PRODUCT MEDIATED REGULATION OF ORNITHINE DECARBOXYLASE

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Because short-lived proteins control such cellular processes as growth differentiation and oncogenesis, we need to understand how they are recognized and degraded. Ornithine decarboxylase (ODC) is among the most labile of cellular proteins. It is a key enzyme in the biosynthesis of polyamines, molecules essential for life. Elaborate and diverse cellular mechanisms control polyamine production, catabolism and transport. Increased intracellular levels of polyamines accelerate degradation of ODC, by triggering production of a second protein, termed antizyme (AZ). AZ binds to ODC and directs its proteolytic destruction by the proteasome, thereby providing a form of feedback control of polyamine pools. This system will serve as a model for studying directed and regulated protein degradation. In collaboration with X-ray crystallographers, we will determine the three-dimensional structure of both ODC and AZ, so as to better understand their structural domains. We will create mutant and chimeric proteins, in order to determine the functional domains that enable AZ to specify ODC as a substrate for degradation and to ascertain whether these domains retain their functional properties when associated with diverse proteins. The proteasome, an intracellular proteolytic molecular machine composed of multiple polypeptides, degrades ODC. We will study the interactions between and among the proteasome, ODC and AZ. We will determine whether AZ escorts ODC to the proteasome or activates the proteasome and will identify the constituents of the proteasome responsible for association with ODC or AZ. Polyamines also promote degradation of ODC in yeast. We will seek and characterize yeast mutants in which this process is disrupted. We will clone the genes for postulated yeast proteins that bind ODC and examines their regulatory role. AZ probably has functions that extend beyond regulation of ODC. To find out whether it has more general cellular effects, we will force its expression. By doing so in cells that are repleted with polyamines, we will determine whether its expression creates phenotypic effects independent of its effect on polyamine production.