RESUMO
BACKGROUND: The SMC5-6 protein complex is involved in the cellular response to DNA damage. It is composed of 6-8 polypeptides, of which Nse1, Nse3 and Nse4 form a tight sub-complex. MAGEG1, the mammalian ortholog of Nse3, is the founding member of the MAGE (melanoma-associated antigen) protein family and Nse4 is related to the EID (E1A-like inhibitor of differentiation) family of transcriptional repressors. METHODOLOGY/PRINCIPAL FINDINGS: Using site-directed mutagenesis, protein-protein interaction analyses and molecular modelling, we have identified a conserved hydrophobic surface on the C-terminal domain of Nse3 that interacts with Nse4 and identified residues in its N-terminal domain that are essential for interaction with Nse1. We show that these interactions are conserved in the human orthologs. Furthermore, interaction of MAGEG1, the mammalian ortholog of Nse3, with NSE4b, one of the mammalian orthologs of Nse4, results in transcriptional co-activation of the nuclear receptor, steroidogenic factor 1 (SF1). In an examination of the evolutionary conservation of the Nse3-Nse4 interactions, we find that several MAGE proteins can interact with at least one of the NSE4/EID proteins. CONCLUSIONS/SIGNIFICANCE: We have found that, despite the evolutionary diversification of the MAGE family, the characteristic hydrophobic surface shared by all MAGE proteins from yeast to humans mediates its binding to NSE4/EID proteins. Our work provides new insights into the interactions, evolution and functions of the enigmatic MAGE proteins.
Assuntos
Proteínas de Transporte/metabolismo , Evolução Molecular , Antígenos Específicos de Melanoma/metabolismo , Proteínas Nucleares/metabolismo , Mapeamento de Interação de Proteínas , Proteínas Repressoras/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Sequência de Aminoácidos , Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Sequência Conservada/fisiologia , Humanos , Antígenos Específicos de Melanoma/química , Antígenos Específicos de Melanoma/genética , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Família Multigênica , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/genética , Ligação Proteica/genética , Proteínas Repressoras/química , Proteínas Repressoras/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genética , Homologia de SequênciaRESUMO
Drug resistance remains a barrier to the effective long term treatment of ovarian cancer. We have established an RNAi-based screen to identify genes which confer resistance to carboplatin or paclitaxel. To validate the screen we showed that siRNA interfering with the apoptosis regulators FLIP and Bcl-X(L) conferred sensitivity to paclitaxel and carboplatin respectively. The expression of 90 genes which have previously been shown to be over-expressed in drug-resistant ovarian cancer was inhibited using siRNA and the impact on sensitivity to carboplatin and paclitaxel was assessed. ENPP2 was identified as a candidate gene causing drug resistance. ENPP2 encodes autotaxin, a phospholipase involved in the synthesis of the survival factor lysophosphatidic acid. siRNA directed to ENPP2 resulted in earlier apoptosis following treatment with carboplatin. 2-carbacyclic phosphatidic acid (ccPA 16:1), a small molecule inhibitor of autotaxin, also accelerated apoptosis induced by carboplatin. Stable ectopic expression of autotaxin in OVCAR-3 cells led to a delay in apoptosis. When serum was withdrawn to remove exogenous LPA, ccPA caused a pronounced potentiation of apoptosis induced by carboplatin in cells expressing autotaxin. These results indicate that autotaxin delays apoptosis induced by carboplatin in ovarian cancer cells.
Assuntos
Antineoplásicos/farmacologia , Apoptose , Carboplatina/farmacologia , Complexos Multienzimáticos/fisiologia , Neoplasias Ovarianas/genética , Fosfodiesterase I/fisiologia , Pirofosfatases/fisiologia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Humanos , Complexos Multienzimáticos/antagonistas & inibidores , Complexos Multienzimáticos/genética , Neoplasias Ovarianas/patologia , Fosfodiesterase I/antagonistas & inibidores , Fosfodiesterase I/genética , Diester Fosfórico Hidrolases , Pirofosfatases/antagonistas & inibidores , Pirofosfatases/genética , Interferência de RNARESUMO
Ectopic expression provides an alternative to RNAi to conduct functional genomic screens. We have determined whether transient expression can be used to identify genes which confer drug resistance. We constructed a bigenic vector that allows high throughput cloning and which also encodes a red fluorescent protein to monitor transfection. Assay conditions were optimized to allow detection of changes in sensitivity to carboplatin or paclitaxel. Following transient expression of MDR-1 and MCJ, changes in the sensitivity of Sk-Ov-3 cells to paclitaxel were detected whereas expression of Src, Bcl-2 and Bcl-X(L) decreased the sensitivity of Sk-Ov-3 cells to carboplatin. This approach may potentially be used as an independent screen or as a method to help rank hit identified in screens utilizing methods such as RNAi.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , Proteínas de Choque Térmico HSP40/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Neoplasias Ovarianas/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP , Animais , Carboplatina/farmacologia , Relação Dose-Resposta a Droga , Feminino , Corantes Fluorescentes/metabolismo , Genes MDR , Genes bcl-2 , Humanos , Proteínas Luminescentes/genética , Neoplasias Ovarianas/tratamento farmacológico , Paclitaxel/farmacologia , Transfecção , Proteína bcl-X/genética , Quinases da Família src/genética , Proteína Vermelha FluorescenteRESUMO
The SMC protein complexes play important roles in chromosome dynamics. The function of the SMC5-6 complex remains unclear, though it is involved in resolution of different DNA structures by recombination. We have now identified and characterized the four non-SMC components of the human complex and in particular demonstrated that the MAGEG1 protein is part of this complex. MAGE proteins play important but as yet undefined roles in carcinogenesis, apoptosis, and brain development. We show that, with the exception of the SUMO ligase hMMS21/hNSE2, depletion of any of the components results in degradation of all the other components. Depletion also confers sensitivity to methyl methanesulfonate. Several of the components are modified by sumoylation and ubiquitination.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Ligases/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/química , Sobrevivência Celular/efeitos dos fármacos , Proteínas Cromossômicas não Histona , Dano ao DNA , Células HeLa , Humanos , Imunoprecipitação , Ligases/química , Espectrometria de Massas , Metanossulfonato de Metila/farmacologia , Dados de Sequência Molecular , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Termodinâmica , Ubiquitinação/efeitos dos fármacosRESUMO
PURPOSE: The effective treatment of ovarian cancer is hampered by the development of drug resistance, which may be mediated by members of the Bcl-2 family of apoptosis regulators. ABT-737 is a recently described inhibitor of members of this family. We investigated whether this compound could sensitize ovarian cancer cells to chemotherapeutic agents. EXPERIMENTAL DESIGN: The sensitivity of ovarian cancer cell lines to ABT-737 in combination with either carboplatin or paclitaxel was tested either in vitro by assessing cell growth/survival and apoptosis or in xenograft studies. RESULTS: As a single agent, ABT-737 inhibited the growth of eight ovarian cancer cell lines, although with relatively poor potency. However, ABT-737, but not a less active enantiomer, increased the sensitivity of several cell lines to carboplatin. The increased sensitivity to carboplatin was accompanied by a decrease in time at which apoptosis was observed when assessed according to the number of attached cells, PARP cleavage, and nucleosome formation. ABT-737 was more effective at sensitizing IGROV-1 cells when ABT-737 was administered after carboplatin. In addition, ABT-737 significantly enhanced the activity of carboplatin in one of three primary cultures derived directly from ascitic tumor cells in patients recently treated with chemotherapy. Small interfering RNA directed to Bcl-X(L) also increased the sensitivity of ovarian cancer cell lines to carboplatin. ABT-737 was also able to augment the inhibition of IGROV-1 tumor xenograft growth beyond that obtained with carboplatin alone. CONCLUSIONS: These data suggest that ABT-737, in combination with carboplatin, may find utility in the treatment of patients with ovarian cancer.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Compostos de Bifenilo/farmacologia , Carboplatina/farmacologia , Nitrofenóis/farmacologia , Neoplasias Ovarianas/tratamento farmacológico , Paclitaxel/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Sulfonamidas/farmacologia , Proteína bcl-X/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Compostos de Bifenilo/administração & dosagem , Carboplatina/administração & dosagem , Linhagem Celular Tumoral , Esquema de Medicação , Sinergismo Farmacológico , Feminino , Humanos , Camundongos , Nitrofenóis/administração & dosagem , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Paclitaxel/administração & dosagem , Piperazinas/administração & dosagem , Piperazinas/farmacologia , Interferência de RNA , RNA Interferente Pequeno/genética , Sulfonamidas/administração & dosagem , Ensaios Antitumorais Modelo de Xenoenxerto , Proteína bcl-X/genéticaRESUMO
Structural maintenance of chromosomes (SMC) proteins play fundamental roles in many aspects of chromosome organization and dynamics. The SMC complexes form unique structures with long coiled-coil arms folded at a hinge domain, so that the globular N- and C-terminal domains are brought together to form a "head." Within the Smc5-Smc6 complex, we previously identified two subcomplexes containing Smc6-Smc5-Nse2 and Nse1-Nse3-Nse4. A third subcomplex containing Nse5 and -6 has also been identified recently. We present evidence that Nse4 is the kleisin component of the complex, which bridges the heads of Smc5 and -6. The C-terminal part of Nse4 interacts with the head domain of Smc5, and structural predictions for Nse4 proteins suggest similar motifs that are shared within the kleisin family. Specific mutations within a predicted winged helix motif of Nse4 destroy the interaction with Smc5. We propose that Nse4 and its orthologs form the delta-kleisin subfamily. We further show that Nse3, as well as Nse5 and Nse6, also bridge the heads of Smc5 and -6. The Nse1-Nse3-Nse4 and Nse5-Nse6 subcomplexes bind to the Smc5-Smc6 heads domain at different sites.
