Methenyltetrahydrofolate Cyclohydrolase Research Articles

Comparative multiomics analysis of cell physiological state after culture in a basket bioreactor

Bioreactors are one of the most important, basic pieces of equipment in the biopharmaceutical industry. Understanding the effects of mechanical damage and other factors on the physiological state of cells during cell matrix culture is the basis for continuously achieving greater efficiency and higher product quality. In this study, Vero cells were used as a model and apoptosis, senescence, transcriptomics, proteomics, and metabolomics were carried out for analysis at the cellular and molecular levels. The results showed that compared with cells cultured in the simulated natural state, the cells cultured in the basket bioreactor displayed no obvious senescence. Additionally, the proportion of early apoptotic cells increased, but the proportions of damaged, late apoptotic and dead cells did not change significantly. The transcription levels of aminoacyl-tRNA synthetase and cyclin D1 and the expression levels of DNA replication licensing factor, methenyltetrahydrofolate cyclohydrolase, arachidonic acid and other metabolites of cells cultured in the basket bioreactor were significantly increased. These results suggest that DNA replication, protein translation and the metabolic activities in cells cultured in basket bioreactors are more active, which is more conducive to cell amplification and target product production. In this study, the growth and physiological state of cells in a basket bioreactor were characterized at the molecular level for the first time. Additionally, a tool to evaluate the physiological state of cells in a bioreactor was established, which can be used to guide the development and optimization of cell matrix culture conditions in industrial production and improve the production efficiency of the target products.

MTHFD2 is a potential oncogene for its strong association with poor prognosis and high level of immune infiltrates in urothelial carcinomas of bladder

BackgroundThe bifunctional methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 2, methenyltetrahydrofolate cyclohydrolase (MTHFD2) has been reported to play an oncogenic role in various types of cancers. However, the function of MTHFD2 in urothelial carcinomas of bladder (UCB) and its association with tumor immune infiltration remains unknown. We aim to examine the suitability of MTHFD2 to be a novel biomarker of bladder cancer and whether MTHFD2 is linked to immune infiltration.MethodsRNA sequencing data and clinical information (bladder cancer samples: normal samples = 414: 19) were downloaded from The Cancer Genome Atlas official website. Western blot analysis was performed to detect MTHFD2 expression in human bladder cancer (BLCA) cells and normal urothelial cell line SV-HUC-1. Associations between MTHFD2 expression and clinicopathological features were analyzed using Mann Whitney U test or Kruskal-Wallis H test. The “survival” and “survminer” packages were utilized to plot Kaplan-Meier survival curves. Moreover, the gene set enrichment analysis (GSEA) was conducted using a clusterProfiler package. The correlation of MTHFD2 expression with immune infiltration level was estimated using the single sample GSEA (ssGSEA) algorithm. Furthermore, associations between MTHFD2 and immune checkpoint genes were evaluated using the correlation analysis.ResultsTranscriptome analysis manifested that MTHFD2 was highly expressed in UCB tissues than normal bladder tissues, which was further confirmed by western blot analysis in human BLCA cells and SV-HUC-1 cells. Moreover, MTHFD2 high expression was significantly associated with the advanced disease progression. Also, the high expression of MTHFD2 was correlated with poor prognosis, and MTHFD2 was considered as an independent prognostic factor for disease specific survival. Furthermore, a number of cancer-related pathways were enriched in MTHFD2 high group, including NF-κB activation, JAK/STAT, and cancer immunotherapy by PD1 blockade. Several immune checkpoint molecules were also strongly associated with MTHFD2 expression, including PDCD1, CD274, CTLA4, CD276, LAG3, HAVCR2, and TIGIT.ConclusionsMTHFD2 expression was remarkably elevated in UCB, suggesting that MTHFD2 could be a promising biomarker for BLCA as well as novel target for anti-cancer immunotherapy since its close association with immune infiltration.

Structural insight into a molecular mechanism of methenyltetrahydrofolate cyclohydrolase from Methylobacterium extorquens AM1

Bioconversion of the C1 compounds into value-added products is one of the CO2-reducing strategies. In particular, because CO2 can be easily converted into formate, the efficient and direct bioconversion of CO2 through formate assimilation is attracting attention. The tetrahydrofolate (THF) cycle is the highly efficient reconstructed formate assimilation pathway, and 5,10-methenyltetrahydrofolate cyclohydrolase (FchA) is an essential enzyme involved in the THF cycle. In this study, a kinetic analysis of FchA from Methylobacterium extorquens AM1 (MeFchA) was performed and revealed that the enzyme has much higher cyclization than hydrolyzation activity, making it an optimal enzyme for formate assimilation. The crystal structure of MeFchA in the apo- and the THF-complexed forms was also determined, revealing that the substrate-binding site of the enzyme has three differently charged regions to stabilize the three differently charged moieties of the formyl-THF substrate. The residues involved in the substrate binding were also verified through site-directed mutagenesis. This study provides a biochemical and structural basis for the molecular mechanism underlying formate assimilation.

Mthfd2 Modulates Mitochondrial Function and DNA Repair to Maintain the Pluripotency of Mouse Stem Cells.

SummaryThe pluripotency of stem cells determines their developmental potential. While the pluripotency states of pluripotent stem cells are variable and interconvertible, the mechanisms underlying the acquisition and maintenance of pluripotency remain largely elusive. Here, we identified that methylenetetrahydrofolate dehydrogenase (NAD+-dependent), methenyltetrahydrofolate cyclohydrolase (Mthfd2) plays an essential role in maintaining embryonic stem cell pluripotency and promoting complete reprogramming of induced pluripotent stem cells. Mechanistically, in mitochondria, Mthfd2 maintains the integrity of the mitochondrial respiratory chain and prevents mitochondrial dysfunction. In the nucleus, Mthfd2 stabilizes the phosphorylation of EXO1 to support DNA end resection and promote homologous recombination repair. Our results revealed that Mthfd2 is a dual-function factor in determining the pluripotency of pluripotent stem cells through both mitochondrial and nuclear pathways, ultimately ensuring safe application of pluripotent stem cells.

Suppression of MTHFD2 in MCF-7 Breast Cancer Cells Increases Glycolysis, Dependency on Exogenous Glycine, and Sensitivity to Folate Depletion.

Methylenetetrahydrofolate dehydrogenase (NAD(P)+ dependent) 2, methenyltetrahydrofolate cyclohydrolase (MTHFD2) is a mitochondrial enzyme involved in folate metabolism. A number of recent studies have highlighted this enzyme as being highly expressed in many solid tumors, including breast cancer, and to be correlated with poor survival. However, the metabolic functions of MTHFD2 in cancer cells have not been well-defined. To investigate the function of MTHFD2 in breast cancer cells, we generated and characterized MCF-7 cells with stable suppression of MTHFD2 expression using a combination of cellular assays and metabolic profiling. Loss of MTHFD2 caused MCF7 cells to become glycine auxotrophs, that is, reliant on exogenous glycine, and more sensitive to exogenous folate depletion. Another prominent metabolic alteration observed as a consequence of MTHFD2 suppression was a more glycolytic phenotype, consistent with widespread modifications of cellular metabolism. Collectively, these data suggest that targeting MTHFD2 activity is likely to influence multiple metabolic pathways in breast cancer and could be combined with a range of antimetabolite therapies.

Is MTHFD1 polymorphism rs 2236225 (c.1958G>A) associated with the susceptibility of NSCL/P? A systematic review and meta-analysis.

To investigate the association between the methylenetetrahydrofolate dehydrogenase 1 (MTHFD1) polymorphism rs 2236225 (c.1958G>A) and susceptibility to non-syndromic cleft of the lip and/or palate (NSCL/P). An extensive literature review has been conducted using PubMed, Web of Science, Cochrane Library, Google Scholar, the China National Knowledge Infrastructure (CNKI), and Wanfang Database for eligible researches. The terms for searching were "cleft lip OR cleft palate OR CLP OR CL/P OR oral facial cleft OR OFC" AND "methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 1 OR methenyltetrahydrofolate cyclohydrolase formyltetrahydrofolate synthetase OR MTHFD1 OR MTHFD". Two independent researchers screened, evaluated and extracted the data of included studies. The pooled odds ratios (OR) with 95% confidence intervals (95% CI) were calculated by random effects model under five gene models. Subgroup, sensitivity analysis and publication bias were also assessed. Ten case-control studies have been included in the systematic review and eight studies have been considered for the meta-analysis. Overall, the MTHFD1 polymorphism rs2236225 and the risk of NSCL/P showed pooled OR (95% CI) of 1.02 (0.86-1.21) under allelic model. A higher degree of heterogeneity was observed in Asian countries (I (2) = 75.6%) compared to non-Asian countries (I (2) = 48.9%). Similar consequence appeared in the subgroup of children (I (2) = 78.6%) compared with that of mothers (I (2) = 0.0%). There was no significant difference in the publication bias by the Begg's funnel plot (P = 0.711) and Egger's regression test (P = 0.746). Our assessment suggested there was no significant association between the MTHFD1 polymorphism rs 2236225 (c.1958G>A) and the susceptibility to NSCL/P. Further investigations using a large sample size and a more advanced technique should be adopted to reach a more precise conclusion in the future.

Physiological role of FolD (methylenetetrahydrofolate dehydrogenase), FchA (methenyltetrahydrofolate cyclohydrolase) and Fhs (formyltetrahydrofolate synthetase) from Clostridium perfringens in a heterologous model of Escherichia coli.

Most organisms possess bifunctional FolD [5,10-methylenetetrahydrofolate (5,10-CH2-THF) dehydrogenase-cyclohydrolase] to generate NADPH and 10-formyltetrahdrofolate (10-CHO-THF) required in various metabolic steps. In addition, some organisms including Clostridium perfringens possess another protein, Fhs (formyltetrahydrofolate synthetase), to synthesize 10-CHO-THF. Here, we show that unlike the bifunctional FolD of Escherichia coli (EcoFolD), and contrary to its annotated bifunctional nature, C. perfringens FolD (CpeFolD) is a monofunctional 5,10-CH2-THF dehydrogenase. The dehydrogenase activity of CpeFolD is about five times more efficient than that of EcoFolD. The 5,10-methenyltetrahydrofolate (5,10-CH+-THF) cyclohydrolase activity in C. perfringens is provided by another protein, FchA (5,10-CH+-THF cyclohydrolase), whose cyclohydrolase activity is ∼ 10 times more efficient than that of EcoFolD. Kinetic parameters for CpeFhs were also determined for utilization of all of its substrates. Both CpeFolD and CpeFchA are required to substitute for the single bifunctional FolD in E. coli. The simultaneous presence of CpeFolD and CpeFchA is also necessary to rescue an E. coli folD deletion strain (harbouring CpeFhs support) for its formate and glycine auxotrophies, and to alleviate its susceptibility to trimethoprim (an antifolate drug) or UV light. The presence of the three clostridial proteins (FolD, FchA and Fhs) is required to maintain folate homeostasis in the cell.

Is MTHFD1 polymorphisms rs 2236225 (c.1958G>A) associated with the susceptibility of NSCL/P? A systematic review and meta-analysis

To investigate the association between the methylenetetrahydrofolate dehydrogenase 1 (MTHFD1) polymorphism rs 2236225 (c.1958G>A) and susceptibility to non-syndromic cleft of the lip and/or palate (NSCL/P). An extensive literature review has been conducted using PubMed, Web of Science, Cochrane Library, Google Scholar, the China National Knowledge Infrastructure (CNKI), and Wanfang Database for eligible researches. The terms for searching were "cleft lip OR cleft palate OR CLP OR CL/P OR oral facial cleft OR OFC" AND "methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 1 OR methenyltetrahydrofolate cyclohydrolase formyltetrahydrofolate synthetase OR MTHFD1 OR MTHFD". Two independent researchers screened, evaluated and extracted the data of included studies. The pooled odds ratios (OR) with 95% confidence intervals (95% CI) were calculated by random effects model under five gene models. Subgroup, sensitivity analysis and publication bias were also assessed. Ten case-control studies have been included in the systematic review and eight studies have been considered for the meta-analysis. Overall, the MTHFD1 polymorphism rs2236225 and the risk of NSCL/P showed pooled OR (95% CI) of 1.02 (0.86-1.21) under allelic model. A higher degree of heterogeneity was observed in Asian countries (I (2) = 75.6%) compared to non-Asian countries (I (2) = 48.9%). Similar consequence appeared in the subgroup of children (I (2) = 78.6%) compared with that of mothers (I (2) = 0.0%). There was no significant difference in the publication bias by the Begg's funnel plot (P = 0.711) and Egger's regression test (P = 0.746). Our assessment suggested there was no significant association between the MTHFD1 polymorphism rs 2236225 (c.1958G>A) and the susceptibility to NSCL/P. Further investigations using a large sample size and a more advanced technique should be adopted to reach a more precise conclusion in the future.

Potential functional gene diversity involved in methanogenesis and methanogenic community structure in Indian buffalo (Bubalus bubalis) rumen.

Understanding the methanogen community structure and methanogenesis from Bubalus bubalis in India may be beneficial to methane mitigation. Our current understanding of the microbial processes leading to methane production is incomplete, and further advancement in the knowledge of methanogenesis pathways would provide means to manipulate its emission in the future. In the present study, we evaluated the methanogenic community structure in the rumen as well as their potential genes involved in methanogenesis. The taxonomic and metabolic profiles of methanogens were assessed by shotgun sequencing of rumen metagenome by Ion Torrent semiconductor sequencing. The buffalo rumen contained representative genera of all the families of methanogens. Members of Methanobacteriaceae were found to be dominant, followed by Methanosarcinaceae, Methanococcaceae, Methanocorpusculaceae, and Thermococcaceae. A total of 60 methanogenic genera were detected in buffalo rumen. Methanogens related to the genera Methanobrevibacter, Methanosarcina, Methanococcus, Methanocorpusculum, Methanothermobacter, and Methanosphaera were predominant, representing >70 % of total archaeal sequences. The metagenomic dataset indicated the presence of genes involved in the methanogenesis and acetogenesis pathways, and the main functional genes were those of key enzymes in the methanogenesis. Sequences related to CoB--CoM heterodisulfide reductase, methyl coenzyme M reductase, f420-dependent methylenetetrahydromethanopterin reductase, and formylmethanofuran dehydrogenase were predominant in rumen. In addition, methenyltetrahydrofolate cyclohydrolase, methylenetetrahydrofolate dehydrogenase, 5,10-methylenetetrahydrofolate reductase, and acetyl-coenzyme A synthetase were also recovered.

Update and new concepts in vitamin responsive disorders of folate transport and metabolism

Derivatives of folic acid are involved in transfer of one-carbon units in cellular metabolism, playing a role in synthesis of purines and thymidylate and in the remethylation of homocysteine to form methionine. Five inborn errors affecting folate transport and metabolism have been well studied: hereditary folate malabsorption, caused by mutations in the gene encoding the proton-coupled folate transporter (SLC46A1); glutamate formiminotransferase deficiency, caused by mutations in the FTCD gene; methylenetetrahydrofolate reductase deficiency, caused by mutations in the MTHFR gene; and functional methionine synthase deficiency, either as the result of mutations affecting methionine synthase itself (cblG, caused by mutations in the MTR gene) or affecting the accessory protein methionine synthase reductase (cblE, caused by mutations in the MTRR gene). Recently additional inborn errors have been identified. Cerebral folate deficiency is a clinically heterogeneous disorder, which in a few families is caused by mutations in the FOLR1 gene. Dihydrofolate reductase deficiency is characterized by megaloblastic anemia and cerebral folate deficiency, with variable neurological findings. It is caused by mutations in the DHFR gene. Deficiency in the trifunctional enzyme containing methylenetetrahydrofolate dehydrogenase, methenyltetrahydrofolate cyclohydrolase and formyltetrahydrofolate synthetase activities, has been identified in a single patient with megaloblastic anemia, atypical hemolytic uremic syndrome and severe combined immune deficiency. It is caused by mutations in the MTHFD1 gene.

Crystal Structure of FolD Bifunctional Protein, a Methylenetetrahydrofolate Dehydrogenase/Cyclohydrolase from Campylobacter jejuni

Tetrahydrofolate (THF) plays a crucial role as a molecule for the production and utilization of monocarbon units in various oxidation states. Enzymes involved in THF metabolism are of pharmaceutical interest, as their function is crucial for amino acid and DNA synthesis. The interconversion of two major one‐carbon donors, methylene and methenyl groups, occurs through the sequential activities of NAD(P)‐dependent methylenetetrahydrofolate dehydrogenase (MTHFD) and methenyltetrahydrofolate cyclohydrolase (MTHFC). These activities frequently coexist as a part of a bifunctional enzyme in prokaryotes. In higher organisms, MTHFR and MTHFC are often linked together with another protein, formyl‐THF synthetase (FTHFS) and operate as a trifunctional enzyme. We have determined the crystal structure of a FolD protein, a predicted MTHFR/MTHFC from Campylobacter jejuni at 2.20 Å resolution. The protein exists as a dimer and each subunit is composed of two α/β domains that assemble to form a wide, largely hydrophobic cleft. Comparison with the structure of the homologous enzyme domain from a trifunctional human protein allowed the identification of a highly conserved cluster of basic residues that most likely participate in the binding of a THF substrate. Exploring the differences between the active sites of bacterial and human enzymes might allow the development of inhibitors specific to the bacterial target. This research was supported by the NIH contract No. HHSN272200700058C, and by the U.S. DOE, Office of Biological and Environmental Research, contract DE‐AC02‐06CH11357.

The MTHFD1 p.Arg653Gln variant alters enzyme function and increases risk for congenital heart defects

Methylenetetrahydrofolate dehydrogenase)methenyltetrahydrofolate cyclohydrolase)formyltetrahydrofolate synthetase (MTHFD1) is a trifunctional enzyme that interconverts tetrahydrofolate (THF) derivatives for nucleotide synthesis. A common variant in MTHFD1, p.Arg653Gln (c.1958G>A), may increase the risk for neural tube defects (NTD). To examine the biological impact of this variant on MTHFD1 function, we measured enzyme activity and stability in vitro and assessed substrate flux in transfected mammalian cells. The purified Arg653Gln enzyme has normal substrate affinity but a 36% reduction in half)life at 42 degrees C. Thermolability is reduced by magnesium adenosine triphosphate and eliminated by the substrate analog folate pentaglutamate, suggesting that folate status may modulate impact of the variant. The mutation reduces the metabolic activity of MTHFD1 within cells: formate incorporation into DNA in murine Mthfd1 knockout cells transfected with Arg653Gln is reduced by 26%+/-7.7% (P<0.05), compared to cells transfected with wild)type protein, indicating a disruption of de novo purine synthesis. We assessed the impact of the variant on risk for congenital heart defects (CHD) in a cohort of Quebec children (158 cases, 110 controls) and mothers of children with heart defects (199 cases, 105 controls). The 653QQ genotype in children is associated with increased risk for heart defects (odds ratio [OR], 2.11; 95% confidence interval [CI], 1.01-4.42), particularly Tetralogy of Fallot (OR, 3.60; 95% CI, 1.38-9.42) and aortic stenosis (OR, 3.13; 95% CI, 1.13-8.66). There was no effect of maternal genotype. Our results indicate that the Arg653Gln polymorphism decreases enzyme stability and increases risk for CHD. Further evaluation of this polymorphism in folate)related disorders and its potential interaction with folate status is warranted.

Thymidylate synthase promoter tandem repeat and MTHFD1 R653Q polymorphisms modulate the risk for migraine conferred by the MTHFR T677 allele

There is growing evidence that folate metabolism is involved in migraine pathophysiology, mainly in migraine with aura. Even though folate metabolism is regulated by a number of enzymes, only two functional polymorphisms have been tested in association studies with migraine. Here, we have explored the possible role in migraine of other folate-metabolizing enzymes which are in close interdependency with 5′,10′-methylenetetrahydrofolate reductase analyzing functional polymorphisms of these enzymes in a case-control study. Individually, thymidylate synthase ( TS), methenyltetrahydrofolate cyclohydrolase formyltetrahydrofolate synthase ( MTHFD1), or methionine synthase ( MS) polymorphisms did not modify the general risk for suffering migraine. Nevertheless, we observed a strong interaction between TS and MTHFR mutated genotypes, which increased over 8-fold the risk for experiencing aura among migraineurs; MTHFD1 and MTHFR mutated genotypes also increased together the risk for migraine in general (OR = 3.08; 95% CI = 1.3–7.4). We conclude that the pathogenetic role of the MTHFR T677 allele in migraine is modulated by functional polymorphisms of TS and MTHFD1.

Analysis of genes of tetrahydrofolate-dependent metabolism from cultivated spirochaetes and the gut community of the termite Zootermopsis angusticollis.

The hindguts of wood-feeding termites are the sites of intense, CO2-reductive acetogenesis. This activity profoundly influences host nutrition and methane emissions. Homoacetogens previously isolated from diverse termites comprised novel taxa belonging to two distinct bacterial phyla, Firmicutes and Spirochates. Little else is known about either the diversity or abundance of homoacetogenic species present in any given termite or the genetic details underlying CO2-reductive acetogenesis by Spirochaetes. A key enzyme of CO2-reductive acetogenesis is formyltetrahydrofolate synthetase (FTHFS). A previously designed primer set was used to amplify FTHFS genes from three isolated termite-gut spirochaetes. Sequencing DNA flanking the FTHFS gene of Treponema strain ZAS-2 revealed genes encoding two acetogenesis-related enzymes, methenyltetrahydrofolate cyclohydrolase and methylenetetrahydrofolate dehydrogenase. Although termite-gut spirochaetes are only distantly related to clostridia at the ribosomal level, their tetrahydrofolate-dependent enzymes appear to be closely related. In contrast, homologous proteins identified in the non-homoacetogenic oral spirochaete Treponema denticola were only distantly related to those from clostridia and the termite-gut treponemes. Having demonstrated their utility with spirochaete pure cultures, the FTHFS primers were used to construct a 91-clone library from the termite-gut community DNA. From this, 19 DNA and eight amino acid FTHFS types were identified. Over 75 % of the retrieved clones formed a novel, coherent cluster with the FTHFS homologues obtained from the termite-gut treponemes. Thus, FTHFS gene diversity in the gut of the termite Zootermopsis angusticollis appears to be dominated by spirochaetes. The homoacetogenic capacity of termite-gut spirochaetes may have been acquired via lateral gene transfer from clostridia.

Microarray analysis of brain RNA in mice with methylenetetrahydrofolate reductase deficiency and hyperhomocysteinemia

Methylenetetrahydrofolate reductase (MTHFR) deficiency is the most common genetic cause of hyperhomocysteinemia, which is associated with increased risk for cardiovascular disease, stroke and possibly other neurological disorders. Microarray analysis of brain RNA from day 14 Mthfr −/− mice revealed several genes with altered expression. Expression changes in inositol 1,4,5-triphosphate receptor, type 1 ( Itpr1), proteolipid protein ( Plp), neurogenic differentiation factor 1 ( Neurod1), S100 calcium binding protein A8 ( S100a8), and methylenetetrahydrofolate dehydrogenase (NAD+ dependent), methenyltetrahydrofolate cyclohydrolase ( Mthfd2) were confirmed by RT-PCR. We propose that neuronal damage by hyperhomocysteinemia may involve disruption of intracellular calcium.

Isolation and Sequencing of a Plant cDNA Encoding a Bifunctional Methylenetetrahydrofolate Dehydrogenase : Methenyltetrahydrofolate Cyclohydrolase Protein

Summary In plant cells, the interconversion of formyl- and methylene-tetrahydrofolates is catalyzed by a bifunctional protein possessing methenyltetrallydrofolate cydohydrolase (EC 3.5.4.9) and methylenetetrahydrofolate dehydrogenase (EC 1.5.1.5) activities. The present work reports the isolation and sequencing of a cDNA that encodes this protein. Polydonal antibodies, raised against purified pea cytosolic dehydrogenase:cyclohydrolase, were used to screen a λgt 11 cDNA expression library, constructed from leaf extracts of this species. The screen identified a phage containing a cDNA insert with an open reading frame encoding a 294 amino acid protein (Mr 31,344). The deduced primary structure of this protein contained most of the conserved regions found in other dehydrogenase:cyclohydrolase proteins including the corresponding domains of the trifunctional C1-THF synthases of mammalian and yeast origins.

A methenyl tetrahydromethanopterin cyclohydrolase and a methenyl tetrahydrofolate cyclohydrolase in Methylobacterium extorquens AM1.

Recently it was found that Methylobacterium extorquens AM1 contains both tetrahydromethanopterin (H4MPT) and tetrahydrofolate (H4F) as carriers of C1 units. In this paper we report that the aerobic methylotroph contains a methenyl H4MPT cyclohydrolase (0.9 U x mg-1 cell extract protein) and a methenyl H4F cyclohydrolase (0.23 U x mg-1). Both enzymes, which were specific for their substrates, were purified and characterized and the encoding genes identified via the N-terminal amino acid sequence. The purified methenyl H4MPT cyclohydrolase with a specific activity of 630 U x mg-1 (Vmax = 1500 U x mg-1; Km = 30 microm) was found to be composed of two identical subunits of molecular mass 33 kDa. Its sequence was approximately 40% identical to that of methenyl H4MPT cyclohydrolases from methanogenic archaea. The methenyl H4F cyclohydrolase with a specific activity of 100 U x mg-1 (Vmax = 330 U x mg-1; Km = 80 microm) was found to be composed of two identical subunits of molecular mass 22 kDa. Its sequence was not similar to that of methenyl H4MPT cyclohydrolases or to that of other methenyl H4F cyclohydrolases. Based on the specific activities in cell extract and from the growth properties of insertion mutants it is suggested that the methenyl H4MPT cyclohydrolase might have a catabolic, and the methenyl-H4F cyclohydrolase an anabolic function in the C1-unit metabolism of M. extorquens AM1.

Nonradioactive Differential Display Cloning of Genes Induced by Homocysteine in Vascular Endothelial Cells

Hyperhomocysteinemia is known to be a risk factor for arteriosclerosis and thrombosis. To elucidate the mechanisms by which homocysteine may promote vascular diseases, we have applied a modified nonradioactive differential display analysis that evaluates changes in gene expression induced by homocysteine treatment of cultured human umbilical vein endothelial cells (HUVECs). We identified six upregulated and one downregulated gene. One upregulated gene was GRP78/BiP, an endoplasmic reticulum (ER)-resident molecular chaperone, suggesting that unfolded proteins would accumulate in the ER because of redox potential changes caused by homocysteine. Another upregulated gene encoded a bifunctional enzyme with activities of methylenetetrahydrofolate dehydrogenase and methenyltetrahydrofolate cyclohydrolase, which is involved in homocysteine metabolism. A third upregulated gene encoded activating transcription factor 4. Homology searches of the remaining four clones failed to retrieve any similar sequences with a known function. We isolated a full-length cDNA of one of the upregulated genes from a HUVEC library. It encoded a novel protein with 394 amino acids, which was termed RTP (reducing agent and tunicamycin-responsive protein). Northern blot analysis revealed that RTP mRNA expression was induced in HUVECs treated with not only homocysteine but also 2-mercaptoethanol and tunicamycin, both of which are known to induce ER stress. RTP mRNA was ubiquitously expressed in human adult organs, and seemed to be regulated in mouse embryogenesis. Consequently, our differential display analysis revealed that homocysteine alters the expressivity of multiple proteins, especially ER stress-responsive ones. This potential ability of homocysteine may be involved in atherogenesis.

Analysis of gene expression in homocysteine-injured vascular endothelial cells: demonstration of GRP78/BiP expression, cloning and characterization of a novel reducing agent-tunicamycin regulated gene.

An elevated plasma level of homocysteine is associated with arteriosclerosis and thrombosis. The mechanisms by which homocysteine may promote vascular diseases have not yet been elucidated. In the present study, we have applied a modified nonradioactive differential display analysis to evaluate changes in gene expression induced by homocysteine treatment of cultured human umbilical vein endothelial cells (HUVEC). We identified six upregulated and one downregulated gene. One upregulated gene was GRP78/BiP, a stress protein, suggesting that unfolded proteins would accumulate in the endoplasmic reticulum because of redox potential changes caused by homocysteine. Another upregulated gene encoded a bifunctional enzyme with activities of methylenetetrahydrofolate dehydrogenase and methenyltetrahydrofolate cyclohydrolase, which is involved in a homocysteine metabolism. A third upregulated gene encoded activating transcription factor 4. The remaining four were uncharacterized genes. We isolated a full-length cDNA of one of the upregulated genes from a HUVEC library. It encoded a novel protein with 394 amino acids, which was termed reducing-agents and tunicamycin-responsive protein (RTP). Northern blot analysis revealed that RTP gene expression was induced in HUVEC after 4 h of incubation with homocysteine. RTP mRNA was also observed in unstimulated cells and induced by not only homocysteine but also 2-mercaptoethanol and tunicamycin. The mRNA was ubiquitously expressed in human tissues. These observations indicate that homocysteine can alter the expressivity of multiple genes, including a stress protein and several novel genes. These responses may contribute to atherogenesis.

Genetic defects of folate and cobalamin metabolism.

Deficient activity of an enzyme can result from a defect in the conversion of the vitamin to a co-enzyme as well from an abnormal apo-enzyme or disturbed binding of coenzyme to enzyme. Conversion of dietary vitamin to intracellular active co-enzyme can be complex and require many physiological and biochemical processes including stomach release of bound vitamin, intestinal uptake, carriers/transport, blood transport, cellular uptake, intracellular release and intracellular compartmentalisation. Disorders of malabsorption (food cobalamin malabsorption, intrinsic factor deficiency and abnormal enterocyte cobalamin processing) and transport proteins (transcobalamin II deficiency, R-binder deficiency) mostly lead to disturbed function of the two cobalamin requiring enzymes, methylmalonyl CoA mutase and methionine synthase. Defects of early steps of intracellular cobalamin (cblF, cbl C/D) result in marked deficiencies of both cobalamin co-enzymes and homocystinuria combined with methylmalonic aciduria. Defective synthesis of adenosyl cobalamin in the cbl A/B defects leads to methylmalonyl CoA mutase. Isolated methionine synthase deficiency is also classified as a cobalamin disorder due to its associated deficient formation of methylcobalamin. Folate disorders include methylene-tetrahydrofolate reductase deficiency and glutamate formimino-transferase deficiency. In addition a hereditary disorder of intestinal folate transport has been described. Less well established are disorders of dihydrofolate reductase, methenyl-tetrahydrofolate cyclohydrolase, and defects of cellular folate uptake.