Melatonergic system‐based two‐gene index is prognostic in human gliomas

Gliomas, the most common primary brain tumors in adults, are classified into four malignancy grades according to morphological features. Recent studies have shown that melatonin treatment induces cytotoxicity in glioma‐initiating cells and reduces the invasion and migration of glioma cell lines, inhibiting the nuclear factor κB (NFκB) oncopathway. Given that C6 rat glioma cells produce melatonin, we investigated the correlation between the capacity of gliomas to synthesize/metabolize melatonin and their overall malignancy. We first characterized the melatonergic system of human gliomas cell lines with different grades of aggressiveness (HOG, T98G, and U87MG) and demonstrated that glioma‐synthesized melatonin exerts an autocrine antiproliferative effect. Accordingly, the sensitivity to exogenous melatonin was higher for the most aggressive cell line, U87MG, which synthesized/accumulated less melatonin. Using The Cancer Genome Atlas RNAseq data of 351 glioma patients, we designed a predictive model of the content of melatonin in the tumor microenvironment, the ASMT:CYP1B1 index, combining the gene expression levels of melatonin synthesis and metabolism enzymes. The ASMT:CYP1B1 index negatively correlated with tumor grade, as well as with the expression of pro‐proliferation and anti‐apoptotic NFκB target genes. More importantly, the index was a grade‐ and histological type‐independent prognostic factor. Even when considering only high‐grade glioma patients, a low ASMT:CYP1B1 value, which suggests decreased melatonin and enhanced aggressiveness, was strongly associated with poor survival. Overall, our data reveal the prognostic value of the melatonergic system of gliomas and provide insights into the therapeutic role of melatonin.     

Caffeic acid O‐methyltransferase is involved in the synthesis of melatonin by methylating N‐acetylserotonin in Arabidopsis

Although a plant N‐acetylserotonin methyltransferase (ASMT) was recently cloned from rice, homologous genes appear to be absent in dicotyledonous plants. To clone an ASMT de novo from a dicotyledonous plant, we expressed eight Arabidopsis thaliana O‐methyltransferase (OMT) cDNAs in Escherichia coli and screened for ASMT activity by measuring melatonin production after the application of 1 mm N‐acetylserotonin (NAS). Among the eight strains harboring the full‐length cDNAs, the OMT3 strain produced high levels of melatonin, suggesting that OMT3 encodes an active ASMT. OMT3 is already known as caffeic acid OMT (COMT), suggesting multiple functions for this enzyme. The purified recombinant A. thaliana COMT (AtCOMT) showed high ASMT activity, catalyzing the conversion of NAS to melatonin. The K_m and V_max values for ASMT activity were 233 μm and 1800 pmol/min/mg protein, while the K_m and V_max values for COMT activity were 103 μm and 564,000 pmol/min/mg protein, respectively. The catalytic efficiency (V_max/K_m) for ASMT activity was 709‐fold lower than for COMT. In vitro, ASMT activity was dramatically decreased by the addition of caffeic acid in a dose‐dependent manner, but the activity of COMT was not altered by NAS. Lastly, the Arabidopsis comt knockout mutant exhibited less production of melatonin than the wild type when Arabidopsis leaves were infiltrated with 1 mm NAS, suggestive of in vivo role of COMT in melatonin biosynthesis in plants.     

Cellular localization and kinetics of the rice melatonin biosynthetic enzymes SNAT and ASMT

Serotonin N‐acetyltransferase (SNAT) and N‐acetylserotonin methyltransferase (ASMT) are the final two enzymes in the melatonin synthesis pathway in plants. Although their corresponding genes have been cloned, their cellular localization and enzymatic characteristics are unknown. Using confocal microscopy, we showed that SNAT protein is localized in chloroplasts, whereas ASMT is expressed in the cytoplasm. In vitro measurement of ASMT enzyme activity revealed a peak of activity in roots, but SNAT enzyme activity was not detected in any plant tissues. This may be attributed in part to an effect of chlorophyll because SNAT enzyme activity was greatly inhibited by chlorophyll in a dose‐dependent manner. Because the SNAT protein of cyanobacteria is thermophilic, we examined the effect of temperature on the activity of the rice SNAT and ASMT enzymes. Purified recombinant rice SNAT and ASMT enzymes had an optimum temperature for activity of 55°C. The K_m and V_max values for SNAT at 55°C were 270 μm and 3.3 nmol/min/mg protein, whereas the K_m and V_max for ASMT were 222 μm and 9 nmol/min/mg protein, respectively. The catalytic efficiency (V_max/K_m) values of SNAT and ASMT were 16‐fold and 4054‐fold higher at 55°C than at 30°C suggestive of increased melatonin production at high temperature in plants.     

Predominance of 2‐hydroxymelatonin over melatonin in plants

The cloning of the gene encoding melatonin 2‐hydroxylase (M2H), which is responsible for the synthesis of 2‐hydroxymelatonin, has expanded the study of melatonin metabolism in plants. Kinetic analysis of M2H enzymatic activity demonstrated that the catalytic efficiency of M2H is much higher than those of other melatonin biosynthetic enzymes such as serotonin N‐acetyltransferase (SNAT) and N‐acetylserotonin O‐methyltransferase (ASMT), suggesting that melatonin metabolism is rapid in plants. To test this prediction, we selected 24 plant species belonging to 16 families and quantified the levels of melatonin and 2‐hydroxymelatonin using liquid chromatography–tandem mass spectrometry (LC‐MS/MS). The melatonin levels in most of the species were <1 ng/g fresh weight (FW), while those in leaves from radish and feverfew were 3.5 and 3.3 ng/g FW, respectively. In contrast, the average levels of 2‐hydroxymelatonin were much higher at 6.2 ng/g FW. The average ratio of 2‐hydroxymelatonin to melatonin in plants was approximately 368:1, indicating that the accumulation of 2‐hydroxymelatonin predominates over that of melatonin. These data were consistent with previous results on the kinetics of the corresponding enzymes, as well as with in vivo melatonin conversion data. Among several melatonin metabolites in plants, the most abundant metabolite was found to be 2‐hydroxymelatonin (99%) followed by 4‐hydroxymelatonin (0.05%), but 6‐hydroxymelatonin was not detected in rice seedlings.     

Chloroplast overexpression of rice caffeic acid O‐methyltransferase increases melatonin production in chloroplasts via the 5‐methoxytryptamine pathway in transgenic rice plants

Recent analyses of the enzymatic features of various melatonin biosynthetic genes from bacteria, animals, and plants have led to the hypothesis that melatonin could be synthesized via the 5‐methoxytryptamine (5‐MT) pathway. 5‐MT is known to be synthesized in vitro from serotonin by the enzymatic action of O‐methyltransferases, including N‐acetylserotonin methyltransferase (ASMT) and caffeic acid O‐methyltransferase (COMT), leading to melatonin synthesis by the subsequent enzymatic reaction with serotonin N‐acetyltransferase (SNAT). Here, we show that 5‐MT was produced and served as a precursor for melatonin synthesis in plants. When rice seedlings were challenged with senescence treatment, 5‐MT levels and melatonin production were increased in transgenic rice seedlings overexpressing the rice COMT in chloroplasts, while no such increases were observed in wild‐type or transgenic seedlings overexpressing the rice COMT in the cytosol, suggesting a 5‐MT transport limitation from the cytosol to chloroplasts. In contrast, cadmium treatment led to results different from those in senescence. The enhanced melatonin production was not observed in the chloroplast COMT lines relative over the cytosol COMT lines although 5‐MT levels were equally induced in all genotypes upon cadmium treatment. The transgenic seedlings with enhanced melatonin in their chloroplasts exhibited improved seedling growth vs the wild type under continuous light conditions. This is the first report describing enhanced melatonin production in chloroplasts via the 5‐MT pathway with the ectopic overexpression of COMT in chloroplasts in plants.     

Molecular cloning of rice serotonin N‐acetyltransferase,the penultimate gene in plant melatonin biosynthesis

Because of the absence of an arylalkylamine N‐acetyltransferase (AANAT) homolog in the plant genome, the proposal was made that a GCN5‐related N‐acetyltransferase superfamily gene (GNAT) could be substituted for AANAT. To clone rice serotonin N‐acetyltransferase (SNAT), we expressed 31 rice GNAT cDNAs in Escherichia coli and screened SNAT activity by measuring N‐acetyltryptamine after application with 1 mm tryptamine. GNAT5 was shown to produce high levels of N‐acetyltryptamine in E. coli, suggesting a possible rice SNAT. To confirm SNAT activity, the GNAT5 protein was purified through affinity purification from E. coli culture. The purified recombinant GNAT5 showed high SNAT enzyme activity catalyzing serotonin into N‐acetylserotonin. The values for K_m and V_max were 385 μm and 282 pmol/min/mg protein, respectively. An in vitro enzyme assay of purified SNAT showed N‐acetylserotonin formation to be proportional to enzyme concentration and time, with peak activity at pH 8.8. High substrate concentrations above 1 mm serotonin inhibited SNAT activity. Finally, the mRNA level of SNAT was higher in shoots than in roots, but it was expressed constitutively, unlike N‐acetylserotonin methyltransferase (ASMT), the terminal enzyme in melatonin synthesis. These results suggest that ASMT rather than SNAT is the rate‐limiting enzyme of melatonin biosynthesis in plants.     

Pan‐cancer genomic analyses reveal prognostic and immunogenic features of the tumor melatonergic microenvironment across 14 solid cancer types

We performed comprehensive genomic analyses of the melatonergic system within the tumor microenvironment and their clinical relevance across a broad spectrum of solid tumors. RNA‐seq data from The Cancer Genome Atlas (TCGA) of 14 solid tumors representing 6658 human samples were analyzed. The tumor melatonergic system was characterized by the rates of melatonin synthesis and metabolism using a two‐gene expression model (melatonin synthesis/metabolism Index). We calculated three indexes according to different melatonin metabolism isoenzymes (Index‐I [ASMT:CYP1A1], Index‐II [ASMT:CYP1A2], and Index‐III [ASMT:CYP1B1]). Samples of each cancer type were classified into two subgroups (high vs low) based on median values. Clinical outcomes, mutational burden, and neoepitope abundance were analyzed and compared. We found that the ability of the tumor microenvironment to synthesize and accumulate melatonin varied across cancer types and negatively correlated with tumor burden. Kaplan‐Meier survival analyses and multivariable modeling showed that the three indexes played different roles across different cancers and harbored prognostic values in breast cancer (adjusted hazard ratio [AHR]_Index‐II = 0.65 [0.44‐0.97]; P = 0.03), cervical cancer (AHR_Index‐I = 0.62 [0.39‐0.98]; P = 0.04), lung squamous cell carcinoma (AHR_Index‐III = 0.75 [0.56‐0.99]; P = 0.04), melanoma (AHR_Index‐I = 0.74 [0.55‐0.98]; P = 0.04), and stomach adenocarcinoma (AHR_Index‐III = 0.68 [0.41‐0.94]; P = 0.02). We further investigated its clinical relevance with tumor immunogenic features (mutational burden and neoantigen abundance), which may predict immunotherapy benefits. We observed significant negative correlations with mutational burden in the majority of tumors (P < 0.05), except cervical cancer, pancreatic adenocarcinoma, and thyroid carcinoma. Our study provides a systematic overview of the oncostatic values of the melatonergic system and highlights the utilization of this simple and promising gene signature as a prognosticator and potential predictor of response to immunotherapy.     

Genetic variations of the melatonin pathway in patients with attention-deficit and hyperactivity disorders

Melatonin is a powerful antioxidant and a synchronizer of many physiological processes. Alteration in melatonin signaling has been reported in a broad range of diseases, but little is known about the genetic variability of this pathway in humans. Here, we sequenced all the genes of the melatonin pathway -AA-NAT, ASMT, MTNR1A, MTNR1B and GPR50 - in 321 individuals from Sweden including 101 patients with attention-deficit/hyperactivity disorder (ADHD) and 220 controls from the general population. We could find several damaging mutations in patients with ADHD, but no significant enrichment compared with the general population. Among these variations, we found a splice site mutation in ASMT (IVS5+2T>C) and one stop mutation in MTNR1A (Y170X) - detected exclusively in patients with ADHD - for which biochemical analyses indicated that they abolish the activity of ASMT and MTNR1A. These genetic and functional results represent the first comprehensive ascertainment of melatonin signaling deficiency in ADHD.     

Low melatonin production by suppression of either serotonin N‐acetyltransferase or N‐acetylserotonin methyltransferase in rice causes seedling growth retardation with yield penalty,abiotic stress susceptibility,and enhanced coleoptile growth under anoxic conditions

Serotonin N‐acetyltransferase (SNAT) and N‐acetylserotonin methyltransferase (ASMT) are the last two key enzymes for melatonin biosynthesis in living organisms. In this study, we demonstrated that transgenic rice (Oryza sativa L.) plants, in which expression of either endogenous SNAT or ASMT was suppressed, had reduced melatonin synthesis, confirming that both SNAT and ASMT are functionally involved in melatonin synthesis. The melatonin‐deficient SNAT rice had retarded seedling growth, which was partially restored by exogenous melatonin application, suggesting melatonin's role in seedling growth. In addition, the plants were more sensitive to various abiotic stresses, including salt and cold, compared with the wild type. Melatonin‐deficient SNAT rice had increased coleoptile growth under anoxic conditions, indicating that melatonin also inversely regulates plant growth under anaerobic conditions with the concomitant high expression of alcohol dehydrogenase genes. Similarly, the melatonin‐deficient ASMT rice exhibited accelerated senescence in detached flag leaves, as well as significantly reduced yield. These loss‐of‐function studies on the melatonin biosynthetic genes confirmed most previous pharmacological reports that melatonin not only promotes plant growth but also mitigates various abiotic stresses.     

Molecular cloning of a plant N-acetylserotonin methyltransferase and its expression characteristics in rice

N-acetylserotonin methyltransferase (ASMT), the last enzyme in the synthesis of melatonin, catalyzes N-acetylserotonin into melatonin. For the first time, we cloned ASMT from rice through the analysis of recombinant Escherichia coli harboring putative rice O-methyltransferase (OMT) cDNAs. In total, 18 full-length cDNAs, which show homology to wheat caffeic acid 3-O-methyltransferase, were expressed in E. coli and induced in the presence of N-acetylserotonin; we then analyzed the production of melatonin. Only recombinant E. coli line 15 showed melatonin synthesis; no other recombinant lines produced melatonin with the addition of N-acetylserotonin in E. coli culture. Line 15 clearly exhibited in vitro ASMT enzyme activity with 0.27 pkat/mg protein. ASMT enzyme activity was inhibited by various related compounds such as N-acetyltryptamine and N-acetyltyrosine. The open reading frame of ASMT consists of 364 amino acids possessing well-conserved motifs found in plant OMT such as S-adenosyl-L-methionine-binding and catalytic sites. Induction patterns of ASMT mRNA were well matched with the production of melatonin in rice leaves during senescence, as well as several stressors.     

An AANAT/ASMT transgenic animal model constructed with CRISPR/Cas9 system serving as the mammary gland bioreactor to produce melatonin‐enriched milk in sheep

Melatonin as a potent antioxidant exhibits important nutritional and medicinal values. To produce melatonin‐enriched milk will benefit the consumers. In this study, a sheep bioreactor which generates melatonin‐enriched milk has been successfully developed by the technology that combined CRISPR/Cas9 system and microinjection. The AANAT and ASMT were cloned from pineal gland of Dorper sheep (Ovis aries). The in vitro studies found that AANAT and ASMT were successfully transferred to the mammary epithelial cell lines and significantly increased melatonin production in the culture medium compared to the nontransgenic cell lines. In addition, the Cas9 mRNA, sgRNA, and the linearized vectors pBC1‐AANAT and pBC1‐ASMT were co‐injected into the cytoplasm of pronuclear embryos which were implanted into ewes by oviducts transferring. Thirty‐four transgenic sheep were generated with the transgenic positive rate being roughly 35% which were identified by Southern blot and sequencing. Seven carried transgenic AANAT, two carried ASMT, and 25 carried both of AANAT and ASMT genes. RT‐PCR and Western blot demonstrated that the lambs expressed these genes in their mammary epithelial cells and these animals produced melatonin‐enriched milk. This is the first report to show a functional AANAT and ASMT transgenic animal model which produce significantly high levels of melatonin milk compared to their wild‐type counterparts. The advanced technologies used in the study laid a foundation for generating large transgenic livestock, for example, the cows, which can produce high level of melatonin milk.     

Kinetic analysis of purified recombinant rice N-acetylserotonin methyltransferase and peak melatonin production in etiolated rice shoots

Abstract: Rice (Oryza sativa) N‐acetylserotonin methyltransferase (osASMT), the last enzyme in the synthesis of melatonin, was expressed in Escherichia coli and purified. We then characterized its enzyme kinetics, which is the first time this has been performed in plants. Purified glutathione S‐transferase (GST)‐fused recombinant osASMT (GST‐osASMT) and GST‐free osASMT showed specific enzyme activities of 6.6 and 12.6 pmol/min per mg protein, respectively. When evaluated by the Lineweaver‐Burk equation, GST‐free osASMT exhibited a K_m of 864 μm . An in vitro enzyme assay of purified osASMT showed melatonin formation to be proportional to the enzyme and substrate concentrations, as well as time. Unlike animal ASMT, high substrate concentrations did not inhibit the activity of osASMT. Finally, melatonin biosynthesis in rice seedlings was affected by light intensity, with etiolated shoots grown in continuous darkness producing more melatonin than shoots grown in continuous light. The level of melatonin in relation to the light intensity closely paralleled the mRNA level of osASMT in the shoots, suggesting that endogenous melatonin is upregulated in darkness, as is the case in animals.     

BCL2 mutations do not confer adverse prognosis in follicular lymphoma patients treated with rituximab

BCL2 mutations have been suggested to confer an adverse prognosis to follicular lymphoma (FL) patients, but their prognostic value has not been assessed in patients treated with a rituximab‐containing regimen. Here we evaluated the prognostic value of BCL2 mutations in a large prospective cohort of 252 patients with FL treated with immunochemotherapy in the PRIMA randomized trial. Using a DNA‐targeted sequencing approach, we detected amino acid altering mutations in 135 patients (54%) and showed that these mutations were probably mediated by the over‐activation of AICDA (activation‐induced cytidine deaminase) in the context of the t(14;18) translocation. The BCL2 variants identified in PRIMA patients affected the BH1, BH2, and BH3 functional motifs at a lower frequency than the N‐terminus and flexible loop domain, with mostly conservative aminoacid changes. With a median follow‐up of 6.7 years, we did not observe any impact of BCL2 mutations either on overall survival or progression‐free survival.     

Cloning and characterization of a serotonin N‐acetyltransferase from a gymnosperm,loblolly pine (Pinus taeda)

Serotonin N‐acetyltransferase (SNAT) is the penultimate enzyme in melatonin biosynthesis in both animals and plants. SNAT catalyzes serotonin into N‐acetylserotonin, an immediate precursor for melatonin biosynthesis by N‐acetylserotonin methyltransferase (ASMT). We cloned the SNAT gene from a gymnosperm loblolly pine (Pinus teada). The loblolly pine SNAT (PtSNAT) gene encodes 255 amino acids harboring a transit sequence with 67 amino acids and shows 67% amino acid identity with rice SNAT when comparing the mature polypeptide regions. Purified recombinant PtSNAT showed peak activity at 55°C with the K_m (428 μm ) and V_max (3.9 nmol/min/mg protein) values. As predicted, PtSNAT localized to chloroplasts. The SNAT mRNA was constitutively expressed in all tissues, including leaf, bud, flower, and pinecone, whereas the corresponding protein was detected only in leaf. In accordance with the exclusive SNAT protein expression in leaf, melatonin was detected only in leaf at 0.45 ng per gram fresh weight. Sequence and phylogenetic analysis indicated that the gymnosperm PtSNAT had high homology with SNATs from all plant phyla (even with cyanobacteria), and formed a clade separated from the angiosperm SNATs, suggestive of direct gene transfer from cyanobacteria via endosymbiosis.