RESUMO
Repair of DNA double-strand breaks is crucial for maintaining genome integrity and is governed by post-translational modifications such as protein ubiquitylation. Here, we establish that the deubiquitylating enzyme USP4 promotes DNA-end resection and DNA repair by homologous recombination. We also report that USP4 interacts with CtIP and the MRE11-RAD50-NBS1 (MRN) complex and is required for CtIP recruitment to DNA damage sites. Furthermore, we show that USP4 is ubiquitylated on multiple sites including those on cysteine residues and that deubiquitylation of these sites requires USP4 catalytic activity and is required for USP4 to interact with CtIP/MRN and to promote CtIP recruitment and DNA repair. Lastly, we establish that regulation of interactor binding by ubiquitylation occurs more generally among USP-family enzymes. Our findings thus identify USP4 as a novel DNA repair regulator and invoke a model in which ubiquitin adducts regulate USP enzyme interactions and functions.
Assuntos
Quebras de DNA de Cadeia Dupla , Modelos Biológicos , Reparo de DNA por Recombinação , Ubiquitina Tiolesterase/metabolismo , Ubiquitinação/fisiologia , Hidrolases Anidrido Ácido , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases , Humanos , Proteína Homóloga a MRE11 , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ubiquitina Tiolesterase/genética , Proteases Específicas de UbiquitinaRESUMO
Protein kinase B (PKB/AKT) has been identified as a promising cancer drug target downstream of PI3 kinase. To find novel inhibitors of PKB/AKT kinase activity for progression as anticancer agents, the authors have used a high-throughput screen based on AlphaScreentrade mark technology. A known kinase inhibitor, the isoquinoline H8, was used as a positive control with mean inhibition in the screen of 43.4% +/- 13.1%. The performance of the screen was highly acceptable with Z' and Z factors of 0.83 +/- 0.07 and 0.75 +/- 0.04, respectively. A number of confirmed hits ( approximately 0.1% hit rate) were identified from 63,500 compounds screened. Five compounds have previously been described as PKB inhibitors, demonstrating the ability of the assay to find authentic inhibitors of the enzyme. Five hits had the potential to interfere with the assay signal and were deemed to be false positives. Two compounds were nonspecific inhibitors of PKB as enzyme inhibition in a filter-based assay was markedly reduced in the presence of 0.01% Triton X100. The authors now include an interference assay during hit confirmation procedures and check compound activity in the presence of Triton X100 in an attempt to eliminate nonspecific aggregators at an early stage.
