title: Identification of the highly reactive cysteine 151 in the chemopreventive agent-sensor Keap1 protein is method-dependent.
abstract: Upregulation of cytoprotective and detoxifying enzyme expression by small molecules is emerging as an important means of preventing carcinogenesis as well as other diseases. A proposed target of these agents is the Kelch-like ECH-associated protein 1 (Keap1). The vast majority of these agents contain electrophilic moieties, which react with a subset of the 27 cysteines of human Keap1. Modification of these cysteines is proposed to result in nuclear accumulation of transcription factor NF-E2-related factor-2 (Nrf2), a Keap1 binding partner, leading to upregulation of cytoprotective enzymes. The electrophilic agent biotinylated iodoacetamide (BIA) has been used by different laboratories to determine the most reactive cysteines in human Keap1, and the different methods used have generated very different results. In particular, our group has found C151 of human Keap1 to be highly reactive, while others have not identified this cysteine as being even weakly reactive. Nevertheless, C151 is the only cysteine of Keap1 shown thus far in the cell environment to be required to sense chemopreventive agents. In this work, we show that the BIA-modified C151 tryptic peptide is reproducibly detected by our method. We also investigated the key differences in the methods that have been used to prepare the protein for modification by BIA. Removal of the reducing agent from Keap1 before the addition of BIA did not significantly change the modification pattern of Keap1. However, treatment of Keap1 using an ultracentrifugation device in one method resulted in approximately 99% of the protein remaining bound to the device at the time of BIA addition. In addition, the resulting pattern of cysteines identified as modified by BIA differed significantly from that obtained by our method. Notably, C151 was no longer detected as modified by BIA. We therefore recommend our method of Keap1 protein preparation for the detection of modified cysteines in proteomic studies.
authors.affiliation: The Center for Pharmaceutical Biotechnology, and Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago 60607, USA.
authors.forename: Aimee L
authors.initials: AL
authors.lastname: Eggler
authors.forename: Yan
authors.initials: Y
authors.lastname: Luo
authors.forename: Richard B
authors.initials: RB
authors.lastname: van Breemen
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Mesecar
mesh_terms: D000975:Antioxidants / Q000378:metabolism; D019921:Biotinylation; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D007460:Iodoacetamide / Q000737:chemistry / Q000494:pharmacology*; D000072019:Kelch-Like ECH-Associated Protein 1; D051267:NF-E2-Related Factor 2 / Q000235:genetics / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D020011:Protective Agents / Q000737:chemistry / Q000494:pharmacology*; D040901:Proteomics / Q000379:methods*; D020218:Response Elements / Q000235:genetics; D053719:Tandem Mass Spectrometry
chemical_list: D000975:Antioxidants; D047908:Intracellular Signaling Peptides and Proteins; C431364:KEAP1 protein, human; D000072019:Kelch-Like ECH-Associated Protein 1; D051267:NF-E2-Related Factor 2; D010455:Peptides; D020011:Protective Agents; D003545:Cysteine; D007460:Iodoacetamide