A Naturally-Occurring Dominant-Negative Inhibitor of Keap1 Competitively against Its Negative Regulation of Nrf2.

Lu Qiu,Yuping Zhu,Meng Wang,Yiguo Zhang,Yuancai Xiang

doi:10.3390/ijms19082150

Lu Qiu, Yuping Zhu + Show 3 more

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https://doi.org/10.3390/ijms19082150

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Abstract

Transcription factor Nrf2 (nuclear factor-erythroid 2-related factor 2) is a master regulator of antioxidant and/or electrophile response elements (AREs/EpREs)-driven genes involved in homeostasis, detoxification, and adaptation to various stresses. The cytoprotective activity of Nrf2, though being oppositely involved in both cancer prevention and progression, is critically controlled by Keap1 (Kelch-like ECH-associated protein 1), which is an adaptor subunit of Cullin 3-based E3 ubiquitin ligase and also is a key sensor for oxidative and electrophilic stresses. Here, we first report a novel naturally-occurring mutant of Keap1, designated Keap1ΔC, which lacks most of its C-terminal Nrf2-interacting domain essential for inhibition of the cap’n’collar (CNC) basic-region leucine zipper (bZIP) factor. This mutant Keap1ΔC is yielded by translation from an alternatively mRNA-spliced variant lacking the fourth and fifth exons, but their coding sequences are retained in the wild-type Keap1 locus (with no genomic deletions). Although this variant was found primarily in the human highly-metastatic hepatoma (MHCC97H) cells, it was widely expressed at very lower levels in all other cell lines examined. Such Keap1ΔC retains no or less ability to inhibit Nrf2, so that it functions as a dominant-negative competitor of Keap1 against its inhibition of Nrf2 due to its antagonist effect on Keap1-mediated turnover of Nrf2 protein.

Highlights

The full-length protein of Keap1 was first identified to consist of 624 amino acids [1]
Structural studies have revealed that only a functional homodimer of Keap1 with each other’s BTB domains is necessary for direct binding to the ETGE and DLG motifs within the Neh2 domain of Nrf2 [14], which acts as a degron targeting the CNC-basic-region leucine zipper (bZIP) protein to ubiquitin-mediated proteasomal degradation pathways
Only the highly-metastatic hepatoma MHCC97H cells gave rise to double cDNA bands of Keap1, at much lower levels than those obtained from all other hepatocellular carcinoma cell lines, which just gave a single band of its full-length

Summary

Introduction

The full-length protein of Keap was first identified to consist of 624 amino acids (aa) [1]. The gene encoding Nrf was cloned in 1994 and later identified as one of the most important members of the CNC-bZIP family [7,8]. It is generally accepted as a master regulator of AREs/EpREs-driven genes, which adaptive cytoprotection against various stresses [9,10]. Structural studies have revealed that only a functional homodimer of Keap with each other’s BTB domains is necessary for direct binding to the ETGE and DLG motifs within the Neh domain of Nrf2 [14], which acts as a degron targeting the CNC-bZIP protein to ubiquitin-mediated proteasomal degradation pathways. When upon stimulation of Keap by oxidative and electrophilic stresses, Nrf is dissociated from Keap1-sequestered confinements to be translocated into the nucleus before transactivating AREs/EpREs-battery genes, such as heme oxygenase-1 (HO-1), glutamate-cysteine ligase modifier subunit (GCLM), and NAD(P)H:quinone dehydrogenase 1 (NQO1) [15,16,17]

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