Medical Research Council Centre for Drug Safety Science, Department of Pharmacology and Therapeutics, the University of Liverpool, Sherrington Buildings, Ashton Street, Liverpool L69 3GE, United Kingdom.
J Biol Chem. 2010 May 28;285(22):16782-8. doi: 10.1074/jbc.M109.096545. Epub 2010 Apr 8.
Nrf2 regulates the expression of numerous cytoprotective genes in mammalian cells. The activity of Nrf2 is regulated by the Cul3 adaptor Keap1, yet little is known regarding mechanisms of regulation of Keap1 itself. Here, we have used immunopurification of Keap1 and mass spectrometry, in addition to immunoblotting, to identify sequestosome 1 (SQSTM1) as a cellular binding partner of Keap1. SQSTM1 serves as a scaffold in various signaling pathways and shuttles polyubiquitinated proteins to the proteasomal and lysosomal degradation machineries. Ectopic expression of SQSTM1 led to a decrease in the basal protein level of Keap1 in a panel of cells. Furthermore, RNA interference (RNAi) depletion of SQSTM1 resulted in an increase in the protein level of Keap1 and a concomitant decrease in the protein level of Nrf2 in the absence of changes in Keap1 or Nrf2 mRNA levels. The increased protein level of Keap1 in cells depleted of SQSTM1 by RNAi was linked to a decrease in its rate of degradation; the half-life of Keap1 was almost doubled by RNAi depletion of SQSTM1. The decreased level of Nrf2 in cells depleted of SQSTM1 by RNAi was associated with decreases in the mRNA levels, protein levels, and function of several Nrf2-regulated cell defense genes. SQSTM1 was dispensable for the induction of the Keap1-Nrf2 pathway, as Nrf2 activation by tert-butylhydroquinone or iodoacetamide was not affected by RNAi depletion of SQSTM1. These findings demonstrate a physical and functional interaction between Keap1 and SQSTM1 and reveal an additional layer of regulation in the Keap1-Nrf2 pathway.
Nrf2 调节哺乳动物细胞中许多细胞保护基因的表达。Nrf2 的活性受 Cul3 适配器 Keap1 调节，但关于 Keap1 自身调节的机制知之甚少。在这里，我们使用 Keap1 的免疫沉淀和质谱分析，以及免疫印迹，鉴定了自噬相关蛋白 1（SQSTM1）是 Keap1 的一种细胞结合伴侣。SQSTM1 在各种信号通路中作为支架，将多泛素化蛋白转运到蛋白酶体和溶酶体降解机制中。在一系列细胞中，SQSTM1 的异位表达导致 Keap1 的基础蛋白水平降低。此外，RNA 干扰（RNAi）敲低 SQSTM1 导致 Keap1 蛋白水平增加，Nrf2 蛋白水平降低，而 Keap1 或 Nrf2 mRNA 水平没有变化。RNAi 敲低 SQSTM1 导致细胞中 Keap1 蛋白水平增加与降解率降低有关；RNAi 敲低 SQSTM1 使 Keap1 的半衰期几乎增加了一倍。RNAi 敲低 SQSTM1 导致细胞中 Nrf2 水平降低与几种 Nrf2 调节的细胞防御基因的 mRNA 水平、蛋白水平和功能降低有关。SQSTM1 对于 Keap1-Nrf2 途径的诱导是可有可无的，因为 tert-butylhydroquinone 或碘乙酰胺对 Nrf2 的激活不受 SQSTM1 RNAi 敲低的影响。这些发现表明 Keap1 和 SQSTM1 之间存在物理和功能相互作用，并揭示了 Keap1-Nrf2 途径的另一个调节层。
