MTHFD2-mediated redox homeostasis promotes gastric cancer progression under hypoxic conditions.

OBJECTIVES: Cancer cells undergo metabolic reprogramming to adapt to high oxidative stress, but little is known about how metabolic remodeling enables gastric cancer cells to survive stress associated with aberrant reactive oxygen species (ROS) production. Here, we aimed to identify the key metabolic enzymes that protect gastric cancer (GC) cells from oxidative stress. METHODS: ROS level was detected by DCFH-DA probes. Multiple cell biological studies were performed to identify the underlying mechanisms. Furthermore, cell-based xenograft and patient-derived xenograft (PDX) model were performed to evaluate the role of MTHFD2 in vivo. RESULTS: We found that overexpression of MTHFD2, but not MTHFD1, is associated with reduced overall and disease-free survival in gastric cancer. In addition, MTHFD2 knockdown reduces the cellular NADPH/NADP+ ratio, colony formation and mitochondrial function, increases cellular ROS and cleaved PARP levels and induces in cell death under hypoxia, a hallmark of solid cancers and a common inducer of oxidative stress. Moreover, genetic or pharmacological inhibition of MTHFD2 reduces tumor burden in both tumor cell lines and patient-derived xenograft-based models. DISCUSSION: our study highlights the crucial role of MTHFD2 in redox regulation and tumor progression, demonstrating the therapeutic potential of targeting MTHFD2.

Dynamic evolution of the mTHF gene family associated with primary metabolism across life.

BACKGROUND: The folate cycle of one-carbon (C1) metabolism, which plays a central role in the biosynthesis of nucleotides and amino acids, demonstrates the significance of metabolic adaptation. We investigated the evolutionary history of the methylenetetrahydrofolate dehydrogenase (mTHF) gene family, one of the main drivers of the folate cycle, across life. RESULTS: Through comparative genomic and phylogenetic analyses, we found that several lineages of Archaea lacked domains vital for folate cycle function such as the mTHF catalytic and NAD(P)-binding domains of FolD. Within eukaryotes, the mTHF gene family diversified rapidly. For example, several duplications have been observed in lineages including the Amoebozoa, Opisthokonta, and Viridiplantae. In a common ancestor of Opisthokonta, FolD and FTHFS underwent fusion giving rise to the gene MTHFD1, possessing the domains of both genes. CONCLUSIONS: Our evolutionary reconstruction of the mTHF gene family associated with a primary metabolic pathway reveals dynamic evolution, including gene birth-and-death, gene fusion, and potential horizontal gene transfer events and/or amino acid convergence.

Optical coherence tomography-guided Brillouin microscopy highlights regional tissue stiffness differences during anterior neural tube closure in the Mthfd1l murine mutant.

Neurulation is a highly synchronized biomechanical process leading to the formation of the brain and spinal cord, and its failure leads to neural tube defects (NTDs). Although we are rapidly learning the genetic mechanisms underlying NTDs, the biomechanical aspects are largely unknown. To understand the correlation between NTDs and tissue stiffness during neural tube closure (NTC), we imaged an NTD murine model using optical coherence tomography (OCT), Brillouin microscopy and confocal fluorescence microscopy. Here, we associate structural information from OCT with local stiffness from the Brillouin signal of embryos undergoing neurulation. The stiffness of neuroepithelial tissues in Mthfd1l null embryos was significantly lower than that of wild-type embryos. Additionally, exogenous formate supplementation improved tissue stiffness and gross embryonic morphology in nullizygous and heterozygous embryos. Our results demonstrate the significance of proper tissue stiffness in normal NTC and pave the way for future studies on the mechanobiology of normal and abnormal embryonic development.

Interference with MTHFD2 induces ferroptosis in ovarian cancer cells through ERK signaling to suppress tumor malignant progression.

Ovarian cancer (OC) is a deadliest gynecological cancer with the highest mortality rate. Methylenetetrahydrofolate dehydrogenase 2 (MTHFD2), a crucial tumor-promoting factor, is over-expressed in several malignancies including OC. The present study aimed to explore the role and mechanisms of MTHFD2 in OC malignant progression. Thus, cell proliferation, cycling, apoptosis, migration, and invasion were evaluated by CCK-8 assay, EdU assay, flow cytometry, wound healing, transwell assay and western blotting. Additionally, glycolysis was assessed by measuring the level of glucose and lactate production, as well as the expressions of GLUT1, HK2 and PKM2. Then the expression of ferroptosis-related proteins and ERK signaling was detected using western blotting. Ferroptosis was detected through the measurement of iron level, GSH, MDA and ROS activities. The results revealed that MTHFD2 was highly expressed in OC cells. Besides, interference with MTHFD2 induced ferroptosis, promoted ROS accumulation, destroyed mitochondrial function, reduced ATP content and inhibited glycolysis in OC cells. Subsequently, we further found that interference with MTHFD2 affected mitochondrial function and glycolysis in OC cells through ERK signaling. Moreover, interference with MTHFD2 affected ferroptosis to inhibit the malignant progression of OC cells. Collectively, our present study disclosed that interference with MTHFD2 induced ferroptosis in OC to inhibit tumor malignant progression through regulating ERK signaling.

MTHFD1 regulates the NADPH redox homeostasis in MYCN-amplified neuroblastoma.

MYCN amplification is an independent poor prognostic factor in patients with high-risk neuroblastoma (NB). Further exploring the molecular regulatory mechanisms in MYCN-amplified NB will help to develop novel therapy targets. In this study, methylenetetrahydrofolate dehydrogenase 1 (MTHFD1) was identified as the differentially expressed gene (DEG) highly expressed in MYCN-amplified NB, and it showed a positive correlation with MYCN and was associated with a poor prognosis of NB patients. Knockdown of MTHFD1 inhibited proliferation and migration, and induced apoptosis of NB cells in vitro. Mouse model experiments validated the tumorigenic effect of MTHFD1 in NB in vivo. In terms of the mechanism, ChIP-qPCR and dual-luciferase reporter assays demonstrated that MTHFD1 was directly activated by MYCN at the transcriptional level. As an important enzyme in the folic acid metabolism pathway, MTHFD1 maintained the NADPH redox homeostasis in MYCN-amplified NB. Knockdown of MTHFD1 reduced cellular NADPH/NADP+ and GSH/GSSG ratios, increased cellular reactive oxygen species (ROS) and triggered the apoptosis of NB cells. Moreover, genetic knockdown of MTHFD1 or application of the anti-folic acid metabolism drug methotrexate (MTX) potentiated the anti-tumor effect of JQ1 both in vitro and in vivo. Taken together, MTHFD1 as an oncogene is a potential therapeutic target for MYCN-amplified NB. The combination of MTX with JQ1 is of important clinical translational significance for the treatment of patients with MYCN-amplified NB.

Downregulation of MTHFD2 Inhibits Proliferation and Enhances Chemosensitivity in Hepatocellular Carcinoma via PI3K/AKT Pathway.

BACKGROUND: Despite the substantial impact of methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) on cancer progression, its significance in the regulation of hepatocellular carcinoma (HCC) cell proliferation and chemosensitivity remains poorly defined. METHODS: We evaluated MTHFD2 expression in a total of 95 HCC tissues by immunohistochemistry and analyzed the association of MTHFD2 with clinicopathologic features. qRT-PCR and Western blotting were conducted to verify MTHFD2 expression levels. Bioinformatics analysis such as gene set enrichment analysis (GSEA) and kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis were used to predict the signaling pathways involved in MTHFD2. In addition, to investigate the anti-tumor effects of MTHFD2 knockdown, Cell Counting Kit-8 (CCK-8) and EdU assays were used. RESULTS: We found that MTHFD2 was frequently upregulated in HCC, and the combination of increased expression of MTHFD2 and Ki67 was associated with poor HCC prognosis. MTHFD2 knockdown significantly inhibited HCC cell proliferation and effectively sensitized HCC cells to sorafenib and lenvatinib. PI3K/AKT pathway was involved in MTHFD2-mediated modulation of proliferation and chemosensitivity. CONCLUSIONS: These findings indicate that MTHFD2 plays an important role in proliferation and chemosensitivity of HCC, indicating that it may serve as a novel pharmacological target for improving HCC therapy.

Expression, Prognostic Value, and Immune Infiltration of MTHFD Family in Bladder Cancer.

BACKGROUND: The Methylenetetrahydrofolate Dehydrogenase (MTHFD) family plays an important role in the development and prognosis of a variety of tumors; however, the role of the MTHFD family in bladder cancer is unclear. METHODS: R software, cBioPortal, GeneMANIA, and online sites such as String-LinkedOmics were used for bioinformatics analysis. RESULTS: MTHFD1/1L/2 was significantly upregulated in bladder cancer tissues compared with normal tissues, high expression of the MTHFD family was strongly associated with poorer clinical grading and staging, and bladder cancer patients with upregulated expression of MTHFD1L/2 had a significantly worse prognosis. Gene function and PPI network analysis revealed that the MTHFD family and related genes play synergistic roles in the development of bladder cancer. 800 co-expressed genes related to the MTHFD family were used for functional enrichment analysis, and the results showed that many genes were associated with various oncogenic pathways such as cell cycle and DNA replication. More importantly, the MTHFD family was closely associated with multiple infiltrating immune lymphocytes, including Treg cells, and immune molecules such as TNFSF9, CD274, and PDCD1. CONCLUSION: Our study shows that MTHFD family genes may be potential prognostic markers and therapeutic targets for patients with bladder cancer.

Targeting MTHFD2 to Exploit Cancer-Specific Metabolism and the DNA Damage Response.

The one-carbon folate enzyme methylenetetrahydrofolate dehydrogenase/cyclohydrolase 2 (MTHFD2) is a promising therapeutic target in cancer. MTHFD2 is upregulated across numerous cancer types, promotes growth and metastasis of cancer, and correlates with poorer survival. Recent studies have developed small-molecule inhibitors to the isozymes MTHFD2 and MTHFD1 that show promise as anticancer agents through different mechanisms. This review discusses the current understanding of the function of MTHFD2 in cancer and the status of inhibitors for treating MTHFD2-overexpressing cancers.

Targeting allosteric binding site in methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) to identify natural product inhibitors via structure-based computational approach.

Cancer has been viewed as one of the deadliest diseases worldwide. Among various types of cancer, breast cancer is the most common type of cancer in women. Methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) is a promising druggable target and is overexpressed in cancerous cells, like, breast cancer. We conducted structure-based modeling on the allosteric site of the enzyme. Targeting the allosteric site avoids the problem of drug resistance. Pharmacophore modeling, molecular docking, HYDE assessment, drug-likeness, ADMET predictions, simulations, and free-energy calculations were performed. The RMSD, RMSF, RoG, SASA, and Hydrogen-bonding studies showed that seven candidates displayed stable behaviour. As per the literature, average superimposed simulated structures revealed a similar protein conformational change in the αE'-ßf' loop, causing its displacement away from the allosteric site. The MM-PBSA showed tight binding of six compounds with the allosteric pocket. The effect of inhibitors interacting in the allosteric site causes a decrease in the binding energy of J49 (active-site inhibitor), suggesting the effect of allosteric binding. The PCA and FEL analysis revealed the significance of the docked compounds in the stable behaviour of the complexes. The outcome can contribute to the development of potential natural products with drug-like properties that can inhibit the MTHFD2 enzyme.

Common Variants in One-Carbon Metabolism Genes (MTHFR, MTR, MTHFD1) and Depression in Gynecologic Cancers.

We investigated the association between methylenetetrahydrofolate reductase (gene MTHFR 677C>T, rs1801133), 5-methyltetrahydrofolate-homocysteine methyltransferase (MTR 2756A>G, rs1805087), and methylenetetrahydrofolate dehydrogenase, cyclohydrolase and formyltetrahydrofolate synthetase 1 (gene MTHFD1 1958G>A, rs2236225)-well-studied functional variants involved in one-carbon metabolism-and gynecologic cancer risk, and the interaction between these polymorphisms and depression. A total of 200 gynecologic cancer cases and 240 healthy controls were recruited to participate in this study. Three single-nucleotide variants (SNVs) (rs1801133, rs1805087, rs2236225) were genotyped using the PCR-restriction fragment length polymorphism method. Depression was assessed in all patients using the Hamilton Depression Scale. Depression was statistically significantly more frequent in women with gynecologic cancers (69.5% vs. 34.2% in controls, p < 0.001). MTHFD1 rs2236225 was associated with an increased risk of gynecologic cancers (in dominant OR = 1.53, p = 0.033, and in log-additive models OR = 1.37, p = 0.024). Moreover, an association was found between depression risk and MTHFR rs1801133 genotypes in the controls but not in women with gynecologic cancers (in codominant model CC vs. TT: OR = 3.39, 95%: 1.49-7.74, p = 0.011). Cancers of the female reproductive system are associated with the occurrence of depression, and ovarian cancer may be associated with the rs2236225 variant of the MTHFD1 gene. In addition, in healthy aging women in the Polish population, the rs1801133 variant of the MTHFR gene is associated with depression.

Association of Maternal Folate Intake and Offspring MTHFD1 and MTHFD2 Genes with Congenital Heart Disease.

Existing evidence supported that congenital heart defect (CHD) was associated with a combination of environmental and genetic factors. Based on this, this study aimed at assessing the association of maternal folic acid supplementation (FAS), genetic variations in offspring methylenetetrahydrofolate dehydrogenase (MTHFD)1 and MTHFD2 genes, and their interactions with CHD and its subtypes. A hospital-based case-control study, including 620 cases with CHD and 620 healthy children, was conducted. This study showed that the absence of FAS was significantly associated with an increased risk of total CHD and its subtypes, such as atrial septal defect (ASD). FAS during the first and second trimesters was associated with a significantly higher risk of CHD in offspring compared to FAS during the three months prior to conception. The polymorphisms of offspring MTHFD1 and MTHFD2 genes at rs2236222, rs11849530, and rs828858 were significantly associated with the risk of CHD. Additionally, a significantly positive interaction between maternal FAS and genetic variation at rs828858 was observed for the risk of CHD. These findings suggested that pregnant women should carefully consider the timing of FAS, and individuals with higher genetic risk may benefit from targeted folic acid supplementation as a preventive measure against CHD.

Folate trapping is lethal to cancer cells.

Regulation of formate flux by a key folate enzyme, MTHFD2 (methylene tetrahydrofolate dehydrogenase 2) in cancer cells remains poorly understood. Green et al. (Nature Metabolism, 2023; 5: 642-659) showed an interesting phenomenon of "folate trapping" toxicity leads to cancer cell kill using a potent inhibitor (TH9619) against the dehydrogenase and cyclohydrolase (DC) activities of cytosolic methylenetetrahydrofolate dehydrogenase 1 (cMTHFD1) and nuclear methylenetetrahydrofolate dehydrogenase 2 (nMTHFD2), but not the mitochondrial MTHFD2 (mTHFD2). But, mMTHFD2 is required for formate flow to cytosol which leads to the trapping of 10-formyl tetrahydrofolate and causes toxicity by TH9619 treatment, to kill cancer cells expressing mMTHFD2. This article opens new avenues to be evaluated for therapeutic benefits of cancer patients where MTHFD2 shows overexpression viz-a-viz breast, prostate, colorectal, acute myeloid leukemia, and other cancer types.

Mitochondrial one-carbon metabolic enzyme MTHFD2 facilitates mammary gland development during pregnancy.

Mitochondrial one-carbon metabolism is crucial for embryonic development and tumorigenesis, as it supplies one-carbon units necessary for nucleotide synthesis and rapid cell proliferation. However, its contribution to adult tissue homeostasis remains largely unknown. To examine its role in adult tissue homeostasis, we specifically investigated mammary gland development during pregnancy, as it involves heightened cell proliferation. We discovered that MTHFD2, a mitochondrial one-carbon metabolic enzyme, is expressed in both luminal and basal/myoepithelial cell layers, with upregulated expression during pregnancy. Using the mouse mammary tumor virus (MMTV)-Cre recombinase system, we generated mice with a specific mutation of Mthfd2 in mammary epithelial cells. While the mutant mice were capable of properly nurturing their offspring, the pregnancy-induced expansion of mammary glands was significantly delayed. This indicates that MTHFD2 contributes to the rapid development of mammary glands during pregnancy. Our findings shed light on the role of mitochondrial one-carbon metabolism in facilitating rapid cell proliferation, even in the context of the adult tissue homeostasis.

Binding Modes of Xanthine-Derived Selective Allosteric Site Inhibitors of MTHFD2.

Methylenetetrahydrofolate dehydrogenase (MTHFD2) is a mitochondrial enzyme involved in 1 C metabolism that is upregulated in various cancer cells, but absent in normal proliferating cells. Xanthine derivatives are the first selective inhibitors of MTHFD2 which bind to its allosteric site. Xanthine derivatives (including the co-crystallized inhibitors) were herein interrogated by molecular/induced-fit docking, MM-GBSA binding free energy calculations and molecular dynamics simulations in both MTHFD2 and MTHFD1 (a close homolog expressed in healthy cells). The gained insights from our in silico protocol allowed us to study binding mode, key protein-ligand interactions and dynamic movement of the allosteric inhibitors, correlating with their experimental binding affinities, biological activities and selectivity for MTHFD2. The reported conformational changes with MTHFD2 upon binding of xanthine derivatives were furthermore evaluated and confirmed by RMSF analyses of the MD simulation trajectories. The results reported herein are expected to benefit in the rational design of selective MTHFD2 allosteric inhibitors.

Formate overflow drives toxic folate trapping in MTHFD1 inhibited cancer cells.

Cancer cells fuel their increased need for nucleotide supply by upregulating one-carbon (1C) metabolism, including the enzymes methylenetetrahydrofolate dehydrogenase-cyclohydrolase 1 and 2 (MTHFD1 and MTHFD2). TH9619 is a potent inhibitor of dehydrogenase and cyclohydrolase activities in both MTHFD1 and MTHFD2, and selectively kills cancer cells. Here, we reveal that, in cells, TH9619 targets nuclear MTHFD2 but does not inhibit mitochondrial MTHFD2. Hence, overflow of formate from mitochondria continues in the presence of TH9619. TH9619 inhibits the activity of MTHFD1 occurring downstream of mitochondrial formate release, leading to the accumulation of 10-formyl-tetrahydrofolate, which we term a 'folate trap'. This results in thymidylate depletion and death of MTHFD2-expressing cancer cells. This previously uncharacterized folate trapping mechanism is exacerbated by physiological hypoxanthine levels that block the de novo purine synthesis pathway, and additionally prevent 10-formyl-tetrahydrofolate consumption for purine synthesis. The folate trapping mechanism described here for TH9619 differs from other MTHFD1/2 inhibitors and antifolates. Thus, our findings uncover an approach to attack cancer and reveal a regulatory mechanism in 1C metabolism.

MTHFD1 is critical for the negative regulation of retinoic acid receptor signalling in anencephaly.

Neural tube defects are the most severe congenital malformations that result from failure of neural tube closure during early embryonic development, and the underlying molecular mechanisms remain elusive. Retinoic acid, an active derivative of vitamin A, is critical for neural system development, and retinoic acid receptor (RAR) signalling malfunctions have been observed in human neural tube defects. However, retinoic acid-retinoic acid receptor signalling regulation and mechanisms in neural tube defects are not fully understood. The mRNA expression of RARs and retinoid X receptors in the different human neural tube defect phenotypes, including 11 pairs of anencephaly foetuses, 10 pairs of hydrocephalus foetuses and nine pairs of encephalocele foetuses, was investigated by NanoString nCounter technology. Immunoprecipitation-mass spectrometry was performed to screen the potential interacting targets of retinoic acid receptor γ. The interactions between proteins were confirmed by co-immunoprecipitation and immunofluorescence laser confocal microscopy. Luciferase and chromatin immunoprecipitation with quantitative real-time polymerase chain reaction assays were used to clarify the underlying mechanism. Moreover, a neural tube defect animal model, constructed using excess retinoic acid, was used for further analysis with established molecular biology technologies. We report that level of retinoic acid receptor γ (RARγ) mRNA was significantly upregulated in the brain tissues of human foetuses with anencephaly. To further understand the actions of retinoic acid receptor γ in neural tube defects, methylenetetrahydrofolate dehydrogenase 1 was identified as a specific retinoic acid receptor γ target from IP-MS screening. Additionally, methylenetetrahydrofolate dehydrogenase 1 negatively regulated retinoic acid receptor γ transcription factor activity. Furthermore, low expression of methylenetetrahydrofolate dehydrogenase 1 and activation of retinoic acid receptor signalling were further determined in human anencephaly and a retinoic acid-induced neural tube defect mouse model. This study reveals that methylenetetrahydrofolate dehydrogenase 1, the rate-determining enzyme in the one-carbon cycle, might be a specific regulator of retinoic acid receptors; these findings provide new insights into the functional linkage between nuclear folate metabolism and retinoic acid receptor signalling in neural tube defect pathology.

The negative effect of G1958A polymorphism on MTHFD1 protein stability and HCC growth.

PURPOSE: Methylenetetrahydrofolate dehydrogenase (MTHFD1), a key enzyme on the folate pathway, has been implicated in the tumor development of distinct types of cancers. The single nucleotide polymorphism (SNP) of 1958G > A mutation in the coding region of MTHFD1 (arginine 653 is mutated into glutamine) has been detected in a significant proportion of clinical samples of hepatocellular carcinoma (HCC). METHODS : Hepatoma cell lines, 97H and Hep3B were used. The expression of MTHFD1 and SNP mutation protein was determined by immunoblotting analysis. The protein ubiquitination of MTHFD1 was detected by immunoprecipitation analysis. The post-translational modification sites and interacting proteins of MTHFD1 in the presence of G1958A SNP were identified by mass spectrometry. Metabolic flux analysis was used to detect the synthesis of relevant metabolites sourced from serine isotope. RESULTS: The present study showed G1958A SNP of MTHFD1, encoding MTHFD1 R653Q, was associated with the attenuated protein stability caused by ubiquitination-mediated protein degradation. Mechanistically, MTHFD1 R653Q displayed an enhanced binding to the E3 ligase TRIM21, which was responsible for the augmented ubiquitination, and MTHFD1 K504 was identified to be the primary ubiquitination site. The subsequent metabolite analysis revealed MTHFD1 R653Q resulted in the repressed flux of serine-derived methyl group into metabolite precursors for purine synthesis, and the compromised purine synthesis was demonstrated to be responsible for the impeded growth capability in MTHFD1 R653Q-expressing cells. Moreover, the suppressive effect of MTHFD1 R653Q expression in tumorigenesis was verified by xenograft analysis, and the relationship between MTHFD1 G1958A SNP and its protein levels was revealed in clinical human liver cancer specimens. CONCLUSION: Our results uncovered an unidentified mechanism underlying of the impact of G1958A SNP on MTHFD1 protein stability and tumor metabolism in HCC. which provides a molecular basis for the according clinical management when considering MTHFD1 as a therapeutic target.

Newcastle Disease Virus Manipulates Mitochondrial MTHFD2-Mediated Nucleotide Metabolism for Virus Replication.

Viruses require host cell metabolic reprogramming to satisfy their replication demands; however, the mechanism by which the Newcastle disease virus (NDV) remodels nucleotide metabolism to support self-replication remains unknown. In this study, we demonstrate that NDV relies on the oxidative pentose phosphate pathway (oxPPP) and the folate-mediated one-carbon metabolic pathway to support replication. In concert with [1,2-13C2] glucose metabolic flow, NDV used oxPPP to promote pentose phosphate synthesis and to increase antioxidant NADPH production. Metabolic flux experiments using [2,3,3-2H] serine revealed that NDV increased one-carbon (1C) unit synthesis flux through the mitochondrial 1C pathway. Interestingly, methylenetetrahydrofolate dehydrogenase (MTHFD2) was upregulated as a compensatory mechanism for insufficient serine availability. Unexpectedly, direct knockdown of enzymes in the one-carbon metabolic pathway, except for cytosolic MTHFD1, significantly inhibited NDV replication. Specific complementation rescue experiments on small interfering RNA (siRNA)-mediated knockdown further revealed that only a knockdown of MTHFD2 strongly restrained NDV replication and was rescued by formate and extracellular nucleotides. These findings indicated that NDV replication relies on MTHFD2 to maintain nucleotide availability. Notably, nuclear MTHFD2 expression was increased during NDV infection and could represent a pathway by which NDV steals nucleotides from the nucleus. Collectively, these data reveal that NDV replication is regulated by the c-Myc-mediated 1C metabolic pathway and that the mechanism of nucleotide synthesis for viral replication is regulated by MTHFD2. IMPORTANCE Newcastle disease virus (NDV) is a dominant vector for vaccine and gene therapy that accommodates foreign genes well but can only infect mammalian cells that have undergone cancerous transformation. Understanding the remodeling of nucleotide metabolic pathways in host cells by NDV proliferation provides a new perspective for the precise use of NDV as a vector or in antiviral research. In this study, we demonstrated that NDV replication is strictly dependent on pathways involved in redox homeostasis in the nucleotide synthesis pathway, including the oxPPP and the mitochondrial one-carbon pathway. Further investigation revealed the potential involvement of NDV replication-dependent nucleotide availability in promoting MTHFD2 nuclear localization. Our findings highlight the differential dependence of NDV on enzymes for one-carbon metabolism, and the unique mechanism of action of MTHFD2 in viral replication, thereby providing a novel target for antiviral or oncolytic virus therapy.

Pairing structural reconstruction with catalytic competence to evaluate the mechanisms of key enzymes in the folate-mediated one-carbon pathway.

Mammalian metabolism comprises a series of interlinking pathways that include two major cycles: the folate and methionine cycles. The folate-mediated metabolic cycle uses several oxidation states of tetrahydrofolate to carry activated one-carbon units to be readily used and interconverted within the cell. They are required for nucleotide synthesis, methylation and metabolism, and particularly for proliferation of cancer cells. Based on the latest progress in genome-wide CRISPR loss-of-function viability screening of 789 cell lines, we focus on the most cancer-dependent enzymes in this pathway, especially those that are hyperactivated in cancer, to provide new insight into the chemical basis for cancer drug development. Since the complete 3D structure of several of these enzymes of the one-carbon pathway in their active form are not available, we used homology modelling integrated with the interpretation of the reaction mechanism. In addition, have reconstructed the most likely scenario for the reactions taking place paired with their catalytic competence that provides a testable framework for this pathway.