title: DNA-protein cross-linking by 1,2,3,4-diepoxybutane.
abstract: 1,2,3,4-diepoxybutane (DEB) is a strongly genotoxic diepoxide hypothesized to be the ultimate carcinogenic metabolite of the common industrial chemical and environmental carcinogen 1,3-butadiene. DEB is a bis-electrophile capable of cross-linking cellular biomolecules to form DNA-DNA and DNA-protein cross-links (DPCs), which are thought to play a central role in its biological activity. Previous studies with recombinant proteins have shown that the biological outcomes of DEB-induced DPCs are strongly influenced by protein identities. The present work combines affinity capture methodology with mass spectrometry-based proteomics and immunological detection to identify the proteins that form DPCs in nuclear extracts from human cervical carcinoma (HeLa) cells. We identified 39 human proteins that form covalent DPCs in the presence of DEB. DNA-protein cross-linking efficiency following treatment with 25 mM DEB was 2-12%, depending on protein identity. High-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI+-MS/MS) analysis of the total proteolytic digests of cross-linked proteins revealed the presence of 1-(S-cysteinyl)-4-(guan-7-yl)-2,3-butanediol conjugates, suggesting that DEB forms DPCs between cysteine thiols within proteins and the N-7 guanine positions within DNA.
authors.affiliation: Department of Medicinal Chemistry and the Masonic Cancer Center, University of Minnesota, Minneapolis, Minnesota 55455, USA.
authors.forename: Erin D
authors.initials: ED
authors.lastname: Michaelson-Richie
authors.forename: Rachel L
authors.initials: RL
authors.lastname: Loeber
authors.forename: Simona G
authors.initials: SG
authors.lastname: Codreanu
authors.forename: Xun
authors.initials: X
authors.lastname: Ming
authors.forename: Daniel C
authors.initials: DC
authors.lastname: Liebler
authors.forename: Colin
authors.initials: C
authors.lastname: Campbell
authors.forename: Natalia Y
authors.initials: NY
authors.lastname: Tretyakova
mesh_terms: D000595:Amino Acid Sequence; D015153:Blotting, Western; D002467:Cell Nucleus / Q000737:chemistry; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents / Q000737:chemistry*; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004852:Epoxy Compounds / Q000737:chemistry*; D006367:HeLa Cells; D006801:Humans; D009687:Nuclear Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D040901:Proteomics / Q000379:methods; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry; D014357:Trypsin / Q000378:metabolism
chemical_list: D003432:Cross-Linking Reagents; D004852:Epoxy Compounds; D009687:Nuclear Proteins; D010446:Peptide Fragments; D011506:Proteins; C007366:diepoxybutane; D004247:DNA; D014357:Trypsin