RESUMEN
Fanconi Anemia (FA) is a rare genetic disorder characterized by developmental defects, bone marrow failure and high predisposition to cancer. The FA DNA repair pathway is required in humans to coordinate repair of DNA interstrand cross-links. The central event in the activation of the pathway is the monoubiquitination of FANCD2 and FANCI by the E2-E3 pair, Ube2T-FANCL, with the central UBC-RWD (URD) domain of FANCL recognizing the substrates. Whole genome sequencing studies of cancer cells from patients identified point mutations in the FANCL URD domain. We analysed 17 such variants of FANCL, including known substrate binding mutants (W212A, W214A and L248A, F252A, L254A, I265A), a FA mutation (R221C) and 14 cancer-associated mutations (F110S, I136V, L149V, L154S, A192G, E215Q, E217K, R221W, T224K, M247V, F252L, N270K, V287G, E289Q) through recombinant expression analysis, thermal shift assay, interaction with FANCD2, in vitro ubiquitination activity, and cellular sensitivity to an interstrand cross-linking agent. We find that the FANCL mutations I136V, L154S, W212A and L214A, R221W, R221C, and V287G are destabilizing, with N270K and E289Q destabilizing the C-terminal helices of the URD domain. The hydrophobic patch mutant (L248A, F252A, L254A, I265A), along with mutations E217K, T224K, and M247V, cause defects in the catalytic function of FANCL. This highlights the C-terminal lobe of the FANCL URD domain as important for the activity and function of FANCL. These mutations which affect the fold and activity of FANCL may contribute to tumorigenesis in these non-FA cancer patients, and this implicates FA genes in general cancer progression.
Asunto(s)
Biomarcadores de Tumor/genética , Neoplasias Óseas/genética , Proteína del Grupo de Complementación L de la Anemia de Fanconi/genética , Anemia de Fanconi/genética , Mutación , Osteosarcoma/genética , Antineoplásicos Alquilantes/farmacología , Biomarcadores de Tumor/metabolismo , Neoplasias Óseas/tratamiento farmacológico , Neoplasias Óseas/metabolismo , Neoplasias Óseas/patología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Análisis Mutacional de ADN , Anemia de Fanconi/diagnóstico , Anemia de Fanconi/metabolismo , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , Proteína del Grupo de Complementación L de la Anemia de Fanconi/metabolismo , Predisposición Genética a la Enfermedad , Humanos , Mitomicina/farmacología , Osteosarcoma/tratamiento farmacológico , Osteosarcoma/metabolismo , Osteosarcoma/patología , Conformación Proteica en Hélice alfa , Pliegue de Proteína , Estabilidad Proteica , Proteolisis , UbiquitinaciónRESUMEN
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.