Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 88
Filtrar
Más filtros

Banco de datos
País/Región como asunto
Tipo del documento
Intervalo de año de publicación
1.
Cell ; 150(5): 909-21, 2012 Aug 31.
Artículo en Inglés | MEDLINE | ID: mdl-22939620

RESUMEN

Some Ts in nuclear DNA of trypanosomes and Leishmania are hydroxylated and glucosylated to yield base J (ß-D-glucosyl-hydroxymethyluracil). In Leishmania, about 99% of J is located in telomeric repeats. We show here that most of the remaining J is located at chromosome-internal RNA polymerase II termination sites. This internal J and telomeric J can be reduced by a knockout of J-binding protein 2 (JBP2), an enzyme involved in the first step of J biosynthesis. J levels are further reduced by growing Leishmania JBP2 knockout cells in BrdU-containing medium, resulting in cell death. The loss of internal J in JBP2 knockout cells is accompanied by massive readthrough at RNA polymerase II termination sites. The readthrough varies between transcription units but may extend over 100 kb. We conclude that J is required for proper transcription termination and infer that the absence of internal J kills Leishmania by massive readthrough of transcriptional stops.


Asunto(s)
Glucósidos/metabolismo , Leishmania/genética , Leishmania/metabolismo , Transcripción Genética , Uracilo/análogos & derivados , Técnicas de Inactivación de Genes , ARN Polimerasa II/metabolismo , ARN Bicatenario/metabolismo , Uracilo/metabolismo
2.
Mol Cell ; 61(3): 405-418, 2016 Feb 04.
Artículo en Inglés | MEDLINE | ID: mdl-26774285

RESUMEN

DNA double-strand break repair by homologous recombination is initiated by the formation of 3' single-stranded DNA (ssDNA) overhangs by a process termed end resection. Although much focus has been given to the decision to initiate resection, little is known of the mechanisms that regulate the ongoing formation of ssDNA tails. Here we report that DNA helicase B (HELB) underpins a feedback inhibition mechanism that curtails resection. HELB is recruited to ssDNA by interacting with RPA and uses its 5'-3' ssDNA translocase activity to inhibit EXO1 and BLM-DNA2, the nucleases catalyzing resection. HELB acts independently of 53BP1 and is exported from the nucleus as cells approach S phase, concomitant with the upregulation of resection. Consistent with its role as a resection antagonist, loss of HELB results in PARP inhibitor resistance in BRCA1-deficient tumor cells. We conclude that mammalian DNA end resection triggers its own inhibition via the recruitment of HELB.


Asunto(s)
Reparación del ADN por Unión de Extremidades , ADN Helicasas/metabolismo , Neoplasias Mamarias Experimentales/enzimología , Animales , Proteína BRCA1/genética , ADN Helicasas/deficiencia , ADN Helicasas/genética , Enzimas Reparadoras del ADN/genética , Enzimas Reparadoras del ADN/metabolismo , Exodesoxirribonucleasas/genética , Exodesoxirribonucleasas/metabolismo , Retroalimentación Fisiológica , Femenino , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Células HeLa , Humanos , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Ftalazinas/farmacología , Piperazinas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Interferencia de ARN , RecQ Helicasas/genética , RecQ Helicasas/metabolismo , Fase S , Factores de Tiempo , Transfección , Proteínas Supresoras de Tumor/genética
3.
Trends Biochem Sci ; 44(2): 125-140, 2019 02.
Artículo en Inglés | MEDLINE | ID: mdl-30446375

RESUMEN

Ever since Garrod deduced the existence of inborn errors in 1901, a vast array of metabolic diseases has been identified and characterized in molecular terms. In 2018 it is difficult to imagine that there is any uncharted backyard left in the metabolic disease landscape. Nevertheless, it took until 2013 to identify the cause of a relatively frequent inborn error, pseudoxanthoma elasticum (PXE), a disorder resulting in aberrant calcification. The mechanism found was not only biochemically interesting but also points to possible new treatments for PXE, a disease that has remained untreatable. In this review we sketch the tortuous road that led to the biochemical understanding of PXE and to new ideas for treatment. We also discuss some of the controversies still haunting the field.


Asunto(s)
Errores Innatos del Metabolismo/genética , Seudoxantoma Elástico/genética , Humanos , Errores Innatos del Metabolismo/metabolismo , Errores Innatos del Metabolismo/terapia , Seudoxantoma Elástico/metabolismo , Seudoxantoma Elástico/terapia
4.
Nat Methods ; 15(2): 134-140, 2018 02.
Artículo en Inglés | MEDLINE | ID: mdl-29256493

RESUMEN

Poly(ADP-ribose) polymerase inhibition (PARPi) is a promising new therapeutic approach for the treatment of cancers that show homologous recombination deficiency (HRD). Despite the success of PARPi in targeting HRD in tumors that lack the tumor suppressor function of BRCA1 or BRCA2, drug resistance poses a major obstacle. We developed three-dimensional cancer organoids derived from genetically engineered mouse models (GEMMs) for BRCA1- and BRCA2-deficient cancers. Unlike conventional cell lines or mammospheres, organoid cultures can be efficiently derived and rapidly expanded in vitro. Orthotopically transplanted organoids give rise to mammary tumors that recapitulate the epithelial morphology and preserve the drug response of the original tumor. Notably, GEMM-tumor-derived organoids can be easily genetically modified, making them a powerful tool for genetic studies of tumor biology and drug resistance.


Asunto(s)
Antineoplásicos/farmacología , Proliferación Celular/efectos de los fármacos , Resistencia a Antineoplásicos , Neoplasias Mamarias Animales/patología , Organoides/patología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Subfamilia B de Transportador de Casetes de Unión a ATP/fisiología , Animales , Proteína BRCA1 , Proteína BRCA2/deficiencia , Femenino , Neoplasias Mamarias Animales/tratamiento farmacológico , Neoplasias Mamarias Animales/metabolismo , Ratones , Ratones Noqueados , Técnicas de Cultivo de Órganos , Organoides/efectos de los fármacos , Organoides/metabolismo , Proteínas Supresoras de Tumor/deficiencia
5.
Nature ; 521(7553): 541-544, 2015 May 28.
Artículo en Inglés | MEDLINE | ID: mdl-25799992

RESUMEN

Error-free repair of DNA double-strand breaks (DSBs) is achieved by homologous recombination (HR), and BRCA1 is an important factor for this repair pathway. In the absence of BRCA1-mediated HR, the administration of PARP inhibitors induces synthetic lethality of tumour cells of patients with breast or ovarian cancers. Despite the benefit of this tailored therapy, drug resistance can occur by HR restoration. Genetic reversion of BRCA1-inactivating mutations can be the underlying mechanism of drug resistance, but this does not explain resistance in all cases. In particular, little is known about BRCA1-independent restoration of HR. Here we show that loss of REV7 (also known as MAD2L2) in mouse and human cell lines re-establishes CTIP-dependent end resection of DSBs in BRCA1-deficient cells, leading to HR restoration and PARP inhibitor resistance, which is reversed by ATM kinase inhibition. REV7 is recruited to DSBs in a manner dependent on the H2AX-MDC1-RNF8-RNF168-53BP1 chromatin pathway, and seems to block HR and promote end joining in addition to its regulatory role in DNA damage tolerance. Finally, we establish that REV7 blocks DSB resection to promote non-homologous end-joining during immunoglobulin class switch recombination. Our results reveal an unexpected crucial function of REV7 downstream of 53BP1 in coordinating pathological DSB repair pathway choices in BRCA1-deficient cells.


Asunto(s)
Roturas del ADN de Doble Cadena , Proteínas Mad2/metabolismo , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Reparación del ADN por Recombinación , Proteínas Adaptadoras Transductoras de Señales , Animales , Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteína BRCA1/deficiencia , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Proteínas de Ciclo Celular , Línea Celular , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Unión al ADN/metabolismo , Resistencia a Antineoplásicos/genética , Histonas/metabolismo , Humanos , Cambio de Clase de Inmunoglobulina/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Mad2/deficiencia , Proteínas Mad2/genética , Ratones , Proteínas Nucleares/metabolismo , Transactivadores/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53 , Ubiquitina-Proteína Ligasas/metabolismo
6.
IUBMB Life ; 72(11): 2241-2259, 2020 11.
Artículo en Inglés | MEDLINE | ID: mdl-32916028

RESUMEN

This article presents a personal and critical review of the history of the malate-aspartate shuttle (MAS), starting in 1962 and ending in 2020. The MAS was initially proposed as a route for the oxidation of cytosolic NADH by the mitochondria in Ehrlich ascites cell tumor lacking other routes, and to explain the need for a mitochondrial aspartate aminotransferase (glutamate oxaloacetate transaminase 2 [GOT2]). The MAS was soon adopted in the field as a major pathway for NADH oxidation in mammalian tissues, such as liver and heart, even though the energetics of the MAS remained a mystery. Only in the 1970s, LaNoue and coworkers discovered that the efflux of aspartate from mitochondria, an essential step in the MAS, is dependent on the proton-motive force generated by the respiratory chain: for every aspartate effluxed, mitochondria take up one glutamate and one proton. This makes the MAS in practice uni-directional toward oxidation of cytosolic NADH, and explains why the free NADH/NAD ratio is much higher in the mitochondria than in the cytosol. The MAS is still a very active field of research. Most recently, the focus has been on the role of the MAS in tumors, on cells with defects in mitochondria and on inborn errors in the MAS. The year 2019 saw the discovery of two new inborn errors in the MAS, deficiencies in malate dehydrogenase 1 and in aspartate transaminase 2 (GOT2). This illustrates the vitality of ongoing MAS research.


Asunto(s)
Aspartato Aminotransferasas/deficiencia , Ácido Aspártico/metabolismo , Malato Deshidrogenasa/deficiencia , Malatos/metabolismo , Errores Innatos del Metabolismo/patología , Mitocondrias/patología , Animales , Aspartato Aminotransferasas/genética , Respiración de la Célula , Humanos , Malato Deshidrogenasa/genética , Errores Innatos del Metabolismo/etiología , Errores Innatos del Metabolismo/metabolismo , Mitocondrias/metabolismo , Mutación
7.
EMBO J ; 34(24): 2993-3008, 2015 Dec 14.
Artículo en Inglés | MEDLINE | ID: mdl-26530471

RESUMEN

Although platinum-based drugs are widely used chemotherapeutics for cancer treatment, the determinants of tumor cell responsiveness remain poorly understood. We show that the loss of subunits LRRC8A and LRRC8D of the heteromeric LRRC8 volume-regulated anion channels (VRACs) increased resistance to clinically relevant cisplatin/carboplatin concentrations. Under isotonic conditions, about 50% of cisplatin uptake depended on LRRC8A and LRRC8D, but neither on LRRC8C nor on LRRC8E. Cell swelling strongly enhanced LRRC8-dependent cisplatin uptake, bolstering the notion that cisplatin enters cells through VRAC. LRRC8A disruption also suppressed drug-induced apoptosis independently from drug uptake, possibly by impairing VRAC-dependent apoptotic cell volume decrease. Hence, by mediating cisplatin uptake and facilitating apoptosis, VRAC plays a dual role in the cellular drug response. Incorporation of the LRRC8D subunit into VRAC substantially increased its permeability for cisplatin and the cellular osmolyte taurine, indicating that LRRC8 proteins form the channel pore. Our work suggests that LRRC8D-containing VRACs are crucial for cell volume regulation by an important organic osmolyte and may influence cisplatin/carboplatin responsiveness of tumors.


Asunto(s)
Antineoplásicos/farmacología , Carboplatino/farmacología , Cisplatino/farmacología , Resistencia a Antineoplásicos , Proteínas de la Membrana/metabolismo , Apoptosis , Tamaño de la Célula , Células HCT116 , Células HEK293 , Humanos , Proteínas de la Membrana/genética , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo
8.
JAMA ; 330(15): 1423-1424, 2023 10 17.
Artículo en Inglés | MEDLINE | ID: mdl-37732817

RESUMEN

In this Viewpoint, Lasker Award winner Piet Borst looks back over a 50-year career in scientific research, including work with trypanosomatids, mechanisms of drug resistance in cancer cells, and inborn errors of metabolism.


Asunto(s)
Distinciones y Premios , Investigación Biomédica , Animales , Humanos , Investigación Biomédica/historia , Historia del Siglo XX , Historia del Siglo XXI , Medicina , Errores Innatos del Metabolismo , Neoplasias/terapia , Países Bajos , Parásitos
9.
Nucleic Acids Res ; 43(4): 2102-15, 2015 Feb 27.
Artículo en Inglés | MEDLINE | ID: mdl-25662217

RESUMEN

Base J (ß-D-glucosyl-hydroxymethyluracil) replaces 1% of T in the Leishmania genome and is only found in telomeric repeats (99%) and in regions where transcription starts and stops. This highly restricted distribution must be co-determined by the thymidine hydroxylases (JBP1 and JBP2) that catalyze the initial step in J synthesis. To determine the DNA sequences recognized by JBP1/2, we used SMRT sequencing of DNA segments inserted into plasmids grown in Leishmania tarentolae. We show that SMRT sequencing recognizes base J in DNA. Leishmania DNA segments that normally contain J also picked up J when present in the plasmid, whereas control sequences did not. Even a segment of only 10 telomeric (GGGTTA) repeats was modified in the plasmid. We show that J modification usually occurs at pairs of Ts on opposite DNA strands, separated by 12 nucleotides. Modifications occur near G-rich sequences capable of forming G-quadruplexes and JBP2 is needed, as it does not occur in JBP2-null cells. We propose a model whereby de novo J insertion is mediated by JBP2. JBP1 then binds to J and hydroxylates another T 13 bp downstream (but not upstream) on the complementary strand, allowing JBP1 to maintain existing J following DNA replication.


Asunto(s)
Glucósidos/análisis , Uracilo/análogos & derivados , Proteínas de Unión al ADN/metabolismo , Glucósidos/metabolismo , Leishmania/genética , Plásmidos/genética , Proteínas Protozoarias/metabolismo , Análisis de Secuencia de ADN , Uracilo/análisis , Uracilo/metabolismo
10.
J Biol Chem ; 290(51): 30429-40, 2015 Dec 18.
Artículo en Inglés | MEDLINE | ID: mdl-26515061

RESUMEN

The ubiquitous efflux transporter ABCC5 (ATP-binding cassette subfamily C member 5) is present at high levels in the blood-brain barrier, neurons, and glia, but its in vivo substrates and function are not known. Using untargeted metabolomic screens, we show that Abcc5(-/-) mice accumulate endogenous glutamate conjugates in several tissues, but brain in particular. The abundant neurotransmitter N-acetylaspartylglutamate was 2.4-fold higher in Abcc5(-/-) brain. The metabolites that accumulated in Abcc5(-/-) tissues were depleted in cultured cells that overexpressed human ABCC5. In a vesicular membrane transport assay, ABCC5 also transported exogenous glutamate analogs, like the classic excitotoxic neurotoxins kainic acid, domoic acid, and NMDA; the therapeutic glutamate analog ZJ43; and, as previously shown, the anti-cancer drug methotrexate. Glutamate conjugates and analogs are of physiological relevance because they can affect the function of glutamate, the principal excitatory neurotransmitter in the brain. After CO2 asphyxiation, several immediate early genes were expressed at lower levels in Abcc5(-/-) brains than in wild type brains, suggesting altered glutamate signaling. Our results show that ABCC5 is a general glutamate conjugate and analog transporter that affects the disposition of endogenous metabolites, toxins, and drugs.


Asunto(s)
Encéfalo/metabolismo , Dipéptidos/farmacocinética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Animales , Dipéptidos/farmacología , Humanos , Ácido Kaínico/análogos & derivados , Ácido Kaínico/farmacocinética , Ácido Kaínico/farmacología , Ratones , Ratones Noqueados , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Proteínas del Tejido Nervioso/genética , Urea/análogos & derivados , Urea/farmacocinética , Urea/farmacología
11.
Arterioscler Thromb Vasc Biol ; 34(9): 1985-9, 2014 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-24969777

RESUMEN

OBJECTIVE: Mutations in ABCC6 underlie the ectopic mineralization disorder pseudoxanthoma elasticum (PXE) and some forms of generalized arterial calcification of infancy, both of which affect the cardiovascular system. Using cultured cells, we recently showed that ATP-binding cassette subfamily C member 6 (ABCC6) mediates the cellular release of ATP, which is extracellularly rapidly converted into AMP and the mineralization inhibitor inorganic pyrophosphate (PPi). The current study was performed to determine which tissues release ATP in an ABCC6-dependent manner in vivo, where released ATP is converted into AMP and PPi, and whether human PXE ptients have low plasma PPi concentrations. APPROACH AND RESULTS: Using cultured primary hepatocytes and in vivo liver perfusion experiments, we found that ABCC6 mediates the direct, sinusoidal, release of ATP from the liver. Outside hepatocytes, but still within the liver vasculature, released ATP is converted into AMP and PPi. The absence of functional ABCC6 in patients with PXE leads to strongly reduced plasma PPi concentrations. CONCLUSIONS: Hepatic ABCC6-mediated ATP release is the main source of circulating PPi, revealing an unanticipated role of the liver in systemic PPi homeostasis. Patients with PXE have a strongly reduced plasma PPi level, explaining their mineralization disorder. Our results indicate that systemic PPi is relatively stable and that PXE, generalized arterial calcification of infancy, and other ectopic mineralization disorders could be treated with PPi supplementation therapy.


Asunto(s)
Transportadoras de Casetes de Unión a ATP/fisiología , Adenosina Trifosfato/metabolismo , Difosfatos/sangre , Hígado/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/fisiología , Adenosina Monofosfato/sangre , Anciano , Animales , Células Cultivadas , Medios de Cultivo Condicionados , Femenino , Células HEK293 , Células HeLa , Hepatocitos/metabolismo , Homeostasis , Humanos , Hígado/irrigación sanguínea , Masculino , Ratones , Persona de Mediana Edad , Seudoxantoma Elástico/genética , Seudoxantoma Elástico/metabolismo , Ratas
12.
FASEB J ; 26(2): 738-47, 2012 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-22034653

RESUMEN

The ATP-binding cassette (ABC) genes encode the largest family of transmembrane proteins. ABC transporters translocate a wide variety of substrates across membranes, but their physiological function is often incompletely understood. We describe a new method to study the substrate spectrum of ABC transporters: We incubate extracts of mouse urine with membrane vesicles prepared from Spodoptera frugiperda Sf9 insect cells overproducing an ABC transporter and determine the compounds transported into the vesicles by LC/MS-based metabolomics. We illustrate the power of this simple "transportomics" approach using ABCC2, a protein present at sites of uptake and elimination. We identified many new substrates of ABCC2 in urine. These included glucuronides of plant-derived xenobiotics, a class of compounds to which humans are exposed on a daily basis. Moreover, we show that the excretion of these compounds in vivo depends on ABCC2: compared to wild-type mice, the urinary excretion of several glucuronides was increased up to 20-fold in Abcc2(-/-) mice. Transportomics has broad applicability, as it is not restricted to urine and can be applied to other ATP-dependent transport proteins as well.


Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Animales , Transporte Biológico Activo , Líquidos Corporales/metabolismo , Glucurónidos/metabolismo , Glucurónidos/orina , Humanos , Cinética , Metaboloma , Ratones , Ratones Noqueados , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/deficiencia , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Fitoestrógenos/metabolismo , Fitoestrógenos/orina , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Xenobióticos/metabolismo , Xenobióticos/orina
13.
Nucleic Acids Res ; 39(13): 5715-28, 2011 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-21415010

RESUMEN

The J-binding protein 1 (JBP1) is essential for biosynthesis and maintenance of DNA base-J (ß-d-glucosyl-hydroxymethyluracil). Base-J and JBP1 are confined to some pathogenic protozoa and are absent from higher eukaryotes, prokaryotes and viruses. We show that JBP1 recognizes J-containing DNA (J-DNA) through a 160-residue domain, DB-JBP1, with 10 000-fold preference over normal DNA. The crystal structure of DB-JBP1 revealed a helix-turn-helix variant fold, a 'helical bouquet' with a 'ribbon' helix encompassing the amino acids responsible for DNA binding. Mutation of a single residue (Asp525) in the ribbon helix abrogates specificity toward J-DNA. The same mutation renders JBP1 unable to rescue the targeted deletion of endogenous JBP1 genes in Leishmania and changes its distribution in the nucleus. Based on mutational analysis and hydrogen/deuterium-exchange mass-spectrometry data, a model of JBP1 bound to J-DNA was constructed and validated by small-angle X-ray scattering data. Our results open new possibilities for targeted prevention of J-DNA recognition as a therapeutic intervention for parasitic diseases.


Asunto(s)
Proteínas de Unión al ADN/química , ADN/química , Glucósidos/química , Proteínas Protozoarias/química , Uracilo/análogos & derivados , Secuencia de Aminoácidos , Arginina/química , Ácido Aspártico/química , Cristalografía por Rayos X , ADN Bacteriano/metabolismo , Proteínas de Unión al ADN/metabolismo , Glucósidos/metabolismo , Lisina/química , Espectrometría de Masas , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Protozoarias/metabolismo , Dispersión del Ángulo Pequeño , Alineación de Secuencia , Uracilo/química , Uracilo/metabolismo , Difracción de Rayos X
14.
Drug Resist Updat ; 15(1-2): 81-9, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-22335919

RESUMEN

Drug resistance is one of the most pressing problems in treating cancer patients today. Local and regional disease can usually be adequately treated, but patients eventually die from distant metastases that have become resistant to all available chemotherapy. Although work on cultured tumor cell lines has yielded a lot of information on potential drug resistance mechanisms, it has proven difficult to translate these results to clinical drug resistance in patients. The controversy regarding the contribution of ABC transporters to drug resistance in patients is one example. The study of genetically engineered mouse models (GEMMs), which closely resemble cancer in human patients, can help to bridge this gap. In models for BRCA1- or BRCA2-associated breast cancer, we observed a substantial synergy between the defect in homology-directed DNA repair and sensitivity to DNA-targeting drugs. Nevertheless, tumors are not easily eradicated and eventually drug resistance develops. In this review we will discuss the use of the new generation mouse models to address major clinical problems, such as mechanisms of drug resistance, predicting chemotherapy response or characterizing the nature of residual tumor cells that escape eradication. Moreover, we will address the contribution of ABC transporters to drug resistance in our model.


Asunto(s)
Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Antineoplásicos/uso terapéutico , Proteína BRCA1/antagonistas & inhibidores , Proteína BRCA2/antagonistas & inhibidores , ADN de Neoplasias/genética , Resistencia a Antineoplásicos/genética , Neoplasias Experimentales/tratamiento farmacológico , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Transporte Biológico/efectos de los fármacos , Reparación del ADN/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Perfilación de la Expresión Génica , Humanos , Ratones , Ratones Transgénicos , Neoplasias Experimentales/genética , Neoplasias Experimentales/metabolismo , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Trasplante Heterólogo
15.
J Am Chem Soc ; 134(32): 13357-65, 2012 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-22775585

RESUMEN

Base J (ß-D-glucosyl-hydroxymethyluracil) was discovered in the nuclear DNA of some pathogenic protozoa, such as trypanosomes and Leishmania, where it replaces a fraction of base T. We have found a J-Binding Protein 1 (JBP1) in these organisms, which contains a unique J-DNA binding domain (DB-JBP1) and a thymidine hydroxylase domain involved in the first step of J biosynthesis. This hydroxylase is related to the mammalian TET enzymes that hydroxylate 5-methylcytosine in DNA. We have now studied the binding of JBP1 and DB-JBP1 to oligonucleotides containing J or glucosylated 5-hydroxymethylcytosine (glu-5-hmC) using an equilibrium fluorescence polarization assay. We find that JBP1 binds glu-5-hmC-DNA with an affinity about 40-fold lower than J-DNA (~400 nM), which is still 200 times higher than the JBP1 affinity for T-DNA. The discrimination between glu-5-hmC-DNA and T-DNA by DB-JBP1 is about 2-fold less, but enough for DB-JBP1 to be useful as a tool to isolate 5-hmC-DNA. Pre-steady state kinetic data obtained in a stopped-flow device show that the initial binding of JBP1 to glucosylated DNA is very fast with a second order rate constant of 70 µM(-1) s(-1) and that JBP1 binds to J-DNA or glu-5-hmC-DNA in a two-step reaction, in contrast to DB-JBP1, which binds in a one-step reaction. As the second (slower) step in binding is concentration independent, we infer that JBP1 undergoes a conformational change upon binding to DNA. Global analysis of pre-steady state and equilibrium binding data supports such a two-step mechanism and allowed us to determine the kinetic parameters that describe it. This notion of a conformational change is supported by small-angle neutron scattering experiments, which show that the shape of JBP1 is more elongated in complex with DNA. The conformational change upon DNA binding may allow the hydroxylase domain of JBP1 to make contact with the DNA and hydroxylate T's in spatial proximity, resulting in regional introduction of base J into the DNA.


Asunto(s)
Proteínas Portadoras/química , Glucósidos/química , Uracilo/análogos & derivados , Proteínas Portadoras/metabolismo , Glucósidos/metabolismo , Cinética , Conformación Molecular , Unión Proteica , Factores de Tiempo , Uracilo/química , Uracilo/metabolismo
16.
Blood ; 115(8): 1632-9, 2010 Feb 25.
Artículo en Inglés | MEDLINE | ID: mdl-19897579

RESUMEN

Cobalamin (Cbl, vitamin B(12)) deficiency in humans is a cause of hematologic and neurologic disorders. We show here that the cellular export of Cbl, in contrast to the carrier- and receptor-dependent cellular import of Cbl, occurs by transmembrane transport of "free" Cbl. Screening of candidate transporters by cellular gene silencing showed a role in cellular Cbl efflux of the ATP-binding cassette (ABC)-drug transporter, ABCC1, alias multidrug resistance protein 1 (MRP1), which is present in the basolateral membrane of intestinal epithelium and in other cells. The ability of MRP1 to mediate ATP-dependent Cbl transport was confirmed by vesicular transport experiments, and a physiologic role of MRP1 in mammalian Cbl homeostasis is indicated by the phenotype of knockout mice with targeted disruption of MRP1. These animals have a reduced concentration of Cbl in plasma and in the storage organs liver and kidney. In contrast, Cbl accumulates in the terminal part of the intestine of these mice, suggesting a functional malabsorption because of a lower epithelial basolateral Cbl efflux. The identification of this Cbl export mechanism now allows the delineation of a coherent pathway for Cbl trafficking from food to the body cells.


Asunto(s)
Mucosa Intestinal/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Vitamina B 12/metabolismo , Adenosina Trifosfato/genética , Adenosina Trifosfato/metabolismo , Animales , Transporte Biológico/fisiología , Células CHO , Cricetinae , Cricetulus , Células HeLa , Humanos , Absorción Intestinal/fisiología , Ratones , Ratones Noqueados , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Ratas , Vitamina B 12/genética , Deficiencia de Vitamina B 12/genética , Deficiencia de Vitamina B 12/metabolismo
17.
Biochem Biophys Res Commun ; 415(3): 468-71, 2011 Nov 25.
Artículo en Inglés | MEDLINE | ID: mdl-22056557

RESUMEN

Vitamin K is a cofactor required for gamma-glutamyl carboxylation of several proteins regulating blood clotting, bone formation and soft tissue mineralization. Vitamin K3 is an important intermediate during conversion of the dietary vitamin K1 to the most abundant vitamin K2 form. It has been suggested that ABCC6 may have a role in transporting vitamin K or its derivatives from the liver to the periphery. This activity is missing in pseudoxanthoma elasticum, a genetic disorder caused by mutations in ABCC6 characterized by abnormal soft tissue mineralization. Here we examined the efflux of the glutathione conjugate of vitamin K3 (VK3GS) from the liver in wild type and Abcc6(-/-) mice, and in transport assays in vitro. We found in liver perfusion experiments that VK3GS is secreted into the inferior vena cava, but we observed no significant difference between wild type and Abcc6(-/-) animals. We overexpressed the human ABCC6 transporter in Sf9 insect and MDCKII cells and assayed its vitamin K3-conjugate transport activity in vitro. We found no measurable transport of VK3GS by ABCC6, whereas ABCC1 transported this compound at high rate in these assays. These results show that VK3GS is not the essential metabolite transported by ABCC6 from the liver and preventing the symptoms of pseudoxanthoma elasticum.


Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Glutatión/metabolismo , Hígado/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Seudoxantoma Elástico/metabolismo , Vitamina K 3/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Animales , Transporte Biológico , Línea Celular , Perros , Humanos , Insectos/citología , Ratones , Ratones Mutantes , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Especificidad por Sustrato
18.
Proc Natl Acad Sci U S A ; 105(44): 17079-84, 2008 Nov 04.
Artículo en Inglés | MEDLINE | ID: mdl-18971340

RESUMEN

Whereas target-specific drugs are available for treating ERBB2-overexpressing and hormone receptor-positive breast cancers, no tailored therapy exists for hormone receptor- and ERBB2-negative ("triple-negative") mammary carcinomas. Triple-negative tumors account for 15% of all breast cancers and frequently harbor defects in DNA double-strand break repair through homologous recombination (HR), such as BRCA1 dysfunction. The DNA-repair defects characteristic of BRCA1-deficient cells confer sensitivity to poly(ADP-ribose) polymerase 1 (PARP1) inhibition, which could be relevant to treatment of triple-negative tumors. To evaluate PARP1 inhibition in a realistic in vivo setting, we tested the PARP inhibitor AZD2281 in a genetically engineered mouse model (GEMM) for BRCA1-associated breast cancer. Treatment of tumor-bearing mice with AZD2281 inhibited tumor growth without signs of toxicity, resulting in strongly increased survival. Long-term treatment with AZD2281 in this model did result in the development of drug resistance, caused by up-regulation of Abcb1a/b genes encoding P-glycoprotein efflux pumps. This resistance to AZD2281 could be reversed by coadministration of the P-glycoprotein inhibitor tariquidar. Combination of AZD2281 with cisplatin or carboplatin increased the recurrence-free and overall survival, suggesting that AZD2281 potentiates the effect of these DNA-damaging agents. Our results demonstrate in vivo efficacy of AZD2281 against BRCA1-deficient breast cancer and illustrate how GEMMs of cancer can be used for preclinical evaluation of novel therapeutics and for testing ways to overcome or circumvent therapy resistance.


Asunto(s)
Antineoplásicos/uso terapéutico , Proteína BRCA1/genética , Neoplasias Mamarias Animales/tratamiento farmacológico , Ftalazinas/uso terapéutico , Piperazinas/uso terapéutico , Compuestos de Platino/uso terapéutico , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Animales , Inhibidores de la Aromatasa/uso terapéutico , Cisplatino/uso terapéutico , Daño del ADN , Reparación del ADN/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Femenino , Neoplasias Mamarias Animales/genética , Neoplasias Mamarias Animales/patología , Ratones , Ratones Transgénicos , Trasplante de Neoplasias , Poli(ADP-Ribosa) Polimerasas/genética
19.
Gastroenterology ; 137(5): 1725-35, 2009 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-19577570

RESUMEN

BACKGROUND & AIMS: The physiologic function of the efflux transporter Multidrug Resistance Protein 3 (MRP3) remains poorly defined. In vitro, MRP3 transports several glucuronidated compounds, but the compounds transported under physiologic conditions are unknown. Knowledge of the compounds transported by MRP3 in vivo would greatly contribute to the elucidation of the physiologic function of this transport protein. METHODS: We used targeted metabolomics to identify substrates of MRP3 in vivo. Liquid chromatography coupled to mass spectrometry was used to specifically screen in plasma and urine of mice for compounds containing a glucuronic acid moiety. RESULTS: We found that several highly abundant compounds containing a glucuronic acid moiety have a much lower abundance in plasma and urine of Mrp3((-/-)) than of wild-type mice. We identified these as phytoestrogen-glucuronides, and we show that MRP3 transports these compounds at high rates and with high affinity in vitro. CONCLUSIONS: We have identified the efflux transporter MRP3 as a major factor in the disposition of phytoestrogens, a class of compounds to which mammals are exposed via food of plant origin. Our targeted metabolomics approach is not restricted to MRP3 but applicable to many other transport proteins for which knockout mouse models are available. Similar screens could be developed for sulpho- and glutathione-conjugates, further increasing the potential of identifying new physiologic transporter substrates.


Asunto(s)
Subfamilia B de Transportador de Casetes de Unión a ATP/fisiología , Glucurónidos/sangre , Glucurónidos/orina , Metabolómica , Fitoestrógenos/metabolismo , Animales , Mucosa Intestinal/metabolismo , Ratones , Ratones Noqueados , Transporte de Proteínas/fisiología
20.
FEBS Lett ; 594(23): 4001-4011, 2020 12.
Artículo en Inglés | MEDLINE | ID: mdl-33111311

RESUMEN

This paper presents a personal, selective, and sometimes critical retrospective of the history of ABC transporters in multidrug resistance (MDR) of cancer cells, overrepresenting discoveries of some early pioneers, long forgotten, and highlights of research in Amsterdam, mainly focussing on discoveries made with disruptions of ABC genes in mice (KO mice) and on the role of ABC transporters in causing drug resistance in a mouse model of mammary cancer. The history is complemented by a list of erroneous concepts often found in papers and grant applications submitted anno 2020.


Asunto(s)
Transportadoras de Casetes de Unión a ATP , Investigación Biomédica , Resistencia a Antineoplásicos , Neoplasias , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Investigación Biomédica/historia , Historia del Siglo XX , Historia del Siglo XXI , Humanos , Ratones , Neoplasias/metabolismo , Países Bajos
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA