Hossain, KR, Turkewitz, DR, Holt, SA, Herson, L, Brown, LJ, Cornell, BA, Curmi, PMG & Valenzuela, SM 2019, 'A conserved GXXXG motif in the transmembrane domain of CLIC proteins is essential for their cholesterol-dependant membrane interaction.', Biochimica et biophysica acta. General subjects, vol. 1863, no. 8, pp. 1243-1253.View/Download from: UTS OPUS or Publisher's site
BACKGROUND:Sterols have been reported to modulate conformation and hence the function of several membrane proteins. One such group is the Chloride Intracellular Ion Channel (CLIC) family of proteins. The CLIC protein family consists of six evolutionarily conserved protein members in vertebrates. These proteins exist as both monomeric soluble proteins and as membrane bound proteins. To date, the structure of their membrane-bound form remains unknown. In addition to several studies indicating cellular redox environment and pH as facilitators of CLIC1 insertion into membranes, we have also demonstrated that the spontaneous membrane insertion of CLIC1 is regulated by membrane cholesterol. METHOD:We have performed Langmuir-film, Impedance Spectroscopy and Molecular Docking Simulations to study the role of this GXXXG motif in CLIC1 interaction with cholesterol. RESULTS:Unlike CLIC1-wild-type protein, the G18A and G22A mutants, that form part of the GXXXG motif, showed much slower initial kinetics and lower ion channel activity compared to the native protein. This difference can be attributed to the significantly reduced membrane interaction and insertion rate of the mutant proteins and/or slower formation of the final membrane configuration of the mutant proteins once in the membrane. CONCLUSION:In this study, our findings uncover the identification of a GXXXG motif in CLIC1, which likely serves as the cholesterol-binding domain, that facilitates the protein's membrane interaction and insertion. Furthermore, we were able to postulate a model by which CLIC1 can autonomously insert into membranes to form functional ion channels. GENERAL SIGNIFICANCE:Members of the CLIC family of proteins demonstrate unusual structural and dual functional properties - as ion channels and enzymes. Elucidating how the CLIC proteins' interact with membranes, thus allowing them to switch between their soluble and membrane form, will provide key information as to a mechanism of moonlighting ac...
Bray, K, Cheung, L, Hossain, KR, Aharonovich, I, Valenzuela, SM & Shimoni, O 2018, 'Versatile multicolor nanodiamond probes for intracellular imaging and targeted labeling', JOURNAL OF MATERIALS CHEMISTRY B, vol. 6, no. 19, pp. 3078-3084.View/Download from: UTS OPUS or Publisher's site
Hossain, KR, Holt, SA, Le Brun, AP, Al Khamici, H & Valenzuela, SM 2017, 'X-ray and Neutron Reflectivity Study Shows That CLIC1 Undergoes Cholesterol-Dependent Structural Reorganization in Lipid Monolayers.', Langmuir, vol. 33, no. 43, pp. 12497-12509.View/Download from: UTS OPUS or Publisher's site
CLIC1 belongs to the ubiquitous family of chloride intracellular ion channel proteins that are evolutionarily conserved across species. The CLICs are unusual in that they exist mainly as soluble proteins but possess the intriguing property of spontaneous conversion from the soluble to an integral membrane-bound form. This conversion is regulated by the membrane lipid composition, especially by cholesterol, together with external factors such as oxidation and pH. However, the precise physiological mechanism regulating CLIC1 membrane insertion is currently unknown. In this study, X-ray and neutron reflectivity experiments were performed to study the interaction of CLIC1 with different phospholipid monolayers prepared using POPC, POPE, or POPS with and without cholesterol in order to better understand the regulatory role of cholesterol in CLIC1 membrane insertion. Our findings demonstrate for the first time two different structural orientations of CLIC1 within phospholipid monolayers, dependent upon the absence or presence of cholesterol. In phospholipid monolayers devoid of cholesterol, CLIC1 was unable to insert into the lipid acyl chain region. However, in the presence of cholesterol, CLIC1 showed significant insertion within the phospholipid acyl chains occupying an area per protein molecule of 6-7 nm(2) with a total CLIC1 thickness ranging from ∼50 to 56 Å across the entire monolayer. Our data strongly suggests that cholesterol not only facilitates the initial docking or binding of CLIC1 to the membrane but also promotes deeper penetration of CLIC1 into the hydrophobic tails of the lipid monolayer.
Al Khamici, H, Hossain, KR, Cornell, BA & Valenzuela, SM 2016, 'Investigating Sterol and Redox Regulation of the Ion Channel Activity of CLIC1 Using Tethered Bilayer Membranes.', Membranes, vol. 6, no. 4, pp. 1-13.View/Download from: UTS OPUS or Publisher's site
The Chloride Intracellular Ion Channel (CLIC) family consists of six conserved proteins in humans. These are a group of enigmatic proteins, which adopt both a soluble and membrane bound form. CLIC1 was found to be a metamorphic protein, where under specific environmental triggers it adopts more than one stable reversible soluble structural conformation. CLIC1 was found to spontaneously insert into cell membranes and form chloride ion channels. However, factors that control the structural transition of CLIC1 from being an aqueous soluble protein into a membrane bound protein have yet to be adequately described. Using tethered bilayer lipid membranes and electrical impedance spectroscopy system, herein we demonstrate that CLIC1 ion channel activity is dependent on the type and concentration of sterols in bilayer membranes. These findings suggest that membrane sterols play an essential role in CLIC1's acrobatic switching from a globular soluble form to an integral membrane form, promoting greater ion channel conductance in membranes. What remains unclear is the precise nature of this regulation involving membrane sterols and ultimately determining CLIC1's membrane structure and function as an ion channel. Furthermore, our impedance spectroscopy results obtained using CLIC1 mutants, suggest that the residue Cys24 is not essential for CLIC1's ion channel function. However Cys24 does appear important for optimal ion channel activity. We also observe differences in conductance between CLIC1 reduced and oxidized forms when added to our tethered membranes. Therefore, we conclude that both membrane sterols and redox play a role in the ion channel activity of CLIC1.
Cranfield, CG, Berry, T, Holt, SA, Hossain, KR, Le Brun, AP, Carne, S, Al Khamici, H, Coster, H, Valenzuela, SM & Cornell, B 2016, 'Evidence of the Key Role of H3O+ in Phospholipid Membrane Morphology', LANGMUIR, vol. 32, no. 41, pp. 10725-10734.View/Download from: UTS OPUS or Publisher's site
Hossain, KR, Al Khamici, H, Holt, SA & Valenzuela, SM 2016, 'Cholesterol Promotes Interaction of the Protein CLIC1 with Phospholipid Monolayers at the Air-Water Interface.', Membranes, vol. 6, no. 1, pp. 1-13.View/Download from: UTS OPUS or Publisher's site
CLIC1 is a Chloride Intracellular Ion Channel protein that exists either in a soluble state in the cytoplasm or as a membrane bound protein. Members of the CLIC family are largely soluble proteins that possess the intriguing property of spontaneous insertion into phospholipid bilayers to form integral membrane ion channels. The regulatory role of cholesterol in the ion-channel activity of CLIC1 in tethered lipid bilayers was previously assessed using impedance spectroscopy. Here we extend this investigation by evaluating the influence of cholesterol on the spontaneous membrane insertion of CLIC1 into Langmuir film monolayers prepared using 1-palmitoyl-2-oleoylphosphatidylcholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-ethanolamine and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine alone or in combination with cholesterol. The spontaneous membrane insertion of CLIC1 was shown to be dependent on the presence of cholesterol in the membrane. Furthermore, pre-incubation of CLIC1 with cholesterol prior to its addition to the Langmuir film, showed no membrane insertion even in monolayers containing cholesterol, suggesting the formation of a CLIC1-cholesterol pre-complex. Our results therefore suggest that CLIC1 membrane interaction involves CLIC1 binding to cholesterol located in the membrane for its initial docking followed by insertion. Subsequent structural rearrangements of the protein would likely also be required along with oligomerisation to form functional ion channels.
Al Khamici, H, Brown, LJ, Hossain, KR, Hudson, AL, Sinclair-Burton, AA, Jane, PMN, Daniel, EL, Hare, JE, Cornell, BA, Curmi, PMG, Davey, MW & Valenzuela, SM 2015, 'Members of the Chloride Intracellular Ion Channel Protein Family Demonstrate Glutaredoxin-Like Enzymatic Activity', PLOS ONE, vol. 10, no. 1.View/Download from: UTS OPUS or Publisher's site
Yepuri, NR, Holt, SA, Moraes, G, Holden, PJ, Hossain, KR, Valenzuela, SM, James, M & Darwish, TA 2014, 'Stereoselective synthesis of perdeuterated phytanic acid, its phospholipid derivatives and their formation into lipid model membranes for neutron reflectivity studies', CHEMISTRY AND PHYSICS OF LIPIDS, vol. 183, pp. 22-33.View/Download from: Publisher's site