RESUMEN
The FANC-BRCA DNA repair pathway is activated in response to interstrand crosslinks formed in DNA. A homozygous mutation in 1 of the 17 Fanconi anemia (FA) genes results in malfunctions of this pathway and development of FA syndrome. The integrity of this protein network is essential for good maintenance of DNA repair process and genome stability. Following the identification of an alternatively splice isoform of FANCE (Fanconi anemia complementation group E) significantly expressed in breast cancer individuals from high-risk non-BRCA1/2 families, we studied the impact of this FANCE splice isoform (FANCEΔ4) on DNA repair processes. We have demonstrated that FANCEΔ4 mRNA was efficiently translated into a functional protein and expressed in normal and breast cancer cell lines. Following treatment with the crosslinking agent mitomycin C, EUFA130 (FANCE-deficient) cells infected with FANCEΔ4 were blocked into G2/M phase, while cell survival was significantly reduced compared with FANCE-infected EUFA130 cells. In addition, FANCEΔ4 did not allow FANCD2 and FANCI monoubiquitination, which represents a crucial step of the FANC-BRCA functional pathway. As observed for FANCE wild-type protein, localization of FANCEΔ4 protein was confined to the nucleus following mitomycin C treatment. Although FANCEΔ4 protein showed interaction with FANCE, FANCEΔ4 did not support normal function of FANCE protein in this pathway and could have deleterious effects on FANCE protein activity. We have demonstrated that FANCEΔ4 seems to act as a regulator of FANCD2 protein expression level by promoting its degradation. This study highlights the importance of an efficient regulation of alternative splicing expression of FA genes for proper DNA repair.
Asunto(s)
Empalme Alternativo , Reparación del ADN , Proteína del Grupo de Complementación E de la Anemia de Fanconi/genética , Anemia de Fanconi/genética , Secuencia de Aminoácidos , Proteína BRCA1/metabolismo , Proteína BRCA2/metabolismo , Supervivencia Celular , Anemia de Fanconi/metabolismo , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/genética , Proteína del Grupo de Complementación E de la Anemia de Fanconi/química , Proteína del Grupo de Complementación E de la Anemia de Fanconi/metabolismo , Proteínas del Grupo de Complementación de la Anemia de Fanconi/metabolismo , Regulación de la Expresión Génica , Células HEK293 , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estructura Terciaria de Proteína , Alineación de SecuenciaRESUMEN
Fanconi anemia (FA) is a genome instability syndrome characterized by bone marrow failure and cellular hypersensitivity to DNA cross-linking agents. In response to DNA damage, the FA pathway is activated through the cooperation of 16 FA proteins. A central player in the pathway is a multisubunit E3 ubiquitin ligase complex or the FA core complex, which monoubiquitinates its substrates FANCD2 and FANCI. FANCE, a subunit of the FA core complex, plays an essential role by promoting the integrity of the complex and by directly recognizing FANCD2. To delineate its role in substrate ubiquitination from the core complex assembly, we analyzed a series of mutations within FANCE. We report that a phenylalanine located at the highly conserved extreme C terminus, referred to as Phe-522, is a critical residue for mediating the monoubiquitination of the FANCD2-FANCI complex. Using the FANCE mutant that specifically disrupts the FANCE-FANCD2 interaction as a tool, we found that the interaction-deficient mutant conferred cellular sensitivity in reconstituted FANCE-deficient cells to a similar degree as FANCE null cells, suggesting the significance of the FANCE-FANCD2 interaction in promoting cisplatin resistance. Intriguingly, ectopic expression of the FANCE C terminus fragment alone in FA normal cells disrupts DNA repair, consolidating the importance of the FANCE-FANCD2 interaction in the DNA cross-link repair.
Asunto(s)
Reparación del ADN , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , Proteína del Grupo de Complementación E de la Anemia de Fanconi/metabolismo , Anemia de Fanconi/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Secuencia de Aminoácidos , Anemia de Fanconi/genética , Proteína del Grupo de Complementación E de la Anemia de Fanconi/química , Proteína del Grupo de Complementación E de la Anemia de Fanconi/genética , Proteína del Grupo de Complementación L de la Anemia de Fanconi/metabolismo , Células HEK293 , Células HeLa , Humanos , Datos de Secuencia Molecular , Fenilalanina/química , Fenilalanina/genética , Fenilalanina/metabolismo , Estructura Terciaria de Proteína , UbiquitinaciónRESUMEN
The eleven Fanconi anemia (FA) proteins cooperate in a novel pathway required for the repair of DNA cross-links. Eight of the FA proteins (A, B, C, E, F, G, L, and M) form a core enzyme complex, required for the monoubiquitination of FANCD2 and the assembly of FANCD2 nuclear foci. Here, we show that, in response to DNA damage, Chk1 directly phosphorylates the FANCE subunit of the FA core complex on two conserved sites (threonine 346 and serine 374). Phosphorylated FANCE assembles in nuclear foci and colocalizes with FANCD2. A nonphosphorylated mutant form of FANCE (FANCE-T346A/S374A), when expressed in a FANCE-deficient cell line, allows FANCD2 monoubiquitination, FANCD2 foci assembly, and normal S-phase progression. However, the mutant FANCE protein fails to complement the mitomycin C hypersensitivity of the transfected cells. Taken together, these results elucidate a novel role of Chk1 in the regulation of the FA/BRCA pathway and in DNA cross-link repair. Chk1-mediated phosphorylation of FANCE is required for a function independent of FANCD2 monoubiquitination.
Asunto(s)
Proteína BRCA1/metabolismo , Proteína del Grupo de Complementación E de la Anemia de Fanconi/metabolismo , Anemia de Fanconi/metabolismo , Proteínas Quinasas/metabolismo , Secuencia de Aminoácidos , Ciclo Celular/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Secuencia Conservada , Daño del ADN , Replicación del ADN/efectos de los fármacos , Resistencia a Medicamentos/efectos de los fármacos , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , Proteína del Grupo de Complementación E de la Anemia de Fanconi/química , Células HeLa , Humanos , Mitomicina/farmacología , Datos de Secuencia Molecular , Proteínas Mutantes/metabolismo , Fosforilación/efectos de los fármacos , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , ARN Interferente Pequeño/metabolismo , Ubiquitina/metabolismoRESUMEN
Fanconi Anaemia (FA) is a cancer predisposition disorder characterized by spontaneous chromosome breakage and high cellular sensitivity to genotoxic agents. In response to DNA damage, a multi-subunit assembly of FA proteins, the FA core complex, monoubiquitinates the downstream FANCD2 protein. The FANCE protein plays an essential role in the FA process of DNA repair as the FANCD2-binding component of the FA core complex. Here we report a crystallographic and biological study of human FANCE. The first structure of a FA protein reveals the presence of a repeated helical motif that provides a template for the structural rationalization of other proteins defective in Fanconi Anaemia. The portion of FANCE defined by our crystallographic analysis is sufficient for interaction with FANCD2, yielding structural information into the mode of FANCD2 recruitment to the FA core complex. Disease-associated mutations disrupt the FANCE-FANCD2 interaction, providing structural insight into the molecular mechanisms of FA pathogenesis.
Asunto(s)
Proteína del Grupo de Complementación E de la Anemia de Fanconi/química , Anemia de Fanconi/genética , Secuencia de Aminoácidos , Cristalografía por Rayos X , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/química , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , Proteína del Grupo de Complementación E de la Anemia de Fanconi/genética , Proteína del Grupo de Complementación E de la Anemia de Fanconi/metabolismo , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Estructura Terciaria de Proteína , Secuencias Repetitivas de Aminoácido , Alineación de Secuencia , Técnicas del Sistema de Dos HíbridosRESUMEN
The Fanconi anemia (FA) protein FANCE is an essential component of the nuclear FA core complex, which is required for monoubiquitination of the downstream target FANCD2, an important step in the FA pathway of DNA cross-link repair. FANCE is predominantly localized in the nucleus and acts as a molecular bridge between the FA core complex and FANCD2, through direct binding of both FANCC and FANCD2. At present, it is poorly understood how the nuclear accumulation of FANCE is regulated and therefore we investigated the nuclear localization of this FA protein. We found that FANCE has a strong tendency to localize in the nucleus, since the addition of a nuclear export signal does not interfere with the nuclear localization of FANCE. We also demonstrate that the nuclear accumulation of FANCE does not rely solely on its nuclear localization signal motifs, but also on FANCC. The other FA proteins are not involved in the nuclear accumulation of FANCE, indicating a tight relationship between FANCC and FANCE, as suggested from their direct interaction. Finally, we show that the region of FANCE interacting with FANCC appears to be different from the region involved in binding FANCD2. This strengthens the idea that FANCE recruits FANCD2 to the core complex, without interfering with the binding of FANCC.