Bruno Ramos-Molina

Antizyme Inhibitor 2 (AZIN2/ODCp) Stimulates Polyamine Uptake in Mammalian Cells

One of the processes that regulate intracellular levels of polyamines in mammalian cells is polyamine uptake. We have measured polyamine uptake in COS7 cells for putrescine, spermidine, and spermine, obtaining Km values of 4.5, 1.0, and 0.8 μm, respectively. Treatment of nonconfluent cells with cycloheximide stimulated polyamine uptake and prevented the inhibitory effect found in cells preloaded with polyamines, suggesting the existence of a feedback repression mechanism mediated by antizymes. Transient transfected cells with mutated antizyme forms of AZ1, AZ2, and AZ3, which do not require frameshifting, showed a total blockade of polyamine uptake. Transfection of COS7 cells with mouse or human AZIN2, a novel member of the antizyme inhibitor family, recently characterized by our group, markedly stimulated polyamine uptake and counteracted the action of any of the three antizymes in co-transfected cells. The stimulatory effect of AZIN2 on polyamine uptake was abrogated when the putative antizyme binding sequence, formed by residues 117–140 in AZIN2, was deleted. Real time reverse transcription-PCR analysis of antizyme inhibitor transcripts revealed that in brain and testes AZIN2 is more expressed than AZIN1, especially in the testes where the relative expression was about 25-fold higher. Collectively, our results clearly indicate that AZIN2 affects polyamine homeostasis not only by increasing ornithine decarboxylase activity but also by stimulating polyamine uptake, through negating the inhibitory effect of the antizymes. This finding may have physiological relevance, mostly in testes where AZ3 and AZIN2 are mainly expressed.

Antizyme inhibitor 2: molecular, cellular and physiological aspects

Polyamines are small organic polycations essential for cell proliferation and survival. Antizymes (AZs) are small proteins regulated by polyamines that inhibit polyamine biosynthesis and uptake in mammalian cells. In addition, antizyme functions are also regulated by antizyme inhibitors, homologue proteins of ornithine decarboxylase lacking enzymatic activity. There are two antizyme inhibitors (AZIN), known as AZIN1 and AZIN2, that bind to AZs and negate their effects on polyamine metabolism. Here, we review different molecular and cellular properties of the novel AZIN2 with particular emphasis on the role that this protein may have in brain and testis physiology. Whereas AZIN1 is ubiquitously found in mammalian tissues, AZIN2 expression appears to be restricted to brain and testis. In transfected cells, AZIN2 is mainly located in the endoplasmic reticulum–Golgi intermediate compartment and in the cis-Golgi network. AZIN2 is a labile protein that is degraded by the proteasome by a ubiquitin-dependent mechanism. Regarding its physiological role, spatial and temporal analyses of AZIN2 expression in the mouse testis suggest that this protein may have a role in spermiogenesis.

Expression of antizyme inhibitor 2 in male haploid germinal cells suggests a role in spermiogenesis

Recently, we have found that the antizyme inhibitor 2, a novel member of the antizyme binding proteins related to polyamine metabolism, was expressed mainly in the adult testes, although its function in testicular physiology is completely unknown. Therefore, in the present work, the spatial and temporal expression of antizyme inhibitor 2, and other genes related to polyamine metabolism were studied in the mouse testis, in an attempt to understand the role of antizyme inhibitor 2 in testicular functions. For that purpose, the temporal expression of different genes, during the first wave of spermatogenesis in postnatal mice, was studied by real-time RT-PCR, and the spatial distribution of transcripts and protein in the adult testis was examined by both RNA in situ hybridization and immunocytochemistry. The results indicated that antizyme inhibitor 2 was specifically expressed in the haploid germinal cells, similarly to antizyme 3, the testis specific antizyme. Conversely, ornithine decarboxylase mRNA was mainly found in the outer part of the seminiferous tubules where spermatogonia and spermatocytes are located. Functional transfection assays and co-immunoprecipitation experiments corroborated that antizyme inhibitor 2 counteracts the negative action of antizyme 3 on polyamine biosynthesis and uptake. All these results indicate that the expression of antizyme inhibitor 2 is postnatally regulated and strongly suggest that antizyme inhibitor 2 may have a role in spermiogenesis.

Subcellular localization of antizyme inhibitor 2 in mammalian cells: Influence of intrinsic sequences and interaction with antizymes

Ornithine decarboxylase (ODC) and the antizyme inhibitors (AZIN1 and AZIN2), regulatory proteins of polyamine levels, are antizyme‐binding proteins. Although it is widely recognized that ODC is mainly a cytosolic enzyme, less is known about the subcellular distribution of AZIN1 and AZIN2. We found that these proteins, which share a high degree of homology in their amino acid sequences, presented differences in their subcellular location in transfected mammalian cells. Whereas ODC was mainly present in the cytosol, and AZIN1 was found predominantly in the nucleus, interestingly, AZIN2 was located in the ER‐Golgi intermediate compartment (ERGIC) and in the cis‐Golgi network, apparently not related to any known cell‐sorting sequence. Our results rather suggest that the N‐terminal region may be responsible for this particular location, since its deletion abrogated the incorporation of the mutated AZIN2 to the ERGIC complex and, on the other hand, the substitution of this sequence for the corresponding sequence in ODC, translocated ODC from cytosol to the ERGIC compartment. Furthermore, the coexpression of AZIN2 with any members of the antizyme family induced a shift of AZIN2 from the ERGIC to the cytosol. These findings underline the complexity of the AZs/AZINs regulatory system, supporting early evidence that relates these proteins with additional functions other than regulating polyamine homeostasis. J. Cell. Biochem. 107: 732–740, 2009.

Differential expression of ornithine decarboxylase antizyme inhibitors and antizymes in rodent tissues and human cell lines

Ornithine decarboxylase antizyme inhibitors, AZIN1 and AZIN2, are regulators and homologous proteins of ornithine decarboxylase (ODC), the rate limiting enzyme in the biosynthesis of polyamines. In this study, we have examined by means of real-time RT-PCR the relative abundance of mRNA of the three ODC paralogs in different rodent tissues, as well as in several cell lines derived from human tumors. With the exception of mouse and rat testes, ODC mRNA was the most expressed gene in all tissues examined (values higher than 60%). AZIN2 was more expressed than AZIN1 in testis, epididymis, brain, adrenal gland and lung, whereas the opposite was found in liver, kidney, heart, intestine and pancreas, as well as in all the cell lines examined. mRNA abundance of the three antizymes (AZ1, AZ2 and AZ3) that interact with ODC and antizyme inhibitors was also analyzed. AZ1 and AZ2 mRNA were ubiquitously expressed, AZ1 mRNA being more abundant than that of AZ2, although the ratio was dependent on the mouse tissue. In carcinoma-derived cells AZ1 was more expressed than AZ2, whereas in neuroblastoma-derived cells AZ2 mRNA was much more abundant than that of AZ1. AZ3 was expressed exclusively in rodent testes, where it was the most abundant of the three antizymes (~80%). This study is the first comparative-quantitative analysis on the expression of antizymes and antizyme inhibitors in different types of mammalian cells.

Antizyme Inhibitor 2 Hypomorphic Mice. New Patterns of Expression in Pancreas and Adrenal Glands Suggest a Role in Secretory Processes

The intracellular levels of polyamines, polycations implicated in proliferation, differentiation and cell survival, are regulated by controlling their biosynthesis, catabolism and transport. Antizymes and antizyme inhibitors are key regulatory proteins of polyamine levels by affecting ornithine decarboxylase, the rate-limiting biosynthetic enzyme, and polyamine uptake. We recently described the molecular function of a novel antizyme inhibitor (AZIN2). However, the physiological function of AZIN2 in mammals is mostly unknown. To gain insight on the tissue expression profile of AZIN2 and to find its possible physiological role, we have generated, transgenic mice with severe Azin2 hypomorphism. This mouse model expresses transgenic bacterial β-D-galactosidase as a reporter gene, under the control of the Azin2 endogenous promoter, what allows a very sensitive and specific detection of the expression of the gene in the different tissues of transgenic mice. The biochemical and histochemical analyses of β-D-galactosidase together with the quantification of Azin2 mRNA levels, corroborated that AZIN2 is mainly expressed in testis and brain, and showed for the first time that AZIN2 is also expressed in the adrenal glands and pancreas. In these tissues, AZIN2 was not expressed in all type of cells, but rather in specific type of cells. Thus, AZIN2 was mainly found in the haploid germinal cells of the testis and in different brain regions such as hippocampus and cerebellum, particularly in specific type of neurons. In the adrenal glands and pancreas, the expression was restricted to the adrenal medulla and to the Langerhans islets, respectively. Interestingly, plasma insulin levels were significantly reduced in the transgenic mice. These results support the idea that AZIN2 may have a role in the modulation of reproductory and secretory functions and that this mouse model might be an interesting tool for the progress of our understanding on the role of AZIN2 and polyamines in specific mammalian cells.

The induction of cardiac ornithine decarboxylase by β2 -adrenergic agents is associated with calcium channels and phosphorylation of ERK1/2.

The role that the induction of cardiac ornithine decarboxylase (ODC), a key enzyme in polyamine biosynthesis, by beta‐adrenergic agents may have in heart hypertrophy is a controversial issue. Besides, the signaling pathways related to cardiac ODC regulation have not been fully elucidated. Here we show that in Balb C mice the stimulation of cardiac ODC activity by adrenergic agents was mainly mediated by β2‐adrenergic receptors, and that this induction was lower in the hypertrophic heart. Interestingly, this stimulation was abolished by the L‐calcium channel antagonists verapamil and nifedipine. In addition, whereas the treatment with β2‐adrenergic agents was associated to both the increases in ODC, ODC‐antizyme inhibitor 1 (AZIN1), c‐fos and c‐myc mRNA levels and the phosphorylation of CREB and MAP kinases ERK1 and ERK2 (ERK1/2), the co‐treatment with L‐calcium channel blockers differentially prevented most of these changes. These results suggest that the stimulation of cardiac ODC by β2‐adrenergic agents is associated with the activation of MAP kinases through the participation of L‐calcium channels, and that by itself p‐CREB does not appear to be sufficient for the transcriptional activation of ODC. In addition, post‐translational mechanisms related with the induction of AZIN1 appear to be related to the increase of cardiac ODC activity. J. Cell. Biochem. 114: 1978–1986, 2013.

Structural and degradative aspects of ornithine decarboxylase antizyme inhibitor 2

Ornithine decarboxylase (ODC) is the key enzyme in the polyamine biosynthetic pathway. ODC levels are controlled by polyamines through the induction of antizymes (AZs), small proteins that inhibit ODC and target it to proteasomal degradation without ubiquitination. Antizyme inhibitors (AZIN1 and AZIN2) are proteins homologous to ODC that bind to AZs and counteract their negative effect on ODC. Whereas ODC and AZIN1 are well‐characterized proteins, little is known on the structure and stability of AZIN2, the lastly discovered member of this regulatory circuit. In this work we first analyzed structural aspects of AZIN2 by combining biochemical and computational approaches. We demonstrated that AZIN2, in contrast to ODC, does not form homodimers, although the predicted tertiary structure of the AZIN2 monomer was similar to that of ODC. Furthermore, we identified conserved residues in the antizyme‐binding element, whose substitution drastically affected the capacity of AZIN2 to bind AZ1. On the other hand, we also found that AZIN2 is much more labile than ODC, but it is highly stabilized by its binding to AZs. Interestingly, the administration of the proteasome inhibitor MG132 caused differential effects on the three AZ‐binding proteins, having no effect on ODC, preventing the degradation of AZIN1, but unexpectedly increasing the degradation of AZIN2. Inhibitors of the lysosomal function partially prevented the effect of MG132 on AZIN2. These results suggest that the degradation of AZIN2 could be also mediated by an alternative route to that of proteasome. These findings provide new relevant information on this unique regulatory mechanism of polyamine metabolism.

Mutational analysis of the antizyme-binding element reveals critical residues for the function of ornithine decarboxylase.

BACKGROUND Ornithine decarboxylase (ODC), the key enzyme in the polyamine biosynthetic pathway, is highly regulated by antizymes (AZs), small proteins that bind and inhibit ODC and increase its proteasomal degradation. Early studies delimited the putative AZ-binding element (AZBE) to the region 117-140 of ODC. The aim of the present work was to study the importance of certain residues of the region 110-142 that includes the AZBE region for the interaction between ODC and AZ1 and the ODC functionality. METHODS Computational analysis of the protein sequences of the extended AZBE site of ODC and ODC paralogues from different eukaryotes was used to search for conserved residues. The influence of these residues on ODC functionality was studied by site directed mutagenesis, followed by different biochemical techniques. RESULTS The results revealed that: a) there are five conserved residues in ODC and its paralogues: K115, A123, E138, L139 and K141; b) among these, L139 is the most critical one for the interaction with AZs, since its substitution decreases the affinity of the mutant protein towards AZs; c) all these conserved residues, with the exception of A123, are critical for ODC activity; d) substitutions of K115, E138 or L139 diminish the formation of ODC homodimers. CONCLUSIONS These results reveal that four of the invariant residues of the AZBE region are strongly related to ODC functionality. GENERAL SIGNIFICANCE This work helps to understand the interaction between ODC and AZ1, and describes various new residues involved in ODC activity, a key enzyme for cell growth and proliferation.

Antizyme Inhibitors in Polyamine Metabolism and Beyond: Physiopathological Implications

The intracellular levels of polyamines, cationic molecules involved in a myriad of cellular functions ranging from cellular growth, differentiation and apoptosis, is precisely regulated by antizymes and antizyme inhibitors via the modulation of the polyamine biosynthetic and transport systems. Antizymes, which are mainly activated upon high polyamine levels, inhibit ornithine decarboxylase (ODC), the key enzyme of the polyamine biosynthetic route, and exert a negative control of polyamine intake. Antizyme inhibitors (AZINs), which are proteins highly homologous to ODC, selectively interact with antizymes, preventing their action on ODC and the polyamine transport system. In this review, we will update the recent advances on the structural, cellular and physiological functions of AZINs, with particular emphasis on the action of these proteins in the regulation of polyamine metabolism. In addition, we will describe emerging evidence that suggests that AZINs may also have polyamine-independent effects on cells. Finally, we will discuss how the dysregulation of AZIN activity has been implicated in certain human pathologies such as cancer, fibrosis or neurodegenerative diseases.