Antizyme inhibitor (AzI) regulates cellular polyamine homeostasis by binding to the polyamine-induced protein, Antizyme (Az), with greater affinity than ornithine decarboxylase (ODC). AzI is highly homologous to ODC but is not enzymatically active. In order to understand these specific characteristics of AzI and its differences from ODC, we determined the 3D structure of mouse AzI to 2.05 Å resolution. Both AzI and ODC crystallize as a dimer. However, fewer interactions at the dimer interface, a smaller buried surface area, and lack of symmetry of the interactions between residues from the two monomers in the AzI structure suggest that this dimeric structure is nonphysiological. In addition, the absence of residues and interactions required for pyridoxal 59-phosphate (PLP) binding suggests that AzI does not bind PLP. Biochemical studies confirmed the lack of PLP binding and revealed that AzI exists as a monomer in solution while ODC is dimeric. Our findings that AzI exists as a monomer and is unable to bind PLP provide two independent explanations for its lack of enzymatic activity and suggest the basis for its enhanced affinity toward Az.Keywords: structure/function studies; protein crystallization; protein structures-new; antizyme inhibitor; antizyme; ornithine decarboxylase Polyamines are small organic polycations that are essential for cell proliferation and play an important role in regulating other fundamental cellular processes. Elevated polyamine levels are observed in rapidly growing cells including transformed cells; thus, polyamine metabolism has been suggested as a potential target for cancer therapy (Pegg 1988;Marton and Pegg 1995;Wallace and Fraser 2004). The range of intracellular polyamines is determined at the lower limit by their absolute requirement for cellular proliferation and at the upper limit by their cytotoxicity (Poulin et al. 1993;Tobias and Kahana 1995), indicating a need for strict regulation of their intracellular concentration. Multiple pathways such as synthesis, uptake, degradation, and efflux regulate cellular polyamine levels. Ornithine decarboxylase (ODC) is the first and rate-limiting enzyme in the polyamine biosynthesis pathway (Pegg 2006). It is a pyridoxal 59-phosphate (PLP)-dependent enzyme that provides the only route for converting ornithine to putrescine. ODC, which is characterized by a short intracellular half-life, is part of an autoregulatory circuit mediated by a polyamine-induced protein, termed Antizyme (Az). An increased intracellular polyamine concentration increases the synthesis of Az by stimulating ribosomal frameshifting (Rom and Kahana 1994;Matsufuji et al. 1995). Az, in turn, binds to transient ODC monomer subunits with high affinity, preventing their re-association into active homodimers and targeting them for rapid degradation by the 26S proteasome (Murakami et al. 1992). Az also regulates polyamine transport across the plasma membrane via an unknown mechanism Mitchell et al. 1994;Suzuki et al. 1994;Sakata et al. 2000). 3 These authors contri...

ODC (ornithine decarboxylase), the first enzyme in the polyamine biosynthesis pathway in mammalian cells, is a labile protein. ODC degradation is stimulated by Az (antizyme), a polyamine-induced protein, which in turn is regulated by an ODC-related protein termed AzI (Az inhibitor). Recently, another ODCp (ODC paralogue) was suggested to function as AzI, on the basis of its ability to increase ODC activity and inhibit Az-stimulated ODC degradation in vitro. We show in the present study that ODCp is indeed capable of negating Az functions, as reflected by its ability to increase ODC activity and polyamine uptake and by its ability to provide growth advantage in stably transfected cells. However, ODCp is less potent than AzI1 in stimulating ODC activity, polyamine uptake and growth rate. The superiority of AzI1 to ODCp in inhibiting the Az-stimulated ODC degradation is also demonstrated using an in vitro degradation assay. We show that the basis for the inferiority of ODCp as an AzI is its lower affinity towards Az (Az1 and Az3). Further, we show here that ODCp, like AzI, is degraded in a ubiquitin-dependent manner, in a reaction that does not require either interaction with Az or the integrity of its C-terminus. Interaction with Az actually stabilizes ODCp by interfering with its ubiquitination. This results in sequestration of Az into a stable complex with ODCp, which is the central feature contributing to the ability of ODCp to function as AzI.

Azs (antizymes) are small polyamine-induced proteins that function as feedback regulators of cellular polyamine homoeostasis. They bind to transient ODC (ornithine decarboxylase) monomeric subunits, resulting in inhibition of ODC activity and targeting ODC to ubiquitin-independent proteasomal degradation. Az3 is a mammalian Az isoform expressed exclusively in testicular germ cells and therefore considered as a potential regulator of polyamines during spermatogenesis. We show here that, unlike Az1 and Az2, which efficiently inhibit ODC activity and stimulate its proteasomal degradation, Az3 poorly inhibits ODC activity and fails to promote ODC degradation. Furthermore, Az3 actually stabilizes ODC, probably by protecting it from the effect of Az1. Its inhibitory effect is revealed only when it is present in excess compared with ODC. All three Azs efficiently inhibit the ubiquitin-dependent degradation of AzI (Az inhibitor) 1 and 2. Az3, similar to Az1 and Az2, efficiently inhibits polyamine uptake. The potential significance of the differential behaviour of Az3 is discussed.

Among several single nucleotide polymorphisms (SNPs) that correlate with fibrosis progression in chronic HCV, a SNP in the antizyme inhibitor (AzI) gene is most strongly associated with slow fibrosis progression. Our aim was to identify the mechanism(s) underlying this observation by exploring the impact of the AzI SNP on hepatic stellate cell (HSC) activity. Seven novel AZIN1 splice variants (“SV2-8”) were PCR-cloned from the LX2 human HSC line. Expression of a minigene in LX2 containing the AZIN1 slow-fibrosis SNP yielded a 1.67 fold increase in AZIN1 splice variant 2 (AZIN1 SV2) mRNA (p= 0.05). In healthy human leukocytes, the SNP variant also correlated with significantly increased SV2 mRNA. Cells (293T) transfected with shRNA complementary to the exonic splicing chaperone SRp40 expressed 30% less SRp40 (p = 0.044) and 43% more AzI SV2 (p = 0.021) than control shRNA-expressing cells, mimicking the effect of the sequence variant. LX2 cells transfected with AZIN1 full-length cDNA expressed 35% less collagen I mRNA (p = 0.09) and 18% less SMA mRNA (p=0.09). Transient transfection of AZIN1 SV2 cDNA into LX2 cells reduced collagen I gene expression by 64% (p = 0.001) and αSMA by 43% (p = 0.005) compared to vector-transfected controls, paralleling changes in protein expression. Both AZIN1 and AZIN-SV2 mRNAs are detectable in normal human liver and reduced in HCV cirrhotic livers. The AZIN1-SV2 acts via a polyamine-independent pathway, as it neither interacts with antizyme nor affects the ability of AZIN1 lacking this variant to neutralize antizyme. Conclusions A SNP variant in the AZIN1 gene leads to enhanced generation of a novel alternative splice form that modifies the fibrogenic potential of HSCs.

Background: Antizyme is a regulator of cell proliferation, inhibiting this process when overexpressed. Results: Antizyme overexpression does not attenuate cell proliferation and viability in cells whose polyamine supply is secured. Conclusion: Antizyme affects cell proliferation and viability only by modulating polyamine metabolism. Significance: This result emphasizes the functional relationship of antizyme to cellular polyamine metabolism.