RESUMO
The Fanconi Anemia (FA) DNA repair pathway is essential for the recognition and repair of DNA interstrand crosslinks (ICL). Inefficient repair of these ICL can lead to leukemia and bone marrow failure. A critical step in the pathway is the monoubiquitination of FANCD2 by the RING E3 ligase FANCL. FANCL comprises 3 domains, a RING domain that interacts with E2 conjugating enzymes, a central domain required for substrate interaction, and an N-terminal E2-like fold (ELF) domain. The ELF domain is found in all FANCL homologues, yet the function of the domain remains unknown. We report here that the ELF domain of FANCL is required to mediate a non-covalent interaction between FANCL and ubiquitin. The interaction involves the canonical Ile44 patch on ubiquitin, and a functionally conserved patch on FANCL. We show that the interaction is not necessary for the recognition of the core complex, it does not enhance the interaction between FANCL and Ube2T, and is not required for FANCD2 monoubiquitination in vitro. However, we demonstrate that the ELF domain is required to promote efficient DNA damage-induced FANCD2 monoubiquitination in vertebrate cells, suggesting an important function of ubiquitin binding by FANCL in vivo.
Assuntos
Reparo do DNA , Proteínas de Drosophila/química , Proteína do Grupo de Complementação L da Anemia de Fanconi/química , Proteínas de Grupos de Complementação da Anemia de Fanconi/química , Ubiquitina/química , Proteínas de Xenopus/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Anemia de Fanconi/genética , Proteína do Grupo de Complementação L da Anemia de Fanconi/genética , Proteína do Grupo de Complementação L da Anemia de Fanconi/metabolismo , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Regulação da Expressão Gênica , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Transdução de Sinais , Ubiquitina/genética , Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitinação , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevisRESUMO
Neuroblastoma, a malignancy of neuroectoderrmal origin, accounts for 15% of childhood cancer deaths. Despite advances in understanding the biology, it remains one of the most difficult paediatric cancers to treat. A major obstacle in the effective treatment of neuroblastoma is the development of multidrug resistance (MDR). There is thus a compelling demand for new treatment strategies for this cancer that can bypass such resistance mechanisms. The pyrrolo-1,5-benzoxazepine (PBOX) compounds are a series of novel microtubule-targeting agents that potently induce apoptosis in various cancer cell lines, ex vivo patient samples and in vivo cancer models. In this study we examined the ability of two members, PBOX-6 and -15, to exhibit anti-cancer effects in a panel of drug sensitive and MDR neuroblastoma cell lines. The PBOX compounds potently reduced the viability of all neuroblastoma cells examined and exhibited a lower fold resistance in MDR cells when compared to standard chemotherapeutics. In addition, the PBOX compounds synergistically enhanced apoptosis induced by etoposide, carboplatin and doxorubicin. Exposure of drug sensitive and resistant cell lines to PBOX-6/carboplatin induced cleavage of Bcl-2, a downregulation of Mcl-1 and a concomitant increase in Bak. Furthermore, activation of caspase-3, -8 and -9 was demonstrated. Finally, gene silencing of Mcl-1 by siRNA was shown to sensitise both drug sensitive and multidrug resistant cells to carboplatin-induced apoptosis demonstrating the importance of Mcl-1 downregulation in the apoptotic pathway mediated by the PBOX compounds in neuroblastoma. In conclusion, our findings indicate the potential of the PBOX compounds in enhancing chemosensitivity in neuroblastoma.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Carboplatina/farmacologia , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neuroblastoma/patologia , Oxazepinas/farmacologia , Pirróis/farmacologia , Antineoplásicos/uso terapêutico , Apoptose/fisiologia , Carboplatina/uso terapêutico , Linhagem Celular Tumoral , Resistência a Múltiplos Medicamentos/fisiologia , Resistencia a Medicamentos Antineoplásicos/fisiologia , Sinergismo Farmacológico , Humanos , Neuroblastoma/tratamento farmacológico , Oxazepinas/uso terapêutico , Pirróis/uso terapêutico , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologiaRESUMO
The genotoxin cisplatin is commonly used in chemotherapy to treat solid tumors, yet our understanding of the mechanism underlying the drug response is limited. In a focused siRNA screen, using an siRNA library targeting genes involved in ubiquitin and ubiquitin-like signaling, we identified the E3 ubiquitin ligase HOIP as a key regulator of cisplatin-induced genotoxicity. HOIP forms, with SHARPIN and HOIL-1L, the linear ubiquitin assembly complex (LUBAC). We show that cells deficient in the HOIP ligase complex exhibit hypersensitivity to cisplatin. This is due to a dramatic increase in caspase-8/caspase-3-mediated apoptosis that is strictly dependent on ATM-, but not ATR-mediated DNA damage checkpoint activation. Moreover, basal and cisplatin-induced activity of the stress response kinase JNK is enhanced in HOIP-depleted cells and, conversely, JNK inhibition can increase cellular resistance to cisplatin and reverse the apoptotic hyperactivation in HOIP-depleted cells. Furthermore, we show that HOIP depletion sensitizes cancer cells, derived from carcinomas of various origins, through an enhanced apoptotic cell death response. We also provide evidence that ovarian cancer cells classified as cisplatin-resistant can regain sensitivity following HOIP downregulation. Cumulatively, our study identifies a HOIP-regulated antiapoptotic signaling pathway, and we envisage HOIP as a potential target for the development of combinatorial chemotherapies to potentiate the efficacy of platinum-based anticancer drugs.