Keap1 is a redox-regulated substrate adaptor protein for a Cul3-dependent ubiquitin ligase complex
Keap1 is a redox-regulated substrate adaptor protein for a Cul3-dependent ubiquitin ligase complex
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DOI：
10.1128/mcb.24.24.10941-10953.2004
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发表时间：
2004-12-01
影响因子：
5.3
通讯作者：
Hannink, M
中科院分区：
文献类型：
--
作者：
Zhang, DD;Lo, SC;Hannink, M
bZIP转录因子Nrf 2控制着一个遗传程序，保护细胞免受氧化损伤并维持细胞氧化还原稳态。Keap 1是一种BTB-Kelch蛋白，是Nrf 2的主要上游调节因子，控制Nrf 2的亚细胞定位和稳态水平。在这份报告中，我们证明了Keap 1作为Cul 3依赖性E3泛素连接酶复合物的底物衔接蛋白的功能。Keap 1与Cul 3和Rbx 1组装成功能性E3泛素连接酶复合物，其靶向位于Nrf 2的N-末端Neh 2结构域中的多个赖氨酸残基，用于体内和体外的泛素缀合。Keap 1依赖的泛素化Nrf 2抑制细胞暴露于醌诱导的氧化应激和萝卜硫素，癌症预防异硫氰酸酯。突变体Keap 1蛋白在Keap 1的BTB结构域内的残基151处含有单个半胱氨酸至丝氨酸取代，其对醌诱导的氧化应激或萝卜硫素的抑制具有显著抗性。抑制Keap 1依赖的Nrf 2泛素化与Keap 1与Cul 3的关联减少相关。醌诱导的氧化应激和萝卜硫素都不会破坏Keap 1和Nrf 2之间的联系。我们的研究结果表明，Keap 1组装成功能性E3泛素连接酶复合物的能力是控制Nrf 2稳态水平的关键决定因素，以应对癌症预防化合物和氧化应激。
The bZIP transcription factor Nrf2 controls a genetic program that protects cells from oxidative damage and maintains cellular redox homeostasis. Keap1, a BTB-Kelch protein, is the major upstream regulator of Nrf2 and controls both the subcellular localization and steady-state levels of Nrf2. In this report, we demonstrate that Keap1 functions as a substrate adaptor protein for a Cul3-dependent E3 ubiquitin ligase complex. Keap1 assembles into a functional E3 ubiquitin ligase complex with Cul3 and Rbx1 that targets multiple lysine residues located in the N-terminal Neh2 domain of Nrf2 for ubiquitin conjugation both in vivo and in vitro. Keap1-dependent ubiquitination of Nrf2 is inhibited following exposure of cells to quinone-induced oxidative stress and sulforaphane, a cancer-preventive isothiocyanate. A mutant Keap1 protein containing a single cysteine-to-serine substitution at residue 151 within the BTB domain of Keap1 is markedly resistant to inhibition by either quinone-induced oxidative stress or sulforaphane. Inhibition of Keap1-dependent ubiquitination of Nrf2 correlates with decreased association of Keap1 with Cul3. Neither quinone-induced oxidative stress nor sulforaphane disrupts association between Keap1 and Nrf2. Our results suggest that the ability of Keap1 to assemble into a functional E3 ubiquitin ligase complex is the critical determinant that controls steady-state levels of Nrf2 in response to cancer-preventive compounds and oxidative stress.