In Vitro Enzymatic Studies Reveal pH and Temperature Sensitive Properties of the CLIC Proteins

Chloride intracellular ion channel (CLIC) proteins exist as both soluble and integral membrane proteins, with CLIC1 capable of shifting between two distinct structural conformations. New evidence has emerged indicating that members of the CLIC family act as moonlighting proteins, referring to the ability of a single protein to carry out multiple functions. In addition to their ion channel activity, CLIC family members possess oxidoreductase enzymatic activity and share significant structural and sequence homology, along with varying overlaps in their tissue distribution and cellular localization. In this study, the 2-hydroxyethyl disulfide (HEDS) assay system was used to characterize kinetic properties, as well as the temperature and pH profiles of three CLIC protein family members (CLIC1, CLIC3, CLIC4). We also assessed the effects of the drugs rapamycin and amphotericin B, on the three CLIC proteins' enzymatic activity in the HEDS assay. Our results demonstrate CLIC1 to be highly heat-sensitive, with optimal enzymatic activity observed at neutral pH7 and at a temperature of 37 degrees C, while CLIC3 had higher oxidoreductase activity in more acidic pH5 and was found to be relatively heat stable. CLIC4, like CLIC1, was temperature sensitive with optimal enzymatic activity observed at 37 degrees C; however, it showed optimal activity in more alkaline conditions of pH8. Our current study demonstrates individual differences in the enzymatic activity between the three CLIC proteins, suggesting each CLIC protein is likely regulated in discrete ways, involving changes in the subcellular milieu and microenvironment.

Automated summary

Chloride intracellular ion channel (CLIC) proteins can exist both in soluble and integral membrane forms, with CLIC1 being able to switch between two different structural conformations. Recent evidence suggests that CLIC family members act as moonlighting proteins, performing multiple functions. In addition to their ion channel activity, CLIC proteins also possess oxidoreductase enzymatic activity and show variability in tissue distribution and cellular localization. This study characterizes the kinetic properties and the temperature and pH profiles of three CLIC family members (CLIC1, CLIC3, CLIC4) using the 2-hydroxyethyl disulfide (HEDS) assay system. The effects of the drugs rapamycin and amphotericin B on the enzymatic activity of the three CLIC proteins in the HEDS assay are also investigated. The results reveal individual differences in enzymatic activity among the three CLIC proteins, suggesting distinct regulation mechanisms involving changes in subcellular milieu and microenvironment.