GTP Cyclohydrolase Research Articles

Procyanidin B2 ameliorates endothelial dysfunction induced by nicotine via the induction of tetrahydrobiopterin synthesis

• Nicotine decreases the expression of DHFR and GCH1 to inhibit BH 4 and NO production. • PCB2 prevents nicotine-induced DHFR and GCH1 loss. • PCB2 improves endothelium-dependent vasorelaxation impaired by nicotine. • PCB2 induces vasorelaxation through a PPARδ-BH 4 dependent manner. Smoking is an independent risk factor for the cardiovascular diseases. Nicotine, a major component of tobacco, is responsible for the impaired endothelial-dependent vasorelaxation in smokers. Procyanidin B2 (PCB2), a natural flavonoid, has been reported to possess several potential beneficial effects on the cardiovascular system. However, whether PCB2 prevents nicotine-induced endothelial dysfunction remains unknown. In this study, we demonstrated that PCB2 improved nicotine-impaired endothelium-dependent vasorelaxation in mouse thoracic aortas. Mechanistically, PCB2 increased the expression levels of dihydrofolate reductase (DHFR) and GTP cyclohydrolase 1 (GCH1), two important enzymes in the synthesis of tetrahydrobiopterin (BH 4 ), in mouse aortas and cultured ECs. GSK0660, a selective antagonist of peroxisome proliferator-activated receptor δ (PPARδ), abolished the PCB2-induced effects on BH 4 synthesis, nitric oxide (NO) production and vasodilation. Collectively, we demonstrated that PCB2 mitigates nicotine-induced endothelial dysfunction through a PPARδ-BH 4 dependent manner, thereby suggesting a novel role of PCB2 in preventing vascular dysfunction caused by nicotine exposure.

Disruption of tetrahydrobiopterin (BH4) biosynthesis pathway affects cuticle pigmentation in Henosepilachna vigintioctopunctata

Tetrahydrobiopterin (BH4) is produced from guanosine triphosphate (GTP) under catalyzation of GTP cyclohydrolase I (GTPCH), 6-pyruvoyltetrahydropterin synthase (PTPS) and sepiapterin reductase (SR), among others. In Drosophila melanogaster, BH4 and other pteridines are required for cuticle tanning and eye pigmentation. In this study, two Hvgtpch (Hvgtpch-a and Hvgtpch-b), an Hvptps and an Hvsr transcripts were identified in a serious defoliator Henosepilachna vigintioctopunctata. Hvgtpch-a and Hvgtpch-b were highly expressed just before and/or right after the molt, in contrast to Hvptps and Hvsr. RNA interference (RNAi) by injection of a dsgtpch targeting the common fragment of Hvgtpch-a and Hvgtpch-b into the third instar larvae caused albino fourth-instar larvae and pupae. Around 80% of the Hvgtpch RNAi larvae failed to pupate. The remaining 20% of Hvgtpch RNAi pupated beetles did not completely remove the larval/pupal exuviae after emerged as adults and eventually died. Depletion of Hvgtpch at the fourth instar stage resulted in under-pigmented pupae and adults, with significantly low pupation and emergence rates. The Hvgtpch RNAi adults rarely moved and fed on plant leaves; they died within a week after emergence. Silence of Hvptps or Hvsr at the third- and fourth-instar stages led to similar but less serious phenotypes, with lowest influence in the Hvsr RNAi ladybirds. Moreover, RNAi of Hvgtpch, Hvptps or Hvsr did not affect coloration of the larval ocelli and pupal/adult compound eyes. Therefore, our results demonstrated that pteridines are involved in melanin formation but not in eye pigmentation in H. vigintioctopunctata. Moreover, our findings will enable the development of a dsgtpch-based pesticide to control H. vigintioctopunctata larvae.

Nitrosative stress induced by homocysteine thiolactone drives vascular cognitive impairments via GTP cyclohydrolase 1 S-nitrosylation in vivo

Backgrounds: Hyperhomocysteinemia (HHcy) is one of risk factors for vascular cognitive impairment (VCI). GTP cyclohydrolase 1 (GCH1) deficiency is critical to oxidative stress in vascular dysfunction. The aim of this study was designed to examine whether HHcy induces VCI through GCH1 S-nitrosylation, a redox-related post-translational modification of cysteine. MethodsThe VCI model was induced by feeding mice homocysteine thiolactone (HTL) for 16 consecutive weeks. The cognitive functions were evaluated by step-down avoidance test, passive avoidance step-through task test, and Morris water maze (MWM) test. Protein S-nitrosylation was assayed using a biotin-switch method. ResultsIn cell-free system, nitric oxide (NO) donor induced GCH1 protein S-nitrosylation and decreased GCH1 activity. In endothelial cells, HTL increased GCH1 S-nitrosylation, reduced tetrahydrobiopterin, and induced oxidative stress, which were attenuated by N-acetyl-cysteine, L-N6-1-Iminoethyl-lysine, mutant of GCH1 cysteine 141 to alanine (MT-GCH1) or gene deletion of inducible NO synthase (iNOS). Further, HTL incubation or iNOS overexpression promoted endothelial cellular senescence, but abolished by exogenous expression of MT-GCH1 or pharmacological approaches including N-acetyl-cysteine, L-sepiapterin, and tempol. In wildtype mice, long-term administration of HTL induced GCH1 S-nitrosylation and vascular stiffness, decreased cerebral blood flow, and damaged the cognitive functions. However, these abnormalities induced by HTL administration were rescued by enforced expression of MT-GCH1 or gene knockout of iNOS. In human subjects, GCH1 S-nitrosylation was increased and cognitive functions were impaired in patients with HHcy. ConclusionThe iNOS-mediated nitrosative stress induced by HTL drives GCH1 S-nitrosylation to induce cerebral vascular stiffness and cognitive impairments.

Abstract 13625: Autophagy Plays a Key Role in the Loss of Nitric Oxide Signaling and Endothelial Dysfunction in Pulmonary Vascular Diseases

Introduction: Congenital heart defects (CHDs) are the most common type of birth defect. Children or infants with CHD often develop pulmonary vascular diseases (PVD) due to increased pulmonary blood flow and pressure. Unfortunately, there is no effective therapy to limit the shared pathophysiologic features of pulmonary endothelial dysfunction and vascular remodeling. Autophagy, a lysosome-dependent proteolytic pathway, enables cells to sequester portions of cytosolic proteins or damaged organelles into autophagosomes and degrade them in autolysosomes. Studies have found that autophagy participates in pulmonary hypertension, acute lung injury, lung cancer, and other pulmonary diseases. Hypothesis: Autophagy plays a key role in the development of pulmonary endothelial dysfunction in PVD associated with CHD. Methods and Results: We utilized lung endothelial cells isolated from an ovine CHD model, created by the fetal placement of an aortopulmonary graft (Shunt). First, we confirmed that autophagy was activated in Shunt pulmonary artery endothelial cells (PAEC), with increased LC3-II/LC3-I ratio (1.45 fold increase, n=3, p<0.05) and reduced p62 levels (64% reduction, n=3, p<0.05). Shunt PAECs were found hyperproliferative (BrdU Assay A 450 : control vs. Shunt: 2.36 ± 0.05 vs . 2.58 ± 0.05, n=9, p<0.01), which can be attenuated by pharmacologic autophagy inhibitor bafilomycin A1 (2.30 ± 0.05, n=9, p<0.01 compared with Shunt) or 3-MA (2.20 ± 0.03, n=9, p<0.001 compared with Shunt). Shunt PAECs exhibited significant endothelial Nitic Oxide Synthase (eNOS) uncoupling by BH4 reduction (31% reduction, n=3, p<0.01). Bafilomycin A1 reversed eNOS uncoupling in Shunt PAECs by BH4 restoration (2.1 fold increase, n=3, p<0.01), resulting in elevated cellular NOx level (1.8 increase fold, n=3, p<0.01). Mechanistically, this was dependent on increased levels of GTP cyclohydrolase I (GCH1) protein in Shunt PAEC. Conclusions: Enhanced autophagy plays a key role in the loss of NO signaling and endothelial dysfunction associated with CHD. We speculate that autophagy might be an effective therapeutic target for pulmonary vascular diseases.

A plant-based mutant huntingtin model-driven discovery of impaired expression of GTPCH and DHFR

Pathophysiology associated with Huntington’s disease (HD) has been studied extensively in various cell and animal models since the 1993 discovery of the mutant huntingtin (mHtt) with abnormally expanded polyglutamine (polyQ) tracts as the causative factor. However, the sequence of early pathophysiological events leading to HD still remains elusive. To gain new insights into the early polyQ-induced pathogenic events, we expressed Htt exon1 (Httex1) with a normal (21), or an extended (42 or 63) number of polyQ in tobacco plants. Here, we show that transgenic plants accumulated Httex1 proteins with corresponding polyQ tracts, and mHttex1 induced protein aggregation and affected plant growth, especially root and root hair development, in a polyQ length-dependent manner. Quantitative proteomic analysis of young roots from severely affected Httex1Q63 and unaffected Httex1Q21 plants showed that the most reduced protein by polyQ63 is a GTP cyclohydrolase I (GTPCH) along with many of its related one-carbon (C1) metabolic pathway enzymes. GTPCH is a key enzyme involved in folate biosynthesis in plants and tetrahydrobiopterin (BH4) biosynthesis in mammals. Validating studies in 4-week-old R6/2 HD mice expressing a mHttex1 showed reduced levels of GTPCH and dihydrofolate reductase (DHFR, a key folate utilization/alternate BH4 biosynthesis enzyme), and impaired C1 and BH4 metabolism. Our findings from mHttex1 plants and mice reveal impaired expressions of GTPCH and DHFR and may contribute to a better understanding of mHtt-altered C1 and BH4 metabolism, and their roles in the pathogenesis of HD.

Phenotypic drug screen uncovers the metabolic GCH1/BH4 pathway as key regulator of EGFR/KRAS-mediated neuropathic pain and lung cancer.

Increased tetrahydrobiopterin (BH4) generated in injured sensory neurons contributes to increased pain sensitivity and its persistence. GTP cyclohydrolase 1 (GCH1) is the rate-limiting enzyme in the de novo BH4 synthetic pathway, and human single-nucleotide polymorphism studies, together with mouse genetic modeling, have demonstrated that decreased GCH1 leads to both reduced BH4 and pain. However, little is known about the regulation of Gch1 expression upon nerve injury and whether this could be modulated as an analgesic therapeutic intervention. We performed a phenotypic screen using about 1000 bioactive compounds, many of which are target-annotated FDA-approved drugs, for their effect on regulating Gch1 expression in rodent injured dorsal root ganglion neurons. From this approach, we uncovered relevant pathways that regulate Gch1 expression in sensory neurons. We report that EGFR/KRAS signaling triggers increased Gch1 expression and contributes to neuropathic pain; conversely, inhibiting EGFR suppressed GCH1 and BH4 and exerted analgesic effects, suggesting a molecular link between EGFR/KRAS and pain perception. We also show that GCH1/BH4 acts downstream of KRAS to drive lung cancer, identifying a potentially druggable pathway. Our screen shows that pharmacologic modulation of GCH1 expression and BH4 could be used to develop pharmacological treatments to alleviate pain and identified a critical role for EGFR-regulated GCH1/BH4 expression in neuropathic pain and cancer in rodents.

Time estimation and arousal responses in dopa-responsive dystonia

Dopa-responsive dystonia (DRD) is caused by an impaired dopamine biosynthesis due to a GTP-cyclohydrolase-1 (GCH1) deficiency, resulting in a combination of dystonia and parkinsonism. However, the effect of GCH1 mutations and levodopa treatment on motor control beyond simple movements, such as timing, action preparation and feedback processing, have not been investigated so far. In an active time estimation task with trial-by-trial feedback, participants indicated a target interval (1200 ms) by a motor response. We compared 12 patients tested (in fixed order) under their current levodopa medication ("ON") and after levodopa withdrawal ("OFF") to matched healthy controls (HC), measured twice to control for repetition effects. We assessed time estimation accuracy, trial-to-trial adjustment, as well as task- and feedback-related pupil-linked arousal responses. Patients showed comparable time estimation accuracy ON medication as HC but reduced performance OFF medication. Task-related pupil responses showed the reverse pattern. Trial-to-trial adjustments of response times were reduced in DRD, particularly OFF medication. Our results indicate differential alterations of time estimation accuracy and task-related arousal dynamics in DRD patients as a function of dopaminergic medication state. A medication-independent alteration of task repetition effects in DRD cannot be ruled out with certainty but is discussed as less likely.

Frequency of Parkinson's Disease Genes and Role of PARK2 in Amyotrophic Lateral Sclerosis: An NGS Study.

Amyotrophic lateral sclerosis (ALS) and Alzheimer’s disease (AD) patients show a higher prevalence of Lewy body disease than the general population. Additionally, parkinsonian features were found in about 30% of ALS patients. We aimed to explore the frequency of Parkinson’s disease (PD)-causative genes in ALS patients, compared to AD and healthy controls (HCs). We used next-generation sequencing multigene panels by analyzing SNCA, LRRK2, PINK1, PARK2, PARK7, SYNJ1, CHCHD2, PLA2G6, GCH1, ATP13A2, DNAJC6 and FBXO genes. GBA gene, a risk factor for PD, was also analyzed. In total, 130 ALS and 100 AD patients were investigated. PD-related genes were found to be altered in 26.2% of ALS, 20% of AD patients and 19.2% of HCs. Autosomal recessive genes were significantly more involved in ALS as compared to AD and HCs (p = 0.021). PARK2 variants were more frequent in ALS than in AD and HCs, although not significantly. However, the p.Arg402Cys variant was increased in ALS than in HCs (p = 0.025). This finding is consistent with current literature, as parkin levels were found to be decreased in ALS animal models and patients. Our results confirm the possible role of PD-related genes as risk modifier in ALS pathogenesis.

Analysis of GCH1 gene variant in a consanguineous Chinese pedigree affected with tetrahydrobiopterin deficiency

To explore the genetic basis for a child featuring tetrahydrobiopterin deficiency and global developmental delay. Clinical and laboratory examinations were carried out for the child. Genomic DNA of the patient was subjected to high-throughput sequencing to identify genetic variants associated with hyperphenylalaninemia. Candidate variants were verified by Sanger sequencing. The result of blood tandem mass spectrometry showed that the Phenylalanine in the blood was 642.7 μmol/l, and the ratio of Phenylalanine/Tyrosine was 5.42. Analysis of urinary pterin: neopterin 0.09 mmol/mol Cr, biopterin 0.04 mmol/mol Cr, biopterin% 77%, which suggested tetrahydrobiopterin deficiency. The parents of the proband were first cousins. DNA sequencing revealed that the proband has harbored homozygous c.353A>T variants in exon 2 of the GCH1 gene, for which his great grandmother, grandfather, mother, uncle, father and elder brother were heterozygous carriers with normal phenotype and no clinical symptoms associated with dopa responsive dystonia. The homozygous c.353A>T variant of the GCH1 gene probably underlay the tetrahydrobiopterin deficiency in this pedigree of consanguineous marriage.

Isolation and In vitro Culture of Bone Marrow-Derived Macrophages for the Study of NO-Redox Biology.

Macrophages are derived from hematopoietic progenitor cells throughout the body, are central to inflammatory processes, and participate in innate and adaptive immune responses. In vitro study of macrophages can be undertaken by ex vivo culture from the peritoneum or through differentiation of myeloid bone marrow progenitor cells to form bone marrow-derived macrophages (BMDMs). A common approach to macrophage differentiation from precursors involves the use of conditioned media from L929 cells (LCM). This media is easy to self-produce but suffers from batch variability, and its constituents are undefined. Similarly, Foetal Bovine Serum (FBS) is used to support growth but contains a vast mixture of undefined molecules that may vary between batches. These methods are not adequate for the study of nitric oxide biology and redox mechanisms as they both contain substantial amounts of small molecules that either interfere with redox mechanisms or supplement levels of cofactors, such as tetrahydrobiopterin (BH4), required for the production of NO from inducible nitric oxide synthase (iNOS). In this report, we present an optimized protocol allowing for control of the NO-redox environment by reducing the levels of exogenous biopterin while maintaining conditions suitable for cell growth and differentiation. Tight control of culture media composition helps ensure experimental reproducibility and facilitates accurate interpretation of results. In this protocol, BMDMs were obtained from a GTP cyclohydrolase (GCH)- deficient mouse model. Culture of BMDMs was performed with media containing either (i) conditioned LCM, or (ii) recombinant M-CSF and GM-CSF to produce minimal artifacts while obtaining BH4 and NO-deficient culture conditions - thus allowing for the reproducible study of NO-redox biology and immunometabolism in vitro.

Isolation and <em>In vitro</em> Culture of Bone Marrow-Derived Macrophages for the Study of NO-Redox Biology

Macrophages are derived from hematopoietic progenitor cells throughout the body, are central to inflammatory processes, and participate in innate and adaptive immune responses. In vitro study of macrophages can be undertaken by ex vivo culture from the peritoneum or through differentiation of myeloid bone marrow progenitor cells to form bone marrow-derived macrophages (BMDMs). A common approach to macrophage differentiation from precursors involves the use of conditioned media from L929 cells (LCM). This media is easy to self-produce but suffers from batch variability, and its constituents are undefined. Similarly, Foetal Bovine Serum (FBS) is used to support growth but contains a vast mixture of undefined molecules that may vary between batches. These methods are not adequate for the study of nitric oxide biology and redox mechanisms as they both contain substantial amounts of small molecules that either interfere with redox mechanisms or supplement levels of cofactors, such as tetrahydrobiopterin (BH4), required for the production of NO from inducible nitric oxide synthase (iNOS). In this report, we present an optimized protocol allowing for control of the NO-redox environment by reducing the levels of exogenous biopterin while maintaining conditions suitable for cell growth and differentiation. Tight control of culture media composition helps ensure experimental reproducibility and facilitates accurate interpretation of results. In this protocol, BMDMs were obtained from a GTP cyclohydrolase (GCH)- deficient mouse model. Culture of BMDMs was performed with media containing either (i) conditioned LCM, or (ii) recombinant M-CSF and GM-CSF to produce minimal artifacts while obtaining BH4 and NO-deficient culture conditions - thus allowing for the reproducible study of NO-redox biology and immunometabolism in vitro.

Naringenin upregulates GTPCH1/eNOS to ameliorate high glucose-induced retinal endothelial cell injury.

Diabetic retinopathy (DR) is a microvascular complication of diabetes, while retinal endothelial cell (REC) dysfunction is considered the primary pathological process of DR. Naringenin, a natural flavonoid compound, exhibits therapeutic potential against multiple types of endothelial cell injury. To the best of our knowledge, however, its effect on REC injury has not been previously investigated. Therefore, the aim of the present study was to investigate the effect of naringenin on high glucose (HG)-induced REC injury and assess the underlying mechanism. To establish a retinal injury model, human (H)RECs were treated with 30 mM glucose. Cell Counting Kit-8 assay and TUNEL staining were used to assess the effects of naringenin on cell proliferation and apoptosis, respectively. Reactive oxygen species (ROS) levels and concentration of tetrahydrobiopterin (BH4), the essential cofactor of endothelial nitric oxide synthase (eNOS), were measured using a ROS detection kit and ELISA, respectively. The transfection efficiency of HRECs with guanosine triphosphate cyclohydrolase-1 (GTPCH1) interfering plasmid was examined by reverse transcription-quantitative PCR. The protein expression levels of Ki67, proliferative cell nuclear antigen (PCNA), eNOS and GTPCH1 were determined by western blot analysis. Compared with the HG-induced group alone, co-treatment with naringenin inhibited HG-induced HREC apoptosis in a dose-dependent manner, increased expression levels of the proliferation-associated proteins Ki67 and PCNA and effectively decreased intracellular ROS levels. Furthermore, naringenin upregulated GTPCH1/eNOS signaling and promoted release of BH4. However, GTPCH1 knockdown partially reversed the ameliorative effect of naringenin on HG-induced HREC injury. In summary, the present study suggested that naringenin effectively inhibited HG-induced HREC apoptosis and intracellular oxidative stress, which may be associated with naringenin-mediated GTPCH1/eNOS upregulation.

Zng1 is a GTP-dependent zinc transferase needed for activation of methionine aminopeptidase.

The evolution of zinc (Zn) as a protein cofactor altered the functional landscape of biology, but dependency on Zn also created an Achilles' heel, necessitating adaptive mechanisms to ensure Zn availability to proteins. A debated strategy is whether metallochaperones exist to prioritize essential Zn-dependent proteins. Here, we present evidence for a conserved family of putative metal transferases in human and fungi, which interact with Zn-dependent methionine aminopeptidase type I (MetAP1/Map1p/Fma1). Deletion of the putative metal transferase in Saccharomyces cerevisiae (ZNG1; formerly YNR029c) leads to defective Map1p function and a Zn-deficiency growth defect. Invitro, Zng1p can transfer Zn2+ or Co2+ to apo-Map1p, but unlike characterized copper chaperones, transfer is dependent on GTP hydrolysis. Proteomics reveal mis-regulation of the Zap1p transcription factor regulon because of loss of ZNG1 and Map1p activity, suggesting that Zng1p is required to avoid a compounding effect of Map1p dysfunction on survival during Zn limitation.

A Putative Guanosine Triphosphate Cyclohydrolase I Named CaGCH1 Is Involved in Hyphal Branching and Fruiting Development in Cyclocybe aegerita.

Guanosine triphosphate (GTP) cyclohydrolase I (GCH1) is the limiting enzyme of the tetrahydrobiopterin (BH4) synthesis pathway. The disruption of gch1 gene may cause conditional lethality due to folic acid auxotrophy in microorganisms, although the function of gch1 in basidiomycetes has not been deciphered so far. In the present study, gch1 expression in Cyclocybe aegerita (cagch1) was downregulated using the RNAi method, which resulted in growth retardation in both solid and liquid medium, with the hyphal tips exhibiting increased branching compared to that in the wild strain. The development of fruiting bodies in the mutant strains was significantly blocked, and there were short and bottle-shaped stipes. The transcriptional profile revealed that the genes of the MAPK pathway may be involved in the regulation of these effects caused by cagch1 knockdown, which provided an opportunity to study the role of gch1 in the development process of basidiomycetes.

Neuropsychiatric and sleep study in autosomal dominant dopa-responsive dystonia

IntroductionAlthough the diurnal fluctuation of motor dysfunction, reversible with small doses of dopamine, is a cornerstone for the phenotype of the autosomal dominant Segawa syndrome, the non-motor symptoms of this neurotransmitter deficiency have still received limited attention. ObjectiveThis study aims to evaluate non-motor symptoms of this dopa-responsive dystonia through an intrafamilial comparative cross-sectional study. MethodsSeventeen individuals with a c.IVS5 + 3insT (c.626 + 3insT) variation in the GTP cyclohydrolase-1 gene (GCH1, HGNC: 4193) and 34 intrafamilial controls were studied using the Beck Depression Inventory-II, the Wiener Matrizen Test 2, the Epworth Sleepiness Scale, the Pittsburgh Sleep Quality Index, the MINI/MINI PLUS Questionnaires, the World Health Organization Quality of Life – BREF Instrument and a drug use assessment questionnaire. ResultsNo significant difference was found between the groups in the prevalence of sleep disorders and in cognitive function. Nevertheless, generalized anxiety disorder (p = 0.050) and attention-deficit/hyperactivity disorder in childhood (p = 0.011) were observed only in individuals without the molecular variation. The group with the GCH1 variation presented a worse perception about how safe they feel in their daily lives (p = 0.034), less satisfaction with themselves (p = 0.049) and with their relationships (p = 0.029), and a higher prevalence of past major depressive episodes before use of L-Dopa (p = 0.046). ConclusionLow dopamine could have been protective against generalized anxiety disorder and attention-deficit/hyperactivity disorder in childhood in Segawa group individuals. The prevalence of depression was higher in individuals with the molecular variant prior to the L-Dopa treatment. Considering it, the penetrance estimates for the variant carriers increased from 58.8% to up to 88% in this large studied family. Additionally, neuropsychiatric tests of all individuals with a molecular diagnosis in an affected family are a valuable instrument for its clinical management.

Marginal BH4 deficiencies, iNOS, and self-perpetuating oxidative stress in post-acute sequelae of Covid-19

The treatment of post-acute sequelae of Covid-19 (PASC) has been informed primarily by symptomatic parallels with other chronic inflammatory syndromes. This manuscript takes a more systemic approach by examining how a marginal deficiency of tetrahydrobiopterin (BH4) resulting from mutations of the GCH1 (GTP cyclohydrolase 1) gene may result in the uncoupling of inducible Nitric Oxide Synthase (iNOS) early in the initial response of the innate immune system to SARS-CoV-2. The resulting production of superoxide instead of nitric oxide leads to a self-perpetuating cycle of oxidative stress with the potential to impair numerous metabolic processes and damage multiple organ systems. This marginal deficiency of BH4 may be exhibited by 30% or more of the patient population that have heterozygous or homozygous mutations of GCH1. As the cycle of oxidative stress continues, there is less BH4 available for other metabolic needs such as 1) resisting increased ferroptosis with its damage to organs, and 2) regulating the deactivation of the hyperinflammatory state. Finally, possible steps are proposed for clinical treatment of the hypothesized oxidative stress involved with PASC.

Co-Overexpression of RIB1 and RIB6 Increases Riboflavin Production in the Yeast Candida famata

Riboflavin or vitamin B2 is a water-soluble vitamin and a precursor of flavin coenzymes, flavin mononucleotide, and flavin adenine dinucleotide, which play a key role as enzyme cofactors in energy metabolism. Candida famata yeast is a promising producer of riboflavin, as it belongs to the group of so-called flavinogenic yeasts, capable of riboflavin oversynthesis under conditions of iron starvation. The role of the particular structural genes in the limitation of riboflavin oversynthesis is not known. To study the impact of overexpression of the structural genes of riboflavin synthesis on riboflavin production, a set of plasmids containing genes RIB1, RIB6, and RIB7 in different combinations was constructed. The transformants of the wild-type strain of C. famata, as well as riboflavin overproducer, were obtained, and the synthesis of riboflavin was studied. It was found that overexpression of RIB1 and RIB6 genes coding for enzymes GTP cyclohydrolase II and 3,4-dihydroxy-2-butanone-4-phosphate synthase, which catalase the initial steps of riboflavin synthesis, elevated riboflavin production by 13–28% relative to the parental riboflavin-overproducing strains.

Novel Therapeutic Savior for Osteosarcoma: The Endorsement of Ferroptosis.

Ferroptosis has recently been discovered as an iron-dependent and non-apoptotic regulated mechanism of cell death. The induction of ferroptosis in tumor cells improves tumor treatment, making it a current research hotspot. Mechanistically, it starts by lipid peroxidation, iron accumulation, reactive oxygen species (ROS) production, and glutathione deprivation, highlighting novel treatment opportunities for many tumors and neurodegenerative disorders. Several tumor cell lines are resistant to ferroptosis inducers, even when the ferroptosis key enzyme glutathione peroxidase 4 (GPX4) is blocked, indicating that other important elements are also involved in this process. Ferroptosis-suppressor-protein 1 (FSP1) was discovered to be one of these elements in addition to a few others such as ferroptotic gatekeepers like GTP cyclohydrolase 1 (GCH1) and dihydroorotate dehydrogenase (DHODH). Osteosarcoma is the most common primary malignant bone tumor observed most frequently in children and adolescents. Several studies demonstrated that ferroptosis plays a critical role in the treatment of osteosarcoma, in particular drug-resistant osteosarcoma cells. We outlined four primary regulators involved in ferroptosis in this article, reviewed previously published studies of ferroptosis in osteosarcoma to provide covert insights about osteosarcoma treatment, and highlighted several critical issues to point out future research possibilities.

M6 A-mediated regulation of PBX1-GCH1 axis promotes gastric cancer proliferation and metastasis by elevating tetrahydrobiopterin levels.

BackgroundMethyltransferase 3 (METTL3)‐mediated N6‐methyladenosine (m6A) RNA modification has been demonstrated to be a potential factor in promoting gastric cancer (GC). METTL3 regulates a series of signaling pathways by modifying various mRNAs. This study aimed to identify novel METTL3‐mediated signaling pathways and explored possible targets for use in the clinical setting of gastric cancer.MethodsTo investigate the proliferation and metastatic capacity of GC cell lines with METTL3 knockdown, a xenograft, lung metastasis, and popliteal lymph node metastasis model was used. The m6A‐modified RNA immunoprecipitation (Me‐RIP) sequence was utilized to explore the target mRNAs of METTL3. Cell counting kit 8 and transwell assays were performed to investigate the promoting function of pre‐B cell leukemia homeobox 1 (PBX1) and GTP cyclohydrolase 1 (GCH1). Western blotting and chromatin immunoprecipitation were employed to confirm the involvement of the METTL3‐PBX1‐GCH1 axis. ELISA and liquid chromatography‐mass spectrometry were used to explore the biological function of tetrahydrobiopterin (BH4).ResultsKnockdown of METTL3 suppressed xenograft tumor growth and lung/lymph node metastasis in vivo. Mechanistically, we found that METTL3 combined with and stabilized PBX1 mRNAs. Chromatin immunoprecipitation (ChIP) and further experiments suggested that PBX1 acted as a transcription factor inducing GCH1 expression. Moreover, the METTL3‐PBX1‐GCH1 axis increased BH4 levels in GC cells, thereby promoting tumor progression.ConclusionsThis study suggested that METTL3 enzymes promote tumor growth and lung/lymph node metastasis via METTL3‐PBX1‐GCH1 axis increasing BH4 levels in GC.

Identification and Functional Analysis of GTP Cyclohydrolase II in Candida glabrata in Response to Nitrosative Stress.

Reactive nitrogen species (RNS) are signal molecules involved in various biological events; however, excess levels of RNS cause nitrosative stress, leading to cell death and/or cellular dysfunction. During the process of infection, pathogens are exposed to nitrosative stress induced by host-derived RNS. Therefore, the nitrosative stress resistance mechanisms of pathogenic microorganisms are important for their infection and pathogenicity, and could be promising targets for antibiotics. Previously, we demonstrated that the RIB1 gene encoding GTP cyclohydrolase II (GCH2), which catalyzes the first step of the riboflavin biosynthesis pathway, is important for nitrosative stress resistance in the yeast Saccharomyces cerevisiae. Here, we identified and characterized the RIB1 gene in the opportunistic pathogenic yeast Candida glabrata. Our genetic and biochemical analyses indicated that the open reading frame of CAGL0F04279g functions as RIB1 in C. glabrata (CgRIB1). Subsequently, we analyzed the effect of CgRIB1 on nitrosative stress resistance by a growth test in the presence of RNS. Overexpression or deletion of CgRIB1 increased or decreased the nitrosative stress resistance of C. glabrata, respectively, indicating that GCH2 confers nitrosative stress resistance on yeast cells. Moreover, we showed that the proliferation of C. glabrata in cultures of macrophage-like cells required the GCH2-dependent nitrosative stress detoxifying mechanism. Additionally, an infection assay using silkworms as model host organisms indicated that CgRIB1 is indispensable for the virulence of C. glabrata. Our findings suggest that the GCH2-dependent nitrosative stress detoxifying mechanism is a promising target for the development of novel antibiotics.