Increased hepatic putrescine levels as a new potential factor related to the progression of metabolic dysfunction-associated steatotic liver disease.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is a chronic liver condition that often progresses to more advanced stages, such as metabolic dysfunction-associated steatohepatitis (MASH). MASH is characterized by inflammation and hepatocellular ballooning, in addition to hepatic steatosis. Despite the relatively high incidence of MASH in the population and its potential detrimental effects on human health, this liver disease is still not fully understood from a pathophysiological perspective. Deregulation of polyamine levels has been detected in various pathological conditions, including neurodegenerative diseases, inflammation, and cancer. However, the role of the polyamine pathway in chronic liver disorders such as MASLD has not been explored. In this study, we measured the expression of liver ornithine decarboxylase (ODC1), the rate-limiting enzyme responsible for the production of putrescine, and the hepatic levels of putrescine, in a preclinical model of MASH as well as in liver biopsies of patients with obesity undergoing bariatric surgery. Our findings reveal that expression of ODC1 and the levels of putrescine, but not spermidine nor spermine, are elevated in hepatic tissue of both diet-induced MASH mice and patients with biopsy-proven MASH compared with control mice and patients without MASH, respectively. Furthermore, we found that the levels of putrescine were positively associated with higher aspartate aminotransferase concentrations in serum and an increased SAF score (steatosis, activity, fibrosis). Additionally, in in vitro assays using human HepG2 cells, we demonstrate that elevated levels of putrescine exacerbate the cellular response to palmitic acid, leading to decreased cell viability and increased release of CK-18. Our results support an association between the expression of ODC1 and the progression of MASLD, which could have translational relevance in understanding the onset of this disease. © 2024 The Pathological Society of Great Britain and Ireland.

Melanoma-associated melanocortin 1 receptor variants confer redox signaling-dependent protection against oxidative DNA damage.

Cutaneous melanoma, a lethal skin cancer, arises from malignant transformation of melanocytes. Solar ultraviolet radiation (UVR) is a major environmental risk factor for melanoma since its interaction with the skin generates DNA damage, either directly or indirectly via oxidative stress. Pheomelanin pigments exacerbate oxidative stress in melanocytes by UVR-dependent and independent mechanisms. Thus, oxidative stress is considered to contribute to melanomagenesis, particularly in people with pheomelanic pigmentation. The melanocortin 1 receptor gene (MC1R) is a major melanoma susceptibility gene. Frequent MC1R variants (varMC1R) associated with fair skin and red or yellow hair color display hypomorphic signaling to the cAMP pathway and are associated with higher melanoma risk. This association is thought to be due to production of photosensitizing pheomelanins as well as deficient induction of DNA damage repair downstream of varMC1R. However, the data on modulation of oxidative DNA damage repair by MC1R remain scarce. We recently demonstrated that varMC1R accelerates clearance of reactive oxygen species (ROS)-induced DNA strand breaks in an AKT-dependent manner. Here we show that varMC1R also protects against ROS-dependent formation of 8-oxodG, the most frequent oxidative DNA lesion. Since the base excision repair (BER) pathway mediates clearance of these DNA lesions, we analyzed induction of BER enzymes in human melanoma cells of varMC1R genotype. Agonist-mediated activation of both wildtype (wtMC1R) and varMC1R significantly induced OGG and APE-1/Ref1, the rate-limiting BER enzymes responsible for repair of 8-oxodG. Moreover, we found that NADPH oxidase (NOX)-dependent generation of ROS was responsible for AKT activation and oxidative DNA damage repair downstream of varMC1R. These observations provide a better understanding of the functional properties of melanoma-associated MC1R alleles and may be useful for the rational development of strategies to correct defective varMC1R responses for efficient photoprotection and melanoma prevention in fair-skinned individuals.

A Side-by-Side Comparison of Wildtype and Variant Melanocortin 1 Receptor Signaling with Emphasis on Protection against Oxidative Damage to DNA.

Common variants of the MC1R gene coding the α-melanocyte stimulating hormone receptor are associated with light skin, poor tanning, blond or red hair, and increased melanoma risk, due to pigment-dependent and -independent effects. This complex phenotype is usually attributed to impaired activation of cAMP signaling. However, several MC1R variants show significant residual coupling to cAMP and efficiently activate mitogenic extracellular signal-regulated kinase 1 and 2 (ERK1/2) signaling. Yet, residual signaling and the key actions of wildtype and variant MC1R have never been assessed under strictly comparable conditions in melanocytic cells of identical genetic background. We devised a strategy based on CRISPR-Cas9 knockout of endogenous MC1R in a human melanoma cell line wildtype for BRAF, NRAS and NF1, followed by reconstitution with epitope-labeled MC1R constructs, and functional analysis of clones expressing comparable levels of wildtype, R151C or D294H MC1R. The proliferation rate, shape, adhesion, motility and sensitivity to oxidative DNA damage were compared. The R151C and D294H RHC variants displayed impaired cAMP signaling, intracellular stability similar to the wildtype, triggered ERK1/2 activation as effectively as the wildtype, and afforded partial protection against oxidative DNA damage, although less efficiently than the wildtype. Therefore, common melanoma-associated MC1R variants display biased signaling and significant genoprotective activity.

MGRN1 as a Phenotypic Determinant of Human Melanoma Cells and a Potential Biomarker.

Mahogunin Ring Finger 1 (MGRN1), a ubiquitin ligase expressed in melanocytes, interacts with the α melanocyte-stimulating hormone receptor, a well-known melanoma susceptibility gene. Previous studies showed that MGRN1 modulates the phenotype of mouse melanocytes and melanoma cells, with effects on pigmentation, shape, and motility. Moreover, MGRN1 knockdown augmented the burden of DNA breaks in mouse cells, indicating that loss of MGRN1 promoted genomic instability. However, data concerning the roles of MGRN1 in human melanoma cells remain scarce. We analyzed MGRN1 knockdown in human melanoma cells. Transient MGRN1 depletion with siRNA or permanent knockdown in human melanoma cells by CRISPR/Cas9 caused an apparently MITF-independent switch to a more dendritic phenotype. Lack of MGRN1 also increased the fraction of human cells in the S phase of the cell cycle and the burden of DNA breaks but did not significantly impair proliferation. Moreover, in silico analysis of publicly available melanoma datasets and estimation of MGRN1 in a cohort of clinical specimens provided preliminary evidence that MGRN1 expression is higher in human melanomas than in normal skin or nevi and pointed to an inverse correlation of MGRN1 expression in human melanoma with patient survival, thus suggesting potential use of MGRN1 as a melanoma biomarker.

Antizyme Inhibitor 2-Deficient Mice Exhibit Altered Brain Polyamine Levels and Reduced Locomotor Activity.

BACKGROUND: Alterations in the neural polyamine system are known to be associated with different brain pathological conditions. In addition, the regulation of enzymes involved in polyamine metabolism such as ornithine decarboxylase (ODC), antizymes (AZs), and antizyme inhibitors (AZINs) is critical during brain development. However, while most studies focus on ODC and AZs, less is known about AZIN expression and function in the brain. Thus, our aim was to analyze the expression pattern of AZIN2 during postnatal development, its brain distribution, and its possible implication in phenotypical alterations. METHODS: The expression pattern of Azin2 and other genes related to polyamine metabolism was analyzed by RT-qPCR. ß-D-galactosidase staining was used to determine the anatomical distribution of AZIN2 in a Azin2 knockout model containing the ßGeo marker. Brain polyamine content was determined by HPLC. The Rota-Rod and Pole functional tests were used to evaluate motor skills in Azin2-lacking mice. RESULTS: Our results showed that expression of genes codifying for AZs and AZINs showed a similar increasing pattern over time that coincided with a decrease in ODC activity and putrescine levels. The analysis of AZIN2 distribution demonstrated that it is strongly expressed in the cerebellum and distributed along the neuron body and dendrites. The ablation of Azin2 showed a decrease in putrescine levels and is related to reduced motor skills. CONCLUSIONS: Our study revealed that AZIN2 expression in the brain is particularly limited to the cerebellum. In addition, the ablation of Azin2 leads to a reduction in putrescine that relates to alterations in motor function, suggesting the role of AZIN2 in the functioning of dopaminergic neurons.

Mahogunin Ring Finger 1 regulates pigmentation by controlling the pH of melanosomes in melanocytes and melanoma cells.

Mahogunin Ring Finger 1 (MGRN1) is an E3-ubiquitin ligase absent in dark-furred mahoganoid mice. We investigated the mechanisms of hyperpigmentation in Mgrn1-null melan-md1 melanocytes, Mgrn1-KO cells obtained by CRISPR-Cas9-mediated knockdown of Mgrn1 in melan-a6 melanocytes, and melan-a6 cells depleted of MGRN1 by siRNA treatment. Mgrn1-deficient melanocytes showed higher melanin content associated with increased melanosome abundance and higher fraction of melanosomes in highly melanized maturation stages III-IV. Expression, post-translational processing and enzymatic activity of the rate-limiting melanogenic enzyme tyrosinase measured in cell-free extracts were comparable in control and MGRN1-depleted cells. However, tyrosinase activity measured in situ in live cells and expression of genes associated with regulation of pH increased upon MGRN1 repression. Using pH-sensitive fluorescent probes, we found that downregulation of MGRN1 expression in melanocytes and melanoma cells increased the pH of acidic organelles, including melanosomes, strongly suggesting a previously unknown role of MGRN1 in the regulation of melanosomal pH. Among the pH regulatory genes upregulated by Mgrn1 knockdown, we identified those encoding several subunits of the vacuolar adenosine triphosphatase V-ATPase (mostly Atp6v0d2) and a calcium channel of the transient receptor potential channel family, Mucolipin 3 (Mcoln3). Manipulation of expression of the Mcoln3 gene showed that overexpression of Mcoln3 played a significant role in neutralization of the pH of acidic organelles and activation of tyrosinase in MGRN1-depleted cells. Therefore, lack of MGRN1 led to cell-autonomous stimulation of pigment production in melanocytes mostly by increasing tyrosinase specific activity through neutralization of the melanosomal pH in a MCOLN3-dependent manner.

Polyamine biosynthesis in Xenopus laevis: the xlAZIN2/xlODC2 gene encodes a lysine/ornithine decarboxylase.

Ornithine decarboxylase (ODC) is a key enzyme in the biosynthesis of polyamines, organic cations that are implicated in many cellular processes. The enzyme is regulated at the post-translational level by an unusual system that includes antizymes (AZs) and antizyme inhibitors (AZINs). Most studies on this complex regulatory mechanism have been focused on human and rodent cells, showing that AZINs (AZIN1 and AZIN2) are homologues of ODC but devoid of enzymatic activity. Little is known about Xenopus ODC and its paralogues, in spite of the relevance of Xenopus as a model organism for biomedical research. We have used the information existing in different genomic databases to compare the functional properties of the amphibian ODC1, AZIN1 and AZIN2/ODC2, by means of transient transfection experiments of HEK293T cells. Whereas the properties of xlODC1 and xlAZIN1 were similar to those reported for their mammalian orthologues, the former catalyzing the decarboxylation of L-ornithine preferentially to that of L-lysine, xlAZIN2/xlODC2 showed important differences with respect to human and mouse AZIN2. xlAZIN2 did not behave as an antizyme inhibitor, but it rather acts as an authentic decarboxylase forming cadaverine, due to its higher affinity to L-lysine than to L-ornithine as substrate; so, in accordance with this, it should be named as lysine decarboxylase (LDC) or lysine/ornithine decarboxylase (LODC). In addition, AZ1 stimulated the degradation of xlAZIN2 by the proteasome, but the removal of the 21 amino acid C-terminal tail, with a sequence quite different to that of mouse or human ODC, made the protein resistant to degradation. Collectively, our results indicate that in Xenopus there is only one antizyme inhibitor (xlAZIN1) and two decarboxylases, xlODC1 and xlLDC, with clear preferences for L-ornithine and L-lysine, respectively.

Dietary and Gut Microbiota Polyamines in Obesity- and Age-Related Diseases.

The polyamines putrescine, spermidine, and spermine are widely distributed polycationic compounds essential for cellular functions. Intracellular polyamine pools are tightly regulated by a complex regulatory mechanism involving de novo biosynthesis, catabolism, and transport across the plasma membrane. In mammals, both the production of polyamines and their uptake from the extracellular space are controlled by a set of proteins named antizymes and antizyme inhibitors. Dysregulation of polyamine levels has been implicated in a variety of human pathologies, especially cancer. Additionally, decreases in the intracellular and circulating polyamine levels during aging have been reported. The differences in the polyamine content existing among tissues are mainly due to the endogenous polyamine metabolism. In addition, a part of the tissue polyamines has its origin in the diet or their production by the intestinal microbiome. Emerging evidence has suggested that exogenous polyamines (either orally administrated or synthetized by the gut microbiota) are able to induce longevity in mice, and that spermidine supplementation exerts cardioprotective effects in animal models. Furthermore, the administration of either spermidine or spermine has been shown to be effective for improving glucose homeostasis and insulin sensitivity and reducing adiposity and hepatic fat accumulation in diet-induced obesity mouse models. The exogenous addition of agmatine, a cationic molecule produced through arginine decarboxylation by bacteria and plants, also exerts significant effects on glucose metabolism in obese models, as well as cardioprotective effects. In this review, we will discuss some aspects of polyamine metabolism and transport, how diet can affect circulating and local polyamine levels, and how the modulation of either polyamine intake or polyamine production by gut microbiota can be used for potential therapeutic purposes.

New insights of polyamine metabolism in testicular physiology: A role of ornithine decarboxylase antizyme inhibitor 2 (AZIN2) in the modulation of testosterone levels and sperm motility.

The specific role of polyamines in the testis physiology is not fully understood. Antizymes (OAZs) and antizyme inhibitors (AZINs) are modulators of ornithine decarboxylase (ODC), a key enzyme in polyamine biosynthesis and polyamine uptake. Although the three known OAZs are expressed in the testis, only OAZ3 is testis specific and has been proven to have an essential role in male fertility. Regarding the two existing AZINs, AZIN2 is the most abundantly expressed member in this gonad. Whereas previous studies suggested that AZIN2 might participate in mouse spermatogenesis, immunohistological analysis of human testicular sections revealed that AZIN2 is also detected in the steroidogenic Leydig cells but not in the germinal epithelium. In the present study, we found a close ontogenic similarity in the mRNA levels of OAZs and AZINs between mice and rats, but an opposite expression pattern of ODC activity. Further analysis of AZIN2 and OAZ3 in the testis of mice with different alterations in spermatogenesis and fertility, induced either genetically or pharmacologically, corroborated that both AZIN2 and OAZ3 are mainly expressed in the haploid germinal cells. Finally, by using transgenic mice with a truncated Azin2 gene fused to the bacterial lacZ gene, we studied the expression of Azin2 in testes, epididymides and spermatozoa. AZIN2 was detected in spermatids and spermatozoa, as well as in Leydig cells, and in epithelial epidydimal cells. Azin2 knock-out male mice were fertile; however, they showed marked decreases in testicular putrescine and plasma and testicular testosterone levels, and a dramatic reduction in the sperm motility. These results suggest an important role for AZIN2 in testicular cells by modulating polyamine concentrations, testosterone synthesis and sperm function. Overall, our data corroborate the relevance of polyamine regulation in testis functions, where both AZIN2 and OAZ3 play fundamental roles.

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).

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. © 2017 Wiley Periodicals, Inc.

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.

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.

Influence of ornithine decarboxylase antizymes and antizyme inhibitors on agmatine uptake by mammalian cells.

Agmatine (4-aminobutylguanidine), a dicationic molecule at physiological pH, exerts relevant modulatory actions at many different molecular target sites in mammalian cells, having been suggested that the administration of this compound may have therapeutic interest. Several plasma membrane transporters have been implicated in agmatine uptake by mammalian cells. Here we report that in kidney-derived COS-7 cell line, at physiological agmatine levels, the general polyamine transporter participates in the plasma membrane translocation of agmatine, with an apparent Km of 44 ± 7 µM and Vmax of 17.3 ± 3.3 nmol h(-1) mg(-1) protein, but that at elevated concentrations, agmatine can be also taken up by other transport systems. In the first case, the physiological polyamines (putrescine, spermidine and spermine), several diguanidines and bis(2-aminoimidazolines) and the polyamine transport inhibitor AMXT-1501 markedly decreased agmatine uptake. In cells transfected with any of the three ornithine decarboxylase antizymes (AZ1, AZ2 and AZ3), agmatine uptake was dramatically reduced. On the contrary, transfection with antizyme inhibitors (AZIN1 and AZIN2) markedly increased the transport of agmatine. Furthermore, whereas putrescine uptake was significantly decreased in cells transfected with ornithine decarboxylase (ODC), the accumulation of agmatine was stimulated, suggesting a trans-activating effect of intracellular putrescine on agmatine uptake. All these results indicate that ODC and its regulatory proteins (antizymes and antizyme inhibitors) may influence agmatine homeostasis in mammalian tissues.

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 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 ß-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 ß-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 ß2 -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 ß2 -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 ß2 -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 ß2 -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.