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

Heba Al Khamici,Mary W Davey,Joanna E Hare,Jane Phui Mun Ng,Elizabeth L Daniel,Amanda L Hudson,Bruce A Cornell,Alxcia A Sinclair-Burton,Khondker R Hossain,Paul M G Curmi,Louise J Brown,Stella M Valenzuela,Luis Eduardo Soares Netto

doi:10.1371/journal.pone.0115699

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.

Highlights

The Chloride Intracellular Channel (CLIC) proteins are highly conserved in vertebrates, with the following six members found in humans: CLIC1 [1], CLIC2 [2], CLIC3 [3], CLIC4 [4, 5], CLIC5 [6] and CLIC6 [7]
Similar consumption of nicotinamide adenine dinucleotide phosphate (NADPH) is observed when CLIC1, CLIC4 and to a lesser extent CLIC2, are substituted for HcTrx-5 in the hydroxyethyl disulphide (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
Members of the CLIC family are soluble proteins capable of spontaneously inserting into lipid membranes - in particular intracellular membranes - to form chloride selective ion channels [8, 14, 20, 54]. Characterization of these proteins has focused on their membrane insertion and their ion channel activity, with no distinct function assigned to their soluble form

Summary

Introduction

The Chloride Intracellular Channel (CLIC) proteins are highly conserved in vertebrates, with the following six members found in humans: CLIC1 [1], CLIC2 [2], CLIC3 [3], CLIC4 [4, 5], CLIC5 [6] and CLIC6 [7]. Knock-out mouse models have been established for CLIC1 [15, 16] CLIC4 [17, 18] and CLIC5 [19] with each demonstrating distinct phenotypes. From such studies, it is postulated that individual CLIC protein members are involved in regulation of processes including cell growth, cell division and apoptosis [18,19,20,21,22] acidification of intracellular organelles [23, 24], formation of stereocilia [19] and development of the organ of Corti [25, 26]

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Results

Conclusion