Newcastle University Biosciences Institute, Newcastle University, Newcastle upon Tyne, United Kingdom.
Newcastle University Biosciences Institute, Newcastle University, Newcastle upon Tyne, United Kingdom
Mol Pharmacol. 2020 Sep;98(3):222-233. doi: 10.1124/mol.119.118893. Epub 2020 Jun 25.
DNA topoisomerase II (TOP2) is required for the unwinding and decatenation of DNA through the induction of an enzyme-linked double-strand break (DSB) in one DNA molecule and passage of another intact DNA duplex through the break. Anticancer drugs targeting TOP2 (TOP2 poisons) prevent religation of the DSB and stabilize a normally transient intermediate of the TOP2 reaction mechanism called the TOP2-DNA covalent complex. Subsequently, TOP2 remains covalently bound to each end of the enzyme-bridged DSB, which cannot be repaired until TOP2 is removed from the DNA. One removal mechanism involves the proteasomal degradation of the TOP2 protein, leading to the liberation of a protein-free DSB. Proteasomal degradation is often regulated by protein ubiquitination, and here we show that inhibition of ubiquitin-activating enzymes reduces the processing of TOP2A- and TOP2B-DNA complexes. Depletion or inhibition of ubiquitin-activating enzymes indicated that ubiquitination was required for the liberation of etoposide-induced protein-free DSBs and is therefore an important layer of regulation in the repair of TOP2 poison-induced DNA damage. TOP2-DNA complexes stabilized by etoposide were shown to be conjugated to ubiquitin, and this was reduced by inhibition or depletion of ubiquitin-activating enzymes. SIGNIFICANCE STATEMENT: There is currently great clinical interest in the ubiquitin-proteasome system and ongoing development of specific inhibitors. The results in this paper show that the therapeutic cytotoxicity of DNA topoisomerase II (TOP2) poisons can be enhanced through combination therapy with ubiquitin-activating enzyme inhibitors or by specific inhibition of the BMI/RING1A ubiquitin ligase, which would lead to increased cellular accumulation or persistence of TOP2-DNA complexes.
DNA 拓扑异构酶 II（TOP2）通过在一个 DNA 分子中诱导酶连接的双链断裂（DSB）并使另一个完整的 DNA 双链穿过断裂来解开和解缠 DNA。针对 TOP2 的抗癌药物（TOP2 毒药）阻止 DSB 的重新连接，并稳定 TOP2 反应机制的通常短暂中间体，称为 TOP2-DNA 共价复合物。随后，TOP2 仍然共价结合在酶桥接的 DSB 的每一端，直到 TOP2 从 DNA 中移除，否则无法修复。一种去除机制涉及 TOP2 蛋白的蛋白酶体降解，导致无蛋白 DSB 的释放。蛋白酶体降解通常受蛋白质泛素化调节，在这里我们表明，抑制泛素激活酶会减少 TOP2A 和 TOP2B-DNA 复合物的处理。泛素激活酶的耗尽或抑制表明泛素化是释放依托泊苷诱导的无蛋白 DSB 所必需的，因此是修复 TOP2 毒药诱导的 DNA 损伤的重要调控层。用依托泊苷稳定的 TOP2-DNA 复合物被缀合到泛素上，并且通过抑制或耗尽泛素激活酶而降低。意义声明：目前，泛素蛋白酶体系统具有很大的临床兴趣，并且正在开发特定的抑制剂。本文的结果表明，通过与泛素激活酶抑制剂联合治疗或通过特异性抑制 BMI/RING1A 泛素连接酶，可增强 DNA 拓扑异构酶 II（TOP2）毒药的治疗细胞毒性，从而导致 TOP2-DNA 复合物的细胞内积累或持续增加。
