Conservation of the Keap1-Nrf2 System: An Evolutionary Journey through Stressful Space and Time

Abstract

The Keap1-Nrf2 system is an evolutionarily conserved defense mechanism against oxidative and xenobiotic stress. Its regulatory mechanisms, e.g., stress-sensing mechanism, proteasome-based regulation of Nrf2 activity and selection of target genes, have been elucidated mainly in mammals. In addition, emerging model animals, such as zebrafish, fruit fly and Caenorhabditis elegans, have been shown to have similar anti-stress systems to mammals, suggesting that analogous defense systems are widely conserved throughout the animal kingdom. Experimental evidence in lower animals provides important information beyond mere laboratory-confined utility, such as regarding how these systems transformed during evolution, which may help characterize the mammalian system in greater detail. Recent advances in genome projects of both model and non-model animals have provided a great deal of useful information toward this end. We herein review the research on Keap1-Nrf2 and its analogous systems in both mammals and lower model animals. In addition, by comparing the amino acid sequences of Nrf2 and Keap1 proteins from various species, we can deduce the evolutionary history of the anti-stress system. This combinatorial approach using both experimental and genetic data will suggest perspectives of approach for researchers studying the stress response.

Keywords: C. elegans Skn-1; Drosophila Cnc; Hydra Nrf; Keap1; Nrf2; anti-stress system; evolutionary history; mouse; yeast Yap1; zebrafish.

Figure 1

Regulatory mechanisms of the transcription factor-based oxidative stress response in eukaryotes. The activation mechanism of: Keap1-Nrf2/Cnc system (A); Yap1 in S. cerevisiae (B); and Skn-1 system in C. elegans (C) are depicted.

Figure 2

Comparison of Nrf family proteins. (A) The domain structures of four Nrf proteins in mammals were compared based on the six Neh domains of Nrf2. Striped color denotes the region that is partially conserved with the Neh4 domain. The ER binding domain (NHB1) that is specific to Nrf1 and -3 is also indicated; (B) Amino acid sequences of Nrf/Cnc transcription factors from mouse (Mm), chicken (Gg), clawed frog (Xt), zebrafish (Dr), ascidian (Ci), sea urchin (Sp), octopus (Ob), fruit fly (Dm), C. elegans (Ce) and Hydra (Hm). The amino acids identical to mouse Nrf2 are shaded in gray. Leucine residues comprising the zipper structure in Neh1, the DLG and ETGE motifs in Neh2, the VFLVPK motif in Neh3, the FxD/ExxxLLxE/D sequence in Neh4, the QxWxELxSxPELQ sequence in Neh5 and the DSGIS and DSAPGS motifs in Neh6 are shaded in yellow. Basic amino acid residues in the Neh1 basic region, lysine residues between the DLG and ETGE motif (ubiquitination sites) and serine/threonine residues (Ser-40, phosphorylation site) in the Neh2 domain are shown in pink, blue and orange letters, respectively.

Figure 3

A summary of the domain structure of the Nrf/Cnc transcription factors. Conservation of the Neh domains was evaluated as follows: ◎, highly conserved; ○, relatively conserved; △, partially conserved; ×, not conserved. Specific motifs were described as “highly conserved” only when the sequences were identical to mouse Nrf2. The amino acid lengths between DLG and ETGE motifs are also shown.

Figure 4

The evolution of Nrf/Cnc transcription factors deduced from amino acid sequences. Gray bars and subscripts (s1 and s2) in Deuterostomes Nrf denote products of alternative splicing.

Figure 5

A comparison of Keap1 proteins: (A) domain structures of Keap1; and (B) amino acid sequences of Keap1 proteins from mouse (Mm), chicken (Gg), anole lizard (Ac), clawed frog (Xt), coelacanth (Lc), zebrafish (Dr), medaka (Ol), green spotted puffer (Tn), ascidian (Ci), sea urchin (Sp), octopus (Ob) and fruit fly (Dm). The amino acids identical to mouse Nrf2 are shaded in gray. The serine residues essential for homodimer formation in the BTB domain, NES consensus sequence in the IVR domain and three reactive cysteine residues are shaded in yellow.

Figure 6

A summary of the cysteine residues of Keap1. The conservation of each cysteine is indicated as follows ○: conserved; △: not conserved but cysteine exists within three amino acids; ×: not conserved. Sensor cysteines are shaded in red, and cysteine residues conserved among Kelch family proteins in mice are shaded in orange.

Figure 7

The evolution of Keap1 proteins deduced from amino acid sequences.