MG53 mediates skeletal muscle-liver cross-talk and enhances alcohol metabolism in alcoholic liver disease

Domain: neuroscience
Published: 2026-02-13
Editorial outcome: major_revision

Abstract
Alcoholic liver disease (ALD) remains a significant clinical challenge with limited therapeutics. It is strongly associated with sarcopenia, which further worsens the prognosis in liver cirrhosis. Zinc-deficiency is a hallmark of ALD, impairing the liver’s antioxidant defenses and alcohol metabolism. Here we identify a chaperone role for MG53/TRIM72 in facilitating crosstalk between zinc and alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH2), acting as a metabolic regulator of muscle-liver communication. In ALD, muscle-derived MG53 is transported to liver and mitigates liver damage. MG53 deficiency exacerbates hepatic zinc-deficiency and impairs ADH and ALDH2 activity, which are reversed by systemic administration of recombinant human MG53 (rhMG53). MG53’s B-box motif coordinates zinc interaction with ADH and ALDH2, enhancing their enzymatic activity to clear toxic alcohol byproducts. We developed a bio-derived nanoparticle for co-delivery of rhMG53 and zinc, offering a promising therapeutic approach for ALD. In vivo findings are applicable to the male sex only.

Introduction
Alcoholic liver disease (ALD) is a progressive condition caused by chronic alcohol consumption, leading to liver pathologies such as steatosis, hepatitis, and cirrhosis1–3. A significant complication of ALD is sarcopenia, which is characterized by the loss of muscle mass and strength4. Sarcopenia greatly exacerbates hepatic encephalopathy and mortality in patients with liver cirrhosis5,6. However, the effect of sarcopenia on ALD has not been clarified. Myokines, signaling molecules produced by skeletal muscle, play a key role in regulating metabolic processes and tissue regeneration, supporting the health of the liver and other vital organs7,8. Understanding the role of myokines in tissue protection and regeneration is essential for developing effective treatments for ALD.

About 15 years ago, our group identified MG53, a muscle-derived zinc-binding protein, as a critical component of the cell membrane repair machinery9. Dubbed a “molecular bandage”, MG53 has been shown to protect a variety of vital organs, including muscle, heart, liver, lung, kidney, eye, and brain, under injurious conditions10–14. Notably, hepatocytes do not contain endogenous MG53 protein, relying instead on muscle-derived MG53 delivered through circulation to aid in liver protection under stress conditions13. While extensive studies have demonstrated MG53’s therapeutic potential as a tissue repair protein, its role in protecting against chronic liver injury and metabolic dysfunction associated with ALD has yet to be explored.

Zinc is a key element in various metabolic processes, including enzyme function, antioxidant defense, and cell membrane repair15–20. Chronic alcohol consumption is known to deplete hepatic zinc levels, leading to impaired liver and muscle health4,21. Zinc deficiency worsens liver damage in ALD by weakening the liver’s antioxidant defenses and reducing its ability to metabolize toxic alcohol byproducts, while also compromising the liver’s regenerative capacity17,22.

In this study, we identify a chaperone role for MG53 in facilitating the crosstalk between zinc and alcohol dehydrogenase (ADH) and aldehyde dehydrogenase 2 (ALDH2), acting as a metabolic regulator of muscle–liver communication. By modulating zinc metabolism in ALD, MG53 helps preserve ADH and ALDH2 activity, thereby enhancing the liver’s metabolic capacity under chronic-plus-binge ethanol exposure. To harness the synergistic effects of MG53 and zinc in combating ALD, we developed a bio-derived nanoparticle for the co-delivery of recombinant human MG53 (rhMG53) and zinc. We demonstrated its safety and efficacy in preserving liver metabolic function in rodent models, offering a promising therapeutic strategy for ALD treatment.

Results
ALD causes MG53 transfer from the circulation to the liver tissues
To investigate the role of sarcopenia in ALD, we first analyzed data from the National Health and Nutrition Examination Survey (NHANES). A total of 1858 participants with complete information were included, of whom 498 were classified as ALD (Fig. S1A, B). Both crude and adjusted analyses showed that sarcopenia was significantly associated with an approximately 2-fold higher risk of ALD (Fig. 1A). Sarcopenia was defined using appendicular skeletal muscle mass normalized to body mass index (ASM/BMI) as an index23. Restricted cubic spline analysis revealed a nonlinear relationship between ASM/BMI and ALD probability (p < 0.001), with a higher risk in male participants (Fig. 1B).

Further to examine the crosstalk between skeletal muscle and liver in ALD, we performed a combined analysis using data from the Human Liver Proteome Database24, The Human Protein Atlas25,26, and liver proteomics from ALD patients. Proteins typically present in normal liver tissue were excluded, leaving a list of 455 exogenous proteins in ALD liver, suggesting these were secreted from other organs and taken up by the liver. Among these, two potential secreted myokines-MG53/TRIM72 and Ankyrin 1 (ANK1) were identified (Fig. 1C). A literature search revealed no published studies linking ANK1 to liver function or the pathogenesis of ALD. Consistently, a 5-year clinical follow-up data (Fig. S1C) showed no significant impact of ANK1 expression on patient survival. In contrast, higher expressions of MG53 were positively correlated with increased survival in patients with alcohol abuse and liver cancer, as demonstrated using the Kaplan–Meier Plotter database (Fig. 1D)27. These findings highlight that MG53 emerged as a primary candidate myokine mediating skeletal muscle–liver communication in ALD.

MG53, a skeletal muscle-derived protein involved in membrane repair, has been shown to protect against acute liver injury and suppress hepatocellular carcinoma13,18,28,29. Since our discovery of MG53 in 20099, extensive studies from our group and others have established that MG53 is predominantly expressed in skeletal and cardiac muscle (see Fig. S1D, taken from the FANTOM5 database). Consistent with previous reports13,29, MG53 is absent in normal liver tissue; however, we detected MG53 in the liver of ALD patients with chronic alcohol abuse and in mice fed with ethanol (EtOH, 2.8% v/v) for 4 weeks (Fig. 1E, F and Fig. S1E, F). The ALD mouse model was successfully established, as confirmed by hematoxylin-eosin (H&E) staining showing hepatic lesions and increased ALT, AST, triglyceride, and cholesterol levels (Fig. S2A–D). This suggests a translocation of MG53 from circulation to the liver, as its concentration decreased in the blood but increased in the liver of both ALD patients and mice (Fig. 1G). Moreover, we found an inverse correlation between serum MG53 levels and liver function markers (serum ALT, AST, and LDH levels) (Fig. 1H and Fig. S2E). Using AAV-mediated muscle-specific expression of MG53-EGFP (AAV9-MHCK7-MG53-EGFP) in mice30,31, we tracked the dynamic transfer of MG53-EGFP from skeletal muscle to other tissues during chronic ethanol exposure. After 4 weeks of ethanol feeding, immunofluorescence revealed MG53-EGFP translocation from skeletal muscle into injured hepatocytes (Fig. S2F). Together, these findings suggest that MG53 acts as a key mediator of skeletal muscle–liver communication, influencing hepatic function and contributing to ALD progression.

MG53 deficiency exacerbates ALD severity, and exogenous rhMG53 ameliorates ALD in mice
To investigate MG53’s role in ALD, MG53 knockout (MG53−/−) and littermate wild-type (WT) mice were fed with ethanol (EtOH) for 4–8 weeks (Figs. S3 and S4). Under physiological conditions, no difference was observed between MG53−/− and WT mice in terms of liver weight/body weight ratio and liver function (Fig. S3A–C), along with increased liver MG53 in ethanol-stimulated WT mice but no significant difference in ethanol intake between the WT and MG53−/− mice (Fig. S3D-E). MG53 deficiency significantly reduced the survival rates over the 8-week observation period (Fig. 2A). Due to the high mortality at 8 weeks, liver function was assessed at the 4-week time-point (Fig. S4A).

In EtOH-fed mice, MG53 deficiency increased liver weight/body weight ratio (Fig. 2B) and muscle loss (Fig. S4B), along with worsened liver function, as indicated by elevated serum ALT, AST, and LDH levels (Fig. 2C and Fig. S4C). H&E staining revealed that EtOH-fed MG53−/− mice displayed severe hepatic lesions, with higher pathological scores and pronounced hepatocyte ballooning, compared with WT mice (Fig. 2D and Fig. S4D). These MG53−/− mice also showed aggravated hepatic steatosis, as evidenced by Oil Red O staining (Fig. 2E and Fig. S4E) and increased liver triglyceride and cholesterol levels (Fig. 2F and Fig. S4F).

Additionally, MG53 deficiency led to heightened oxidative stress, mitochondrial injury, and inflammation injury, as measured by DHE fluorescence (Fig. 2G and Fig. S4G), MPO activities (Fig. S4H), and transmission electron microscopy (TEM) (Fig. 2H and Fig. S4I), alongside elevated levels of inflammation markers with TNF-α, IL-6, and IL-1β levels (Fig. 2I and Fig. S4J). The EtOH diet also significantly exacerbated fibrosis in MG53−/− mice at 4-weeks post EtOH treatment, as shown by Sirius red staining (Fig. 2J and Fig. S4K) and increased collagen I and IV expression (Fig. 2K and Fig. S4L).

To further explore MG53’s protective role in ALD, a rescue study was conducted by administering recombinant human MG53 protein (rhMG53, 1mg/kg with 1µg/h) via micro-osmotic pump to MG53−/− mice (Fig. S4A). Replenishment of rhMG53 improved survival rate (Fig. 2A), reduced the skeletal muscle loss (Fig. S4B), mitigated hepatic injury (Fig. 2B, C), steatosis, inflammation, and fibrosis (Fig. 2D–K and Fig. S4), supporting the important function of MG53 in protection against ALD in mice.

ADH and ALDH2 are MG53-interacting proteins that control alcohol metabolism in ALD
We performed mass spectrometry to identify potential MG53-binding proteins in the liver of ALD mice after immunoprecipitation with MG53 antibody (Fig. 3A). A total of 417 potential MG53-binding proteins were detected, and after merging with 9749 ALD-related genes, 205 candidate targets were selected for further analysis.

GO enrichment analysis of molecular functions, cell components, and biological processes revealed that these proteins are predominantly involved in alcohol metabolism, mitochondrial function, and membrane-related pathways (Fig. 3B). Of the top 10 KEGG pathways, “alcoholic liver disease” was highlighted as potentially playing a critical role in MG53’s protective effects against ALD (Fig. 3C).

In the “alcoholic liver disease” pathway, ADH and ALDH are two key enzymes responsible for alcohol metabolism (Fig. 3D). Our data demonstrate that ADH and ALDH2 are direct binding partners of MG53, a finding confirmed through co-immunoprecipitation (Fig. 3E and Fig. S5A, B) in liver and extra-liver, including gut (Fig. S5C, D). These enzymes, essential for antioxidant defense and liver protection under stress, are regulated by cellular zinc levels17,22. Extensive research shows that hepatic zinc deficiency, a hallmark of ALD, impairs the function of both enzymes32,33. Notably, the ability of ADH and ALDH2 to metabolize toxic alcohol byproducts is zinc-dependent17,22.

Spectroscopic analysis revealed a progressive decline in hepatic zinc levels associated with chronic EtOH exposure34,35. Interestingly, MG53−/− mice exhibited more severe zinc deficiency in the liver compared with WT mice after 4 weeks of EtOH exposure. However, this zinc deficiency was ameliorated in MG53−/− mice following rhMG53 administration (Fig. 3F).

Given the pivotal role of ADH and ALDH2 in alcohol metabolism, we measured serum ethanol and acetaldehyde levels. In MG53−/− mice, ethanol and acetaldehyde concentrations were significantly higher compared to controls, but these levels were markedly reduced by rhMG53 replenishment (Fig. 3G, H). This suggests that MG53 exerts a protective effect against ALD by binding with ADH and ALDH, enhancing their activities and improving alcohol metabolism, thus reducing EtOH-induced liver injury. We also treated cultured AML12 liver cells with rhMG53 in the presence of EtOH for 24h (Fig. S5E–G). As the concentration of EtOH escalates, there is a corresponding exacerbation of cellular lipid droplet accumulation and injury in AML12 cells, as indicated by the increased fluorescent intensity of Nile red (Fig. S5E) and the marked reduction in cell viability (Fig. S5F). EtOH treatment (400 mM) for 24h also caused significant ROS elevation, evidenced by increased fluorescent intensity of DHE (Fig. S5H) and increased levels of LDH in the culture medium (Fig. S5I), indicating cellular damage. However, the addition of rhMG53 (10µg/mL) significantly mitigated the harmful effects of EtOH on AML12 cells. This was reflected in the substantially improved cell viability (Fig. S5G), reduction of DHE fluorescence (Fig. S5H), and decreased LDH release (Fig. S5I). Further immunofluorescence analysis confirmed that rhMG53 treatment increases intracellular zinc levels and enhances its co-localization with ADH and ALDH2 in EtOH-stimulated AML12 cells (Fig. S5J). Indeed, in AML12 cells subjected to EtOH treatment, rhMG53 significantly increased the enzymatic activity of both ADH and ALDH2 (Fig. 3I, J).

We systemically administered Cy5.5-labeled rhMG53 to mice and observed markedly elevated levels of Cy5.5-rhMG53 in the livers of ALD mice (after 4-week exposure to EtOH), but not in control mice on a normal diet (Fig. 3K). Immunofluorescence staining revealed clear co-localization of rhMG53 with both ADH and ALDH2 (Fig. 3K) in livers from ALD mice. Furthermore, consistent with previous reports of MG53 localizing to injured cell membrane13,18, we observed that rhMG53 could enter the cytosol and co-localize with ADH and ALDH2 in AML12 cells treated with EtOH (Fig. 3L), which also co-precipitated in rhMG53-treated primary mouse hepatic parenchymal cells (Fig. S5K). These findings confirm the intracellular action of rhMG53 to enhance the enzymatic activities of ADH and ALDH2 under ALD conditions.

MG53 coordinates with zinc recruitment to enhance ADH/ALDH2 function in ALD
To investigate the mechanisms underlying MG53’s regulation of ADH/ALDH2 functions, we obtained the structure coordinates of MG53, ADH, and ALDH2 from the AlphaFold Protein Structure Database and performed molecular docking analysis to model their interactions. These studies revealed that three key residues in MG53-Glu87, Asp100, and Arg101 are critical for its interaction with ADH, while Leu103 is essential for MG53’s interaction with ALDH2 (Fig. 4A).

To further assess the stability of the MG53/ADH or MG53/ALDH2 complexes, we conducted molecular dynamics simulations (Fig. 4B and Fig. S6A–D). The Gibbs free energy landscape demonstrated stable complex formation for both MG53/ADH and MG53/ALDH2 (Fig. 4B and Fig. S6E). During the 100ns simulations, rapid binding of ADH and ALDH2 to MG53 occurred within the initial 20ns, with complex stabilization observed throughout the remainder of the simulation (Fig. 4C). The specific interactions between rhMG53 and ADH, and rhMG53 and ALDH2, were confirmed by Bio-Layer Interferometry (BLI) assay (Fig. 4D). These data confirm the stable combination between rhMG53 and ADH/ALDH2.

Guided by the molecular docking studies (Fig. 4A), we performed site-directed mutagenesis to mutate the three critical residues in MG53 (Glu87, Asp100, and Arg101) to alanines, generating the mutant rhMG53-3A protein using our established E. coli fermentation protocol (Fig. S7A, B)10. BLI assays confirmed that the rhMG53-3A mutant lost its specific binding with ADH (Fig. 4E). While the wild-type rhMG53 protein enhanced the enzymatic activity of ADH in an in vitro biochemical assay, the rhMG53-3A mutant was ineffective in stimulating ADH function (Fig. 4F). Similarly, when Leu103 was mutated to alanine, the rhMG53-1A mutant lost its interaction with ALDH2 in the BLI assay (Fig. 4G), and was unable to enhance ALDH2 enzymatic activity in the in vitro conditions (Fig. 4H). These results indicate that the stimulating effects of rhMG53 on ADH and ALDH2 are dependent on its interaction with these enzymes.

Domain homology analysis identified two zinc-binding domains in MG53, located in the RING finger and B-box motifs (Fig. S7C)18,36,37. Mutations of Cys29 to Leucine (C29L) in the RING finger and Cys105 to Serine (C105S) in the B-box motif are known to disrupt MG53’s zinc-binding capacity18. We generated the rhMG53-C29L/C105S mutant protein (Fig. S7). Interestingly, the mutant rhMG53-C29L/C105S protein, which lost its zinc-binding ability18, showed significantly reduced capacity to activate both ADH and ALDH2, compared to the wild-type rhMG53 protein (Fig. 4I, J). Moreover, rhMG53-C29L/C105S was ineffective in protecting AML12 cells from EtOH-induced injury, as shown by the minimum impact on cell viability (Fig. 4K) and LDH release (Fig. 4L). We performed additional experiments to evaluate the rhMG53-C29L/C105S mutant in the ALD mouse model. MG53−/− mice were subjected to a 4-week chronic-plus-binge ethanol feeding and treated with rhMG53-C29L/C105S via micro-osmotic pump (1mg/kg with 1µg/h). Unlike wild-type rhMG53, the mutant protein had no significant effect on alcohol-induced mortality or liver injury in MG53−/− mice (Fig. S7D–F).

Collectively, these findings reveal that MG53 acts as a chaperone by coordinating the transfer of zinc to ADH and ALDH2, thereby boosting their enzymatic function in metabolizing toxic alcohol byproducts under ALD conditions.

Developing a bio-nanoparticle for co-delivery of rhMG53 and zinc to treat ALD
Given the crucial role of MG53 and its interaction with zinc in combating ALD, we developed bio-derived MG53@Zn nanoparticles (MG53@Zn-NPs) as a delivery platform for the co-delivery of rhMG53 and zinc (Fig. 5A). We reasoned that this targeted co-delivery system could not only enhance liver regeneration through MG53’s tissue-protective properties but also improve the liver’s metabolic capacity to clear toxic alcohol byproducts by boosting the MG53-zinc-ADH/ALDH2 axis.

The MG53@Zn-NPs were synthesized using a desolvation protocol, involving the sequential mixing of the rhMG53/Zn complex with human serum albumin (HSA) (Fig. 5A). The resulting nanoparticles had a multi-leaf shape and an average size of ~250 nm (Fig. 5B, C). When tested in cultured AML12 cells, Cy5.5-labeled rhMG53@Zn-NPs was taken by AML12 cells in a time-dependent manner (Fig. 5D) and provided superior protective effects against EtOH-induced injury (400 mM, 24h) compared to rhMG53 alone, as evidenced by improved cell viability and reduced LDH release (Fig. 5E, F). Additionally, MG53@Zn-NP enhanced oxidative stress resistance in AML12 cells, as demonstrated by reduced DHE fluorescence (Fig. 5G) and MDA levels, along with increased SOD and GSH levels (Fig. S8A). These nanoparticles also lowered lipid oxidation, as indicated by decreased Nile red fluorescence (Fig. 5H).

Systemic administration of MG53@Zn-NP facilitated targeted delivery of the MG53/Zn complex to the liver. Following tail vein injection (1mg/kg with rhMG53 equivalent) in normal mice, we observed prominent accumulation of Cy5.5-labeled rhMG53@Zn-NPs in the liver (Fig. 5I), confirmed by immunofluorescence staining and immunoblotting of Zn2+ and rhMG53 of hepatocytes (Fig. 5J and Fig. S8B). Liver frozen sections incubated with a Zn2+ probe further revealed a marked increase in intracellular Zn2+ fluorescence, with clear co-localization of the Zn2+ signal and Cy5.5-labeled nanoparticles by confocal microscopy (Fig. 5J). The half-life of rhMG53 in the liver was approximately 5 days, after intramuscular injection of MG53@Zn-NPs (Fig. 5K). We also conducted repetitive dosing of MG53@Zn-NPs in WT mice (1mg/kg every 5 days for 30 days) and observed no adverse effects on vital organs (Fig. 5L and Fig. S8C). Before in vivo studies, we tested MG53@Zn-NPs in EtOH-stressed AML12 cells, where they restored ADH and ALDH2 activities that were diminished by EtOH exposure, surpassing endogenous ADH/ALDH2 activity levels (Fig. 6A). In EtOH-fed WT mice, more than 60% of the animals died within 8 weeks (Fig. 6B, red; Fig. S9A), but MG53@Zn-NPs notably improved the survival of the mice (Fig. 6B, purple).

To show the long-term effect of MG53@Zn-NPs on liver structure and function, we took 8-week treatment as the observation time-point, which is unlike that in MG53−/− mice. MG53@Zn-NPs reduced liver weight/body weight ratio (Fig. 6C), and serum AST, ALT, and LDH levels (Fig. 6D and Fig. S9B), indicating a reduction in EtOH-induced liver injury. Histological analysis showed improved hepatocyte integrity and steatosis in treated mice (Fig. 6E, F and Fig. S9C, D), with a mitigation of EtOH-induced triglyceride and cholesterol elevation (Fig. 6G and Fig. S9E).

Spectroscopic analysis of liver tissue revealed that MG53@Zn-NPs significantly restored zinc levels in EtOH-fed mice, bringing them close to normal (Fig. 6H). MG53@Zn-NPs also reduced oxidative stress (measured by DHE fluorescence) (Fig. 6I), improved the liver’s antioxidant function as evidenced by decreased serum levels of EtOH and aldehyde (Fig. 6J-K). Additionally, TNF-α, IL-6, and IL-1β, elevated by EtOH-induced inflammation, were markedly reduced by MG53@Zn-NPs (Fig. 6L and Fig. S9F), contributing to the long-term benefits of MG53@Zn-NPs in alleviating EtOH-induced liver fibrosis (Fig. 6M and Fig. S9G), as quantified by the changes in collagen I and IV as biomarkers of fibrosis (Fig. 6N and Fig. S9H). Importantly, MG53@Zn-NPs showed superior therapeutic efficacy for ALD compared to treatment with rhMG53 alone (1mg/kg with 1μg/h via micro-osmotic pump) (Fig. 6 and Fig. S9).

Methods
Patients
Human samples were collected from Daping Hospital (Chongqing, China), with the study receiving approval from the Daping Hospital Institutional Ethics Review Board (ID223). Blood samples from ALD patients were obtained from individuals with a history of alcohol abuse spanning more than 5 years, who continued alcohol consumption at the time of sampling. Control blood samples were obtained from inpatients with no history of alcohol abuse and normal liver function. Liver biopsy tissues were obtained from patients with alcoholic hepatitis or cirrhosis, who had a similar history of alcohol abuse. Normal liver samples were obtained from non-pathological para-carcinoma tissues from patients undergoing surgical resection for hepatic carcinoma. All participants were fully informed and provided consent before sample collection. Liver tissues were either immediately submerged in liquid nitrogen for protein extraction and stored at −80°C, or preserved in 4% paraformaldehyde for a minimum of 24h before being flash-frozen or embedded in paraffin. Blood samples were processed by centrifugation at 16,000 × g for 15 min at room temperature to collect serum, which was subsequently stored at −80°C. This study strictly adhered to the ethical guidelines outlined in the World Medical Association’s Declaration of Helsinki for research involving human participants.

Animals
All animal study protocols were approved by the Institutional Animal Care and Use Committee of the Third Military Medical University (Chongqing, China) (Certification No: AMUWEC20191177). Eight-week-old C57BL/6 mice were purchased from Vital River Laboratories (Beijing, China). MG53 knockout (MG53−/−) mice and their wild-type (WT) littermates were generated as described previously9,56. Mice were housed in individually ventilated cages, maintained under a 12:12h light–dark cycle at 22–24°C and 40–60% humidity, with free access to food and water. The animal facility maintains a thermoneutral micro-environment, eliminating hypothermia as a confounder. All procedures were performed under anesthesia, and every effort was made to minimize suffering. Mice were euthanized by cervical dislocation, and blood, liver, skeletal muscle, heart, spleen, lung, and kidney were harvested for further analysis.

To establish the ALD mouse model, mice were fed a Lieber–DeCarli ethanol-containing diet (purchased from XiaoShuYouTai Biotechnology Co., Ltd., Beijing, China), which involved increasing ethanol concentrations over time based on the NIAAA model57. The diet began with 0.75% ethanol for 3 days, followed by 1.5% for 4 days, and with 2.8% EtOH for either 4 or 8 weeks, finally followed by gavage of a single dose of ethanol (28.5%, 20 μL/g body weight) 9h before being euthanized. Mice in the control group were given an isocaloric Lieber–DeCarli pair-fed diet. The rhMG53 protein from Novoprotein (Suzhou, China) was administered via micro-osmotic pump (ALZET, 1004, Beijing, China) at a dose of 1mg/kg body weight. Mutated rhMG53 plasmids were constructed in the MiaoLing Plasmid Platform (MiaoLing Biology, Wuhan, China), and the mutant proteins (rhMG53-1A, rhMG53-3A, and rhMG53-C29L/C105S) were produced by Novoprotein.

Recombinant viruses and infection
Muscle-restricted gene transfer was accomplished with AAV9 vectors in which the MHCK7 promoter drives either MG53-EGFP or a control sequence (AAV9-MHCK7-Control and AAV9-MHCK7-Trim72-EGFP; HanBio, Shanghai)30,31. After isoflurane induction, the lower-limb fur of 8-week-old male C57BL/6J mice was clipped, and the skin was wiped with 70% ethanol. The same viral dose (1 × 1012 vg/ml, 10 μl per site) was slowly introduced into 4 sites—quadriceps femoris and gastrocnemius of both legs.

Histological analysis and transmission electron microscopy
Tissues were fixed in 4% paraformaldehyde for 24h and embedded in paraffin. Thin sections (4 μm) were prepared using a microtome. For pathological observation, paraffin sections of liver, heart, spleen, lung, and kidney samples were stained with hematoxylin-eosin (H&E). To evaluate hepatic fibrosis, liver sections were stained with Sirius red. For analyzing hepatic lipid accumulation, fresh liver tissues were embedded in a Tissue-Tek optimal cutting temperature (OCT) compound (SAKURA, Torrance, California) and rapidly frozen. These samples were then sectioned to 8-μm using a cryotome (Sakura Finetek) and stained with Oil Red O. All histological staining kits were purchased from Solarbio Life Science (Beijing, China), and staining was performed according to the manufacturer’s instructions. The pathological features of liver tissues were assessed and scored as described previously58,59.

For transmission electron microscopy (TEM), fresh liver tissues were trimmed into 2mm × 2mm samples and fixed in 2% glutaraldehyde at 4°C overnight. Regions were excised randomly using a glass scribe and mounted for ultramicrotomy. After negative staining, the sections were observed, and images were taken using a transmission electron microscope (Fischione Instruments).

Biochemical assays
The levels and activities of ALT, AST, LDH, cholesterol, triglyceride, MPO, SOD, GSH, MDA, and ethanol were quantified using specific assay kits from Solarbio Life Science, in accordance with the manufacturer’s protocols. These include Glutamic-pyruvic Transaminase Activity Assay Kit, Glutamic-oxalacetic Transaminase Activity Assay Kit, Lacate Dehydrogenase (LDH) Activity Assay Kit, Triglyceride Content Assay Kit, Total Cholesterol Content Assay Kit, Myeloperoxidase (MPO) Activity Assay Kit, Superoxide Dismutase (SOD) Activity Assay Kit, Glutathione Peroxidase (GPX) Activity Assay Kit, and Malondialdehyde (MDA) Content Assay Kit. Serum acetaldehyde was measured using the Acetaldehyde Assay Kit from Sigma-Aldrich, following the manufacturer’s instructions. Absorbance readings were measured at the corresponding wavelength using a spectrophotometer (Thermo Scientific).

Hepatic zinc detection
Liver tissues (about 100mg wet weight per sample) were placed in 15mL clean centrifuge tubes and dissolved with aqua regia. The treated samples were then introduced into the injector of an Inductively Coupled Plasma Mass Spectrometry (PerkinElmer Nexion 1000, Waltham, MA). Following plasma ignition, the ion source was optimized to ensure full ionization of the sample elements, allowing them to enter the mass spectrometer. In the mass spectrometer, zinc ions were selectively screened and quantified based on their respective nucleus-mass ratio (m/z) for spectroscopic analysis. The ion signals detected by the mass spectrometer were subsequently transmitted to a computer for data collection and storage.

Mass spectrometry (MS)
Liver tissues were obtained from C57BL/6 mice after an EtOH-fed diet for 4 weeks. To detect liver proteins, the total protein of liver tissues was prepared and quantified using the BCA Protein Assay Kit (Beyotime Biotechnology), then diluted to 1 μg/μL with 200 μg of proteins per sample. Proteins were reduced with DTT and alkylated with IAA, then precipitated with acetone and redissolved in ammonium bicarbonate. Trypsin digestion was performed overnight at 37°C. The peptides were desalted, resuspended in 0.1% FA, and separated by UltiMate 3000 RSLC nanoLC. Elution was performed with a gradient of solvents A and B. The eluate was analyzed by Orbitrap Exploris 480 MS in data-dependent mode. MS and MS/MS spectra were acquired with specific resolutions and settings. Data were processed using Proteome Discoverer software with the Mouse UniProt Protein Database. Peptides were identified using the SEQUEST algorithm with specified parameters, including trypsin as the protease, mass tolerances, and a false discovery rate of 1%.

According to the instructions of the Protein A/G Immunoprecipitation Kit (Beaverbio, Suzhou, China), the total proteins of liver tissues were extracted and incubated with Beaver Beads™ MG53 antibody (Proteintech, Wuhan, China) immunomagnetic beads at 4°C overnight to form the MG53 antibody-antigen complex. Following magnetic separation, the beads containing the complex were washed and stored in immunoprecipitation washing buffer. Samples of MG53 antibody-antigen complex were analyzed using a Q Exactive mass spectrometer (Thermo Finnigan). Proteome Discoverer 1.4 software (Thermo Scientific) was used to analyze raw MS files and generate a data matrix that consisted of the retention time, mass-to-charge ratio (m/z) values, and peak intensity. Specific peptides were used to recognize protein. Bioinformatic analyses were performed by Bioinformatics online (https://www.bioinformatics.com.cn/).

Co-immunoprecipitation
Protein was extracted from mouse liver tissues as described above, and incubated with specific antibodies, gently rotating at 4°C overnight. The immunocomplex was captured by adding Beaver Beads™ immunomagnetic beads and rotating gently at 4°C for 3h. The mixture was then centrifuged at 2500 × g for 5 min at 4°C to obtain the precipitate. The precipitate was washed 3 times with ice-cold RIPA buffer, and the immunocomplex was separated from the beads by resuspending and boiling, followed by centrifugation to collect the supernatant. The supernatant was subsequently analyzed via SDS-PAGE, followed by western blotting with the appropriate antibodies. Rabbit anti-MG53, rabbit anti-ADH, and rabbit anti-ALDH2 antibodies were purchased from Proteintech.

Molecular docking
Protein structures of MG53, ADH, and ALDH2 were obtained from AlphaFold Protein Structure Database. These structures were introduced to the Molecular Operating Environment (MOE) 2019.01 software to predict the binding interactions of MG53 and ADH, as well as MG53 and ALDH2. At the end of the docking procedure, 3D structural maps and planar representations were created, with binding sites marked. To assess the interaction and stability of protein complexes, a molecular dynamics simulation was performed using Gromacs 2022.3 software over a period of 100ns. Stability was evaluated using Gibbs energy landscapes, Root Mean Square Deviation (RMSD), Root Mean Square Fluctuation (RMSF), Radius of Gyration, the number of hydrogen bonds, and Solution Accessible Surface Area (SASA), and MM/GBSA of contributions (Nine Chapters Calculation, Sichuan, China).

Protein–protein interaction assay
Biotin labeling. Initially, ADH and ALDH2 proteins were dissolved in phosphate-buffered saline with 0.02% Tween 20 (TBST) to achieve the appropriate concentrations. Streptavidin (SA) biotin was then added to 100 μl protein solutions at a molar ratio of 3:1 relative to the protein and incubated at room temperature for 30 min to ensure adequate conjugation. Labeled protein solutions were purified using a desalting column to remove unbound biotin.

Bio-layer interferometry assay. SA sensors were immersed in solutions containing biotin-labeled ADH or ALDH2 proteins to immobilize the proteins on the sensor surface. The sensors were then incubated in solutions with varying concentrations of rhMG53 (or mutated) proteins to conduct binding assays. Real-time monitoring of interference spectral changes induced by the binding of rhMG53 (or mutated) proteins to the sensors was performed. Experimental procedures were carried out on the Octet RED96 instrument, utilizing consumables recommended by SARTORIUS (Beijing, China). A biotinylation kit was purchased from Genemore (Suzhou, China). Purified ADH protein and ALDH2 protein were purchased from CUSABIO (Wuhan, China), and purified rhMG53 (including mutated) proteins were purchased from Novoprotein.

Cell culture and viability assay
AML12 cells, mouse hepatic parenchymal cells, and the corresponding media were purchased from Pricella Biotechnology (Wuhan, China). Cells were maintained at 37°C in a humidified cell incubator with 5% CO2, and the culture medium was replaced every two days. Following the fourth passage, cells at 60–70% confluency were used for experiments. To establish the ALD cell model, AML12 cells were treated with 50-, 100-, 200-, 400-, and 800-mM EtOH for 24h to determine the appropriate ethanol concentration. For the therapeutic observation, cells cultured with ethanol were supplemented with a gradient concentration of rhMG53 (2.5–20 μg/mL) or rhMG53@Zn-NPs for 24h. For cell viability assessment, cells were plated in 96-well plates at a density of 5 × 103 cells/well and cultured overnight to allow for adherence. Cells were then pretreated with various interventions for 24h at 37°C. Cell viability was estimated using the cell counting kit-8 (CCK-8) colorimetric assay (Solarbio Life Science), and LDH release was detected by LDH Cytotoxicity Assay Kit (Beyotime), according to the manufacturer’s instructions.

Immunohistochemistry and immunofluorescence
Primary antibodies included mouse anti-MG53, rabbit anti-MG53, rabbit anti-CK18 (HUABIO, Zhejiang, China), rabbit anti-ADH, rabbit anti-ALDH2, and rabbit anti-Alb (all from Proteintech). Secondary antibodies included goat anti-rabbit and goat anti-mouse (Proteintech). For tissue staining, samples were fixed in 4% paraformaldehyde for 24h, embedded in paraffin, and sectioned to 4-μm thickness. Paraffin-embedded sections were deparaffinized with deparaffinization buffer (Solarbio Life Science).

For immunohistochemical (IHC) staining, liver sections were immersed in 10 mM citric acid buffer (pH 6.0) for 30 min and treated with 3% H2O2 for 30 min to quench endogenous peroxidase activity. Sections were blocked in QuickBlock™ Blocking Buffer for Immune Staining (Beyotime Biotechnology) for 1h to prevent nonspecific antibody binding. Primary antibodies (1:100) were applied in a humidified chamber at 4°C overnight, followed by secondary antibodies (1:100) for 1h at room temperature. In each step, liver sections were rinsed with phosphate buffer saline (PBS) three times for 5 min each. Images were acquired using a SLIDEVIEW VS200 microscope (Olympus, Japan).

For tissue immunofluorescence (IF) staining, liver sections were immersed in 50× Citrate Antigen Retrieval Solution (pH 6.0) (Beyotime Biotechnology) for 30 min and cooled naturally to room temperature to retrieval antigen, and then blocked in QuickBlock™ Blocking Buffer for Immune Staining for 1h to prevent nonspecific antibody binding. MG53, CK18, ADH, ALDH2, and Alb staining were performed by incubating with the corresponding primary antibodies (1:100) in a humidified chamber at 4°C overnight, followed by incubation with secondary antibodies (1:100) for 1h at room temperature. In each step, liver sections were cleaned with PBS for 3 times × 5 min. Finally, sections were sealed with Antifade Mounting Medium with DAPI (Beyotime Biotechnology). Images were acquired using a Confocal Laser Scanning Microscope (Olympus).

For cellular immunofluorescence staining, AML12 cells were cultured in 24-well plates with cell slides at a density of 1 × 104 cells/mL. After treatment, the culture medium was removed, and cells were fixed with 4% paraformaldehyde for 30 min. Blocking and antibody incubation were performed with the above method, with PBS rinses performed three times for 5 min each. Finally, cell slides were sealed on the object slide with Antifade Mounting Medium with DAPI. Images were acquired using a Confocal Laser Scanning Microscope.

Cellular zinc level measurement
AML12 cells were cultured in 24-well plates covered with cell slides at a density of 1 × 104 cells/mL. After treatment, the culture medium was removed, and 4% paraformaldehyde was added to fix cells for 30 min, and washed with PBS for 3 times × 5 min. Cell slides were incubated with 25 μM zinquin ethyl ester (MedChemExpress, Shanghai, China) in PBS for 30 min at 37°C, and washed with PBS for 3 times × 5 min. Finally, cell slides were taken out and sealed on the object slide with Antifade Mounting Medium with DAPI. Images were acquired using a Confocal Laser Scanning Microscope at 368 nm excitation.

ROS level measurement in liver tissue and AML12 cells
For ROS measurement, liver tissues were embedded in OCT compound, rapidly frozen, and sectioned into 8-μm-thick slides. The sections were cleaned with PBS for 3 times × 5 min. AML12 cells were also cultured in 24-well plates covered with cell slides and treated as necessary. Both cleaned frozen sections and cell slides were incubated with Dihydroethidium (DHE) solution (Beyotime Biotechnology) in a dark chamber at 37°C for 30 min. After washing with PBS for 3 times × 5 min, samples were mounted with Antifade Mounting Medium with DAPI. Images were acquired using a confocal laser scanning microscope.

Enzyme activity assay
The enzyme activity of hepatic ADH and ALDH2 was evaluated using the Alcohol Dehydrogenase Activity Assay Kit and Acetaldehyde Dehydrogenase Activity Assay Kit from Solarbio Life Science.

Enzyme-linked immunosorbent assay (ELISA)
Serum levels of IL-6, TNF-α, and IL-1β were determined by the mouse IL-6 ELISA kit, the mouse TNF-α ELISA kit, and the mouse IL-1β ELISA kit, separately, purchased from Solarbio Life Science. Measurement processes followed the manufacturer’s protocols.

Western blot
The total protein of liver tissues, serum, and AML12 cells was prepared and quantified using the BCA Protein Assay Kit (Beyotime Biotechnology). Equal amounts of protein samples were subjected to 4–20% SDS-PAGE and transferred to polyvinylidene fluoride membranes. The membranes were blocked with 5% nonfat milk-TBST at room temperature for 30 min and were washed with TBST for 3 times 5 min, and then were incubated with primary antibodies at 4°C overnight, followed by incubating with fluorescent secondary antibodies in the dark at room temperature for 1h, washed with TBST for 3 times × 5 min. The loading serum protein was determined by staining with ponceau S (MeilunBio, Shanghai, China) for photography and washed with TBST 3 times × 5 min. The primary antibodies include rabbit anti-MG53 (1:1000, Proteintech), rabbit anti-ADH (1:1000, Proteintech), rabbit anti-ALDH2 (1:1000, Proteintech), and mouse anti-GAPDH (1:1000, Proteintech). The secondary antibodies include goat anti-rabbit IRDye 800 and goat anti-mouse IR Dye 800 (1:10,000, Li-Cor Biosciences, Beijing, China). The protein-bound complexes were detected by the Odyssey Infrared Imaging System (LI-Cor Biosciences), and the band intensities were measured by ImageJ software.

Quantitative real-time PCR
Total RNA samples of liver tissues were extracted using TRIzol reagent. Two μg of total RNA was used to synthesize the first-strand cDNA (TAKARA, Kusatsu, Japan) for reverse transcription, and the fluorescence was detected using the CFX96 Real-Time PCR Detection System (Bio-Rad, San Diego, CA) according to the manufacturer’s instructions. The expression of the target gene was normalized by GAPDH mRNA as an internal control.

Synthesis and characterization of MG53@Zn nanoparticles
To synthesize MG53@Zn-NPs, 1 mL of rhMG53 (2 mg/mL) was mixed with 0.2 mL zinc acetate solution (14 mg/mL), followed by stirring at 4°C for 4h. The mixture was then dialyzed with 8000–14,000 molecular weight dialysis bags (Solarbio Life Science) in deionized water at 4°C for 12h, resulting in MG53@Zn solution. Next, 400 μL human serum albumin (HSA, 10 mg/mL) was combined with 100 μL MG53@Zn solution. The pH was adjusted to 9 by adding 2–8 μL NaOH (1M), followed by the addition of 4 times the volume of anhydrous ethanol (2 mL), at a drop rate of about 1 mL/min, while stirring at 4°C for 10 min. Subsequently, 25 μL of NHS-TK-NHS linker solution (10 mg/ml, xarxbio, Shanxi, China) solution (10 mg/mL) was added, and the reaction was allowed to continue under stirring at 4°C for 2h. After dialysis in deionized water at 4°C for 12h, the final MG53@Zn-NPs were obtained.

The particle size and distribution of MG53@Zn-NPs were measured using Dynamic Light Scattering in Zeta Size Nanoseries (Malvern Panalytical, UK). To visualize the nanoparticles, the solution was dropped onto the surface of the 4mm × 4mm silicon wafer, dried naturally, and then imaged using a scanning electron microscope (SEM) (Fischione Instruments). For additional characterization, the NPs solution was applied to copper meshes (200-mesh), air-dried, and analyzed using TEM (FEI TECNAI G2 F20, Fischione Instruments), while the elemental composition was examined by XFlash 6T-30 detector (Bruker), generating specific element peak maps.

In vivo distribution and in vitro uptake of MG53@Zn-NPs
For in vivo distribution analysis, C57BL/6J mice were intravenously injected with MG53@Zn-NPs labeled with Cy5.5, a near-infrared dye. The bio-distribution of these NPs within the mice was detected and visualized by Small Animal In Vivo Imaging (SAIVI™). After imaging, the mice were euthanized with anesthesia, and their heart, livers, spleens, lungs, and kidneys were harvested to assess organ-specific distribution. The livers were further imaged to evaluate the localization of the MG53@Zn-NPs. For the in vitro experiments, AML12 cells were co-cultured with Cy5.5-labelled MG53@Zn-NPs for 0, 1, 3, 6, 12, and 24h. After each time-point, cells were fixed and stained, then observed and imaged by a Confocal Laser Scanning Microscope to assess NP uptake over time.

Available data
Potential crosstalk proteins between skeletal muscle and alcohol-injured liver were revealed by combining ALD-related proteins, muscle-specific proteins, and secreted proteins, and then eliminating proteins detected in a normal liver. ALD-related proteins were obtained from the Human Liver Proteome Database (http://www.liverproteome.org/) and liver samples of ALD patients from our center (https://wwwn.cdc.gov/nchs/nhanes/Default.aspx)24. Muscle-specific proteins, secreted proteins, and liver-detected proteins were obtained from The Human Protein Atlas (https://www.proteinatlas.org/)25,26. The associations between expression levels of potential proteins and survival rates of liver cancer patients with alcohol abuse were evaluated on the Kaplan–Meier Plotter database (http://kmplot.com)41. Liver cancer patients with alcohol abuse were split by auto select best cutoff in 60 months follow-up threshold to produce the overall survival analysis27. The two patient cohorts with different expression levels of potential proteins are compared by a Kaplan–Meier survival plot, and the hazard ratio with 95% confidence intervals and log-rank P-value are calculated.

Epidemiological analysis of NHANES data
Study population: The data used in this study were derived from the National Health and Nutrition Examination Survey (NHANES) 2017–2018, a cross-sectional survey conducted in the United States. NHANES collects comprehensive data on the health and nutritional status of non-institutionalized U.S. children and adults through household interviews, mobile examination centers (MEC), blood samples, and physical examinations60. Inclusion criteria: All participants from the NHANES 2017–2018 survey. Exclusion criteria: (1) Pregnant individuals; (2) Individuals under 18 years of age; (3) Missing diagnosis of sarcopenia; (4) Missing records of alcohol consumption; (5) Missing data on liver damage diagnosis. The NHANES protocol received ethical approval from the National Centre for Health Statistics (NCHS) review board, with written informed consent provided by all participants.

Definition of ALD. The NHANES 2017–2018 survey incorporated vibration-controlled transient elastography (VCTE) to measure liver stiffness and the controlled attenuation parameter (CAP) to quantify hepatic fat content, providing an estimate of liver fibrosis. VCTE has been widely validated for assessing liver steatosis and fibrosis. CAP values range from 100 to 400 dB/m, with higher values indicating increased hepatic fat content61.

In this study, alcohol consumption was categorized into four levels: never drinking, light drinking, moderate drinking, and heavy drinking. “Never drinking” was defined as consuming alcohol fewer than 12 times in lifetime; “Former drinking” was defined as consuming alcohol more than 12 times a year and did not drink last year, or did not drink last year but drank more than 12 times in lifetime; “Mild drinking” included women consuming up one drink per day and men consuming up to two drinks per day; “Moderate drinking” included women who drank at least two times per day and men who drank at least three times per day, or those who consumed alcohol at least two days per month in binge patterns; “Heavy drinking” was defined as women consuming at least three drinks per day and men consuming at least four drinks per day, or those who binged rank five or more days per month. Binge drinking was defined as consuming four or more drinks per day in women and five or more drinks per day in men62. For the definition of ALD in this study, participants were required to meet all three of the following criteria: (1) Alcohol consumption of at least mild drinking; (2) CAP score ≥248 dB/m (to indicate hepatic steatosis)63; (3) Elevated alanine aminotransferase (ALT) or aspartate aminotransferase (AST) levels (women >19 U/L or men >29 U/L62). Participants who did not meet these diagnostic criteria were classified as non-ALD.

Definition of sarcopenia. The NHANES 2017–2018 survey utilized dual-energy X-ray absorptiometry (DXA) to measure body composition, a standard method for diagnosing sarcopenia. DXA-derived appendicular skeletal muscle mass (ASM), the sum of lean mass in the arms and legs, was used to define sarcopenia. In this study, the skeletal muscle mass index (ASM/BMI = ASM/BMI), as proposed by the National Institutes of Health (NIH) Foundation, was used for sarcopenia diagnosis. Specifically, an ASM/BMI < 0.789 for men and < 0.512 for women was considered indicative of sarcopenia23.

Covariates. Body mass index (BMI) was calculated as weight (kg) divided by height squared (m²), and categorized into three groups: <25.0 kg/m², 25.0–30.0 kg/m², and ≥30.0 kg/m². Ethnicity was classified based on self-reported data from NHANES and included Mexican American, non-Hispanic Black, non-Hispanic White, other Hispanic, and other races (including non-Hispanic and multi-racial individuals). Marital status was categorized as married, divorced, widowed, or never married. Education was categorized into three levels: less than high school, high school graduate, and college graduate or higher. Household income was divided into three categories based on the poverty income ratio (PIR): <1.0, 1.0–3.0, and ≥3.0. Smoking status was categorized into three groups: never smoker (smoked fewer than 100 cigarettes in a lifetime), former smoker (smoked ≥100 cigarettes but currently not smoking), and current smoker (smoked ≥100 cigarettes and currently smoking either daily or occasionally).

Statistical analysis
For NHANES data. Given the complex multi-stage (stratified and clustered) sampling design of NHANES, all analyses were weighted using the “Survey” package in R, applying the 2017–2018 “WT MEC 2YR-Full Sample 2 Year MEC Examination Weights” to adjust for survey design. For covariates with missing values, such as marital status, education, and smoking, multiple imputation was performed using the “Mice” package. Comparisons of baseline characteristics were conducted using independent-samples t-tests for continuous variables and χ2 tests for categorical variables. The relationship between sarcopenia and ALD was analyzed using two-sided logistic regression. Both unadjusted and adjusted models were used, adjusting for age, sex, race, marital status, smoking status, and alcohol consumption. Odds ratios (OR) and 95% confidence intervals (CI) were estimated. Nonlinear associations between ASM/BMI (as a continuous variable) and the prevalence of ALD were analyzed using two-sided restricted cubic splines (RCS) with a likelihood ratio test. All statistical analyses were performed using R software (version 4.4.1), which is available at http://www.R-project.org. A p-value of <0.05 was considered statistically significant.

For experimental data. Data were expressed as mean ± standard deviation (SD). An unpaired Student’s t-test was used to compare two groups. For three or more group comparisons, one-way ANOVA with Tukey’s test or two-way ANOVA with Sidak’s test was performed. Survival curve with Log-rank (Mantel–Cox) test was performed for Kaplan–Meier survival analysis. Two-tailed linear regression with Pearson’s Correlation Coefficient was used to determine the correlation between two different variables. All data are representative of more than 3 independent experiments. GraphPad Prism 9 was used for statistical analyses. Results were considered to be statistically significant when p < 0.05.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this manuscript, Shu et al established MG53 as a key mediator of muscle-liver communication in ALD, integrated zinc homeostasis and alcohol metabolism. And proposed a bio-derived nanoparticle platform for dual delivery of MG53 and zinc, addressed zinc deficiency and enzyme dysfunction synergistically. It offered a potential strategy for ALD treatments. Although the conclusion is largely supported by the data, some concerns should be addressed.\nSpecific comments:\n1. In Figure 1, plasma MG53 decreases while hepatic MG53 increases in ALD patients and ethanol-fed mice. Does MG53 expression change in other tissues other than the liver under these conditions?\n2. While MG53 is highly expressed in cardiac and skeletal muscle , and the study claims MG53 is \"muscle-derived,\" the manuscript lacks direct evidence demonstrating that MG53 in ALD patients or mice originates specifically from muscle tissue. Meanwhile, the manuscript does not quantify MG53 levels in muscle tissues (e.g., quadriceps, heart) of ALD mice or patients to correlate with liver MG53 accumulation.\n3. The manuscript demonstrates that MG53 knockout (MG53⁻/⁻) exacerbates ALD severity, while exogenous rhMG53 ameliorates it. The authors should address why MG53 deficiency leads to opposing effects on liver injury and metabolic pathways (e.g., increased oxidative stress but reduced ADH/ALDH2 activity). It is suggested that the dual role of MG53 should be discussed in the discussion.\n4. The manuscript identifies ADH and ALDH2 as MG53-interacting proteins via mass spectrometry and co-immunoprecipitation. However, the criteria for prioritizing these enzymes over other candidates (e.g., from the 205 overlapping proteins in Fig. 3A) are unclear.\n5. Figure 1D, sample size?", "reviewer_2": "1. Line 118, “Given that ANK1 had no impact on survival rates (fig. S1C), we excluded it from further consideration. Therefore, MG53 emerged as the primary candidate myokine facilitating skeletal muscle-liver communication in ALD.” Why dose the ANK1 have no impact on survival rates?\n2. As the concentration of EtOH escalates, there is a corresponding exacerbation of cellular lipid droplet accumulation and injury in AML12 cells, as indicated by the increased fluorescent intensity of Nile red (fig. S5D) and the marked reduction in cell viability (fig. S5E). should be “As the concentration of EtOH escalates, there is a corresponding exacerbation of cellular lipid droplet accumulation and injury in AML12 cells, as indicated by the increased fluorescent intensity of Nile red (fig. S5C) and the marked reduction in cell viability (fig. S5D).”\n3. EtOH treatment (400mM) for 24 hr also caused significant ROS elevation, evidenced by increased fluorescent intensity of DHE (fig. S5G) and increased levels of LDH in the culture medium (fig. S5H)….” Should be “EtOH treatment (400mM) for 24 hr also caused significant ROS elevation, evidenced by increased fluorescent intensity of DHE (fig. S5F) and increased levels of LDH in the culture medium (fig. S5G)”.\n4. This was reflected in the substantially improved cell viability (fig. S5F), reduction of DHE fluorescence (fig. S5G), and decreased LDH release (fig. S5H). should be “This was reflected in the substantially improved cell viability (fig. S5E), reduction of DHE fluorescence (fig. S5F), and decreased LDH release (fig. S5G).”\n5. Line 197, “We systemically administrated Cy5.5-labeled rhMG53 to mice and observed markedly elevated level of Cy5.5-rhMG53 in ALD mice (after 4-week exposure of EtOH), but not in control mice on a normal diet.” Where is the result of elevated level of Cy5.5-rhMG53 in ALD mice? In Figure 3K? Liver tissue?\n6. Line 255, “Systemic administration of MG53@Zn-NP facilitated targeted delivery of the MG53/Zn complex to the liver”, what is the targeting mechanism?\n7. How about oral administration of rhMG53@Zn-NPs in alleviation ALD? Dose it work for reduction of ALD?", "reviewer_3": "In this manuscript, the authors identified that muscle-derived MG53 is transported to the liver, and MG53 ameliorates alcohol-induced liver injury by enhancing ADH and ALDH2 activity. MG53’s B box motif coordinates zinc interaction with ADH and ALDH2, thereby enhancing their enzymatic activity. Finally, the authors developed a bio-derived nanoparticle for co-delivery of rhMG53 and zinc that ameliorates ALD.\nHere are some major concerns:\n1. In the paper, the authors reported that MG53 reduces alcohol-induced mortality. It is well known that alcohol-mediated mortality in mice is due to hypothermia not due to liver injury. Thus, the protective effect of MG53 on mortality is not due to protection against liver injury.\n2. The authors suggest that muscle-derived MG53 is transported into the liver, then interacted with ADH and ALDH2 to enhance the enzyme activity. However, ALDH2 is expressed in many other organs in addition to the liver. For example, a gut-liver ALDH2 axis, rather than liver ALDH2 alone, plays a key role in promoting acetaldehyde metabolism (PMID: 38902331). The authors should check whether MG53 also interacts with gut ALDH2.\n3. The authors show that MG53 KO mice had much higher serum ethanol and aldehyde levels than wt mice post alcohol feeding. Given the different ethanol metabolism, did wt and MG53 KO mice consumed different amounts of ethanol diet?\n4. The authors proposed that MG53 is transported into hepatocytes, what are the underlying mechanisms by which MG53 is transported into hepatocytes. ALDH2 is located in the mitochondria, what are the underlying mechanisms by which MG53 is transported into mitochondria to interact with ALDH2?\n5. The ethanol concentration in one fig. is mmol/L, but in another fig is mmol/ml; the serum aldehyde concentration in pair-fed is more than 50 micromol/ml, this is not correct. The basal levels should be undatable.\n6. The authors used the NIAAA model as described in the materials, but in the result section, the authors stated chronic alcohol feeding. The authors should state chronic-plus-binge ethanol feeding.", "reviewer_4": "In this manuscript, the authors investigated the role of MG53, a protein derived from muscle, in a murine model of alcoholic liver disease (ALD). Through transcriptomic and proteomic data analysis, they identified MG53 as a potential metabolic regulator of muscle-liver communication. Subsequent studies showed that MG53 improves survival and attenuates liver injury in mice by knocking out the MG53 gene or supplementing with exogenous MG53 protein in ALD. Mechanistically, the B-box motif of MG53 coordinates zinc ions to interact with the ethanol-metabolizing enzymes ADH and ALDH2, thereby enhancing their enzymatic activities and mitigating ethanol toxicity. Finally, the authors developed nanoparticles loaded with MG53 protein and zinc ions, which exhibited improved liver targeting and conferred enhanced protection in ALD models.\nOverall, this work presents a logical progression of findings with significant potential clinical therapeutic implications. However, some concerns those should be further addressed for the sake of strengthening the reliability of these findings and the main conclusions.\nMajor concerns:\n1. In Figure 2, the authors demonstrate a significant protective effect with rhMG53 treatment. To further characterize its role, Could the authors also administer rhMG53 mutants (such as either rhMG53-3A, rhMG53-1A, or rhMG53-C29L/C105S, as mentioned in Figure.4) to MG53-/- mice in the ALD model, followed by assessment of survival rates and hepatic injury.\n2. In Figure 3A and E, the authors conclude that MG53 binds directly to ADH/ALDH2 based on mass spectrometry and co-IP assays using liver tissue lysates. However, since lysates represent a complex mixture of proteins from multiple cell types, these data cannot confirm whether the interaction occurs within the same cell type. To address this limitation, the authors should perform co-IP assays in hepatocyte cell lines or isolated primary hepatocytes to validate the binding between MG53 and ADH/ALDH2 in a hepatocyte-specific context.\n3. In Figure 5J, the authors should quantify zinc ion levels in hepatocytes following supplementation with the MG53@Zn nanoparticle.\nMinor concerns:\n1. The model image shows MG53 in muscle entering the liver via circulation. In Figure 1G, did muscle loss occur in ALD patients or EtOH-fed mice, and was there a change in MG53 content in muscle? In addition, the control group in the immunoblotting experiment to detect MG53 in liver did not have lane background in lower panels in Figure 1G, and the authors need to provide original images without modification.\n2. In Figure 2, the authors administered rhMG53 treatment, which should have provided the relevant evidence to ensure liver delivery of rhMG53 in injury site.\n3. The authors should clarify what symbols, such as */#, represent for in most of figures, and specify which groups of samples were compared to carried on statistical analysis in most of figures and the corresponding p-values of the grouped samples."}, "author_response": "Response to Editors and Reviewers\nReviewer #1 (Remarks to the Author):\nIn this manuscript, Shu et al established MG53 as a key mediator of muscle-liver communication in ALD,\nintegrated zinc homeostasis and alcohol metabolism. And proposed a bio-derived nanoparticle platform for\ndual delivery of MG53 and zinc, addressed zinc deficiency and enzyme dysfunction synergistically. It offered\na potential strategy for ALD treatments. Although the conclusion is largely supported by the data, some\nconcerns should be addressed.\nSpecific comments:\n1. In Figure 1, plasma MG53 decreases while hepatic MG53 increases in ALD patients and ethanol-fed mice.\nDoes MG53 expression change in other tissues other than the liver under these conditions?\nResponse: Thank you for raising this important question. To examine MG53 expression in other tissues during\nALD, we performed additional studies in ethanol-fed mice (4-week treatment model). Western blot analysis\nrevealed a tissue-specific redistribution of MG53 following ethanol exposure. First, cardiac and skeletal\nmuscle tissues showed markedly reduced MG53 levels, suggesting alcohol suppresses MG53 synthesis at its\nprimary source (muscle/heart) (Data only for Review Fig. 1A-B). Second, plasma MG53 levels were\nconsistently decreased, likely reflecting both reduced synthesis in muscle and enhanced uptake by injured\ntissues such as liver and intestine (Fig. 1G). Third, we observed a significant increase of MG53 in intestinal\ntissue, consistent with its redistribution to injury-prone organs alongside the liver (Data only for Review Fig.\n1C-D; revised Fig. S5C).\nThese findings highlight alcohol’s multi-faceted effects on MG53 - reducing synthesis in muscle, lowering\ncirculating levels, and promoting uptake into liver and intestine to preserve tissue integrity under ethanol stress.\nWe are preparing a separate study to address the mechanisms by which alcohol suppresses MG53 expression\nand how muscle-derived MG53 supports gut function under chronic alcohol exposure. For now, we include\nthese new results in the Data for Review file (Data only for Review Fig. 1) for the editor and reviewers’\nconsideration.\n2. While MG53 is highly expressed in cardiac and skeletal muscle, and the study claims MG53 is \"muscle-\nderived,\" the manuscript lacks direct evidence demonstrating that MG53 in ALD patients or mice originates\nspecifically from muscle tissue. Meanwhile, the manuscript does not quantify MG53 levels in muscle tissues\n(e.g., quadriceps, heart) of ALD mice or patients to correlate with liver MG53 accumulation.\nResponse: We appreciate the reviewer’s insightful question. Since our discovery of MG53 in 2009, extensive\nstudies from our group and others have established that MG53 is predominantly expressed in skeletal and\ncardiac muscle, while liver lacks endogenous expression. To address this point specifically, we now include\nin revised Fig. S1D (FANTOM5 database) evidence of abundant MG53 expression in muscle but not liver.\nFurthermore. we performed new experiments using a muscle-specific MG53-EGPF fusion (AAV9-MHCK7-\nTrim72-EGFP, HanBio Co., LTD, Shanghai) in mice (Cell Metab. 2025;37:824; Cell Metab. 2023;35:2028).\nAfter 4 weeks of ethanol feeding, immunofluorescence revealed MG53-EGFP translocation from skeletal\nmuscle to injured hepatocytes (revised Fig. S2F). Western blot analysis confirmed an inverse relationship:\nMG53 decreased by 53% in quadriceps and 38% in heart tissue, while increasing in liver compared with pair-\nfed controls (Data only for Review Fig. 1; revised Fig. S5C). Because portion of these muscle expression\ndata for MG53 are part of another unpublished manuscript, we hope the Reviewer would agree that we only\nput the data in Data for Review only. Collectively, these results provide direct evidence that hepatic MG53 in\nALD derives from striated muscle.\n3. The manuscript demonstrates that MG53 knockout (MG53⁻/⁻) exacerbates ALD severity, while exogenous\nrhMG53 ameliorates it. The authors should address why MG53 deficiency leads to opposing effects on liver\ninjury and metabolic pathways (e.g., increased oxidative stress but reduced ADH/ALDH2 activity). It is\nsuggested that the dual role of MG53 should be discussed in the discussion.\nResponse: Thank you very much for your helpful comment and suggestion. As indicated by other references,\nROS is from multiple pathways, including ADH/ALDH2 and CYP2E1 (BMJ. 2023;383:e077090). Chronic\nalcohol consumption shifts alcohol metabolism from the ADH-dependent pathway to the cytochrome P450\nsystem via CYP2E1, increasing ROS production and causing Zn2+ deficiency (Hepatology. 1988;8:1605-9;\nNat Rev Dis Primers. 2018;4:16). As the references indicated, ROS production is higher in CYP2E1-P450\nthan ADH-dependent pathway, which leads to seemingly like opposing effects after chronic alcohol\nconsumption, i.e., increased oxidative stress but reduced ADH/ALDH2 activity, in fact, this seemingly like\nopposing effects were augmented by MG53 deficiency in our present study. We have expanded the Discussion\nin the revised manuscript (see in page 15, lines 5-13).\n4. The manuscript identifies ADH and ALDH2 as MG53-interacting proteins via mass spectrometry and co-\nimmunoprecipitation. However, the criteria for prioritizing these enzymes over other candidates (e.g., from\nthe 205 overlapping proteins in Fig. 3A) are unclear.\nResponse: The criteria for prioritizing these enzymes over other candidates was guided by a systematic,\nbioinformatics-driven approach. After identifying potential MG53-binding proteins in ALD, we performed\nKEGG pathway enrichment analysis, which highlighted the “Alcoholic liver disease” pathway as the most\nsignificantly enriched. Within this pathway, ADH, ALDH2, and CYP2E1 represent the core enzymes\nresponsible for ethanol metabolism. We prioritized ADH (coverage: 58%) and ALDH2 (coverage: 41%) over\nCYP2E1 (coverage: 14%) and other candidates, as ADH and ALDH2 are the primary alcohol-metabolizing\nenzymes and critical determinants of ethanol clearance and detoxification. Their stronger coverage with MG53\nfurther supports their predominant role in ALD pathogenesis (Annu Rev Pathol. 2023;18:411–38). For clarity,\na schematic model has been added to the revised manuscript (revised Fig. 3A).\n5. Figure 1D, sample size?\nResponse: We have now clearly stated the sample size in the revised figure legends, and the revised the\nimage with sample size in revised Fig. 1D.\nReviewer #2 (Remarks to the Author):\n1. Line 118, “Given that ANK1 had no impact on survival rates (fig. S1C), we excluded it from further\nconsideration. Therefore, MG53 emerged as the primary candidate myokine facilitating skeletal muscle-liver\ncommunication in ALD.” Why does ANK1 have no impact on survival rates?\nResponse: Thank you for this important question. The 5-year clinical follow up data shown in revised Fig.\nS1C were derived from published data base, which did not show a significant effect of ANK1 on patient\nsurvival. In addition, our literature search in PubMed found no published studies linking ANK1 to liver\nfunction or the pathogenesis of ALD. While the potential role of ANK1 in ALD warrants future investigation,\nthe current evidence supports MG53 as the primary candidate myokine mediating skeletal muscle-liver\ncommunication.\n2. As the concentration of EtOH escalates, there is a corresponding exacerbation of cellular lipid droplet\naccumulation and injury in AML12 cells, as indicated by the increased fluorescent intensity of Nile red (fig.\nS5D) and the marked reduction in cell viability (fig. S5E). should be “As the concentration of EtOH escalates,\nthere is a corresponding exacerbation of cellular lipid droplet accumulation and injury in AML12 cells, as\nindicated by the increased fluorescent intensity of Nile red (fig. S5C) and the marked reduction in cell viability\n(fig. S5D).”\nResponse: We are grateful for your specific recommendation, and have revised our manuscript accordingly.\n3. EtOH treatment (400mM) for 24 hr also caused significant ROS elevation, evidenced by increased\nfluorescent intensity of DHE (fig. S5G) and increased levels of LDH in the culture medium (fig. S5H)….”\nShould be “EtOH treatment (400mM) for 24 hr also caused significant ROS elevation, evidenced by increased\nfluorescent intensity of DHE (fig. S5F) and increased levels of LDH in the culture medium (fig. S5G)”.\nResponse: We are grateful for your specific recommendation, and have revised our manuscript accordingly.\n4. This was reflected in the substantially improved cell viability (fig. S5F), reduction of DHE fluorescence (fig.\nS5G), and decreased LDH release (fig. S5H). should be “This was reflected in the substantially improved cell\nviability (fig. S5E), reduction of DHE fluorescence (fig. S5F), and decreased LDH release (fig. S5G).”\nResponse: Again, thank you for your valuable suggestions. We have revised our manuscript accordingly.\n5. Line 197, “We systemically administrated Cy5.5-labeled rhMG53 to mice and observed markedly elevated\nlevel of Cy5.5-rhMG53 in ALD mice (after 4-week exposure of EtOH), but not in control mice on a normal\ndiet.” Where is the result of elevated level of Cy5.5-rhMG53 in ALD mice? In Figure 3K? Liver tissue?\nResponse: We apologize for lack of clarity in the text. The figure number is included, and the tissue is\nindicated in the revised manuscript. The sentence is revised as “We systemically administrated Cy5.5-labeled\nrhMG53 to mice and observed markedly elevated level of Cy5.5-rhMG53 in the livers of ALD mice (after 4-\nweek exposure of EtOH), but not in control mice on a normal diet (Fig. 3K).”\n6. Line 255, “Systemic administration of MG53@Zn-NP facilitated targeted delivery of the MG53/Zn complex\nto the liver”, what is the targeting mechanism?\nResponse: The hepatotropic behavior of MG53@Zn-NPs is governed by the innate biology of the albumin\nshell, which is a natural ligand for receptors highly expressed on various liver cells, including the FcRn\nreceptor on hepatocytes and the SPARC receptor on hepatic stellate cells (Expert Opin Drug Deliv.\n2015;12:793-812). The albumin-NPs target liver has been included in recent published papers (J Control\nRelease. 2025;382:113705; Artif Cells Nanomed Biotechnol. 2017;45:740-747; Artif Cells Nanomed\nBiotechnol. 2019;47:154-165). We have clarified this mechanism in the revised Discussion section (see page\n16, lines 15-20).\n7. How about oral administration of rhMG53@Zn-NPs in alleviation ALD? Dose it work for reduction of\nALD?\nResponse: We appreciate the reviewer’s suggestion. While oral administration remains the preferred route for\ntherapeutics due to its non-invasiveness and high patient compliance, the application for protein therapeutics\nis severely constrained by gastrointestinal barriers, including chemical degradation, mucus entrapment,\nepithelial impermeability, first-pass metabolism, and gut microbiota interactions. Advanced systems to deliver\nproteins using engineered probiotics, lipid-based, polymer-based, inorganic, and hybrid nanomaterials could\novercome some of these barriers, but the clinical translation is limited by regulatory, manufacturing, and\nbiological challenges (Adv Drug Deliv Rev. 2025;222:115603; Pharmacol Res. 2025;218:107844; Pharmacol\nRes. 2020;158:104685). Therefore, oral administration of rhMG53/HSA proteins remains a significant\nchallenge, and exploring innovative oral delivery platforms constitutes a very valuable direction for our future\nresearch.\nReviewer #3 (Remarks to the Author):\nIn this manuscript, the authors identified that muscle-derived MG53 is transported to the liver, and MG53\nameliorates alcohol-induced liver injury by enhancing ADH and ALDH2 activity. MG53’s B box motif\ncoordinates zinc interaction with ADH and ALDH2, thereby enhancing their enzymatic activity. Finally, the\nauthors developed a bio-derived nanoparticle for co-delivery of rhMG53 and zinc that ameliorates ALD.\nHere are some major concerns:\n1. In the paper, the authors reported that MG53 reduces alcohol-induced mortality. It is well known that\nalcohol-mediated mortality in mice is due to hypothermia not due to liver injury. Thus, the protective effect of\nMG53 on mortality is not due to protection against liver injury.\nResponse: We appreciate the reviewer’s comment regarding the mechanisms of alcohol-mediated mortality\nin mice. Indeed, a single high-dose binge (20–40% v/v ethanol) can cause death through CNS depression and\nhypothermia (Hepatology 2015;62:1086–100; Nat Genet 1996;14:98–101). However, our study employed a\nchronic ethanol protocol, where hypothermia is not considered the predominant cause of mortality (J Hepatol\n2016;64:1378–87; Gut 2023;72:1959–70; Hepatology 2023;78:878–95; Acta Pharm Sin B 2023;13:3906–18).\nTo further exclude this possibility, we now provided detail of our experimental procedure in the revised\nmanuscript to specifically indicate that all experiments were conducted in individually ventilated cages under\nthermoneutral conditions (see Data only for Review Fig. 2). Thus, the mortality observed in our model is\nunlikely attributable to hypothermia, supporting our conclusion that MG53 protects against alcohol-induced\ninjury.\n2. The authors suggest that muscle-derived MG53 is transported into the liver, then interacted with ADH and\nALDH2 to enhance the enzyme activity. However, ALDH2 is expressed in many other organs in addition to the\nliver. For example, a gut-liver ALDH2 axis, rather than liver ALDH2 alone, plays a key role in promoting\nacetaldehyde metabolism (PMID: 38902331). The authors should check whether MG53 also interacts with\ngut ALDH2.\nResponse: Thanks a lot for your insightful comments. We agree that extra-hepatic ALDH2, especially in the\nintestine, is a critical component of systemic ethanol/acetaldehyde clearance, and that the gut-liver axis is\ncentral to ALD pathogenesis (Nat Metab. 2024;6:1380-1396). Consistent with this concept, we detected\naccumulation of MG53 in ethanol-stimulated murine gut (revised Fig. S5C). Please see also our response to\nquestion 1 raised by reviewer #1. We further performed ALDH2 co-immunoprecipitation on intestinal tissues\nfrom chronically ethanol-fed mice. The results revealed MG53 interacts with gut ALDH2 (revised Fig. S5D).\n3. The authors show that MG53 KO mice had much higher serum ethanol and aldehyde levels than wt mice\npost alcohol feeding. Given the different ethanol metabolism, did wt and MG53 KO mice consumed different\namounts of ethanol diet?\nResponse: We used the same protocol and facility as that in published paper (refer: Nat Protoc. 2013;8:627-\n37), the intake food amount was checked by feeding bottles with scales. We confirm that there was no\nsignificant difference in ethanol intake between the WT and MG53 KO mice throughout the study, with the\nrecords on the 4th week as representation (please see revised Fig. S3E).\n4. The authors proposed that MG53 is transported into hepatocytes, what are the underlying mechanisms by\nwhich MG53 is transported into hepatocytes. ALDH2 is located in the mitochondria, what are the underlying\nmechanisms by which MG53 is transported into mitochondria to interact with ALDH2?\nResponse: Thank you for raising this insightful question. Our previous studies demonstrated that MG53\nlocalizes to mitochondria through binding to cardiolipin, thereby preserving mitochondrial integrity (Redox\nBiol. 2022;54:102357; Redox Biol. 2025;83:103663). We have also shown that MG53 can traverse the\nsinusoidal endothelium and enter hepatocytes to protect against liver injury (J Hepatol. 2022;76:558-567; J\nCell Mol Med. 2017;21:2503-2513). To further explore the mechanisms of MG53 uptake, we reanalyzed our\nco-immunoprecipitation/mass spectrometry data and found that MG53 associates with the hepatocyte-specific\nasialoglycoprotein receptor-1 (ASGR1), a well-characterized mediator of protein endocytosis (Cell.\n1983;32:277-87). These new findings suggest that MG53 may be internalized via ASGR1-mediated clathrin-\ndependent endocytosis, providing a plausible pathway for its entry into hepatocytes and subsequent\nmitochondrial targeting. Given the significance of this mechanism, we provide these preliminary data in Data\nonly for Review Fig. 3 and plan to present a comprehensive study on this pathway in a future manuscript.\nPlease also see our response to reviewer #1.\n5. The ethanol concentration in one fig. is mmol/L, but in another fig is mmol/m（l Fig3G）; the serum aldehyde\nconcentration in pair-fed is more than 50 micromol/ml, this is not correct. The basal levels should be undatable.\nResponse: Thank you so much for your meticulous review. The unit in the manuscript was wrongly indicated;\nthe unit for ethanol concentration should be “mmol/L”, and the unit for acetaldehyde concentration should be\n“μmol/L”. We agree with the Reviewer’s point that the basal levels should be undatable, due to technical\nlimitation – we have acknowledged this in our revised manuscript.\n6. The authors used the NIAAA model as described in the materials, but in the result section, the authors stated\nchronic alcohol feeding. The authors should state chronic-plus-binge ethanol feeding.\nResponse: Thank you for this precise and accurate comment. We sincerely apologize for the inconsistency in\nour terminology. We have carefully revised the manuscript throughout the Results and any other relevant\nsections to consistently and accurately describe our method as “chronic-plus-binge ethanol feeding”.\nReviewer #4 (Remarks to the Author):\nIn this manuscript, the authors investigated the role of MG53, a protein derived from muscle, in a murine\nmodel of alcoholic liver disease (ALD). Through transcriptomic and proteomic data analysis, they identified\nMG53 as a potential metabolic regulator of muscle-liver communication. Subsequent studies showed that\nMG53 improves survival and attenuates liver injury in mice by knocking out the MG53 gene or supplementing\nwith exogenous MG53 protein in ALD. Mechanistically, the B-box motif of MG53 coordinates zinc ions to\ninteract with the ethanol-metabolizing enzymes ADH and ALDH2, thereby enhancing their enzymatic\nactivities and mitigating ethanol toxicity. Finally, the authors developed nanoparticles loaded with MG53\nprotein and zinc ions, which exhibited improved liver targeting and conferred enhanced protection in ALD\nmodels. Overall, this work presents a logical progression of findings with significant potential clinical\ntherapeutic implications. However, some concerns those should be further addressed for the sake of\nstrengthening the reliability of these findings and the main conclusions.\nMajor concerns:\n1. In Figure 2, the authors demonstrate a significant protective effect with rhMG53 treatment. To further\ncharacterize its role, Could the authors also administer rhMG53 mutants (such as either rhMG53-3A,\nrhMG53-1A, or rhMG53-C29L/C105S, as mentioned in Figure.4) to MG53-/- mice in the ALD model, followed\nby assessment of survival rates and hepatic injury.\nResponse: Following your recommendation, we performed additional experiments to evaluate the rhMG53-\nC29L/C105S mutant in our ALD mouse model. MG53-/- mice were subjected to a 4-week chronic-plus-binge\nethanol feeding and treated with rhMG53-C29L/C105S via micro-osmotic pump (1 mg/kg with 1 µg/hr).\nUnlike wild type rhMG53, the mutant protein had no significantly effect on alcohol-induced mortality or liver\ninjury in MG53-/- mice. These new data are presented in the revised Fig. S7D-F (see in page 12, lines 13-18).\n2. In Figure 3A and E, the authors conclude that MG53 binds directly to ADH/ALDH2 based on mass\nspectrometry and co-IP assays using liver tissue lysates. However, since lysates represent a complex mixture\nof proteins from multiple cell types, these data cannot confirm whether the interaction occurs within the same\ncell type. To address this limitation, the authors should perform co-IP assays in hepatocyte cell lines or\nisolated primary hepatocytes to validate the binding between MG53 and ADH/ALDH2 in a hepatocyte-specific\ncontext.\nResponse: Thanks a lot for your valuable suggestion. We have performed additional co-immunoprecipitation\nexperiments using lysates from mouse hepatic parenchymal cells (CP-M033, Procell, Wuhan) treated with\nethanol (400 mM for 24 hours). The results demonstrate that endogenous ADH and ALDH2 co-precipitate\nwith MG53 in the rhMG53 treatment group following ethanol stimulation (revised Fig. S5K) (see in page 11,\nline 9).\n3. In Figure 5J, the authors should quantify zinc ion levels in hepatocytes following supplementation with the\nMG53@Zn nanoparticle.\nResponse: Thank you for valuable suggestion. As indicated in Figure 6H in the original manuscript, which\nshowed the hepatic Zn2+ levels were significantly increased in MG53@Zn-NPs group, detected by Inductively\nCoupled Plasma Mass Spectrometry. To further show the exogenous Zn2+ from MG53@Zn-NPs, we used liver\nfrozen sections to incubate with Zn2+ probe. Confocal microscopy images revealed a significant increase in\nintracellular Zn2+ fluorescence in hepatocytes of mice treated with MG53@Zn-NPs, with clear co-localization\nof the Zn2+ signal with the Cy5.5 fluorescence from the labeled nanoparticle (revised Fig. 5J). These data\nhave been incorporated into the revised manuscript (see in page 13, lines 15-17).\nMinor concerns:\n1. The model image shows MG53 in muscle entering the liver via circulation. In Figure 1G, did muscle loss\noccur in ALD patients or EtOH-fed mice, and was there a change in MG53 content in muscle? In addition,\nthe control group in the immunoblotting experiment to detect MG53 in liver did not have lane background in\nlower panels in Figure 1G, and the authors need to provide original images without modification.\nResponse: As noted in our response to reviewer #1, we now provide new data showing That alcohol\nconsumption significantly reduces MG53 expression in both skeletal and cardiac muscle, and that rhMG53\ntreatment mitigates skeletal muscle loss in ALD model (revised Fig. S4B) (see in page 9, lines 2-3).\nConsistently, MG53 levels in muscle were lower in ALDH model to controls (Data only for Review Fig. 1A).\nBecause these muscle-specific MG53 data are part of a separate unpublished study, we have included them\nhere only in the “Data only for Review.”\nRegarding to the Figure 1G, we changed the image contrast, which showed the lane background in the revised\nimage (revised Fig. 1G), and the original full scan of Figure 1G was provided in Source Data-Unprocessed\nwestern blots.\n2. In Figure 2, the authors administered rhMG53 treatment, which should have provided the relevant evidence\nto ensure liver delivery of rhMG53 in injury site.\nResponse: To verify that rhMG53 reaches the injured liver, we administered Cy5.5-labelled rhMG53 to ALD\nmice. As shown in Figure 3K in the original manuscript, fluorescence imaging revealed accumulation of\nCy5.5-rhMG53 in the alcohol-injured livers, demonstrating efficient, injury-targeted delivery of rhMG53 to\nthe liver (see in page 11, lines 3-5).\n3. The authors should clarify what symbols, such as */#, represent for in most of figures, and specify which\ngroups of samples were compared to carried on statistical analysis in most of figures and the corresponding\np-values of the grouped samples.\nResponse: We thank the reviewer for this helpful suggestion. In the revised manuscript, all figure legends\nhave been updated to clearly define the statistical symbols (e.g., *, #), specify the groups compared in each\nanalysis, and provide the corresponding p-value ranges."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors answered my concerns.", "reviewer_2": "All my questions have been fully addressed by the authors, so the paper may be accepted without further revisions.", "reviewer_3": "the authors have adequately addressed my concerns", "reviewer_4": "The authors have addressed the most of my comments. However, there were the left minor concerns on the quality the immunostaining and the amount of Zinc ions in vehicle control in revised Figure 5J. First, the quality of immunofluorescence staining of zinc ions was quite lower in left-lower panel shown in revised Figure 5J, it seemed that there were some nonspecific staining signals from the background shown in the image. The authors need to replace it with one higher-quality zinc ion staining image for Figure 5J to validate their findings.\nIn addition, according to there data shown in revised Figure 6H, it seemed that the amount of hepatic Zinc ions from the vehicle control in wet liver could be comparable with that detected in the sample from MG53@Zn. However, there were barely specific signal of the immunofluorescence staining for Zinc ions in the upper left panel in vehicle control presented in revised Figure 5J, could the author explain the reason in discussed session?"}, "author_response": "Response to Editors and Reviewers\nReviewer #4 (Remarks to the Author):\n1. The authors have addressed the most of my comments. However, there were the left minor concerns on the\nquality the immunostaining and the amount of Zinc ions in vehicle control in revised Figure 5J. First, the\nquality of immunofluorescence staining of zinc ions was quite lower in left-lower panel shown in revised\nFigure 5J, it seemed that there were some nonspecific staining signals from the background shown in the\nimage. The authors need to replace it with one higher-quality zinc ion staining image for Figure 5J to validate\ntheir findings.\nResponse: According to the Reviewer’s suggestion, we replace the zinc ion immunofluorescence staining\nwith a representative one. It should be noticed, due to the endogenous zinc ion in the normal liver (Am J Pathol.\nstaining.\n2. In addition, according to there data shown in revised Figure 6H, it seemed that the amount of hepatic Zinc\nions from the vehicle control in wet liver could be comparable with that detected in the sample from MG53@Zn.\nHowever, there were barely specific signal of the immunofluorescence staining for Zinc ions in the upper left\npanel in vehicle control presented in revised Figure 5J, could the author explain the reason in discussed\nsession?\nResponse: Thanks a lot for your consideration. In fact, the amount of hepatic zinc ions from the vehicle control\nand MG53@Zn (for 24hr) in revised Figure 5J was not comparable with that detected in the sample (for 8\nweeks) in revised Figure 6H. In Figure 6H, after 8-weeks chronic treatment duration, the hepatic Zinc ions\naccumulations in mice received MG53@Zn with pair-fed diet were significantly higher than that in mice\nreceived vehicle with pair-fed diet."}]

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