Figure 1.
Conserved G-site motif in members of the CLIC family.
Multiple sequence alignment of human proteins: CLIC 1-6, GST-omega and Grx1-3. Highlighted in grey is the glutaredoxin/thioredoxin active site motif (G-site) (Accession numbers: CLIC1 (CAG46868), CLIC2 (CAG03948), CLIC3 (CAG46863.1), CLIC4 (CAG38532), CLIC5 (AAF66928), CLIC6 (NP_444507), GST-omega (AAF73376), Grx-1 (BAAO4769), Grx-2 (AAK83089) and Grx-3 (AAH0528289) obtained from ClustalW.
Figure 2.
Oxidoreductase activity of the CLIC proteins.
Oxidoreductase enzymatic activity was measured using 5 uM of CLIC proteins or HcTrx-5 or Grx-1, 250 uM NADPH, 1 mM HEDS and 50 nM GR. The reaction was initiated by the addition of 1 mM GSH and the absorbance of NADPH was monitored at A340 nm. Reaction conditions: 5 mM potassium phosphate with 1 mM EDTA, pH 7, at 37°C. (A) Activity of CLIC1, CLIC2 and CLIC4 compared to HcTrx-5 and Grx-1 (positive controls). (B) Activity of 5 uM CLIC1 dimer compared to 5 uM CLIC1 monomer. Error bars represent the S.E. of at least three independent measurements.
Figure 3.
Glutathione-dependant activity of the CLIC proteins.
(A) The reaction mixture contained 2 mM EDTA in 0.1 M Tris-HCl (pH 7.5), 5 uM reduced CLIC1, CLIC2 or CLIC4 (WT) protein, 200 uM NADPH, 750 uM HEDS, 50 nM TrxR and 5 uM Trx-1 (included as a positive control). (B) Insulin disulfide reductase assay to determine catalytic activity of Trx-1 and CLIC1 based on solution turbidity monitored by A650 nm over 30 minutes.
Figure 4.
Comparison of the oxidoreductase activity of CLIC1 (WT) monomer and CLIC1-Cys mutants.
(A) The reaction contained 1 mM EDTA in 5 mM potassium phosphate (pH 7), 250 uM NADPH, 50 nM GR, 1 mM HEDS and 5 uM of reduced CLIC1 (WT), CLIC1-C24A, CLIC1-C24S or CLIC1-C59A. The mixture was incubated for 5 mins at 37°C before initiation of the reaction with the addition of 1 mM GSH followed by monitoring NADPH absorbance at A340 nm. Error bars represent the S.E. of at least three experimental repeats. (B) A reaction of 5 uM of CLIC1 (WT) reduced monomer or CLIC1-C59A protein, 250 uM NADPH, HEDS (0, 0.25, 0.5, 1, 2, 4 or 6 mM) and 50 nM GR. The reaction was initiated by the addition of 1 mM GSH and the absorbance of NADPH was monitored at A340 nm. The reaction conditions where 5 mM potassium phosphate with 1 mM EDTA, pH 7, at 37°C.
Figure 5.
Sodium selenite and dehydroascorbic acid as substrates for CLIC1.
(A) The oxidoreductase enzymatic reaction using sodium selenite as a substrate was performed in 0.1 mM Tris-HCl (pH 7.5) with 1 mM EDTA containing 200 uM NADPH, 50 nM GR,15 uM sodium selenite, 0.1 mg/mL BSA and 5 uM CLIC1(WT) reduced monomer or 5 uM Grx-1 as a control. The reaction was initiated by the addition of 50 uM GSH at 20°C with consumption of NADPH measured at A340 nm. Error bars represent the S.E. of at least three experimental repeats. (B) The reaction was performed in 0.1 mM Tris-HCl (pH 7.5) with 1 mM EDTA containing 200 uM NADPH, 50 nM GR, 5 uM CLIC1 (WT) reduced monomer and sodium selenite (0, 1, 2, 4, 8 or 16 uM). The initiation of the reaction was achieved by adding 50 uM GSH at 20°C where the consumption of NADPH was measured at A340 nm. (C) The oxidoreductase enzymatic reaction using DHAR as a substrate was performed in 137 mM sodium phosphate buffer (pH 7.5) containing 2 mM EDTA, 0.35 mM NADPH, 50 nM GR, 2 mM GSH and 1 mM DHA. The reaction was initiated after addition of 5 uM reduced CLIC1, CLIC4 or HcTrx-5 (as control). Consumption of NADPH was measured at A340 nm. Error bars represent the S.E. of at least three experimental repeats. (D) DHAR activity of the CLIC proteins was determined using 137 mM sodium phosphate buffer (pH 7.5) with 2 mM EDTA, 0.35 mM NADPH, 50 nM GR, 2 mM GSH and DHA (0, 0.25, 0.5,1, 2, 4 or 6 uM). The reaction was initiated after the addition of 5 uM CLIC1 (WT) protein and the NADPH consumption was monitored at A340 nm.
Figure 6.
Effect of chloride ion channel inhibitor drugs on the oxidoreductase enzymatic activity of CLIC1.
5 uM of CLIC1 reduced (WT) or HcTrx-5 protein was incubated with 10 uM IAA-94, A9C, DIDS or Saxitoxin for ∼1 hour prior use of the protein in the assay. The enzyme assay mixture contained 250 uM NADPH, 1 mM HEDS, 50 nM GR in 5 mM potassium phosphate buffer with 1 mM EDTA, pH 7, at 37°C. The consumption of NADPH was monitored at A340 nm post addition of 1 mM GSH. Error bars shown represent the S.E. of at least three experimental measurements.
Figure 7.
Effect of cholesterol on the enzymatic activity of CLIC1.
5 uM of CLIC1 monomer (WT) protein was incubated with 0.4, 0.8 and 1.6 mM cholesterol for ∼1 hour prior to its addition to a reaction mixture of 250 uM NADPH, 1 mM HEDS, 50 nM GR in 5 mM potassium phosphate buffer with 1 mM EDTA, pH 7, at 37°C. The consumption of NADPH was monitored at A340 nm post addition of 1 mM GSH. Control included all the reaction components including 1.6 mM cholesterol, except with no CLIC1 protein. The error bars shown represent the S.E. of at least three experimental measurements.