Use of mg53 protein modification in diagnosis and treatment of metabolic-related diseases

Abstract

The present disclosure relates to the field of biological medicine, and particularly relates to application of MG53 protein modification in diagnosis and treatment of metabolic related diseases. The present disclosure provides application of a substance for detecting MG53 protein modification in preparation of a reagent for diagnosing metabolic related diseases, a product for diagnosing metabolic related diseases, and application of a substance for inhibiting MG53 protein modification in preparation of a medicine for preventing or treating metabolic related diseases. The present disclosure provides a new direction for diagnosis and effective treatment of metabolic related diseases, and has a broad application prospect in clinic.

Description

Technical Field

[0001] This disclosure relates to the biomedical field, specifically to the application of MG53 protein modification in the diagnosis and treatment of metabolic-related diseases. Background Technology

[0002] MG53 (Mitsugumin53), also known as TRIM72, is a member of the TRIM protein family, primarily expressed in skeletal and cardiac muscle, and possesses multiple biological functions. First, MG53 promotes the repair of skeletal and cardiac cell membrane damage. Second, it acts as a scaffold protein supporting the formation of pro-cell survival complexes during myocardial ischemia preconditioning and postconditioning. Third, as an E3 ubiquitin ligase, MG53 can inhibit the insulin signaling pathway and myogenesis by mediating the ubiquitination and degradation of its substrates. Finally, as a transcription factor, MG53 can induce diabetic cardiomyopathy by upregulating the transcription of target genes. Studies have shown that dysregulation of MG53 protein regulation is associated with the development and progression of related diseases.

[0003] Protein acetylation has become a key post-translational modification in cellular regulation. It refers to the enzyme-catalyzed transfer of an acetyl group from acetyl-CoA (Ac-CoA) to the ε-amino group of a specific lysine residue side chain. Acetyltransferases (KATs) catalyze the addition of an acetyl group to the ε-amino group of a protein substrate's lysine residue. The reverse reaction, deacetylation, is mediated by deacetylases (KDACs), catalyzing the removal of the acetyl group. The attachment of an acetyl group to the amino group of lysine changes its positively charged state to neutral while simultaneously increasing the size of the amino acid side chain, thereby influencing the bias of protein secondary structure and its interactions with other biomolecules.

[0004] Ubiquitination is one of the most common post-translational modifications of proteins involved in regulating cell signal transduction and homeostasis. Ubiquitin (Ub) is a small protein composed of only 76 amino acids, widely distributed in eukaryotes. At the molecular level, ubiquitin can be viewed as an intracellular signal, reversibly linked to substrate proteins to regulate a range of cellular biological functions. Ubiquitination modifications are widely involved in the regulation of protein structure and function, playing crucial roles in cell signal transduction, DNA damage repair, and apoptosis.

[0005] In eukaryotic cells, acetylation is one of the most common covalent protein modifications, ranking alongside phosphorylation. Initially, it was thought to be an epigenetic modification mechanism that regulates chromatin looseness and DNA accessibility by modifying histones. Later, many studies found that acetylation can also occur on non-histone proteins, thereby regulating protein activity or stability, subcellular localization, and protein-protein interactions. Furthermore, as an important post-translational modification, acetylation may interact with other types of modifications, jointly participating in the functional regulation of proteins. Acetylation is a reversible reaction and is widely involved in protein regulation processes in response to metabolic changes. Some metabolic intermediates, such as oxidized nicotinamide adenine dinucleotide (NAD+) and Ac-CoA, directly or indirectly participate in the acetylation chemical reaction process. Therefore, acetylation modification, to some extent, reflects the metabolic responses of cellular nutritional status and is linked to protein regulation related to energy storage and cellular homeostasis. Protein acetylation is particularly associated with many common metabolic diseases, including obesity, type 2 diabetes, neurodegenerative diseases, and cancer, as well as some rare diseases such as mitochondrial diseases and lipid metabolism disorders. Therefore, there is a need for products that can be used to diagnose and treat metabolic-related diseases. Summary of the Invention

[0006] This disclosure provides the application of a substance that detects MG53 expression or its modification in a sample in the preparation of a reagent for diagnosing metabolic-related diseases, a product for diagnosing metabolic-related diseases, the application of a substance that inhibits MG53 expression or its modification in a subject in the preparation of a drug for preventing or treating metabolic-related diseases, a system and / or device for detecting and diagnosing metabolic-related diseases in a subject, and a biomarker for diagnosing metabolic-related diseases.

[0007] According to one aspect of this disclosure, there is an application of detecting MG53 expression or substances modified thereon in a sample in the preparation of reagents for diagnosing metabolic-related diseases.

[0008] According to another aspect of this disclosure, there is an application of detecting MG53 expression or substances modified therefrom in a sample for the diagnosis of metabolic-related diseases.

[0009] According to another aspect of this disclosure, a method for detecting MG53 expression or its modification in a sample is provided, the method comprising the step of detecting the sample to be tested using a substance capable of detecting MG53 expression or its modification in the sample.

[0010] In some embodiments, the reagent includes one or more of the following: 1) Reagents for detecting MG53 expression in samples; 2) Reagents for detecting MG53 protein modification in samples.

[0011] In some embodiments, the MG53 expression includes expression at the DNA, RNA, and / or protein levels.

[0012] In some embodiments, the MG53 protein modification includes one or more of ubiquitination, acetylation, phosphorylation, glycosylation, methylation, biotinylation, SUMOylation, crotonylation, succinylation, malonylation, hydroxybutyrylation, and lactylation.

[0013] In some embodiments, the MG53 protein modification includes ubiquitination and / or acetylation. In a preferred embodiment, the MG53 protein modification is a modification of lysine (Lys, K) at position 317 of the amino acid sequence shown in SEQ ID NO: 11.

[0014] Those skilled in the art will understand that although this disclosure uses the amino acid sequence shown in SEQ ID NO: 11 as an example sequence to detect the modification at lysine position 317, the lysine at this position in the MG53 protein is highly conserved across different species. Therefore, it can be expected that similar modifications are present at lysine position 317 corresponding to the amino acid sequence shown in SEQ ID NO: 11 in the MG53 protein or its homologous sequences (e.g., monkeys, mice, rats, rabbits).

[0015] In some embodiments, the detection includes immunoassay, mass spectrometry, chromatography, spectroscopy, and / or PCR amplification.

[0016] In some embodiments, the reagent includes an antibody or antigen-binding fragment thereof that specifically binds to the MG53 protein, an antibody or antigen-binding fragment thereof that specifically binds to the modified MG53 protein, primers that specifically amplify the encoding nucleotide sequence of MG53, and / or probes that specifically recognize the encoding nucleotide sequence of MG53.

[0017] In some implementations, the sample includes a cell sample, a tissue sample, or a body fluid sample.

[0018] In some embodiments, the tissue sample includes fresh tissue, paraffin sections, and their processed forms.

[0019] In some embodiments, the body fluid sample includes blood, plasma, serum, lymph, pleural fluid, cerebrospinal fluid, synovial fluid, ascites, saliva, lymph, or other bodily fluids.

[0020] In some embodiments, the metabolic-related diseases include diabetes, non-alcoholic fatty liver disease, hyperlipidemia, atherosclerosis, cardiovascular disease, hypertension, obesity, and insulin resistance syndrome.

[0021] In some implementations, the diabetes includes type 1 diabetes, type 2 diabetes, and gestational diabetes.

[0022] In some implementations, the diagnosis includes: 1) When the expression level of MG53 in the sample is elevated compared with that of healthy subjects, the subjects are judged to have metabolic-related diseases; 2) If the acetylation modification level of MG53 protein in the sample is elevated compared to healthy subjects, the subject is judged to have a metabolic-related disease; and / or 3) If the ubiquitination level of MG53 protein in the sample is reduced compared with that of healthy subjects, the subject is judged to have a metabolic-related disease.

[0023] According to another aspect of this disclosure, a product for diagnosing metabolic-related diseases is provided, the product comprising the reagents of this disclosure capable of detecting the expression level of MG53 or its modification level in a sample.

[0024] In some embodiments, the reagent includes one or more of the following: 1) Reagents for detecting MG53 expression in samples; 2) Reagents for detecting MG53 protein modification in samples.

[0025] In some embodiments, the reagent includes an antibody or antigen-binding fragment thereof that specifically binds to the MG53 protein, an antibody or antigen-binding fragment thereof that specifically binds to the modified MG53 protein, primers that specifically amplify the encoding nucleotide sequence of MG53, and / or probes that specifically recognize the encoding nucleotide sequence of MG53.

[0026] In some embodiments, the metabolic-related diseases include diabetes, non-alcoholic fatty liver disease, hyperlipidemia, atherosclerosis, cardiovascular disease, hypertension, obesity, and / or insulin resistance syndrome.

[0027] In some embodiments, the diabetes includes type 1 diabetes, type 2 diabetes, and / or gestational diabetes.

[0028] In some embodiments, the product includes a test kit, a test chip, and / or a test strip.

[0029] According to another aspect of this disclosure, the use of substances that inhibit MG53 expression or its modifications in subjects in the preparation of medicaments for the prevention or treatment of metabolic-related diseases is provided.

[0030] According to another aspect of this disclosure, the use of substances that inhibit MG53 expression or its modifications in subjects in medicaments for the prevention or treatment of metabolic-related diseases is provided.

[0031] According to another aspect of this disclosure, a method for preventing or treating metabolic-related diseases is provided, the method comprising administering to a subject a drug that inhibits MG53 expression or a modification thereof in the subject.

[0032] In some embodiments, the drug includes one or more of the following: 1) Drugs that inhibit MG53 expression in subjects; 2) Drugs that inhibit MG53 protein modification in subjects.

[0033] In some embodiments, the MG53 expression includes MG53 expression levels at the DNA, RNA, and / or protein levels.

[0034] In some embodiments, the MG53 protein modification includes ubiquitination and / or acetylation. In a preferred embodiment, the MG53 protein modification is a modification of lysine residue 317 corresponding to the amino acid sequence shown in SEQ ID NO: 11.

[0035] In some embodiments, the detection includes immunoassay, mass spectrometry, chromatography, spectroscopy, and / or PCR amplification.

[0036] In some embodiments, the drug comprises an antibody or antigen-binding fragment thereof that specifically binds to the MG53 protein, an antibody or antigen-binding fragment thereof that specifically binds to a modified MG53 protein, a compound or small nucleic acid molecule that inhibits MG53 expression.

[0037] In some embodiments, the metabolic-related diseases include diabetes, non-alcoholic fatty liver disease, hyperlipidemia, atherosclerosis, cardiovascular disease, hypertension, obesity, and / or insulin resistance syndrome.

[0038] In some embodiments, the diabetes includes type 1 diabetes, type 2 diabetes, and / or gestational diabetes.

[0039] In some embodiments, the subjects include mammals. In some embodiments, the subjects include humans, non-human primates (e.g., chimpanzees), dogs, mice, rats, guinea pigs, rabbits, cats, and alpacas.

[0040] According to another aspect of this disclosure, a system and / or apparatus for diagnosing metabolic-related diseases in a subject is provided, characterized in that the system and / or apparatus comprises: Computer-readable media containing instructions; A processor is used to execute the instructions and process the input information using bioinformatics methods; The input module is used to input data on MG53 expression or its modifications in the sample; and The output module is used to output whether the subject has a metabolic-related disease.

[0041] In some embodiments, the MG53 expression includes MG53 expression levels at the DNA, RNA, and / or protein levels.

[0042] In some embodiments, the MG53 modification includes MG53 protein modification.

[0043] In some embodiments, the MG53 modification includes one or more of ubiquitination, acetylation, phosphorylation, glycosylation, methylation, biotinylation, SUMOylation, crotonylation, succinylation, malonylation, hydroxybutyrylation, and lactylation.

[0044] In some embodiments, the MG53 protein modification includes ubiquitination and / or acetylation.

[0045] In some embodiments, the MG53 protein is modified at lysine position 317, corresponding to the amino acid sequence shown in SEQ ID NO: 11.

[0046] In some implementations, the diagnosis includes: 1) When the expression level of MG53 in the sample is elevated compared with that of healthy subjects, the subjects are judged to have metabolic-related diseases; 2) If the acetylation modification level of MG53 protein in the sample is elevated compared to healthy subjects, the subject is judged to have a metabolic-related disease; and / or 3) If the ubiquitination level of MG53 protein in the sample is reduced compared with that of healthy subjects, the subject is judged to have a metabolic-related disease.

[0047] According to another aspect of this disclosure, a biomarker for diagnosing metabolic-related diseases is provided, said biomarker comprising a modified MG53 protein.

[0048] In some embodiments, the biomarker includes MG53 protein with a modified lysine residue at position 317.

[0049] In some embodiments, the biomarker includes the MG53 protein with acetylation and / or ubiquitination modification at lysine position 317.

[0050] The beneficial effects of this disclosure are: The diagnostic biomarker of MG53 protein with modified lysine at position 317 disclosed herein can be used for the diagnosis, treatment and prevention of metabolic-related diseases, and can help in the later development of precision medicine products. Attached Figure Description

[0051] Figure 1 The K317 mutation enhances the stability of MG53 protein. (A) Immunoblotting analysis of the stability of MG53-WT (MG53 wild-type) and MG53-K317R proteins in cells treated with actinomycete ketone. (B) Quantitative statistical graph of protein levels in MG53-WT and MG53-K317R mutants after different treatment times with actinomycete ketone. (C) Intracellular ubiquitination levels of MG53-WT and MG53-K317R mutants. Protein expression levels were standardized to match the expression level of the internal control β-actin. Data are presented as mean ± standard error (mean ± SEM). p < 0.001, ns indicates no significant difference. For each immunoprecipitation assay, the sample loading represented 5% of the total sample volume. All experiments were repeated at least 3 times, and similar results were obtained.

[0052] Figure 2 The validation of the MG53-K317 site-specific acetylation antibody is shown. (A) Immunoblot detection of antibody binding to acetylated and non-acetylated peptides. (B) Immunoblot detection of acetylation signal at the K317 site of exogenously overexpressed MG53 protein in HEK293T cells. (C) Immunoblot detection of acetylation signal at the K317 site of exogenously overexpressed MG53 protein in C2C12 myotube cells. (D) Immunoblot detection of acetylation signal at the K317 site of endogenously expressed MG53 protein in differentiated C2C12 myotube cells. (E) Immunoblot detection of acetylation signal at the K317 site of endogenously expressed MG53 protein in skeletal muscle of C57BL / 6J wild-type mice. All antibody validation experiments were repeated at least three times, and similar results were obtained.

[0053] Figure 3 The specificity of the peptide competitive binding assay for detecting acetylated antibodies at the MG53-K317 site is shown. (A) Immunoblotting analysis of the inhibitory effect of pre-incubation of acetylated peptides on antibody binding to MG53 protein on the membrane. (B) Immunoblotting analysis of the inhibitory effect of pre-incubation of non-acetylated peptides on antibody binding to MG53 protein on the membrane. All antibody validation experiments were repeated at least three times, yielding similar results.

[0054] Figure 4This study shows the downregulation of endogenous K317 acetylation and MG53 protein levels in C2C12 cells under starvation treatment. (A) Immunoblotting analysis of endogenous K317 acetylation and MG53 protein levels in C2C12 myotube cells under starvation treatment. (B) Quantitative statistical graph of K317 acetylation levels under starvation treatment. (C) Quantitative statistical graph of MG53 protein levels under starvation treatment. (D) Quantitative statistical graph of endogenous MG53 mRNA levels in C2C12 cells under starvation treatment. In the experiment, MG53 protein expression levels were standardized to the level of the internal control β-actin, and K317 acetylation levels were standardized to the level of total MG53 protein. MG53 mRNA values ​​were standardized to the level of the internal control 18S. Data are presented as mean ± standard error (mean ± SEM). This indicates that p < 0.05 and n = 7 for each group.

[0055] Figure 5 This study demonstrates that glucose regulates the acetylation level at the K317 site and the stability of the MG53 protein in a concentration-dependent manner.

[0056] (A) Immunoblotting analysis of K317 acetylation and MG53 protein levels at different time points after cyclohexane treatment under different sugar concentrations. (B) Comparison of intracellular MG53 protein stability under different sugar concentrations, presented as a broken-line graph. MG53 protein expression levels were standardized to match the expression level of the internal control β-actin. Data are presented as mean ± standard error (mean ± SEM). This indicates that at this time point, the high-sugar control group had a p < 0.05 compared to the sugar-free control group, and # indicates that the low-sugar (5mM) control group had a p < 0.05 compared to the sugar-free control group. This indicates that the high-sugar control group had a p < 0.01 compared to the sugar-free control group, and each group had n = 3.

[0057] Figure 6 The study demonstrated that deacetylase inhibitors enhance the stability of MG53 protein under starvation conditions.

[0058] (A) Immunoblotting analysis of K317 acetylation and MG53 protein levels at different time points after pretreatment with deacetylase inhibitors and cyclohexane treatment. (B) A line graph comparing the stability of MG53 protein under different sugar concentrations under deacetylase inhibitor treatment. MG53 protein expression levels were standardized to match the expression level of the internal control β-actin. Data are presented as mean ± standard error (mean ± SEM). This indicates that at this time point, the high-glucose level was significantly higher than the sugar-free control group (p < 0.05). The values ​​indicate that at this time point, the high-sugar treatment was <0.01 compared to the sugar-free control group (p < 0.01), # indicates that the sugar-free treatment with inhibitors was <0.05 compared to the sugar-free control group (p < 0.05), and ## indicates that the sugar-free treatment with inhibitors was <0.01 compared to the sugar-free control group (p < 0.01). Each group had n = 3, and ns indicates no significant difference. The solvent represents the control group treated with a solvent that did not contain nicotinamide or trogopterin A.

[0059] Figure 7 The ubiquitination levels of MG53 protein under different sugar concentrations and deacetylase inhibitor treatments are shown. For each immunoprecipitation experiment, the sample loading represented 5% of the total sample volume. Gluc: glucose. The experiments were repeated at least three times, and similar results were obtained.

[0060] Figure 8 It shows db / db Acetylation at K317 site and MG53 protein levels in skeletal muscle of mice and HFD mice. (A) Immunoblotting detection db / db Acetylation at K317 site and MG53 protein levels in mouse skeletal muscle. (B) db / db Quantitative statistical graph of acetylation levels at the K317 site in mouse skeletal muscle. (C) db / db Quantitative statistical graphs of MG53 protein levels in mouse skeletal muscle. (D) Immunoblotting detection of K317 acetylation and MG53 protein levels in HFD mouse skeletal muscle. (E) Quantitative statistical graphs of K317 acetylation levels in HFD mouse skeletal muscle. (F) Quantitative statistical graphs of MG53 protein levels in HFD mouse skeletal muscle. In the experiment, MG53 protein expression levels were standardized to the expression level of the internal control GAPDH, and K317 acetylation levels were standardized to the total MG53 protein level. Data are presented as mean ± standard error (mean ± SEM). This means p < 0.001 and n = 6.

[0061] Figure 9The following figures illustrate the levels of K317 acetylation and MG53 protein in the skeletal muscle of monkeys with metabolic syndrome and obese patients. (A) Immunoblotting detection of K317 acetylation and MG53 protein levels in the skeletal muscle of monkeys with metabolic syndrome. (B) Quantitative statistical graph of K317 acetylation level in the skeletal muscle of monkeys with metabolic syndrome. (C) Quantitative statistical graph of MG53 protein level in the skeletal muscle of monkeys with metabolic syndrome. (D) Immunoblotting detection of K317 acetylation and MG53 protein levels in the skeletal muscle of obese patients. (E) Quantitative statistical graph of K317 acetylation level in the skeletal muscle of obese patients. (F) Quantitative statistical graph of MG53 protein level in the skeletal muscle of obese patients. MG53 protein expression levels were standardized against the internal control GAPDH, and K317 acetylation levels were standardized against the total MG53 protein content. Data are presented as mean ± standard error (mean ± SEM). This indicates that p < 0.01, and n = 6 for each group.

[0062] Figure 10 The study showed K317 acetylation and MG53 protein levels in the skeletal muscle of mice subjected to starvation and chronic exercise.

[0063] (A) Immunoblotting detection of K317 acetylation and MG53 protein levels in skeletal muscle of starved mice. (B) Quantitative statistical graph of K317 acetylation levels in skeletal muscle of starved mice. (C) Quantitative statistical graph of MG53 protein levels in skeletal muscle of starved mice. (D) Immunoblotting detection of K317 acetylation and MG53 protein levels in skeletal muscle of chronically exercised mice. (E) Quantitative statistical graph of K317 acetylation levels in skeletal muscle of chronically exercised mice. (F) Quantitative statistical graph of MG53 protein levels in skeletal muscle of chronically exercised mice. In the experiment, MG53 protein expression levels were standardized to the expression level of the internal control GAPDH, and K317 acetylation levels were standardized to the total MG53 protein level. Data are presented as mean ± standard error (mean ± SEM). This means p < 0.05n = 6 - 9.

[0064] Figure 11 The degradation ubiquitination levels of MG53 protein in the skeletal muscle of diabetic mice, high-fat diet-induced obese mice, and starved mice are shown. (A) Immunoblotting detection db / dbDegradative ubiquitination level of MG53 protein in skeletal muscle of diabetic mice. (B) Immunoblotting detection of degradative ubiquitination level of MG53 protein in skeletal muscle of HFD mice. (C) Immunoblotting detection of degradative ubiquitination level of MG53 protein in skeletal muscle of starved mice. For each immunoprecipitation experiment, the sample loading represented 5% of the total sample volume. All experiments were repeated at least 3 times, and similar results were obtained. Detailed Implementation

[0065] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts of this disclosure. Such structures and techniques have also been described in many publications.

[0066] definition Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa.

[0067] Unless the context clearly indicates otherwise, the terms “a” and “an” as used herein include plural references. For example, reference to “a cell” includes multiple such cells and equivalents known to those skilled in the art, etc.

[0068] As used herein, the term "about" indicates a range of ±20% of the following value. In some embodiments, the term "about" indicates a range of ±10% of the following value. In some embodiments, the term "about" indicates a range of ±5% of the following value.

[0069] Metabolic syndrome (MetS) refers to a group of metabolic disorders including insulin resistance, central obesity, dyslipidemia, and hypertension, which can increase the risk of cardiovascular disease and type 2 diabetes. Insulin resistance (IR) is the core pathogenic mechanism of metabolic syndrome.

[0070] The following examples are provided to aid in understanding the invention. However, it should be understood that these examples are for illustrative purposes only and do not constitute any limitation. The actual scope of protection of the invention is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the invention. The reagents and / or kits used in the following examples are commercially available or can be synthesized by known methods.

[0071] It should be noted that, unless specific conditions are specified in the examples, experimental conditions should be performed according to standard conditions, manufacturer recommendations, or publicly reported experimental conditions. Reagents or instruments whose manufacturers are not specified are all commercially available, standard products. For reagents whose manufacturers are specified, similar products from other manufacturers are substitutes.

[0072] Unless otherwise specified, the quantitative experiments in the following examples were all repeated three times, and the results were averaged. Example

[0073] 1. Experimental Materials 1.1 Laboratory animals and cell lines The animals used in this study included C57BL / 6J wild-type mice (+ / +) and diabetic mice. db / db High-fat diet (HFD) induced mouse model, etc. Treatment methods for high-fat diet (HFD) mice and control (Chow) mice: Six-week-old male C57BL / 6J mice were fed a 60% fat diet (Research DIETS, catalog number D1249i) for 8-12 weeks. The successful establishment of the model was verified by monitoring changes in body weight, blood lipids, and adipose tissue. The control Chow mice were fed a standard diet (SPF-F02-001).

[0074] Spontaneous metabolic syndrome monkeys (MS) were normally fed and spontaneously developed obesity and metabolic syndrome. Control monkeys (Ctrl) were healthy control monkeys. Both were provided by the Peking University Animal Center (see Song, Ruisheng et al. "Centralrole of E3 ubiquitin ligase MG53 in insulin resistance and metabolic disorders." Nature, vol. 494,7437 (2013): 375-9.).

[0075] Obese patients with skeletal muscle (Obese, BMI ≥ 28 kg / m²) 2 The skeletal muscle (Lean) and normal body type control were obtained from Peking University Third Hospital.

[0076] Induction methods for chronic exercise mice (Exe) and control groups (Sed): 19-20 week old C57 BL / 6 mice were randomly divided into a sedentary control group or a chronic exercise group according to their body weight. Both groups were allowed free access to food, with 5 mice per cage. The ambient temperature was controlled at 25°C, and the light cycle was 12 hours of light and 12 hours of darkness. Subsequently, the control group received no treatment, while the experimental group received exercise training every night between 6-10 pm. The exercise conditions on the treadmill (SANS BioInstrument, SA102) were as follows: In the first week, the exercise time was gradually increased from 5 minutes to 10 minutes. The initial treadmill conditions were: speed 5 m / min, duration 2 minutes; speed 7 m / min, duration 2 minutes (second round); speed 9 m / min, duration 1 minute (third round). The speed in the third round was gradually increased from 9 m / min to 10 m / min. Starting from the second week, the speed is 7 m / min for 2 minutes (first round); the speed is 9 m / min for 2 minutes (second round); and the speed is 10 m / min for 35 minutes (third round). 40 minutes / day, 5 days a week, for a total of 5-6 weeks.

[0077] Treatment of mice starved for 48 hours (Fasted) and normally fed mice (Fed): 19-20 week old C57 BL / 6 mice were fasted for 48 hours and given free access to water; normally fed mice were allowed to eat regular feed without any food restrictions for 48 hours.

[0078] MG53 knockout mice (mg53) - / - (Mouse model) See Song, Ruisheng et al. “Central roleof E3 ubiquitin ligase MG53 in insulin resistance and metabolic disorders.” Nature, vol. 494, 7437 (2013): 375-9.

[0079] All laboratory animals were housed at the Peking University Laboratory Animal Center (AAALAC internationally accredited), under conditions of 12 hours of light / 12 hours of darkness, at a room temperature of 23.0±0.1℃, with adequate water and food provided. All animal-related procedures were authorized by the Peking University Laboratory Animal Management and Use Committee and fully complied with the requirements of the "Guidelines for the Management and Use of Laboratory Animals" (NIH, 1985).

[0080] The cell lines used in this study included human embryonic kidney cells 293T (HEK293T) and mouse myoblasts (C2C12), both purchased from the ATCC (American Type Culture Collection) cell bank.

[0081] 1.2 Experimental reagents and consumables The experimental reagents and consumables used in this study are detailed in Table 1.

[0082] Table 1. Information on experimental reagents and consumables

[0083]

[0084] Other reagents: Inorganic and organic reagents such as NaCl, KCl, NaF, sodium pyrophosphate, and D-Glucose are domestically produced (purchased from Beijing Reagent Factory) or imported (purchased from Sigma-Aldrich); Ethanol, acetone, methanol, chloroform, and isopropanol are domestically produced analytical grade reagents (purchased from Beijing Reagent Factory).

[0085] The antibodies used in this study are detailed in Table 2. The preparation and validation methods for the Anti-MG53-K317ac antibody are described in Example 1.

[0086] Table 2 Antibody Information

[0087] The plasmids and viruses used in this study are detailed in Table 3.

[0088] Table 3 Plasmid and Virus Information

[0089] 2. Experimental Methods Solution preparation 1) PBS: 8.0 g NaCl, 0.20 g KCl, 1.44 g Na2HPO4, 0.24 g KH2PO4, dissolved in 800 mL ultrapure water, pH adjusted to 7.4 with HCl, and water added to a final volume of 1 L.

[0090] 2) 0.125% trypsin: 0.125 g trypsin, 0.02 g EDTA·2Na·2H2O, bring the volume to 100 mL with 0.1 M pH=7.4 PBS, filter and dispense through a 0.22 μm filter membrane.

[0091] 3) Double antibiotics: 0.6 g penicillin and 1 g streptomycin, dissolved in 10 mL deionized water. Sterilize by filtration using a disposable 0.22 μm filter in a clean bench, then aliquot and store at -20℃. When using, add to cell culture medium at a ratio of 1:1000.

[0092] 4) Cell cryopreservation solution: Prepare the cell cryopreservation solution by mixing the three reagents in a volume ratio of DMEM medium: FBS: DMSO = 7:2:1.

[0093] 5) Cell lysis buffer: Add saturated sodium fluoride and sodium pyrophosphate solutions to RIPA lysis buffer at ratios of 3:100 and 1:500, respectively. Before use, add Cocktail at a ratio of 1:100, then add PMSF and MG132 to achieve final concentrations of 1 mM and 20 μM, respectively. If used for acetylation signal detection, an appropriate amount of deacetylase inhibitor should be added.

[0094] 6) Co-immunoprecipitation (Co-IP) lysis buffer: Weigh 2.922 g NaCl and 3.5745 g HEPES powder, add 2.5 mL of NP-40, and dilute with pure water to 500 mL to prepare a solution. Before use, add Cocktail and 1 mM PMSF at a ratio of 1:100.

[0095] 7) 1.5 M Tris-HCl (pH 8.8): Weigh 18.15 g of Tris base, adjust the pH to 8.8 with 6 M HCl, and bring the volume to 100 mL with pure water.

[0096] 8) 1.0 M Tris-HCl (pH 6.8): Weigh 12.1 g of Tris base, adjust the pH to 6.8 with 6M HCl, and bring the volume to 100 mL with pure water.

[0097] 9) 10% Ammonium Persulfate (10% APS): Weigh 100 mg of ammonium persulfate and dissolve it in 1 mL of double-distilled water. Prepare and use immediately.

[0098] 10) Preparation of 8% polyacrylamide separating gel (20 mL): 1.5 M Tris-HCl (pH 8.8), 5.0 mL; 10% SDS, 200 μL; 10% APS, 200 μL; 30% acrylamide, 5.3 mL; deionized water, 6.3 mL; TEMED, 12 μL. Pour the gel immediately after preparation.

[0099] 11) Preparation of 5% polyacrylamide stacking gel (10 mL): 1 M Tris-HCl (pH 6.8), 1.25 mL; 10% SDS, 100 μL; 10% APS, 100 μL; 30% acrylamide, 1.7 mL; deionized water, 6.8 mL; TEMED, 10 μL. Pour the gel immediately after preparation.

[0100] 12) 5×SDS-PAGE gel electrophoresis buffer: Weigh 15.1 g Tris base, 94 g glycine, and 5 g SDS, add double-distilled water to 1 L, adjust the pH to 8.3, and store at room temperature for later use.

[0101] 13) 20× Electroporation Buffer: Weigh 58 g g of glycine and 116 g of Tris base and dissolve them in 1 L of double-distilled water. Mix thoroughly and store at room temperature for later use.

[0102] 14) 10× TBS: Weigh 60.6 g Tris base and 87.7 g NaCl, dissolve in 1 L of double-distilled water, and adjust the pH to 7.4 with 6 M HCl. To prepare TBST, take 100 mL of the above 10× TBS solution, dilute with double-distilled water to 1 L, and add 1 mL of Tween-20 to bring the final concentration to 0.1%.

[0103] 15) 5% blocking solution: 5 g of skim milk powder dissolved in 100 mL of TBST.

[0104] 16) LB liquid medium: 10.0 g tryptone for bacterial culture, 5.0 g yeast extract, 10.0 g NaCl, add deionized water and stir until completely dissolved, then bring the volume to 1 L and autoclave for 20 min.

[0105] 17) LB solid medium: 2.0 g tryptone, 1.0 g yeast extract, 2.0 g NaCl, 3.0 g agar powder, add deionized water and stir until completely dissolved. Adjust the volume to 200 mL and autoclave for 20 min. Remove from heat and allow to cool to approximately 45℃. Add ampicillin (100 mg / mL) or kanamycin (50 mg / mL) at a ratio of 1:1000. Mix well and pour into sterile plates. After solidification, invert and store at 4℃.

[0106] 18) 4% Paraformaldehyde (PFA): 700 mL deionized water, 100 mL 10×PBS, 4.0 g PFA, heat to 65℃, dissolve, adjust pH to 7.4, bring volume to 1 L, filter, and store at 4℃.

[0107] 2.1 Molecular Experiments 1) Plasmid construction (taking Myc-MG53 plasmid as an example, primer sequence information can be found in 2.5 "PCR Primer Table") a) Polymerase Chain Reaction (PCR) amplification of the target gene. Based on the target gene, select appropriate species' cDNA (obtained by reverse transcription after extracting mRNA from the corresponding cells or tissues) or existing plasmids as templates, and perform a PCR reaction to obtain the target gene fragment. The reaction system is as follows.

[0108] Table 4

[0109] b) Briefly centrifuge to bring the liquid on the PCR tube wall to the bottom, then set the PCR program to perform the PCR reaction. The PCR program is as follows.

[0110] Table 5

[0111] c) Vector linearization by enzyme digestion. Prepare the following enzyme digestion reaction system and digest overnight at 37°C.

[0112] Table 6

[0113] d) Gel recovery of PCR products. PCR products were detected by running a 1% agarose gel. After excising the target band, the PCR products were recovered and purified using a gel recovery kit.

[0114] e) Recombining the vector and fragment. After inactivating the endonuclease by heating at 65°C for 20 min, the recovered DNA fragment and linearized vector were recombined using the ClonExpress II One Step Cloning Kit (Novizan), as shown in the following system.

[0115] Table 7

[0116] Gently blow the mixture with the pipette tip, briefly centrifuge, react at 37°C for 30 minutes, and then immediately place it on ice to cool.

[0117] f) Transformation and plating of the recombinant product. Add 5 μL of the ligation product to 50 μL of DH5α competent cells, incubate on ice for 30 min, then heat shock at 42°C for 60 s, and cool on ice for 2-3 min. Add the bacterial culture to the resistant medium, spread evenly to ensure full absorption, and incubate at 37°C inverted overnight.

[0118] g) Select single colonies for sequencing. Select several single colonies, transfer them to LB medium containing ampicillin, and then sequence them. Select positive colonies for subsequent large-scale sequencing.

[0119] h) Plasmid extraction. Inoculate the positive bacterial culture at a ratio of 1:1000 into LB medium containing ampicillin and incubate overnight. Extract the plasmid according to the steps in the Axygen plasmid extraction kit. After determining the plasmid concentration using Nanodrop 2000, store at -20°C for later use.

[0120] 2) Construction of site-directed mutagenesis plasmids (taking the construction of Myc-MG53-K317R plasmid as an example) a) Primer design. First, identify the nucleotide sequence and position of the amino acid to be mutated in the cDNA sequence. Using the amino acid codon table as a reference, design the mutation sequence. Then, select 5-7 bp upstream of the mutation position, the mutated nucleotide sequence, and 15-20 bp downstream of the mutation position, and combine them into the final primer sequence.

[0121] b) Site-directed mutagenesis PCR. Using the corresponding WT wild-type plasmid as a template, PCR reactions were designed according to the KAPA HiFi hot-start high-fidelity enzyme instructions for amplification (see 2.5 PCR primer table for primer sequence information).

[0122] c) Electrophoresis detection. A 1% agarose gel is used to detect whether the target band has been amplified.

[0123] d) Dpn I digestion of PCR products. Prepare the following digestion reaction system and digest overnight at 37°C to remove the template plasmid.

[0124] Table 8

[0125] e) Transformation and plating. After inactivating Dpn I enzyme by digestion at 85℃ for 20 min, the product is transformed into DH5α competent cells, plated, and single clones are picked for sequencing.

[0126] f) Select positive clones according to the steps described above, shake the bacterial culture and extract the mutant plasmid.

[0127] 2.2 Cell Experiments 1) Cell line resuscitation, culture, passage, and cryopreservation a) Cell line thawing. Remove the frozen cells from the liquid nitrogen container and thaw them in a 37°C water bath. Transfer the cell-containing cryopreservation solution to a 50 mL centrifuge tube containing culture medium, centrifuge at 800 rpm for 5 min, discard the supernatant, resuspend the cell pellet in DMEM medium containing 10% FBS, mix well, evenly spread in cell culture dishes, shake well, and incubate in a 37°C incubator.

[0128] b) Cell culture. After culturing for a period of time, remove the old culture medium, add freshly prepared DMEM medium containing 10% FBS preheated at 37°C, and place in a cell culture incubator for further culture.

[0129] c) Cell passage. When cells reach 80-90% confluence, wash them once with pre-warmed PBS, remove any residual PBS, add 1 mL of 0.125% Trypsin digestion solution, gently mix, and digest at 37°C for 1 min. Add culture medium to stop the digestion. Transfer the digested cell suspension to a sterile centrifuge tube, centrifuge at 800 rpm for 5 min, discard the supernatant, add freshly prepared cell culture medium, carefully resuspend the cells, aliquot into culture dishes, mix well, and incubate in a 37°C cell culture incubator.

[0130] d) Cell cryopreservation. When cells reach 80-90% confluence, aspirate the cell culture medium from the dish, wash once with pre-warmed PBS, aspirate any remaining PBS, add Trypsin to digest the cells, add culture medium to stop digestion, thoroughly pipette the cells, transfer to a 50 mL centrifuge tube, centrifuge at 800 rpm for 5 min, and discard the supernatant. Resuspend the cells in the prepared cell cryopreservation solution, aliquot into cryovials (1 mL per tube), place in a cryopreservation box, and store at -80°C for 24 hours, then transfer to liquid nitrogen for long-term storage.

[0131] 2) C2C12 myoblast differentiation a) Culture of C2C12 myoblasts. Cells were passaged in DMEM medium containing 10% FBS and cultured in a 37°C cell culture incubator.

[0132] b) Induction of C2C12 differentiation. When the cells reach 70% confluence, the culture medium is replaced with DMEM medium containing 2% DES. Cell differentiation is induced for more than 4 days (the medium is changed every 1-2 days), and the cells are cultured in a 37°C constant temperature cell culture incubator.

[0133] 3) Plasmid transfection a) Change the medium before transfection. When the confluence of cells reaches 70-80%, replace the medium with fresh DMEM medium that does not contain FBS.

[0134] b) Prepare the transfection system. Dilute the plasmid (μg): transfection reagent (μL) to a certain volume of DMEM according to the ratio of plasmid (μg): transfection reagent (μL) = 1:3. Mix them separately and let them stand at room temperature for 5 min. Then mix the plasmid and transfection reagent dilutions, gently pipette to mix, and let them stand at room temperature for 20 min.

[0135] c) Transfecting cells. Add the transfection system to the cells, gently shake the culture medium, and incubate the cells in a 37°C incubator.

[0136] d) Transfection and medium change. After 5-8 hours of transfection, remove the medium and add freshly prepared DMEM medium containing 10% FBS. Place the cells in a 37°C incubator for culture.

[0137] 4) Cell RNA extraction (using a 60 mm culture dish as an example) a) Cell lysis. Aspirate the cell culture medium, add 1 mL of Trizol lysis buffer, and repeatedly pipette the cells to completely detach them from the bottom of the dish. Transfer the cell lysis buffer to a 1.5 mL EP centrifuge tube. Vortex thoroughly and incubate at room temperature for 5 min to separate nucleic acids and proteins.

[0138] b) Chloroform extraction. Add 200 μL of chloroform, shake vigorously for 15 s to mix, incubate at room temperature for 10 min, centrifuge at 12,000 rpm for 15 min at 4°C, and transfer the uppermost colorless layer to a new 1.5 mL EP centrifuge tube.

[0139] c) Isopropanol precipitation. Add 500 μL of isopropanol, invert to mix, precipitate at room temperature for 20 min, centrifuge at 12,000 rpm for 10 min at 4℃, discard the supernatant, and retain the white precipitate.

[0140] d) Ethanol washing. Add 1 mL of 75% ethanol to wash the precipitate, centrifuge at 12,000 rpm for 5 min at 4°C, discard the ethanol, and repeat the above steps once.

[0141] e) RNA dissolution. After the precipitate has air-dried, add 60 μL of DEPC water and heat at 55-60℃ for 15 min to fully dissolve the RNA.

[0142] f) RNA concentration determination. RNA concentration was measured using Nanodrop 2000. The ratio of OD260 to OD280 of the RNA sample should be between 1.9 and 2.0, and no degradation should be detected during gel electrophoresis.

[0143] 5) RNA reverse transcription Reverse transcription was performed using abm's 5×All-In-One RT MasterMix kit.

[0144] a) Removal of genomic DNA (gDNA). Prepare the following system.

[0145] Table 9

[0146] After incubating at room temperature for 5 min, add 2 μL of AccuRT Reaction Stopper (5×) and mix well.

[0147] b) Reverse transcription reaction. Continue by adding the following reagents.

[0148] Table 10

[0149] After incubating at 25°C for 10 min, transfer to 42°C for 15 min (for downstream qPCR) or 50 min (for downstream PCR reaction).

[0150] c) Reaction inactivation. Inactivate the reaction at 85℃ for 5 min, and store the reversed sample at -20℃ for subsequent experiments.

[0151] 6) Quantitative real-time PCR (qPCR) The qPCR primer sequence information used in this experiment can be found in 2.5 "qPCR Primer Table".

[0152] a) cDNA sample dilution. The reversed sample was diluted 10-fold with ddH2O.

[0153] b) Prepare the reaction system. The system is as follows.

[0154] Table 11

[0155] c) Quantitative PCR reaction. The reaction conditions are as follows: Table 12

[0156] 7) Cell protein extraction and concentration determination, protein sample preparation (using a 60 mm culture dish as an example) a) Collect cells. Aspirate the culture medium, wash once with pre-cooled PBS, aspirate the PBS again, and add 200 μL of RIPA lysis buffer. Use a cell scraper to scrape the cells and transfer them into a 1.5 mL centrifuge tube.

[0157] b) Sonication. Disrupt the cells three times with an ultrasonic disruptor, 5 seconds each time, to ensure complete cell lysis. Centrifuge at 12,000 rpm for 10 minutes at 4°C. Transfer the supernatant to new centrifuge tubes, aliquot, and freeze at -80°C.

[0158] c) Protein concentration determination. Protein concentration was determined according to the Thermo BCA Protein Assay method. The absorbance of the sample was measured at 562 nm using a microplate reader, and the protein concentration was calculated based on the protein standard curve.

[0159] d) Protein sample preparation. Take the same total volume of sample solution as the measured protein concentration, and add 4× loading buffer (final concentrations of each component: 200 mM Tris-HCl (pH 6.8); 8% (w / v) SDS; 40% (w / v) glycerol; 0.4% (w / v) bromophenol blue; ultrapure water to a final volume of 10 mL, aliquot and store at -20℃ protected from light; add 100 μL β-ME per 900 μL of stock solution before loading), mix well, and heat at 100℃ for 5 min for denaturation. 8) Detection of ubiquitin modification in Co-IP cell lysate a) Cell pretreatment. Before receiving the samples, HEK293T cells that had been transfected with the corresponding plasmid were treated with 10 μM MG132 for 12 hours.

[0160] b) Cell lysis. Collect cell proteins from the Co-IP cell lysis buffer and lyse the cells as described above.

[0161] c) Immunoprecipitation. Antibodies are used to immunoprecipitate target proteins.

[0162] d) Ubiquitin signal detection. SDS-PAGE gel chromatography was performed, and ubiquitin signals were detected using ubiquitin molecular antibodies.

[0163] 9) Immune dot blot a) Sample preparation. Dissolve the peptide fragment in PBS and prepare serially diluted peptide samples according to the amount of protein required for loading, with each sample having the same volume (1-2 μL).

[0164] b) Spotting. Take a clean nitrocellulose filter membrane (NC membrane), cut a corner to mark the front and back sides, and use a small pipette tip box as a holder (for easy spotting at equal intervals) to spot 1-2 μL of sample onto the NC membrane.

[0165] c) Dry the sample. Place the membrane in a clean box (to prevent dust from falling onto the membrane) and let it stand at room temperature for 20-30 minutes to allow the liquid to dry completely.

[0166] d) Sealing. Sealing with 5% skim milk (prepared with TBST) at room temperature for 1 hour.

[0167] e) Primary antibody incubation. Recover the blocking buffer, wash the membrane three times with TBST for 5 min each time. Dilute the primary antibody in skim milk at a certain ratio and incubate overnight on a plate shaker at 4°C.

[0168] f) Secondary antibody incubation. After recovering the primary antibody, wash the membrane three times with TBST, 5 min each time. Dilute the secondary antibody in blocking buffer at a ratio of 1:10000 and incubate on a plate shaker at room temperature for 1 hour.

[0169] g) Exposure. Wash the membrane three times with TBST, 5 min each time. Prepare ECL luminescent solution according to the ratio of A:B solution = 1:1. Place the NC membrane in a ChemiDocXRS imager for chemiluminescence detection. After adding an appropriate amount of luminescent solution, acquire the image.

[0170] h) Grayscale analysis. Image Lab software is used to process images and perform swimlane grayscale value analysis.

[0171] 10) Western Blot a) SDS-PAGE electrophoresis. Assemble a vertical double-layer electrophoresis glass plate. Place the prepared SDS-PAGE gel into the holder inside the tank, and add 1X electrophoresis buffer to both the inside and outside of the tank. Carefully remove the comb from the upper gel and add the protein samples into the corresponding wells in a specific order. Connect the electrophoresis apparatus to the power supply, adjust the power supply voltage to 70 V, and start electrophoresis. When the bromophenol blue leading edge enters the separating gel, increase the voltage to 110 V and continue electrophoresis until the bromophenol blue reaches the bottom of the separating gel.

[0172] b) Transfer. Remove the double-layered glass plate, carefully remove the gel, and use an electroporation tank to transfer the separated sample protein onto a polyvinylidene fluoride (PVDF) membrane. The transfer conditions are a constant current of 270 mA and a transfer time of 2 hours.

[0173] c) Blocking. After the transfer, the PVDF membrane was blocked with 5% skim milk (prepared with TBST) at room temperature for 1 hour.

[0174] d) Primary antibody incubation. Recover the blocking buffer, wash the membrane three times with TBST for 5 min each time. Dilute the primary antibody in antibody dilution buffer at a certain ratio and incubate overnight on a plate shaker at 4°C.

[0175] e) Secondary antibody incubation. After recovering the primary antibody, wash the membrane three times with TBST, 5 min each time. Dilute the secondary antibody in blocking buffer at a ratio of 1:10000 and incubate on a plate shaker at room temperature for 1 hour.

[0176] f) Exposure. Wash the membrane three times with TBST, 5 min each time. Prepare ECL luminescent solution according to the ratio of A:B solution = 1:1. Perform chemiluminescence detection on the PVDF membrane in a ChemiDoc XRS imager. After adding an appropriate amount of luminescent solution, acquire the image.

[0177] g) Grayscale analysis. Image Lab software is used to process images and perform swimlane grayscale value analysis.

[0178] 11) Acetylated peptide-antibody competition assay a) Antibody preparation. Dilute the MG53-K317 acetylated antibody (anti-MG53-K317ac) in antibody dilution buffer at a ratio of 1:2500.

[0179] b) Prepare peptide gradient solutions. Calculate the required amount of peptides according to the peptide:antibody ratios of 10:1, 100:1, 1000:1, and 10000:1, and dilute with PBS to prepare acetylated and non-acetylated peptide gradient solutions (keeping the final volume as consistent as possible).

[0180] c) Co-incubation of antibody and peptide. Mix peptide gradient solution with antibody dilution solution, add an equal volume of PBS as a control, and incubate at 4°C for 4 hours.

[0181] d) SDS-PAGE. Prepare multiple aliquots of the lysis buffer of HEK293T sample overexpressing Myc-MG53 and run them on SDS-PAGE, following the same procedure as Western blot.

[0182] e) Antibody incubation. The converted PVDF membrane was cut into small pieces, and different small membranes were incubated with different antibody-peptide incubation solutions overnight at 4°C.

[0183] f) The remaining steps are the same as the subsequent steps of Western blot.

[0184] 2.3 Animal Experiments 1) Animal tissue sampling a) Blood collection. One eyeball of the mouse was removed and a blood sample was collected. The whole blood was allowed to stand at room temperature for 30 min, then centrifuged at 3,000 rpm for 10 min at room temperature. The supernatant was collected and stored at -80°C.

[0185] b) Other tissue sampling. Mice were euthanized by vertebral dislocation, and skeletal muscles including the gastrocnemius, soleus, quadriceps femoris, and tibialis anterior were collected. Adhesive hairs were washed off with physiological saline, dried, flash-frozen in liquid nitrogen, and then stored at -80°C.

[0186] 2) Tissue RNA extraction a) Tissue grinding. Take 20-30 mg of gastrocnemius muscle (tissue from the same location) into a 1.5 mL EP tube, add 1 mL of Trizol and two clean 3 mm stainless steel beads. Set the TissueLyser II to 30 Hz and grind for 2 min. Remove the adapter, rotate the tube rack so that the EP tube closest to the instrument is on the outermost side, reassemble, and grind again at the same frequency for 2 min.

[0187] b) Lysis. Vortex thoroughly and let stand at room temperature for 10 min to separate nucleic acids and proteins.

[0188] c) RNA extraction. Subsequent steps are the same as for cellular RNA extraction.

[0189] 3) Extraction of tissue proteins a) Tissue homogenization. Take 20-30 mg of gastrocnemius muscle into a 1.5 mL EP tube, add 300 μL of prepared RIPA lysis buffer and two clean stainless steel beads. Homogenize using the same method as described above.

[0190] b) Lysis. After grinding, the sample was placed on a shaker at 4°C for 1 hour for lysis. After removal, it was centrifuged at 12,000 rpm at 4°C for 10 minutes, and the supernatant was collected into a new EP tube.

[0191] c) Concentration determination and sample preparation. Subsequent steps are the same as for cell protein extraction.

[0192] 4) Extraction of tissue metabolites a) Preparation. Pre-cool 80% methanol and clean the tissue homogenizer. Before grinding begins, rinse the homogenizer with pre-cooled 80% methanol to prevent the tissue homogenate from adhering to the homogenizer wall.

[0193] b) Tissue grinding. Transfer 300-400 mg of gastrocnemius muscle tissue (from the same location in mice) to a homogenizer, add 300-400 µL of 80% methanol, and grind in an ice bath until no particles remain.

[0194] c) Metabolite extraction. Transfer the homogenate to a clean EP tube, vortex for 15 seconds, and place on a shaker at 4°C for 30 minutes. Remove and place on ice, vortex for 15 seconds, repeat 3 times, sonicate for 2-3 seconds, pause for 4 seconds, and repeat 4-6 times. After sonication, vortex for 15 seconds and place on ice.

[0195] d) Protein concentration determination. The BCA method is used to determine protein concentration. Since protein is insoluble in 80% methanol, each sample must be shaken thoroughly before addition. It is recommended to take three replicates to reduce bias.

[0196] e) Collect the metabolite solution. After measuring the concentration, centrifuge at 12,000 rpm and 4°C for 10 min, transfer the supernatant to a new clean EP tube, freeze at -80°C or quick-freeze in liquid nitrogen, and send it to the metabolomics platform for instrumental measurement.

[0197] 2.4 Data Analysis Statistical methods All data are expressed as mean ± standard error. Differences between two groups were analyzed using Student's t-test. When comparing multiple groups, one-way ANOVA or repeated measures one-way ANOVA was used, with further analysis using the Bonferroni test. p < 0.05 was considered statistically significant, and p < 0.01 was considered highly significant.

[0198] 2.5 Sequence Information Table 13 PCR Primer Information

[0199] Table 14 qPCR primer information

[0200] Table 15 Amino acid sequences of human MG53 and human MG53-K317R

[0201] Example 1: The K317 site of MG53 protein affects the stability of MG53 protein. Through research on post-translational modifications of human MG53 protein, the inventors discovered that post-translational modifications of lysine (K) at position 317 of the MG53 protein (including acetylation and ubiquitination) are related to the stability of the MG53 protein.

[0202] This study constructed a single-site mutant of MG53, MG53-K317R, by mutating only the lysine at position 317 to arginine. Both MG53-WT and MG53-K317R mutant proteins were overexpressed in HEK293T cells. Protein stability experiments confirmed that, compared to MG53-WT, the MG53-K317R mutant showed some stability to MG53 protein after treatment with actinomycete ketone (with a final concentration of 50 μg / mL in the culture medium). Figure 1 A, Figure 1 B). Furthermore, the MG53-K317R mutant exhibits lower levels of intracellular ubiquitination compared to the MG53-WT mutant. Figure 1 C). The above results confirm that the K317 site mediates the ubiquitination and degradation of MG53, thereby affecting the protein stability of MG53. In summary, the K317 site of the MG53 protein is crucial for the proteasome-mediated degradation of MG53, and mutations at this site can enhance the intracellular stability of the MG53 protein.

[0203] To investigate the effect of acetylation modification at the MG53-K317 site on the stability of MG53 protein, a site-specific monoclonal antibody (MG53-K317ac) targeting the acetylation at the K317 site was prepared. The preparation and screening process included: (1) synthesizing an acetylated peptide containing the K317 site (SDQK(ac)APPAGEDTR, SEQ ID No: 13) and using it as an immunogen to induce corresponding antibody production in rabbit spleen cells; (2) isolating rabbit spleen B lymphocytes and fusing them with immortalized myeloma cells cultured in vitro to form hybridoma cells; (3) selectively culturing and screening hybridoma cells that secrete antibodies; (4) cloning hybridoma cells, screening monoclonal antibodies, and ensuring the uniformity of the antibodies secreted by them; (5) preparing large quantities of monoclonal antibodies that meet the requirements.

[0204] After multiple screening and purification steps, this study finally screened out the monoclonal antibody MG53-K317ac, and performed a series of validations on the purified antibody to ensure that it met the requirements. First, the acetylated and non-acetylated (SDQKAPPAGEDTR, SEQ ID No: 14) peptide solutions were serially diluted, and equal volumes of protein solution were evenly dropped onto a nitrocellulose filter membrane (NC membrane). After drying, the antibody was incubated and exposed for development using the Western blot method. Dot blot results showed that the antibody specifically recognized the purified acetylated peptides and exhibited a dose-dependent effect according to the sample loading gradient, without binding to peptides with the same sequence but not acetylated. Figure 2 A).

[0205] Secondly, this study overexpressed MG53-WT and MG53-K317R mutant proteins in HEK293T and C2C12 cells, respectively. Immunoblotting results confirmed that the MG53-K317ac antibody specifically recognized MG53-WT but not the MG53-K317R mutant, which cannot be acetylated. Figure 2 B, Figure 2 C).

[0206] In induced differentiated C2C12 myotube cells, this antibody also showed strong specificity for endogenously expressed MG53 protein. Figure 2D); More importantly, when myotube cells were treated with the deacetylase inhibitor nicotinamide (NIC; the final concentration of NIC was increased to 200 mM) and trichostatin A (TSA; the final concentration of TSA was increased to 200 nM) to enhance the endogenous MG53 protein acetylation level, the signal of MG53 protein acetylated at the K317 site detected by this antibody was significantly enhanced. Figure 2 D). Additionally, this antibody can specifically recognize MG53 protein endogenously expressed in the muscle of wild-type mice, but not in MG53 knockout mice (mg53). - / - No signal was detected in the skeletal muscle of the mouse model. Figure 2 E).

[0207] Furthermore, the results of the peptide competitive binding antibody experiment showed that if the antibody was pre-incubated with the peptide, the acetylated peptide could inhibit the subsequent binding of the antibody to the MG53 protein on the PVDF membrane in a concentration-dependent manner. Figure 3 A), but pre-incubation of the antibody with non-acetylated peptides does not have this effect. Figure 3 B) demonstrates that the antibody can specifically recognize MG53 protein acetylated at the K317 site.

[0208] Based on the above experimental results, the screened MG53-K317ac monoclonal antibody can specifically recognize MG53 protein acetylated at the K317 site.

[0209] Example 2: Glucose-regulated acetylation modification of the K317 site of MG53 protein Both ubiquitination and acetylation are known to occur on the ε-amino group of lysine residues in proteins. When one or more lysine sites can be acetylated and ubiquitinated, there is competition between the two. Acetylation at a particular site may interfere with ubiquitination, preventing the protein from being recognized and degraded by the proteasome, thus stabilizing the protein. Considering the regulation of protein acetylation by cellular energy metabolism, this study investigated whether cellular energy state is related to acetylation at the MG53-K317 site and MG53 ubiquitination, as well as the stability of the MG53 protein.

[0210] First, differentiated C2C12 myotube cells were incubated in sugar-free medium (Wuhan Procell: catalog number PM150322) to simulate starvation treatment. Cells were cultured in sugar-free medium, and starvation treatment was initiated. Results showed that compared to the non-starved group (0hr), 8 hours of starvation treatment induced acetylation at the K317 site (…). Figure 4 A, Figure 4 B) and MG53 total protein level ( Figure 4 A, Figure 4 The significant downregulation of C) and the even greater downregulation of acetylation levels at K317 site led to a significant decrease in the proportion of MG53-K317ac in MG53. Figure 4 B), while the mRNA level of MG53 remained unchanged at this time. Figure 4 D) indicates that the decrease in MG53 protein levels is due to post-transcriptional regulation. These data suggest that glucose starvation treatment reduces MG53-K317 acetylation levels, accompanied by a decrease in MG53 protein expression levels, suggesting a possible correlation between K317 acetylation levels and MG53 protein levels.

[0211] To further confirm the effect of cellular energy status on K317 acetylation and MG53 protein stability, the inventors incubated differentiated C2C12 myotube cells with media containing different glucose concentrations (0 mM, 5 mM, and 25 mM glucose), and simultaneously tested the stability of MG53 protein using actinomycin (CHX) treatment (CHX added to the culture medium at a final concentration of 50 μg / mL). The results showed that the addition of glucose-free medium significantly reduced the K317 acetylation level compared to the control group. In 25 mM high-glucose medium (Procell: PM150210), the K317 acetylation level remained essentially unchanged, while in 5 mM low-glucose medium (Procell: PM150220), the K317 acetylation level was between the two. Figure 5 A). Importantly, the higher the sugar concentration in the culture medium, the more stable the MG53 protein tends to be. Figure 5 A, Figure 5 B), and the stability of MG53 protein in 5 mM medium is between that under sugar-free and high-sugar culture conditions, indicating that glucose regulates MG53-K317 acetylation level and MG53 protein stability in a concentration-dependent manner.

[0212] In contrast, pretreatment with the deacetylase inhibitor nicotinamide (NIC) and trolactam A (TSA) (with a final concentration of 200 mM NIC and 200 mM TSA added to the culture medium) for 8 hours resulted in no further decrease in acetylation levels at the K317 site under sugar-free incubation conditions. Simultaneously, the MG53 protein tended to stabilize, comparable to that under 25 mM high-glucose incubation conditions. Figure 6 A, Figure 6 B). This suggests that high-glucose treatment may regulate the stability of the MG53 protein by mediating acetylation at the K317 site.

[0213] To investigate the relationship between glucose concentration and MG53 ubiquitination, this study incubated differentiated C2C12 myotube cells in culture media with different glucose contents. To eliminate interference from other types of ubiquitination signals, a specific antibody against the Lys48 ubiquitin chain was used to detect MG53 degradation-related ubiquitination levels. The results showed that glucose upregulated K317 acetylation levels in a concentration-dependent manner while decreasing MG53 protein degradation-related ubiquitination modifications. Figure 7 This is consistent with the aforementioned results showing that high glucose treatment enhances the acetylation level at the K317 site and the stability of the MG53 protein. However, when deacetylase inhibitors are added, the acetylation level at the K317 site is significantly increased in the treated groups at different glucose concentrations compared to the untreated control group, while the ubiquitination level related to MG53 degradation is significantly decreased; furthermore, at different glucose concentrations, the acetylation level at the K317 site remains relatively high, while the corresponding ubiquitination level is relatively low. Figure 7 These findings are consistent with the aforementioned results showing that deacetylase inhibitors enhance the stability of MG53 protein at low glucose concentrations.

[0214] In summary, high glucose treatment can stabilize MG53 protein by enhancing acetylation at the K317 site in cells, thereby inhibiting its ubiquitination and subsequent degradation.

[0215] Example 3: Changes in MG53-K317 acetylation modification levels in an animal model (1) Acetylation of MG53-K317 site in animal models of metabolic disorders MG53 is known to play an important role in metabolism. In this embodiment, the acetylation level of MG53 protein at the K317 site was detected in a mouse model of metabolic disorder.

[0216] The results showed that in mouse models associated with insulin resistance and metabolic syndrome, including db / db In diabetic mice and high-fat diet-induced obese mice, the relative levels of K317 acetylation modification in muscle tissue were also increased to varying degrees, accompanied by a significant increase in MG53 protein levels. Figure 8 ).

[0217] Similarly, in the skeletal muscle of monkeys with metabolic syndrome and obese patients, MG53 protein levels were elevated, along with a trend towards increased K317 acetylation levels. Figure 9 ).

[0218] In mice starved for 48 hours and mice subjected to chronic exercise, MG53 protein levels were significantly decreased, and acetylation modification at the K317 site in muscle tissue also showed a slight downregulation trend. Figure 10This indicates a certain degree of positive correlation between the acetylation level at the K317 site and the protein content of MG53 in the pathophysiological model, indirectly supporting the possibility that acetylation at the K317 site may be involved in the regulation of changes in MG53 protein levels caused by different energy states.

[0219] (2) Ubiquitination-dependent degradation of MG53 protein in animal models of metabolic disorders At the same time, this study also detected db / db The levels of ubiquitination associated with MG53 protein degradation in the skeletal muscle of diabetic mice, high-fat diet (HFD) induced obese mice, and starved mice. Immunoblotting results showed that... db / db The levels of MG53 ubiquitination in the skeletal muscle of both mice and HFD mice were decreased to some extent. Figure 11 A, Figure 11 B), while the MG53 ubiquitination level in the skeletal muscle of starved mice was slightly increased ( Figure 11 C), which is consistent with the aforementioned results.

[0220] In summary, the K317 site of the MG53 protein plays a crucial role in the regulation of post-translational modifications. Ubiquitination at K317 is involved in the degradation of MG53, while acetylation at this site enhances MG53 protein stability by competitively inhibiting ubiquitination. These two sites thus jointly regulate MG53 protein stability and influence its nucleoplasmic distribution and downstream functions within the cell. Since protein acetylation is closely related to cellular energy state, the switching between acetylation and ubiquitination at K317 also participates in the regulation of MG53 protein homeostasis by glucose, and provides a molecular mechanism for changes in MG53 protein levels in some metabolically related pathophysiological models.

[0221] In addition, this study also examined the acetylation and ubiquitination of the K317 site of MG53 protein in patients clinically diagnosed with metabolic syndrome (e.g., diabetes) and healthy individuals. The study found that the acetylation level of the K317 site of MG53 protein was significantly higher in patients with metabolic syndrome (e.g., diabetes) than in healthy individuals, while the ubiquitination level of the K317 site of MG53 protein was significantly lower in healthy individuals.

[0222] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. The application of substances modified with MG53 protein in the detection sample in the preparation of reagents for diagnosing metabolic-related diseases, characterized in that, The MG53 protein is modified by ubiquitination and / or acetylation at lysine position 317 of the amino acid sequence shown in SEQ ID NO: 11, and the metabolic-related disease is diabetes, obesity, or metabolic syndrome. The sample is a skeletal muscle tissue sample, and the diabetes is type 2 diabetes.

2. The application according to claim 1, characterized in that, The reagents include those for detecting MG53 protein modification in samples.

3. The application according to claim 1, characterized in that, The reagents include antibodies or antigen-binding fragments thereof that specifically bind to MG53 protein, antibodies or antigen-binding fragments thereof that specifically bind to modified MG53 protein, primers that specifically amplify the encoding nucleotide sequence of MG53, and / or probes that specifically recognize the encoding nucleotide sequence of MG53.

4. The application according to claim 1, characterized in that, The detection methods include immunoassay, mass spectrometry, chromatography, spectroscopy, and / or PCR amplification.

5. The application according to claim 3, characterized in that, The tissue samples include fresh tissue, paraffin sections, and their processed forms.

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