9603740 Stanley Selective protein degradation is an important cellular process contributing to abnormal protein removal, cell-cycle progression, stress responses, and normal growth regulation, yet little is known about the molecular determinants of such selectivity, particularly differences in protein structure or proteolytic systems that might account for the many-fold range of protein half-lives in cells. Approaches using in vitro degradation systems or in vivo destabilization of mutated versions of normally stable proteins may not represent physiological degradation pathways or targeting signals. This project will use the polyamine biosynthetic enzyme S-adenosylmethionine decarboxylase (SAMDC), a cytosolic enzyme which is normally rapidly degraded, as a model for the subclass of proteins which are rapidly turned over in the cytosol, and examine its degradation in cell culture under a variety of conditions designed to determine the intracellular effectors of this degradation, the proteolytic system(s) which degrade SAMDC, and the targeting signal(s) in the SAMDC structure which cause its rapid degradation. Using specific inhibitors of the other enzymes of the polyamine biosynthetic pathway, as well as inhibitors of the production of the SAMDC substrate S-adenosylmethionine, the investigator will determine which of these substrate products serve as endogenous effectors of SAMDC degradation. An additional goal of the research will be to determine the cellular proteolytic systems which normally degrade SAMDC, using specific inhibitors of the various known intracellular proteolytic activities to look for SAMDC-stabilizing effects in vivo. Finally, potential targeting signals in the SAMDC structure will be examined by creating deletions and site-specific mutations, expressing these mutant SAMDCs in mammalian cells, and measuring the resulting half-lives. Candidate regions/residues will be chosen in several ways, including a comparison of the half lives (in mammalian cells) of expressed SAMDCs fro m other species (which have regions of similarity as well as dissimilarity with the mammalian enzyme), computer predictions of surface-accessible regions and experimental determination of surface accessible acidic and basic residues. Selective protein degradation has become recognized as a critical process which contributes to the regulation of the amount of specific proteins present in cells at any time, but very little is known about how this selectivity occurs. Unstable proteins as a class include many proteins known to be involved in cell growth, so understanding the processes involved in this selection for instability is quite important. The present research focuses on a particular unstable, growth-related protein, SAMDC, with a goal of defining what actually rapidly degrades SAMDC in cells, as well as what biochemical aspect of the SAMDC structure makes it a target for this rapid degradation. The information gained from these studies will help to elucidate the basis of targeting proteins for degradation, and will lay the groundwork for future studies of how this degradation process is controlled.
