Cell culture analysis of the regulatory frameshift event required for the expression of mammalian antizymes.

BACKGROUND: Antizyme is a critical regulator of cellular polyamine levels due to its effect on polyamine transport and its ability to target ornithine decarboxylase for degradation. Antizyme expression is autoregulatory, through dependence on an unusual +1 translational frameshift mechanism that responds to polyamine levels. RESULTS: HEK293 cells were depleted of polyamines by treatment with an ornithine decarboxylase inhibitor, difluoromethylornithine (DFMO), and grown in the presence or absence of exogenous polyamines prior to the analysis of ribosomal frameshifting levels. Results obtained using an optimized dual luciferase assay system reveal a 10-fold dynamic range of frameshifting, which correlates positively with polyamine addition. Polyamine addition to cells, which have not been pre-treated with DFMO, also resulted in an increase in antizyme frameshifting but to a lesser degree (1.3 to 1.5-fold). In addition, the constructs with the 3' deletion were more responsive to stimulation by polyamine addition than those with the 5' deletion. CONCLUSIONS: The observed regulation of antizyme frameshifting demonstrates the efficiency of a polyamine homeostatic mechanism, and illustrates the utility of a quantifiable cell-based assay for the analysis of polyamines or their analogues on translational frameshifting.

Antizyme regulates the degradation of ornithine decarboxylase in fission yeast Schizosaccharomyces pombe. Study in the spe2 knockout strains.

The mechanism of the regulatory degradation of ornithine decarboxylase (ODC) by polyamines was studied in fission yeast, Schizosaccharomyces pombe. To regulate cellular spermidine experimentally, we cloned and disrupted S-adenosylmethionine decarboxylase gene (spe2) in S. pombe. The null mutant of spe2 was devoid of spermidine and spermine, accumulated putrescine, and contained a high level of ODC. Addition of spermidine to the culture medium resulted in rapid decrease in the ODC activity caused by the acceleration of ODC degradation, which was dependent on de novo protein synthesis. A fraction of ODC forming an inactive complex concomitantly increased. The accelerated ODC degradation was prevented either by knockout of antizyme gene or by selective inhibitors of proteasome. Thus, unlike budding yeast, mammalian type antizyme-mediated ODC degradation by proteasome is operating in S. pombe.

Methylation of the ribosyl moiety at position 34 of selenocysteine tRNA[Ser]Sec is governed by both primary and tertiary structure.

The selenocysteine (Sec) tRNA[Ser]Sec population in higher vertebrates consists of two major isoacceptors that differ from each other by a single nucleoside modification in the wobble position of the anticodon (position 34). One isoacceptor contains 5-methylcarboxymethyluridine (mcmU) in this position, whereas the other contains 5-methylcarboxymethyluridine-2'-O-methylribose (mcmUm). The other modifications in these tRNAs are N6-isopentenyladenosine (i6A), pseudouridine (psi), and 1-methyladenosine (m1A) at positions 37, 55, and 58, respectively. As methylation of the ribose at position 34 is influenced by the intracellular selenium status and the presence of this methyl group dramatically alters tertiary structure, we investigated the effect of the modifications at other positions as well as tertiary structure on its formation. Mutations were introduced within a synthetic gene encoded in an expression vector, transcripts generated and microinjected into Xenopus oocytes, and the resulting tRNA products analyzed for the presence of modified bases. The results suggest that efficient methylation of mcmU to yield mcmUm requires the prior formation of each modified base and an intact tertiary structure, whereas formation of modified bases at other positions, including mcmU, is not as stringently connected to precise primary and tertiary structure. These results, along with the observations that methylation of mcmU is enhanced in the presence of selenium and that this methyl group affects tertiary structure, further suggest that the mcmUm isoacceptor must have a role in selenoprotein synthesis different from that of the mcmU isoacceptor.

Conservation of polyamine regulation by translational frameshifting from yeast to mammals.

Regulation of ornithine decarboxylase in vertebrates involves a negative feedback mechanism requiring the protein antizyme. Here we show that a similar mechanism exists in the fission yeast Schizosaccharomyces pombe. The expression of mammalian antizyme genes requires a specific +1 translational frameshift. The efficiency of the frameshift event reflects cellular polyamine levels creating the autoregulatory feedback loop. As shown here, the yeast antizyme gene and several newly identified antizyme genes from different nematodes also require a ribosomal frameshift event for their expression. Twelve nucleotides around the frameshift site are identical between S.pombe and the mammalian counterparts. The core element for this frameshifting is likely to have been present in the last common ancestor of yeast, nematodes and mammals.

Degradation of ornithine decarboxylase by the 26S proteasome.

Ornithine decarboxylase (ODC) is a key enzyme in polyamine biosynthesis. Turnover of ODC is extremely rapid and highly regulated, and is accelerated when polyamine levels increase. Polyamine-stimulated ODC degradation is mediated by association with antizyme (AZ), an ODC inhibitory protein induced by polyamines. ODC, in association with AZ, is degraded by the 26S proteasome in an ATP-dependent, but ubiquitin-independent, manner. The 26S proteasome irreversibly inactivates ODC prior to its degradation. The inactivation, possibly due to unfolding, is coupled to sequestration of ODC within the 26S proteasome. This process requires AZ and ATP, but not proteolytic activity of the 26S proteasome. The carboxyl-terminal region of ODC presumably exposed by interaction with AZ plays a critical role for being trapped by the 26S proteasome. Thus, the degradation pathway of ODC proceeds as a sequence of multiple distinct processes, including recognition, sequestration, unfolding, translocation, and ultimate degradation mediated by the 26S proteasome.

Two zebrafish (Danio rerio) antizymes with different expression and activities.

Cellular polyamines are regulated by a unique feedback mechanism involving ornithine decarboxylase (ODC) antizyme. The synthesis of mammalian antizyme requires a programmed translational frameshift event induced by polyamines. Antizyme represses ODC, a key enzyme for polyamine synthesis, through accelerating enzyme degradation by the 26 S proteasome. Antizyme also inhibits the cellular uptake of polyamines. In the present study we isolated two distinct zebrafish (Danio rerio) antizyme cDNA clones (AZS and AZL) from an embryonic library. Their sequences revealed that both clones required translational frameshifting for expression. Taking account of +1 frameshifting, AZS and AZL products were 214 and 218 residues long respectively and shared 51.8% amino acid identity. In rabbit reticulocyte lysates, both mRNA species were translated through spermidine-induced frameshifting. The presence of the two antizyme mRNA species in embryos, adult fish and a cultured cell line was confirmed by Northern blot analysis. The ratio of AZS mRNA to AZL mRNA in the adult fish was 1.8-fold higher than in the embryos. Whole-mount hybridization in situ demonstrated that both mRNA species are expressed in every tissue in embryo, but predominantly in the central nervous system and the eyes. Bacterial expression products of both cDNA species inhibited ODC activity, but only the AZS product accelerated ODC degradation in vitro. These results show that both zebrafish antizymes are induced by polyamines but their mRNA species are expressed differently during development. The difference in activities on ODC degradation suggests their functional divergence.

Dietary induction of ornithine decarboxylase in male mouse kidney.

In male mouse kidney, ornithine decarboxylase (ODC) is induced after feeding, and the induction depends on dietary protein content. 24 h after feeding with 50% casein-containing meal, ODC activity and amount of immunoreactive ODC protein increased more than 10-fold, ODC mRNA level increased 2-fold, and the ODC half-life extended 7-fold. The renal ODC induction after feeding is, therefore, due mainly to stabilization of ODC protein. Urinary excretion of putrescine increased in response to the ODC induction, but the renal polyamine contents scarcely changed. Consistently, the level of antizyme, a polyamine-inducible protein, determined as the ODC-antizyme complex level, scarcely changed after feeding, and the antizyme/ODC ratio in the kidney largely decreased, resulting in the stabilization of ODC protein. The present results suggest that the strong excretion system of the kidney for newly synthesized polyamines enables renal ODC escape from antizyme-mediated feedback regulation.

ATP-Dependent inactivation and sequestration of ornithine decarboxylase by the 26S proteasome are prerequisites for degradation.

The 26S proteasome is a eukaryotic ATP-dependent protease, but the molecular basis of its energy requirement is largely unknown. Ornithine decarboxylase (ODC) is the only known enzyme to be degraded by the 26S proteasome without ubiquitinylation. We report here that the 26S proteasome is responsible for the irreversible inactivation coupled to sequestration of ODC, a process requiring ATP and antizyme (AZ) but not proteolytic activity. Neither the 20S proteasome (catalytic core) nor PA700 (the regulatory complex) by itself contributed to this ODC inactivation. Analysis with a C-terminal mutant ODC revealed that the 26S proteasome recognizes the C-terminal degradation signal of ODC exposed by attachment of AZ, and subsequent ATP-dependent sequestration of ODC in the 26S proteasome causes irreversible inactivation, possibly unfolding, of ODC and dissociation of AZ. These processes may be linked to the translocation of ODC into the 20S proteasomal inner cavity, centralized within the 26S proteasome, for degradation.

Antiproliferative effect of IL-1 is mediated by p38 mitogen-activated protein kinase in human melanoma cell A375.

The role of p38 mitogen-activated protein kinase (MAPK) in IL-1-induced growth inhibition was investigated using IL-1-sensitive human melanoma A375-C2-1 cells and IL-1-resistant A375-R8 cells. In both cells, p38 MAPK was activated by IL-1. A selective inhibitor for p38 MAPK, SB203580, almost completely recovered the IL-1-induced growth inhibition in A375-C2-1 cells. IL-1-induced IL-6 production was also suppressed by SB203580. However, the reversal effect of SB203580 was not due to the suppression of IL-6 production because the SB203580 effect was still observed in the presence of exogenous IL-6. Down-regulation of ornithine decarboxylase (ODC) activity as well as its protein level has been shown to be essential for IL-1-induced growth inhibition. SB203580 also reversed the IL-1-induced down-regulation of ODC activity and intracellular polyamine levels without affecting ODC mRNA levels in A375-C2-1 cells. In IL-1-resistant R8 cells, however, IL-1 only slightly suppressed ODC activity. In A375-C2-1 cells, the mRNA expression level of antizyme (AZ), a regulatory factor of ODC activity, has been shown to be up-regulated by IL-1. IL-1-induced up-regulation of AZ mRNA level was not affected by SB203580. These findings demonstrate that p38 MAPK plays an important role in IL-1-induced growth inhibition in A375 cells through down-regulating ODC activity without affecting the level of ODC mRNA and AZ mRNA. In IL-1-resistant A375-R8 cells, IL-1 signaling pathway is deficient between p38 MAPK activation and down-regulation of ODC activity.

Overexpression and purification of recombinant eRF1 proteins of rabbit and Tetrahymena thermophila.

The polypeptide release factor (eRF1) gene was cloned from rabbit and its overexpression and purification system was established in parallel with that of the eRF1 gene of Tetrahymena thermophila that has been cloned recently in this laboratory. The rabbit eRF1 (Ra-eRF1) is composed of 437 amino acids and is completely identical to human eRF1 though 3% distinct in the nucleotide sequence. This is in sharp contrast to Tetrahymena eRF1 (Tt-eRF1) that is only 57% identical to human eRF1. The recombinant Ra-eRF1 was marked with a histidine tag, overexpressed, and purified to homogeneity by two-step chromatography using Ni-NTA-agarose and Mono Q columns. In contrast to Ra-eRF1, Tt-eRF1 formed aggregates upon overexpression in Escherichia coli, hence it was purified under denaturing conditions, and used to raise rabbit antibody. The resulting anti-Tt-eRF1 antibody proved useful for examining conditions for soluble Tt-eRF1 in test cells. Finally, a soluble Tt-eRF1 fraction was purified from Saccharomyces cerevisiae transformed with the Tt-eRF1 expression plasmid by three steps of affinity and anion exchange chromatography. The cloned Ra-eRF1 gene complemented a temperature-sensitive allele in the eRF1 gene, sup45 (ts), of S. cerevisiae, though the complementation activity was significantly impaired by the histidine tag, whereas Tt-eRF1 failed to complement the sup45 (ts) allele.

Programmed frameshifting in the synthesis of mammalian antizyme is +1 in mammals, predominantly +1 in fission yeast, but -2 in budding yeast.

The coding sequence for mammalian ornithine decarboxylase antizyme is in two different partially overlapping reading frames with no independent ribosome entry to the second ORF. Immediately before the stop codon of the first ORF, a proportion of ribosomes undergo a quadruplet translocation event to shift to the +1 reading frame of the second and main ORF. The proportion that frameshifts is dependent on the polyamine level and, because the product antizyme is a negative regulator of intracellular polyamine levels, the frameshifting acts to complete an autoregulatory circuit by sensing polyamine levels. An mRNA element just 5' of the shift site and a 3' pseudoknot are important for efficient frameshifting. Previous work has shown that a cassette with the mammalian shift site and associated signals directs efficient shifting in the budding yeast Saccharomyces cerevisiae at the same codon to the correct frame, but that the shift is -2 instead of +1. The product contains an extra amino acid corresponding to the shift site. The present work shows efficient frameshifting also occurs in the fission yeast, Schizosaccharomyces pombe. This frameshifting is 80% +1 and 20% -2. The response of S. pombe translation apparatus to the mammalian antizyme recoding signals is more similar to that of the mammalian system than to that of S. cerevisiae. S. pombe provides a good model system for genetic studies on the mechanism of at least this type of programmed mammalian frameshifting.

Agmatine suppresses proliferation by frameshift induction of antizyme and attenuation of cellular polyamine levels.

Polyamines are required for entry and progression of the cell cycle. As such, augmentation of polyamine levels is essential for cellular transformation. Polyamines are autoregulated through induction of antizyme, which represses both the rate-limiting polyamine biosynthetic enzyme ornithine decarboxylase and cellular polyamine transport. In the present study we demonstrate that agmatine, a metabolite of arginine via arginine decarboxylase (an arginine pathway distinct from that of the classical polyamines), also serves the dual regulatory functions of suppressing polyamine biosynthesis and cellular polyamine uptake through induction of antizyme. The capacity of agmatine to induce antizyme is demonstrated by: (a) an agmatine-dependent translational frameshift of antizyme mRNA to produce a full-length protein and (b) suppression of agmatine-dependent inhibitory activity by either anti-antizyme IgG or antizyme inhibitor. Furthermore, agmatine administration depletes intracellular polyamine levels to suppress cellular proliferation in a transformed cell line. This suppression is reversible with polyamine supplementation. We propose a novel regulatory pathway in which agmatine acts as an antiproliferative molecule and potential tumor suppressor by restricting the cellular polyamine supply required to support growth.

Cloning and sequencing of a human cDNA encoding ornithine decarboxylase antizyme inhibitor.

We report here cloning and sequencing human antizyme inhibitor from a human kidney cDNA library. Amino acid sequence deduced from the nucleotide sequence shows 92.9% identity to that of rat antizyme inhibitor. Northern blot analysis reveals that antizyme inhibitor is expressed in human liver.

Characterization of the human antizyme gene.

Antizyme is a polyamine-inducible protein involved in feedback regulation of cellular polyamine levels. Recently, we isolated genomic clones for the human antizyme gene and determined its chromosomal location (Matsufuji et al., Genomics 38 (1996) 112-114). In the present study, we report complete nucleotide sequence and organization of the human antizyme gene. The organizations of human and rat genes are very similar, but their introns show divergency in terms of the length and nucleotide sequence. Luciferase reporter assay revealed that the 5'-flanking region of the human gene had a strong transcriptional activity in NIH-3T3 with and without addition of spermidine. The promoter was also effective in transfected COS7 and HeLa cells. A 223-bp region at the proximity of the transcriptional start points carries several regulatory sequence motifs including a TATA box, CAAT boxes and GC boxes, and was shown to be important for the strong transcriptional activity.

Reading two bases twice: mammalian antizyme frameshifting in yeast.

Programmed translational frameshifting is essential for the expression of mammalian ornithine decarboxylase antizyme, a protein involved in the regulation of intracellular polyamines. A cassette containing antizyme frameshift signals is found to direct high-level (16%) frameshifting in yeast, Saccharomyces cerevisiae. In contrast to +1 frameshifting in the mammalian system, in yeast the same frame is reached by -2 frameshifting. Two bases are read twice. The -2 frameshifting is likely to be mediated by slippage of mRNA and re-pairing with the tRNA in the P-site. The downstream pseudoknot stimulates frameshifting by 30-fold compared with 2.5-fold in reticulocyte lysates. When the length of the spacer between the shift site and the pseudoknot is extended by three nucleotides, +1 and -2 frameshifting become equal.

Superinduction of mouse epidermal ornithine decarboxylase activity by repeated 12-o-tetradecanoylphorbol-13-acetate treatments.

A correlation of the levels of epidermal protein kinase C (PKC) isozymes, steady state levels of ornithine decarboxylase (ODC) mRNA, and ODC antizyme with the induction of ornithine decarboxylase (ODC) activity by a second repeat 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment to mouse skin was determined. A single application of TPA to female CD-1 mouse skin leads to a dramatic induction of ODC activity (approximately 3 nmol CO2/60 min/mg protein) which peaks at about 5 h after treatment. However, a superinduction of ODC activity (approximately 13 CO2/60 min/mg protein) is observed upon the second TPA application at 48 or 72 h after the first TPA treatment. Prior application of a tumor initiating dose of 7,12-dimethylbenz[a]anthracine to mouse skin did not influence the degree of induction of ODC by a repeat TPA treatment. Western Blot analyses using antibodies specific to PKC alpha, beta, gamma, delta and epsilon indicate detectable levels of PKC alpha, beta, delta and epsilon in mouse epidermal extracts. A time course of the effects of a single topical application of 20 nmol of TPA to the mouse skin indicate that none of PKC isozymes (alpha, beta, gamma, delta and epsilon) were completely downregulated at times (72 h) when ODC was overinduced by TPA. TPA-induced steady state levels of ODC mRNA did not correlate with the degree of superinduction of ODC activity by TPA. The second TPA treatment, 72 h after the first TPA treatment, which leads to superinduction of ODC activity did not decrease the levels of the ODC-antizyme. The results indicate that superinduction of mouse epidermal ODC activity is regulated in part post-transcriptionally and may not be the result of either a loss of PKC isoform(s) or a decrease in the levels of ODC antizyme.

Cloning of antizyme inhibitor, a highly homologous protein to ornithine decarboxylase.

The degradation of ornithine decarboxylase (ODC) catalyzed by the 26 S proteasome is accelerated by antizyme, an ODC inhibitory protein induced by polyamines. Previously, we have found another possible regulatory protein of ODC degradation, antizyme inhibitor. Antizyme inhibitor binds to the antizyme with a higher affinity than that of ODC, releasing ODC from ODC-antizyme complex. We report here the cDNA sequence of rat heart antizyme inhibitor. The deduced sequence of the protein is highly similar to, but distinct from, sequences of ODCs from various species. Antizyme inhibitor contains amino acid residues required for formation of active sites of ODC, but it completely lacks ODC activity. Antizyme inhibitor has no homology with peptide sequence in the mammalian ODC carboxyl terminus, which is needed for rapid turnover of ODC. It inhibits antizyme-dependent ODC degradation, but, unlike ODC, its degradation is not accelerated by antizyme.

Ornithine decarboxylase antizyme: a novel type of regulatory protein.

Antizyme plays an important regulatory role in the synthesis of ornithine decarboxylase (ODC), a key enzyme of polyamine synthesis in higher animals. As well as inactivating polyamine uptake, antizyme is induced by polyamine-enhanced translational frameshifting, and binds to ODC, accelerating its ATP-dependent degradation, a process catalysed by the 26S proteasome.