Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Cloridrato de Bendamustina/administração & dosagem , Comorbidade , Humanos , Leucemia Linfocítica Crônica de Células B/diagnóstico , Leucemia Linfocítica Crônica de Células B/mortalidade , Rituximab/administração & dosagem , Resultado do TratamentoRESUMO
AML1-ETO is the chimeric protein product of t(8;21) in acute myeloid leukemia. The ETO portion of the fusion protein includes the nervy homology region (NHR) 3 domain, which shares homology with A-kinase anchoring proteins and interacts with the regulatory subunit of type II cAMP-dependent protein kinase A (PKA(RIIα)). We determined the solution structure of a complex between the AML1-ETO NHR3 domain and PKA(RIIα). Based on this structure, a key residue in AML1-ETO for PKA(RIIα) association was mutated. This mutation did not disrupt AML1-ETO's ability to enhance the clonogenic capacity of primary mouse bone marrow cells or its ability to repress proliferation or granulocyte differentiation. Introduction of the mutation into AML1-ETO had minimal impact on in vivo leukemogenesis. Therefore, the NHR3-PKA(RIIα) protein interaction does not appear to significantly contribute to AML1-ETO's ability to induce leukemia.
Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core/genética , Subunidade RIIalfa da Proteína Quinase Dependente de AMP Cíclico/química , Leucemia Mieloide Aguda/metabolismo , Proteínas de Fusão Oncogênica/química , Proteínas Proto-Oncogênicas/genética , Fatores de Transcrição/genética , Animais , Sítios de Ligação/genética , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Diferenciação Celular , Subunidade alfa 2 de Fator de Ligação ao Core/química , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Subunidade RIIalfa da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Humanos , Leucemia Mieloide Aguda/etiologia , Leucemia Mieloide Aguda/genética , Camundongos , Mutação , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/metabolismo , Proteína 1 Parceira de Translocação de RUNX1 , Fatores de Transcrição/química , Fatores de Transcrição/metabolismoRESUMO
AML1-ETO and TEL-AML1 are chimeric proteins resulting from the t(8;21)(q22;q22) in acute myeloid leukemia, and the t(12;21)(p13;q22) in pre-B-cell leukemia, respectively. The Runt domain of AML1 in both proteins mediates DNA binding and heterodimerization with the core binding factor beta (CBFbeta) subunit. To determine whether CBFbeta is required for AML1-ETO and TEL-AML1 activity, we introduced amino acid substitutions into the Runt domain that disrupt heterodimerization with CBFbeta but not DNA binding. We show that CBFbeta contributes to AML1-ETO's inhibition of granulocyte differentiation, is essential for its ability to enhance the clonogenic potential of primary mouse bone marrow cells, and is indispensable for its cooperativity with the activated receptor tyrosine kinase TEL-PDGFbetaR in generating acute myeloid leukemia in mice. Similarly, CBFbeta is essential for TEL-AML1's ability to promote self-renewal of B cell precursors in vitro. These studies validate the Runt domain/CBFbeta interaction as a therapeutic target in core binding factor leukemias.
Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core/fisiologia , Subunidade beta de Fator de Ligação ao Core/fisiologia , Proteínas de Fusão Oncogênica/fisiologia , Animais , Diferenciação Celular/genética , Proliferação de Células , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Subunidade beta de Fator de Ligação ao Core/química , Subunidade beta de Fator de Ligação ao Core/genética , Subunidade beta de Fator de Ligação ao Core/metabolismo , Granulócitos/metabolismo , Granulócitos/fisiologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Modelos Moleculares , Mutação/fisiologia , Células NIH 3T3 , Proteínas de Fusão Oncogênica/genética , Ligação Proteica , Estrutura Terciária de Proteína/genética , Estrutura Terciária de Proteína/fisiologia , Proteína 1 Parceira de Translocação de RUNX1 , TransfecçãoRESUMO
The signal transduction pathway leading from the insulin receptor to stimulate the fusion of vesicles containing the glucose transporter GLUT4 with the plasma membrane in adipocytes and muscle cells is not completely understood. Current evidence suggests that in addition to the Rab GTPase-activating protein AS160, at least one other substrate of Akt (also called protein kinase B), which is as yet unidentified, is required. Sec8 is a component of the exocyst complex that has been previously implicated in GLUT4 trafficking. In the present study, we report that insulin stimulates the phosphorylation of Sec8 on Ser-32 in 3T3-L1 adipocytes. On the basis of the sequence around Ser-32 and the finding that phosphorylation is inhibited by the PI3K (phosphoinositide 3-kinase) inhibitor wortmannin, it is likely that Akt is the kinase for Ser-32. We examined the possible role of Ser-32 phosphorylation in the insulin-stimulated trafficking of GLUT4, as well as the TfR (transferrin receptor), to the plasma membrane by determining the effects of overexpression of the non-phosphorylatable S32A mutant of Sec8 and the phosphomimetic S32E mutant of Sec8. Substantial overexpression of both mutants had no effect on the amount of GLUT4 or TfR at the cell surface in either the untreated or insulin-treated states. These results indicate that insulin-stimulated phosphorylation of Sec8 is not part of the mechanism by which insulin enhances the fusion of vesicles with the plasma membrane.
Assuntos
Adipócitos/efeitos dos fármacos , Proteínas de Transporte/metabolismo , Hipoglicemiantes/farmacologia , Insulina/farmacologia , Fosforilação/efeitos dos fármacos , Células 3T3-L1 , Adipócitos/metabolismo , Animais , Carbocianinas/metabolismo , Proteínas de Transporte/genética , Células Cultivadas , Meios de Cultura Livres de Soro , Eletroporação , Epitopos/metabolismo , Exocitose/efeitos dos fármacos , Técnica Direta de Fluorescência para Anticorpo , Corantes Fluorescentes/metabolismo , Genes Reporter , Transportador de Glucose Tipo 4/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hemaglutininas/metabolismo , Proteínas de Membrana , Camundongos , Plasmídeos/genética , Fatores de TempoRESUMO
Menin binds to the N terminus of the chromatin-remodeling histone methyltransferase MLL and is essential for the transforming activity of at least several oncogenic MLL fusion proteins. In this issue, Yokoyama and Cleary (2008) show that menin's essential, and perhaps only, contribution to leukemia is to tether a third protein, LEDGF--a chromatin-associated protein implicated in leukemia and several other disease states--to MLL. Thus, this study identifies a new, critical player in leukemias caused by MLL fusion proteins and defines the biochemical function of menin in the MLL complex.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Transformação Celular Neoplásica/metabolismo , Leucemia/metabolismo , Células Progenitoras Mieloides/metabolismo , Proteína de Leucina Linfoide-Mieloide/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , Regulação Leucêmica da Expressão Gênica , Histona Metiltransferases , Histona-Lisina N-Metiltransferase/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Leucemia/enzimologia , Leucemia/genética , Leucemia/patologia , Neoplasia Endócrina Múltipla Tipo 1/genética , Neoplasia Endócrina Múltipla Tipo 1/metabolismo , Mutação , Células Progenitoras Mieloides/enzimologia , Proteína de Leucina Linfoide-Mieloide/genética , Ligação Proteica , Proteínas Metiltransferases , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/genética , Fatores de Transcrição/genética , Transcrição Gênica , Proteínas Supressoras de Tumor/genéticaRESUMO
The two subunits of core binding factor (Runx1 and CBFbeta) play critical roles in hematopoiesis and are frequent targets of chromosomal translocations found in leukemia. The binding of the CBFbeta-smooth muscle myosin heavy chain (SMMHC) fusion protein to Runx1 is essential for leukemogenesis, making this a viable target for treatment. We have developed inhibitors with low micromolar affinity which effectively block binding of Runx1 to CBFbeta. NMR-based docking shows that these compounds bind to CBFbeta at a site displaced from the binding interface for Runx1, that is, these compounds function as allosteric inhibitors of this protein-protein interaction, a potentially generalizable approach. Treatment of the human leukemia cell line ME-1 with these compounds shows decreased proliferation, indicating these are good candidates for further development.
Assuntos
Sítio Alostérico , Proliferação de Células/efeitos dos fármacos , Subunidade alfa 2 de Fator de Ligação ao Core/antagonistas & inibidores , Subunidade beta de Fator de Ligação ao Core/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Leucemia/patologia , Sítios de Ligação , Linhagem Celular Tumoral , Subunidade alfa 2 de Fator de Ligação ao Core/química , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Subunidade beta de Fator de Ligação ao Core/química , Subunidade beta de Fator de Ligação ao Core/metabolismo , Inibidores Enzimáticos/química , Transferência Ressonante de Energia de Fluorescência , Hematopoese/genética , Hematopoese/fisiologia , Humanos , Leucemia/metabolismo , Espectroscopia de Ressonância Magnética , Cadeias Pesadas de Miosina/química , Cadeias Pesadas de Miosina/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Miosinas de Músculo Liso/química , Miosinas de Músculo Liso/metabolismo , Translocação Genética/genética , Translocação Genética/fisiologiaRESUMO
AML1/ETO results from the t(8;21) associated with 12%-15% of acute myeloid leukemia. The AML1/ETO MYND domain mediates interactions with the corepressors SMRT and N-CoR and contributes to AML1/ETO's ability to repress proliferation and differentiation of primary bone marrow cells as well as to enhance their self renewal in vitro. We solved the solution structure of the MYND domain and show it to be structurally homologous to the PHD and RING finger families of proteins. We also determined the solution structure of an MYND-SMRT peptide complex. We demonstrated that a single amino acid substitution that disrupts the interaction between the MYND domain and the SMRT peptide attenuated AML1/ETO's effects on proliferation, differentiation, and gene expression.
Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Proteínas Repressoras/metabolismo , Animais , Células da Medula Óssea , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Expressão Gênica , Humanos , Camundongos , Modelos Moleculares , Mutação , Proteínas Nucleares/genética , Correpressor 1 de Receptor Nuclear , Ligação Proteica , Estrutura Terciária de Proteína , Proteína 1 Parceira de Translocação de RUNX1 , Proteínas Repressoras/genéticaRESUMO
Monoallelic RUNX1 mutations cause familial platelet disorder with predisposition for acute myelogenous leukemia (FPD/AML). Sporadic mono- and biallelic mutations are found at high frequencies in AML M0, in radiation-associated and therapy-related myelodysplastic syndrome and AML, and in isolated cases of AML M2, M5a, M3 relapse, and chronic myelogenous leukemia in blast phase. Mutations in RUNX2 cause the inherited skeletal disorder cleidocranial dysplasia (CCD). Most hematopoietic missense mutations in Runx1 involve DNA-contacting residues in the Runt domain, whereas the majority of CCD mutations in Runx2 are predicted to impair CBFbeta binding or the Runt domain structure. We introduced different classes of missense mutations into Runx1 and characterized their effects on DNA and CBFbeta binding by the Runt domain, and on Runx1 function in vivo. Mutations involving DNA-contacting residues severely inactivate Runx1 function, whereas mutations that affect CBFbeta binding but not DNA binding result in hypomorphic alleles. We conclude that hypomorphic RUNX2 alleles can cause CCD, whereas hematopoietic disease requires more severely inactivating RUNX1 mutations.
Assuntos
Displasia Cleidocraniana/genética , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 2 de Fator de Ligação ao Core/genética , DNA/metabolismo , Doenças Hematológicas/genética , Modelos Moleculares , Mutação de Sentido Incorreto/genética , Estrutura Terciária de Proteína , Animais , Contagem de Células Sanguíneas , Western Blotting , Subunidade beta de Fator de Ligação ao Core/metabolismo , Primers do DNA , Citometria de Fluxo , Transferência Ressonante de Energia de Fluorescência , Espectroscopia de Ressonância Magnética , Camundongos , Espectrometria de Fluorescência , Técnicas do Sistema de Duplo-HíbridoRESUMO
Fluorescent-labeled derivatives of the Antennapedia-derived cell-penetating peptide penetratin, and of the simpler but similarly charged peptides R(6)GC-NH(2) and K(6)GC-NH(2), are shown to be able to translocate into large unilamellar lipid vesicles in the presence of a transbilayer potential (inside negative). Vesicles with diverse lipid compositions, and combining physiological proportions of neutral and anionic lipids, are able to support substantial potential-dependent uptake of all three cationic peptides. The efficiency of peptide uptake under these conditions is strongly modulated by the vesicle lipid composition, in a manner that suggests that more than one mechanism of peptide uptake may operate in different systems. Remarkably, peptide uptake is accompanied by only minor perturbations of the overall barrier function of the lipid bilayer, as assessed by assays of vesicle leakiness under the same conditions. Fluorescence microscopy of living CV-1 and HeLa cells incubated with the labeled peptides shows that the peptides accumulate in peripheral vesicular structures at early times of incubation, consistent with an initial endosomal localization as recently reported, but gradually accumulate in the cytoplasm and nucleus during more extended incubations (several hours). Our findings indicate that these relatively hydrophilic, polybasic cell-penetrating peptides can translocate through lipid bilayers by a potential- and composition-dependent pathway that causes only minimal perturbation to the overall integrity and barrier function of the bilayer.
