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.     

Ubiquitin-independent mechanisms of mouse ornithine decarboxylase degradation are conserved between mammalian and fungal cells

The polyamine biosynthetic enzyme ornithine decarboxylase (ODC) is degraded by the 26 S proteasome via a ubiquitin-independent pathway in mammalian cells. Its degradation is greatly accelerated by association with the polyamine-induced regulatory protein antizyme 1 (AZ1). Mouse ODC (mODC) that is expressed in the yeast Saccharomyces cerevisiae is also rapidly degraded by the proteasome of that organism. We have now carried out in vivo and in vitro studies to determine whether S. cerevisiae proteasomes recognize mODC degradation signals. Mutations of mODC that stabilized the protein in animal cells also did so in the fungus. Moreover, the mODC degradation signal was able to destabilize a GFP or Ura3 reporter in GFP-mODC and Ura3-mODC fusion proteins. Co-expression of AZ1 accelerated mODC degradation 2-3-fold in yeast cells. The degradation of both mODC and the endogenous yeast ODC (yODC) was unaffected in S. cerevisiae mutants with various defects in ubiquitin metabolism, and ubiquitinylated forms of mODC were not detected in yeast cells. In addition, recombinant mODC was degraded in an ATP-dependent manner by affinity-purified yeast 26 S proteasomes in the absence of ubiquitin. Degradation by purified yeast proteasomes was sensitive to mutations that stabilized mODC in vivo, but was not accelerated by recombinant AZ1. These studies demonstrate that cell constituents required for mODC degradation are conserved between animals and fungi, and that both mammalian and fungal ODC are subject to proteasome-mediated proteolysis by ubiquitin-independent mechanisms.     

Characterization of a counterpart to Mammalian ornithine decarboxylase antizyme in prokaryotes

The degradation of mammalian ornithine decarboxylase (ODC) (EC 4.1.1.17) by 26 S proteasome, is accelerated by the ODC antizyme (AZ), a trigger protein involved in the specific degradation of eukaryotic ODC. In prokaryotes, AZ has not been found. Previously, we found that in Selenomonas ruminantium, a strictly anaerobic and Gram-negative bacterium, a drastic degradation of lysine decarboxylase (LDC; EC 4.1.1.18), which has decarboxylase activities toward both L-lysine and L-ornithine with similar K(m) values, occurs upon entry into the stationary phase of cell growth by protease together with a protein of 22 kDa (P22). Here, we show that P22 is a direct counterpart of eukaryotic AZ by the following evidence. (i) P22 synthesis is induced by putrescine but not cadaverine. (ii) P22 enhances the degradation of both mouse ODC and S. ruminantium LDC by a 26 S proteasome. (iii) S. ruminantium LDC degradation is also enhanced by mouse AZ replacing P22 in a cell-free extract from S. ruminantium. (iv) Both P22 and mouse AZ bind to S. ruminantium LDC but not to the LDC mutated in its binding site for P22 and AZ. In this report, we also show that P22 is a ribosomal protein of S. ruminantium.     

Polyamines regulate their synthesis by inducing expression and blocking degradation of ODC antizyme

Polyamines are essential organic cations with multiple cellular functions. Their synthesis is controlled by a feedback regulation whose main target is ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis. In mammals, ODC has been shown to be inhibited and targeted for ubiquitin-independent degradation by ODC antizyme (AZ). The synthesis of mammalian AZ was reported to involve a polyamine-induced ribosomal frameshifting mechanism. High levels of polyamine therefore inhibit new synthesis of polyamines by inducing ODC degradation. We identified a previously unrecognized sequence in the genome of Saccharomyces cerevisiae encoding an orthologue of mammalian AZ. We show that synthesis of yeast AZ (Oaz1) involves polyamine-regulated frameshifting as well. Degradation of yeast ODC by the proteasome depends on Oaz1. Using this novel model system for polyamine regulation, we discovered another level of its control. Oaz1 itself is subject to ubiquitin-mediated proteolysis by the proteasome. Degradation of Oaz1, however, is inhibited by polyamines. We propose a model, in which polyamines inhibit their ODC-mediated biosynthesis by two mechanisms, the control of Oaz1 synthesis and inhibition of its degradation.     

Antizyme targets cyclin D1 for degradation. A novel mechanism for cell growth repression

Overproduction of the ornithine decarboxylase (ODC) regulatory protein ODC-antizyme has been shown to correlate with cell growth inhibition in a variety of different cell types. Although the exact mechanism of this growth inhibition is not known, it has been attributed to the effect of antizyme on polyamine metabolism. Antizyme binds directly to ODC, targeting ODC for ubiquitin-independent degradation by the 26 S proteasome. We now show that antizyme induction also leads to degradation of the cell cycle regulatory protein cyclin D1. We demonstrate that antizyme is capable of specific, noncovalent association with cyclin D1 and that this interaction accelerates cyclin D1 degradation in vitro in the presence of only antizyme, cyclin D1, purified 26 S proteasomes, and ATP. In vivo, antizyme up-regulation induced either by the polyamine spermine or by antizyme overexpression causes reduction of intracellular cyclin D1 levels. The antizyme-mediated pathway for cyclin D1 degradation is independent of the previously characterized phosphorylation- and ubiquitination-dependent pathway, because antizyme up-regulation induces the degradation of a cyclin D1 mutant (T286A) that abrogates its ubiquitination. We propose that antizyme-mediated degradation of cyclin D1 by the proteasome may provide an explanation for the repression of cell growth following antizyme up-regulation.     

Antizyme, a mediator of ubiquitin-independent proteasomal degradation

Ornithine decarboxylase (ODC) is among the small set of proteasome substrates that is not ubiquitinated. It is instead degraded in conjunction with the protein antizyme (AZ). ODC and AZ are participants in a regulatory circuit that restricts pools of polyamines, the downstream products of ODC enzymatic activity. Functional studies using directed mutagenesis have identified regions of ODC and AZ required for the process of ODC degradation. Within ODC, there is a region that is required for AZ binding which lies on the surface of an alpha-beta barrel forming one domain of the ODC monomer. A carboxy-terminal ODC domain is needed for both AZ-dependent and AZ-independent degradation. Within AZ, the carboxy-terminal half molecule is sufficient for binding to ODC, but an additional domain found within the AZ amino terminus must be present for stimulation of ODC degradation by the proteasome. Recently, the AZs have been found to consist of an ancient gene family. Within vertebrate species, multiple isoforms are found, with distinct functions that remain to be sorted out. Although AZ homologs have been found in some yeast species, homology searches have failed to identify an AZ homolog in Saccharomyces cerevisiae. Nevertheless, the close parallel between polyamine-induced ODC degradation in S. cerevisiae and in animal cells suggests that this organism will also be found to harbor an AZ-like protein.     

Determinants of proteasome recognition of ornithine decarboxylase,a ubiquitin-independent substrate

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. We used purified components to study the structural elements required for proteasomal recognition of this ubiquitin-independent substrate. Our 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.     

Crystal structure of human ornithine decarboxylase at 2.1 A resolution: structural insights to antizyme binding

The polyamines spermidine and spermine are ubiquitous and required for cell growth and differentiation in eukaryotes. Ornithine decarboxylase (ODC, EC 4.1.1.17) 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 which targets ODC for proteolysis by the 26S proteasome. The crystal structure of ornithine decarboxylase from human liver has been determined to 2.1 A resolution by molecular replacement using truncated mouse ODC (Delta425-461) as the search model and refined to a crystallographic R-factor of 21.2% and an R-free value of 28.8%. 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.     

Subcellular localization and phosphorylation of antizyme 2

Antizymes (AZs) are polyamine‐induced proteins that negatively regulate cellular polyamine synthesis and uptake. Three antizyme isoforms are conserved among mammals. AZ1 and AZ2 have a broad tissue distribution, while AZ3 is testis specific. Both AZ1 and AZ2 inhibit ornithine decarboxylase (ODC) activity by binding to ODC monomer and target it to the 26S proteasome at least in vivo. Both also inhibit extra‐cellular polyamine uptake. Despite their being indistinguishable by these criteria, we show here using enhanced green fluorescent protein (EGFP)‐AZ2 fusion protein that in mammalian cells, the subcellular location of AZ2 is mainly in the nucleus, and is different from that of AZ1. The C‐terminal part of AZ2 is necessary for the nuclear distribution. Within a few hours, a shift in the distribution of EGFP‐AZ2 fusion protein from cytoplasm to the nucleus or from nucleus to cytoplasm is observable in NIH3T3 cells. In addition, we found that in cells a majority of AZ2, but not AZ1, is phosphorylated at Ser‐186, likely by protein kinase CK2. There may be a specific function of AZ2 in the nucleus. J. Cell. Biochem. 108: 1012–1021, 2009. © 2009 Wiley‐Liss, Inc.     

Ubiquitin dependent and independent protein degradation in the regulation of cellular polyamines

Summary. Protein degradation mediated by the ubiquitin/proteasome system is the major route for the degradation of cellular proteins. In this pathway the ubiquitination of the target proteins is manifested via the concerted action of several enzymes. The ubiquinated proteins are then recognized and degraded by the 26S proteasome. There are few reports of proteins degraded by the 26S protesome without ubiquitination, with ornithine decarboxylase being the most notable representative of this group. Interestingly, while the degradation of ODC is independent of ubiquitination, the degradation of other enzymes of the polyamine biosynthesis pathway is ubiquitin dependent. The present review describes the degradation of enzymes and regulators of the polyamine biosynthesis pathway.     

The antizyme family: polyamines and beyond

The family of antizymes functions as regulators of polyamine homeostasis. They are a class of small, inhibitory proteins, whose expression is regulated by a unique ribosomal frameshift mechanism. They have been shown to inhibit cell proliferation and possess anti-tumor activity. Antizymes bind ornithine decarboxylase (ODC), the key enzyme of polyamine biosynthesis. They inhibit its enzymatic activity and promote the ubiquitin-independent degradation of ODC by the 26S proteasome. In addition, they also negatively regulate polyamine transport. Antizyme-mediated, ubiquitin-independent degradation of ODC is conserved from yeast to man. But recent data suggest that this degradation pathway might not be restricted to ODC alone and could involve newly discovered antizyme binding partners. Interestingly, antizyme proteins have been strictly preserved over a vast evolutionary timeframe. Antizymes consequently represent an important class of proteins that regulate cell growth and metabolism by a diverse set of mechanisms that include protein degradation, inhibition of enzyme activity, small molecule transport and other, potentially not yet discovered properties.     

Effects of hyperactive Janus kinase 2 signaling in mammary epithelial cells

Ornithine decarboxylase (ODC), the first rate-limiting enzyme in the polyamine biosynthesis is one of the most rapidly degraded proteins in eukaryotic cells. Mammalian ODC is a notable exception to the widely accepted dogma that ubiquitination is always required for targeting a protein to degradation by the 26S proteasome. However, while it is well established that in mammalian cells degradation of ODC is ubiquitin independent, the requirement of ubiquitination for degradation of ODC in yeast cells remained undetermined. We have investigated ODC degradation in three mutant strains of Saccharomyces cerevisiae in which ubiquitin-dependent protein degradation activity is severely compromised. While yeast ODC was rapidly degraded in all these mutant strains the degradation of N-end rule substrates was inhibited. A mutant mouse ODC that fails to interact with Az was rapidly degraded in yeast cells but was stable in mammalian cells suggesting that interaction with a mammalian Az like yeast protein is not necessary for the degradation of ODC in yeast cells. Deletion analysis revealed that sequences from its unique N-terminus are involved in targeting yeast ODC to rapid degradation in yeast cells.     

Mechanisms of degradation of the fungal ornithine decarboxylase

Ornithine decarboxylase (ODC) is the first enzyme in polyamine biosynthesis in numerous living organisms, from bacteria to mammalian cells. Its control is under negative feedback regulation by the end products of the pathway. In dimorphic fungi, ODC activity and therefore polyamine concentrations are related to the morphogenetic process. From the fission yeast Schizosaccharomyces pombe to human, polyamines induce antizyme synthesis which in turn inactivates ODC. This is hydrolyzed by the 26S proteasome without ubiquitination. The regulatory mechanism of antizyme on polyamines is conserved, although to date no antizyme homology has been identified in some fungal species. The components that are responsible for regulating polyamine levels in cells and the current knowledge of ODC regulation in dimorphic fungi are presented in this review. ODC degradation is of particular interest because inhibitors of this pathway may lead to the discovery of novel antifungal drugs.     

Regulation of ornithine decarboxylase and polyamine import by hypoxia in pulmonary artery endothelial cells

In rat lung and cultured lung vascular cells, hypoxia decreases ornithine decarboxylase (ODC) activity and increases polyamine import. In this study, we used rat cultured pulmonary artery endothelial cells to explore the mechanism of hypoxia-induced reduction in ODC activity and determined whether this event was functionally related to the increase in polyamine import. Two strategies known to suppress proteasome-mediated ODC degradation, lactacystin treatment and use of cells expressing a truncated ODC incapable of interacting with the proteasome, prevented the hypoxia-induced decrease in ODC activity. Interestingly, though, cellular abundance of the 24-kDa antizyme, a known physiological accelerator of ODC degradation, was not increased by hypoxia. These observations suggest that an antizyme-independent ODC degradation pathway contributes to hypoxia-induced reductions of ODC activity. When reductions in ODC activity in hypoxia were prevented by the proteasome inhibitor strategies, hypoxia failed to increase polyamine transport. The induction of polyamine transport in hypoxic pulmonary artery endothelial cells thus seems to require decreased ODC activity as an initiating event.     

Mechanisms of Protein Degradation: An Odyssey with ODC

Intracellular proteolysis plays an important role in regulating fundamental cellularprocesses such as cell cycle, immune and inflammation responses, development,differentiation, and transformation. The ubiquitin-proteasome system accounts for thedegradation of the majority of cellular short-lived proteins. This system involves theconjugation of multiple ubiquitin residues to the target protein and its recognition by the26S proteasome through the poly-ubiquitin chain. Studies on the degradation of ornithinedecarboxylase (ODC) demonstrated that poly-ubiquitin is not the only signal recognizedby the 26S proteasome. The recognition of ODC by the 26S proteasome is mediated byinteraction with a polyamine-induced protein termed, antizyme (Az). While thedegradation of ODC is ubiquitin-independent, the degradation of its regulator Az, and ofantizyme-inhibitor (AzI), an ODC homologous protein that regulates Az availability, areubiquitin dependent. Interestingly, ODC undergoes another type of ubiquitin-independentdegradation by the 20S proteasome that is regulated by NAD(P)H quinoneoxidoreductase 1 (NQO1). Considering the prevalence of the ubiquitin system in theprocess of cellular protein degradation it is rather remarkable that a key cellular enzymeis subjected to two different proteolytic pathways that are different from the ubiquitindependent one. This exceptional behavior of ODC provides us with valuable insightsregarding protein degradation in general.     

In vitro study of proteolytic degradation of rat histidine decarboxylase.

Mammalian ornithine decarboxylase (ODC) is a very unstable protein which is degraded in an ATP-dependent manner by proteasome 26S, after making contact with the regulatory protein antizyme. PEST regions are sequences described as signals for protein degradation. The C-terminal PEST region of mammalian ODC is essential for its degradation by proteasome 26S. Mammalian histidine decarboxylase (HDC) is also a short-lived protein. The full primary sequence of mammalian HDC contains PEST-regions at both the N- and C-termini. Rat ODC and different truncated and full versions of rat HDC were expressed in vitro. In vitro degradation of rat ODC and rat 1-512 HDC were compared. Like ODC, rat 1-512 HDC is degraded mainly by an ATP-dependent mechanism. However, antizyme has no effect on the degradation of 1-512 HDC. The use of the inhibitors MG-132 and lactacystine significantly inhibited the degradation of 1-512 HDC, suggesting that a ubiquitin-dependent, proteasome 26S proteolytic pathway is involved. Results obtained with the different modifications of rat HDC containing all three PEST regions (full version, 1-656 HDC), only the N-terminal PEST region (1-512 HDC), or no PEST region (69-512 HDC), indicate that the N-terminal (1-69) fragment, but not the C-terminal fragment, determines that the HDC protein is a proteasome substrate in vitro.     

The N-terminal unstructured domain of yeast ODC functions as a transplantable and replaceable ubiquitin-independent degron

Ornithine decarboxylase (ODC), a homodimeric enzyme with a rate-limiting function in polyamine biosynthesis, is subject to a feedback control involving its selective proteolysis. Targeting of ODC monomers to the proteasome is mediated by ODC antizyme (OAZ), the expression of which is induced by high levels of polyamines. Here, we report our analysis of the N-terminal degron in Saccharomyces cerevisiae ODC and the mechanism of its antizyme-dependent targeting. This ∼ 45-residue domain of ODC [termed ODC degradation signal (ODS)] is essential for degradation of ODC. Extensive mutagenesis indicated that it is not a specific sequence within ODS that is important but, rather, its unstructured nature. Consistent with this conclusion, ODS could be functionally replaced by an unrelated unstructured domain. We show that increasing the distance of ODS to the rest of the ODC protein reduced the dependence on Oaz1 for targeting, indicating that exposure of ODS is critical for its function. Disruption of ODC dimers by introducing interface mutations, in contrast, was insufficient for targeting. Binding of Oaz1 to ODC monomers is thus required to activate ODS. Fusion of ODS to the N terminus of Ura3 was sufficient to convert it into a ubiquitin-independent substrate of the proteasome. By contrast, ODS failed to destabilize maltose-binding protein or dihydrofolate reductase, indicating that this degron only operates in an appropriate structural context that enables rapid unfolding.     

Discrimination between ubiquitin-dependent and ubiquitin-independent proteolytic pathways by the 26S proteasome subunit 5a.

The 26S proteasome subunit 5a binds polyubiquitin chains and has previously been shown to inhibit the degradation of mitotic cyclins. Presumably inhibition results from S5a binding and preventing recognition of Ub-cyclin conjugates by the 26S proteasome. Here we show that S5a does not inhibit the degradation of full-length ornithine decarboxylase (ODC) consistent with previous reports that the enzyme is degraded in an antizyme-dependent, but ubiquitin-independent reaction. S5a does, however, inhibit degradation of short ODC translation products generated by internal initiation events. Because in vitro translation often produces some shortened products, the existence of ubiquitin conjugated to a 35S-labeled protein is not necessarily evidence that the full-length protein is a substrate of the Ub-dependent proteolytic pathway.     

Ornithine decarboxylase-antizyme is rapidly degraded through a mechanism that requires functional ubiquitin-dependent proteolytic activity.

Antizyme is a polyamine-induced cellular protein that binds to ornithine decarboxylase (ODC), and targets it to rapid ubiquitin-independent degradation by the 26S proteasome. However, the metabolic fate of antizyme is not clear. We have tested the stability of antizyme in mammalian cells. In contrast with previous studies demonstrating stability in vitro in a reticulocyte lysate-based degradation system, in cells antizyme is rapidly degraded and this degradation is inhibited by specific proteasome inhibitors. While the degradation of ODC is stimulated by the presence of cotransfected antizyme, degradation of antizyme seems to be independent of ODC, suggesting that antizyme degradation does not occur while presenting ODC to the 26S proteasome. Interestingly, both species of antizyme, which represent initiation at two in-frame initiation codons, are rapidly degraded. The degradation of both antizyme proteins is inhibited in ts20 cells containing a thermosensitive ubiquitin-activating enzyme, E1. Therefore we conclude that in contrast with ubiquitin-independent degradation of ODC, degradation of antizyme requires a functional ubiquitin system.