Fiche publication
Date publication
février 2015
Auteurs
Membres identifiés du Cancéropôle Est :
Dr CORDONNIER Agnès
Tous les auteurs :
Ahmed-Seghir S, Pouvelle C, Despras E, Cordonnier A, Sarasin A, Kannouche PL
Lien Pubmed
Résumé
Xeroderma pigmentosum variant (XP-V) is a rare genetic disease, characterized by sunlight sensitivity and predisposition to cutaneous malignancies. XP-V is caused by a deficiency in DNA polymerase eta (Poleta) that plays a pivotal role in translesion synthesis by bypassing UV-induced pyrimidine dimers. Previously we identified a new Poleta variant containing two missense mutations, one mutation within the bipartite NLS (T692A) and a second mutation on the stop codon (X714W) leading to a longer protein with an extra 8 amino acids (721 instead of 713 AA). First biochemical analysis revealed that this Poleta missense variant was barely detectable by western blot. As this mutant is extremely unstable and is nearly undetectable, a definitive measure of its functional deficit in cells has not been explored. Here we report the molecular and cellular characterization of this missense variant. In cell free extracts, the extra 8 amino acids in the C-terminal of Poleta721 only slightly reduce the bypass efficiency through CPD lesions. In vivo, Poleta721 accumulates in replication factories and interacts with mUb-PCNA albeit at lower level than Poletawt. XP-V cells overexpressing Poleta721 were only slightly UV-sensitive. Altogether, our data strongly suggest that Poleta721 is functional and that the patient displays a XP-V phenotype because the mutant protein is excessively unstable. We then investigated the molecular mechanisms involved in this excessive proteolysis. We showed that Poleta721 is degraded by the proteasome in an ubiquitin-dependent manner and that this proteolysis is independent of the E3 ligases, CRL4cdt2 and Pirh2, reported to promote Poleta degradation. We then demonstrated that the extra 8 amino acids of Poleta721 do not act as a degron but rather induce a conformational change of the Poleta C-terminus exposing its bipartite NLS as well as a sequence close to its UBZ to the ubiquitin/proteasome system. Interestingly we showed that the clinically approved proteasome inhibitor, Bortezomib restores the levels of Poleta721 suggesting that this might be a therapeutic approach to preventing tumor development in certain XP-V patients harboring missense mutations.
Référence
DNA Repair (Amst). 2015 Feb 26. pii: S1568-7864(15)00053-1
