Human Sepiapterin Reductase Research Articles

Evaluation of the silkworm lemon mutant as an invertebrate animal model for human sepiapterin reductase deficiency.

Human sepiapterin reductase (SR) deficiency is an inherited disease caused by SPR gene mutations and is a monoamine neurotransmitter disorder. Here, we investigated whether the silkworm lemon mutant could serve as a model of SR deficiency. A point mutation in the BmSPR gene led to a five amino acid deletion at the carboxyl terminus in the lemon mutant. In addition, classical phenotypes seen in SR deficient patients were observed in the lemon mutant, including a normal phenylalanine level, a decreased dopamine and serotonin content, and an increased neopterin level. A recovery test showed that the replenishment of l-dopa significantly increased the dopamine level in the lemon mutant. The silkworm lemon mutant also showed negative behavioural abilities. These results suggest that the silkworm lemon mutant has an appropriate genetic basis and meets the biochemical requirements to be a model of SR deficiency. Thus, the silkworm lemon mutant can serve as a candidate animal model of SR deficiency, which may be helpful in facilitating accurate diagnosis and effective treatment options of SR deficiency.

Repurposing of Tranilast for Potential Neuropathic Pain Treatment by Inhibition of Sepiapterin Reductase in the BH4 Pathway.

Tetrahydrobiopterin (BH4) is a cofactor in the production of various signaling molecules including nitric oxide, dopamine, adrenaline, and noradrenaline. BH4 levels are critical for processes associated with cardiovascular function, inflammation, mood, pain, and neurotransmission. Increasing pieces of evidence suggest that BH4 is upregulated in chronic pain. Sepiapterin reductase (SPR) catalyzes both the reversible reduction of sepiapterin to dihydrobiopterin (BH2) and 6-pyruvoyl-tetrahydrobiopterin to BH4 within the BH4 pathway. Therefore, inhibition of SPR by small molecules can be used to control BH4 production and ultimately alleviate chronic pain. Here, we have used various in silico and in vitro experiments to show that tranilast, licensed for use in bronchial asthma, can inhibit sepiapterin reduction by SPR. Docking and molecular dynamics simulations suggest that tranilast can bind to human SPR (hSPR) at the same site as sepiapterin including S157, one of the catalytic triad residues of hSPR. Colorimetric assays revealed that tranilast was nearly twice as potent as the known hSPR inhibitor, N-acetyl serotonin. Tranilast was able to inhibit hSPR activity both intracellularly and extracellularly in live cells. Triple quad mass spectrophotometry of cell lysates showed a proportional decrease of BH4 in cells treated with tranilast. Our results suggest that tranilast can act as a potent hSPR inhibitor and therefore is a valid candidate for drug repurposing in the treatment of chronic pain.

Inhibition of Sepiapterin Mediated Formation of Dihydrobiopterin and Chemical Redox Cycling by Sulfa Drugs

Sepiapterin reductase (SPR) catalyzes the reduction of sepiapterin to dihydrobiopterin (BH2), a precursor for tetrahydrobiopterin (BH4), a cofactor critical for nitric oxide biosynthesis and alkylglycerol and aromatic amino acid metabolism. SPR also mediates chemical redox cycling, catalyzing one electron reduction of redox active chemicals including quinones; rapid reaction of these radicals with molecular oxygen generates reactive oxygen species (ROS). Using recombinant human SPR, sulfonamide and sulfonylurea based sulfa drugs were found to be potent non‐competitive inibitors of both sepiapterin reduction and redox cycling. The most active inhibitors of sepiapterin reduction (IC50's = 31‐180 nM) were sulfasalazine, sulfathiazole, sulfapyridine, sulfamethoxazole and chlorpropamide. Higher concentrations of the sulfa drugs (IC50's = 0.37‐19.4 µM) were required to inhibit redox cycling. In PC12 cells, which generate catecholamine and monoamine neurotransmitters via BH4‐dependent amino acid hydroxylases, sulfa drugs inhibited both BH2/BH4 biosynthesis and redox cycling. This resulted in decreased production of dopamine and dopamine metabolites, 3, 4‐dihydroxyphenylacetic acid and homovanillic acid, and 5‐hydroxytryptamine. This was reversed by BH4. These data suggest that SPR and BH4‐dependent enzymes, are ‘off‐targets' for sulfa drugs, which may underlie their side effects. AR055073, ES005022.

Sulfa drugs inhibit sepiapterin reduction and chemical redox cycling by sepiapterin reductase.

Sepiapterin reductase (SPR) catalyzes the reduction of sepiapterin to dihydrobiopterin (BH2), the precursor for tetrahydrobiopterin (BH4), a cofactor critical for nitric oxide biosynthesis and alkylglycerol and aromatic amino acid metabolism. SPR also mediates chemical redox cycling, catalyzing one-electron reduction of redox-active chemicals, including quinones and bipyridinium herbicides (e.g., menadione, 9,10-phenanthrenequinone, and diquat); rapid reaction of the reduced radicals with molecular oxygen generates reactive oxygen species (ROS). Using recombinant human SPR, sulfonamide- and sulfonylurea-based sulfa drugs were found to be potent noncompetitive inhibitors of both sepiapterin reduction and redox cycling. The most potent inhibitors of sepiapterin reduction (IC50s = 31-180 nM) were sulfasalazine, sulfathiazole, sulfapyridine, sulfamethoxazole, and chlorpropamide. Higher concentrations of the sulfa drugs (IC50s = 0.37-19.4 μM) were required to inhibit redox cycling, presumably because of distinct mechanisms of sepiapterin reduction and redox cycling. In PC12 cells, which generate catecholamine and monoamine neurotransmitters via BH4-dependent amino acid hydroxylases, sulfa drugs inhibited both BH2/BH4 biosynthesis and redox cycling mediated by SPR. Inhibition of BH2/BH4 resulted in decreased production of dopamine and dopamine metabolites, 3,4-dihydroxyphenylacetic acid and homovanillic acid, and 5-hydroxytryptamine. Sulfathiazole (200 μM) markedly suppressed neurotransmitter production, an effect reversed by BH4. These data suggest that SPR and BH4-dependent enzymes, are "off-targets" of sulfa drugs, which may underlie their untoward effects. The ability of the sulfa drugs to inhibit redox cycling may ameliorate ROS-mediated toxicity generated by redox active drugs and chemicals, contributing to their anti-inflammatory activity.

Sepiapterin Reductase Mediates Chemical Redox Cycling in Lung Epithelial Cells

In the lung, chemical redox cycling generates highly toxic reactive oxygen species that can cause alveolar inflammation and damage to the epithelium, as well as fibrosis. In this study, we identified a cytosolic NADPH-dependent redox cycling activity in mouse lung epithelial cells as sepiapterin reductase (SPR), an enzyme important for the biosynthesis of tetrahydrobiopterin. Human SPR was cloned and characterized. In addition to reducing sepiapterin, SPR mediated chemical redox cycling of bipyridinium herbicides and various quinones; this activity was greatest for 1,2-naphthoquinone followed by 9,10-phenanthrenequinone, 1,4-naphthoquinone, menadione, and 2,3-dimethyl-1,4-naphthoquinone. Whereas redox cycling chemicals inhibited sepiapterin reduction, sepiapterin had no effect on redox cycling. Additionally, inhibitors such as dicoumarol, N-acetylserotonin, and indomethacin blocked sepiapterin reduction, with no effect on redox cycling. Non-redox cycling quinones, including benzoquinone and phenylquinone, were competitive inhibitors of sepiapterin reduction but noncompetitive redox cycling inhibitors. Site-directed mutagenesis of the SPR C-terminal substrate-binding site (D257H) completely inhibited sepiapterin reduction but had minimal effects on redox cycling. These data indicate that SPR-mediated reduction of sepiapterin and redox cycling occur by distinct mechanisms. The identification of SPR as a key enzyme mediating chemical redox cycling suggests that it may be important in generating cytotoxic reactive oxygen species in the lung. This activity, together with inhibition of sepiapterin reduction by redox-active chemicals and consequent deficiencies in tetrahydrobiopterin, may contribute to tissue injury.

Abstract 448: Tetrahydrobiopterin Rescues Mitochondrial Dysfunction by Regulating Mitochondrial Biogenesis and Oxidative Phosphorylation

The multifunctional cofactor tetrahydrobiopterin (BH 4 ) has antioxidant effects, and low levels of BH 4 expression are implicated in various cardiovascular diseases involving mitochondrial dysfunction[1]. We investigated the role of BH 4 in the regulation of cardiac mitochondrial function using sepiapterin reductase knockout ( Spr– ) mice as a model of BH 4 deficiency[2]. Based on systematic data-integrative analysis of the mitochondrial proteome, we found that BH 4 deficiency resulted in significant remodeling of oxidative phosphorylation, which impaired mitochondrial membrane potential, oxygen consumption, and ATP production, and induced oxidative stress in mitochondria DNA, leading to cardiac contractile dysfunction. Moreover, the expression of major regulators of mitochondrial biogenesis and respiration, such as PGC1-α and mt TFA, was reduced by BH 4 deficiency. Finally, we confirmed that BH 4 supplementation could rescue cardiac mitochondrial biogenesis and oxidative phosphorylation, which demonstrates the therapeutic potential of BH 4 for the treatment of cardiovascular diseases involving mitochondrial dysfunction. Reference [1] Kim, H.K., Ha, S.H., and Han, J. (2010). Potential therapeutic applications of tetrahydrobiopterin: from inherited hyperphenylalaninemia to mitochondrial diseases. Ann N Y Acad Sci 1201 , 177-182. [2] Yang, S., Lee, Y.J., Kim, J.M., Park, S., Peris, J., Laipis, P., Park, Y.S., Chung, J.H., and Oh, S.P. (2006). A murine model for human sepiapterin-reductase deficiency. Am J Hum Genet 78 , 575-587.

The Silkworm Mutant lemon (lemon lethal) Is a Potential Insect Model for Human Sepiapterin Reductase Deficiency

Tetrahydrobiopterin (BH4) is an essential cofactor for aromatic acid hydroxylases, which control the levels of monoamine neurotransmitters. BH4 deficiency has been associated with many neuropsychological disorders. An inherited defect in BH4 biosynthesis is caused by the deficiency of sepiapterin reductase (SPR), which catalyzes the biosynthesis of BH4 from guanosine triphosphate at the terminal step. The human SPR gene has been mapped at the PARK3 locus, which is related to the onset of Parkinson disease. In this study, we report that mutant strains, lemon (lem) and its lethal allele lemon lethal (lem(1)) with yellow body coloration, of the silkworm Bombyx mori could be used as the first insect model for human SPR deficiency diseases. We demonstrated that mutations in the SPR gene (BmSpr) were responsible for the irregular body coloration of lem and lem(l). Moreover, biochemical analysis revealed that SPR activity in lem(l) larvae was almost completely diminished, resulting in a lethal phenotype that the larvae cannot feed and that die immediately after the first ecdysis. Oral administration of BH4 and dopamine to lem(l) larvae effectively increased their survival rates and feeding abilities. Our data demonstrate that BmSPR plays a crucial role in the generation of BH4, and monoamine neurotransmitters in silkworms and the lem (lem(l)) mutant strains will be an invaluable resource to address many questions regarding SPR and BH4 deficiencies.

Localization of sepiapterin reductase in the human brain

Sepiapterin reductase (SPR) is the enzyme that catalyzes the final step of the synthesis of tetrahydrobiopterin (BH4), the cofactor for phenylalanine hydroxylase, tyrosine hydroxylase (TH), tryptophan hydroxylase, and nitric oxide synthase (NOS). Although SPR is essential for synthesizing BH4, the distribution of SPR in the human brain has not yet been clarified. In the present study, we purified recombinant human SPR from cDNA, raised an antibody against human SPR (hSPR), and examined the localization of SPR protein and SPR activity. Human brain homogenates from the substantia nigra (SN), caudate nucleus (CN), gray and white matters of the cerebral cortex (CTX), and dorsal and ventral parts of the medulla oblongata (MO) were subjected to Western blot analysis with anti-hSPR antibody or with anti-TH antibody. Whereas TH protein showed a restricted localization, being mainly detected in the SN and CN, SPR protein was detected in all brain regions examined. SPR activity was relatively high compared with the activity of GTP cyclohydrolase I (GCH), the rate-limiting biosynthetic enzyme of BH4, and was more widely distributed than GCH activity. Immunohistochemistry revealed SPR immunoreactivity in pyramidal neurons in the cerebral CTX, in a small number of striatal neurons, and in neurons of the hypothalamic and brain stem monoaminergic fields and olivary nucleus. Double-staining immunohistochemistry showed that TH and SPR were colocalized in the SN dopamine neurons. Localization of SPR immunoreactive neurons corresponded to monoamine or NOS neuronal fields, and also to the areas where no monoamine or NOS neurons were located. The results indicate that there might be a BH4 biosynthetic pathway where GCH is not involved and that SPR might have some yet unidentified function(s) in addition to BH4 biosynthesis.

Mutational analysis of sites in sepiapterin reductase phosphorylated by Ca 2+/calmodulin-dependent protein kinase II

Sepiapterin reductase (SPR) catalyzes the last step in the pathway of tetrahydrobiopterin biosynthesis in tissues. SPR is phosphorylated by Ca 2+-dependent protein kinases, which indicates that Ca 2+-activated protein kinases may play a role in the regulation of SPR in vivo. Phosphorylation sites of rat sepiapterin reductase (rSPR) by Ca 2+/calmodulin-dependent protein kinase II were determined in the present study. Using specific monoclonal anti-phospho-Ser and -Thr antibodies, we found that only Ser residues of rSPR were phosphorylated. We constructed several point mutants of SPR by systematically replacing the three Ser residues by Ala ones. These mutants showed that all three Ser residues, i.e. S46, S196, and S214, of rSPR were phosphorylated. We also recognized that only Ser-213 of human SPR was phosphorylated. Each of these serine residues in SPR was found in the consensus sequence (Arg-X-X-Ser/Thr) of the phosphorylation site.

Genomic Organization and Chromosomal Localization of the Human Sepiapterin Reductase Gene

Sepiapterin reductase (SPR) catalyzes the final step of the biosynthetic pathway of tetrahydrobiopterin, which is an essential cofactor for aromatic amino acid hydroxylases and nitric oxide synthases. To aid the analysis of any possible human diseases caused by mutations in SPR, we have cloned and characterized the human SPR gene. The gene is composed of three exons spanning approximately 4 kilobases. The transcriptional starting point was determined around the cytosine nucleotide at position −81 by primer extension and RT-PCR analyses. There was no typical TATA-box within 300 bp from the transcriptional starting point. We found the Sp1-binding consensus sequence in the 5′-flanking region. The human SPR gene was mapped to chromosome band 2p13 by fluorescencein situhybridization.

Detection of a Novel Sepiapterin Reductase mRNA: Assay of mRNA in Various Cells and Tissues of Various Species

Fragments of cDNA coding for rat, murine, and human sepiapterin reductase (SR) were amplified by PCR via primer positioning close to the reported 3′-end of the coding region in the rat enzyme. They were sequenced and used as probes for mRNA detection. Northern blot analysis detected two mRNA species for SR. Their sizes were 1.3 and 2.1 kb for rat, 1.3 and 2.3 kb for mouse, and 1.6 and 2.1 kb for human cell lines. Comparison of rat cell lines and rat tissues indicated that in tissues only the 1.3-kb species is present. Washing of the Northern blots under different stringency conditions indicated a more stable interaction of the 1.3-kb mRNA species with the cDNA probe as compared to the 2.3-kb species. The 1.3-kb species corresponds to the reported 28.2-kDa molecular mass of rat SR monomer. SR mRNA expression is absent in the human NK-like cell line YT and in the murine erythroleukemia subclone B8/3, which both lack SR activity. Moreover, the relative mRNA expression correlates with the enzymatic activities of different cell lines within the same species. This indicates that SR activity is regulated by its steady state mRNA levels.

Cloning and sequencing of cDNA encoding human sepiapterin reductase: An enzyme involved in tetrahydrobiopterin biosynthesis

A full-length cDNA clone for sepiapterin reductase, an enzyme involved in tetrahydrobiopterin biosynthesis, was isolated from a human liver cDNA library by plaque hybridization. The nucleotide sequence of hSPR 8–25, which contained an entire coding region of the enzyme, was determined. The clone encoded a protein of 261 amino acids with a calculated molecular mass of 28,047 daltons. The predicted amino acid sequence of human sepiapterin reductase showed a 74% identity with the rat enzyme. We further found a striking homology between human SPR and carbonyl reductase, estradiol 17β-dehydrogenase, and 3β-hydroxy-5-ene steroid dehydrogenase, especially in their N-terminal region.

Cloning and Sequencing of cDNA Encoding Human Sepiapterin Reductase

Cloning and Sequencing of cDNA Encoding Human Sepiapterin Reductase

Purification and Characterization of a Carbonyl Reductase from Human Liver, which is Competent in the Reduction of 6-Pyruvoyl-Tetrahydropterin

Summary An enzyme which reduces 6-pyruvoyl-tetrahydropterin has been purified to apparent homogeneity from human liver. It consists of a single polypeptide chain with a molecular weight of 35 kDa, has an isoelectric point of 5.9 ± 0.1 and contains no glycosyl residues. The pure enzyme has a specific activity of 450 mU/mg protein at pH 7.0 in 10 mM potassium phosphate buffer. It converts 6-pyruvoyl-tetrahydropterin to 6-lactoyltetrahydropterin by transfer of the pro 4R-hydrogen of NADPH to form the side chain -OH at position C(2') of the substrate. Km values are 1.8 J..lM for 6-pyruvoyl-tetrahydropterin and 5.5 J..lM for NADPH. Polyclonal antibodies raised against the purified enzyme recognize 6-pyruvoyl-tetrahydropterin reductase in Western blot and ELISA but do not cross-react with human sepiapterin reductase. The enzyme appears to be identical with aldose reductase.