Aromatic Amino Acid Hydroxylases Research Articles

Aromatic Amino Acid Hydroxylases as Off-Targets of Histone Deacetylase Inhibitors.

The aromatic amino acid hydroxylases (AAAHs) phenylalanine hydroxylase, tyrosine hydroxylase, and tryptophan hydroxylases 1 and 2 are structurally related enzymes that contain an active site iron atom and depend on tetrahydrobiopterin (BH4) as cosubstrate. Due to their important roles in synthesis of serotonin, dopamine, noradrenaline, and adrenaline and their involvement in cardiovascular, neurological, and endocrine disorders, AAAHs have been targeted by substrate analogs, iron chelators, and allosteric ligands. Phenylalanine hydroxylase is also off-target of the histone deacetylase (HDAC) inhibitor panobinostat. To systematically explore the binding of HDAC inhibitors to AAAHs, we screened a library of 307 HDAC inhibitors and structural analogs against tryptophan hydroxylase 1 using a fluorescence-based thermal stability assay, followed by activity assays. Selected hits were enzymatically tested against all four purified human AAAHs. Cellular thermal shift assay was performed for phenylalanine hydroxylase. We show that panobinostat and structurally related compounds such as TB57, which similarly to panobinostat also contains a cinnamoyl hydroxamate, bind to human AAAHs and inhibit these enzymes with high selectivity within the class (panobinostat inhibition (IC50): phenylalanine hydroxylase (18 nM) > tyrosine hydroxylase (450 nM) > tryptophan hydroxylase 1 (1960 nM). This study shows that panobinostat and related hydroxamic acid type HDAC inhibitors inhibit all AAAHs at therapeutically relevant concentrations. Our results warrant further investigations of the off-target relevance of HDAC inhibitors intended for clinical use and provide directions for new dual HDAC/AAAH and selective AAAH inhibitors. These findings may also provide a new mechanistic link between regulation of histone modification, AAAH function, and monoaminergic neurotransmission.

Characterization of Dnajc12 knockout mice, a model of hypodopaminergia.

Homozygous DNAJC12 c.79-2A>G (p. V27Wfs*14) loss-of-function mutations were first reported as a cause of young-onset Parkinson's disease. However, bi-allelic autosomal recessive pathogenic variants in DNAJC12 may lead to an alternative constellation of neurological features including infantile dystonia, developmental delay, intellectual disability and neuropsychiatric disorders. DNAJC12 is understood to co-chaperone aromatic amino acid hydroxylases to enhance the synthesis of biogenic amines. In vitro , we confirm overexpressed DNAJC12 forms a complex with tyrosine hydroxylase, the rate-limiting enzyme in dopamine (DA) synthesis. Now we describe a conditional knockout mouse (cDKO) in which loxP sites flanking Dnajc12 exon 2 enable its excision by cre-recombinase to create a constitutive Dnajc12 knock out (DKO). At three months of age, DKO animals exhibit reduced locomotion and exploratory behavior in automated open-field testing. DKO mice also manifest increased plasma phenylalanine levels, a cardinal feature of patients with DNAJC12 pathogenic variants. In striatal tissue, total DA and serotonin, and their metabolites, are reduced. Biochemical alterations in synaptic proteins and tyrosine hydroxylase are also apparent, with enhanced phosphorylation of pSer31 and pSer40 sites that may reflect biological compensation. Electrically-evoked striatal DA is reduced. Most immediately, cDKO and DKO mice present models to develop and refined therapeutic approaches for the treatment of DNAJC12 dystonia and parkinsonism. These models may also enable the pleiotropic functions of biogenic amines (including DA) to be individually investigated in the brain and periphery.

Mouse models for inherited monoamine neurotransmitter disorders.

Several mouse models have been developed to study human defects of primary and secondary inherited monoamine neurotransmitter disorders (iMND). As the field continues to expand, current defects in corresponding mouse models include enzymes and a molecular co-chaperone involved in monoamine synthesis and metabolism (PAH, TH, PITX3, AADC, DBH, MAOA, DNAJC6), tetrahydrobiopterin (BH4) cofactor synthesis and recycling (adGTPCH1/DRD, arGTPCH1, PTPS, SR, DHPR), and vitamin B6 cofactor deficiency (ALDH7A1), as well as defective monoamine neurotransmitter packaging (VMAT1, VMAT2) and reuptake (DAT). No mouse models are available for human DNAJC12 co-chaperone and PNPO-B6 deficiencies, disorders associated with recessive variants that result in decreased stability and function of the aromatic amino acid hydroxylases and decreased neurotransmitter synthesis, respectively. More than one mutant mouse is available for some of these defects, which is invaluable as different variant-specific (knock-in) models may provide more insights into underlying mechanisms of disorders, while complete gene inactivation (knock-out) models often have limitations in terms of recapitulating complex human diseases. While these mouse models have common phenotypic traits also observed in patients, reflecting the defective homeostasis of the monoamine neurotransmitter pathways, they also present with disease-specific manifestations with toxic accumulation or deficiency of specific metabolites related to the specific gene affected. This review provides an overview of the currently available models and may give directions toward selecting existing models or generating new ones to investigate novel pathogenic mechanisms and precision therapies.

LC-MS/MS methods for direct measurement of sepiapterin and tetrahydrobiopterin in human plasma and clinical applications.

Aim: Tetrahydrobiopterin (BH4), a natural cofactor of aromatic amino acid hydroxylases, and sepiapterin, a natural precursor of BH4, are endogenously present in human plasma. This is the first report on methods for direct quantification of sepiapterin and BH4 in human plasma by LC-MS/MS for pharmacokinetic assessment. Materials & methods: The analytes in plasma were harvested from blood that were treated with 10% ascorbic acid (AA) to a final concentration of 1% AA. Results & conclusion: The quantification methods were validated for calibration ranges of 0.75-500ng/ml and 0.5-500ng/ml for sepiapterin and BH4, respectively. Quantification of analytes was challenging due to their susceptibility to redox reactions. The validated methods were utilized successfully to support clinical development of sepiapterin.

In Vitro and In Vivo Chaperone Effect of (R)-2-amino-6-(1R, 2S)-1,2-dihydroxypropyl)-5,6,7,8-tetrahydropterin-4(3H)-one on the C1473G Mutant Tryptophan Hydroxylase 2.

Tryptophan hydroxylase 2 (TPH2) is the key and rate-limiting enzyme of serotonin (5-HT) synthesis in the mammalian brain. The 1473G mutation in the Tph2 gene decreases TPH2 activity in the mouse brain by twofold. (R)-2-amino-6-(1R, 2S)-1,2-dihydroxypropyl)-5,6,7,8-tetrahydropterin-4(3H)-one (BH4) is a pharmacological chaperone for aromatic amino acid hydroxylases. In the present study, chaperone effects of BH4 on the mutant C1473G TPH2 were investigated in vitro and in vivo. In vitro BH4 increased the thermal stability (T50 value) of mutant and wild-type TPH2 molecules. At the same time, neither chronic (twice per day for 7 days) intraperitoneal injection of 48.3 mg/kg of BH4 nor a single intraventricular administration of 60 μg of the drug altered the mutant TPH2 activity in the brain of Balb/c mice. This result indicates that although BH4 shows a chaperone effect in vitro, it is unable to increase the activity of mutant TPH2 in vivo.

Peripheralized sepiapterin reductase inhibition as a safe analgesic therapy.

The development of novel analgesics for chronic pain in the last 2decades has proven virtually intractable, typically failing due to lack of efficacy and dose-limiting side effects. Identified through unbiased gene expression profiling experiments in rats and confirmed by human genome-wide association studies, the role of excessive tetrahydrobiopterin (BH4) in chronic pain has been validated by numerous clinical and preclinical studies. BH4 is an essential cofactor for aromatic amino acid hydroxylases, nitric oxide synthases, and alkylglycerol monooxygenase so a lack of BH4 leads to a range of symptoms in the periphery and central nervous system (CNS). An ideal therapeutic goal therefore would be to block excessive BH4 production, while preventing potential BH4 rundown. In this review, we make the case that sepiapterin reductase (SPR) inhibition restricted to the periphery (i.e., excluded from the spinal cord and brain), is an efficacious and safe target to alleviate chronic pain. First, we describe how different cell types that engage in BH4 overproduction and contribute to pain hypersensitivity, are themselves restricted to peripheral tissues and show their blockade is sufficient to alleviate pain. We discuss the likely safety profile of peripherally restricted SPR inhibition based on human genetic data, the biochemical alternate routes of BH4 production in various tissues and species, and the potential pitfalls to predictive translation when using rodents. Finally, we propose and discuss possible formulation and molecular strategies to achieve peripherally restricted, potent SPR inhibition to treat not only chronic pain but other conditions where excessive BH4 has been demonstrated to be pathological.

Enzyme Promiscuity in Serotonin Biosynthesis, From Bacteria to Plants and Humans.

Serotonin is a phylogenetically ancient compound found in animals, plants, and some bacteria. In eukaryotes, serotonin is synthesized from the aromatic amino acid tryptophan via the key enzymes aromatic amino acid hydroxylase (AAAH) and aromatic amino acid decarboxylase (AAAD). Serotonin is also an intermediate in the melatonin biosynthetic pathway and is involved in several vital functions. In humans, serotonin is produced in the gut and in the brain, is critical in the regulation of multiple body functions, and its depletion has been implicated in multiple neurological disorders including depression and Alzheimer’s disease, as well as other peripheral conditions namely irritable bowel syndrome and fibromyalgia. The serotonin biosynthetic pathway is well described in eukaryotes, but very little is known about this pathway in bacteria. Evidence points to similar pathways since eukaryote-like AAAH and AAAD (and their genes) have been identified in multiple bacteria, even though serotonin production has not yet been detected in most species. Although data on bacterial tryptophan decarboxylase genes are very limited and no bacterial tryptophan hydroxylase genes have been identified to date, evidence suggests that serotonin production in bacteria might occur through different AAAH and AAAD. Substrate promiscuity in these enzymes has been previously reported and seems to be the key aspect in bacterial serotonin synthesis. Considering the human gut microbiota as a potential source of serotonin, further investigation on its biosynthetic pathways in microbes might lead to important discoveries, which may ultimately foster the development of new therapeutic strategies to treat serotonin depletion-related disorders in humans.

Structural studies of a novel auxiliary-domain-containing phenylalanine hydroxylase from Bacillus cereus ATCC 14579.

Phenylalanine hydroxylase (PAH), which belongs to the aromatic amino-acid hydroxylase family, is involved in protein synthesis and pyomelanine production through the hydroxylation of phenylalanine to tyrosine. In this study, the crystal structure of PAH from Bacillus cereus ATCC 14579 (BcPAH) with an additional 280 amino acids in the C-terminal region was determined. The structure of BcPAH consists of three distinct domains: a core domain with two additional inserted α-helices and two novel auxiliary domains: BcPAH-AD1 and BcPAH-AD2. Structural homologues of BcPAH-AD1 and BcPAH-AD2 are known to be involved in mRNA regulation and protein-protein interactions, and thus it was speculated that BcPAH might utilize the auxiliary domains for interaction with its partner proteins. Furthermore, phylogenetic tree analysis revealed that the three-domain PAHs, including BcPAH, are completely distinctive from both conventional prokaryotic PAHs and eukaryotic PAHs. Finally, biochemical studies of BcPAH showed that BcPAH-AD1 might be important for the structural integrity of the enzyme and that BcPAH-AD2 is related to enzyme stability and/or activity. Investigations into the intracellular functions of the two auxiliary domains and the relationship between these functions and the activity of PAH are required.

4-Arylthiosemicarbazide Derivatives as Toxoplasmic Aromatic Amino Acid Hydroxylase Inhibitors and Anti-inflammatory Agents.

Approximately one-third of the human population is infected with the intracellular cosmopolitan protozoan Toxoplasma gondii (Tg), and a specific treatment for this parasite is still needed. Additionally, the increasing resistance of Tg to drugs has become a challenge for numerous research centers. The high selectivity of a compound toward the protozoan, along with low cytotoxicity toward the host cells, form the basis for further research, which aims at determining the molecular targets of the active compounds. Thiosemicarbazide derivatives are biologically active organic compounds. Previous studies on the initial preselection of 58 new 4-arylthiosemicarbazide derivatives in terms of their anti-Tg activity and selectivity made it possible to select two promising derivatives for further research. One of the important amino acids involved in the proliferation of Tg and the formation of parasitophorous vacuoles is tyrosine, which is converted by two unique aromatic amino acid hydroxylases to levodopa. Enzymatic studies with two derivatives (R: para-nitro and meta-iodo) and recombinant aromatic amino acid hydroxylase (AAHs) obtained in the E. coli expression system were performed, and the results indicated that toxoplasmic AAHs are a molecular target for 4-arylthiosemicarbazide derivatives. Moreover, the drug affinity responsive target stability assay also confirmed that the selected compounds bind to AAHs. Additionally, the anti-inflammatory activity of these derivatives was tested using THP1-Blue™ NF-κB reporter cells due to the similarity of the thiosemicarbazide scaffold to thiosemicarbazone, both of which are known NF-κB pathway inhibitors.

EP011: Diagnosis of DNAJC12-deficient hyperphenylalaninemia offers targeted therapeutic options to counteract neurotransmitter deficiency

DNAJC12-related hyperphenylalaninemia is a recently described inborn error of metabolism (IEM) associated with hyperphenylalaninemia and neurotransmitter deficiency, caused by biallelic pathogenic variants in the DNAJC12 gene. Clinical features include global developmental delay, intellectual disability, autism spectrum disorder, and dystonia. DNAJC12-encoded protein functions as a chaperone facilitating the proper folding of the biopterin-dependent aromatic amino acid hydroxylases, including the phenylalanine, tyrosine, and tryptophan hydroxylases.

The Critical Role of Tetrahydrobiopterin (BH4) Metabolism in Modulating Radiosensitivity: BH4/NOS Axis as an Angel or a Devil.

Ionizing radiation and radioactive materials have been widely used in industry, medicine, science and military. The efficacy of radiotherapy and adverse effects of normal tissues are closed related to cellular radiosensitivity. Molecular mechanisms underlying radiosensitivity are of significance to tumor cell radiosensitization as well as normal tissue radioprotection. 5,6,7,8-Tetrahydrobiopterin (BH4) is an essential cofactor for nitric oxide synthases (NOS) and aromatic amino acid hydroxylases, and its biosynthesis involves de novo biosynthesis and a pterin salvage pathway. In this review we overview the role of BH4 metabolism in modulating radiosensitivity. BH4 homeostasis determines the role of NOS, affecting the production of nitric oxide (NO) and oxygen free radicals. Under conditions of oxidative stress, such as UV-radiation and ionizing radiation, BH4 availability is diminished due to its oxidation, which subsequently leads to NOS uncoupling and generation of highly oxidative free radicals. On the other hand, BH4/NOS axis facilitates vascular normalization, a process by which antiangiogenic therapy corrects structural and functional flaws of tumor blood vessels, which enhances radiotherapy efficacy. Therefore, BH4/NOS axis may serve as an angel or a devil in regulating cellular radiosensitivity. Finally, we will address future perspectives, not only from the standpoint of perceived advances in treatment, but also from the potential mechanisms. These advances have demonstrated that it is possible to modulate cellular radiosensitivity through BH4 metabolism.

Inhibition of Tryptophan Hydroxylases and Monoamine Oxidase-A by the Proton Pump Inhibitor, Omeprazole-In Vitro and In Vivo Investigations.

Serotonin (5-HT) is a hormone and neurotransmitter that modulates neural activity as well as a wide range of other physiological processes including cardiovascular function, bowel motility, and platelet aggregation. 5-HT synthesis is catalyzed by tryptophan hydroxylase (TPH) which exists as two distinct isoforms; TPH1 and TPH2, which are responsible for peripheral and central 5-HT, respectively. Due to the implication of 5-HT in a number of pathologies, including depression, anxiety, autism, sexual dysfunction, irritable bowel syndrome, inflammatory bowel disease, and carcinoid syndrome, there has been a growing interest in finding modulators of these enzymes in recent years. We thus performed high-throughput screening (HTS) using a fluorescence-based thermal shift assay (DSF) to search the Prestwick Chemical Library containing 1,280 compounds, mostly FDA-approved drugs, for TPH1 binders. We here report the identification of omeprazole, a proton pump inhibitor, as an inhibitor of TPH1 and TPH2 with low micromolar potency and high selectivity over the other aromatic amino acid hydroxylases. The S-enantiomer of omeprazole, esomeprazole, has recently also been described as an inhibitor of monoamine oxidase-A (MAO-A), the main enzyme responsible for 5-HT degradation, albeit with lower potency compared to the effect on TPH1 and TPH2. In order to investigate the net effect of simultaneous inhibition of TPH and MAO-A in vivo, we administered high-dose (100 mg/kg) omeprazole to CD-1 mice for 4 days, after which the animals were subjected to the tail suspension test. Finally, central (whole brain) and peripheral (serum) 5-HT content was measured using liquid chromatography-mass spectrometry (LC-MS). Omeprazole treatment significantly increased 5-HT concentrations, both in brain and in serum, and reduced the time spent immobile in the tail suspension test relative to vehicle control. Thus, the MAO-A inhibition afforded by high-dose omeprazole appears to overcome the opposing effect on 5-HT produced by inhibition of TPH1 and TPH2. Further modification of proton pump inhibitor scaffolds may yield more selective modulators of 5-HT metabolism.

Autoxidation and photooxidation of tetrahydrobiopterin: a theoretical study

Pterins are naturally occurring pigments and enzyme cofactors widespread in living organisms. Tetrahydrobiopterin (H4Bip) is a coenzyme of aromatic amino acid hydroxylases, NO-synthases, and alkylglycerol monooxygenases. This coenzyme is prone to oxidation in the presence of molecular oxygen, a so-called autoxidation. The reactions participating in H4Bip autoxidation are well known. However, our study is an attempt to evaluate theoretically the feasibility of reactions participating in autoxidation. To do so, we have calculated the Gibbs free energy of elementary reactions between H4Bip, its derivatives, molecular oxygen, and reactive oxygen species (ROS). In the last few years, we have established the photosensitized oxidation of H4Bip experimentally. Thus, we have also evaluated the feasibility of H4Bip photooxidation reactions, which may occur according to both type-I and type-II photosensitized oxidation. We calculated Fukui indices for H4Bip and found particular atoms in the molecule that interact with nucleophiles (for example, singlet oxygen 1O2) and radicals (in particular, molecular oxygen 3O2). Therefore, we evaluated the probability of H4Bip autoxidation reactions, photooxidation reactions, and the reactivity of particular atoms in H4Bip molecule using the theoretical methods of quantum chemistry.

Discovery and biological characterization of a novel scaffold for potent inhibitors of peripheral serotonin synthesis.

Aim: Tryptophan hydroxylase 1 (TPH1) catalyzes serotonin synthesis in peripheral tissues. Selective TPH1 inhibitors may be useful for treating disorders related to serotonin dysregulation. Results &methodology: Screening using a thermal shift assay for TPH1 binders yielded Compound 1 (2-(4-methylphenyl)-1,2-benzisothiazol-3(2H)-one), which showed high potency (50% inhibition at 98±30nM) and selectivity for inhibiting TPH over related aromatic amino acid hydroxylases in enzyme activity assays. Structure-activity relationships studies revealed several analogs of 1 showing comparable potency. Kinetic studies suggested a noncompetitive mode of action of 1, with regards to tryptophan and tetrahydrobiopterin. Computational docking studies and live cell assays were also performed. Conclusion: This TPH1 inhibitor scaffold may be useful for developing new therapeutics for treating elevated peripheral serotonin.

Phase I clinical evaluation of CNSA-001 (sepiapterin), a novel pharmacological treatment for phenylketonuria and tetrahydrobiopterin deficiencies, in healthy volunteers

Tetrahydrobiopterin (BH4) is the natural cofactor of aromatic amino acid hydroxylases and essential for degradation of phenylalanine and synthesis of catecholamines and serotonin. It can be synthesized either de novo from GTP or through the salvage pathway from sepiapterin. Sepiapterin, a natural precursor of BH4, is a more stable molecule and is transported more efficiently across cellular membranes, thus having potentially significant advantage over BH4 as a pharmacological agent for diseases associated with BH4-deficient conditions.We report the results of a first-in-humans, randomized, double-blind, placebo-controlled, dose-ranging, Phase I clinical trial in 83 healthy volunteers of CNSA-001, a novel formulation of sepiapterin. Single oral doses of 2.5–80 mg/kg CNSA-001 caused dose-related increases in plasma sepiapterin (mean Cmax 0.58–2.92 ng/mL) and BH4 (mean Cmax 57–312 ng/mL). Maximum plasma concentrations were achieved in about 1–2 h (sepiapterin) or about 4 h (BH4) after CNSA-001 oral intake.Increases in plasma BH4 were substantially larger in absolute terms and on a dose-for-dose basis following treatment with CNSA-001 vs. sapropterin dihydrochloride, a synthetic form of BH4. The pharmacokinetics of plasma sepiapterin and BH4 were similar before and after seven days of repeat daily dosing with CNSA-001 at 5, 20 or 60 mg/kg indicating little or no drug accumulation. Oral administration of CNSA-001 resulted in higher concentrations of sepiapterin in fasted vs. fed subjects, but overall BH4 plasma exposure following CNSA-001 intake increased by 1.7–1.8-fold in fed subjects. CNSA-001 was well tolerated, with no clear dose-relationship for adverse events (AE), no serious AE and no study discontinuations for AE. These data indicate that CNSA-001 is rapidly and efficiently converted to BH4 in humans supporting further clinical evaluation of CNSA-001 for the management of PKU, primary BH4 deficiencies and other diseases associated with deficient BH4 metabolism.

Phenylalanine hydroxylase variants interact with the co-chaperone DNAJC12.

DNAJC12, a type III member of the HSP40/DNAJ family, has been identified as the specific co-chaperone of phenylalanine hydroxylase (PAH) and the other aromatic amino acid hydroxylases. DNAJ proteins work together with molecular chaperones of the HSP70 family to assist in proper folding and maintenance of intracellular stability of their clients. Autosomal recessive mutations in DNAJC12 were found to reduce PAH levels, leading to hyperphenylalaninemia (HPA) in patients without mutations in PAH. In this work, we investigated the interaction of normal wild-type DNAJC12 with mutant PAH in cells expressing several PAH variants associated with HPA in humans, as well as in the Enu1/1 mouse model, homozygous for the V106A-Pah variant, which leads to severe protein instability, accelerated PAH degradation and mild HPA. We found that mutant PAH exhibits increased ubiquitination, instability, and aggregation compared with normal PAH. In mouse liver lysates, we showed that DNAJC12 interacts with monoubiquitin-tagged PAH. This form represented a major fraction of PAH in the Enu1/1 but was also present in liver of wild-type PAH mice. Our results support a role of DNAJC12 in the processing of misfolded ubiquitinated PAH by the ubiquitin-dependent proteasome/autophagy systems and add to the evidence that the DNAJ proteins are important players both for proper folding and degradation of their clients.

Progress on study of guanosine triphosphate cyclohydrolase 1

Guanosine triphosphate cyclohydrolase 1 (GTPCH1) is a protein encoded by the GCH1 gene, which catalyze GTP to tetrahydrofolinine (BH4) under physiological condition. BH4 is a coenzyme of aromatic amino acid hydroxylase and a cofactor of nitric oxide synthases. BH4 involves in the synthesis of various hormones and neurotransmitters and plays an important role in a series of pathophysiological processes in vivo. Recent studies showed that GTPCH1 is involved in the pathogenesis of neuropathic pain, dopa-responsive dystonia, cancer and cardiovascular diseases. In this review, we will discuss the role of GTPCH1 in those diseases mentioned above. Key words: GTP cyclohydrolase; Dystonia, dopa-responsive; Cardiovascular diseases; Neoplasms; Neuralgia; Review

Reversible S-glutathionylation of human 6-pyruvoyl tetrahydropterin synthase protects its enzymatic activity

6-Pyruvoyl tetrahydropterin synthase (PTS) converts 7,8-dihydroneopterin triphosphate into 6-pyruvoyltetrahydropterin and is a critical enzyme for the de novo synthesis of tetrahydrobiopterin, an essential cofactor for aromatic amino acid hydroxylases and nitric-oxide synthases. Neopterin derived from 7,8-dihydroneopterin triphosphate is secreted by monocytes/macrophages, and is a well-known biomarker for cellular immunity. Because PTS activity in the cell can be a determinant of neopterin production, here we used recombinant human PTS protein to investigate how its activity is regulated, especially depending on redox conditions. Human PTS has two cysteines: Cys-43 at the catalytic site and Cys-10 at the N terminus. PTS can be oxidized and consequently inactivated by H2O2 treatment, oxidized GSH, or S-nitrosoglutathione, and determining the oxidized modifications of PTS induced by each oxidant by MALDI-TOF MS, we show that PTS is S-glutathionylated in the presence of GSH and H2O2S-Glutathionylation at Cys-43 protected PTS from H2O2-induced irreversible sulfinylation and sulfonylation. We also found that PTS expressed in HeLa and THP-1 cells is reversibly modified under oxidative stress conditions. Our findings suggest that PTS activity and S-glutathionylation is regulated by the cellular redox environment and that reversible S-glutathionylation protects PTS against oxidative stress.

Tetrahydrobiopterin improves hippocampal nitric oxide-linked long-term memory

Tetrahydrobiopterin (BH4) is synthesized by the combined action of three metabolic pathways, namely de novo synthesis, recycling, and salvage pathways. The best-known function of BH4 is its mandatory action as a natural cofactor of the aromatic amino acid hydroxylases and nitric oxide synthases. Thus, BH4 is essential for the synthesis of nitric oxide, a retrograde neurotransmitter involved in learning and memory. We investigated the effect of BH4 (4–4000 pmol) intracerebroventricular administration on aversive memory, and on BH4 metabolism in the hippocampus of rodents. Memory-related behaviors were assessed in Swiss and C57BL/6 J mice, and in Wistar rats. It was consistently observed across all rodent species that BH4 facilitates aversive memory acquisition and consolidation by increasing the latency to step-down in the inhibitory avoidance task. This effect was associated with a reduced threshold to generate hippocampal long-term potentiation process. In addition, two inhibitors of memory formation (N(ω)-nitro-L-arginine methyl ester - L-Name – and dizocilpine - MK-801 -) blocked the enhanced effect of BH4 on memory, while the amnesic effect was not rescue by the co-administration of BH4 or a cGMP analog (8-Br-cGMP). The data strongly suggest that BH4 enhances aversive memory by activating the glutamatergic neurotransmission and the retrograde activity of NO. It was also demonstrated that BH2 can be converted into BH4 by activating the BH4 salvage pathway under physiological conditions in the hippocampus. This is the first evidence showing that BH4 enhances aversive memory and that the BH4 salvage pathway is active in the hippocampus.

Regulation of Tetrahydrobiopterin (BH4) Synthesis in the Nematode C. elegans

5,6,7,8‐tetrahydrobiopterin (BH4) is a cofactor required by the aromatic amino acid hydroxylases (AAAHs), nitric oxide synthases, and alkylglycerol monooxygenase (Werner et al., 2011, Biochem. J. 438: 397). Among the AAAHs are the rate‐limiting synthetic enzymes for serotonin and dopamine synthesis, and for conversion of phenylalanine (Phe) to tyrosine. In humans, the activity of phenylalanine hydroxylase (PAH) is essential for Phe catabolism; dysfunction of PAH, either from mutations in the PAH gene or in genes required for BH4 synthesis, results in phenylketonuria. Regulation of BH4 levels is important for many other physiological functions. The first and rate‐limiting step in BH4 de novo synthesis is performed by GTP Cyclohydrolase I (GTPCH1). Like in many biosynthesis pathways, this first enzyme is regulated by ‘end product feedback inhibition’ whereby BH4 inhibits GTPCH1 activity. In mammals, BH4 does not bind to GTPCH1 alone, but requires an additional protein called GTPCH1 feedback regulatory protein (GFRP) to inhibit GTPCH1. Crystal structures of rat GTPCH1 and GFRP with the BH4 analog BH2 show that the biopterin binds at the interface between the proteins (Maita et al., 2004, J Biol Chem 279: 51534). Phe increases BH4 production via GTPCH1 stimulation, also in a GFRP‐dependent fashion, similarly binding at the interface between GTPCH1 and GFRP. We have recently described the function and expression of genes required for BH4 synthesis and regeneration in the nematode C. elegans (Loer et al., 2015, Genetics 200: 237). Here we present work on the function of the gene gfrp‐1, which encodes a ortholog of mammalian GFRP, in the regulation of BH4 synthesis in C. elegans. Sequence analysis and structural predictions suggest the worm GFRP will function like the mammalian protein, binding both BH4 and Phe. In mammals, GTPCH1 and GFRP can also bind the selective inhibitor 2,4‐Diamino‐6‐hydroxypyrimidine (DAHP), which is structurally similar to both GTP (substrate) and BH4 (pathway end product). At low concentrations, DAHP apparently inhibits GTPCH1 like BH4 by binding with GFRP; this inhibition is GFRP‐dependent. At higher concentrations, DAHP acts as a competitive inhibitor, binding at the GTPCH1 active site like GTP; this inhibition is GFRP‐independent (Xie et al., 1998, J Biol Chem 273: 21091). Using a reduction‐of‐function mutant in the worm GTPCH1 gene [cat‐4(e3015) – S148N], we find that DAHP reduces BH4 production, as assessed by a significant reduction in serotonin immunoreactivity in whole worms. Treatment with Phe significantly increases serotonin levels in cat‐4(e3015) worms. We are currently assessing the effects of mutations in gfrp‐1 on serotonin levels (hence BH4 levels) in single mutants and cat‐4; gfrp‐1 double mutants, and whether the effects of DAHP and Phe on BH4 synthesis are GFRP‐dependent.Support or Funding InformationSupported by a Univ of San Diego Faculty Research Grant and an endowment from the Fletcher Jones Foundation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.