RESUMO
Macrophages are professional phagocytes that are essential for host defense and tissue homeostasis. Proper membrane trafficking and degradative functions of the endolysosomal system are known to be critical for the function of these cells. We have found that PIKfyve, the kinase that synthesizes the endosomal phosphoinositide phosphatidylinositol-3,5-bisphosphate, is an essential regulator of lysosomal biogenesis and degradative functions in macrophages. Genetically engineered mice lacking PIKfyve in their myeloid cells (PIKfyvefl/fl LysM-Cre) develop diffuse tissue infiltration of foamy macrophages, hepatosplenomegaly, and systemic inflammation. PIKfyve loss in macrophages causes enlarged endolysosomal compartments and impairs the lysosomal degradative function. Moreover, PIKfyve deficiency increases the cellular levels of lysosomal proteins. Although PIKfyve deficiency reduced the activation of mTORC1 pathway and was associated with increased cleavage of TFEB proteins, this does not translate into transcriptional activation of lysosomal genes, suggesting that PIKfyve modulates the abundance of lysosomal proteins by affecting the degradation of these proteins. Our study shows that PIKfyve modulation of lysosomal degradative activity and protein expression is essential to maintain lysosomal homeostasis in macrophages.
Assuntos
Lisossomos/metabolismo , Macrófagos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Animais , Endossomos/metabolismo , Feminino , Homeostase/fisiologia , Inflamação/metabolismo , Masculino , Camundongos , Camundongos Knockout , Células Mieloides/metabolismo , Fagossomos/metabolismo , Fosfatidilinositol 3-Quinases/deficiência , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatidilinositóis/metabolismo , Transporte ProteicoRESUMO
Hereditary folate malabsorption (HFM) is an autosomal recessive disorder, recently shown to be due to loss-of-function mutations of the proton-coupled folate transporter (PCFT-SLC46A1), resulting in systemic and central nervous system folate deficiency. Data is emerging on the spectrum of PCFT mutations associated with this disorder. In this report, novel mutations are described in three subjects with HFM: A335D/N68Kfs (c.1004C>A/c.204-205delCC), compound heterozygous mutations, and two homozygous PCFT mutations, G338R (c.1012G>C) and E9Gfs (c.17-18insC). Functional assessment of A335D and G338R PCFT mutants transfected into folate transporter-deficient HeLa R1-11 cells indicated a complete loss of transport activity. There were neurological deficiencies in two of the families reported; in particular, late-onset seizures. The importance of early diagnosis and treatment to achieve physiological cerebrospinal fluid folate levels is emphasized.
Assuntos
Síndromes de Malabsorção/genética , Mutação/genética , Transportador de Folato Acoplado a Próton/genética , Substituição de Aminoácidos , Sequência de Bases , Criança , Consanguinidade , Estudos de Associação Genética , Doenças Genéticas Inatas/genética , Doenças Genéticas Inatas/metabolismo , Genótipo , Células HeLa , Humanos , Masculino , Linhagem , Transportador de Folato Acoplado a Próton/metabolismo , Adulto JovemRESUMO
The proton-coupled folate transporter (PCFT; SLC46A1) mediates folate transport into enterocytes in the proximal small intestine; pcft loss-of-function mutations are the basis for hereditary folate malabsorption. The current study explored the roles of Asp residues in PCFT function. A novel, homozygous, loss-of-function mutation, D156Y, was identified in a child of Pakistani origin with hereditary folate malabsorption. Of the 6 other conserved Asp residues, only one, D109, is shown to be required for function. D156Y, along with a variety of other substitutions at this site (Trp, Phe, Val, Asn, or Lys), lacked function due to instability of the PCFT protein. Substantial function was preserved with Glu, Gly, and, to a lesser extent, with Ser, Thr, and Ala substitutions. This correlated with PCFT bio-tinylated at the cell surface. In contrast, all D109 mutants, including D109E, lacked function irrespective of pH (4.5, 5.5, and 7.4) or substrate concentration (0.5-100 µM), despite surface expression comparable to wild-type PCFT. Hence, D156 plays a critical role in PCFT protein stability, and D109, located in the first intracellular loop between the second and third transmembrane domains, is absolutely required for PCFT function.
Assuntos
Ácido Aspártico , Síndromes de Malabsorção/genética , Mutação de Sentido Incorreto , Transportador de Folato Acoplado a Próton/genética , Substituição de Aminoácidos , Criança , Análise Mutacional de DNA , Ácido Fólico/metabolismo , Homozigoto , Humanos , Recém-Nascido , Paquistão , Estabilidade ProteicaRESUMO
Recently, this laboratory identified a proton-coupled folate transporter (PCFT), with optimal activity at low pH. PCFT is critical to intestinal folate absorption and transport into the central nervous system because there are loss-of-function mutations in this gene in the autosomal recessive disorder, hereditary folate malabsorption. The current study addresses the role PCFT might play in another transport pathway, folate receptor (FR)-mediated endocytosis. FRalpha cDNA was transfected into novel PCFT(+) and PCFT(-) HeLa sublines. FRalpha was shown to bind and trap folates in vesicles but with minimal export into the cytosol in PCFT(-) cells. Cotransfection of FRalpha and PCFT resulted in enhanced folate transport into cytosol as compared with transfection of FRalpha alone. Probenecid did not inhibit folate binding to FR, but inhibited PCFT-mediated transport at endosomal pH, and blocked FRalpha-mediated transport into the cytosol. FRalpha and PCFT co-localized to the endosomal compartment. These observations (i) indicate that PCFT plays a role in FRalpha-mediated endocytosis by serving as a route of export of folates from acidified endosomes and (ii) provide a functional role for PCFT in tissues in which it is expressed, such as the choroid plexus, where the extracellular milieu is at neutral pH.
Assuntos
Proteínas de Transporte/metabolismo , Citosol/metabolismo , Endocitose/fisiologia , Endossomos/metabolismo , Ácido Fólico/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Receptores de Superfície Celular/metabolismo , Transporte Biológico Ativo/efeitos dos fármacos , Transporte Biológico Ativo/fisiologia , Proteínas de Transporte/genética , Endossomos/genética , Receptores de Folato com Âncoras de GPI , Ácido Fólico/genética , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Absorção Intestinal/efeitos dos fármacos , Absorção Intestinal/genética , Síndromes de Malabsorção/genética , Síndromes de Malabsorção/metabolismo , Proteínas de Membrana Transportadoras/genética , Erros Inatos do Metabolismo/genética , Erros Inatos do Metabolismo/metabolismo , Probenecid/farmacologia , Transportador de Folato Acoplado a Próton , Receptores de Superfície Celular/genética , Uricosúricos/farmacologiaRESUMO
We report 2 sequential homozygous mutations in the recently cloned proton-coupled folate transporter (PCFT) gene, resulting in the absence of this protein, in a 27-year-old woman with hereditary folate malabsorption, normal in all respects having completed higher education, who has been treated with parenteral 5-formyltetrahydrofolate since infancy.
Assuntos
Ácido Fólico/metabolismo , Síndromes de Malabsorção/genética , Proteínas de Membrana Transportadoras/genética , Mutação , RNA Mensageiro/genética , Adulto , Northern Blotting , Western Blotting , Análise Mutacional de DNA , Éxons , Feminino , Seguimentos , Humanos , Infusões Parenterais , Leucovorina/administração & dosagem , Leucovorina/uso terapêutico , Síndromes de Malabsorção/tratamento farmacológico , Síndromes de Malabsorção/metabolismo , Proteínas de Membrana Transportadoras/biossíntese , Transportador de Folato Acoplado a Próton , Complexo Vitamínico B/administração & dosagem , Complexo Vitamínico B/uso terapêuticoRESUMO
We studied seven children with CNS folate deficiency (CFD). All cases exhibited psychomotor retardation, regression, cognitive delay, and dyskinesia; six had seizures; four demonstrated neurological abnormalities in the neonatal period. Two subjects had profound neurological abnormalities that precluded formal behavioral testing. Five subjects received ADOS and ADI-R testing and met diagnostic criteria for autism or autism spectrum disorders. They exhibited difficulties with transitions, insistence on sameness, unusual sensory interests, and repetitive behaviors. Those with the best language skills largely used repetitive phrases. No mutations were found in folate transporter or folate enzyme genes. These findings demonstrate that autistic features are salient in CFD and suggest that a subset of children with developmental regression, mental retardation, seizures, dyskinesia, and autism may have CNS folate abnormalities.
Assuntos
Transtorno Autístico/diagnóstico , Transtorno Autístico/fisiopatologia , Encéfalo/fisiopatologia , Deficiências do Desenvolvimento/diagnóstico , Deficiências do Desenvolvimento/fisiopatologia , Discinesias/diagnóstico , Discinesias/fisiopatologia , Epilepsia/diagnóstico , Epilepsia/fisiopatologia , Deficiência de Ácido Fólico/diagnóstico , Deficiência de Ácido Fólico/fisiopatologia , Deficiência Intelectual/diagnóstico , Deficiência Intelectual/fisiopatologia , Regressão Psicológica , Adolescente , Transtorno Autístico/psicologia , Criança , Pré-Escolar , Deficiências do Desenvolvimento/psicologia , Discinesias/psicologia , Epilepsia/psicologia , Feminino , Ácido Fólico/líquido cefalorraquidiano , Deficiência de Ácido Fólico/psicologia , Humanos , Lactente , Recém-Nascido , Deficiência Intelectual/psicologia , Masculino , Gravidez , Transtornos Psicomotores/diagnóstico , Transtornos Psicomotores/fisiopatologia , Transtornos Psicomotores/psicologia , Valores de Referência , Tetra-Hidrofolatos/líquido cefalorraquidianoRESUMO
Pemetrexed is a new generation antifolate approved for the treatment of mesothelioma and non-small cell lung cancer. Caffeine is known to augment radiation or chemotherapeutic drug-induced cell killing. The current study addresses the impact of caffeine on the activity of pemetrexed in mesothelioma cell lines. Caffeine enhanced pemetrexed activity in all four mesothelioma cell lines tested (H2052, H2373, H28 and MSTO-211H). Caffeine sensitized H2052 cells in a dose- and schedule-dependent manner, and was associated with a markedly decreased clonogenic survival. Caffeine sensitization occurred only in cells subjected to pulse, but not continuous, exposure to pemetrexed. Similar pemetrexed sensitization was also observed with the clinically better tolerated caffeine analog, theobromine. Pemetrexed sensitization by caffeine was associated with an increase in pemetrexed-induced phosphorylation of ataxia-telangiectasia-mutated (ATM) and Chk1. These data indicate that caffeine and its analog, theobromine, may be a useful approach to enhance pemetrexed-based chemotherapy.
Assuntos
Cafeína/farmacologia , Antagonistas do Ácido Fólico/farmacologia , Glutamatos/farmacologia , Guanina/análogos & derivados , Mesotelioma/tratamento farmacológico , Proteínas Mutadas de Ataxia Telangiectasia , Cafeína/administração & dosagem , Proteínas de Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Quinase 1 do Ponto de Checagem , Proteínas de Ligação a DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Relação Dose-Resposta a Droga , Esquema de Medicação , Antagonistas do Ácido Fólico/administração & dosagem , Glutamatos/administração & dosagem , Guanina/administração & dosagem , Guanina/farmacologia , Humanos , Pemetrexede , Fosforilação/efeitos dos fármacos , Proteínas Quinases/efeitos dos fármacos , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , Teobromina/farmacologia , Proteínas Supressoras de Tumor/efeitos dos fármacos , Proteínas Supressoras de Tumor/metabolismoRESUMO
This laboratory recently identified a human gene that encodes a novel folate transporter [Homo sapiens proton-coupled folate transporter (HsPCFT); SLC46A1] required for intestinal folate absorption. This study focused on mouse (Mus musculus) PCFT (MmPCFT) and rat (Rattus norvegicus) PCFT (RnPCFT) and addresses their secondary structure, specificity, tissue expression, and regulation by dietary folates. Both rodent PCFT proteins traffic to the cell membrane with the NH(2)- and COOH-termini accessible to antibodies targeted to these domains only in permeabilized HeLa cells. This, together with computer-based topological analyses, is consistent with a model in which rodent PCFT proteins likely contain 12 transmembrane domains. Transport of [(3)H]folates was optimal at pH 5.5 and decreased with increasing pH due to an increase in K(m) and a decrease in V(max). At pH 7.0, folic acid and methotrexate influx was negligible, but there was residual (6S)5-methyltetrahydrofolate transport. Uptake of folates in PCFT-injected Xenopus oocytes was electrogenic and pH dependent. Folic acid influx K(m) values of MmPCFT and RnPCFT, assessed electrophysiologically, were 0.7 and 0.3 microM at pH 5.5 and 1.1 and 0.8 microM at pH 6.5, respectively. Rodent PCFTs were highly specific for monoglutamyl but not polyglutamyl methotrexate. MmPCFT mRNA was highly expressed in the duodenum, proximal jejunum, liver, and kidney with lesser expression in the brain and other tissues. MmPCFT protein was localized to the apical brush-border membrane of the duodenum and proximal jejunum. MmPCFT mRNA levels increased approximately 13-fold in the proximal small intestine in mice fed a folate-deficient vesus folate-replete diet, consistent with the critical role that PCFT plays in intestinal folate absorption.
Assuntos
Proteínas de Transporte de Ânions/metabolismo , Membrana Celular/metabolismo , Antagonistas do Ácido Fólico/metabolismo , Deficiência de Ácido Fólico/metabolismo , Ácido Fólico/metabolismo , Intestino Delgado/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Transporte de Ânions/química , Proteínas de Transporte de Ânions/genética , Modelos Animais de Doenças , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Cinética , Masculino , Potenciais da Membrana , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/genética , Metotrexato/análogos & derivados , Metotrexato/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microvilosidades/metabolismo , Dados de Sequência Molecular , Oócitos , Ácido Poliglutâmico/análogos & derivados , Ácido Poliglutâmico/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Transporte Proteico , Transportador de Folato Acoplado a Próton , RNA Mensageiro/metabolismo , Ratos , Tetra-Hidrofolatos/metabolismo , XenopusRESUMO
Hereditary folate malabsorption (HFM) is a rare autosomal recessive disorder caused by impaired intestinal folate absorption and impaired folate transport into the central nervous system. Recent studies in 1 family revealed that the molecular basis for this disorder is a loss-of-function mutation in the PCFT gene encoding a proton-coupled folate transporter. The current study broadens the understanding of the spectrum of alterations in the PCFT gene associated with HFM in 5 additional patients. There was no racial, ethnic, or sex pattern. A total of 4 different homozygous mutations were detected in 4 patients; 2 heterozygous mutations were identified in the fifth patient. Mutations involved 4 of the 5 exons, all at highly conserved amino acid residues. A total of 4 of the mutated transporters resulted in a complete loss of transport function, primarily due to decreased protein stability and/or defects in membrane trafficking, while 2 of the mutated carriers manifested residual function. Folate transport at low pH was markedly impaired in transformed lymphocytes from 2 patients. These findings further substantiate the role that mutations in PCFT play in the pathogenesis of HFM and will make possible rapid diagnosis and treatment of this disorder in infants, and prenatal diagnosis in families that carry a mutated gene.
Assuntos
Ácido Fólico/metabolismo , Síndromes de Malabsorção/genética , Proteínas de Membrana Transportadoras/genética , Mutação , Sequência de Aminoácidos , Northern Blotting , Western Blotting , Estudos de Casos e Controles , Pré-Escolar , Análise Mutacional de DNA , Feminino , Imunofluorescência , Células HeLa , Heterozigoto , Humanos , Lactente , Mucosa Intestinal/metabolismo , Linfócitos/efeitos dos fármacos , Linfócitos/metabolismo , Masculino , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Linhagem , Reação em Cadeia da Polimerase , Transportador de Folato Acoplado a PrótonRESUMO
Folates are essential nutrients that are required for one-carbon biosynthetic and epigenetic processes. While folates are absorbed in the acidic milieu of the upper small intestine, the underlying absorption mechanism has not been defined. We now report the identification of a human proton-coupled, high-affinity folate transporter that recapitulates properties of folate transport and absorption in intestine and in various cell types at low pH. We demonstrate that a loss-of-function mutation in this gene is the molecular basis for hereditary folate malabsorption in a family with this disease. This transporter was previously reported to be a lower-affinity, pH-independent heme carrier protein, HCP1. However, the current study establishes that a major function of this gene product is proton-coupled folate transport required for folate homeostasis in man, and we have thus amended the name to PCFT/HCP1.