RESUMO
Mutations in the gene encoding ATP-binding cassette transporter 1 ( ABC1) have been reported in Tangier disease (TD), an autosomal recessive disorder that is characterized by almost complete absence of plasma high-density lipoprotein (HDL), deposition of cholesteryl esters in the reticulo-endothelial system (RES) and aberrant cellular lipid trafficking. We demonstrate here that mice with a targeted inactivation of Abc1 display morphologic abnormalities and perturbations in their lipoprotein metabolism concordant with TD. ABC1 is expressed on the plasma membrane and the Golgi complex, mediates apo-AI associated export of cholesterol and phospholipids from the cell, and is regulated by cholesterol flux. Structural and functional abnormalities in caveolar processing and the trans-Golgi secretory pathway of cells lacking functional ABC1 indicate that lipid export processes involving vesicular budding between the Golgi and the plasma membrane are severely disturbed.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Membrana Celular/metabolismo , Glicoproteínas/genética , Glicoproteínas/metabolismo , Complexo de Golgi/metabolismo , Metabolismo dos Lipídeos , Doença de Tangier/genética , Doença de Tangier/metabolismo , Transportador 1 de Cassete de Ligação de ATP , Animais , Apoptose , Plaquetas/metabolismo , Colesterol/sangue , Colesterol/metabolismo , HDL-Colesterol/sangue , Fibroblastos/metabolismo , Genótipo , Humanos , Mucosa Intestinal/patologia , Mucosa Intestinal/ultraestrutura , Intestino Delgado/patologia , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Fosfolipídeos/metabolismo , Triglicerídeos/sangueRESUMO
ATP-binding-cassette transporter 1 (ABC1) has been implicated in processes related to membrane-lipid turnover. Here, using in vivo loss-of-function and in vitro gain-of-function models, we show that ABC1 promotes Ca2+-induced exposure of phosphatidylserine at the membrane, as determined by a prothrombinase assay, membrane microvesiculation and measurement of transbilayer redistribution of spin-labelled phospholipids. That ABC1 promotes engulfment of dead cells is shown by the impaired ability of ABC1-deficient macrophages to engulf apoptotic preys and by the acquisition of phagocytic behaviour by ABC1 transfectants. Release of membrane phospholipids and cholesterol to apo-AI, the protein core of the cholesterol-shuttling high-density lipoprotein (HDL) particle, is also ABC1-dependent. We propose that both the efficiency of apoptotic-cell engulfment and the efflux of cellular lipids depend on ABC1-induced perturbation of membrane phosphatidylserine turnover. Transient local exposure of anionic phospholipids in the outer membrane leaflet may be sufficient to alter the general properties of the membrane and thus influence discrete physiological functions.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Apoptose , Glicoproteínas/metabolismo , Fagocitose , Fosfatidilserinas/metabolismo , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Anexina A5/metabolismo , Apolipoproteína A-I/metabolismo , Cálcio/farmacologia , Membrana Celular/química , Membrana Celular/metabolismo , Células Cultivadas , Colesterol/metabolismo , Glicoproteínas/genética , Células HeLa , Humanos , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Macrófagos/citologia , Macrófagos/imunologia , Camundongos , Camundongos Knockout , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Marcadores de Spin , Tromboplastina/metabolismo , Timo/citologia , TransfecçãoRESUMO
Pediatric acute myeloid leukemia (AML) is a rare disease whose prognosis is highly variable according to factors such as chromosomal abnormalities. Recurrent genomic rearrangements are detected in half of pediatric AML by karyotype. NUcleoPorin 98 (NUP98) gene is rearranged with 31 different fusion partner genes. These rearrangements are frequently undetected by conventional cytogenetics, as the NUP98 gene is located at the end of the chromosome 11 short arm (11p15). By screening a series of 574 pediatric AML, we detected a NUP98 rearrangement in 22 cases (3.8%), a frequency similar to CBFB-MYH11 fusion gene (4.0%). The most frequent NUP98 fusion gene partner is NSD1. These cases are homogeneous regarding their biological and clinical characteristics, and associated with bad prognosis only improved by bone marrow transplantation. We detailed the biological characteristics of these AML by exome sequencing which demonstrated few recurrent mutations (FLT3 ITD, WT1, CEBPA, NBPF14, BCR and ODF1). The analysis of the clonal structure in these cases suggests that the mutation order in the NUP98-rearranged pediatric AML begins with the NUP98 rearrangement leading to epigenetic dysregulations then followed by mutations of critical hematopoietic transcription factors and finally, activation of the FLT3 signaling pathway.
Assuntos
Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Translocação Genética , Alelos , Biomarcadores Tumorais , Proteínas Estimuladoras de Ligação a CCAAT/genética , Criança , Pré-Escolar , Epigênese Genética , Exoma , Feminino , Regulação Leucêmica da Expressão Gênica , Frequência do Gene , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Hibridização in Situ Fluorescente , Lactente , Recém-Nascido , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/mortalidade , Masculino , Mutação , Proteínas de Fusão Oncogênica/genética , Prognóstico , Transdução de Sinais , Proteínas WT1/genética , Tirosina Quinase 3 Semelhante a fms/metabolismoRESUMO
UNLABELLED: ESSENTIALS: The role of ATP-binding cassette transporter 1 (ABCA1) in platelet functions is poorly characterized. We studied the impact of ABCA1 deficiency on platelet responses in a mouse model and two Tangier patients. ABCA1-deficient platelets exhibit reduced positive feedback loop mechanisms. This reduced reactivity is dependent on external environment and independent of hematopoietic ABCA1. BACKGROUND: The ATP-binding cassette transporter ABCA1 is required for the conversion of apolipoprotein A-1 to high-density lipoprotein (HDL), and its defect causes Tangier disease, a rare disorder characterized by an absence of HDL and accumulation of cholesterol in peripheral tissues. The role of ABCA1 in platelet functions remains poorly characterized. OBJECTIVE: To determine the role of ABCA1 in platelet functions and to clarify controversies concerning its implication in processes as fundamental as platelet phosphatidylserine exposure and control of platelet membrane lipid composition. METHODS AND RESULTS: We studied the impact of ABCA1 deficiency on platelet responses in a mouse model and in two Tangier patients. We show that platelets in ABCA1-deficient mice are slightly larger in size and exhibit aggregation and secretion defects in response to low concentrations of thrombin and collagen. These platelets have normal cholesterol and major phospholipid composition, granule morphology, or calcium-induced phosphatidylserine exposure. Interestingly, ABCA1-deficient platelets display a reduction in positive feedback loop mechanisms, particularly in thromboxane A2 (TXA2) production. Hematopoietic chimera mice demonstrated that defective eicosanoids production, particularly TXA2, was primarily dependent on external environment and not on the hematopoietic ABCA1. Decreased aggregation and production of TXA2 and eicosanoids were also observed in platelets from Tangier patients. CONCLUSIONS: Absence of ABCA1 and low HDL level induce reduction of platelet reactivity by decreasing positive feedback loops, particularly TXA2 production through a hematopoietic ABCA1-independent mechanism.
Assuntos
Transportador 1 de Cassete de Ligação de ATP/deficiência , Plaquetas/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Doença de Tangier/sangue , Transportador 1 de Cassete de Ligação de ATP/sangue , Transportador 1 de Cassete de Ligação de ATP/genética , Animais , Plaquetas/patologia , Tamanho Celular , Modelos Animais de Doenças , Retroalimentação Fisiológica , Feminino , Predisposição Genética para Doença , Transplante de Células-Tronco Hematopoéticas , Hemostasia , Humanos , Lipoproteínas HDL/sangue , Masculino , Camundongos Endogâmicos DBA , Camundongos Knockout , Pessoa de Meia-Idade , Fenótipo , Agregação Plaquetária , Doença de Tangier/genética , Doença de Tangier/patologia , Trombose/sangue , Trombose/genética , Tromboxano A2/metabolismo , Fatores de TempoRESUMO
We describe here a subclass of mammalian ABC transporters, the ABCA subfamily. This is a unique group that, in contrast to any other human ABC transporters, lacks a structural counterpart in yeast. The structural hallmark of the ABCA subfamily is the presence of a stretch of hydrophobic amino acids thought to span the membrane within the putative regulatory (R) domain. As for today, four ABCA transporters have been fully characterised but 11 ABCA-encoding genes have been identified. ABCA-specific motifs in the nucleotide binding folds can be detected when analysing the conserved sequences among the different members. These motifs may reveal functional constraints exclusive to this group of ABC transporters.
Assuntos
Transportadores de Cassetes de Ligação de ATP/classificação , Proteínas de Caenorhabditis elegans , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/genética , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/genética , Proteínas de Transporte/genética , Oftalmopatias/genética , Genoma , Glicoproteínas/genética , Humanos , Invertebrados , Dados de Sequência Molecular , Nucleotídeos/químicaRESUMO
ALDR is one of the four genes encoding an ATP Binding Cassette (ABC) hemi-transporter of the peroxisomal membrane so far identified in mammalian cells. The best known of these is X-ALD, whose dysfunction has been causally associated with X-linked adrenoleukodystrophy. ALDR and X-ALD protein product are closely related and we show here that this striking conservation is maintained at the genomic level. Although extending to a larger genomic region, the organisation of the mouse ALDR gene mirrors exactly that of X-ALD. This supports further the hypothesis that among the four known peroxisomal ABC hemi-transporters ALDRP is the most likely candidate as a modifier contributing to the phenotypic variability of X-linked adrenoleukodystrophy.
Assuntos
Transportadores de Cassetes de Ligação de ATP , Adrenoleucodistrofia/genética , Éxons , Proteínas/genética , Subfamília D de Transportador de Cassetes de Ligação de ATP , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA Complementar , Camundongos , Dados de Sequência Molecular , Mapeamento por Restrição , Homologia de Sequência de Aminoácidos , Cromossomo XRESUMO
B-cell acute lymphoblastic leukemia (B-ALL) is often associated with chromosomal translocations leading to the deregulation of proto-oncogenes. MicroRNAs can also be affected by chromosomal alterations and thus contribute to carcinogenesis. The microRNA, miR-125b-1, is overexpressed in B-ALL cases with the t(11;14)(q24;q32) translocation; therefore, we sought to determine the role of this microRNA in B-cell fate. We used murine pre-BI cells alongside murine and human leukemic B-cell lines to show that miR-125b expression enhances proliferation by targeting B-cell regulator of immunoglobulin heavy-chain transcription (Bright)/ARID3a, an activator of immunoglobulin heavy-chain transcription. Accordingly, this target gene was downregulated in B-ALL patients with the t(11;14)(q24;q32) translocation. Repression of Bright/ARID3a blocked differentiation and conferred a survival advantage to Ba/F3 cells under interleukin-3 starvation. In addition, overexpression of miR-125b protected pre-BI and leukemic B-cell lines from apoptosis by blockade of caspase activation by a mechanism that was independent of p53 and BAK1. In summary, miR-125b can act as an oncogene in B-ALL by targeting ARID3a and mediating its repression, thus leading to a blockage in differentiation, increased proliferation and inhibition of apoptosis.
Assuntos
Proteínas de Ligação a DNA/fisiologia , MicroRNAs/fisiologia , Células Precursoras de Linfócitos B/fisiologia , Fatores de Transcrição/fisiologia , Diferenciação Celular , Proliferação de Células , Cromossomos Humanos Par 11 , Cromossomos Humanos Par 14 , Humanos , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Células Precursoras de Linfócitos B/citologia , Translocação Genética , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/fisiologiaRESUMO
Myeloproliferative neoplasms are frequently associated with aberrant constitutive tyrosine kinase (TK) activity resulting from chimaeric fusion genes or point mutations such as BCR-ABL1 or JAK2 V617F. We report here the cloning and functional characterization of two novel fusion genes BCR-RET and FGFR1OP-RET in chronic myelomonocytic leukemia (CMML) cases generated by two balanced translocations t(10;22)(q11;q11) and t(6;10)(q27;q11), respectively. The two RET fusion genes leading to the aberrant activation of RET, are able to transform hematopoietic cells and skew the hematopoietic differentiation program towards the monocytic/macrophage lineage. The RET fusion genes seem to constitutively mimic the same signaling pathway as RAS mutations frequently involved in CMML. One patient was treated with Sorafenib, a specific inhibitor of the RET TK function, and demonstrated cytological and clinical remissions.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Leucemia Mielomonocítica Crônica/patologia , Monócitos/citologia , Proteínas Proto-Oncogênicas c-ret/genética , Sequência de Bases , Primers do DNA , Humanos , Hibridização in Situ Fluorescente , Leucemia Mielomonocítica Crônica/genética , Mutação Puntual , Reação em Cadeia da Polimerase/métodos , Translocação GenéticaRESUMO
Adult and child B-cell progenitor acute lymphoblastic leukemia (BCP-ALL) differ in terms of incidence and prognosis. These disparities are mainly due to the molecular abnormalities associated with these two clinical entities. A genome-wide analysis using oligo SNP arrays recently demonstrated that PAX5 (paired-box domain 5) is the main target of somatic mutations in childhood BCP-ALL being altered in 38.9% of the cases. We report here the most extensive analysis of alterations of PAX5 coding sequence in 117 adult BCP-ALL patients in the unique clinical protocol GRAALL-2003/GRAAPH-2003. Our study demonstrates that PAX5 is mutated in 34% of adult BCP-ALL, mutations being partial or complete deletion, partial or complete amplification, point mutation or fusion gene. PAX5 alterations are heterogeneous consisting in complete loss in 17%, focal deletions in 10%, point mutations in 7% and translocations in 1% of the cases. PAX5 complete loss and PAX5 point mutations differ. PAX5 complete loss seems to be a secondary event and is significantly associated with BCR-ABL1 or TCF3-PBX1 fusion genes and a lower white blood cell count.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteínas de Ligação a DNA/genética , Proteínas de Fusão bcr-abl/genética , Fator de Transcrição PAX5/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Proteínas Proto-Oncogênicas/genética , Adolescente , Adulto , Antineoplásicos/uso terapêutico , Benzamidas , Ensaios Clínicos Fase II como Assunto , Dosagem de Genes , Rearranjo Gênico do Linfócito T/genética , Genômica , Haplótipos , Humanos , Mesilato de Imatinib , Cadeias Pesadas de Imunoglobulinas/genética , Imunofenotipagem , Pessoa de Meia-Idade , Estudos Multicêntricos como Assunto , Piperazinas/uso terapêutico , Mutação Puntual , Fator de Transcrição 1 de Leucemia de Células Pré-B , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamento farmacológico , Prognóstico , Estudos Prospectivos , Pirimidinas/uso terapêutico , Adulto JovemAssuntos
Biomarcadores Tumorais/genética , Cromossomos Humanos Par 7/genética , Cromossomos Humanos Par 9/genética , Proteínas de Fusão Oncogênica/genética , Fator de Transcrição PAX5/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/classificação , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Proteínas/genética , Translocação Genética/genética , Biomarcadores Tumorais/sangue , Contagem de Células Sanguíneas , Pré-Escolar , Proteínas do Citoesqueleto , Feminino , Humanos , Leucemia-Linfoma Linfoblástico de Células Precursoras B/sangue , Leucemia-Linfoma Linfoblástico de Células Precursoras B/terapia , Fatores de TranscriçãoRESUMO
We report here the genomic and transcriptional characterization in mouse and man of a novel transporter of the ABCA subclass, named ABCA7. As it is the case for other ABCA genes, the predicted protein encoded by ABCA7 is a full symmetric transporter, highly conserved across species. The ABCA7 gene maps to human chromosome 19 and to the homologous region at band B4-C1 on mouse chromosome 10. The preferential expression of ABCA7 in the spleen, thymus, and fetal liver is consistent with the finding, in both human and mouse promoter, of sites targeted by lymphomyeloid-specific transcription factors. This suggests that ABCA7 may play a pivotal role in the developmental specification of hematopoietic cell lineages.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Éxons/genética , Íntrons/genética , Regiões Promotoras Genéticas/genética , Transportadores de Cassetes de Ligação de ATP/química , Sequência de Aminoácidos , Animais , Linhagem Celular , Cromossomos Humanos Par 19/genética , Sequência Conservada/genética , DNA Complementar/genética , Humanos , Hibridização in Situ Fluorescente , Fígado/embriologia , Fígado/metabolismo , Camundongos , Dados de Sequência Molecular , Especificidade de Órgãos , RNA Mensageiro/análise , RNA Mensageiro/genética , Mapeamento de Híbridos Radioativos , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , Baço/metabolismo , Timo/metabolismoRESUMO
Recently, the human ATP-binding cassette transporter-1 (ABC1) gene has been demonstrated to be mutated in patients with Tangier disease. To investigate the role of the ABC1 protein in an experimental in vivo model, we used gene targeting in DBA-1J embryonic stem cells to produce an ABC1-deficient mouse. Expression of the murine Abc1 gene was ablated by using a nonisogenic targeting construct that deletes six exons coding for the first nucleotide-binding fold. Lipid profiles from Abc1 knockout (-/-) mice revealed an approximately 70% reduction in cholesterol, markedly reduced plasma phospholipids, and an almost complete lack of high density lipoproteins (HDL) when compared with wild-type littermates (+/+). Fractionation of lipoproteins by FPLC demonstrated dramatic alterations in HDL cholesterol (HDL-C), including the near absence of apolipoprotein AI. Low density lipoprotein (LDL) cholesterol (LDL-C) and apolipoprotein B were also significantly reduced in +/- and -/- compared with their littermate controls. The inactivation of the Abc1 gene led to an increase in the absorption of cholesterol in mice fed a chow or a high-fat and -cholesterol diet. Histopathologic examination of Abc1-/- mice at ages 7, 12, and 18 mo demonstrated a striking accumulation of lipid-laden macrophages and type II pneumocytes in the lungs. Taken together, these findings demonstrate that Abc1-/- mice display pathophysiologic hallmarks similar to human Tangier disease and highlight the capacity of ABC1 transporters to participate in the regulation of dietary cholesterol absorption.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Células Espumosas/citologia , Glicoproteínas/genética , Lipoproteínas HDL/deficiência , Mutação , Transportador 1 de Cassete de Ligação de ATP , Animais , Sequência de Bases , Colesterol/sangue , Primers do DNA , Humanos , Lipoproteínas HDL/sangue , Camundongos , Camundongos KnockoutRESUMO
Tangier disease is characterized by low serum high density lipoproteins and a biochemical defect in the cellular efflux of lipids to high density lipoproteins. ABC1, a member of the ATP-binding cassette family, recently has been identified as the defective gene in Tangier disease. We report here the organization of the human ABC1 gene and the identification of a mutation in the ABC1 gene from the original Tangier disease kindred. The organization of the human ABC1 gene is similar to that of the mouse ABC1 gene and other related ABC genes. The ABC1 gene contains 49 exons that range in size from 33 to 249 bp and is over 70 kb in length. Sequence analysis of the ABC1 gene revealed that the proband for Tangier disease was homozygous for a deletion of nucleotides 3283 and 3284 (TC) in exon 22. The deletion results in a frameshift mutation and a premature stop codon starting at nucleotide 3375. The product is predicted to encode a nonfunctional protein of 1,084 aa, which is approximately half the size of the full-length ABC1 protein. The loss of a Mnl1 restriction site, which results from the deletion, was used to establish the genotype of the rest of the kindred. In summary, we report on the genomic organization of the human ABC1 gene and identify a frameshift mutation in the ABC1 gene of the index case of Tangier disease. These results will be useful in the future characterization of the structure and function of the ABC1 gene and the analysis of additional ABC1 mutations in patients with Tangier disease.