RESUMO
The proton-coupled folate transporter (PCFT, SLC46A1) is required for folate intestinal absorption and transport across the choroid plexus. Recent work has identified a F392V mutation causing hereditary folate malabsorption. However, the residue properties responsible for this loss of function remains unknown. Using site-directed mutagenesis, we observed complete loss of function with charged (Lys, Asp, and Glu) and polar (Thr, Ser, and Gln) Phe-392 substitutions and minimal function with some neutral substitutions; however, F392M retained full function. Using the substituted-cysteine accessibility method (with N-biotinyl aminoethyl methanethiosulfonate labeling), Phe-392 mutations causing loss of function, although preserving membrane expression and trafficking, also resulted in loss of accessibility of the substituted cysteine in P314C-PCFT located within the aqueous translocation pathway. F392V function and accessibility of the P314C cysteine were restored by insertion of a G305L (suppressor) mutation. A S196L mutation localized in proximity to Gly-305 by homology modeling was inactive. However, when inserted into the inactive F392V scaffold, function was restored (mutually compensatory mutations), as was accessibility of the P314C cysteine residue. Reduced function, documented with F392H PCFT, was due to a 15-fold decrease in methotrexate influx Vmax, accompanied by a decreased influx Kt (4.5-fold) and Ki (3-fold). The data indicate that Phe-392 is required for rapid oscillation of the carrier among its conformational states and suggest that this is achieved by dampening affinity of the protein for its folate substrates. F392V and other inactivating Phe-392 PCFT mutations lock the protein in its inward-open conformation. Reach (length) and hydrophobicity of Phe-392 appear to be features required for full activity.
Assuntos
Transportador de Folato Acoplado a Próton/metabolismo , Sequência de Aminoácidos , Animais , Transporte Biológico , Cisteína/química , Cisteína/metabolismo , Deficiência de Ácido Fólico/patologia , Células HeLa , Humanos , Cinética , Síndromes de Malabsorção/patologia , Metotrexato/metabolismo , Mutagênese Sítio-Dirigida , Estrutura Terciária de Proteína , Transportador de Folato Acoplado a Próton/química , Transportador de Folato Acoplado a Próton/genéticaRESUMO
The proton-coupled folate transporter (PCFT) mediates intestinal absorption of folates and their transport from blood to cerebrospinal fluid across the choroid plexus. Substitutions at Asp-109 in the first intracellular loop between the first and second transmembrane domains (TMDs) abolish PCFT function, but protein expression and trafficking to the cell membrane are retained. Here, we used site-directed mutagenesis, the substituted-cysteine accessibility method, functional analyses, and homology modeling to determine whether the D109A substitution locks PCFT in one of its conformational states. Cys-substituted residues lining the PCFT aqueous translocation pathway and accessible in WT PCFT to the membrane-impermeable cysteine-biotinylation reagent, MTSEA-biotin, lost accessibility when introduced into the D109A scaffold. Substitutions at Gly-305 located exofacially within the eighth TMD, particularly with bulky residues, when introduced into the D109A scaffold largely restored function and MTSEA-biotin accessibility to Cys-substituted residues within the pathway. Likewise, Ser-196 substitution in the fifth TMD, predicted by homology modeling to be in proximity to Gly-305, also partially restored function found in solute transporters, is critical to oscillation of the carrier among its conformational states. Substitutions at Asp-109 and Gly-112 lock PCFT in an inward-open conformation, resulting in the loss of function. However, the integrity of the locked protein is preserved, indicated by the restoration of function after insertion of a second "unlocking" mutation. and accessibility. Similarly, the inactivating G112K substitution within the first intracellular loop was partially reactivated by introducing the G305L substitution. These data indicate that the first intracellular loop, with a sequence identical to "motif A" (GXXXDXXGR(R/K)).
Assuntos
Transportador de Folato Acoplado a Próton/metabolismo , Cisteína/química , Glicina/metabolismo , Células HeLa , Humanos , Cinética , Mutação , Conformação Proteica , Transportador de Folato Acoplado a Próton/químicaRESUMO
Hereditary folate malabsorption (HFM) is an autosomal recessive disease caused by mutations in SLC46A1 encoding the proton-coupled folate transporter (PCFT). HFM patients present with various clinical features including megaloblastic anemia, thrombocytopenia, combined immunodeficiency and neurodevelopmental disorders. In this study, we report the same deep intronic mutation of c.1166-285â¯Tâ¯>â¯G shared by four unrelated Japanese patients with HFM. This mutation was shown to generate a cryptic splice donor site for a 168-bp insertion of intron 3 sequences, leading to premature termination in the middle of this insertion. This mutation could be a founder mutation in the Japanese population, but also could be a hot-spot and could be present in undiagnosed HFM patients worldwide because of the difficulty to detect this mutation.
Assuntos
Deficiência de Ácido Fólico/genética , Síndromes de Malabsorção/genética , Transportador de Folato Acoplado a Próton/genética , Povo Asiático/genética , Feminino , Humanos , Lactente , Masculino , MutaçãoRESUMO
5-fluorouracil in combination with the folate leucovorin is the cornerstone in treatment of colorectal cancer. Transport of leucovorin into cells, and subsequent metabolic action, require expression of several genes. The aim was to analyze if tumoral expression of genes putatively involved in leucovorin transport, polyglutamation, or metabolism was associated with outcome of patients with stage III colorectal cancer treated with adjuvant chemotherapy. A total of 363 stage III colorectal cancer patients who received adjuvant bolus 5-fluorouracil + leucovorin alone, or in combination with oxaliplatin according to Nordic bolus regimes were included. Expression of 11 folate pathway genes was determined in tumors using quantitative real-time polymerase chain reaction and related to disease-free survival. The median follow-up time was 5 years. During follow-up, 114 (31%) patients suffered from recurrent disease. A high tumoral expression of the genes SLC46A1/PCFT, SLC19A1/RFC-1, ABCC3/MRP3, GGH, and MTHFD1L, which are involved in folate transport, polyglutamation, or metabolism, was associated with longer disease-free survival of the patients. Each of these genes either encodes mitochondrial enzymes or is being regulated by mitochondrial transcription factors. Expression of the SLC46A1/PCFT gene was most strongly associated with disease-free survival, regardless of treatment regimen. In conclusion, the results show that expression of folate pathway genes are associated with outcome of colorectal cancer patients treated with adjuvant 5-fluorouracil in combination with leucovorin. A prospective study needs to be conducted to determine if expression of these genes can be used to predict response to leucovorin and other folates that are now being tested in clinical studies. Moreover, there seems to be a link between folate metabolism and mitochondrial biogenesis and respiration that deserves further exploration.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biomarcadores Tumorais/genética , Respiração Celular , Neoplasias Colorretais/genética , Neoplasias Colorretais/mortalidade , Ácido Fólico/genética , Biogênese de Organelas , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias Colorretais/tratamento farmacológico , Feminino , Fluoruracila/administração & dosagem , Seguimentos , Perfilação da Expressão Gênica , Humanos , Leucovorina/administração & dosagem , Masculino , Pessoa de Meia-Idade , Oxaliplatina/administração & dosagem , Prognóstico , Estudos Retrospectivos , Taxa de Sobrevida , Adulto JovemRESUMO
Cerebral folate deficiency is typically defined as a deficiency of the major folate species 5-methyltetrahydrofolate in the cerebrospinal fluid (CSF) in the presence of normal peripheral total folate levels. However, it should be noted that cerebral folate deficiency is also often used to describe conditions where CSF 5-MTHF is low, in the presence of low or undefined peripheral folate levels. Known defects of folate transport are deficiency of the proton coupled folate transporter, associated with systemic as well as cerebral folate deficiency, and deficiency of the folate receptor alpha, leading to an isolated cerebral folate deficiency associated with intractable seizures, developmental delay and/or regression, progressive ataxia and choreoathetoid movement disorders. Inborn errors of folate metabolism include deficiencies of the enzymes methylenetetrahydrofolate reductase, dihydrofolate reductase and 5,10-methenyltetrahydrofolate synthetase. Cerebral folate deficiency is potentially a treatable condition and so prompt recognition of these inborn errors and initiation of appropriate therapy is of paramount importance. Secondary cerebral folate deficiency may be observed in other inherited metabolic diseases, including disorders of the mitochondrial oxidative phosphorylation system, serine deficiency, and pyridoxine dependent epilepsy. Other secondary causes of cerebral folate deficiency include the effects of drugs, immune response activation, toxic insults and oxidative stress. This review describes the absorption, transport and metabolism of folate within the body; analytical methods to measure folate species in blood, plasma and CSF; inherited and acquired causes of cerebral folate deficiency; and possible treatment options in those patients found to have cerebral folate deficiency.
Assuntos
Encefalopatias Metabólicas Congênitas/diagnóstico , Epilepsia/diagnóstico , Deficiência de Ácido Fólico/diagnóstico , Ácido Fólico/uso terapêutico , Tetra-Hidrofolatos/deficiência , Encéfalo/patologia , Encefalopatias Metabólicas Congênitas/líquido cefalorraquidiano , Encefalopatias Metabólicas Congênitas/tratamento farmacológico , Encefalopatias Metabólicas Congênitas/genética , Diagnóstico Diferencial , Epilepsia/líquido cefalorraquidiano , Epilepsia/tratamento farmacológico , Epilepsia/genética , Receptor 1 de Folato/genética , Deficiência de Ácido Fólico/líquido cefalorraquidiano , Deficiência de Ácido Fólico/tratamento farmacológico , Deficiência de Ácido Fólico/genética , Humanos , Tetra-Hidrofolatos/líquido cefalorraquidianoRESUMO
The human proton coupled folic acid transporter PCFT is the major import route for dietary folates. Mutations in the gene encoding PCFT cause hereditary folic acid malabsorption, which manifests itself by compromised folate absorption from the intestine and also in impaired folate transport into the central nervous system. Since its recent discovery, PCFT has been the subject of numerous biochemical studies aiming at understanding its structure and mechanism. One major focus has been its oligomeric state, with some reports supporting oligomers and others a monomer. Here, we report the overexpression and purification of recombinant PCFT. Following detergent screening, n-Dodecyl ß-D-maltoside (DDM) and lauryl maltose neopentyl glycol (LMNG) were chosen for further work as they exhibited the most optimal solubilization. We found that purified detergent solubilized PCFT was able to bind folic acid, thus indicating a functionally active protein. Size exclusion chromatography showed that PCFT in DDM was polydisperse; the LMNG preparation was clearly monodisperse but with shorter retention time than the major DDM peak. To assess the oligomeric state negative stain electron microscopy was performed which showed a particle with the size of a PCFT dimer.
Assuntos
Transportador de Folato Acoplado a Próton/química , Animais , Detergentes , Ácido Fólico/metabolismo , Glucosídeos , Glicóis , Humanos , Ligantes , Microscopia Eletrônica , Modelos Moleculares , Multimerização Proteica , Estrutura Quaternária de Proteína , Transportador de Folato Acoplado a Próton/metabolismo , Transportador de Folato Acoplado a Próton/ultraestrutura , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Células Sf9 , Solubilidade , SpodopteraRESUMO
The substituted cysteine accessibility method (SCAM) is widely used to study the structure and function of channels, receptors and transporters. In its usual application, a cysteine residue is introduced into a protein which lacks native cysteines following which the accessibility of the residue to the aqueous compartment is assessed. Implicit, and generally assumed, is that if the cysteine-substituted residue is not available to react with sulfhydryl reagents it is not exposed to the extracellular compartment or within the aqueous translocation pathway. We demonstrate here, in a Hela-derived cell line, that some cysteine-substituted residues of the proton-coupled folate transporter (PCFT, SLC46A1) that are inaccessible to 2-((biotinoyl)amino)ethyl methanethiosulfonate are glutathionylated by biotinylated glutathione ethyl ester in the absence of an oxidizing agent. Intramolecular disulfide formation involving cysteine-substituted residues was also identified in some instances. These posttranslational modifications limit the accessibility of the cysteine residues to sulfhydryl-reactive reagents and can have a profound impact on the interpretation of SCAM but may not alter function. When a posttranslationally modified residue is used as a reference extracellular control, the high level of exposure required for detection on Western blot results in erroneous detection of otherwise inaccessible intracellular cysteine-substituted residues. The data indicate that in the application of SCAM, when a cysteine-substituted residue does not appear to be accessible to sulfhydryl-reactive reagents, the possibility of a posttranslational modification should be excluded. The data explain the discrepancies in the assessment, and confirm the localization, of the first intracellular loop of PCFT.
Assuntos
Substituição de Aminoácidos/fisiologia , Cisteína/química , Cisteína/metabolismo , Glutationa/química , Glutationa/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Sítios de Ligação , Células HeLa , Humanos , Ligação Proteica , Engenharia de Proteínas/métodos , Relação Estrutura-AtividadeRESUMO
BACKGROUND: Thymidylate synthase (TS) has a predictive role in pemetrexed treatment of mesothelioma; however, additional chemoresistance mechanisms are poorly understood. Here, we explored the role of the reduced-folate carrier (RFC/SLC19A1) and proton-coupled folate transporter (PCFT/SLC46A1) in antifolate resistance in mesothelioma. PATIENTS AND METHODS: PCFT, RFC and TS RNA and PCFT protein levels were determined by quantitative RT-PCR of frozen tissues and immunohistochemistry of tissue-microarrays, respectively, in two cohorts of pemetrexed-treated patients. Data were analyzed by t-test, Fisher's/log-rank test and Cox proportional models. The contribution of PCFT expression and PCFT-promoter methylation to pemetrexed activity were evaluated in mesothelioma cells and spheroids, through 5-aza-2'-deoxycytidine-mediated demethylation and siRNA-knockdown. RESULTS: Pemetrexed-treated patients with low PCFT had significantly lower rates of disease control, and shorter overall survival (OS), in both the test (N = 73, 11.3 versus 20.1 months, P = 0.01) and validation (N = 51, 12.6 versus 30.3 months, P = 0.02) cohorts. Multivariate analysis confirmed PCFT-independent prognostic role. Low-PCFT protein levels were also associated with shorter OS. Patients with both low-PCFT and high-TS levels had the worst prognosis (OS, 5.5 months), whereas associations were neither found for RFC nor in pemetrexed-untreated patients. PCFT silencing reduced pemetrexed sensitivity, whereas 5-aza-2'-deoxycytidine overcame resistance. CONCLUSIONS: These findings identify for the first time PCFT as a novel mesothelioma prognostic biomarker, prompting prospective trials for its validation. Moreover, preclinical data suggest that targeting PCFT-promoter methylation might eradicate pemetrexed-resistant cells characterized by low-PCFT expression.
Assuntos
Biomarcadores Tumorais/metabolismo , Resistencia a Medicamentos Antineoplásicos , Mesotelioma/patologia , Pemetrexede/uso terapêutico , Neoplasias Pleurais/patologia , Transportador de Folato Acoplado a Próton/metabolismo , Proteína Carregadora de Folato Reduzido/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Proliferação de Células/efeitos dos fármacos , Feminino , Antagonistas do Ácido Fólico/uso terapêutico , Seguimentos , Humanos , Técnicas Imunoenzimáticas , Masculino , Mesotelioma/tratamento farmacológico , Mesotelioma/metabolismo , Pessoa de Meia-Idade , Neoplasias Pleurais/tratamento farmacológico , Neoplasias Pleurais/metabolismo , Prognóstico , Taxa de Sobrevida , Timidilato Sintase/metabolismo , Células Tumorais CultivadasRESUMO
The proton-coupled folate transporter (PCFT) mediates folate absorption across the brush-border membrane of the proximal small intestine and is required for folate transport across the choroid plexus into the cerebrospinal fluid. In this study, the functional role and accessibility of the seven PCFT Trp residues were assessed by the substituted-cysteine accessibility method. Six Trp residues at a lipid-aqueous interface tolerated Cys substitution in terms of protein stability and function. W85C, W202C, and W213C were accessible to N-biotinyl aminoethylmethanethiosulfonate; W48C and W299C were accessible only after treatment with dithiotreitol (DTT), consistent with modification of these residues by an endogenous thiol-reacting molecule and their extracellular location. Neither W107C nor W333C was accessible (even after DTT) consistent with their cytoplasmic orientation. Biotinylation was blocked by pemetrexed only for the W48C (after DTT), W85C, W202C residues. Function was impaired only for the W299C PCFT mutant located in the 4th external loop between the 7th and 8th transmembrane helices. Despite its aqueous location, function could only be fully preserved with Phe and, to a lesser extent, Ala substitutions. There was a 6.5-fold decrease in the pemetrexed influx Vmax and a 3.5- and 6-fold decrease in the influx Kt and Ki, respectively, for the W299S PCFT. The data indicate that the hydrophobicity of the W299 residue is important for function suggesting that during the transport cycle this residue interacts with the lipid membrane thereby impacting on the oscillation of the carrier and, indirectly, on the folate binding pocket.
Assuntos
Membrana Celular/metabolismo , Ácido Fólico/metabolismo , Transportador de Folato Acoplado a Próton/metabolismo , Sítios de Ligação , Biotinilação , Membrana Celular/efeitos dos fármacos , Cisteína , Antagonistas do Ácido Fólico/farmacologia , Genótipo , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Cinética , Mutagênese Sítio-Dirigida , Mutação , Pemetrexede/farmacologia , Fenótipo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Domínios Proteicos , Estabilidade Proteica , Transportador de Folato Acoplado a Próton/química , Transportador de Folato Acoplado a Próton/efeitos dos fármacos , Transportador de Folato Acoplado a Próton/genética , Relação Estrutura-Atividade , Transfecção , TriptofanoRESUMO
PURPOSE: In Caco-2 cells, folate uptake via the proton-coupled folate transporter (PCFT) increases significantly by a 3-day treatment with 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). Additionally, mRNA content and protein expression of the transporter OATP1A2 were increased up to ninefold with 1,25(OH)2D3. We investigated whether these in vitro findings can be confirmed in humans in vivo. METHODS: Ten healthy volunteers (six women) received 5 mg folic acid orally once before and once together with the last intake of a 10-day course of 0.5 µg 1,25(OH)2D3 orally. One hundred twenty milligrams fexofenadine, an OATP1A2 substrate, was taken in 1 day before the first folic acid intake, and again on the ninth day of 1,25(OH)2D3 intake. Duodenal biopsies were taken for transporter mRNA assessments once before and once on the ninth or tenth day of the vitamin D3 course. Serum folic acid and fexofenadine concentrations were quantified with a chemiluminescence immunoassay and LC-MS/MS, respectively. Pharmacokinetics were compared between periods with standard bioequivalence approaches. RESULTS: While geometric mean folic acid AUC0-2h, which mainly reflects absorption, was 0.403 and 0.414 mg/L·h before and after the vitamin D3 course (geometric mean ratio (GMR), 1.027; 90 % confidence interval (90 % CI), 0.788-1.340), the geometric mean fexofenadine AUC0-2h was 1.932 and 2.761 mg/L·h, respectively (GMR, 1.429; 90 % CI, 0.890-2.294). PCFT- and OATP1A2-mRNA expressions in duodenal biopsies were essentially unchanged. CONCLUSIONS: No significant changes in folic acid and fexofenadine absorption were observed after a 10-day course of 1,25(OH)2D3 in humans in vivo. This study underlines the importance of confirming in vitro findings in vivo in humans.
Assuntos
Ácido Fólico/farmacocinética , Absorção Intestinal/efeitos dos fármacos , Terfenadina/análogos & derivados , Vitamina D/análogos & derivados , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Administração Oral , Adulto , Duodeno/efeitos dos fármacos , Duodeno/metabolismo , Feminino , Ácido Fólico/sangue , Voluntários Saudáveis , Humanos , Masculino , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Transportadores de Ânions Orgânicos/genética , Transportador de Folato Acoplado a Próton/genética , RNA Mensageiro/metabolismo , Terfenadina/sangue , Terfenadina/farmacocinética , Vitamina D/farmacologia , Adulto JovemRESUMO
The proton-coupled folate transporter (PCFT) mediates intestinal folate absorption and transport of folates across the choroid plexus. This study focuses on the role of Tyr residues in PCFT function. The substituted Cys-accessibility method identified four Tyr residues (Y291, Y362, Y315, and Y414) that are accessible to the extracellular compartment; three of these (Y291, Y362, and Y315) are located within or near the folate binding pocket. When the Tyr residues were replaced with Cys or Ala, these mutants showed similar (up to 6-fold) increases in influx Vmax and Kt/Ki for [(3)H]methotrexate and [(3)H]pemetrexed. When the Tyr residues were replaced with Phe, these changes were moderated or absent. When Y315A PCFT was used as representative of the mutants and [(3)H]pemetrexed as the transport substrate, this substitution did not increase the efflux rate constant. Furthermore, neither influx nor efflux mediated by Y315A PCFT was transstimulated by the presence of substrate in the opposite compartment; however, substantial bidirectional transstimulation of transport was mediated by wild-type PCFT. This resulted in a threefold greater efflux rate constant for cells that express wild-type PCFT than for cells that express Y315 PCFT under exchange conditions. These data suggest that these Tyr residues, possibly through their rigid side chains, secure the carrier in a high-affinity state for its folate substrates. However, this may be achieved at the expense of constraining the carrier's mobility, thereby decreasing the rate at which the protein oscillates between its conformational states. The Vmax generated by these Tyr mutants may be so rapid that further augmentation during transstimulation may not be possible.
Assuntos
Ácido Fólico/metabolismo , Absorção Intestinal/fisiologia , Transportador de Folato Acoplado a Próton/metabolismo , Tirosina/química , Sequência de Aminoácidos , Substituição de Aminoácidos , Sítios de Ligação/genética , Transporte Biológico/genética , Linhagem Celular Tumoral , Glutamatos/metabolismo , Guanina/análogos & derivados , Guanina/metabolismo , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Absorção Intestinal/genética , Metotrexato/metabolismo , Modelos Moleculares , Pemetrexede , Estrutura Terciária de Proteína , Transportador de Folato Acoplado a Próton/genéticaRESUMO
Folates are essential to the maintenance of normal life activities in almost all organisms. Proton-coupled folate transporter (PCFT), belonging to the major facilitator superfamily, is one of the three major folate transporter types widely studied in mammals. However, information about plant PCFTs is limited. Here, a genome-wide identification of maize PCFTs was performed, and two PCFTs, ZmMFS_1-62 and ZmMFS_1-73, were functionally investigated. Both proteins contained the typical 12 transmembrane helixes with N- and C-termini located in the cytoplasm, and were localized in the plasma membrane. Molecular docking analysis indicated their binding activity with folates via hydrogen bonding. Interference with ZmMFS_1-62 and ZmMFS_1-73 in maize seedlings through virus-induced gene silencing disrupted folate homeostasis, mainly in the roots, and reduced tolerance to drought and salt stresses. Moreover, a molecular chaperone protein, ZmHSP20, was found to interact with ZmMFS_1-62 and ZmMFS_1-73, and interference with ZmHSP20 in maize seedlings also led to folate disruption and increased sensitivity to drought and salt stresses. Overall, this is the first report of functional identification of maize PCFTs, which play essential roles in salt and drought stress tolerance, thereby linking folate metabolism with abiotic stress responses in maize.
Assuntos
Secas , Proteínas de Plantas , Transportador de Folato Acoplado a Próton , Zea mays , Zea mays/metabolismo , Zea mays/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Transportador de Folato Acoplado a Próton/metabolismo , Transportador de Folato Acoplado a Próton/genética , Ácido Fólico/metabolismo , Tolerância ao Sal/genética , Regulação da Expressão Gênica de Plantas , Resistência à SecaRESUMO
A large percentage (~60%) of prescription drugs and new molecular entities are designed for oral delivery, which requires passage through a semi-impervious membrane bilayer in the gastrointestinal wall. Passage through this bilayer can be dependent on membrane transporters that regulate the absorption of nutrients or endogenous substrates. Several investigations have provided links between nutrient, endogenous substrate, or drug absorption and the activity of certain membrane transporters. This knowledge has been key in the development of new therapeutics that can alleviate various symptoms of select diseases, such as cholestasis and diabetes. Despite this progress, recent studies revealed potential clinical dangers of unintended altered nutrient or endogenous substrate disposition due to the drug-mediated disruption of intestinal transport activity. This review outlines reports of glucose, folate, thiamine, lactate, and bile acid (re)absorption changes and consequent adverse events as examples. Finally, the need to comprehensively expand research on intestinal transporter-mediated drug interactions to avoid the unwanted disruption of homeostasis and diminish therapeutic adverse events is highlighted.
RESUMO
Genitourinary cancers comprise of a heterogenous group of cancers of which renal cell carcinoma, urothelial bladder carcinoma, and prostate adenocarcinoma are the most commonly encountered subtypes. A lot of research is ongoing using various strategies for exploration of novel biomarkers for genitourinary cancers. These biomarkers would not reduce the need for invasive diagnostic techniques but also could be used for early and accurate diagnosis to improve the clinical management required for the disease. Moreover, selecting the appropriate treatment regimen for the responsive patients based on these biomarkers would reduce the treatment toxicity as well as cost. Biomarkers identified using various advanced techniques like next generation sequencing and proteomics, which have been classified as immunological biomarkers, tissue-specific biomarkers and liquid biomarkers. Immunological biomarkers include markers of immunological pathways such as CTLA4, PD-1/PDl-1, tissue biomarkers include tissue specific molecules such as PSA antigen and liquid biomarkers include biomarkers detectable in urine, circulating cells etc. The purpose of this review is to provide a brief introduction to the most prevalent genitourinary malignancies, including bladder, kidney, and prostate cancers along with a major focus on the novel diagnostic biomarkers and the importance of targeting them prior to genitourinary cancers treatment. Understanding these biomarkers and their potential in diagnosis of genitourinary cancer would not help in early and accurate diagnosis as mentioned above but may also lead towards a personalized approach for better diagnosis, prognosis and specified treatment approach for an individual.
RESUMO
PURPOSE: The aim of study was to investigate the relationship between folate concentration and expression of folate-associated genes in tumour, mucosa and plasma of patients with colorectal cancer, after intraoperative administration of bolus leucovorin (LV). METHODS: Eighty patients were randomized into four groups to receive 0, 60, 200, or 500 mg/m2 LV, respectively. Tissue and plasma folate concentrations were assessed by LC-MS/MS. Gene expression of ABCC3/MRP3, FPGS, GGH, MTHFD1L, SLC46A1/PCFT, and SLC19A1/RFC-1 was determined using quantitative PCR. RESULTS: The folate concentration in tumour increased with increasing dosage of LV. Half of the patients treated with 60 mg/m2 did not reach a level above the levels of untreated patients. A significant correlation between folate concentration in tumour and mucosa was found in untreated patients, and in the group treated with 60 mg/m2 LV. The 5-MTHF/LV ratio correlated negatively with folate concentration in mucosa, whereas a positive correlation was found in tumour of patients who received 200 or 500 mg/m2 LV. A positive correlation was found between folate concentration and expression of all genes, except MTHFD1L, in patients who received LV. There was a negative correlation between 5-MTHF concentration in plasma of untreated patients and expression of GGH and SLC46A1/PCFT in tumour. CONCLUSIONS: The results indicate the possibility of using the individual plasma 5-MTHF/LV ratio after LV injection as a surrogate marker for tissue folate concentration. Expression of several folate-associated genes is associated with folate concentration in tissue and plasma and may become useful when predicting response to LV treatment.
Assuntos
Neoplasias Colorretais/cirurgia , Cuidados Intraoperatórios , Leucovorina/administração & dosagem , Leucovorina/sangue , Tetra-Hidrofolatos , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias Colorretais/sangue , Neoplasias Colorretais/metabolismo , Relação Dose-Resposta a Droga , Feminino , Perfilação da Expressão Gênica , Humanos , Injeções Intravenosas , Masculino , Pessoa de Meia-Idade , Tetra-Hidrofolatos/sangue , Tetra-Hidrofolatos/genéticaRESUMO
BACKGROUND: Folate plays a fundamental role for fetal development, participating in cell division, embryogenesis, and fetal growth. The fetus depends on maternal supply of folate across the placenta. The objective of this study was to compare the expression of Folate Receptor-α (FR-α), Reduced Folate Carrier (RFC), and Proton Coupled Folate Transporter (PCFT) in placentas from pregnancies complicated with birth defects (BD) and controls. METHODS: Case-control study, including placentas of BD-complicated pregnancies (n = 25) and a control group (n = 25). We determined the placental expression of FR-α, RFC, and PCFT by immunohistochemistry. Optical density was measured to obtain a relative quantification of the expression. RESULTS: The expression of PCFT was greater in placentas from pregnancies complicated with BD than in those from the control group (p < .01). The expression of FR-α and RFC was not different between groups. CONCLUSION: The expression of PCFT in placentas from BD-complicated pregnancies is increased, possibly as an adaptive response to increase the folate flux at the maternal-fetal interface.
Assuntos
Transportadores de Ácido Fólico/genética , Ácido Fólico/metabolismo , Placenta/metabolismo , Adulto , Estudos de Casos e Controles , Anormalidades Congênitas/fisiopatologia , Feminino , Receptor 1 de Folato/análise , Receptor 1 de Folato/metabolismo , Transportadores de Ácido Fólico/metabolismo , Humanos , Imuno-Histoquímica , Gravidez , Complicações na Gravidez , Transportador de Folato Acoplado a Próton/análise , Transportador de Folato Acoplado a Próton/metabolismo , Proteína Carregadora de Folato Reduzido/metabolismoRESUMO
The proton-coupled folate transporter (PCFT-SLC46A1) is the mechanism by which folates are absorbed across the brush-border membrane of the small intestine. The transporter is also expressed in the choroid plexus and is required for transport of folates into the cerebrospinal fluid. Loss of PCFT function, as occurs in the autosomal recessive disorder "hereditary folate malabsorption" (HFM), results in a syndrome characterized by severe systemic and cerebral folate deficiency. Folate-receptor alpha (FRα) is expressed in the choroid plexus, and loss of function of this protein, as also occurs in an autosomal recessive disorder, results solely in "cerebral folate deficiency" (CFD), the designation for this disorder. This paper reviews the current understanding of the functional and structural properties and regulation of PCFT, an electrogenic proton symporter, and contrasts PCFT properties with those of the reduced folate carrier (RFC), an organic anion antiporter, that is the major route of folate transport to systemic tissues. The clinical characteristics of HFM and its treatment, based upon the thirty-seven known cases with the clinical syndrome, of which thirty have been verified by genotype, are presented. The ways in which PCFT and FRα might interact at the level of the choroid plexus such that each is required for folate transport from blood to cerebrospinal fluid are considered along with the different clinical presentations of HFM and CFD.
Assuntos
Deficiência de Ácido Fólico/metabolismo , Síndromes de Malabsorção/metabolismo , Transportador de Folato Acoplado a Próton/metabolismo , Animais , Transporte Biológico , Deficiência de Ácido Fólico/líquido cefalorraquidiano , Humanos , Absorção Intestinal , Síndromes de Malabsorção/líquido cefalorraquidiano , Modelos Moleculares , Transportador de Folato Acoplado a Próton/químicaRESUMO
BACKGROUND: Adequate folate levels are essential for successful pregnancy outcomes. We aimed to study the relationship between placental mRNA and protein levels of folate transporters to birth weight. METHODS: Placental folate transporters (FOLR1, RFC1 and HCP1/PCFT) mRNA and protein levels in basal (BP) and chorionic plate (CP) of small (SGA), appropriate (AGA) and large (LGA) for gestational age term infants (≥37 weeks gestation, n = 111) were determined by real-time PCR and Western blot respectively. RESULTS: FOLR1 and HCP1/PCFT mRNA were lower in both plates of SGA and LGA placentas compared to AGA (p < 0.01) and RFC1 mRNA was lower only in CP (p < 0.02). RFC1 protein levels were lower in BP of SGA (p < 0.05) and LGA (p < 0.01), and FOLR1 protein levels were lower in CP of SGA (p < 0.02) and LGA (p < 0.01) groups compared to AGA. HCP1/PCFT protein levels remained unchanged in all groups. CONCLUSION: Placentas of SGA and LGA groups showed a reduced mRNA expression and protein levels of folate transporters, with some differences depending on the location within the placenta (BP or CP). This suggests the presence of specific placental regulation mechanisms in gene expression that may be associated to birth weight.
Assuntos
Peso ao Nascer , Transportadores de Ácido Fólico/genética , Placenta/metabolismo , Nascimento a Termo/genética , Adolescente , Adulto , Peso ao Nascer/genética , Feminino , Desenvolvimento Fetal/genética , Macrossomia Fetal/genética , Macrossomia Fetal/metabolismo , Transportadores de Ácido Fólico/metabolismo , Expressão Gênica , Humanos , Recém-Nascido , Recém-Nascido Pequeno para a Idade Gestacional/metabolismo , Masculino , Gravidez , Nascimento a Termo/metabolismo , Adulto JovemRESUMO
Heme (iron-protoporphyrin IX) is an essential co-factor involved in multiple biological processes: oxygen transport and storage, electron transfer, drug and steroid metabolism, signal transduction, and micro RNA processing. However, excess free-heme is highly toxic due to its ability to promote oxidative stress and lipid peroxidation, thus leading to membrane injury and, ultimately, apoptosis. Thus, heme metabolism needs to be finely regulated. Intracellular heme amount is controlled at multiple levels: synthesis, utilization by hemoproteins, degradation and both intracellular and intercellular trafficking. This review focuses on recent findings highlighting the importance of controlling intracellular heme levels to counteract heme-induced oxidative stress. The contributions of heme scavenging from the extracellular environment, heme synthesis and incorporation into hemoproteins, heme catabolism and heme transport in maintaining adequate intracellular heme content are discussed. Particular attention is put on the recently described mechanisms of heme trafficking through the plasma membrane mediated by specific heme importers and exporters. Finally, the involvement of genes orchestrating heme metabolism in several pathological conditions is illustrated and new therapeutic approaches aimed at controlling heme metabolism are discussed.