Target fishing approach to unveil the mechanism of action of the C17 corrector in sarcoglycanopathies

Muscular dystrophies are a collection of genetic diseases characterized by gradual muscle weakness and degeneration. Sarcoglycanopathies belong to the family of limb-girdle muscular dystrophy. Most of the sarcoglycanopathy cases show early onset and rapid progression with loss of ambulation during adolescence. Elevated serum kinase levels and the replacement of contractile tissue with fibrotic or adipose tissue are other common features of the disorder. Sarcoglycanopathies are caused by mutations in the SGCA, SGCB, SGCG, and SGCD genes coding for α-, β-, γ- and δ-sarcoglycan (SG), respectively. These four proteins form a tetramer (SG-complex) that is part of the dystrophin-glycoprotein complex (DGC) that is present at muscle sarcolemma and provides mechanical support during myofiber contraction. The genetic defects in one of the sarcoglycans lead to a reduction of the mutated protein, as well as of the wild type subunits. Most of the mutations are missense mutations that result in a folding defective SG that is recognized and degraded by the quality control system of the endoplasmic reticulum (ERAD pathway), a ubiquitin-proteasome-dependent process. The process is operated by membrane-embedded ubiquitin ligase complexes, which ubiquitinates, and retro translocates the mutated protein in the cytosol where the proteasome degrades them. Due to the fact that sarcoglycan proteins are folding defective, helping their folding process or interfering with the degradative pathway might help to rescue these proteins. One of the promising approaches for sarcoglycanopathies, recently proven in vitro and in vivo, is the use of CFTR correctors, which are small molecules used to treat cystic fibrosis. These molecules have been developed to restore the cell surface expression of the mutated chloride channel known as cystic fibrosis transmembrane conductance regulator (CFTR) protein acting as a pharmaco-chaperone that promotes folding and trafficking of the defective protein. Several studies have demonstrated that CFTR correctors are also effective on α-SG mutants. Among these CFTR molecules, the C17 corrector exhibited remarkable efficacy on different SG-mutants as well as other proteins different from CFTR. Considering the structural and functional difference between SGs and CFTR, it was hypothesized that the C17 corrector could act as a proteostasis regulator for α-SG mutants. Considering the promising results achieved so far, the present research aims to investigate the mechanism of action of the C17 corrector and to identify its possible binding partner(s). To this intent, a target fishing approach was applied based on affinity chromatography, followed by mass spectrometry (MS) analysis for the identification of potential interactors. The C17 has been chemically derivatized to obtain immobilized baits to use in affinity chromatography. The analysis highlighted some components of the ERAD pathway even when stringent conditions were applied. Another technique that was employed is photo-affinity labeling coupled with click chemistry. To see how treatment of C17 affects the interaction of α-SG with components of the ERAD pathway, we have carried out experiments of co-affinity purification (Co-AP) and, co-immunoprecipitation (Co-IP) using HEK293 cells stably expressing mutated αSG and primary myotubes from a sarcoglycanopathy patient, respectively. Thanks to these techniques, a number of polypeptides including members of the ERAD pathway emerged as possible interactors. Concurrently, we also employed modification-free approaches such as Drug Affinity Responsive Target Stability (DARTS) and Cellular Thermal Shift Assay (CETSA) that revealed consistent results with protein-protein interaction studies. From the combination of these approaches, it will be possible to identify and validate the mechanism of action by which CFTR correctors are effective in rescuing the α-SG mutants.