Diffusion dynamics of the Keap1-Cullin3 interaction in single live cells
Diffusion dynamics of the Keap1-Cullin3 interaction in single live cells
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DOI：
10.1016/j.bbrc.2013.02.065
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发表时间：
2013-03-29
影响因子：
3.1
通讯作者：
Dinkova-Kostova, Albena T.
中科院分区：
文献类型：
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作者：
Baird, Liam;Dinkova-Kostova, Albena T.
转录因子 NF-E2 p45 相关因子 2 (Nrf2) 调节编码药物解毒、抗炎和代谢酶的基因网络的表达，以及参与细胞氧化还原稳态调节的蛋白质。在基础条件下，Kelch 样 ECH 相关蛋白 1 (Keap1) 通过与基于 Cullin3 (Cul3) 的 Rbx1 E3 泛素连接酶结合，靶向 Nrf2 进行泛素化和蛋白酶体降解。各种小分子（诱导剂）激活 Nrf2，导致细胞保护基因表达上调。诱导剂对 Keap1 的特定半胱氨酸残基进行化学修饰，最终失去靶向 Nrf2 降解的能力。 Keap1-Cul3 复合物通过诱导剂解离是一种可能的机制，但缺乏单个活细胞的证据。为了研究 Keap1-Cul3 相互作用的扩散动力学和诱导剂的影响，我们使用 Keap1-EGFP 和 mCherry-Cul3 融合蛋白开发了基于光漂白后定量荧光恢复 (FRAP) 的系统。我们证明 Keap1-EGFP 和 mCherry-Cul3 在单个活细胞中相互作用。暴露于 4 种不同类型的小分子诱导剂（齐墩果烷三萜 CDDO、异硫氰酸萝卜硫素、磺氧基硫代氨基甲酸酯 STCA 和氧化剂过氧化氢）1 小时，这些诱导剂以 Keap1 内不同的半胱氨酸传感器为目标，其效力相差近 4000 倍，不会解离 Keap1-Cul3 复合物。由于诱导剂会引起 Keap1 的构象变化，因此我们得出结论，构象的变化而不是与 Cul3 的解离使 Keap1 的阻遏功能失活，从而导致 Nrf2 稳定。 (c) 2013 Elsevier Inc. 保留所有权利。
Transcription factor NF-E2 p45-related factor 2 (Nrf2) regulates the expression of a network of genes encoding drug-detoxification, anti-inflammatory, and metabolic enzymes, as well as proteins involved in the regulation of cellular redox homeostasis. Under basal conditions, Kelch-like ECH associated protein 1 (Keap1) targets Nrf2 for ubiquitination and proteasomal degradation via association with Cullin3 (Cul3)-based Rbx1 E3 ubiquitin ligase. Various small molecules (inducers) activate Nrf2 leading to upregulation of cytoprotective gene expression. Inducers chemically modify specific cysteine residues of Keap1 which ultimately loses its ability to target Nrf2 for degradation. Dissociation of the Keap1-Cul3 complex by inducers is one possible mechanism, but evidence in single live cells is lacking. To investigate the diffusion dynamics of the Keap1-Cul3 interaction and the effect of inducers, we developed a quantitative fluorescence recovery after photobleaching (FRAP)-based system using Keap1-EGFP and mCherry-Cul3 fusion proteins. We show that Keap1-EGFP and mCherry-Cul3 interact in single live cells. Exposure for 1 h to small-molecule inducers of 4 different types, the oleanane triterpenoid CDDO, the isothiocyanate sulforaphane, the sulfoxythiocarbamate STCA, and the oxidant hydrogen peroxide which target distinct cysteine sensors within Keap1 with potencies which differ by nearly 4000-fold, does not dissociate the Keap1-Cul3 complex. As inducers cause conformational changes in Keap1, we conclude that changes in conformation rather than dissociation from Cul3 inactivate the repressor function of Keap1 leading to Nrf2 stabilization. (c) 2013 Elsevier Inc. All rights reserved.