ABSTRACT
Previous studies have demonstrated that α-synuclein (α-SYN) is closely associated with rapid eye movement sleep behavior disorder (RBD) related to several neurodegenerative disorders. However, the exact molecular mechanisms are still rarely investigated. In the present study, we found that in the α-SYNA53T induced RBD-like behavior mouse model, the melatonin level in the plasma and pineal gland were significantly decreased. To elucidate the underlying mechanism of α-SYN-induced melatonin reduction, we investigated the effect of α-SYN in melatonin biosynthesis. Our findings showed that α-SYN reduced the level and activity of melatonin synthesis enzyme acetylserotonin O-methyltransferase (ASMT) in the pineal gland and in the cell cultures. In addition, we found that microtubule-associated protein 1 light chain 3 beta (LC3B) as an important autophagy adapter is involved in the degradation of ASMT. Immunoprecipitation assays revealed that α-SYN increases the binding between LC3B and ASMT, leading to ASMT degradation and a consequent reduction in melatonin biosynthesis. Collectively, our results demonstrate the molecular mechanisms of α-SYN in melatonin biosynthesis, indicating that melatonin is an important molecule involved in the α-SYN-associated RBD-like behaviors, which may provide a potential therapeutic target for RBD of Parkinson's disease.
Subject(s)
Melatonin , Pineal Gland , Mice , Animals , Melatonin/metabolism , Acetylserotonin O-Methyltransferase/chemistry , Acetylserotonin O-Methyltransferase/metabolism , alpha-Synuclein/metabolism , Pineal Gland/metabolismABSTRACT
KEY MESSAGE: This study provides novel insights into the evolution, diversification, and functions of melatonin biosynthesis genes in Prunus species, highlighting their potential role in regulating bud dormancy and abiotic stresses. The biosynthesis of melatonin (MEL) in plants is primarily governed by enzymatic reactions involving key enzymes such as serotonin N-acetyltransferase (SNAT), tryptamine 5-hydroxylase (T5H), N-acetylserotonin methyltransferase (ASMT) and tryptophan decarboxylase (TDC). In this study, we analyzed Melatonin genes in four Prunus species such as Prunus avium (Pavi), Prunus pusilliflora (Ppus), Prunus serulata (Pser), and Prunus persica (Pper) based on comparative genomics approach. Among the four Prunus species, a total of 29 TDCs, 998 T5Hs, 16 SNATs, and 115 ASMTs within the genome of four Prunus genomes. A thorough investigation of melatonin-related genes was carried out using systematic biological methods and comparative genomics. Through phylogenetic analysis, orthologous clusters, Go enrichment, syntenic relationship, and gene duplication analysis, we discovered both similarities and variations in Melatonin genes among these Prunus species. Additionally, our study revealed the existence of unique subgroup members in the Melatonin genes of these species, which were distinct from those found in Arabidopsis genes. Furthermore, the transcriptomic expression analysis revealed the potential significance of melatonin genes in bud dormancy regulation and abiotic stresses. Our extensive results offer valuable perspectives on the evolutionary patterns, intricate expansion, and functions of PavMEL genes. Given their promising attributes, PavTDCs, PavT5H, PavNAT, and three PavASMT genes warrant in-depth exploration as prime candidates for manipulating dormancy in sweet cherry. This was done to lay the foundation for future explorations into the structural and functional aspects of these factors in Prunus species. This study offers significant insights into the functions of ASMT, SNAT, T5H, and TDC genes and sheds light on their roles in Prunus avium. Moreover, it established a robust foundation for further exploration functional characterization of melatonin genes in fruit species.
Subject(s)
Arabidopsis , Melatonin , Prunus avium , Prunus , Prunus avium/genetics , Prunus avium/metabolism , Prunus/genetics , Prunus/metabolism , 5-Methoxytryptamine , Melatonin/genetics , Melatonin/metabolism , Phylogeny , Acetylserotonin O-Methyltransferase/chemistry , Acetylserotonin O-Methyltransferase/genetics , Acetylserotonin O-Methyltransferase/metabolism , Arabidopsis/genetics , Genomics , Stress, Physiological/geneticsABSTRACT
BACKGROUND: As an important reproductive hormone, melatonin plays an important role in regulating the reproductive activities of sheep and other mammals. Hu sheep is a breed favoring for meat, with prolific traits. In order to explore the relationship between melatonin and reproductive function of Hu sheep, 7,694,759 SNPs were screened out through the whole genome sequencing analysis from high and low melatonin production Hu sheep. RESULTS: A total of 68,673 SNPs, involving in 1126 genes, were identified by ED association analysis. Correlation analysis of SNPs of AANAT/ASMT gene and MTNR1A/MTNR1B gene were carried out. The melatonin level of CG genotype 7,981,372 of AANAT, GA genotype 7,981,866 of ASMT and GG genotype 17,355,171 of MTNR1A were higher than the average melatonin level of 1.64 ng/mL. High melatonin Hu sheep appear to have better multiple reproductive performance. CONCLUSIONS: By using different methods, three SNPs which are associated with high melatonin production trait have been identified in Hu sheep. These 3 SNPs are located in melatonin synthetase AANAT/ASMT and receptor MTNR1A, respectively. Considering the positive association between melatonin production and reproductive performance in ruminants, these three SNPs can be served as the potential molecular markers for breading Hu sheep with the desirable reproductive traits.
Subject(s)
Melatonin , Sheep/genetics , Animals , Melatonin/genetics , Polymorphism, Single Nucleotide , Phenotype , Genotype , Bread , MammalsABSTRACT
In fish, the skin is directly exposed to multiple environmental stressors and provides the first line of defense against harmful external factors. It turned out that cortisol and melatonin (Mel) are involved in fish cutaneous stress response system (CSRS) similar to mammalian. This study investigates the mode of action of CSRS in two teleost species of different biology and skin characteristics, the three-spined stickleback and the European flounder, after exposure to oxidative stress induced by a potassium dichromate solution. The cutaneous stress response system presents different ways of action in two studied species: Mel concentration increases in the skin of both species, but cortisol concentration increases in the skin only in sticklebacks. Data suggest that stickleback skin cells can produce cortisol. However, cortisol is not involved in the response to oxidative stress in flounders. In stickleback skin, two genes encoding AANAT and ASMT/HIOMT (enzymes involved in Mel synthesis), aanat1a and asmt2, are expressed, but in flounder skin, only one, asmtl. Because gene expression does not change in stickleback skin after exposure to stress, the source of increased Mel is probably outside the skin. A lack of expression of the gene encoding AANAT in flounder skin strongly suggests that Mel is transported to the skin by the bloodstream from other sites of synthesis. Pigment dispersion in the skin after exposure to oxidative stress is found only in sticklebacks.
Subject(s)
Flounder , Melatonin , Smegmamorpha , Animals , Flounder/metabolism , Hydrocortisone , Smegmamorpha/genetics , Fishes/metabolism , Oxidative Stress , Arylalkylamine N-Acetyltransferase/genetics , Mammals/metabolismABSTRACT
Melatonin is a pleiotropic molecule with a variety of biological functions, which include its immunoregulatory action in mammals. Brucellosis is a worldwide endemic zoonotic disease caused by the Brucella, which not only causes huge economic losses for the livestock industry but also impacts human health. To target this problem, in current study, two marker-free transgenic sheep overexpressing melatonin synthetic enzyme ASMT (acetylserotonin O-methyltransferase) gene were generated and these melatonin enrich transgenic sheep were challenged by Brucella infection. The results showed that the serum melatonin concentration was significantly higher in transgenic sheep than that of wild type (726.92 ± 70.6074 vs 263.10 ± 34.60 pg/mL, P < .05). Brucella challenge test showed that two thirds (4/6) of the wild-type sheep had brucellosis, while none of the transgenic sheep were infected. Whole-blood RNA-seq results showed that differential expression genes (DEGs) were significantly enriched in natural killer cell-mediated cytotoxicity, phagosome, antigen processing, and presentation signaling pathways in overexpression sheep. The DEGs of toll-like receptors (TLRs) and NOD-like receptors (NLRs) families were verified by qPCR and it showed that TLR1, TLR2, TLR7, CD14, NAIP, and CXCL8 expression levels in overexpression sheep were significantly higher and NLRP1, NLRP3, and TNF expression levels were significantly lower than those of wild type. The rectal feces were subjected to 16S rDNA amplicon sequencing, and the microbial functional analysis showed that the transgenic sheep had significantly lower abundance of microbial genes related to infectious diseases compared to the wild type, indicating overexpression animals are likely more resistant to infectious diseases than wild type. Furthermore, exogenous melatonin treatment relieved brucellosis inflammation by upregulating anti-inflammatory cytokines IL-4 and downregulating pro-inflammatory IL-2, IL-6, and IFN-γ. Our preliminary results provide an informative reference for the study of the relationship between melatonin and brucellosis.
Subject(s)
Acetylserotonin O-Methyltransferase/genetics , Brucellosis/genetics , Brucellosis/immunology , Gastrointestinal Microbiome , Signal Transduction/immunology , Acetylserotonin O-Methyltransferase/metabolism , Animals , Animals, Genetically Modified , Brucellosis/prevention & control , Feces/microbiology , Gastrointestinal Microbiome/genetics , Inflammation Mediators/immunology , Melatonin/therapeutic use , Sheep/immunologyABSTRACT
BACKGROUND: Transgenic animal production is an important means of livestock breeding and can be used to model pharmaceutical applications. METHODS: In this study, to explore the biological activity of endogenously produced melatonin, Acetylserotonin-O-methyltransferase (ASMT)-overexpressed melatonin-enriched dairy goats were successfully generated through the use of pBC1-ASMT expression vector construction and prokaryotic embryo microinjection. RESULTS: These transgenic goats have the same normal phenotype as the wild-type goats (WT). However, the melatonin levels in their blood and milk were significantly increased (p < 0.05). In addition, the quality of their milk was also improved, showing elevated protein content and a reduced somatic cell number compared to the WT goats. No significant changes were detected in the intestinal microbiota patterns between groups. When the animals were challenged by the intravenous injection of E. coli, the ASMT-overexpressed goats had a lower level of pro-inflammatory cytokines and higher anti-inflammatory cytokines compared to the WT goats. Metabolic analysis uncovered a unique arachidonic acid metabolism pattern in transgenic goats. CONCLUSIONS: The increased melatonin production due to ASMT overexpression in the transgenic goats may have contributed to their improved milk quality and enhanced the anti-inflammatory ability compared to the WT goats.
Subject(s)
Acetylserotonin O-MethyltransferaseABSTRACT
1. Melatonin is an indole hormone that, among its myriad biological functions, regulates circadian and seasonal rhythms in animals. The ASMT gene plays an essential role in melatonin synthesis. However, in chickens, little is known about the regulatory elements governing its transcription.2. The following study identified the transcription start site of the chicken ASMT gene by 5'-RACE. Then, the proximal minimal promoter was identified using a series of 5' truncations of the ASMT promoter (e.g. -3502/+17, -2698/+17, -2003/+17, -1378/+17, and -254/+17). Site-directed mutagenesis, overexpression, and electrophoretic mobility shift assay (EMSA) were applied to show that the transcription factor Oct-1 binds to the promoter region of ASMT.3. The translation start site was located 19 bp upstream from the translational start site. The luciferase reporter assay confirmed that the core promoter of chicken ASMT gene was in the -254/+17 region. Using site-directed mutagenesis, overexpression, and EMSA, Oct-1 bound the promoter of ASMT.4. Overall, Oct1 plays an important role in the transcriptional regulation of chicken ASMT gene.
Subject(s)
Chickens , Melatonin , Animals , Chickens/genetics , Base Sequence , Gene Expression Regulation , Promoter Regions, GeneticABSTRACT
Acetylserotonin O-methyltransferase (ASMT) is a key enzyme in the synthesis of melatonin. Although melatonin has been shown to exhibit anticancer activity and prevents endocrine resistance in breast cancer, the role of ASMT in breast cancer progression remains unclear. In this retrospective study, we analyzed gene expression profiles in 27 data sets on 7244 patients from 11 countries. We found that ASMT expression was significantly reduced in breast cancer tumors relative to healthy tissue. Among breast cancer patients, those with higher levels of ASMT expression had better relapse-free survival outcomes and longer metastasis-free survival times. Following treatment with tamoxifen, patients with greater ASMT expression experienced longer periods before relapse or distance recurrence. Motivated by these results, we devised an ASMT gene signature that can correctly identify low-risk cases with a sensitivity and specificity of 0.997 and 0.916, respectively. This signature was robustly validated using 23 independent breast cancer mRNA array data sets from different platforms (consisting of 5800 patients) and an RNAseq data set from TCGA (comprising 1096 patients). Intriguingly, patients who are classified as high-risk by the signature benefit from adjuvant chemotherapy, and those with grade II tumors who are classified as low-risk exhibit improved overall survival and distance relapse-free outcomes following endocrine therapy. Together, our findings more clearly elucidate the roles of ASMT, provide strategies for improving the efficacy of tamoxifen treatment and help to identify those patients who may maximally benefit from adjuvant or endocrine therapies.
Subject(s)
Acetylserotonin O-Methyltransferase/genetics , Breast Neoplasms/drug therapy , Sequence Analysis, RNA/methods , Tamoxifen/therapeutic use , Up-Regulation , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Databases, Genetic , Female , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Humans , Neoplasm Grading , Oligonucleotide Array Sequence Analysis , Retrospective Studies , Survival Analysis , Treatment OutcomeABSTRACT
Terrestrialization is one of the most momentous events in the history of plant life, which leads to the subsequent evolution of plant diversity. The transition species, in this process, had to acquire a range of adaptive mechanisms to cope with the harsh features of terrestrial environments compared to that of aquatic habitat. As an ancient antioxidant, a leading regulator of ROS signaling or homeostasis, and a presumed plant master regulator, melatonin likely assisted plants transition to land and their adaption to terrestrial ecosystems. N-acetylserotonin methyltransferases (ASMT) and caffeic acid O-methyltransferases (COMT), both in the O-methyltransferase (OMT) family, catalyze the core O-methylation reaction in melatonin biosynthesis. How these two enzymes with close relevance evolved in plant evolutionary history and whether they participated in plant terrestrialization remains unknown. Using combined phylogenetic evidence and protein structure analysis, it is revealed that COMT likely evolved from ASMT by gene duplication and subsequent divergence. Newly emergent COMT gained a significantly higher ASMT activity to produce greater amounts of melatonin for immobile plants to acclimate to the stressful land environments after evolving from the more environmentally-stable aquatic conditions. The COMT genes possess more conserved substrate-binding sites at the amino acid level and more open protein conformation compared to ASMT, and getting a new function to catalyze the lignin biosynthesis. This development directly contributed to the dominance of vascular plants among the Earth's flora and prompted plant colonization of land. Thus, ASMT, together with its descendant COMT, might play key roles in plant transition to land. The current study provides new insights into plant terrestrialization with gene duplication contributing to this process along with well-known horizontal gene transfer.
Subject(s)
Acetylserotonin O-Methyltransferase , Melatonin , Acetylserotonin O-Methyltransferase/genetics , Ecosystem , Methyltransferases/genetics , Phylogeny , Serotonin/analogs & derivativesABSTRACT
In this article, we attempt to classify a potential dimorphism of melatonin production. Thus, a new concept of "reserve or maximum capacity of melatonin synthetic function" is introduced to explain the subtle dimorphism of melatonin production in mammals. Considering ASMT/ASMTL genes in the pseudoautosomal region of sex chromosomes with high prevalence of mutation in males, as well as the sex bias of the mitochondria in which melatonin is synthesized, we hypothesize the existence of a dimorphism in melatonin production to favor females, which are assumed to possess a higher reserve capacity for melatonin synthesis than males. Under physiological conditions, this subtle dimorphism is masked by the fact that cells or tissues only need baseline melatonin production, which can be accomplished without exploiting the full potential of melatonin's synthetic capacity. This capacity is believed to exceed the already remarkable nocturnal increase as observed within the circadian cycle. However, during aging or under stressful conditions, the reserve capacity of melatonin's synthetic function is required to be activated to produce sufficiently high levels of melatonin for protective purposes. Females seem to possess a higher reserve/maximum capacity for producing more melatonin than males. Thus, this dimorphism of melatonin production becomes manifest and detectable under these conditions. The biological significance of the reserve/maximum capacity of melatonin's synthetic function is to improve the recovery rate of organisms from injury, to increase resistance to pathogen infection, and even to enhance their chances of survival by maximizing melatonin production under stressful conditions. The higher reserve/maximum capacity of melatonin synthesis in females may also contribute to the dimorphism in longevity, favoring females in mammals.
Subject(s)
Melatonin/metabolism , Acetylserotonin O-Methyltransferase/genetics , Acetylserotonin O-Methyltransferase/metabolism , Animals , Biosynthetic Pathways , Female , Humans , Male , Melatonin/genetics , Methyltransferases/genetics , Methyltransferases/metabolism , Mitochondria/genetics , Mitochondria/metabolism , Sex CharacteristicsABSTRACT
Melatonin synthesis is controlled by aralkylamine N-acetyltransferase (AANAT: EC 2.3.1.87) acetylating serotonin (5-hydroxytryptamine; 5-HT) to N-acetylserotonin (NAS), and N-acetylserotonin O-methyltransferase (ASMT: EC 2.1.1.4) methylating NAS to melatonin (Mel; N-acetyl-5-methoxytryptamine). We examined the levels of expression of the aanat and asmt genes, Mel concentrations as well as AANAT isozyme activity in the eyeball (with retina) and skin of the three-spined stickleback (Gasterosteus aculeatus), at noon and midnight. We found mRNA of four genes (aanat1a, snat, asmt and asmt2) in the eyeball, and two (aanat1a and asmt2) in the skin. The presence of two transcripts of genes encoding AANAT and two of ASMT in the eyeball at noon and midnight, suggests activity of AANAT and ASMT isozymes in metabolic pathways besides "the way to melatonin", all the more so because day/night changes in Mel concentration do not follow the changes in either the expression of genes or the activity of AANAT. The high effectiveness of noon NAS synthesis in the eyeball at low substrate concentrations, which is not reflected in high Mel production, suggests the function of eye NAS beyond that of a precursor to the biosynthesis of Mel. The inhibition of AANAT isozyme activity by product observed in the eyeball may be one of the mechanisms of 5-HT husbanding in the eye (retina). The presence of transcripts of genes encoding both AANAT and ASMT and the activity of AANAT, at noon and midnight, supports a local Mel synthesis in the sticklebacks' skin.
Subject(s)
Acetylserotonin O-Methyltransferase/metabolism , Arylalkylamine N-Acetyltransferase/metabolism , Melatonin/metabolism , Smegmamorpha/metabolism , Acetylserotonin O-Methyltransferase/genetics , Animals , Arylalkylamine N-Acetyltransferase/genetics , Eye/metabolism , Skin/metabolism , Smegmamorpha/genetics , Smegmamorpha/growth & developmentABSTRACT
Melatonin regulates the seasonal reproduction in photoperiodic sensitive animals. Its function in plants reproduction has not been extensively studied. In the current study, the effects of melatonin on the apple tree flowering have been systematically investigated. For consecutive 2-year monitoring, it was found that the flowering was always associated with the drop of melatonin level in apple tree. Melatonin application before flowering postponed apple tree flowering with a dose-dependent manner. The increased melatonin levels at a suitable range also resulted in more flowering. The data indicated that similar to the animals, the melatonin also serves as the signal of the environmental light to regulate the plant reproduction. It was mainly the blue and far-red light to regulate the gene expression of melatonin synthetic enzymes and melatonin production in plants. The seasonal alterations of the blue and far-red lights coordinated well with the changes of the melatonin levels and led to decreased melatonin level before flowering. The mechanism studies showed that melatonin per se inhibits all the four flowering pathways in apple. The results not only provide the basic knowledge for melatonin research, but also uncover melatonin as a chemical message of light signal to mediate plant reproduction. This information can be potentially used to control flowering period and prolong the harvest time, helpfully to open a new avenue for increasing crop yield by melatonin application.
Subject(s)
Flowers/metabolism , Malus/metabolism , Melatonin/metabolism , Plant Growth Regulators/metabolism , Light , Photoperiod , SeasonsABSTRACT
Seasonal changes impact the melatonin production and immuno-activities in vertebrates. This is believed due to the photoperiodic alterations of the different seasons which impact the functions of pineal gland. The short photoperiod promotes pineal melatonin production. As a result, during the winter, animals have significantly higher levels of melatonin than in summer. However, the seasonal changes also include temperature changes. This factor has never been systemically investigated in animals. In the current study, we observed that increased temperature had limited influence on melatonin production. In contrast, cold temperature is the major factor to induce melatonin production in hamsters. Cold temperature per se can upregulate the expressions of melatonin synthetic gene AANAT and ASMT, which are the important enzymes for melatonin biosynthesis. The elevated melatonin levels induced by the cold exposure in hamster in turn, improve the immuno-responses of the animals with increased levels of IL1, 6, and 10 as well CD3. In addition, melatonin as a potent antioxidant and thermogenic agent would improve the survival chance of animals during cold weather.
Subject(s)
Cold Temperature , Immunity , Melatonin/biosynthesis , Seasons , Animals , Arylalkylamine N-Acetyltransferase/genetics , Arylalkylamine N-Acetyltransferase/metabolism , Cricetinae , Gene Expression Regulation , Melatonin/blood , Membrane Proteins/genetics , Membrane Proteins/metabolism , Pineal Gland/metabolismABSTRACT
Inflammatory bowel diseases, particularly ulcerative colitis (UC) and lymphocytic colitis (LC), affect many people. The role of melatonin in the pathogenesis of UC is precisely determined, whereas in LC it remains unknown. The aim of this study was to compare the expression of the melatonin-synthesizing enzymes tryptophan hydroxylase (TPH1), arylalkylamine-N-acetyltransferase (AANAT), and N-acetylserotonin methyltransferase (ASMT) in the colonic mucosa and urinary excretion of 6-sulfatoxymelatonin in patients with ulcerative and lymphocytic colitis. The study included 30 healthy subjects (group C), 30 patients with severe ulcerative colitis (group UC), and 30 patients with lymphocytic colitis (group LC). The diagnosis was based on endoscopic, histological, and laboratory examinations. Biopsy specimens were collected from right, transverse, and left parts of the colon. The levels of mRNA expression, TPH1, AANAT, and ASMT were estimated in the colonic mucosa with RT-PCR. The urine concentration of aMT6s was determined by the photometric method. The expression of TPH1, AANAT, and ASMT in colonic mucosa in UC and LC patients was significantly higher than in healthy subjects. Significant differences were found in the urinary aMT6s excretion: group C-13.4 ± 4.8 µg/24 h, group UC-7.8 ± 2.6 µg/24 h (p < 0.01), group LC-19.2 ± 6.1 µg/24 h (p < 0.01). Moreover, a negative correlation was found between fecal calprotectin and MT6s-in patients with UC - r = -0.888 and with LC - r = -0.658. These results indicate that patients with UC and those with LC may display high levels of melatonin-synthesizing enzymes in their colonic mucosa, which could possibly be related to increased melatonin synthesis as an adaptive antioxidant activity.
Subject(s)
Acetylserotonin O-Methyltransferase/metabolism , Arylalkylamine N-Acetyltransferase/metabolism , Colitis, Lymphocytic/metabolism , Colitis, Ulcerative/metabolism , Intestinal Mucosa/metabolism , Melatonin/metabolism , Tryptophan Hydroxylase/metabolism , Adult , Biopsy , Colitis, Lymphocytic/pathology , Colitis, Ulcerative/pathology , Female , Humans , Intestinal Mucosa/pathology , Male , Middle AgedABSTRACT
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.
Subject(s)
Acetylserotonin O-Methyltransferase/genetics , Arylalkylamine N-Acetyltransferase/genetics , CRISPR-Cas Systems/genetics , Mammary Glands, Animal/metabolism , Melatonin/metabolism , Sheep/metabolism , Acetylserotonin O-Methyltransferase/metabolism , Animals , Animals, Genetically Modified/genetics , Animals, Genetically Modified/metabolism , Arylalkylamine N-Acetyltransferase/metabolism , Cloning, Molecular , Female , Melatonin/analysis , Melatonin/chemistry , Melatonin/genetics , Milk/chemistry , Milk/metabolism , Sheep/geneticsABSTRACT
Acetylserotonin methyltransferase (ASMT) is the last enzyme of melatonin biosynthesis and may play a rate-limiting role in the melatonin production of plants. In this study, systematic analysis of the ASMT gene family in tomato (Solanum lycopersicum Mill) has been presented by the integration of the structural features, phylogenetic relationships, exon/intron configuration, and expression profile during growth and development, as well as biotic stresses. The results revealed that the tomato genome encoded a minimum of 14 members, containing three probable encoded pseudogenes. Chromosome mapping indicated that the family had probably expanded via tandem duplication events. Genome-wide RNA-seq and qRT-PCR based gene expression analysis revealed that almost half of the SlASMT genes were expressed in at least one of the experimental stages studied and also showed differential accumulation. Furthermore, the tandem duplicated SlASMT genes showed differential expression levels, which indicated probable functional divergence during the course of the evolution. Finally, this study also determined that some SlASMT genes were induced by multiple pathogens. The results suggested that these genes could be involved in tomato plant response to biotic stresses.
Subject(s)
Acetylserotonin O-Methyltransferase/genetics , Solanum lycopersicum/genetics , Acetylserotonin O-Methyltransferase/metabolism , Chromosome Mapping , Gene Expression Regulation, Plant , Genome, Plant , Solanum lycopersicum/metabolism , Melatonin/biosynthesis , Phylogeny , Stress, PhysiologicalABSTRACT
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.
Subject(s)
Acetylserotonin O-Methyltransferase , Brain Neoplasms , Cytochrome P-450 CYP1B1 , Genes, Neoplasm , Glioma , Melatonin , Neoplasm Proteins , Acetylserotonin O-Methyltransferase/genetics , Acetylserotonin O-Methyltransferase/metabolism , Animals , Brain Neoplasms/diagnosis , Brain Neoplasms/genetics , Brain Neoplasms/metabolism , Brain Neoplasms/mortality , Cell Line, Tumor , Cytochrome P-450 CYP1B1/genetics , Cytochrome P-450 CYP1B1/metabolism , Glioma/diagnosis , Glioma/genetics , Glioma/metabolism , Glioma/mortality , Humans , Melatonin/biosynthesis , Melatonin/genetics , NF-kappa B/metabolism , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Prognosis , RatsABSTRACT
The N-acetylserotonin O-methyltransferase (ASMT) gene encodes the enzyme that catalyzes the conversion of N-acetylserotonin to melatonin as the last step in melatonin biosynthesis. The first plant ASMT gene to be cloned was from rice. An orthologous gene encoding a protein with ASMT activity and only 39.7% amino acid sequence identity to the rice ASMT protein was recently isolated from apple (Malus zumi). The low homology of the apple ASMT sequence prompted us to screen the Arabidopsis genome for a homologous ASMT gene. The At4g35160 gene exhibited the highest sequence identity (31%) to the rice ASMT gene, followed by the At1g76790 gene with 29% sequence identity. We purified recombinant proteins expressed from the two Arabidopsis genes. The At4g35160 recombinant protein exhibited ASMT enzyme activity, but the At1g76790 recombinant protein did not; thus, we designated At4g35160 as an Arabidopsis thaliana ASMT (AtASMT) gene. The AtASMT protein catalyzed the conversion of N-acetylserotonin to melatonin and serotonin to 5-methoxytryptamine with Vmax values of 0.11 and 0.29 pkat/mg protein, respectively. However, AtASMT exhibited no caffeic acid O-methyltransferase activity, suggesting that its function was highly specific to melatonin synthesis. AtASMT transcripts were induced by cadmium treatment in Arabidopsis followed by increased melatonin synthesis. Similar to other ASMT proteins, AtASMT was localized in the cytoplasm and its ectopic overexpression in rice resulted in increased ASMT enzyme activity and melatonin production, indicating the involvement of AtASMT in melatonin synthesis.
Subject(s)
Acetylserotonin O-Methyltransferase , Arabidopsis Proteins , Arabidopsis , Melatonin/biosynthesis , Acetylserotonin O-Methyltransferase/biosynthesis , Acetylserotonin O-Methyltransferase/chemistry , Acetylserotonin O-Methyltransferase/genetics , Arabidopsis/enzymology , Arabidopsis/genetics , Arabidopsis Proteins/biosynthesis , Arabidopsis Proteins/chemistry , Arabidopsis Proteins/genetics , Cloning, Molecular , Melatonin/chemistry , Melatonin/genetics , Oryza/enzymology , Oryza/genetics , Plants, Genetically Modified/enzymology , Plants, Genetically Modified/genetics , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/geneticsABSTRACT
This study investigated the maturational stage (immature and mature ovaries) differences of mRNA expression of melatonin-forming enzymes (Aanat and Asmt), melatonin membrane receptors (Mt1 and Mt2) and putative nuclear (Rorα) receptors, and clock genes (Clock, Bmal1, Per1, Per2, Cry1, Cry2) in cumulus-oocyte complexes (COC) from weaning Wistar rats. We also examined the effects of pinealectomy and of melatonin pharmacological replacement on the daily expression of these genes in COC. qRT-PCR analysis revealed that in oocytes, the mRNA expression of Asmt, Mt2, Clock, Bmal1, Per2, and Cry1 were higher (P < 0.05) in immature ovaries than in the mature ones. In cumulus cells, the same pattern of mRNA expression for Asmt, Aanat, Rorα, Clock, Per1, Cry1, and Cry2 genes was observed. In oocytes, pinealectomy altered the daily mRNA expression profiles of Asmt, Mt1, Mt2, Clock, Per1, Cry1, and Cry2 genes. In cumulus cells, removal of the pineal altered the mRNA expression profiles of Mt1, Mt2, Rorα, Aanat, Asmt, Clock, Bmal1, Per2, Cry1, and Cry2 genes. Melatonin treatment partially or completely re-established the daily mRNA expression profiles of most genes studied. The mRNA expression of melatonin-related genes and clock genes in rat COC varies with the maturational stage of the meiotic cellular cycle in addition to the hour of the day. This suggests that melatonin might act differentially in accordance with the maturational stage of cumulus/oocyte complex. In addition, it seems that circulating pineal melatonin is very important in the design of the daily profile of mRNA expression of COC clock genes and genes related to melatonin synthesis and action.
Subject(s)
Cumulus Cells/metabolism , Melatonin/metabolism , Oocytes/metabolism , Pineal Gland/surgery , Animals , Arylalkylamine N-Acetyltransferase/metabolism , Circadian Rhythm/physiology , Female , Period Circadian Proteins/metabolism , Rats , Rats, Wistar , Receptors, Melatonin/metabolismABSTRACT
Melatonin is highly produced in the placenta where it protects against molecular damage and cellular dysfunction arising from hypoxia/re-oxygenation-induced oxidative stress as observed in primary cultures of syncytiotrophoblast. However, little is known about melatonin and its receptors in the human placenta throughout pregnancy and their role in villous trophoblast development. The purpose of this study was to determine melatonin-synthesizing enzymes, arylalkylamine N-acetyltransferase (AANAT) and hydroxyindole O-methyltransferase (HIOMT), and melatonin receptors (MT1 and MT2) expression throughout pregnancy as well as the role of melatonin and its receptors in villous trophoblast syncytialization. Our data show that the melatonin generating system is expressed throughout pregnancy (from week 7 to term) in placental tissues. AANAT and HIOMT show maximal expression at the 3rd trimester of pregnancy. MT1 receptor expression is maximal at the 1st trimester compared to the 2nd and 3rd trimesters, while MT2 receptor expression does not change significantly during pregnancy. Moreover, during primary villous cytotrophoblast syncytialization, MT1 receptor expression increases, while MT2 receptor expression decreases. Treatment of primary villous cytotrophoblast with an increasing concentration of melatonin (10 pM-1 mM) increases the fusion index (syncytium formation; 21% augmentation at 1 mM melatonin vs. vehicle) and ß-hCG secretion (121% augmentation at 1 mM melatonin vs. vehicle). This effect of melatonin appears to be mediated via its MT1 and MT2 receptors. In sum, melatonin machinery (synthetizing enzymes and receptors) is expressed in human placenta throughout pregnancy and promotes syncytium formation, suggesting an essential role of this indolamine in placental function and pregnancy well-being.