Cloning and characterization of the human 5,10-methenyltetrahydrofolate synthetase-encoding cDNA

Dayan, Alain; Bertrand, Richard; Beauchemin, M.; Chahla, Dounia; Mamo, Aline; Filion, Mario; Skup, Daniel; Massie, Bernard; Jolivet, Jacques

doi:10.1016/0378-1119(95)00321-v

Cited by 28 publications

(26 citation statements)

References 21 publications

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“…Identification and Cloning of Yeast MTHFS-An ORF (YER183c) with homology to human MTHFS was identified in the Saccharomyces Genome Databank using a BLAST search against the human MTHFS sequence (30). When aligned with human and rabbit MTHFS using the ClustalW algorithm (31) (Fig.…”

Section: Resultsmentioning

confidence: 99%

Cloning and Characterization of Methenyltetrahydrofolate Synthetase from Saccharomyces cerevisiae

Holmes¹,

Appling

2002

Journal of Biological Chemistry

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The folate derivative 5-formyltetrahydrofolate (folinic acid; 5-CHO-THF) was discovered over 40 years ago, but its role in metabolism remains poorly understood. Only one enzyme is known that utilizes 5-CHO-THF as a substrate: 5,10-methenyltetrahydrofolate synthetase (MTHFS). A BLAST search of the yeast genome using the human MTHFS sequence revealed a 211-amino acid open reading frame (YER183c) with significant homology. The yeast enzyme was expressed in Escherichia coli, and the purified recombinant enzyme exhibited kinetics similar to previously purified MTHFS. No new phenotype was observed in strains disrupted at MTHFS or in strains additionally disrupted at the genes encoding one or both serine hydroxymethyltransferases (SHMT) or at the genes encoding one or both methylenetetrahydrofolate reductases. However, when the MTHFS gene was disrupted in a strain lacking the de novo folate biosynthesis pathway, folinic acid (5-CHO-THF) could no longer support the folate requirement. We have thus named the yeast gene encoding methenyltetrahydrofolate synthetase FAU1 (folinic acid utilization). Disruption of the FAU1 gene in a strain lacking both 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) transformylase isozymes (ADE16 and ADE17) resulted in a growth deficiency that was alleviated by methionine. Genetic analysis suggested that intracellular accumulation of the purine intermediate AICAR interferes with a step in methionine biosynthesis. Intracellular levels of 5-CHO-THF were determined in yeast disrupted at FAU1 and other genes encoding folate-dependent enzymes. In fau1 disruptants, 5-CHO-THF was elevated 4-fold over wild-type yeast. In yeast lacking MTHFS along with both AICAR transformylases, 5-CHO-THF was elevated 12-fold over wild type. 5-CHO-THF was undetectable in strains lacking SHMT activity, confirming SHMT as the in vivo source of 5-CHO-THF. Taken together, these results indicate that S. cerevisiae harbors a single, nonessential, MTHFS activity. Growth phenotypes of multiply disrupted strains are consistent with a regulatory role for 5-CHO-THF in one-carbon metabolism and additionally suggest a metabolic interaction between the purine and methionine pathways.Although 5-formyl-tetrahydrofolate (5-CHO-THF 1 ; folinic acid or leucovorin) is the most stable derivative of the reduced folates (1), it does not have a known direct role as a one-carbon donor. Leucovorin has been employed extensively as a rescue agent in chemotherapeutic protocols, but the physiological role of this folate derivative remains poorly understood. 5-CHO-THF can arise nonenzymatically from the hydrolysis of 5,10-methenyl-THF (5,10-CH ϩ -THF) under mild conditions (2). For this reason, it was considered likely that the 5-CHO-THF detected in cell extracts was an artifact of preparation rather than a normal component of folate metabolism. More recent studies designed to prevent the possibility of artifactual conversion concluded that 5-CHO-THF is indeed a naturally occurring metabolite (3). The biological source of 5-CHO-THF, however,...

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Section: Resultsmentioning

confidence: 99%

Cloning and Characterization of Methenyltetrahydrofolate Synthetase from Saccharomyces cerevisiae

Holmes¹,

Appling

2002

Journal of Biological Chemistry

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“…On the other hand, utilization of 5-CHO-THF requires its conversion to 5,10 methenyl-THF, a reaction mediated by methenyltetrahydrofolate synthetase (36). This is the only known step through which 5-CHO-THF can enter the folate cycle and become available for folate-dependent biosynthetic reactions.…”

Section: Discussionmentioning

confidence: 99%

The inverse relationship between reduced folate carrier function and Pemetrexed activity in a human colon cancer cell line

Chattopadhyay

Zhao

Krupenko

et al. 2006

Molecular Cancer Therapeutics

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Pemetrexed, a new generation antifolate recently approved for the treatment of mesothelioma and non -small cell lung cancer, is an excellent substrate for the reduced folate carrier (RFC). To explore the carrier's effect on pemetrexed activity, RFC was inactivated in HCT-15 colon cancer cells by mutagenesis and PT632 selective pressure. A clone (PT1) was obtained with a glycine to arginine substitution at amino acid 401, resulting in the loss of RFC function. PT1 cells were resistant to PT632 (178-fold), methotrexate (4-fold), and ZD1694 (Tomudex, raltitrexed; 20-fold), but were 3-fold collaterally sensitive to pemetrexed when grown in 25 nmol/L of 5-formyltetrahydrofolate. PT1 cells transfected with wild-type RFC had antifolate sensitivities comparable to that of wild-type HCT-15 cells, indicating that the RFC mutation was the sole basis for resistance. Folate pools were contracted in PT1 cells by 32% or 60%, as measured by radiolabeling intracellular folates or by an enzyme binding assay, respectively. This was reflected in marked (6.5-fold) collateral sensitivity to trimetrexate. The initial uptake of pemetrexed in PT1 cells was markedly reduced (f85%) but intracellular pemetrexed levels increased to f60% and f70% to that of wild-type cells after 2 hours and 6 days, respectively. There was increased pemetrexed inhibition of glycinamide ribonucleotide transformylase and, to a lesser extent, thymidylate synthase in PT1 cells growing in 5-formyltetrahydrofolate based on nucleoside protection analyses. Hence, loss of RFC function leads to collateral sensitivity to pemetrexed in HCT-15 cells, likely due to cellular folate pool contraction resulting in partial preservation of pemetrexed polyglutamylation and increased target enzyme inhibition. [Mol Cancer Ther 2006;5(2):438 -49]

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“…The predicted Lost polypeptide contains a putative 5-formyltetrahydrofolate cyclo-ligase domain that is 35% identical to human 5-FTHF cyclo-ligase domains. FTHF cyclo-ligases are essential components of folate metabolism in humans (Dayan et al, 1995;Anguera et al, 2003), but it is currently unknown if and how this domain functions in Drosophila. Notably, there are no conserved protein-protein or RNA-binding domains that would shed light on a role for Lost in mRNA localization.…”

Section: Purification and Identification Of Lostmentioning

confidence: 99%

A late phase of germ plasm accumulation during Drosophila oogenesis requires Lost and Rumpelstiltskin

et al. 2011

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SUMMARYAsymmetric mRNA localization is an effective mechanism for establishing cellular and developmental polarity. Posterior localization of oskar in the Drosophila oocyte targets the synthesis of Oskar to the posterior, where Oskar initiates the assembly of the germ plasm. In addition to harboring germline determinants, the germ plasm is required for localization and translation of the abdominal determinant nanos. Consequently, failure of oskar localization during oogenesis results in embryos lacking germ cells and abdominal segments. oskar accumulates at the oocyte posterior during mid-oogenesis through a well-studied process involving kinesin-mediated transport. Through live imaging of oskar mRNA, we have uncovered a second, mechanistically distinct phase of oskar localization that occurs during late oogenesis and results in amplification of the germ plasm. Analysis of two newly identified oskar localization factors, Rumpelstiltskin and Lost, that are required specifically for this late phase of oskar localization shows that germ plasm amplification ensures robust abdomen and germ cell formation during embryogenesis. In addition, our results indicate the importance of mechanisms for adapting mRNAs to utilize multiple localization pathways as necessitated by the dramatic changes in ovarian physiology that occur during oogenesis.

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Cloning and characterization of the human 5,10-methenyltetrahydrofolate synthetase-encoding cDNA

Cited by 28 publications

References 21 publications

Cloning and Characterization of Methenyltetrahydrofolate Synthetase from Saccharomyces cerevisiae

Cloning and Characterization of Methenyltetrahydrofolate Synthetase from Saccharomyces cerevisiae

The inverse relationship between reduced folate carrier function and Pemetrexed activity in a human colon cancer cell line

A late phase of germ plasm accumulation during Drosophila oogenesis requires Lost and Rumpelstiltskin

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