Biochemical characterisation of the enzymatic activity of chloride intracellular ion channel proteins

Moghaddasi, Saba

Biochemical characterisation of the enzymatic activity of chloride intracellular ion channel proteins

Moghaddasi, Saba

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Publication Type:: Thesis
Issue Date:: 2018

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Field	Value	Language
dc.contributor.author	Moghaddasi, Saba
dc.date.accessioned	2019-01-09T01:45:20Z
dc.date.available	2019-01-09T01:45:20Z
dc.date.issued	2018
dc.identifier.uri	http://hdl.handle.net/10453/129361
dc.description	University of Technology Sydney. Faculty of Science.	en_AU
dc.description.abstract	The chloride intracellular ion channel (CLIC) family, are the group of unusual proteins that exist either in monomeric soluble or integral membrane form. This family is comprised of six protein members in humans, CLIC1-CLIC6. CLIC1 is classified as a “metamorphic” protein, which means it can exist in two stable tertiary conformations. The CLIC proteins can spontaneously insert into phospholipid membranes from their soluble state where they act as ion channel proteins. In addition, the CLIC proteins have structural similarities with both the Glutathione-S-Transferases and glutaredoxin enzyme families. Recently it has been demonstrated by in vitro assay systems that the monomeric CLIC proteins have similar “oxidoreductase” activity to the Glutaredoxin family. Therefore, following on from these new discoveries, further detailed characterization of their enzymatic activity was required, which was the main purpose of this research project. The first objective of this thesis was to determine the important structural regions of CLICs that could be sensitive to the various environmental conditions. In that regards, a comparative study was conducted on both “Histidine tagged” and “non-tagged” CLIC proteins in order to check whether the “6 Polyhistidine tag” and “imidazole” compound that are routinely used in the preparation and purification of the recombinant CLIC proteins, interferes with the functional activity of CLIC1 and CLIC3 proteins. Indeed, the results indicate that the His tag altered the enzymatic function by lowering its catalytic activity. In addition, the imidazole compound was also found to interfere with CLIC’s catalytic activity by acting as a second substrate that led to inaccurate assay measurements. As such, it is recommended that removal of both the “His-tag” and “imidazole” be done in order to avoid any interference in subsequent enzyme characterization studies. The project then proceeded to determine the optimal conditions for the enzymatic activity of the CLIC1 and 3 enzymes, across a range of pH and temperatures. CLIC3 was found to be heat resistant compared to CLIC1, which demonstrated heat sensitivity at higher temperatures. CLIC1 was seen to decrease in it activity and stability at increased temperature. The optimal thermal activity of both proteins obtained was 37°C in the HEDS enzyme assay. However, these studies revealed that the optimal catalytic activity of CLIC3 is obtained under acidic conditions (around pH 6), in contrast to CLIC1 that was optimally active at under more physiological conditions (pH 7). Furthermore, this study focused on exploring alternate new substrates for CLIC enzymes. It was found that soluble CLIC3 and CLIC1, demonstrate an affinity to a number of substrates including Imidazole, DHA and sodium selenite. The inhibitory effect of the ion channel blocker, drug IAA94 on the oxidoreductase activity of these two proteins was also examined, which showed that it inhibited the enzymatic activity of CLIC3 similar to CLIC1. The current study also sought to define the kinetic profile of oxidoreductase catalytic activity of these CLIC proteins. Based on our characterization study, kinetic constants (Vmax, Km) for both CLIC1 and CLIC3 were determined. By comparing these catalytic efficiencies, it was evident that both CLIC1 and CLIC3 obey a Michaelis-Menten kinetics module with Vmax values of (2.026, 3.33 μM/min) and Km value of (2.503, 0.9941 mM) respectively. Also, this characteristic study revealed that both CLIC1 and CLIC3 enzymes are following first-order kinetic reactions with a Vmax value of (1.4mM/min) and (0.7mM/min), respectively. These combined in vitro studies revealed the distinct enzymatic activity and profile of each protein member. Such information is critical in beginning to unravel the newly described enzymatic activity of these proteins and will allow the discrete roles within cells to be assigned to each of these proteins. Future studies determining intracellular function will need to take into account the role of pH and local environmental conditions, along with consideration of relevant substrates and likely protein targets for the enzymatic oxidoreductase activity of the CLIC proteins.	en_AU
dc.format	Thesis (MSc)
dc.language.iso	en_AU	en_AU
dc.relation	https://opus.lib.uts.edu.au/bitstream/10453/129361/7/02whole.pdf
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	au.edu.uts.lib/ppc
dc.rights	info:eu-repo/semantics/openAccess
dc.subject	Chloride intracellular ion channel proteins
dc.subject	Enzymatic activity
dc.subject	Biochemical characterisation
dc.title	Biochemical characterisation of the enzymatic activity of chloride intracellular ion channel proteins	en_AU
dc.type	Thesis	en_AU
utslib.copyright.status	open_access

Abstract:

The chloride intracellular ion channel (CLIC) family, are the group of unusual proteins that exist either in monomeric soluble or integral membrane form. This family is comprised of six protein members in humans, CLIC1-CLIC6. CLIC1 is classified as a “metamorphic” protein, which means it can exist in two stable tertiary conformations. The CLIC proteins can spontaneously insert into phospholipid membranes from their soluble state where they act as ion channel proteins. In addition, the CLIC proteins have structural similarities with both the Glutathione-S-Transferases and glutaredoxin enzyme families. Recently it has been demonstrated by in vitro assay systems that the monomeric CLIC proteins have similar “oxidoreductase” activity to the Glutaredoxin family. Therefore, following on from these new discoveries, further detailed characterization of their enzymatic activity was required, which was the main purpose of this research project. The first objective of this thesis was to determine the important structural regions of CLICs that could be sensitive to the various environmental conditions. In that regards, a comparative study was conducted on both “Histidine tagged” and “non-tagged” CLIC proteins in order to check whether the “6 Polyhistidine tag” and “imidazole” compound that are routinely used in the preparation and purification of the recombinant CLIC proteins, interferes with the functional activity of CLIC1 and CLIC3 proteins. Indeed, the results indicate that the His tag altered the enzymatic function by lowering its catalytic activity. In addition, the imidazole compound was also found to interfere with CLIC’s catalytic activity by acting as a second substrate that led to inaccurate assay measurements. As such, it is recommended that removal of both the “His-tag” and “imidazole” be done in order to avoid any interference in subsequent enzyme characterization studies. The project then proceeded to determine the optimal conditions for the enzymatic activity of the CLIC1 and 3 enzymes, across a range of pH and temperatures. CLIC3 was found to be heat resistant compared to CLIC1, which demonstrated heat sensitivity at higher temperatures. CLIC1 was seen to decrease in it activity and stability at increased temperature. The optimal thermal activity of both proteins obtained was 37°C in the HEDS enzyme assay. However, these studies revealed that the optimal catalytic activity of CLIC3 is obtained under acidic conditions (around pH 6), in contrast to CLIC1 that was optimally active at under more physiological conditions (pH 7). Furthermore, this study focused on exploring alternate new substrates for CLIC enzymes. It was found that soluble CLIC3 and CLIC1, demonstrate an affinity to a number of substrates including Imidazole, DHA and sodium selenite. The inhibitory effect of the ion channel blocker, drug IAA94 on the oxidoreductase activity of these two proteins was also examined, which showed that it inhibited the enzymatic activity of CLIC3 similar to CLIC1. The current study also sought to define the kinetic profile of oxidoreductase catalytic activity of these CLIC proteins. Based on our characterization study, kinetic constants (Vmax, Km) for both CLIC1 and CLIC3 were determined. By comparing these catalytic efficiencies, it was evident that both CLIC1 and CLIC3 obey a Michaelis-Menten kinetics module with Vmax values of (2.026, 3.33 μM/min) and Km value of (2.503, 0.9941 mM) respectively. Also, this characteristic study revealed that both CLIC1 and CLIC3 enzymes are following first-order kinetic reactions with a Vmax value of (1.4mM/min) and (0.7mM/min), respectively. These combined in vitro studies revealed the distinct enzymatic activity and profile of each protein member. Such information is critical in beginning to unravel the newly described enzymatic activity of these proteins and will allow the discrete roles within cells to be assigned to each of these proteins. Future studies determining intracellular function will need to take into account the role of pH and local environmental conditions, along with consideration of relevant substrates and likely protein targets for the enzymatic oxidoreductase activity of the CLIC proteins.

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