RESUMEN

Mammalian spermatogenesis comprises three successive phases: mitosis phase, meiosis phase, and spermiogenesis. During spermiogenesis, round spermatid undergoes dramatic morphogenesis to give rise to mature spermatozoon, including the condensation and elongation of nucleus, development of acrosome, formation of flagellum, and removal of excessive cytoplasm. Although these transformations are well defined at the morphological level, the mechanisms underlying these intricate processes are largely unknown. Here, we report that Iqcg, which was previously characterized to be involved in a chromosome translocation of human leukemia, is highly expressed in the spermatogenesis of mice and localized to the manchette in developing spermatids. Iqcg knockout causes male infertility, due to severe defects of spermiogenesis and resultant total immobility of spermatozoa. The axoneme in the Iqcg knockout sperm flagellum is disorganized and hardly any typical ("9+2") pattern of microtubule arrangement could be found in Iqcg knockout spermatids. Iqcg interacts with calmodulin in a calcium dependent manner in the testis, suggesting that Iqcg may play a role through calcium signaling. Furthermore, cilia structures in the trachea and oviduct, as well as histological appearances of other major tissues, remain unchanged in the Iqcg knockout mice, suggesting that Iqcg is specifically required for spermiogenesis in mammals. These results might also provide new insights into the genetic causes of human infertility.

Asunto(s)

Proteínas de Unión a Calmodulina/metabolismo , Flagelos/metabolismo , Espermatozoides/citología , Animales , Calcio/metabolismo , Calmodulina/metabolismo , Proteínas de Unión a Calmodulina/deficiencia , Proteínas de Unión a Calmodulina/genética , Proteínas del Citoesqueleto , Regulación de la Expresión Génica , Técnicas de Inactivación de Genes , Humanos , Masculino , Ratones , Fenotipo , Espermatogénesis , Testículo/metabolismo , Testículo/fisiología

RESUMEN

We previously reported a fusion protein NUP98-IQCG in an acute leukaemia, which functions as an aberrant regulator of transcriptional expression, yet the structure and function of IQCG have not been characterized. Here we use zebrafish to investigate the role of iqcg in haematopoietic development, and find that the numbers of haematopoietic stem cells and multilineage-differentiated cells are reduced in iqcg-deficient embryos. Mechanistically, IQCG binds to calmodulin (CaM) and acts as a molecule upstream of CaM-dependent kinase IV (CaMKIV). Crystal structures of complexes between CaM and IQ domain of IQCG reveal dual CaM-binding footprints in this motif, and provide a structural basis for a higher CaM-IQCG affinity when deprived of calcium. The results collectively allow us to understand IQCG-mediated calcium signalling in haematopoiesis, and propose a model in which IQCG stores CaM at low cytoplasmic calcium concentrations, and releases CaM to activate CaMKIV when calcium level rises.

Asunto(s)

Calmodulina/metabolismo , Proteínas de Pez Cebra/metabolismo , Animales , Proteína Quinasa Tipo 4 Dependiente de Calcio Calmodulina/metabolismo , Proliferación Celular , Técnicas de Silenciamiento del Gen , Proteínas HSP70 de Choque Térmico/metabolismo , Hematopoyesis , Pez Cebra , Proteínas de Pez Cebra/genética

RESUMEN

LDL-receptor-related protein 6 (LRP6), alongside Frizzled receptors, transduces Wnt signaling across the plasma membrane. The LRP6 ectodomain comprises four tandem ß-propeller-EGF-like domain (PE) pairs that harbor binding sites for Wnt morphogens and their antagonists including Dickkopf 1 (Dkk1). To understand how these multiple interactions are integrated, we combined crystallographic analysis of the third and fourth PE pairs with electron microscopy (EM) to determine the complete ectodomain structure. An extensive inter-pair interface, conserved for the first-to-second and third-to-fourth PE interactions, contributes to a compact platform-like architecture, which is disrupted by mutations implicated in developmental diseases. EM reconstruction of the LRP6 platform bound to chaperone Mesd exemplifies a binding mode spanning PE pairs. Cellular and binding assays identify overlapping Wnt3a- and Dkk1-binding surfaces on the third PE pair, consistent with steric competition, but also suggest a model in which the platform structure supports an interplay of ligands through multiple interaction sites.

Asunto(s)

Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/química , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/metabolismo , Proteínas Wnt/metabolismo , Vía de Señalización Wnt , Cristalografía por Rayos X , Células HEK293 , Humanos , Modelos Moleculares , Estructura Terciaria de Proteína

RESUMEN

Abnormal epigenetic regulation has been implicated in oncogenesis. We report here the identification of somatic mutations by exome sequencing in acute monocytic leukemia, the M5 subtype of acute myeloid leukemia (AML-M5). We discovered mutations in DNMT3A (encoding DNA methyltransferase 3A) in 23 of 112 (20.5%) cases. The DNMT3A mutants showed reduced enzymatic activity or aberrant affinity to histone H3 in vitro. Notably, there were alterations of DNA methylation patterns and/or gene expression profiles (such as HOXB genes) in samples with DNMT3A mutations as compared with those without such changes. Leukemias with DNMT3A mutations constituted a group of poor prognosis with elderly disease onset and of promonocytic as well as monocytic predominance among AML-M5 individuals. Screening other leukemia subtypes showed Arg882 alterations in 13.6% of acute myelomonocytic leukemia (AML-M4) cases. Our work suggests a contribution of aberrant DNA methyltransferase activity to the pathogenesis of acute monocytic leukemia and provides a useful new biomarker for relevant cases.

Asunto(s)

ADN (Citosina-5-)-Metiltransferasas/genética , Leucemia Monocítica Aguda/enzimología , Leucemia Monocítica Aguda/genética , Mutación Missense , Adulto , Anciano , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Secuencia de Bases , Biomarcadores de Tumor/genética , Secuencia Conservada , ADN (Citosina-5-)-Metiltransferasas/química , Metilación de ADN/genética , ADN Metiltransferasa 3A , ADN Complementario/genética , Exones , Femenino , N-Metiltransferasa de Histona-Lisina , Humanos , Estimación de Kaplan-Meier , Leucemia Mieloide Aguda/genética , Masculino , Persona de Mediana Edad , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteína de la Leucemia Mieloide-Linfoide/genética , Pronóstico , Homología de Secuencia de Aminoácido

RESUMEN

Arsenic, an ancient drug used in traditional Chinese medicine, has attracted worldwide interest because it shows substantial anticancer activity in patients with acute promyelocytic leukemia (APL). Arsenic trioxide (As2O3) exerts its therapeutic effect by promoting degradation of an oncogenic protein that drives the growth of APL cells, PML-RARalpha (a fusion protein containing sequences from the PML zinc finger protein and retinoic acid receptor alpha). PML and PML-RARalpha degradation is triggered by their SUMOylation, but the mechanism by which As2O3 induces this posttranslational modification is unclear. Here we show that arsenic binds directly to cysteine residues in zinc fingers located within the RBCC domain of PML-RARalpha and PML. Arsenic binding induces PML oligomerization, which increases its interaction with the small ubiquitin-like protein modifier (SUMO)-conjugating enzyme UBC9, resulting in enhanced SUMOylation and degradation. The identification of PML as a direct target of As2O3 provides new insights into the drug's mechanism of action and its specificity for APL.

Asunto(s)

Arsénico/metabolismo , Arsenicales/metabolismo , Arsenicales/farmacología , Proteínas Nucleares/metabolismo , Proteínas de Fusión Oncogénica/metabolismo , Óxidos/metabolismo , Óxidos/farmacología , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Trióxido de Arsénico , Línea Celular , Humanos , Leucemia Promielocítica Aguda/tratamiento farmacológico , Leucemia Promielocítica Aguda/genética , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutación , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas de Fusión Oncogénica/química , Proteínas de Fusión Oncogénica/genética , Oxazinas/metabolismo , Proteína de la Leucemia Promielocítica , Conformación Proteica , Multimerización de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Receptores de Ácido Retinoico/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Receptor alfa de Ácido Retinoico , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Factores de Transcripción/química , Factores de Transcripción/genética , Proteínas Supresoras de Tumor/química , Proteínas Supresoras de Tumor/genética , Ubiquitinación , Dedos de Zinc

RESUMEN

To explore the genetic abnormalities that cooperate with AML1-ETO (AE) fusion gene to cause acute myeloid leukemia (AML) with t(8;21), we screened a number of candidate genes and identified 11 types of mutations in C-KIT gene (mC-KIT), including 6 previously undescribed ones among 26 of 54 (48.1%) cases with t(8;21). To address a possible chronological order between AE and mC-KIT, we showed that, among patients with AE and mC-KIT, most leukemic cells at disease presentation harbored both genetic alteration, whereas in three such cases investigated during complete remission, only AE, but not mC-KIT, could be detected by allele-specific PCR. Therefore, mC-KIT should be a subsequent event on the basis of t(8;21). Furthermore, induced expression of AE in U937-A/E cells significantly up-regulated mRNA and protein levels of C-KIT. This may lead to an alternative way of C-KIT activation and may explain the significantly higher C-KIT expression in 81.3% of patients with t(8;21) than in patients with other leukemias. These data strongly suggest that t(8;21) AML follows a stepwise model in leukemogenesis, i.e., AE represents the first, fundamental genetic hit to initiate the disease, whereas activation of the C-KIT pathway may be a second but also crucial hit for the development of a full-blown leukemia. Additionally, Gleevec suppressed the C-KIT activity and induced proliferation inhibition and apoptosis in cells bearing C-KIT N822K mutation or overexpression, but not in cells with D816 mC-KIT. Gleevec also exerted a synergic effect in apoptosis induction with cytarabine, thus providing a potential therapeutic for t(8;21) leukemia.

Asunto(s)

Antineoplásicos/farmacología , Cromosomas Humanos Par 21 , Cromosomas Humanos Par 8 , Leucemia Mieloide Aguda/genética , Mutación , Proteínas de Fusión Oncogénica/genética , Piperazinas/farmacología , Proteínas Proto-Oncogénicas c-kit/genética , Pirimidinas/farmacología , Factores de Transcripción/genética , Translocación Genética , Adolescente , Adulto , Apoptosis/efectos de los fármacos , Benzamidas , Niño , Preescolar , Subunidad alfa 2 del Factor de Unión al Sitio Principal , Femenino , Humanos , Mesilato de Imatinib , Leucemia Mieloide Aguda/tratamiento farmacológico , Masculino , Persona de Mediana Edad , Proteína 1 Compañera de Translocación de RUNX1

RESUMEN

The t(8;21) translocation is one of the most frequent chromosome abnormalities in acute myeloid leukemia. This translocation creates a fusion between the acute myelogenous leukemia 1 (AML1, a transcription factor) gene on chromosome 21 and the eight-twenty-one (ETO, a zinc finger nuclear protein) gene on chromosome 8, leading to the repression of certain AML1 target genes. We cloned NHR3 domain coding fragment into vector pGEX-6p-1 using PCR and obtained recombinant plasmid pGEX-6p-1-NHR3, which can be induced to stably overexpress fusion protein in E. coli. Through the purification on GST affinity chromatography column and PreScission protease cleavage, a large amount of NHR3 protein with high purity was obtained. In order to avoid possible interference of some strong negative charged molecules, NHR3 protein was further purified by Mono Q anion exchange chromatography. The NHR3 crystals were grown with hanging drop/vapor diffusion method and the first crystals appeared after four weeks at 18 degrees in 0.2 M Tris-sodium citrate dihydrate, 0.1 M sodium cacodylate, pH 6.5, and 30% iso-propanol (V/V). ESI mass spectrum showed that the molecular weight of this domain was in agreement with its primary structure sequence prediction, and circular dichroism spectral data (190-250 nm) showed that NHR3 had a well-defined secondary structure of 25.9% alpha-helix, 23.2% random coil and 50.9% turn, which was consistent with GOV4 software prediction.

Asunto(s)

Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/aislamiento & purificación , Factores de Transcripción/genética , Factores de Transcripción/aislamiento & purificación , Secuencia de Bases , Dicroismo Circular , Clonación Molecular , Subunidad alfa 2 del Factor de Unión al Sitio Principal , Cristalización , ADN de Neoplasias/genética , Expresión Génica , Humanos , Leucemia Mieloide Aguda/genética , Proteínas de Fusión Oncogénica/química , Estructura Terciaria de Proteína , Proteína 1 Compañera de Translocación de RUNX1 , Espectrometría de Masa por Ionización de Electrospray , Factores de Transcripción/química