title: Identification of two reactive cysteine residues in the tumor suppressor protein p53 using top-down FTICR mass spectrometry.
abstract: The tumor suppressor p53 is a redox-regulated transcription factor involved in cell cycle arrest, apoptosis and senescence in response to multiple forms of stress, as well as many other cellular processes such as DNA repair, glycolysis, autophagy, oxidative stress and differentiation. The discovery of cysteine-targeting compounds that cause re-activation of mutant p53 and the death of tumor cells in vivo has emphasized the functional importance of p53 thiols. Using a combination of top-down and middle-down FTICR mass spectrometry, we show that of the 10 Cys residues in the core domain of wild-type p53, Cys182 and Cys277 exhibit a remarkable preference for modification by the alkylating reagent N-ethylmaleimide. The assignment of Cys182 and Cys277 as the two reactive Cys residues was confirmed by site-directed mutagenesis. Further alkylation of p53 beyond Cys182 and Cys277 was found to trigger co-operative modification of the remaining seven Cys residues and protein unfolding. This study highlights the power of top-down FTICR mass spectrometry for analysis of the cysteine reactivity and redox chemistry in multiple cysteine-containing proteins.
authors.affiliation: SIRCAMS, School of Chemistry, University of Edinburgh, West Mains Road, Edinburgh EH9 3JJ, UK.
authors.forename: Jenna
authors.initials: J
authors.lastname: Scotcher
authors.forename: David J
authors.initials: DJ
authors.lastname: Clarke
authors.forename: Stefan K
authors.initials: SK
authors.lastname: Weidt
authors.forename: C Logan
authors.initials: CL
authors.lastname: Mackay
authors.forename: Ted R
authors.initials: TR
authors.lastname: Hupp
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Sadler
authors.forename: Pat R R
authors.initials: PR
authors.lastname: Langridge-Smith
mesh_terms: D000477:Alkylating Agents; D000595:Amino Acid Sequence; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry* / Q000378:metabolism; D005033:Ethylmaleimide; D005583:Fourier Analysis; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D058767:Protein Unfolding; D013696:Temperature; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry* / Q000378:metabolism
chemical_list: D000477:Alkylating Agents; D010446:Peptide Fragments; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; D003545:Cysteine; D005033:Ethylmaleimide