Ornithine decarboxylase antizyme
The mechanism of the regulatory degradation of ornithine decarboxylase (ODC) by polyamines was studied in fission yeast, Schizosaccharomyces pombe. To regulate cellular spermidine experimentally, S-adenosylmethionine decarboxylase gene (spe2) was cloned and disrupted 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 (Chattopadhyay, 2001).
The effect of spermidine on the degradation of ornithine decarboxylase has been investigated in S. cerevisiae. In S. cerevisiae, as in other eukaryotic cells, the rate of degradation of ornithine decarboxylase, measured either enzymatically or immunologically, is increased by the addition of spermidine to a yeast culture. It is noteworthy that this effect of added spermidine is found even when the experiments are conducted with strains in which the ornithine decarboxylase is overexpressed several hundred-fold more than the wild-type level. The effect of added spermidine in the overexpressed SPE1 strains is best seen in spe2 mutants in which the initial intracellular spermidine is very low or absent. Experiments with cycloheximide show that new protein synthesis is required to effect the breakdown of the ornithine decarboxylase. These results indicate that S. cerevisiae contains an antizyme-like mechanism for the control of the level of ornithine decarboxylase by spermidine, even though, as contrasted with other eukaryotic cells, no specific antizyme homolog has been detected either in in vitro experiments or in the S. cerevisiae genome (Gupta, 2001).
Conventional reading of the sequence of ornithine decarboxylase-antizyme mRNA (a protein that modulates the rate of ornithine decarboxylase degradation) results in premature termination at an in-frame termination codon (stop-1), located shortly after the initiation codon. Antizyme expression is demonstrated to require that ribosomes shift from the first open reading frame (termed ORF0) to a second +1 open reading frame (ORF1). These studies show that this frame-shifting, which occurs at maximal efficiency of approximately 20%, is stimulated by polyamines and requires the functional integrity of the stop codon (stop-1) of ORF0. By introducing in-frame deletions, it has been shown that an 87-nt segment surrounding stop-1 enhances frame-shifting efficiency, whereas the 6 nt located just upstream of stop-1 are absolutely essential for this process. Because this segment does not contain sequences that were previously characterized as shifty segments, these results suggest that another mechanism of frame-shifting is involved in mediating antizyme expression (Rom, 1994).
Rat antizyme gene expression requires programmed, ribosomal frameshifting. A novel autoregulatory mechanism enables modulation of frameshifting according to the cellular concentration of polyamines. Antizyme binds to, and destabilizes, ornithine decarboxylase, a key enzyme in polyamine synthesis. Rapid degradation ensues, thus completing a regulatory circuit. In vitro experiments with a fusion construct using reticulocyte lysates demonstrate polyamine-dependent expression with a frameshift efficiency of 19% at the optimal concentration of spermidine. The frameshift is +1 and occurs at the codon just preceding the terminator of the initiating frame. Both the termination codon of the initiating frame and a pseudoknot downstream in the mRNA have a stimulatory effect. The shift site sequence, UCC-UGA-U, is not similar to other known frameshift sites. The mechanism does not seem to involve re-pairing of peptidyl-tRNA in the new frame but rather reading or occlusion of a fourth base (Matsufuji, 1995).
Regulation of ornithine decarboxylase in vertebrates involves a negative feedback mechanism requiring the protein antizyme. 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. 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 (Ivanov, 2000a).
Degradation of ornithine decarboxylase, a key enzyme in polyamine biosynthesis, is accelerated by the binding of antizyme, an ornithine decarboxylase inhibitory protein induced by polyamines. The effects of a series of deletion mutants of rat antizyme have been studied. The results indicated that two regions of antizyme, one internal (amino acids 122-144) and the other near the C-terminus (amino acids 211-218) are necessary for its binding to ornithine decarboxylase and inhibition of its activity, and an additional internal region (amino acids 88-118, especially 113-118) is necessary for its destabilization (Ichiba, 1994).
The polyamines spermidine and spermine are ubiquitous and required for cell growth and differentiation in eukaryotes. Ornithine decarboxylase performs the first step in polyamine biosynthesis, the decarboxylation of ornithine to putrescine. Elevated polyamine levels can lead to down-regulation of ODC activity by enhancing the translation of antizyme mRNA, resulting in subsequent binding of antizyme to ODC monomers; this targets ODC for proteolysis by the 26S proteasome. The crystal structure of ornithine decarboxylase from human liver has been determined to 2.1 Å resolution by molecular replacement using truncated mouse ODC. The human ODC model includes several regions that are disordered in the mouse ODC crystal structure, including one of two C-terminal basal degradation elements that have been demonstrated to independently collaborate with antizyme binding to target ODC for degradation by the 26S proteasome. The crystal structure of human ODC suggests that the C terminus, which contains basal degradation elements necessary for antizyme-induced proteolysis, is not buried by the structural core of homodimeric ODC as previously proposed. Analysis of the solvent-accessible surface area, surface electrostatic potential, and the conservation of primary sequence between human ODC and Trypanosoma brucei ODC provides clues to the identity of potential protein-binding-determinants in the putative antizyme binding element in human ODC (Almrud, 2000).
Ornithine decarboxylase (ODC), a key enzyme in polyamine biosynthesis, is the most rapidly turned over mammalian enzyme. Its degradation is accelerated by ODC antizyme, an inhibitory protein induced by polyamines. This is a new type of enzyme regulation and may be a model for selective protein degradation. Using a cell-free degradation system, it has been demonstrated that immunodepletion of proteasomes from cell extracts causes almost complete loss of ATP- and antizyme-dependent degradation of ODC. In addition, purified 26S proteasome complex, but not the 20S proteasome, catalyses ODC degradation in the absence of ubiquitin. These results strongly suggest that the 26S proteasome, widely viewed as specific for ubiquitin-conjugated proteins, is the main enzyme responsible for ODC degradation. The 26S proteasome may therefore have a second role in ubiquitin-independent proteolysis (Murakami, 1992).
Regulated degradation of ornithine decarboxylase (ODC) is mediated by its association with the inducible protein antizyme. The N terminus of antizyme (NAZ), although unneeded for the interaction with ODC, must be present to induce degradation. Covalently grafting NAZ to ODC confers lability that normally results from the non-covalent association of native antizyme and ODC. To determine whether NAZ could act similarly as a modular functional domain when grafted to other proteins, it was fused to a region of cyclin B (amino acids 13-90) capable of undergoing degradation or to cyclin B (amino acids 13-59), which is not subject to degradation. The association with NAZ made both NAZ-cyclin B13-90 and NAZ-cyclin B13-59 unstable. Furthermore, NAZ and cyclin B 13-59 are together able to induce in vitro degradation of Trypanosoma brucei ODC, a stable protein. The ODC-antizyme complex binds to the 26 S protease but not the 20 S proteasome, consistent with the observation that ODC degradation is mediated by the 26 S protease. The association is independent of NAZ, suggesting that NAZ does not act as a recognition signal (Li, 1996).
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. 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 (Murakami, 1999).
The antizyme family consists of closely homologous proteins believed to regulate cellular polyamine pools. Antizyme1, the first described, negatively regulates ornithine decarboxylase, the initial enzyme in the biosynthetic pathway for polyamines. Antizyme1 targets ornithine decarboxylase for degradation and inhibits polyamine transport into cells, thereby diminishing polyamine pools. A polyamine-stimulated ribosomal frameshift is required for decoding antizyme1 mRNA. Recently, additional novel conserved members of the antizyme family have been described. Antizyme2, like antizyme1, binds to ornithine decarboxylase and inhibits polyamine transport. Using a baculovirus expression system in cultured Sf21 insect cells, both antizymes were found to accelerate ornithine decarboxylase degradation. Expression of either antizyme1 or 2 in Sf21 cells also diminished their uptake of the polyamine spermidine. Both forms of antizyme can therefore function as negative regulators of polyamine production and transport. However, in contrast to antizyme1, antizyme2 has negligible ability to stimulate degradation of ornithine decarboxylase in a rabbit reticulocyte lysate (Zhu, 1999).
The bone morphogenetic proteins (BMPs) regulate early embryogenesis and morphogenesis of multiple organs, such as bone, kidney, limbs, and muscle. Smad1 is one of the key signal transducers of BMPs and is responsible for transducing receptor activation signals from the cytoplasm to the nucleus, where Smad1 serves as a transcriptional regulator of various BMP-responsive genes. Based upon the ability of Smad1 to bind multiple proteins involved in proteasome-mediated degradation pathway, whether Smad1 could be a substrate for proteasome was investigated. Smad1 is targeted to proteasome for degradation in response to BMP type I receptor activation. The targeting of Smad1 to proteasome involves not only the receptor activation-induced Smad1 ubiquitination but also the targeting functions of the ornithine decarboxylase antizyme and the proteasome beta subunit HsN3. These studies provide the first evidence for BMP-induced proteasomal targeting and degradation of Smad1 and also reveal new players and novel mechanisms involved in this important aspect of Smad1 regulation and function (Gruendler, 2001).
Smad1 binds to multiple proteins involved in proteasome-mediated degradation pathways such as HsN3, antizyme (Az), and ubiquitin. HsN3 is one of the seven subunits of the 20 S proteasome, the catalytic core of the 26 S proteasome. HsN3 was also shown to be involved in the targeting of p105 NF-kappaB subunit to proteasome for processing. Ubiquitin is well known for its role in covalently modifying proteasomal substrates for ubiquitin-dependent degradation. Az is a protein previously known to bind ornithine decarboxylase (ODC), the rate-limiting enzyme for polyamine synthesis. Interestingly, its physical interaction with ODC is necessary and sufficient for targeting ODC to the 26 S proteasome for ubiquitin-independent degradation. Thus, ubiquitin and Az are two types of proteasome targeting proteins that mark proteins for both ubiquitin-dependent and ubiquitin-independent degradation by the 26 S proteasome. Currently, it is not clear how proteasome recognizes ubiquitinated proteins or Az-bound ODC. The ability of Smad1 to bind to ubiquitin and Az as well as HsN3, which is a proteasome component, suggests an interesting link between Smad1 and the proteasome targeting events involving ubiquitin, Az and HsN3. Studies were carried out to test whether the physical interaction between Smad1 and proteins involved in proteasomal degradation pathways (HsN3, Az, and Ub) may lead to: (1) proteasomal degradation of Smad1 or (2) proteasomal degradation of Smad1 interacting proteins. Concomitant with these studies, recent studies by others in the field have demonstrated several important roles of proteasomal degradation in regulating the protein levels of Smads and Smad-interacting proteins (28-35). In the signaling pathways of BMPs, it has been shown that Smad1 interacts with an ubiquitin E3 ligase, Smurf1, which regulates proteasomal degradation of Smad1 independent of BMP type I receptor activation. This study provides the first evidence that proteasomal degradation of Smad1 is also induced upon the activation by the BMP type I receptor. Furthermore, the data reveal novel roles of two Smad1 interactors, Az and HsN3, in proteasomal targeting and degradation of Smad1, in addition to Smad1 ubiquitination (Gruendler, 2001 and references therein).
Ornithine decarboxylase (ODC) is regulated by its metabolic products through a feedback loop that employs a second protein, antizyme 1 (AZ1). AZ1 accelerates the degradation of ODC by the proteasome. Purified components were used to study the structural elements required for proteasomal recognition of this ubiquitin-independent substrate. These results demonstrate that AZ1 acts on ODC to enhance the association of ODC with the proteasome, not the rate of its processing. Substrate-linked or free polyubiquitin chains compete for AZ1-stimulated degradation of ODC. ODC-AZ1 is therefore recognized by the same element(s) in the proteasome that mediate recognition of polyubiquitin chains. The 37 C-terminal amino acids of ODC harbor an AZ1-modulated recognition determinant. Within the ODC C terminus, three subsites are functionally distinguishable. The five terminal amino acids (ARINV, residues 457-461) collaborate with residue C441 to constitute one recognition element, and AZ1 collaborates with additional constituents of the ODC C terminus to generate a second recognition element (Zhang, 2003).
The antizymes constitute a conserved gene family with at least three mammalian orthologs. 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, the properties of AZ1 and AZ2 and protein chimeras composed of elements of the two were characterized. 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 (Chen, 2003).
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 it has been demonstrated 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: (1) an agmatine-dependent translational frameshift of antizyme mRNA to produce a full-length protein and (2) 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. A novel regulatory pathway is proposed in which agmatine acts as an antiproliferative molecule and potential tumor suppressor by restricting the cellular polyamine supply required to support growth (Satriano, 1998).
Cyclical phases of accumulation and depletion of polyamines occur during cell-cycle progression. Regulatory ornithine decarboxylase (ODC) catalyses the first step of polyamine biosynthesis. Ornithine decarboxylase antizyme (OAZ), induced by high polyamine levels, inhibits ODC activity and prevents extracellular polyamine uptake. Spermidine/spermine N1-acetyltransferase (SSAT) regulates the polyamine degradation/excretion pathway. 24 h transient transfection of immortalized human prostatic epithelial cells (PNT1A and PNT2) with antisense ODC RNA or OAZ cDNA, or both, while effectively causing marked decreases of ODC activity and polyamine (especially putrescine) concentrations, results in accumulation of cells in the S phase of the cell cycle. Transfection with SSAT cDNA leads to more pronounced decreases in spermidine and spermine levels and results in accumulation of cells in the G2/M phases. Transfection with all three constructs together produces maximal depletion of all polyamines, accompanied by accumulation of PNT1A cells in the S phase and PNT2 cells in the G0/G1 and G2/M phases. Accumulation of PNT1A cells in the S phase progressively increased at 15, 18 and 24 h of transfection with antisense ODC and/or OAZ cDNA. At 24 h, the DNA content was always reduced, as a possible outcome of altered chromosome condensation. A direct link between polyamine metabolism, cell proliferation and chromatin structure is thus proposed (Scorcioni, 2001).
Studies with mice overproducing ornithine decarboxylase have demonstrated the importance of polyamine homeostasis for normal mammalian spermatogenesis. The present study introduces a likely key player in the maintenance of proper polyamine homeostasis during spermatogenesis. Antizyme 3 is a paralog of mammalian ornithine decarboxylase antizymes. Like its previously described counterparts, antizymes 1 and 2, it inhibits ornithine decarboxylase, which catalyzes the synthesis of putrescine. Earlier work has shown that the coding sequences for antizymes 1 and 2 are in two different, partially overlapping reading frames. Ribosomes translate the first reading frame, and just before the stop codon for that frame, they shift to the second reading frame to synthesize a trans-frame product. The efficiency of this frameshifting depends on polyamine concentration, creating an autoregulatory circuit. Antizyme 3 cDNA has the same arrangement of reading frames and a potential shift site with definite, although limited, homology to its evolutionarily distant antizyme 1 and 2 counterparts. In contrast to antizymes 1 and 2, which are widely expressed throughout the body, antizyme 3 transcription is restricted to testis germ cells. Expression starts early in spermiogenesis and finishes in the late spermatid phase (Ivanov, 2000b).
ODC mRNA and protein are present at some level in all testicular cells (Leydig, Sertoli, spermatogenic cells, etc.), but the levels of expression vary greatly. ODC expression during sperm development increases sharply (from earlier background levels) and peaks in late pachytene spermatocytes and early round spermatids. In later stages of spermatid development and in spermatozoa, ODC expression falls back to background levels. By contrast, no antizyme 3 mRNA expression is detected before early spermatid stages of spermatogenesis or in any of the nonspermatogenic cells of testes. Antizyme 3 mRNA expression is maximal during the middle and late stages (stages VIII through XII) of spermatid development, and then it disappears again in the spermatozoa. It appears that the antizyme 3 wave of expression follows the wave of high ODC expression during spermatogenesis. This pattern of expression of the two genes implies that the physiological role of antizyme 3 is to quickly 'extinguish' (prevent overaccumulation of) ODC activity after the stage at which ODC plays its role in spermatogenesis, most likely late spermatocytic/early spermatidal phase. By the same type of experiments (in situ hybridization), antizyme 1 expression is essentially nondetectable in testicular tissues (specifically during spermatogenesis). This finding is consistent with observations demonstrating that of 50 human tissues examined, antizyme 1 mRNA is least abundant in testis. Because antizyme 2 mRNA was found to be even less abundant in testis by the same experiment (dot-blot analysis), its expression there would also be expected to be below the sensitivity threshold of in situ hybridization. From the fact that antizyme 3 is expressed only in testis and then only in the spermatid phase, whereas antizymes 1 and 2 are only negligibly (if at all) expressed during spermatogenesis, it is concluded that antizyme 3 has evolved specifically to provide spatial and temporal regulation of ODC during spermatogenesis (Ivanov, 2000b).
The role of ODC and, by extension, polyamines in spermatogenesis is not clear. Several functions have been proposed. These functions include possible roles in DNA synthesis and packaging during meiosis or regulation of transcription in haploid spermatogenic cells. Whatever their roles in spermatogenesis, there is a good indication of what might be the physiological consequences of extending high levels of ODC expression past its normal stage in sperm development. The main phenotype of transgenic mice overproducing ODC is male infertility and an associated alteration in seminiferous epithelial morphology. In such animals there is a significant reduction of mature spermatozoa. In fact, this deleterious effect is observed in all postmeiotic cells. Perhaps, in these cells the normally occurring antizyme 3 is 'swamped out' by the extra ODC, which leads to excessive polyamine (putrescine) accumulation and premature cell death. If this hypothesis is correct, mammals (mice in particular) lacking this gene should be male infertile with a specific morphology of the seminiferous epithelium (i.e., normal Sertoli cells, spermatogonia, and spermatocytes, but reduced or absent spermatids and spermatozoa). If this phenotype is confirmed, the antizyme 3 gene will become a candidate for heritable forms of human male infertility with similar testicular morphology (certain types of Sertoli cell only syndrome). These findings are consistent with the conclusion that polyamines play an important and special role during late meiosis and early spermiogenesis (Ivanov, 2000b).
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