A NEW GROUP OF MOONLIGHTING PROTEINS - THE CHLORIDE INTRACELLULAR ION CHANNEL PROTEINS DEMONSTRATE GLUTAREDOXIN-LIKE ENZYMATIC ACTIVITY — The Association Specialists
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A NEW GROUP OF MOONLIGHTING PROTEINS - THE CHLORIDE INTRACELLULAR ION CHANNEL PROTEINS DEMONSTRATE GLUTAREDOXIN-LIKE ENZYMATIC ACTIVITY (345)

Heba Al Khamici 1 , Khondker R Hossain 1 , Elizabeth Danial 2 , Amanda L Hudson 3 , Mary W Davey 1 , Paul Curmi 4 5 , Louise J Brown 2 , Stella M Valenzuela 1
  1. Medical and Molecular Biosciences, UTS, Sydney, NSW, Australia
  2. Chemistry and Bimolecular Sciences, Macquarie University, Sydney, NSW, Australia
  3. Australia and the Centre for Health Technologies, UTS, Sydney, NSW, Australia
  4. School of Physics, UNSW, Sydney, NSW, Australia
  5. Centre for Applied Medical Research, St Vincent's Hospital, Sydney, NSW, Australia

Background and Aims: The Intracellular Ion Channel (CLIC) protein family consists of six evolutionarily conserved protein members in vertebrates. These proteins are unusual, existing as both monomeric soluble proteins and as membrane bound proteins, where they function as chloride selective channels. Structural studies identified high levels of homology between the CLICs and glutathione-S-transferase superfamily of enzymes, in particular the GST-omega. We have for the first time demonstrated that CLIC protein family members have glutaredoxin-like enzymatic activity. Our novel findings provide evidence for the functional activity of the soluble form of these proteins, which is autonomous to their known ion channel activity, therefore classing them as a new group of ‘moonlighting proteins’.
Methods: All recombinant CLIC wild type and mutant proteins were purified by Ni-NTA followed by size exclusion chromatography. HEDS enzyme assays were performed using potassium phosphate buffer (pH 7) with 2mM EDTA, HEDS, glutathione reductase (GR) or thioredoxin reductase (TR), GSH, and NADPH. Substrates used included dehydroascorbic acid and sodium selenite. NADPH absorbance was measured at 340nm.
Results: CLIC proteins demonstrated typical glutaredoxin and GST-omega class enzymatic activity in the HEDS assay. They were positive in the presence of GR and reduced GSH, but showed no activity in the presence of TR. CLIC1 mutants C24A & C24S were not active in HEDS assay, but C59A showed activity indicating Cys24 is essential for enzymatic activity of CLIC1. The specific chloride channel blocker drugs IAA94 and A9C also inhibited enzymatic function of CLIC1; however cholesterol did not have any effect on CLIC1enzymatic activity. Sodium selenite and dehydroascorbic acid were found to be substrates for CLIC1 in the HEDS assay.
Conclusion: CLIC proteins resemble the glutaredoxins in the HEDS enzyme assay. These findings reveal that CLIC proteins may function as oxidoreductases and are likely to be involved in antioxidant cellular defense processes.