title: Oxidation of p53 through DNA charge transport involves a network of disulfides within the DNA-binding domain.
abstract: Transcription factor p53 plays a critical role in the cellular response to stress stimuli. We have seen that p53 dissociates selectively from various promoter sites as a result of oxidation at long-range through DNA-mediated charge transport (CT). Here, we examine this chemical oxidation and determine the residues in p53 that are essential for oxidative dissociation, focusing on the network of cysteine residues adjacent to the DNA-binding site. Of the eight mutants studied, only the C275S mutation shows decreased affinity for the Gadd45 promoter site. However, both mutations C275S and C277S result in substantial attenuation of oxidative dissociation, with C275S causing the most severe attenuation. Differential thiol labeling was used to determine the oxidation states of cysteine residues within p53 after DNA-mediated oxidation. Reduced cysteines were iodoacetamide-labeled, whereas oxidized cysteines participating in disulfide bonds were (13)C2D2-iodoacetamide-labeled. Intensities of respective iodoacetamide-modified peptide fragments were analyzed by mass spectrometry. A distinct shift in peptide labeling toward (13)C2D2-iodoacetamide-labeled cysteines is observed in oxidized samples, confirming that chemical oxidation of p53 occurs at long range. All observable cysteine residues trend toward the heavy label under conditions of DNA CT, indicating the formation of multiple disulfide bonds among the cysteine network. On the basis of these data, it is proposed that disulfide formation involving C275 is critical for inducing oxidative dissociation of p53 from DNA.
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology , Pasadena, California 91125, United States.
authors.forename: Kathryn N
authors.initials: KN
authors.lastname: Schaefer
authors.forename: Wendy M
authors.initials: WM
authors.lastname: Geil
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Sweredoski
authors.forename: Annie
authors.initials: A
authors.lastname: Moradian
authors.forename: Sonja
authors.initials: S
authors.lastname: Hess
authors.forename: Jacqueline K
authors.initials: JK
authors.lastname: Barton
mesh_terms: D003545:Cysteine / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D004220:Disulfides / Q000378:metabolism*; D004583:Electrons; D024202:Electrophoretic Mobility Shift Assay; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics; D007700:Kinetics; D013058:Mass Spectrometry; D050505:Mutant Proteins / Q000378:metabolism; D010084:Oxidation-Reduction; D011401:Promoter Regions, Genetic; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship; D013438:Sulfhydryl Compounds / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry* / Q000378:metabolism*; D000097642:GADD45 Proteins
chemical_list: D003545:Cysteine; D004247:DNA; D004220:Disulfides; D047908:Intracellular Signaling Peptides and Proteins; D050505:Mutant Proteins; D013438:Sulfhydryl Compounds; D016159:Tumor Suppressor Protein p53; D000097642:GADD45 Proteins