Polyamines are small aliphatic polycations present in all living cells. Polyamines are essential for cellular viability and are involved in regulating fundamental cellular processes, most notably cellular growth and proliferation. Being such central regulators of fundamental cellular functions, the intracellular polyamine concentration is tightly regulated at the levels of synthesis, uptake, excretion and catabolism. ODC (ornithine decarboxylase) is the first key enzyme in the polyamine biosynthesis pathway. ODC is characterized by an extremely rapid intracellular turnover rate, a trait that is central to the regulation of cellular polyamine homoeostasis. The degradation rate of ODC is regulated by its end-products, the polyamines, via a unique autoregulatory circuit. At the centre of this circuit is a small protein called Az (antizyme), whose synthesis is stimulated by polyamines. Az inactivates ODC and targets it to ubiquitin-independent degradation by the 26S proteasome. In addition, Az inhibits uptake of polyamines. Az itself is regulated by another ODC-related protein termed AzI (antizyme inhibitor). AzI is highly homologous with ODC, but it lacks ornithine-decarboxylating activity. Its ability to serve as a regulator is based on its high affinity to Az, which is greater than the affinity Az has to ODC. As a result, it interferes with the binding of Az to ODC, thus rescuing ODC from degradation and permitting uptake of polyamines.
The polyamines spermidine, spermine, and their diamine precursor, putrescine, are ubiquitous physiological cations. The polyamines fulfil a crucial role in regulating various fundamental cellular processes, most notably cell growth and proliferation. Depletion of cellular polyamines results in cessation of cellular proliferation, which can be completely reverted upon re-addition of exogenous polyamines. Although their explicit mechanism of action is still unknown, it is likely that their evenly distributed positive charges, which are capable of interacting with negative charges present in various cellular macromolecules, plays a key role in their ability to regulate cellular processes. Interactions with polyamines increase the melting temperature and condensation state of DNA. Polyamines can convert B-DNA into Z-DNA and A-DNA and affect binding of some transcription factors to DNA. Polyamine depletion inhibits both DNA and protein synthesis. The effect on protein synthesis may be mediated, at least in part, by an unusual covalent modification, termed hypusination, of the putative translation initiation factor eIF-5A (eukaryotic initiation factor 5A) . Polyamines have also been demonstrated to act as activators and inhibitors of various cation channels [2–5] and have been suggested to modulate synthesis of nitric oxide . However, the most notable feature of polyamines is their involvement in regulating proliferation of normal and malignant cells . Depletion of cellular polyamine levels affects the expression of a number of growth-regulated/regulating genes. However, it is unknown whether these are the only changes in gene expression caused by changes in polyamine levels, and which of these changes affect growth, and which are actually an outcome of the growth arrest. Cell viability is affected both when polyamines are limiting and when they are present at excessive levels. Therefore their intracellular concentration must be maintained within a narrow optimal concentration. This is achieved by maintaining a balance between synthesis, catabolism, uptake and excretion. ODC (ornithine decarboxylase) is a highly regulated key enzyme in the polyamine biosynthesis pathway. ODC is subject to regulation by its end-products, the polyamines, through an autoregulatory circuit. At the centre of this circuit is a polyamine-induced protein termed Az (antizyme), which is itself regulated by an inactive ODC-related protein termed AzI (antizyme inhibitor).
ODC is a PLP (pyridoxal phosphate)-dependent enzyme that decarboxylates ornithine to form putrescine. In its active form, ODC exists as a homodimer with a two-fold symmetry. The monomer retains no enzymatic activity. The homodimer contains two symmetrical active sites located at the interface between the two subunits, with residues from each subunit contributing to the formation of each active site (for a review, see ). The association between the two subunits is rather weak, leading to a high rate of association and dissociation, a situation that, as will be described below, is central for its regulation.
ODC is a highly regulated enzyme that responds rapidly and dramatically to a variety of growth-promoting stimuli. The increase in ODC activity is correlated with an increase in the level of the enzyme protein. The increase in the amount of ODC is regulated at the levels of transcription, transcript stability and translatability. A key element enabling regulation by these regulatory mechanisms is the rapid turnover rate of ODC. In fact, ODC is one of the most rapidly degraded proteins in eukaryotic cells. Interestingly, ODC is not degraded at a constant rate, but rather its degradation rate is regulated by the intracellular concentration of polyamines. As mentioned above, two proteins regulate the degradation rate of ODC. The first, Az, whose synthesis is stimulated by polyamines, targets ODC to ubiquitin-independent degradation by the 26S proteasome. The second, AzI, negates the ability of Az to promote ODC degradation. Interestingly, Az and AzI also affect the process of polyamine uptake through the plasma membrane. However, neither the exact mechanism of polyamine uptake, nor the way it is affected by Az, are presently known.
Az is a small protein originally described as a polyamine-induced ODC inhibitory activity. It is positioned at the centre of an autoregulatory circuit that regulates cellular polyamine levels.
Az is encoded by two independent ORFs (open reading frames; ORF1 and ORF2) [9,10]. Translation that starts at one of two possible in-frame initiation codons is terminated shortly thereafter at an in-frame stop codon. In order to produce a functional full-length Az, the ribosomes that scan ORF1 must be subverted to the +1 reading frame (ORF2) (Figure 1). Comparison of the sequence of the mature functional Az protein with the sequence of the encoding mRNA reveals that the actual frameshifting event occurs while the scanning ribosome encounters the last codon of ORF1. In most cases, the ribosome slips one nucleotide forward, encoding the first amino acid (aspartic acid, GAU) of ORF2. Polyamines stimulate the efficiency of this +1 frameshifting event. The reliance of frameshifting efficiency on the polyamine concentration serves as an intracellular sensing mechanism for the free intracellular polyamine pool.
The most important segment of Az mRNA that is required for frameshifting is the sequence at which frameshifting occurs. This segment contains two elements, the stop codon that halts the scanning ribosome, and the sequence just upstream to it that programmes the repositioning of the ribosome. Frameshifting efficiency remains unaffected when the sequence of the stop codon is converted into the sequence of one of the other stop codons, but it is severely inhibited when the stop codon is replaced by a sense codon. The importance of the stop function is further emphasized by recent demonstrations that the yeast prion [PSI+], which represents a non-functional aggregated conformation of the translational release factor eRF3, or inactivation of eRF3 by interaction with the interferon-induced RNAse L, increase frameshifting efficiency [11,12]. The mechanism by which polyamines stimulate frameshifting efficiency is still unknown.
Mechanism of ODC inactivation and stimulation of ODC degradation by Az
Az was originally identified as an ODC inhibitory activity that is stimulated by an increase in the intracellular polyamine concentration . Upon its cloning, it was demonstrated that the affinity of Az for ODC subunits is significantly higher than the affinity of ODC subunits for each other . This, together with the rapid and frequent association and dissociation of ODC subunits, enables efficient trapping of transient ODC monomers by Az, forming a tightly bound heterodimer. The strong association of ODC monomers with Az prevents their re-association to form active ODC homodimers. Although ODC inactivation is a clear function of Az, in most cases it is only an intermediate step in the process of targeting ODC subunits to ubiquitin-independent degradation by the 26S proteasome  (Figure 2). Interaction with Az seems to impose conformational alteration on ODC, resulting in the exposure of an ODC segment encompassing its most C-terminal 37 amino acids. This C-terminal segment functions as the proteasome recognition signal . Exposure of the C-terminal targeting signal enhances the interaction of ODC with the proteasome without stimulating proteasomal activity. Another segment of ODC, encompassing amino acids 117–140, is also essential for stimulating ODC degradation, since it is required for binding to Az . Trypanosoma brucei ODC, which is a stable protein, lacks sequences that parallel the C-terminal segment of the mammalian enzyme and it does not bind Az [17,18]. Although refractory to Az binding, trypanosome ODC is converted into a rapidly degraded protein when the C-terminal segment of the mammalian enzyme is appended to its C-terminus, probably because, in the context of the trypanosome enzyme, the C-terminal mammalian segment is exposed without requiring interaction with Az. Interestingly, although, like the trypanosome enzyme, yeast ODC also lacks a C-terminal targeting segment, it is rapidly degraded in yeast cells in an Az-dependent manner . It appears that yeast ODC is targeted to degradation by an N-terminal segment that can be replaced by the C-terminal targeting segment of the mammalian enzyme. At present, it is not known whether, like the C-terminal segment of the mammalian enzyme, the N-terminal yeast segment can also function as a dominant transferrable degradation signal.
Segments of Az that affect its ability to stimulate ODC degradation
Az also has two segments that are required for its ability to stimulate ODC degradation . A large segment encompassing the C-terminal half of the molecule is important for the ability to bind ODC. Although the binding mediated by this sequence is sufficient to inactivate ODC, it is insufficient for stimulating ODC degradation. A smaller N-terminal segment is required to induce ODC degradation. However, the mechanism by which this sequence affects the degradation process is still unknown.
The structure of Az in solution was determined using NMR methods . Az was found to contain eight β-strands and two α-helices, with the strands forming a mixture of parallel and antiparallel β-sheets. A significant proportion of the residues that are highly conserved among Azs were found on the surface. Some of these residues form a negatively charged patch that might interact with an electropositive surface located in the putative Az-binding site of ODC.
Az inhibits polyamine uptake
In addition to regulating ODC activity and degradation, Az inhibits uptake of polyamines and stimulates their excretion  (Figure 3). The mechanism by which Az regulates transport of polyamines across the plasma membrane is mostly unknown, as is the basic process of polyamine transport in eukaryotic cells. Interestingly, Az has only a minor effect on polyamine uptake in yeast cells.
Az targets additional proteins that are unrelated to the polyamine metabolism for degradation
Although components of polyamine metabolism are the natural targets of Az, a number of studies suggested that Az might also target for degradation proteins that do not belong to the polyamine metabolic pathway or to its regulation (for a review, see ). It was suggested that Az, together with the proteasome β subunit HsN3, mediates the targeting of the signal transducer Smad1 to the proteasome [24,25]. Az was also demonstrated to interact and stimulate the degradation of two growth-related proteins, cyclin D1 and Aurora A [26,27]. Since, in contrast with ODC, whose degradation is always ubiquitin-independent, these proteins are also degraded in a ubiquitin-dependent manner, it is not clear when and under what physiological conditions each of these degradation pathways operates.
Az is a rapidly degraded protein
It is widely accepted that Az acts catalytically; namely, it is recycled to support additional rounds of ODC degradation . Compatible with a catalytic mode of action, Az is not degraded together with ODC when presenting ODC to the proteasome (Figure 2). Nevertheless, Az is a rapidly degraded protein . Interestingly, the degradation of Az is ubiquitin-dependent , and in yeast it is inhibited by polyamines. It is unclear how Az escapes degradation while escorting ODC to the proteasome, and how it is recognized as a substrate for ubiquitination.
Forced Az overexpression interferes with cellular proliferation and tumour development
The ability of Az to stimulate degradation of ODC and possibly of other growth-regulating proteins, together with its ability to inhibit polyamine uptake, strongly suggest that Az may inhibit cellular proliferation (Figure 3). Therefore it is not surprising that Az acts as a negative regulator of cellular proliferation and of tumour development both when expressed in cultured cells and in transgenic mice [29,30]. In some experimental systems, artificial Az overexpression provokes apoptotic cell death. It is therefore not surprising that in all studies performed in cultured cells, Az was expressed in an inducible manner. When expressed in transgenic animals, Az interferes with tumour development.
A family of Az proteins
The Az species described above, termed Az1, appears to be the prototype of a family of Azs containing three members characterized to date. A second member of this family, termed Az2, is extremely interesting as it has a tissue distribution similar to that of Az1, but it is expressed at much lower levels . Az2 is more conserved evolutionarily than Az1, suggesting that it is likely to have a significant biological role. Its minority co-existence with Az1 suggests that its role might be different from that of Az1. In support of this possibility, is a recent demonstration that the level of Az2, but not of Az1, changes in neuroblastomas . Although Az2 inhibits ODC activity, stimulates its degradation in cells and inhibits polyamine uptake as efficiently as Az1, it fails to promote ODC degradation in an in vitro reaction. The reasons and the significance of this odd behaviour are presently unknown. Also, it is presently unknown whether Az2 might possess additional functions not shared by Az1.
A third member of the antizyme family, Az3, is unique in being tissue-specific, expressed predominantly in testis. It is observed only in haploid germinal cells [33,34]. Interestingly, the localization of Az3 is different from that of ODC, and it is found mainly in the outer part of the seminiferous tubules where spermatogonia and spermatocytes are located . This is compatible with the observation that Az3 does not target ODC to degradation and therefore might have a different role. Such an alternative role may be reflected by its interaction with the germ-cell-specific protein, gametogenetin protein-1. However, the functional consequence for this interaction has not yet been demonstrated.
Subcellular localization of Az1 is determined by alternative utilization of initiation codons
As mentioned above, Az1 mRNA contains two AUG codons that can initiate translation. Although translation generally starts at the first AUG codon, translation of Az1 is initiated predominantly at the second initiation codon because it is situated within a superior sequence context for translation initiation. Nevertheless, some initiations occur at the first AUG as well, and the products of the two initiation events are observed both in vivo and in vitro as 24.5 and 29 kDa isoforms. The segment located between the two initiation codons contains a positive amphiphathic helix that is part of a mitochondrial localization signal [36,37]. Thus only the minor long form is localized to the mitochondria in transfected cells and imported into mitochondria in an in vitro assay . Although both forms stimulate ODC degradation and inhibit polyamine uptake through the plasma membrane, neither affects polyamine uptake by rat liver mitochondria . Az1 also contains two independent nuclear export signals . One is N-terminal, overlapping the mitochondrial localization signal, whereas the other resides in the central part of the protein. The relevance of the central signal is uncertain as it is functional only in the context of an N-terminal truncation. In agreement with the existence of such signals, shuttling of Az between the cytoplasm and nucleus has been documented [39–41].
AzI was originally described as an activity capable of inhibiting Az functions . Although it is highly homologous with ODC, AzI is a distinct protein that lacks ornithine-decarboxylating activity and inhibits all members of the Az family. Recently it was demonstrated that expression of AzI might be repressed by polyamines via utilization of upstream ORFs initiating at non-canonical initiation codons .
Structural features explain the ability of AzI to negate Az functions
Recent studies have demonstrated that, to a large extent, the ability of AzI to inhibit Az is an outcome of its structure . Although, like ODC, AzI crystallizes as a dimer, fewer interactions at the dimer interface, a smaller buried surface area, and lack of symmetry of the interactions between residues from the two monomers suggest that, under physiological conditions, AzI is actually a monomer . Indeed, biochemical studies have confirmed that AzI exists as a monomer, whereas ODC is dimeric. As a monomer, AzI is more available for interaction with Az compared with ODC subunits that are in a constant state of association/dissociation. Since it is unlikely that greater availability is the only reason for this higher affinity, it is possible that the sequence of these two proteins may also be of importance for determining the affinity. Based on comparison between the sequence of mouse ODC, which binds Az, and trypanosome ODC, which does not bind Az, the segment encompassing amino acids 117–140 was suggested as the putative Az-binding site of mouse ODC . However, based on comparison between the structures of human and trypanosome ODC, it was suggested that the Az-binding segment might actually be larger .
Since the active site of ODC is formed at the interface between the two monomers , the monomeric existence of AzI helps to explain why it lacks ornithine-decarboxylating activity. The observation that AzI is unable to bind PLP provides an additional and independent explanation for the lack of enzymatic activity .
AzI is degraded via the ubiquitin system
Like ODC, AzI is a rapidly degraded protein. However, in contrast with the ubiquitin-independent degradation of ODC, AzI is degraded in an Az-independent, ubiquitin-dependent manner . In contrast with the interaction of Az with ODC, which greatly stimulates ODC degradation, interaction with Az actually stabilizes AzI by interfering with its ubiquitination. Since both partners of the Az–AzI complex are stabilized, it is tempting to suggest that AzI buffers Az by maintaining it in a stable complex. It will be of interest to identify conditions that might cause disintegration of this complex.
AzI stimulates cellular proliferation
Since AzI negates the ability of Az to stimulate ODC degradation and inhibit polyamine uptake, it is expected to elicit a growth-stimulatory effect (Figure 3). Indeed, several lines of evidence provide support to this notion (for a review, see ). AzI mRNA is rapidly induced in quiescent cells following growth stimulation by serum-derived growth factors. The induction of AzI mRNA precedes that of ODC mRNA, suggesting that AzI synthesis might protect ODC from Az. DNA array analysis has demonstrated increased levels of AzI mRNA in gastric tumours compared with nearby healthy tissue .
The human AzI gene is located on chromosome 8q22.3, and amplification of this region is associated with several tumours. Ectopic expression of AzI leads to increased proliferation and to cellular transformation. Conversely, silencing of AzI using siRNA (small interfering RNA) is associated with inhibition of ODC activity and with reduced cell proliferation. However, it is unclear whether the growth-promoting role of AzI is executed only through manipulating polyamine metabolism, since AzI has been demonstrated to stabilize cyclin D1 even in the absence of its Az-binding segment . The most compelling evidence demonstrating that AzI has a true and meaningful physiological role was recently provided through the demonstration that mice lacking functional AzI die at birth, exhibiting abnormal liver morphology that is accompanied by increased ODC degradation and perturbed biosynthesis of putrescine and spermidine .
Accumulating evidence obtained mainly from databases suggests the existence of AzI isoforms that result from differences in coding and non-coding regions, resulting from alternative splicing and differential utilization of polyadenylation signals. Although in some of these isoforms the coding region remains unaffected, there are isoforms that exhibit differences in the coding region, as well. It is presently unknown whether the isoforms with alterations in the coding region are able to regulate polyamine metabolism, or whether they are involved in regulating other cellular processes.
Recent studies have demonstrated that mammalian cells contain another type of AzI, termed ODCp (ODC paralogue) or AzI-2 . AzI-2 is expressed only in brain and testis [35,52] and, like AzI-1, it lacks ODC activity. Like AzI-1, AzI-2 is also rapidly degraded in a ubiquitin-dependent manner and, when artificially overexpressed, it stimulates ODC activity, polyamine uptake and cellular proliferation, although less efficiently than AzI-1 [53,54]. The spatial expression pattern of AzI-2 is similar to that of Az3, both being expressed in the haploid germinal cells. In addition, AzI-2 and Az3 are expressed at minimal levels during the first 3 postnatal weeks, and are highly induced at the fourth week . These results suggest that AzI-2 and Az3 may have a role in spermiogenesis.
Although significant progress has been made in our understanding of the regulation of the cellular polyamine metabolism, there are still aspects that require additional clarification. These include (i) the characterization of the physical interactions of Az with ODC and AzI; (ii) the role of the N-terminal segment of Az, which enables its ability to degrade ODC; (iii) characterization of the specific components of the ubiquitin system that mediate the degradation of Az and AzI; and (iv) determination of their possible regulation by polyamines. A major task will be the detailed characterization of the process of polyamine transport across the plasma membrane and the role Az plays in regulating this process.
Because of the importance of polyamines for cellular growth, polyamine metabolism has become a target of therapeutic efforts in battling cancer and other hyperproliferative diseases. Despite initial enthusiasm, inhibitors of key enzymes in the polyamine biosynthesis pathway were found to be rather ineffective owing to the ability of cells to overcome their effect by accumulating polyamines from their environment. This dominant effect of the uptake activity suggested the therapeutic use of toxic structural analogues of polyamines that cross the plasma membrane through the polyamine uptake system. Since the effectiveness of such toxic polyamine analogues also depends on the level of AzI, which negates Az function in the treated cells, an additional challenge will be to match optimal treatment, namely the choice of synthesis inhibitors versus polyamine analogues or their combination, to the composition of the molecular players, especially ODC and AzI, in the treated cells.
• ODC, the first rate-limiting enzyme in the biosynthesis of polyamines, is characterized by an extremely rapid degradation rate.
• ODC is degraded in a ubiquitin-independent manner.
• The degradation of ODC is mediated by a polyamine-induced protein termed Az that traps transient ODC monomers preventing their reassociation to form active dimers, and targets them to ubiquitin-independent degradation by the 26S proteasome.
• In addition to stimulating ODC degradation, Az inhibits uptake and stimulates excretion of polyamines via an, as yet unresolved, mechanism.
• In addition to affecting ODC degradation and polyamine transport, Az stimulates the degradation of other growth-regulating proteins.
• Forced Az overexpression inhibits cellular proliferation, tumour development and, in some systems, leads to apoptotic death.
• Az is regulated by an inactive ODC-related protein, termed AzI, which binds Az with high affinity and keeps it in a stable complex.
• Like ODC, Az and AzI are rapidly degraded proteins but, unlike ODC, their degradation is ubiquitin-dependent.
• Az is not degraded together with ODC while presenting it to the 26S proteasome.
• In contrast with its ability to stimulate ODC degradation, interaction with Az stabilizes AzI by inhibiting its ubiquitination.
• Forced AzI overexpression negates Az functions, and therefore stimulates cellular proliferation and transformation.
Research in the Kahana laboratory on the regulation of polyamine metabolism is supported by grants from the Israel Academy of Science and Humanities, The Leo and Julia Forchheimer Center for Molecular Genetics, The M.D. Moross Institute for Cancer Research and The Y. Leon Benoziyo Institute for Molecular Medicine at the Weizmann Institute of Science. C.K is the incumbent of the Jules J. Mallon Professorial chair in Biochemistry.
- © The Authors Journal compilation © 2009 Biochemical Society