Chuanxi Cai

MG53 nucleates assembly of cell membrane repair machinery

Dynamic membrane repair and remodelling is an elemental process that maintains cell integrity and mediates efficient cellular function. Here we report that MG53, a muscle-specific tripartite motif family protein (TRIM72), is a component of the sarcolemmal membrane-repair machinery. MG53 interacts with phosphatidylserine to associate with intracellular vesicles that traffic to and fuse with sarcolemmal membranes. Mice null for MG53 show progressive myopathy and reduced exercise capability, associated with defective membrane-repair capacity. Injury of the sarcolemmal membrane leads to entry of the extracellular oxidative environment and MG53 oligomerization, resulting in recruitment of MG53-containing vesicles to the injury site. After vesicle translocation, entry of extracellular Ca2+ facilitates vesicle fusion to reseal the membrane. Our data indicate that intracellular vesicle translocation and Ca2+-dependent membrane fusion are distinct steps involved in the repair of membrane damage and that MG53 may initiate the assembly of the membrane repair machinery in an oxidation-dependent manner.

Enhancing Muscle Membrane Repair by Gene Delivery of MG53 Ameliorates Muscular Dystrophy and Heart Failure in δ-Sarcoglycan-deficient Hamsters

Muscular dystrophies (MDs) are caused by genetic mutations in over 30 different genes, many of which encode for proteins essential for the integrity of muscle cell structure and membrane. Their deficiencies cause the muscle vulnerable to mechanical and biochemical damages, leading to membrane leakage, dystrophic pathology, and eventual loss of muscle cells. Recent studies report that MG53, a muscle-specific TRIM-family protein, plays an essential role in sarcolemmal membrane repair. Here, we show that systemic delivery and muscle-specific overexpression of human MG53 gene by recombinant adeno-associated virus (AAV) vectors enhanced membrane repair, ameliorated pathology, and improved muscle and heart functions in δ-sarcoglycan (δ-SG)-deficient TO-2 hamsters, an animal model of MD and congestive heart failure. In addition, MG53 overexpression increased dysferlin level and facilitated its trafficking to muscle membrane through participation of caveolin-3. MG53 also protected muscle cells by activating cell survival kinases, such as Akt, extracellular signal-regulated kinases (ERK1/2), and glycogen synthase kinase-3β (GSK-3β) and inhibiting proapoptotic protein Bax. Our results suggest that enhancing the muscle membrane repair machinery could be a novel therapeutic approach for MD and cardiomyopathy, as demonstrated here in the limb girdle MD (LGMD) 2F model.

Nonmuscle myosin IIA facilitates vesicle trafficking for MG53-mediated cell membrane repair

Repair of injury to the plasma membrane is an essential mechanism for maintenance of cellular homeostasis and integrity that involves coordinated movement of intracellular vesicles to membrane injury sites to facilitate patch formation. We have previously identified MG53 as an essential component of the cell membrane repair machinery. In order for MG53 and intracellular vesicles to translocate to membrane injury sites, motor proteins must be involved. Here, we show that nonmuscle myosin type IIA (NM‐IIA) interacts with MG53 to regulate vesicle trafficking during cell membrane repair. In cells that are deficient for NM‐IIA expression, MG53 cannot translocate to acute injury sites, whereas rescue of NM‐IIA expression in these cells can restore MG53‐mediated membrane repair. Compromised cell membrane repair is observed in cells with RNAi‐mediated knockdown of NM‐IIA expression, or following pharmacological alteration of NM‐IIA motor function. Together, our data reveal NM‐IIA as a key cytoskeleton motor protein that facilitates vesicle trafficking during MG53‐mediated cell membrane repair.—Lin, P., Zhu, H., Cai, C., Wang, X., Cao, C., Xiao, R., Pan, Z., Weisleder, N., Takeshima, H., Ma, J. Nonmuscle myosin IIA facilitates vesicle trafficking for MG53‐mediated cell membrane repair. FASEB J. 26, 1875‐1883 (2012). www.fasebj.org

Zinc Binding to MG53 Protein Facilitates Repair of Injury to Cell Membranes

Background: MG53, a zinc finger protein, is essential to cell membrane repair. It is not known whether zinc contributes to MG53-mediated membrane repair. Results: Chelation of Zn2+ or mutation of Zn2+-binding motifs in MG53 affects membrane repair. Conclusion: Zn2+ binding to MG53 is required for membrane repair. Significance: This study establishes a base for Zn2+ interaction with MG53 in protection against injury to the cell membrane. Zinc is an essential trace element that participates in a wide range of biological functions, including wound healing. Although Zn2+ deficiency has been linked to compromised wound healing and tissue repair in human diseases, the molecular mechanisms underlying Zn2+-mediated tissue repair remain unknown. Our previous studies established that MG53, a TRIM (tripartite motif) family protein, is an essential component of the cell membrane repair machinery. Domain homology analysis revealed that MG53 contains two Zn2+-binding motifs. Here, we show that Zn2+ binding to MG53 is indispensable to assembly of the cell membrane repair machinery. Live cell imaging illustrated that Zn2+ entry from extracellular space is essential for translocation of MG53-containing vesicles to the acute membrane injury sites for formation of a repair patch. The effect of Zn2+ on membrane repair is abolished in mg53−/− muscle fibers, suggesting that MG53 functions as a potential target for Zn2+ during membrane repair. Mutagenesis studies suggested that both RING and B-box motifs of MG53 constitute Zn2+-binding domains that contribute to MG53-mediated membrane repair. Overall, this study establishes a base for Zn2+ interaction with MG53 in protection against injury to the cell membrane.

The Amino-terminal Peptide Of Bax Perturbs Intracellular Ca2+ Homeostasis To Enhance Apoptosis In Prostate Cancer Cells

Targeting the interconnected cellular pathways controlling apoptosis and regulation of Ca2+ homeostasis are two avenues for treatment of human cancers. During apoptosis, proteolytic cleavage of Bax at the amino-terminus generates a truncated Bax of ∼18 kDa (p18Bax) and an amino-terminal peptide of ∼3 kDa (p3Bax). Extensive studies have shown that p18Bax behaves like a BH3 protein with enhanced pro-apoptotic function over the full-length Bax (p21Bax), little is known about the function of p3Bax in apoptosis. We have previously shown that Bax and Ca2+ synergistically amplifying apoptosis signaling (Pan, et al. J Biol Chem 276: 32257, 2001), and that store-operated Ca2+ entry (SOCE) contributes to Bax-mediated apoptosis in prostate cancer cells (Li, et al. J Cell Physiol 216: 172, 2008). Here we test if p3Bax can contribute to regulation of Ca2+ signaling during apoptosis, through a membrane penetrating peptide (TAT) to facilitate delivery of recombinant p3Bax into NRP-154 cells, a prostate epithelial cell line with tumorigenic capacity. We find that TAT-p3Bax fusion peptide can enhance thapsigargin-induced apoptosis in NRP-154 cells, elevate SOCE activity and increase IP3 sensitive intracellular Ca2+ stores. Our data indicates that p3Bax can modulate the entry of extracellular Ca2+, and thus regulate the amplification of apoptosis in prostate cancer cells. Another unique observation of this study is that TAT-p3Bax is not toxic to NRP-Bax cells under resting conditions, it only enhances the process of apoptosis initiated by exposure to TG. This is particularly important when considering the exogenous p3Bax peptide as a therapeutic agent for prostate cancer. In such a case, p3Bax would not produce cytotoxic effects in cells with normal Ca2+ homeostasis, it could be used in combination with other cytotoxic agents to amplify apoptosis in targeted cancer cells.

MG53 Nucleates Assembly Of Cell Membrane Repair Machinery

Dynamic membrane repair is essential not only for long-term maintenance of cellular integrity but also for recovery from acute cell injury. While compromised membrane repair contributes to various pathological states, including muscular dystrophy, heart failure and neurodegeneration, the associated molecular machinery is largely unknown. We have recently found MG53, a muscle-specific tri-partite motif family protein (TRIM72), is a component of the sarcolemmal membrane-repair machinery. Mice null for MG53 exhibit progressive myopathy, reduced exercise capability and defective membrane-repair capacity. MG53 interacts with phosphatidylserine to associate with intracellular vesicles that display trafficking to and fusion with sarcolemmal membranes. Injury of the sarcolemmal membrane leads to MG53 oligomerization in an oxidation-dependent manner that results in recruitment of MG53-containing vesicles to the injury site. A conserved cysteine residue (C242) is involved in oxidation-mediated oligomerization of MG53, and is critical for MG53 function in membrane repair. The response of MG53-mediated membrane patching is rapid, occurring on the order of seconds after injury, indicating that MG53 mediates the acute repair process following cellular damage. While MG53-mediated vesicle accumulation at the injury site does not require entry of extracellular Ca, Ca entry does facilitate vesicle fusion with the plasma membrane to complete the formation of a repair patch. Our data indicate that intracellular vesicle translocation and Ca-dependent membrane fusion are distinct steps involved in repair of membrane damage, and that MG53 may act as a sensor for oxidation to nucleate the assembly of the membrane repair machinery.