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1.
FEBS J ; 291(2): 308-322, 2024 01.
Artigo em Inglês | MEDLINE | ID: mdl-37700610

RESUMO

d-Serine plays vital physiological roles in the functional regulation of the mammalian brain, where it is produced from l-serine by serine racemase and degraded by d-amino acid oxidase. In the present study, we identified a new d-serine metabolizing activity of serine hydroxymethyltransferase (SHMT) in bacteria as well as mammals. SHMT is known to catalyze the conversion of l-serine and tetrahydrofolate (THF) to glycine and 5,10-methylenetetrahydrofolate, respectively. In addition, we found that human and Escherichia coli SHMTs have d-serine dehydratase activity, which degrades d-serine to pyruvate and ammonia. We characterized this enzymatic activity along with canonical SHMT activity. Intriguingly, SHMT required THF to catalyze d-serine dehydration and did not exhibit dehydratase activity toward l-serine. Furthermore, SHMT did not use d-serine as a substrate in the canonical hydroxymethyltransferase reaction. The d-serine dehydratase activities of two isozymes of human SHMT were inhibited in the presence of a high concentration of THF, whereas that of E. coli SHMT was increased. The pH and temperature profiles of d-serine dehydratase and serine hydroxymethyltransferase activities of these three SHMTs were partially distinct. The catalytic efficiency (kcat /Km ) of dehydratase activity was lower than that of hydroxymethyltransferase activity. Nevertheless, the d-serine dehydratase activity of SHMT was physiologically important because d-serine inhibited the growth of an SHMT deletion mutant of E. coli, ∆glyA, more than that of the wild-type strain. Collectively, these results suggest that SHMT is involved not only in l- but also in d-serine metabolism through the degradation of d-serine.


Assuntos
Escherichia coli , Glicina Hidroximetiltransferase , Animais , Humanos , Glicina Hidroximetiltransferase/genética , Glicina Hidroximetiltransferase/química , Escherichia coli/genética , Escherichia coli/metabolismo , Tetra-Hidrofolatos , Metiltransferases , Serina , Hidroliases/genética , Mamíferos/metabolismo
2.
Mol Neurobiol ; 61(1): 434-449, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-37624487

RESUMO

Ischemic stroke is a heterogeneous brain injury with complex pathophysiology and it is also a time sensitive neurological injury disease. At present, the treatment options for ischemic stroke are still limited. 6S-5-methyltetrahydrofolate-calcium (MTHF-Ca) is the calcium salt of the predominant form of dietary folate in circulation. MTHF-Ca has potential neuroprotective effect on neurocytes, but whether it can be used for ischemic stroke treatment remains unknown. We established zebrafish ischemic stroke model through photothrombotic method to evaluate the protective effect of MTHF-Ca on the ischemic brain injury of zebrafish. We demonstrated that MTHF-Ca reduced the brain damage by reducing motor dysfunction and neurobehavioral defects of zebrafish with telencephalon infarction injury. MTHF-Ca counteracted oxidative damages after Tel injury by increasing the activities of GSH-Px and SOD and decreasing the content of MDA. RNA-seq and RT-qPCR results showed that MTHF-Ca played a neuroprotective role by alleviating neuroinflammation, inhibiting blood coagulation, and neuronal apoptosis processes. Overall, we have demonstrated that MTHF-Ca has neuroprotective effect in ischemic stroke and can be used as a potential treatment for ischemic stroke.


Assuntos
Lesões Encefálicas , AVC Isquêmico , Fármacos Neuroprotetores , Acidente Vascular Cerebral , Tetra-Hidrofolatos , Animais , Peixe-Zebra , Cálcio , Infarto , Acidente Vascular Cerebral/complicações , Acidente Vascular Cerebral/tratamento farmacológico
3.
Front Endocrinol (Lausanne) ; 14: 1273580, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-38116318

RESUMO

Background: Most studies have explored the relationship between serum total folate and nonalcoholic fatty liver disease (NAFLD) in adults, but there has been no study on the relationship between different folate forms and hepatic steatosis or liver stiffness in adolescents. Objective: To investigate the association of different folate forms with hepatic steatosis or liver stiffness in adolescents, and further explore the intermediary role of BMI in this relationship. Methods: The cross-sectional study included 549 participants from the 2017-2018 National Health and Nutrition Inspection Survey (NHANES) survey cycle who had complete data. Four folate data (red blood cell folate, serum total folate, 5-methyl-tetrahydrofolate and folic acid) were included in our study. Controlled attenuation parameters (CAP) and liver stiffness came from the results of liver ultrasound transient elastography. We used linear regression to analyze the relationship between different forms of folate and CAP or liver stiffness, and logistic regression to analyze the relationship between different forms of folate and NAFLD or significant fibrosis. We also used restricted cubic splines to analyze the nonlinear relationship between different forms of folate and NAFLD or significant fibrosis. Finally, we used regression-based intermediary analysis to distinguish the direct and BMI-mediated effects of folate on CAP or liver stiffness. All the analyses adjusted the relevant covariates. Results: The means of CAP and liver hardness in this study were 223.02dB/m and 5.03kPa, respectively. We found that in model 2, there was a negative correlation between serum total folate (ß: -18.53; 95%CI: -29.32 to -7.73) or 5-methyltetrahydrofolate (ß: -14.13; 95%CI: -28.98 to -7.86) and CAP. However, when the BMI was further adjusted in model 3, this negative correlation no longer existed (serum total folate: ß: -8.36; 95%CI: -17.69 to 0.97; 5-methyltetrahydrofolate: ß: -8.05; 95%CI: -17.19 to 1.09). Similarly, we found a negative correlation between serum total folate or 5-Methyl-tetrahydrofolate and liver stiffness in model 2. There was no significant correlation between red blood cell folate or folic acid and CAP or liver stiffness in either model 2 or model 3. The nonlinear relationship between different folate forms and NAFLD or significant fibrosis was not significant. It is estimated that 76% of the total association between serum total folate and CAP is mediated by BMI. The mediating proportion of BMI in the total correlation between serum total folate and liver stiffness was 50%. Similarly, we found that BMI significantly mediated the relationship between 5-Methyl-tetrahydrofolate and CAP or liver stiffness, with a mediating ratio of 77% and 49%, respectively. Conclusion: Our results show that serum total folate or 5-Methyl-tetrahydrofolate are negatively correlated with hepatic steatosis or liver stiffness in adolescents, and BMI plays major mediating role in this relationship. Our findings emphasize the importance of monitoring the concentration of serum folate, not just the serum total folate concentration.


Assuntos
Hepatopatia Gordurosa não Alcoólica , Adulto , Humanos , Adolescente , Estados Unidos/epidemiologia , Hepatopatia Gordurosa não Alcoólica/epidemiologia , Índice de Massa Corporal , Ácido Fólico , Inquéritos Nutricionais , Estudos Transversais , Cirrose Hepática/epidemiologia , Tetra-Hidrofolatos
4.
Essays Biochem ; 67(5): 853-863, 2023 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-37449444

RESUMO

Methionine synthases (MetH) catalyse the methylation of homocysteine (Hcy) with 5-methyl-tetrahydrofolate (5, methyl-THF) acting as methyl donor, to form methionine (Met) and tetrahydrofolate (THF). This function is performed by two unrelated classes of enzymes that differ significantly in both their structures and mechanisms of action. The genomes of plants and many fungi exclusively encode cobalamin-independent enzymes (EC.2.1.1.14), while some fungi also possess proteins from the cobalamin-dependent (EC.2.1.1.13) family utilised by humans. Methionine synthase's function connects the methionine and folate cycles, making it a crucial node in primary metabolism, with impacts on important cellular processes such as anabolism, growth and synthesis of proteins, polyamines, nucleotides and lipids. As a result, MetHs are vital for the viability or virulence of numerous prominent human and plant pathogenic fungi and have been proposed as promising broad-spectrum antifungal drug targets. This review provides a summary of the relevance of methionine synthases to fungal metabolism, their potential as antifungal drug targets and insights into the structures of both classes of MetH.


Assuntos
5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase , Antifúngicos , Humanos , 5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/química , 5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/metabolismo , Virulência , Tetra-Hidrofolatos/metabolismo , Vitamina B 12/metabolismo , Vitamina B 12/farmacologia , Metionina/metabolismo
5.
Anal Bioanal Chem ; 415(21): 5151-5163, 2023 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-37347300

RESUMO

Climate change directs the focus in biotechnology increasingly on one-carbon metabolism for fixation of CO2 and CO2-derived chemicals (e.g. methanol, formate) to reduce our reliance on both fossil and food-competing carbon sources. The tetrahydrofolate pathway is involved in several one-carbon fixation pathways. To study such pathways, stable isotope-labelled tracer analysis performed with mass spectrometry is state of the art. However, no such method is currently available for tetrahydrofolate vitamers. In the present work, we established a fit-for-purpose extraction method for the methylotrophic yeast Komagataella phaffii that allows access to intracellular methyl- and methenyl-tetrahydrofolate (THF) with demonstrated stability over several hours. To determine isotopologue distributions of methyl-THF, LC-QTOFMS provides a selective fragment ion with suitable intensity of at least two isotopologues in all samples, but not for methenyl-THF. However, the addition of ion mobility separation provided a critical selectivity improvement allowing accurate isotopologue distribution analysis of methenyl-THF with LC-IM-TOFMS. Application of these new methods for 13C-tracer experiments revealed a decrease from 83 ± 4 to 64 ± 5% in the M + 0 carbon isotopologue fraction in methyl-THF after 1 h of labelling with formate, and to 54 ± 5% with methanol. The M + 0 carbon isotopologue fraction of methenyl-THF was reduced from 83 ± 2 to 78 ± 1% over the same time when using 13C-methanol labelling. The labelling results of multiple strains evidenced the involvement of the THF pathway in the oxygen-tolerant reductive glycine pathway, the presence of the in vivo reduction of formate to formaldehyde, and the activity of the spontaneous condensation reaction of formaldehyde with THF in K. phaffii.


Assuntos
Dióxido de Carbono , Metanol , Carbono/metabolismo , Tetra-Hidrofolatos/metabolismo , Espectrometria de Massas , Formiatos
6.
Environ Microbiol Rep ; 15(5): 339-351, 2023 10.
Artigo em Inglês | MEDLINE | ID: mdl-37150590

RESUMO

To inactivate the Wood-Ljungdahl pathway in the acetogenic model bacterium Acetobacterium woodii, the genes metVF encoding two of the subunits of the methylene-tetrahydrofolate reductase were deleted. As expected, the mutant did not grow on C1 compounds and also not on lactate, ethanol or butanediol. In contrast to a mutant in which the first enzyme of the pathway (hydrogen-dependent CO2 reductase) had been genetically deleted, cells were able to grow on fructose, albeit with lower rates and yields than the wild-type. Growth was restored by addition of an external electron sink, glycine betaine + CO2 or caffeate. Resting cells pre-grown on fructose plus an external electron acceptor fermented fructose to two acetate and four hydrogen, that is, performed hydrogenogenesis. Cells pre-grown under fermentative conditions on fructose alone redirected carbon and electrons to form lactate, formate, ethanol as well as hydrogen. Apparently, growth on fructose alone induced enzymes for mixed acid fermentation (MAF). Transcriptome analyses revealed enzymes potentially involved in MAF and a quantitative model for MAF from fructose in A. woodii is presented.


Assuntos
Dióxido de Carbono , Frutose , Fermentação , Frutose/metabolismo , Dióxido de Carbono/metabolismo , Oxirredutases/metabolismo , Etanol , Hidrogênio/metabolismo , Lactatos , Tetra-Hidrofolatos/metabolismo
7.
J Genet ; 1022023.
Artigo em Inglês | MEDLINE | ID: mdl-37204128

RESUMO

Ischemic stroke, a prevalent neurological disease, is the major reason of serious disability and death worldwide. Methylenetetrahydrofolate reductase (MTHFR) gene polymorphisms increase homocysteine levels which also raise the risk of vascular diseases. Angiotensin-converting enzyme (ACE) gene polymorphisms can cause vascular reorganization and disrupt arterial wall stability. The aim of this study was to explore how the MTHFR and ACE gene polymorphisms are related to acute ischemic stroke. A total of 200 individuals (102 acute ischemic stroke patients and 98 healthy controls) were included in this case-control research. MTHFR gene C677T (rs1801133) and A1298C (rs1801131) polymorphisms were studied through polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) assays, ACE gene I/D polymorphism (rs1799752) was studied by PCR. The MTHFR C667T and ACE I/D polymorphisms did not show any statistically meaningful differences between healthy controls and acute ischemic stroke patients (P>0.05). However, compared to healthy controls, acute ischemic stroke patients had almost nine-fold higher prevalence of the CC genotype based on the MTHFR A1298C polymorphism (P=0.024, OR=8.8, 95%Cl=1.27-208.2). Additionally, individuals with acute ischemic stroke had greater frequencies of the combined genotypes of MTHFR and ACE gene polymorphisms in the forms of CC/CC (C667T/A1298C), CC/ DD (A1298C/ACE I/D) and CC/CC/DD (C677T/A1298C/ACE I/D) (P = 0.027, P = 0.015 and P = 0.037, respectively). A statistically significant correlation was assessed between MTHFR gene A1298C polymorphism and acute ischemic stroke. Additionally, it was discovered that the genotype combinations of CC/CC (C667T/A1298C), CC/DD (A1298C/ACE I/D) and CC/CC/DD (C677T/A1298C/ ACE I/D) have risk-increasing effects on acute ischemic stroke. To employ these genetic variations as alternative treatments for ischemic stroke, these findings should be validated by more research.


Assuntos
AVC Isquêmico , Metilenotetra-Hidrofolato Redutase (NADPH2) , Humanos , Estudos de Casos e Controles , Frequência do Gene , Predisposição Genética para Doença , Genótipo , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Peptidil Dipeptidase A/genética , Polimorfismo de Nucleotídeo Único , Tetra-Hidrofolatos/genética
8.
Nat Metab ; 5(4): 642-659, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-37012496

RESUMO

Cancer cells fuel their increased need for nucleotide supply by upregulating one-carbon (1C) metabolism, including the enzymes methylenetetrahydrofolate dehydrogenase-cyclohydrolase 1 and 2 (MTHFD1 and MTHFD2). TH9619 is a potent inhibitor of dehydrogenase and cyclohydrolase activities in both MTHFD1 and MTHFD2, and selectively kills cancer cells. Here, we reveal that, in cells, TH9619 targets nuclear MTHFD2 but does not inhibit mitochondrial MTHFD2. Hence, overflow of formate from mitochondria continues in the presence of TH9619. TH9619 inhibits the activity of MTHFD1 occurring downstream of mitochondrial formate release, leading to the accumulation of 10-formyl-tetrahydrofolate, which we term a 'folate trap'. This results in thymidylate depletion and death of MTHFD2-expressing cancer cells. This previously uncharacterized folate trapping mechanism is exacerbated by physiological hypoxanthine levels that block the de novo purine synthesis pathway, and additionally prevent 10-formyl-tetrahydrofolate consumption for purine synthesis. The folate trapping mechanism described here for TH9619 differs from other MTHFD1/2 inhibitors and antifolates. Thus, our findings uncover an approach to attack cancer and reveal a regulatory mechanism in 1C metabolism.


Assuntos
Metilenotetra-Hidrofolato Desidrogenase (NADP) , Neoplasias , Metilenotetra-Hidrofolato Desidrogenase (NADP)/genética , Metilenotetra-Hidrofolato Desidrogenase (NADP)/metabolismo , Ácido Fólico/metabolismo , Formiatos , Purinas , Tetra-Hidrofolatos
9.
Artigo em Inglês | MEDLINE | ID: mdl-36919686

RESUMO

Chronic hepatitis B infection caused by Hepatitis B virus (HBV), influences over two billion people worldwide despite having an effective vaccine. With a total prevalence of 4.57%, there are 3.3 million estimated HBV carriers in Türkiye. Methylene-tetrahydrofolate reductase (MTHFR) arrange folate metabolism through nucleic acid synthesis and DNA methylation. C677T (rs1801133, p.Ala222Val) and A1298C (rs1801131, p.Glu429Ala) polymorphisms of MTHFR gene have effect of reducing the activity of enzyme. We purposed to investigate the correlation between C677T and A1298C polymorphisms of MTHFR gene with HBV infection in a Turkish population. One hundred eighteen HBV-infected participants and ninety healthy controls were incorporated in this research. The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay was applied to discover the genotypes of MTHFR polymorphisms. We demonstrated that T allele and CT + TT genotype frequencies of C677T polymorphism were significantly increased in HBV-infected participants than healthy controls [p = 0.015, OR (95% Cl) = 1.7 (1.11-2.79) and p = 0.020, OR (95% Cl) = 1.9 (1.10-3.42), respectively). No significant associations were noted concerning the A1298C polymorphism (p > 0.05). CC-AA composite genotype was observed to be significantly elevated in healthy controls than HBV-infected participants (32.2% vs. 13.6%, p = 0.001). In addition, the frequency of T-C haplotype was found to be considerably higher in the patient group than control group (15.8% vs 11.8%, p = 0.018). In conclusion, we found that T allele of C677T polymorphism poses a risk factor for HBV infection. We also discovered a protective impact of the CC-AA composite genotype against HBV infection and a risk effect of the T-A haplotype for HBV-infection.


Assuntos
Vírus da Hepatite B , Hepatite B , Humanos , Predisposição Genética para Doença , Genótipo , Fatores de Risco , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Estudos de Casos e Controles , Tetra-Hidrofolatos/genética , Polimorfismo de Nucleotídeo Único , Frequência do Gene
10.
J Biomol Struct Dyn ; 41(23): 14497-14509, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36883866

RESUMO

In all species, dihydrofolate reductase (DHFR) is an essential enzyme that regulates the cellular amount of tetrahydrofolate. Human DHFR (hDHFR) activity inhibition results in tetrahydrofolate depletion and cell death. This property has made hDHFR a therapeutic target for cancer. Methotrexate is a well-known hDHFR inhibitor, but its administration has shown some light to severe adverse effects. Therefore, we aimed to find new potential hDHFR inhibitors using structure-based virtual screening, ADMET prediction, molecular docking, and molecular dynamics simulations. Here, we used the PubChem database to find all compounds with at least 90% structural similarity to known natural DHFR inhibitors. To explore their interaction pattern and estimate their binding affinities, the screened compounds (2023) were subjected to structure-based molecular docking against hDHFR. The fifteen compounds that showed higher binding affinity to the hDHFR than the reference compound (methotrexate) displayed important molecular orientation and interactions with key residues in the enzyme's active site. These compounds were subjected to Lipinski and ADMET prediction. PubChem CIDs: 46886812 and 638190 were identified as putative inhibitors. In addition, molecular dynamics simulations revealed that the binding of compounds (CIDs: 46886812 and 63819) stabilized the hDHFR structure and caused minor conformational changes. Our findings suggest that two compounds (CIDs: 46886812 and 63819) could be promising potential inhibitors of hDHFR in cancer therapy.Communicated by Ramaswamy H. Sarma.


Assuntos
Antagonistas do Ácido Fólico , Neoplasias , Humanos , Metotrexato/farmacologia , Metotrexato/química , Tetra-Hidrofolato Desidrogenase/química , Simulação de Acoplamento Molecular , Antagonistas do Ácido Fólico/farmacologia , Antagonistas do Ácido Fólico/química , Tetra-Hidrofolatos
11.
Nucleic Acids Res ; 51(2): 952-965, 2023 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-36620887

RESUMO

In bacteria, expression of folate-related genes is controlled by the tetrahydrofolate (THF) riboswitch in response to specific binding of THF and its derivatives. Recently, a second class of THF riboswitches, named THF-II, was identified in Gram-negative bacteria, which exhibit distinct architecture from the previously characterized THF-I riboswitches found in Gram-positive bacteria. Here, we present the crystal structures of the ligand-bound THF-II riboswitch from Mesorhizobium loti. These structures exhibit a long rod-like fold stabilized by continuous base pair and base triplet stacking across two helices of P1 and P2 and their interconnecting ligand-bound binding pocket. The pterin moiety of the ligand docks into the binding pocket by forming hydrogen bonds with two highly conserved pyrimidines in J12 and J21, which resembles the hydrogen-bonding pattern at the ligand-binding site FAPK in the THF-I riboswitch. Using small-angle X-ray scattering and isothermal titration calorimetry, we further characterized the riboswitch in solution and reveal that Mg2+ is essential for pre-organization of the binding pocket for efficient ligand binding. RNase H cleavage assay indicates that ligand binding reduces accessibility of the ribosome binding site in the right arm of P1, thus down-regulating the expression of downstream genes. Together, these results provide mechanistic insights into translation regulation by the THF-II riboswitch.


Assuntos
Bactérias , Riboswitch , Pareamento de Bases , Ligantes , Conformação de Ácido Nucleico , Tetra-Hidrofolatos/metabolismo , Bactérias/genética
12.
Methods Enzymol ; 680: 85-100, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36710024

RESUMO

Formate-tetrahydrofolate ligase catalyzes reversible, ATP-dependent conversion of tetrahydrofolate and formate to 10-formyltetrahydrofolate, simultaneously releasing ADP and inorganic phosphate. This enzyme has traditionally been assayed in the direction of 10-CHO-tetrahydrofolate formation by lowering pH of the reaction post-incubation, thus converting the product of the reaction to 5,10-methenyltetrahydrofolate, which is then quantified spectrophotometrically. To increase sensitivity of the product detection, which is particularly useful when determining the kinetic parameters of the enzyme with polyglutamylated substrates, we have replaced the spectrophotometric detection with HPLC separation and fluorescence detection. In addition to the modified enzyme assay protocol, we are also providing protocols for producing recombinant formate-tetrahydrofolate ligase from Arabidopsis in Escherichia coli cells, producing crude Arabidopsis leaf and root extracts suitable for assaying this enzyme, and for synthesis of polyglutamylated tetrahydrofolate substrates.


Assuntos
Arabidopsis , Formiato-Tetra-Hidrofolato Ligase , Formiato-Tetra-Hidrofolato Ligase/metabolismo , Ligases/metabolismo , Cromatografia Líquida de Alta Pressão , Arabidopsis/metabolismo , Tetra-Hidrofolatos , Cinética
13.
Lab Med ; 54(1): 23-29, 2023 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-36036632

RESUMO

Human immunodeficiency virus type 1 (HIV-1) infection varies substantially among individuals. One of the factors influencing viral infection is genetic variability. Methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism is a genetic factor that has been correlated with different types of pathologies, including HIV-1. The MTHFR gene encodes the MTHFR enzyme, an essential factor in the folate metabolic pathway and in maintaining circulating folate and methionine at constant levels, thus preventing the homocysteine accumulation. Several studies have shown the role of folate on CD4+ T lymphocyte count among HIV-1 subjects. In this case-control study we aimed to determine the association between the MTHFR C677T polymorphism and HIV-1 infection susceptibility, AIDS development, and therapeutic outcome among Moroccans. The C677T polymorphism was genotyped by polymerase chain reaction followed by fragment length polymorphism digestion in 214 participants living with HIV-1 and 318 healthy controls. The results of the study revealed no statistically significant association between MTHFR C677T polymorphism and HIV-1 infection (P > .05). After dividing HIV-1 subjects according to their AIDS status, no significant difference was observed between C677T polymorphism and AIDS development (P > .05). Furthermore, regarding the treatment response outcome, as measured by HIV-1 RNA viral load and CD4+ T cell counts, no statistically significant association was found with MTHFR C677T polymorphism. We conclude that, in the genetic context of the Moroccan population, MTHFR C677T polymorphism does not affect HIV-1 infection susceptibility, AIDS development, or response to treatment. However, more studies should be done to investigate both genetic and nutritional aspects for more conclusive results.


Assuntos
Síndrome de Imunodeficiência Adquirida , Infecções por HIV , HIV-1 , Humanos , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Marrocos/epidemiologia , Estudos de Casos e Controles , Predisposição Genética para Doença , Genótipo , Ácido Fólico , Infecções por HIV/epidemiologia , Infecções por HIV/genética , Tetra-Hidrofolatos/genética
14.
Medicina (Kaunas) ; 58(11)2022 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-36363550

RESUMO

Background and ObjectivesEpilepsy is a chronic brain disease, with inherent and noninherent factors. Although over 20 anti-seizure medications (ASMs) are commercially available, nearly one-third of patients develop drug-resistant epilepsy. We evaluated the association between the clinical features and the methyl tetrahydrofolate (MTHFR) rs1801133 polymorphism and ASMs response among pediatric patients with epilepsy. Materials and Methods This was a multicenter, retrospective, case-control study of 101 children with epilepsy and 59 healthy children in Jeddah. The MTHFR rs1801133 polymorphism was genotyped using the real-time polymerase chain reaction TaqMan Genotyping Assay. Results Among the patients with epilepsy, 56 and 45 showed good and poor responses to ASMs, respectively. No significant genetic association was noted between the single-nucleotide polymorphism (SNP) rs1801133 within the MTHFR gene and the response to ASMs. However, a significant association was noted between reports of drug-induced toxicity and an increase in allele A frequencies. The MTHFR rs1801133 genotype was significantly associated with the development of electrolyte disturbance among good and poor responders to ASMs. Conclusion This is the first pharmacogenetic study of MTHFR in patients with epilepsy in Saudi Arabia that found no significant association between the MTHFR SNP rs1801133 and gene susceptibility and drug responsiveness. A larger sample size is needed for testing gene polymorphisms in the future.


Assuntos
Predisposição Genética para Doença , Metilenotetra-Hidrofolato Redutase (NADPH2) , Humanos , Criança , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Estudos de Casos e Controles , Estudos Retrospectivos , Arábia Saudita , Polimorfismo de Nucleotídeo Único/genética , Genótipo , Tetra-Hidrofolatos/genética
15.
Food Funct ; 13(21): 10923-10936, 2022 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-36205648

RESUMO

In the present study, mice with high-fat-diet-induced obesity were used in investigating the anti-obesity effects of an aqueous extract and isoquercitrin from Apocynum venetum L. The aqueous extract and the signal molecule isoquercitrin significantly reduced the body weight gain, food intake, water consumption, and fasting blood glucose, plasma triglyceride and total cholesterol levels of the obese mice. Furthermore, the mechanism of action of isoquercitrin was explored through RT-PCR analyses and uptake experiments of adenosine 5'-monophosphate-activated protein kinase (AMPK) and sterol regulatory-element binding protein (SREBP-1c) inhibitors and glucose. The indexes of SREBP-1c, fatty acid synthase (FAS), stearoyl-CoA desaturase-1 (SCD), and cluster of differentiation 36 (CD36) in obese mice significantly increased but returned to normal levels after the administration of isoquercitrin. Meanwhile, the anti-obesity effect of isoquercitrin was diminished by the inhibitors of AMPK and SREBP-1c. In addition, intestinal glucose uptake in normal mice was significantly inhibited after the oral administration of isoquercitrin. Moreover, 2D gel electrophoresis based proteome-wide cellular thermal shift assay (CETSA) showed that the potential target proteins of isoquercitrin were C-1-tetrahydrofolate synthase, carbonyl reductase, and glutathione S-transferase P. These results suggested that isoquercitrin produces an anti-obesity effect by targeting the above-mentioned proteins and regulating the AMPK/SREBP-1c signaling pathway and potentially prevents obesity and obesity-related metabolic disorders.


Assuntos
Apocynum , Proteínas de Ligação a Elemento Regulador de Esterol , Camundongos , Animais , Camundongos Obesos , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Proteínas de Ligação a Elemento Regulador de Esterol/metabolismo , Proteínas de Ligação a Elemento Regulador de Esterol/farmacologia , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Apocynum/metabolismo , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Fígado/metabolismo , Ácido Graxo Sintases/genética , Ácido Graxo Sintases/metabolismo , Obesidade/tratamento farmacológico , Obesidade/metabolismo , Transdução de Sinais , Tetra-Hidrofolatos/metabolismo , Tetra-Hidrofolatos/farmacologia , Camundongos Endogâmicos C57BL , Metabolismo dos Lipídeos
16.
Clin Chim Acta ; 537: 96-104, 2022 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-36261072

RESUMO

BACKGROUND: Little known about folates status in folate deficiency patients. This study aims to establish liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay-specific reference intervals (RIs) for serum 5-Methyltetrahydrofolate (5MeTHF), folic acid (FA), 5-Formyltetrahydrofolate (5FoTHF), and total folate (TFOL), and investigate the folates status in FA-supplemented folate deficient patients. METHODS: Sera from 120 reference subjects were selected and measured. An LC-MS/MS method for serum 5MeTHF, FA, and 5FoTHF was employed. RIs were derived based on the CLSI C28-A3. Serum folate levels of 38 FA-supplemented folate deficiency patients were analyzed. RESULTS: RIs (median) for 5MeTHF, FA, 5FoTHF, and TFOL were 3.83-62.33 nmol/L (12.27 nmol/L), 0.30-0.92 nmol/L (0.49 nmol/L), <0.73 nmol/L (0.00 nmol/L), and 4.17-63.47 nmol/L (12.66 nmol/L), respectively. Approximately 53 % (20/38), 74 % (28/38), and 63 % (24/38) of patients presented high levels of 5MeTHF, FA, and TFOL, respectively, which far exceeded the upper reference limit (URL) of the corresponding RIs. A half (18/38) of patients showed simultaneously higher 5MeTHF and FA levels which were beyond the URLs of RIs. Near one-third (11/38) of patients exhibited extremely high FA levels which exceeded the 100-fold URL of RIs. The highest levels can be 539 nmol/L for FA, 364 nmol/L for 5MeTHF, and 686 nmol/L for TFOL. CONCLUSIONS: LC-MS/MS assay-specific RIs were established for folates vitamers without age or gender partitioning. Abnormally high levels of unmetabolized FA and 5MeTHF were observed in quite a few FA-supplemented patients. Considering the adverse risks caused by folates excess, we appeal for a justifiable and individualized FA supplementation. We also recommend establishing LC-MS/MS assay-specific RIs for routine monitoring of folates status.


Assuntos
Ácido Fólico , Espectrometria de Massas em Tandem , Humanos , Cromatografia Líquida , Tetra-Hidrofolatos , Suplementos Nutricionais
17.
Int J Mol Sci ; 23(19)2022 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-36232579

RESUMO

The serine hydroxymethyltransferase (SHMT; E.C. 2.1.2.1) is involved in the interconversion of serine/glycine and tetrahydrofolate (THF)/5,10-methylene THF, playing a key role in one-carbon metabolism, the de novo purine pathway, cellular methylation reactions, redox homeostasis maintenance, and methionine and thymidylate synthesis. GmSHMT08 is the soybean gene underlying soybean cyst nematode (SCN) resistance at the Rhg4 locus. GmSHMT08 protein contains four tetrahydrofolate (THF) cofactor binding sites (L129, L135, F284, N374) and six pyridoxal phosphate (PLP) cofactor binding/catalysis sites (Y59, G106, G107, H134, S190A, H218). In the current study, proteomic analysis of a data set of protein complex immunoprecipitated using GmSHMT08 antibodies under SCN infected soybean roots reveals the presence of enriched pathways that mainly use glycine/serine as a substrate (glyoxylate cycle, redox homeostasis, glycolysis, and heme biosynthesis). Root and leaf transcriptomic analysis of differentially expressed genes under SCN infection supported the proteomic data, pointing directly to the involvement of the interconversion reaction carried out by the serine hydroxymethyltransferase enzyme. Direct site mutagenesis revealed that all mutated THF and PLP sites at the GmSHMT08 resulted in increased SCN resistance. We have shown the involvement of PLP sites in SCN resistance. Specially, the effect of the two Y59 and S190 PLP sites was more drastic than the tested THF sites. This unprecedented finding will help us to identify the biological outcomes of THF and PLP residues at the GmSHMT08 and to understand SCN resistance mechanisms.


Assuntos
Cistos , Nematoides , Animais , Carbono , Glicina/metabolismo , Glicina Hidroximetiltransferase/química , Glioxilatos , Heme , Metionina/genética , Nematoides/genética , Doenças das Plantas/genética , Proteômica , Purinas , Fosfato de Piridoxal/metabolismo , Serina/genética , Tetra-Hidrofolatos/genética , Tetra-Hidrofolatos/metabolismo , Transcriptoma
18.
Expert Opin Ther Pat ; 32(10): 1079-1095, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-36189616

RESUMO

INTRODUCTION: Dihydrofolate reductase (DHFR) plays an important role in the biosynthesis of amino acid and folic acid. It participates by reducing dihydrofolate to tetrahydrofolate, in the presence of nicotinamide dinucleotide phosphate cofactor, and has been verified by various clinical studies to use DHFR as a target for the treatment of cancer and various bacterial infections. AREA COVERED: In this review, we have disclosed patents of synthetics and natural DHFR inhibitors with diaminopyrimidine and quinazoline nucleus from 2001. Additionally, this review highlights the clinical progression of numerous DHFR inhibitors received from the last five years. EXPERT OPINION: From 2001 to 2021, numerous active chemical scaffolds have been introduced and are exposed as lead candidates that have entered clinical trials as potent DHFR inhibitors. Moreover, researchers have paid considerable attention to the development of a new class of DHFR inhibitors with higher selectivity and potency. This development includes synthesis of synthetic as well as natural compounds that are potent DHFR inhibitors. On the basis of literature review, we can anticipate that there are a huge number of novel active molecules available for the future that could possess superior abilities to target this enzyme with a profound pharmacological profile.


Assuntos
Antagonistas do Ácido Fólico , Humanos , Antagonistas do Ácido Fólico/farmacologia , Antagonistas do Ácido Fólico/química , Tetra-Hidrofolato Desidrogenase/química , Tetra-Hidrofolato Desidrogenase/metabolismo , Patentes como Assunto , Ácido Fólico , Aminoácidos , Tetra-Hidrofolatos , Quinazolinas , Niacinamida , Fosfatos
19.
Methods Mol Biol ; 2546: 311-319, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36127600

RESUMO

We describe a simple stable isotope dilution method for accurate and precise measurement of cerebrospinal fluid (CSF) 5-methyltetrahydrofolate (5-MTHF) as a clinical diagnostic test. 5-MTHF is the main biologically active form of folate and is involved in the regulation of homocysteine and numerous methylation reactions, including synthesis of neurotransmitters, lipids, DNA, and RNA. Measurement of 5-MTHF in CSF provides diagnostic information regarding disorders affecting folate metabolism within the central nervous system, in particular inborn errors of folate metabolism and cerebral folate deficiency. Determination of 5-MTHF in CSF (50 µL) was performed utilizing high-performance liquid chromatography coupled with electrospray positive ionization tandem mass spectrometry (HPLC-ESI-MS/MS). 5-MTHF in CSF is determined by a 1:2 dilution with internal standard (5-MTHF-13C5) and injected directly onto the HPLC-ESI-MS/MS system. Each assay is quantified using a five-point standard curve (25-400 nM) and has an analytical measurement range of 3-1000 nM.


Assuntos
Ácido Fólico , Espectrometria de Massas em Tandem , Cromatografia Líquida , DNA , Homocisteína , Isótopos , Lipídeos , RNA , Espectrometria de Massas em Tandem/métodos , Tetra-Hidrofolatos
20.
Microb Biotechnol ; 15(11): 2758-2772, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36070350

RESUMO

L-5-Methyltetrahydrofolate (L-5-MTHF) is the only biologically active form of folate in the human body. Production of L-5-MTHF by using microbes is an emerging consideration for green synthesis. However, microbes naturally produce only a small amount of L-5-MTHF. Here, Escherichia coli BL21(DE3) was engineered to increase the production of L-5-MTHF by overexpressing the intrinsic genes of dihydrofolate reductase and methylenetetrahydrofolate (methylene-THF) reductase, introducing the genes encoding formate-THF ligase, formyl-THF cyclohydrolase and methylene-THF dehydrogenase from the one-carbon metabolic pathway of Methylobacterium extorquens or Clostridium autoethanogenum and disrupting the gene of methionine synthase involved in the consumption and synthesis inhibition of the target product. Thus, upon its native pathway, an additional pathway for L-5-MTHF synthesis was developed in E. coli, which was further analysed and confirmed by qRT-PCR, enzyme assays and metabolite determination. After optimizing the conditions of induction time, temperature, cell density and concentration of IPTG and supplementing exogenous substances (folic acid, sodium formate and glucose) to the culture, the highest yield of 527.84 µg g-1 of dry cell weight for L-5-MTHF was obtained, which was about 11.8 folds of that of the original strain. This study paves the way for further metabolic engineering to improve the biosynthesis of L-5-MTHF in E. coli.


Assuntos
Infecções por Escherichia coli , Escherichia coli , Humanos , Escherichia coli/genética , Escherichia coli/metabolismo , Tetra-Hidrofolatos/metabolismo , Tetra-Hidrofolatos/farmacologia , Ácido Fólico/metabolismo , Ácido Fólico/farmacologia
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