Mouse Ornithine Decarboxylase-like Gene Encodes an Antizyme Inhibitor Devoid of Ornithine and Arginine Decarboxylating Activity

Abstract

Ornithine decarboxylase (ODC), a key enzyme in the biosynthesis of polyamines, is a labile protein that is regulated by interacting with antizymes (AZs), a family of polyamine-induced proteins. Recently, a novel human gene highly homologous to ODC, termed ODC-like or ODC-paralogue (ODCp), was cloned, but the studies aimed to determine its function rendered contradictory results. We have cloned the mouse orthologue of human ODCp and studied its expression and possible function. mRNA of mouse Odcp was found in the brain and testes, showing a conserved expression pattern with regard to the human gene. Transfection of mouse Odcp in HEK 293T cells elicited an increase in ODC activity, but no signs of arginine decarboxylase activity were evident. On the other hand, whereas the ODCp protein was mainly localized in the mitochondrial/membrane fraction, ODC activity was found in the cytosolic fraction and was markedly decreased by small interfering RNA against human ODC. Co-transfection experiments with combinations of Odc, Az1, Az2, Az3, antizyme inhibitor (Azi), and Odcp genes showed that ODCp mimics the action of AZI, rescuing ODC from the effects of AZs and prevented ODC degradation by the proteasome. A direct interaction between ODCp and AZs was detected by immunoprecipitation experiments. We conclude that mouse ODCp has no intrinsic decarboxylase activity, but it acts as a novel antizyme inhibitory protein (AZI2).The polyamines spermidine and spermine and their precursor putrescine are ubiquitous polycations implicated in the growth, differentiation, and death of eukaryotic cells (1-4). Intracellular levels of polyamines are tightly regulated through multiple mechanisms affecting their biosynthesis, catabolism, and transport (5-9). In mammalian cells, putrescine synthesis, the first step in the polyamine biosynthetic pathway, is mediated by ornithine decarboxylase (ODC) 2 (EC 4.1.1.17) through the decarboxylation of L-ornithine. This enzyme is subject to a complex regulation by transcriptional, translational, and posttranslational mechanisms (10 -16). At the post-translational level, ODC is finely regulated by a family of inhibitory proteins called antizymes (AZ) (15,17,18). AZ1, the first described member of the family, binds to ODC monomers preventing the formation of active ODC homodimers and promoting the degradation of ODC through the 26 S proteasome in a ubiquitinindependent manner (19 -21). Synthesis of AZ is influenced by polyamines through the stimulation of ribosomal frameshifting (22,23). Moreover, the action of AZ on ODC function is also mediated by a protein called antizyme inhibitor (AZI). This protein, having a sequence highly similar to that of ODC, is devoid of ornithine decarboxylating activity; however, it can activate ODC by competing for AZ, because AZI binds to AZ with high affinity preventing or decreasing the formation of the ODC-AZ complex (24, 25). In addition, AZ1 and AZ2 not only decrease polyamine biosynthesis but also prevent the accumulation of excess polyamines by inhibiting or suppre...

Antizyme Inhibitor 2 (AZIN2/ODCp) Stimulates Polyamine Uptake in Mammalian Cells

“…These findings corroborate our previous claim that ODCp or ODC-like acts as an antizyme inhibitor, not only because it binds to the three antizymes and stimulates ODC activity (29) but also by negating the repressing effect of any of the three antizymes on polyamine uptake. Moreover, the fact that the deletion in AZIN2 of the sequence equivalent to the antizyme binding domain, existing in mouse ODC (37) and in AZIN1 (38,39), abolished the stimulatory effect of AZIN2 on polyamine uptake also supports that this domain is critical for the binding of AZIN2 to the antizyme molecule.…”

“…These results have later been corroborated for human ODCp by others (30). AZIN2/ODCp is mainly expressed in testes and brain both in mice and human (29,31), and by both functional and co-immunoprecipitation experiments, we demonstrate that this protein is able to interact with the three antizyme isoforms as is also shown for the former antizyme inhibitor, the AZIN1 (32). As stated above, AZ3 is a testis-specific protein that is believed to participate in the maintenance of appropriate levels of polyamines during spermiogenesis (22).…”

“…We recently showed that a new paralogue gene of ODC, named ODCp or ODC-like, that had been postulated to code for arginine decarboxylase (28) was devoid of ornithine or arginine decarboxylase activity but acted as an antizyme inhibitor; consequently we proposed to name it antizyme inhibitor 2 (AZIN2) (29). These results have later been corroborated for human ODCp by others (30).…”

“…The FLAG epitope was introduced to the N terminus of AZIN2 as described previously (29). AZIN2 with a streptavidin epitope (STrEP Tag) in its C terminus was obtained by subcloning the ORF of AZIN2 into the pEXPR-IBA 103 vector (Novagen) using the following primers: STag, forward, 5Ј-CTGTCTAGA (XbaI)ATGGCTGG CTATCTGAGTG-3Ј, and reverse, 5Ј-GCCGGATCC(BamHI)-CATGATGCTTGCTGGGGTG-3Ј).…”

“…Cloning of AZ1, AZ2, AZ3, AZIN1, and AZIN2-The desired mouse genes were cloned into the expression vector pcDNA3 (Invitrogen) following standard procedures (34) and using the primers described elsewhere (29). Antizyme constructs with an appropriate deletion of one nucleotide in the frameshifting site, for full-length and functional expression, were obtained by the QuickChange site-directed mutagenesis kit.…”