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Comparative Study
, 102 (29), 10070-5

Modifying Specific Cysteines of the Electrophile-Sensing Human Keap1 Protein Is Insufficient to Disrupt Binding to the Nrf2 Domain Neh2

Affiliations
Comparative Study

Modifying Specific Cysteines of the Electrophile-Sensing Human Keap1 Protein Is Insufficient to Disrupt Binding to the Nrf2 Domain Neh2

Aimee L Eggler et al. Proc Natl Acad Sci U S A.

Abstract

The risks of cancer and other degenerative diseases caused by reactive oxygen species and electrophiles can be reduced by the up-regulation of detoxifying enzymes. A major mechanism whereby these protective enzymes are induced occurs through activation of the antioxidant response element (ARE) by the oxidative-stress sensor protein Kelch-like ECH-associated protein 1 (Keap1) and the transcription factor NF-E2-related factor 2 (Nrf2). Under basal conditions, Keap1 sequesters Nrf2 in the cytoplasm by binding to its Neh2 domain. Chemical inducers such as sulforaphane are known to react with Keap1 cysteine residues, thereby promoting Nrf2 nuclear accumulation and hence ARE activation. A widely accepted model for Nrf2 nuclear accumulation is that modification of Keap1 cysteines leads directly to dissociation of the Keap1-Nrf2 complex. This model is based on studies with mouse proteins and has served as the experimental basis and hypothesis for numerous investigations. Through a combination of chemical, mass spectrometry, and isothermal titration calorimetry methods, we have tested the direct-dissociation model using a series of ARE inducers: sulforaphane, isoliquiritigenin, 15-deoxy-Delta12,14-prostaglandin-J2, menadione, 1-Cl-2,4-dinitrobenzene, and biotinylated iodoacetamide. Surprisingly, these data suggest that the direct disruption model for Keap1-Nrf2 is incorrect. The relative reactivity of human Keap1 cysteines was determined. In addition to the same five cysteines identified for mouse Keap1, two highly reactive and previously unobserved cysteines were identified. Based on these results, a model is proposed that should aid in the understanding of Keap1-Nrf2 signaling mechanisms.

Figures

Fig. 1.
Fig. 1.
Primary sequence representation of the mouse and human Keap1 proteins and the most readily modified cysteines. (A) The cysteines in mouse Keap1 most readily modified by dexamethasone 21-mesylate (12) are shown in green. All cysteines shown were modified by BIA in this study, both green- and red-labeled, and those in red were found uniquely in this study. Those labeled with an asterisk were reported to be important in the regulation of Nrf2 ubiquitination (5). (B) The order of reactivity of the cysteines determined in this study is shown, along with the ratio of BIA to Keap1 required to obtain those modifications.
Fig. 2.
Fig. 2.
Keap1-Neh2 titration showing a binding stoichiometry of 2:1 Keap1:Neh2. Reaction conditions corresponding to lanes 2-10 included 15 μM Keap1 and increasing concentrations of Neh2 from 3.3 to 9.2 μM as shown. Lane 1 corresponds to an incubation containing 9.2 μM Neh2 but with Keap1 omitted. The asterisk in lane 7 indicates the faint band of Keap1 not bound to Neh2 located just above the asterisk.
Fig. 3.
Fig. 3.
Native EMSA gels for Keap1 modified with sulforaphane (SUL). (A) Keap1 was modified with increasing concentrations of sulforaphane, and its ability to bind to Neh2 was tested. Reaction conditions corresponding to lanes 2-10 include 15 μM Keap1 and 0.4 mM TCEP. Keap1 was modified with sulforaphane at the concentrations in excess of Keap1 as shown before Neh2 addition, except for lane 10, in which Neh2 and Keap1 were prebound before sulforaphane addition. Lanes 1 and 6-10 contain 7.5 μM Neh2. (B) The Keap1-Neh2 binding stoichiometry does not change when Keap1 is preincubated with sulforaphane. Reaction conditions corresponding to lanes 2-9 include 15 μM Keap1 and increasing concentrations of Neh2 from 5 to 7.5 μM as shown. Where indicated, Keap1 was preincubated with 180 μM sulforaphane. Lane 1 contains 7.5 μM Neh2.
Fig. 4.
Fig. 4.
Native EMSA of Keap1 and Neh2 with Keap1 incubated with various Michael reaction acceptor electrophiles alone or in the presence of Neh2. Reaction conditions corresponding to all lanes include 15 μM Keap1, and, in lanes 2, 5, 8, and 11, Keap1 was preincubated with the electrophiles indicated before addition of 7.5 μM Neh2. In the reactions corresponding to lanes 3, 6, 9, and 12, Neh2 was prebound to Keap1 before addition of the electrophile. The average number of modifications per Keap1 molecule as determined by MALDI-TOF is indicated. nd, Not determined; isoliquirit, isoliquiritigenin.
Fig. 5.
Fig. 5.
ITC profiles of Neh2 with unmodified or modified Keap1. Representative profiles are shown for 39.5 μM Neh2 titrated into 1.5 μM Keap1, unmodified or modified with BIA or CDNB as labeled. (Upper) ITC thermographs. (Lower) The fitted binding isotherms.
Fig. 6.
Fig. 6.
A model depicting how modification of Keap1 C151 by ARE inducers could lead to Nrf2 nuclear accumulation. (A) Under basal conditions, Keap1 forms a bridge between Cul3 and Nrf2, leading to ubiquitination of the Neh2 domain of Nrf2. Upon introduction of electrophiles, modification of Keap1 C151 leads to a change in the conformation of the BTB domain by means of perturbing the homodimerization site, disrupting Neh2 ubiquitination, and causing ubiquitination of Keap1. Modification of Keap1 cysteines by electrophiles does not lead to disruption of the Keap1-Nrf2 complex. Rather, the switch of ubiquitination from Nrf2 to Keap1 leads to Nrf2 nuclear accumulation. (B) Structural representation of the putative Keap1 BTB domain homodimerization interface and BTB-Cul3 interaction interface. The PLZF BTB domain was aligned structurally with Skp1 (Fig. 11, which is published as supporting information on the PNAS web site), with which it shares significant structural similarity, to map the Cul3-BTB interaction site. The sequences of the PLZF and Keap1 BTB domains were aligned (Fig. 11) to obtain the locations of the Keap1 residues illustrated. The location of Keap1 C151 (PLZF D98) is mapped as a cysteine. The locations of the Keap1 residues 125-127 and 162-164, in the putative Cul3 binding region, are shown colored in yellow on the PLZF backbone.

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