Investigating the oxidoreductase activity of members of the chloride intracellular ion channel protein family

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The chloride intracellular ion channel (CLICs) proteins are atypical anion selective channel proteins, as they are principally soluble proteins, with some members now known to also demonstrate enzymatic activity. Structural studies demonstrate that the CLIC proteins share strong structural homology with members of the glutathione-S-transferase superfamily of enzymes, in particular the omega glutathione-S-transferase (GST-Ω) members. The discovery that the CLIC proteins have the functional ability to act as glutathione dependent oxidoreductase enzymes, also suggested a role for them as cell protective proteins and antioxidants. Therefore, this PhD project aimed to further define the functional activity of the CLIC proteins, in particular CLIC3 as one of more recently identified members. The principal findings of this PhD project include demonstrating for the first time that CLIC3 has glutathione dependant oxidoreductase activity via its dithiol active enzymatic site. In this study, we directly contributed to the finding that the extracellular activity of transglutaminase-2 (TGM2) is regulated by CLIC3’s oxidoreductase activity, and their interaction was dependent upon the redox environment. Furthermore, our 𝘪𝘯 𝘷𝘪𝘵𝘳𝘰 studies of CLIC3, were key in helping demonstrate a critical role for secreted CLIC3 in cancer metastasis and tumour cell invasiveness. To further investigate this oxidoreductase activity of the CLIC proteins we used a bacterial cell model to probe their ability to protect cells against oxidative assault. Recombinant CLIC proteins were expressed in bacterial 𝘌. 𝘤𝘰𝘭𝘪 cells, followed by their exposure to the oxidising agent hydrogen peroxide (H2O2). Expression of CLIC1 by the 𝘌. 𝘤𝘰𝘭𝘪 cells was found to provide increased tolerance of up to 5mM H2O2, while CLIC3 afforded some protection, with no difference seen for cells expressing CLIC4. This work for the first time demonstrates CLIC1 protein acting in antioxidant cellular protection. The final part of this PhD project pursued the study of the CLIC proteins’ glutathionylation activity. Given their close resemblance to the GST-omega-1 proteins, we hypothesised that members of the CLIC family would like them, be capable of deglutathionylation activity. Using a synthetic peptide substrate, our 𝘪𝘯 𝘷𝘪𝘵𝘳𝘰 studies demonstrate members of the CLIC family have significant deglutathionylation activity supporting a major role for them in the cellular gluathionylation cycle. In conclusion, our results provided new insights into the functions of the CLIC proteins as soluble enzymes, with functions in cellular antioxidant protective mechanisms. Most importantly, it points to their role as post-translational regulators of target proteins via their deglutathionylation activity.
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