RESUMEN
The MLL gene at chromosome band 11q23 is commonly involved in reciprocal translocations detected in acute leukemias. A number of experiments show that the resulting MLL fusion genes directly contribute to leukemogenesis. Among the many known MLL fusion partners, AF4 is relatively common, particularly in acute lymphoblastic leukemia in infants. The AF4 protein interacts with the product of another gene, AF9, which is also fused to MLL in acute leukemias. Based on mapping studies of the AF9-binding domain of AF4, we have developed a peptide, designated PFWT, which disrupts the AF4-AF9 interaction in vitro and in vivo. We provide evidence that this peptide is able to inhibit the proliferation of leukemia cells with t(4;11) chromosomal translocations expressing MLL-AF4 fusion genes. Further, we show that this inhibition is mediated through apoptosis. Importantly, the peptide does not affect the proliferative capacity of hematopoietic progenitor cells. Our findings indicate that the AF4-AF9 protein complex is a promising new target for leukemia therapy and that the PFWT peptide may serve as a lead compound for drug development.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Leucemia/patología , Proteínas Nucleares/metabolismo , Oligopéptidos/farmacología , Secuencia de Aminoácidos , Antineoplásicos/síntesis química , Línea Celular Tumoral , Cromosomas Humanos Par 11 , Cromosomas Humanos Par 4 , Sistemas de Liberación de Medicamentos , Humanos , Leucemia/tratamiento farmacológico , Leucemia/genética , Proteína de la Leucemia Mieloide-Linfoide , Proteínas Nucleares/efectos de los fármacos , Oligopéptidos/síntesis química , Proteínas de Fusión Oncogénica , Unión Proteica/efectos de los fármacos , Transfección , Translocación GenéticaRESUMEN
The MLL gene is involved in translocations associated with both acute lymphoblastic and acute myelogenous leukemia. These translocations fuse MLL with one of over 30 partner genes. Collectively, the MLL partner genes do not share a common structural motif or biochemical function. We have identified a protein interaction between the two most common MLL fusion partners AF4 and AF9. This interaction is restricted to discrete nuclear foci we have named 'AF4 bodies'. The AF4 body is non-nucleolar and is not coincident with any known nuclear structures we have examined. The AF4-AF9 interaction is maintained by the MLL-AF4 fusion protein, and expression of the MLL-AF4 fusion can alter the subnuclear localization of AF9. In view of other research indicating that other MLL fusion partners also interact with one another, these results suggest that MLL fusion partners may participate in a web of protein interactions with a common functional goal. The disruption of this web of interactions by fusion with MLL may be important to leukemogenesis.
Asunto(s)
Núcleo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/metabolismo , Proto-Oncogenes , Factores de Transcripción , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Glutatión Transferasa/metabolismo , Células HeLa/patología , N-Metiltransferasa de Histona-Lisina , Humanos , Proteína de la Leucemia Mieloide-Linfoide , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Señales de Localización Nuclear , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Plásmidos , Saccharomyces cerevisiae , Fracciones Subcelulares , Factores de Elongación Transcripcional , Técnicas del Sistema de Dos Híbridos , Dedos de ZincRESUMEN
Polycomb group (PcG) proteins assemble to form large multiprotein complexes involved in gene silencing. Evidence suggests that PcG complexes are heterogeneous with respect to both protein composition and specific function. MPc3 is a recently described mouse Polycomb (Pc) protein that shares structural homology with at least two other Pc proteins, M33 and MPc2. All three Pc proteins bind another PcG protein, RING1, through a conserved carboxy-terminal C-box motif. Here, data are presented demonstrating that MPc3 also interacts with AF9, a transcriptional activator implicated in the development of acute leukemias. The carboxy-terminus of AF9 is fused to the MLL protein in leukemias characterized by t(9;11)(p22;q23) chromosomal translocations. Importantly, it is the carboxy-terminus of AF9 to which MPc3 binds. The AF9 binding site of MPc3 maps to a central, non-conserved, region of the polypeptide sequence. In contrast to MPc3, data indicate that the Pc protein M33 does not interact with AF9. This finding suggests a potentially unique role for MPc3 in linking a PcG silencing complex to a transcriptional activator protein.
Asunto(s)
Cromosomas Humanos Par 11 , Cromosomas Humanos Par 9 , Proteínas de Unión al ADN/metabolismo , Leucemia Bifenotípica Aguda/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Fusión Oncogénica/metabolismo , Proto-Oncogenes , Proteínas Represoras/metabolismo , Transactivadores/metabolismo , Factores de Transcripción , Translocación Genética , Células 3T3 , Animales , Sitios de Unión , Núcleo Celular/metabolismo , Proteínas de Unión al ADN/genética , N-Metiltransferasa de Histona-Lisina , Humanos , Ratones , Proteínas de Transporte de Membrana Mitocondrial , Proteína de la Leucemia Mieloide-Linfoide , Proteínas Nucleares/genética , Proteínas de Fusión Oncogénica/genética , Complejo Represivo Polycomb 1 , Proteínas del Grupo Polycomb , Pruebas de Precipitina , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Represoras/genética , Saccharomyces cerevisiae , Transactivadores/genética , Técnicas del Sistema de Dos HíbridosRESUMEN
A new mouse Polycomb (Pc) gene, MPc3, has been identified. The MPc3 protein contains the highly conserved chromodomain and carboxy-terminal COOH box of other known Pc proteins from diverse species. Like other Pc proteins, MPc3 physically interacts with the RING finger proteins RING1A and dinG/RING1B. MPc3 maps to the distal arm of mouse chromosome 11 (11E2), a region that contains other known Pc genes in addition to several disease loci that may be linked to abnormal Pc gene function.
Asunto(s)
Proteínas Represoras/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Mapeo Cromosómico , ADN/química , ADN/genética , ADN Complementario/química , ADN Complementario/genética , Proteínas de Unión al ADN/metabolismo , Embrión de Mamíferos/metabolismo , Exones , Regulación del Desarrollo de la Expresión Génica , Hibridación Fluorescente in Situ , Intrones , Ratones , Ratones Endogámicos , Proteínas de Transporte de Membrana Mitocondrial , Datos de Secuencia Molecular , Complejo Represivo Polycomb 1 , Proteínas del Grupo Polycomb , Unión Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Represoras/metabolismo , Saccharomyces cerevisiae/genética , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Técnicas del Sistema de Dos HíbridosRESUMEN
The target of the immunosuppressants cyclosporin A(CsA) and FK506 is calcineurin, a highly conserved protein phosphatase that is required for T-cell activation and the regulation of ion homeostasis in yeast. Here we identify two genes, PMR2B and LIC4 which, when overexpressed, suppress the cation-sensitive phenotype of yeast cells lacking calcineurin. PMR2B encodes a Na+/Li+-specific plasma membrane pump and is similar to PMR2A, whose expression is known to be regulated by calcineurin. LIC4 (lithium comvertas) encodes a novel 33-kDa protein with no identity to known proteins. LIC4 overexpression suppresses the Li+-sensitive phenotype of calcineurin mutants but not the defect in recovery from pheromone arrest or viability of calcineurin dependent mutants, indicating a specific role in cation homeostasis. Similarly, lic4 mutations increase the Li+ sensitivity of both wild-type and calcineurin mutant strains, and reduce expression of pmr2A in calcineurin mutant strains, indicating that calcineurin and Lic4 may regulate parallel cation homeostatic pathways. lic4 mutations also exacerbate the Li+-sensitive phenotype of hal3 mutant strains, and overexpression of either Lic4 or Hal3 suppresses the salt sensitivity of mutant strains lacking calcineurin, Hal3, or Lic4, either singly or in combination. Taken together, these observations suggest that calcineurin, Hal3, and Lic4 cooperatively regulate the response of yeast cells to cation stress. Lic4 is phosphoprotein in vivo and a calcineurin substrate in vitro. By indirect and direct immunofluorescence detection of HA- and GFP-tagged proteins, Lic4 is localized in the nucleus in wild-type cells but predominantly cytoplasmic in cells lacking calcineurin. Taken together, our findings support a model in which calcineurin and Lic4 are components of signalling cascades that regulate cation stress responses in yeast.
Asunto(s)
Calcineurina/metabolismo , Proteínas de Ciclo Celular , Proteínas Fúngicas/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Calmodulina , Cationes , Proteínas Fúngicas/genética , Dosificación de Gen , Genes Fúngicos , Homeostasis , Litio , Mutagénesis , Proteínas Nucleares/genética , Fenotipo , Fosfoproteínas/genética , Saccharomyces cerevisiae/genética , Especificidad por SustratoRESUMEN
Calcineurin is a serine-threonine specific Ca(2+)-calmodulin-activated protein phosphatase that is conserved from yeast to humans. Remarkably, this enzyme is the common target for two novel and structurally unrelated immunosuppressive antifungal drugs, cyclosporin A and FK506. Both drugs form complexes with abundant intracellular binding proteins, cyclosporin A with cyclophilin A and FK506 with FKBP 12, which bind to and inhibit calcineurin. The X-ray structure of an FKPB12-FK506-calcineurin AB ternary complex reveals that FKBP12-FK506 binds in a hydophobic groove between the calcineurin A catalytic and the regulatory B subunit, in accord with biochemical and genetic studies on inhibitor action. Calcineurin plays a key role in regulating the transcription factor NF-AT during T-cell activation, and in mediating responses of microorganisms to cation stress. These findings highlight the potential of yeast genetic studies to define novel drug targets and elucidate conserved elements of signal transduction cascades.
Asunto(s)
Inhibidores de la Calcineurina , Calcineurina/química , Calcineurina/fisiología , Proteínas Nucleares , Ciclosporina/química , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Modelos Biológicos , Modelos Químicos , Modelos Moleculares , Factores de Transcripción NFATC , Tacrolimus/química , Factores de Transcripción/metabolismo , Levaduras/metabolismoRESUMEN
Experimentally-induced mutations in the C3HC4 RING finger domain of the Bmi-1 oncoprotein block its ability to induce lymphomas in mice. In this report, the role of the Bmi-1 RING finger in mediating protein-protein interactions is examined using the yeast two-hybrid system. Bmi-1 interacts directly with the RING finger protein dinG/RING1B. Heterodimerization of the two proteins requires the intact RING finger structures of both Bmi-1 and dinG. Although the RING finger domains are necessary for dimerization, they are not sufficient for this process as residues outside the C3HC4 motif are also required. Thus, binding specificity may be partly conferred by residues outside the RING motif. Both Bmi-1 and dinG interact with the Polyhomeotic protein MPh2 through binding domains apart from the RING finger. The data suggest a model whereby Bmi-1, dinG, and MPh2 form a stable heterotrimeric complex in which each protein contributes to the binding of the others.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila , Proteínas Nucleares/metabolismo , Nucleoproteínas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Represoras , Dedos de Zinc/fisiología , Animales , Proteínas de Unión al ADN/genética , Humanos , Ratones , Datos de Secuencia Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/fisiología , Nucleoproteínas/genética , Complejo Represivo Polycomb 1 , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/fisiología , Relación Estructura-Actividad , Ubiquitina-Proteína LigasasRESUMEN
The mammalian P-glycoprotein (Pgp) is a approximately 170-kDa membrane protein that mediates multidrug resistance in many chemotherapy-resistant tumors by effluxing toxic compounds from the cell. Pgp homologs are expressed in many organisms, from bacteria to yeast and mammals. Previous studies established a model system to analyze the function of murine, human, and Plasmodium falciparum Pgp by heterologous expression in the yeast Saccharomyces cerevisiae. However, such studies have been hampered by the inherent resistance of yeast cells to chemotherapeutic agents. We find that an erg6 mutation, which blocks the final synthetic step of the membrane sterol ergosterol, renders yeast sensitive to anthracyclines and dactinomycin, clinically relevant Pgp substrates. We demonstrate that expression of the murine mdr3 gene confers dactinomycin resistance in both the erg6 mutant yeast strain and in an erg6 rad52 DNA repair mutant yeast strain. Similarly, murine mdr3 expression confers resistance to the immunosuppressants cyclosporin A (CsA) and FK506 in a CsA-FK506-sensitive vph6 mutant yeast strain. CsA and FK506 are known to partially overcome Pgp-mediated drug resistance, suggesting the targets of these drugs might regulate Pgp function. We find that both murine mdr3 and the yeast Pgp homolog STE6 function in yeast mutants lacking the CsA target proteins cyclophilin A and calcineurin. In contrast, murine mdr3 function was severely compromised in yeast mutants lacking the FK506/rapamycin target protein FKBP12. Both wild-type FKBP12 and an F43Y FKBP12 mutant with reduced prolyl isomerase activity supported mdr3 function. Our results support the model that immunosuppressants reverse multidrug resistance by competing with other Pgp substrates but reveal that inhibition of FKBP12-dependent Pgp function may also contribute to reversal of multidrug resistance by FK506 and rapamycin.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Proteínas Portadoras/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Choque Térmico/metabolismo , Saccharomyces cerevisiae/metabolismo , Isomerasas de Aminoácido/metabolismo , Animales , Calcineurina , Proteínas de Unión a Calmodulina/metabolismo , Proteínas Portadoras/genética , Ciclosporina/farmacología , Proteínas de Unión al ADN/genética , Dactinomicina/farmacología , Resistencia a Múltiples Medicamentos/genética , Proteínas de Choque Térmico/genética , Humanos , Inmunosupresores/farmacología , Metiltransferasas/genética , Metiltransferasas/metabolismo , Ratones , Mutación , Isomerasa de Peptidilprolil , Fosfoproteínas Fosfatasas/metabolismo , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Tacrolimus/metabolismo , Tacrolimus/farmacología , Proteínas de Unión a TacrolimusRESUMEN
We have characterized a Saccharomyces cerevisiae mutant strain that is hypersensitive to cyclosporin A (CsA) and FK506, immunosuppressants that inhibit calcineurin, a serine-threonine-specific phosphatase (PP2B). A single nuclear mutation, designated cev1 for calcineurin essential for viability, is responsible for the CsA-FK506-sensitive phenotype. The peptidyl-prolyl cis-trans isomerases cyclophilin A and FKBP12, respectively, mediate CsA and FK506 toxicity in the cev1 mutant strain. We demonstrate that cev1 is an allele of the VPH6 gene and that vph6 mutant strains fail to assemble the vacuolar H(+)-ATPase (V-ATPase). The VPH6 gene was mapped on chromosome VIII and is predicted to encode a 181-amino acid (21 kD) protein with no identity to other known proteins. We find that calcineurin is essential for viability in many mutant strains with defects in V-ATPase function or vacuolar acidification. In addition, we find that calcineurin modulates extracellular acidification in response to glucose, which we propose occurs via calcineurin regulation of the plasma membrane H(+)-ATPase PMA1. Taken together, our findings suggest calcineurin plays a general role in the regulation of cation transport and homeostasis.
Asunto(s)
Proteínas de Unión a Calmodulina/fisiología , Proteínas Fúngicas/genética , Genes Fúngicos , Fosfoproteínas Fosfatasas/fisiología , ATPasas de Translocación de Protón/deficiencia , Saccharomyces cerevisiae/genética , Vacuolas/enzimología , Secuencia de Bases , Calcineurina , Proteínas de Unión a Calmodulina/antagonistas & inhibidores , Ciclosporina/farmacología , Proteínas Fúngicas/fisiología , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , ATPasas de Translocación de Protón/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/fisiología , Tacrolimus/farmacologíaRESUMEN
The immunophilin-immunosuppressant complexes cyclophilin-cyclosporin A (CsA) and FKBP12-FK506 inhibit the phosphatase calcineurin to block T-cell activation. Although cyclophilin A, FKBP12, and calcineurin are highly conserved from yeast to man, none had previously been shown to be essential for viability. We find that CsA-sensitive yeast strains are FK506 hypersensitive and demonstrate that calcineurin is required for viability in these strains. Mutants lacking cyclophilin A or FKBP12 are resistant to CsA or FK506, respectively. Thus, both the immunosuppressive and the antifungal actions of CsA and FK506 result from calcineurin inhibition by immunophilin-drug complexes. In yeast strains in which calcineurin is not essential, calcineurin inhibition or mutation of calcineurin confers hypersensitivity to LiCl or NaCl, suggesting that calcineurin regulates cation transport.