Improving cell membrane repair to treat muscular dystrophy

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Project Summary Duchenne Muscular Dystrophy (DMD) is a fatal degenerative muscle disease caused by mutations in the X- linked dystrophin gene. DMD is the most prevalent form of muscular dystrophy with an incidence of 1:5,000 live male births. Dystrophin mutations lead to the loss of protein expression and compromised sarcolemmal membrane integrity. Injury from mechanical stress often leads to muscle fiber death that eventually overwhelms the regenerative capacity of the muscle. Repeated injury severely reduces skeletal muscle strength contributing to early death by the second to fourth decade of life. Current treatment options can prolong survival and improve quality of life; however, they are not curative. Therefore, there remains a need for novel therapies that target the molecular mechanisms underlying disease pathology. Previous studies determined that the tripartite motif protein 72/mitsugumin 53 (TRIM72/MG53) is essential for proper cell membrane repair in skeletal and cardiac muscle through its binding of phosphatidylserine (PS). Loss of MG53 function has been associated with the development of muscular dystrophy and increased susceptibility to cardiac injury. MG53 is a muscle-enriched TRIM family E3 ubiquitin ligase protein that our laboratory previously reported can increase membrane repair by overexpression or by exogenous application of recombinant MG53 (rhMG53) to ameliorate disease pathology in multiple models of muscular dystrophy. This fellowship project aims to interrogate the role of rhMG53 protein domains in this observed effect on membrane repair. My preliminary data suggest that the E3 ubiquitin ligase activity is not required for the enhanced membrane repair phenotype. This observation supports the hypothesis that the canonical TRIM E3 ubiquitin ligase activity of MG53 is not required for therapeutic membrane repair capacity, therefore therapeutic effects may be achieved by compact versions of the MG53 protein that can bind PS. In this fellowship application I propose to test this hypothesis using three specific and independent aims. Aim 1 screens rhMG53 mutant protein constructs to identify rhMG53 protein domains that are essential for membrane repair. Aim 2 defines the mechanistic role of phosphatidylserine binding to rhMG53 by identifying protein domain binding partners. Aim 3 evaluates the therapeutic potential of identified rhMG53 mutant protein constructs when exogenously delivered to intact muscle fibers ex vivo. These studies will provide an excellent training experience by elucidating previously uncharacterized MG53 protein interactions that will enable the development of novel protein therapeutics for DMD. These experimental efforts will be coupled with additional skill development activities to produce an integrated training experience to allow my continued development toward a career as an independent investigator in the neuromuscular disease field.