Discovery of a spermatogenesis stage-specific ornithine decarboxylase antizyme: Antizyme 3

Antizyme-1 (AZ1) is a protein that negatively regulates polyamine synthesis by inhibiting the key synthetic enzyme ornithine decarboxylase and targeting it for degradation by the 26 S proteasome. Recent work shows that antizyme protein translocates to the nucleus during mouse development (Gritli-Linde, A., Nilssom, J., Bohlooly, Y. M., Heby, O., and Linde, A. (2001) Dev. Dyn. 220, 259 -275). However, the significance and mechanism of this phenomenon remain unclear. In this study, we expressed AZ1 fused with enhanced green fluorescent protein (EGFP) to study its localization in a living cell. We found that EGFP-AZ1 was predominantly localized in the cytoplasm and that treatment with leptomycin B, a specific inhibitor of chromosomal region maintenance 1 (CRM1) induced nuclear accumulation of EGFP-AZ1 in Chinese hamster ovary and NIH3T3 cells. Two independent nuclear export signal (NES) sequences, each containing essential hydrophobic residues, were identified in the 50 N-terminal amino acid residues and in the central part of AZ1. The activity of the second NES was inhibited by an N-terminal adjacent region and was only revealed in N-terminal truncated constructs. Both NESs were active when fused to an artificial nuclear protein SV40-NLS-EGFP-EGFP. The ability of AZ1 to shuttle between the nucleus and the cytoplasm suggests that it has a novel function in the nucleus.Polyamines are ubiquitous organic cations with tightly regulated cellular concentrations and are essential for cell growth and development (1, 2). Antizyme (AZ) 1 was first described as an inhibitor of ornithine decarboxylase (ODC), a key enzyme in polyamine biosynthesis (3). When cellular polyamine levels rise, AZ is induced and then binds to the monomeric form of ODC (4, 5), which makes the enzyme a preferential substrate for proteolysis by the 26 S proteasome without ubiquitination (6). AZ also inhibits the uptake of extracellular polyamines (7,8). There are at least three independent antizyme isoforms conserved among mammals. AZ1 and AZ2 have a wide tissue distribution, whereas AZ3 is testis-specific (9, 10). Expression of AZs is induced by a unique translational frameshift mechanism (11). For AZ1, when cellular polyamine levels are low, the polypeptide corresponding to amino acid residues 1-68 is produced and translation is terminated at the following UGA codon (11). When cellular polyamine levels are high, ϩ1 frameshifting occurs after the 68th codon is decoded, resulting in the full-length antizyme protein (amino acid residues 1-227). All of the known regulatory activities of AZ1 are present in amino acid residues 69 -227 (12). A second AUG exists in AZ1 mRNA at the 34th codon, and this second AUG is also utilized as initiation codon (11, 13). The two resulting translation products have different half-lives (14). Furthermore, it has been suggested that the N-terminal region of the translation product from the first AUG contains a putative mitochondrial targeting signal and thus the distribution of AZ1 may depend on this region (15). It is curre...

Section: Az1 Contains Two Nuclear Exportmentioning

confidence: 99%

“…There are at least three independent antizyme isoforms conserved among mammals. AZ1 and AZ2 have a wide tissue distribution, whereas AZ3 is testis-specific (9,10). Expression of AZs is induced by a unique translational frameshift mechanism (11).…”

mentioning

confidence: 99%

Identification of Nuclear Export Signals in Antizyme-1

Murai

Murakami

Matsufuji

2003

“…Mammalian cells express three characterized members of the Az family of proteins (6,16). While one of them, Az3, is testis-specific and observed only in haploid germinal cells (17,18), the other two, the prototypical Az1 and Az2, are ubiquitously expressed, with Az1 being expressed at much higher levels (5,6,19).…”

mentioning

confidence: 99%

Antizyme Affects Cell Proliferation and Viability Solely through Regulating Cellular Polyamines

Bercovich

Snapir²,

Keren-Paz

et al. 2011

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.

“…AZ1 and antizyme 2 (AZ2) are found in all tissues, with AZ1 transcripts generally present in 10-to 20-fold greater amounts than AZ2 (11). AZ3 is found only in the testes and is expressed solely during postmeiotic stages of spermatogenesis (12).…”

mentioning

confidence: 99%

Structural Elements of Antizymes 1 and 2 Are Required for Proteasomal Degradation of Ornithine Decarboxylase

Chen

MacDonald

Coffino

2002

The antizymes constitute a conserved gene family with at least three mammalian orthologs. As described previously, in a degradation system utilizing rabbit reticulocyte lysate, antizyme 1 (AZ1) accelerates proteasomal ornithine decarboxylase (ODC) degradation, but antizyme 2 (AZ2) does not. To examine the relationship between antizyme structure and function, we further characterized the properties of AZ1 and AZ2 and protein chimeras composed of elements of the two. AZ1 binds to ODC with about a 3-fold higher potency than AZ2, but this cannot account for their distinct degradative activities. The dissimilar degradative capacity of AZ1 and AZ2 is also observed using purified proteasomes. A series of reciprocal AZ1/ AZ2 chimeras was used to determine the sequence elements needed to direct ODC degradation. An element contained within amino acids 130 -145 of AZ1 is essential for this function. Constructs in which amino acids 130 -145 were exchanged between the antizymes confirmed the critical nature of this region. Within this region, amino acids 131 and 145 proved responsible for the functional difference between the two forms of AZ.