Mitochondrial regulation of ferroptosis.

Ferroptosis is a form of iron-dependent regulated cell death driven by uncontrolled lipid peroxidation. Mitochondria are double-membrane organelles that have essential roles in energy production, cellular metabolism, and cell death regulation. However, their role in ferroptosis has been unclear and somewhat controversial. In this Perspective, I summarize the diverse metabolic processes in mitochondria that actively drive ferroptosis, discuss recently discovered mitochondria-localized defense systems that detoxify mitochondrial lipid peroxides and protect against ferroptosis, present new evidence for the roles of mitochondria in regulating ferroptosis, and outline outstanding questions on this fascinating topic for future investigations. An in-depth understanding of mitochondria functions in ferroptosis will have important implications for both fundamental cell biology and disease treatment.

Ionizing radiation induces BH4 deficiency by downregulating GTP-cyclohydrolase 1, a novel target for preventing and treating radiation enteritis.

Radiation enteritis (RE) is a common side effect after radiotherapy for abdominal cancer. RE pathogenesis is complicated, with no drugs available for prevention or treatments. Intestinal ischemia is a key factor in the occurrence and development of enteritis. The effect of ionizing radiation (IR) on intestinal ischemia is unknown. Deficiency of tetrahydrobiopterin (BH4) produced by GTP-cyclohydrolase 1 (Gch1) is important in ischemic diseases. This study focused on the relationship of Gch1/BH4 between intestinal ischemia in radiation enteritis. BH4 levels were analyzed by high-performance liquid chromatography in humans and rats after radiotherapy. Intestinal blood perfusion was measured by laser doppler flow imaging. Vascular ring tests determined the diastolic functions of rat mesenteric arteries. Gene, protein, and immunohistochemical staining experiments and inhibitor interventions were used to investigate Gch1 and endothelial NOS (eNOS) in rat mesenteric arteries and endothelial cells. The results showed that IR decreased BH4 levels in patients and rats after radiotherapy and decreased intestinal blood perfusion in rats. The degree of change in intestinal ischemia was consistent with intestinal villus injury. Gch1 mRNA and protein levels and nitric oxide (NO) production significantly decreased, while eNOS uncoupling in arterial and vascular endothelial cells strongly increased. BH4 supplementation improved eNOS uncoupling and NO levels in vascular endothelia after IR. The results of this study showed that downregulation of Gch1 in intestinal blood vessels after IR is an important target in RE. BH4 supplementation may prevent intestinal ischemia and improve vascular endothelial function after IR. These findings have clinical significance for the prevention and treatment of RE.

Structure of GTP cyclohydrolase I from Listeria monocytogenes, a potential anti-infective drug target.

A putative open reading frame encoding GTP cyclohydrolase I from Listeria monocytogenes was expressed in a recombinant Escherichia coli strain. The recombinant protein was purified and was confirmed to convert GTP to dihydroneopterin triphosphate (Km = 53 µM; vmax = 180 nmol mg-1 min-1). The protein was crystallized from 1.3 M sodium citrate pH 7.3 and the crystal structure was solved at a resolution of 2.4 Š(Rfree = 0.226) by molecular replacement using human GTP cyclohydrolase I as a template. The protein is a D5-symmetric decamer with ten topologically equivalent active sites. Screening a small library of about 9000 compounds afforded several inhibitors with IC50 values in the low-micromolar range. Several inhibitors had significant selectivity with regard to human GTP cyclohydrolase I. Hence, GTP cyclohydrolase I may be a potential target for novel drugs directed at microbial infections, including listeriosis, a rare disease with high mortality.

Pharmacological inhibition of guanosine triphosphate cyclohydrolase1 elevates tyrosine phosphorylation of caveolin1 and cellular senescence.

The role of 2,4-diamino-6-hydroxypyrimidine (DAHP), on cellular-senescence remains unclear as differential effects of DAHP have been reported in cardiovascular and cerebrovascular systems. We investigated the effect of pharmacologically-induced guanosine-triphosphate-cyclohydrolase1 (GTPCH1)-inhibition, through DAHP, on cellular-senescence in experimentally-induced diabetic and non-diabetic Wistar rats. Cellular-senescence was evaluated through senescence-associated events, namely, cell-cycle-arrest of peripheral blood mononuclear cells (PBMNCs); myocardial DNA fragmentation, total antioxidant capacity (TAC), telomerase-activity, nicotinamide adenine dinucleotide (NAD+)-content and tyrosine14-phosphorylation of caveolin1 (pY14) in similarly-aged, pubertal Wistar rats with streptozotocin (STZ) and/or DAHP. Oxidative stress (OS) indices such as myocardial biopterin concentrations (tetrahydrobiopterin-BH4 and dihydrobiopterin-BH2) and plasma total nitrite and nitrate (NOx) were determined. DAHP, per se, exhibited distinct senescence; in addition, in STZ+DAHP (the cardiomyopathy model), there was a marked accumulation of cells in G0G1 phase, as evidenced through flow-cytometry analysis, as-well-as fragmented DNA, than the respective controls suggesting the DAHP-mediated onset of senescence in circulating cells and the myocardium, with or without STZ. Concentrations of BH4 and BH2, and NOx were impaired in STZ and/or DAHP, indicating elevated OS in the treatment groups. In the independent treatment groups or the combination treatment, typical senescence indicators including myocardial telomerase-activity, NAD+-content and TAC were significantly reduced, while there was a marked elevation in the concentrations of pY14 as compared to the respective controls, reinforcing the occurrence of senescence in PBMNCs and the myocardium. We postulate that DAHP promotes early onset of cellular-senescence, potentially through OS-mediated cellular events in diabetic or non-diabetic models.

Mast cell tetrahydrobiopterin contributes to itch in mice.

GTP cyclohydrolase (GCH1) governs de novo synthesis of the enzyme cofactor, tetrahydrobiopterin (BH4), which is essential for biogenic amine production, bioactive lipid metabolism and redox coupling of nitric oxide synthases. Overproduction of BH4 via upregulation of GCH1 in sensory neurons is associated with nociceptive hypersensitivity in rodents, and neuron-specific GCH1 deletion normalizes nociception. The translational relevance is revealed by protective polymorphisms of GCH1 in humans, which are associated with a reduced chronic pain. Because myeloid cells constitute a major non-neuronal source of BH4 that may contribute to BH4-dependent phenotypes, we studied here the contribution of myeloid-derived BH4 to pain and itch in lysozyme M Cre-mediated GCH1 knockout (LysM-GCH1-/- ) and overexpressing mice (LysM-GCH1-HA). Unexpectedly, knockout or overexpression in myeloid cells had no effect on nociceptive behaviour, but LysM-driven GCH1 knockout reduced, and its overexpression increased the scratching response in Compound 48/80 and hydroxychloroquine-evoked itch models, which involve histamine and non-histamine dependent signalling pathways. Mechanistically, GCH1 overexpression increased BH4, nitric oxide and hydrogen peroxide, and these changes were associated with increased release of histamine and serotonin and degranulation of mast cells. LysM-driven GCH1 knockout had opposite effects, and pharmacologic inhibition of GCH1 provided even stronger itch suppression. Inversely, intradermal BH4 provoked scratching behaviour in vivo and BH4 evoked an influx of calcium in sensory neurons. Together, these loss- and gain-of-function experiments suggest that itch in mice is contributed by BH4 release plus BH4-driven mediator release from myeloid immune cells, which leads to activation of itch-responsive sensory neurons.

Cigarette smoke constituents cause endothelial nitric oxide synthase dysfunction and uncoupling due to depletion of tetrahydrobiopterin with degradation of GTP cyclohydrolase.

Cigarette smoking (CS) is a well-established risk factor for cardiovascular disease (CVD). Endothelial dysfunction (ED) with loss of nitric oxide (NO) production is a central mechanism leading to the advent of CVD. Despite many prior studies of this major health problem, the exact mechanism by which CS induces ED is not well understood. This study examines the mechanism by which CS induces ED with altered endothelial NO synthase (eNOS) function in aortic endothelial cells (AECs). Exposure of AECs to cigarette smoke extract (CSE) resulted in a marked decrease in NO production with concomitant increase in superoxide (O2.-) generation and accumulation of 4-hydroxy-2-nonenal protein adducts. CSE exposure led to depletion of the essential eNOS cofactor tetrahydrobiopterin (BH4) as well as total biopterin levels and decreased the expression level of guanosine triphosphate cyclohydrolase (GTPCH), the rate limiting enzyme in BH4 biosynthesis. Moreover, exposure of AECs to CSE increased the level of ubiquitinated proteins and increased 26 S proteasomal activity in a concentration-dependent manner. Pre-treatment with MG132, a 26 S proteasome inhibitor, partially prevented CSE-induced loss of BH4, total biopterin, GTPCH, and increased NO production following CSE exposure, indicating a role of the ubiquitin-proteasome system in CSE-induced eNOS dysfunction. In conclusion, CSE-induced eNOS dysfunction and uncoupling occurs due to BH4 depletion with BH4de novo synthesis limited by diminished GTPCH expression.

Reactive species balance via GTP cyclohydrolase I regulates glioblastoma growth and tumor initiating cell maintenance.

Background: Depending on the level, differentiation state, and tumor stage, reactive nitrogen and oxygen species inhibit or increase cancer growth and tumor initiating cell maintenance. The rate-limiting enzyme in a pathway that can regulate reactive species production but has not been thoroughly investigated in glioblastoma (GBM; grade IV astrocytoma) is guanosine triphosphate (GTP) cyclohydrolase 1 (GCH1). We sought to define the role of GCH1 in the regulation of GBM growth and brain tumor initiating cell (BTIC) maintenance. Methods: We examined GCH1 mRNA and protein expression in patient-derived xenografts, clinical samples, and glioma gene expression datasets. GCH1 levels were modulated using lentiviral expression systems, and effects on cell growth, self-renewal, reactive species production, and survival in orthotopic patient-derived xenograft models were determined. Results: GCH1 was expressed in GBMs with elevated but not exclusive RNA and protein levels in BTICs in comparison to non-BTICs. Overexpression of GCH1 in GBM cells increased cell growth in vitro and decreased survival in an intracranial GBM mouse model. In converse experiments, GCH1 knockdown with short hairpin RNA led to GBM cell growth inhibition and reduced self-renewal in association with decreased CD44 expression. GCH1 was critical for controlling reactive species balance, including suppressing reactive oxygen species production, which mediated GCH1 cell growth effects. In silico analyses demonstrated that higher GCH1 levels in glioma patients correlate with higher glioma grade, recurrence, and worse survival. Conclusions: GCH1 expression in established GBMs is pro-tumorigenic, causing increased growth due, in part, to promotion of BTIC maintenance and suppression of reactive oxygen species.

Mechanism and catalytic strategy of the prokaryotic-specific GTP cyclohydrolase-IB.

Guanosine 5'-triphosphate (GTP) cyclohydrolase-I (GCYH-I) catalyzes the first step in folic acid biosynthesis in bacteria and plants, biopterin biosynthesis in mammals, and the biosynthesis of 7-deazaguanosine-modified tRNA nucleosides in bacteria and archaea. The type IB GCYH (GCYH-IB) is a prokaryotic-specific enzyme found in many pathogens. GCYH-IB is structurally distinct from the canonical type IA GCYH involved in biopterin biosynthesis in humans and animals, and thus is of interest as a potential antibacterial drug target. We report kinetic and inhibition data of Neisseria gonorrhoeae GCYH-IB and two high-resolution crystal structures of the enzyme; one in complex with the reaction intermediate analog and competitive inhibitor 8-oxoguanosine 5'-triphosphate (8-oxo-GTP), and one with a tris(hydroxymethyl)aminomethane molecule bound in the active site and mimicking another reaction intermediate. Comparison with the type IA enzyme bound to 8-oxo-GTP (guanosine 5'-triphosphate) reveals an inverted mode of binding of the inhibitor ribosyl moiety and, together with site-directed mutagenesis data, shows that the two enzymes utilize different strategies for catalysis. Notably, the inhibitor interacts with a conserved active-site Cys149, and this residue is S-nitrosylated in the structures. This is the first structural characterization of a biologically S-nitrosylated bacterial protein. Mutagenesis and biochemical analyses demonstrate that Cys149 is essential for the cyclohydrolase reaction, and S-nitrosylation maintains enzyme activity, suggesting a potential role of the S-nitrosothiol in catalysis.

Shear stress-mediated upregulation of GTP cyclohydrolase/tetrahydrobiopterin pathway ameliorates hypertension-related decline in reendothelialization capacity of endothelial progenitor cells.

OBJECTIVES: Guanosine triphosphate cyclohydrolase/tetrahydrobiopterin (GTPCH)/(BH4) pathway has been proved to regulate the function of endothelial progenitor cells (EPCs) in deoxycorticosterone acetate-salt hypertensive mice, indicating that GTPCH/BH4 pathway may be an important repair target for hypertension-related endothelial injury. Shear stress is an important nonpharmacologic strategy to modulate the function of EPCs. Here, we investigated the effects of laminar shear stress on the GTPCH/BH4 pathway and endothelial repair capacity of circulating EPCs in hypertension. METHOD: Laminar shear stress was loaded on the human EPCs from hypertensive patients and normotensive patients. The in-vitro function, in-vivo reendothelialization capacity and GTPCH/BH4 pathway of human EPCs were evaluated. RESULTS: Both in-vitro function and reendothelialization capacity of EPCs were lower in hypertensive patients than that in normotensive patients. The GTPCH/BH4 pathway of EPCs was downregulated in hypertensive patients. Shear stress increased in-vitro function and reendothelialization capacity of EPCs from hypertensive patients and normotensive patients. Furthermore, shear stress upregulated the expression of GTPCH I and levels of BH4, nitric oxide, and cGMP of EPCs, and reduced thrombospondin-1 expression. With treatment of GTPCH knockdown or nitroarginine methyl ester inhibition, shear stress-induced increased levels of BH4, nitric oxide and cGMP of EPCs was suppressed. When GTPCH/BH4 pathway of EPCs was blocked, the effects of shear stress on in-vitro function and reendothelialization capacity of EPCs were inhibited. CONCLUSION: The study demonstrates for the first time that shear stress-induced upregulation of the GTPCH/BH4 pathway ameliorates hypertension-related decline in endothelial repair capacity of EPCs. These findings provide novel nonpharmacologic therapeutic approach for hypertension-related endothelial repair.

Evidence of a role for GTP cyclohydrolase-1 in visceral pain.

BACKGROUND: The enzyme guanosine triphosphate-cyclohydrolase-1 (GCH-1) is a rate limiting step in the de novo synthesis of tetrahydrobiopterin (BH4) a co-factor in monoamine synthesis and nitric oxide production. GCH-1 is strongly implicated in chronic pain based on data generated using the selective GCH-1 inhibitor 2,4-diamino-6-hydroxypyrimidine (DAHP), and studies which have identified a pain protective GCH-1 haplotype associated with lower BH4 production and reduced pain. METHODS: To investigate the role for GCH-1 in visceral pain we examined the effects of DAHP on pain behaviors elicited by colorectal injection of mustard oil in rats, and the pain protective GCH-1 haplotype in healthy volunteers characterized by esophageal pain sensitivity before and after acid injury, and assessed using depression and anxiety questionnaires. KEY RESULTS: In rodents pretreatment with DAHP produced a substantial dose related inhibition of pain behaviors from 10 to 180 mg/kg i.p. (p < 0.01 to 0.001). In healthy volunteers, no association was seen between the pain protective GCH-1 haplotype and the development of hypersensitivity following injury. However, a substantial increase in baseline pain thresholds was seen between first and second visits (26.6 ± 6.2 mA) in subjects who sensitized to esophageal injury and possessed the pain protective GCH-1 haplotype compared with all other groups (p < 0.05). Furthermore the same subjects who sensitized to acid and possessed the haplotype, also had significantly lower depression scores (p < 0.05). CONCLUSIONS & INFERENCES: The data generated indicate that GCH-1 plays a role in visceral pain processing that requires more detailed investigation.

Attenuation of biopterin synthesis prevents Escherichia coli K1 invasion of brain endothelial cells and the development of meningitis in newborn mice.

Elevated levels of pterins and nitric oxide (NO) are observed in patients with septic shock and bacterial meningitis. We demonstrate that Escherichia coli K1 infection of human brain microvascular endothelial cells (HBMECs) induces the expression of guanosine triphosphate cyclohydrolase (GCH1), the rate-limiting enzyme in pterin synthesis, thereby elevating levels of biopterin. DAHP (2,4-diamino hydroxyl pyrimidine), a specific inhibitor of GCH1, prevented biopterin and NO production and invasion of E. coli K1 in HBMECs. GCH1 interaction with Ecgp96, the receptor for outer membrane protein A of E. coli K1, also increases on infection, and suppression of Ecgp96 expression prevents GCH1 activation and biopterin synthesis. Pretreatment of newborn mice with DAHP prevented the production of biopterin and the development of meningitis. These results suggest a novel role for biopterin synthesis in the pathogenesis of E. coli K1 meningitis.

Inhibition of GTP cyclohydrolase attenuates tumor growth by reducing angiogenesis and M2-like polarization of tumor associated macrophages.

GTP cyclohydrolase (GCH1) is the key-enzyme to produce the essential enzyme cofactor, tetrahydrobiopterin. The byproduct, neopterin is increased in advanced human cancer and used as cancer-biomarker, suggesting that pathologically increased GCH1 activity may promote tumor growth. We found that inhibition or silencing of GCH1 reduced tumor cell proliferation and survival and the tube formation of human umbilical vein endothelial cells, which upon hypoxia increased GCH1 and endothelial NOS expression, the latter prevented by inhibition of GCH1. In nude mice xenografted with HT29-Luc colon cancer cells GCH1 inhibition reduced tumor growth and angiogenesis, determined by in vivo luciferase and near-infrared imaging of newly formed blood vessels. The treatment with the GCH1 inhibitor shifted the phenotype of tumor associated macrophages from the proangiogenic M2 towards M1, accompanied with a shift of plasma chemokine profiles towards tumor-attacking chemokines including CXCL10 and RANTES. GCH1 expression was increased in mouse AOM/DSS-induced colon tumors and in high grade human colon and skin cancer and oppositely, the growth of GCH1-deficient HT29-Luc tumor cells in mice was strongly reduced. The data suggest that GCH1 inhibition reduces tumor growth by (i) direct killing of tumor cells, (ii) by inhibiting angiogenesis, and (iii) by enhancing the antitumoral immune response.

Inhibition of GTP cyclohydrolase reduces cancer pain in mice and enhances analgesic effects of morphine.

Noncoding polymorphisms of the GTP cyclohydrolase gene (GCH1) reduce the risk for chronic pain in humans suggesting GCH1 inhibitors as analgesics. We assessed the effects of the GCH1 inhibitor diaminohydroxypyrimidine (DAHP) on nociception and inflammation in a mouse melanoma and a sarcoma cancer pain model, and its co-effects with morphine in terms of analgesic efficacy and respiratory depression. GCH1 inhibition did not reduce the tumor-evoked nociceptive hypersensitivity of the tumor-bearing paw. However, DAHP reduced melanoma- and sarcoma-evoked systemic hyperalgesia as determined by analyzing contralateral paws. GCH1 inhibition increased the inflammatory edema and infiltration with polymorphonuclear leukocytes surrounding the tumor but reduced the tumor-evoked microglia activation in the spinal cord suggesting that an increase of the local immune attack against the tumor may avoid general pain hypersensitivity. When used in combination with morphine at high or low doses, GCH1 inhibition increased and prolonged the analgesic effects of the opioid. It did not, however, increase the respiratory depression caused by morphine. Conversely, the GCH1-product, tetrahydrobiopterin, caused hyperalgesia, antagonized antinociceptive effects of morphine, and aggravated morphine-evoked respiratory depression, the latter mimicked by a cGMP analog suggesting that respiratory effects were partly mediated through the BH4-NO-cGMP pathway. The observed effects of GCH1 inhibition in the tumor model and its enhancement of morphine-evoked antinociception without increase of morphine toxicity suggest that GCH1 inhibitors might be useful as co-therapeutics for opioids in cancer patients.

GCH1, BH4 and pain.

Understanding and consequently treating neuropathic pain effectively is a challenge for modern medicine, as unlike inflammation, which can be controlled relatively well, chronic pain due to nerve injury is refractory to most current therapeutics. Here we define a target pathway for a new class of analgesics, tetrahydrobiopterin (BH4) synthesis and metabolism. BH4 is an essential co-factor in the synthesis of serotonin, dopamine, epinephrine, norepinephrine and nitric oxide and as a result, its availability influences many systems, including neurons. Following peripheral nerve damage, levels of BH4 are dramatically increased in sensory neurons, consequently this has a profound effect on the physiology of these cells, causing increased activity and pain hypersensitivity. These changes are principally due to the upregulation of the rate limiting enzyme for BH4 synthesis GTP Cyclohydrolase 1 (GCH1). A GCH1 pain-protective haplotype which decreases pain levels in a variety of settings, by reducing the levels of endogenous activation of this enzyme, has been characterized in humans. Here we define the control of BH4 homeostasis and discuss the consequences of large perturbations within this system, both negatively via genetic mutations and after pathological increases in the production of this cofactor that result in chronic pain. We explain the nature of the GCH1 reduced-function haplotype and set out the potential for a ' BH4 blocking' drug as a novel analgesic.

An important role of increase in tetrahydrobiopterin via H2O2-JAK2 signalling pathway in late phase of ischaemic preconditioning.

The goal of this study was to elucidate whether there is an increase in myocardial tetrahydrobiopterin (BH4), which is a cofactor for nitric oxide synthase, during the late phase of ischaemic preconditioning (IPC) leading to cardioprotection against myocardial infarction and, if so, to examine the induction mechanisms of BH4 synthesis. Rats were preconditioned with four cycles of 3 min left main coronary artery (LCA) occlusion followed by 10 min reperfusion. Twenty-four hours later, the rats were subjected to 20 min ischaemia by LCA ligation and 2 h reperfusion, and the infarct size was determined by 2,3,5-triphenyltetrazolium chloride staining. The IPC protocol reduced the infarct size, and increased the BH4 content and expression of GTP-cyclohydrolase I (GTPCH), which is the rate-limiting enzyme for BH4 synthesis. Administration of a GTPCH inhibitor attenuated both the reduction in infarct size and the increase in BH4 levels. Moreover, the increase in BH4 content was reduced by administration of catalase or a Janus tyrosine kinase-2 (JAK2) inhibitor. These observations suggest that upregulation of BH4 synthesis in the heart contributes to an acquisition of ischaemic tolerance in late IPC, and the increase in myocardial BH4 content seems to be mediated by the induction of GTPCH via the H(2)O(2)-JAK2 pathway.

Tetrahydrobiopterin (BH4), a cofactor for nNOS, restores gastric emptying and nNOS expression in female diabetic rats.

Gastroparesis is a debilitating disease predominantly affecting young women. Recently, dysregulation of neuronal nitric oxide synthase (nNOS) in myenteric plexus neurons has been implicated for delayed solid gastric emptying/gastroparesis in diabetic patients. In this study, we have explored the role of tetrahydrobiopterin (BH4), a major cofactor for nNOS activity and NO synthesis in diabetic gastroparesis. Diabetes was induced with single injection of streptozotocin (55 mg/kg body wt, ip) in female rats, with experiments performed on week 3 or 9 following induction, with or without 3-wk BH4 supplementation. Gastric pyloric BH4 levels were significantly decreased in diabetic female rats compared with control (18.6 +/- 1.45 vs. 31.0 +/- 2.31 pmol/mg protein). In vitro studies showed that 2,4-diamino-6-hydroxypyrimidine (DAHP), an inhibitor of BH4 synthesis, significantly decreased gastric NO release and nitrergic relaxation. Three-week dietary supplementation of BH4 either from day 1 or week 6 significantly attenuated diabetes-induced delayed gastric emptying for solids (3 wk: BH4, 67 +/- 6.7 vs. diabetic, 36.05 +/- 7.09; 9 wk: BH4, 57 +/- 8.45 vs. diabetic, 33 +/- 9.91) and diabetes-induced reduction in pyloric nNOS-alpha protein expression in female rats. Supplementation of BH4 significantly restored gastric nNOS-alpha dimerization in 9-wk-old diabetic female rats. In addition, BH4 treatment reversed (17.23 +/- 5.81 vs. 42.0 +/- 2.70 mmHg x s) the diabetes-induced changes in intragastric pressures (IGP) and gastric pyloric nitrergic relaxation (-0.62 +/- 0.01 vs. -0.22 +/- 0.07). BH4 deficiency plays a critical role in diabetes-induced alterations including delayed solid gastric emptying, increased IGP, reduced pyloric nitrergic relaxation, and nNOS-alpha expression in female rats. Supplementation of BH4 accelerates gastric emptying by restoring nitrergic system in diabetic female rats. Therefore, BH4 supplementation is a potential therapeutic option for female patients of diabetic gastroparesis.

GTP cyclohydrolase I phosphorylation and interaction with GTP cyclohydrolase feedback regulatory protein provide novel regulation of endothelial tetrahydrobiopterin and nitric oxide.

RATIONALE: GTP cyclohydrolase I (GTPCH-1) is the rate-limiting enzyme involved in de novo biosynthesis of tetrahydrobiopterin (BH(4)), an essential cofactor for NO synthases and aromatic amino acid hydroxylases. GTPCH-1 undergoes negative feedback regulation by its end-product BH(4) via interaction with the GTP cyclohydrolase feedback regulatory protein (GFRP). Such a negative feedback mechanism should maintain cellular BH(4) levels within a very narrow range; however, we recently identified a phosphorylation site (S81) on human GTPCH-1 that markedly increases BH(4) production in response to laminar shear. OBJECTIVE: We sought to define how S81 phosphorylation alters GTPCH-1 enzyme activity and how this is modulated by GFRP. METHODS AND RESULTS: Using prokaryotically expressed proteins, we found that the GTPCH-1 phospho-mimetic mutant (S81D) has increased enzyme activity, reduced binding to GFRP and resistance to inhibition by GFRP compared to wild-type GTPCH-1. Using small interfering RNA or overexpressing plasmids, GFRP was shown to modulate phosphorylation of GTPCH-1, BH(4) levels, and NO production in human endothelial cells. Laminar, but not oscillatory shear stress, caused dissociation of GTPCH-1 and GFRP, promoting GTPCH-1 phosphorylation. We also found that both GTPCH-1 phosphorylation and GFRP downregulation prevents endothelial NO synthase uncoupling in response to oscillatory shear. Finally oscillatory shear was associated with impaired GTPCH-1 phosphorylation and reduced BH(4) levels in vivo. CONCLUSIONS: These studies provide a new mechanism for regulation of endothelial GTPCH-1 by its phosphorylation and interplay with GFRP. This mechanism allows for escape from GFRP negative feedback and permits large amounts of BH(4) to be produced in response to laminar shear stress.