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Molecular Mechanisms for MG53-Mediated Cell Membrane Repair

Moonsun Hwang
M.S. Biological Science
Korea Advanced Institute of Science and Technology (KAIST) - 1998

Thesis Advisor: Jianjie Ma, Ph.D.
Graduate Program in Physiology & Integrated Biology

School of Public Health
Conference Room 258

Thursday, October 21, 2010
11:00 a.m.


The plasma membrane is a biological barrier between the intracellular and extracellular environments and the maintenance of membrane continuity is essential to cell viability. During our normal lifetime, our bodies have to experience the considerable mechanical stresses, leading to tears in the plasma membrane. Disruptions of plasma membranes are widespread, common, and normal events in many tissues, and cells restore the integrity of membrane by active repair mechanism. Recently, we cloned a new member of membrane repair machinery, MG53 which is skeletal and cardiac muscle specific TRIM family protein (TRIM72). Genetic ablation of MG53 showed progressive myopathy and defective membrane repair capacity, suggesting that MG53 mediates membrane resealing in muscle. In contrast to previously reported repair machineries, MG53 does not require extracellular Ca2+ for its repair function. To understand molecular mechanism underlying MG53-mediated membrane repair, I studied MG53 with three different approaches. First, we characterized the biochemical properties of MG53 using site-directed mutagenesis and live cell imaging. We found that MG53 proteins forms intermolecular disulfide-bond between Cys242 residues, and C242A mutation completely compromised MG53-mediated translocation of intracellular vesicles toward sites of membrane disruption. Furthermore, alkylating reagent treatments showed that oxidation of Cys242 to cystine was induced by exposing to extracellular oxidative environment during membrane disruption. Additionally, cross-linking experiments showed that one leucine-zipper motif (LZ1 - L176/L183/L190/V197) is critical for MG53 dimerization while the other (LZ2 - L205/L212/L219/L226) is not essential. Live cell imaging studies demonstrate that the movement of LZ1 and LZ2 mutants to membrane injury sites is compromised and the LA1/2 double mutant is completely ineffective in translocation toward the injury sites. Second, for the structural analysis and therapeutic application, we optimized MG53 protein expression in E.coli system by screening different vector system and strains, and in silico design. While wild type cDNA did not express untagged MG53 protein at the detectable level in colloidal blue staining, redesigned hMG53 cDNA showed drastically increased protein expression level. Third, we tried to identify the interacting partner with MG53 by yeast two hybrid screening and co-purification of interacting molecules. We found several potential candidates to be further characterized by other biochemical methods; Kif11 and Rad50 from co-purification and DMAP1, -actinin3 and CSN6 from yeast two hybrid screening. This study characterized intermolecular interaction between MG53 proteins and defined the triggering signal of MG53-mediated repair process. In addition to the characterization of biochemical properties, optimized recombinant protein expression system and interacting partners would help to understand the MG53-mediated membrane repair. A complete understanding of the molecular mechanism of the membrane resealing will provide the framework for the therapeutic application for wound healing.

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