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.     

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.     

A new perspective on ornithine decarboxylase regulation: prevention of polyamine toxicity is the overriding theme

The polyamines are essential cellular components for growth. Control of a key regulated enzyme of polyamine biosynthesis, ornithine decarboxylase (ODC), as a function of growth, is an area of intense interest. A unique regulatory property of ODC is the short half-life of the protein, which has been suggested to be an important factor in rapid activation of polyamine biosynthesis after cells are mitogenically stimulated. In this paper, it is argued that the biological significance of the short half-life of ODC is unrelated to the rate of its induction to a new steady state by growth factors, which is in fact limited by the relatively long half-life of the ODC mRNA. Instead, I suggest that the rapid turnover of ODC protein becomes of significance when cells cease growth and expeditious downregulation of the enzyme is important in preventing polyamine overproduction, which would result in cytotoxicity in the arrested cells. Although mitogenic activation of ODC expression has been studied extensively, there is very little known about the mechanisms controlling downregulation of polyamine biosynthesis during the arrest of animal cell growth. These considerations suggest that this would be a fertile area of future inquiry.     

Inhibition of the yeast-mycelial transition and the phorogenesis of Mucorales by diamino butanone

Diamino butanone (DAB), a competitive inhibitor of ornithine decarboxylase (ODC) a key enzyme in polyamine biosynthesis, inhibited the yeast to hyphae transition in Mucor rouxii, induced by transfer from anaerobiosis to aerobiosis, but not the opposite phenomenon. Addition of DAB to anaerobic yeast cells brought about a decrease in ODC and polyamine levels. In these conditions, the aerobic shift produced only a weak increase in ODC activity and no change in polyamine levels. DAB also blocked phorogenesis in M. rouxii and in Phycomyces blakesleeanus. At the effective concentrations DAB did not affect cell growth of either fungus. It is suggested that low, constant levels of ODC and polyamines are necessary for cell growth, and that high transient levels are required during the differentiative steps. DAB, at the concentrations used, affects this last process, but does not interfere with the maintenance level of polyamines.     

Cell-cycle-dependent regulation of ornithine decarboxylase activity in the unicellular green alga Chlamydomonas reinhardtii

Polyamines play an important role in the control of cell growth and cell division. In the unicellular green alga Chlamydomonas reinhardtii as in animal cells, biosynthesis of the 3 commonly occurring polyamines (putrescine, spermidine and spermine) is dependent on the activity of ornithine decarboxylase (ODC, EC 4.1.1.17) catalyzing the formation of putrescine, which is the precursor of the other two polyamines. Therefore, we have investigated the regulation of ODC activity during the cell cycle of Clamydomonas reinhardtii using synchronized cultures. A 2.5–3-fold increase in ODC activity was observed during the transition to the cell division phase. This up-regulation of ODC activity was not due to an increased level of ODC-mRNA as revealed by northern-blot analyses, but correlated with an increased half-life of this particular enzyme (from 1.1 to 3.2 h). Addition of the DNA topoisomerase II inhibitor nalidixic acid during the second half of the growth period caused a transient decrease of ODC activity and a considerable delay of cell divisions. After cell division, a down-regulation of ODC activity was observed which was faster in the dark than in the light and also correlated with changes of the ODC half-life.     

Destabilization of ornithine decarboxylase by transfected antizyme gene expression in hepatoma tissue culture cells.

The degradation of ornithine decarboxylase (ODC) is stimulated by polyamines in a protein synthesis-dependent manner. It has been suggested that antizyme, an ODC-inhibiting protein induced by polyamines, is involved in the process of polyamine-stimulated ODC decay. In this study, we investigated the direct effect of antizyme on ODC decay in hepatoma tissue culture (HTC) cells. A truncated rat antizyme cDNA, Z1, was inserted into an expression vector at a site under the control of a glucocorticoid-inducible promoter and transfected into HTC cells. In the transfected cells dexamethasone increased the amount of Z1 mRNA and induced active antizyme in the absence of exogenous polyamines. When dexamethasone was added to cells with a high level of ODC, rapid decays of ODC activity and protein were elicited after a lag time. Cycloheximide abolished the effect of dexamethasone. These effects of dexamethasone were not observed in control HTC cells transfected with the chloramphenicol acetyltransferase gene. This study indicated that, once induced, antizyme stimulated ODC degradation independently of polyamines and strongly supported our previous hypothesis that the ODC decay-accelerating action of polyamines is mediated by antizyme.     

Structural elements of ornithine decarboxylase required for intracellular degradation and polyamine-dependent regulation.

Mammalian ornithine decarboxylase (ODC), a key enzyme in polyamine biosynthesis, is rapidly degraded in cells, an attribute important to the regulation of its activity. Mutant and chimeric ODCs were created to determine the structural requirements for two modes of proteolysis. Constitutive degradation requires the carboxy terminus and is independent of intracellular polyamines. Truncation of five or more carboxy-terminal amino acids prevents this mode of degradation, as do several internal deletions within the 37 carboxy-most amino acids that spare the last five residues. Polyamine-dependent degradation of ODC requires a distinct region outside the carboxy terminus. The ODC of a parasite, Trypanosoma brucei, is structurally very similar to mouse ODC but lacks the carboxy-terminal domain; it is not a substrate for either pathway. The regulatory properties of enzymatically active chimeric proteins incorporating regions of the two ODCs support the conclusion that distinct domains of mouse ODC confer constitutive degradation and polyamine-mediated regulation. Mouse ODC contains two PEST regions. The first was not required for either form of degradation; major deletions within the second ablated constitutive degradation. When mouse and T. brucei ODC RNAs were translated in vitro in a reticulocyte lysate system, the effects of polyamine concentration on ODC protein production and activity were similar for the two mRNAs, which contradicts claims that this system accurately reflects the in vivo effects of polyamines on responsive ODCs.     

Similarities in ornithine decarboxylase regulation in intact and enucleated 3T3 cells

The role of the cytoplasm in the regulation of ornithine decarboxylase (ODC) has been examined in enucleated 3T3 cells (cytoplasts). ODC activity can be increased 15–25-fold by a cytoplasmic mechanism(s) in enucleates prepared from growing cells by treatment which lead to 50–75-fold increases in intact growing cells. Since the enzyme activity simultaneously becomes less stable in these cytoplasts as in whole cells, it appears this increase is due either to activation of pre-existing enzyme or increased synthesis. A biphasic increase during the first 20 h after stimulus is seen in cytoplasts prepared from growing cells which have been stimulated for the prior 5 h. The second increase in activity does not appear to be due to decreased degradation or conversion to a more active form. These results are analogous to those reported previously for intact growing cells in experiments which employed metabolic inhibitors, and similarly suggest that there is cytoplasmic control of ODC synthesis. Medium polyamines reduce ODC activity in cytoplasts with kinetics and characteristics similar to those previously reported for intact cells. These data are also most consistent with regulation of synthesis at the translational level.     

The development of competence in resting B cells. The induction of cyclic AMP and ornithine decarboxylase activity after direct contact between B and T helper cells.

In the present communication, an experimental approach is utilized that facilitates the study of biochemical processes induced in B cells after their interaction with Th cells. In this approach, Th cell clones are stimulated for 18 h upon anti-CD3-coated plates, fixed with paraformaldehyde, and added at a 2 to 3:1 ratio to small, resting B cells (isolated from Percoll gradients). Th cells not stimulated on anti-CD3-coated plates, but fixed with paraformaldehyde, serve as controls for these experiments. The activated, fixed Th cells induce a transient, sixfold increase in B cell levels of cAMP, as well as an increase in B cell expression of ornithine decarboxylase (ODC) activity. This enzyme initiates the synthesis of polyamines and has been shown to be increased as cells enter the growth phase. In addition, previous studies have shown that the cellular levels of ODC activity are controlled by a multi-tiered regulatory cascade. To examine this aspect, polyclonally stimulated B cells were studied. Such cells demonstrated a gradual increase in ODC mRNA levels that peaked between 6 and 15 h and can be partially explained by a three- to fourfold increase in mRNA stability but not by changes in the enzyme affinity for substrate. The increase in ODC mRNA occurs in the absence of protein synthesis, suggesting that the ODC gene is a member of the immediate/early gene family. Finally, the early increase in ODC mRNA was enhanced in cells in which cAMP levels were artificially elevated, suggesting the possibility that the cAMP-dependent signaling pathway participates during the regulation of this gene expression. The significance of these experimental results concerning the process of B cell activation is discussed.     

The Plasmodium falciparum bifunctional ornithine decarboxylase, S-adenosyl-L-methionine decarboxylase, enables a well balanced polyamine synthesis without domain-domain interaction

In the human malaria parasite Plasmodium falciparum (Pf), polyamines are synthesized by a bifunctional enzyme that possesses both ornithine decarboxylase (ODC) and S-adenosyl-l-methionine decarboxylase (AdoMetDC) activities. The mature enzyme consists of the heterotetrameric N-terminal AdoMetDC and the C-terminal dimeric ODC, which results in the formation of a heterotetrameric complex. For the native bifunctional protein a half-life longer than 2 h was determined, which is in contrast to the extreme short half-life of its mammalian monofunctional counterparts. The biological advantage of the plasmodial bifunctional ODC/AdoMetDC might be that the control of polyamine synthesis is achieved by only having to regulate the abundance and activity of one protein. An interesting feature in the regulation of the bifunctional protein is that putrescine inhibits PfODC activity approximately 10-fold more efficiently than the mammalian ODC activity, and in contrast to the mammalian AdoMetDC the activity of the PfAdoMetDC domain is not stimulated by the diamine. To analyze post-translational processing, polymerization, and domain-domain interactions, several mutant proteins were generated that have single mutations in either the PfODC or PfAdoMetDC domains. The exchange of amino acids essential for the activity of one domain had no effect on the enzyme activity of the other domain. Even prevention of the post-translational cleavage of the AdoMetDC domain or ODC dimerization and thus the interference with the folding of the protein hardly affected the activity of the partner domain. In addition, inhibition of the activity of the PfODC domain had no effect on the activity of the PfAdoMetDC domain and vice versa. These results demonstrate that no domain-domain interactions occur between the two enzymes of the bifunctional PfODC/AdoMetDC and that both enzymatic activities are operating as independent catalytic sites that do not affect each other.     

Inhibition of the yeast-mycelial transition and the phorogenesis of Mucorales by diamino butanone

Diamino butanone (DAB), a competitive inhibitor of ornithine decarboxylase (ODC) a key enzyme in polyamine biosynthesis, inhibited the yeast to hyphae transition in Mucor rouxii, induced by transfer from anaerobiosis to aerobiosis, but not the opposite phenomenon. Addition of DAB to anaerobic yeast cells brought about a decrease in ODC and polyamine levels. In these conditions, the aerobic shift produced only a weak increase in ODC activity and no change in polyamine levels. DAB also blocked phorogenesis in M. rouxii and in Phycomyces blakesleeanus. At the effective concentrations DAB did not affect cell growth of either fungus. It is suggested that low, constant levels of ODC and polyamines are necessary for cell growth, and that high transient levels are required during the differentiative steps. DAB, at the concentrations used, affects this last process, but does not interfere with the maintenance level of polyamines.Abbreviations ODC ornithine decarboxylase - DAB 1,4-diamino butanone     

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.     

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.     

Effect of ATP depletion and phenanthroline on the spermidine-mediated decay of ornithine decarboxylase in erythroleukemia cells

Addition of spermidine to Friend erythroleukemia cells caused a rapid decay of ornithine decarboxylase (ODC) activity and the accumulation of a ODC-antizyme complex. The induction of antizyme only partially accounted for the decrease of ODC activity by a direct inhibition of the enzyme. However, the antizyme induction was accompanied by a marked reduction of the half-life of ODC. Shift of the cells to an ATP-depleting medium prevented the spermidine-elicited decay of ODC activity as well as the accumulation of ODC-antizyme complex. However, ODC appeared to be stabilized even when ATP depletion was performed 40 min after spermidine addition, in the presence of high levels of antizyme. Similar results were obtained by treating the cells with phenanthroline, a heavy metal chelator and protease inhibitor. These findings indicate that ATP and some metalloprotease(s) may be involved in the degradation pathway of ODC, even in the presence of high levels of polyamines.     

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.     

Degradation of ornithine decarboxylase in mammalian cells is ATP dependent but ubiquitin independent

Ornithine decarboxylase (ODC), a key enzyme in the biosynthesis of polyamines in mammalian cells is characterized by an extremely short half-life. In the present study, ODC degradation was investigated in 653-1 mouse myeloma cells that overproduce ODC and in ts85 cells that are thermosensitive for conjunction of ubiquitin to target proteins. Addition of 2-deoxyglucose and dinitrophenol (agents that efficiently deplete cellular ATP) to the growth medium of these cells inhibited ODC degradation. In contrast, chloroquine and leupeptin, inhibitors of intralysosomal proteolysis, did not affect ODC degradation. Shifting ts85 cells to 42 degrees C (a non-permissive temperature that inhibited conjugation of ubiquitin to target proteins) did not prevent ODC degradation. The ATP-dependent degradation of ODC in 653-1 cells was inhibited substantially by N alpha-tosyl-L-lysine chloromethane (TosPheMeCl), iodoacetamide and o-phenanthroline. These results suggest that ODC degradation occurs via a non-lysosomal. ATP-requiring and ubiquitin-independent cellular proteolytic mechanism, and that serine proteases and enzymes containing sulphydryl groups and metalloenzyme(s) may be involved in this process.