Development of Novel Engineered Recombinant Proteins that Improve Cell Membrane Repair Response

Abstract

Muscular dystrophies are a group of genetic diseases hallmarked by progressive muscle weakness and degeneration. Duchenne Muscular Dystrophy (DMD) is a fatal X-linked disease and the most prevalent from of the muscular dystrophy, occurring in 1:5,000 live male births. DMD is caused by mutations in the dystrophin gene, which is critical for the structural integrity of muscle cell plasma membrane (sarcolemma). Current treatments can prolong survival and improve quality of life; however, offer no cure. Therefore, there remains a need for novel therapies that address the underlying molecular cause of the disease. Previous studies have shown that the tripartite motif protein 72/mitsugumin 53 (TRIM72/MG53) is critical for an effective cell membrane response after injury. Our previous results and those of other research groups showed that by overexpressing MG53 or exogenously delivering recombinant MG53 protein (rhMG53) we can increase membrane repair capacity in many different cell types and improve pathology in multiple models of muscular dystrophy. TRIM72/MG53 mediates both of these effects on membrane repair by binding to phosphatidylserine (PS) at membrane injury sites. However, there is a poor understanding of the structural basis of PS binding by the TRIM72/MG53 protein and how this contributes to the membrane repair effects of the protein. In these studies, we conducted a systematic analysis of the MG53 canonical protein domains to determine the minimal functional unit required to increase membrane repair. We generated a panel of nine deletion and fusion protein constructs for human MG53. We transfected human embryonic kidney (HEK293) cells with our mutant panel and confirmed protein expression. We found that at least four mutant protein constructs could increase membrane repair. We generated recombinant protein of our top four candidates and measured membrane repair kinetics using a laser injury assay. We identified one candidate that modulated membrane resealing in mouse muscle fibers. These data suggest that the therapeutic effect on membrane repair can be achieved by a modified version of native MG53 protein.