Protein regulating activity of mitochondrial calcium ion channel and application thereof in prevention and treatment of obesity and related diseases

Abstract

The present application relates to a protein for regulating the activity of a mitochondrial calcium channel and its application in preventing and treating obesity and related diseases, and belongs to the field of adipose tissue and related metabolism. The technical problem to be solved by the present application is how to prevent and / or treat obesity or obesity-related diseases. To solve the above technical problem, the present application provides a protein, which is an EMRE-UCP1 protein, wherein the EMRE-UCP1 protein is a protein fused from a mitochondrial calcium ion transport regulatory protein and a uncoupling protein-1. To solve the above technical problem, the present application provides a method for drug screening, wherein the method comprises using the transgenic animal cell line, or the transgenic animal tissue, or the transgenic animal organ of B11) to perform drug screening, and the drug is a drug for preventing and / or treating obesity or obesity-related diseases.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of adipose tissue and related metabolism, in particular to a protein regulating the activity of mitochondrial calcium channel and its application in preventing and treating obesity and related diseases. BACKGROUND

[0002] Obesity is a global epidemic, causing many health risks and has become a global health problem. Obesity is closely related to many metabolic diseases, such as diabetes, non-alcoholic fatty liver disease, hypertension and atherosclerosis. The occurrence of obesity is mainly due to the energy intake greater than energy consumption, including excessive diet, low energy consumption and lack of physical exercise. The excess energy is stored in the form of triglycerides in adipose tissue, causing abnormal accumulation of fat, forming obesity. Therefore, one of the main solutions to the problem of obesity is to regulate the balance of energy intake and output, and reduce the accumulation of energy in the body.

[0003] At the body level, two basic ways can be used to combat obesity: reducing energy / food intake or increasing energy consumption. Most strategies targeting energy / food intake have been proven to be unsuccessful, and existing related drugs can cause cardiovascular and even mental side effects. Therefore, finding a method to increase energy consumption by enhancing body heat production has become the development direction of new weight loss drugs.

[0004] Adipose tissue is mainly composed of adipocytes, and if specific expression of exogenous proteins in adipocytes can be achieved, it is of great significance for studying the function of adipocytes and even treating some diseases related to adipocytes. However, tools for specific expression of exogenous proteins in adipocytes have not been developed. SUMMARY

[0005] The technical problem to be solved by the present application is how to prevent and / or treat obesity or obesity-related diseases.

[0006] To solve the above technical problem, the first aspect of the present application provides a protein, which can be an EMRE-UCP1 protein, wherein the EMRE-UCP1 protein is a protein fused from an essential MCU regulator (EMRE) and a uncoupling protein-1 (UCP1).

[0007] Further, the present application provides a biological material related to the above-mentioned protein, which is selected from any one of the following:

[0008] B1) a nucleic acid molecule encoding the EMRE-UCP1 protein;

[0009] B2) an expression cassette containing the nucleic acid molecule of B1);

[0010] B3) a recombinant expression vector containing the nucleic acid molecule of B1) or containing the expression cassette of B2);

[0011] B4) a recombinant microorganism expressing the EMRE-UCP1 protein;

[0012] B5) a transgenic animal cell line containing the recombinant microorganism of B4), or a transgenic animal tissue containing the recombinant microorganism of B4), or a transgenic animal organ containing the recombinant microorganism of B4).

[0013] Further, the above-mentioned protein can be derived from an animal, for example a mammal.

[0014] The mammal can be a human, and can also be a non-human mammal, for example a mouse.

[0015] In an embodiment of the present application, the EMRE protein and uncoupling protein-1 (UCP1) are derived from a mouse.

[0016] The mitochondrial calcium uniporter regulator protein (EMRE protein) and uncoupling protein-1 (UCP1) can be connected by a linker (connecting peptide), the amino acid (21) sequence of the linker is: GSGSGSGSAAGSGSGSGSGSG (SEQ ID No. 7 108-128); the nucleotide sequence is: GGT TCA GGC TCT GGA AGT GGG TCC GCG GCC GGA AGT GGT AGT GGC TCC GGT TCC GGA TCC GGT (SEQ ID No. 8 322-384).

[0017] The NCBI reference sequence of the amino acid sequence of the EMRE protein is XP_036015460.1 (21-SEP-2020), and the encoding gene is mouse Emre, the NCBI reference sequence of the nucleotide sequence of the open reading frame (ORF) of the mouse Emre is XM_036159567.1 (21-SEP-2020);

[0018] The NCBI reference sequence of the amino acid sequence of the UCP1 protein is NP_033489.1 (19-JUN-2022), and the encoding gene is mouse Ucp1, the NCBI reference sequence of the nucleotide sequence of the open reading frame (ORF) of the mouse Ucp1 is NM_009463.3 (19-JUN-2022);

[0019] The amino acid sequence of the EMRE-UCP1 protein is SEQ ID No. 7, and the nucleotide sequence is SEQ ID No. 8

[0020] The above proteins can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0021] The above protein may also be tagged with a protein tag, which refers to a polypeptide or protein fused with the target protein using DNA in vitro recombination technology to facilitate the expression, detection, tracing and / or purification of the target protein. The protein tag may be a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, or a

[0022] , GST tag and / or SUMO tag, etc.

[0023] The nucleic acid molecule can be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA, siRNA, shRNA, sgRNA, miRNA or antisense RNA.

[0024] Furthermore, in the above-mentioned biological material, the promoter for driving the nucleic acid molecule in the expression cassette B2) or the recombinant expression vector B3) may be an ADP promoter, and the ADP promoter may be P1) or P2):

[0025] P1) The nucleotide sequence is the DNA molecule of SEQ ID No. 2, positions 156-1797;

[0026] P2) The nucleotide sequence is the DNA molecule of SEQ ID No. 1.

[0027] Furthermore, in the above-mentioned biological materials, the recombinant expression vector described in B3) may be an adeno-associated virus vector.

[0028] Furthermore, in the above-mentioned biological materials, the recombinant microorganism described in B4) may be an adeno-associated virus.

[0029] In order to solve the above technical problems, in a second aspect, the present invention provides an application, which can be any of the following:

[0030] A1) Use of the EMRE-UCP1 protein or a biomaterial related to the EMRE-UCP1 protein to promote or enhance the metabolic level of an organism and / or an organ and / or a tissue and / or a cell;

[0031] A2) Use of the EMRE-UCP1 protein or a biomaterial related to the EMRE-UCP1 protein for promoting or enhancing calcium ion channel (MCU) activity in an organism and / or organ and / or tissue and / or cell;

[0032] A3) Use of the above EMRE-UCP1 protein or biological material associated with the EMRE-UCP1 protein in promoting or increasing the metabolic level of the body and / or organ and / or tissue and / or cell;

[0033] A4) Use of the above EMRE-UCP1 protein or biological material associated with the EMRE-UCP1 protein in promoting or increasing the insulin response level of the body and / or organ and / or tissue and / or cell;

[0034] A5) Use of the above EMRE-UCP1 protein or biological material associated with the EMRE-UCP1 protein in promoting or increasing the sugar metabolic rate of the body and / or organ and / or tissue and / or cell;

[0035] A6) Use of the above EMRE-UCP1 protein or biological material associated with the EMRE-UCP1 protein in inhibiting or reducing the fat accumulation of the body and / or organ and / or tissue and / or cell;

[0036] A7) Use of the above EMRE-UCP1 protein or biological material associated with the EMRE-UCP1 protein in the preparation of a product for promoting or increasing the metabolic level of the body and / or organ and / or tissue and / or cell;

[0037] A8) Use of the above EMRE-UCP1 protein or biological material associated with the EMRE-UCP1 protein in the preparation of a product for promoting or increasing the calcium ion channel activity of the body and / or organ and / or tissue and / or cell;

[0038] A9) Use of the above EMRE-UCP1 protein or biological material associated with the EMRE-UCP1 protein in the preparation of a product for promoting or increasing the oxygen consumption rate of the body and / or organ and / or tissue and / or cell;

[0039] A10) Use of the above EMRE-UCP1 protein or biological material associated with the EMRE-UCP1 protein in the preparation of a product for promoting or increasing the insulin response level of the body and / or organ and / or tissue and / or cell;

[0040] A11) Use of the above EMRE-UCP1 protein or biological material associated with the EMRE-UCP1 protein in the preparation of a product for promoting or increasing the sugar metabolic rate of the body and / or organ and / or tissue and / or cell;

[0041] A12) Use of the above EMRE-UCP1 protein or biological material associated with the EMRE-UCP1 protein in the preparation of a product for inhibiting or reducing the fat accumulation of the body and / or organ and / or tissue and / or cell;

[0042] A13) Use of the above-mentioned EMRE-UCP1 protein or biological material related to the EMRE-UCP1 protein in the manufacture of a product for preventing and / or treating obesity;

[0043] A14) Use of the MICU1 protein and biological material related to the MICU1 protein in the drug screening for preventing and / or treating obesity;

[0044] A15) Use of the MICU1 protein and biological material related to the MICU1 protein in the manufacture of a product for drug screening for preventing and / or treating obesity;

[0045] A16) Use of the MICU1 protein and biological material related to the MICU1 protein in regulating the metabolic level of the body and / or organ and / or tissue and / or cell;

[0046] A17) Use of the MICU1 protein and biological material related to the MICU1 protein in regulating the calcium ion absorption of the body and / or organ and / or tissue and / or cell;

[0047] A18) Use of the MICU1 protein and biological material related to the MICU1 protein in regulating the oxygen consumption rate of the body and / or organ and / or tissue and / or cell;

[0048] A19) Use of the MICU1 protein and biological material related to the MICU1 protein in regulating the insulin response level of the body and / or organ and / or tissue and / or cell;

[0049] A20) Use of the MICU1 protein and biological material related to the MICU1 protein in regulating the sugar metabolism rate of the body and / or organ and / or tissue and / or cell;

[0050] A21) Use of the MICU1 protein and biological material related to the MICU1 protein in regulating the fat accumulation of the body and / or organ and / or tissue and / or cell;

[0051] A22) Use of the MICU1 protein and biological material related to the MICU1 protein in the manufacture of a product for regulating the metabolic level of the body and / or organ and / or tissue and / or cell;

[0052] A23) Use of the MICU1 protein and biological material related to the MICU1 protein in the manufacture of a product for regulating the calcium ion absorption of the body and / or organ and / or tissue and / or cell;

[0053] A24) Use of MICU1 protein and biological material related to said MICU1 protein for the preparation of a product for modulating the oxygen consumption rate of an organism and / or of an organ and / or of a tissue and / or of a cell;

[0054] A25) Use of MICU1 protein and biological material related to said MICU1 protein for the preparation of a product for modulating the insulin responsive level of an organism and / or of an organ and / or of a tissue and / or of a cell;

[0055] A26) Use of MICU1 protein and biological material related to said MICU1 protein for the preparation of a product for modulating the sugar metabolism rate of an organism and / or of an organ and / or of a tissue and / or of a cell;

[0056] A27) Use of MICU1 protein and biological material related to said MICU1 protein for the preparation of a product for modulating the fat accumulation of an organism and / or of an organ and / or of a tissue and / or of a cell;

[0057] A28) Use of MICU1 protein and biological material related to said MICU1 protein for the preparation of a product for preventing and / or treating obesity;

[0058] Said biological material related to said MICU1 protein is selected from any one of the following B7) to B17):

[0059] B7) Nucleic acid molecule encoding said MICU1 protein;

[0060] B8) Expression cassette containing the nucleic acid molecule of B7);

[0061] B9) Recombinant expression vector containing the nucleic acid molecule of B7) or recombinant expression vector containing the expression cassette of B8);

[0062] B10) Recombinant virus expressing said MICU1 protein;

[0063] B11) Transgenic animal cell line containing the recombinant virus of B10), or transgenic animal tissue containing the recombinant virus of B10), or transgenic animal organ containing the recombinant virus of B10);

[0064] B12) RNA molecule inhibiting or reducing the expression of the gene encoding said MICU1 protein or RNA molecule inhibiting or reducing the activity or content of said MICU1 protein;

[0065] B13) Nucleic acid molecule expressing the RNA molecule of B12);

[0066] B14) Expression cassette containing the nucleic acid molecule of B13);

[0067] B15) a recombinant expression vector containing the nucleic acid molecule of B13) or a recombinant expression vector containing the expression cassette of B14);

[0068] B16) a recombinant virus expressing the RNA molecule of B12);

[0069] B17) a transgenic animal cell line containing the recombinant virus of B16), or a transgenic animal tissue containing the recombinant virus of B16), or a transgenic animal organ containing the recombinant virus of B16).

[0070] In the present application, the MICU1 protein is mitochondrial calcium uptake protein 1 (MICU1).

[0071] The MICU1 protein mentioned above can be derived from an animal, for example a mammal.

[0072] The mammal can be a human, or a non-human mammal, for example a mouse.

[0073] In an embodiment of the present application, the EMRE protein and uncoupling protein-1 (UCP1) are derived from a mouse.

[0074] The NCBI reference sequence of the amino acid sequence of the MICU1 protein is NP_659071.1 (19-JUN-2022), the encoding gene of which is mouse Micu1, the NCBI reference sequence of the nucleotide sequence of the open reading frame (ORF) of mouse Micu1 is NM_144822.3 (19-JUN-2022), and the NCBI reference sequence of the nucleotide sequence of the Micu1 genome is NC_000076.7 (59538385-5969956).

[0075] In the present application, the promotion or improvement of the calcium ion channel activity of the body and / or organ and / or tissue and / or cell can specifically be the promotion or improvement of the calcium ion absorption efficiency of the body and / or organ and / or tissue and / or cell.

[0076] To solve the above technical problems, in a third aspect, the present application provides a medicament for preventing and / or treating obesity or obesity-related diseases, the medicament containing at least one of the following components:

[0077] 1) the EMRE-UCP1 protein mentioned above;

[0078] 2) the biological material related to the EMRE-UCP1 protein mentioned above;

[0079] 3) the biological material related to the MICU1 protein mentioned above.

[0080] In the present application, when preparing the medicine for preventing and / or treating obesity or obesity-related diseases, a carrier material can also be added.

[0081] The carrier materials include, but are not limited to, water-soluble carrier materials (such as polyethylene glycol, polyvinyl pyrrolidone, organic acids, etc.), poorly soluble carrier materials (such as ethyl cellulose, cholesterol stearate, etc.), and enteric carrier materials (such as cellulose acetate phthalate and carboxymethyl ethyl cellulose, etc.). These materials can be used to make a variety of dosage forms, including but not limited to tablets, capsules, dripping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal preparations, buccal tablets, suppositories, freeze-dried powder injections, etc. The formulations can be conventional preparations, sustained-release preparations, controlled-release preparations, and various microparticle delivery systems. In order to make unit dosage forms into tablets, various carriers well known in the art can be widely used. Examples of carriers include diluents and absorbents, such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, aluminum silicate, etc.; wetting agents and binders, such as water, glycerol, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, honey, glucose solution, acacia slurry, gelatin slurry, sodium carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, polyvinyl pyrrolidone, etc.; disintegrants. , such as dried starch, alginate, agar powder, brown seaweed starch, sodium bicarbonate with citric acid, calcium carbonate, polyoxyethylene, sorbitan fatty acid esters, sodium lauryl sulfate, methylcellulose, ethylcellulose, etc.; disintegration inhibitors, such as sucrose, tristearin, cocoa butter, hydrogenated oil, etc.; absorption enhancers, such as quaternary ammonium salts, sodium lauryl sulfate, etc.; lubricants, such as talc, silicon dioxide, corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol, etc. Tablets can also be further prepared as coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or bilayer tablets and multilayer tablets. To prepare the unit dosage form into a pill, a wide variety of carriers known in the art can be used. Examples of carriers include diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oils, polyvinylpyrrolidone, kaolin, and talc; binders such as gum arabic, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste, or flour paste; and disintegrants such as agar powder, dried starch, alginates, sodium lauryl sulfate, methylcellulose, and ethylcellulose. To prepare unit dosage forms as suppositories, a wide variety of carriers known in the art can be used. Examples of carriers include polyethylene glycol, lecithin, cocoa butter, higher alcohols, higher alcohol esters, gelatin, and semi-synthetic glycerides. To prepare unit dosage forms as injectable preparations, such as solutions, emulsions, lyophilized powder injections, and suspensions, all diluents commonly used in the art can be used, including water, ethanol, polyethylene glycol, 1,3-propylene glycol, ethoxylated isostearyl alcohol, polyoxyethylene isostearyl alcohol, and polyoxyethylene sorbitan fatty acid esters. In addition, in order to prepare an isotonic injection, an appropriate amount of sodium chloride, glucose or glycerol may be added to the injection preparation. In addition, conventional solubilizers, buffers, pH adjusters, etc. may also be added.In addition, if necessary, colorants, preservatives, perfumes, flavorings, sweeteners or other materials may be added to the pharmaceutical preparations.

[0082] The above dosage forms can be administered by injection, including subcutaneous injection, intravenous injection, intramuscular injection and intracavitary injection.

[0083] To solve the above technical problems, in a fourth aspect, the present invention provides a method for drug screening, which comprises using the transgenic animal cell line, or transgenic animal tissue, or transgenic animal organ described in B11) above to screen drugs, wherein the drugs may be drugs for preventing and / or treating obesity or obesity-related diseases.

[0084] The screening method involves observing whether the location of the MICU1 protein in the mitochondria changes after drug administration to provide a predictive or auxiliary predictive judgment. Specifically, if MICU1 in brown adipocytes of mice is translocated from the cristae membrane inside the mitochondria to the inner boundary membrane of the mitochondria after drug administration, the drug to be screened may be a drug or candidate for use in preventing and / or treating obesity or obesity-related diseases.

[0085] The drug treats and / or prevents obesity and related metabolic diseases by inhibiting the expression of MICU1 protein in brown fat cells or affecting the mitochondrial localization of MICU1, or increasing the activity of mitochondrial calcium ion channel MCU.

[0086] In one embodiment of the present invention, norepinephrine (NE) treatment causes MICU1 protein to be transferred from the cristae membrane inside the mitochondria to the inner boundary membrane of the mitochondria.

[0087] To solve the above technical problems, in a fifth aspect, the present invention provides a method for regulating the metabolic level of a body and / or organ and / or tissue and / or cell, the method comprising M1) and M2):

[0088] M1) regulating the activity or content of the EMRE-UCP1 protein in the recipient's body and / or organ and / or tissue and / or cell to regulate the metabolic level of the body and / or organ and / or tissue and / or cell;

[0089] M2) regulates the activity or content of MICU1 (Mitochondrial calcium uptake protein 1) protein in the body and / or organs and / or tissues and / or cells of the receptor to regulate the metabolic level of the body and / or organs and / or tissues and / or cells.

[0090] Further, in the above-mentioned method, M1) the regulation of the EMRE-UCP1 protein activity or content in the body and / or organ and / or tissue and / or cell of the receptor is to increase or promote or up-regulate the EMRE-UCP1 protein activity or content in the body and / or organ and / or tissue and / or cell of the receptor, so as to promote or increase the metabolic level of the body and / or organ and / or tissue and / or cell.

[0091] M2) the regulation of the MICU1 protein activity or content in the body and / or organ and / or tissue and / or cell of the receptor is to inhibit or reduce or down-regulate the MICU1 protein activity or content in the body and / or organ and / or tissue and / or cell of the receptor, so as to promote or increase the metabolic level of the body and / or organ and / or tissue and / or cell.

[0092] Further, in the above-mentioned method, M1) the EMRE-UCP1 protein activity or content in the body and / or organ and / or tissue and / or cell of the receptor is increased or promoted or up-regulated by introducing a recombinant virus expressing the EMRE-UCP1 into the body and / or organ and / or tissue and / or cell of the receptor.

[0093] M2) the MICU1 protein activity or content in the body and / or organ and / or tissue and / or cell of the receptor is inhibited or reduced or down-regulated by introducing the recombinant virus of B14) into the body and / or organ and / or tissue and / or cell of the receptor.

[0094] Further, in the above-mentioned method, the body can be a mammal, the tissue can be adipose tissue, and the cell can be an adipocyte.

[0095] In the present application, the mammal can be a mouse,

[0096] The adipose tissue can be brown adipose tissue, subcutaneous adipose tissue, perigonadal adipose tissue, and liver tissue.

[0097] Further, the adipocyte can be a brown adipocyte.

[0098] The application or method provided by the present application can be for the purpose of diagnosis and treatment of diseases, or for the purpose of diagnosis and treatment of non-diseases.

[0099] In the present application, the obesity-related diseases include hypertension, hyperglycemia, hyperlipidemia, endocrine disorders, etc. caused by obesity.

[0100] In the present application, the increase or promotion or up-regulation of the metabolic level of the body and / or organ and / or tissue and / or cell mainly manifests as:

[0101] ① the oxygen consumption and heat production of the body and / or organ and / or tissue and / or cell are increased;

[0102] ② the insulin response level of the body and / or organ and / or tissue and / or cell is improved, that is, the blood glucose is reduced more obviously after injection of insulin;

[0103] ③ the glucose tolerance of the body and / or organ and / or tissue and / or cell is improved, and the glucose can be consumed more quickly;

[0104] ④ under high-fat feeding conditions, the fat accumulation amount of the control group is reduced, mainly manifested as that the weights of brown adipose tissue, subcutaneous adipose tissue, gonadal adipose tissue and liver tissue are lower than those of the control group;

[0105] ⑤ the norepinephrine (NE) induced brown adipocyte mitochondrial calcium absorption can be enhanced.

[0106] The beneficial technical effects obtained by the present application are as follows:

[0107] 1. The present application fuses the natural proteins EMRE and UCP1 to construct a new protein, which can interact with the endogenous calcium channel MCU, enhance the MCU activity, increase the mitochondrial calcium absorption, and increase the heat production effect of the body. The expression of the tandem protein can significantly improve the heat production capacity of mouse brown adipose tissue, increase the energy output of the body, thereby effectively resisting obesity, improving the insulin sensitivity and glucose tolerance induced by obesity in mice. Therefore, the tandem protein or its truncated form is expected to develop into a protein drug for treating obesity and related metabolic diseases.

[0108] 2. The present application provides 1.1K-ADP carrier and 1.6K-ADP carrier or 1.1K-ADP promoter and 1.6K-ADP promoter for specifically overexpressing exogenous proteins in mouse adipocytes or adipose tissue, which are used for scientific research or treatment of fat and fat-related diseases.

[0109] 3. The present application provides a method for screening biological agents or drugs for preventing and treating obesity and related diseases by taking the MICU1 protein in brown adipocytes or the activity of the mitochondrial calcium channel MCU as a target. The drugs inhibit the expression of the MICU1 protein in brown adipocytes or affect the mitochondrial localization of MICU1, or improve the activity of the mitochondrial calcium channel MCU to treat and / or prevent obesity and related metabolic diseases.

[0110] 4. The content of the MICU1 protein in brown adipocytes is reduced or the function of the MICU1 protein is reduced to prevent and / or treat obesity and diseases related to obesity. BRIEF DESCRIPTION OF DRAWINGS

[0111] Figure 1Schematic diagram of the design of the target fragment in pAAV-1.1K-ADP-GFP-Flag and pAAV-1.6K-ADP-GFP-Flag.

[0112] Figure 2 Plasmid map of pAAV-CMV-GFP-Flag.

[0113] Figure 3 Plasmid map of pAAV-1.1K-ADP-GFP-Flag.

[0114] Figure 4 Plasmid map of pAAV-1.6K-ADP-GFP-Flag.

[0115] Figure 5 Calculation formula of AAV titer.

[0116] Figure 6 ADP promoter tissue expression specificity immunoblotting detection.

[0117] Figure 7 Immunoblotting analysis of the expression activity of 1.6K-ADP and 1.1K-ADP promoters in three types of adipose tissues.

[0118] Figure 8 Model display of the interaction of tandem protein MICU1 and endogenous MCU.

[0119] Figure 9 CLAMS detection of norepinephrine (NE) induced oxygen consumption rate of mice, n = 6.

[0120] Figure 10 Under the stimulation of norepinephrine (NE) (1.5 μM), the mitochondrial calcium absorption of primary mature brown adipocytes was monitored by GCaMP5-mt. The primary mature brown adipocytes were isolated from BAT of WT mice overexpressing UCP1, EMRE, EMRE (S85W)-UCP1 or EMRE-UCP1, n = 25 cells.

[0121] Figure 11 Body weight growth curve of mice under high-fat feeding, n = 8.

[0122] Figure 12 Insulin tolerance test was performed on mice fed with high-fat diet for 12 weeks, n = 8.

[0123] Figure 13 Glucose tolerance test was performed on mice fed with high-fat diet for 14 weeks, n = 8.

[0124] Figure 14Body fat was measured using a DEXA body fat analyzer after 16 weeks of high-fat feeding. (a) DEXA analysis of body fat content in mice after 16 weeks of high-fat feeding, n = 8. (b) Adipose tissue and liver weights in mice after 16 weeks of high-fat feeding, n = 8.

[0125] Figure 15 is the knockout efficiency of MICU1 in brown adipose tissue of Micu1-BKO mice.

[0126] Figure 16 Norepinephrine (NE)-induced oxygen consumption in control and Micu1-BKO mice was measured by CLAMS, n=6.

[0127] Figure 17 Figure 2 is the mitochondrial calcium uptake induced by norepinephrine (NE) in primary mature brown adipocytes from Ctrl and Micu1-BKO mice, n=25 cells.

[0128] Figure 18 Immunofluorescence staining was used to analyze the localization of MICU1-Flag in mitochondria of primary mature brown adipocytes after mice were treated with norepinephrine (NE). Figure 18 (a) is a fluorescence observation photo. Figure 18 Middle (b) shows the fluorescence intensity and the distance from the mitochondrial center.

[0129] Figure 19 Body weight changes of control and Micu1-BKO mice under high-fat diet, n=6.

[0130] Figure 20 Figure 2 shows the body fat content of control and Micu1-BKO mice fed a high-fat diet, where (a) shows the body fat content of mice fed a high-fat diet for 16 weeks, n = 6. (b) shows the tissue weights of three types of fat and liver in mice fed a high-fat diet for 16 weeks, n = 6.

[0131] Figure 21 Figure 2 shows the insulin sensitivity and glucose tolerance of control and Micu1-BKO mice under high-fat diet, where (a) is the insulin tolerance test of mice fed a high-fat diet for 12 weeks, n = 6. (b) is the glucose tolerance test of mice fed a high-fat diet for 14 weeks, n = 6. DETAILED DESCRIPTION

[0132] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0133] The experimental methods in the following examples are all routine methods, unless otherwise specified, which are carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained commercially.

[0134] The above content of the application will be further described in detail in the form of preferred embodiments, but it does not constitute a limitation on the application.

[0135] The experimental methods in the following examples are all routine methods, unless otherwise specified.

[0136] The test materials used in the following examples, unless otherwise specified, can be purchased through commercial channels.

[0137] The sequence of the mouse Adiponectin gene is available at genbank accession number NM_001177800.2 (24-JUL-2022), and the encoded amino acid sequence is available at genbank accession number NP_001171271.1 (24-JUL-2022).

[0138] All mice were raised in standard independent ventilation cages (IVC) with a temperature of 22±1℃, humidity of 40%-60%, 12-hour day-night alternation, free access to water and food, and no pathogens in the environment. Unless otherwise specified, standard ordinary feed (purchased from Jiangsu Cooperation Pharmaceutical Biological Engineering Co., Ltd.) was fed. All mice were raised in accordance with the regulations of the Animal Management and Use Committee of Peking University.

[0139] The pAAV-CAG-GFP-Flag vector is preserved in the laboratory and is disclosed in the literature "Yao J, Wu D, Zhang C, Yan T, Zhao Y, Shen H, Xue K, Huang X, Wang Z, Qiu Y. Macrophage IRX3 promotes diet-induced obesity and metabolic inflammation. Nat Immunol. 2021 Oct;22(10):1268-1279. doi: 10.1038 / s41590-021-01023-y." The above biological material can be obtained from the applicant by the public, and the obtained biological material can only be used for experimental verification in this application and cannot be used for other purposes.

[0140] Example 1, adipocyte-specific overexpression of exogenous protein 1.6K-ADP / 1.1K-ADP promoter and vector construction

[0141] 1.1, Obtaining 1.6k-ADP / 1.1K-ADP promoter region

[0142] In the mouse genome, the first adenosine ribonucleotide of the ATG codon of the Adiponectin gene (adipoq or ADP for short) is 0bp, the DNA fragment from -8446bp to -9572bp upstream is the 1.1K-ADP promoter, and its nucleotide sequence is SEQ ID No. 1; the DNA fragment from -8446bp to -1088bp upstream is the 1.6K-ADP promoter, and its nucleotide sequence is SEQ ID No. 2 156-1797.

[0143] Table 1: 1.6k-ADP / 1.1K-ADP promoter sequence

[0144]

[0145]

[0146] 1.2, Constructing overexpression vector

[0147] A small piece of mouse tail was obtained using scissors and placed in a 1.5ml centrifuge tube, 200uL of genomic extraction buffer (10mM Tris-HCl, pH 8.0, 50mM NaCl, 25mM EDTA, pH 8.0, 0.5% SDS) and 2uL of proteinase K were added, and the tissue was digested at 55°C for 4 hours. Then 5M NaCl was added to the reaction system to a final concentration of 1.5M NaCl, and it was placed on ice for 15 minutes. After centrifugation at 13000rpm, 4°C for 15 minutes, 400uL of supernatant was taken to a new 1.5mL centrifuge tube, 2.5 times the volume of absolute ethanol was added, and it was precipitated at -20°C for 20 minutes. After centrifugation at 13000rpm, 4°C for 15 minutes, the precipitate was dissolved with 20uL of water to obtain the mouse genome.

[0148] The 1.1K-ADP fragment is amplified by PCR (polymerase chain reaction) using primer pair 1.1K-ADP-F / ADP-R with mouse genome as template; the DNA fragment containing 1.6K-ADP is obtained by PCR (polymerase chain reaction) using primer pair 1.6K-ADP-F / ADP-R; then the obtained DNA fragment is cloned into the pAAV-CMV-GFP-Flag vector to obtain two plasmids, pAAV-1.1K-ADP-GFP-Flag and pAAV-1.6K-ADP-GFP-Flag, and the construction process is as follows: the DNA fragment containing 1.1K-ADP and the DNA fragment containing 1.6K-ADP obtained by primer amplification, and the vector pAAV-CMV-GFP-Flag are double digested with MluI and AgeI, then the DNA fragment containing 1.1K-ADP obtained by enzyme digestion is connected with the enzyme-digested vector pAAV-CMV-GFP-Flag to obtain the recombinant vector pAAV-1.1K-ADP-GFP-Flag, which is a recombinant vector obtained by replacing the small fragment between the MluI and AgeI enzyme digestion recognition sites of the vector pAAV-CMV-GFP-Flag with the 1.1K-ADP promoter shown in SEQ ID No. 1, maintaining other sequences of pAAV-CMV-GFP-Flag unchanged, and realizing the replacement of the CMV promoter in the vector pAAV-CMV-GFP-Flag with the 1.1K-ADP promoter; similarly, the DNA fragment containing 1.6K-ADP obtained by enzyme digestion is connected with the enzyme-digested vector pAAV-CMV-GFP-Flag to obtain the recombinant vector pAAV-1.6K-ADP-GFP-Flag, which is a recombinant vector obtained by replacing the small fragment between the MluI and AgeI enzyme digestion recognition sites of the vector pAAV-CMV-GFP-Flag with the 1.6K-ADP promoter shown in SEQ ID No. 2, maintaining other sequences of pAAV-CMV-GFP-Flag unchanged, and realizing the replacement of the CMV promoter in the vector pAAV-CMV-GFP-Flag with the 1.6K-ADP promoter. The full nucleotide sequence of the recombinant vector pAAV-1.6K-ADP-GFP-Flag is SEQ ID No. 2, and the recombinant vector pAAV-1.1K-ADP-GFP-Flag is obtained by replacing the 1.6K-ADP shown in SEQ ID No. 2 with the 1.1K-ADP shown in SEQ ID No. 1. The design of the adipocyte-specific overexpression of exogenous protein 1.6K-ADP / 1.1K-ADP promoter and vector is shown in Figures 1 to 4 wherein Figure 1Schematic diagram of the design of the target fragment in pAAV-1.1K-ADP-GFP-Flag and pAAV-1.6K-ADP-GFP-Flag, Figure 2 pAAV-CMV-GFP-Flag plasmid map, Figure 3 pAAV-1.1K-ADP-GFP-Flag plasmid map, Figure 4 pAAV-1.6K-ADP-GFP-Flag plasmid map.

[0149] The nucleotide sequence of the primer pair is as follows:

[0150] 1.1K-ADP-F: 5'-TGCGGCCGC ACGCGT GGTATATACTTAAGGAGTCT-3' (SEQ ID No. 3, the MluI enzyme digestion recognition site is marked with an underline);

[0151] ADP-R: 5'-CATGGTGGC ACCGGT TGCCCCAACCATGTTTCCAA-3' (SEQ ID No. 4, the AgeI enzyme digestion recognition site is marked with an underline);

[0152] 1.6K-ADP-F: 5'-TGCGGCCGC ACGCGT TGGGAAGTGTCCAGGGCCAT-3' (SEQ ID No. 5, the MluI enzyme digestion recognition site is marked with an underline).

[0153] 1.3, AAV virus packaging, purification, titer determination

[0154] Prepare 10 plates (15 cm in diameter cell culture plates) of 293T cells (CGMCC, 8.00020), the cell density is greater than 90%, and the deltaF6 plasmid (Addgene #112867), RC2 / 8 plasmid (Addgene #112864), and pAAV-1.1K-ADP-GFP-Flag plasmid are respectively transfected with PEI at a dosage of 200 ug, 70 ug, and 70 ug. After 24 hours, replace the fresh complete culture medium, and after 60 hours, blow and centrifuge to obtain the cell precipitate. Then, resuspend the precipitate with 2.5 ml of AAV lysis buffer (150 mM NaCl, 20 mM Tris pH 8.0, high-temperature high-pressure sterilized and stored at 4°C) to obtain the pAAV-1.1K-ADP-GFP-Flag virus stock solution.

[0155] Another 10 dishes (15 cm in diameter) of 293T cells were prepared, with a cell density of more than 90%, and the deltaF6 plasmid, RC2 / 8 plasmid, and pAAV-1.6K-ADP-GFP-Flag plasmid were transfected with PEI at a dosage of 200 ug, 70 ug, and 70 ug, respectively. After 24 hours, the complete culture medium was replaced, and after 60 hours, the cell precipitate was obtained by blowing and centrifugation. Then, the precipitate was resuspended in 2.5 ml of AAV lysis buffer (150 mM NaCl, 20 mM Tris pH 8.0, sterilized at high temperature and high pressure and stored at 4°C) to obtain the pAAV-1.6K-ADP-GFP-Flag virus stock solution.

[0156] The preparation method of the Vec virus stock solution (also called pAAV-1.6K-ADP virus stock solution) is the same as described above, except that the pAAV-1.6K-ADP-GFP-Flag plasmid is digested with AgeI and EcoRV to remove the GFP-Flag on the pAAV-ADP-GFP-Flag vector, and the connection is smoothed to become a pAAV-1.6K-ADP plasmid as a control virus. The other methods remain unchanged.

[0157] The virus stock solution described above was repeatedly frozen and thawed in liquid nitrogen and a 37°C water bath for 3 times until the cells were completely broken to release virus particles. Benzonase (working concentration 50 U / ml) was added and digested at 37°C for 15 minutes. The supernatant (containing released virus particles) was obtained by centrifugation at 4°C and 4000 rpm for 30 minutes.

[0158] Optiseal centrifuge tubes (361625, Beckman) were used to configure the Optiprep density gradient (6 ml-6 ml-5 ml-4 ml) at 17%-25%-40%-60%. The supernatant containing the virus was carefully placed on the upper layer of the 17% density layer, and the centrifuge tube was filled with AAV lysis buffer (150 mM NaCl, 20 mM Tris pH 8.0, sterilized at 4°C). The centrifuge tube was placed in a ultracentrifuge at 53000 rpm, 14°C for 2 hours and 4 minutes. The virus particles were mainly distributed in the 40% density layer. The liquid in this layer was sucked using a syringe and transferred to a centrifugal collection tube (Millipore, Amacon 100k-UFC 910096). The virus was concentrated by centrifugation at 3500 rpm for 10 minutes. Then, the PBS containing F188 (POLOMAXER: Sigma, 10000 times dilution) was added to fill the centrifuge tube. The PBS containing F188 was added again after the supernatant was discarded. The virus was finally concentrated in a volume of 500 ul after three times.

[0159] Take 5ul concentrated virus liquid with 10xdnase buffer, 2ul dnase (Takara, 5u / ul), 38ul water in centrifugal tube, digest the genomic DNA after 37℃ water bath for 15 minutes, transfer to 95℃ for 10 minutes to inactivate Dnase; then, 1ul protease K (20mg / ml), 5ul 10xdnase buffer, 44ddH2O, mix evenly, transfer to 95℃ for 10 minutes to inactivate protease K after 37℃ water bath for 15 minutes to obtain total viral genome.

[0160] The plasmid pAAV-1.6K-ADP-GFP-Flag is configured into a plasmid solution with a concentration gradient of 10, 1, 0.1, 0.01, 0.001, 0.0001, 0.00001, 0.000001 ng / ul, which is used to make a standard curve.

[0161] The extracted total viral genome and the plasmid solution with a gradient concentration are used as templates for reverse transcription according to the 5x All-In-One RT MasterMix kit instructions of abm company. The obtained cDNA is diluted with 100ul ultrapure water. Real-time fluorescent quantitative PCR uses a 96-well plate, with 2 replicate wells for each sample. Each well is added according to the EvaGreen 2x qPCR MasterMix-ROX kit instructions of abm company. Real-time quantitative detection is performed using the StepOnePlus instrument of ABI company. The AAV titer is calculated according to the AAV titer calculation formula shown in Figure 5 The AAV titer is calculated according to the AAV titer calculation formula shown in

[0162] 1.4, AAV virus in situ injection

[0163] The mouse is anesthetized, injected with analgesic agent, and the dorsal (brown fat location) hair is shaved off with an electric shaver. After disinfecting with 75% alcohol, the skin is cut open with scissors to expose the brown adipose tissue, and the AAV virus is uniformly injected into the brown adipose tissue using a microsyringe, with an injection dose of 2x10^ 11 Vp, then use wound clips to clamp the wound to normal skin state, and apply iodine to the wound.

[0164] The mouse is anesthetized, injected with analgesic agent, and the ventral (subcutaneous fat location) hair is shaved off with an electric shaver. After disinfecting with 75% alcohol, the skin is cut open with scissors to expose the subcutaneous fat tissue, and the AAV virus is uniformly injected into the subcutaneous fat tissue using a microsyringe, with an injection dose of 2x10^ 11 Vp, then use wound clips to clamp the wound to normal skin state, and apply iodine to the wound.

[0165] The mice were anesthetized, injected with a pain relieving agent, the abdomen (approximately the location of the intestines) was shaved with an electric shaver, and after being disinfected with 75% alcohol, the skin and peritoneum were cut with scissors to a length of about 1 cm, the epididymal adipose tissue was exposed, and the tissue was uniformly injected with AAV virus using a microsyringe at a dose of 2 x 10^ 11 Vp, and then the peritoneum and skin wounds were sutured to normal using sterilized needles and thread, and the wounds were applied with iodine.

[0166] 1.5, Tissue extraction and immunoblotting

[0167] The mice were sacrificed, and the subcutaneous adipose tissue (scWAT), brown adipose tissue (BAT), inguinal adipose tissue (eWAT), liver (Liver), muscle (Muscle), heart (Heart), spleen (Spleen), lung (Lung), kidney (Kidney), and hypothalamus (Hypothalamus) were obtained and stored in liquid nitrogen.

[0168] RIPA lysis solution (RIPA lysis solution: 50 mM Tris-HCl pH = 7.5, 450 mM sodium chloride, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate; protease inhibitor components in RIPA lysis solution: 5 mM NaF, 1 mM Na3VO4, 1 mM PMSF, 1 μg / ml leupeptin, 0.2 μg / ml aprotinin) was prepared. The tissue was placed on ice, and 300 uL of pre-cooled RIPA lysis solution was added with pre-cooled steel beads. Then the tissue was transferred to a tissue grinder and ground at 45 HZ for 180 seconds, and then removed and lysed on ice for 30 minutes. After that, after centrifugation at 12000 rpm for 15 minutes, the supernatant was taken to a new 1.5 mL centrifuge tube to obtain the total protein extract.

[0169] Quantification of total protein: The total protein extract was subjected to total protein quantification analysis using a BCA kit.

[0170] Western blotting of proteins: HSP90 was used as an internal control protein to calibrate the loading amount of proteins in different groups of mice adipose tissue. The loading buffer was added to the total protein extract, heated at 95°C for 10 minutes to obtain the protein loading sample. After the protein loading sample was cooled, 30 μg was loaded into a 10% polyacrylamide gel for SDS-PAGE electrophoresis, and then the protein was electrotransferred to an NC membrane, and the NC membrane was blocked with 5% skim milk. After blocking, the membrane was washed with TBST for 3 times, 5 minutes each time. After washing the membrane, the primary antibody diluent (10000 times dilution of Flag antibody, 10000 times dilution of GFP antibody, 10000 times dilution of HSP90 antibody) was incubated at 4°C for 12 hours. Then washed with TBST for 3 times, 5 minutes each time, and the corresponding goat anti-mouse IgG (H+L) secondary antibody or goat anti-rabbit IgG (H+L) secondary antibody was added and incubated at room temperature for 1 hour. Then the membrane was washed with TBST for 3 times, 5 minutes each time. Finally, the Super ECL Detection Reagent chemiluminescence supersensitive color developing kit from Superbio Technology Co., Ltd. was incubated for 10 seconds, and the picture was taken on the 5200 automatic chemiluminescence / fluorescence image analysis system from Tian Neng Technology Co., Ltd. The experimental results are shown in Figure 6 With Figure 7 .

[0171] Figure 6 The results show that after subcutaneous adipose tissue (scWAT) injection of AAV-1.1K-ADP-GFP-Flag (shown as ADP (1.1K)) and AAV-1.6K-ADP-GFP-Flag (shown as ADP (1.6K)) viruses, the FLAG protein is only specifically expressed in the subcutaneous adipose tissue (scWAT), with a small amount of expression in the brown adipose tissue (BAT), but no expression is detected in the inguinal adipose tissue (eWAT), liver (Liver), muscle (Muscle), heart (Heart), spleen (Spleen), lung (Lung), kidney (Kidney) and hypothalamus (Hypothalamus), which indicates that AAV-1.1K-ADP-GFP-Flag and AAV-1.6K-ADP-GFP-Flag have very good tissue specificity, and only have high expression in the subcutaneous adipose tissue where they are injected in situ.

[0172] Figure 7The results show that after in situ injection of the Vec virus (shown as Vec, no expression of exogenous protein and Flag tag), AAV-1.1K-ADP-GFP-Flag (shown as ADP (1.1K)) and AAV-1.6K-ADP-GFP-Flag (shown as ADP (1.6K)) viruses into brown adipose tissue (BAT), subcutaneous adipose tissue (scWAT) and inguinal adipose tissue (eWAT), respectively, the FLAG protein and the GFP protein can be detected in the expression of the three adipose tissues, which shows that the 1.6K-ADP promoter and the 1.1K-ADP promoter have expression activity in the three adipose tissues. And in the three adipose tissues, the expression amount of the FLAG protein and the GFP protein in the ADP (1.6K) group is slightly higher than that in the ADP (1.1K) group, which shows that the activity of the 1.6K-ADP promoter is slightly higher than that of the 1.1K-ADP promoter. Therefore, in the following examples, the recombinant plasmid pAAV-1.6K-ADP-GFP-Flag, abbreviated as pAAV-ADP-GFP-Flag, is mainly used.

[0173] Example 2, tandem protein EMRE-UCP1 enhances norepinephrine-induced oxygen consumption in mice

[0174] 2.1, The open reading frame (ORF) of mouse Mcu, Emre, Micu1 and Ucp1 was cloned from the cDNA of brown adipose tissue (BAT) to obtain the corresponding sequence. At the same time, the mtGFP sequence was synthesized. The above sequence (the open reading frame (ORF) of Mcu, Emre, Micu1 and Ucp1 and the mtGFP sequence) was replaced with a small fragment between the BglII and SalI enzyme digestion sites of the pShuttle-IRES-hrGFP-1 (Agilent technologies, #240081) plasmid. Then the sequence fragment with FLAG cloned on the pShuttle-IRES-hrGFP-1 vector was cloned into the pAAV-ADP-GFP-Flag vector to replace the fragment between the AgeI and EcoRV (remove the original GFP-Flag). The pAAV-ADP-expression sequence-Flag vector was obtained.

[0175] Taking EMRE as an example, the ORF of EMRE is cloned into the plasmid pShuttle-IRES-hrGFP-1, and the ORF of EMRE is used to replace the small fragment between the BglII and SalI enzyme digestion sites of the plasmid pShuttle-IRES-hrGFP-1. Then the EMRE-Flag fragment on the plasmid is cloned into the pAAV-ADP-GFP-Flag vector to replace the fragment between the AgeI and EcoRV. The recombinant expression plasmid pAAV-ADP-EMRE-Flag is obtained.

[0176] wherein the NCBI Reference Sequence for the nucleotide sequence of mouse Mcu open reading frame (ORF) is NM_001033259.4 (03-JUL-2022) and the NCBI Reference Sequence for the amino acid sequence of MCU protein is NP_001028431.2 (03-JUL-2022).

[0177] The NCBI Reference Sequence for the nucleotide sequence of mouse Emre open reading frame (ORF) is XM_036159567.1 (21-SEP-2020) and the NCBI Reference Sequence for the amino acid sequence of EMRE protein is XP_036015460.1 (21-SEP-2020).

[0178] The NCBI Reference Sequence for the nucleotide sequence of mouse Micul open reading frame (ORF) is NM_144822.3 (19-JUN-2022) and the NCBI Reference Sequence for the amino acid sequence of MICU1 protein is NP_659071.1 (19-JUN-2022). The NCBI Reference Sequence for the nucleotide sequence of Micul gene is NC_000076.7 (59538385-5969956).

[0179] The NCBI Reference Sequence for the nucleotide sequence of mouse Ucpl open reading frame (ORF) is NM_009463.3 (19-JUN-2022) and the NCBI Reference Sequence for the amino acid sequence of UCP1 protein is NP_033489.1 (19-JUN-2022).

[0180] The nucleotide sequence of mtGFP is SEQ ID No. 6.

[0181] Table 2: Nucleotide sequence of mtGFP

[0182]

[0183] 2.2, AAV plasmid construction of tandem protein EMRE-UCP1

[0184] The three sequences of Emre, linker and Ucp1 were connected in turn by the method of overlap PCR. The amino acid sequence of the linker (21) is: GSGSGSGSAAGSGSGSGSGSG (SEQ ID No. 7, 108-128); the nucleotide sequence is: GGT TCA GGC TCT GGA AGT GGG TCC GCG GCC GGA AGT GGT AGT GGC TCC GGT TCC GGA TCC GGT (SEQ ID No. 8, 322-384). Finally, the recombinant vector pAAV-ADP-EMRE-UCP1-Flag was obtained, and the assembly process was the same as that of pAAV-ADP-EMRE-Flag in 2.1. The assembly process of EMRE(S85W)-UCP1 is the same, except that the nucleotide sequence of the coding gene of EMRE(S85W)-UCP1 is AGC replaced by TGG at SEQ ID No. 8, 253-255.

[0185] EMRE(S85W)-UCP1 or mtGFP cannot interact with endogenous MCU (S85W mutation can destroy the interaction between EMRE and MCU). See the schematic diagram of the tandem protein Figure 8 .

[0186] Table 3: Amino acid sequence and coding sequence of EMRE-UCP1 protein

[0187]

[0188]

[0189] 2.3, pAAV-CAG-GCaMP5mt plasmid construction

[0190] The nucleotide sequence encoding GCaMP5mt was cloned into the AAV master plasmid vector pAAV-CAG-GFP-Flag to obtain the recombinant expression plasmid pAAV-CAG-GCaMP5mt, in which CAG is a constitutive enhancer promoter similar to CMV. The constructed AAV master plasmid pAAV-CAG-GCaMP5mt is used for AAV (serotype 2 / 8) packaging, and the process is referred to in Example 1.

[0191] The recombinant plasmid pAAV-CAG-GCaMP5mt is a recombinant expression plasmid obtained by replacing the fragment between the Age I and Hind III enzyme cutting sites of the plasmid pAAV-CAG-GFP-Flag with a DNA molecule encoding GCaMP5mt, while keeping other nucleotide sequences of the plasmid pAAV-CAG-GFP-Flag unchanged. The nucleotide sequence of the DNA molecule encoding GCaMP5mt is SEQ ID No. 9.

[0192] Table 4: Nucleotide sequence encoding GCaMP5mt

[0193]

[0194]

[0195] 2.4, pAAV-ADP-mtGFP-Flag, pAAV-ADP-UCP1-Flag, pAAV-ADP-EMRE-Flag, pAAV-ADP-EMRE(S85W)-UCP1-Flag, pAAV-ADP-EMRE-UCP1-Flag and pAAV-CAG-GCaMP5mt were packaged into AAV respectively, and the AAV packaging process was referred to Example 1. The obtained AAV was injected into the brown adipose tissue of each group of mice respectively, and the process was the same as Example 1. The injection amount was 5x10 11 vp / mouse. After three weeks, the mice were anesthetized and placed in the CLAMS comprehensive experimental animal monitoring system at 32°C, and the overall oxygen consumption rate of the mice was monitored. After the basal oxygen consumption rate was stable, the mice were injected with norepinephrine (NE, 1 mg / kg) subcutaneously between the scapulae to stimulate the thermogenesis of brown adipose tissue. After 60-90 minutes of norepinephrine (NE) injection, the mice began to wake up, at which time the oxygen consumption recording was stopped.

[0196] The results are shown in Figure 9 As shown in the results, after overexpression of mtGFP, UCP1, EMRE, EMRE(S85W)-UCP1 or EMRE-UCP1 in the brown adipose tissue (BAT) of wild-type mice by site-specific injection of AAV, it was found that compared with other overexpressed proteins, EMRE-UCP1 could significantly increase the norepinephrine-induced oxygen consumption of mice and enhance the thermogenic capacity of brown adipose tissue (BAT).

[0197] Example 3, tandem protein EMRE-UCP1 enhances norepinephrine-induced calcium ion absorption of brown adipose tissue mitochondria

[0198] 3.1, AAV virus packaging

[0199] 1) The plasmids pAAV-ADP-UCP1-Flag, pAAV-ADP-EMRE-Flag, pAAV-ADP- EMRE(S85W)-UCP1-Flag, pAAV-ADP-EMRE-UCP1-Flag, pAAV-CAG-GCaMP5mt were packaged into AAV respectively, the virus packaging process is the same as Example 1.3, the only difference is that the pAAV series of plasmids are replaced by the above plasmids, and the corresponding virus stock solutions are prepared respectively.

[0200] 2) The AAV-ADP-UCP1-Flag, pAAV-ADP-EMRE-Fla, pAAV-ADP-EMRE(S85W)- UCP1-Flag, pAAV-ADP-EMRE-UCP1-Flag packaged AAV (injection amount is 5x10 11 vp / mouse) were combined with pAAV-CAG-GCaMP5mt (5x10 11 vp / mouse) respectively and injected into the brown adipose tissue of each group of mice, the injection process is the same as Example 1. Mature brown adipocytes were extracted and adherent cultured for calcium measurement experiment after 3 weeks of injection.

[0201] 3.2, Isolation and culture of mature brown adipocytes

[0202] Interscapular brown adipose tissue was quickly isolated and minced from mice and digested in digestion buffer (Hank’s Balanced Salt Solution (HBSS, Corning, 21-023-CV) plus 4% free fatty acid-BSA (Sigma, S25762) and 2 mg / ml Collagenase II (Sigma-Aldrich, V900892)) for 30 min at 37°C (100 rpm). After digestion, cells were washed three times with Krebs Ringer Bicarbonate Buffer (KRBMB, 120 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 0.4 mM K2HPO4, 10 mM Glucose, 15 mM NaHCO3, and 20 mM HEPES) containing 4% free fatty acid-BSA, 30 g centrifugation for 5 min each time, and the supernatant and other cells at the bottom were discarded with a long needle tube. The washed cells were resuspended in mature brown adipocyte culture medium (DMEM / F-12, Hyclone, SH30023.02, plus 10% FBS) which was previously CO2- charged for 2 hours in a CO2incubator to facilitate cell attachment and survival in a sealed dish. The resuspended cells were placed in a cell culture dish (norepinephrine (NE) ST, 801002) coated with collagen IV (Sigma-Aldrich, C5533) at the bottom, and filled with culture medium. The dish was carefully sealed with a dish cover to avoid air bubbles. The sealed dish was inverted so that the mature brown adipocytes were suspended and attached to the collagen IV-coated glass. The cells were left to attach in a CO2incubator for 12 hours, and then the successfully attached mature brown adipocytes were used for calcium imaging or immunofluorescence experiments.

[0203] 3.3, Measurement of mitochondrial calcium uptake in mature brown adipocytes

[0204] Before imaging, the attached mature brown adipocytes were gently washed three times with pre-warmed HBSS. 100 μΐ of HBSS was added first, and the dish was placed in a live cell workstation of a DeltaVision Elite microscope (Applied Precision) for imaging. The incubation condition of the live cell workstation was 37°C with 5% CO2. The imaging condition was Plan Fluor 10x inverted air objective, excitation light at 488 nm, one image per second for 720 seconds. 100 μΐ of HBSS containing 3 μΜ norepinephrine was added at 120 seconds into the imaging to give a final concentration of 1.5 μΜ. The imaging data were processed with Volocity software, and the intensity of GCaMP5-mt in each cell was calculated after background fluorescence was subtracted.

[0205] Results are shown in Figure 10 Figure 6. Expression of tandem protein EMRE-UCP1 in brown adipocyte mitochondria can significantly enhance norepinephrine-induced mitochondrial calcium uptake, while proteins UCP1, EMRE or EMRE(S85W)-UCP1 do not affect norepinephrine (NE)-induced mitochondrial calcium uptake. Figure 10 In the figure, UCP1, EMRE and EMRE(S85W)-UCP1 are coincident, and the fluorescence density F / F0 after adding NE is less than 1.5, and the fluorescence density F / F0 of EMRE-UCP1 after adding NE is not less than 1.5. This result shows that the EMRE-UCP1 protein complex can improve the mitochondrial calcium uptake activity of brown adipocytes under norepinephrine (NE) stimulation.

[0206] Example 4, Tandem protein EMRE-UCP1 improves high-fat diet-induced obesity and related metabolic defects

[0207] 4.1, pAAV-ADP-mtGFP-Flag, pAAV-ADP-EMRE(S85W)-UCP1-Flag, and pAAV-ADP-EMRE-UCP1-Flag were packaged into AAV, respectively. The AAV was injected into the brown adipose tissue of each group of mice, and the process was the same as in Example 1. The injection amount was 5x10 11 vp / mouse. After 2 weeks of injection, high-fat feeding was started, and insulin tolerance test was performed at 12 weeks of high-fat feeding, glucose tolerance test was performed at 14 weeks, and body fat measurement was performed after 16 weeks, and the weights of three kinds of fat and liver were measured.

[0208] The body weight change curve is shown in Figure 11 Compared with the control group, the mice expressing the tandem protein EMRE-UCP1 grew more slowly in body weight under high-fat feeding.

[0209] 4.2, Insulin tolerance test (ITT),

[0210] The operation is as follows: the mice in each group were fasted for 6 hours, then 1 U / kg body weight of insulin was injected intraperitoneally, and the blood glucose of the tail tip was measured with a blood glucose meter at 15, 30, 60, 90 and 120 minutes after injection, respectively. The results are shown in Figure 12 .

[0211] Figure 12 The results show that the blood glucose of each group of mice is significantly reduced after injection of insulin, but compared with the control groups overexpressing mtGFP and EMRE(S85W)-UCP1, the blood glucose of the mice overexpressing the EMRE-UCP1 tandem protein is reduced more obviously, indicating that the response to insulin is better.

[0212] 4.3 Glucose tolerance test (GTT)

[0213] The operation is as follows: each group of mice is fasted for 14 hours overnight, then intraperitoneally injected with 2g / kg body weight of D(+)-glucose, and the blood glucose at the tail tip is measured with a blood glucose meter before injection (i.e. 0 min) and 15, 30, 60, 120 and 150 minutes after injection, respectively. The results are shown in Figure 13 .

[0214] Figure 13 The results show that the blood glucose of each group of mice has a significant increase 15 minutes after injection of glucose, but 30 minutes later, the increase in the overexpression of EMRE-UCP1 tandem protein is lower, and a more obvious downward trend appears, which indicates that the mice have better ability to control blood glucose.

[0215] 4.4 Body fat measurement

[0216] After 16 weeks of high-fat feeding, the body fat of the mice was measured by DEXA body fat instrument. Then the mice were sacrificed, and the adipose tissue including brown adipose tissue, subcutaneous adipose tissue, and perigonadal adipose tissue, and liver tissue were taken out and weighed, respectively.

[0217] Figure 14 The results show that the body fat content of the mice expressing EMRE-UCP1 is significantly lower than that of the control mice, and the weight of the three adipose tissues and liver is also lower than that of the control mice.

[0218] Example 5: Brown fat-specific knockout of Micul enhances norepinephrine-induced oxygen consumption in mice

[0219] 5.1 Obtaining of Micul-BKO mice: Micul-gRNA (CTA GTT CTG CCA ACG CAG AA) was cloned into the vector pAAV-U6-gRNA (Addgene #91947, cut the vector with BspQI enzyme) to obtain pAAV-U6-Micul-gRNA, and the constructed AAV master plasmid was used for AAV (serotype 2 / 8) packaging, the process is the same as in Example 1. pAAV-U6-gRNA was used as a control gRNA plasmid for AAV (serotype 2 / 8) packaging, the process is the same as in Example 1.

[0220] Rosa26-LSL-Cas9 mice (Jaxlab, 026175) and AdiponectinCre mice (Jaxlab, 010803) were mated to obtain offspring Rosa26-LSL-Cas9; AdiponectinCre mice, which can specifically express Cas9 protein in adipocytes.

[0221] AAV was injected into the BAT of Rosa26-LSL-Cas9; AdiponectinCre mice to specifically knock out Micul in BAT. The injection dose of gRNA was 1x10 12 vp, significant knock-out effect was achieved 3 weeks after virus injection.

[0222] Western-blot (WB) was used to detect the knock-out effect of MICU1, with HSP90 as a reference. The primary antibody was Rabbit anti-MICU1 (Sigma, Cat. #HPA037479), Mouse anti-HSP90a / b (Santa Cruz Biotech, Cat. #sc-7947), and the secondary antibody was Goat anti-mouse IgG:HRP, Thermo Scientific, Cat. #32430, Goat anti-rabbit IgG:HRP, Thermo Scientific, Cat. #31460. The WB results of the knock-out efficiency of MICU1 in the BAT of Micul-BKO mice are shown in Figure 5. Figure 15 , Figure 15 Micul-BKO represents MICU1 knock-out mice, and Ctrl represents wild-type mice (Ctrl). The results show that the MICU1 protein has been successfully knocked out in the Micul-BKO mice.

[0223] 5.2, After the mice were anesthetized, they were placed in the CLAMS comprehensive experimental animal monitoring system, and the overall oxygen consumption rate of the mice was monitored at 32°C. After the basal oxygen consumption rate was stable, the mice were subcutaneously injected with norepinephrine (NE, 1 mg / kg) between the shoulder blades to stimulate brown adipose tissue thermogenesis. After 60-90 minutes of norepinephrine (NE) injection, the mice began to wake up, at which time the oxygen consumption recording was stopped.

[0224] The results are shown in Figure 6. Figure 16 The medium gray square icon represents the Micul-BKO mice, and the black circle icon represents the wild-type mice (Ctrl). The results show that the Micul-BKO mice can significantly increase the oxygen consumption induced by norepinephrine and enhance the thermogenic capacity of BAT compared to the control gRNA. Figure 16

[0225] Example 6, Brown fat-specific knock-out of Micul enhances norepinephrine-induced mitochondrial calcium uptake in brown adipocytes

[0226] The preparation method of the Micul-BKO mice is the same as that of Example 5.

[0227] ​The isolation and culture of mature brown adipocytes were the same as in Example 3.

[0228] The measurement of mitochondrial calcium ion uptake in mature brown adipocytes was the same as in Example 3.

[0229] The results are as follows Figure 17 As shown: When stimulated by norepinephrine (NE), BAT mitochondria of Micu1-BKO mice exhibited stronger mitochondrial calcium ion uptake, indicating that Micu1 negatively regulates the activity of MCU channels in brown adipocytes.

[0230] Example 7: Brown fat-specific knockout of Micu1 protects against high-fat-induced obesity and related metabolic defects

[0231] Micu1-BKO mice were obtained as in Example 5.

[0232] Two weeks after virus injection, high-fat feeding was started. An insulin tolerance test was performed at 12 weeks of high-fat feeding, a glucose tolerance test was performed at 14 weeks, and body fat was measured after 16 weeks, and the three types of fat and liver were removed and weighed. The specific procedures were the same as in Example 4.

[0233] Among them, the weight change curve is as follows Figure 19 , compared with the control group, Micu1-BKO mice gained weight more slowly under high-fat feeding. Figure 20 The results showed that the body fat content of Micu1-BKO mice was significantly lower than that of control mice, and the weights of the three adipose tissues and liver were also lower than those of control mice. Figure 21 The results showed that ITT and GTT experiments indicated that Micu1-BKO mice had better insulin sensitivity and glucose tolerance under high-fat diet.

[0234] Example 7: Changes in Micu1 Mitochondrial Localization Under Norepinephrine Treatment

[0235] Preparation of brown fat overexpressing MICU1-FLAG mice: The open reading frame of Micu1 was cloned into the AAV master plasmid vector pAAV-ADP-MCS-Flag (same as Example 2), and the constructed AAV master plasmid was used for AAV (serotype 2 / 8) packaging, and the packaging process was the same as Example 1. The plasmid pAAV-ADP-MICU1-Flag was packaged into AAV, and the virus packaging process was the same as Example 1.3, the only difference being that the plasmid of the pAAV series was replaced with the above plasmid, and the corresponding virus stock solution was prepared respectively. AAV-ADP-MICU1-FLAG (injection volume was 5x10 11 vp / mouse) were injected into the brown adipose tissue of wild-type mice, and the injection process was the same as in Example 1. Three weeks after the injection, mature brown adipocytes were extracted and cultured for immunofluorescence experiments.

[0236] Immunofluorescence examination of Micul mitochondrial localization: Mature brown adipocytes were isolated from MICU1-FLAG expressing brown fat and plated on glass-bottomed dishes. Cells were washed with PBS and fixed with 4% paraformaldehyde and 0.1% glutaraldehyde in PBS for 30 min. Cells were permeabilized in PBS containing 3% BSA and 0.1% Triton X-100 for 1 h at room temperature. Primary antibody was diluted 1:200 in PBS containing 3% BSA and 0.1% Triton X-100 and cells were incubated with primary antibody overnight at 4°C. Cells were washed 3 times with PBS containing 0.3% BSA and 0.01% Triton X-100 before addition of fluorescent secondary antibody (Alexa Fluor 488 labeled goat anti-mouse antibody, Invitrogen, A11029, 1:400) for 1 h at room temperature. Cells were washed 3 times with PBS containing 0.3% BSA and 0.01% Triton X-100 and cells on the glass slides were covered with mounting buffer before the glass slides were sealed to the glass slides with nail polish. Samples were imaged with a DeltaVision OMX SR-3D-SIM (Applied Precision) using a 60x oil objective and data acquisition software SoftWoRx (Guo et al., 2020).

[0237] Results are shown in Figure 18 Figure 6: Noradrenaline (NE) treatment causes translocation of MICU1 from the crista membrane to the inner boundary membrane of mitochondria.

[0238] The present application has been described in detail. For those skilled in the art, without departing from the purpose and scope of the present application, and without unnecessary experiments, the present application can be implemented in a wider range under the same parameters, concentrations and conditions. Although the present application gives a special example, it should be understood that further improvements can be made to the present application. In summary, according to the principle of the present application, this application intends to include any change, use or improvement of the present application, including changes made by conventional techniques known in the art, which deviates from the scope disclosed in the present application.

Claims

1. A protein, characterized in that: The protein is EMRE-UCP1 protein, which is a protein formed by the fusion of mitochondrial calcium ion transport regulatory protein and uncoupling protein-1. The amino acid sequence of the EMRE-UCP1 protein is shown in SEQ ID No.

7.

2. A nucleic acid molecule, characterized in that: The nucleic acid molecule is a nucleic acid molecule encoding the EMRE-UCP1 protein according to claim 1.

3. An expression cassette, characterized in that: The expression cassette is an expression cassette containing the nucleic acid molecule according to claim 2.

4. The expression cassette according to claim 3, characterized in that: The promoter for driving the nucleic acid molecule in the expression cassette is an ADP promoter, and the ADP promoter is P1) or P2): P1) a DNA molecule having a nucleotide sequence of positions 156-1797 of SEQ ID No. 2; P2) The nucleotide sequence is a DNA molecule of SEQ ID No.

1.

5. A recombinant vector, characterized in that: The recombinant vector is a recombinant expression vector containing the nucleic acid molecule according to claim 2 or the expression cassette according to claim 3 or 4.

6. The recombinant vector according to claim 5, characterized in that: The recombinant vector is a recombinant adeno-associated virus vector.

7. A recombinant microorganism, characterized in that: The recombinant microorganism is a recombinant microorganism that expresses the EMRE-UCP1 protein according to claim 1.

8. The recombinant microorganism according to claim 7, characterized in that: The recombinant microorganism is a recombinant adeno-associated virus.

9. Application, characterized in that: The application is any of the following: A1) Use of the EMRE-UCP1 protein or a biomaterial related to the EMRE-UCP1 protein according to claim 1 for promoting or enhancing norepinephrine-induced calcium channel activity in brown adipocytes, wherein the use is for non-disease treatment and / or diagnosis purposes; A2) Use of the EMRE-UCP1 protein or a biomaterial related to the EMRE-UCP1 protein according to claim 1 for promoting or enhancing the oxygen consumption rate of brown adipocytes induced by norepinephrine, wherein the use is for non-disease treatment and / or diagnosis purposes; A3) Use of the EMRE-UCP1 protein or a biomaterial related to the EMRE-UCP1 protein according to claim 1 to promote or enhance the insulin response level of cells, wherein the use is for non-disease treatment and / or diagnosis purposes; A4) Use of the EMRE-UCP1 protein or a biomaterial related to the EMRE-UCP1 protein according to claim 1 for promoting or increasing the glucose metabolism rate of cells, where the use is for purposes other than disease treatment and / or diagnosis; A5) Use of the EMRE-UCP1 protein or a biomaterial related to the EMRE-UCP1 protein according to claim 1 for inhibiting or reducing fat accumulation in cells, wherein the use is for purposes other than disease treatment and / or diagnosis; A6) Use of the EMRE-UCP1 protein or a biomaterial related to the EMRE-UCP1 protein according to claim 1 in the preparation of a product for promoting or enhancing norepinephrine-induced calcium channel activity in brown adipocytes; A7) Use of the EMRE-UCP1 protein or a biomaterial related to the EMRE-UCP1 protein according to claim 1 in the preparation of a product for promoting or enhancing norepinephrine-induced oxygen consumption rate; A8) Use of the EMRE-UCP1 protein or a biomaterial related to the EMRE-UCP1 protein according to claim 1 in the preparation of a product that promotes or enhances the insulin response level of an organism and / or organ and / or tissue and / or cell; A9) Use of the EMRE-UCP1 protein or a biomaterial related to the EMRE-UCP1 protein according to claim 1 in the preparation of a product that promotes or increases the rate of glucose metabolism in an organism and / or an organ and / or a tissue and / or a cell; A10) Use of the EMRE-UCP1 protein or a biomaterial related to the EMRE-UCP1 protein according to claim 1 in the preparation of a product for inhibiting or reducing fat accumulation in an organism and / or an organ and / or a tissue and / or a cell; A11) Use of the EMRE-UCP1 protein or a biomaterial related to the EMRE-UCP1 protein according to claim 1 in the preparation of a product for preventing and / or treating obesity; The biological material is the nucleic acid molecule according to claim 2, the expression cassette according to claim 3 or 4, the recombinant vector according to claim 5 or 6, or / and the recombinant microorganism according to claim 7 or 8; The application is achieved by introducing the gene encoding the EMRE-UCP1 protein into an organism and / or an organ and / or a tissue and / or a cell.

10. A drug for preventing and / or treating obesity or obesity-related diseases, comprising the protein of claim 1 and / or a biological material related to the protein, wherein the biological material is the nucleic acid molecule of claim 2, the expression cassette of claim 3 or 4, the recombinant vector of claim 5 or 6, or the recombinant microorganism of claim 7 or 8.

11. A method for increasing cellular metabolic levels, the method comprising increasing the content of the EMRE-UCP1 protein of claim 1 in a recipient cell to increase the metabolic level of the recipient cell, wherein the method is for non-disease treatment and / or diagnosis purposes, and the metabolic level is at least one of the following (1) to (5): (1) Norepinephrine-induced calcium channel activity in brown adipocytes; (2) Norepinephrine-induced oxygen consumption rate of brown adipocytes; (3) The cellular insulin response level; (4) Cellular sugar metabolism rate; (5) The amount of fat accumulated in cells; The increasing of the EMRE-UCP1 protein content in the cell is achieved by introducing the gene encoding the EMRE-UCP1 protein into the cell.

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