Understanding CLIC1s membrane insertion mechanism

CLICs are a family of metamorphic human proteins that exist in a range of cell types and are responsible for a host of pathologies . The membrane translocation of the CLIC family is still far from completely solved. Questions remain as to the structures the proteins assume in membranes and a how they convert to their membrane form. To tackle this problem different CLIC1 constructs have been experimented with, this being CLIC1 with differently attached His and GFP tags as well as CLIC1 mutants. SAXS and insertion assays revealed that C-terminally attached tags increased CLIC1s membrane insertion when compared to WTCLIC1. A mechanism that, in the current literature, mainly focuses on the N-terminal domain of the protein being structurally altered to allow for CLIC1s membrane insertion. Experiments, based on changes in CLIC1s transmembrane region, also revealed that the CLIC1 mutant R29A prevents the membrane insertion of the protein, while W35A increases the proteins inserting capabilities. This work has also uncovered a new hypothesis of a phosphorylation-based insertion of CLICs, discovered using phosphomimic mutants. The main identified phosphorylation site found in CLIC1 was T222. The phosphomimic mutant T222E incited numerous structural changes to the protein, this being identified by NMR, that ultimately lead to increased insertion of the protein. Further studies will need to be carried out to confirm phosphorylation as a driver for CLICs membrane insertion, but this discovery could open the possibility for the use of phosphorylation inhibitors in the treatment of CLIC related diseases.