Mathematical kinetic modelling followed by in vitro and in vivo assays reveal the bifunctional rice GTPCHII/DHBPS enzymes and demonstrate the key roles of OsRibA proteins in the vitamin B2 pathway.

BACKGROUND: Riboflavin is the precursor of several cofactors essential for normal physical and cognitive development, but only plants and some microorganisms can produce it. Humans thus rely on their dietary intake, which at a global level is mainly constituted by cereals (> 50%). Understanding the riboflavin biosynthesis players is key for advancing our knowledge on this essential pathway and can hold promise for biofortification strategies in major crop species. In some bacteria and in Arabidopsis, it is known that RibA1 is a bifunctional protein with distinct GTP cyclohydrolase II (GTPCHII) and 3,4-dihydroxy-2-butanone-4-phosphate synthase (DHBPS) domains. Arabidopsis harbors three RibA isoforms, but only one retained its bifunctionality. In rice, however, the identification and characterization of RibA has not yet been described. RESULTS: Through mathematical kinetic modeling, we identified RibA as the rate-limiting step of riboflavin pathway and by bioinformatic analysis we confirmed that rice RibA proteins carry both domains, DHBPS and GTPCHII. Phylogenetic analysis revealed that OsRibA isoforms 1 and 2 are similar to Arabidopsis bifunctional RibA1. Heterologous expression of OsRibA1 completely restored the growth of the rib3∆ yeast mutant, lacking DHBPS expression, while causing a 60% growth improvement of the rib1∆ mutant, lacking GTPCHII activity. Regarding OsRibA2, its heterologous expression fully complemented GTPCHII activity, and improved rib3∆ growth by 30%. In vitro activity assays confirmed that both OsRibA1 and OsRibA2 proteins carry GTPCHII/DHBPS activities, but that OsRibA1 has higher DHBPS activity. The overexpression of OsRibA1 in rice callus resulted in a 28% increase in riboflavin content. CONCLUSIONS: Our study elucidates the critical role of RibA in rice riboflavin biosynthesis pathway, establishing it as the rate-limiting step in the pathway. By identifying and characterizing OsRibA1 and OsRibA2, showcasing their GTPCHII and DHBPS activities, we have advanced the understanding of riboflavin biosynthesis in this staple crop. We further demonstrated that OsRibA1 overexpression in rice callus increases its riboflavin content, providing supporting information for bioengineering efforts.

Mechanisms underlying the efficacy and limitation of dopa and tetrahydrobiopterin therapies for the deficiency of GTP cyclohydrolase 1 revealed in a novel mouse model.

Dopa and tetrahydrobiopterin (BH4) supplementation are recommended therapies for the dopa-responsive dystonia caused by GTP cyclohydrolase 1 (GCH1, also known as GTPCH) deficits. However, the efficacy and mechanisms of these therapies have not been intensively studied yet. In this study, we tested the efficacy of dopa and BH4 therapies by using a novel GTPCH deficiency mouse model, Gch1KI/KI, which manifested infancy-onset motor deficits and growth retardation similar to the patients. First, dopa supplementation supported Gch1KI/KI mouse survival to adulthood, but residual motor deficits and dwarfism remained. Interestingly, RNAseq analysis indicated that while the genes participating in BH4 biosynthesis and regeneration were significantly increased in the liver, no significant changes were observed in the brain. Second, BH4 supplementation alone restored the growth of Gch1KI/KI pups only in early postnatal developmental stage. High doses of BH4 supplementation indeed restored the total brain BH4 levels, but brain dopamine deficiency remained. While total brain TH levels were relatively increased in the BH4 treated Gch1KI/KI mice, the TH in the striatum were still almost undetectable, suggesting differential BH4 requirements among brain regions. Last, the growth of Gch1KI/KI mice under combined therapy outperformed dopa or BH4 therapy alone. Notably, dopamine was abnormally high in more than half, but not all, of the treated Gch1KI/KI mice, suggesting the existence of variable synergetic effects of dopa and BH4 supplementation. Our results provide not only experimental evidence but also novel mechanistic insights into the efficacy and limitations of dopa and BH4 therapies for GTPCH deficiency.

De novo p.Glu61Ter mutation in GCH1 in a Moroccan patient with dopa-responsive dystonia: a case report.

Dopa-responsive dystonia (DRD) is a hereditary movement disorder due to a selective nigrostriatal dopamine deficiency. It is characterized by onset in childhood or adolescence with marked diurnal fluctuation with or without Parkinsonian features, and is caused by mutations in GCH1 gene. We report in this study the clinical and genetic features of the first DRD Moroccan patient. Using a gene panel sequencing, we identified a heterozygous nonsense variant p. Glu61Ter in GCH1. A subsequent targeted segregation analysis by Sanger sequencing validated the presence of the mutation in the patient, which was found to have occurred de novo. The objective of this study is to report the first description of DRD in Morocco, and highlights the importance of new generation sequencing technology in the reduction of medical wandering and the management of hereditary diseases.

Three-Dimensional Gait Analysis as a Biomarker for GTP Cyclohydrolase 1-Deficient Dopa-Responsive Dystonia.

BACKGROUND: GTP-cyclohydrolase 1-deficient dopa-responsive dystonia (GTPCH1-deficient DRD) typically presents in childhood with dystonic posture of the lower extremities, gait impairment, and a significant response to levodopa. We performed three-dimensional gait analysis (3DGA) to quantitatively assess the gait characteristics and changes associated with levodopa treatment in patients with GTPCH1-deficient DRD. METHODS: Three levodopa-treated patients with GTPCH1-deficient DRD underwent 3DGA twice, longitudinally. Changes were evaluated for cadence; gait speed; step length; gait deviation index; kinematic data of the pelvis, hip, knee, and ankle joints; and foot progression angle. RESULTS: Levodopa treatment increased the cadence and gait speed in one of three patients and increased the gait deviation index in two of three patients. The kinematic data for each joint exhibited different characteristics, with some improvement observed in each of the three patients. There was consistent marked improvement in the abnormal foot progression angle; one patient had excessive external rotation of one foot, another had excessive bilateral internal rotation, and the other had excessive internal rotation of one foot and excessive external rotation of the opposite foot, all of which improved. CONCLUSION: The 3DGA findings demonstrate that the gait pathology and recovery process in GTPCH1-deficient DRD vary from case to case. Changes in the foot progression angle and gait deviation index can enable the effects of treatment to be more easily evaluated.

Long non­coding RNA lung cancer­associated transcript 1 regulates ferroptosis via microRNA­34a­5p­mediated GTP cyclohydrolase 1 downregulation in lung cancer cells.

Ferroptosis, a recently discovered type of programmed cell death triggered by excessive accumulation of iron­dependent lipid peroxidation, is linked to several malignancies, including non­small cell lung cancer. Long non­coding RNAs (lncRNAs) are involved in ferroptosis; however, data on their role and mechanism in cancer therapy remains limited. Therefore, the aim of the present study was to identify ferroptosis­associated mRNAs and lncRNAs in A549 lung cancer cells treated with RAS­selective lethal 3 (RSL3) and ferrostatin­1 (Fer­1) using RNA sequencing. The results demonstrated that lncRNA lung cancer­associated transcript 1 (LUCAT1) was significantly upregulated in lung adenocarcinoma and lung squamous cell carcinoma tissues. Co­expression analysis of differentially expressed mRNAs and lncRNAs suggested that LUCAT1 has a crucial role in ferroptosis. LUCAT1 expression was markedly elevated in A549 cells treated with RSL3, which was prevented by co­incubation with Fer­1. Functionally, overexpression of LUCAT1 facilitated cell proliferation and reduced the occurrence of ferroptosis induced by RSL3 and Erastin, while inhibition of LUCAT1 expression reduced cell proliferation and increased ferroptosis. Mechanistically, downregulation of LUCAT1 resulted in the downregulation of both GTP cyclohydrolase 1 (GCH1) and ferroptosis suppressor protein 1 (FSP1). Furthermore, inhibition of LUCAT1 expression upregulated microRNA (miR)­34a­5p and then downregulated GCH1. These results indicated that inhibition of LUCAT1 expression promoted ferroptosis by modulating the downregulation of GCH1, mediated by miR­34a­5p. Therefore, the combination of knocking down LUCAT1 expression with ferroptosis inducers may be a promising strategy for lung cancer treatment.

Tetrahydrobiopterin metabolism attenuates ROS generation and radiosensitivity through LDHA S-nitrosylation: novel insight into radiogenic lung injury.

Genotoxic therapy triggers reactive oxygen species (ROS) production and oxidative tissue injury. S-nitrosylation is a selective and reversible posttranslational modification of protein thiols by nitric oxide (NO), and 5,6,7,8-tetrahydrobiopterin (BH4) is an essential cofactor for NO synthesis. However, the mechanism by which BH4 affects protein S-nitrosylation and ROS generation has not been determined. Here, we showed that ionizing radiation disrupted the structural integrity of BH4 and downregulated GTP cyclohydrolase I (GCH1), which is the rate-limiting enzyme in BH4 biosynthesis, resulting in deficiency in overall protein S-nitrosylation. GCH1-mediated BH4 synthesis significantly reduced radiation-induced ROS production and fueled the global protein S-nitrosylation that was disrupted by radiation. Likewise, GCH1 overexpression or the administration of exogenous BH4 protected against radiation-induced oxidative injury in vitro and in vivo. Conditional pulmonary Gch1 knockout in mice (Gch1fl/fl; Sftpa1-Cre+/- mice) aggravated lung injury following irradiation, whereas Gch1 knock-in mice (Gch1lsl/lsl; Sftpa1-Cre+/- mice) exhibited attenuated radiation-induced pulmonary toxicity. Mechanistically, lactate dehydrogenase (LDHA) mediated ROS generation downstream of the BH4/NO axis, as determined by iodoacetyl tandem mass tag (iodoTMT)-based protein quantification. Notably, S-nitrosylation of LDHA at Cys163 and Cys293 was regulated by BH4 availability and could restrict ROS generation. The loss of S-nitrosylation in LDHA after irradiation increased radiosensitivity. Overall, the results of the present study showed that GCH1-mediated BH4 biosynthesis played a key role in the ROS cascade and radiosensitivity through LDHA S-nitrosylation, identifying novel therapeutic strategies for the treatment of radiation-induced lung injury.

Dopa-responsive Dystonia with a Novel Initiation Codon Mutation in the GCH1 Gene Misdiagnosed as Cerebral Palsy

Dopa-responsive dystonia (DRD) is a clinical syndrome characterized by childhood-onset dystonia and a dramatic response to relatively low doses of levodopa. However, patients with DRD can be misdiagnosed as cerebral palsy or spastic diplegia due to phenotypic variation. Here we report a young woman with DRD who were severely disabled and misdiagnosed as cerebral palsy for over 10 yr. A small dose of levodopa restored wheelchair-bound state to normality. However, thoracolumbar scoliosis has remained as a sequel due to late detection of DRD. Genetic analysis by using PCR-direct sequencing revealed a novel initiation codon mutation (c.1A>T; p.Met1Leu) in GTP cyclohydrolase 1 (GCH1) gene. Although it is known that DRD can be misdiagnosed as cerebral palsy, this case reinforces the importance of differential diagnosis of DRD from cerebral palsy.

Predisposition of genetic disease by modestly decreased expression of GCH1 mutant allele

Recently it was shown that single nucleotide polymorphisms (SNPs) can explain individual variation because of the small changes of the gene expression level and that the 50% decreased expression of an allele might even lead to predisposition to cancer. In this study, we found that a decreased expression of an allele might cause predisposition to genetic disease. Dopa responsive dystonia (DRD) is a dominant disease caused by mutations in GCH1 gene. The sequence analysis of the GCH1 in a patient with typical DRD symptoms revealed two novel missense mutations instead of a single dominant mutation. Family members with either of the mutations did not have any symptoms of DRD. The expression level of a R198W mutant allele decreased to about 50%, suggesting that modestly decreased expression caused by an SNP should lead to predisposition of a genetic disease in susceptible individuals.

A novel missense mutation pattern of the GCH1 gene in dopa-responsive dystonia

Dopa-responsive dystonia (DRD) is an inherited metabolic disorder now classified as DYT5 with two different biochemical defects: autosomal dominant GTP cyclohydrolase 1 (GCH1) deficiency or autosomal recessive tyrosine hydroxylase deficiency. We report the case of a 10-years-old girl with progressive generalized dystonia and gait disorder who presented dramatic response to levodopa. The phenylalanine to tyrosine ratio was significantly higher after phenylalanine loading test. This condition had two different heterozygous mutations in the GCH1 gene: the previously reported P23L mutation and a new Q182E mutation. The characteristics of the DRD and the molecular genetic findings are discussed.

Distonia dopa-responsiva (DRD), classificada como DYT5, é um erro inato do metabolismo que pode ser causado por dois diferentes tipos de defeito bioquímico: deficiência de GTP ciclo-hidrolase 1 (GCH1) (autossômica dominante) ou de tirosina hidroxilase (autossômica recessiva). Descrevemos o caso de menina de 10 anos com distonia generalizada progressiva e alteração da marcha com importante melhora após uso de levodopa. A relação fenilalanina/tirosina estava aumentada após teste de sobrecarga com fenilalanina. O estudo molecular mostrou que o paciente apresenta uma combinação hererozigótica de mutação no gene GCH1: a já conhecida mutação P23L e uma nova mutação Q182E. Discutem-se as características da DRD e as alterações genéticas possíveis.

Diferentes caras de la distonía sensible a la levodopa / No disponible

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