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1.
Methods Enzymol ; 658: 161-190, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34517946

RESUMO

The RNA methyltransferase (MTase) complex METTL3-METTL14 transfers methyl groups from S-adenosyl-l-methionine (AdoMet) to the N6-position of adenosines within its consensus sequence, the DRACH motif (D=A, G, U; R=A, G; H=A, C, U). Interestingly, this MTase complex shows remarkable promiscuity regarding the cosubstrate. This can be exploited to install nonnatural modifications, like clickable or photocaging groups. Clickable groups are widely used for subsequent functionalization and open a broad range of possibilities for downstream applications. Here, we elaborate on click chemistry for coupling of RNA to biotin to enrich MTase targets via streptavidin-coated magnetic beads. Importantly, after clicking and coupling to beads the modification becomes sterically demanding and stalls reverse transcriptases, leading to termination adjacent to the MTase target site. Using radioactively labeled primers in the reverse transcription, the modified position can be precisely identified on a sequencing gel via phosphor imaging.


Assuntos
Metiltransferases , RNA , Adenosina , Metionina , Metiltransferases/genética , S-Adenosilmetionina
2.
Se Pu ; 39(10): 1118-1127, 2021 Oct.
Artigo em Chinês | MEDLINE | ID: mdl-34505434

RESUMO

The late endosomal/lysosomal adaptor MAPK and mTOR activator 1 (LAMTOR1) is an important regulator protein in the response to energy stress. Public gene expression data shows that the expression of LAMTOR1 is abnormally high in nonalcoholic steatohepatitis (NASH) and hepatocellular carcinoma (HCC); hence, LAMTOR1 may play an important role in the development of NASH and HCC. Therefore, exploring the LAMTOR1 regulatory mechanism in the progression of NASH and malignant transformation of liver inflammation may be crucial for translational medicine. First, a NASH mouse model was established by feeding a methionine choline-deficient (MCD) diet. Hematoxylin-eosin staining of liver tissues showed successful modeling of inflammatory injury in the mouse liver. Immunoblot analysis confirmed LAMTOR1- and LAMTOR1-mediated protein expression in LAMTOR1 specifically depleted mouse livers. Subsequently, metabolic profiling of liver tissues was performed using an ultra-performance liquid chromatography-time-of-flight mass spectrometry strategy. Based on the retention time, m/z value, and tandem mass spectra, 134 metabolites were identified. Among these, the levels of 45 metabolite were significantly influenced by hepatic LAMTOR1 depletion. According to the metabolomics results, uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) was significantly upregulated in LAMTOR1-depleted (LAMTOR1LKO) hepatocyte tissues. As the final product of the hexosamine biosynthetic pathway (HBP), alteration in UDP-GlcNAc levels may regulate LAMTOR1 and metabolic regulatory genes downstream of HBP. Moreover, there was an obvious increase in the levels of several methylation-related metabolites. Thus, upregulated S-adenosylmethionine, S-adenosylhomocysteine, and N6,N6,N6-trimethyl-L-lysine indicated that LAMTOR1 may regulate the process of DNA or protein methylation. In addition, downregulation of 9-oxo-octadecadienoate, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) was also observed in LAMTOR1LKO mice liver tissues. Alterations in polyunsaturated fatty acids, such as EPA and DHA, link LAMTOR1 to inflammatory and immune processes, which are known to play important roles in NASH pathogenesis. These metabolic disorders demonstrated that LAMTOR1 significantly contributed to the metabolic mechanism of NASH. Furthermore, gene expression correlations were analyzed to interpret the regulatory role of LAMTOR1 from the perspective of genetic networks. Owing to a paucity of liver whole-transcriptome studies in NASH, correlation analysis was performed based on HCC transcriptome data from public databases. First, a negatively regulated relationship was observed between LAMTOR1 and MAT1A (R=-0.47). MAT1A encodes methionine adenosyltransferase 1A, an essential enzyme that catalyzes the formation of S-adenosylmethionine. Based on the upregulation of UDP-GlcNAc under hepatocyte LAMTOR1 depletion, it was predicted that LAMTOR1 positively influenced MGAT1 (R=0.47), a gene encoding alpha-1,3-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase. Together with changes in succinyladenosine caused by hepatocyte LAMTOR1 deletion, predicted correlation results showed that LAMTOR1 may also participate in the pathogenesis through the positive regulatory relationship with ADSL (R=0.59). The ADSL gene provides instructions for making an enzyme called adenylosuccinate lyase, which can dephosphorylate the substrate succinyladenosine. In this study, LAMTOR1 was identified to specifically regulate multiple key metabolic pathways based on both NASH mouse models and gene expression correlations. These results illustrate the important role of LAMTOR1 in the progression of NASH and malignant transformation of liver inflammation, which provides a theoretical basis for the diagnosis and treatment of NASH or possible NASH-driven HCC.


Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Animais , Cromatografia Líquida , Modelos Animais de Doenças , Inflamação , Fígado , Espectrometria de Massas , Metionina , Camundongos
3.
Enzyme Microb Technol ; 150: 109881, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34489034

RESUMO

Natural fluorinated products are rare and attract great attention. The de novo fluorometabolites biosynthetic pathway in microbes has been studied. It is revealed that the carbon-fluorine (C-F) bond is formed by an exotic enzyme called fluorinase (FLA) when using fluorine ions and S-adenosyl-l-methionine (SAM) as substrates. However, the resource of the precursor SAM is still elusive. To solve this, a novel methionine adenosyltransferase from Streptomyces xinghaiensis (SxMAT) was identified and characterized. We proved that SAM was enzymatically synthesized by SxMAT, an enzyme that mediated the reaction between adenosine triphosphate (ATP) and l-methionine (l-Met) with 99% diastereoisomeric excess (d.e.) and 80% yield. Such high diastereoselectivity had never been reported before. SxMAT was a Co2+-dependent metalloenzyme. The results showed that the metal cobalt ion contributes to the activity and selectivity of SxMAT. Molecular docking was performed to reveal its catalytic mechanism. The optimal temperature and pH were 55 °C and 8.5, respectively. Lastly, a two-step tandem enzymatic reaction using SxMAT and FLA both from S. xinghaiensis to generate 5'-fluoro-deoxyadenosine (5'-FDA) was performed. This implied that SxMAT may be present in this fluorometabolites biosynthetic route. These results suggested that SxMAT could be a useful biocatalyst for the synthesis of optically pure (S)-S-adenosyl-l-methionine, an important nutraceutical. In addition, SxMAT will probably play an important role in the biosynthetic pathway of fluorinated natural products in bacteria.


Assuntos
Metionina Adenosiltransferase , S-Adenosilmetionina , Vias Biossintéticas , Metionina/metabolismo , Metionina Adenosiltransferase/genética , Metionina Adenosiltransferase/metabolismo , Simulação de Acoplamento Molecular , S-Adenosilmetionina/metabolismo , Streptomyces
4.
Proc Natl Acad Sci U S A ; 118(39)2021 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-34556577

RESUMO

Proteins achieve efficient energy storage and conversion through electron transfer along a series of redox cofactors. Multiheme cytochromes are notable examples. These proteins transfer electrons over distance scales of several nanometers to >10 µm and in so doing they couple cellular metabolism with extracellular redox partners including electrodes. Here, we report pump-probe spectroscopy that provides a direct measure of the intrinsic rates of heme-heme electron transfer in this fascinating class of proteins. Our study took advantage of a spectrally unique His/Met-ligated heme introduced at a defined site within the decaheme extracellular MtrC protein of Shewanella oneidensis We observed rates of heme-to-heme electron transfer on the order of 109 s-1 (3.7 to 4.3 Å edge-to-edge distance), in good agreement with predictions based on density functional and molecular dynamics calculations. These rates are among the highest reported for ground-state electron transfer in biology. Yet, some fall 2 to 3 orders of magnitude below the Moser-Dutton ruler because electron transfer at these short distances is through space and therefore associated with a higher tunneling barrier than the through-protein tunneling scenario that is usual at longer distances. Moreover, we show that the His/Met-ligated heme creates an electron sink that stabilizes the charge separated state on the 100-µs time scale. This feature could be exploited in future designs of multiheme cytochromes as components of versatile photosynthetic biohybrid assemblies.


Assuntos
Grupo dos Citocromos c/metabolismo , Citocromos/metabolismo , Elétrons , Heme/metabolismo , Histidina/metabolismo , Metionina/metabolismo , Shewanella/metabolismo , Grupo dos Citocromos c/química , Citocromos/química , Transporte de Elétrons , Heme/química , Histidina/química , Metionina/química , Simulação de Dinâmica Molecular , Nanofios , Oxirredução
5.
Nat Commun ; 12(1): 5416, 2021 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-34518544

RESUMO

Hypoxia is the most prominent feature in human solid tumors and induces activation of hypoxia-inducible factors and their downstream genes to promote cancer progression. However, whether and how hypoxia regulates overall mRNA homeostasis is unclear. Here we show that hypoxia inhibits global-mRNA decay in cancer cells. Mechanistically, hypoxia induces the interaction of AGO2 with LUBAC, the linear ubiquitin chain assembly complex, which co-localizes with miRNA-induced silencing complex and in turn catalyzes AGO2 occurring Met1-linked linear ubiquitination (M1-Ubi). A series of biochemical experiments reveal that M1-Ubi of AGO2 restrains miRNA-mediated gene silencing. Moreover, combination analyses of the AGO2-associated mRNA transcriptome by RIP-Seq and the mRNA transcriptome by RNA-Seq confirm that AGO2 M1-Ubi interferes miRNA-targeted mRNA recruiting to AGO2, and thereby facilitates accumulation of global mRNAs. By this mechanism, short-term hypoxia may protect overall mRNAs and enhances stress tolerance, whereas long-term hypoxia in tumor cells results in seriously changing the entire gene expression profile to drive cell malignant evolution.


Assuntos
Proteínas Argonauta/genética , Regulação Neoplásica da Expressão Gênica , Homeostase/genética , Metionina/genética , RNA Mensageiro/genética , Ubiquitinação , Células A549 , Proteínas Argonauta/metabolismo , Hipóxia Celular , Linhagem Celular Tumoral , Inativação Gênica , Células HEK293 , Células HeLa , Humanos , Hipóxia , Metionina/metabolismo , MicroRNAs/genética , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Células PC-3 , Estabilidade de RNA/genética , RNA Mensageiro/metabolismo
6.
BMC Cancer ; 21(1): 982, 2021 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-34470601

RESUMO

BACKGROUND: B vitamins and methionine are essential substrates in the one-carbon metabolism pathway involved in DNA synthesis and methylation. They may have essential roles in cancer development. We aimed to evaluate the associations of dietary intakes of vitamin B12, vitamin B6, folate, and methionine with the risk of esophageal cancer (EC) using data from the Japan Public Health Center-based Prospective Study. METHODS: We included 87,053 Japanese individuals who completed a food frequency questionnaire and were followed up from 1995-1998 to 2013 and 2015. Hazard ratios (HRs) and 95% confidence intervals (CIs) were calculated by Cox proportional-hazard regression across quintiles of dietary intakes of B vitamins and methionine. RESULTS: After 1,456,678 person-years of follow-up, 427 EC cases were documented. The multivariable HR (95% CI) of incident EC in the highest versus lowest quintile of dietary intake of vitamin B12 was 1.75 (1.13-2.71; p-trend=0.01). Stratification analysis based on alcohol consumption showed that higher dietary intakes of vitamin B12 and methionine were associated with an increased risk of EC among never-drinkers; HRs (95% CIs) were 2.82 (1.18-6.74; p-trend=0.009; p-interaction=0.18) and 3.45 (1.32-9.06; p-trend=0.003; p-interaction 0.02) for vitamin B12 and methionine, respectively. Meanwhile, there was no association between vitamin B12 and methionine intake with the risk of EC among drinkers. There were no associations between dietary intake of folate or vitamin B6 and the risk of EC. CONCLUSION: Dietary intake of vitamin B12 was positively associated with the risk of EC in the Japanese population.


Assuntos
Consumo de Bebidas Alcoólicas/fisiopatologia , Neoplasias Esofágicas/epidemiologia , Ácido Fólico/administração & dosagem , Metionina/administração & dosagem , Vitamina B 12/administração & dosagem , Vitamina B 6/administração & dosagem , Vitaminas/administração & dosagem , Idoso , Ingestão de Alimentos , Neoplasias Esofágicas/metabolismo , Feminino , Seguimentos , Humanos , Japão/epidemiologia , Masculino , Pessoa de Meia-Idade , Prognóstico , Estudos Prospectivos , Saúde Pública , Fatores de Risco
7.
Molecules ; 26(17)2021 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-34500777

RESUMO

Human neutrophil elastase (HNE) is a uniquely destructive serine protease with the ability to unleash a wave of proteolytic activity by destroying the inhibitors of other proteases. Although this phenomenon forms an important part of the innate immune response to invading pathogens, it is responsible for the collateral host tissue damage observed in chronic conditions such as chronic obstructive pulmonary disease (COPD), and in more acute disorders such as the lung injuries associated with COVID-19 infection. Previously, a combinatorially selected activity-based probe revealed an unexpected substrate preference for oxidised methionine, which suggests a link to oxidative pathogen clearance by neutrophils. Here we use oxidised model substrates and inhibitors to confirm this observation and to show that neutrophil elastase is specifically selective for the di-oxygenated methionine sulfone rather than the mono-oxygenated methionine sulfoxide. We also posit a critical role for ordered solvent in the mechanism of HNE discrimination between the two oxidised forms methionine residue. Preference for the sulfone form of oxidised methionine is especially significant. While both host and pathogens have the ability to reduce methionine sulfoxide back to methionine, a biological pathway to reduce methionine sulfone is not known. Taken together, these data suggest that the oxidative activity of neutrophils may create rapidly cleaved elastase "super substrates" that directly damage tissue, while initiating a cycle of neutrophil oxidation that increases elastase tissue damage and further neutrophil recruitment.


Assuntos
Imunidade Inata , Elastase de Leucócito/metabolismo , Metionina/análogos & derivados , Neutrófilos/imunologia , Biocatálise , COVID-19/imunologia , COVID-19/patologia , COVID-19/virologia , Domínio Catalítico/genética , Ensaios Enzimáticos , Interações Hospedeiro-Patógeno/imunologia , Humanos , Elastase de Leucócito/antagonistas & inibidores , Elastase de Leucócito/genética , Pulmão/imunologia , Pulmão/patologia , Pulmão/virologia , Metionina/metabolismo , Simulação de Dinâmica Molecular , Infiltração de Neutrófilos , Neutrófilos/enzimologia , Oxirredução/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Doença Pulmonar Obstrutiva Crônica/imunologia , Doença Pulmonar Obstrutiva Crônica/patologia , SARS-CoV-2/imunologia , Especificidade por Substrato/imunologia
8.
J Anim Sci ; 99(9)2021 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-34333630

RESUMO

Grain-based ingredients are replaced in part by pulse ingredients in grain-free pet foods. Pulse ingredients are lower in methionine and cysteine, amino acid (AA) precursors to taurine synthesis in dogs. Although recent work has investigated plasma and whole blood taurine concentrations when feeding grain-free diets, supplementation of a grain-free diet with various nutrients involved in the biosynthesis of taurine has not been evaluated. This study aimed to investigate the effects of supplementing a complete grain-free dry dog food with either methionine (MET), taurine (TAU), or methyl donors (choline) and methyl receivers (creatine and carnitine; CCC) on postprandial AA concentrations. Eight healthy Beagle dogs were fed one of the three treatments or the control grain-free diet (CON) for 7 d in a 4 × 4 Latin square design. On day 7, cephalic catheters were placed and one fasted sample (0 min) and a series of nine post-meal blood samples were collected at 15, 30, 60, 90, 120, 180, 240, 300, and 360 min. Data were analyzed as repeated measures using the PROC GLIMMIX function in SAS (Version 9.4). Dogs fed MET had greater plasma and whole blood methionine concentrations from 30 to 360 min after a meal (P < 0.0001) and greater plasma homocysteine concentrations from 60 to 360 min after a meal (P < 0.0001) compared with dogs fed CON, TAU, and CCC. Dogs fed TAU had greater plasma taurine concentrations over time compared with dogs fed CON (P = 0.02) but were not different than dogs fed MET and CCC (P > 0.05). In addition, most AAs remained significantly elevated at 6 h post-meal compared with fasted samples across all treatments. Supplementation of creatine, carnitine, and choline in grain-free diets may play a role in sparing the methionine requirement without increasing homocysteine concentrations. Supplementing these nutrients could also aid in the treatment of disease that causes metabolic or oxidative stress, including cardiac disease in dogs, but future research is required.


Assuntos
Metionina , Taurina , Animais , Dieta/veterinária , Suplementos Nutricionais , Cães , Grão Comestível , Homocisteína
9.
Phytomedicine ; 91: 153666, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34339944

RESUMO

BACKGROUND: Metabolic-associated fatty liver disease (MAFLD) is a spectrum of liver disorders. Nonalcoholic steatohepatitis (NASH) is defined as a more serious process of MAFLD with liver inflammation. PURPOSE: This study aims to observe the alleviation of Yinhuang granule (YHG), a Chinese patent medicine, on methionine and choline-deficient diet (MCD)-induced MAFLD in mice. METHODS: Network pharmacology was used to analyze the improving effect of YHG on MAFLD and possible targets. MAFLD was induced in mice by MCD diet feeding for 6 weeks. In the last 2 weeks, the mice were orally given with YHG (400, 800 mg/kg) every day. Biochemical parameters of serum and liver, as well as hepatic gene expression were detected. RESULTS: Network pharmacology showed that YHG could improve MAFLD, inflammation, liver fibrosis, and oxidative stress. In animal experiments, YHG reduced hepatocellular damage and hepatic lipids accumulation which induced by MCD. In terms of liver inflammation, YHG attenuated MCD-induced liver inflammation in mice. YHG also inhibited the activation of hepatic stellate cells (HSCs) and alleviated liver fibrosis in MCD-fed mice. Through nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway, YHG alleviated liver oxidative stress injury in mice which induced by MCD. CONCLUSION: YHG ameliorated MCD-induced MAFLD in mice by reducing hepatic lipids accumulation, alleviating liver oxidative, inflammatory injury and attenuating hepatic fibrosis.


Assuntos
Medicamentos de Ervas Chinesas/farmacologia , Hepatopatia Gordurosa não Alcoólica , Animais , Dieta , Modelos Animais de Doenças , Fígado/metabolismo , Cirrose Hepática/tratamento farmacológico , Cirrose Hepática/metabolismo , Metionina , Camundongos , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Hepatopatia Gordurosa não Alcoólica/metabolismo , Estresse Oxidativo
10.
Nutrients ; 13(8)2021 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-34444768

RESUMO

Dietary protein restriction and dietary methionine restriction (MR) produce a comparable series of behavioral, physiological, biochemical, and transcriptional responses. Both dietary regimens produce a similar reduction in intake of sulfur amino acids (e.g., methionine and cystine), and both diets increase expression and release of hepatic FGF21. Given that FGF21 is an essential mediator of the metabolic phenotype produced by both diets, an important unresolved question is whether dietary protein restriction represents de facto methionine restriction. Using diets formulated from either casein or soy protein with matched reductions in sulfur amino acids, we compared the ability of the respective diets to recapitulate the metabolic phenotype produced by methionine restriction using elemental diets. Although the soy-based control diets supported faster growth compared to casein-based control diets, casein-based protein restriction and soy-based protein restriction produced comparable reductions in body weight and fat deposition, and similar increases in energy intake, energy expenditure, and water intake. In addition, the prototypical effects of dietary MR on hepatic and adipose tissue target genes were similarly regulated by casein- and soy-based protein restriction. The present findings support the feasibility of using restricted intake of diets from various protein sources to produce therapeutically effective implementation of dietary methionine restriction.


Assuntos
Dieta com Restrição de Proteínas , Metionina/metabolismo , Tecido Adiposo/metabolismo , Aminoácidos Essenciais , Aminoácidos Sulfúricos , Animais , Peso Corporal , Caseínas , Ingestão de Alimentos , Ingestão de Energia , Metabolismo Energético/fisiologia , Fatores de Crescimento de Fibroblastos/metabolismo , Expressão Gênica , Humanos , Fígado/metabolismo , Masculino , Metionina/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas de Soja , Transcriptoma
11.
Animal ; 15(9): 100326, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34371467

RESUMO

Chromium may regulate dairy cow metabolism; a chelated formation of chromium methionine (Cr-Met) is available to the feed industry. The objective of this study was to investigate the effect of Cr-Met supplementation on lactation performance, hepatic respiratory rate and anti-oxidative capacity in early-lactating Holstein dairy cows. 64 multiparous cows were assigned to 16 blocks based on parity and milk yield and then the four cows in a block were randomly allocated to four treatment groups with 0, 4, 8 or 16 g/d of Cr-Met per cow supplemented to a basal diet. Cows were moved from an open dry lot to a naturally ventilated tie stall barn 2 weeks before treatment to adapt to this facility, fed and milked at 0630, 1400, and 1930 h every day. The experiment lasted for 12 weeks. Milk yield and composition were recorded weekly. Dry matter intake was measured every 2 weeks for a total of six times throughout the trial. The plasma variables were measured in weeks 4, 8 and 12 of the experiment. Supplementation of Cr-Met did not affect DM intake of cows. As the supplementation of Cr-Met increased, yields of milk, fat, energy corrected milk (P < 0.01) and lactose (P = 0.01) increased in a linear manner. In terms of plasma variables, insulin concentration decreased in a linear manner with Cr-Met supplementation. As for variables relating to hepatic respiration rate, concentrations of pyruvate and NAD in the plasma were increased in quadratic manners, and lactic dehydrogenase activity was linearly increased as Cr-Met feeding levels increased. Moreover, plasma glutathione peroxidase and superoxide dismutase activity were increased in a linear manner. In conclusion, our study suggested that Cr-Met supplementation improved lactation performance of early-lactating dairy cows through enhancing antioxidant capacity and hepatic cellular respiration.


Assuntos
Lactação , Metionina , Animais , Bovinos , Cromo , Dieta/veterinária , Suplementos Nutricionais , Feminino , Leite , Gravidez , Taxa Respiratória
12.
J Chromatogr A ; 1654: 462449, 2021 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-34399143

RESUMO

In both biologics quality control experiments and protein post-translational modification studies, the analytical system used is not supposed to bring any artefactual modifications which could impair the results. In this work, we investigated oxidation of methionine-containing peptides during reversed-phase (RP) chromatographic separation. We first used a synthetic methionine-containing peptide to evaluate this artefactual phenomenon and then considered more complex samples (i.e., plasma and HeLa protein digests). The methionine oxidation levels of the peptides were systematically assessed and compared for the long-term use of the analytical column, the sample trapping time, the gradient length, the sample load and the nature of the stationary phase (HSS T3 from Waters, YMC Triart C18 from YMC Europe GmbH and BEH130 C18 from Waters). In addition to the oxidation of methionine in solution, we observed on the HSS T3 and the BEH130 stationary phases an additional broad peak corresponding to an on-column oxidized species. Considering the HSS T3 phase, our results highlight that the on-column oxidation level significantly increases with the age of the analytical column and the gradient length and reaches 56 % when a 1-year-old column set is used with a 180 min-long LC method. These levels go to 0 % and 18 % for the YMC Triart C18 and the BEH130 C18 phases respectively. Interestingly, the on-column oxidation proportion decreases as the injected sample load increases suggesting the presence of a discrete number of oxidation sites within the stationary phase of the analytical column. Those findings observed in different laboratories using distinct set of columns, albeit to varying degrees, strengthen the need for a standard of methionine-containing peptide that could be used as a quality control to appraise the status of the liquid chromatographic columns.


Assuntos
Cromatografia de Fase Reversa , Metionina , Peptídeos , Cromatografia de Fase Reversa/instrumentação , Cromatografia de Fase Reversa/normas , Metionina/metabolismo , Oxirredução , Peptídeos/metabolismo , Controle de Qualidade
13.
Elife ; 102021 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-34409939

RESUMO

NmMetQ is a substrate-binding protein (SBP) from Neisseria meningitidis that has been identified as a surface-exposed candidate antigen for meningococcal vaccines. However, this location for NmMetQ challenges the prevailing view that SBPs in Gram-negative bacteria are localized to the periplasmic space to promote interaction with their cognate ABC transporter embedded in the bacterial inner membrane. To elucidate the roles of NmMetQ, we characterized NmMetQ with and without its cognate ABC transporter (NmMetNI). Here, we show that NmMetQ is a lipoprotein (lipo-NmMetQ) that binds multiple methionine analogs and stimulates the ATPase activity of NmMetNI. Using single-particle electron cryo-microscopy, we determined the structures of NmMetNI in the presence and absence of lipo-NmMetQ. Based on our data, we propose that NmMetQ tethers to membranes via a lipid anchor and has dual function and localization, playing a role in NmMetNI-mediated transport at the inner membrane and moonlighting on the bacterial surface.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Bactérias/metabolismo , Lipoproteínas/metabolismo , Metionina/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Microscopia Crioeletrônica , Lipoproteínas/química , Lipoproteínas/genética , Neisseria meningitidis/metabolismo , Periplasma , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína
14.
J Anim Sci ; 99(10)2021 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-34448863

RESUMO

While the raw pet food market continues to grow, the risk of bacterial contamination in these types of diets is a major concern, with Salmonella enterica and Listeria monocytogenes being the most frequently associated pathogens in raw pet food product recalls. dl-Methionine is included in some commercial feline kibble and canned diets to improve protein quality; however, an alternative to this is a liquid methionine supplement, 2-hydroxy-4-(methylthio)-butanoic acid (HMTBa), which is also an organic acid. 2-Hydroxy-4-(methylthio)-butanoic acid has previously demonstrated similar efficacy to formic acid against pathogens in a liquid environment and may be a good candidate to inhibit S. enterica and L. monocytogenes in raw ground meat. First, the minimum inhibitory concentration and minimum bactericidal concentration of HMTBa against these pathogens under laboratory growth conditions were determined by measuring growth of pathogens over 36 h when exposed to 10 concentrations of HMTBa (0.10% to 1.00%) mixed with tryptic soy broth. 2-Hydroxy-4-(methylthio)-butanoic acid included at ≥0.50% was bactericidal to S. enterica and L. monocytogenes (P < 0.05). Next, five levels of HMTBa (0.50% to 1.25%) were included in raw ground meat mixtures inoculated with cocktails of S. enterica or L. monocytogenes, and contamination levels were determined at four timepoints: immediately, and after refrigerated storage (4 °C) at 24, 48, and 72 h after removal from freezer (24 h at -20 °C). 2-Hydroxy-4-(methylthio)-butanoic acid included as 1.25% of the meat mixture reduced S. enterica and L. monocytogenes compared with the control (P < 0.05); however, it did not result in total kill of either of these pathogens. Following this, feeding behaviors of seven domestic cats were assessed when offered a raw chicken diet treated with or without 1.25% HMTBa for 5 d each, after which a 2-d 2-choice preference test was conducted. Cats demonstrated a preference for raw diets without HMTBa, but still readily consumed diets with 1.25% HMTBa, suggesting that such a diet was still palatable to them.


Assuntos
Ração Animal , Listeria , Ração Animal/análise , Animais , Ácido Butírico , Gatos , Dieta/veterinária , Carne , Metionina , Salmonella
15.
Curr Protoc ; 1(8): e213, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34370893

RESUMO

Protein methyltransferases (PMTs) regulate many aspects of normal and disease processes through substrate methylation, with S-adenosyl-L-methionine (SAM) as a cofactor. It has been challenging to elucidate cellular protein lysine and arginine methylation because these modifications barely alter physical properties of target proteins and often are context dependent, transient, and substoichiometric. To reveal bona fide methylation events associated with specific PMT activities in native contexts, we developed the live-cell Bioorthogonal Profiling of Protein Methylation (lcBPPM) technology, in which the substrates of specific PMTs are labeled by engineered PMTs inside living cells, with in situ-synthesized SAM analogues as cofactors. The biorthogonality of this technology is achieved because these SAM analogue cofactors can only be processed by the engineered PMTs-and not native PMTs-to modify the substrates with distinct chemical groups. Here, we describe the latest lcBPPM protocol and its application to reveal proteome-wide methylation and validate specific methylation events. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Live-cell labeling of substrates of protein methyltransferases GLP1 and PRMT1 with lcBPPM-feasible enzymes and SAM analogue precursors Support Protocol: Gram-scale synthesis of Hey-Met Basic Protocol 2: Click labeling of lcBPPM cell lysates with a biotin-azide probe Alternate Protocol: Click labeling of small-scale lcBPPM cell lysates with a TAMRA-azide dye for in-gel fluorescence visualization Basic Protocol 3: Enrichment of biotinylated lcBPPM proteome with streptavidin beads Basic Protocol 4: Proteome-wide identification of lcBPPM targets with mass spectrometry Basic Protocol 5: Validation of individual lcBPPM targets by western blot.


Assuntos
Metionina , S-Adenosilmetionina , Humanos , Metilação , Proteínas Metiltransferases/metabolismo , Proteína-Arginina N-Metiltransferases/genética , Proteoma/metabolismo , Proteínas Repressoras , S-Adenosilmetionina/metabolismo
16.
Theriogenology ; 173: 102-111, 2021 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-34365138

RESUMO

The objective of the present study was to evaluate the effect of feeding rumen-protected methionine (RPM) during the peripartal period and early lactation on mRNA gene expression profiles of uterine cytological smear and endometrial samples of Holstein cows (n = 20). Treatments consisted of a supplementation with RPM [MET; n = 11; RPM at a rate of 0.08 % of DM: Lys:Met = 2.8:1, (Smartamine® M Adisseo, Alpharetta, GA, USA)] and no supplementation (CON; n = 9; Lys:Met = 3.5:1). Uterine cytology smears and endometrial samples were collected at 15, 30, and 73 days in milk (DIM) and analyzed for expression of genes related with metabolism, inflammation, and methionine metabolism. Regarding the cytological smear samples, RPM supplementation tended to increase mRNA expression of methionine adenosyltransferase 1 alpha (MAT1A) and increased the mRNA expression of fibroblast growth factor 7 (FGF7), with an effect of time for the latter. On the other hand, RPM decreased mRNA expression for glucose transporter 4 (GLUT4), interleukin 1 beta (IL-1ß), interleukin 6 (IL-6), interleukin 8 (IL-8), prostaglandin E synthase 3 (PTGES3), translocator protein 18 kDa (TSPO), mucin 1 (MUC1) and superoxide dismutase (SOD1) in cytological smear samples. There was an effect of time for all variables except MAT1A, with decreasing expression over time. There was a TRT × TIME interaction for GLUT4 mRNA expression, with higher GLUT4 mRNA expression for cows fed CON than for cows fed RPM at time 15 and a tendency to higher expression for cows fed CON on time 30 when compared with cows fed RPM. For uterine tissue samples, feeding RPM increased the mRNA expression of lecithin-cholesterol acyltransferase (LCAT), S-adenosyl-l-homocysteine hydrolase (SAAH), FGF7, GLUT4, and apolipoproteins 3 (APOL3), with an effect of time for APOL3 where its expression increased over time. There was a tendency for cows fed RPM to have decreased IL1ß mRNA expression. In conclusion, feeding RPM during transition period and early lactation is beneficial for uterine immune response and metabolism in early lactation as indicated by the favorable expressions of genes affecting the uterine immunometabolism during such a challenging period.


Assuntos
Metionina , Período Periparto , Animais , Bovinos , Dieta/veterinária , Suplementos Nutricionais , Feminino , Expressão Gênica , Lactação , Leite
17.
J Anim Sci ; 99(9)2021 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-34432053

RESUMO

An N-balance experiment was conducted to test the hypothesis that d-Methionine (d-Met) has the same bioavailability and efficacy as l-Methionine (l-Met) when fed to weanling pigs. A Met-deficient basal diet containing 0.24% standardized ileal digestible (SID) Met was formulated. Six additional diets were formulated by adding 0.036%, 0.072%, or 0.108% d-Met or l-Met to the basal diet, and these diets, therefore, contained 77%, 87%, or 97% of the requirement for SID Met. Fifty-six barrows (10.53 ± 1.17 kg) were housed in metabolism crates and allotted to the seven diets with eight replicate pigs per diet. Feces and urine were collected quantitatively with 7-d adaptation and 5-d collection periods. Blood and tissue samples from pigs fed the basal diet and pigs fed diets containing 0.108% supplemental Met were collected on the last day. Results indicated that N retention (%) linearly increased (P < 0.01) as supplemental d-Met or l-Met increased in diets. Based on N retention (%) as a response, the linear slope-ratio regression estimated the bioavailability of d-Met relative to l-Met to be 101% (95% confidence interval: 57%-146%). The villus height and crypt depth in the jejunum were not affected by the Met level or Met source. Total antioxidant capacity or thiobarbituric acid reactive substance concentrations in plasma or tissue samples from pigs fed the control diet or diets containing 0.108% supplemental d-Met or l-Met were not different. Abundance of mRNA for some AA transporters analyzed in intestinal mucosa of pigs also did not differ. Therefore, it is concluded that d-Met and l-Met are equally bioavailable for weanling pigs.


Assuntos
Ração Animal , Metionina , Sistemas de Transporte de Aminoácidos , Ração Animal/análise , Fenômenos Fisiológicos da Nutrição Animal , Animais , Antioxidantes , Dieta/veterinária , Digestão , Íleo , Nitrogênio , RNA Mensageiro , Suínos
18.
J Dairy Sci ; 104(10): 11210-11225, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34304872

RESUMO

Our primary objective was to evaluate the effect of feeding rumen-protected Met (RPM) in the pre- and postpartum total mixed ration (TMR) on pregnancy per artificial insemination (AI) and pregnancy loss in multiparous Holstein cows. We also evaluated multiple secondary reproductive physiological outcomes before and after AI, including uterine health, ovarian cyclicity, response to synchronization of ovulation, and markers of embryo development and size. A total of 470 multiparous Holstein cows [235 at the University of Wisconsin (UW) and 235 at Cornell University (CU)] were used for this experiment. Experimental treatment diets were applied at the pen level (2 and 4 close-up pens at CU and UW, respectively, and 12 and 6 postfresh pens at CU and UW, respectively); thus, pen was the experimental unit, and cow was the observational unit. Cows were enrolled and randomly assigned to be fed the experimental treatment diets at approximately 4 wk before parturition until 67 d of gestation [147 d in milk (DIM)] after their first service. Close-up dry cow and replicated lactation pens were randomly assigned to treatment diets: RPM, prepartum = 2.83% (UW) and 2.85% (CU), postpartum = 2.58% (UW) and 2.65% (CU); and control (CON), prepartum = 2.30% (UW) and 2.22% (CU), postpartum = 2.09% (UW) and 2.19% (CU; Met as percentage of metabolizable protein). Vaginal discharge and uterine cytology (percentage of polymorphonuclear leucocytes) were evaluated at 35 ± 3 DIM. Cows received timed AI (TAI) at 80 ± 3 DIM after synchronization of ovulation with the Double-Ovsynch protocol. Ovarian cyclicity status, response to synchronization of ovulation, and luteal function were determined by measuring circulating concentrations of progesterone at 35 and 49 ± 3 DIM, 48 and 24 h before TAI, and 8, 18, 22, 25, and 29 d after TAI. Interferon-stimulated gene expression in white blood cells were compared on 18 d after TAI (CU only) and pregnancy-specific protein B concentrations at 22, 25, 29, 32, and 67 d after TAI. Pregnancy status was determined using pregnancy-specific protein B at 25 and 29 d after TAI, and by transrectal ultrasonography at 32, 39, and 67 d after TAI. Embryo and amniotic vesicle size were determined at 32 and 39 d after TAI. Pregnancy per AI (25 d: 64.7 vs. 64.0%, 32 d: 54.3 vs. 55.1% for CON and RPM, respectively) and pregnancy loss (25 to 67 d: 22.6 vs. 19.2% for CON and RPM, respectively) for synchronized cows did not differ. The proportion of cows with purulent vaginal discharge (CON = 7.7 vs. RPM = 4.6%) and cytological endometritis (CON = 20.8 vs. RPM = 23.6%) did not differ. Cyclicity status, ovarian responses to the synchronization protocol, and synchronization rate also did not differ. In addition, fold change for interferon-stimulated genes, concentrations of pregnancy-specific protein B, and embryo size were not affected by treatments. In conclusion, feeding RPM in the pre- and postpartum TMR at the amounts used in this experiment did not affect uterine health, cyclicity, embryo development, or reproductive efficiency in dairy cows.


Assuntos
Sincronização do Estro , Rúmen , Animais , Bovinos , Dinoprosta , Feminino , Hormônio Liberador de Gonadotropina , Inseminação Artificial/veterinária , Lactação , Metionina , Período Pós-Parto , Gravidez , Progesterona
19.
Nan Fang Yi Ke Da Xue Xue Bao ; 41(7): 1037-1043, 2021 Jul 20.
Artigo em Chinês | MEDLINE | ID: mdl-34308853

RESUMO

OBJECTIVE: To study the effect of parathyroid hormone-related protein (PTHrP) on nonalcoholic fatty liver disease (NAFLD) induced by methionine choline-deficient diet (MCD) in mice. METHODS: Twelve male C57BL/6J mice were randomized into blank control group, vehicle group and PTHrP group (n=4). The mice in vehicle group and PTHrP group received injections of a control adeno-associated virus (AAV) vector and an AVV vector carrying PTHrP (AAV-PTHrP) gene, respectively, followed one week later by MCD feeding for 3 weeks; the mice in the blank control were fed a normal diet for 4 weeks. Body weight changes of the mice were monitored during the experiment. At the end of the experiment, liver tissues were harvested from the mice for histological analysis using HE staining, oil red O staining, and Sirius red staining. The levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), triglyceride, and free fatty acids (FFAs) in the liver and serum were detected to assess hepatic impairment and lipid metabolism of the mice. Cell models of NAFLD were established in mouse and human normal liver cells by treatment with 250 µmol/L FFAs for 24 h, and the effect of AAV-PTHrP on lipid deposition and viability of the cells were tested using Oil Red O and Nile red staining and CCK8 assay. RESULTS: Treatment with AAV-PTHrP, as compared with the control AVV vector, caused more rapid reduction of body weight in mice with MCD feeding and significantly increased the levels of AST (P < 0.05), ALT (P < 0.05), triglyceride (P < 0.01) and FFA (P < 0.05) in the liver and the scores of NAS (P < 0.01) and SAF (P < 0.05). HE and Oil red O staining of the liver tissue revealed obvious lipid deposition after MCD feeding, which was more serious in PTHrP group. In the cell experiment, FFAs induced steatosis in both mouse and human hepatocytes, and treatment with PTHrP increased the accumulation of lipid droplets and lowered the viability of the cell model of NAFLD (P < 0.01 or 0.05). CONCLUSION: PTHrP may aggravate MCD-induced NAFLD in mice by promoting the deposition of lipid droplets in the hepatocytes.


Assuntos
Hepatopatia Gordurosa não Alcoólica , Animais , Colina , Dieta , Modelos Animais de Doenças , Fígado , Masculino , Metionina , Camundongos , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/etiologia , Proteína Relacionada ao Hormônio Paratireóideo
20.
J Agric Food Chem ; 69(28): 7932-7937, 2021 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-34232654

RESUMO

l-Methionine is an essential bioactive amino acid with high commercial value for diverse applications. Sustained attentions have been paid to efficient and economical preparation of l-methionine. In this work, a novel method for l-methionine production was established using O-acetyl-homoserine (OAH) and 3-methylthiopropionaldehyde (MMP) as substrates by catalysis of the yeast OAH sulfhydrylase MET17. The OAH sulfhydrylase gene Met17 was cloned from Saccharomyces cerevisiae S288c and overexpressed in Escherichia coli BL21. A 49 kDa MET17 was detected in the supernatant of the recombinant E. coli strain BL21-Met17 lysate with IPTG induction, which exhibited the biological activity of l-methionine biosynthesis from OAH and MMP. The recombinant MET17 was then purified from E. coli BL21-Met17 and used for in vitro biosynthesis of l-methionine. The maximal conversion rate (86%) of OAH to l-methionine catalyzed by purified MET17 was achieved by optimization of the molar ratio of OAH to MMP. The method proposed in this study provides a possible novel route for the industrial production of l-methionine.


Assuntos
Metionina , Saccharomyces cerevisiae , Aldeídos , Carbono-Oxigênio Liases , Catálise , Escherichia coli/genética , Homosserina , Cinética , Propionatos , Saccharomyces cerevisiae/genética
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