Exogenously added recombinant CLIC proteins provide antioxidant protection to cells in culture

Chloride intracellular ion channels (CLICs) are a family of six human proteins that exist as both soluble and integral membrane proteins and are expressed across a range of different tissues throughout the body. CLIC1 and CLIC4 act as moonlighting proteins, exhibiting oxidoreductase enzymatic activity in addition to their membrane ion channel activity. Transient siRNA knockdown of either CLIC1 or CLIC4 in primary human dermal fibroblast (HDF), human epidermal keratinocyte (HKE) cells and in the stable murine fibroblast cell line, NIH/3T3, showed significant reduction in cell viability. Conversely, NIH/3T3 cells over-expressing CLIC1 or CLIC4 demonstrated that both proteins assist in protecting the cells from hydrogen peroxide (H₂O₂)-induced oxidative damage, resulting in reduced cell death and reduced Reactive Oxygen Species (ROS) generation. While the opposite effect was seen in cells where these proteins had been silenced using siRNA. We have also now demonstrated that by exogenously adding recombinant CLIC (rCLIC) proteins to either HDF or HKE cells in culture, both rCLIC1 and rCLIC4 proteins provided cellular antioxidant protection to the fibroblast and keratinocyte cells against H₂O₂-induced oxidative damage. Our study also demonstrates rCLIC1 and rCLIC4’s ability to act as skin cell protective antioxidant agents, arises from their oxidoreductase enzymatic activity. Our findings also showed exogenous addition of rCLIC1 or rCLIC4 to skin cells resulted in similar or greater protection against H₂O₂-induced oxidative damage when compared to other well-known endogenous antioxidants like glutaredoxin (Grx), Glutathione S-transferase-Omega (GST-Ω) and the antioxidant drug, N-acetylcysteine (NAC).