Analysis of Protein Adduction Kinetics and the Effects of Protein Adduction on C-Jun N-Terminal Kinase Signaling

Persistent Link:
http://hdl.handle.net/10150/194247
Title:
Analysis of Protein Adduction Kinetics and the Effects of Protein Adduction on C-Jun N-Terminal Kinase Signaling
Author:
Orton, Christopher R.
Issue Date:
2006
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:
Defining the mechanics and consequences of protein adduction is crucial to understanding the toxicity of reactive electrophiles. Application of tandem mass spectrometry and data analysis algorithms enables detection and mapping of chemical adducts at the level of amino acid sequence. Nevertheless, detection of adducts does not indicate relative reactivity of different sites. In this dissertation I describe a method to measure the kinetics of competing adduction reactions at different sites on the same protein using quantitative mass spectrometry. Adducts are formed by electrophiles at Cys-14 and Cys-47 on the metabolic enzyme glutathione-S-transferase P1-1 and accompanied by a loss of enzymatic activity. Relative quantitation of protein adducts was done by tagging N-termini of peptide digests with isotopically labeled phenyl isocyanate and tracking the ratio of light-tagged peptide adducts to heavy-tagged reference samples. This method was used to measure rate constants for adduction at both positions with two different model electrophiles, IAB and BMCC. The results indicate that Cys-47 was approximately 2-3-fold more reactive toward both electrophiles than was Cys-14. This result was consistent with the relative reactivity of these electrophiles in a complex proteome system. Quantitative analyses of protein modifications provide a means of determining the reactivity and selectivity of damaging protein modifications in chemical toxicity.Another area of study explored in this dissertation is looking at the effects of protein alkylation on activating cellular signaling pathways, specifically the JNK signaling pathway. Protein adduction has been shown to be selective between different alkylating agents. It would then be reasonable to think this selectivity of adduction translates to selectivity of downstream consequences or cellular events directly tied to specific adductions. My work will show how treatment of HEK293 cells with either IAB or BMCC leads to differences in activation of JNK signaling. In addition, I've been able to show a difference in selectivity of a number of adducted targets by each alkylating agent, which are directly involved in regulation of the JNK signaling pathway. These studies illustrate not only the significance of protein adduction, but the importance for continual research to better understand their behavior in living systems.
Type:
text; Electronic Dissertation
Keywords:
Proteomics; Protein Adduction Kinetics; Quantitative Mass Spectrometry; Glutathione S-transferase; c-Jun N-terminal Kinase Signaling
Degree Name:
PhD
Degree Level:
doctoral
Degree Program:
Pharmacology & Toxicology; Graduate College
Degree Grantor:
University of Arizona
Committee Chair:
Liebler, Daniel C.; Vaillancourt, Richard R.

Full metadata record

DC FieldValue Language
dc.language.isoENen_US
dc.titleAnalysis of Protein Adduction Kinetics and the Effects of Protein Adduction on C-Jun N-Terminal Kinase Signalingen_US
dc.creatorOrton, Christopher R.en_US
dc.contributor.authorOrton, Christopher R.en_US
dc.date.issued2006en_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.abstractDefining the mechanics and consequences of protein adduction is crucial to understanding the toxicity of reactive electrophiles. Application of tandem mass spectrometry and data analysis algorithms enables detection and mapping of chemical adducts at the level of amino acid sequence. Nevertheless, detection of adducts does not indicate relative reactivity of different sites. In this dissertation I describe a method to measure the kinetics of competing adduction reactions at different sites on the same protein using quantitative mass spectrometry. Adducts are formed by electrophiles at Cys-14 and Cys-47 on the metabolic enzyme glutathione-S-transferase P1-1 and accompanied by a loss of enzymatic activity. Relative quantitation of protein adducts was done by tagging N-termini of peptide digests with isotopically labeled phenyl isocyanate and tracking the ratio of light-tagged peptide adducts to heavy-tagged reference samples. This method was used to measure rate constants for adduction at both positions with two different model electrophiles, IAB and BMCC. The results indicate that Cys-47 was approximately 2-3-fold more reactive toward both electrophiles than was Cys-14. This result was consistent with the relative reactivity of these electrophiles in a complex proteome system. Quantitative analyses of protein modifications provide a means of determining the reactivity and selectivity of damaging protein modifications in chemical toxicity.Another area of study explored in this dissertation is looking at the effects of protein alkylation on activating cellular signaling pathways, specifically the JNK signaling pathway. Protein adduction has been shown to be selective between different alkylating agents. It would then be reasonable to think this selectivity of adduction translates to selectivity of downstream consequences or cellular events directly tied to specific adductions. My work will show how treatment of HEK293 cells with either IAB or BMCC leads to differences in activation of JNK signaling. In addition, I've been able to show a difference in selectivity of a number of adducted targets by each alkylating agent, which are directly involved in regulation of the JNK signaling pathway. These studies illustrate not only the significance of protein adduction, but the importance for continual research to better understand their behavior in living systems.en_US
dc.typetexten_US
dc.typeElectronic Dissertationen_US
dc.subjectProteomicsen_US
dc.subjectProtein Adduction Kineticsen_US
dc.subjectQuantitative Mass Spectrometryen_US
dc.subjectGlutathione S-transferaseen_US
dc.subjectc-Jun N-terminal Kinase Signalingen_US
thesis.degree.namePhDen_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplinePharmacology & Toxicologyen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.chairLiebler, Daniel C.en_US
dc.contributor.chairVaillancourt, Richard R.en_US
dc.contributor.committeememberLiebler, Daniel C.en_US
dc.contributor.committeememberVaillancourt, Richard R.en_US
dc.contributor.committeememberCherrington, Nathan J.en_US
dc.contributor.committeememberRegan, John W.en_US
dc.contributor.committeememberLau, Serrine S.en_US
dc.identifier.proquest1839en_US
dc.identifier.oclc659746364en_US
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