Step In Serotonin Biosynthesis Research Articles

Advances in the microbial synthesis of the neurotransmitter serotonin.

Serotonin, as a monoamine neurotransmitter, modulates the activity of the nervous system. Due to its importance in the coordination of movement and regulation of mood, impairments in the synthesis and homeostasis of serotonin are involved in numerous disorders, including depression, Parkinson's disease, and anxiety. Currently, serotonin is primarily obtained via natural extraction. But this method is time-consuming and low yield, as well as unstable supply of raw materials. With the development of synthetic biology, researchers have established the method of microbial synthesis of serotonin. Compared with natural extraction, microbial synthesis has the advantages of short production cycle, continuous production, not limited by season and source, and environment-friendly; hence, it has garnered considerable research attention. However, the yield of serotonin is still too low to industrialization. Therefore, this review provides the latest progress and examples that illustrate the synthesis pathways of serotonin as well as proposes strategies for increasing the production of serotonin. KEY POINTS: • Two biosynthesis pathways of serotonin are introduced. • L-tryptophan hydroxylation is the rate-limiting step in serotonin biosynthesis. • Effective strategies are proposed to improve serotonin production.

Structural characterization of human tryptophan hydroxylase 2 reveals that L-Phe is superior to L-Trp as the regulatory domain ligand

Tryptophan hydroxylase 2 (TPH2) catalyzes the rate-limiting step in serotonin biosynthesis in the brain. Consequently, regulation of TPH2 is relevant for serotonin-related diseases, yet the regulatory mechanism of TPH2 is poorly understood and structural and dynamical insights are missing. We use NMR spectroscopy to determine the structure of a 47 N-terminally truncated variant of the regulatory domain (RD) dimer of human TPH2 in complex with L-Phe, and show that L-Phe is the superior RD ligand compared with the natural substrate, L-Trp. Using cryo-EM, we obtain a low-resolution structure of a similarly truncated variant of the complete tetrameric enzyme with dimerized RDs. The cryo-EM two-dimensional (2D) class averages additionally indicate that the RDs are dynamic in the tetramer and likely exist in a monomer-dimer equilibrium. Our results provide structural information on the RD as an isolated domain and in the TPH2 tetramer, which will facilitate future elucidation of TPH2's regulatory mechanism.

Predictive analysis of Tryptophan Hydroxylase 2 (TPH2) missense mutations in psychiatric disorders

Psychiatric disorders are syndromes characterized by cognitive disturbance and behavioral dysfunction, which affect over 800 million people worldwide. It is considered a major public health problem responsible for severe distress with significant impairment in social and working relationships. In the United States and Canada, psychiatric disorders are considered the main cause of disability in young individuals, in addition to being a key factor underlying suicide. Missense mutations in tryptophan hydroxylase 2 enzyme (TPH2) are associated with the development of psychiatric disorders. TPH2 catalyzes the first step of serotonin biosynthesis, a neurotransmitter that plays a central role in the regulation of emotional behavior and cognition. These mutations lead to TPH2 dysfunction with impaired enzymatic activity, which ultimately results in abnormally low levels of serotonin in the brain. Despite the importance of missense mutations in TPH2 to the development of psychiatric disorders, most of them have not yet been characterized, so their effects are still unknown. In this study, we characterized the impact of missense mutations in TPH2 using prediction algorithms and evolutionary conservation analysis. We also used a penalty system to prioritize the most likely harmful mutations of TPH2 by combining the predictive analyses, evolutionary conservation, literature review, and alterations in physicochemical properties upon amino acid substitution. Three hundred and eighty-four missense mutations of TPH2 were compiled from the literature and databases. Our predictive analysis pointed to a high rate of deleterious and destabilizing predictions for the TPH2 mutations. These mutations mainly affect conserved and, possibly, functionally important residues. Among the uncharacterized mutations of TPH2, variants V295E, R441C T311P, Y281C, R441S, S383F, P308S, Y281H, and E363G received the highest penalties, thus, being the most likely deleterious and, consequently, important targets for future investigation. Our findings may guide the design of clinical and laboratory experiments, optimizing time and resources.

Gene Expression Profiling and Assessment of Vitamin D and Serotonin Pathway Variations in Patients With Irritable Bowel Syndrome.

Background/AimsIrritable bowel syndrome (IBS) is a multifaceted disorder that afflicts millions of individuals worldwide. IBS is currently diagnosed based on the presence/duration of symptoms and systematic exclusion of other conditions. A more direct manner to identify IBS is needed to reduce healthcare costs and the time required for accurate diagnosis. The overarching objective of this work is to identify gene expression-based biological signatures and biomarkers of IBS.MethodsGene transcripts from 24 tissue biopsy samples were hybridized to microarrays for gene expression profiling. A combination of multiple statistical analyses was utilized to narrow the raw microarray data to the top 200 differentially expressed genes between IBS versus control subjects. In addition, quantitative polymerase chain reaction was employed for validation of the DNA microarray data. Gene ontology/pathway enrichment analysis was performed to investigate gene expression patterns in biochemical pathways. Finally, since vitamin D has been shown to modulate serotonin production in some models, the relationship between serum vitamin D and IBS was investigated via 25-hydroxyvitamin D (25[OH]D) chemiluminescence immunoassay.ResultsA total of 858 genetic features were identified with differential expression levels between IBS and asymptomatic populations. Gene ontology enrichment analysis revealed the serotonergic pathway as most prevalent among the differentially expressed genes. Further analysis via real-time polymerase chain reaction suggested that IBS patient-derived RNA exhibited lower levels of tryptophan hydroxylase-1 expression, the enzyme that catalyzes the rate-limiting step in serotonin biosynthesis. Finally, mean values for 25(OH)D were lower in IBS patients relative to non-IBS controls.ConclusionsValues for serum 25(OH)D concentrations exhibited a trend towards lower vitamin D levels within the IBS cohort. In addition, the expression of select IBS genetic biomarkers, including tryptophan hydroxylase 1, was modulated by vitamin D. Strikingly, the direction of gene regulation elicited by vitamin D in colonic cells is “opposite” to the gene expression profile observed in IBS patients, suggesting that vitamin D may help “reverse” the pathological direction of biomarker gene expression in IBS. Thus, our results intimate that IBS pathogenesis and pathophysiology may involve dysregulated serotonin production and/or vitamin D insufficiency.

TPH2 in the ventral tegmental area of the male rat brain

This study surveyed the distribution of tryptophan hydroxylase 2 (TPH2) mRNA, protein, and enzymatic activity throughout the male Sprague–Dawley rat brain. TPH2 is the genetic isoform of TPH that catalyzes the rate-limiting step in serotonin biosynthesis within the central nervous system. Although cell bodies of serotonergic neurons are located mainly in the raphe, serotonin-containing axons innervate many regions of the brain. In the present study, we assessed the levels of mRNA, protein expression, and enzyme activity of TPH2 in the rat raphe, ventral tegmental area (VTA), substantia nigra, hippocampus, cerebellum, dorsal striatum, nucleus accumbens, amygdala, and medial prefrontal cortex to more fully understand the distribution of this enzyme throughout the central nervous system. The pineal gland was used as a control tissue that expresses TPH1 (the peripheral enzyme), but not TPH2. As expected, the raphe showed the highest brain TPH2 activity and protein expression. In the contrast to other reports, however, the VTA followed the raphe as the region with the second-highest amount of TPH2 activity, mRNA and protein expression. There were significantly lower TPH activities and levels of TPH2 protein in the other regions. In addition, TPH2 immunocytochemistry demonstrated the presence of TPH-positive cell bodies within the VTA. The results of this study indicate that TPH2 and serotonergic signaling may play an important role in the mesolimbic/mesocortical reward pathway.

Tissue-Specific Activities of an Immune Signaling Module Regulate Physiological Responses to Pathogenic and Nutritional Bacteria in C. elegans

Microbes represent both an essential source of nutrition and a potential source of lethal infection to the nematode Caenorhabditis elegans. Immunity in C. elegans requires a signaling module comprised of orthologs of the mammalian Toll-interleukin-1 receptor (TIR) domain protein SARM, the mitogen-activated protein kinase kinase kinase (MAPKKK) ASK1, and MAPKK MKK3, which activates p38 MAPK. We determined that the SARM-ASK1-MKK3 module has dual tissue-specific roles in the C. elegans response to pathogens--in the cell-autonomous regulation of innate immunity and the neuroendocrine regulation of serotonin-dependent aversive behavior. SARM-ASK1-MKK3 signaling in the sensory nervous system also regulates egg-laying behavior that is dependent on bacteria provided as a nutrient source. Our data demonstrate that these physiological responses to bacteria share a common mechanism of signaling through the SARM-ASK1-MKK3 module and suggest the co-option of ancestral immune signaling pathways in the evolution of physiological responses to microbial pathogens and nutrients.

Functional properties of missense variants of human tryptophan hydroxylase 2

Tryptophan hydroxylase 2 (TPH2) catalyzes the rate-limiting step in serotonin biosynthesis in the nervous system. Several variants of human TPH2 have been reported to be associated with a spectrum of neuropsychiatric disorders such as unipolar major depression, bipolar disorder, suicidality, and attention-deficit/hyperactivity disorder (ADHD). We used three different expression systems: rabbit reticulocyte lysate, Escherichia coli, and human embryonic kidney cells, to identify functional effects of all human TPH2 missense variants reported to date. The properties of mutants affecting the regulatory domain, that is, p.Leu36Val, p.Leu36Pro, p.Ser41Tyr, and p.Arg55Cys, were indistinguishable from the wild-type (WT). Moderate loss-of-function effects were observed for mutants in the catalytic and oligomerization domains, that is, p.Pro206Ser, p.Ala328Val, p.Arg441His, and p.Asp479Glu, which were manifested via stability and solubility effects, whereas p.Arg303Trp had severely reduced solubility and was completely inactive. All variants were tested as substrates for protein kinase A and were found to have similar phosphorylation stoichiometries. A standardized assay protocol as described here for activity and solubility screening should also be useful for determining properties of other TPH2 variants that will be discovered in the future.

Molecular cloning, characterization and expression of two tryptophan hydroxylase (TPH-1 and TPH-2) genes in the hypothalamus of Atlantic croaker: Down-regulation after chronic exposure to hypoxia

Recently we discovered that hypoxia causes marked impairment of reproductive neuroendocrine function in Atlantic croaker, a marine teleost, which is due to a decline in hypothalamic serotonergic activity. As a first step in understanding the molecular responses of the hypothalamic serotonergic system to hypoxia, we cloned and characterized the genes for the enzymes regulating the rate-limiting step in serotonin biosynthesis, tryptophan hydroxylase (TPH-1 and TPH-2) in the croaker brain. The full-length croaker TPH-1 and TPH-2 cDNAs contain open reading frames encoding proteins with 479 and 487 amino acids, respectively, which are highly homologous to the TPH-1 (76–93%) and TPH-2 (64–92%) proteins of other vertebrates. Croaker TPH-1 and TPH-2 mRNA expression was detected throughout the brain but was greatest in the hypothalamic region. Both Northern blot analysis and real-time PCR showed that TPH-1 (transcript size ∼2.1 kb) and TPH-2 (∼1.9 kb) mRNA levels were significantly decreased in the hypothalami of croaker exposed for 2 weeks to hypoxic conditions compared with those in fish exposed to normoxic conditions. Immunohistochemistry of hypothalamic neurons with TPH antibodies showed reduced expression of TPHs in hypoxia-exposed fish compared with normoxic fish. Western blot analysis confirmed that hypoxia caused a marked decline in hypothalamic TPH protein levels, which was associated with decreases in hypothalamic TPH enzyme activity and 5-hydroxytryptophan levels. These results suggest that TPH is a major site of hypoxia-induced down-regulation of serotonergic function in croaker brains. Moreover, they provide the first evidence that hypoxia decreases the expression of TPH transcripts in vertebrate brains.

Amino-terminal analysis of tryptophan hydroxylase: protein kinase phosphorylation occurs at serine-58.

Tryptophan hydroxylase (TPH) catalyzes the rate-limiting and committed step in serotonin biosynthesis. Within this enzyme, two distinct domains have been hypothesized to exist, an amino-terminal regulatory domain and a carboxyl-terminal catalytic domain. In the present experiments, the functional boundary between the putative domains was defined using deletion mutagenesis. A full-length cDNA clone for rabbit TPH was engineered for expression in bacteria. Five amino-terminal deletions were constructed using PCR, i.e., Ndelta50, Ndelta60, Ndelta90, Ndelta106, and Ndelta116 (referring to the number of amino acids deleted from the amino terminus). Enzymatic activity was determined for each mutant after expression in bacteria. Whereas deletion of 116 amino acids (Ndelta116) abolished enzyme activity, all of the other amino-terminal deletions exhibited increased specific activity relative to the recombinant wild-type TPH. The ability of the cyclic AMP-dependent protein kinase (PKA) to phosphorylate members of the deletion series was also examined. Deletion of the first 60 amino-terminal residues abolished the ability of the enzyme to serve as a substrate for PKA, yet the native and Ndelta50 enzymes were phosphorylated. Moreover, a serine-58 point mutant (S58A) was not phosphorylated by PKA. In conclusion, the first 106 amino acids comprise a regulatory domain that is phosphorylated by PKA at serine-58. In addition, the boundary between regulatory and catalytic domains is analogous to the domain structure observed for the related enzyme tyrosine hydroxylase.

A 6.1 kb 5′ upstream region of the mouse tryptophan hydroxylase gene directs expression of E. coli lacZ to major serotonergic brain regions and pineal gland in transgenic mice

Tryptophan hydroxylase (TPH) catalyzes the first step of serotonin biosynthesis in serotonergic neurons and neuroendocrine cells. Serotonin influences diverse vital physiological functions and is thought to play an important role in several human psychiatric disorders. To localize DNA element(s) important for serotonergic tissue-specific expression of TPH, 6.1 kb of the 5′ flanking region of the mouse TPH gene was fused to the coding region of the E. coli lacZ gene, and expression of the resulting fusion gene was analyzed in transgenic mice. The 6.1 kb of 5′ flanking sequence was able to direct the expression of a lacZ reporter gene to serotonergic tissues in six lines of transgenic mice. A high level of lacZ expression in transgenic mice carrying the fusion gene was detected in the pineal gland as well as a moderate level of lacZ expression in serotonergic brain regions such as the median and dorsal raphe nuclei, the nuclei raphe magnus and raphe pallidus. In contrast, a smaller 5′ flanking sequence of 1.1 kb directed no detectable serotonergic tissue-specific lacZ expression in five lines of transgenic mice. These results presented in this paper suggest first that DNA elements critical to serotonergic tissue-specific expression reside between −6.1 kb and −1.1 kb of 5′ flanking region of the mouse TPH gene, but second that this region confers a restricted tissue-specific expression.

Characterization of a tryptophan hydroxylating enzyme from malignant mouse mast cell

An enzyme has been partially purified from malignant mouse mast cells which catalyzes the hydroxylation of tryptophan and phenylalanine. This enzyme exhibits a marked dependency on ferrous ion and a tetrahydropteridine at an early stage of purification. The properties of this hydroxylase have been examined and the enzyme found to be distinct from liver phenylalanine hydroxylase. Catechol compounds and p-chlorophenylalanine are inhibitors of this enzyme. The discovery of potent inhibitors of this rate-limiting step in serotonin biosynthesis is of obvious importance.