The Differential Contribution of TRIM72/MG53 Protein Domains in Plasma Membrane Repair

Abstract

Duchenne Muscular Dystrophy (DMD) is a fatal X‐linked genetic disease that is hallmarked by progressive muscle weakness and degeneration. DMD is the most common form of all the muscular dystrophies, affecting 1:5,000 live male births. DMD is caused by mutations in the dystrophin gene, which ablate the expression of dystrophin resulting in compromised structural integrity of the muscle cell plasma membrane, also known as the sarcolemma. There is still an unmet need for novel therapies that address the underlying molecular cause of the disease. Previous work has shown that the tripartite motif protein 72/mitsugumin 53 (TRIM72/MG53) is critical for an effective cell membrane repair response after injury. Our previous results demonstrated that overexpressing TRIM72/MG53 or exogenously delivering recombinant human MG53 protein (rhMG53) can increase membrane repair capacity in many different cell types and improve pathology in multiple animal models of muscular dystrophy. TRIM72/MG53 mediates this effect on membrane repair by binding phosphatidylserine (PS) at membrane injury sites. However, there is a poor understanding of the structural basis of PS binding by TRIM72/MG53 and how this contributes to the membrane repair effects of the protein when expressed endogenously or delivered exogenously. Here we aimed to address this knowledge gap through structure/function analysis. In these studies, we examined the mechanistic basis for MG53 binding to PS by conducting a systematic analysis of MG53 canonical protein domains. We generated a catalytic mutant of TRIM72/MG53 (hMG53(ΔE3)) to delineate its native E3 ubiquitin ligase enzymatic activity and its effect on membrane repair. To identify which protein domains are required for the effect on membrane repair, we generated a panel of nine deletion and fusion protein constructs of human TRIM72/MG53. We confirmed protein expression by transient transfection of human embryonic kidney (HEK293) cells and found that specific protein domains mediated the subcellular location of the protein mutant. Moreover, we evaluated ensemble membrane repair capacity by overexpressing our mutant panel in mouse neuroblastoma (N2a) cells and measuring lactate dehydrogenase (LDH) release after damage with ~500 micrometer glass beads. We found that hMG53(ΔE3) displays comparable subcellular localization to wild‐type hMG53 and improves membrane resealing when endogenously expressed in HEK293 cells or made available exogenously as recombinant protein. We observed that hMG53(ΔE3) can also bind phosphatidylserine (PS)‐coated beads. We found that at least two deletion constructs display enhanced membrane repair response comparable to full length TRIM72/MG53. Taken together, these data suggest that the therapeutic effect of TRIM72/MG53 on membrane repair involves specific protein domains but does not require its E3 ligase enzymatic activity.