Effects of Spermine Synthase Deficiency in Mesenchymal Stromal Cells Are Rescued by Upstream Inhibition of Ornithine Decarboxylase.

Despite the well-known relevance of polyamines to many forms of life, little is known about how polyamines regulate osteogenesis and skeletal homeostasis. Here, we report a series of in vitro studies conducted with human-bone-marrow-derived pluripotent stromal cells (MSCs). First, we show that during osteogenic differentiation, mRNA levels of most polyamine-associated enzymes are relatively constant, except for the catabolic enzyme spermidine/spermine N1-acetyltransferase 1 (SAT1), which is strongly increased at both mRNA and protein levels. As a result, the intracellular spermidine to spermine ratio is significantly reduced during the early stages of osteoblastogenesis. Supplementation of cells with exogenous spermidine or spermine decreases matrix mineralization in a dose-dependent manner. Employing N-cyclohexyl-1,3-propanediamine (CDAP) to chemically inhibit spermine synthase (SMS), the enzyme catalyzing conversion of spermidine into spermine, also suppresses mineralization. Intriguingly, this reduced mineralization is rescued with DFMO, an inhibitor of the upstream polyamine enzyme ornithine decarboxylase (ODC1). Similarly, high concentrations of CDAP cause cytoplasmic vacuolization and alter mitochondrial function, which are also reversible with the addition of DFMO. Altogether, these studies suggest that excess polyamines, especially spermidine, negatively affect hydroxyapatite synthesis of primary MSCs, whereas inhibition of polyamine synthesis with DFMO rescues most, but not all of these defects. These findings are relevant for patients with Snyder-Robinson syndrome (SRS), as the presenting skeletal defects-associated with SMS deficiency-could potentially be ameliorated by treatment with DFMO.

Potentilla sericea stress-responsive spermine synthase PsSPMS enhances cadmium tolerance in Arabidopsis thaliana.

Potentilla sericea is resistant and tolerates rough management. It is an excellent garden groundcover for ecological restoration and soil consolidation for slope protection. Polyamines have functions such as promoting tissue growth and physiological resistance, while spermine synthase catalyzes the production of spermine. The PsSPMS gene from Potentilla sericea was cloned and transformed into Arabidopsis thaliana to study the response of transgenic Arabidopsis thaliana to cadmium stress. The results showed that the contents of spermidine, spermine as well as glutathione were higher in PsSPMS overexpressing Arabidopsis thaliana than the control, while the contents of putrescine were less than the control. Net photosynthetic rate, stomatal conductance, chlorophyll content, water use efficiency, electron transfer rate, PSII-related parameters, proline content, superoxide dismutase, and glutathione reductase activities were higher in PsSPMS overexpressing Arabidopsis thaliana than the control, while malondialdehyde, superoxide anion, and hydrogen peroxide contents were lower than the control. Correlation analysis showed significant differences between the indicators (P < 0.05 and P < 0.01). Expression of AtSPMS, AtSPD3, AtGSH2 and AtGR in transgenic Arabidopsis thaliana was higher than that of the control. Therefore, this study provides a genetic reference for the cultivation of cadmium-tolerant plants through genetic engineering and lays the foundation for further research on cadmium-tolerant Potentilla sericea.

Difluoromethylornithine rebalances aberrant polyamine ratios in Snyder-Robinson syndrome.

Snyder-Robinson syndrome (SRS) results from mutations in spermine synthase (SMS), which converts the polyamine spermidine into spermine. Affecting primarily males, common manifestations of SRS include intellectual disability, osteoporosis, hypotonia, and seizures. Symptom management is the only treatment. Reduced SMS activity causes spermidine accumulation while spermine levels are reduced. The resulting exaggerated spermidine:spermine ratio is a biochemical hallmark of SRS that tends to correlate with symptom severity. Our studies aim to pharmacologically manipulate polyamine metabolism to correct this imbalance as a therapeutic strategy for SRS. Here we report the repurposing of 2-difluoromethylornithine (DFMO), an FDA-approved inhibitor of polyamine biosynthesis, in rebalancing spermidine:spermine ratios in SRS patient cells. Mechanistic in vitro studies demonstrate that, while reducing spermidine biosynthesis, DFMO also stimulates the conversion of spermidine into spermine in hypomorphic SMS cells and induces uptake of exogenous spermine, altogether reducing the aberrant ratios. In a Drosophila SRS model characterized by reduced lifespan, DFMO improves longevity. As nearly all SRS patient mutations are hypomorphic, these studies form a strong foundation for translational studies with significant therapeutic potential.

The Role of Spermidine Synthase (SpdS) and Spermine Synthase (Sms) in Regulating Triglyceride Storage in Drosophila.

Polyamines are small organic cations that are important for several biological processes such as cell proliferation, cell cycle progression, and apoptosis. The dysregulation of intracellular polyamines is often associated with diseases such as cancer, diabetes, and developmental disorders. Although polyamine metabolism has been well studied, the effects of key enzymes in the polyamine pathway on lipid metabolism are not well understood. Here, we determined metabolic effects resulting from the absence of spermidine synthase (SpdS) and spermine synthase (Sms) in Drosophila. While SpdS mutants developed normally and accumulated triglycerides, Sms mutants had reduced viability and stored less triglyceride than the controls. Interestingly, when decreasing SpdS and Sms, specifically in the fat body, triglyceride storage increased. While there was no difference in triglycerides stored in heads, thoraxes and abdomen fat bodies, abdomen fat body DNA content increased, and protein/DNA decreased in both SpdS- and Sms-RNAi flies, suggesting that fat body-specific knockdown of SpdS and Sms causes the production of smaller fat body cells and triglycerides to accumulate in non-fat body tissues of the abdomen. Together, these data provide support for the role that polyamines play in the regulation of metabolism and can help enhance our understanding of polyamine function in metabolic diseases.

Exogenous spermine-induced expression of SlSPMS gene improves salinity-alkalinity stress tolerance by regulating the antioxidant enzyme system and ion homeostasis in tomato.

The study tested the function of exogenous spermine (Spm) in resisting salinity-alkalinity stress in tomato seedlings and found that tomato Spm synthase gene (SlSPMS) was involved in this regulation. The tomato seedlings cultivated in normal conditions or salinity-alkalinity conditions were irrigated with 100 ml one strength Hoagland nutrient solution 100 ml mixed solution (5 ml 300 mmol/L NaCl, 45 ml 300 mmol/L Na2SO4, 45 ml 300 mmol/L NaHCO3, and 5 ml 300 mmol/L Na2CO3 (pH = 8.90)) every 2 days, respectively. The 0.5 mM Spm pretreatment improved superoxide dismutase (SOD; EC 1.15.1.1) activity, catalase (CAT; EC 1.11.1.6) activity, ascorbate peroxidase (APX; EC 1.11.1.11) activity, and glutathione reductase (GR; EC 1.6.4.2) activity and decreased endogenous hydrogen peroxide (H2O2) content, malondialdehyde (MDA) content, and relative electrical conductivity (REC) in tomato leaves. Na+ content declined and K+ concentration rose in tomato seedlings when pre-treated with Spm. However the results showed that under salinity-alkalinity stress, silencing of SlSPMS with virus-induced gene silencing had lower antioxidant enzyme activities and higher Na+ content and lower K+ content than normal tomato seedlings, meaning that they had low salinity-alkalinity tolerance. Exogenous Spm could not reconstruct the tolerance to salinity-alkalinity stress in SlSPMS gene-silencing tomato seedlings. Taken together, exogenous Spm could induce the expression level of SlSPMS, which regulated the antioxidant enzyme system and ion homeostasis in tomato seedlings living in salinity-alkalinity environment, thereby improving the ability of tomato seedlings to resist salinity-alkalinity stress.

(R,R)-1,12-Dimethylspermine can mitigate abnormal spermidine accumulation in Snyder-Robinson syndrome.

Snyder-Robinson syndrome (SRS) is an X-linked intellectual disability syndrome caused by a loss-of-function mutation in the spermine synthase (SMS) gene. Primarily affecting males, the main manifestations of SRS include osteoporosis, hypotonic stature, seizures, cognitive impairment, and developmental delay. Because there is no cure for SRS, treatment plans focus on alleviating symptoms rather than targeting the underlying causes. Biochemically, the cells of individuals with SRS accumulate excess spermidine, whereas spermine levels are reduced. We recently demonstrated that SRS patient-derived lymphoblastoid cells are capable of transporting exogenous spermine and its analogs into the cell and, in response, decreasing excess spermidine pools to normal levels. However, dietary supplementation of spermine does not appear to benefit SRS patients or mouse models. Here, we investigated the potential use of a metabolically stable spermine mimetic, (R,R)-1,12-dimethylspermine (Me2SPM), to reduce the intracellular spermidine pools of SRS patient-derived cells. Me2SPM can functionally substitute for the native polyamines in supporting cell growth while stimulating polyamine homeostatic control mechanisms. We found that both lymphoblasts and fibroblasts from SRS patients can accumulate Me2SPM, resulting in significantly decreased spermidine levels with no adverse effects on growth. Me2SPM administration to mice revealed that Me2SPM significantly decreases spermidine levels in multiple tissues. Importantly, Me2SPM was detectable in brain tissue, the organ most affected in SRS, and was associated with changes in polyamine metabolic enzymes. These findings indicate that the (R,R)-diastereomer of 1,12-Me2SPM represents a promising lead compound in developing a treatment aimed at targeting the molecular mechanisms underlying SRS pathology.

Association Between Plasma Diacetylspermine and Tumor Spermine Synthase With Outcome in Triple-Negative Breast Cancer.

BACKGROUND: MYC is an oncogenic driver of development and progression in triple-negative breast cancer (TNBC). Ornithine decarboxylase, the rate-limiting enzyme in polyamine metabolism, is a transcriptional target of MYC. We therefore hypothesized that a plasma polyamine signature may be predictive of TNBC development and progression. METHODS: Using liquid chromatography mass spectrometry, polyamine levels were determined in plasma samples from newly diagnosed patients with TNBC (n = 87) and cancer-free controls (n = 115). Findings were validated in plasma samples from an independent prospective cohort of 54 TNBC, 55 estrogen receptor negative (ER-) and progesterone receptor negative (PR-) and HER2 positive (HER2+), and 73 ER+ case patients, and 30 cancer-free control subjects. Gene expression data and clinical data for 921 and 2359 breast cancer tumors were obtained from The Cancer Genome Atlas repository and the Oncomine database, respectively. Relationships between plasma diacetylspermine (DAS) and tumor spermine synthase (SMS) mRNA expression with metastasis-free survival and overall survival were determined using Cox proportional hazard models; Fisher exact tests were used to assess risk of distant metastasis in relation to tumor SMS mRNA expression. RESULTS: An increase in plasma DAS, a catabolic product of spermine mediated through SMS, was observed in the TNBC subtype of breast cancer. Plasma levels of DAS in TNBC associated with increased risk of metastasis (plasma DAS value ≥ 1.16, hazard ratio = 3.06, 95% confidence interval [CI] = 1.15 to 8.13, two-sided P = .03). SMS mRNA expression in TNBC tumor tissue was also found to be predictive of poor overall survival (top 25th percentile hazard ratio = 2.06, 95% CI = 1.04 to 4.08, one-sided P = .04) and increased risk of distant metastasis in TNBC (comparison of lowest SMS quartile [reference] to highest SMS quartile relative risk = 1.90, 95% CI = 0.97 to 4.06, one-sided Fisher exact test P=.03). CONCLUSIONS: Metabolomic profiling identified plasma DAS as a predictive marker for TNBC progression and metastasis.

Modeling Snyder-Robinson Syndrome in multipotent stromal cells reveals impaired mitochondrial function as a potential cause for deficient osteogenesis.

Patients with Snyder-Robinson Syndrome (SRS) exhibit deficient Spermidine Synthase (SMS) gene expression, which causes neurodevelopmental defects and osteoporosis, often leading to extremely fragile bones. To determine the underlying mechanism for impaired bone formation, we modelled the disease by silencing SMS in human bone marrow - derived multipotent stromal cells (MSCs) derived from healthy donors. We found that silencing SMS in MSCs led to reduced cell proliferation and deficient bone formation in vitro, as evidenced by reduced mineralization and decreased bone sialoprotein expression. Furthermore, transplantation of MSCs in osteoconductive scaffolds into immune deficient mice shows that silencing SMS also reduces ectopic bone formation in vivo. Tag-Seq Gene Expression Profiling shows that deficient SMS expression causes strong transcriptome changes, especially in genes related to cell proliferation and metabolic functions. Similarly, metabolome analysis by mass spectrometry, shows that silencing SMS strongly impacts glucose metabolism. This was consistent with observations using electron microscopy, where SMS deficient MSCs show high levels of mitochondrial fusion. In line with these findings, SMS deficiency causes a reduction in glucose consumption and increase in lactate secretion. Our data also suggests that SMS deficiency affects iron metabolism in the cells, which we hypothesize is linked to deficient mitochondrial function. Altogether, our studies suggest that SMS deficiency causes strong transcriptomic and metabolic changes in MSCs, which are likely associated with the observed impaired osteogenesis both in vitro and in vivo.

Myosin Va interacts with the exosomal protein spermine synthase.

Myosin Va (MyoVa) is an actin-based molecular motor that plays key roles in the final stages of secretory pathways, including neurotransmitter release. Several studies have addressed how MyoVa coordinates the trafficking of secretory vesicles, but why this molecular motor is found in exosomes is still unclear. In this work, using a yeast two-hybrid screening system, we identified the direct interaction between the globular tail domain (GTD) of MyoVa and four protein components of exosomes: the WD repeat-containing protein 48 (WDR48), the cold shock domain-containing protein E1 (CSDE1), the tandem C2 domain-containing protein 1 (TC2N), and the enzyme spermine synthase (SMS). The interaction between the GTD of MyoVa and SMS was further validated in vitro and displayed a Kd in the low micromolar range (3.5 ± 0.5 µM). SMS localized together with MyoVa in cytoplasmic vesicles of breast cancer MCF-7 and neuroblastoma SH-SY5Y cell lines, known to produce exosomes. Moreover, MYO5A knockdown decreased the expression of SMS gene and rendered the distribution of SMS protein diffuse, supporting a role for MyoVa in SMS expression and targeting.

The Spermine Synthase OsSPMS1 Regulates Seed Germination, Grain Size, and Yield.

Polyamines, including putrescine, spermidine, and spermine, play essential roles in a wide variety of prokaryotic and eukaryotic organisms. Rice (Oryza sativa) contains four putative spermidine/spermine synthase (SPMS)-encoding genes (OsSPMS1, OsSPMS2, OsSPMS3, and OsACAULIS5), but none have been functionally characterized. In this study, we used a reverse genetic strategy to investigate the biological function of OsSPMS1 We generated several homozygous RNA interference (RNAi) and overexpression (OE) lines of OsSPMS1 Phenotypic analysis indicated that OsSPMS1 negatively regulates seed germination, grain size, and grain yield per plant. The ratio of spermine to spermidine was significantly lower in the RNAi lines and considerably higher in the OE lines than in the wild type, suggesting that OsSPMS1 may function as a SPMS. S-Adenosyl-l-methionine is a common precursor of polyamines and ethylene biosynthesis. The 1-aminocyclopropane-1-carboxylic acid (ACC) and ethylene contents in seeds increased significantly in RNAi lines and decreased in OE lines, respectively, compared with the wild type. Additionally, the reduced germination rates and growth defects of OE lines could be rescued with ACC treatment. These data suggest that OsSPMS1 affects ethylene synthesis and may regulate seed germination and plant growth by affecting the ACC and ethylene pathways. Most importantly, an OsSPMS1 knockout mutant showed an increase in grain yield per plant in a high-yield variety, Suken118, suggesting that OsSPMS1 is an important target for yield enhancement in rice.

Crystal structure of thermospermine synthase from Medicago truncatula and substrate discriminatory features of plant aminopropyltransferases.

Polyamines are linear polycationic compounds that play a crucial role in the growth and development of higher plants. One triamine (spermidine, SPD) and two tetraamine isomers (spermine, SPM, and thermospermine, TSPM) are obtained by the transfer of the aminopropyl group from decarboxylated S-adenosylmethionine to putrescine and SPD. These reactions are catalyzed by the specialized aminopropyltransferases. In that respect, plants are unique eukaryotes that have independently evolved two enzymes, thermospermine synthase (TSPS), encoded by the gene ACAULIS5, and spermine synthase, which produce TSPM and SPM, respectively. In this work, we structurally characterize the ACAULIS5 gene product, TSPS, from the model legume plant Medicago truncatula (Mt). Six crystal structures of MtTSPS - one without ligands and five in complexes with either reaction substrate (SPD), reaction product (TSPM), or one of three cofactor analogs (5'-methylthioadenosine, S-adenosylthiopropylamine, and adenosine) - give detailed insights into the biosynthesis of TSPM. Combined with small-angle X-ray scattering data, the crystal structures show that MtTSPS is a symmetric homotetramer with an interdomain eight-stranded ß-barrel. Such an assembly and the presence of a hinge-like feature between N-terminal and C-terminal domains give the protein additional flexibility which potentially improves loading substrates and discarding products after the catalytic event. We also discuss the sequence and structural features around the active site of the plant aminopropyltransferases that distinguish them from each other and determine their characteristic substrate discrimination.

A Trans-omics Mathematical Analysis Reveals Novel Functions of the Ornithine Metabolic Pathway in Cancer Stem Cells.

Bioinformatics and computational modelling are expected to offer innovative approaches in human medical science. In the present study, we performed computational analyses and made predictions using transcriptome and metabolome datasets obtained from fluorescence-based visualisations of chemotherapy-resistant cancer stem cells (CSCs) in the human oesophagus. This approach revealed an uncharacterized role for the ornithine metabolic pathway in the survival of chemotherapy-resistant CSCs. The present study fastens this rationale for further characterisation that may lead to the discovery of innovative drugs against robust CSCs.

Cotton S-adenosylmethionine decarboxylase-mediated spermine biosynthesis is required for salicylic acid- and leucine-correlated signaling in the defense response to Verticillium dahliae.

MAIN CONCLUSION: Cotton S-adenosylmethionine decarboxylase-, rather than spermine synthase-, mediated spermine biosynthesis is required for salicylic acid- and leucine-correlated signaling in the defense response to Verticillium dahliae. Spermine (Spm) signaling is correlated with plant resistance to the fungal pathogen Verticillium dahliae. We identified genes for key rate-limiting enzymes in the biosynthesis of Spm, namely S-adenosylmethionine decarboxylase (GhSAMDC) and Spm synthase (GhSPMS). These were found by screening suppression subtractive hybridization and cDNA libraries of cotton (Gossypium) species tolerant to Verticillium wilt. Both were induced early and strongly by inoculation with V. dahliae and application of plant hormones. Silencing of GhSPMS or GhSAMDC in cotton leaves led to a significant accumulation of upstream substrates and, ultimately, enhanced plant susceptibility to Verticillium infection. Exogenous supplementation of Spm to the silenced cotton plants improved resistance. When compared with the wild type (WT), constitutive expression of GhSAMDC in Arabidopsis thaliana was associated with greater Verticillium wilt resistance and higher accumulations of Spm, salicylic acid, and leucine during the infection period. By contrast, transgenic Arabidopsis plants that over-expressed GhSPMS were unexpectedly more susceptible than the WT to V. dahliae and they also had impaired levels of putrescine (Put) and salicylic acid (SA). The susceptibility exhibited in GhSPMS-overexpressing Arabidopsis plants was partially reversed by the exogenous supply of Put or SA. In addition, the responsiveness of those two transgenic Arabidopsis lines to V. dahliae was associated with an alteration in transcripts of genes involved in plant resistance to epidermal penetrations and amino acid signaling. Together, these results suggest that GhSAMDC-, rather than GhSPMS-, mediated spermine biosynthesis contributes to plant resistance against V. dahliae through SA- and leucine-correlated signaling.

Molecular Cloning and Characterization of Spermine Synthesis Gene Associated with Cold Tolerance in Tea Plant (Camellia sinensis).

Spermine synthase (SPMS, EC 2.5.1.22), enzyme of spermine (Spm) biosynthesis, has been shown to be related to stress response. In this study, attempts were made to clone and characterize a gene encoding SPMS from tea plant (Camellia sinensis). The effect of exogenous application of Spm in C. sinensis subjected to low-temperature stress was also investigated. A full-length SPMS complementary DNA (cDNA) (CsSPMS) with an open reading frame of 1113 bp was cloned using reverse transcription-PCR and rapid amplification of cDNA ends (RACE) techniques from cultivar "Yingshuang". The CsSPMS gene, which encoded a 371 amino acid polypeptide, in four cultivars is highly homologous. Quantitative real-time PCR indicated that the CsSPMS gene shows tissue-specific expression, mainly in the leaf and root of tea plant. The expression analysis demonstrated that the CsSPMS gene is quickly induced by cold stress and had similar trends in four cultivars. Spm-supplemented "Baicha" cultivar contains higher endogenous polyamines compared to the control, coupling with higher expression levels of ADC and SPMS. In addition, activities of peroxidase (POD), superoxide dismutase (SOD), catalase (CAT), as well as free proline content in the Spm-supplemented samples were higher than the control during the experiment course or at a given time point, indicating that Spm exerted a positive effect on antioxidant systems. Moreover, Agrobacterium-mediated expression of CsSPMS in tobacco leaves showed relatively higher cold tolerance. Taken together, these findings will enhance the understanding of the relationships among CsSPMS gene regulatory, polyamines accumulation, and cold tolerance in tea plant.

Impaired osteoblast and osteoclast function characterize the osteoporosis of Snyder - Robinson syndrome.

BACKGROUND: Snyder-Robinson Syndrome (SRS) is an X-linked intellectual disability disorder also characterized by osteoporosis, scoliosis, and dysmorphic facial features. It is caused by mutations in SMS, a ubiquitously expressed gene encoding the polyamine biosynthetic enzyme spermine synthase. We hypothesized that the tissue specificity of SRS arises from differential sensitivity to spermidine toxicity or spermine deficiency. METHODS: We performed detailed clinical, endocrine, histopathologic, and morphometric studies on two affected brothers with a spermine synthase loss of function mutation (NM_004595.4:c.443A > G, p.Gln148Arg). We also measured spermine and spermidine levels in cultured human bone marrow stromal cells (hBMSCs) and fibroblasts using the Biochrom 30 polyamine protocol and assessed the osteogenic potential of hBMSCs. RESULTS: In addition to the known tissue-specific features of SRS, the propositi manifested retinal pigmentary changes, recurrent episodes of hyper- and hypoglycemia, nephrocalcinosis, renal cysts, and frequent respiratory infections. Bone histopathology and morphometry identified a profound depletion of osteoblasts and osteoclasts, absence of a trabecular meshwork, a low bone volume and a thin cortex. Comparison of cultured fibroblasts from affected and unaffected individuals showed relatively small changes in polyamine content, whereas comparison of cultured osteoblasts identified marked differences in spermidine and spermine content. Osteogenic differentiation of the SRS-derived hBMSCs identified a severe deficiency of calcium phosphate mineralization. CONCLUSIONS: Our findings support the hypothesis that cell specific alterations in polyamine metabolism contribute to the tissue specificity of SRS features, and that the low bone density arises from a failure of mineralization.

A maize spermine synthase 1 PEST sequence fused to the GUS reporter protein facilitates proteolytic degradation.

Polyamines are low molecular weight aliphatic compounds involved in various biochemical, cellular and physiological processes in all organisms. In plants, genes involved in polyamine biosynthesis and catabolism are regulated at transcriptional, translational, and posttranslational level. In this research, we focused on the characterization of a PEST sequence (rich in proline, glutamic acid, serine, and threonine) of the maize spermine synthase 1 (ZmSPMS1). To this aim, 123 bp encoding 40 amino acids of the C-terminal region of the ZmSPMS1 enzyme containing the PEST sequence were fused to the GUS reporter gene. This fusion was evaluated in Arabidopsis thaliana transgenic lines and onion monolayers transient expression system. The ZmSPMS1 PEST sequence leads to specific degradation of the GUS reporter protein. It is suggested that the 26S proteasome may be involved in GUS::PEST fusion degradation in both onion and Arabidopsis. The PEST sequences appear to be present in plant spermine synthases, mainly in monocots.

A Y328C missense mutation in spermine synthase causes a mild form of Snyder-Robinson syndrome.

Snyder-Robinson syndrome (SRS, OMIM: 309583) is an X-linked intellectual disability (XLID) syndrome, characterized by a collection of clinical features including facial asymmetry, marfanoid habitus, hypertonia, osteoporosis and unsteady gait. It is caused by a significant decrease or loss of spermine synthase (SMS) activity. Here, we report a new missense mutation, p.Y328C (c.1084A>G), in SMS in a family with XLID. The affected males available for evaluation had mild ID, speech and global delay, an asthenic build, short stature with long fingers and mild kyphosis. The spermine/spermidine ratio in lymphoblasts was 0.53, significantly reduced compared with normal (1.87 average). Activity analysis of SMS in the index patient failed to detect any activity above background. In silico modeling demonstrated that the Y328C mutation has a significant effect on SMS stability, resulting in decreased folding free energy and larger structural fluctuations compared with those of wild-type SMS. The loss of activity was attributed to the increase in conformational dynamics in the mutant which affects the active site geometry, rather than preventing dimer formation. Taken together, the biochemical and in silico studies confirm the p.Y328C mutation in SMS is responsible for the patients having a mild form of SRS and reveal yet another molecular mechanism resulting in a non-functional SMS causing SRS.

The polyamine spermine protects Arabidopsis from heat stress-induced damage by increasing expression of heat shock-related genes.

It is known that the polyamine (PA) biosynthetic pathway is modulated at the transcriptional level during abiotic stresses. Here we studied the expression of PA biosynthetic pathway genes upon exposure to heat shock (HS) in Arabidopsis and showed that the spermine (Spm) synthase gene (SPMS) and S-adenosylmethionine decarboxylase 2 gene are induced at the earliest stage, followed by the induction of the arginine decarboxylase 2 gene. Correspondingly, Spm content increased linearly upon HS, and putrescine (Put) and spermidine (Spd) content also increased but not thermospermine (T-Spm) content. Exogenously applied Spm had a potential to protect Arabidopsis plants from HS-induced damage. Such protection was also observed to the same extent with T-Spm and by Spd to a lesser extent but not by Put. Then we tested whether altered endogenous Spm content affects sensitivity to HS using both transgenic plants overexpressing SPMS and a Spm deficient (spms) mutant plant. The result revealed that the higher the Spm content the higher the thermotolerance. Even in the spms plant, representative genes encoding heat shock proteins (HSPs) and heat shock transcription factors were upregulated upon HS, while the expression of such genes was increased in a positively correlated manner with Spm content. Furthermore four kinds of HSPs (HSP101, HSP90, HSP70 and HSP17.6) were detected proportionally with the levels of their respective transcripts upon HS. We propose that Spm increases the HS response at transcriptional and translational levels and protects host plants from HS-induced damage.

Spermine synthase overexpression in vivo does not increase susceptibility to DMBA/TPA skin carcinogenesis or Min-Apc intestinal tumorigenesis.

Numerous studies have demonstrated a link between elevated polyamine biosynthesis and neoplastic growth, but the specific contribution of spermine synthase to epithelial tumor development has never been explored in vivo. Mice with widespread overexpression of spermine synthase (CAG-SpmS) exhibit decreased spermidine levels, increased spermine and a significant rise in tissue spermine:spermidine ratio. We characterized the response of CAG-SpmS mice to two-stage skin chemical carcinogenesis as well as spontaneous intestinal carcinogenesis induced by loss of the Apc tumor suppressor in Apc (Min) (/+) (Min) mice. CAG-SpmS mice maintained the canonical increases in ornithine decarboxylase (ODC) activity, polyamine content and epidermal thickness in response to tumor promoter treatment of the skin. The induction of S-adenosylmethionine decarboxylase (AdoMetDC) activity and its product decarboxylated AdoMet were impaired in CAG-SpmS mice, and the spermine:spermidine ratio was increased 3-fold in both untreated and 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated skin. The susceptibility to 7,12-dimethylbenz[a]anthracene (DMBA)/TPA skin carcinogenesis was not altered in CAG-SpmS mice, and SpmS overexpression did not modify the previously described tumor resistance of mice with targeted antizyme expression or the enhanced tumor response in mice with targeted spermidine/spermine-N ( 1) -acetyltransferase expression. CAG-SpmS/Min mice also exhibited elevated spermine:spermidine ratios in the small intestine and colon, yet their tumor multiplicity and size was similar to Min mice. Therefore, studies in two of the most widely used tumorigenesis models demonstrate that increased spermine synthase activity and the resulting elevation of the spermine:spermidine ratio does not alter susceptibility to tumor development initiated by c-Ha-Ras mutation or Apc loss.

Global gene expression profiling of the polyamine system in suicide completers.

In recent years, gene expression, genetic association, and metabolic studies have implicated the polyamine system in psychiatric conditions, including suicide. Given the extensive regulation of genes involved in polyamine metabolism, as well as their interconnections with the metabolism of other amino acids, we were interested in further investigating the expression of polyamine-related genes across the brain in order to obtain a more comprehensive view of the dysregulation of this system in suicide. To this end, we examined the expression of genes related to polyamine metabolism across 22 brain regions in a sample of 29 mood-disordered suicide completers and 16 controls, and identified 14 genes displaying differential expression. Among these, altered expression of spermidine/spermine N1-acetyltransferase, spermine oxidase, and spermine synthase, has previously been observed in brains of suicide completers, while the remainder of the genes represent novel findings. In addition to genes with direct involvement in polyamine metabolism, including S-adenosylmethionine decarboxylase, ornithine decarboxylase antizymes 1 and 2, and arginase II, we identified altered expression of several more distally related genes, including aldehyde dehydrogenase 3 family, member A2, brain creatine kinase, mitochondrial creatine kinase 1, glycine amidinotransferase, glutamic-oxaloacetic transaminase 1, and arginyl-tRNA synthetase-like. Many of these genes displayed altered expression across several brain regions, strongly implying that dysregulated polyamine metabolism is a widespread phenomenon in the brains of suicide completers. This study provides a broader view of the nature and extent of the dysregulation of the polyamine system in suicide, and highlights the importance of this system in the neurobiology of suicide.