title: Identification and characterization of the localization and expression of CLIC4 under redox conditions in mammalian cells
Creators: name: Sadeghinia, Shaghayegh
Publisher: name: University of Glasgow
Year: 2022

description: Chloride intracellular ion channel-4 (CLIC4) is a member of the CLIC family of proteins which were originally identified as channels of intracellular membranes permeable to ion chloride ions. Its expression and localization to intracellular membrane is sensitive to oxidative stress. This sensitivity of CLIC4 is indicated with its physiological redox regulatory function either as an oxidoreductase enzyme or an ion channel in response to decrease the cellular glutathione level, ROS accumulation, and consequently non-native disulfide formation in the cells. The pathways for disulfide formation are well characterized. However, the understanding of redox state of CLIC4 and possibility to participate in reductive pathway to removing the non-native disulfide bond is still limited and whether CLIC4 as a membranebinding protein with oxidoreductase activity might be needed in the reduction pathway either in the cytosol or ER, are the questions we need to address. In this project the oxidative stress induced by TNF-α and CLIC4 in response to TNF-α can be re-localized to the ER from the cytosol. The ER is a host for disulfide formation within folding proteins entering the mammalian secretory pathway. The consequence of oxidative stress in the ER is the accumulation of misfolded and unfolded proteins. The mammalian cells have a family of oxidoreductase that is thought to be isomerised non-native disulfide bonds. This reductive catalytic activity of oxidoreductases is maintained via a reductive pathway. For CLIC4 to act as an oxidoreductase for performing isomerisation or reduction reactions, it must be preserved in a reduced position. Here, by mass spectrometry, using purified proteins and ER microsomal membrane following TNF-α induced oxidative stress, we illustrate CLIC4 is predominantly placed in a reduced state in the intact cells, demonstrating a reductive pathway is prepared in mammalian cells and CLIC4 can be involved with this pathway as an oxidoreductase through its either enzyme catalytic activity or ion channel activity. In this project, the glutathione has identified to be responsible for the reduction of CLIC4 during oxidative stress. Furthermore, when inhibitors of glutathione synthesis or reductase are added to the cells, CLIC4 is not reduced. The results demonstrate that glutathione plays a direct role in the isomerisation of disulfide bonds by maintaining CLIC4 in a reduced state. To confirm the reduction effect of GSH on CLIC4 and overall microsomal membrane protein in response to TNF-α, we have applied a cysteine-reactive tandem mass tag (Iodo-TMT) to differentially label cysteine residues and analyse the overall protein expression level and redox state into one-step analysis. The individually labeled samples have been pooled in differential combinations to create multiple six-plex samples to determine the effect of GSH on cysteine oxidation and overall protein expression in the microsomal membrane. The result highlights the redox status of CLIC4 under reduction of GSH was confirmed with MS/LC, and the TMT-labeling detected the redox state of the overall microsomal membrane proteins with respect to cysteine oxidation and protein expression. This study is important because CLIC4 as either a membrane binding protein or an ion channel can be considered as a mis-component in reductive pathway.