Intracellular protein degradation plays a number of critically important roles in the normal physiology and in the pathology of cellular protein metabolism. This highly regulated process is a key determinant of the quality and quantity of cellular protein; it serves to selectively remove metabolically or genetically damaged proteins from the cell, and controls levels of individual cellular proteins (including those with important regulatory functions), as well as the net growth or atrophy of tissues. Despite the importance of intracellular protein degradation, the biochemical mechanisms by which it occurs are poorly defined. The long term objectives of this research program are to provide a comprehensive understanding of both the biochemical mechanisms of intracellular protein degradation and the physiological regulation of this process in intact cells and tissues. The specific goal of the proposed research is to determine the biochemical mechanisms of regulation of the multicatalytic protease, the proteasome, by two newly discovered activator proteins, termed PA28 and PA700. Each proteasome activator has been purified and paretically characterized. PA28 and PA700 each activates the proteasomes's three distinct peptidase activities, and together they activate the proteasome's action of PA700, requires hydrolysis of ATP. The first portion of this work will define the biochemical mechanisms regulatory processes involved in each of these actions. The second portion of the work will determine whether the proteasome, PA700, and/or PA28 are components of a large """"""""26S protease"""""""" known to degrade ubiquitinated proteins. The third portion of the work will determine the primary structures of PA28 and PA700 by recombinant DNA methodologies. This essential information will be used for the final portion of the work, which will establish detailed structure/function relationships for PA28. This analysis will use synthetic peptide corresponding to functional domains of PA28, as well as native and mutant PA28 proteins expressed in E. coli. These studies will provide important information about the biochemical function and regulation of the proteasome, and will serve as the basis for exploration of its role in physiological and pathological states of intracellular protein degradation.
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