Department of Biology and Genetics Institute, University of Florida, Gainesville, Florida, USA.
Department of Environmental Biology, College of Bioscience and Biotechnology, Chubu University, Kasugai, Aichi, Japan.
Mol Cell Biol. 2014 Aug;34(16):3156-67. doi: 10.1128/MCB.00114-14. Epub 2014 Jun 9.
SKN-1/Nrf transcription factors activate cytoprotective genes in response to reactive small molecules and strongly influence stress resistance, longevity, and development. The molecular mechanisms of SKN-1/Nrf regulation are poorly defined. We previously identified the WD40 repeat protein WDR-23 as a repressor of Caenorhabditis elegans SKN-1 that functions with a ubiquitin ligase to presumably target the factor for degradation. However, SKN-1 activity and nuclear accumulation are not always correlated, suggesting that there could be additional regulatory mechanisms. Here, we integrate forward genetics and biochemistry to gain insights into how WDR-23 interacts with and regulates SKN-1. We provide evidence that WDR-23 preferentially regulates one of three SKN-1 variants through a direct interaction that is required for normal stress resistance and development. Homology modeling predicts that WDR-23 folds into a β-propeller, and we identify the top of this structure and four motifs at the termini of SKN-1c as essential for the interaction. Two of these SKN-1 motifs are highly conserved in human Nrf1 and Nrf2 and two directly interact with target DNA. Lastly, we demonstrate that WDR-23 can block the ability of SKN-1c to interact with DNA sequences of target promoters identifying a new mechanism of regulation that is independent of the ubiquitin proteasome system, which can become occupied with damaged proteins during stress.
SKN-1/Nrf 转录因子响应反应性小分子激活细胞保护基因，并强烈影响应激抗性、寿命和发育。SKN-1/Nrf 调节的分子机制尚未完全定义。我们之前确定 WD40 重复蛋白 WDR-23 为 Caenorhabditis elegans SKN-1 的抑制剂，它与泛素连接酶一起作用，可能将该因子靶向降解。然而，SKN-1 的活性和核积累并不总是相关的，这表明可能存在其他调节机制。在这里，我们整合正向遗传学和生物化学来深入了解 WDR-23 如何与 SKN-1 相互作用和调节。我们提供的证据表明，WDR-23 通过直接相互作用优先调节三种 SKN-1 变体之一，这是正常应激抗性和发育所必需的。同源建模预测 WDR-23 折叠成 β-桨叶，我们确定了该结构的顶部和 SKN-1c 末端的四个基序是相互作用所必需的。这两个 SKN-1 基序在人类 Nrf1 和 Nrf2 中高度保守，并且直接与靶 DNA 相互作用。最后，我们证明 WDR-23 可以阻断 SKN-1c 与靶启动子 DNA 序列相互作用的能力，从而确定了一种新的独立于泛素蛋白酶体系统的调节机制，该系统在应激过程中可能会被受损蛋白占据。
