DYSTROPHIN PROTEIN COMPLEX ASSEMBLY IN LIVING CELLS

The Duchenne and Limb Girdle Muscular Dystrophies (DMD, LGMD) are a heterogeneous group of genetic disorders. Primary mutations in the dystrophin gene result in the absence of the protein in DMD, and mutations in any one of four sarcoglycan (á, â, ä, ã) genes results in a loss of the entire sarcoglycan complex in LGMD. Mutations of the á-sarcoglycan gene are clinically the most frequently observed, and of these cases, one-third have a missense substitution of a cysteine for an arginine at residue 77 (R77C) of the á-sarcoglycan protein. The function of á-sarcoglycan and the implications of the R77C mutation on protein traffic are currently unknown. Here a model system has been developed to study dystrophin protein complex (DPC) assembly in living cells. We report that a minidystrophin gene construct, currently the most promising avenue for adeno-associated virus mediated gene therapy, properly assembles and integrates into the DPC in vivo, utilizing similar mechanisms as wild type dystrophin. We also demonstrate by a variety of assays that in the absence of sarcoglycan complex assembly, á-sarcoglycan is recycled from the plasma membrane. Furthermore, I provide evidence that R77C, the most commonly occurring LGMD mutation, causes a fundamental defect in protein biosynthesis, trapping the mutant protein in the endoplasmic reticulum in vitro and in vivo. Additionally, I show through re-introduction of selected sarcoglycans that the sarcoglycans are able to associate intracellularly to form specific sub-complexes. Central to sarcoglycan complex assembly is the formation of a â-ä-core complex which promotes the deposition of both the core complex and á-sarcoglycan at the plasma membrane, as seen clinically in the microscopic pathology of some cases of LGMD-2C (ã-sarcoglycan deficiency). Taken together these data show the DPC follows a systematic and sequential assembly process, where proper integration, delivery and deposition of each protein into the complex is dependent on several protein-protein associations that in turn allow appropriate trafficking and assembly at the plasma membrane. The multi-factorial reconstruction of the DPC must therefore be carefully evaluated when treating the muscular dystrophies in humans