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Genetic regulators and environmental stimuli modulate T cell activation in autoimmunity and cancer. The enzyme co-factor tetrahydrobiopterin (BH4) is involved in the production of monoamine neurotransmitters, the generation of nitric oxide, and pain 1 , 2 . Here we uncover a link between these processes, identifying a fundamental role for BH4 in T cell biology. We find that genetic inactivation of GTP cyclohydrolase 1 (GCH1, the rate-limiting enzyme in the synthesis of BH4) and inhibition of sepiapterin reductase (the terminal enzyme in the synthetic pathway for BH4) severely impair the proliferation of mature mouse and human T cells. BH4 production in activated T cells is linked to alterations in iron metabolism and mitochondrial bioenergetics. In vivo blockade of BH4 synthesis abrogates T-cell-mediated autoimmunity and allergic inflammation, and enhancing BH4 levels through GCH1 overexpression augments responses by CD4- and CD8-expressing T cells, increasing their antitumour activity in vivo. Administration of BH4 to mice markedly reduces tumour growth and expands the population of intratumoral effector T cells. Kynurenine—a tryptophan metabolite that blocks antitumour immunity—inhibits T cell proliferation in a manner that can be rescued by BH4. Finally, we report the development of a potent SPR antagonist for possible clinical use. Our data uncover GCH1, SPR and their downstream metabolite BH4 as critical regulators of T cell biology that can be readily manipulated to either block autoimmunity or enhance anticancer immunity. Tetrahydrobiopterin (BH4) is an enzyme co-factor that is involved in the nervous system; it is shown here to also function in T cell activation and proliferation, with roles in autoimmunity, allergic inflammation and cancer.

Ferroptosis is an iron-dependent cell death with the accumulation of lipid peroxidation and dysfunction of antioxidant systems. As the critical regulator, glutathione peroxidase 4 (GPX4) has been demonstrated to be down-regulated in amyotrophic lateral sclerosis (ALS). However, the mechanism of ferroptosis in ALS remains unclear. In this research, bioinformatics analysis revealed a high correlation between ALS, ferroptosis, and Speedy/RINGO cell cycle regulator family member A (SPY1). Lipid peroxidation of ferroptosis in hSOD1G93A cells and mice was generated by TFR1-imported excess free iron, decreased GSH, mitochondrial membrane dysfunction, upregulated ALOX15, and inactivation of GCH1, GPX4. SPY1 is a “cyclin-like” protein that has been proved to enhance the viability of hSOD1G93A cells by inhibiting DNA damage. In our study, the decreased expression of SPY1 in ALS was resulted from unprecedented ubiquitination degradation mediated by MDM2 (a nuclear-localized E3 ubiquitin ligase). Further, SPY1 was identified as a novel ferroptosis suppressor via alleviating lipid peroxidation produced by dysregulated GCH1/BH4 axis (a resistance axis of ferroptosis) and transferrin receptor protein 1 (TFR1)-induced iron. Additionally, neuron-specific overexpression of SPY1 significantly delayed the occurrence and prolonged the survival in ALS transgenic mice through the above two pathways. These results suggest that SPY1 is a novel target for both ferroptosis and ALS.

Background Parkinson’s disease is a highly prevalent neurodegenerative disorder. Hyposecretion of dopamine (DA) is the end result in the pathology of Parkinson’s disease. Unfortunately, safe and efficient therapeutic drugs are deficient. Tyrosine hydroxylase is the rate-limiting enzyme for DA synthesis, could hydroxylate tyrosine and generate levodopa with tetrahydrobiopterin (BH4) as an indispensable coenzyme. Furthermore, BH4 was confirmed to confer neuroprotection against Parkinson’s disease. Thus, regulation of BH4 synthesis was verified to become a promising therapeutic strategy for Parkinson’s disease. Results We demonstrate that artemisinin effectively produced neuroprotection against Parkinson’s disease in rats. Integrated analysis of midbrain proteomics and non-targeted metabolomics suggests that artemisinin might target adenylate cyclase 5 (Adcy5) to increase GTP cyclohydrolase 1 (Gch1, BH4 synthetase) expression to further boost BH4 synthesis. To verify this hypothesis, molecular docking experiments demonstrate that ART could directly bind to Adcy5. Artemisinin increases Adcy5 and Gch1 expressions and BH4 production both in vivo and in vitro. Further rescue experiments demonstrate that artemisinin-generated DA neuroprotection and hypersecretion of DA and BH4 disappears after inhibition of Adcy5 or Gch1 in vitro. Additionally, suppression of Adcy5 aggravates Parkinson’s disease manifestation, decreases midbrain DA and BH4 production and down-regulated Gch1 expression in vivo. Conclusions Artemisinin mediates neuroprotection against Parkinson’s disease via regulation of Adcy5-Gch1-BH4 axis in rats. These findings present a beneficial potential for future application of artemisinin on Parkinson’s disease treatment. Graphical Abstract

Folates are indispensable co-factors for one-carbon metabolism in all organisms. In humans, suboptimal folate intake results in serious disorders. One promising strategy for improving human folate status is to enhance folate levels in food crops by metabolic engineering. In this study, we cloned two GmGCHI (GTP cyclohydrolase I) genes (Gm8gGCHI and Gm3gGCHI) and one GmADCS (aminodeoxychorismate synthase) gene from soybean, which are responsible for synthesizing the folate precursors pterin and p-aminobenzoate, respectively. We initially confirmed their functions in transgenic Arabidopsis plants and found that Gm8gGCHI increased pterin and folate production more than Gm3gGCHI did. We then co-expressed Gm8gGCHI and GmADCS driven by endosperm-specific promoters in maize and wheat, two major staple crops, to boost their folate metabolic flux. A 4.2-fold and 2.3-fold increase in folate levels were observed in transgenic maize and wheat grains, respectively. To optimize wheat folate enhancement, codon-optimized Gm8gGCHI and tomato LeADCS genes under the control of a wheat endosperm-specific glutenin promoter (1Dx5) were co-transformed. This yielded a 5.6-fold increase in folate in transgenic wheat grains (Gm8gGCHI⁺/LeADCS⁺). This two-gene co-expression strategy therefore has the potential to greatly enhance folate levels in maize and wheat, thus improving their nutritional value.

Neuropathic pain is a chronic pain condition that is primarily caused by underlying neurological damage and dysfunction. Recent studies have identified microRNAs (miRNAs) as a key factor in the treatment of neuropathic pain. To explore the effects of miR-133a-3p on neuroinflammation and neuropathic pain via GTP cyclohydrolase (GCH1), and its underlying mechanisms. In vitro models were constructed using BV-2 cells that had been treated with lipopolysaccharide, followed by treatment with either miR-133a-3p mimic or GCH1 viral knockdown/overexpression. The expression of miR-133a-3p and GCH1 in BV-2 cells was quantified by RT-qPCR. The degree of neuroinflammation was quantified using an enzyme-linked immunosorbent assay (ELISA). The targeting relationship between miR-133a-3p and GCH1 was confirmed by western blot and dual luciferase reporter assay. A chronic constriction injury model was employed to induce neuropathic pain in rats, and the mechanical withdrawal threshold (MWT) was quantified. Immunofluorescence was used to demonstrate alterations in microglial cells. The expression of miR-133a–3p was found to be decreased in lipopolysaccharide-induced BV-2 cells. The overexpression of miR-133a–3p was observed to inhibit the expression of IL-1β, IL-6, TNF-α and iNOS, which was attributed to a reduction in GCH1.Nevertheless, OE-GCH1 could partially reverse the downregulation by miR-133a-3p of the expression of inflammatory factors. In animal experiments, intrathecal injection of AVV-miR-133a-3p was observed to alleviate mechanical nociceptive abnormalities induced by activated microglia. Furthermore, miR-133a-3p ameliorated neuroinflammation in the spinal cord of chronic constriction injury rats. In summary, miR-133a-3p improves neuroinflammation and neuropathic pain by binding to GCH1. The binding of miR-133a-3p to GCH1 has been demonstrated to improve neuroinflammation and neuropathic pain.This insight will facilitate the development of new methods to effectively treat neuropathic pain.

Ferroptosis, a recently discovered type of programmed cell death triggered by excessive accumulation of iron-dependent lipid peroxidation, is linked to several malignancies, including non-small cell lung cancer. Long non-coding 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 ferroptosis-associated mRNAs and lncRNAs in A549 lung cancer cells treated with RAS-selective lethal 3 (RSL3) and ferrostatin-1 (Fer-1) using RNA sequencing. The results demonstrated that lncRNA lung cancer-associated transcript 1 (LUCAT1) was significantly upregulated in lung adenocarcinoma and lung squamous cell carcinoma tissues. Co-expression 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 co-incubation with Fer-1. 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)-34a-5p and then downregulated GCH1. These results indicated that inhibition of LUCAT1 expression promoted ferroptosis by modulating the downregulation of GCH1, mediated by miR-34a-5p. Therefore, the combination of knocking down LUCAT1 expression with ferroptosis inducers may be a promising strategy for lung cancer treatment.

Guanosine triphosphate (GTP) cyclohydrolase I (GCH1) catalyzes the conversion of GTP to dihydroneopterin triphosphate (H2NTP), the initiating step in the biosynthesis of tetrahydrobiopterin (BH4). Besides other roles, BH4 functions as cofactor in neurotransmitter biosynthesis. The BH4 biosynthetic pathway and GCH1 have been identified as promising targets to treat pain disorders in patients. The function of mammalian GCH1s is regulated by a metabolic sensing mechanism involving a regulator protein, GCH1 feedback regulatory protein (GFRP). GFRP binds to GCH1 to form inhibited or activated complexes dependent on availability of cofactor ligands, BH4 and phenylalanine, respectively. We determined high-resolution structures of human GCH1−GFRP complexes by cryoelectron microscopy (cryo-EM). Cryo-EM revealed structural flexibility of specific and relevant surface lining loops, which previously was not detected by X-ray crystallography due to crystal packing effects. Further, we studied allosteric regulation of isolated GCH1 by X-ray crystallography. Using the combined structural information, we are able to obtain a comprehensive picture of the mechanism of allosteric regulation. Local rearrangements in the allosteric pocket upon BH4 binding result in drastic changes in the quaternary structure of the enzyme, leading to a more compact, tense form of the inhibited protein, and translocate to the active site, leading to an open, more flexible structure of its surroundings. Inhibition of the enzymatic activity is not a result of hindrance of substrate binding, but rather a consequence of accelerated substrate binding kinetics as shown by saturation transfer difference NMR (STD-NMR) and site-directed mutagenesis. We propose a dissociation rate controlled mechanism of allosteric, noncompetitive inhibition.

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.

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 ( Gch1 fl/fl ; Sftpa1-Cre +/− mice) aggravated lung injury following irradiation, whereas Gch1 knock-in mice ( Gch1 lsl/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. Radiation-induced Lung Injury Mitigated by GCH1-Mediated ROS Regulation Radiation therapy for cancer can harm healthy tissues, causing swelling and oxidative stress (an imbalance between free radicals and antioxidants in your body). This research examined the part of a molecule named tetrahydrobiopterin (BH4) in this. The scientists discovered that radiation therapy decreases the amount of BH4 in the body, which then leads to a rise in harmful reactive oxygen species (ROS - molecules that can damage cells). However, when BH4 amounts were artificially boosted, this lowered ROS levels and shielded against radiation-caused harm. This implies that BH4 might be used as a treatment to guard against the damaging side effects of radiation therapy. More research is required to further investigate this potential in clinic. This summary was initially drafted using artificial intelligence, then revised and fact-checked by the author.

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 (Htt ex1 ) with a normal (21), or an extended (42 or 63) number of polyQ in tobacco plants. Here, we show that transgenic plants accumulated Htt ex1 proteins with corresponding polyQ tracts, and mHtt ex1 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 Htt ex1 Q63 and unaffected Htt ex1 Q21 plants showed that the most reduced protein by polyQ63 is a GTP cyclohydrolase I (GTPCH) along with many of its related one-carbon (C 1 ) metabolic pathway enzymes. GTPCH is a key enzyme involved in folate biosynthesis in plants and tetrahydrobiopterin (BH 4 ) biosynthesis in mammals. Validating studies in 4-week-old R6/2 HD mice expressing a mHtt ex1 showed reduced levels of GTPCH and dihydrofolate reductase (DHFR, a key folate utilization/alternate BH 4 biosynthesis enzyme), and impaired C 1 and BH 4 metabolism. Our findings from mHtt ex1 plants and mice reveal impaired expressions of GTPCH and DHFR and may contribute to a better understanding of mHtt-altered C 1 and BH 4 metabolism, and their roles in the pathogenesis of HD.