Manuela Cervelli

Polyamine oxidase, a hydrogen peroxide-producing enzyme, is up-regulated by light and down-regulated by auxin in the outer tissues of the maize mesocotyl.

Exogenously supplied auxin (1-naphthaleneacetic acid) inhibited light-induced activity increase of polyamine oxidase (PAO), a hydrogen peroxide-producing enzyme, in the outer tissues of maize (Zea mays) mesocotyl. The same phenomenon operates at PAO protein and mRNA accumulation levels. The wall-bound to extractable PAO activity ratio was unaffected by auxin treatment, either in the dark or after light exposure. Ethylene treatment did not affect PAO activity, thus excluding an effect of auxin via increased ethylene biosynthesis. The auxin polar transport inhibitorsN 1-naphthylphthalamic acid or 2,3,5-triiodobenzoic acid caused a further increase of PAO expression in outer tissues after light treatment. The small increase of PAO expression, normally occurring in the mesocotyl epidermis during plant development in the dark, was also inhibited by auxin, although to a lesser extent with respect to light-exposed tissue, and was stimulated by N 1-naphthylphthalamic acid or 2,3,5-triiodobenzoic acid, thus suggesting a complex regulation of PAO expression. Immunogold ultrastructural analysis in epidermal cells revealed the association of PAO with the secretory pathway and the cell walls. The presence of the enzyme in the cell walls of this tissue greatly increased in response to light treatment. Consistent with auxin effects on light-induced PAO expression, the hormone treatment inhibited the increase in immunogold staining both intraprotoplasmically and in the cell wall. These results suggest that both light and auxin finely tune PAO expression during the light-induced differentiation of the cell wall in the maize mesocotyl epidermal tissues.

Heterologous expression and characterization of mouse spermine oxidase

Polyamine oxidases are key enzymes responsible of the polyamine interconversion metabolism in animal cells. Recently, a novel enzyme belonging to this class of enzymes has been characterized for its capability to oxidize preferentially spermine and designated as spermine oxidase. This is a flavin adenine dinucleotide-containing enzyme, and it has been expressed both in vitro and in vivo systems. The primary structure of mouse spermine oxidase (mSMO) was deduced from a cDNA clone (Image Clone 264769) recovered by a data base search utilizing the human counterpart of polyamine oxidases, PAOh1. The open reading frame predicts a 555-amino acid protein with a calculatedM r of 61,852.30, which shows a 95.1% identity with PAOh1. To understand the biochemical properties of mSMO and its structure/function relationship, the mSMO cDNA has been subcloned and expressed in secreted and secreted-tagged forms intoEscherichia coli BL21 DE3 cells. The recombinant enzyme shows an optimal pH value of 8.0 and is able to oxidize rapidly spermine to spermidine and 3-aminopropanal and fails to act upon spermidine and N 1-acetylpolyamines. The purified recombinant-tagged form enzyme (M r∼68,000) has K m and k catvalues of 90 μm and 4.5 s−1, respectively, using spermine as substrate at pH 8.0. Molecular modeling of mSMO protein based on maize polyamine oxidase three-dimensional structure suggests that the general features of maize polyamine oxidase active site are conserved in mSMO.

Spermine Metabolism and Anticancer Therapy

The natural polyamines (PA), putrescine (PUT), spermidine (SPD) and spermine (SPM) are ubiquitous constituents of eukaryotic cells. The increase of PA in malignant and proliferating cells attracted the interest of scientists during last decades, addressing PA depletion as a new strategy to inhibit cell growth. Selective enzyme inhibitors were developed for decreasing PA metabolism and to act as chemotherapeutic anticancer agents. Indeed, the complexity of the PA homoeostasis overcomes the PA perturbation by a single enzyme to take effect therapeutically. Recently, an increasing interest has been posed on spermine-oxidase (SMO), the only catabolic enzyme able to specifically oxidise SPM. Interestingly, the absence of SPM is compatible with life, but its accumulation and degradation is lethal. Augmented SMO activity provokes an oxidative stress rendering cells prone to die, and appears to be important in the cell differentiation pathway. Extra-cellular SPM is cytotoxic, but its analogues are capable of inhibiting cell growth at low concentrations, most likely by intracellular SPM depletion. These pivotal roles seem to evoke the biological processes of stress response, wherein balance is mandatory to live or to die. Thus, altering SPM metabolism could allow a multi-tasking therapeutic strategy, addressed not only to inhibit PA metabolism. Several tetramines are presently in early phases (I and II) of clinical trials, and it will be a matter of a few more years to understand whether SPM-related therapeutic approaches would be of benefit for composite treatment protocols of cancer.

Isolation and characterization of three polyamine oxidase genes from Zea mays.

Isolation and sequencing of three genes, MPAO1, MPAO2 and MPAO3, coding for polyamine oxidase (PAO) from maize (Zea mays) are reported here. Gene organization is extremely conserved among these copies, being composed of eight exons and seven introns. Furthermore, these genes encode for a protein of an almost identical amino acid sequence. These data suggest that the three MPAO copies have been derived from gene duplication of a common ancestor gene. Long inverted repeat sequences, also present in other maize genes, have been found within the second intron. Promoter sequences of MPAO1 and MPAO2 genes have been analysed for putative cis-acting elements. According to genomic Southern blot analysis, the MPAO gene family in maize and other monocots is represented by a small number of copies. Northern and western blot analysis have revealed a tissue-specific accumulation of both MPAO mRNA and protein.

A role for spermine oxidase as a mediator of reactive oxygen species production in HIV-Tat-induced neuronal toxicity

Chronic oxidative stress, which occurs in brain tissues of HIV-infected patients, is involved in the pathogenesis of HIV-associated dementia. Oxidative stress can be induced by HIV-1-secreted proteins, either directly or indirectly through the release of cytotoxic factors. In particular, HIV-1 Tat is able to induce neuronal death by interacting with and activating the polyamine-sensitive subtype of the NMDA receptor (NMDAR). Here, we focused on the role of polyamine catabolism in Tat-induced oxidative stress in human neuroblastoma (SH-SY5Y) cells. First, Tat was found to induce reactive oxygen species production and to affect cell viability in SH-SY5Y cells, these effects being mediated by spermine oxidase (SMO). Second, Tat was observed to increase SMO activity as well as decreasing the intracellular spermine levels. Third, Tat-induced SMO activation was completely prevented by the NMDAR antagonist MK-801, clearly indicating an involvement of NMDAR stimulation. Finally, pretreatment of cells with N-acetylcysteine, a scavenger of H₂O₂, and with MK-801 was able to completely inhibit reactive oxygen species formation and to restore cell viability. Altogether, these data strongly suggest a role for polyamine catabolism-derived H₂O₂ in neurotoxicity as elicited by Tat-stimulated NMDAR.

Probing mammalian spermine oxidase enzyme-substrate complex through molecular modeling, site-directed mutagenesis and biochemical characterization

Spermine oxidase (SMO) and acetylpolyamine oxidase (APAO) are FAD-dependent enzymes that are involved in the highly regulated pathways of polyamine biosynthesis and degradation. Polyamine content is strictly related to cell growth, and dysfunctions in polyamine metabolism have been linked with cancer. Specific inhibitors of SMO and APAO would allow analyzing the precise role of these enzymes in polyamine metabolism and related pathologies. However, none of the available polyamine oxidase inhibitors displays the desired characteristics of selective affinity and specificity. In addition, repeated efforts to obtain structural details at the atomic level on these two enzymes have all failed. In the present study, in an effort to better understand structure–function relationships, SMO enzyme–substrate complex has been probed through a combination of molecular modeling, site-directed mutagenesis and biochemical studies. Results obtained indicate that SMO binds spermine in a similar conformation as that observed in the yeast polyamine oxidase FMS1-spermine complex and demonstrate a major role for residues His82 and Lys367 in substrate binding and catalysis. In addition, the SMO enzyme–substrate complex highlights the presence of an active site pocket with highly polar characteristics, which may explain the different substrate specificity of SMO with respect to APAO and provide the basis for the design of specific inhibitors for SMO and APAO.

Spermine oxidase (SMO) activity in breast tumor tissues and biochemical analysis of the anticancer spermine analogues BENSpm and CPENSpm

BackgroundPolyamine metabolism has a critical role in cell death and proliferation representing a potential target for intervention in breast cancer (BC). This study investigates the expression of spermine oxidase (SMO) and its prognostic significance in BC. Biochemical analysis of Spm analogues BENSpm and CPENSpm, utilized in anticancer therapy, was also carried out to test their property in silico and in vitro on the recombinant SMO enzyme.MethodsBC tissue samples were analyzed for SMO transcript level and SMO activity. Students t test was applied to evaluate the significance of the differences in value observed in T and NT samples. The structure modeling analysis of BENSpm and CPENSpm complexes formed with the SMO enzyme and their inhibitory activity, assayed by in vitro experiments, were examined.ResultsBoth the expression level of SMO mRNA and SMO enzyme activity were significantly lower in BC samples compared to NT samples. The modeling of BENSpm and CPENSpm complexes formed with SMO and their inhibition properties showed that both were good inhibitors.ConclusionsThis study shows that underexpression of SMO is a negative marker in BC. The SMO induction is a remarkable chemotherapeutical target. The BENSpm and CPENSpm are efficient SMO inhibitors. The inhibition properties shown by these analogues could explain their poor positive outcomes in Phases I and II of clinical trials.

Molecular evolution of the polyamine oxidase gene family in Metazoa

BackgroundPolyamine oxidase enzymes catalyze the oxidation of polyamines and acetylpolyamines. Since polyamines are basic regulators of cell growth and proliferation, their homeostasis is crucial for cell life. Members of the polyamine oxidase gene family have been identified in a wide variety of animals, including vertebrates, arthropodes, nematodes, placozoa, as well as in plants and fungi. Polyamine oxidases (PAOs) from yeast can oxidize spermine, N1-acetylspermine, and N1-acetylspermidine, however, in vertebrates two different enzymes, namely spermine oxidase (SMO) and acetylpolyamine oxidase (APAO), specifically catalyze the oxidation of spermine, and N1-acetylspermine/N1-acetylspermidine, respectively. Little is known about the molecular evolutionary history of these enzymes. However, since the yeast PAO is able to catalyze the oxidation of both acetylated and non acetylated polyamines, and in vertebrates these functions are addressed by two specialized polyamine oxidase subfamilies (APAO and SMO), it can be hypothesized an ancestral reference for the former enzyme from which the latter would have been derived.ResultsWe analysed 36 SMO, 26 APAO, and 14 PAO homologue protein sequences from 54 taxa including various vertebrates and invertebrates. The analysis of the full-length sequences and the principal domains of vertebrate and invertebrate PAOs yielded consensus primary protein sequences for vertebrate SMOs and APAOs, and invertebrate PAOs. This analysis, coupled to molecular modeling techniques, also unveiled sequence regions that confer specific structural and functional properties, including substrate specificity, by the different PAO subfamilies. Molecular phylogenetic trees revealed a basal position of all the invertebrates PAO enzymes relative to vertebrate SMOs and APAOs. PAOs from insects constitute a monophyletic clade. Two PAO variants sampled in the amphioxus are basal to the dichotomy between two well supported monophyletic clades including, respectively, all the SMOs and APAOs from vertebrates. The two vertebrate monophyletic clades clustered strictly mirroring the organismal phylogeny of fishes, amphibians, reptiles, birds, and mammals. Evidences from comparative genomic analysis, structural evolution and functional divergence in a phylogenetic framework across Metazoa suggested an evolutionary scenario where the ancestor PAO coding sequence, present in invertebrates as an orthologous gene, has been duplicated in the vertebrate branch to originate the paralogous SMO and APAO genes. A further genome evolution event concerns the SMO gene of placental, but not marsupial and monotremate, mammals which increased its functional variation following an alternative splicing (AS) mechanism.ConclusionsIn this study the explicit integration in a phylogenomic framework of phylogenetic tree construction, structure prediction, and biochemical function data/prediction, allowed inferring the molecular evolutionary history of the PAO gene family and to disambiguate paralogous genes related by duplication event (SMO and APAO) and orthologous genes related by speciation events (PAOs, SMOs/APAOs). Further, while in vertebrates experimental data corroborate SMO and APAO molecular function predictions, in invertebrates the finding of a supported phylogenetic clusters of insect PAOs and the co-occurrence of two PAO variants in the amphioxus urgently claim the need for future structure-function studies.

The analysis of rRNA sequence-structure in phylogenetics: An application to the family Pectinidae (Mollusca: Bivalvia)

Several studies pointed out the relevance of integrating secondary structure information in sequence analysis and phylogenetics, both in terms of phylogenetic resolution and of marker suitability for phylogenetic reconstruction at higher taxonomic-rank. In this study we explore in a phylogenetic framework the primary and secondary structure information from nuclear (ITS2) and mitochondrial (16S) ribosomal DNA sequences from the Pectinidae, commonly known as scallops. Primary sequences were analysed under neighbour-joining, maximum parsimony, maximum likelihood, and Bayesian approaches. The individual RNA secondary structures were analysed alone and with primary sequences employing a combined model of sequence-structure evolution. The information from primary sequences and secondary structure of the ITS2 are concordant and provide good phylogenetic resolution, while the mitochondrial marker 16S fails to resolve the relationships between the major clades and shows a lack of structural signals. Our phylogenetic reconstruction provided evidence for the monophyly of the subfamily Pectininae and the tribes Aequipectinini and Pectinini while the subfamily Chlamydinae, although recovered in some analyses, did not receive good support. The secondary structure analysis of the derived pectinid ITS2 rRNA sequence revealed three striking differences between Pectininae and Chlamydinae subfamilies: (a) Chlamydinae ITS2 rRNA folding shows the typical four domains architecture, while the one of Pectininae only three; (b) the Pectinidae basal DI pairing shows a different sequence-structure consensus between Pectininae and Chlamydinae; (c) the Pectininae DIII domain holds a specific short secondary stem (Pec STEM). Furthermore, the scallop ITS2 rRNA folding analysis has shown the presence of a conserved sequence motif (invariably located on apical portion of the DIII domain) which emerges as a common feature across Bivalvia. The combined sequence-structure approach employed in this study, corroborates the deep significance of including the secondary structure information in phylogenetic analysis both as combined sequence-structure alignment as well as pointing out conserved elements of the RNA folding.

HIV-Tat Induces the Nrf2/ARE Pathway through NMDA Receptor-Elicited Spermine Oxidase Activation in Human Neuroblastoma Cells

Previously, we reported that HIV-Tat elicits spermine oxidase (SMO) activity upregulation through NMDA receptor (NMDAR) stimulation in human SH-SY5Y neuroblastoma cells, thus increasing ROS generation, which in turn leads to GSH depletion, oxidative stress, and reduced cell viability. In several cell types, ROS can trigger an antioxidant cell response through the transcriptional induction of oxidative stress-responsive genes regulated by the nuclear factor erythroid 2-related factor 2 (Nrf2). Here, we demonstrate that Tat induces both antioxidant gene expression and Nrf2 activation in SH-SY5Y cells, mediated by SMO activity. Furthermore, NMDAR is involved in Tat-induced Nrf2 activation. These findings suggest that the NMDAR/SMO/Nrf2 pathway is an important target for protection against HIV-associated neurocognitive disorders.