulatory protein complexes. These experiments will test the hypothesis that PA28's cellular function is to promote degradation both of large peptides directly and of large peptides generated by the action of other proteasome complexes Finally, cellular substrates for the PA28 proteasome complex will be identified with a screening strategy using expression cloning. These highly integrated studies will define cellular role of PA28 and the possible biochemical bases for them. Therefore, this project will greatly increase the understanding of the mechanisms and regulation of intracellular protein degradation.
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