Cellular Study of the Human Chloride Intracellular Ion Channel Proteins: Exploring their Physiological Role and Potential as Novel Targets for Treating Disease

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Chloride Intracellular Ion Channels (CLIC) are a family of six, highly conserved enigmatic proteins. They are expressed as soluble proteins, however under specific conditions they can spontaneously insert into cell membranes, where they function as selective chloride channels. To date, their ion channel form has been the main focus of researchers’ attention. However, in vitro studies have more recently demonstrated that soluble CLICs possess oxidoreductase enzymatic activity. Furthermore, they share strong similarities to the glutaredoxin enzymes and the GST omega proteins. Armed with this information and the emerging evidence of the CLICs enzymatic activity, this project aimed to uncover the role of CLICs in human physiology, with the goal to reveal the broader role of these proteins inside cells, thus disclosing potential uses as therapeutic targets in the future. This study succeeded in demonstrating in situ an involvement of soluble CLICs in the glutathionylation/deglutathionylation pathway. The effects of environmental pH and cell-cycle stage as factors influencing such enzymatic activity were explored, while changes in temperature were shown to impact the overall enzymatic activity based on the level of cellular CLIC expression. Interestingly, when trying to identify specific targets for CLIC-mediated S-glutathionylation activity, a link to respiratory conditions arose. This study revealed for the first time a strong overexpression of CLIC4 in the asthmatic lung, suggesting participation of this protein in the disease mechanism. We also analysed the effects of altered CLIC expression on cell recovery following oxidative stress. A reduction in CLIC1 renders the cells more susceptible to such stress, while extraneously added recombinant CLIC1 provided protective effects on the cells’ stress recovery, however addition of recombinant CLIC4 had an overall detrimental effect on the cells. A second part of this study focused on CLICs’ as diagnostic markers for disease. The aim was to investigate their expression on exosomes shed from cancerous cells, and to profile conductance changes due to their presence in such vesicles, by using impedance spectroscopy and tethered membranes. The long-term aim being to develop a diagnostic screening platform targeted at a variety of cancers that overexpress CLICs. Our study showed changes in levels of CLIC4 carried within exosomes shed from ovarian cancer cells, warranting further studies. This PhD project has expanded our understanding around the nature and activity of CLICs, and the novel discoveries in this thesis support our hypothesis, that CLICs play a fundamental role in the physiology of cells, while also providing novel insights into their functional properties in human diseases.
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