Solution NMR studies reveal novel CLIC4 chaperone activity with respect to profilin-1.

Abstract

Despite its name, chloride intracellular channel 4 (CLIC4) is a soluble enzyme that can catalyze glutathione-dependent thiol transfers. It is unclear how this enzymatic activity relates to its biological role in remodeling the actin cytoskeleton. Previous studies have shown that CLIC4 interacts with profilin-1, a critical regulator of actin polymerization. We confirmed a direct interaction, 170 ± 20 μM by microscale thermophoresis. However, using solution nuclear magnetic resonance (NMR) spectroscopy, we demonstrate that CLIC4 has a higher affinity for profilin when it is unfolded. Amide hydrogen exchange studies demonstrate that the N- and C-terminal helices of profilin readily unfold, whereas the central β-beta sheet core remains relatively intact. Interaction with CLIC4 increases amide exchange rates throughout profilin, indicating preferential stabilization of partially unfolded intermediates. We note that profilin is glutathionylated in proteomic studies of cells exposed to oxidative stress. Profilin is unfolded by glutathionylation of buried cysteine residues in its N- or C-terminal helices, and this disrupts its ability to bind actin, as measured by pyrene-actin polymerization assay. We are currently attempting to demonstrate that CLIC4 can reverse glutathionylation of profilin-1 and promote its refolding in cells and in vitro.