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

Zinc is an essential trace element that participates in a wide range of biological functions, including wound healing. Although Zn(2+) deficiency has been linked to compromised wound healing and tissue repair in human diseases, the molecular mechanisms underlying Zn(2+)-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 Zn(2+)-binding motifs. Here, we show that Zn(2+) binding to MG53 is indispensable to assembly of the cell membrane repair machinery. Live cell imaging illustrated that Zn(2+) 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 Zn(2+) on membrane repair is abolished in mg53(-/-) muscle fibers, suggesting that MG53 functions as a potential target for Zn(2+) during membrane repair. Mutagenesis studies suggested that both RING and B-box motifs of MG53 constitute Zn(2+)-binding domains that contribute to MG53-mediated membrane repair. Overall, this study establishes a base for Zn(2+) interaction with MG53 in protection against injury to the cell membrane.

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

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.

MG53 constitutes a primary determinant of cardiac ischemic preconditioning.

BACKGROUND: Ischemic heart disease is the greatest cause of death in Western countries. The deleterious effects of cardiac ischemia are ameliorated by ischemic preconditioning (IPC), in which transient ischemia protects against subsequent severe ischemia/reperfusion injury. IPC activates multiple signaling pathways, including the reperfusion injury salvage kinase pathway (mainly PI3K-Akt-glycogen synthase kinase-3beta [GSK3beta] and ERK1/2) and the survivor activating factor enhancement pathway involving activation of the JAK-STAT3 axis. Nevertheless, the fundamental mechanism underlying IPC is poorly understood. METHODS AND RESULTS: In the present study, we define MG53, a muscle-specific TRIM-family protein, as a crucial component of cardiac IPC machinery. Ischemia/reperfusion or hypoxia/oxidative stress applied to perfused mouse hearts or neonatal rat cardiomyocytes, respectively, causes downregulation of MG53, and IPC can prevent ischemia/reperfusion-induced decrease in MG53 expression. MG53 deficiency increases myocardial vulnerability to ischemia/reperfusion injury and abolishes IPC protection. Overexpression of MG53 attenuates whereas knockdown of MG53 enhances hypoxia- and H(2)O(2)-induced cardiomyocyte death. The cardiac protective effects of MG53 are attributable to MG53-dependent interaction of caveolin-3 with phosphatidylinositol 3 kinase and subsequent activation of the reperfusion injury salvage kinase pathway without altering the survivor activating factor enhancement pathway. CONCLUSIONS: These results establish MG53 as a primary component of the cardiac IPC response, thus identifying a potentially important novel therapeutic target for the treatment of ischemic heart disease.

Human zinc fingers as building blocks in the construction of artificial transcription factors.

We describe methods for generating artificial transcription factors capable of up- or downregulating the expression of genes whose promoter regions contain the target DNA sequences. To accomplish this, we screened zinc fingers derived from sequences in the human genome and isolated 56 zinc fingers with diverse DNA-binding specificities. We used these zinc fingers as modular building blocks in the construction of novel, sequence-specific DNA-binding proteins. Fusion of these zinc-finger proteins with either a transcriptional activation or repression domain yielded potent transcriptional activators or repressors, respectively. These results show that the human genome encodes zinc fingers with diverse DNA-binding specificities and that these domains can be used to design sequence-specific DNA-binding proteins and artificial transcription factors.

Recombinant MG53 protein modulates therapeutic cell membrane repair in treatment of muscular dystrophy.

Mitsugumin 53 (MG53), a muscle-specific TRIM family protein, is an essential component of the cell membrane repair machinery. Here, we examined the translational value of targeting MG53 function in tissue repair and regenerative medicine. Although native MG53 protein is principally restricted to skeletal and cardiac muscle tissues, beneficial effects that protect against cellular injuries are present in nonmuscle cells with overexpression of MG53. In addition to the intracellular action of MG53, injury to the cell membrane exposes a signal that can be detected by MG53, allowing recombinant MG53 protein to repair membrane damage when provided in the extracellular space. Recombinant human MG53 (rhMG53) protein purified from Escherichia coli fermentation provided dose-dependent protection against chemical, mechanical, or ultraviolet-induced damage to both muscle and nonmuscle cells. Injection of rhMG53 through multiple routes decreased muscle pathology in the mdx dystrophic mouse model. Our data support the concept of targeted cell membrane repair in regenerative medicine, and present MG53 protein as an attractive biological reagent for restoration of membrane repair defects in human diseases.

Market segmentation of health information use on the Internet in Korea.

BACKGROUND: Internet is the common way to access all kind of information, thus it becomes to have highly influential place on the range of health care as well. More and more people have been using Internet to get the latest information on the health-related matters. However, it is very important but not easy to find the correct data and select it efficiently among the enormous amount of information. To identify the information seekers and understand their needs or purposes, they would be very effective for the providers to give high-quality health information for the Internet users. OBJECTIVES: The purpose of this study is to reveal the patterns of utilization of health information on the Internet. The subjects of the survey were randomly selected among the local residents by their age and gender. It was conducted in Gangwon, the province and Incheon, the metropolitan city at the same time. To investigate the similar amount of population, 0.5% of Gangwon and 0.3% of Incheon dwellers were chosen. METHODS: 10,325 people were responded to the telephone poll and the investigation had been conducted from July to October 2006. 8656 from 10,325 completed the survey and 1665 (19.2%) of them had used Internet to get health information in last 1-year. The use of health information on the Internet was classified into four categories as follows: general health tips, disease specific information, shopping for health commodities, and selection of hospitals. The questionnaire included articles regarding socio-demographic characteristics such as age, gender, income, education, dwelling area, health status and behaviors such as cigarette smoking and alcohol consumption. RESULTS: Of all 1665 respondents, 726 (17.2%) men (total 4210) and 939 (21.1%) women (total 4446) had used Internet for health information in last 1-year. The health information that people wanted from Internet was as follows: general health tips (64.2%), disease specific information (32.0%), shopping for health commodities (23.7%), and selection of hospitals (19.3%). The people of higher education and higher income level tended to use Internet more often for health information. Metropolitan city dwellers used health information on the Internet more than people from province did. Personal health status appeared to be the most important factor to search the information about general health tips from the Internet. Healthy people (68.3%) used the Internet more than people with diseases (44.4%) who used Internet most frequently for disease specific information (62.6%). Residence area was the most important factor of online shopping for health commodities. While 31.8% of city dwellers used the Internet, only 19.0% of metropolitan or county dwellers used it for the same purpose. Determinant factors for the utilization of the Internet for hospital selection were residence area, age, and health examination. CONCLUSIONS: We found that each section of health information has its own clients concerning their personal interests and status of health and life style. That is why customized market segmentation is strongly recommended for their loyal customers. Depends on the information seekers' health status, it should be decided which information would be more useful for them between general health tips and specific disease. On the other hand, the area where the information seekers live can be more influential on terms of shopping for health commodities. These results will be a great help to start to understand the closer atmosphere of the ideal health information market to provide more valuable services in more various use.

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 Ca(2+) facilitates vesicle fusion to reseal the membrane. Our data indicate that intracellular vesicle translocation and Ca(2+)-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.

Mutations in JPH2-encoded junctophilin-2 associated with hypertrophic cardiomyopathy in humans.

Junctophilin-2 (JPH2) is a cardiac specific member of the junctophilins, a newly characterized family of junctional membrane complex proteins important in physically approximating the plasmalemmal L-type calcium channel and the sarcoplasmic reticulum ryanodine receptor for calcium-induced calcium release. JPH2 knockout mice showed disrupted calcium transients, altered junctional membrane complex formation, cardiomyopathy, and embryonic lethality. Furthermore, JPH2 gene expression is down-regulated in murine cardiomyopathy models. To this end, we explored JPH2 as a novel candidate gene for the pathogenesis of hypertrophic cardiomyopathy (HCM) in humans. Using polymerase chain reaction, denaturing high performance liquid chromatography, and direct DNA sequencing, comprehensive open reading frame/splice site mutational analysis of JPH2 was performed on DNA obtained from 388 unrelated patients with HCM. HCM-associated JPH2 mutations were engineered and functionally characterized using immunocytochemistry, cell morphometry measurements, and live cell confocal calcium imaging. Three novel HCM-susceptibility mutations: S101R, Y141H and S165F, which localize to key functional domains, were discovered in 3/388 unrelated patients with HCM and were absent in 1000 ethnic-matched reference alleles. Functionally, each human mutation caused (i) protein reorganization of junctophilin-2, (ii) perturbations in intracellular calcium signaling, and (iii) marked cardiomyocyte hyperplasia. The molecular and functional evidence implicates defective junctophilin-2 and disrupted calcium signaling as a novel pathogenic mechanism for HCM and establishes HCM as the first human disease associated with genetic defects in JPH2. Whether susceptibility for other cardiomyopathies, such as dilated cardiomyopathy, can be conferred by mutations in JPH2 warrants investigation.