Effect of vitamin B6 availability on serine hydroxymethyltransferase in MCF-7 cells

Folate-activated one-carbon units are derived from serine through the activity of the pyridoxal-phosphate (PLP)-dependent isozymes of serine hydroxymethyltransferase (SHMT). The effect of vitamin B(6) availability on the activity and expression of the human mitochondrial and cytoplasmic SHMT isozymes was investigated in human MCF-7 cells. Cells were cultured for 6 months in vitamin B(6) replete (4.9 microM pyridoxine) minimal essential medium (alphaMEM) or vitamin B(6)-deficient medium containing 49, 4.9 or 0.49 nM pyridoxine. Total cellular PLP levels and SHMT activity were reduced 72% and 7%, respectively, when medium pyridoxine was decreased from 4.9 microM to 49 nM. Cells cultured in medium containing 4.9 nM pyridoxine exhibited 75%, 27% and 60% reduced levels of PLP, SHMT activity and S-adenosylmethionine, respectively, compared to cells cultured in alphaMEM. Cytoplasmic SHMT activity and protein levels, but not mRNA levels, were decreased in cells cultured in vitamin B(6) deficient medium, whereas mitochondrial SHMT activity and protein levels were less sensitive to vitamin B(6) availability. PLP bound to cytoplasmic SHMT with a K(d)=850 nM, a value two orders of magnitude lower than previously reported for the bovine cytoplasmic SHMT isozyme. Collectively, these data indicate that vitamin B(6) restriction decreases the activity and stability of SHMT, and that the cytoplasmic isozyme is more sensitive to vitamin B(6) deficiency than the mitochondrial isozyme in MCF-7 cells.     

Competition between sumoylation and ubiquitination of serine hydroxymethyltransferase 1 determines its nuclear localization and its accumulation in the nucleus

Serine hydroxymethyltransferase 1 (SHMT1) expression limits rates of de novo dTMP synthesis in the nucleus. Here we report that SHMT1 is ubiquitinated at the small ubiquitin-like modifier (SUMO) consensus motif and that ubiquitination at that site is required for SHMT1 degradation. SHMT1 protein levels are cell cycle-regulated, and Ub-SHMT1 levels are lowest at S phase when SHMT1 undergoes SUMO modification and nuclear transport. Mutation of the SUMO consensus motif increases SHMT1 stability. SHMT1 interacts with components of the proteasome in both the nucleus and cytoplasm, indicating that degradation occurs in both compartments. Ubc13-mediated ubiquitination is required for SHMT1 nuclear export and increases stability of SHMT1 within the nucleus, whereas Ubc9-mediated modification with Sumo2/3 is involved in nuclear degradation. These data demonstrate that SUMO and ubiquitin modification of SHMT1 occurs on the same lysine residue and determine the localization and accumulation of SHMT1 in the nucleus.     

Inhibition of 5,10-methenyltetrahydrofolate synthetase

The interaction of 5-formyltetrahydrofolate analogs with murine methenyltetrahydrofolate synthetase (MTHFS) was investigated using steady-state kinetics, molecular modeling, and site-directed mutagenesis. MTHFS catalyzes the irreversible cyclization of 5-formyltetrahydrofolate to 5,10-methenyltetrahydrofolate. Folate analogs that cannot undergo the rate-limiting step in catalysis were inhibitors of murine MTHFS. 5-Formyltetrahydrohomofolate was an effective inhibitor of murine MTHFS (K(i)=0.7 microM), whereas 5-formyl,10-methyltetrahydrofolate was a weak inhibitor (K(i)=10 microM). The former, but not the latter, was slowly phosphorylated by MTHFS. 5-Formyltetrahydrohomofolate was not a substrate for murine MTHFS, but was metabolized when the MTHFS active site Y151 was mutated to Ala. MTHFS active site residues do not directly facilitate N10 attack on the on the N5-iminium phosphate intermediate, but rather restrict N10 motion around N5. Inhibitors specifically designed to block N10 attack appear to be less effective than the natural 10-formyltetrahydrofolate polyglutamate inhibitors.     

Crystal structure of bifunctional 5,10-methylenetetrahydrofolate dehydrogenase/cyclohydrolase from Thermoplasma acidophilum

Folate co-enzymes play a pivotal role in one-carbon transfer cellular processes. Many eukaryotes encode the tri-functional tetrahydrofolate dehydrogenase/cyclohydrolase/synthetase (deh/cyc/syn) enzyme, which consists of a N-terminal bifunctional domain (deh/cyc) and a C-terminal monofunctional domain (syn). Here, we report the first analogous archeal enzyme structures, for the bifunctional methylenetetrahydrofolate dehydrogenase/cyclohydrolase from Thermoplasma acidophilum (TaMTHFDC) as the native protein and also as its NADP complex. The TaMTHFDC structure is a dimer with a polar interface, as well as a NADP binding site that shows minor conformational change. The orientations of the residues in the NADP binding site do not change on ligand binding, incorporating three water molecules which are hydrogen bonded with phosphate groups of NADP in the structure of the complex. Our structural information will contribute to an improved understanding of the basis of THF and one-carbon metabolism.     

S-Adenosyl methionine synthetase 1 limits fat storage in Caenorhabditis elegans

Cytosolic lipid droplets are versatile, evolutionarily conserved organelles that are important for the storage and utilization of lipids in almost all cell types. To obtain insight into the physiological importance of lipid droplet size, we isolated and characterized a new S-adenosyl methionine synthetase 1 (SAMS-1)-deficient Caenorhabditis elegans mutant, which have enlarged lipid droplets throughout its life cycle. We found that the sams-1 mutant showed a markedly reduced body size and progeny number; impaired synthesis of phosphatidylcholine, a major membrane phospholipid; and elevated expression of key lipogenic genes, such as dgat-2, resulting in the accumulation of triacylglyceride in fewer, but larger, lipid droplets. The sams-1 mutant store more than 50 % (wild type: 10 %) of its intestinal fat in large lipid droplets, ≥10 μm³ in size. In response to starvation, SAMS-1 deficiency causes reduced depletion of a subset of lipid droplets located in the anterior intestine. Given the importance of liberation of fatty acids from lipid droplets, we propose that the physiological function of SAMS-1, a highly conserved enzyme involved in one-carbon metabolism, is the limitation of fat storage to ensure proper growth and reproduction.

Electronic supplementary material

The online version of this article (doi:10.1007/s12263-014-0386-6) contains supplementary material, which is available to authorized users.     

Whole-genome RNAi screen identifies methylation-related genes influencing lipid metabolism in Caenorhabditis elegans

Lipid droplets (LDs) are highly conserved multifunctional cellular organelles and aberrant lipid storage in LDs can lead to many metabolic diseases. However, the molecular mechanisms governing lipid dynamic changes remain elusive, and the high-throughput screen of genes influencing LD morphology was limited by lacking specific LD marker proteins in the powerful genetic tool Caenorhabditis elegans. In this study, we established a new method to conduct whole-genome RNAi screen using LD resident protein DHS-3 as a LD marker, and identified 78 genes involved in significant LD morphologic changes. Among them, mthf-1, as well as a series of methylation-related genes, was found dramatically influencing lipid metabolism. SREBP-1 and SCD1 homologs in C. elegans were involved in the lipid metabolic change of mthf-1(RNAi) worms, and the regulation of ATGL-1 also contributed to it by decreasing triacylglycerol (TAG) hydrolysis. Overall, this study not only identified important genes involved in LD dynamics, but also provided a new tool for LD study using C. elegans, with implications for the study of lipid metabolic diseases.     

Mouse betaine-homocysteine S-methyltransferase deficiency reduces body fat via increasing energy expenditure and impairing lipid synthesis and enhancing glucose oxidation in white adipose tissue

Betaine-homocysteine S-methyltransferase (BHMT) catalyzes the synthesis of methionine from homocysteine. In our initial report, we observed a reduced body weight in Bhmt(-/-) mice. We initiated this study to investigate the potential role of BHMT in energy metabolism. Compared with the controls (Bhmt(+/+)), Bhmt(-/-) mice had less fat mass, smaller adipocytes, and better glucose and insulin sensitivities. Compared with the controls, Bhmt(-/-) mice had increased energy expenditure, with no changes in food intake, fat uptake or absorption, or in locomotor activity. The reduced adiposity in Bhmt(-/-) mice was not due to hyperthermogenesis. Bhmt(-/-) mice failed to maintain a normal body temperature upon cold exposure because of limited fuel supplies. In vivo and ex vivo tests showed that Bhmt(-/-) mice had normal lipolytic function. The rate of (14)C-labeled fatty acid incorporated into [(14)C]triacylglycerol was the same in Bhmt(+/+) and Bhmt(-/-) gonadal fat depots (GWAT), but it was 62% lower in Bhmt(-/-) inguinal fat depots (IWAT) compared with that of Bhmt(+/+) mice. The rate of (14)C-labeled fatty acid oxidation was the same in both GWAT and IWAT from Bhmt(+/+) and Bhmt(-/-) mice. At basal level, Bhmt(-/-) GWAT had the same [(14)C]glucose oxidation as did the controls. When stimulated with insulin, Bhmt(-/-) GWAT oxidized 2.4-fold more glucose than did the controls. Compared with the controls, the rate of [(14)C]glucose oxidation was 2.4- and 1.8-fold higher, respectively, in Bhmt(-/-) IWAT without or with insulin stimulus. Our results show for the first time a role for BHMT in energy homeostasis.     

Enzymology of one-carbon metabolism in methanogenic pathways

Methanoarchaea, the largest and most phylogenetically diverse group in the Archaea domain, have evolved energy-yielding pathways marked by one-carbon biochemistry featuring novel cofactors and enzymes. All of the pathways have in common the two-electron reduction of methyl-coenzyme M to methane catalyzed by methyl-coenzyme M reductase but deviate in the source of the methyl group transferred to coenzyme M. Most of the methane produced in nature derives from acetate in a pathway where the activated substrate is cleaved by CO dehydrogenase/acetyl-CoA synthase and the methyl group is transferred to coenzyme M via methyltetrahydromethanopterin or methyltetrahydrosarcinapterin. Electrons for reductive demethylation of the methyl-coenzyme M originate from oxidation of the carbonyl group of acetate to carbon dioxide by the synthase. In the other major pathway, formate or H2 is oxidized to provide electrons for reduction of carbon dioxide to the methyl level and reduction of methyl-coenzyme to methane. Methane is also produced from the methyl groups of methanol and methylamines. In these pathways specialized methyltransferases transfer the methyl groups to coenzyme M. Electrons for reduction of the methyl-coenzyme M are supplied by oxidation of the methyl groups to carbon dioxide by a reversal of the carbon dioxide reduction pathway. Recent progress on the enzymology of one-carbon reactions in these pathways has raised the level of understanding with regard to the physiology and molecular biology of methanogenesis. These advances have also provided a foundation for future studies on the structure/function of these novel enzymes and exploitation of the recently completed sequences for the genomes from the methanoarchaea Methanobacterium thermoautotrophicum and Methanococcus jannaschii.     

A novel mitochondrial C1-tetrahydrofolate synthetase is upregulated in human colon adenocarcinoma

To seek the genes involved in the development of colorectal cancer, we analyzed the microarray gene expression profiles of human normal and cancerous colon tissues using the BioExpress database platform. Through the analysis we found one gene named DKFZp586G1517 that was upregulated in colon adenocarcinomas. The full-length cDNA of the DKFZp586G1517 cloned by polymerase chain reaction (PCR) encodes a protein with 978 amino acids, which is homologous to the human cytosolic C(1)-tetrahydrofolate synthetase and contains a mitochondrial target signal at N-terminus. The gene product expressed in 293 cells was localized in mitochondria and processed at the predicted signal cleavage site, supporting the idea that DKFZp586G1517 is a novel mitochondrial C(1)-tetrahydrofolate synthetase (mtC(1)-THFS). The overexpression of mtC(1)-THFS in 293 cells stimulated the colony formation. These results suggest that mtC(1)-THFS may participate in the progression of colorectal cancer by conferring growth advantage and could be a new molecular target for cancer therapy.