Protein degradation is a fundamental cellular process that is believed to be involved in cellular regulation, differentiation and development. Defects in this process are associated with certain tissue degenerative diseases such as muscular dystrophy and may be involved in senescence. The mechanisms by which proteins are degraded in vivo are essentially unknown. The long-range goals are to understand what features of a protein signal its destruction, what enzymes are involved and how this process can be regulated. Ultimately, we wish to understand how these processes are integrated into the cellular control systems. As a first step in understanding these processes, we are studying the mechanism by which fragments of the well-characterized enzyme, Beta-galactosidase, are degraded in the bacterium E. coli. (1) We have demonstrated that in vivo the first step in degradation of large fragments created by nonsense mutations in lacZ is a specific endoproteolytic attack. We will characterize the site of attack and identify and characterize the enzyme(s) responsible. A rapid peptide mapping scheme will be developed that involves SDS PAGE, double-label techniques and HPLC procedures and an in vitro assay system will be developed in which monoclonal antibodies will be used as probes. (2) The fate of the fragments produced in this first cleavage event are unknown. We will determine whether the fragments are degraded intracellularly by an ordered pathway, by random proteolytic cleavages or excreted from the cell. (3) Our kinetic studies suggest that the initiation of degradation may be autoregulated. We will test various models that could account for such autoregulation. (4) Finally, we will address the question of what features of a protein signal its destruction. This problem will be approached both by in vitro structural perturbation studies and by genetic manipulations of the Beta-galactosidase fragments. Hopefully, these studies on a simple, well-defined system will provide insight into similar events in more complex cellular systems.
