Ubiquitylation-dependent regulation of NEIL1 by Mule and TRIM26 is required for the cellular DNA damage response.
Ubiquitylation-dependent regulation of NEIL1 by Mule and TRIM26 is required for the cellular DNA damage response.
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DOI：
10.1093/nar/gkw959
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发表时间：
2017-01-25
影响因子：
14.9
通讯作者：
Parsons JL
中科院分区：
文献类型：
--
作者：
Edmonds MJ;Carter RJ;Nickson CM;Williams SC;Parsons JL
核酸内切酶VIII样蛋白1（NEIL 1）是一种DNA糖基化酶，参与启动碱基切除修复途径，这是修复DNA碱基损伤的主要细胞机制。在这里，我们已经从人类细胞中纯化了主要的E3泛素连接酶，负责通过泛素化调节NEIL 1。有趣的是，我们已经鉴定了催化NEIL 1多聚泛素化的两种酶，Mcl-1泛素连接酶E3（Mule）和三重基序26（TRIM 26）。我们证明，这些酶能够在体外多泛素化NEIL 1，并且都催化相同的C-末端赖氨酸残基内的NEIL 1的泛素化。Mule或TRIM 26的siRNA介导的敲低导致NEIL 1的稳定，表明这些酶在调节细胞NEIL 1稳态蛋白水平中是重要的。类似地，缺乏泛素化残基的突变NEIL 1蛋白在体内比野生型蛋白更稳定。我们还表明，细胞NEIL 1蛋白诱导电离辐射（IR）的反应，虽然这是专门发生在一个骡子依赖的方式。最后，我们表明，NEIL 1的稳定，特别是TRIM 26 siRNA后，有助于细胞抵抗IR。这突出了Mule和TRIM 26在维持NEIL 1稳态水平，以及细胞DNA损伤反应所需的重要性。
Endonuclease VIII-like protein 1 (NEIL1) is a DNA glycosylase involved in initiating the base excision repair pathway, the major cellular mechanism for repairing DNA base damage. Here, we have purified the major E3 ubiquitin ligases from human cells responsible for regulation of NEIL1 by ubiquitylation. Interestingly, we have identified two enzymes that catalyse NEIL1 polyubiquitylation, Mcl-1 ubiquitin ligase E3 (Mule) and tripartite motif 26 (TRIM26). We demonstrate that these enzymes are capable of polyubiquitylating NEIL1 in vitro, and that both catalyse ubiquitylation of NEIL1 within the same C-terminal lysine residues. An siRNA-mediated knockdown of Mule or TRIM26 leads to stabilisation of NEIL1, demonstrating that these enzymes are important in regulating cellular NEIL1 steady state protein levels. Similarly, a mutant NEIL1 protein lacking residues for ubiquitylation is more stable than the wild type protein in vivo. We also demonstrate that cellular NEIL1 protein is induced in response to ionising radiation (IR), although this occurs specifically in a Mule-dependent manner. Finally we show that stabilisation of NEIL1, particularly following TRIM26 siRNA, contributes to cellular resistance to IR. This highlights the importance of Mule and TRIM26 in maintaining steady state levels of NEIL1, but also those required for the cellular DNA damage response.