Palmitoylation and Glutathionylation of Key Sarcolemmal Signalling Proteins in Cardiac Muscle

  • Fiona B. Ashford

    Student thesis: Doctoral ThesisDoctor of Philosophy

    Abstract

    Cysteine residues are susceptible to a variety of different post-translational modifications (PTMs), such as the addition of a 16-carbon fatty acid palmitate known as palmitoylation and the addition of the tripeptide glutathione, known as glutathionylation. Palmitoylation can regulate many processes such as intracellular membrane targeting and membrane interactions. Glutathione acts as a redox buffer and antioxidant to prevent proteins from irreversible oxidative damage. Protein glutathionylation is a critical signalling event in cardiovascular disease due to its ability to regulate many physiological processes involved in cardiac homeostasis. However, only a handful of these proteins have been identified, therefore using isolated Adult Rat Ventricular Myocytes (ARVM) under physiological conditions, glutathionylated proteins were identified using LC-MS/MS and cross referenced with the palmitoylproteome of ARVM. This identified 129 proteins that are both palmitoylated and glutathionylated in ARVM.

    Novel glutathionylated proteins identified under physiological conditions include, signalling proteins G alpha s (Gas) and G alpha i (Gai) and the scaffolding protein, Caveolin 3 (Cav3). Cav3 acts as a negative regulator binding many proteins eg. Ga proteins, in the caveolae to maintain efficient signalling within the cardiomyocyte. Interaction and co localisation of Cav3 and G protein a subunits is disrupted following an diamide induced increase in glutathionylation. However, sucrose gradient fractionation reveals no gross change in localisation within the caveolae, suggesting caveolae are still intact. Ischemia Reperfusion (IR) decreases palmitoylation and increases glutathionylation of Cav3 in whole heart, which may influence caveolae structure and Ga protein co-localisation with Cav3.

    Mutation of Cysteine 19 to an Alanine (C19A) in transiently transfected HEK cells, identified a decrease in glutathionylation, possibly through a structural change. This is partially restored with Cysteine 19 and 72 mutated to alanine (C19,72A). Suggesting glutathionylation causes a structural change in Cav3, which may be responsible for the dissociation of Ga subunits. This work demonstrates that there is the potential for glutathionylation to remodel signalling microdomains at the level of recruitment (such as Cav3) and the activity of individual constituents, such as G-protein alpha s and i subunits.
    Date of Award2017
    Original languageEnglish
    SponsorsBritish Heart Foundation
    SupervisorWill Fuller (Supervisor) & Colin Henderson (Supervisor)

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