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Members of the chloride intracellular ion channel protein family demonstrate glutaredoxin-like enzymatic activity.

Al Khamici H, Brown LJ, Hossain KR, Hudson AL, Sinclair-Burton AA, Ng JP, Daniel EL, Hare JE, Cornell BA, Curmi PM, Davey MW, Valenzuela SM - PLoS ONE (2015)

Bottom Line: We demonstrate that CLIC proteins have glutaredoxin-like glutathione-dependent oxidoreductase enzymatic activity.CLIC1 was shown to reduce sodium selenite and dehydroascorbate in a glutathione-dependent manner.These same compounds inhibit CLIC1 oxidoreductase activity.

View Article: PubMed Central - PubMed

Affiliation: School of Medical and Molecular Biosciences, University of Technology Sydney, Sydney, New South Wales 2007, Australia.

ABSTRACT
The Chloride Intracellular Ion Channel (CLIC) family consists of six evolutionarily conserved proteins in humans. Members of this family are unusual, existing as both monomeric soluble proteins and as integral membrane proteins where they function as chloride selective ion channels, however no function has previously been assigned to their soluble form. Structural studies have shown that in the soluble form, CLIC proteins adopt a glutathione S-transferase (GST) fold, however, they have an active site with a conserved glutaredoxin monothiol motif, similar to the omega class GSTs. We demonstrate that CLIC proteins have glutaredoxin-like glutathione-dependent oxidoreductase enzymatic activity. CLICs 1, 2 and 4 demonstrate typical glutaredoxin-like activity using 2-hydroxyethyl disulfide as a substrate. Mutagenesis experiments identify cysteine 24 as the catalytic cysteine residue in CLIC1, which is consistent with its structure. CLIC1 was shown to reduce sodium selenite and dehydroascorbate in a glutathione-dependent manner. Previous electrophysiological studies have shown that the drugs IAA-94 and A9C specifically block CLIC channel activity. These same compounds inhibit CLIC1 oxidoreductase activity. This work for the first time assigns a functional activity to the soluble form of the CLIC proteins. Our results demonstrate that the soluble form of the CLIC proteins has an enzymatic activity that is distinct from the channel activity of their integral membrane form. This CLIC enzymatic activity may be important for protecting the intracellular environment against oxidation. It is also likely that this enzymatic activity regulates the CLIC ion channel function.

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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.
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pone-0115699-g002: 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.

Mentions: HEDS, 2-hydroxyethyl disulphide, is a low molecular weight compound found to act as a specific and sensitive substrate, suitable for use in assaying glutaredoxin enzymatic activity [35], [36], [49]. The HEDS assay was therefore employed in the current study to test for similar enzymatic activity by members of the CLIC protein family. As seen in Fig. 2A, consumption of NADPH increases (resulting in a decreased A340 nm) in the presence of the positive controls HcTrx-5 and Grx-1, well-known glutathione-dependent oxidoreductases. Similar consumption of NADPH is observed when CLIC1, CLIC4 and to a lesser extent CLIC2, are substituted for HcTrx-5 in the HEDS assay. This indicates that all three proteins reduced the HEDS substrate when coupled with glutathione (GSH) and glutathione reductase (GR) in the presence of NADPH. However CLIC2 is less active than CLIC1 and CLIC4.


Members of the chloride intracellular ion channel protein family demonstrate glutaredoxin-like enzymatic activity.

Al Khamici H, Brown LJ, Hossain KR, Hudson AL, Sinclair-Burton AA, Ng JP, Daniel EL, Hare JE, Cornell BA, Curmi PM, Davey MW, Valenzuela SM - PLoS ONE (2015)

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4291220&req=5

pone-0115699-g002: 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.
Mentions: HEDS, 2-hydroxyethyl disulphide, is a low molecular weight compound found to act as a specific and sensitive substrate, suitable for use in assaying glutaredoxin enzymatic activity [35], [36], [49]. The HEDS assay was therefore employed in the current study to test for similar enzymatic activity by members of the CLIC protein family. As seen in Fig. 2A, consumption of NADPH increases (resulting in a decreased A340 nm) in the presence of the positive controls HcTrx-5 and Grx-1, well-known glutathione-dependent oxidoreductases. Similar consumption of NADPH is observed when CLIC1, CLIC4 and to a lesser extent CLIC2, are substituted for HcTrx-5 in the HEDS assay. This indicates that all three proteins reduced the HEDS substrate when coupled with glutathione (GSH) and glutathione reductase (GR) in the presence of NADPH. However CLIC2 is less active than CLIC1 and CLIC4.

Bottom Line: We demonstrate that CLIC proteins have glutaredoxin-like glutathione-dependent oxidoreductase enzymatic activity.CLIC1 was shown to reduce sodium selenite and dehydroascorbate in a glutathione-dependent manner.These same compounds inhibit CLIC1 oxidoreductase activity.

View Article: PubMed Central - PubMed

Affiliation: School of Medical and Molecular Biosciences, University of Technology Sydney, Sydney, New South Wales 2007, Australia.

ABSTRACT
The Chloride Intracellular Ion Channel (CLIC) family consists of six evolutionarily conserved proteins in humans. Members of this family are unusual, existing as both monomeric soluble proteins and as integral membrane proteins where they function as chloride selective ion channels, however no function has previously been assigned to their soluble form. Structural studies have shown that in the soluble form, CLIC proteins adopt a glutathione S-transferase (GST) fold, however, they have an active site with a conserved glutaredoxin monothiol motif, similar to the omega class GSTs. We demonstrate that CLIC proteins have glutaredoxin-like glutathione-dependent oxidoreductase enzymatic activity. CLICs 1, 2 and 4 demonstrate typical glutaredoxin-like activity using 2-hydroxyethyl disulfide as a substrate. Mutagenesis experiments identify cysteine 24 as the catalytic cysteine residue in CLIC1, which is consistent with its structure. CLIC1 was shown to reduce sodium selenite and dehydroascorbate in a glutathione-dependent manner. Previous electrophysiological studies have shown that the drugs IAA-94 and A9C specifically block CLIC channel activity. These same compounds inhibit CLIC1 oxidoreductase activity. This work for the first time assigns a functional activity to the soluble form of the CLIC proteins. Our results demonstrate that the soluble form of the CLIC proteins has an enzymatic activity that is distinct from the channel activity of their integral membrane form. This CLIC enzymatic activity may be important for protecting the intracellular environment against oxidation. It is also likely that this enzymatic activity regulates the CLIC ion channel function.

Show MeSH