Mthfd1 Is an Essential Gene in Mice and Alters Biomarkers of Impaired One-carbon Metabolism

Cytoplasmic folate-mediated one carbon (1C) metabolism functions to carry and activate single carbons for the de novo synthesis of purines, thymidylate, and for the remethylation of homocysteine to methionine. C1 tetrahydrofolate (THF) synthase, encoded by Mthfd1, is an entry point of 1Cs into folate metabolism through its formyl-THF synthetase (FTHFS) activity that catalyzes the ATP-dependent conversion of formate and THF to 10-formyl-THF. Disruption of FTHFS activity by the insertion of a gene trap vector into the Mthfd1 gene results in embryonic lethality in mice. Mthfd1^gt/+ mice demonstrated lower hepatic adenosylmethionine levels, which is consistent with formate serving as a source of 1Cs for cellular methylation reactions. Surprisingly, Mthfd1^gt/+ mice exhibited decreased levels of uracil in nuclear DNA, indicating enhanced de novo thymidylate synthesis, and suggesting that serine hydroxymethyltransferase and FTHFS compete for a limiting pool of unsubstituted THF. This study demonstrates the essentiality of the Mthfd1 gene and indicates that formate-derived 1Cs are utilized for de novo purine synthesis and the remethylation of homocysteine in liver. Further, the depletion of cytoplasmic FTHFS activity enhances thymidylate synthesis, affirming the competition between thymidylate synthesis and homocysteine remethylation for THF cofactors.Folate-mediated one-carbon (1C)³ metabolism is compartmentalized in the cytoplasm, mitochondria, and nucleus of mammalian cells (1). In the cytoplasm, 1C metabolism functions to carry and chemically activate single carbons for the de novo synthesis of purines, thymidylate, and for the remethylation of homocysteine to methionine (2) (see Fig. 1). Methionine can be adenosylated to form S-adenosylmethionine (AdoMet), the major cellular methyl group donor required for the methylation of DNA, RNA, histones, small molecules, and lipids. Nuclear 1C metabolism functions to synthesize thymidylate from dUMP and serine during S phase through the small ubiquitin-like modifier-dependent translocation of cytoplasmic serine hydroxymethyltransferase (cSHMT), dihydrofolate reductase, and thymidylate synthase into the nucleus (3).Open in a separate windowFIGURE 1.Folate-mediated one-carbon metabolism occurs in the mitochondria, nucleus, and cytoplasm. Mitochondrial-derived formate traverses to the cytoplasm where it is incorporated into the folate-activated one-carbon pool through the activity of FTHFS and utilized in the synthesis of purines, thymidylate, and the methylation of homocysteine to methionine. Methionine can be converted to a methyl donor through its adenosylation to AdoMet. Thymidylate biosynthesis occurs in the cytoplasm and nucleus. The one-carbon unit is labeled in bold. GCS, glycine cleavage system; mSHMT, mitochondrial serine hydroxymethyltransferase; mMTHFD, mitochondrial methylenetetrahydrofolate dehydrogenase; mMTHFC, mitochondrial methenyltetrahydrofolate cyclohydrolase; mFTHFS, mitochondrial formyltetrahydrofolate synthetase; MTHFD, methylenetetrahydrofolate dehydrogenase; MTHFC, methenyltetrahydrofolate cyclohydrolase; FTHFS, formyltetrahydrofolate synthetase; MTHFR, methylenetetrahydrofolate reductase; TS, thymidylate synthase; DHFR, dihydrofolate reductase; and cSHMT, cytoplasmic serine hydroxymethyltransferase.Serine, through its conversion to glycine by SHMT, is a primary source of 1Cs for nucleotide and methionine synthesis (4). SHMT generates 1Cs in the cytoplasm, mitochondria, and nucleus, although the generation of 1Cs through SHMT activity in the cytoplasm is not essential in mice, indicating the essentiality of mitochondria-derived 1Cs for cytoplasmic 1C metabolism (5). In mitochondria, the hydroxymethyl group of serine and the C2 carbon of glycine are transferred to tetrahydrofolate (THF) to generate 5,10-methylene-THF by the mitochondrial isozyme of SHMT and the glycine cleavage system, respectively (6). The 1C carried by methylene-THF is oxidized and hydrolyzed to generate formate by the NAD-dependent methylene-THF dehydrogenase (MTHFD) and methenyl-THF cyclohydrolase (MTHFC) activities encoded by a single gene, Mthfd2 (7), and 10-formyl-THF synthetase (FTHFS) activity, encoded by Mthfd1L (8) (see Fig. 1).In the cytoplasm, the product of the Mthfd1 gene, C1THF synthase, is a trifunctional enzyme that contains NADP-dependent MTHFD and MTHFC activities on the N-terminal domain of the protein, and FTHFS activity on the C-terminal domain (9). These three activities collectively catalyze the interconversion of THF, 10-formyl-THF, 5,10-methenyl-THF, and 5,10-methylene-THF (10) (Fig. 1). The ATP-dependent FTHFS activity of C1THF synthase condenses mitochondria-derived formate with THF to form 10-formyl-THF, which is required for the de novo synthesis of purines (9). The MTHFC and MTHFD activities convert 10-formyl-THF to methylene-THF (11). Methylene-THF is utilized in the de novo synthesis of thymidylate or, alternatively, can be irreversibly reduced by methylene-THF reductase to 5-methyl-THF, which is used in the remethylation of homocysteine to methionine (12).Impairments in 1C metabolism, due to insufficient folate cofactors and/or single nucleotide polymorphisms in genes that encode folate-dependent enzymes, are associated with numerous pathologies and developmental anomalies, including cancers, cardiovascular disease, and neural tube defects. The causal mechanisms underlying the folate-pathology relationship(s) remains to be established. However, a number of hypotheses have been proposed related to the role of 1C metabolism in genome stability and gene expression. Decreased thymidylate synthesis results in increased uracil misincorporation into DNA and decreased rates of cell division, causing double strand breaks in DNA and genomic instability (13). Decreased AdoMet synthesis alters methylation patterns in CpG islands in DNA and can result in histone hypomethylation, which can alter gene expression (2). Proliferating cells also require the de novo synthesis of purines to maintain rates of DNA synthesis (14).It has been shown that the gene product of Mthfd2, mitochondrial MTHFC/MTHFD is essential in mice, and Mthfd2 deficiency results in embryonic lethality (15). This protein is required for the generation of formate from serine in the mitochondria of embryonic cells. Here, we have investigated the essentiality of the Mthfd1 gene in mice and the effect of altered Mthfd1 gene expression on biomarkers of cytoplasmic 1C metabolism. Our data demonstrate that Mthfd1 is an essential gene in mice and that Mthfd1-deficient mice are a model for the study of folate-associated pathologies.     

Benzo[g]quinazolin-based scaffold derivatives as dual EGFR/HER2 inhibitors

Targeting EGFR has proven to be beneficial in the treatment of several types of solid tumours. So, a series of novel 2-(4-oxo-3-(4-sulfamoylphenyl)-3,4-dihydrobenzo[g]quinazolin-2-ylthio)-N-substituted acetamide 5–19 were synthesised from the starting material 4-(2-mercapto-4-oxobenzo[g]quinazolin-3(4H)-yl) benzenesulfonamide 4, to be evaluated as dual EGFR/HER2 inhibitors. The target compounds 5–19, were screened for their cytotoxic activity against A549 lung cancer cell line. The percentage inhibition of EGFR enzyme was measured and compared with erlotinib as the reference drug. Compounds 6, 8, 10, and 16 showed excellent EGFR inhibitory activity and were further selected for screening as dual EGFR/HER2 inhibitors. The four selected compounds showed IC₅₀ ranging from 0.009 to 0.026?µM for EGFR and 0.021 to 0.069?µM for the HER2 enzyme. Compound 8 was found to be the most potent in this study with IC₅₀ 0.009 and 0.021?µM for EGFR and HER2, respectively.     

Modeling of the aorta artery aneurysms and renal artery stenosis using cardiovascular electronic system

Background  

The aortic aneurysm is a dilatation of the aortic wall which occurs in the saccular and fusiform types. The aortic aneurysms can rupture, if left untreated. The renal stenosis occurs when the flow of blood from the arteries leading to the kidneys is constricted by atherosclerotic plaque. This narrowing may lead to the renal failure. Previous works have shown that, modelling is a useful tool for understanding of cardiovascular system functioning and pathophysiology of the system. The present study is concerned with the modelling of aortic aneurysms and renal artery stenosis using the cardiovascular electronic system.     

Genetic polymorphism of <Emphasis Type="Italic">GSTT1</Emphasis> may be under natural selection in a population chronically exposed to natural sour gas

Objective In order to examine whether chronic exposure to natural sour gas containing sulfur compounds act as natural selection force on genetic polymorphisms of glutathione S-transferase M1 (GSTM1) and T1 (GSTT1), the present study was done. Methods The study was performed on two groups of healthy individuals of Masjid-i-Sulaiman (Khozestan province, southwest of Iran) citizens with the mean ages of 47.5 ± 12.4 (36 male and 58 female) and 16.3 ± 2.4 (47 male and 140 female) that were considered as first and second generation, respectively. The GSTT1 and GSTM1 genotypes were determined using a PCR-based method. Results The genotypic frequencies of GSTM1 did not change significantly (χ² = 0.085, df = 1, P = 0.770). The frequency of the GSTT1 null genotype was 52.1% in the first generation and reached to 36.4% in the second generation. There was significant difference between two generations for the GSTT1 polymorphism (χ² = 6.397, df = 1, P = 0.011). Conclusion It was suggested that the GSTT1 polymorphism may be under natural selection because of probably favored ability of GSTT1-active genotype to survival and reproduction.     

Fluorescence of phytochrome adducts with synthetic locked chromophores

We performed steady state fluorescence measurements with phytochromes Agp1 and Agp2 of Agrobacterium tumefaciens and three mutants in which photoconversion is inhibited. These proteins were assembled with the natural chromophore biliverdin (BV), with phycoerythrobilin (PEB), which lacks a double bond in the ring C-D-connecting methine bridge, and with synthetic bilin derivatives in which the ring C-D-connecting methine bridge is locked. All PEB and locked chromophore adducts are photoinactive. According to fluorescence quantum yields, the adducts may be divided into four different groups: wild type BV adducts exhibiting a weak fluorescence, mutant BV adducts with about 10-fold enhanced fluorescence, adducts with locked chromophores in which the fluorescence quantum yields are around 0.02, and PEB adducts with a high quantum yield of around 0.5. Thus, the strong fluorescence of the PEB adducts is not reached by the locked chromophore adducts, although the photoconversion energy dissipation pathway is blocked. We therefore suggest that ring D of the bilin chromophore, which contributes to the extended π-electron system of the locked chromophores, provides an energy dissipation pathway that is independent on photoconversion.     

Viral Population Estimation Using Pyrosequencing

The diversity of virus populations within single infected hosts presents a major difficulty for the natural immune response as well as for vaccine design and antiviral drug therapy. Recently developed pyrophosphate-based sequencing technologies (pyrosequencing) can be used for quantifying this diversity by ultra-deep sequencing of virus samples. We present computational methods for the analysis of such sequence data and apply these techniques to pyrosequencing data obtained from HIV populations within patients harboring drug-resistant virus strains. Our main result is the estimation of the population structure of the sample from the pyrosequencing reads. This inference is based on a statistical approach to error correction, followed by a combinatorial algorithm for constructing a minimal set of haplotypes that explain the data. Using this set of explaining haplotypes, we apply a statistical model to infer the frequencies of the haplotypes in the population via an expectation–maximization (EM) algorithm. We demonstrate that pyrosequencing reads allow for effective population reconstruction by extensive simulations and by comparison to 165 sequences obtained directly from clonal sequencing of four independent, diverse HIV populations. Thus, pyrosequencing can be used for cost-effective estimation of the structure of virus populations, promising new insights into viral evolutionary dynamics and disease control strategies.     

Consanguineous marriages in Afghanistan

The present cross-sectional study was done in order to illustrate the prevalence and types of consanguineous marriages among Afghanistan populations. Data on types of marriages were collected using a simple questionnaire. The total number of couples in the study was 7140 from the following provinces: Badakhshan, Baghlan, Balkh, Bamyan, Kabul, Kunduz, Samangan and Takhar. Consanguineous marriages were classified by the degree of relationship between couples: double first cousins, first cousins, first cousins once removed, second cousins and beyond second cousins. The coefficient of inbreeding (F) was calculated for each couple and the mean coefficient of inbreeding (α) estimated for each population. The proportion of consanguineous marriages in the country was 46.2%, ranging from 38.2% in Kabul province to 51.2% in Bamyan province. The equivalent mean inbreeding coefficient (α) was 0.0277, and ranged from 0.0221 to 0.0293 in these two regions. There were significant differences between provinces for frequencies of different types of marriages (p<0.001). First cousin marriages (27.8%) were the most common type of consanguineous marriages, followed by double first cousin (6.9%), second cousin (5.8%), beyond second cousin (3.9%) and first cousin once removed (1.8%). There were significant differences between ethnic groups for the types of marriages (χ2=177.6, df=25, p<0.001). Tajiks (Soni) and Turkmens (also Pashtuns) showed the lowest (α=0.0250) and highest (α=0.0297) mean inbreeding coefficients, respectively, among the ethnic groups in Afghanistan. The study shows that Afghanistan's populations, like other Islamic populations, have a high level of consanguinity.     

Micellisation and immunoreactivities of dimeric β-caseins

Bovine β-casein (β-CN) is a highly amphiphilic micellising phospho-protein showing chaperone-like activity in vitro. Recently, existence of multiple sequential epitopes on β-CN polypeptide chain in both hydrophilic–polar (ψ) and hydrophobic–apolar domains (φ) has been evidenced. In order to clarify specific contribution of polar and apolar domains in micellisation process and in shaping immunoreactivity of β-CN, its dimeric/bi-amphiphilic “quasi palindromic” forms covalently connected by a disulfide bond linking either N-terminal (C4 β-CND) or C-terminal domain (C208 β-CND) were produced and studied. Depending on the C- or N-terminal position of inserted cysteine, each dimeric β-CN contains one polar/apolar region at the centre and two external hydrophobic/hydrophilic ends. Consequently, such casein dimers have radically different polarities/hydrophobicities on their outside surfaces. Dynamic light scattering (DLS) measurements indicate that these dimeric casein molecules form micelles of different sizes depending on arrangement of polar fragments of the β-CN mutants in their constrained dimers. Non-aggregated dimers have different hydrodynamic diameters that could be explained by their different geometries. Measurements of fluorescence showed more hydrophobic environment of Trp residues of C208 β-CND, while in similar experimental conditions Trp residues of C4 β-CND and native β-CN were more exposed to the polar medium. Both fluorescence and DLS studies showed greater propensity for micellisation of the dimeric β-CNs, suggesting that the factors inducing the formation of micelles are stronger in the bi-amphiphilic dimers. 1-Anilino-naphthalene-8-sulfonate (ANS) binding studies showed different binding of ANS by these dimers as well as different exposition of ANS binding (hydrophobic) regions in the micellar states. The differences in fluorescence resonance energy transfer (FRET) profiles of C4 β-CND and C208 β-CND can be explained by differences of distances and/or by differences of relative orientations of the donor (Trp) and acceptor (ANS), as well as by differences in quenching properties of the disulfide bridges and intra-molecular hydrophobic interactions. The immunoreactivity assays showed somewhat lower IgE response to C208 β-CND than to C4 β-CND. Thus, dimerization of C208 β-CN, connecting two C-terminal hydrophobic domains of two monomers doubling long-range hydrophobic interactions, possibly may hide a part of epitopes in the hydrophobic interface/core of C208 β-CND that is consistent with the results of DLS and fluorescence studies. The obtained results indicate structural differences of dimers – possibly the formation of Y- and U-shaped structures for C208 β-CND and C4 β-CND, respectively. This study not only demonstrated the importance of the organization of polar and hydrophobic regions during micellisation of the constrained and oriented β-CN dimers but also confirmed a possible role of C-terminal hydrophobic domain in the immunoreactivity profile of native β-CN.     

Estradiol-dependent regulation of angiopoietin expression in breast cancer cells

Angiopoietin-1 (Ang-1) is a ligand for Tie-2 receptors and a promoter of angiogenesis. Angiogenesis plays an important role in breast cancer, as it is one of the critical events required for tumors to grow and metastasize. In this study, we investigated the influence of estradiol (E2) on the expression of angiopoietins in breast cancer cell lines. Ang-1 mRNA and protein expressions were significantly higher in estrogen receptor-negative (ERα-) breast cancer cells than in estrogen receptor-positive (ERα+) cells. Exposure of ERα+ cells to E2 resulted in further reductions of Ang-1 levels. In mouse mammary pads inoculated with breast cancer cells, both tumor size and Ang-1 production were significantly lower in ERα+ cell-derived xenografts, as compared to those derived from ERα- cells. Reduction of circulating levels of E2 by ovariectomy eliminated this response. Overall, these results indicate that Ang-1 mRNA and protein expressions: (1) negatively correlate with the level of ERα in breast cancer cell lines; (2) are downregulated by E2 in an ERα dependent manner; and (3) positively correlate with the degree of angiogenesis in vivo. We conclude that Ang-1 is an important modulator of growth and progression of ERα- breast cancers.