Folylpolyglutamate synthetase mRNA G-quadruplexes regulate its cell protrusion localization and enhance a cancer cell invasive phenotype upon folate repletion.

BACKGROUND: Folates are crucial for the biosynthesis of nucleotides and amino acids, essential for cell proliferation and development. Folate deficiency induces DNA damage, developmental defects, and tumorigenicity. The obligatory enzyme folylpolyglutamate synthetase (FPGS) mediates intracellular folate retention via cytosolic and mitochondrial folate polyglutamylation. Our previous paper demonstrated the association of the cytosolic FPGS (cFPGS) with the cytoskeleton and various cell protrusion proteins. Based on these recent findings, the aim of the current study was to investigate the potential role of cFPGS at cell protrusions. RESULTS: Here we uncovered a central role for two G-quadruplex (GQ) motifs in the 3'UTR of FPGS mediating the localization of cFPGS mRNA and protein at cell protrusions. Using the MBSV6-loop reporter system and fluorescence microscopy, we demonstrate that following folate deprivation, cFPGS mRNA is retained in the endoplasmic reticulum, whereas upon 15 min of folate repletion, this mRNA is rapidly translocated to cell protrusions in a 3'UTR- and actin-dependent manner. The actin dependency of this folate-induced mRNA translocation is shown by treatment with Latrunculin B and inhibitors of the Ras homolog family member A (RhoA) pathway. Upon folate repletion, the FPGS 3'UTR GQs induce an amoeboid/mesenchymal hybrid cell phenotype during migration and invasion through a collagen gel matrix. Targeted disruption of the 3'UTR GQ motifs by introducing point mutations or masking them by antisense oligonucleotides abrogated cell protrusion targeting of cFPGS mRNA. CONCLUSIONS: Collectively, the GQ motifs within the 3'UTR of FPGS regulate its transcript and protein localization at cell protrusions in response to a folate cue, inducing cancer cell invasive phenotype. These novel findings suggest that the 3'UTR GQ motifs of FPGS constitute an attractive druggable target aimed at inhibition of cancer invasion and metastasis.

Methotrexate Provokes Disparate Folate Metabolism Gene Expression and Alternative Splicing in Ex Vivo Monocytes and GM-CSF- and M-CSF-Polarized Macrophages.

Macrophages constitute important immune cell targets of the antifolate methotrexate (MTX) in autoimmune diseases, including rheumatoid arthritis. Regulation of folate/MTX metabolism remains poorly understood upon pro-inflammatory (M1-type/GM-CSF-polarized) and anti-inflammatory (M2-type/M-CSF-polarized) macrophages. MTX activity strictly relies on the folylpolyglutamate synthetase (FPGS) dependent intracellular conversion and hence retention to MTX-polyglutamate (MTX-PG) forms. Here, we determined FPGS pre-mRNA splicing, FPGS enzyme activity and MTX-polyglutamylation in human monocyte-derived M1- and M2-macrophages exposed to 50 nmol/L MTX ex vivo. Moreover, RNA-sequencing analysis was used to investigate global splicing profiles and differential gene expression in monocytic and MTX-exposed macrophages. Monocytes displayed six-eight-fold higher ratios of alternatively-spliced/wild type FPGS transcripts than M1- and M2-macrophages. These ratios were inversely associated with a six-ten-fold increase in FPGS activity in M1- and M2-macrophages versus monocytes. Total MTX-PG accumulation was four-fold higher in M1- versus M2-macrophages. Differential splicing after MTX-exposure was particularly apparent in M2-macrophages for histone methylation/modification genes. MTX predominantly induced differential gene expression in M1-macrophages, involving folate metabolic pathway genes, signaling pathways, chemokines/cytokines and energy metabolism. Collectively, macrophage polarization-related differences in folate/MTX metabolism and downstream pathways at the level of pre-mRNA splicing and gene expression may account for variable accumulation of MTX-PGs, hence possibly impacting MTX treatment efficacy.

Maternal genotypes of folate/one-carbon metabolism gene variants and nonsyndromic cleft lip with or without cleft palate risk in Chile.

The aim of this study was to evaluate, in a case-control design, the association between maternal genotypes for variants in 23 genes involved in folate/one-carbon metabolism and nonsyndromic cleft lip with or without cleft palate (NSCL/P) in a Chilean population. After applying several filters to an Illumina array, we extracted 175 single nucleotide polymorphisms (SNPs) from 150 mothers of NSCL/P cases and 150 control women. Association was evaluated using computed odds ratio (OR) with a 95% confidence interval (95% CI) in additive, recessive, and dominant models. After multiple comparison correction, only SNP rs4451422 (A>C), located 237 bp downstream of the gene encoding the human folylpolyglutamate synthetase (FPGS), maintained a significant association with NSCL/P in the offspring (OR 3.03; 95% CI 1.69-5.26). The variant rs4451422 is associated with a decrease in FPGS expression according to database annotation. Our results lead to a new hypothesis that a lower activity of FPGS enzyme reduces intracellular folate levels and increases the risk of an offspring having NSCL/P.

ß-catenin promotes MTX resistance of leukemia cells by down-regulating FPGS expression via NF-κB.

BACKGROUND: Aberrant activation of ß-catenin has been shown to play important roles in the chemoresistance of acute lymphoblastic leukemia (ALL), but the involvement and mechanism of ß-catenin in methotrexate (MTX) resistance is poorly understood. In the present study, we demonstrate a critical role of ß-catenin-NF-κB-FPGS pathway in MTX resistance in the human T-lineage ALL cell lines. METHODS: Lentivirus sh-ß-catenin was used to silence the expression of ß-catenin. Flow cytometry was performed to detect apoptosis after MTX treatment. Western blot, real-time PCR, Co-immunoprecipitation (Co-IP), Chromatin immunoprecipitation (ChIP), Re-ChIP, and Luciferase assay were utilized to investigate the relationship among ß-catenin, nuclear factor (NF)-κB, and folypoly-γ-glutamate synthetase (FPGS). RESULTS: Depletion of ß-catenin significantly increased the cytotoxicity of MTX. At the molecular level, knockdown of ß-catenin caused the increase of the protein level of FPGS and NF-κB p65. Furthermore, ß-catenin complexed with NF-κB p65 and directly bound to the FPGS promoter to regulate its expression. In addition, ß-catenin repression prolonged the protein turnover of FPGS. CONCLUSIONS: Taken together, our results demonstrate that ß-catenin may contribute to MTX resistance in leukemia cells via the ß-catenin-NF-κB-FPGS pathway, posing ß-catenin as a potential target for combination treatments during ALL therapy.

Plastidial Folate Prevents Starch Biosynthesis Triggered by Sugar Influx into Non-Photosynthetic Plastids of Arabidopsis.

Regulation of sucrose-starch interconversion in plants is important to maintain energy supplies necessary for viability and growth. Arabidopsis mutants were screened for aberrant responses to sucrose to identify candidates with a defect in the regulation of starch biosynthesis. One such mutant, fpgs1-4, accumulated substantial amounts of starch in non-photosynthetic cells. Dark-grown mutant seedlings exhibited shortened hypocotyls and accumulated starch in etioplasts when supplied with exogenous sucrose/glucose. Similar starch accumulation from exogenous sucrose was observed in mutant chloroplasts, when photosynthesis was prevented by organ culture in darkness. Molecular genetic analyses revealed that the mutant was defective in plastidial folylpolyglutamate synthetase, one of the enzymes engaged in folate biosynthesis. Active folate derivatives are important biomolecules that function as cofactors for a variety of enzymes. Exogenously supplied 5-formyl-tetrahydrofolate abrogated the mutant phenotypes, indicating that the fpgs1-4 mutant produced insufficient folate derivative levels. In addition, the antifolate agents methotrexate and 5-fluorouracil induced starch accumulation from exogenously supplied sucrose in dark-grown seedlings of wild-type Arabidopsis. These results indicate that plastidial folate suppresses starch biosynthesis triggered by sugar influx into non-photosynthetic cells, demonstrating a hitherto unsuspected link between plastidial folate and starch metabolism.

Arabidopsis plastidial folylpolyglutamate synthetase is required for nitrogen metabolism under nitrate-limited condition in darkness.

Folates play an important role in plant metabolism. Here we report a T-DNA insertion mutant (atdfb-3) of the plastidial folylpolyglutamate synthetase gene (AtDFB) was defective in folate metabolism and nitrogen metabolism under nitrate-limited conditions in darkness. Exogenous applied 5-formyl-tetrahydrofolate (5-F-THF) completely restored nitrogen content, soluble protein, total amino acids, individual amino acids including Glu, Gln, Asp, Asn, Pro, Arg and Met, nitrate, and endogenous 5-F-THF in atdfb-3 to the wild-type level. At the same time the application of 5-F-THF partially restored the content of Ser and nitrite in the mutant. Taken together, these results indicated that intact folate metabolism was necessary for nitrogen metabolism in Arabidopsis thaliana under nitrate-limited condition in darkness, providing novel insights into function of folate.

Folylpolyglutamate synthetase splicing alterations in acute lymphoblastic leukemia are provoked by methotrexate and other chemotherapeutics and mediate chemoresistance.

Methotrexate (MTX), a folate antagonist which blocks de novo nucleotide biosynthesis and DNA replication, is an anchor drug in acute lymphoblastic leukemia (ALL) treatment. However, drug resistance is a primary hindrance to curative chemotherapy in leukemia and its molecular mechanisms remain poorly understood. We have recently shown that impaired folylpolyglutamate synthetase (FPGS) splicing possibly contributes to the loss of FPGS activity in MTX-resistant leukemia cell line models and adult leukemia patients. However, no information is available on the possible splicing alterations in FPGS in pediatric ALL. Here, using a comprehensive PCR-based screen we discovered and characterized a spectrum of FPGS splicing alterations including exon skipping and intron retention, all of which proved to frequently emerge in both pediatric and adult leukemia patient specimens. Furthermore, an FPGS activity assay revealed that these splicing alterations resulted in loss of FPGS function. Strikingly, pulse-exposure of leukemia cells to antifolates and other chemotherapeutics markedly enhanced the prevalence of several FPGS splicing alterations in antifolate-resistant cells, but not in their parental antifolate-sensitive counterparts. These novel findings suggest that an assortment of deleterious FPGS splicing alterations may constitute a mechanism of antifolate resistance in childhood ALL. Our findings have important implications for the rational overcoming of drug resistance in individual leukemia patients.

Mammalian mitochondrial and cytosolic folylpolyglutamate synthetase maintain the subcellular compartmentalization of folates.

Folylpoly-γ-glutamate synthetase (FPGS) catalyze the addition of multiple glutamates to tetrahydrofolate derivatives. Two mRNAs for the fpgs gene direct isoforms of FPGS to the cytosol and to mitochondria in mouse and human tissues. We sought to clarify the functions of these two compartmentalized isoforms. Stable cell lines were created that express cDNAs for the mitochondrial and cytosolic isoforms of human FPGS under control of a doxycycline-inducible promoter in the AUXB1 cell line. AUXB1 are devoid of endogenous FPGS activity due to a premature translational stop at codon 432 in the fpgs gene. Loss of folates was not measurable from these doxycycline-induced cells or from parental CHO cells over the course of three CHO cell generations. Likewise, there was no detectable transfer of folate polyglutamates either from the cytosol to mitochondria, or from mitochondria to the cytosol. The cell line expressing cytosolic FPGS required exogenous glycine but not thymidine or purine, whereas cells expressing the mitochondrial isoform required exogenous thymidine and purine but not glycine for optimal growth and survival. We concluded that mitochondrial FPGS is required because folate polyglutamates are not substrates for transport across the mitochondrial membrane in either direction and that polyglutamation not only traps folates in the cytosol, but also in the mitochondrial matrix.

Evaluation of Intracellular Metabolism of Methotrexate in Hepatocytes and Embryonic Kidney Cells based on Folylpolyglutamate Synthetase and Gamma-Glutamyl Hydrolase Expression.

BACKGROUND: Methotrexate (MTX) is a common folic acid antagonist in clinical medicine, easily inducing a common adverse side effect of liver and kidney injury. It has been found that the expression of Folylpolyglutamate Synthetase (FPGS) and gamma-Glutamyl Hydrolase (GGH) may be closely related to that of related proteins to affect the intracellular metabolism of MTX. OBJECTIVE: The relationship between FPGS/GGH and MTXPGs accumulation in liver and kidney cells was explored by adjusting the expression of FPGS and GGH in cells using UPLC-MS/MS quantitative technology. METHOD: Based on UPLC-MS/MS quantitative techniques, the relationship between MTXPGs accumulation and FPGS/GGH in hepatocytes and embryonic kidney cells was explored by adjusting the expression of FPGS and GGH, and the effect of FPGS/GGH on the intracellular toxicity of MTX was comprehensively analyzed. RESULT: The results showed that the difference in methotrexate polyglutamates (MTXPGs) accumulation in liver and kidney cells was related to the difference in FPGS and GGH expression. The expression of FPGS interacted with that of GGH. These results suggest that the protein abundance ratio of FPGS to GGH (FPGS/GGH) has more potential to be used as a predictor of MTX efficacy than the FPGS or GGH single protein index. This can effectively avoid liver and kidney damage caused by MTX and guides the rational use of drugs in MTX. CONCLUSION: The results prove that there is a positive correlation between the FPGS/GGH and the accumulation of MTXPGS in liver and kidney cells. Summarily, the FPGS/GGH is expected to be a predictor for MTXPGs accumulation and provides an effective method to evaluate the toxicity caused by MTX.

Silencing Folylpolyglutamate Synthetase1 (FPGS1) in Switchgrass (Panicum virgatum L.) Improves Lignocellulosic Biofuel Production.

Switchgrass (Panicum virgatum L.) is a lignocellulosic perennial grass with great potential in bioenergy field. Lignocellulosic bioenergy crops are mostly resistant to cell wall deconstruction, and therefore yield suboptimal levels of biofuel. The one-carbon pathway (also known as C1 metabolism) is critical for polymer methylation, including that of lignin and hemicelluloses in cell walls. Folylpolyglutamate synthetase (FPGS) catalyzes a biochemical reaction that leads to the formation of folylpolyglutamate, an important cofactor for many enzymes in the C1 pathway. In this study, the putatively novel switchgrass PvFPGS1 gene was identified and its functional role in cell wall composition and biofuel production was examined by RNAi knockdown analysis. The PvFPGS1-downregulated plants were analyzed in the field over three growing seasons. Transgenic plants with the highest reduction in PvFPGS1 expression grew slower and produced lower end-of-season biomass. Transgenic plants with low-to-moderate reduction in PvFPGS1 transcript levels produced equivalent biomass as controls. There were no significant differences observed for lignin content and syringyl/guaiacyl lignin monomer ratio in the low-to-moderately reduced PvFPGS1 transgenic lines compared with the controls. Similarly, sugar release efficiency was also not significantly different in these transgenic lines compared with the control lines. However, transgenic plants produced up to 18% more ethanol while maintaining congruent growth and biomass as non-transgenic controls. Severity of rust disease among transgenic and control lines were not different during the time course of the field experiments. Altogether, the unchanged lignin content and composition in the low-to-moderate PvFPGS1-downregulated lines may suggest that partial downregulation of PvFPGS1 expression did not impact lignin biosynthesis in switchgrass. In conclusion, the manipulation of PvFPGS1 expression in bioenergy crops may be useful to increase biofuel potential with no growth penalty or increased susceptibility to rust in feedstock.

High gamma-glutamyl hydrolase and low folylpolyglutamate synthetase expression as prognostic biomarkers in patients with locally advanced gastric cancer who were administrated postoperative adjuvant chemotherapy with S-1.

PURPOSE: The enzymes gamma-glutamyl hydrolase (GGH) and folylpolyglutamate synthetase (FPGS) regulate intracellular folate concentrations needed for cell proliferation, DNA synthesis, and repair. High GGH expression affects 5-FU thymidylate synthase (TS) inhibition and is a risk factor for various malignancies. Here, the clinical significance of GGH and FPGS expression was investigated in Stage II/III gastric cancer patients undergoing postoperative adjuvant chemotherapy with S-1. METHODS: Surgical specimens of cancer tissue and adjacent normal mucosa, obtained from 253 patients with previously untreated gastric cancer, were examined. GGH and FPGS mRNA expression was measured by qPCR to evaluate their clinicopathological significance in gastric cancer patients after curative resection. RESULTS: While FPGS expression showed no significant differences between the cancerous and normal samples, GGH expression was higher in cancer tissue than in adjacent normal mucosa. High GGH expression was correlated with age, histological type, and vascular invasion. Overall survival (OS) of patients with high GGH mRNA expression was significantly poorer than of patients with low GGH expression. Multivariate analysis showed that high GGH expression was an independent prognostic factor of OS (HR: 2.58, 95% CI 1.29-5.16). Patients who received S-1 adjuvant treatment showed a significantly poor OS between high GGH/low FPGS and low GGH/high FPGS. Patients without adjuvant treatment showed no significant difference. CONCLUSION: GGH expression was significantly higher in gastric cancer tissue than in adjacent normal mucosa. High GGH and low FPGS expression is a useful independent predictor of poor outcomes in stage II/III gastric cancer patients undergoing postoperative adjuvant chemotherapy with S-1.

Combining loss of function of FOLYLPOLYGLUTAMATE SYNTHETASE1 and CAFFEOYL-COA 3-O-METHYLTRANSFERASE1 for lignin reduction and improved saccharification efficiency in Arabidopsis thaliana.

BACKGROUND: Downregulation of genes involved in lignin biosynthesis and related biochemical pathways has been used as a strategy to improve biofuel production. Plant C1 metabolism provides the methyl units used for the methylation reactions carried out by two methyltransferases in the lignin biosynthetic pathway: caffeic acid 3-O-methyltransferase (COMT) and caffeoyl-CoA 3-O-methyltransferase (CCoAOMT). Mutations in these genes resulted in lower lignin levels and altered lignin compositions. Reduced lignin levels can also be achieved by mutations in the C1 pathway gene, folylpolyglutamate synthetase1 (FPGS1), in both monocotyledons and dicotyledons, indicating a link between the C1 and lignin biosynthetic pathways. To test if lignin content can be further reduced by combining genetic mutations in C1 metabolism and the lignin biosynthetic pathway, fpgs1ccoaomt1 double mutants were generated and functionally characterized. RESULTS: Double fpgs1ccoaomt1 mutants had lower thioacidolysis lignin monomer yield and acetyl bromide lignin content than the ccoaomt1 or fpgs1 mutants and the plants themselves displayed no obvious long-term negative growth phenotypes. Moreover, extracts from the double mutants had dramatically improved enzymatic polysaccharide hydrolysis efficiencies than the single mutants: 15.1% and 20.7% higher than ccoaomt1 and fpgs1, respectively. The reduced lignin and improved sugar release of fpgs1ccoaomt1 was coupled with changes in cell-wall composition, metabolite profiles, and changes in expression of genes involved in cell-wall and lignin biosynthesis. CONCLUSION: Our observations demonstrate that additional reduction in lignin content and improved sugar release can be achieved by simultaneous downregulation of a gene in the C1 (FPGS1) and lignin biosynthetic (CCOAOMT) pathways. These improvements in sugar accessibility were achieved without introducing unwanted long-term plant growth and developmental defects.

Loss of function of folylpolyglutamate synthetase 1 reduces lignin content and improves cell wall digestibility in Arabidopsis.

BACKGROUND: One-carbon (C1) metabolism is important for synthesizing a range of biologically important compounds that are essential for life. In plants, the C1 pathway is crucial for the synthesis of a large number of secondary metabolites, including lignin. Tetrahydrofolate and its derivatives, collectively referred to as folates, are crucial co-factors for C1 metabolic pathway enzymes. Given the link between the C1 and phenylpropanoid pathways, we evaluated whether folylpolyglutamate synthetase (FPGS), an enzyme that catalyzes the addition of a glutamate tail to folates to form folylpolyglutamates, can be a viable target for reducing cell wall recalcitrance in plants. RESULTS: Consistent with its role in lignocellulosic formation, FPGS1 was preferentially expressed in vascular tissues. Total lignin was low in fpgs1 plants leading to higher saccharification efficiency of the mutant. The decrease in total lignin in fpgs1 was mainly due to lower guaiacyl (G) lignin levels. Glycome profiling revealed subtle alterations in the cell walls of fpgs1. Further analyses of hemicellulosic polysaccharides by NMR showed that the degree of methylation of 4-O-methyl glucuronoxylan was reduced in the fpgs1 mutant. Microarray analysis and real-time qRT-PCR revealed that transcripts of a number of genes in the C1 and lignin pathways had altered expression in fpgs1 mutants. Consistent with the transcript changes of C1-related genes, a significant reduction in S-adenosyl-l-methionine content was detected in the fpgs1 mutant. The modified expression of the various methyltransferases and lignin-related genes indicate possible feedback regulation of C1 pathway-mediated lignin biosynthesis. CONCLUSIONS: Our observations provide genetic and biochemical support for the importance of folylpolyglutamates in the lignocellulosic pathway and reinforces previous observations that targeting a single FPGS isoform for down-regulation leads to reduced lignin in plants. Because fpgs1 mutants had no dramatic defects in above ground biomass, selective down-regulation of individual components of C1 metabolism is an approach that should be explored further for the improvement of lignocellulosic feedstocks.