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1.
Cochrane Database Syst Rev ; 10: CD012649, 2021 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-34661903

RESUMO

BACKGROUND: Arsenic is a common environmental toxin. Exposure to arsenic (particularly its inorganic form) through contaminated food and drinking water is an important public health burden worldwide, and is associated with increased risk of neurotoxicity, congenital anomalies, cancer, and adverse neurodevelopment in children. Arsenic is excreted following methylation reactions, which are mediated by folate. Provision of folate through folic acid supplements could facilitate arsenic methylation and excretion, thereby reducing arsenic toxicity. OBJECTIVES: To assess the effects of provision of folic acid (through fortified foods or supplements), alone or in combination with other nutrients, in lessening the burden of arsenic-related health outcomes and reducing arsenic toxicity in arsenic-exposed populations. SEARCH METHODS: In September 2020, we searched CENTRAL, MEDLINE, Embase, 10 other international databases, nine regional databases, and two trials registers. SELECTION CRITERIA: Randomised controlled trials (RCTs) and quasi-RCTs comparing the provision of folic acid (at any dose or duration), alone or in combination with other nutrients or nutrient supplements, with no intervention, placebo, unfortified food, or the same nutrient or supplements without folic acid, in arsenic-exposed populations of all ages and genders. DATA COLLECTION AND ANALYSIS: We used standard methodological procedures expected by Cochrane. MAIN RESULTS: We included two RCTs with 822 adults exposed to arsenic-contaminated drinking water in Bangladesh. The RCTs compared 400 µg/d (FA400) or 800 µg/d (FA800) folic acid supplements, given for 12 or 24 weeks, with placebo. One RCT, a multi-armed trial, compared FA400 plus creatine (3 g/d) to creatine alone. We judged both RCTs at low risk of bias in all domains. Due to differences in co-intervention, arsenic exposure, and participants' nutritional status, we could not conduct meta-analyses, and therefore, provide a narrative description of the data. Neither RCT reported on cancer, all-cause mortality, neurocognitive function, or congenital anomalies. Folic acid supplements alone versus placebo Blood arsenic. In arsenic-exposed individuals, FA likely reduces blood arsenic concentrations compared to placebo (2 studies, 536 participants; moderate-certainty evidence). For folate-deficient and folate-replete participants who received arsenic-removal water filters as a co-intervention, FA800 reduced blood arsenic levels more than placebo (percentage change (%change) in geometric mean (GM) FA800 -17.8%, 95% confidence intervals (CI) -25.0 to -9.8; placebo GM -9.5%, 95% CI -16.5 to -1.8; 1 study, 406 participants). In one study with 130 participants with low baseline plasma folate, FA400 reduced total blood arsenic (%change FA400 mean (M) -13.62%, standard error (SE) ± 2.87; placebo M -2.49%, SE ± 3.25), and monomethylarsonic acid (MMA) concentrations (%change FA400 M -22.24%, SE ± 2.86; placebo M -1.24%, SE ± 3.59) more than placebo. Inorganic arsenic (InAs) concentrations reduced in both groups (%change FA400 M -18.54%, SE ± 3.60; placebo M -10.61%, SE ± 3.38). There was little to no change in dimethylarsinic acid (DMA) in either group. Urinary arsenic. In arsenic-exposed individuals, FA likely reduces the proportion of total urinary arsenic excreted as InAs (%InAs) and MMA (%MMA) and increases the proportion excreted as DMA (%DMA) to a greater extent than placebo (2 studies, 546 participants; moderate-certainty evidence), suggesting that FA enhances arsenic methylation. In a mixed folate-deficient and folate-replete population (1 study, 352 participants) receiving arsenic-removal water filters as a co-intervention, groups receiving FA had a greater decrease in %InAs (within-person change FA400 M -0.09%, 95% CI -0.17 to -0.01; FA800 M -0.14%, 95% CI -0.21 to -0.06; placebo M 0.05%, 95% CI 0.00 to 0.10), a greater decrease in %MMA (within-person change FA400 M -1.80%, 95% CI -2.53 to -1.07; FA800 M -2.60%, 95% CI -3.35 to -1.85; placebo M 0.15%, 95% CI -0.37 to 0.68), and a greater increase in %DMA (within-person change FA400 M 3.25%, 95% CI 1.81 to 4.68; FA800 M 4.57%, 95% CI 3.20 to 5.95; placebo M -1.17%, 95% CI -2.18 to -0.17), compared to placebo. In 194 participants with low baseline plasma folate, FA reduced %InAs (%change FA400 M -0.31%, SE ± 0.04; placebo M -0.13%, SE ± 0.04) and %MMA (%change FA400 M -2.6%, SE ± 0.37; placebo M -0.71%, SE ± 0.43), and increased %DMA (%change FA400 M 5.9%, SE ± 0.82; placebo M 2.14%, SE ± 0.71), more than placebo. Plasma homocysteine: In arsenic-exposed individuals, FA400 likely reduces homocysteine concentrations to a greater extent than placebo (2 studies, 448 participants; moderate-certainty evidence), in the mixed folate-deficient and folate-replete population receiving arsenic-removal water filters as a co-intervention (%change in GM FA400 -23.4%, 95% CI -27.1 to -19.5; placebo -1.3%, 95% CI -5.3 to 3.1; 1 study, 254 participants), and participants with low baseline plasma folate (within-person change FA400 M -3.06 µmol/L, SE ± 3.51; placebo M -0.05 µmol/L, SE ± 4.31; 1 study, 194 participants). FA supplements plus other nutrient supplements versus nutrient supplements alone In arsenic-exposed individuals who received arsenic-removal water filters as a co-intervention, FA400 plus creatine may reduce blood arsenic concentrations more than creatine alone (%change in GM FA400 + creatine -14%, 95% CI -22.2 to -5.0; creatine -7.0%, 95% CI -14.8 to 1.5; 1 study, 204 participants; low-certainty evidence); may not change urinary arsenic methylation indices (FA400 + creatine: %InAs M 13.2%, SE ± 7.0; %MMA M 10.8, SE ± 4.1; %DMA M 76, SE ± 7.8; creatine: %InAs M 14.8, SE ± 5.5; %MMA M 12.8, SE ± 4.0; %DMA M 72.4, SE ±7.6; 1 study, 190 participants; low-certainty evidence); and may reduce homocysteine concentrations to a greater extent (%change in GM FA400 + creatinine -21%, 95% CI -25.2 to -16.4; creatine -4.3%, 95% CI -9.0 to 0.7; 1 study, 204 participants; low-certainty evidence) than creatine alone. AUTHORS' CONCLUSIONS: There is moderate-certainty evidence that FA supplements may benefit blood arsenic concentration, urinary arsenic methylation profiles, and plasma homocysteine concentration versus placebo. There is low-certainty evidence that FA supplements plus other nutrients may benefit blood arsenic and plasma homocysteine concentrations versus nutrients alone. No studies reported on cancer, all-cause mortality, neurocognitive function, or congenital anomalies. Given the limited number of RCTs, more studies conducted in diverse settings are needed to assess the effects of FA on arsenic-related health outcomes and arsenic toxicity in arsenic-exposed adults and children.


Assuntos
Arsênio , Adulto , Criança , Creatina , Suplementos Nutricionais , Ácido Fólico , Alimentos Fortificados , Humanos
2.
Proc Natl Acad Sci U S A ; 114(20): E4095-E4102, 2017 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-28461497

RESUMO

Clinical vitamin B12 deficiency can result in megaloblastic anemia, which results from the inhibition of DNA synthesis by trapping folate cofactors in the form of 5-methyltetrahydrofolate (5-methylTHF) and subsequent inhibition of de novo thymidylate (dTMP) biosynthesis. In the cytosol, vitamin B12 functions in the remethylation of homocysteine to methionine, which regenerates THF from 5-methylTHF. In the nucleus, THF is required for de novo dTMP biosynthesis, but it is not understood how 5-methylTHF accumulation in the cytosol impairs nuclear dTMP biosynthesis. The impact of vitamin B12 depletion on nuclear de novo dTMP biosynthesis was investigated in methionine synthase-null human fibroblast and nitrous oxide-treated HeLa cell models. The nucleus was the most sensitive cellular compartment to 5-methylTHF accumulation, with levels increasing greater than fourfold. Vitamin B12 depletion decreased de novo dTMP biosynthesis capacity by 5-35%, whereas de novo purine synthesis, which occurs in the cytosol, was not affected. Phosphorylated histone H2AX (γH2AX), a marker of DNA double-strand breaks, was increased in vitamin B12 depletion, and this effect was exacerbated by folate depletion. These studies also revealed that 5-formylTHF, a slow, tight-binding inhibitor of serine hydroxymethyltransferase (SHMT), was enriched in nuclei, accounting for 35% of folate cofactors, explaining previous observations that nuclear SHMT is not a robust source of one-carbons for de novo dTMP biosynthesis. These findings indicate that a nuclear 5-methylTHF trap occurs in vitamin B12 depletion, which suppresses de novo dTMP biosynthesis and causes DNA damage, accounting for the pathophysiology of megaloblastic anemia observed in vitamin B12 and folate deficiency.


Assuntos
Instabilidade Genômica , Glicina Hidroximetiltransferase/metabolismo , Tetra-Hidrofolatos/metabolismo , Timidina Monofosfato/biossíntese , Deficiência de Vitamina B 12/metabolismo , Dano ao DNA , Feminino , Fibroblastos/metabolismo , Células HeLa , Humanos , Lactente , Deficiência de Vitamina B 12/genética
3.
Proc Natl Acad Sci U S A ; 114(12): E2319-E2326, 2017 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-28265077

RESUMO

Arsenic exposure increases risk for cancers and is teratogenic in animal models. Here we demonstrate that small ubiquitin-like modifier (SUMO)- and folate-dependent nuclear de novo thymidylate (dTMP) biosynthesis is a sensitive target of arsenic trioxide (As2O3), leading to uracil misincorporation into DNA and genome instability. Methylenetetrahydrofolate dehydrogenase 1 (MTHFD1) and serine hydroxymethyltransferase (SHMT) generate 5,10-methylenetetrahydrofolate for de novo dTMP biosynthesis and translocate to the nucleus during S-phase, where they form a multienzyme complex with thymidylate synthase (TYMS) and dihydrofolate reductase (DHFR), as well as the components of the DNA replication machinery. As2O3 exposure increased MTHFD1 SUMOylation in cultured cells and in in vitro SUMOylation reactions, and increased MTHFD1 ubiquitination and MTHFD1 and SHMT1 degradation. As2O3 inhibited de novo dTMP biosynthesis in a dose-dependent manner, increased uracil levels in nuclear DNA, and increased genome instability. These results demonstrate that MTHFD1 and SHMT1, which are key enzymes providing one-carbon units for dTMP biosynthesis in the form of 5,10-methylenetetrahydrofolate, are direct targets of As2O3-induced proteolytic degradation, providing a mechanism for arsenic in the etiology of cancer and developmental anomalies.


Assuntos
Aminoidrolases/antagonistas & inibidores , Núcleo Celular/metabolismo , Formiato-Tetra-Hidrofolato Ligase/antagonistas & inibidores , Metilenotetra-Hidrofolato Desidrogenase (NADP)/antagonistas & inibidores , Complexos Multienzimáticos/antagonistas & inibidores , Óxidos/toxicidade , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/antagonistas & inibidores , Timidina Monofosfato/biossíntese , Aminoidrolases/genética , Aminoidrolases/metabolismo , Animais , Trióxido de Arsênio , Arsenicais , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/enzimologia , Núcleo Celular/genética , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Fibroblastos/metabolismo , Formiato-Tetra-Hidrofolato Ligase/genética , Formiato-Tetra-Hidrofolato Ligase/metabolismo , Instabilidade Genômica/efeitos dos fármacos , Glicina Hidroximetiltransferase/genética , Glicina Hidroximetiltransferase/metabolismo , Humanos , Metilenotetra-Hidrofolato Desidrogenase (NADP)/genética , Metilenotetra-Hidrofolato Desidrogenase (NADP)/metabolismo , Camundongos , Camundongos Knockout , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Proteólise , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Sumoilação , Timidilato Sintase/genética , Timidilato Sintase/metabolismo , Uracila/metabolismo
4.
Annu Rev Nutr ; 38: 219-243, 2018 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-30130467

RESUMO

Despite unequivocal evidence that folate deficiency increases risk for human pathologies, and that folic acid intake among women of childbearing age markedly decreases risk for birth defects, definitive evidence for a causal biochemical pathway linking folate to disease and birth defect etiology remains elusive. The de novo and salvage pathways for thymidylate synthesis translocate to the nucleus of mammalian cells during S- and G2/M-phases of the cell cycle and associate with the DNA replication and repair machinery, which limits uracil misincorporation into DNA and genome instability. There is increasing evidence that impairments in nuclear de novo thymidylate synthesis occur in many pathologies resulting from impairments in one-carbon metabolism. Understanding the roles and regulation of nuclear de novo thymidylate synthesis and its relationship to genome stability will increase our understanding of the fundamental mechanisms underlying folate- and vitamin B12-associated pathologies.


Assuntos
Núcleo Celular/metabolismo , Ácido Fólico/metabolismo , Animais , Ciclo Celular , Regulação da Expressão Gênica/fisiologia , Humanos , Timidina Monofosfato/metabolismo
5.
Proc Natl Acad Sci U S A ; 112(2): 400-5, 2015 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-25548164

RESUMO

An inborn error of metabolism associated with mutations in the human methylenetetrahydrofolate dehydrogenase 1 (MTHFD1) gene has been identified. The proband presented with SCID, megaloblastic anemia, and neurologic abnormalities, but the causal metabolic impairment is unknown. SCID has been associated with impaired purine nucleotide metabolism, whereas megaloblastic anemia has been associated with impaired de novo thymidylate (dTMP) biosynthesis. MTHFD1 functions to condense formate with tetrahydrofolate and serves as the primary entry point of single carbons into folate-dependent one-carbon metabolism in the cytosol. In this study, we examined the impact of MTHFD1 loss of function on folate-dependent purine, dTMP, and methionine biosynthesis in fibroblasts from the proband with MTHFD1 deficiency. The flux of formate incorporation into methionine and dTMP was decreased by 90% and 50%, respectively, whereas formate flux through de novo purine biosynthesis was unaffected. Patient fibroblasts exhibited enriched MTHFD1 in the nucleus, elevated uracil in DNA, lower rates of de novo dTMP synthesis, and increased salvage pathway dTMP biosynthesis relative to control fibroblasts. These results provide evidence that impaired nuclear de novo dTMP biosynthesis can lead to both megaloblastic anemia and SCID in MTHFD1 deficiency.


Assuntos
Metilenotetra-Hidrofolato Desidrogenase (NADP)/deficiência , Metilenotetra-Hidrofolato Desidrogenase (NADP)/genética , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Timidina Monofosfato/biossíntese , Substituição de Aminoácidos , Anemia Megaloblástica/genética , Anemia Megaloblástica/metabolismo , Linhagem Celular , Núcleo Celular/metabolismo , Códon sem Sentido , Dano ao DNA , Fibroblastos/metabolismo , Humanos , Redes e Vias Metabólicas , Metilenotetra-Hidrofolato Desidrogenase (NADP)/química , Antígenos de Histocompatibilidade Menor , Proteínas Mutantes/química , Fenótipo , Mutação Puntual , Imunodeficiência Combinada Severa/genética , Imunodeficiência Combinada Severa/metabolismo
6.
Adv Exp Med Biol ; 963: 143-168, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28197911

RESUMO

Protein modification with the small ubiquitin-related modifier (SUMO) can affect protein function, enzyme activity, protein-protein interactions, protein stability, protein targeting and cellular localization. SUMO influences the function and regulation of metabolic enzymes within pathways, and in some cases targets entire metabolic pathways by affecting the activity of transcription factors or by facilitating the translocation of entire metabolic pathways to subcellular compartments. SUMO modification is also a key component of nutrient- and metabolic-sensing mechanisms that regulate cellular metabolism. In addition to its established roles in maintaining metabolic homeostasis, there is increasing evidence that SUMO is a key factor in facilitating cellular stress responses through the regulation and/or adaptation of the most fundamental metabolic processes, including energy and nucleotide metabolism. This review focuses on the role of SUMO in cellular metabolism and metabolic disease.


Assuntos
Metabolismo Energético , Transdução de Sinais , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Sumoilação , Ubiquitina-Proteína Ligases/metabolismo , Animais , Homeostase , Humanos , Mitocôndrias/metabolismo , Estresse Fisiológico
7.
J Biol Chem ; 289(43): 29642-50, 2014 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-25213861

RESUMO

Folate-mediated one-carbon metabolism is a metabolic network of interconnected pathways that is required for the de novo synthesis of three of the four DNA bases and the remethylation of homocysteine to methionine. Previous studies have indicated that the thymidylate synthesis and homocysteine remethylation pathways compete for a limiting pool of methylenetetrahydrofolate cofactors and that thymidylate biosynthesis is preserved in folate deficiency at the expense of homocysteine remethylation, but the mechanisms are unknown. Recently, it was shown that thymidylate synthesis occurs in the nucleus, whereas homocysteine remethylation occurs in the cytosol. In this study we demonstrate that methylenetetrahydrofolate dehydrogenase 1 (MTHFD1), an enzyme that generates methylenetetrahydrofolate from formate, ATP, and NADPH, functions in the nucleus to support de novo thymidylate biosynthesis. MTHFD1 translocates to the nucleus in S-phase MCF-7 and HeLa cells. During folate deficiency mouse liver MTHFD1 levels are enriched in the nucleus >2-fold at the expense of levels in the cytosol. Furthermore, nuclear folate levels are resistant to folate depletion when total cellular folate levels are reduced by >50% in mouse liver. The enrichment of folate cofactors and MTHFD1 protein in the nucleus during folate deficiency in mouse liver and human cell lines accounts for previous metabolic studies that indicated 5,10-methylenetetrahydrofolate is preferentially directed toward de novo thymidylate biosynthesis at the expense of homocysteine remethylation during folate deficiency.


Assuntos
Núcleo Celular/metabolismo , Coenzimas/metabolismo , Deficiência de Ácido Fólico/enzimologia , Ácido Fólico/metabolismo , Metilenotetra-Hidrofolato Desidrogenase (NADP)/metabolismo , Timidina Monofosfato/biossíntese , Animais , Pontos de Checagem do Ciclo Celular , Linhagem Celular , DNA/metabolismo , Dieta , Feminino , Deficiência de Ácido Fólico/patologia , Formiatos/sangue , Técnicas de Silenciamento de Genes , Glicina Hidroximetiltransferase/metabolismo , Humanos , Fígado/enzimologia , Masculino , Metionina/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Transporte Proteico , Purinas/biossíntese , Fase S , Uracila/metabolismo
8.
J Biol Chem ; 288(20): 14522-14530, 2013 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-23572525

RESUMO

Mutations in the PLEKHG4 (puratrophin-1) gene are associated with the heritable neurological disorder autosomal dominant spinocerebellar ataxia. However, the biochemical functions of this gene product have not been described. We report here that expression of Plekhg4 in the murine brain is developmentally regulated, with pronounced expression in the newborn midbrain and brainstem that wanes with age and maximal expression in the cerebellar Purkinje neurons in adulthood. We show that Plekhg4 is subject to ubiquitination and proteasomal degradation, and its steady-state expression levels are regulated by the chaperones Hsc70 and Hsp90 and by the ubiquitin ligase CHIP. On the functional level, we demonstrate that Plekhg4 functions as a bona fide guanine nucleotide exchange factor (GEF) that facilitates activation of the small GTPases Rac1, Cdc42, and RhoA. Overexpression of Plekhg4 in NIH3T3 cells induces rearrangements of the actin cytoskeleton, specifically enhanced formation of lamellopodia and fillopodia. These findings indicate that Plekhg4 is an aggregation-prone member of the Dbl family GEFs and that regulation of GTPase signaling is critical for proper cerebellar function.


Assuntos
Regulação Enzimológica da Expressão Gênica , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Actinas/metabolismo , Sequência de Aminoácidos , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Células COS , Chlorocebus aethiops , Citoesqueleto/metabolismo , Modelos Animais de Doenças , Escherichia coli/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Camundongos , Dados de Sequência Molecular , Mutação , Células NIH 3T3 , Pseudópodes/metabolismo , Células de Purkinje/metabolismo , Homologia de Sequência de Aminoácidos , Ataxias Espinocerebelares/metabolismo , Ubiquitina/metabolismo
9.
J Virol ; 87(24): 13598-608, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24109216

RESUMO

In most retroviruses, plasma membrane (PM) association of the Gag structural protein is a critical step in viral assembly, relying in part on interaction between the highly basic Gag MA domain and the negatively charged inner leaflet of the PM. Assembly is thought to begin with Gag dimerization followed by multimerization, resulting in a hexameric lattice. To directly address the role of multimerization in membrane binding, we fused the MA domains of Rous sarcoma virus (RSV) and HIV-1 to the chemically inducible dimerization domain FK506-binding protein (FKBP) or to the hexameric protein CcmK4 from cyanobacteria. The cellular localization of the resulting green fluorescent protein (GFP)-tagged chimeric proteins was examined by fluorescence imaging, and the association of the proteins with liposomes was quantified by flotation in sucrose gradients, following synthesis in a reticulocyte extract or as purified proteins. Four lipid compositions were tested, representative of liposomes commonly reported in flotation experiments. By themselves, GFP-tagged RSV and HIV-1 MA proteins were largely cytoplasmic, but both hexamerized proteins were highly concentrated at the PM. Dimerization led to partial PM localization for HIV-1 MA. These in vivo effects of multimerization were reproduced in vitro. In flotation analyses, the intact RSV and HIV-1 Gag proteins were more similar to multimerized MA than to monomeric MA. RNA is reported to compete with acidic liposomes for HIV-1 Gag binding, and thus we also examined the effects of RNase treatment or tRNA addition on flotation. tRNA competed with liposomes in the case of some but not all lipid compositions and ionic strengths. Taken together, our results further underpin the model that multimerization is critical for PM association of retroviral Gag proteins. In addition, they suggest that the modulation of membrane binding by RNA, as previously reported for HIV-1, may not hold for RSV.


Assuntos
Membrana Celular/virologia , Produtos do Gene gag/química , Produtos do Gene gag/metabolismo , Infecções por HIV/virologia , HIV-1/metabolismo , Vírus do Sarcoma de Rous/metabolismo , Sarcoma Aviário/virologia , Produtos do Gene gag do Vírus da Imunodeficiência Humana/química , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo , Animais , Linhagem Celular , Citoplasma/virologia , Produtos do Gene gag/genética , HIV-1/química , HIV-1/genética , Humanos , Multimerização Proteica , Estrutura Terciária de Proteína , Codorniz , Vírus do Sarcoma de Rous/química , Vírus do Sarcoma de Rous/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética
10.
J Exp Med ; 218(9)2021 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-34283207

RESUMO

Type 2 inflammation is associated with epithelial cell responses, including goblet cell hyperplasia, that promote worm expulsion during intestinal helminth infection. How these epithelial responses are regulated remains incompletely understood. Here, we show that mice deficient in the prostaglandin D2 (PGD2) receptor CRTH2 and mice with CRTH2 deficiency only in nonhematopoietic cells exhibited enhanced worm clearance and intestinal goblet cell hyperplasia following infection with the helminth Nippostrongylus brasiliensis. Small intestinal stem, goblet, and tuft cells expressed CRTH2. CRTH2-deficient small intestinal organoids showed enhanced budding and terminal differentiation to the goblet cell lineage. During helminth infection or in organoids, PGD2 and CRTH2 down-regulated intestinal epithelial Il13ra1 expression and reversed Type 2 cytokine-mediated suppression of epithelial cell proliferation and promotion of goblet cell accumulation. These data show that the PGD2-CRTH2 pathway negatively regulates the Type 2 cytokine-driven epithelial program, revealing a mechanism that can temper the highly inflammatory effects of the anti-helminth response.


Assuntos
Citocinas/metabolismo , Mucosa Intestinal/parasitologia , Prostaglandina D2/metabolismo , Receptores Imunológicos/metabolismo , Receptores de Prostaglandina/metabolismo , Infecções por Strongylida/parasitologia , Animais , Feminino , Gastroenterite/parasitologia , Gastroenterite/patologia , Células Caliciformes/patologia , Interações Hospedeiro-Parasita/fisiologia , Mucosa Intestinal/patologia , Masculino , Camundongos Endogâmicos C57BL , Nippostrongylus/patogenicidade , Organoides , Receptores Imunológicos/genética , Receptores de Prostaglandina/genética , Infecções por Strongylida/patologia
11.
Mol Cell Biol ; 27(5): 1809-22, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17178836

RESUMO

The dbl proto-oncogene product is a prototype of a growing family of guanine nucleotide exchange factors (GEFs) that stimulate the activation of small GTP-binding proteins from the Rho family. Mutations that result in the loss of proto-Dbl's amino terminus produce a variant with constitutive GEF activity and high oncogenic potential. Here, we show that proto-Dbl is a short-lived protein that is kept at low levels in cells by efficient ubiquitination and degradation. The cellular fate of proto-Dbl is regulated by interactions with the chaperones Hsc70 and Hsp90 and the protein-ubiquitin ligase CHIP, and these interactions are mediated by the spectrin domain of proto-Dbl. We show that CHIP is the E3 ligase responsible for ubiquitination and proteasomal degradation of proto-Dbl, while Hsp90 functions to stabilize the protein. Onco-Dbl, lacking the spectrin homology domain, cannot bind these regulators and therefore accumulates in cells at high levels, leading to persistent stimulation of its downstream signaling pathways.


Assuntos
Fatores de Troca do Nucleotídeo Guanina/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas Oncogênicas de Retroviridae/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Células COS , Chlorocebus aethiops , Proteínas de Choque Térmico HSC70/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Camundongos , Modelos Biológicos , Mutação , Células NIH 3T3 , Complexo de Endopeptidases do Proteassoma/metabolismo , Estrutura Terciária de Proteína , Proteínas Oncogênicas de Retroviridae/genética , Espectrina/química , Spodoptera/citologia , Transfecção
12.
J Exp Med ; 216(6): 1268-1279, 2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-30975892

RESUMO

Type 2 inflammation drives the clearance of gastrointestinal helminth parasites, which infect over two billion people worldwide. Basophils are innate immune cells that support host-protective type 2 inflammation during murine infection with the helminth Trichuris muris However, the mechanisms required for basophil function and gene expression regulation in this context remain unclear. We show that during T. muris infection, basophils localized to the intestine and up-regulated Notch receptor expression, rendering them sensitive to Notch signals that rapidly regulate gene expression programs. In vitro, Notch inhibition limited basophil cytokine production in response to cytokine stimulation. Basophil-intrinsic Notch signaling was required for T. muris-elicited changes in genome-wide basophil transcriptional programs. Mice lacking basophil-intrinsic functional Notch signaling had impaired worm clearance, decreased intestinal type 2 inflammation, altered basophil localization in the intestine, and decreased CD4+ T helper 2 cell responses following infection. These findings demonstrate that Notch is required for basophil gene expression and effector function associated with helminth expulsion during type 2 inflammation.


Assuntos
Basófilos/imunologia , Inflamação/patologia , Receptores Notch/metabolismo , Transdução de Sinais , Animais , Ceco/parasitologia , Feminino , Regulação da Expressão Gênica , Inflamação/complicações , Interleucinas/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Trichuris/fisiologia , Regulação para Cima
13.
Biochimie ; 126: 27-30, 2016 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-26853819

RESUMO

Disruptions in folate-mediated one-carbon metabolism (FOCM) are associated with risk for several pathologies including developmental anomalies such as neural tube defects and congenital heart defects, diseases of aging including cognitive decline, neurodegeneration and epithelial cancers, and hematopoietic disorders including megaloblastic anemia. However, the causal pathways and mechanisms that underlie these pathologies remain unresolved. Because folate-dependent anabolic pathways are tightly interconnected and best described as a metabolic network, the identification of causal pathways and associated mechanisms of pathophysiology remains a major challenge in identifying the contribution of individual pathways to disease phenotypes. Investigations of genetic mouse models and human inborn errors of metabolism enable a more precise dissection of the pathways that constitute the FOCM network and enable elucidation of causal pathways associated with NTDs. In this overview, we summarize recent evidence that the enzyme MTHFD1 plays an essential role in FOCM in humans and in mice, and that it determines the partitioning of folate-activated one carbon units between the folate-dependent de novo thymidylate and homocysteine remethylation pathways through its regulated nuclear localization. We demonstrate that impairments in MTHFD1 activity compromise both homocysteine remethylation and de novo thymidylate biosynthesis, and provide evidence that MTHFD1-associated disruptions in de novo thymidylate biosynthesis lead to genome instability that may underlie folate-associated immunodeficiency and birth defects.


Assuntos
Instabilidade Genômica , Metilenotetra-Hidrofolato Desidrogenase (NADP)/metabolismo , Antígenos de Histocompatibilidade Menor/metabolismo , Timidina Monofosfato/biossíntese , Animais , Anormalidades Congênitas/enzimologia , Anormalidades Congênitas/genética , Ácido Fólico/biossíntese , Ácido Fólico/genética , Homocisteína/biossíntese , Homocisteína/genética , Humanos , Síndromes de Imunodeficiência/enzimologia , Síndromes de Imunodeficiência/genética , Erros Inatos do Metabolismo/enzimologia , Erros Inatos do Metabolismo/genética , Metilenotetra-Hidrofolato Desidrogenase (NADP)/genética , Camundongos , Antígenos de Histocompatibilidade Menor/genética
14.
Rare Dis ; 3(1): e1112479, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-27123375

RESUMO

Human mutations in MTHFD1 have recently been identified in patients with severe combined immunodeficiency (SCID). SCID results from inborn errors of metabolism that cause impaired T- and B-cell proliferation and function. One of the most common causes of SCID is adenosine deaminase (ADA) deficiency, which ultimately inhibits DNA synthesis and cell division. MTHFD1 has been shown to translocate to the nucleus during S-phase of the cell cycle; this localization is critical for synthesis of thymidyate (dTMP or the "T" base in DNA) and subsequent progression through the cell cycle and cell proliferation. Identification of MTHFD1 mutations that are associated with SCID highlights the potential importance of adequate dTMP synthesis in the etiology of SCID.

15.
J Am Soc Nephrol ; 16(8): 2279-87, 2005 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15958725

RESUMO

Aldosterone plays a central role in Na+ homeostasis by controlling Na+ reabsorption in the aldosterone-sensitive distal nephron involving the epithelial Na+ channel (ENaC). Part of the effects of aldosterone is mediated by serum and glucocorticoid-induced kinase 1 (Sgk1), a Ser/Thr kinase whose expression is rapidly induced by aldosterone and that increases in heterologous expression systems ENaC cell surface abundance and activity. Previous work in Xenopus laevis oocytes suggested that Sgk1 phosphorylates specific residues (Ser212 and Ser328) on the ubiquitin-protein ligase Nedd4-2, an enzyme that directly interacts with ENaC and negatively controls channel density at the plasma membrane. It further indicated that phosphorylation of Nedd4-2 led to impairment of ENaC/Nedd4-2 interaction and consequently to more channels at the cell surface. These data suggested a novel mode of aldosterone-dependent action, yet this was not demonstrated formally in epithelial cells that physiologically express ENaC. Here it is shown, with the use of an anti-phospho-Ser328-mNedd4-2 antibody, that 2 to 6 h of aldosterone treatment induces an increase in Nedd4-2 phosphorylation, both in a mouse cortical collecting duct cell line (mpkCCDcl4) and in kidneys of adrenalectomized rats. This augmentation, which is accompanied by a raise in Sgk1 expression and transepithelial Na+ transport, is sensitive to phosphatidylinositol-3 kinase inhibition, as is Sgk1 phosphorylation and Na+ transport. Hence, these data provide evidence in cortical collecting duct cells in vitro and in vivo that Sgk1-dependent phosphorylation of Nedd4-2 is part of the aldosterone response.


Assuntos
Aldosterona/farmacologia , Proteínas Imediatamente Precoces/biossíntese , Túbulos Renais Coletores/citologia , Túbulos Renais Coletores/metabolismo , Proteínas Serina-Treonina Quinases/biossíntese , Sódio/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Adenoviridae/metabolismo , Aldosterona/metabolismo , Animais , Linhagem Celular , Criopreservação , DNA Complementar/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte , Proteínas Imediatamente Precoces/metabolismo , Rim/metabolismo , Camundongos , Ubiquitina-Proteína Ligases Nedd4 , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação , Plasmídeos/metabolismo , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Ratos , Retroviridae/metabolismo , Sódio/química , Fatores de Tempo , Ubiquitina-Proteína Ligases/fisiologia , Proteínas de Xenopus , Xenopus laevis
16.
Am J Physiol Renal Physiol ; 283(3): F377-87, 2002 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-12167587

RESUMO

The epithelial Na(+) channel (ENaC), located in the apical membrane of renal aldosterone-responsive epithelia, plays an essential role in controlling the Na(+) balance of extracellular fluids and hence blood pressure. As of now, ENaC is the only Na(+) transport protein for which genetic evidence exists for its involvement in the genesis of both hypertension (Liddle's syndrome) and hypotension (pseudohypoaldosteronism type 1). The regulation of ENaC involves a variety of hormonal signals (aldosterone, vasopressin, insulin), but the molecular mechanisms behind this regulation are mostly unknown. Two regulatory proteins have gained interest in recent years: the ubiquitin-protein ligase neural precursor cell-expressed, developmentally downregulated gene 4 isoform Nedd4-2, which negatively controls ENaC cell surface expression, and serum glucocorticoid-inducible kinase 1 (Sgk1), which is an aldosterone- and insulin-dependent, positive regulator of ENaC density at the plasma membrane. Here, we summarize present ideas about Sgk1 and Nedd4-2 and the lines of experimental evidence, suggesting that they act sequentially in the regulatory pathways governed by aldosterone and insulin and regulate ENaC number at the plasma membrane.


Assuntos
Proteínas de Ligação ao Cálcio/fisiologia , Ligases/fisiologia , Proteínas Nucleares , Proteínas Serina-Treonina Quinases/fisiologia , Canais de Sódio/fisiologia , Sódio/metabolismo , Ubiquitina-Proteína Ligases , Aldosterona/farmacologia , Animais , Transporte Biológico , Interações Medicamentosas , Complexos Endossomais de Distribuição Requeridos para Transporte , Canais Epiteliais de Sódio , Epitélio/metabolismo , Humanos , Proteínas Imediatamente Precoces , Rim/metabolismo , Ubiquitina-Proteína Ligases Nedd4 , Fosforilação
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