Regulatory flexibility in the Nrf2-mediated stress response is conferred by conformational cycling of the Keap1-Nrf2 protein complex
Regulatory flexibility in the Nrf2-mediated stress response is conferred by conformational cycling of the Keap1-Nrf2 protein complex
复制标题
DOI：
10.1073/pnas.1305687110
复制
发表时间：
2013-09-17
影响因子：
11.1
通讯作者：
Dinkova-Kostova, Albena T.
中科院分区：
文献类型：
--
作者：
Baird, Liam;Lleres, David;Dinkova-Kostova, Albena T.
转录因子NF-E2 p45相关因子2 （Nrf2）是细胞保护基因的主要调节因子，由二聚体kelch样ECH相关蛋白1 （Keap1）控制，Keap1是Cullin3/RING-box蛋白1泛素连接酶的底物衔接蛋白，通常靶向Nrf2泛素化和降解，但在响应亲电试剂和氧化剂（诱导剂）时失去这种能力。通过使用重组蛋白和细胞群，概述了Keap1调控Nrf2的一些一般特征。然而，这两种蛋白如何在单细胞水平上相互作用目前尚不清楚。我们现在报告了一种基于多光子荧光寿命成像显微镜的定量Forster共振能量转移系统的发展，并将其用于研究单个活细胞中Nrf2和Keap1之间的相互作用。通过使用这种方法，我们发现在稳态条件下，Keap1和Nrf2之间的相互作用遵循一个循环，在这个循环中，复合物依次采用两种不同的构象：“开放”，Nrf2与Keap1的单个分子相互作用，然后是“封闭”，Nrf2与Keap1二聚体的两个成员结合。诱导剂通过使复合物在封闭构象中积累而不释放Nrf2来破坏这个循环。因此，游离的Keap1不能再生，新合成的Nrf2被稳定下来。基于这些发现，我们提出了一个我们命名为“keap1介导的Nrf2降解的循环顺序附着和再生模型”的模型。这种先前未预料到的动态允许对环境变化的快速转录反应，并可以适应多种调节模式。
The transcription factor NF-E2 p45-related factor 2 (Nrf2), a master regulator of cytoprotective genes, is controlled by dimeric Kelch-like ECH associated protein 1 (Keap1), a substrate adaptor protein for Cullin3/RING-box protein 1 ubiquitin ligase, which normally targets Nrf2 for ubiquitination and degradation but loses this ability in response to electrophiles and oxidants (inducers). By using recombinant proteins and populations of cells, some of the general features of the regulation of Nrf2 by Keap1 have been outlined. However, how the two proteins interact at a single-cell level is presently unknown. We now report the development of a quantitative Forster resonance energy transfer-based system using multiphoton fluorescence lifetime imaging microscopy and its application for investigating the interaction between Nrf2 and Keap1 in single live cells. By using this approach, we found that under homeostatic conditions, the interaction between Keap1 and Nrf2 follows a cycle in which the complex sequentially adopts two distinct conformations: "open," in which Nrf2 interacts with a single molecule of Keap1, followed by " closed," in which Nrf2 binds to both members of the Keap1 dimer. Inducers disrupt this cycle by causing accumulation of the complex in the closed conformation without release of Nrf2. As a consequence, free Keap1 is not regenerated, and newly synthesized Nrf2 is stabilized. On the basis of these findings, we propose a model we have named the "cyclic sequential attachment and regeneration model of Keap1-mediated degradation of Nrf2." This previously unanticipated dynamism allows rapid transcriptional responses to environmental changes and can accommodate multiple modes of regulation.