Identification and Characterization of Quinone-Thioether Protein Adducts In Vivo

Persistent Link:
http://hdl.handle.net/10150/193748
Title:
Identification and Characterization of Quinone-Thioether Protein Adducts In Vivo
Author:
Labenski, Matthew Thomas
Issue Date:
2008
Publisher:
The University of Arizona.
Rights:
Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
Abstract:
Quinones represent an important class of endogenous compounds such as neurotransmitters and coenzyme Q10, electrophilic xenobiotics and environmental toxicants that have known reactivity based on their ability to redox cycle and generate oxidative stress, as well as to alkylate target proteins. 1,4-Benzoquinone (BQ) is a reactive quinone that we have used to help predict target residue covalent binding by such compounds. Hydroquinone glutathione conjugates (HQ-GSH) cause renal cell necrosis by producing reactive oxygen species (ROS) and by adducting proteins preferentially localized in the S3 segment of the renal proximal tubules. In vitro experimentation using model peptides and proteins have identified cysteine, lysine, arginine, and glutamic acid as amino acids targeted for quinone-thioether adduction. By mimicking a standard protein digestion protocol (100 mM ammonium bicarbonate pH 7.5, or 50 mM Tris-HCl pH 7.5), we demonstrated that cysteine-BQ adducts are unstable. Taken together, these results indicate that BQ-adduct formation on cysteine residues may be a transient interaction, where physiological conditions may play a role in adduct stability. In vivo experimentation following administration of 2-(glutathion-S-yl)HQ (MGHQ, 400 μmol/kg, iv, 2 hr) to Long Evans rats identified the specific site of quinone-thioether protein adduction on a number of proteins. Urinary proteins were isolated, and either trypsin digested en masse and analyzed by multi-dimensional protein identification technology (MuDPIT) or, following SDS-PAGE, single immunopositive bands were excised, trypsin digested and analysed by LC-MSMS. Following site-specific identification of adducts, 3-dimensional protein modeling of adducts on the protein was performed as a way to reveal the potential structural consequence of the modification on 3D structure. The outer stripe of the outer medulla (OSOM) is the target site of protein adduction caused by quinone-thioethers. Using a 2DGE-Western blot approach, in combination with an extensive knowledge of quinol-thioether chemistry, LC-MSMS, and the latest MSMS analysis software, we identified the specific amino acid site of adduction on 17 unique peptides from 34 target proteins within the OSOM. Of the 22 bands analyzed, adducted peptides were identified in 11 of them. Many of the target proteins identified have previously been identified as a target of other electrophiles, producing additional evidence that such protein adduction is selective rather than random. The site-specific identification of covalently adducted proteins is a prerequisite for understanding the biological significance of chemical-induced PTMs and the subsequent toxicological response.
Type:
text; Electronic Dissertation
Keywords:
nephrotoxicity; protein adduction; Quinone-thioether; reactive oxygen species
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Pharmacology & Toxicology; Graduate College
Degree Grantor:
University of Arizona
Advisor:
Lau, Serrine S
Committee Chair:
Lau, Serrine S

Full metadata record

DC FieldValue Language
dc.language.isoENen_US
dc.titleIdentification and Characterization of Quinone-Thioether Protein Adducts In Vivoen_US
dc.creatorLabenski, Matthew Thomasen_US
dc.contributor.authorLabenski, Matthew Thomasen_US
dc.date.issued2008en_US
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en_US
dc.description.abstractQuinones represent an important class of endogenous compounds such as neurotransmitters and coenzyme Q10, electrophilic xenobiotics and environmental toxicants that have known reactivity based on their ability to redox cycle and generate oxidative stress, as well as to alkylate target proteins. 1,4-Benzoquinone (BQ) is a reactive quinone that we have used to help predict target residue covalent binding by such compounds. Hydroquinone glutathione conjugates (HQ-GSH) cause renal cell necrosis by producing reactive oxygen species (ROS) and by adducting proteins preferentially localized in the S3 segment of the renal proximal tubules. In vitro experimentation using model peptides and proteins have identified cysteine, lysine, arginine, and glutamic acid as amino acids targeted for quinone-thioether adduction. By mimicking a standard protein digestion protocol (100 mM ammonium bicarbonate pH 7.5, or 50 mM Tris-HCl pH 7.5), we demonstrated that cysteine-BQ adducts are unstable. Taken together, these results indicate that BQ-adduct formation on cysteine residues may be a transient interaction, where physiological conditions may play a role in adduct stability. In vivo experimentation following administration of 2-(glutathion-S-yl)HQ (MGHQ, 400 μmol/kg, iv, 2 hr) to Long Evans rats identified the specific site of quinone-thioether protein adduction on a number of proteins. Urinary proteins were isolated, and either trypsin digested en masse and analyzed by multi-dimensional protein identification technology (MuDPIT) or, following SDS-PAGE, single immunopositive bands were excised, trypsin digested and analysed by LC-MSMS. Following site-specific identification of adducts, 3-dimensional protein modeling of adducts on the protein was performed as a way to reveal the potential structural consequence of the modification on 3D structure. The outer stripe of the outer medulla (OSOM) is the target site of protein adduction caused by quinone-thioethers. Using a 2DGE-Western blot approach, in combination with an extensive knowledge of quinol-thioether chemistry, LC-MSMS, and the latest MSMS analysis software, we identified the specific amino acid site of adduction on 17 unique peptides from 34 target proteins within the OSOM. Of the 22 bands analyzed, adducted peptides were identified in 11 of them. Many of the target proteins identified have previously been identified as a target of other electrophiles, producing additional evidence that such protein adduction is selective rather than random. The site-specific identification of covalently adducted proteins is a prerequisite for understanding the biological significance of chemical-induced PTMs and the subsequent toxicological response.en_US
dc.typetexten_US
dc.typeElectronic Dissertationen_US
dc.subjectnephrotoxicityen_US
dc.subjectprotein adductionen_US
dc.subjectQuinone-thioetheren_US
dc.subjectreactive oxygen speciesen_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplinePharmacology & Toxicologyen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.advisorLau, Serrine Sen_US
dc.contributor.chairLau, Serrine Sen_US
dc.contributor.committeememberMonks, Terrence Jen_US
dc.contributor.committeememberTsaprailis, Georgeen_US
dc.contributor.committeememberWondrak, Georgen_US
dc.contributor.committeememberChen, Qinen_US
dc.identifier.proquest10048en_US
dc.identifier.oclc659750469en_US
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