Ana Lambertos

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.

An exercise in brain genoarchitectonics: Analysis of AZIN2-Lacz expressing neuronal populations in the mouse hindbrain

We examined in detail the distribution of AZIN2 (antizyme inhibitor 2) expression in the adult mouse hindbrain and neighboring spinal cord. AZIN2, similar to previously known AZIN1, is a recently‐discovered, a functional paralog of ornithine decarboxylase (ODC). Due to their structural similarity to ODC, both AZIN1 and AZIN2 counteract the inhibitory action of 3 known antizymes (AZ1‐3) on the ODC synthesis of polyamines, thus increasing intracytoplasmic levels of polyamines. AZIN2 is strongly, but heterogeneously, expressed in the brain. Our study uses a mouse line carrying an AZIN2‐LacZ construct, and, in our topographic analysis of AZIN2‐positive structures, we intend to share new knowledge about the rhombomeric segmentation of the hindbrain (a function of Hox paralogs and other genes). The observed labeled cell populations predominantly coincide with known cholinergic and glutamatergic cells, but occasionally also correspond to GABAergic, and possibly glycinergic cells. Some imperfectly known hindbrain populations stood out in unprecedented detail, and some axonal tracts were also differentially stained.

A novel role for antizyme inhibitor 2 as a regulator of serotonin and histamine biosynthesis and content in mouse mast cells

Antizymes and antizyme inhibitors are key regulatory proteins of polyamine levels by affecting ornithine decarboxylase and polyamine uptake. Our previous studies indicated a metabolic interplay among polyamines, histamine and serotonin in mast cells, and demonstrated that polyamines are present in mast cell secretory granules, being important for histamine storage and serotonin levels. Recently, the novel antizyme inhibitor-2 (AZIN2) was proposed as a local regulator of polyamine biosynthesis in association with mast cell serotonin-containing granules. To gain insight into the role of AZIN2 in the biosynthesis and storage of serotonin and histamine, we have generated bone marrow derived mast cells (BMMCs) from both wild-type and transgenic Azin2 hypomorphic mice, and have analyzed polyamines, serotonin and histamine contents, and some elements of their metabolisms. Azin2 hypomorphic BMMCs did not show major mast cell phenotypic alterations as judged by morphology and specific mast cell proteases. However, compared to wild-type controls, these cells showed reduced spermidine and spermine levels, and diminished growth rate. Serotonin levels were also reduced, whereas histamine levels tended to increase. Accordingly, tryptophan hydroxylase-1 (TPH1; the key enzyme for serotonin biosynthesis) mRNA expression and protein levels were reduced, whereas histidine decarboxylase (the enzyme responsible for histamine biosynthesis) enzymatic activity was increased. Furthermore, microphtalmia-associated transcription factor, an element involved in the regulation of Tph1 expression, was reduced. Taken together, our results show, for the first time, an element of polyamine metabolism –AZIN2–, so far described as exclusively devoted to the control of polyamine concentrations, involved in regulating the biosynthesis and content of other amines like serotonin and histamine.

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.

The mouse Gm853 gene encodes a novel enzyme: Leucine decarboxylase

Ornithine decarboxylase (ODC) is a key enzyme in the biosynthesis of polyamines. ODC-antizyme inhibitors (AZINs) are homologous proteins of ODC, devoid of enzymatic activity but acting as regulators of polyamine levels. The last paralogue gene recently incorporated into the ODC/AZINs family is the murine Gm853, which is located in the same chromosome as AZIN2, and whose biochemical function is still unknown. By means of transfection assays of HEK293T cells with a plasmid containing the coding region of Gm853, we show here that unlike ODC, GM853 was a stable protein that was not able to decarboxylate l-ornithine or l-lysine and that did not act as an antizyme inhibitor. However, GM853 showed leucine decarboxylase activity, an enzymatic activity never described in animal cells, and by acting on l-leucine (Km=7.03×10-3M) it produced isopentylamine, an aliphatic monoamine with unknown function. The other physiological branched-chain amino acids, l-valine and l-isoleucine were poor substrates of the enzyme. Gm853 expression was mainly detected in the kidney, and as Odc, it was stimulated by testosterone. The conservation of Gm853 orthologues in different mammalian species, including primates, underlines the possible biological significance of this new enzyme. In this study, we describe for the first time a mammalian enzyme with leucine decarboxylase activity, therefore proposing that the gene Gm853 and its protein product should be named as leucine decarboxylase (Ldc, LDC).

Tissue-specific regulation of potassium homeostasis by high doses of cationic amino acids

The administration of l-arginine hydrochloride has been used for testing pituitary secretion in humans, and as an experimental model for induction of acute pancreatitis in rats and mice. Whereas in the first case, the administration of the amino acid is associated with hiperkalemia, in the model of acute pancreatitis no data are available on possible changes in potassium homeostasis. The present study shows that the acute administration to mice of l-arginine hydrochloride or other cationic amino acids almost duplicate plasma potassium levels. This effect was associated to a marked decrease of tissue potassium in both pancreas and liver. No changes were found in other tissues. These changes cannot be ascribed to the large load of chloride ions, since similar effects were produced when l-ornithine aspartate was administered. The changes in potassium levels were dependent on the dose. The displacement of intracellular potassium from the liver and pancreas to the extracellular compartment appears to be dependent on the entry of the cationic amino acid, since the administration of an equivalent dose of alfa-difluoromethyl ornithine HCl (DFMO), a non physiological analog of l-ornithine, which is poorly taken by the tissues in comparison with the physiological cationic amino acids, did not produce any change in potassium levels in pancreas and liver. The analyses of the expression of cationic amino acid transporters (CAT) suggest that the CAT-2 transporter may be implicated in the potassium/cationic amino acid interchange in liver and pancreas. The possible physiological or pathological relevance of these findings is discussed.