Department of Pathology, College of Medicine, King Khalid University, Abha, Saudi Arabia.
Forensic Medicine and Clinical Toxicology Department, Faculty of Medicine, Mansoura University, Mansoura, Egypt.
Neoplasma. 2018 Sep 19;65(5):708-719. doi: 10.4149/neo_2018_171020N673. Epub 2018 Sep 4.
Ku70 protein in hetero-trimeric complex with Ku80 and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) represents a critical component of the nonhomologous-end-joining (NHEJ), the major machinery of DSBs repair in mammalian cells. It has been previously shown that modulation of Ku70 acetylation by histone deacetylases (HDAC) inhibitors induced sensitization of cancer cells to chemotherapeutic agents. However, the effects of such modulation on the repair of Ionizing Radiation (IR)-induced DSBs and the importance of dynamic equilibrium of acetylation/deacetylation have not been studied in details. To address these questions aceto-blocking and aceto-mimicking mutants were designed by replacing Ku70 lysine residues K317, K331 and K338 with arginine and glutamine respectively via site-directed mutagenesis. Transformed human embryonic lung fibroblasts MRC5VA were transfected to create stables cells lines over-expressing Ku70 mutant proteins. Clonogenic survival and γ-H2AX foci assays were performed to study the impact of these mutants on DNA repair proficiency of MRC5VA cells in response to IR. We report here that both Ku70 aceto-blocking and aceto-mimicking mutants rendered MRC5VA cells more susceptible to IR in terms of clonogenic survival and γH2AX foci. Moreover, modelling the possible interactions and structural impact of these aceto-blocking and aceto-mimicking mutants with DNA substrate showed that mimicking acetylation/deacetylation of K331 and K338 could directly compromise KU-DNA interactions, whereas K317 may have a more subtle role via forming a salt bridge with E330 thus optimising the positioning of the helix containing both K331 and K338 residues on the DNA. Our data indicates that dynamic equilibrium of acetylation/deacetylation of Ku70 lysine residues K317, K331 and K338 is critical for optimal repair of IR-induced DSBs, and may offer a novel therapeutic approach for cancer treatment.
Ku70 蛋白与 Ku80 和 DNA 依赖性蛋白激酶催化亚基（DNA-PKcs）形成异三聚体复合物，是哺乳动物细胞中非同源末端连接（NHEJ）的关键组成部分，也是双链断裂（DSBs）修复的主要机制。先前的研究表明，组蛋白去乙酰化酶（HDAC）抑制剂对 Ku70 乙酰化的调节会导致癌细胞对化疗药物的敏感性增加。然而，这种调节对电离辐射（IR）诱导的 DSBs 修复的影响，以及乙酰化/去乙酰化的动态平衡的重要性尚未被详细研究。为了解决这些问题，通过定点突变，将 Ku70 的赖氨酸残基 K317、K331 和 K338 分别替换为精氨酸和谷氨酰胺，设计了乙酰化阻断和乙酰化模拟突变体。通过转染转化的人胚肺成纤维细胞 MRC5VA 来创建稳定过表达 Ku70 突变蛋白的细胞系。通过集落形成存活和 γ-H2AX 焦点测定来研究这些突变对 MRC5VA 细胞在 IR 反应中 DNA 修复能力的影响。我们在这里报告，Ku70 乙酰化阻断和乙酰化模拟突变体都使 MRC5VA 细胞对 IR 更加敏感，表现在集落形成存活和 γH2AX 焦点方面。此外，模拟这些乙酰化阻断和乙酰化模拟突变体与 DNA 底物的可能相互作用和结构影响表明，模拟 K331 和 K338 的乙酰化/去乙酰化可能直接破坏 KU-DNA 相互作用，而 K317 可能通过与 E330 形成盐桥来发挥更微妙的作用，从而优化包含 K331 和 K338 残基的螺旋在 DNA 上的定位。我们的数据表明，Ku70 赖氨酸残基 K317、K331 和 K338 的乙酰化/去乙酰化的动态平衡对于 IR 诱导的 DSBs 的最佳修复至关重要，并且可能为癌症治疗提供一种新的治疗方法。
