Harnessing the Newly Discovered Antioxidant Capabilities of the Moonlighting CLIC Proteins-Investigating both their intra and extracellular actions

Turkewitz, Daniel Ryan

Harnessing the Newly Discovered Antioxidant Capabilities of the Moonlighting CLIC Proteins-Investigating both their intra and extracellular actions

Turkewitz, Daniel Ryan

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Publication Type:: Thesis
Issue Date:: 2020

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Field	Value	Language
dc.contributor.author	Turkewitz, Daniel Ryan
dc.date.accessioned	2020-11-17T23:33:18Z
dc.date.available	2020-11-17T23:33:18Z
dc.date.issued	2020
dc.identifier.uri	http://hdl.handle.net/10453/144103
dc.description	University of Technology Sydney. Faculty of Science.	en_AU
dc.description.abstract	Chloride Intracellular Ion Channel (CLIC) proteins are a unique set of ion channels that can exist as both soluble and integral membrane proteins, with CLIC1 also capable of shifting between two distinct conformations. New evidence has emerged to suggest members of the CLIC family act as moonlighting proteins, referring to the ability of a single protein to carry out multiple functions. It has now been demonstrated that CLIC family members have additional oxidoreductase enzymatic activity, and thus within biological systems, these CLICs may function as either membrane spanning ion channels and/or display enzymatic oxidoreductase activities. This is an exciting period in the study of these proteins, with the hope of revealing their seemingly broad cellular roles, in order to then exploit these activities for health and therapeutic benefits. The research undertaken and presented in this thesis covers Molecular Docking Simulations that support empirical studies of the regulation of CLIC protein membrane interactions by cholesterol, as well as other sterols, via a GXXXG binding motif. The research also examined the effects of a 6 x Histidine amino acid tag at the amino terminus of the CLIC proteins on their enzymatic activity, as well as, determining the optimal pH and temperature ranges for peak activity. The main study, however, focused on exploring CLIC’s role as putative antioxidant protective proteins in skin. This was done in collaboration with our industry partner BOD Australia Pty Ltd. The study used skin fibroblast cells as a model cell system. Our findings demonstrate for the first time that overexpressing CLIC1 or CLIC4 in NIH/3T3 fibroblast cells resulted in higher levels of glutaredoxin-like oxidoreductase activity, a significant decrease in levels of ROS and increased viability when challenged with either hydrogen peroxide titrations or UVC light exposure. We also demonstrate that the addition of recombinant CLIC1 or CLIC4 to primary skin fibroblasts in culture, provided cellular protective effects when administered before challenging the cells with hydrogen peroxide. Furthermore, it was shown that recombinant CLIC1 and CLIC4 proteins remain enzymatically active when stored at either room temperature or +4℃ for seven days. Overall, this thesis provides both proof of concept as well as potential for further experiments such as utilising additional CLIC proteins and CLIC combinations to provide cellular protection against UV damage and against other external stressors.	en_AU
dc.format	Thesis (PhD)
dc.language.iso	en_US	en_US
dc.rights	info:eu-repo/semantics/embargoedAccess
dc.rights	The author owns the copyright in this thesis including all reproduction and reuse rights for the work. The work may not be altered without the permission of the copyright owner. Attribution is essential when quoting or paraphrasing from this thesis.
dc.rights	info:eu-repo/semantics/embargoedAccess
dc.title	Harnessing the Newly Discovered Antioxidant Capabilities of the Moonlighting CLIC Proteins-Investigating both their intra and extracellular actions	en_AU
dc.type	Thesis
utslib.copyright.status	embargoed	*

Abstract:

Chloride Intracellular Ion Channel (CLIC) proteins are a unique set of ion channels that can exist as both soluble and integral membrane proteins, with CLIC1 also capable of shifting between two distinct conformations. New evidence has emerged to suggest members of the CLIC family act as moonlighting proteins, referring to the ability of a single protein to carry out multiple functions. It has now been demonstrated that CLIC family members have additional oxidoreductase enzymatic activity, and thus within biological systems, these CLICs may function as either membrane spanning ion channels and/or display enzymatic oxidoreductase activities. This is an exciting period in the study of these proteins, with the hope of revealing their seemingly broad cellular roles, in order to then exploit these activities for health and therapeutic benefits. The research undertaken and presented in this thesis covers Molecular Docking Simulations that support empirical studies of the regulation of CLIC protein membrane interactions by cholesterol, as well as other sterols, via a GXXXG binding motif. The research also examined the effects of a 6 x Histidine amino acid tag at the amino terminus of the CLIC proteins on their enzymatic activity, as well as, determining the optimal pH and temperature ranges for peak activity. The main study, however, focused on exploring CLIC’s role as putative antioxidant protective proteins in skin. This was done in collaboration with our industry partner BOD Australia Pty Ltd. The study used skin fibroblast cells as a model cell system. Our findings demonstrate for the first time that overexpressing CLIC1 or CLIC4 in NIH/3T3 fibroblast cells resulted in higher levels of glutaredoxin-like oxidoreductase activity, a significant decrease in levels of ROS and increased viability when challenged with either hydrogen peroxide titrations or UVC light exposure. We also demonstrate that the addition of recombinant CLIC1 or CLIC4 to primary skin fibroblasts in culture, provided cellular protective effects when administered before challenging the cells with hydrogen peroxide. Furthermore, it was shown that recombinant CLIC1 and CLIC4 proteins remain enzymatically active when stored at either room temperature or +4℃ for seven days. Overall, this thesis provides both proof of concept as well as potential for further experiments such as utilising additional CLIC proteins and CLIC combinations to provide cellular protection against UV damage and against other external stressors.

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http://hdl.handle.net/10453/144103