Regulation of all members of the antizyme family by antizyme inhibitor.
ODC (ornithine decarboxylase) is the rate-limiting enzyme in polyamine biosynthesis. Polyamines are essential for cellular growth and differentiation but enhanced ODC activity is associated with cell transformation. Post-translationally, ODC is negatively regulated through members of the antizyme family. Antizymes inhibit ODC activity, promote ODC degradation through the 26 S proteasome and regulate polyamine transport. Besides the ubiquitously expressed antizymes 1 and 2, there is the tissue-specific antizyme 3 and an yet uncharacterized antizyme 4. Antizyme 1 has been shown to be negatively regulated through the AZI (antizyme inhibitor) that binds antizyme 1 with higher affinity compared with ODC. In the present study, we show by yeast two- and three-hybrid protein-protein interaction studies that AZI interacts with all members of the antizyme family and is capable of disrupting the interaction between each antizyme and ODC. In a yeast-based ODC complementation assay, we show that human ODC is able to complement fully the function of the yeast homologue of ODC. Co-expression of antizymes resulted in ODC inhibition and cessation of yeast growth. The antizyme-induced growth inhibition could be reversed by addition of putrescine or by the co-expression of AZI. The protein interactions could be confirmed by immunoprecipitation of the human ODC-antizyme 2-AZI complexes. In summary, we conclude that human AZI is capable of acting as a general inhibitor for all members of the antizyme family and that the previously not yet characterized antizyme 4 is capable of binding ODC and inhibiting its enzymic activity similar to the other members of the antizyme family.
Mangold
Ursula
U
Center for Functional Genomics, Aventis Pharma GmbH, 82152 Martinsried, Germany. Ursula.Mangold@childrens.harvard.edu
Leberer
Ekkehard
E
D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D004791:Enzyme Inhibitors / Q000378:metabolism*; D005816:Genetic Complementation Test; D006801:Humans; D047468:Immunoprecipitation; D046912:Multiprotein Complexes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009928:Organ Specificity; D009955:Ornithine Decarboxylase / Q000235:genetics / Q000378:metabolism; D065108:Ornithine Decarboxylase Inhibitors; D011485:Protein Binding; D011506:Proteins / Q000037:antagonists & inhibitors* / Q000145:classification* / Q000235:genetics / Q000378:metabolism; D011700:Putrescine / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D020798:Two-Hybrid System Techniques
D002352:Carrier Proteins; D004791:Enzyme Inhibitors; D046912:Multiprotein Complexes; D065108:Ornithine Decarboxylase Inhibitors; D011506:Proteins; C031017:ornithine decarboxylase antizyme; C039317:ornithine decarboxylase antizyme inhibitor; D009955:Ornithine Decarboxylase; D011700:Putrescine