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Objectives. Biogenic polyamines are widely present in nature. They are characteristic of both protozoan cells and multicellular organisms. These compounds have a wide range of biological functions and are necessary for normal growth and development of cells. Violation of polyamine homeostasis can cause significant abnormalities in cell functioning, provoking various pathological processes, including oncological and neuropsychiatric diseases. The impact on the “polyamine pathway” is an attractive basis for the creation of many pharmacological agents with a diverse spectrum of action. The purpose of this review is to summarize the results of the studies devoted to understanding the biological activity of compounds of the polyamine series, comparing their biological action with action on certain molecular targets. Due to the structural diversity of this group of substances, it is impossible to fully reflect the currently available data in one review. Therefore, in this work, the main attention is paid to the derivatives, acyclic saturated polyamines.Results. The following aspects are considered: biological functionality, biosynthesis and catabolism, cell transport, and localization of biogenic polyamines in the living systems. Structural analogs and derivatives of biogenic polyamines with antitumor, neuroprotective, antiarrhythmic, antiparasitic, antibacterial, and other biological activities are represented; the relationship between biological activity and the target of exposure is reflected. It was found that the nature of the substituent, the number of cationic centers, and the length of the polyamine chain have a great influence on the nature of the effect.Conclusions. At present, the use of polyamine structures is restrained by cytotoxicity and nonspecific toxic effects on the central nervous system. Further research in the field of biochemistry, cell transport, and a deeper understanding of receptor interaction mechanisms will help making polyamines as the basis for potential drug formulation.

Polyamines are common intracellular metabolites of nearly all cells, and their conservation across a vast diversity of cells suggests critical roles for these compounds in cellular physiology. Most intracellular polyamines are associated with RNA and, subsequently, polyamines have significant effects on transcription and translation. Putrescine and spermidine are the most common polyamines in bacteria. Intracellular polyamine pools in bacteria are tightly controlled by both de novo synthesis and transport. Polyamine homeostasis is emerging as a critical parameter of multiple pathways and physiology with substantial impact on bacterial pathogenesis, including the important human pathogenStreptococcus pneumoniae. Modulation of polyamine metabolism in pneumococci is an important regulator of central metabolism. It has broad effects on virulence factors such as capsule as well as stress responses that ultimately impact the survival of pneumococcus in a host. Polyamine transport protein as a single antigen or in combination with other pneumococcal proteins is shown to be an efficacious immunogen that protects against nasopharyngeal colonization, and invasive disease. A comprehensive description of polyamine metabolic pathways and their intersection with pneumococcal pathogenesis will undoubtedly point to novel approaches for treatment and prevention of pneumococcal disease.

Spermidine is important for the hypothalamic control of pituitary secretion of hormones involved in neuroendocrine functions in mammals. In this study, the effect of exogenous spermidine on the expression of genes and proteins related to polyamine metabolism and polyamine levels was examined. The results indicated that treatment with spermidine at 0.05 mg/g (BW) significantly increased the levels of Oaz1 mRNA and protein expression and decreased putrescine content in mouse hypothalamus (p < 0.05). The administration with spermidine at 0.10 mg/g significantly increased the levels of Oaz1, Oaz2, and Odc expression in mouse hypothalamus (p < 0.05). Treatment with spermidine at 0.05 mg/g significantly increased the levels of Ssat mRNA expression and reduced the level of Smo mRNA expression in mouse hypothalamus (p < 0.05). Putrescine concentrations in the hypothalamus after the administration of spermidine at 0.10 and 0.15 mg/g were significantly higher than those in the control group (p < 0.05). The concentration of both spermidine and spermine in the hypothalamus after the administration of spermidine at 0.15 mg/g was decreased significantly (p < 0.05). In summary, our results indicate that exogenous spermidine affects polyamine homeostasis in the mouse hypothalamus by modulating the expression of genes and proteins related to polyamine metabolism.

Polyamines are actively involved in diverse processes, including fruit ripening and stress responses. The aim of this study was to investigate the role of storage temperature on polyamine metabolism of tomato fruit, which were either harvested at the turning stage or left to mature on-plant. The applied temperatures (5, 10 and 25 °C) and storage duration (7 d) are regularly employed in real-world scenarios. The metabolic profile of polyamines (putrescine, spermidine and spermine), gene transcription of the enzymes mediating polyamine biosynthesis and catabolism, protein accumulation of the putrescine synthesis enzyme and the putrescine-produced H2O2 were evaluated. Putrescine was the major polyamine in all cases, and its content generally increased during ripening, as well as in chilled fruit (stored at 5 °C). Increases in arginine decarboxylase protein content and in arginine decarboxylase transcription of both attached fruit and detached ones stored at either 10 or 25 °C showed that putrescine accumulation was mainly driven via the arginine decarboxylase biosynthetic pathway. Interestingly, putrescine catabolism by copper-containing amine oxidase was favored in parallel with increases in arginine decarboxylase and ornithine decarboxylase transcripts in fruit stored at 5 °C. However, the arginine decarboxylase protein accumulation suggests that ornithine decarboxylase is mainly responsible for putrescine accumulation at 5 °C. This study indicates that storage temperature modifies the homeostasis of polyamines in tomato fruit, which in turn orchestrates ripening-associated physiological processes.

Polyamines spermidine and spermine, and their diamine precursor putrescine, are essential for normal cellular functions in both pro- and eukaryotes. Cellular polyamine levels are regulated by biosynthesis, degradation and transport. Transport of dietary and luminal bacterial polyamines in gastrointestinal (GI) tissues plays a significant role in tissue polyamine homeostasis. We have reported that caveolin-1 play an inhibitory role in polyamine uptake in GI tissues. We investigated the mechanism of caveolin-1-regulated polyamine transport. We found that glutathione S-transferase Π(GSTΠ) was secreted from caveolin-1 knockdown cells and stimulated spermidine transport in human colon-derived HCT116 cells. GSTΠ secreted in the medium increased S-glutathionylated protein level in the plasma membrane fraction. Proteomic analysis revealed that actin was S-glutathionylated by GSTΠ. Immunofluorescence microscopy demonstrated that actin filaments around plasma membrane were S-glutathionylated in caveolin-1 knockdown cells. Inhibition of actin remodeling by jasplakinolide caused a decrease in polyamine uptake activity. These data support a model in which caveolin-1 negatively regulates polyamine uptake by inhibiting GSTΠ secretion, which stimulates actin remodeling and endocytosis.

Background Tau stabilizes microtubules; however, in Alzheimer’s disease (AD) and tauopathies, tau becomes hyperphosphorylated, aggregates, and results in neuronal death. Our group recently uncovered a unique interaction between polyamine metabolism and tau fate. Polyamines exert an array of physiological effects that support neuronal function and cognitive processing. Specific stimuli can elicit a polyamine stress response (PSR), resulting in altered central polyamine homeostasis. Evidence suggests that elevations in polyamines following a short-term stressor are beneficial; however, persistent stress and subsequent PSR activation may lead to maladaptive polyamine dysregulation, which is observed in AD, and may contribute to neuropathology and disease progression. Methods Male and female mice harboring tau P301L mutation (rTg4510) were examined for a tau-induced central polyamine stress response (tau-PSR). The direct effect of tau-PSR byproducts on tau fibrillization and oligomerization were measured using a thioflavin T assay and a N2a split superfolder GFP-Tau (N2a-ssGT) cell line, respectively. To therapeutically target the tau-PSR, we bilaterally injected caspase 3-cleaved tau truncated at aspartate 421 (AAV9 Tau ΔD421) into the hippocampus and cortex of spermidine/spermine-N 1-acetyltransferase (SSAT), a key regulator of the tau-PSR, knock out (SSAT-/-), and wild type littermates, and the effects on tau neuropathology, polyamine dysregulation, and behavior were measured. Lastly, cellular models were employed to further examine how SSAT repression impacted tau biology. Results Tau induced a unique tau-PSR signature in rTg4510 mice, notably in the accumulation of acetylated spermidine. In vitro, higher-order polyamines prevented tau fibrillization but acetylated spermidine failed to mimic this effect and even promoted fibrillization and oligomerization. AAV9 Tau ΔD421 also elicited a unique tau-PSR in vivo, and targeted disruption of SSAT prevented the accumulation of acetylated polyamines and impacted several tau phospho-epitopes. Interestingly, SSAT knockout mice presented with altered behavior in the rotarod task, the elevated plus maze, and marble burying task, thus highlighting the impact of polyamine homeostasis within the brain. Conclusion These data represent a novel paradigm linking tau pathology and polyamine dysfunction and that targeting specific arms within the polyamine pathway may serve as new targets to mitigate certain components of the tau phenotype.

The objective of this study was to investigate the effects of exogenous glutathione (GSH) on the ion balance and micro-distribution, polyamine metabolism (PA) levels, activity of key enzymes, expression of PA synthesis and metabolism genes in tomato (Solanum lycopersicum L. cv. Zhongshu No. 4) seedlings under salt (NaCl) stress (100 mM). we found that NaCl stress caused damage to tomato, including seedling growth inhibition, imbalanced intracellular ion homeostasis caused by the accumulation of Na+ and Cl− in roots and leaves, and reduced transport capacity of K+, Ca2+, and Mg2+ from roots to leaves. Salt stress also led to a significant increase in polyamine levels in leaves. BSO (L-Buthionine-sulfoximine, an inhibitor of key GSH synthesis enzyme gamma- glutamylcysteine synthetase) application under salt stress exacerbated growth inhibition and ion imbalance in tomato seedlings and induced further accumulation of polyamine. However, the application of exogenous GSH reduced the Na+ and Cl- content of roots and leaves of plants under salt stress and NB (NaCl + BSO) treatments, regulated ion homeostasis and microdomain distribution, inhibited the selective transport of Na+, and promoted the selective transport of K+ and Ca2+ from root to leaf, thus alleviating the ion imbalance and poisoning caused by salt stress. In addition, exogenous GSH also down-regulated the activities and transcriptional levels of the PA synthesis- and degradation-related key enzymes ADC (arginine decarboxylase), SAMDC (S-adenosylmethionine decarboxylase), and DAO (diamine oxidase) under salt stress and NB treatments, and up-regulated the activities and the transcriptional levels of PAO (PA oxidase) and ODC (ornithine decarboxylase), thereby reducing the level of PAs and promoting the transformation of polyamines between different morphologies. These findings suggest that exogenous GSH applied to salt-stressed tomato confers salinity tolerance in tomato seedlings by modulating ion absorption, microdomain distribution, and the ion selective transport ratio and by regulating polyamine synthesis and metabolism to maintain cellular ions and polyamine homeostasis. This is the first study to provide evidence of the interaction between GSH and PAs in plant adaptive responses to salinity.

Polyamines are essential metabolites found in all organisms. Intracellular polyamine levels are tightly maintained by biosynthesis, degradation, uptake and excretion processes that involve regulatory mechanisms – such as the antizyme inhibitory protein – that are conserved across the kingdoms of life, indicating that polyamine levels are critical to cell function. Nonetheless, the biochemical roles of polyamines and their involvement in numerous fundamental cellular processes including aging, cell cycle progression and growth only become apparent when polyamine homeostasis is perturbed. Thus, while polyamines are present in most cells and essential for cell growth, their biochemical functions are largely enigmatic. Studies in fungi have contributed to our basic understanding of polyamines, and might continue to bridge knowledge gaps regarding polyamine metabolism and cell function. Moreover, when considering the impact of fungi – directly or indirectly, for good or for ill – on human society, closing gaps in our understanding of polyamine functions in fungal physiology is an important goal in itself that might lead to the discovery of new targets for enhancing beneficial fungal interactions and diminishing those detrimental to crop and human health. To facilitate progress towards this prospect, here we appraise what is known about polyamine metabolism in fungi, how prevalent polyamines impact fungal physiology and metabolism, and how the levels of each polyamine are maintained in the fungal cell – thus pointing to how they might be perturbed.

Polyamines (PAs) are indispensable polycations ubiquitous to all living cells. Among their many critical functions, PAs contribute to the oxidative balance of the cell. Beginning with studies by the Tabor laboratory in bacteria and yeast, the requirement for PAs as protectors against oxygen radical–mediated damage has been well established in many organisms, including mammals. However, PAs also serve as substrates for oxidation reactions that produce hydrogen peroxide (H(2)O(2)) both intra- and extracellularly. As intracellular concentrations of PAs can reach millimolar concentrations, the H(2)O(2) amounts produced through their catabolism, coupled with a reduction in protective PAs, are sufficient to cause the oxidative damage associated with many pathologies, including cancer. Thus, the maintenance of intracellular polyamine homeostasis may ultimately contribute to the maintenance of oxidative homeostasis. Again, pioneering studies by Tabor and colleagues led the way in first identifying spermine oxidase in Saccharomyces cerevisiae. They also first purified the extracellular bovine serum amine oxidase and elucidated the products of its oxidation of primary amine groups of PAs when included in culture medium. These investigations formed the foundation for many polyamine-related studies and experimental procedures still performed today. This Minireview will summarize key innovative studies regarding PAs and oxidative damage, starting with those from the Tabor laboratory and including the most recent advances, with a focus on mammalian systems.

Polyamines regulate many important biological processes including gene expression, intracellular signaling, and biofilm formation. Their intracellular concentrations are tightly regulated by polyamine transport systems and biosynthetic and catabolic pathways. Spermidine/spermine N-acetyltransferases (SSATs) are catabolic enzymes that acetylate polyamines and are critical for maintaining intracellular polyamine homeostasis. These enzymes belong to the Gcn5-related N-acetyltransferase (GNAT) superfamily and adopt a highly conserved fold found across all kingdoms of life. SpeG is an SSAT protein found in a variety of bacteria, including the human pathogen Vibrio cholerae. This protein adopts a dodecameric structure and contains an allosteric site, making it unique compared to other SSATs. Currently, we have a limited understanding of the critical structural components of this protein that are required for its allosteric behavior. Therefore, we explored the importance of two key regions of the SpeG protein on its kinetic activity. To achieve this, we created various constructs of the V. cholerae SpeG protein, including point mutations, a deletion, and chimeras with residues from the structurally distinct and non-allosteric human SSAT protein. We measured enzyme kinetic activity toward spermine for ten constructs and crystallized six of them. Ultimately, we identified specific portions of the allosteric loop and the β6-β7 structural elements that were critical for enzyme kinetic activity. These results provide a framework for further study of the structure/function relationship of SpeG enzymes from other organisms and clues toward the structural evolution of members of the GNAT family across domains of life.

Polyamine uptake transporter (PUT) plays important roles in polyamine homeostasis, but knowledge regarding PUT family genes in sweet orange (Citrus sinensis) remains elusive. Herein, our study aimed to perform a genome-wide identification of the PUT gene family in C. sinensis. A total of eight putative PUT genes (CsPUT1-CsPUT8) were identified in the sweet orange genome and distributed on three chromosomes. The CsPUT genes were divided into two major groups according to the phylogenetic tree analysis, with high similarities in protein domains and gene structure organization. The CsPUT genes were differentially expressed in different tissues, with the highest transcript levels being in the flowers and roots. Interestingly, the CsPUT genes were significantly induced by polyamines, putrescine, spermidine and spermine, indicating that CsPUT were possibly associated with intracellular polyamine transport and uptake. In addition, CsPUT showed differential expression in callus treated with ABA, cold, salt or osmotic shock. CsPUT4 was selected as a candidate for functional analysis of PUT. Overexpression of CsPUT4 elevated endogenous polyamine content and led to enhanced cold tolerance in transgenic callus cultures. Overall, these data provide valuable information for better understanding the potential biological functions of PUT genes in future.

Soil arbuscular mycorrhizal fungi (AMF) enhance the tolerance of plants against soil moisture deficit stress (SMDS), but the underlying mechanisms are still not fully understood. Polyamines (PAs) as low-molecular-weight, aliphatic polycations have strong roles in abiotic stress tolerance of plants. We aimed to investigate the effect of AMF (Funneliformis mosseae) inoculation on PAs, PA precursors, activities of PA synthases and degrading enzymes, and concentration of reactive oxygen species in the roots of trifoliate orange (Poncirus trifoliata) subjected to 15 days of SMDS. Leaf water potential and total chlorophyll levels were comparatively higher in AMF-inoculated than in non-AMF-treated plants exposed to SMDS. Mycorrhizal plants recorded a significantly higher concentration of precursors of PA synthesis such as L-ornithine, agmatine, and S-adenosyl methionine, besides higher putrescine and cadaverine and lower spermidine during the 15 days of SMDS. AMF colonization raised the PA synthase (arginine decarboxylase, ornithine decarboxylase, spermidine synthase, and spermine synthase) activities and PA-degrading enzymes (copper-containing diamine oxidase and FAD-containing polyamine oxidase) in response to SMDS. However, mycorrhizal plants showed a relatively lower degree of membrane lipid peroxidation, superoxide anion free radical, and hydrogen peroxide than non-mycorrhizal plants, whereas the difference between them increased linearly up to 15 days of SMDS. Our study concluded that AMF regulated PA homeostasis in roots of trifoliate orange to tolerate SMDS.

Aspergillus fumigatusis a well-known opportunistic pathogen that causes invasive aspergillosis (IA) infections, which have high mortality rates in immunosuppressed individuals. Long-term antifungal drug azole use in clinical treatment and agriculture results in loss of efficacy or drug resistance. Drug resistance is related to cellular metabolites and the corresponding gene transcription. In this study, through untargeted metabolomics and transcriptomics under itraconazole (ITC) treatment, we identified two plasma membrane-localized polyamine regulatorstpo3anddur3, which were important for polyamine homeostasis and susceptibility to ITC inA. fumigatus. In the absence oftpo3and/ordur3, the levels of cytoplasmic polyamines had a moderate increase, which enhanced the tolerance ofA. fumigatusto ITC. In comparison, overexpression oftpo3ordur3induced a drastic increase in polyamines, which increased the sensitivity ofA. fumigatusto ITC. Further analysis revealed that polyamines concentration-dependently affected the susceptibility ofA. fumigatusto ITC by scavenging reactive oxygen species (ROS) at a moderate concentration and promoting the production of ROS at a high concentration rather than regulating drug transport. Moreover, inhibition of polyamine biosynthesis reduced the intracellular polyamine content, resulted in accumulation of ROS and enhanced the antifungal activity of ITC. Interestingly,A. fumigatusproduces much lower levels of ROS under voriconazole (VOC) treatment than under ITC-treatment. Accordingly, our study established the link among the polyamine regulatorstpo3anddur3, polyamine homeostasis, ROS content, and ITC susceptibility inA. fumigatus.

Robust and effective T cell-mediated immune responses require proper allocation of metabolic resources through metabolic pathways to sustain the energetically costly immune response. As an essential class of polycationic metabolites ubiquitously present in all living organisms, the polyamine pool is tightly regulated by biosynthesis and salvage pathway. We demonstrated that arginine is a major carbon donor and glutamine is a minor carbon donor for polyamine biosynthesis in T cells. Accordingly, the dependence of T cells can be partially relieved by replenishing the polyamine pool. In response to the blockage of de novo synthesis, T cells can rapidly restore the polyamine pool through a compensatory increase in polyamine uptake from the environment, indicating a layer of metabolic plasticity. Simultaneously blocking synthesis and uptake depletes the intracellular PA pool, inhibits T cell proliferation, suppresses T cell inflammation, indicating the potential therapeutic value of targeting the polyamine for managing inflammatory and autoimmune diseases.

Polyamine homeostasis determines T cell function., Robust and effective T cell–mediated immune responses require proper allocation of metabolic resources through metabolic pathways to sustain the energetically costly immune response. As an essential class of polycationic metabolites ubiquitously present in all living organisms, the polyamine pool is tightly regulated by biosynthesis and salvage pathway. We demonstrated that arginine is a major carbon donor and glutamine is a minor carbon donor for polyamine biosynthesis in T cells. Accordingly, the dependence of T cells can be partially relieved by replenishing the polyamine pool. In response to the blockage of biosynthesis, T cells can rapidly restore the polyamine pool through a compensatory increase in extracellular polyamine uptake, indicating a layer of metabolic plasticity. Simultaneously blocking synthesis and uptake depletes the intracellular polyamine pool, inhibits T cell proliferation, and suppresses T cell inflammation, indicating the potential therapeutic value of targeting the polyamine pool for managing inflammatory and autoimmune diseases.

Naphthalimides, such as amonafide and mitonafide in clinical trials, have been developed as antitumor agents for orthotopic tumor. However, the serious side effects in cancer patients limit their applications. Herein, a new class of polyamine-based naphthalimide conjugates 5a-5c, 7a-7b, and 11a-11b with and without the alkylation of the distant nitrogen in the polyamine chain were synthesized and the mechanism was determined. Compared with amonafide, dinitro-naphthalimide conjugate 5c with a 4,3-cyclopropyl motif preferentially accumulates in cancer cells and minimizes side effects in vitro and in vivo. More importantly, 5c at the dosage of as low as 3 mg/kg (57.97%) displays better antitumor effects than the positive control amonafide (53.27%) at 5 mg/kg in vivo. And a remarkably elevated antitumor activity and a reduced toxicity are also observed for 5c at 5 mg/kg (65.90%). The upregulated p53 and the apoptotic cells (73.50%) indicate that the mechanism of 5c to induce apoptosis may result from its enhanced DNA damage. Further investigation indicates that in addition to target DNA, 5c can modulate the polyamine homeostasis by upregulating polyamine oxidase (PAO) in a different way from that of amonafide. And also by targeting PTs overexpressed in most of cancer cells, 5c downregulates the contents of Put, Spd, and Spm, which are in favor of suppressing fast-growing tumor cells. Our study implies a promising strategy for naphthalimide conjugates to treat hepatic carcinoma with notable activities and reduced toxicities at a low dosage.

The biogenic polyamines, spermine (Spm) and spermidine, are organic polycations present in millimolar concentrations in all eukaryotic cells participating in the regulation of vital cellular functions including proliferation and differentiation. The design and biochemical evaluation of polyamine analogues are cornerstones of polyamine research. Here we synthesized and studied novel C-methylated Spm analogues: 2,11-dimethylspermine (2,11-Me2Spm), 3,10-dimethylspermine (3,10-Me2Spm), 2-methylspermine, and 2,2-dimethylspermine. The tested analogues overcame growth arrest induced by a 72 h treatment with α-difluoromethylornithine, an ornithine decarboxylase (ODC) inhibitor, and entered into DU145 cells via the polyamine transporter. 3,10-Me2Spm was a poor substrate of spermine oxidase and spermidine/spermine-N1-acetyltransferase (SSAT) when compared with 2,11-Me2Spm, thus resembling 1,12-dimethylspermine, which lacks the substrate properties required for the SSAT reaction. The antizyme (OAZ1)-mediated downregulation of ODC and inhibition of polyamine transport are crucial in the maintenance of polyamine homeostasis. Interestingly, 3,10-Me2Spm was found to be the first Spm analogue that did not induce OAZ1 and, consequently, was a weak downregulator of ODC activity in DU145 cells.

Spermine, spermidine and putrescine polyamines are naturally occurring ubiquitous positively charged amines and are essential metabolites for biological functions in our life. These compounds play a crucial role in many cell processes, including cellular proliferation, growth, and differentiation. Intracellular levels of polyamines depend on their biosynthesis, transport and degradation. Polyamine levels are high in cancer cells, which leads to the promotion of tumor growth, invasion and metastasis. Targeting polyamine metabolism as an anticancer strategy is considerably rational. Due to compensatory mechanisms, a single strategy does not achieve satisfactory clinical effects when using a single agent. Combination regimens are more clinically promising for cancer chemoprevention because they work synergistically with causing little or no adverse effects due to each individual agent being used at lower doses. Moreover, bioactive substances have advantages over single chemical agents because they can affect multiple targets. In this review, we discuss anticancer strategies targeting polyamine metabolism and describe how combination treatments and effective natural active ingredients are promising therapies. The existing research suggests that polyamine metabolic enzymes are important therapeutic targets and that combination therapies can be more effective than monotherapies based on polyamine depletion.

Polyamines are small, naturally occurring polycations that are essential for normal cell growth and development. Maintaining a homeostatic level of both the polyamine pathway enzymes and the levels of the polyamines putrescine, spermidine and spermine is important because the dysregulation of the polyamine pathway has been associated with physical anomalies including cancer, Parkinson's disease, Snyder-Robinson syndrome and aging in mammals. To date, little is understood regarding how the polyamine pathway maintains polyamine homeostasis in the fly, an emerging model for polyamine studies. The aim of these studies was to better characterize how the downregulation or ablation of the polyamine biosynthetic enzymes ornithine decarboxylase, spermidine synthase and spermine synthase influence the expression of the genes coding for other polyamine pathway enzymes and the levels of the polyamines themselves utilizing the model organism, Drosophila melanogaster. We demonstrate the complexities of the polyamine metabolic pathway and show that downregulation of any of these polyamine pathway enzymes can lead to an array of changes in other parts of the pathway as well as alter the cellular levels of polyamines themselves. These data highlight the complex nature of regulation of this pathway and underscore the need to better understand its self-regulation in this and other model systems.

Free polyamine (PA) titers in plants may be regulated through reversible conjugate formation and/or through modulation of their synthesis, transport and degradation. PA signaling involves the well-acknowledged signaling molecule, nitric oxide (NO), which functions in diverse biological processes. Present investigations demonstrate the influence of salt stress (120 mM NaCl) and exogenous NO donor (250 µM Diethylenetriamine, DETA) on PA homeostasis of 2 d old, etiolated sunflower (Helianthus annuus L.) seedling cotyledons as a long-distance signaling response. Significantly enhanced intracellular spermine (Spm) accumulation was observed in seedling cotyledons under salt stress and in response to NO donor, the increase being more pronounced in seedlings treated with NO, evidently as a result of upregulation of the PA biosynthetic enzymes – arginine decarboxylase (ADC) and S-adenosylmethionine decarboxylase (SAMDC) – as revealed by Western blot and confocal imaging (CLSM). Moreover, salt stress induced the activity of polyamine oxidase (PAO), a PA catabolic enzyme, while NO lowered its activity in salt-stressed seedling cotyledons. NO, thus, appears to assist the seedlings in adapting to salt stress by positively regulating PA homeostasis through regulation of PA distribution between free, conjugated and bound forms, increased accumulation of PA biosynthetic enzymes and lowering the rate of PA catabolism.

The polyamines putrescine, spermidine and spermine participate in a variety of cellular processes in all organisms. Many studies have shown that these polycations are important for plant immunity, as well as for the virulence of diverse fungal phytopathogens. However, the polyamines’ roles in the pathogenesis of phytopathogenic bacteria have not been thoroughly elucidated to date. To obtain more information on this topic, we assessed the changes in polyamine homeostasis during the infection of tomato plants by Pseudomonas syringae. Our results showed that polyamine biosynthesis and catabolism are activated in both tomato and bacteria during the pathogenic interaction. This activation results in the accumulation of putrescine in whole leaf tissues, as well as in the apoplastic fluids, which is explained by the induction of its synthesis in plant cells and also on the basis of its excretion by bacteria. We showed that the excretion of this polyamine by P. syringae is stimulated under virulence-inducing conditions, suggesting that it plays a role in plant colonization. However, no activation of bacterial virulence traits or induction of plant invasion was observed after the exogenous addition of putrescine. In addition, no connection was found between this polyamine and plant defence responses. Although further research is warranted to unravel the biological functions of these molecules during plant-bacterial interactions, this study contributes to a better understanding of the changes associated with the homeostasis of polyamines during plant pathogenesis. Fil: Vilas, Juan Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina Fil: Romero, Fernando Matias. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina Fil: Rossi, Franco Rubén. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina Fil: Marina, María. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina Fil: Maiale, Santiago Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina Fil: Calzadilla, Pablo Ignacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina Fil: Pieckenstain, Fernando Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina Fil: Ruiz, Oscar Adolfo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina Fil: Gárriz, Andrés. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina

Loss-of-function mutations of the spermine synthase gene (SMS) result in Snyder-Robinson Syndrome (SRS), a recessive X-linked syndrome characterized by intellectual disability, osteoporosis, hypotonia, speech abnormalities, kyphoscoliosis, and seizures. As SMS catalyzes the biosynthesis of the polyamine spermine from its precursor spermidine, SMS deficiency causes a lack of spermine with an accumulation of spermidine. As polyamines, spermine and spermidine play essential cellular roles that require tight homeostatic control to ensure normal cell growth, differentiation, and survival. Using patient-derived lymphoblast cell lines, we sought to comprehensively investigate the effects of SMS deficiency on polyamine homeostatic mechanisms including polyamine biosynthetic and catabolic enzymes, derivatives of the natural polyamines, and polyamine transport activity. In addition to decreased spermine and increased spermidine in SRS cells, ornithine decarboxylase activity and its product putrescine were significantly decreased. Treatment of SRS cells with exogenous spermine revealed that polyamine transport was active, as the cells accumulated spermine, decreased their spermidine level, and established a spermidine-to-spermine ratio within the range of wild type cells. SRS cells also demonstrated elevated levels of tissue transglutaminase, a change associated with certain neurodegenerative diseases. These studies form a basis for further investigations into the leading biochemical changes and properties of SMS-mutant cells that potentially represent therapeutic targets for the treatment of Snyder-Robinson Syndrome.

Polyamines are a family of low molecular weight organic cations produced in part by the coordinated actions of arginase II (Arg II) and ornithine decarboxylase (Odc). Although gill polyamine homeostasis is affected by acute transfer to fresh water, little is known of its function in fish osmoregulation. The current study investigated the role of polyamines in the compensatory response of hypoosmotic challenge in the euryhaline fish, Fundulus grandis. Adult F. grandis were acclimated to 5 ppt water, transferred abruptly to 5, 2, 1, 0.5 and 0.1 ppt water, and assessed for osmoregulatory function, gill morphology, and polyamine homeostasis. The plasma osmolality, Na(+) concentration, and Cl(-) concentration were only significantly reduced during exposure to salinities at or below 0.5 ppt, although these effects were transient except in the 0.1 ppt treatment. The phenotype of mitochondrion-rich cells (MRCs) shifted from a seawater-type to a freshwater-type only at salinities that also produced a plasma osmotic disturbance. Hypoosmotic exposure increased the concentrations of putrescine, spermidine, and spermine in the gill over the entire 7 day period. Exposure to 0.1 ppt water also transiently increased gill caspase-3 activity and gill mRNA levels of the immediate-early response genes, c-fos and c-myc, thus tightly associating polyamines with gill remodeling during freshwater acclimation. Furthermore, arginase II and ornithine decarboxylase mRNA levels were most highly expressed in MRCs, and these levels were further increased only in the 0.1 ppt treatment. Reduction of gill polyamine levels following administration of the Odc inhibitor, alpha-dl-difluoromethylornithine (DFMO), inhibited gill caspase-3 activity, but surprisingly reduced the magnitude of the plasma osmotic imbalance elicited by exposure to 0.1 ppt water. We used isolated opercular epithelia mounted on Ussing chambers to assess the influence of polyamines on the attenuating response of hypotonic shock on active Cl(-) secretion. Spermidine partially reduced the decrease of short-circuit current (Isc) and membrane conductance (Gt) produced by hypotonic exposure. These data suggest polyamines blunt the hypotonic inhibition of NaCl secretion and may lead to early apoptosis of seawater ionocytes and their replacement by FW-type ionocytes.

Polyamines are primordial, small organic polycations present in almost all cells, but their roles in bacteria are poorly understood. sym‐Homospermidine is the dominant polyamine in the filamentous, N2‐fixing, heterocyst‐forming cyanobacterium Anabaena sp. PCC 7120. Synthesis of homospermidine was dependent on speA (encoding arginine decarboxylase), speB (agmatinase) and speY (deoxyhypusine synthase homologue), which in bacteria is an unprecedented pathway. Inactivation of any of these genes impaired diazotrophic growth. Heterocyst differentiation in the speA mutant was blocked at an early step, after induction of the regulatory gene hetR but before production of heterocyst‐specific glycolipids (HGL). In contrast, the speY mutant produced HGL and showed slow diazotrophic growth. Analysis of fusions to green fluorescent protein revealed that SpeA (like SpeB previously described) accumulates at higher levels in vegetative cells than in heterocysts, and that SpeY accumulates in vegetative cells but also at significant levels in heterocysts. The homospermidine biosynthetic pathway is therefore active primarily in vegetative cells but the last step can be completed in heterocysts. Our findings indicate an important role for polyamines in the diazotrophic biology of Anabaena. Furthermore, inactivation of a gene cluster (potADB) encoding a polyamine ABC transporter disrupted diazotrophic growth, corroborating the importance of polyamine homeostasis in Anabaena., Work was supported by grants number BFU2014‐56757‐P and BFU2017‐88202‐P from Agencia Estatal de Investigación, Spain, co‐financed by the European Regional Development Grant (EF) and by a seed grant from UT Southwestern Medical Center (AJM).

Polyamines are organic polycations essential for cell growth and differentiation; their aberrant accumulation is often associated with diseases, including many types of cancer. To maintain polyamine homeostasis, the catalytic activity and protein abundance of ornithine decarboxylase (ODC), the committed enzyme for polyamine biosynthesis, are reciprocally controlled by the regulatory proteins antizyme isoform 1 (Az1) and antizyme inhibitor (AzIN). Az1 suppresses polyamine production by inhibiting the assembly of the functional ODC homodimer and, most uniquely, by targeting ODC for ubiquitin-independent proteolytic destruction by the 26S proteasome. In contrast, AzIN positively regulates polyamine levels by competing with ODC for Az1 binding. The structural basis of the Az1-mediated regulation of polyamine homeostasis has remained elusive. Here we report crystal structures of human Az1 complexed with either ODC or AzIN. Structural analysis revealed that Az1 sterically blocks ODC homodimerization. Moreover, Az1 binding triggers ODC degradation by inducing the exposure of a cryptic proteasome-interacting surface of ODC, which illustrates how a substrate protein may be primed upon association with Az1 for ubiquitin-independent proteasome recognition. Dynamic and functional analyses further indicated that the Az1-induced binding and degradation of ODC by proteasome can be decoupled, with the intrinsically disordered C-terminal tail fragment of ODC being required only for degradation but not binding. Finally, the AzIN-Az1 structure suggests how AzIN may effectively compete with ODC for Az1 to restore polyamine production. Taken together, our findings offer structural insights into the Az-mediated regulation of polyamine homeostasis and proteasomal degradation.

The polyamines are important for a variety of cellular functions, including cell growth. Their intracellular concentrations are controlled by a complex network of regulatory mechanisms, in which antizyme (Az) has a key role. Az reduces the cellular polyamine content by down-regulating both the enzyme catalysing polyamine biosynthesis, ornithine decarboxylase (ODC), and the uptake of polyamines. The activity of Az is repressed by the binding of a protein, named Az inhibitor (AzI), which is an enzymatically inactive homologue of ODC. Two forms of AzI have been described: AzI1, which is ubiquitous, and AzI2 which is expressed in brain and testis. In the present study, we have investigated the role of AzI1 in polyamine homeostasis and cell proliferation in breast cancer cells. The results obtained showed that the cellular content of AzI increased transiently after induction of cell proliferation by diluting cells in fresh medium. Inhibition of polyamine biosynthesis induced an even larger increase in the cellular AzI content, which remained significantly elevated during the 7-day experimental period. However, this increase was not a consequence of changes in cell cycle progression, as demonstrated by flow cytometry. Instead, the increase appeared to correlate with the cellular depletion of polyamines. Moreover, induced overexpression of AzI resulted in an increased cell proliferation with a concomitant increase in ODC activity and putrescine content. During mitosis, AzI1 was localised in a pattern that resembled that of the two centrosomes, confirming earlier observations. Taken together, the results indicate that AzI fulfils an essential regulatory function in polyamine homeostasis and cell proliferation.

The leucine responsive regulatory protein (Lrp) is a global transcription factor that regulates the expression of genes involved in amino acid metabolism. To identify metabolic pathways and related genes under the control of Lrp in the acetic acid bacterium Komagataeibacter europaeus , the Kelrp null mutant (KGMA7110), which requires supplementation of all 20 amino acids for normal growth, was cultivated in minimal media containing or lacking particular amino acids. The results confirmed that KGMA7110 was auxotrophic for methionine and its catabolites S -adenosylmethionine (SAM) and spermidine (SPD). Quantitative reverse-transcription PCR analysis revealed lower metK (SAM synthetase) and mdtI (SPD efflux pump) expression in KGMA7110 than in wild-type KGMA0119. By contrast, these genes were significantly up-regulated in the Kelrp mutant lacking the putative C-terminal ligand-sensing domain (KGMA7203), indicating abnormal regulation of target genes by the Ke Lrp variant in KGMA7203. KGMA7110 (0.69 ± 0.27 μM) and KGMA7203 (4.90 ± 0.61 μM) excreted lower and higher quantities of SPD, respectively, than KGMA0119 (2.28 ± 0.26 μM). This was attributed to imbalanced carbon flow caused by Kelrp disruption that respectively attenuated and stimulated metK and mdtI expression. These findings indicate that Ke Lrp plays a key role in SAM biosynthesis and intracellular polyamine homeostasis in K. europaeus .

Amine oxidase copper-containing 1 (AOC1; formerly known as amiloride-binding protein 1) is a secreted glycoprotein that catalyzes the degradation of putrescine and histamine. Polyamines and their diamine precursor putrescine are ubiquitous to all organisms and fulfill pivotal functions in cell growth and proliferation. Despite the importance of AOC1 in regulating polyamine breakdown, very little is known about the molecular mechanisms that control its expression. We report here that the Wilms tumor protein, WT1, which is necessary for normal kidney development, activates transcription of the AOC1 gene. Expression of a firefly luciferase reporter under control of the proximal AOC1 promoter was significantly enhanced by co-transfection of a WT1 expression construct. Binding of WT1 protein to a cis-regulatory element in the AOC1 promoter was confirmed by electrophoretic mobility shift assay and chromatin immunoprecipitation. Antisense inhibition of WT1 protein translation strongly reduced Aoc1 transcripts in cultured murine embryonic kidneys and gonads. Aoc1 mRNA levels correlated with WT1 protein in several cell lines. Double immunofluorescent staining revealed a co-expression of WT1 and AOC1 proteins in the developing genitourinary system of mice and rats. Strikingly, induced changes in polyamine homeostasis affected branching morphogenesis of cultured murine embryonic kidneys in a developmental stage-specific manner. These findings suggest that WT1-dependent control of polyamine breakdown, which is mediated by changes in AOC1 expression, has a role in kidney organogenesis.

Although intracellular polyamine levels are highly regulated, it is unclear whether intracellular putrescine (PUT), spermidine (SPD), or spermine (SPM) levels act as a sensor to regulate their synthesis or uptake. Polyamines have been shown to induce AZ1 expression through a unique +1 frameshifting mechanism. However, under physiological conditions which particular polyamine induces AZ1, and thereby ODC activity, is unknown due to their inter-conversion. In this study we demonstrate that SPD regulates AZ1 expression under physiological conditions in IEC-6 cells. PUT and SPD showed potent induction of AZ1 within 4 h in serum-starved confluent cells grown in DMEM (control) medium. Unlike control cells, PUT failed to induce AZ1 in cells grown in DFMO containing medium; however, SPD caused a robust AZ1 induction in these cells. SPM showed very little effect on AZ1 expression in both the control and polyamine-depleted cells. Only SPD induced AZ1 when S-adenosylmethionine decarboxylase (SAMDC) and/or ODC were inhibited. Surprisingly, addition of DENSpm along with DFMO restored AZ1 induction by putrescine in polyamine-depleted cells suggesting that the increased SSAT activity in response to DENSpm converted SPM to SPD, leading to the expression of AZ1. This study shows that intracellular SPD levels controls AZ1 synthesis.

Introduction Polyamines play a fundamental role during embryogenesis by regulating cell growth and proliferation and by interacting with RNA, DNA and protein. The polyamine pools are regulated by metabolism and uptake from exogenous sources. The use of certain inhibitors of polyamine synthesis causes similar defects to those seen in alcohol exposure e.g. retarded embryo growth and endothelial cell sprouting. Methods CD-1 mice received two intraperitoneal injections of 3 g/kg ethanol at 4 h intervals 8.75 days post coitum (dpc). The fetal head, trunk, yolk sac and placenta were collected at 9.5 and 12.5 dpc and polyamine concentrations were determined. Results No measurable quantity of polyamines could be detected in the embryo head at 9.5 dpc, 12 h after ethanol exposure. Putrescine was not detectable in the trunk of the embryo at that time, whereas polyamines in yolk sac and placenta were at control level. Polyamine deficiency was associated with slow cell growth, reduction in endothelial cell sprouting, an altered pattern of blood vessel network formation and consequently retarded migration of neural crest cells and growth restriction. Discussion Our results indicate that the polyamine pools in embryonic and extraembryonic tissues are developmentally regulated. Alcohol administration, at the critical stage, perturbs polyamine levels with various patterns, depending on the tissue and its developmental stage. The total absence of polyamines in the embryo head at 9.5 dpc may explain why this stage is so vulnerable to the development of neural tube defect, and growth restriction, the findings previously observed in fetal alcohol syndrome.

The translational efficiency of an mRNA can be modulated by elements located in the 5'-untranslated region. The flavin-containing polyamine oxidases catabolize oxidative deamination of spermidine and spermine, thus contributing to polyamine homeostasis as well as diverse biological processes through their reaction products. In this study, we characterized the uORF of AtPAO2 gene using the GUS reporter gene. Transgenic lines harboring the native AtPAO2 promoter or the constitutive CaMV 35S promoter show that the uORF negatively affects GUS expression. Exogenous applications of PAs positively modulate GUS expression, thus alleviating the negative effect of AtPAO2 uORF, while treatments with MGBG inhibitor show an opposite effect. Our data suggest that AtPAO2 uORF regulatory mechanism is modulated by polyamines. In addition, we present a comparative in silico study of the uORFs identified in several plant transcripts encoding polyamine oxidases in both mono- and dicotyledonous plants as well as in the Bryophyte Physcomitrella patens. The polyamine oxidase uORF-encoded peptides are conserved among families and share conserved features such as their position, length, and amino acid sequence. Our findings provide new insights into the regulatory mechanism of polyamine oxidase genes and encourage further exploration to assess the biological significance of uORFs in the polyamine catabolic pathway.

Ornithine decarboxylase antizyme 1 (OAZ1) regulates both polyamine biosynthesis and transport and acts as a tumor suppressor because of its functions in the regulation of cell proliferation, apoptosis and growth. However, the roles of OAZ1 in the regulation of polyamine metabolism, cell proliferation, apoptosis and hormone receptor expression in granulosa cells (GCs) from geese have not been determined. To define the roles of OAZ1 in primary GCs, we constructed and transfected an Oaz1 overexpression vector with a thymidine deletion into primary GCs. Oaz1 overexpression induced 65 and 30% decreases in the putrescine and spermidine contents, respectively, 225% increase in the spermine content and up-regulated the expression levels of genes associated with polyamine metabolism (but Oaz2). The Ccnd1, Pcna, Bax and Caspase 3 mRNA expression levels in GCs overexpressing Oaz1 were significantly higher than the expression levels in the vehicle group (p

Polyamine metabolism is intimately linked to the physiological state of the cell. Low polyamines levels promote growth cessation, while increased concentrations are often associated with rapid proliferation or cancer. Delicately balanced biosynthesis, catabolism, uptake and excretion are very important for maintaining the intracellular polyamine homeostasis, and deregulated polyamine metabolism is associated with imbalanced metabolic red/ox state. Although many cellular targets of polyamines have been described, the precise molecular mechanisms in these interactions are largely unknown. Polyamines are readily interconvertible which complicate studies on the functions of the individual polyamines. Thus, non-metabolizable polyamine analogues, like carbon-methylated analogues, are needed to circumvent that problem. This review focuses on methylated putrescine, spermidine and spermine analogues in which at least one hydrogen atom attached to polyamine carbon backbone has been replaced by a methyl group. These analogues allow the regulation of both metabolic and catabolic fates of the parent molecule. Substituting the natural polyamines with methylated analogue(s) offers means to study either the functions of an individual polyamine or the effects of altered polyamine metabolism on cell physiology. In general, gem-dimethylated analogues are considered to be non-metabolizable by polyamine catabolizing enzymes spermidine/spermine-N¹-acetyltransferase and acetylpolyamine oxidase and they support short-term cellular proliferation in many experimental models. Monomethylation renders the analogues chiral, offering some advantage over gem-dimethylated analogues in the specific regulation of polyamine metabolism. Thus, methylated polyamine analogues are practical tools to meet existing biological challenges in solving the physiological functions of polyamines.

Polyamines are ubiquitous organic cations implicated in many physiological processes. Because they are positively charged at physiological pH, carrier-mediated systems are necessary for effective membrane permeation, but the identity of specific polyamine transporter proteins in eukaryotic cells remains unclear. Polyspecific organic cation transporters (OCTs) interact with many natural and xenobiotic monovalent cations and have been reported to transport dicationic compounds, including the short polyamine putrescine. In this study, we used Xenopus oocytes expressing mammalian OCT1 (SLC22A1), OCT2 (SLC22A2), or OCT3 (SLC22A3) to assess binding and transport of longer-chain polyvalent polyamines. In OCT-expressing oocytes, [(3)H]MPP(+) uptake rates were 15- to 35-fold higher than in noninjected oocytes, whereas those for [(3)H]spermidine increased more modestly above the background, up to 3-fold. This reflected up to 20-fold lower affinity for spermidine than for MPP(+); thus, K(0.5) for MPP(+) was ~50 μM in OCT1, ~170 μM in OCT2, and ~60 μM in OCT3, whereas for spermidine, K(0.5) was ~1 mM in OCT1, OCT2, and OCT3. J(max) values for MPP(+) and spermidine were within the same range, suggesting that both compounds are transported at a similar turnover rate. To gain further insight into OCT substrate specificity, we screened a selection of structural polyamine analogues for effect on [(3)H]MPP(+) uptake. In general, blocking potency increased with overall hydrophobic character, which indicates that, as for monovalent cations, hydrophobicity is a major requirement for recognition in polyvalent OCT substrates and inhibitors. Our results demonstrate that the natural polyamines are low affinity, but relatively high turnover, substrates for OCTs. The identification of OCTs as polyamine transport systems may contribute to further understanding of the mechanisms involved in polyamine homeostasis and aid in the design of polyamine-like OCT-targeted drugs.

Changes in the levels of polyamines are correlated with the activation or repression of developmental response pathways, but the role of polyamine transporters in the regulation of polyamine homeostasis and thus indirectly gene expression, has not been previously addressed. Here we show that the A. thaliana and rice transporters AtPUT5 and OsPUT1 were localized to the ER, while the AtPUT2, AtPUT3, and OsPUT3 were localized to the chloroplast by transient expression in N. benthamiana. A. thaliana plants that were transformed with OsPUT1 under the control the PUT5 promoter were delayed in flowering by 16days. In contrast, put5 mutants flowered four days earlier than WT plants. The delay of flowering was associated with significantly higher levels of spermidine and spermidine conjugates in the leaves prior to flowering. A similar delay in flowering was also noted in transgenic lines with constitutive expression of either OsPUT1 or OsPUT3. All three transgenic lines had larger rosette leaves, thicker flowering stems, and produced more siliques than wild type plants. In contrast, put5 plants had smaller leaves, thinner flowering stems, and produced fewer siliques. Constitutive expression of PUTs was also associated with an extreme delay in both plant senescence and maturation rate of siliques. These experiments provide the first genetic evidence of polyamine transport in the timing of flowering, and indicate the importance of polyamine transporters in the regulation of flowering and senescence pathways.

Plant polyamines are catabolized by two classes of amine oxidases, the copper amine oxidases (CuAOs) and the flavin adenine dinucleotide (FAD)-dependent polyamine oxidases (PAOs). These enzymes differ to each other in substrate specificity, catalytic mechanism and subcellular localization. CuAOs and PAOs contribute to several physiological processes both through the control of polyamine homeostasis and as sources of biologically-active reaction products. CuAOs and PAOs have been found at high level in the cell wall of several species belonging to Fabaceae and Poaceae families, respectively, especially in tissues fated to undertake extensive wall loosening/stiffening events and/or in cells undergoing programmed cell death (PCD). Apoplastic CuAOs and PAOs have been shown to play a key role as a source of H2O2 in light- or developmentally-regulated differentiation events, thus influencing cell wall architecture and maturation as well as PCD. Moreover, growing evidence suggests a key role of intracellular CuAOs and PAOs in several facets of plant development. Here, we discuss recent advances in understanding the contribution of different CuAOs/PAOs, as well as their cross-talk with different intracellular and extracellular metabolic pathways, in tissue differentiation and organ development.

MicroRNAs (miRNAs) are non-coding regulatory molecules that play important roles in a variety of biological processes. Although a number of cold-responsive miRNAs have been computationally identified, functions and mechanisms of most miRNAs are not well understood. Herein, the function of trifoliate orange [Poncirus trifoliata (L.) Raf.] miRNA396b (ptr-miR396b) in cold tolerance and its potential regulatory module were investigated. Compared with the wild type (WT), transgenic lemon (Citrus limon) plants overexpressing ptr-MIR396b, the precursor of ptr-miR396b, displayed enhanced cold tolerance. Ptr-miR396b was experimentally confirmed to guide the cleavage of 1-aminocyclopropane-1-carboxylic acid oxidase (ACO). The overexpressing lines exhibited a reduction in ACO transcript levels and ethylene content compared with the WT, and the expression pattern of ACO was opposite to that of ptr-miR396b in response to cold stress. In addition, the transgenic lines exhibited higher levels of free polyamines and mRNA abundance of polyamine biosynthetic genes than WT plants under cold treatment, consistent with reduced reactive oxygen species (ROS) accumulation in the former. Taken together, this study demonstrates that ptr-miR396b positively regulates cold tolerance through reducing ACO transcript levels, thereby repressing ethylene synthesis and simultaneously promoting polyamine synthesis, leading to enhanced ROS scavenging. Identification of the ptr-miR396b-ACO regulatory module provides new insights into the molecular mechanism underlying the reduction of ethylene production under cold.

The ubiquitous polyamine spermidine is indispensable for eukaryotic growth and cell proliferation. A conserved vital function of spermidine across eukaryotes is conferred by its aminobutyl group that is transferred to a single lysine in translation factor eIF5A to form the essential hypusine post-translational modification required for cellular translation. In direct contrast, although spermidine is absolutely essential for growth of alpha-proteobacterial plant pathogen Agrobacterium tumefaciens, we have found, by employing a suite of natural polyamines and synthetic methylated spermidine analogues together with spermidine biosynthetic mutants, that it is solely the 1,3-diaminopropane moiety of spermidine that is required for growth. Indeed, any polyamine containing an intact terminal 1,3-diaminopropane moiety can replace spermidine for growth, including the simple diamine 1,3-diaminopropane itself, a paradigm shift in understanding polyamine function in bacteria. We have identified for the first time a spermidine retroconversion activity in bacteria, producing diamine putrescine from triamine spermidine, however, exogenously-supplied tetraamine spermine is resistant to retroconversion. When spermidine levels are pharmacologically decreased, synthesis of spermine from spermidine is induced via the same biosynthetic enzymes, carboxyspermidine dehydrogenase and carboxyspermidine decarboxylase that produce spermidine from putrescine, the first identification of a spermine biosynthetic pathway in bacteria. This also suggests that spermidine represses spermine biosynthesis, but when spermidine levels decrease, it is then converted by carboxyspermidine dehydrogenase and decarboxylase enzymes to spermine, which is resistant to retroconversion and constitutes a sequestered pool of protected 1,3-diaminopropane modules required for growth. We also identify an efficient N-acetylspermidine deacetylase activity, indicative of a sophisticated bacterial polyamine homeostasis system.

Experimental pancreatitis is associated with activation of polyamine catabolism. The polyamine analog bismethylspermine (Me 2 Spm) can ameliorate pancreatic injury. We investigated the roles of polyamine catabolism in remote organs during pancreatitis and explored the mechanism of polyamine catabolism by administering Me 2 Spm. Acute pancreatitis was induced by an infusion of 2 or 6% taurodeoxycholate before Me 2 Spm administration. Blood, urine and tissues were sampled at 24 and 72 h to assess multi-organ injury and polyamine catabolism. The effect of Me 2 Spm on mortality in experimental pancreatitis was tested separately. Liver putrescine levels were elevated following liver injury. Me 2 Spm increased the activity of spermidine/spermine N 1 -acetyltransferase (SSAT) and depleted the spermidine, spermine or putrescine levels. Lung putrescine levels increased, and SSAT and spermine decreased following lung injury. Me 2 Spm enhanced the activity of SSAT and decreased the spermidine and spermine levels. Renal injury was manifested as an increase in creatinine or a decrease in urine output. Decreases in kidney SSAT, spermidine or spermine and an increase in putrescine were found during pancreatitis. In the 2% taurodeoxycholate model, Me 2 Spm decreased urine output and raised plasma creatinine levels. Me 2 Spm increased SSAT and decreased polyamines. Excessive Me 2 Spm accumulated in the kidney, and greater amounts were found in the 6% taurodeoxycholate model in which this mortality was not reduced by Me 2 Spm. In the 2% taurodeoxycholate model, Me 2 Spm dose-dependently induced mortality at 72 h. Like pancreatic injury, remote organ injury in pancreatitis is associated with increased putrescine levels. However, Me 2 Spm could not ameliorate multi-organ injury. Me 2 Spm administration was associated with significant renal toxicity and induced mortality, suggesting that the current dose is too high and needs to be modified.

Pseudomonas putida S12 is a promising platform organism for the biological production of substituted aromatic compounds due to its extreme tolerance towards toxic chemicals. Solvent or aromatic stress tolerance may be due to membrane modifications and efflux pumps; however in general, polyamines have also been implicated in stressed cells. Previous transcriptomics results of P. putida strains producing an aromatic compound, or being exposed to the solvent toluene, indicated differentially expressed genes involved in polyamine transport and metabolism. Therefore, the metabolism of the polyamine, putrescine was investigated in P. putida S12, as no putrescine degradation pathways have been described for this strain. Via transcriptome analysis various, often redundant, putrescine-induced genes were identified as being potentially involved in putrescine catabolism via oxidative deamination and transamination. A series of knockout mutants were constructed in which up to six of these genes were sequentially deleted, and although putrescine degradation was affected in some of these mutants, complete elimination of putrescine degradation in P. putida S12 was not achieved. Evidence was found for the presence of an alternative pathway for putrescine degradation involving γ-glutamylation. The occurrence of multiple putrescine degradation routes in the solvent-tolerant P. putida S12 is indicative of the importance of controlling polyamine homeostasis, as well as of the high metabolic flexibility exhibited by this microorganism. © 2011 Elsevier B.V.

The yeast QDR3 gene encodes a plasma membrane drug : H+ antiporter of the DHA1 family that was described as conferring resistance against the drugs quinidine, cisplatin and bleomycin and the herbicide barban, similar to its close homologue QDR2. In this work, a new physiological role for Qdr3 in polyamine homeostasis is proposed. QDR3 is shown to confer resistance to the polyamines spermine and spermidine, but, unlike Qdr2, also a determinant of resistance to polyamines, Qdr3 has no apparent role in K+ homeostasis. QDR3 transcription is upregulated in yeast cells exposed to spermine or spermidine dependent on the transcription factors Gcn4, which controls amino acid homeostasis, and Yap1, the main regulator of oxidative stress response. Yap1 was found to be a major determinant of polyamine stress resistance in yeast and is accumulated in the nucleus of yeast cells exposed to spermidine-induced stress. QDR3 transcript levels were also found to increase under nitrogen or amino acid limitation; this regulation is also dependent on Gcn4. Consistent with the concept that Qdr3 plays a role in polyamine homeostasis, QDR3 expression was found to decrease the intracellular accumulation of [3H]spermidine, playing a role in the maintenance of the plasma membrane potential in spermidine-stressed cells.

Polyamines spermine and spermidine are highly regulated, ubiquitous aliphatic cations that maintain DNA structure and function as immunomodulators and as antioxidants. Polyamine homeostasis is disrupted after brain injuries, with concomitant generation of toxic metabolites that may contribute to secondary injuries. To test the hypothesis of increased brain polyamine catabolism after traumatic brain injury (TBI), we determined changes in catabolic enzymes and polyamine levels in the rat brain after lateral controlled cortical impact TBI. Spermine oxidase (SMO) catalyzes the degradation of spermine to spermidine, generating H2O2 and aminoaldehydes. Spermidine/spermine-N(1)-acetyltransferase (SSAT) catalyzes acetylation of these polyamines, and both are further oxidized in a reaction that generates putrescine, H2O2, and aminoaldehydes. In a rat cortical impact model of TBI, SSAT mRNA increased subacutely (6-24 h) after TBI in ipsilateral cortex and hippocampus. SMO mRNA levels were elevated late, from 3 to 7 days post-injury. Polyamine catabolism increased as well. Spermine levels were normal at 6 h and decreased slightly at 24 h, but were normal again by 72 h post-injury. Spermidine levels also decreased slightly (6-24 h), then increased by approximately 50% at 72 h post-injury. By contrast, normally low putrescine levels increased up to sixfold (6-72 h) after TBI. Moreover, N-acetylspermidine (but not N-acetylspermine) was detectable (24-72 h) near the site of injury, consistent with increased SSAT activity. None of these changes were seen in the contralateral hemisphere. Immunohistochemical confirmation indicated that SSAT and SMO were expressed throughout the brain. SSAT-immunoreactivity (SSAT-ir) increased in both neuronal and nonneuronal (likely glial) populations ipsilateral to injury. Interestingly, bilateral increases in cortical SSAT-ir neurons occurred at 72 h post-injury, whereas hippocampal changes occurred only ipsilaterally. Prolonged increases in brain polyamine catabolism are the likely cause of loss of homeostasis in this pathway. The potential for simple therapeutic interventions (e.g., polyamine supplementation or inhibition of polyamine oxidation) is an exciting implication of these studies.

MYC genes are deregulated in a plurality of human cancers. Through direct and indirect mechanisms, the MYC network regulates the expression of > 15% of the human genome, including both protein-coding and noncoding RNAs. This complexity has complicated efforts to define the principal pathways mediating MYC's oncogenic activity. MYC plays a central role in providing for the bioenergetic and biomass needs of proliferating cells, and polyamines are essential cell constituents supporting many of these functions. The rate-limiting enzyme in polyamine biosynthesis, ODC, is a bona fide MYC target, as are other regulatory enzymes in this pathway. A wealth of data link enhanced polyamine biosynthesis to cancer progression, and polyamine depletion may limit the malignant transformation of preneoplastic lesions. Studies with transgenic cancer models also support the finding that the effect of MYC on tumor initiation and progression can be attenuated through the repression of polyamine production. High-risk neuroblastomas (an often lethal embryonal tumor in which MYC activation is paramount) deregulate numerous polyamine enzymes to promote the expansion of intracellular polyamine pools. Selective inhibition of key enzymes in this pathway, e.g., using DFMO and/or SAM486, reduces tumorigenesis and synergizes with chemotherapy to regress tumors in preclinical models. Here, we review the potential clinical application of these and additional polyamine depletion agents to neuroblastoma and other advanced cancers in which MYC is operative. (Clin Cancer Res 2009;15(19):5956–61)

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.

The polyamines spermidine and spermine and their diamine precursor putrescine are naturally occurring, polycationic alkylamines that are essential for eukaryotic cell growth. The requirement for and the metabolism of polyamines are frequently dysregulated in cancer and other hyperproliferative diseases, thus making polyamine function and metabolism attractive targets for therapeutic intervention. Recent advances in our understanding of polyamine function, metabolic regulation, and differences between normal cells and tumour cells with respect to polyamine biology, have reinforced the interest in this target-rich pathway for drug development.

Polyamines are small aliphatic amines found in all living organisms except some Archaea. In plants, putrescine, spermidine, and spermine are major components which are not only involved in fundamental cellular processes, for example cell proliferation, differentiation, and programmed cell death, but also in adaptive responses to environmental stress. In this article we review plant polyamine research focusing on recent studies.

Purpose: Deregulated MYC drives oncogenesis in many tissues yet direct pharmacologic inhibition has proven difficult. MYC coordinately regulates polyamine homeostasis as these essential cations support MYC functions, and drugs that antagonize polyamine sufficiency have synthetic-lethal interactions with MYC. Neuroblastoma is a lethal tumor in which the MYC homologue MYCN, and ODC1, the rate-limiting enzyme in polyamine synthesis, are frequently deregulated so we tested optimized polyamine depletion regimens for activity against neuroblastoma. Experimental Design: We used complementary transgenic and xenograft-bearing neuroblastoma models to assess polyamine antagonists. We investigated difluoromethylornithine (DFMO; an inhibitor of Odc, the rate-limiting enzyme in polyamine synthesis), SAM486 (an inhibitor of Amd1, the second rate-limiting enzyme), and celecoxib (an inducer of Sat1 and polyamine catabolism) in both the preemptive setting and in the treatment of established tumors. In vitro assays were performed to identify mechanisms of activity. Results: An optimized polyamine antagonist regimen using DFMO and SAM486 to inhibit both rate-limiting enzymes in polyamine synthesis potently blocked neuroblastoma initiation in transgenic mice, underscoring the requirement for polyamines in MYC-driven oncogenesis. Furthermore, the combination of DFMO with celecoxib was found to be highly active, alone, and combined with numerous chemotherapy regimens, in regressing established tumors in both models, including tumors harboring highest risk genetic lesions such as MYCN amplification, ALK mutation, and TP53 mutation with multidrug resistance. Conclusions: Given the broad preclinical activity demonstrated by polyamine antagonist regimens across diverse in vivo models, clinical investigation of such approaches in neuroblastoma and potentially other MYC-driven tumors is warranted. Clin Cancer Res; 22(17); 4391–404. ©2016 AACR.