The dynamic turnover of cellular proteins has been recognized for a long time, but the biochemical mechanisms which carry out the selective degradation of intracellular proteins have begun to be elucidated only recently. A major part of selective protein breakdown appears to be carried out by the ubiquitin proteolytic pathway. Studies indicated that the covalent conjugation of the polypeptide ubiquitin to proteins is the initial signal event in protein breakdown. We have characterized several enzymatic steps in the formation and breakdown of ubiquitin-protein conjugates, but major questions concerning the mode of action of the ubiquitin proteolytic pathway remained obscure. We intend to delineate some of the as yet unexplored enzymatic reactions in the formation and breakdown of ubiquitin conjugates. We wish to focus on the question: What determines the specificity of the ubiquitin conjugation system for the commitment of a certain protein for degradation. Recent results indicate that the availability of a free and exposed NH2-terminal Alpha-NH2 group of the protein substrate is an important determinant. We shall characterize the structure of different ubiquitin-protein conjugates to determine whether ubiquitin conjugation to the Alpha-NH2 group is the initial event which allows the subsequent conjugation of multiple molecules of ubiquitin to Epsilon-NH2 groups of lysine residues. We shall examine what other features of protein structure (besides the free NH2-terminus) are recognized by the uniquitin conjugation system. The selectivity of protein breakdown may further be increased by a correction system, which removes ubiquitin from incorrectly conjugated proteins not destined for degradation. We shall examine the possible role of ubiquitin-protein lyases in such a correction mechanism, in an assay system coupled to purified conjugate-forming enzymes. Since protein breakdown carries out important cellular functions, such as the regulation of enzyme levels and the removal of abnormal proteins, the elucidation of its mechanisms and selectivity are of basic importance and may have further implications in the understanding of diseases of abnormal protein catabolism.
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