Biosynthesis Of Tetrahydrobiopterin Research Articles

Tetrahydrobiopterin and alkylglycerol monooxygenase substantially alter the murine macrophage lipidome

Tetrahydrobiopterin is a cofactor synthesized from GTP with well-known roles in enzymatic nitric oxide synthesis and aromatic amino acid hydroxylation. It is used to treat mild forms of phenylketonuria. Less is known about the role of tetrahydrobiopterin in lipid metabolism, although it is essential for irreversible ether lipid cleavage by alkylglycerol monooxygenase. Here we found intracellular alkylglycerol monooxygenase activity to be an important regulator of alkylglycerol metabolism in intact murine RAW264.7 macrophage-like cells. Alkylglycerol monooxygenase was expressed and active also in primary mouse bone marrow-derived monocytes and "alternatively activated" M2 macrophages obtained by interleukin 4 treatment, but almost missing in M1 macrophages obtained by IFN-γ and lipopolysaccharide treatment. The cellular lipidome of RAW264.7 was markedly changed in a parallel way by modulation of alkylglycerol monooxygenase expression and of tetrahydrobiopterin biosynthesis affecting not only various ether lipid species upstream of alkylglycerol monooxygenase but also other more complex lipids including glycosylated ceramides and cardiolipins, which have no direct connection to ether lipid pathways. Alkylglycerol monooxygenase activity manipulation modulated the IFN-γ/lipopolysaccharide-induced expression of inducible nitric oxide synthase, interleukin-1β, and interleukin 1 receptor antagonist but not transforming growth factor β1, suggesting that alkylglycerol monooxygenase activity affects IFN-γ/lipopolysaccharide signaling. Our results demonstrate a central role of tetrahydrobiopterin and alkylglycerol monooxygenase in ether lipid metabolism of murine macrophages and reveal that alteration of alkylglycerol monooxygenase activity has a profound impact on the lipidome also beyond the class of ether lipids.

Dopamine-Responsive Growth-Hormone Deficiency and Central Hypothyroidism in Sepiapterin Reductase Deficiency.

Sepiapterin reductase (SR) deficiency is a rare autosomal recessively inherited error of tetrahydrobiopterin (BH4) biosynthesis, resulting in disturbed dopaminergic and serotonergic neurotransmission. The clinical phenotype is characterized by dopa-responsive movement disorders including muscular hypotonia, dystonia, and parkinsonism. Due to the rarity of the disease, the phenotype of SR deficiency is far from being completely understood. Here, we report a 7-year-old boy, who was referred for diagnostic evaluation of combined psychomotor retardation, spastic tetraplegia, extrapyramidal symptoms, and short stature. Due to discrepancy between motor status and mental condition, analyses of biogenic amines and pterins in CSF were performed, leading to the diagnosis of SR deficiency. The diagnosis was confirmed by a novel homozygous mutation c.530G>C; p.(Arg177Pro) in exon 2 of the SPR gene. Because of persistent short stature, systematic endocrinological investigations were initiated. Insufficient growth-hormone release in a severe hypoglycemic episode after overnight fasting confirmed growth-hormone deficiency as a cause of short stature. In addition, central hypothyroidism was present. A general hypothalamic affection could be excluded. Since dopamine is known to regulate growth-hormone excretion, IGF-1, IGF-BP3, and peripheral thyroid hormone levels were monitored under L-dopa/carbidopa supplementation. Both growth-hormone-dependent factors and thyroid function normalized under treatment. This is the first report describing growth-hormone deficiency and central hypothyroidism in SR deficiency. It extends the phenotypic spectrum of the disease and identifies dopamine depletion as cause for the endocrinological disturbances.

Molecular Characterization of QDPR Gene in Iranian Families with BH4 Deficiency: Reporting Novel and Recurrent Mutations.

Newborn screening for PKU has been in practice in Iran since 2007. Some hyperphenylalaninemia cases have tetrahydrobiopterin (BH4) biosynthesis deficiency/disorder. Several genes including QDPR (encodes DHPR enzyme, the necessary cofactor for PAH activity) have been associated with the BH4. Mutations have been previously described in the QDPR gene. The incidence of BH4 deficiency is expected to be higher in Iran due to high rate of consanguineous marriages.We identified a total of 93 BH4-deficient families. A multiplex set of STR markers linked to 4 genes responsible for the BH4 deficiency (i.e., GCH1, PCBD1, PTS, and QDPR genes) was used to quickly determine which gene may be responsible to cause the disease. Mutation analysis of QDPR gene revealed some known and novel mutations. Our findings show that no common mutation predominates, and they are scattered in the gene in our population.

Regulation of iNOS function and cellular redox state by macrophage Gch1 reveals specific requirements for tetrahydrobiopterin in NRF2 activation

Inducible nitric oxide synthase (iNOS) is a key enzyme in the macrophage inflammatory response, which is the source of nitric oxide (NO) that is potently induced in response to proinflammatory stimuli. However, the specific role of NO production, as distinct from iNOS induction, in macrophage inflammatory responses remains unproven. We have generated a novel mouse model with conditional deletion of Gch1, encoding GTP cyclohydrolase 1 (GTPCH), an essential enzyme in the biosynthesis of tetrahydrobiopterin (BH4) that is a required cofactor for iNOS NO production. Mice with a floxed Gch1 allele (Gch1fl/fl) were crossed with Tie2cre transgenic mice, causing Gch1 deletion in leukocytes (Gch1fl/flTie2cre). Macrophages from Gch1fl/flTie2cre mice lacked GTPCH protein and de novo biopterin biosynthesis. When activated with LPS and IFNγ, macrophages from Gch1fl/flTie2cre mice induced iNOS protein in a manner indistinguishable from wild-type controls, but produced no detectable NO, as judged by L-citrulline production, EPR spin trapping of NO, and by nitrite accumulation. Incubation of Gch1fl/flTie2cre macrophages with dihydroethidium revealed significantly increased production of superoxide in the presence of iNOS expression, and an iNOS-independent, BH4-dependent increase in other ROS species. Normal BH4 levels, nitric oxide production, and cellular redox state were restored by sepiapterin, a precursor of BH4 production by the salvage pathway, demonstrating that the effects of BH4 deficiency were reversible. Gch1fl/flTie2cre macrophages showed only minor alterations in cytokine production and normal cell migration, and minimal changes in basal gene expression. However, gene expression analysis after iNOS induction identified 78 genes that were altered between wild-type and Gch1fl/flTie2cre macrophages. Pathway analysis identified decreased NRF2 activation, with reduced induction of archetypal NRF2 genes (gclm, prdx1, gsta3, nqo1, and catalase) in BH4-deficient Gch1fl/flTie2cre macrophages. These findings identify BH4-dependent iNOS regulation and NO generation as specific requirements for NRF2-dependent responses in macrophage inflammatory activation.

Anxiety- and depression-like phenotype of hph-1 mice deficient in tetrahydrobiopterin

Decreased tetrahydrobiopterin (BH4) biosynthesis has been implicated in the pathophysiology of anxiety and depression. The aim of this study was therefore to characterise the phenotype of homozygous hph-1 (hph) mice, a model of BH4 deficiency, in behavioural tests of anxiety and depression as well as determine hippocampal monoamine and plasma nitric oxide levels. In the elevated zero maze test, hph mice displayed increased anxiety-like responses compared to wild-type mice, while the marble burying test revealed decreased anxiety-like behaviour. This was particularly observed in male mice. In the tail suspension test, hph mice of both sexes displayed increased depression-like behaviours compared to wild-type counterparts, whereas the forced swim test showed a trend towards increased depression-like behaviours in male hph mice, but significant decrease in depression-like behaviours in female mice. This study provides the first evidence that congenital BH4 deficiency regulates anxiety- and depression-like behaviours. The altered responses observed possibly reflect decreased hippocampal serotonin and dopamine found in hph mice compared to wild-type mice, but also reduced nitric oxide formation. We propose that the hph-1 mouse may be a novel tool to investigate the role of BH4 deficiency in anxiety and depression.

Possible involvement of a tetrahydrobiopterin in photoreception for UV-B-induced anthocyanin synthesis in carrot.

Our previous studies of action spectra for UV-B-induced anthocyanin accumulation in cultured carrot cells indicated that a reduced form of pterin, possibly tetrahydrobiopterin, contributes to UV-B photoreception. In this report, we provide additional evidence for the involvement of pterin in UV-B light sensing. UV-B-induced phenylalanine ammonia-lyase (PAL) activity was considerably suppressed by N-acetylserotonin (an inhibitor of tetrahydrobiopterin biosynthesis), and this suppression was partially recovered by adding biopterin or tetrahydrobiobiopterin. In addition, protein(s) specifically bound to biopterin were detected by radiolabeling experiments in N-acetylserotonin-treated cells. Furthermore, diphenyleneiodonium, a potent inhibitor of electron transfer, completely suppressed UV-B-induced PAL activity. These results suggest the occurrence of an unidentified UV-B photoreceptor (other than UVR8, the tryptophan-based UV-B sensor originally identified in Arabidopsis) with reduced pterin in carrot cells. After reexamining published action spectra, we suggest that anthocyanin synthesis is coordinately regulated by these two UV-B sensors.

A functional polymorphism of the GTP cyclohydrolase 1 gene predicts attention performance

Guanosine triphosphate cyclohydrolase 1 (GCH1) is the rate-limiting enzyme for the biosynthesis of tetrahydrobiopterin, a cofactor for aromatic amino acid hydroxylases and nitric oxide synthases. As monoamine neurotransmitters are synthesized by the reactions catalyzed by tyrosine hydroxylase and tryptophan hydroxylase, alterations in the content of tetrahydrobiopterin affect the monoamine levels in the brain. Here, we examined the possible association of a functional single-nucleotide polymorphism (SNP) of the GCH1 gene, rs841 (C+243T), with attentional function as assessed by the Continuous Performance Test-Identical Pairs version (CPT-IP) in healthy individuals. We found that homozygous T/T genotype carriers of rs841 scored lower performance on the CPT-IP test. Our data suggest that alterations in GCH1 activity affect attentional function, especially sustained attention and vigilance.

Mitochondrial Dysfunction and Congenital Heart Disease

The pulmonary vasculature in infants born with congenital heart defects that cause increased Pulmonary Blood Flow (PBF) is subjected to pathologic mechanical forces, including chronically increased shear stress, resulting in early functional abnormalities of the vascular endothelium. These functional abnormalities occur prior to the development of well-described morphologic changes. However, little is known about the factors that transduce the abnormal shear forces associated with increased PBF into abnormal vascular function and reactivity. Recently the disruption of mitochondrial function has been identified as a new mechanism that leads to the development of pulmonary endothelial dysfunction. This is a complex process that involves post-translational regulation of multiple proteins and the mitochondrial redistribution of uncoupled endothelial nitric oxide synthase (eNOS) resulting in the disruption of carnitine metabolism and subsequently mitochondrial bioenergetics. As both eNOS and GTP cyclohydrolase I, the rate limiting enzyme in tetrahydrobiopterin (BH4) biosynthesis, are chaperoned by hsp90 this results in a “feed-forward” signaling cascade in which eNOS becomes progressively more uncoupled resulting in pulmonary endothelial dysfunction. This review will discuss the current knowledge in the field, the limitations in our understanding this complex process, and the potential for targeting mitochondrial function in the treatment of children born with congenital heart defects that result in increased pulmonary blood flow.

Novel Interaction of Ornithine Decarboxylase with Sepiapterin Reductase Regulates Neuroblastoma Cell Proliferation

Ornithine decarboxylase (ODC) is the sentinel enzyme in polyamine biosynthesis. Both ODC and polyamines regulate cell division, proliferation, and apoptosis. Sepiapterin reductase (SPR) catalyzes the last step in the biosynthesis of tetrahydrobiopterin (BH4), an essential cofactor of nitric oxide synthase, and has been implicated in neurological diseases but not yet in cancer. In this study, we present compelling evidence that native ODC and SPR physically interact, and we defined the individual amino acid residues involved in both enzymes using in silico protein–protein docking simulations. The resulting heterocomplex is a surprisingly compact structure, featuring two energetically and structurally equivalent binding modes both in monomer and in dimer conformations. The novel interaction between ODC and SPR proteins was confirmed under physiological conditions by co-immunoprecipitation and co-localization in neuroblastoma (NB) cells. Importantly, we showed that siRNA (small interfering RNA)-mediated knockdown of SPR expression significantly reduced endogenous ODC enzyme activity in NB cells, thus demonstrating the biological relevance of the ODC–SPR interaction. Finally, in a cohort of 88 human NB tumors, we found that high SPR mRNA expression correlated significantly with poor survival prognosis using a Kaplan–Meier analysis (log-rank test, P=5×10−4), suggesting an oncogenic role for SPR in NB tumorigenesis. In conclusion, we showed that ODC binds SPR and thus propose a new concept in which two well-characterized biochemical pathways converge via the interaction of two enzymes. We identified SPR as a novel regulator of ODC enzyme activity and, based on clinical evidence, present a model in which SPR drives ODC-mediated malignant progression in NB.

Increase in tetrahydrobiopterin concentration with aging in the cerebral cortex of the senescence-accelerated mouse prone 10 strain caused by abnormal regulation of tetrahydrobiopterin biosynthesis

6R-L-Erythro-5,6,7,8-tetrahydrobiopterin (BH4) is an essential cofactor for tyrosine hydroxylase (TH) activity and is a risk factor for cognitive decline and brain atrophy. Previous studies have shown that the decline in TH activity in the cerebral cortex of senescence-accelerated mouse prone 10 (SAMP10) mice is caused, at least in part, by a decrease in Fe, ferritin, and TH phosphorylation. We determined the concentrations of BH4 and the enzymes GTP cyclohydrolase-1,6-pyruvoyltetrahydropterin synthase and sepiapterin reductase (SPR) in the de novo pathway of BH4 biosynthesis. Dihydrofolate reductase (DHFR), which converts BH2 to BH4 in the salvage pathway of BH4 synthesis was also determined in the cerebral cortex of SAM mice at 3 and 12months of age. The BH4 concentration was measured by HPLC, and the protein levels of enzymes involved in BH4 synthesis were measured by western blot analysis. At 12months of age, BH4 concentration in the cerebral cortex of SAMP10 mice showed significantly higher values as compared to that of control mice. Further, the protein level of SPR in SAMP10 mice was significantly higher than that in SAMR1 mice at 3 and 12months of age. In contrast to SPR, the protein level of DHFR in SAMP10 mice was significantly lower than that in SAMR1 mice. These results indicate that abnormal regulation of BH4 metabolism occurs in the cerebral cortex of SAMP10 where the dysfunction of the salvage pathway of BH4 synthesis may cause overproduction of BH4 through the de novo pathway, which is considered characteristic in the cerebral cortex of SAMP10 with aging. Therefore, there is a possibility that the excess amounts of BH4 lead to age-related brain dysfunction in the cerebral cortex of SAMP10.

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.

Tetrahydrobiopterin Biosynthesis as an Off-Target of Sulfa Drugs

The introduction of sulfa drugs for the chemotherapy of bacterial infections in 1935 revolutionized medicine. Although their mechanism of action is understood, the molecular bases for most of their side effects remain obscure. Here, we report that sulfamethoxazole and other sulfa drugs interfere with tetrahydrobiopterin biosynthesis through inhibition of sepiapterin reductase. Crystal structures of sepiapterin reductase with bound sulfa drugs reveal how structurally diverse sulfa drugs achieve specific inhibition of the enzyme. The effect of sulfa drugs on tetrahydrobiopterin-dependent neurotransmitter biosynthesis in cell-based assays provides a rationale for some of their central nervous system-related side effects, particularly in high-dose sulfamethoxazole therapy of Pneumocystis pneumonia. Our findings reveal an unexpected aspect of the pharmacology of sulfa drugs and might translate into their improved medical use.

Chronic Estrogen Deficiency Causes Gastroparesis by Altering Neuronal Nitric Oxide Synthase Function

Gastroparesis affects predominantly females; however, the biological basis for this gender bias is completely unknown. Several lines of evidence suggest that nitrergic dependent stomach motility function is reduced in diabetic gastroparesis and that nNOS is estrogen-regulated. The purpose of this study was to investigate whether reduced levels of estradiol-17β (E2) down-regulates tetrahydrobiopterin (BH4, a cofactor for nNOS dimerization and enzyme activity) biosynthesis and therefore nNOS mediated gastric motility would be impaired in a mouse model of chronic estrogen deficiency, follicle stimulating hormone receptor knock-out female mice (FORKO). In-bred 12-week-old female FORKO mice were obtained from our FORKO breeding colony. Gastric emptying was measured in overnight fasting mice. Nitrergic relaxation (AUC/mg tissue) was measured at 2 Hz through electric field stimulation using gastric antrum strips prepared from WT and FORKO mice. Protein expression for nNOSα, BH4 biosynthesis enzymes (GCH-1, DHFR) and estrogen receptors (α, β) were measured in gastric antrum by western blotting. Levels of BH4 and oxidized BH2, B biopterin levels were determined by HPLC. In FORKO, compared to wild type (WT) stomachs we indentified (1) reduced (%) gastric emptying (64 ± 2.5 vs. 77.6 ± 0.88), (2) greater reduction in nitregic relaxation (-0.13 ± 0.012 vs. -0.28 ± 0.012), (3) increased nNOS dimerization (0.48 ± 0.02 vs. 0.34 ± 0.05), (4) decreased NO release whether measured at 24 h (0.6 ± 0.04 vs. 1.7 ± 0.22, p < 0.05) or at 48 h (3.4 ± 0.26 vs. 5.0 ± 0.15, p < 0.05) of incubation, (5) decreased GCH-1 (1.9 ± 0.06 vs. 2.3 ± 0.04), DHFR (1.8 ± 0.14 vs. 2.4 ± 0.07) and ERα (2.7 ± 0.4 vs. 3.9 ± 0.4) and (6) increased oxidized biopterin levels and decreased ratio of BH4 versus BH2 + B. We conclude that chronic estrogen deficiency negatively modifies the function of both BH4 and nNOS thereby contributing to the development of gastroparesis in a FORKO mouse model.

The neurobiology of tetrahydrobiopterin biosynthesis: A model for regulation of GTP cyclohydrolase I gene transcription within nigrostriatal dopamine neurons

Within the brain, the reduced pteridine cofactor 6R-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4) is absolutely required for the synthesis of the monoamine (MA) neurotransmitters dopamine (DA), norepinephrine, epinephrine (E), and serotonin (5-HT), the novel gaseous neurotransmitter nitric oxide and the production of yet to be identified 1-O-alkylglycerol-derived lipids. GTP cyclohydrolase I (GTPCH) catalyzes the first and limiting step in the BH4 biosynthetic pathway, which is now thought to involve up to eight different proteins supporting six alternate de novo and two alternate salvage pathways. Gene expression analysis across different regions of the human brain shows the abundance of transcripts coding for all eight of these proteins to be highly correlated with each other and to be enriched within human MA neurons. The potential for multiple routes for BH4 synthesis therefore exists within the human brain. GTPCH expression is particularly heterogeneous across different populations of human and rodent MA-containing neurons, with low expression levels and therefore BH4 being a characteristic of nigrostriatal DA (NSDA) neurons. Basic knowledge of how GCH1 gene transcription is controlled within NSDA neurons may explain the distinctive susceptibility of these neurons to human genetic mutations that result in BH4 deficiency. A model for cyclic adenosine monophosphate-dependent GCH1 transcription is described that involves a unique combination of DNA regulatory sequences and transcription factors. This model proposes that low levels of GCH1 transcription within NSDA neurons are driven by their distinctive physiology, suggesting that pharmacological manipulation of GCH1 gene transcription can be used to modify BH4 levels and therefore DA synthesis in the basal ganglia.

Association of monoamine-synthesizing genes with the depression tendency and personality in chronic fatigue syndrome patients

AimsTyrosine hydroxylase (TH) and GTP cyclohydrolase I (GCH) are the rate-limiting enzymes for the biosynthesis of catecholamines and tetrahydrobiopterin (BH4), respectively. Since catecholamines and BH4 are thought to be involved in the pathophysiology of CFS, we explored the genetic factors that influence CFS development and examined the possible association between the SNPs of the TH and GCH genes and the various characteristics of CFS patients. Main methodsAfter drawing venous blood from CFS patients and controls, genomic DNA was then extracted from whole blood in accordance with standard procedures. Digestion patterns of the PCR products were used for genotyping the SNPs of GCH (rs841; C+243T) and TH (rs10770141; C−824T). We also performed questionnaires consisting of fatigue-scale and temperament and character inventory scale (TCI) to CFS patients. Key findingsOur results demonstrated that the allele differences for the GCH and TH SNPs were not associated with CFS patients. We did find that the GCH gene with the C+243T polymorphism affected harm avoidance, while the TH gene with the C−824T polymorphism affected persistence in the CFS patients. The concept of persistence has been linked to specific personality, such as perfectionism, in CFS. SignificanceOur results suggest that the biosynthetic pathways of the monoamine neurotransmitters that are mediated by TH and GCH might be associated with the CFS clinical findings, because persistence is one of the typical personality traits observed in CFS and patients with major depressive disorder exhibit a higher harm avoidance score.

De Novo Biosynthesis of Tetrahydrobiopterin is Critical in Protecting against Ionizing Radiation

De Novo Biosynthesis of Tetrahydrobiopterin is Critical in Protecting against Ionizing Radiation

Abstract 2495: Sepiapterin reductase interacts with Ornithine Decarboxylase and regulates neuroblastoma proliferation

Abstract Ornithine Decarboxylase (ODC) is a key, rate-limiting enzyme in mammalian polyamine biosynthesis that is upregulated in various types of cancer including neuroblastoma (NB). MYCN is a transcription factor and proto-oncogene that has been shown to be a key prognostic factor associated with advanced stage NB. ODC is a transcriptional target of MYCN, and has been found to play a critical role in NB progression. Previous studies have shown that inhibition of ODC through drugs such as alpha-difluoromethylornithine (DFMO) depletes intracellular polyamine pools and induces G1 cell cycle arrest. Our previous studies have shown that the anti-proliferative effect of DFMO in NB involves upregulation of the cyclin dependent kinase inhibitor p27 and downregulation of MYCN. In a search for novel regulators of ODC, we recently discovered that sepiapterin reductase (SPR), a key enzyme in the biosynthesis of tetrahydrobiopterin, binds to ODC and may regulate ODC activity through a novel mechanism. Co-immunoprecipitation experiments using NB cell lysates show that ODC interacts with SPR. In addition, immunofluorescence staining and laser-scanning confocal microscopy confirm the co-localization of ODC and SPR. Furthermore, downregulation of SPR expression using siRNA targeting SPR results in significant inhibition of NB proliferation, without affecting ODC protein levels. These results suggest that the interaction between SPR and ODC may be critically important to ODC activity and function and may constitute a novel mechanism for the regulation of NB proliferation. This interaction may play a critical role in fundamental physiological and pathophysiological processes including the development and progression of NB. Understanding the novel interaction between ODC and SPR might play a crucial role for the development of new drugs targeting SPR for the treatment of NB and other MYC/ODC-driven diseases. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2495. doi:1538-7445.AM2012-2495

The Protein Partners of GTP Cyclohydrolase I in Rat Organs

ObjectiveGTP cyclohydrolase I (GCH1) is the rate-limiting enzyme for tetrahydrobiopterin biosynthesis and has been shown to be a promising therapeutic target in ischemic heart disease, hypertension, atherosclerosis and diabetes. The endogenous GCH1-interacting partners have not been identified. Here, we determined endogenous GCH1-interacting proteins in rat.Methods and ResultsA pulldown and proteomics approach were used to identify GCH1 interacting proteins in rat liver, brain, heart and kidney. We demonstrated that GCH1 interacts with at least 17 proteins including GTP cyclohydrolase I feedback regulatory protein (GFRP) in rat liver by affinity purification followed by proteomics and validated six protein partners in liver, brain, heart and kidney by immunoblotting. GCH1 interacts with GFRP and very long-chain specific acyl-CoA dehydrogenase in the liver, tubulin beta-2A chain in the liver and brain, DnaJ homolog subfamily A member 1 and fatty aldehyde dehydrogenase in the liver, heart and kidney and eukaryotic translation initiation factor 3 subunit I (EIF3I) in all organs tested. Furthermore, GCH1 associates with mitochondrial proteins and GCH1 itself locates in mitochondria.ConclusionGCH1 interacts with proteins in an organ dependant manner and EIF3I might be a general regulator of GCH1. Our finding indicates GCH1 might have broader functions beyond tetrahydrobiopterin biosynthesis.

Mutation spectrum of and founder effects affecting the PTS gene in East Asian populations

The enzyme 6-pyruvoyl-tetrahydropterin synthase (PTPS, gene symbol: PTS) is involved in the second step of the de novo biosynthesis of tetrahydrobiopterin (BH4), which is a vital cofactor of nitric oxide synthases and three types of aromatic amino acid hydroxylases; the latter are important enzymes in the production of neurotransmitters. We conducted a study of PTS mutations in East Asia, including Taiwan, Mainland China, Japan, South Korea, the Philippines, Thailand and Malaysia. A total of 43 mutations were identified, comprising 22 previously reported mutations and 21 new discovered mutations. Among these, the c.155A>G, c.259C>T, c. 272A>G, c.286G>A and c.84-291A>G mutations were the most common PTS mutations in East Asia, while the c.58T>C and c.243G>A mutations were, respectively, specific to Filipinos and Japanese originating from Okinawa. Further studies demonstrated that each of the mutations listed above was in linkage disequilibrium to a specific allele of polymorphic microsatellite marker, D11S1347. These results suggest the presence of founder effects that have affected these frequent mutations in East Asia populations. In this context, D11S1347 should become one of the most reliable polymorphic markers for use in prenatal diagnosis among PTPS deficient families, especially where mutations are yet to be identified.

Membrane transport of sepiapterin and dihydrobiopterin by equilibrative nucleoside transporters : A plausible gateway for the salvage pathway of tetrahydrobiopterin biosynthesis

Membrane transport of sepiapterin and dihydrobiopterin by equilibrative nucleoside transporters : A plausible gateway for the salvage pathway of tetrahydrobiopterin biosynthesis