Chemical-Induced Post-Translational Modifications and the Consequent Structural and Functional Alterations

Persistent Link:
http://hdl.handle.net/10150/195794
Title:
Chemical-Induced Post-Translational Modifications and the Consequent Structural and Functional Alterations
Author:
Fisher, Ashley Anne
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:
Chemical-induced post-translational modifications (PTMs) can alter the structure of proteins, with consequences that may alter protein function, including interference with protein-protein interactions, subcellular protein compartmentalization, and disruption of cellular signaling pathways. To identify the impact of PTMs on the structure and function of protein targets in vitro and in vivo, electrophiles with known toxicity were utilized. Hydroquinone, and its thioether metabolites, cause renal proximal tubular cell necrosis and nephrocarcinogenicity in rats. The adverse effects of these chemicals are in part a result of their oxidation to 1,4-benzoquinones (BQ). Cytochrome c and caspase-7 have been studied as model proteins to identify site-specific adductions and the resulting structural and functional consequences associated with apoptosis. BQ and 2-(N-acetylcystein-S-yl)benzoquinone (NAC-BQ) preferentially bind to solvent-exposed lysine-rich regions within cytochrome c, and specific glutamic acid residues within cytochrome c are novel sites of NAC-BQ adduction. Furthermore, the microenvironment at the site of adduction governs both the initial specificity and the structure of the final adduct. Solvent accessibility and local pKa of the adducted and neighboring amino acids contribute to the selectivity of adduction. Post-adduction chemistry subsequently alters the nature of the final adduct. BQ induced PTMs in cytochrome c produce changes in the structure sufficient to inhibit its ability to initiate caspase-3 activation in native lysates, and its ability to promote Apaf-1 oligomerization into an apoptosome complex, in a purely reconstituted system.Quinone-thioether-protein adduct stability is also dependent upon physiological conditions. Adduct formation on cysteine residues under physiological conditions may be transient, whilst remaining capable of impacting cell signaling events, and of thus contributing to the toxic response elicited by these compounds. Indeed, in vitro analysis of caspase-7 revealed that cysteine residues within the protein are transiently modified with BQ, including the active site thiolate anion. In vitro and in vivo analysis of quinone-thioether adduction on caspase proteins also provided evidence that these catalytic proteins may be in vivo quinone-thioether targets, and could contribute to a mechanistic understanding of the necrotic mode of cell death initiated by quinone-thioether exposure. In summary, mass spectroscopic, molecular modeling, and biochemical approaches collectively confirm that electrophile-protein adducts produce structural changes that influence biological function. Identification of such chemical-induced PTMs on target proteins can provide critical mechanistic understanding of their role in response to environmental chemicals and the associated disease progression. Furthermore, because quinones are a well-known class of electrophilic species and the quinone moiety exits in a number of chemotherapeutic agents, identification of these PTMs will provide insight into the field of drug development and the role electrophilic drug metabolite-PTMs may play in unwanted drug-induced toxicities.
Type:
text; Electronic Dissertation
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Pharmaceutical Sciences; 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.titleChemical-Induced Post-Translational Modifications and the Consequent Structural and Functional Alterationsen_US
dc.creatorFisher, Ashley Anneen_US
dc.contributor.authorFisher, Ashley Anneen_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.abstractChemical-induced post-translational modifications (PTMs) can alter the structure of proteins, with consequences that may alter protein function, including interference with protein-protein interactions, subcellular protein compartmentalization, and disruption of cellular signaling pathways. To identify the impact of PTMs on the structure and function of protein targets in vitro and in vivo, electrophiles with known toxicity were utilized. Hydroquinone, and its thioether metabolites, cause renal proximal tubular cell necrosis and nephrocarcinogenicity in rats. The adverse effects of these chemicals are in part a result of their oxidation to 1,4-benzoquinones (BQ). Cytochrome c and caspase-7 have been studied as model proteins to identify site-specific adductions and the resulting structural and functional consequences associated with apoptosis. BQ and 2-(N-acetylcystein-S-yl)benzoquinone (NAC-BQ) preferentially bind to solvent-exposed lysine-rich regions within cytochrome c, and specific glutamic acid residues within cytochrome c are novel sites of NAC-BQ adduction. Furthermore, the microenvironment at the site of adduction governs both the initial specificity and the structure of the final adduct. Solvent accessibility and local pKa of the adducted and neighboring amino acids contribute to the selectivity of adduction. Post-adduction chemistry subsequently alters the nature of the final adduct. BQ induced PTMs in cytochrome c produce changes in the structure sufficient to inhibit its ability to initiate caspase-3 activation in native lysates, and its ability to promote Apaf-1 oligomerization into an apoptosome complex, in a purely reconstituted system.Quinone-thioether-protein adduct stability is also dependent upon physiological conditions. Adduct formation on cysteine residues under physiological conditions may be transient, whilst remaining capable of impacting cell signaling events, and of thus contributing to the toxic response elicited by these compounds. Indeed, in vitro analysis of caspase-7 revealed that cysteine residues within the protein are transiently modified with BQ, including the active site thiolate anion. In vitro and in vivo analysis of quinone-thioether adduction on caspase proteins also provided evidence that these catalytic proteins may be in vivo quinone-thioether targets, and could contribute to a mechanistic understanding of the necrotic mode of cell death initiated by quinone-thioether exposure. In summary, mass spectroscopic, molecular modeling, and biochemical approaches collectively confirm that electrophile-protein adducts produce structural changes that influence biological function. Identification of such chemical-induced PTMs on target proteins can provide critical mechanistic understanding of their role in response to environmental chemicals and the associated disease progression. Furthermore, because quinones are a well-known class of electrophilic species and the quinone moiety exits in a number of chemotherapeutic agents, identification of these PTMs will provide insight into the field of drug development and the role electrophilic drug metabolite-PTMs may play in unwanted drug-induced toxicities.en_US
dc.typetexten_US
dc.typeElectronic Dissertationen_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplinePharmaceutical Sciencesen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.advisorLau, Serrine S.en_US
dc.contributor.chairLau, Serrine S.en_US
dc.contributor.committeememberLau, Serrineen_US
dc.contributor.committeememberMonks, Terrence J.en_US
dc.contributor.committeememberHurley, Laurenceen_US
dc.contributor.committeememberYang, Danzhouen_US
dc.contributor.committeememberOlenyuk, Bogdanen_US
dc.identifier.proquest10165en_US
dc.identifier.oclc659750749en_US
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