PRODUCT MEDIATED REGULATION OF ORNITHINE DECARBOXYLASE

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Proteasome-mediated protein degradation is a central process in the regulation of cell function. Ornithine decarboxylase (ODC), a key enzyme in polyamine production, participates in a unique form of feedback regulation, whereby polyamines promote its destruction by inducing the protein antizyme (AZ), which targets ODC for degradation by the proteasome. Specific Aims: 1) Determine the three-dimensional structure of ODC and AZ, and test the function of their elements in degradation. The studies proposed will establish the structure of full length mouse ODC, its complex with the portion of AZ that binds ODC, full length AZ alone and the heterodimer of both full-length proteins. Comparison of AZ to a newly-identified AZ homolog will be used to resolve the structural determinate(s) of degradative function. The x-ray model will be used to understand the structure of the ODC C-terminal domain and how and whether this changes in response to AZ. This information will be exploited to design recombinant forms of the ODC terminus to determine whether these associate with the proteasome, using direct assays of association and functional competition assays. To distinguish whether AZ acts solely as an ODC carrier, or instead or additionally is an activator of the proteasome, AZ will be tested for trans-acting function. 2) Characterize the stepwise elements of ODC/AZ- proteasome interactions. The components of the proteasome that interact with AZ and ODC will be identified. Proteasomes co- incubated with AZ/ODC or related substrates will be examined by high-resolution electron microscopy to localize their position. Chemical crosslinking of ODC and/or AZ to proteasomes will be used to identify protein-protein contacts. Yeast mutants will be used to isolate sub-components of the 26S proteasome and their ability to degrade ODC will be analyzed. Forms of ODC that contain a bulky obstruction that impedes proteasomal entry will be made and used to test the orientation and processivity of degradation. 3) Determine the cellular components that modulate ODC degradation using yeast genetics. All of the features of polyamine-induced ODC degradation that we have tested are identical in animal cells and yeast, suggesting that the latter will be an informative experimental system for studying properties common to both. We have assembled and validated the means to perform genetic screens to isolate yeast mutants altered in their regulation of ODC by polyamines. Genes that complement regulatory mutants will be identified and the function of their products assessed. These studies will reveal cellular components important for regulated degradation.