Redox-dependent oligomerization through a leucine zipper motif is essential for MG53-mediated cell membrane repair

Abstract

We recently discovered that MG53, a muscle-specific tripartite motif (TRIM) family protein, functions as a sensor of oxidation to nucleate the assembly of cell membrane repair machinery. Our data showed that disulfide bond formation mediated by Cys242 is critical for MG53-mediated translocation of intracellular vesicles toward the injury sites. Here we test the hypothesis that leucine zipper motifs in the coiled-coil domain of MG53 constitute an additional mechanism that facilitates oligomerization of MG53 during cell membrane repair. Two leucine zipper motifs in the coiled-coil domain of MG53 (LZ1 - L176/L183/L190/V197 and LZ2 - L205/L212/L219/L226) are highly conserved across the different animal species. Chemical cross-linking studies show that LZ1 is critical for MG53 homodimerization, whereas LZ2 is not. Mutations of the conserved leucines into alanines in LZ1, not in LZ2, diminish the redox-dependent oligomerization of MG53. Live cell imaging studies demonstrate that the movement of green fluorescent protein (GFP)-tagged MG53 mutants (GFP-LA1 and GFP-LA2) is partially compromised in response to mechanical damage of the cell membrane, and the GFP-LA1/2 double mutant is completely ineffective in translocation toward the injury sites. In addition to the leucine zipper-mediated intermolecular interaction, redox-dependent cross talk between MG53 appears to be an obligatory step for cell membrane repair, since in vivo modification of cysteine residues with alkylating reagents can prevent the movement of MG53 toward the injury sites. Our data show that oxidation of the thiol group of Cys242 and leucine zipper-mediated interaction among the MG53 molecules both contribute to the nucleation process for MG53-mediated cell membrane repair.

repair of injury to the plasma membrane is an important aspect of normal cellular physiology, and disruption of this process can result in pathophysiology in many human diseases, including muscular dystrophy, cardiovascular disease, neurodegeneration, ischemic stroke, and traumatic injuries (2, 20). It is well known that cell membrane repair requires translocation of intracellular vesicles to the injury site that involves coordinated function of multiple intracellular components (10, 17). Recent studies have identified several molecular components involved in membrane repair, particularly those specific to cardiac and skeletal muscles (10). Bansal et al. (1) showed that dysferlin plays an important role in maintenance of sarcolemmal membrane integrity. Several mutations in the dysferlin gene have been linked to human muscular dystrophy (9). Recently, our laboratory discovered that MG53 is an essential component of the membrane repair machinery in skeletal and cardiac muscle, because MG53 ablation results in defective sarcolemmal membrane repair, progressive skeletal myopathy (3), and increased vulnerability of the heart to stress and ischemia-reperfusion-induced injury (5, 29).

As a tripartite motif (TRIM) protein family member, MG53 contains the prototypical TRIM domain at the amino-terminus consisting of the RING finger, B-box, and coiled-coil (CC) domain (18, 25). MG53 also contains a PRYSPRY domain at the carboxyl-terminus (3, 15). We found that MG53 can act as a sensor of oxidation to nucleate recruitment of intracellular vesicles to the injury site for membrane patch formation. MG53 can also interact with dysferlin to facilitate its membrane repair function, and altered interaction between MG53, dysferlin, and caveolin-3 is associated with membrane repair defects in muscular dystrophy (4).

While our previous data showed that disulfide bond formation at Cys242 is critical for MG53 oligomerization and the initiation of cell membrane repair (3), additional mechanisms must be involved to facilitate oligomerization of MG53 and assembly of the repair machinery (30). In this study, we examined the contribution of the CC domain to MG53 function in membrane repair. We present evidence that two leucine zipper motifs in the CC domain have differential functions for redox-dependent oligomerization of MG53 and are indispensible for nucleation of the cell membrane repair apparatus. We found that disruption of MG53 oligomerization through chemical modification of cysteine residues before membrane injury could disrupt the MG53-mediated membrane repair process. Since changes in cellular redox-state are associated with many human diseases, targeting the redox-dependent MG53 oligomerization could be a potential therapeutic avenue for prevention or treatment of tissue damage in human diseases.

MATERIALS AND METHODS

Expression vectors and cloning.

The green fluorescent protein (GFP)-MG53 and FLAG-mRFP-MG53 expression vectors have been described and were originally characterized elsewhere (3). MG53 variants were generated by PCR-based site-directed mutagenesis method (14). Hemagglutinin (HA)-tagged MG53 wild type and mutants were cloned into pHM6 (Roche) and pIRESneo3 (Clontech) at NotI and KpnI sites via PCR amplification. Mouse MG53 wild-type cDNA was cloned into Escherichia coli expression vector, pMAL-p2 (NEB) via PCR amplification at SalI and XbaI restriction sites. For the His₆-MG53 expressing baculovirus, mouse MG53 cDNA was cloned into baculovirus vector, pAcHLT-C (Pharmingen) via PCR amplification at XhoI and NcoI sites.

Cell culture.

Human embryonic kidney (HEK)293, HEK293T, and C2C12 cells were maintained in Dulbecco's modified Eagle's medium (Sigma) supplemented with 10% fetal bovine serum (Cellgro), 100 U/ml penicillin (Sigma), and 10 mg/ml streptomycin (Sigma). Transient transfection of cDNA plasmids was performed using GeneJammar (Stratagene) according to the manufacturer's manual. For generation of stable cell lines, HEK293 cells were transfected with pIRESneo3 (Clontech) constructs and grown in the complete media. At 24 h after transfection, 500 μg/ml of G418 (Invitrogen) was added to media. The medium was replenished every 2 to 3 days. Cells were split once the cells surviving selection formed a confluent monolayer. Neonatal myoblasts were derived from mg53^−/− neonatal mouse pups using established techniques (24). For transient transfections, primary myoblasts or C2C12 cells were plated at 70% confluence in glass-bottom ΔT dishes (Bioptech) and transfected using GeneJammer reagent (Stratagene) following the manufacturer's manual. Cells were visualized by live cell confocal imaging at 24 h after transfection at times indicated for individual experiments. Differentiation of myoblasts into myotubes was induced at 90–95% confluence by replacement of growth medium with low-serum medium, consisting of DMEM supplemented with 2% horse serum (Cellgro). All cell lines were maintained in a humidified, 37°C incubator with 5% CO₂.

Recombinant protein purification.

His₆-tagged recombinant MG53 protein was expressed in Sf9 cells infected with the baculovirus expression system (Pharmingen). His₆-MG53 protein was purified using the Ni-affinity column (Qiagen), according to the manufacturer's instructions. To purify MG53 as the maltose-binding protein (MBP)-fusion protein, expression plasmid DNA was transformed into E. coli JM109. The fusion protein was purified from bacterial lysate using amylose affinity column according to the manufacturer's directions (New England Biolabs).

Preparation of protein extract and Western blotting.

Western blotting was performed according to standard protocol (27). Whole cell extracts of culture cells were prepared in modified RIPA buffer containing 50 mM Tris-Cl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 1 mM PMSF, and protease inhibitor cocktail (Sigma). Lysates were cleared by centrifugation at 16,000 g at 4°C for 15 min. For the preparation of protein samples, cell lysates were mixed with equal volumes of 2× SDS sample loading buffer containing 125 mM Tris-Cl, pH 6.8, 4% SDS, 20% (vol/vol) glycerol, and 0.004% bromophenol blue with or without the reducing agent DTT (100 mM), and heated at 95°C for 8 min. Protein samples were separated on SDS-polyacrylamide gels, proteins were transferred onto polyvinylidene difluoride (PVDF) membrane (Millipore) using transfer buffer containing 25 mM Trizma base, 192 mM glycine, and 20% methanol at 4°C at 40 V overnight, and the membranes were incubated in TBST (containing 100 mM Tris-Cl, pH 7.5, 150 mM NaCl, and 0.1% Tween 20) for blocking, washing, and incubation of antibodies. Antibodies used included the following: monoclonal anti-GFP (1:3,000; Invitrogen); monoclonal anti-FLAG (1:10,000; Sigma); monoclonal horseradish peroxidase (HRP)-conjugated anti-HA3F10 (1:4,000; Roche); and polyclonal HRP-conjugated secondary antibodies (1:20,000; Pierce Biotechnology). Proteins were detected with the ECL plus kit (GE Healthcare).

Chemical cross-linking.

Chemical cross-linking protocol was modified from previous TRIM family protein studies (13, 19, 22, 28). To stabilize oligomeric structure of MG53 proteins in SDS-PAGE, we applied the irreversible chemical cross-linker glutaraldehyde (GA; Sigma) followed by immunoprecipitation. While chemical cross-linking did not affect protein band patterns, it was performed along with immunoprecipitation to standardize expression levels. The HA-tagged or FLAG-tagged forms of MG53 and variants were expressed transiently or stably in HEK293 cells. Cells were washed in phosphate-buffered saline (PBS) and lysed in ice-cold NP-40 lysis buffer containing 140 mM NaCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, 1% NP-40, pH 7.4, 1 mM phenylmethylsulfonyl fluoride (PMSF), and protease inhibitor cocktail (Sigma) on ice. Lysates were centrifuged at 14,000 rpm for 15 min at 4°C. Lysates were cross-linked with varying concentrations (0, 0.1, 1, and 2 mM) of GA for 5 min at room temperature. The reaction mixtures were quenched with 100 mM Tris-Cl, pH 7.4, and the volume was adjusted to 800 μl with modified RIPA buffer containing 50 mM Tris-Cl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 1 mM PMSF, and protease inhibitor cocktail (Sigma). The cross-linked lysates were incubated with 1 μg of anti-HA antibody 3F10 (Roche) or 1 μg of anti-FLAG antibody (Sigma) and protein G-agarose beads (BioBRL) at 4°C for 4 h. Beads were washed with modified RIPA buffer three times and supernatants were completely removed. Precipitated proteins were eluted by addition of 1× SDS sample loading buffer and heating at 95°C for 8 min. Proteins were analyzed with standard Western blotting methods described above.

Cellular microelectrode penetration.

To monitor intracellular trafficking of fluorescent fusion proteins, C2C12 cells was cultured in glass-bottom dishes (Bioptechs) and transfected with plasmid DNA using standard techniques. At 24 h after transfection, complete media were changed to buffered salt solution (BSS; 140 mM NaCl, 2.8 mM KCl, 2 mM MgCl₂, 12 mM glucose, and 10 mM HEPES, pH 7.4) containing 2 mM Ca²⁺, and cells were mechanically damaged using a micropipette attached to a micromanipulator (Narishige). Fluorescence images were captured using a Bio-Rad 2100 Radiance laser-scanning confocal microscope with a ×40 1.3 numerical aperture oil immersion objective. To calculate the fluorescence intensity, we used an area of ∼100 μm² for GFP fluorescence directly adjacent to the injury site. To allow for statistical analysis from different experiments, data are presented as fluorescence intensity relative to the value before injury (ΔF/F₀).

Dye exclusion experiments.

For the dye exclusion experiments, isolated neonatal mg53^−/− myoblasts were transfected with the GFP-tagged form of MG53 or mutants and differentiated into myotubes with 2% horse serum. Extracellular media were changed to BSS containing 2 mM Ca²⁺, and 2.5 μM FM4–64 dye (Molecular Probes) was added just before each damage experiment. To induce damage to myotubes, a 5×5 pixel area of the plasma membrane was irradiated at maximum power (Enterprise, 80 mW, 351/364 nm) for 5 s using a Zeiss-LSM 510 confocal microscope equipped with a ×63 water immersion lens (1.3 numerical aperture). Images were captured at 5-s intervals. To calculate the fluorescence intensity, we used an area of ∼200 μm² for FM4–64 fluorescence directly adjacent to the injury site. To allow for statistical analysis from different experiments, data are presented as fluorescence intensity relative to the value before injury (ΔF/F₀).

RESULTS

MG53 forms a homodimer.

Our previous study showed that changes in redox state can influence the oligomeric structure of MG53 expressed in adult skeletal muscle and differentiated C2C12 myotubes (3). Here we used either Sf9 insect cells to produce recombinant MG53 protein that contains a polyhistidine immunoaffinity tag (His₆-MG53), or E. coli to produce MG53 fusion protein containing MBP at the amino-terminus, to test whether the redox-dependent oligomerization is an intrinsic property of MG53. As shown in Fig. 1A, higher-molecular-weight oligomers of MBP-MG53 fusion proteins were prevalent under oxidized conditions (in the absence of DTT), with increasing concentrations of DTT leading to progressive deoligomerization of MBP-MG53 into dimer (∼200 kDa) and monomers (97 kDa). Similarly, the His₆-MG53 protein also shifted from oligomers under oxidized condition into monomers under reduced conditions (Fig. 1B). Together with our previous study (3), these results showed additional evidence that redox-dependent oligomerization is an intrinsic property of MG53 that does not depend on other cellular factors.

Fig. 1.

MG53 forms homodimers. A: purified recombinant maltose-binding protein (MBP)-MG53 from Escherichia coli showed deoligomerization at various concentrations of DTT (0, 0.01, 0.1, 1.0, 10, and 100 mM) on 3–15% gradient acrylamide gel. MBP-MG53 proteins were detected by Western blotting with anti-MG53 monoclonal antibody. Molecular mass of MBP-MG53 is 96 kDa. m, Monomer. B: purified recombinant His₆-tagged MG53 from Sf9 showed oligomerization in the absence of DTT on 3–15% gradient acrylamide gel. Purified MG53 proteins were detected by colloidal blue staining. Molecular mass of MG53 is 53 kDa. C: FLAG-mRFP-MG53 and hemagglutinin (HA)-tagged forms of MG53 wild type were transiently transfected in human embryonic kidney (HEK)293T cells together. The cell lysates were cross-linked with glutaraldehyde (GA), immunoprecipitated with anti-HA (top) or anti-FLAG (bottom), and detected with anti-HA-horseradish peroxidase (HRP). D and E: the predicted migration patterns of proteins in these experiments. The fusion proteins are depicted with FLAG tag (small black square), mRFP (big white square), HA tag (small oval), and MG53 (big oval). Because the cross-linking reactions could not cross-link all MG53 proteins, HA-tagged monomeric MG53 protein (m) was detected.

Previous studies showed that homodimer or homotrimer formation of other TRIM family proteins plays important roles in their cellular functions (8, 23, 26). Studies by other investigators used chemical cross-linking reagents to explore the various oligomerization properties of TRIM family proteins (13, 19). Here we followed their approach to test whether MG53 proteins form homodimers or trimers using MG53 fusion proteins linked to different affinity tags. HEK293T cells were cotransfected with FLAG-mRFP-MG53, with a molecular size of 80 kDa, and HA-MG53 with a molecular size of 54 kDa. After cross-linking with GA, proteins were precipitated with either anti-HA or anti-FLAG antibody and then probed with anti-HA-HRP for immunoblot analysis (Fig. 1C). Figure 1, D and E, shows the predicted patterns of cross-linked protein bands in cases of dimerization and trimerization, respectively. If MG53 formed dimers, anti-HA would precipitate a 133-kDa dimer and a 53-kDa monomer, and anti-FLAG would precipitate protein bands of the same size, as shown in Fig. 1C. On the other hand, if MG53 were to form trimers, anti-HA would precipitate homotrimers of 160 kDa plus heterotrimers of 196 kDa and 216 kDa, which did not appear in Fig. 1C. In addition, anti-FLAG would precipitate two heterotrimers and a monomer, which are also absent in Fig. 1C. Note that anti-FLAG could precipitate FLAG-tagged homodimers or trimers, but they would not be detected with the anti-HA antibody. Clearly, our data shown in Fig. 1C are consistent with MG53 forming a dimeric structure under these experimental conditions. Since the apparent molecular size for the homo- and heterodimers of the tagged MG53 proteins is slightly larger than the predicted molecular size (Fig. 1C), it is possible that other cellular factors could form heteromeric structure with MG53 and contribute to the different MG53 oligomerization patterns observed in Fig. 1C. Future studies to resolve if a binding protein exists in the oligomeric complex of MG53 should reveal more insights into the mechanism for MG53-mediated cell membrane repair.

A leucine zipper motif is critical for homodimer formation of MG53.

In many TRIM family proteins, the coiled-coil (CC) domain is known to meditate homo-oligomerization (25). To test the role of the CC domain in MG53 oligomerization, we generated a CC deletion mutant of MG53 (HA-ΔCC) and HA-tagged CC (HA-CC) constructs (Fig. 2A). As shown in Fig. 2B, with the full-length HA-MG53, cross-linking with GA led to a dimeric protein band of ∼120 kDa (left). The HA-CC protein showed dimer and higher oligomers upon treatment with GA (middle), whereas the HA-ΔCC deletion construct did not show an oligomerization pattern following identical treatment with GA (right). This result demonstrated that the CC domain is critical for MG53 oligomerization.

Fig. 2.

LZ1 in coiled-coil domain is essential for dimerization. A: schematic diagram of domain structures of MG53 wild type and indicated mutants. R, RING finger domain; B, B-box2 domain; CC, coiled-coil domain; P, PRY domain; S, SPRY domain; LZ, leucine zipper motif; H, HA-epitope. B and C: HEK293 cells stably expressing HA-tagged MG53 or its mutants were lysed and cross-linked with various concentrations of GA (0, 0.2, 1, and 2 mM). Each reaction was precipitated with anti-HA antibody and samples were resolved on 6% or 10% SDS-acrylamide gel. Proteins were detected with HRP-conjugated HA antibody.

The CC domain of MG53 contains two conserved leucine zipper motifs (LZ1 and LZ2) as shown in Fig. 2A. To elucidate the role of LZ1 and LZ2 in MG53 function, three mutant constructs were generated by converting the conserved leucines into alanines: LA1 (A176/A183/A190), LA2 (A212/A219/A226), and double mutant LA1/2 (Fig. 2A). We generated stable HEK293 cell lines that express the HA-tagged form of these various leucine zipper mutants. The oligomeric structures of HA-LA1, HA-LA2, and HA-LA1/2 were probed through chemical cross-linking with different concentrations of GA. As shown in Fig. 2C, the HA-LA1 protein displayed compromised dimeric structure (left), whereas the HA-LA2 protein appeared to be normal (middle) compared with the wild-type HA-MG53 (Fig. 2B, left). The HA-LA1/2 double mutant was completely defective in protein dimerization in response to GA treatment (Fig. 2C, right). These results indicate that LZ1 motif is a major site for MG53 dimerization, while LZ2 is not essential for the MG53 homodimerization,

Effects of leucine zipper mutations on MG53-mediated cell membrane repair.

The functional impact of these LZ mutations on MG53-mediated cell membrane repair was examined using live cell imaging by confocal microscopy. We first examined the subcellular localization of the GFP-tagged MG53 mutants transiently expressed in C2C12 myoblasts. Consistent with our previous immunohistochemical staining of MG53 in skeletal muscle (3), GFP-tagged wild-type MG53 (GFP-WT) displayed targeting to the plasma membrane, intracellular vesicle localization, as well as distribution in the cytosol (Fig. 3A). Interestingly, mutation in either the LZ1 or the LZ2 motifs led to alteration of the subcellular distribution of GFP-LA1, GFP-LA2, or GFP-LA1/2, since all three mutants display predominantly cytosolic distribution when transiently expressed in C2C12 cells (Fig. 3A). In more than 90% of the cells observed under confocal microscopy, we consistently found that the mutant proteins displayed cytosolic distributions (n = 32 for LA1, 22 for LA2, and 17 for LA1/2, experiments tested with C2C12 cells), which is in sharp contrast to the membrane-tethering property of the wild-type GFP-MG53 protein. While a full understanding of the role of LZ1 and LZ2 in targeting MG53 to membrane surfaces would require extensive additional studies, live cell imaging demonstrated compromised movement of these MG53 mutants in response to acute damage to the plasma membrane. As shown in Fig. 3B, penetration of a microelectrode into the C2C12 cells caused rapid translocation of GFP-wild type (GFP-WT) toward the acute injury site (top), consistent with our published studies (3, 4). While both GFP-LA1 and GFP-LA2 could sense the mechanical damage in C2C12 cells and move to injury sites, their efficiencies of translocation were significantly compromised (Fig. 3C). Moreover, the GFP-LA1/2 double mutant was completely defective in cell membrane repair as it could not traffic to injury sites. These results indicate that LZ-mediated MG53 intermolecular interactions are essential for MG53-mediated vesicle translocation toward the acute membrane injury site.

Fig. 3.

Inefficient targeting of LZ mutants on the injury sites. A: green fluorescent protein (GFP)-tagged forms of MG53 and its leucine zipper mutants were transiently transfected into C2C12 myoblasts. Wild-type protein localized at the cytoplasm and plasma membrane, but its leucine zipper mutants were principally localized in cytoplasm. The images represent >17 independent transfection experiments. B: representative images of C2C12 cells that were transfected with GFP-tagged forms of MG53 and mutants after microelectrode penetration. C: time course of GFP fluorescence signal at injury sites following microelectrode penetration in C2C12 cells. Data represent means ± SE (n = 12).

To furthermore dissect the role of leucine zipper motifs in membrane repair function, we performed the FM4–64 dye exclusion assay using mg53^−/− myotubes as a homologous reconstitution system (1). The various GFP-tagged MG53 constructs were transfected into the mg53^−/− myotubes, and the membranes were damaged with UV laser in the presence of FM4–64 dye. As shown in Fig. 4, expression of GFP-WT could effectively prevent entry of FM4–64 dye into the mg53^−/− myotube following UV irradiation, which is consistent with our previous study that showed restoration of membrane repair defects with expression of the wild-type MG53. Clearly, the LA1/2 mutant is ineffective in restoration of membrane repair defects, as more FM4–64 dye entry into the mg53^−/− myotubes was observed following UV irradiation (Fig. 4A). Interestingly, transient expression of GFP-LA2 could restore membrane repair function to similar degree as GFP-WT, whereas transient of GFP-LA1 produced intermediate membrane repair defects between GFP-WT and GFP-LA1/2 (Fig. 4B). These results are consistent with our observation shown in Fig. 3 and further suggest that the two LZ motifs likely play differential functions in the membrane repair process mediated by MG53.

Fig. 4.

LZ1 and LZ2 function differently in MG53-mediated cell membrane repair. A: mg53^−/− myotubes transfected with different GFP-tagged MG53 mutants were assayed for FM4–64 dye entry following UV laser damage to the plasma membrane. Representative images of mg53^−/− myotubes transfected with the different constructs (WT, LZ1, LZ2, and LZ1/2) are shown under control conditions (left, 0 s before UV irradiation), and 100 s post- UV irradiation (right). Arrowheads indicate injury sites. B: summary data for FM4–64 dye entry into mg53^−/− myotubes following UV irradiation are presented for cells transfected with the different mutant constructs. Data represent means ± SE (n = 28, GFP-WT; n = 18, GFP-LA1; n = 17, GFP-LA2; n = 24, GFP-LA1/2).

The leucine zipper motif modulates the redox-dependent oligomerization of MG53.

Our previous studies identified Cys242 as a critical residue in the redox-dependent nucleation process for MG53-mediated membrane repair (3). Here we found that the disulfide bond formation through Cys242 is not required for MG53 homodimerization, because normal dimers could be observed with HA-C242A using the GA cross-linking reagent (Fig. 5A). To assay whether the LA1 and LA2 mutation affects the redox-dependent oligomerization of MG53, we transfected GFP- or HA-tagged MG53 mutants into HEK293T cells and performed Western blot analysis under reducing and nonreducing conditions. Consistent with our previous study, we found that GFP-WT and HA-WT MG53 protein formed dimers in the absence of DTT (Fig. 5, B and C, top), and addition of DTT could convert these dimers into monomers (bottom). GFP-C242A and HA-C242A were defective in dimer formation even in the absence of DTT. While GFP-LA2 and HA-LA2 maintained the redox-dependent protein dimerization similar to the GFP-WT and HA-WT proteins, GFP-LA1 and HA-LA1 displayed significant defects in dimer formation under oxidized conditions. Such defects were more obvious with the GFP-LA1/2 and HA-LA1/2 constructs. Together, these results revealed that the LZ motif-mediated intermolecular interaction can modulate the efficiency of redox-dependent MG53 oligomerization.

Fig. 5.

LZ1 motif is required for disulfide bond formation. A: HEK293 cells stably expressing the HA-tagged C242A were lysed and cross-linked with various concentrations of GA (0, 0.2, 1, and 2 mM). Each reaction was precipitated with anti-HA antibody, and samples were resolved on 6% SDS-acrylamide gel. Proteins were detected with HRP-conjugated HA antibody. B and C: Western blot analysis of mouse skeletal muscle or HEK293T cell lysates from cells transfected with GFP-tagged forms of MG53 and its mutants (B) or HA-tagged MG53 and its mutants (C). MG53 proteins were detected with rabbit polyclonal anti-mMG53 antibody. Under nonreducing conditions, MG53 proteins showed high-molecular-weight bands (top), and addition of 100 mM DTT to samples abolished oligomers (bottom). *Nonspecific protein bands.

In vivo chemical modification of cysteine residues leads to impairment of MG53-mediated cell membrane repair.

To further test whether the redox-dependent MG53 oligomerization is an obligatory process for MG53-mediated cell membrane repair, we performed the following series of experiments. We used an alkylating reagent, N-ethylmaleiamide (NEM), to modify the sulfhydryl groups in MG53 (11) by adding it to the cell lysis buffer before disruption of HEK293T cells. As shown in Fig. 6A, treatment with NEM completely blocked disulfide bond formation between MG53 in all constructs tested. Since NEM is a membrane-permeable reagent (12), we next treated cells with NEM at different time points to determine when disulfide bonds were formed before cell permeabilization. When NEM was added to the culture media 10 min before cell lysis, it completely blocked the disulfide bond formation between MG53 molecules (Fig. 6B, lane 3-4). Similar to Fig. 6A, the presence of NEM in the cell lysis buffer also prevented MG53 oligomerization (Fig. 6B, lane 5). However, when NEM was added after cell lysis, it did not affect the apparent disulfide bridge between MG53 molecules (Fig. 6B, lane 6). These results provide direct evidence that the active sulfhydryl groups (−SH) of MG53 are free in intact cells and can form disulfide bridges following cell membrane disruption.

Fig. 6.

Sulfhydryl groups of Cys242 residues are free inside of cells. A: the alkylating reagent N-ethylmaleiamide (NEM) was added to cell lysate. HA-tagged forms of MG53 wild type or indicated mutants were transiently transfected in HEK293T cells or stably expressed in HEK293 cells. HEK293 cells stably expressing the HA-tagged form of MG53 wild type or indicated mutants were analyzed in both reducing and nonreducing conditions. Cell lysates were prepared with RIPA buffer in the absence (left) or presence (right) of 10 mM of NEM. MG53 proteins were detected with anti-HA antibody. *Nonspecific protein bands. B: HA-tagged MG53 was transiently transfected in HEK293T cells, and NEM was added at the different time points as indicated. NEM was added to cell culturing media, lysis buffer, or cleared cell lysates. Samples were separated by nonreducing (−DTT) or reducing (+DTT) SDS-PAGE, and MG53 proteins were detected with anti-HA-HRP antibody. C: time course of GFP fluorescence signal at injury sites following microelectrode penetration into C2C12 cells in the presence of NEM (1 mM). Data represent means ± SE (n = 12). D: schematic diagram of MG53 oligomerization during cell membrane repair. At the resting state, MG53 proteins are brought to close proximity through interaction with the leucine zipper motif(s). Acute injury of the plasma membrane leads to exposure of MG53 to an oxidized environment and induces disulfide bond formation between Cys242 residues and subsequent formation of an oligomeric structure required for the nucleation process of MG53-mediated cell membrane repair.

We next examined the impact of NEM pretreatment on MG53-mediated cell membrane repair. For this purpose, we transiently transfected GFP-MG53 into C2C12 cells and performed microelectrode penetration experiments in the presence of 1 mM NEM. As shown in Fig. 6C, preincubation of HEK293T cells with NEM could prevent translocation of GFP-MG53 toward the acute injury sites. Thus, NEM treatment could mimic the effect of C242A mutation via its occupation of the critical sulfhydryl group, which could prevent oligomerization of MG53 and lead to compromised cell membrane repair.

DISCUSSION

In this study, we demonstrate that MG53 can form homo-oligomers via its conserved leucine zipper motifs in the coiled-coil domain. Although the two leucine zippers both contribute to the nucleation process during MG53-mediated cell membrane repair, they have differential functions in mediating the intermolecular interactions among the MG53 molecules. We also show that the active thiol groups of MG53 are accessible to modification by an alkylating reagent, and blocking activation of these sulfhydryl groups can prevent redox-dependent oligomerization of MG53. Overall, our data support the hypothesis that oxidation of the thiol group of Cys242 and leucine zipper mediated interaction among the MG53 subunits both contribute to the nucleation process for MG53-mediated cell membrane repair.

Previous studies from other investigators showed that other TRIM family proteins could form dimers (6, 28) or trimers (23, 26), and the coiled-coil domain appeared to be essential for the intermolecular interaction among the TRIM family proteins (16, 25, 28, 31). Like other TRIM family proteins, the coiled-coil domain of MG53 is essential for homodimerization, and, specifically, a leucine zipper motif, LZ1 (L176/L183/L190/V197), works as a major interaction site. Furthermore, this homodimerization of MG53 protein is critical for Cys242-mediated intermolecular disulfide bond formation and MG53-mediated repair function. There is no report of disulfide bond formation in other TRIM family proteins, and amino acid sequence analysis showed that no conserved cysteine residues were observed (data not shown). Therefore, this is a unique property of MG53 among the TRIM family proteins.

Although LZ2 motif (L205/L212/L219/L226) is not involved in homodimerization, LZ2 motif is required for MG53 repair function and its role is different from LZ1 in MG53 repair function. Indeed, double mutation of both LZ1 and LZ2 motifs leads to complete disruption of MG53 membrane repair function. Further study is warranted to better understand the role of LZ2 in MG53-mediated repair function. Our available data do not exclude the possibility that the LZ2 domain could be involved in heteromeric interaction of MG53 with other interacting partners, such as the anchoring protein that could tether MG53-containing vesicles to the acute injury sites (32).

On the basis of the results shown here, we propose a model for MG53 function during cell membrane repair (Fig. 6D). Under resting conditions with intact cell membrane structure, MG53 exists as a dimeric structure that involves leucine zipper-mediated intermolecular interaction. The leucine zipper-mediated MG53 protein interaction also allows for accessibility of active sulfhydryl groups (e.g., Cys242), which are critical for formation of a larger oligomeric complex during the nucleation process of cell membrane repair. Acute injury of the plasma membrane creates transient changes in the redox-state near the injury site, where disulfide formation through Cys242 can provide the structural base for formation of an oligomeric structure that is essential for cell membrane repair. Future study is required to dissect the differential function of the two leucine zipper motifs in MG53, and whether heteromeric interaction with other binding partners contributes to the overall MG53-mediated membrane repair response.

Our studies also revealed that occupation of the active thiol (−SH) group of Cys242 with chemical reagent can influence the redox-dependent oligomerization of MG53 and impact its cell membrane repair function. This result is consistent with our previous study that showed transient exposure of cells with low concentration (2 μM) of thimerisal could enhance MG53-mediated cell membrane repair, whereas sustained exposure of cells with high concentrations of thimerisal could prevent MG53 function in membrane repair. Our recent studies also showed that preincubation of cardiomyocytes with 2 mM H₂O₂ could disrupt MG53 translocation to the acute membrane injury site (29), suggesting that prolonged incubation of membrane-permeable oxidants could oxidize all available Cys242 residues and render MG53 insensitive to oxidative signaling generated by membrane disruption and entry of the external environment.

Increased cellular oxidative stress is a common phenotype with many chronic human diseases, such as heart failure, muscle atrophy, diabetes, and neurodegeneration (7, 21). Overproduction of reactive oxidative species could potentially affect the oxidation state of MG53 and reduce its membrane repair capacity. Thus, targeting the intracellular redox state using antioxidant approaches could have beneficial effects in tissue repair and regeneration. Our identification of the differential function of the two leucine zipper domains in MG53 could provide an attractive target for molecular intervention to modulate endogenous cell membrane repair function in skeletal and cardiac muscle. This could prove to be useful for treatment of human diseases associated with reduced regenerative capacity and/or altered cardiovascular function.

GRANTS

This work was supported by National Institutes of Health grants to J. Ma, N. Weisleder, and H. Takeshima.

DISCLOSURES

J. Ma and N. Weisleder are cofounders for TRIM-edicine, Inc., a university spin-off biotechnology company that is developing recombinant MG53 protein as a therapeutic reagent for regenerative medicine.