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Glutathione Transferase P1 is Modified by Palmitate Open Access

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Other title
Subject/Keyword
Glutathione Transferase P1
Fatty acylation
Palmitoylation
Type of item
Thesis
Degree grantor
University of Alberta
Author or creator
Marensi, Vanessa
Supervisor and department
Leslie, Elaine (Physiology and Laboratory Medicine and Pathology)
Examining committee member and department
Collier, Abby (Pharmaceutical Science)
Lemieux, Joanne (Biochemistry)
Casey, Joseph (Biochemistry)
Lehner, Richard (Cell Biology and Pediatrics)
Department
Department of Physiology
Specialization

Date accepted
2017-04-26T10:27:23Z
Graduation date
2017-11:Fall 2017
Degree
Doctor of Philosophy
Degree level
Doctoral
Abstract
The tripeptide glutathione (GSH, γ-Glu-Cys-Gly) is the most abundant non-protein thiol in mammalian cells. The thiol group within the Cys residue forms a nucleophilic center and neutralizes electronegative charges conferring antioxidant activity to GSH. The addition of GSH to electrophiles can be spontaneous but the majority require catalysis from glutathione transferases (GSTs). GSTP1 is mostly known for its role in detoxification. In addition, GSTP1 has been recognized to have non-catalytic activities regulating cell proliferation, apoptosis, and signalling. GSTP1 is involved in a wide range of diseases, suggesting it has multiple functions. Regulated subcellular localization is important for protein function, especially when a protein is involved in multiple cellular processes. GSTP1 has been shown previously to strongly associate with the plasma membrane. Protein palmitoylation increases hydrophobicity and affinity for cell membranes. Palmitate forms a covalent thioester bond with Cys residues in proteins. Palmitoyl transferases (PATs) catalyze the addition of palmitate to protein, and this occurs most commonly at Cys residues in a basic environment (e.g., Cys residues flanked by polybasic amino acids). In this thesis the mechanism by which GSTP1 associates with the plasma membrane was investigated. GSTP1 was identified as a palmitoylated protein. Subcellular localization of GSTP1 was also characterized. Investigation of lipidated proteins can be quite challenging, we mostly utilized bioorthogonal labelling and click chemistry to identify palmitoylated GSTP1 and screen for palmitoylation sites in MCF7 and analyzed by western blotting and immunohistochemistry, the latter was applied in conjunction with proximity ligation assay. Surprisingly, GSTP1 is a palmitoylated protein that is palmitoylated on at least one non-Cys residue and the palmitoylation is resistant to NaOH treatment. Moreover, incorporation of the fatty acid by GSTP1 requires long metabolic labelling with palmitate, suggesting that modification is not as dynamic as classic palmitoylation of proteins such as, non-receptor tyrosine kinase lck, the Ras proteins and the postsynaptic density protein PSD-95. In addition, we characterized palmitoyl acyl transferase (PAT)-independent palmitoylation of human GSTP1 (purified from E. coli) in vitro and identified Cys48, Cys102, and Lys103 as PAT-independent palmitoylation sites using liquid chromatography tandem mass spectrometry (LC-MS/MS). To further characterize the association of GSTP1 with cellular membranes, subcellular fractions of MCF7-GSTP1 cells were analysed. Palmitoylated GSTP1 is present not only in the membrane but also in the cytosol. Moreover, the GSTP1 in the membrane does not conjugate 1-chloro-2,4-dinitrobenzene (CDNB) with GSH. GSTP1 isolated from E. coli, and MCF7 cells grown under fatty acid free (FAF) or fetal bovine serum (FBS) conditions were palmitoylated in vitro and incubated with plasma membrane-enriched vesicles. GSTP1 associated with cellular membranes without in vitro palmitoylation, suggesting that another mechanism might be important for this association. Investigation of the role of membrane association and lipidation of GSTP1 provides new insight into the cell biology of this multifunctional protein.
Language
English
DOI
doi:10.7939/R36H4D43M
Rights
This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for the purpose of private, scholarly or scientific research. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.
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