Lambertos A and Peñafiel R (2019), Polyamine biosynthesis in Xenopus laevis: the x...

XB-ART-56279

PLoS One 2019 Sep 03;149:e0218500. doi: 10.1371/journal.pone.0218500.

Show Gene links Show Anatomy links

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

Lambertos A , Peñafiel R .

???displayArticle.abstract???
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.

???displayArticle.pubmedLink??? 31509528
???displayArticle.pmcLink??? PMC6738921
???displayArticle.link??? PLoS One

Species referenced: Xenopus laevis
Genes referenced: azin1 azin2 cad mapk1 odc1
GO keywords: polyamine biosynthetic process

???attribute.lit??? ???displayArticles.show???

	Fig 1. Genetic structure of mouse and human ODC paralogues, and their comparison with their Xenopus orthologues. Note that exons 7 and 8 in ODC and AZIN1 are fused in only one exon in AZIN2 (blue boxes). Data obtained from Ensembl (www.ensembl.org).
	Fig 2. Comparison of the amino acid sequences of mouse ODC, xlODC1, xlAZIN1 and xlAZIN2 using ClustalW program for multiple sequence alignment. Asterisks represent amino acid identity; colon and dots represent amino acid similarity between the proteins. Grey background indicates amino acid residues associated with the catalytic activity of mODC that are conserved in the Xenopus laevis homologues. In red: substitutions in these critical residues in xlAZIN1.
	Fig 3. Expression of xlODC1, xlAZIN1 and xlAZIN2 in HEK293T transfected cells. HEK293T cells were transfected with the corresponding constructs of Flag-xlODC1, Flag-xlAZIN1, Flag-xlAZIN2 or empty vector, as indicated in the Experimental Procedures. (A) Top: ODC activity measured in the cell lysates. Bottom: Western blot analysis of the proteins detected using anti-Flag or anti-ERK2 antibodies. Results are expressed as meanÂ±SE, and are representative of three experiments. (*) P<0.001 vs pcDNA3.1 or F-xlODC1. (B) Influence of 1mM alfa-difluoromethylornithine (DFMO) on the ornithine decarboxylase activity of xlODC1 and xlAZIN2 cell lysates. DFMO was added 5h before collecting the cells. () P<0.01.
	Fig 4. Analysis of the products formed by HEK293T cells transfected with different constructs. After 16 h of transfection, the culture media was aspirated and the cells collected. An aliquot of the media was concentrated and resuspended in perchloric acid 0.4 M, whereas the cells were homogenized in the same acid (200 Î¼l per well). After centrifugation at 12,000 Ã—g for 15 min, the supernatants were dansylated and analyzed by HPLC as described in the Experimental section. (A) Overlapped HPLC chromatogram traces of the dansylated extracts from cells transfected with xlAZIN2 (red line) or with the empty vector pcDNA 3.1 (blue line). Hexanediamine (Hxd) and heptanediamine (Hpd) were used as internal standards. Put: putrescine; Cad: cadaverine; Spd: spermidine; Spm: spermine. (B) Overlapped HPLC chromatogram traces corresponding to the dansylated polyamines present in the culture media of cells transfected with xlAZIN2 (red line) or empty vector (blue line). (C) Comparison of the polyamines found in the culture media of cells transfected with xlAZIN2 (red line) with those of xlODC1, mODC and mAZIN2 (blue line).
	Fig 5. Cadaverine concentration and Cad/Put ratio in HEK 293T cells transfected with xlAZIN2 or other homologues. Polyamine levels were analyzed by HPLC in cell homogenates and culture media 16 h after transfection. Cad/Put ratio in cell homogenates (A) or in the culture media (B). Cadaverine concentration in cell homogenates (C) or in the culture media (D). (*) P<0.001 vs the other columns; () P<0.01 vs the other columns.
	Fig 6. Influence of AZ1 on protein levels of xlODC1 and xlAZIN2. (A) Western blot of lysates of HEK293T cells co-transfected with xlODC1 and different combinations of AZ1 and xlAZIN2. (B) Western blot and ODC activity of lysates of cells co-transfected with Flag-xlAZIN2 and pcDNA3.1 or AZ1. (***) P<0.001 vs pcDNA3.1 or F-xlAZIN2+AZ1. C) Western blot of lysates of HEK293T cells transfected with xlAZIN1-Flag alone or in combination with AZ1.
	Fig 7. Subcellular location of xlODC1 and xlAZIN2 in transfected cells. Laser scanning confocal micrographs of HEK293T cells transfected with xlODC1, xlAZIN2, mODC or mAZIN2 fused to the Flag epitope. After transfections, cells were fixed, permeabilized and stained with anti-Flag antibody and ALEXA anti-mouse and nuclear DAPI staining, and then examined in a confocal microscope. Flag-proteins are shown in green and nuclei in blue.
	Fig 8. Protein stability of xlAZIN2 and xlODC1 in transfected cells. After 16 h of transfection, either with xlAZIN2 or xlODC1, cells were incubated with 200 Î¼M cycloheximide (CHX), harvested at the indicated times, and lysed in buffer containing a protease inhibitor cocktail. (A) Left: Western blot analysis of xlAZIN2 protein using the anti-Flag antibody; right: decay of ODC activity. (B) Similar experiments with xlODC1. Half-lives of xlAZIN2 and xlODC1 in the transfected cells were calculated by linear regression analysis (GraphPad software). (C) HEK293T cells transfected with xlAZIN2 or xlAZIN2+AZ1were incubated for 5 h with or without the proteasomal inhibitor MG132 (50 Î¼M). xlAZIN2 protein was determined as in (A). ERK2 was used as a loading control.
	Fig 9. Influence of the C-terminal region of xlAZIN2 in the degradative process induced by AZs. HEK293T cells were transfected with: (A) xlAZIN2, (B) xlAZIN2 lacking the 21 C-terminal residues (xlAZIN2-Î”C) or (C) with a construct coding for a chimeric protein with the substitution of the 21 C-terminal residues of xlAZIN2 by the C-terminal segment of mouse AZIN2 (xlAZIN2-mAZIN2). In parallel, each one of the constructs was co-transfected with members of the AZ family (AZ1, AZ2, and AZ3). Western blots were probed with anti-Flag antibody. On the right side, schematic representations of xlAZIN2 and the two mutated proteins.
	Fig 10. Protein stability of the mutated forms of xlAZIN2. (A) After 16 h of transfection with xlAZIN2-Î”C, cells were incubated with 200 Î¼M cycloheximide (CHX), harvested at the indicated times, and lysed in buffer containing a protease inhibitor cocktail. Top: western blot analysis of xlAZIN2-Î”C at different times after CHX addition; bottom: changes in ODC activity after CHX treatment. (B) Influence of the proteasomal inhibitor MG132 (50 Î¼M) on the effect of AZ1 on xlAZIN2-Î”C protein in HEK293T transfected cells. (C) Influence of the proteasomal inhibitor MG132 (50 Î¼M) on the effect of AZ1 on xlAZIN2-mAZIN2 protein in HEK293T transfected cells.
	S1. Comparison of the amino acid sequences of Xenopus laevis AZIN2 (xlAZIN2) and Xenopus tropicalis AZIN2 (xtAZIN2) using ClustalW program for multiple sequence alignment. Asterisks represent amino acid identity; colon and dots represent amino acid similarity between the proteins. Grey background indicates amino acid residues associated with the catalytic activity of mODC that are conserved in the Xenopus homologues.
	S2. Sequences of the C-terminal region of mODC and its paralogues and Xenopus laevis orthologous proteins. (A) Scheme of the C-terminal region of mODC, where C represents the ~70 amino acid residues, and S1 and S2 the two subregions that may be important for proteasomal degradation of ODC induced by AZ1. (B) Detailed sequence of the C-terminal region of mODC and its different paralogues and orthologues. Sequences corresponding to S1 (residues 391â€“420) and S2 (residues 441â€“461) are underlined.
	S3. Spermidine and spermine levels in cells transfected with xlAZIN2, xlODC1, mODC or mAZIN2. Polyamines were analyzed by HPLC in cell homogenates 16 h after transfection. Neither spermidine (Spd) nor spermine (Spm) were detected in the culture media.
	S4. Calculation of Km and Vm by nonlinear regression analysis of the Michaelis-Menten curves of xlAZIN2 and xlODC1. The GraphPad Prism calculator was used and correlation coefficient (R2) for each curve are given. Values of Km and Vm are shown in Table 2.
	S5. Effect of the supplementation of the culture media with different basic amino acids on cadaverine levels in cells transfected with xlAZIN2. Transfected cells were supplemented with 1 mM L-lysine, L-ornithine, L-arginine or L-histidine. Amino acid concentration in DMEM non-supplemented media: 0.797 mM L-lysine, 0.389 mM L-arginine and 0.20 mM L-histidine. () P<0.05 vs control; (*) P<0.01 vs control. C: control (non-supplemented media).
	S6. Influence of xlAZIN1 on the inhibitory action of AZ1 on xlAZIN2 (A) or mODC (B). Subcellular location of xlAZIN1 in transfected cells (C). HEK293T cells were transfected with different constructs and ODC activity was measured in cell homogenates 16 h after transfection. The amount of AZ1 plasmid in the transfection assays was 1/10 of the other plasmids. xlAZIN1 showed a cytosolic location, similar to that of xlAZIN2 or mODC shown in Fig 7. (*) P<0.001 vs the other columns; () P<0.01 vs the other columns.

References [+] :

Baby, Presence of ornithine decarboxylase antizyme in primary cultured hepatocytes of the frog Xenopus laevis. 1991, Pubmed, Xenbase

Baby, Presence of ornithine decarboxylase antizyme in primary cultured hepatocytes of the frog Xenopus laevis. 1991, Pubmed , Xenbase
Bae, The old and new biochemistry of polyamines. 2018, Pubmed
Bassez, Post-transcriptional regulation of ornithine decarboxylase in Xenopus laevis oocytes. 1990, Pubmed , Xenbase
Bercovich, Degradation of antizyme inhibitor, an ornithine decarboxylase homologous protein, is ubiquitin-dependent and is inhibited by antizyme. 2004, Pubmed
Berko, The direction of protein entry into the proteasome determines the variety of products and depends on the force needed to unfold its two termini. 2012, Pubmed
Cao, Tissue-specific expression of an Ornithine decarboxylase paralogue, XODC2, in Xenopus laevis. 2001, Pubmed , Xenbase
Coffino, Regulation of cellular polyamines by antizyme. 2001, Pubmed
Coleman, Effect of mutations at active site residues on the activity of ornithine decarboxylase and its inhibition by active site-directed irreversible inhibitors. 1993, Pubmed
Erales, Ubiquitin-independent proteasomal degradation. 2014, Pubmed
Gerner, Polyamines and cancer: old molecules, new understanding. 2004, Pubmed
Ghoda, Prevention of rapid intracellular degradation of ODC by a carboxyl-terminal truncation. 1989, Pubmed
Ghoda, Structural elements of ornithine decarboxylase required for intracellular degradation and polyamine-dependent regulation. 1992, Pubmed
Ghoda, Trypanosome ornithine decarboxylase is stable because it lacks sequences found in the carboxyl terminus of the mouse enzyme which target the latter for intracellular degradation. 1990, Pubmed
Gödderz, The N-terminal unstructured domain of yeast ODC functions as a transplantable and replaceable ubiquitin-independent degron. 2011, Pubmed
Gupta, Effect of spermidine on the in vivo degradation of ornithine decarboxylase in Saccharomyces cerevisiae. 2001, Pubmed
Hamana, Occurrence of sym-homospermidine in the Japanese newt, Cynops pyrrhogaster pyrrhogaster. 1979, Pubmed
Igarashi, Modulation of cellular function by polyamines. 2010, Pubmed
Igarashi, Polyamines: mysterious modulators of cellular functions. 2000, Pubmed
Ivanov, Recurrent emergence of catalytically inactive ornithine decarboxylase homologous forms that likely have regulatory function. 2010, Pubmed
Kahana, Antizyme and antizyme inhibitor, a regulatory tango. 2009, Pubmed
Kahana, Protein degradation, the main hub in the regulation of cellular polyamines. 2016, Pubmed
Kahana, The antizyme family for regulating polyamines. 2018, Pubmed
Kanerva, Human ornithine decarboxylase paralogue (ODCp) is an antizyme inhibitor but not an arginine decarboxylase. 2008, Pubmed
Kidron, Functional classification of amino acid decarboxylases from the alanine racemase structural family by phylogenetic studies. 2007, Pubmed
Kurabuchi, Changes in polyamine content during limb regeneration in adult Xenopus laevis. 1983, Pubmed , Xenbase
Lambertos, 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. 2018, Pubmed
Li, Regulated degradation of ornithine decarboxylase requires interaction with the polyamine-inducible protein antizyme. 1992, Pubmed
Li, Degradation of ornithine decarboxylase: exposure of the C-terminal target by a polyamine-inducible inhibitory protein. 1993, Pubmed
López-Contreras, Antizyme inhibitor 2: molecular, cellular and physiological aspects. 2010, Pubmed
López-Contreras, Subcellular localization of antizyme inhibitor 2 in mammalian cells: Influence of intrinsic sequences and interaction with antizymes. 2009, Pubmed
López-Contreras, Expression of antizyme inhibitor 2 in male haploid germinal cells suggests a role in spermiogenesis. 2009, Pubmed
López-Contreras, Mouse ornithine decarboxylase-like gene encodes an antizyme inhibitor devoid of ornithine and arginine decarboxylating activity. 2006, Pubmed
López-Garcia, Antizyme inhibitor 2 hypomorphic mice. New patterns of expression in pancreas and adrenal glands suggest a role in secretory processes. 2013, Pubmed
Mangold, Antizyme inhibitor: mysterious modulator of cell proliferation. 2006, Pubmed
Mangold, The antizyme family: polyamines and beyond. 2005, Pubmed
Matsufuji, Autoregulatory frameshifting in decoding mammalian ornithine decarboxylase antizyme. 1995, Pubmed
Matsuzaki, A possible role of cadaverine in the biosynthesis of polyamines in the Japanese newt testis. 1981, Pubmed
Miller-Fleming, Remaining Mysteries of Molecular Biology: The Role of Polyamines in the Cell. 2015, Pubmed
Minois, Molecular basis of the 'anti-aging' effect of spermidine and other natural polyamines - a mini-review. 2014, Pubmed
Murakami, Degradation of ornithine decarboxylase by the 26S proteasome. 2000, Pubmed
Murakami, Ornithine decarboxylase is degraded by the 26S proteasome without ubiquitination. 1992, Pubmed
Osborne, An appraisal of the developmental importance of polyamine changes in early Xenopus embryos. 1993, Pubmed , Xenbase
Osborne, Polyamine levels during Xenopus laevis oogenesis: a role in oocyte competence to meiotic resumption. 1989, Pubmed , Xenbase
Osborne, Expression and post-transcriptional regulation of ornithine decarboxylase during early Xenopus development. 1991, Pubmed , Xenbase
Pegg, Functions of Polyamines in Mammals. 2016, Pubmed
Pegg, Regulation of ornithine decarboxylase. 2006, Pubmed
Pegg, Decarboxylation of ornithine and lysine in rat tissues. 1979, Pubmed
Porat, Yeast antizyme mediates degradation of yeast ornithine decarboxylase by yeast but not by mammalian proteasome: new insights on yeast antizyme. 2008, Pubmed
Prakash, An unstructured initiation site is required for efficient proteasome-mediated degradation. 2004, Pubmed
Ramos-Molina, Structural and degradative aspects of ornithine decarboxylase antizyme inhibitor 2. 2014, Pubmed
Ramos-Molina, Mutational analysis of the antizyme-binding element reveals critical residues for the function of ornithine decarboxylase. 2013, Pubmed
Ramos-Molina, Differential expression of ornithine decarboxylase antizyme inhibitors and antizymes in rodent tissues and human cell lines. 2012, Pubmed
Ramos-Molina, Antizyme Inhibitors in Polyamine Metabolism and Beyond: Physiopathological Implications. 2018, Pubmed
Rasila, Expression of ODC Antizyme Inhibitor 2 (AZIN2) in Human Secretory Cells and Tissues. 2016, Pubmed
Rom, Polyamines regulate the expression of ornithine decarboxylase antizyme in vitro by inducing ribosomal frame-shifting. 1994, Pubmed
Rosenberg-Hasson, Characterization of sequences involved in mediating degradation of ornithine decarboxylase in cells and in reticulocyte lysate. 1991, Pubmed
Seiler, Liquid chromatographic methods for assaying polyamines using prechromatographic derivatization. 1983, Pubmed
Snapir, ODCp, a brain- and testis-specific ornithine decarboxylase paralogue, functions as an antizyme inhibitor, although less efficiently than AzI1. 2008, Pubmed
Tang, Role of ornithine decarboxylase antizyme inhibitor in vivo. 2009, Pubmed
Tobias, Intersubunit location of the active site of mammalian ornithine decarboxylase as determined by hybridization of site-directed mutants. 1993, Pubmed
Tsirka, Dominant negative mutants of ornithine decarboxylase. 1992, Pubmed
Wu, Structural basis of antizyme-mediated regulation of polyamine homeostasis. 2015, Pubmed
Zhang, Determinants of proteasome recognition of ornithine decarboxylase, a ubiquitin-independent substrate. 2003, Pubmed