New insights into the mechanism of Keap1-Nrf2 interaction based on cancer-associated mutations
Introduction
The Keap1-Nrf2 signaling pathway plays critical roles both in oxidative stress and cancer pathogenesis [1], [2], [3]. Keap1 is a negative regulatory protein of Nrf2 [4]. The activation of Nrf2 induces the cellular defenses via initiating the transcription of cytoprotective genes, which is essential for cells to resist adverse environments [5]. Keap1 works with Nrf2 and Cul3-E3 ubiquitin ligase through the Kelch domain to form Nrf2-Keap1-Cul3 complex, then this complex transfers ubiquitin to Nrf2, resulting in Nrf2 degradation [6]. Under basal condition, one Nrf2 binding with a Keap1 homodimer through exploiting two distinct binding motifs (named DLG and ETGE) within the Neh2 domain. This binding mode allows Nrf2 to be effectively ubiquitinated [7]. ETGE and DLG motifs bind to Keap1 in a very similar fashion, but the binding affinity of ETGE motif is obviously stronger. It is currently believed that acidic residues define the differential binding affinities of the ETGE and DLG motifs [8]. However, subsequent research found the binding characteristic of DLGex to Keap1 is distinct from ETGE motif [9]. Under oxidative stress, specific cysteines in Keap1 are modified by oxidants, which disrupts the weak binding of Keap1 with Nrf2-DLG motif, resulting in Nrf2 cannot be degraded and translocate into the nucleus to initiate cytoprotective transcription [10], [11], [12]. However, if Nrf2 is over-activated, it will cause tumor deterioration [13].
Many somatic mutations within Keap1 or Nrf2 have been reported in various types of cancer tissues and cell lines [14], [15], which are closely related to cancer poor prognosis [16], [17]. Some mutations in Keap1 could impair Nrf2 degradation [18]. Meanwhile, mutations in Nrf2 DLG or ETGE motifs can prevent Nrf2 from the inhibitory effect of Keap1, leading to aberrant cellular Nrf2 accumulation and promote tumor [19]. Previous studies also found that G364C and G430C mutants of Keap1 in lung cancer can cause the activation of Nrf2 [20]. However, little research has been conducted on the binding characteristic of these mutants to the target, and the internal relationship between Keap1-Nrf2 mutants and cancer is unclear. In addition, the currently research on the interaction between Keap1 and Nrf2-ETGE motif mainly focuses on the binding center [21], and their interaction need further study.
To deal with this difficulty, we focused on the key residues in ETGE motif which affect the binding ability to Keap1. We clarified the key role of Thr80 and Pro85 on ETGE motif through resolved the structure of the Kelch domain with Nrf2 25-mer peptide. Moreover, we found mutants in both positions could affect the binding with Keap1. Especially, we demonstrated the effect of mutations in both two positions on the Keap1-Nrf2 signaling pathway in cells. The mutations all lead to the up-regulation of Heme Oxygenase-1(HO-1) and NAD(P)H quinone dehydrogenase 1(NQO1). Besides, we designed a series of mutants in Keap1-Kelch domain to confirm the binding change of mutants to Nrf2 and found that some cancer-related mutations of Keap1 did not affect on the binding to Nrf2. Our study provides a theoretical basis for the research on the interaction of Keap1-Nrf2 and cancer occurrence.
Section snippets
Plasmid constructions
A gene encoding mouse Keap1-Kelch domain (aa 309 to 624) and Nrf2-Neh2 domain (aa 33–98) from mouse cDNA were inserted into the pET-28a(+) and pET-15b(+) respectively. Nrf2 and Keap1 gene were obtained from human cDNA and inserted into Nrf2-pCDNA3.1(+). The Kelch-pET-28a, Neh2-pET15b and Nrf2-pCDNA3.1 mutant plasmids were obtained by PCR method. The PCR product was cleaved by Dpn I restriction enzymes. Then the reaction mixture was subsequently transformed into the strain E. coli DH5α. The
The functional sequence of ETGE motif with more than 12 residues
More than 5000 somatic mutations in Keap1-Nrf2 have been recorded in the Catalogue of Somatic Mutations in Cancer (COSMIC) database. We surveyed the COSMIC database and found that cancer-related Nrf2 mutations are concentrated on DLG and ETGE motif. Meanwhile, through comparing Nrf2 sequences from different species, we found that both motifs are very conservative (Fig. 1A). Furthermore, the activation of Nrf2 is regulated by many kinases. Both threonine and serine on the Nrf2 are potential
Discussion
Previous research has proposed that Keap1 exerts an inhibitory effect through the binding of the Kelch domain with the DLG Motif and ETGE motif within the Neh2 domain of Nrf2 [7], [9]. Both DLG and ETGE peptide possesses a hairpin conformation, but ETGE motif has a stronger affinity with Kelch [24]. Under oxidative stress, the binding of ETGE motif and Kelch will not be destroyed [9]. Most of the mutations in ETGE motif will cause damage to the binding to Keap1 [25]. For example, mutation of
Abbreviations
- ITC
isothermal titration calorimetry
- DSF
differential scanning fluorimetry
- EMSA
electrophoretic mobility shift assay
- WB
western blot
- HO-1
heme oxygenase-1
- NQO1
NAD(P)H quinone dehydrogenase 1
- IPTG
isopropyl β-D-thiogalactoside
- WT
wild type
- Tm
melting temperature
- RT-PCR
reverse-transcription polymerase chain reaction
- COSMIC
catalogue of somatic mutations in cancer
- CGC
cancer gene census
Funding
This work was supported by the National Natural Science Foundation of China (81930114), Guangdong Key Laboratory for Translational Cancer Research of Chinese Medicine (No. 2018B030322011).
CRediT authorship contribution statement
Conceptualization, C.W.; Data curation, L.C.; Methodology, H.W.; Software, S.L.; Validation, Z.L.; Original draft, C.W..
Declaration of competing interest
The authors declare that they have no conflicts of interests.
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