Members of the chloride intracellular ion channel protein family demonstrate catalyse protein deglutathionylation

Ali HA1, Hossain KR1, D’Amario C1, Jiang L1 and Valenzuela SM1,2

  1. School of Life Sciences, University of Technology Sydney, Sydney, NSW 2007, Australia,.
  2. Centre for Health Technologies, University of Technology Sydney, Sydney, NSW 2007Australia.

The chloride intracellular ion channel (CLICs) proteins are atypical anion selective channel proteins, with some members also known to have enzymatic activity. Human CLIC proteins found in most tissues and cells. (1).We have recently shown that members of CLIC proteins have intrinsic enzymatic activity (2, 3). Also, we have evidence that the expression of CLIC proteins by bacterial cells could provide increased tolerance to oxidative stress (unpublished study). Our current study demonstrates for the first time that CLICs possesses a significant deglutathionylation activity with a model peptide substrate. CLIC1 dependent glutathionylation of cellular proteins can also detect in cultured cell lines. In contrast, our study found that the expression of CLIC1 in cultured CHO-K1 cell lines promotes the rapid glutathionylation of cellular proteins. The deglutathionylation activity was determined via an in vitro assay using a glutathionylated model synthetic peptide as a substrate. Deglutathionylation of CLIC proteins was assessed using a real-time fluorescence-based method. CLIC proteins were found to have a significant deglutathionylation activity. Mutating the active site cysteine residue in CLICs largely eliminated their deglutathionylation activity. Furthermore, mutating one of the only two charged residues: arginine 29 (R29A) or lysine 37 (K37A) in the putative transmembrane region of CLIC1 resulted in a reduction of its deglutathionylation activity. The discovery that CLIC proteins participate in the glutathionylation cycle and targets specific proteins could explain the association of CLICs with a diverse range of clinical disorders and provide a novel therapeutic target. REFERENCES 1. Valenzuela, S. M. etal.,(1997). Molecular cloning and expression of a chloride ion channel of cell nuclei.J Biol Chem 272, 12575-12582. 2. Al Khamici, H. etal., (2015). Members of the chloride intracellular ion channel protein family demonstrate glutaredoxin-like enzymatic activity.PLoS One 10, e115699. 3. Juan R. Hernandez-Fernaud etal, (2017).Secreted CLIC3 drives cancer progression through its glutathione- dependent oxidoreductase activity. Received 11 Aug 2016. Accepted 6 Dec 2016. Published 15 Feb 2017. http://www.nature.com/naturecommunications.