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1.
Science ; 382(6670): eabp9201, 2023 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-37917677

RESUMO

One-carbon metabolism is an essential branch of cellular metabolism that intersects with epigenetic regulation. In this work, we show how formaldehyde (FA), a one-carbon unit derived from both endogenous sources and environmental exposure, regulates one-carbon metabolism by inhibiting the biosynthesis of S-adenosylmethionine (SAM), the major methyl donor in cells. FA reacts with privileged, hyperreactive cysteine sites in the proteome, including Cys120 in S-adenosylmethionine synthase isoform type-1 (MAT1A). FA exposure inhibited MAT1A activity and decreased SAM production with MAT-isoform specificity. A genetic mouse model of chronic FA overload showed a decrease n SAM and in methylation on selected histones and genes. Epigenetic and transcriptional regulation of Mat1a and related genes function as compensatory mechanisms for FA-dependent SAM depletion, revealing a biochemical feedback cycle between FA and SAM one-carbon units.


Assuntos
Carbono , Cisteína , Epigênese Genética , Formaldeído , Metionina Adenosiltransferase , S-Adenosilmetionina , Animais , Camundongos , Carbono/metabolismo , Epigênese Genética/efeitos dos fármacos , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/metabolismo , S-Adenosilmetionina/antagonistas & inibidores , S-Adenosilmetionina/metabolismo , Formaldeído/metabolismo , Formaldeído/toxicidade , Exposição Ambiental , Metionina Adenosiltransferase/antagonistas & inibidores , Metionina Adenosiltransferase/genética , Metionina Adenosiltransferase/metabolismo , Cisteína/metabolismo , Humanos , Células Hep G2
2.
J Nat Prod ; 83(9): 2809-2813, 2020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32830503

RESUMO

Arsinothricin [AST (1)], a new broad-spectrum organoarsenical antibiotic, is a nonproteinogenic analogue of glutamate that effectively inhibits glutamine synthetase. We report the chemical synthesis of an intermediate in the pathway to 1, hydroxyarsinothricin [AST-OH (2)], which can be converted to 1 by enzymatic methylation catalyzed by the ArsM As(III) S-adenosylmethionine methyltransferase. This is the first report of semisynthesis of 1, providing a source of this novel antibiotic that will be required for future clinical trials.


Assuntos
Antibacterianos/síntese química , Arsenicais/síntese química , Antibacterianos/farmacologia , Arsenicais/farmacologia , Cromatografia Líquida de Alta Pressão , Inibidores Enzimáticos/farmacologia , Glutamato-Amônia Ligase/antagonistas & inibidores , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , S-Adenosilmetionina/antagonistas & inibidores
3.
Nat Commun ; 9(1): 2261, 2018 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-29891918

RESUMO

The folate and methionine cycles are crucial for biosynthesis of lipids, nucleotides and proteins, and production of the methyl donor S-adenosylmethionine (SAM). 5,10-methylenetetrahydrofolate reductase (MTHFR) represents a key regulatory connection between these cycles, generating 5-methyltetrahydrofolate for initiation of the methionine cycle, and undergoing allosteric inhibition by its end product SAM. Our 2.5 Å resolution crystal structure of human MTHFR reveals a unique architecture, appending the well-conserved catalytic TIM-barrel to a eukaryote-only SAM-binding domain. The latter domain of novel fold provides the predominant interface for MTHFR homo-dimerization, positioning the N-terminal serine-rich phosphorylation region near the C-terminal SAM-binding domain. This explains how MTHFR phosphorylation, identified on 11 N-terminal residues (16 in total), increases sensitivity to SAM binding and inhibition. Finally, we demonstrate that the 25-amino-acid inter-domain linker enables conformational plasticity and propose it to be a key mediator of SAM regulation. Together, these results provide insight into the molecular regulation of MTHFR.


Assuntos
Metilenotetra-Hidrofolato Redutase (NADPH2)/química , Metilenotetra-Hidrofolato Redutase (NADPH2)/metabolismo , Sequência de Aminoácidos , Domínio Catalítico , Cristalografia por Raios X , Evolução Molecular , Humanos , Cinética , Espectrometria de Massas , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Modelos Moleculares , Simulação de Dinâmica Molecular , NADP/metabolismo , Fosforilação , Domínios Proteicos , Dobramento de Proteína , Estrutura Quaternária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , S-Adenosilmetionina/antagonistas & inibidores , S-Adenosilmetionina/metabolismo , Espalhamento a Baixo Ângulo , Difração de Raios X
4.
Mol Nutr Food Res ; 62(7): e1701070, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29453804

RESUMO

SCOPE: Probiotics may influence one-carbon (C1) metabolism, neurotransmitters, liver function markers, or behavior. METHODS AND RESULTS: Male adult Flinders Sensitive Line rats (model of depression, FSL; n = 22) received Lactobacillus helveticus R0052 and Bifidobacterium longum R0175 (109 or 1010 colony-forming units per day) or vehicle for 10 weeks. The controls, Flinders Resistant Line rats (FRL, n = 8), only received vehicle. C1-related metabolites were measured in plasma, urine, and different tissues. Monoamine concentrations were measured in plasma, hippocampus, and prefrontal cortex. Vehicle-treated FSL rats had higher plasma concentrations of betaine, choline, and dimethylglycine, but lower plasma homocysteine and liver S-adenosylmethionine (SAM) than FRLs. FSL rats receiving high-dose probiotics had lower plasma betaine and higher liver SAM compared to vehicle-treated FSL rats. FSLs had higher concentrations of norepinephrine, dopamine, and serotonin than FRLs across various brain regions. Probiotics decreased plasma dopamine in FSLs in a dose-dependent manner. There were no detectable changes in liver function markers or behavior. CONCLUSIONS: Probiotics reduced the flow of methyl groups via betaine, increased liver SAM, and decreased plasma dopamine and norepinephrine. Since these changes in methylation and catecholamine pathways are known to be involved in several diseases, future investigation of the effect of probiotics is warranted.


Assuntos
Antidepressivos/uso terapêutico , Bifidobacterium longum/crescimento & desenvolvimento , Depressão/terapia , Hipocampo/metabolismo , Lactobacillus helveticus/crescimento & desenvolvimento , Córtex Pré-Frontal/metabolismo , Probióticos/uso terapêutico , Animais , Antidepressivos/administração & dosagem , Antidepressivos/efeitos adversos , Comportamento Animal , Biomarcadores/sangue , Biomarcadores/metabolismo , Biomarcadores/urina , Depressão/sangue , Depressão/metabolismo , Depressão/urina , Dopamina/sangue , Dopamina/metabolismo , Antagonistas de Dopamina/administração & dosagem , Antagonistas de Dopamina/efeitos adversos , Antagonistas de Dopamina/uso terapêutico , Liofilização , Homocisteína/antagonistas & inibidores , Homocisteína/sangue , Fígado/metabolismo , Masculino , Metilação , Neurônios/metabolismo , Norepinefrina/antagonistas & inibidores , Norepinefrina/sangue , Norepinefrina/metabolismo , Probióticos/administração & dosagem , Probióticos/efeitos adversos , Distribuição Aleatória , Ratos Mutantes , S-Adenosilmetionina/antagonistas & inibidores , S-Adenosilmetionina/metabolismo
5.
Acta Crystallogr D Biol Crystallogr ; 68(Pt 3): 253-60, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22349227

RESUMO

The biological activity of catechol neurotransmitters such as dopamine in the synapse is modulated by transporters and enzymes. Catechol-O-methyltransferase (COMT; EC 2.1.1.6) inactivates neurotransmitters by catalyzing the transfer of a methyl group from S-adenosylmethionine to catechols in the presence of Mg²âº. This pathway also inactivates L-DOPA, the standard therapeutic for Parkinson's disease. Depletion of catechol neurotransmitters in the prefrontal cortex has been linked to schizophrenia. The inhibition of COMT therefore promises improvements in the treatment of these diseases. The concept of bisubstrate inhibitors for COMT has been described previously. Here, ribose-modified bisubstrate inhibitors were studied. Three high-resolution crystal structures of COMT in complex with novel ribose-modified bisubstrate inhibitors confirmed the predicted binding mode but displayed subtle alterations at the ribose-binding site. The high affinity of the inhibitors can be convincingly rationalized from the structures, which document the possibility of removing and/or replacing the ribose 3'-hydroxyl group and provide a framework for further inhibitor design.


Assuntos
Inibidores de Catecol O-Metiltransferase , Catecóis/antagonistas & inibidores , Desoxirribose/antagonistas & inibidores , Dopamina/metabolismo , Levodopa/farmacologia , Ribose/antagonistas & inibidores , S-Adenosilmetionina/antagonistas & inibidores , Sítios de Ligação , Catecol O-Metiltransferase/química , Catecol O-Metiltransferase/metabolismo , Catecóis/metabolismo , Cristalografia por Raios X , Dopamina/farmacologia , Desenho de Fármacos , Levodopa/metabolismo , Modelos Moleculares , Doença de Parkinson/tratamento farmacológico
6.
Biochem Pharmacol ; 83(7): 969-76, 2012 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-22274639

RESUMO

Thiopurine S-methyltransferase (TPMT; EC 2.1.1.67) plays a pivotal role in thiopurine treatment outcomes. However, little has been known about its intracellular regulation. Here, we describe the effect of fluctuations in physiological levels of S-adenosyl-L-methionine (SAM) and related metabolites on TPMT activity levels in cell lines and erythrocytes from healthy donors. We determined higher TPMT activity in wild-type TPMT*1/*1 individuals with high SAM concentrations (n=96) compared to the low SAM level group (n=19; P<0.001). These findings confirm the results of our in vitro studies, which demonstrated that the restriction of L-methionine (Met) in cell growth media reversibly decreased TPMT activity and protein levels. Selective inhibition of distinct components of Met metabolism was used to demonstrate that SAM is implicitly responsible for direct post-translational TPMT stabilization. The greatest effect of SAM-mediated TPMT stabilization was observed in the case of wild-type TPMT*1 and variant *3C allozymes. In addition to TPMT genotyping, SAM may serve as an important biochemical marker in individualization of thiopurine therapy.


Assuntos
Metiltransferases/metabolismo , Processamento de Proteína Pós-Traducional , S-Adenosilmetionina/metabolismo , Técnicas de Cultura de Células , Meios de Cultura , Inibidores Enzimáticos/farmacologia , Eritrócitos/enzimologia , Ácido Fólico/metabolismo , Ácido Fólico/farmacologia , Genótipo , Células HEK293 , Células Hep G2 , Humanos , Isoenzimas , Metionina/metabolismo , Metionina/farmacologia , Metionina Adenosiltransferase/metabolismo , Metionina Adenosiltransferase/farmacologia , Metiltransferases/antagonistas & inibidores , Metiltransferases/genética , Estabilidade Proteica , S-Adenosilmetionina/antagonistas & inibidores , S-Adenosilmetionina/farmacologia
7.
Org Biomol Chem ; 9(22): 7814-21, 2011 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-21952734

RESUMO

Protein arginine N-methyltransferases (PRMTs) selectively replace N-H for N-CH(3) at substrate protein guanidines, a post-translational modification important for a range of biological processes, such as epigenetic regulation, signal transduction and cancer progression. Selective chemical probes are required to establish the dynamic function of individual PRMTs. Herein, model inhibitors designed to occupy PRMT binding sites for an arginine substrate and S-adenosylmethionine (AdoMet) co-factor are described. Expedient access to such compounds by modular synthesis is detailed. Remarkably, biological evaluation revealed some compounds to be potent inhibitors of PRMT1, but inactive against CARM1. Docking studies show how prototype compounds may occupy the binding sites for a co-factor and arginine substrate. Overlay of PRMT1 and CARM1 binding sites suggest a difference in a single amino acid that may be responsible for the observed selectivity.


Assuntos
Arginina/metabolismo , Inibidores Enzimáticos/síntese química , Processamento de Proteína Pós-Traducional , Proteína-Arginina N-Metiltransferases/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/metabolismo , S-Adenosilmetionina/metabolismo , Arginina/antagonistas & inibidores , Sítios de Ligação , Cristalografia por Raios X , Inibidores Enzimáticos/farmacologia , Epigênese Genética , Escherichia coli , Humanos , Metilação , Modelos Moleculares , Peso Molecular , Plasmídeos , Ligação Proteica , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , Proteína-Arginina N-Metiltransferases/química , Proteína-Arginina N-Metiltransferases/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/química , Proteínas Repressoras/genética , S-Adenosilmetionina/antagonistas & inibidores , Especificidade por Substrato , Transformação Bacteriana
8.
Hepatology ; 52(1): 105-14, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20578266

RESUMO

UNLABELLED: Deletion of glycine N-methyltransferase (GNMT), the main gene involved in liver S-adenosylmethionine (SAM) catabolism, leads to the hepatic accumulation of this molecule and the development of fatty liver and fibrosis in mice. To demonstrate that the excess of hepatic SAM is the main agent contributing to liver disease in GNMT knockout (KO) mice, we treated 1.5-month-old GNMT-KO mice for 6 weeks with nicotinamide (NAM), a substrate of the enzyme NAM N-methyltransferase. NAM administration markedly reduced hepatic SAM content, prevented DNA hypermethylation, and normalized the expression of critical genes involved in fatty acid metabolism, oxidative stress, inflammation, cell proliferation, and apoptosis. More importantly, NAM treatment prevented the development of fatty liver and fibrosis in GNMT-KO mice. Because GNMT expression is down-regulated in patients with cirrhosis, and because some subjects with GNMT mutations have spontaneous liver disease, the clinical implications of the present findings are obvious, at least with respect to these latter individuals. Because NAM has been used for many years to treat a broad spectrum of diseases (including pellagra and diabetes) without significant side effects, it should be considered in subjects with GNMT mutations. CONCLUSION: The findings of this study indicate that the anomalous accumulation of SAM in GNMT-KO mice can be corrected by NAM treatment leading to the normalization of the expression of many genes involved in fatty acid metabolism, oxidative stress, inflammation, cell proliferation, and apoptosis, as well as reversion of the appearance of the pathologic phenotype.


Assuntos
Fígado Gorduroso/prevenção & controle , Glicina N-Metiltransferase/genética , Cirrose Hepática/prevenção & controle , Niacinamida/uso terapêutico , S-Adenosilmetionina/metabolismo , Animais , Fígado Gorduroso/genética , Deleção de Genes , Expressão Gênica , Cirrose Hepática/genética , Camundongos , Camundongos Knockout , S-Adenosilmetionina/antagonistas & inibidores
9.
J Med Chem ; 52(19): 5967-73, 2009 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-19739644

RESUMO

S-adenosylmethionine (AdoMet) lies at an intersection of nucleotide and amino acid metabolism and performs a multitude of metabolic functions. AdoMet formation is catalyzed by S-adenosylmethionine synthetase (ATP: L-methionine S-adenosyltransferase (MAT)), which is a target for development of anticancer and antimicrobial agents. High affinity MAT inhibitors have been found through computational docking of more than 200000 compounds for predicted binding to the crystallographically defined nucleotide binding region of the enzyme's active site. Two of the top scoring candidate compounds had IC(50) values less than 10 nM, more than 10000-fold lower than the substrates' K(M) values. The compounds are structurally unrelated to the natural ligands of the enzyme. The enzyme is protected from inhibition by ATP, but not by methionine, consistent with binding at the adenosyl region of the active site. These results validate in silico screening as a robust approach to the discovery of inhibitors of this chemotherapeutically relevant enzyme.


Assuntos
Simulação por Computador , Avaliação Pré-Clínica de Medicamentos/métodos , Metionina Adenosiltransferase/antagonistas & inibidores , S-Adenosilmetionina/antagonistas & inibidores , Domínio Catalítico , Cristalografia por Raios X , Descoberta de Drogas/métodos , Escherichia coli/efeitos dos fármacos , Concentração Inibidora 50 , Ligação Proteica , Conformação Proteica , S-Adenosilmetionina/biossíntese , Saccharomyces cerevisiae/efeitos dos fármacos
10.
Eur J Clin Microbiol Infect Dis ; 27(11): 1131-6, 2008 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18574605

RESUMO

The antifungal properties of 25-azalanosterol was investigated. Compared to normal antifungal reagents, fluoconazole, clotrimazole and voriconazole, it exhibited significant anti-Candida activity (the minimum inhibitory concentration [MIC] ranges were 0.125-8, 0.5-8 and 0.5-32 microg/mL against C. albicans, C. krusei and C. glabrata, respectively), but showed little toxicity to mice liver cells at clinical dosage after 24 h of exposure, with the lowest lactate dehydrogenase and the highest ED(50) compared to four other azoles antifungal agents. 25-Azalanosterol inhibited the incorporation of [methyl-(3)H(3)] AdoMet into the C-24 of ergosterol in whole cells of C. albicans. Thus, 25-azalanosterol, as an inhibitor of the growth of C. albicans in vitro, may have considerable potential as a new class of anti-Candida agent that lacks toxic side effects in the mammalian host.


Assuntos
Antifúngicos/farmacologia , Candida/efeitos dos fármacos , Lanosterol/análogos & derivados , Lanosterol/farmacologia , Animais , Antifúngicos/toxicidade , Linhagem Celular , Hepatócitos/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Lanosterol/toxicidade , Camundongos , Testes de Sensibilidade Microbiana , S-Adenosilmetionina/antagonistas & inibidores
11.
J Nutr Biochem ; 19(6): 384-91, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17869084

RESUMO

An important aspect in alcohol abuse-associated immune suppression is the loss of T helper CD4(+) lymphocytes, leading to impairment of multiple immune functions. Our work has shown that ethanol can sensitize CD4(+) T lymphocytes to caspase-3-dependent activation-induced cell death (AICD). It has been demonstrated that the formation of S-adenosylmethionine (SAMe) catalyzed by methionine adenosyltransferase (MAT) II is essential for CD4(+) T-cell activation and proliferation. Since ethanol is known to affect SAMe metabolism in hepatocytes, we investigated the effect of ethanol on MAT II activity/expression, SAMe biosynthesis and cell survival in CD4(+) T lymphocytes. We demonstrate for the first time that ethanol at a physiologically relevant concentration (25 mM) substantially decreased the enzymatic activity of MAT II in T lymphocytes. Ethanol was observed to decrease the transcription of MAT2A, which encodes the catalytic subunit of MAT II and is vital for MAT II activity and SAMe biosynthesis. Furthermore, correspondent to its effect on MAT II, ethanol decreased intracellular SAMe levels and enhanced caspase-3-dependent AICD. Importantly, restoration of intracellular SAMe levels by exogenous SAMe supplementation considerably decreased both caspase-3 activity and apoptotic death in T lymphocytes. In conclusion, our data show that MAT II and SAMe are critical molecular components essential for CD4(+) T-cell survival that are affected by ethanol, leading to enhanced AICD. Furthermore, these studies provide a clinical paradigm for the development of much needed therapy using SAMe supplementation in the treatment of immune dysfunction induced by alcohol abuse.


Assuntos
Apoptose/efeitos dos fármacos , Linfócitos T CD4-Positivos/efeitos dos fármacos , Caspase 3/fisiologia , Etanol/farmacologia , Tolerância Imunológica/efeitos dos fármacos , Metionina Adenosiltransferase/antagonistas & inibidores , S-Adenosilmetionina/antagonistas & inibidores , Caspase 3/efeitos dos fármacos , Humanos , Células Jurkat , RNA Mensageiro/metabolismo , S-Adenosilmetionina/biossíntese , S-Adenosilmetionina/farmacologia
12.
Proc Natl Acad Sci U S A ; 104(16): 6678-83, 2007 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-17426150

RESUMO

Saccharomyces cerevisiae is an ideal host from which to obtain high levels of posttranslationally modified eukaryotic proteins for x-ray crystallography. However, extensive replacement of methionine by selenomethionine for anomalous dispersion phasing has proven intractable in yeast. We report a general method to incorporate selenomethionine into proteins expressed in yeast based on manipulation of the appropriate metabolic pathways. sam1(-) sam2(-) mutants, in which the conversion of methionine to S-adenosylmethionine is blocked, exhibit reduced selenomethionine toxicity compared with wild-type yeast, increased production of protein during growth in selenomethionine, and efficient replacement of methionine by selenomethionine, based on quantitative mass spectrometry and x-ray crystallography. The structure of yeast tryptophanyl-tRNA synthetase was solved to 1.8 A by using multiwavelength anomalous dispersion phasing with protein that was expressed and purified from the sam1(-) sam2(-) strain grown in selenomethionine. Six of eight selenium residues were located in the structure.


Assuntos
S-Adenosilmetionina/antagonistas & inibidores , S-Adenosilmetionina/biossíntese , Saccharomyces cerevisiae/metabolismo , Selenometionina/farmacocinética , Cristalografia por Raios X , S-Adenosilmetionina/química , Saccharomyces cerevisiae/química , Selenometionina/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
13.
Drug Metab Dispos ; 32(9): 1015-22, 2004 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-15319344

RESUMO

The metabolism of MK-0767, (+/-)-5-[(2,4-dioxothiazolidin-5-yl)methyl]-2-methoxy-N-[[(4-trifluoromethyl) phenyl]methyl]benzamide, a thiazolidinedione (TZD)-containing peroxisome proliferator-activated receptor alpha/gamma agonist, was studied in liver microsomes and hepatocytes from humans and rat, dog, and rhesus monkey, to characterize the enzyme(s) involved in its metabolism. The major site of metabolism is the TZD ring, which underwent opening catalyzed by CYP3A4 to give the mercapto derivative, M22. Other metabolites formed in NADPH-fortified liver microsomes included the TZD-5-OH derivative (M24), also catalyzed by CYP3A4, and the O-desmethyl derivative (M28), whose formation was catalyzed by CYP2C9 and CYP2C19. Metabolite profiles from hepatocyte incubations were different from those generated with NADPH-fortified microsomal incubations. In addition to M22, M24, and M28, hepatocytes generated several S-methylated metabolites, including the methyl mercapto (M25), the methyl sulfoxide amide (M16), and the methyl sulfone amide (M20) metabolites. Addition of the methyl donor, S-adenosyl methionine, in addition to NADPH, to microsomal incubations enhanced the turnover and resulted in metabolite profiles similar to those in hepatocyte incubations. Collectively, these results indicated that methyltransferases played a major role in the metabolism of MK-0767. Using enzyme-specific inhibitors, it was concluded that microsomal thiol methyltransferases play a more important role than the cytosolic thiopurine methyltransferase. Baculovirus-expressed human flavin-containing monooxygenase 3, as well as CYP3A4, oxidized M25 to M16, whereas further oxidation of M16 to M20 was catalyzed mainly by CYP3A4. Esterases were involved in the formation of the methyl sulfone carboxylic acids, minor metabolites detected in hepatocytes.


Assuntos
Hidrocarboneto de Aril Hidroxilases/fisiologia , Esterases/fisiologia , Metiltransferases/fisiologia , Oxigenases de Função Mista/fisiologia , PPAR alfa/agonistas , PPAR alfa/metabolismo , PPAR gama/agonistas , PPAR gama/metabolismo , Tiazóis/metabolismo , Animais , Hidrocarboneto de Aril Hidroxilases/classificação , Hidrocarboneto de Aril Hidroxilases/efeitos dos fármacos , Benzilaminas/farmacologia , Radioisótopos de Carbono , Citosol/efeitos dos fármacos , Citosol/enzimologia , Cães , Avaliação Pré-Clínica de Medicamentos/métodos , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Cetoconazol/farmacologia , Macaca mulatta , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia , Oxigenases de Função Mista/química , NADP/antagonistas & inibidores , NADP/metabolismo , PPAR alfa/farmacologia , PPAR gama/farmacologia , Ratos , S-Adenosilmetionina/antagonistas & inibidores , S-Adenosilmetionina/metabolismo , Compostos de Sulfidrila/metabolismo , Sulfóxidos/metabolismo , Temperatura , Tiazóis/farmacologia , Tiazolidinedionas/química , Tiazolidinedionas/metabolismo , Tiazolidinedionas/farmacologia
14.
FEMS Microbiol Lett ; 237(2): 179-86, 2004 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-15321660

RESUMO

Pneumocystis is a parasitic fungus causing pneumonia in immunosuppressed mammals and S-adenosylmethionine a key intermediary metabolite for all cells. Other than a species of Rickettsia bacteria and an aberrant strain of the protozoan Amoeba proteus, Pneumocystis is the only cell known unable to synthesize AdoMet; it must extract this key compound from its host. This was discovered using a culture system and confirmed by observing depletion of AdoMet in the plasma of infected animals. Depletion also occurs in patients with Pneumocystis pneumonia (PcP), a phenomenon suggested as a basis for a method for diagnosis and evaluation of response to therapy. Preliminary data indicate that deliberate reduction of host lung AdoMet by nicotine treatment is therapeutic in the rat model of Pneumocystis pneumonia.


Assuntos
Pneumocystis/metabolismo , Pneumonia por Pneumocystis/tratamento farmacológico , S-Adenosilmetionina/metabolismo , Animais , Humanos , Metionina Adenosiltransferase/genética , Nicotina/uso terapêutico , Pneumocystis/crescimento & desenvolvimento , Pneumonia por Pneumocystis/sangue , Pneumonia por Pneumocystis/metabolismo , Ratos , S-Adenosilmetionina/antagonistas & inibidores , S-Adenosilmetionina/fisiologia
15.
Antimicrob Agents Chemother ; 47(9): 2966-70, 2003 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-12937003

RESUMO

We studied the antiproliferative effects of three azasterol analogs [piperidyl-2-yl-5alpha-pregnan-3beta,20(R)-diol (AZA-1), 22-piperidin-2-yl-pregnan-22(S),3beta-diol (AZA-2), and 22-piperidin-3-yl-pregnan-22(S),3beta-diol (AZA-3)] and their effects on the lipid composition of the pathogenic yeastlike phase of the dimorphic fungus Paracoccidioides brasiliensis. Inhibition was 100% for AZA-1 at 5 microM, 62% for AZA-2 at 10 microM, and 100% for AZA-3 at 0.5 microM. The analogs inhibited different stages of the sterol biosynthesis pathway.


Assuntos
Metiltransferases/farmacologia , Oxirredutases/farmacologia , Paracoccidioides/efeitos dos fármacos , S-Adenosilmetionina/antagonistas & inibidores , Meios de Cultura , Paracoccidioides/crescimento & desenvolvimento , Relação Estrutura-Atividade
16.
Am J Physiol Gastrointest Liver Physiol ; 280(1): G130-8, 2001 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-11123206

RESUMO

Ornithine decarboxylase (ODC) is feedback regulated by polyamines. ODC antizyme mediates this process by forming a complex with ODC and enhancing its degradation. It has been reported that polyamines induce ODC antizyme and inhibit ODC activity. Since exogenous polyamines can be converted to each other after they are taken up into cells, we used an inhibitor of S-adenosylmethionine decarboxylase, diethylglyoxal bis(guanylhydrazone) (DEGBG), to block the synthesis of spermidine and spermine from putrescine and investigated the specific roles of individual polyamines in the regulation of ODC in intestinal epithelial crypt (IEC-6) cells. We found that putrescine, spermidine, and spermine inhibited ODC activity stimulated by serum to 85, 46, and 0% of control, respectively, in the presence of DEGBG. ODC activity increased in DEGBG-treated cells, despite high intracellular putrescine levels. Although exogenous spermidine and spermine reduced ODC activity of DEGBG-treated cells close to control levels, spermine was more effective than spermidine. Exogenous putrescine was much less effective in inducing antizyme than spermidine or spermine. High putrescine levels in DEGBG-treated cells did not induce ODC antizyme when intracellular spermidine and spermine levels were low. The decay of ODC activity and reduction of ODC protein levels were not accompanied by induction of antizyme in the presence of DEGBG. Our results indicate that spermine is the most, and putrescine the least, effective polyamine in regulating ODC activity, and upregulation of antizyme is not required for the degradation of ODC protein.


Assuntos
Células Epiteliais/enzimologia , Intestinos/citologia , Mitoguazona/análogos & derivados , Ornitina Descarboxilase/metabolismo , Poliaminas/farmacocinética , S-Adenosilmetionina/metabolismo , Animais , Linhagem Celular , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Inibidores Enzimáticos/farmacologia , Células Epiteliais/citologia , Proteínas Fetais/farmacologia , Intestinos/enzimologia , Mitoguazona/farmacologia , Putrescina/farmacocinética , Ratos , S-Adenosilmetionina/antagonistas & inibidores , Espermidina/farmacocinética , Espermina/farmacocinética
17.
Pharmacol Biochem Behav ; 66(4): 841-9, 2000 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-10973524

RESUMO

Injection of the endogenous methyl donor, S-adenosyl methionine (SAM), into rat brain induces Parkinson's disease (PD)-like symptoms possibly by stimulating deleterious protein methylation. Gel-filtration chromatography of rat brain extracts treated with [3H-methyl]-SAM revealed the presence of radioactive peaks with apparent molecular weights of about 5 kDa. Treatment with guanidine HCl altered the elution volumes of the labeled peaks. Lyophilized peak fractions released volatile 3H-methanol on incubation with NaOH, indicating the presence of carboxyl methyl esters. Because prenylated proteins are avid methyl acceptors at the terminal carboxylic acid groups, 1 micromol S-farnesylcysteine (FC) analogs blocked the SAM-induced tremors in the experimental rats. FC analogs did not only reverse the associated rigidity, abnormal posture, and hypokinesia, but stimulated hyperactivity in the animals. This amphetamine-like effect was monitored for 20 min in an animal activity monitor and movement times between 400 +/- 100 and 560 +/- 125 s covering distances between 78 +/- 29 to 125 +/- 35 m were recorded for rats treated with FC analogs with or without SAM. Control animals moved only for 60 +/- 13 s covering about 6 +/- 1 m, indicating a 7-9-fold and 13-21-fold increase in duration of movement and distance covered, respectively. N-Acetyl-S-farnesylcysteine (AFC) potentiated amphetamine-induced ipsiversive rotation of 6-hydroxydopamine-lesioned rats from 390 +/- 130 to 830 +/- 110, with AFC alone having no significant effect on net rotation compared to controls. These data indicate that intracerebroventricular injection of SAM may induce PD symptoms by interfering with the methylation/demethylation homeostasis of prenylated proteins that function in the dopaminergic and other signaling pathways, and that the FC analogs may counteract the SAM effects by acting synergistically on events subsequent to neurotransmitter release.


Assuntos
Cisteína/análogos & derivados , Doença de Parkinson Secundária/prevenção & controle , S-Adenosilmetionina/antagonistas & inibidores , Anfetamina/farmacologia , Animais , Estimulantes do Sistema Nervoso Central/farmacologia , Cromatografia em Gel , Cisteína/farmacologia , Liofilização , Masculino , Metilação , Atividade Motora/efeitos dos fármacos , Proteínas do Tecido Nervoso/análise , Proteínas do Tecido Nervoso/metabolismo , Oxidopamina , Doença de Parkinson Secundária/induzido quimicamente , Ratos , Ratos Sprague-Dawley , S-Adenosilmetionina/toxicidade , Comportamento Estereotipado/efeitos dos fármacos , Simpatectomia Química , Tremor/induzido quimicamente , Tremor/prevenção & controle
18.
Amino Acids ; 18(4): 319-27, 2000.
Artigo em Inglês | MEDLINE | ID: mdl-10949915

RESUMO

The relationship between cellular glutathione (GSH), protein-SH levels, and lactate dehydrogenase (LDH), with respect to the effect of polyamines on the cytoprotective ability of L-cysteine and L-methionine, the most important components in the sulfur amino acid metabolic pathway, in carbon tetrachloride (CCl4)-induced toxicity in isolated rat hepatocytes was studied. CCl4 induced a LDH release and decreased cellular thiols and polyamines levels but treatment with L-cysteine and L-methionine reversed these decreases. Treating with methylglyoxal bis-(guanylhydrazone), MGBG, an irreversible inhibitor of S-adenosylmethionine decarboxylase, which is a key enzyme in spermidine and spermine biosynthesis, and therefore used to deplete cellular polyamines, prevented the protective effect of L-cysteine and L-methionine, but the addition of exogenous polyamines inhibited the influence of MGBG. These results suggest that the cytoprotective effect of L-cysteine and L-methionine in CCl4-induced toxicity were via maintenance of cellular polyamines, GSH and protein-SH concentrations and prevention of LDH leakage.


Assuntos
Tetracloreto de Carbono/toxicidade , Cisteína/farmacologia , Citoproteção/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Metionina/farmacologia , Poliaminas/metabolismo , Compostos de Sulfidrila/metabolismo , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Glutationa/metabolismo , Glutationa Transferase/química , Glutationa Transferase/metabolismo , Hepatócitos/citologia , Hepatócitos/metabolismo , L-Lactato Desidrogenase/metabolismo , Masculino , Maleatos/farmacologia , Mitoguazona/farmacologia , Ratos , Ratos Sprague-Dawley , S-Adenosilmetionina/antagonistas & inibidores
19.
EMBO J ; 18(11): 3164-72, 1999 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-10357827

RESUMO

The replication complexes of all positive strand RNA viruses of eukaryotes are associated with membranes. In the case of Semliki Forest virus (SFV), the main determinant of membrane attachment seems to be the virus-encoded non-structural protein NSP1, the capping enzyme of the viral mRNAs, which has guanine-7-methyltransferase and guanylyltransferase activities. We show here that both enzymatic activities of SFV NSP1 are inactivated by detergents and reactivated by anionic phospholipids, especially phosphatidylserine. The region of NSP1 responsible for binding to membranes as well as to liposomes was mapped to a short segment, which is conserved in the large alphavirus-like superfamily of viruses. A synthetic peptide of 20 amino acids from the putative binding site competed with in vitro synthesized NSP1 for binding to liposomes containing phosphatidylserine. These findings suggest a molecular mechanism by which RNA virus replicases attach to intracellular membranes and why they depend on the membranous environment.


Assuntos
Membrana Celular/metabolismo , Nucleotidiltransferases/metabolismo , Fosfolipídeos/metabolismo , Vírus da Floresta de Semliki/enzimologia , Proteínas não Estruturais Virais/metabolismo , Sequência de Aminoácidos , Ânions/metabolismo , Ligação Competitiva , Membrana Celular/química , Sequência Conservada/genética , Detergentes/farmacologia , Ativação Enzimática/efeitos dos fármacos , Escherichia coli/citologia , Escherichia coli/genética , Escherichia coli/metabolismo , Lipossomos/antagonistas & inibidores , Lipossomos/metabolismo , Dados de Sequência Molecular , Mutação , Nucleotidiltransferases/antagonistas & inibidores , Nucleotidiltransferases/química , Nucleotidiltransferases/genética , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/metabolismo , Fosfatidilserinas/metabolismo , Fosfatidilserinas/farmacologia , Fosfolipídeos/farmacologia , Ligação Proteica/efeitos dos fármacos , Biossíntese de Proteínas , Conformação Proteica/efeitos dos fármacos , Proteínas Recombinantes/análise , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , S-Adenosilmetionina/antagonistas & inibidores , S-Adenosilmetionina/metabolismo , Vírus da Floresta de Semliki/genética , Solubilidade/efeitos dos fármacos , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética
20.
Artigo em Inglês | MEDLINE | ID: mdl-9473009

RESUMO

The polyamines putrescine, spermine, and spermidine, present in all living cells, have been implicated in the replication of some herpesviruses and retroviruses, and elevated levels of these polyamines have been found in the lymphocytes of patients infected with HIV-1. We have examined the effect of HIV-1 infection on polyamine pools in cell culture. HIV-1 did not significantly affect the polyamine pools in CEM cells. Consistent with this observation, inhibitors of the two key enzymes of this pathway, ornithine decarboxylase and S-adenosylmethionine decarboxylase, did not prevent viral-induced cytopathic effects (CPE) in this cell line. Our results indicate that inhibitors of this pathway will not be therapeutically useful in the treatment of AIDS.


Assuntos
HIV-1/crescimento & desenvolvimento , Poliaminas/análise , Linfócitos T/virologia , Linhagem Celular , Efeito Citopatogênico Viral/efeitos dos fármacos , Células-Tronco Hematopoéticas/virologia , Humanos , Inibidores da Ornitina Descarboxilase , Putrescina/análise , S-Adenosilmetionina/antagonistas & inibidores , Espermidina/análise , Espermina/análise
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