Thesis
Analysis of protein palmitoylation in adipocytes
- Creator
- Rights statement
- Awarding institution
- University of Strathclyde
- Date of award
- 2014
- Thesis identifier
- T13978
- Qualification Level
- Qualification Name
- Department, School or Faculty
- Abstract
- Blood glucose homeostasis is highly regulated and is essential for survival. A key process is the insulin-stimulated recruitment of the facilitative glucose transporter GLUT4 to the plasma membrane in adipocytes and muscle cells; defects in this pathway can cause insulin resistance and type 2 diabetes. The insulin signalling and GLUT4 trafficking pathways in these cells have been extensively characterised, and a prominent role for protein phosphorylation has been uncovered. In contrast, relatively little is known about the role of other post-translational modifications (PTMs) in these pathways. The aim of this study was to expand existing knowledge of how palmitoylation, a PTM involving reversible attachment of fatty acids onto cysteine residues, affects components of the insulin signalling and GLUT4 trafficking pathways. For this, palmitoylated proteins were isolated from 3T3-L1 adipocytes by resin-assisted capture of S-acylated proteins (acyl-RAC), and screened to identify novel palmitoylated components of the insulin signalling and GLUT4 trafficking pathways. This approach successfully identified the following novel palmitoylated proteins: GLUT4, insulin-responsive aminopeptidase (IRAP) and caveolin-2. Furthermore, click-chemistry confirmed palmitoylation of caveolin-2 and IRAP and also enabled identification of the palmitoylation sites on these proteins. Palmitoylation has been shown to regulate proteins in many different ways, in particular by modulating protein trafficking, protein stability and protein-protein interactions. Mutation of the palmitoylation sites in caveolin-2 and IRAP had no obvious effect on protein localisation. However, palmitoylation-deficient mutants of caveolin-2 exhibited: (i) a deficit in conversion of monomeric caveolin-2 into low molecular weight oligomeric complexes, and (ii) decreased oligomer stability, revealed by a loss of SDS-resistant caveolin-2 complexes. Overall, this work has identified novel palmitoylated proteins in 3T3-L1 adipocytes, mapped the palmitoylation sites of these proteins, and determined the effect of this PTM on the localisation and oligomeric status of these proteins. These findings have thus expanded existing knowledge on the potential regulation of insulin signalling and GLUT4 trafficking pathways by PTMs.
- Resource Type
- DOI
- Date Created
- 2014
- Former identifier
- 1218772
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