Covalent Modification at Cys151 Dissociates the Electrophile Sensor Keap1 from the Ubiquitin Ligase CUL3

Abstract

The regulation of cellular stress responses to electrophiles and oxidants is mediated by the transcription factor NF-E2-related factor 2 (Nrf2), which, in turn, is regulated by CUL-E3 (CUL3) ligase-mediated ubiquitylation. The Kelch-like ECH-associated protein 1 (Keap1) serves as an adapter between CUL3 and Nrf2. We used the model electrophile N-iodoacetyl-N-biotinylhexylenediamine (IAB) to define the relationship among the adduction of Keap1 cysteine residues, structure, and function. Exposure of Keap1 to IAB in Vitro was accompanied by progressive loss of protein secondary structure, as monitored by CD spectroscopy and a loss of the ability to associate with recombinant CUL3. Dissociation of Keap1 from CUL3 in Vitro was dependent upon C151 in Keap1. A quantitative mass spectrometry-based kinetic analysis of adduction in HEK293 cells expressing FLAG-Keap1 revealed that Cys151 was one of the most reactive residues in ViVo and that it was required for IAB-mediated dissociation of the Keap1-CUL3 interaction. These results demonstrate that Cys151 adduction confers a critical alkylation sensor function upon Keap1, making Keap1 unique among BTB CUL3 adapter proteins.

Chemistry of the Cysteine Sensors in Kelch-Like ECH-Associated Protein 1

“…3 in Ref. (49)], which demonstrate a marked change in the molar ellipticity spectrum of the C151S mutant. Changes in this region of the spectrum typically reflect alterations in a-helical or b-sheet content and possibly tertiary structure.…”

“…Recently, a partial circular dichroism spectrum of molar ellipticity against wavelength for WT Keap1 and the C151S mutant was published in an attempt to measure differences in structural changes in response to the alkylator biotinylated iodoacetamide (IAB) (49). Unremarked upon, but of special interest here, are the two spectra at t ¼ 0 [ Fig.…”

“…A number of reports attest to a general stabilization of Nrf2 as a result of electrophilic=oxidative assault (15,33,42). Attendant with this is the formation of highmolecular-weight forms of Keap1 (HMWK) (49,63), which are ubiquitylated and possibly oxidatively crosslinked, and in some cases, a decrease in levels of Keap1 generally and an increase in nuclear localization of Nrf2. There are also reports indicating a comigration of Keap1 and Nrf2 to the nucleus under stress, whereas this is not observed under other conditions (16,42,59).…”

“…It has also been reported that Nrf2 ubiquitylation by Keap1 occurs in the nucleus and it is the disruption of this complex in the nucleus by electrophiles=oxidants that leads to phase 2 enzyme upregulation (42). There are reports that some electrophiles=oxidants mediate dissociation of Nrf2 from Keap1 (35,44), whereas others report that electrophiles induce Cul3 dissociation from Keap1, thereby preventing Nrf2 ubiquitylation (9,12,49). Others suggest that Keap1-bound cytoplasmic Nrf2 is irretrievably destined for destruction (60).…”

Electrophilic tuning of the chemoprotective natural product sulforaphane

“…A number of mass spectroscopic studies have examined recombinant Keap1 labeling in vitro by dexamethasone mesylate (15), iodoacetamide-biotin (40)(41)(42)(43), and several other agents (44). Iodoacetamide-biotin has also been studied in cells (41,42).…”