Modifying specific cysteines of the electrophile-sensing human Keap1 protein is insufficient to disrupt binding to the Nrf2 domain Neh2

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-⌬12,14-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.antioxidant response element ͉ sulforaphane ͉ 15-deoxy-⌬12,14-prostaglandin J2 ͉ oxidative stress ͉ ubiquitination

Acrolein: Sources, metabolism, and biomolecular interactions relevant to human health and disease

“…Human Keap1 is structurally organized into five major domains: the N-terminal domain (amino acids 1-60); the "Broad complex, tramtrack and bric-a-brac" BTB domain (amino acids 61-179); the central intervening IVR domain (amino acids 180-314); a double glycine-rich domain comprising six Kelch repeat motifs (amino acids 315-359, 361-410, 412-457, 459-504, 506-551, and 553-598); and a C-terminal domain (amino acids 599-624). The BTB and IVR domains are required for the redox-and electrophile-sensitive regulation of Nrf2 through a series of reactive cysteines [113][114][115]. Human Keap1 has 27 cysteine residues (while rodent Keap1 has 25), nine of which are predicted to be particularly reactive due to their location adjacent to basic amino acids.…”

“…It appeared that the reactivities of the cysteines in human Keap1 are different from the corresponding cysteines in mouse Keap1 [112][113][114][115]117]. Despite some uncertainty regarding the exact order of the reactivity of the cysteine residues towards different electrophiles, Cys-151 (located in the BTB domain) was identified as the most reactive nucleophilic site in human Keap1 [113,117]. It is conceivable that multiple cysteines in Keap1 can be modified by different inducers and it is likely that sites of sulfhydryl modification may vary among the different chemical compound classes and across species [118].…”

“…Furthermore, modification experiments of diverse Keap1-Nrf2 complexes indicated that sulfhydryl alkylation by different Michael acceptors did not lead per se to Keap1-Nrf2 complex dissociation and Nrf2 translocation. To account for these unexpected findings, Mesecar and colleagues [113] recently proposed an alternative model for Keap1-Nrf2-mediated cell signaling. In their model, nuclear accumulation of Nrf2 is achieved via the following events: (i) alkylation of Cys-151 by electrophiles leads to disruption of the homodimerization site and to conformational changes in the BTB domain of Keap1, which would result in altered accessibility of the site of ubiquitination in Nrf2 and thus decreased proteosomal degradation, and (ii) increased ubiquitination and turnover of Keap1.…”

Sites of alkylation of human Keap1 by natural chemoprevention agents

“…In their studies of murine Keap1, Dinkova-Kostova et al [2] used MALDI-TOF to identify modified peptides in a tryptic digest, but LC-MS/MS was used to confirm the sequence and to determine the site of modification in only two of the four modified peptides. Our previous study [30] in which human Keap1 modifications were mapped indicated that C151, C288, and C297 are the most reactive residues toward iodoacetyl-N-biotinyl hexylene diamine. A similar study was reported by Hong et al [13], but their data suggested that C169, C241, and C288 but not C151 were reactive toward human Keap1.…”

“…The details of the cloning, expression, and purification of human Keap1 are described elsewhere [30]. BMCC was purchased from Pierce (Rockford, IL), and trypsin was purchased from Promega (Madison, WI).…”