title: The frequency of 1,4-benzoquinone-lysine adducts in cytochrome c correlate with defects in apoptosome activation.
abstract: Electrophile-mediated post-translational modifications (PTMs) are known to cause tissue toxicities and disease progression. These effects are mediated via site-specific modifications and structural disruptions associated with such modifications. 1,4-Benzoquinone (BQ) and its quinone-thioether metabolites are electrophiles that elicit their toxicity via protein arylation and the generation of reactive oxygen species. Site-specific BQ-lysine adducts are found on residues in cytochrome c that are necessary for protein-protein interactions, and these adducts contribute to interferences in its ability to facilitate apoptosome formation. To further characterize the structural and functional impact of these BQ-mediated PTMs, the original mixture of BQ-adducted cytochrome c was fractionated by liquid isoelectric focusing to provide various fractions of BQ-adducted cytochrome c species devoid of the native protein. The fractionation process separates samples based on their isoelectric point (pI), and because BQ adducts form predominantly on lysine residues, increased numbers of BQ adducts on cytochrome c correlate with a lower protein pI. Each fraction was analyzed for structural changes, and each was also assayed for the ability to support apoptosome-mediated activation of caspase-3. Circular dichroism revealed that several of the BQ-adducted cytochrome c species maintained a slightly more rigid structure in comparison to native cytochrome c. BQ-adducted cytochrome c also failed to activate caspase-3, with increasing numbers of BQ-lysine adducts corresponding to a greater inability to activate the apoptosome. In summary, the specific site of the BQ-lysine adducts, and the nature of the adduct, are important determinants of the subsequent structural changes to cytochrome c. In particular, adducts at sites necessary for protein-protein interactions interfere with the proapoptotic function of cytochrome c.
authors.affiliation: Department of Pharmacology and Toxicology, Southwest Environmental Health Sciences Center, College of Pharmacy, The University of Arizona, Tucson, Arizona 85721, USA.
authors.forename: Ashley A
authors.initials: AA
authors.lastname: Fisher
authors.forename: Matthew T
authors.initials: MT
authors.lastname: Labenski
authors.forename: Srinivas
authors.initials: S
authors.lastname: Malladi
authors.forename: John D
authors.initials: JD
authors.lastname: Chapman
authors.forename: Shawn B
authors.initials: SB
authors.lastname: Bratton
authors.forename: Terrence J
authors.initials: TJ
authors.lastname: Monks
authors.forename: Serrine S
authors.initials: SS
authors.lastname: Lau
mesh_terms: D000818:Animals; D053478:Apoptosomes / Q000187:drug effects* / Q000378:metabolism*; D016227:Benzoquinones / Q000737:chemistry / Q000633:toxicity*; D053148:Caspase 3 / Q000378:metabolism; D002853:Chromatography, Liquid; D002942:Circular Dichroism / Q000379:methods; D045304:Cytochromes c / Q000737:chemistry*; D018736:DNA Adducts; D006736:Horses; D007525:Isoelectric Focusing / Q000379:methods; D008239:Lysine / Q000378:metabolism*; D008958:Models, Molecular; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D020836:Protein Structure, Quaternary; D053719:Tandem Mass Spectrometry
chemical_list: D053478:Apoptosomes; D016227:Benzoquinones; D018736:DNA Adducts; D045304:Cytochromes c; D053148:Caspase 3; D008239:Lysine