WO2018024110A1 - Mg53突变体及其制备方法和用途 - Google Patents

Mg53突变体及其制备方法和用途 Download PDF

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WO2018024110A1
WO2018024110A1 PCT/CN2017/093640 CN2017093640W WO2018024110A1 WO 2018024110 A1 WO2018024110 A1 WO 2018024110A1 CN 2017093640 W CN2017093640 W CN 2017093640W WO 2018024110 A1 WO2018024110 A1 WO 2018024110A1
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seq
mutant
amino acid
acid sequence
serine
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PCT/CN2017/093640
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English (en)
French (fr)
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WO2018024110A8 (zh
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肖瑞平
吕凤祥
张岩
郭寺乐
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北京大学
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Priority claimed from CN201610847346.4A external-priority patent/CN107868125A/zh
Application filed by 北京大学 filed Critical 北京大学
Priority to EP17836292.7A priority Critical patent/EP3492491A4/en
Priority to JP2019505531A priority patent/JP7144402B2/ja
Priority to SG11201900459RA priority patent/SG11201900459RA/en
Priority to CA3031856A priority patent/CA3031856C/en
Priority to KR1020197005658A priority patent/KR102483242B1/ko
Priority to CN201780003946.5A priority patent/CN108473546B/zh
Publication of WO2018024110A1 publication Critical patent/WO2018024110A1/zh
Publication of WO2018024110A8 publication Critical patent/WO2018024110A8/zh
Priority to HK18114549.7A priority patent/HK1255559A1/zh
Priority to US16/262,833 priority patent/US11306296B2/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/104—Aminoacyltransferases (2.3.2)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y203/00—Acyltransferases (2.3)
    • C12Y203/02—Aminoacyltransferases (2.3.2)

Definitions

  • the present application relates to the field of biomedicine, and in particular, the present invention relates to an MG53 mutant, a pharmaceutical composition comprising the MG53 mutant, a nucleic acid encoding the MG53 mutant, a preparation method of the MG53 mutant, and MG53
  • Mitsugumin 53 also known as TRIM72, is a member of the Tripartite motif-containing Proteins (TRIM) family.
  • MG53 consists of an N-terminal TRIM domain and a C-terminal SPRY domain.
  • the TRIM domain consists of successively linked Ring, B-box, and coiled-coil domains (see Chuanxi Cai et al., the Journal of Biological Chemistry, Vol. 284(5), 3314-3322 (2009)).
  • MG53 is mainly expressed in striated muscle and plays an important role in the steady state maintenance of skeletal muscle and heart and even the whole body.
  • MG53 has a cell repair function and a cardioprotective function (see, for example, Chuanxi Cai et al., Nature Cell Biology, Vol. 11, 56-64 (2009); CN101797375B).
  • IPC ischemic preconditioning
  • PostC ischemic postconditioning
  • MG53 is also one of the important protective molecules, and the two ends of MG53 can be Caveolin-3 and p85-PI3K kinases bind to each other to form a complex and activate the downstream reperfusion injury salvage kinase (RISK) pathway to achieve myocardial protection (see, Chun-Mei Cao et al., Circulation 121, 2565-2574, (2010)).
  • RISK reperfusion injury salvage kinase
  • MG53 has a cell repair function and a cardioprotective function
  • E3 ubiquitin ligase activity is involved in the regulation of insulin resistance and the development of metabolic syndrome.
  • the Ring domain of the TRIM domain at the N-terminus of MG53 has E3 ubiquitin ligase activity and can bind to and mediate insulin receptor (IR) and insulin receptor substrate-1 (IRS1).
  • IR insulin receptor
  • IRS1 insulin receptor substrate-1
  • wild type MG53 can cause side effects of metabolic diseases such as insulin resistance, obesity, diabetes, hypertension, and dyslipidemia while repairing cells and protecting the heart. These side effects are undesirable
  • the present application relates to a MG53 mutant, a pharmaceutical composition comprising the MG53 mutant, a nucleic acid encoding the MG53 mutant, a preparation method of the MG53 mutant, and a MG53 mutant prepared for treating heart disease, diabetes
  • a MG53 mutant a pharmaceutical composition comprising the MG53 mutant, a nucleic acid encoding the MG53 mutant, a preparation method of the MG53 mutant, and a MG53 mutant prepared for treating heart disease, diabetes
  • diabetes Uses in cerebrovascular diseases, diabetic ocular complications, diabetic neuropathy, diabetic foot, kidney disease, drugs associated with diseases associated with cellular and/or tissue damage, in particular, the MG53 mutant has cell repair function and/ At the same time as the cardioprotective function, it can avoid or reduce the metabolic side effects such as insulin resistance, obesity, diabetes, hypertension, and dyslipidemia caused by wild-type MG53.
  • the application relates to a MG53 mutant, wherein the MG53 mutant has at least one serine deletion and/or in the coiled-coil-SPRY region of the wild-type MG53 based on the amino acid sequence of wild-type MG53 Or mutated to any other non-serine or threonine amino acid.
  • the coiled-coil-SPRY region is located in amino acid region 122-477 of the amino acid sequence of wild type MG53.
  • the wild type MG53 is derived from an animal, preferably from a mammal, eg, a human, mouse, rat, monkey, pig, dog, and the like.
  • the amino acid sequence of wild type MG53 is SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6.
  • the MG53 mutant has at least one serine mutated to a non-polar amino acid in the coiled-coil-SPRY region of the wild type MG53 based on the amino acid sequence of wild type MG53.
  • the non-polar amino acid is selected from the group consisting of glycine, alanine, leucine, isoleucine, valine, proline, phenylalanine, methionine And tryptophan.
  • the non-polar amino acid is alanine.
  • the MG53 mutant has at least one serine mutation in the coiled-coil-SPRY region of the wild-type MG53 based on the amino acid sequence of wild-type MG53, in addition to serine and threonine Polar amino acids outside.
  • the pole The amino acid is selected from the group consisting of glutamine, cysteine, asparagine, tyrosine, aspartic acid, glutamic acid, lysine, arginine and histidine.
  • the polar amino acid is cysteine.
  • the deletion or mutation of the serine is located at positions 150, 189, 211, 214, 246, 255 of the amino acid sequence of wild type MG53, SEQ ID NO: 1. 269th, 296th, 297th, 301st, 305th, 306th, 307th, 314th, 341th, 377th, 405th, 418th, 425th Bit or one or more of the 430th bit.
  • the serine that is deleted or mutated is located at positions 150, 189, 211, 214, 246, 255 of the amino acid sequence SEQ ID NO: 139 of wild type MG53, 269th, 296th, 297th, 301st, 305th, 306th, 307th, 314th, 341th, 377th, 405th, 418th, 425th Bit or one or more of the 430th bit.
  • the deletion or mutation of the serine is located at positions 150, 189, 211, 214, 246, 255 of the amino acid sequence SEQ ID NO: 140 of wild type MG53, 269th, 296th, 297th, 301st, 305th, 306th, 307th, 314th, 341th, 377th, 405th, 418th, 425th Bit or one or more of the 430th bit.
  • the serine that is deleted or mutated is located at positions 150, 189, 211, 214, 246, 255 of the amino acid sequence SEQ ID NO: 141 of wild type MG53, 269th, 296th, 297th, 301st, 305th, 306th, 307th, 314th, 341th, 377th, 405th, 418th, 425th Bit or one or more of the 430th bit.
  • the serine that is deleted or mutated is located at positions 150, 189, 211, 214, 246, 255 of the amino acid sequence SEQ ID NO: 142 of wild type MG53, One or more of the 269th bits.
  • the deletion or mutation of the serine is located at positions 188, 189, 210, 211, 214, 246 of the amino acid sequence SEQ ID NO: 2 of wild type MG53, 253th, 255th, 269th, 296th, 297th, 301st, 305th, 306th, 307th, 314th, 341th, 367th, 377th
  • One or more of the bits, 418th, 430th, or 440th bits are located at positions 188, 189, 210, 211, 214, 246 of the amino acid sequence SEQ ID NO: 2 of wild type MG53, 253th, 255th, 269th, 296th, 297th, 301st, 305th, 306th, 307th, 314th, 341th, 367th, 377th.
  • the deletion or mutation of the serine is located at positions 150, 188, 189, 210, 211, 214 of the amino acid sequence of wild type MG53, SEQ ID NO:3, 246th, 253th, 255th, 269th, 296th, 297th, 301st, 305th, 307th, 314th, 341th, 367th, 377th
  • the serine that is deleted or mutated is located at positions 150, 189, 211, 214, 246, 255 of the amino acid sequence SEQ ID NO: 4 of wild type MG53, 269, 296, 297, 301, 305, 306, 307, 341, 377, 405, 418, 425, 430 Bit or one or more of the 464th bit.
  • the serine that is deleted or mutated is located at positions 150, 189, 211, 214, 246, 255 of the amino acid sequence of wild type MG53, SEQ ID NO:5, 269th, 296th, 301st, 305th, 307th, 314th, 341th, 377th, 411th, 418th, 425th, 430th or 474th One or more of the bits.
  • the deletion or mutation of the serine is located at positions 150, 189, 211, 214, 246, 255 of the amino acid sequence of wild type MG53, SEQ ID NO: 6. 269, 296, 297, 301, 305, 307, 314, 341, 367, 377, 418, 425 or 430 One or more of the bits.
  • the deletion or mutation of the serine is located in the amino acid sequence of wild type MG53 (eg, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO : 5, SEQ ID NO: 6, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, or SEQ ID NO: 142) at positions 150, 188-189, 210-211 , 214th, 246th, 253th-255th, 269th, 296th-297th, 301st, 305-307th, 314th, 341th, 367th, 377th
  • One or more of the 405th, 411th, 418th, 425th, 430th, 440th, 464th, or 474th positions are examples of wild type MG53 (eg, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO : 5, SEQ ID NO: 6, SEQ ID NO:
  • the deletion or mutation of the serine is located in the amino acid sequence of wild type MG53 (eg, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO : 189, 211, 214, 246 of SEQ ID NO: 6, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, or SEQ ID NO: 142)
  • SEQ ID NO: 1 amino acid sequence of wild type MG53
  • the deletion or mutation of the serine is located in the amino acid sequence of wild type MG53 (eg, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO : 211, 214, 246, 253-, SEQ ID NO: 6, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, or SEQ ID NO: 142)
  • SEQ ID NO: 1 amino acid sequence of wild type MG53
  • the deletion or mutation of the serine is located in the amino acid sequence of wild type MG53 (eg, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO : 211, 214, 246, 255 of SEQ ID NO: 6, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, or SEQ ID NO: 142)
  • wild type MG53 eg, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO : 211, 214, 246, 255 of SEQ ID NO: 6, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, or SEQ ID NO: 142
  • the deletion or mutation of the serine is located in the amino acid sequence of wild type MG53 (eg, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO : 5, one or more of positions 253-255 of SEQ ID NO: 6, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, or SEQ ID NO: 142).
  • the serine that is deleted or mutated is located at position 253 of the amino acid sequence of wild type MG53.
  • the serine that is deleted or mutated is located at position 255 of the amino acid sequence of wild type MG53.
  • the serine that is deleted or mutated is located at position 255 of the amino acid sequence of wild type MG53 SEQ ID NO:1.
  • the deletion or mutation of the serine is at position 255 of amino acid sequence SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141 or SEQ ID NO: 142 of wild type MG53.
  • the MG53 mutant has two or more serine mutations. In certain embodiments, the two or more serine mutations of the MG53 mutant comprise a serine mutation at one or more of positions 253-255. In certain embodiments, the two of the MG53 mutants One or more serine mutations include a serine mutation at position 253. In certain embodiments, the two or more serine mutations of the MG53 mutant comprise a serine mutation at position 255. In certain embodiments, the two or more serine mutations of the MG53 mutant comprise a serine mutation at positions 253 and 255.
  • the position of serine in the wild type MG53 protein of different species may be different, and thus the deletion or mutation of the serine site may also be different.
  • the deletion or mutation of the serine is within 1 to 10 amino acids, 1 to 5 amino acids upstream or downstream of the corresponding serine site in the amino acid sequence of wild type MG53 set forth in SEQ ID NO: 1. Within, or within 1 to 3 amino acids.
  • the amino acid sequence of the MG53 mutant is SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO: 12.
  • the amino acid sequence of the MG53 mutant is the amino acid sequence set forth in SEQ ID NO:7. And a SEQ ID NO: 10, 12.
  • One of the amino acid sequences set forth in SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150 has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence homology, and the MG53 mutant has the function of cell repair and/or cardioprotection while avoiding the wild type MG53 Metabolic side effects.
  • the present application is directed to a pharmaceutical composition
  • a pharmaceutical composition comprising the MG53 mutant and a pharmaceutically acceptable carrier.
  • the present application relates to an isolated nucleic acid comprising a nucleic acid sequence encoding the amino acid sequence of the MG53 mutant.
  • the nucleic acid comprises any one of the nucleic acid sequences set forth in SEQ ID NOs: 13-18. In certain embodiments, the nucleic acid comprises any one of the nucleic acid sequences set forth in SEQ ID NOs: 151-154.
  • the present application relates to an expression vector comprising a nucleic acid sequence encoding an amino acid sequence of the MG53 mutant.
  • the nucleic acid comprises any one of the nucleic acid sequences set forth in SEQ ID NOs: 13-18. In certain embodiments, the nucleic acid comprises any one of the nucleic acid sequences set forth in SEQ ID NOs: 151-154.
  • the present application relates to a host cell comprising an expression vector as described herein.
  • the present application relates to a method of producing the MG53 mutant, comprising: determining one or more positions of a serine to be mutated, and a full length of a plasmid comprising a nucleic acid sequence encoding a wild type MG53 amino acid sequence The sequence is subjected to site-directed mutagenesis at the position, and the site-directed mutant plasmid is transfected into a host cell, and the host cell is expressed to produce the MG53 mutant.
  • the site-directed mutagenesis comprises the steps of: (1) determining the nucleotide position of the amino acid in the cDNA sequence that requires site-directed mutagenesis, modifying the nucleotide sequence of the mutation position based on the amino acid after the mutation, and The primers of the sequence containing the mutation position of 20-40 bp were cut out; (2) the wild type MG53 plasmid was used as a template, and the primer designed in the step (1) was used for PCR reaction, and the PCR product was subjected to agarose gel electrophoresis, and The PCR product is purified; (3) the purified PCR product in step (2) is digested with a nucleic acid restriction endonuclease, and the digested product is ligated with a suitable plasmid expression vector, and the ligated product is subjected to bacterial sensing.
  • the site-directed mutagenesis further comprises the steps of: (4) picking the clone obtained in step (3) using the primer designed in the step (1) for colony PCR identification, and performing agarose gel electrophoresis on the PCR product. Detection, followed by DNA sequencing, identified positive clones with the site-directed mutagenesis.
  • the present application relates to the preparation of the MG53 mutant for the treatment of heart disease, diabetic cerebrovascular disease, diabetic ocular complications, diabetic neuropathy, diabetic foot, kidney disease, and cell and/or tissue damage related diseases.
  • the drug avoids or simultaneously treats a heart disease, a diabetic cerebrovascular disease, a diabetic ocular complication, a diabetic neuropathy, a diabetic foot, a kidney disease, a disease associated with cell and/or tissue damage. Reduces side effects such as insulin resistance, obesity, diabetes, high blood pressure, and dyslipidemia.
  • the heart disease is a disease associated with myocardial damage, including but not limited to, diabetic heart disease, myocardial ischemia, cardiac ischemia/reperfusion injury, myocardial infarction, heart failure, arrhythmia, Heart rupture, angina pectoris, myocarditis, coronary heart disease, pericarditis.
  • the diabetic cerebrovascular disease includes, but is not limited to, cerebral arteriosclerosis, ischemic cerebrovascular disease, cerebral hemorrhage, brain atrophy, cerebral infarction.
  • the diabetic ocular complications include, but are not limited to, diabetic retinopathy, diabetic cataract, uveitis associated with diabetes, blindness.
  • the diabetic neuropathy includes, but is not limited to, diabetic peripheral neuropathy.
  • the renal disease includes, but is not limited to, acute glomerulonephritis, chronic glomerulonephritis, nephrotic syndrome, acute kidney injury, diabetic nephropathy, and the like.
  • the disease associated with cell and/or tissue damage includes, but is not limited to, cells of the kidney, brain, lung, liver, heart, spleen, digestive tract and/ Or tissue-related diseases, diseases related to skin damage, such as brain damage, lung injury, spleen injury, spleen rupture, gastric ulcer, gastritis, gastric perforation, gastrointestinal mucosal injury, trauma, burns, ulcers, mucositis, asthma , chronic obstructive pulmonary disease (COPD), stroke, skin aging, etc.
  • diseases related to skin damage such as brain damage, lung injury, spleen injury, spleen rupture, gastric ulcer, gastritis, gastric perforation, gastrointestinal mucosal injury, trauma, burns, ulcers, mucositis, asthma , chronic obstructive pulmonary disease (COPD), stroke, skin aging, etc.
  • COPD chronic obstructive pulmonary disease
  • the present application relates to the preparation of the polypeptide of SEQ ID NO: 7 for the treatment of heart disease, diabetic cerebrovascular disease, diabetic ocular complications, diabetic neuropathy, diabetic foot, kidney disease, and cells. And/or use in drugs for tissue damage related diseases.
  • the present application relates to polypeptides set forth in SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, or SEQ ID NO: 150 for use in the preparation of a heart disease, diabetic cerebrovascular disease, Use in diabetic ocular complications, diabetic neuropathy, diabetic foot, kidney disease, drugs associated with diseases associated with cellular and/or tissue damage.
  • the present application relates to a method of treating a heart disease, a diabetic cerebrovascular disease, a diabetic ocular complication, a diabetic neuropathy, a diabetic foot, a kidney disease, a disease associated with cell and/or tissue damage, including administering a desired
  • the subject is therapeutically effective in an amount of the MG53 mutant.
  • the MG53 mutant is in the treatment of heart disease, diabetic cerebrovascular disease, diabetic ocular complications, diabetic neuropathy, diabetic foot, kidney disease, and diseases associated with cellular and/or tissue damage, Metabolic side effects such as insulin resistance, obesity, diabetes, hypertension, dyslipidemia, and the like are avoided or reduced.
  • the heart disease is a disease associated with myocardial damage, including but not limited to, diabetic heart disease, myocardial ischemia, cardiac ischemia/reperfusion injury, myocardial infarction, heart failure, arrhythmia, Heart rupture, angina pectoris, myocarditis, coronary heart disease, pericarditis.
  • the diabetic cerebrovascular disease includes, but is not limited to, cerebral arteriosclerosis, ischemic cerebrovascular disease, cerebral hemorrhage, brain atrophy, cerebral infarction.
  • the diabetic ocular complications include, but are not limited to, diabetic retinopathy, diabetic cataract, uveitis associated with diabetes, blindness.
  • the diabetic neuropathy includes, but is not limited to, diabetic peripheral neuropathy.
  • the renal disease includes, but is not limited to, acute glomerulonephritis, chronic glomerulonephritis, nephrotic syndrome, acute kidney injury, diabetic nephropathy, and the like.
  • the disease associated with cell and/or tissue damage includes, but is not limited to, diseases associated with kidney, brain, lung, liver, heart, spleen, cells of the digestive tract, and/or tissue damage, skin Damage-related diseases, such as brain damage, lung injury, spleen injury, spleen rupture, gastric ulcer, gastritis, gastric perforation, gastrointestinal mucosal injury, trauma, burns, ulcers, mucositis, asthma, chronic obstructive pulmonary disease (COPD) ), stroke, skin aging, etc.
  • diseases associated with kidney, brain, lung, liver, heart, spleen, cells of the digestive tract, and/or tissue damage skin Damage-related diseases, such as brain damage, lung injury, spleen injury, spleen rupture, gastric ulcer, gastritis, gastric perforation, gastrointestinal mucosal injury, trauma, burns, ulcers, mucositis, asthma, chronic obstructive pulmonary disease (COPD) ), stroke, skin aging, etc
  • the present application relates to a method of treating heart disease, diabetic cerebrovascular disease, diabetic ocular complications, diabetic neuropathy, diabetic foot, kidney disease, diseases associated with cellular and/or tissue damage, This includes administering to a subject in need thereof a therapeutically effective amount of the polypeptide of SEQ ID NO: 7.
  • the present application relates to a method of treating heart disease, diabetic cerebrovascular disease, diabetic ocular complications, diabetic neuropathy, diabetic foot, kidney disease, diseases associated with cellular and/or tissue damage, including A therapeutically effective amount of a polypeptide of SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149 or SEQ ID NO: 150 is administered to a subject in need thereof.
  • the present application relates to MG53 mutants for use in the treatment of heart disease, diabetic cerebrovascular disease, diabetic ocular complications, diabetic neuropathy, diabetic foot, kidney disease, diseases associated with cellular and/or tissue damage.
  • the MG53 mutant is in the treatment of heart disease, diabetic cerebrovascular disease, diabetic ocular complications, diabetic neuropathy, diabetic foot, kidney disease, and diseases associated with cellular and/or tissue damage, Metabolic side effects such as insulin resistance, obesity, diabetes, high blood pressure, and dyslipidemia are avoided.
  • the heart disease is a disease associated with myocardial damage, including but not limited to, diabetic heart disease, myocardial ischemia, cardiac ischemia/reperfusion injury, myocardial infarction, heart failure, arrhythmia, Heart rupture, angina pectoris, myocarditis, coronary heart disease, pericarditis.
  • the diabetic cerebrovascular disease includes, but is not limited to, cerebral arteriosclerosis, ischemic cerebrovascular disease, cerebral hemorrhage, brain atrophy, cerebral infarction.
  • the diabetic ocular complications include, but are not limited to, diabetic retinopathy, diabetic cataract, uveitis associated with diabetes, blindness.
  • the diabetic neuropathy includes, but is not limited to, diabetic peripheral neuropathy.
  • the renal disease includes, but is not limited to, acute glomerulonephritis, chronic glomerulonephritis, nephrotic syndrome, acute kidney injury, diabetic nephropathy, and the like.
  • the disease associated with cell and/or tissue damage includes, but is not limited to, diseases associated with kidney, brain, lung, liver, heart, spleen, cells of the digestive tract, and/or tissue damage, skin Damage-related diseases, such as brain damage, lung injury, spleen injury, spleen rupture, gastric ulcer, gastritis, gastric perforation, gastrointestinal mucosal injury, trauma, burns, ulcers, mucositis, asthma, chronic obstructive pulmonary disease (COPD) ), stroke, skin aging, etc.
  • diseases associated with kidney, brain, lung, liver, heart, spleen, cells of the digestive tract, and/or tissue damage skin Damage-related diseases, such as brain damage, lung injury, spleen injury, spleen rupture, gastric ulcer, gastritis, gastric perforation, gastrointestinal mucosal injury, trauma, burns, ulcers, mucositis, asthma, chronic obstructive pulmonary disease (COPD) ), stroke, skin aging, etc
  • the present application relates to SEQ ID NO for treating heart disease, diabetic cerebrovascular disease, diabetic ocular complications, diabetic neuropathy, diabetic foot, kidney disease, diseases associated with cellular and/or tissue damage. : The polypeptide shown in 7. In certain embodiments, the present application relates to SEQ ID NO for treating heart disease, diabetic cerebrovascular disease, diabetic ocular complications, diabetic neuropathy, diabetic foot, kidney disease, diseases associated with cellular and/or tissue damage. : 147, SEQ ID NO: 148, SEQ ID NO: 149 or SEQ ID NO: 150.
  • Figure 1 shows a molecular structure pattern of wild type MG53.
  • Figure 2 shows the amino acid sequence of human wild type MG53 SEQ ID NO: 1 and its encoding nucleic acid sequence SEQ ID NO: 19.
  • Figure 3 shows the amino acid sequence of mouse wild type MG53 SEQ ID NO: 2 and its encoding nucleic acid sequence SEQ ID NO: 20.
  • Figure 4 shows the amino acid sequence of rat wild type MG53 SEQ ID NO: 3 and its encoding nucleic acid sequence SEQ ID NO:21.
  • Figure 5 shows the amino acid sequence of monkey wild type MG53 SEQ ID NO: 4 and its encoding nucleic acid sequence SEQ ID NO: 22.
  • Figure 6 shows the amino acid sequence of porcine wild-type MG53 SEQ ID NO: 5 and its encoding nucleic acid sequence SEQ ID NO: 23.
  • Figure 7 shows the amino acid sequence of dog wild type MG53 SEQ ID NO: 6 and its encoding nucleic acid sequence SEQ ID NO: 24.
  • Figure 8 shows the amino acid sequence of human MG53 S255A mutant SEQ ID NO: 7 and its encoding nucleic acid sequence SEQ ID NO: 13.
  • Figure 9 shows the amino acid sequence of mouse MG53 S255A mutant SEQ ID NO: 8 and its encoding nucleic acid sequence SEQ ID NO: 14.
  • Figure 10 shows the amino acid sequence of the rat MG53 S255A mutant SEQ ID NO: 9 and its encoding nucleic acid sequence SEQ ID NO: 15.
  • Figure 11 shows the amino acid sequence of the monkey MG53 S255A mutant SEQ ID NO: 10 and its encoding nucleic acid sequence SEQ ID NO: 16.
  • Figure 12 shows the amino acid sequence of the porcine MG53 S255A mutant SEQ ID NO: 11 and its encoding nucleic acid sequence SEQ ID NO: 17.
  • Figure 13 shows the amino acid sequence of the dog MG53 S255A mutant SEQ ID NO: 12 and its encoding nucleic acid sequence SEQ ID NO: 18.
  • Figure 14 shows the amount of intracellular ATP and LDH released after adenovirus overexpression of mouse wild type MG53 and mouse MG53 S255A mutant in primary cultured cardiomyocytes of neonatal rats.
  • the above figure shows the use of adenovirus overexpressing mouse wild-type MG53 and mouse MG53S255A mutants in primary cultured cardiomyocytes of neonatal rats, and detecting intracellular ATP content after hypoxia-reoxygenation treatment
  • Adv- ⁇ -gal represents a control virus expressing galactosidase but not expressing mouse wild type MG53 and mouse MG53 S255A mutant
  • Adv-MG53-myc represents an adenovirus expressing mouse wild-type MG53
  • the following figure shows the use of adenovirus overexpressing mouse wild-type MG
  • Figure 15 shows that mouse MG53 S255A mutation does not affect the activation of AKT by mouse wild-type MG53.
  • the above figure shows the use of adenovirus overexpressing mouse wild type MG53 and mouse MG53 S255A mutants in primary cultured cardiomyocytes of neonatal rats, and measuring the amount of p-AKT 473 in cells by Western Blot.
  • ⁇ -gal represents a control adenovirus expressing galactosidase but not mouse wild type MG53 and mouse MG53 S255A mutant
  • MG53 is an adenovirus expressing mouse wild type MG53
  • S255A is a mouse MG53 S255A mutation.
  • adenovirus of the body is the lower panel is the statistical diagram of the above figure
  • Adv ⁇ -gal represents the control adenovirus expressing galactosidase but not the mouse wild type MG53 and MG53 S255A mutant
  • Adv MG53 indicates the expression of mouse wild type MG53 Adenovirus
  • Figure 16 shows that mouse MG53 S255A mutation inhibits MG53-mediated substrate degradation.
  • the upper panel of Figure 16A is a plasmid overexpressing IRS1 and mouse wild-type MG53, mouse MG53 S255A mutant, or mouse MG53-D-RING truncated body in HEK293T cell line, and Western Blot is used to detect the protein content of IRS1.
  • CON indicates an empty vector plasmid control that does not express mouse wild type MG53, mouse MG53 S255A mutant, mouse MG53-D-RING truncation, MG53 indicates a vector plasmid expressing mouse wild type MG53, and S255A indicates expression mouse
  • the vector plasmid of the MG53 S255A mutant, D-RING indicates the vector plasmid expressing the mouse RING domain excised MG53 truncation MG53-D-RING; the lower panel of Fig.
  • the upper panel of Figure 16B is an overexpression of IR and mouse wild-type MG53, mouse MG53 S255A mutant, or mouse MG53-D-RING truncation in the HEK293T cell line.
  • Plasmid Western Blot detects the protein content of IR
  • CON indicates empty vector plasmid control that does not express mouse wild-type MG53, mouse MG53 S255A mutant, mouse MG53-D-RING truncation
  • MG53 indicates expression of wild mouse Vector plasmid of type MG53
  • S255A indicates the carrier substance expressing mouse MG53 S255A mutant
  • D-RING represented by the expression
  • Figure 17 shows that the human MG53 S255A mutant inhibits MG53-mediated substrate degradation.
  • the above figure is a plasmid that overexpresses IRS1 and human wild-type MG53, human MG53 S255A mutant, or human MG53-D-RING truncated body in HEK293T cell line, Western Blot detects the protein content of IRS1, and CON indicates that human wild type is not expressed.
  • MG53 human MG53 S255A mutant, empty vector plasmid control of human MG53-D-RING truncation
  • MG53 indicates a vector plasmid expressing human wild-type MG53
  • Figure 18 shows that the mouse MG53 S255A mutant inhibits the binding of MG53 to the substrate IRS1.
  • Figure 18A shows that the substrate IRS1 of mouse wild-type MG53 and MG53 is co-expressed in the HEK293T cell line, or the mouse MG53 S255A mutant and the substrate IRS1 of MG53 are co-expressed, and then the IRS1 protein is immunoprecipitated and subjected to Western Blot assay to detect MG53-IRS1. The content of the complex.
  • MG53 in Figure 18A represents mouse wild-type MG53, MG53 S255A represents mouse MG53 S255A mutant, IRS1 represents mouse insulin receptor substrate;
  • Figure 18B in surface iso-stereo resonance (SPR) assay, immobilized purified protein IRS1 The binding strength of the purified protein mouse wild type MG53 or mouse MG53 S255A mutant in the mobile phase was examined.
  • MG53 in Fig. 18B indicates mouse wild type MG53, MG53-S255A indicates mouse MG53 S255A mutant, and Rat IRS1 indicates rat insulin receptor substrate.
  • Figure 19 shows the effect of mouse MG53 S255A mutant on the amount of IRS1 protein.
  • the top panel shows the plasmids that overexpress the IRS1, Ub, MG53, and MG53 S255A mutants in the HEK293T cell line, and the protein content of IRS1 was detected by Western Blot.
  • Ub represents ubiquitin
  • MG53 represents mouse wild-type MG53
  • MG53-S255A represents mouse MG53 S255A mutant
  • the lower panel is a statistical diagram of the above experiment
  • CON indicates that mouse wild-type MG53 and mouse MG53 S255A mutant are not expressed
  • Empty vector plasmid control MG53 indicates a vector plasmid expressing mouse wild type MG53
  • Figure 20 shows the amino acid sequence of human MG53 subtype SEQ ID NO: 139 (the corresponding NCBI number is BAD18630.1) and its encoding nucleic acid sequence SEQ ID NO: 143.
  • Figure 21 shows the amino acid sequence of human MG53 subtype SEQ ID NO: 140 (corresponding NCBI number XP_016878743.1) and its encoding nucleic acid sequence SEQ ID NO: 144.
  • Figure 22 shows the amino acid sequence of human MG53 subtype SEQ ID NO: 141 (corresponding NCBI numbering is BAC03506.1) and its encoding nucleic acid sequence SEQ ID NO: 145.
  • Figure 23 shows the amino acid sequence of human MG53 subtype SEQ ID NO: 142 (the corresponding NCBI number is AAH33211.1) and its encoding nucleic acid sequence SEQ ID NO: 146.
  • Figure 24 shows the amino acid sequence of human MG53 subtype S255A mutant SEQ ID NO: 147 and its encoding nucleic acid sequence SEQ ID NO: 151.
  • Figure 25 shows the amino acid sequence of human MG53 subtype S255A mutant SEQ ID NO: 148 and its encoding nucleic acid sequence SEQ ID NO: 152.
  • Figure 26 shows the amino acid sequence of human MG53 subtype S255A mutant SEQ ID NO: 149 and its encoding nucleic acid sequence SEQ ID NO: 153.
  • Figure 27 shows the amino acid sequence of human MG53 subtype S255A mutant SEQ ID NO: 150 and its encoding nucleic acid sequence SEQ ID NO: 154.
  • Figure 28 is a diagram showing the naming of amino acid positions of the full-length MG53 protein and its fragments of each species in the present application.
  • the application relates to a MG53 mutant, wherein the MG53 mutant has at least one serine deletion and/or in the coiled-coil-SPRY region of the wild-type MG53 based on the amino acid sequence of wild-type MG53 Or mutated to any other non-serine or threonine amino acid.
  • wild-type MG53 or wild-type MG53 protein as used herein refers to the native sequence of a full-length MG53 protein expressed in a subject or a fragment thereof.
  • the MG53 protein is a multifunctional protein whose structure is shown in Figure 1.
  • the full-length MG53 protein of each species is slightly different in length, but usually has about 477 amino acids, consisting of an N-terminal TRIM domain and a C-terminal SPRY domain.
  • the TRIM domain consists of Rings, B-box, coiled-coil domain (RBCC) composition.
  • MG53 protein is one of the important components of membrane repair, and plays an important role in preconditioning protection and postconditioning adaptation of ischemia-reperfusion injury.
  • MG53 protein may also cause insulin resistance and metabolic syndrome.
  • the structure, function, and interaction with other proteins of MG53 have been reported in detail in the art (see, for example, Chuanxi Cai et al., Journal of Biological Chemistry, 284(5), 3314-3322, (2009); Xianhua Wang et al., Circulation Research 107, 76-83, (2010); Eun Young Park et al., Proteins, 790-795 (2009)).
  • subject as used in this application includes both human and non-human animals.
  • Non-human animals include all vertebrates, such as mammals and non-mammals.
  • Subject may also be livestock animals such as cattle, pigs, sheep, poultry and horses; or rodents such as rats, mice; or primates such as baboons, monkeys; or domestic animals such as dogs and Cat.
  • the "object” can be male or female and can be old, adult, adolescent, child or infant.
  • the human "object” can be a mixture of Caucasians, Africans, Asians, Semitics, or other ethnic or ethnic backgrounds.
  • the wild type MG53 is preferably derived from a mammal, eg, a human, a donkey, a monkey, a mouse, a rat, a pig, a dog, and the like.
  • a mammal eg., a human, a donkey, a monkey, a mouse, a rat, a pig, a dog, and the like.
  • One skilled in the art can obtain the amino acid sequence of wild-type MG53 of each species through an open channel (e.g., the National Center for Biotechnology Information (NCBI), the amino acid sequence of wild-type MG53 of each species is incorporated herein by reference.
  • the amino acid sequence of wild type MG53 is SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO:
  • the wild type MG53 full-length protein corresponding to human, mouse, rat, monkey, pig, dog, and the like, respectively.
  • protein protein
  • polypeptide and “peptide” as used herein are used interchangeably and refer to a polymer of amino acids.
  • the protein, polypeptide or peptide described herein may contain a natural amino acid, or may contain a non-natural amino acid, or an analog or mimetic of an amino acid.
  • the proteins, polypeptides or peptides described herein can be obtained by any method known in the art such as, but not limited to, by natural isolation, recombinant expression, chemical synthesis, and the like.
  • coiled-coil-SPRY domain is a corresponding domain in the amino acid sequence of wild type MG53.
  • the coiled-coil domain ie, coiled-coil domain
  • the coiled-coil domain is present in most TRIM family proteins that mediate homology or heterologous binding between TRIM family members or between TRIM members and other proteins. Complexes such as dimers, multimers, etc., thereby exerting a cell repair function (see, for example, Ozato et al., Nature Review Immunology, 8: 849-860 (2008); Sanchez S. et al., PNAS, 111: 2494-2499 (2014)).
  • the SPRY domain is located at the C-terminus of the amino acid sequence of wild type MG53, and is usually located in the amino acid region 288-477 of wild type MG53.
  • the SPRY domain described in the present application includes a PRY motif and a SPRY motif.
  • the SPRY domain is evolutionarily very conserved and is expressed from fungi to higher animals (see, for example, Ozato et al., Nature Review Immunology, 8: 849-860 (2008)).
  • TRIM domain ie, the Ring-B–box-Coiled-Coil domain
  • MG53 exhibits a conserved primary structure with these TRIM subfamily proteins (see, for example, WO 2009/073808).
  • the specific amino acid region locus corresponding to the Coiled-coil-SPRY domain may vary slightly in different species, but one of skill in the art can pass prior art in the art (eg, as disclosed in NCBI) and/or routine experimentation. Methods The specific amino acid region sites corresponding to the coaled-coil-SPRY domain of wild type MG53 of different species were obtained.
  • the coiled-coil-SPRY domain in the present application refers to amino acids 122-477 of wild-type MG53 or a region similar in structure thereto.
  • the structurally similar region can be a region comprising 70%, 80%, or 90% of the contiguous amino acid sequence of amino acids 122-477.
  • the N-terminal start site of the structurally similar region may grow or be shortened by about 1, 2, 3, 4, 5, than the N-terminus of the 122-477 amino acid region, 6 , 7 , 8 , 9 , 10 , 15 or 20 amino acids , and / or its C - terminal termination site may grow or shorten by about 1 from the C - terminus of the 122 - 477 amino acid region, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 amino acids.
  • the coiled-coil-SPRY domain in the present application refers to the coaled-coil-SPRY domain in human wild-type MG53 (ie, SEQ ID NO: 1), which corresponds to SEQ ID NO: amino acid 122-477.
  • MG53 mutant or "MG53 protein mutant” as used in this application refers to a MG53 protein variant or fragment in which the native amino acid sequence of the wild type MG53 protein is modified. Such modifications include, but are not limited to, one or more amino acid deletions and/or substitutions.
  • the MG53 mutant of the present application has at least one serine deletion and/or mutation to any of the wild-type MG53 in the coiled-coil-SPRY domain based on the amino acid sequence of wild-type MG53. Other non-serine or threonine amino acids.
  • At least one serine refers to one or more serines. For example, it refers to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 in the coiled-coil-SPRY domain of wild type MG53. , 13, 14, 15, 16, 16, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or more Serine.
  • another non-serine or threonine amino acid refers to any other natural amino acid, substituted natural amino acid, unnatural amino acid, substituted unnatural amino acid other than serine, threonine or Any combination.
  • the name of the natural amino acid is indicated in this application as a standard one-letter or three-letter code.
  • Natural amino acids include non-polar amino acids and polar amino acids. Unless otherwise indicated, any of the amino acids described herein can be in the D- or L-configuration.
  • At least one serine (Ser or S) within the coiled-coil-SPRY domain of wild-type MG53 is deleted or mutated to a non-polar amino acid.
  • the non-polar amino acid includes glycine (Gly or G), alanine (Ala or A), leucine (Leu or L), isoleucine (Ile or I), proline (Val or V) Proline (Pro or P), phenylalanine (Phe or F), methionine (Met or M), tryptophan (Trp or W).
  • at least one serine within the coiled-coil-SPRY domain of wild-type MG53 is deleted or mutated to glycine or alanine.
  • At least one serine within the coiled-coil-SPRY domain of wild-type MG53 is deleted or mutated to alanine. More preferably, in certain embodiments, at least one serine within the coiled-coil-SPRY domain of the wild type MG53 is mutated to alanine.
  • At least one serine within the coiled-coil-SPRY domain of wild-type MG53 is deleted or mutated to a polar amino acid.
  • the polar amino acid includes glutamine (Gln or Q), cysteine (Cys or C), asparagine (Asn or N), tyrosine (Tyr or Y), aspartic acid (Asp or D), glutamic acid (Glu or E), lysine (Lys or K), arginine (Arg or R), histidine (His or H).
  • at least one serine within the coiled-coil-SPRY domain of wild-type MG53 is deleted or mutated to cysteine or histidine.
  • At least one serine within the coiled-coil-SPRY domain of wild-type MG53 is deleted or mutated to cysteine. More preferably, in certain embodiments, at least one serine within the coiled-coil-SPRY domain of wild-type MG53 is mutated to cysteine.
  • the serine mutations described herein include a serine substituted with one or more other amino acids other than serine or threonine, for example, one serine can be 1, 2, 3, 4 , 5, 6, 7, 8, 9, or 10 other amino acid substitutions other than serine or threonine. If there are two If one or more serines are substituted, each serine may be independently substituted with one or more other amino acids other than serine or threonine.
  • the specific serine sites of wild-type MG53 may vary from species to species, once the amino acid sequence of wild-type MG53 and the amino acid region corresponding to the coiled-coil-SPRY domain of a species are known, the field The skilled person will be able to determine the specific amino acid position corresponding to the serine in the coiled-coil-SPRY domain.
  • the amino acid sequence of human wild type MG53 is set forth in SEQ ID NO: 1
  • the coiled-coil-SPRY domain corresponds to amino acids 122-477 of SEQ ID NO: 1.
  • one skilled in the art can determine that the specific amino acid positions corresponding to serine in the coiled-coil-SPRY domain of human wild type MG53 are located at positions 150 and 189 of SEQ ID NO: 1, respectively. No. 211, 214, 246, 255, 269, 296, 297, 301, 305, 306, 307, 314, 341 Bit, 377th, 405th, 418th, 425th, 430th.
  • the deletion or mutation of the serine is located at positions 150, 189, 211, 214, 246, 255 of the amino acid sequence of human wild type MG53 SEQ ID NO: 1. 269, 296, 297, 301, 305, 306, 307, 314, 341, 377, 405, 418, One or more of 425 bits and 430th bits.
  • the MG53 protein described herein comprises a full-length MG53 protein, or a truncation of the full-length MG53 protein (ie, a truncated fragment of the full-length MG53 protein), or is either a full-length MG53 protein or Its truncated body is a mutant having one or more amino acid mutations or additions or deletions.
  • the MG53 protein subtype is The amino acid sequence is sequence-aligned with the amino acid sequence of the full-length MG53 protein (for example, SEQ ID NO: 1), and a spacer is introduced in the relevant amino acid sequence as necessary to maximize the number of identical amino acids, and the amino acid of the MG53 subtype
  • the number of positions of the first amino acid of the sequence is specified as the number of amino acid positions of the full-length MG53 protein corresponding to the amino acid, such that when referring to the amino acid position of wild type MG53 of a certain species, whether it is the full-length MG53 protein or its truncation
  • the body refers to the number of sites in the amino acid sequence of the full-length MG53 protein.
  • the full-length MG53 protein of a certain species has n amino acids, and the corresponding amino acid position is the 1-nth position.
  • the amino acid sequence of MG53 subtype 1 contains a truncated amino acid corresponding to the mn amino acid of the full-length sequence compared to the amino acid sequence of the full-length MG53 protein, and then the first amino acid sequence of the amino acid sequence of MG53 subtype 1 is The number of loci is designated as the mth position, and the number of positions of the second amino acid sequence of the amino acid sequence of MG53 subtype 1 is designated as the m+1th position, and so on, and the last amino acid sequence of the amino acid sequence of MG53 subtype 1 is derived.
  • the amino acid sequence of MG53 subtype 2 contains a truncated body corresponding to the amino acid sequence of position 1-s of the full-length sequence compared to the amino acid sequence of the full-length MG53 protein, and then the amino acid sequence of MG53 subtype 2 is The number of sites of the first amino acid is designated as the first position, the number of sites of the second amino acid sequence of the amino acid sequence of MG53 subtype 2 is designated as the second position, and so on, and the last one of the amino acid sequences of MG53 subtype 2 is derived.
  • the amino acid position is designated as the sth position.
  • the amino acid sequence of MG53 subtype 3 contains a truncated amino acid corresponding to the pq amino acid of the full-length sequence as compared with the amino acid sequence of the full-length MG53 protein, and then the amino acid sequence of MG53 subtype 3 is the first.
  • the number of amino acid sites is designated as the p-th position
  • the number of the second amino acid sequence of the amino acid sequence of MG53 subtype 3 is designated as the p+1th position, and so on, and the last amino acid sequence of MG53 subtype 3 is designated.
  • the amino acid position is designated as the qth position.
  • SEQ ID NO: 140 is one of the human wild-type MG53 subtypes, which has 333 amino acids corresponding to amino acid fragments 145-477 of human wild-type MG53 full-length sequence SEQ ID NO: 1.
  • the first amino acid (methionine) site of the amino acid sequence of SEQ ID NO: 140 is designated as position 145 of SEQ ID NO: 140
  • the second amino acid position is designated as 146 of SEQ ID NO: 140.
  • the position, and so on, specifies the last amino acid position of the amino acid sequence of SEQ ID NO: 140 as position 477 of SEQ ID NO:140.
  • SEQ ID NO: 141 is one of the human wild-type MG53 subtypes, which also has 333 amino acids, compared to the human wild type MG53 full length sequence SEQ ID NO: 1 145-477 amino acids only one
  • the amino acid is different, that is, the 315th position of SEQ ID NO: 1 is glutamic acid, and the corresponding position of SEQ ID NO: 141 is glycine, in which case the first amino acid sequence of the amino acid sequence of SEQ ID NO: 141 is The (methionine) site is designated as position 145 of SEQ ID NO: 141, the second amino acid position is designated as position 146 of SEQ ID NO: 141, and so on, and the amino acid sequence of SEQ ID NO: 141 is the last One amino acid position was designated as position 477 of SEQ ID NO:141.
  • SEQ ID NO: 142 is one of the human wild-type MG53 subtypes, which has 269 amino acids corresponding to amino acid residues 1-269 of SEQ ID NO: 1 of human wild-type MG53 full-length sequence
  • the first amino acid (methionine) site of the amino acid sequence of SEQ ID NO: 142 is designated as position 1 of SEQ ID NO: 142
  • the second amino acid sequence of SEQ ID NO: 142 is designated.
  • the amino acid position is designated as position 2 of SEQ ID NO: 142, and so on, and the last amino acid position of the amino acid sequence of SEQ ID NO: 142 is designated as position 269 of SEQ ID NO: 142.
  • the serine that is deleted or mutated is located at positions 150, 189, 211, 214, 246, 214 of the amino acid sequence of human wild type MG53 subtype SEQ ID NO: 139 255, 269, 296, 297, 301, 305, 306, 307, 314, 341, 377, 405, 418 One or more of the 425th or 430th positions.
  • the serine that is deleted or mutated is located at position 150, 189, 211, 214, 246, 214 of the amino acid sequence of human wild type MG53 subtype SEQ ID NO: 140 255, 269, 296, 297, 301, 305, 306, 307, 314, 341, 377, 405, 418 One or more of the 425th or 430th positions.
  • the deletion or mutation of the serine is located at positions 150, 189, 211, 214, 246, 214 of the amino acid sequence of human wild type MG53 subtype SEQ ID NO: 141 255, 269, 296, 297, 301, 305, 306, 307, 314, 341, 377, 405, 418 One or more of the 425th or 430th positions.
  • the serine that is deleted or mutated is located at position 150, 189, 211, 214, 246, 214 of the amino acid sequence of human wild type MG53 subtype SEQ ID NO: 142 One or more of 255 bits and 269th bit.
  • the deletion or mutation of the serine is located at positions 188, 189, 210, 211, 214, 246 of the amino acid sequence of mouse wild type MG53 SEQ ID NO:2. Position, 253th, 255th, 269th, 296th, 297th, 301st, 305th, 306th, 307th, 314th, 341th, 367th, One or more of the 377th, 418th, 430th, and 440th positions.
  • the serine that is deleted or mutated is located at positions 150, 188, 189, 210, 211, 214 of amino acid sequence SEQ ID NO: 3 of rat wild type MG53.
  • Bit, 246th, 253th, 255th, 269th, 296th, 297th, 301st, 305th, 307th, 314th, 341th, 367th, 377th One or more of the bits, 418th, 430th, 440th, 464th, and 474th bits.
  • the deletion or mutation of the serine is at position 150, 189, 211, 214, 246, 255 of the amino acid sequence of SEQ ID NO: 4 of the wild type MG53 of monkey. 269, 296, 297, 301, 305, 306, 307, 341, 377, 405, 418, 425, One or more of 430 bits and 464th.
  • the deletion or mutation of the serine is located at positions 150, 189, 211, 214, 246, 255 of the amino acid sequence of porcine wild-type MG53 SEQ ID NO:5. , 269th, 296th, 301st, 305th, 307th, 314th, 341th, 377th, 411th, 418th, 425th, 430th, One or more of the 474 bits.
  • the deletion or mutation of the serine is at position 150, 189, 211, 214, 246, 255 of the amino acid sequence of SEQ ID NO: 6 of the dog wild type MG53. 269, 296, 297, 301, 305, 307, 314, 341, 367, 377, 418, 425, One or more of the 430 bits.
  • the serine site of the wild-type MG53 protein is highly conserved between species.
  • the amino acid sequence of each wild type MG53 protein is 189th, 211th, 214th, 246th, The 255th, 269th, 296th, 301st, 305th, 307th, 341th, 377th, 418th, and 430th positions are all serine.
  • the serine that is deleted or mutated is located at positions 189, 211, 214, 246, 255 of the amino acid sequence of wild-type MG53 (eg, SEQ ID NOs: 1-6)
  • the bits, the 269th, the 296th, the 301st, the 305th, the 307th, the 341st, the 377th, the 418th, the 430th are bits, the 269th, the 296th, the 301st, the 305th, the 307th, the 341st, the 377th, the 418th, the 430th.
  • the deletion or mutation of the serine is located in the amino acid sequence of wild type MG53 (eg, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO :5, SEQ ID NO: 6, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, or SEQ ID NO: 142) 150th, 188-189, 210-211, 214, 246, 253-255, 269, 296-297, 301, 305-307, 314, 341, 367, 377, 405, 411, 418, 425, 430, 440, 464 or One or more of the 474 bits.
  • wild type MG53 eg, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO :5, SEQ ID NO: 6, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, or SEQ ID NO: 142
  • 150th 188
  • the deletion or mutation of the serine is located in the amino acid sequence of wild type MG53 (eg, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO : 189, 211, 214, 246 of SEQ ID NO: 6, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, or SEQ ID NO: 142)
  • SEQ ID NO: 1 amino acid sequence of wild type MG53
  • the deletion or mutation of the serine is located in the amino acid sequence of wild type MG53 (eg, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO : 211, 214, 246, 253-, SEQ ID NO: 6, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, or SEQ ID NO: 142)
  • SEQ ID NO: 1 amino acid sequence of wild type MG53
  • the deletion or mutation of the serine is located in the amino acid sequence of wild type MG53 (eg, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO : 211, 214, 246, 255 of SEQ ID NO: 6, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, or SEQ ID NO: 142)
  • wild type MG53 eg, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO : 211, 214, 246, 255 of SEQ ID NO: 6, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, or SEQ ID NO: 142
  • the deletion or mutation of the serine is located in the amino acid sequence of wild type MG53 (eg, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO : 5, one or more of positions 253-255 of SEQ ID NO: 6, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, or SEQ ID NO: 142).
  • the serine that is deleted or mutated is located at position 253 of the amino acid sequence of wild type MG53.
  • the serine that is deleted or mutated is located at position 255 of the amino acid sequence of wild type MG53.
  • the serine that is deleted or mutated is located at position 255 of the amino acid sequence of wild type MG53 SEQ ID NO:1. In certain embodiments, the deletion or mutation of the serine is at position 255 of the amino acid sequence of wild type MG53 of SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141 or SEQ ID NO: 142.
  • the MG53 mutant has two or more serine mutations. In certain embodiments, the two or more serine mutations of the MG53 mutant comprise a serine mutation at one or more of positions 253-255. In certain embodiments, the two or more serine mutations of the MG53 mutant comprise a serine mutation at position 253. In certain embodiments, the two or more serine mutations of the MG53 mutant comprise a serine mutation at position 255. In certain embodiments, the two or more serine mutations of the MG53 mutant comprise a serine mutation at positions 253 and 255.
  • the position of serine in the wild type MG53 protein of different species may be different, and thus the deletion or mutation of the serine site may also be different.
  • the deletion or mutation of the serine is within 1 to 10 amino acids, 1 to 5 amino acids upstream or downstream of the corresponding serine site in the amino acid sequence of wild type MG53 set forth in SEQ ID NO: 1. Within, or within 1 to 3 amino acids.
  • the amino acid sequence of the MG53 mutant is SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO: 12. SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149 or SEQ ID NO: 150. More preferably, in certain embodiments, the amino acid sequence of the MG53 mutant is SEQ ID NO:7. And a SEQ ID NO: 10, 12.
  • One of the sequences set forth in SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150 has at least 70%, at least 75%, at least 80%, at least 85%, at least 90 %, at least 95%, at least 98%, or at least 99% amino acid sequence homology, and the MG53 mutant has cell repair function and/or cardioprotective function while avoiding the wild type MG53 Metabolic side effects.
  • the percentage of "sequence homology" is defined as the sequence alignment of the candidate amino acid sequence with the comparative amino acid sequence for the amino acid sequence, and the introduction of the interval if necessary, so that the same number of amino acids is maximized, and on this basis.
  • the percentage of identical amino acids between two amino acid sequences; for nucleic acid sequences The nucleic acid sequence is sequence-aligned with the control nucleic acid sequence, and a spacer is introduced as necessary to maximize the number of identical nucleotides, and on this basis, the percentage of identical nucleotides between the two nucleic acid sequences is calculated.
  • the percentage of homology can be determined by comparison in a variety of ways known in the art. For example, sequence comparisons can be made using publicly available tools such as BLASTp (National Center for Biotechnology Information (NCBI): http://blast.ncbi.nlm.nih.gov/Blast.cgi, also See, Altschul SF et al., J. Mol. Biol., 215: 403-410 (1990); Stephen F.
  • NCBI National Center for Biotechnology Information
  • SEQ ID NOs: 7-12, SEQ ID NOs: 147-150, and MG53 mutants having amino acid sequence homology to SEQ ID NOs: 7-12, SEQ ID NOs: 147-150 are It has cell repair function and/or cardioprotective function while avoiding or reducing the metabolic side effects caused by wild-type MG53.
  • the inventors of the present application have surprisingly discovered that at least one serine in the coiled-coil-SPRY domain of wild-type MG53 is deleted or mutated to any other amino acid other than serine or threonine, thereby avoiding or reducing the Metabolic side effects, but do not affect the cell repair function and/or cardioprotective function of MG53.
  • the above results may be due to the fact that phosphorylation of the serine site in the coiled-coil-SPRY domain of wild-type MG53 can significantly regulate the E3 ubiquitin ligase function of MG53, and through the substrate IR ⁇ and IRS1 regulates the regulation of MG53 on the insulin signaling system, but phosphorylation of the serine site in this domain does not regulate the cellular repair function and/or cardioprotective function of MG53.
  • cell repair function means that in the event of cell damage, particularly in acute cell damage, the wild-type MG53 or MG53 mutant can repair and repair the cell membrane of the damaged cell, optionally, by activation.
  • Signaling pathways eg, the RISK pathway
  • a wild type MG53 or MG53 mutant of the present application can repair a living cell, a cell in vitro, or a cell in vivo.
  • the wild type MG53 or MG53 mutant of the present application can also Repair different types of cells, such as, but not limited to, cardiomyocytes, striated muscle cells, skeletal muscle cells, renal proximal tubular epithelial cells, alveolar epithelial cells, digestive tract epithelial cells (eg, oral epithelial cells, esophageal epithelial cells, stomach) Epithelial cells, duodenal epithelial cells, intestinal epithelial cells, jejunal epithelial cells, ileal epithelial cells, colonic epithelial cells), mucosal cells (eg, oral mucosal cells, nasal mucosal cells, gastric mucosal cells, intestinal mucosal cells, colonic mucosa) Cells, duodenal mucosal cells, skin cells (eg, epidermal cells, epithelial cells, dermal cells, endothelial cells), vascular cells (eg, vascular wall cells, vascular endotheli
  • the wild-type MG53 or MG53 mutants provided herein can repair cardiomyocytes, skeletal muscle cells, striated muscle cells, renal proximal tubular epithelial cells, alveolar epithelial cells, and the like.
  • the cellular repair function of the MG53 mutants described herein can be determined using methods well known in the art. For example, by overexpressing wild-type MG53 and MG53 mutants described herein using adenovirus in suckling rat cardiomyocytes (NRVM), cells are stimulated with hypoxia to detect cell viability (eg, by MTT assay, medium) ATP and LDH concentration assays, TUNEL staining assays, etc. (for specific procedures, see Zhang.T et al.
  • the "cardioprotective function" as used in the present application means that in the case of myocardial injury, particularly in acute myocardial injury, the wild-type MG53 or MG53 mutant can repair the damage of the myocardial cell membrane, optionally, by activating the myocardium.
  • the cell's associated signaling pathway eg, the RISK pathway
  • the cardioprotective function of the MG53 mutants described herein can be determined using methods well known in the art.
  • NRVM suckling rat cardiomyocytes
  • the control group for example, empty cells that do not express wild-type MG53, MG53 mutants
  • Observed indicators eg, intracellular ATP levels, release levels of LDH, etc.
  • the cardiomyocytes are incubated with wild-type MG53 and the MG53 mutant protein described herein, respectively, using various stimuli (eg, hypoxia, H).
  • MG53 cell viability was evaluated according to the present application mutant cardioprotective body.
  • Cell viability can be determined by the MTT cell count, LDH, ATP measurement or TUNEL staining.
  • metabolic side effects refers to a disease or discomfort caused by a metabolic disorder other than the therapeutic purpose that occurs after a therapeutic amount of a drug is applied, including but not limited to, insulin resistance, obesity. , diabetes, high blood pressure, dyslipidemia, etc. Without wishing to be bound by any theory, it is believed that the severity of the metabolic side effects can be assessed by measuring the E3 ubiquitin ligase function of MG53.
  • the wild-type MG53 when at least one serine in the coiled-coil-SPRY domain of wild-type MG53 is deleted or mutated to any other non-serine or threonine amino acid, the wild-type MG53 is inhibited by at least 40%, at least 50 %, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or even 100% of the E3 ubiquitin ligase function, thereby Avoid or reduce the metabolic side effects produced by wild-type MG53, but do not affect the cell repair function and/or cardioprotective function of MG53.
  • “Avoiding or reducing the metabolic side effects produced by wild-type MG53” means that no metabolic side effects or metabolic side effects produced by wild-type MG53 are at least 40% less than the corresponding metabolic side effects produced by wild-type MG53, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or even 100%.
  • “Does not affect the cell repair function and/or cardioprotective function of MG53” means that the cell repair function and/or cardioprotective function of MG53 is not affected at all or the cell repair function and/or cardioprotective function of MG53 is reduced by up to 5%. Up to 10%, up to 15%, up to 20%, up to 25%, up to 30%, and the like.
  • the MG53 mutants provided herein comprise an analog thereof.
  • the MG53 mutant analog refers to a polypeptide having functional or structural features substantially similar to all or part of the MG53 mutant provided herein, but the amino acid sequence of the MG53 mutant analog is wild type The amino acid sequence of MG53 differs at least at one amino acid position.
  • the MG53 mutant analog may be a partial fragment, derivative or variant of the MG53 mutant and may comprise chemical or biological modifications.
  • the MG53 mutant analog may have conservative substitutions, additions, deletions, insertions, truncations, modifications (eg, phosphorylation, glycosylation, labeling, etc.) of one or more amino acids of the MG53 mutant, or any combination thereof.
  • MG53 mutant analogs may include naturally occurring variants of MG53 mutants and artificially produced variants, such as by recombinant methods or chemical synthesis. The obtained artificial polypeptide sequence.
  • the MG53 mutant analog may comprise non-naturally occurring amino acid residues.
  • the analogs of the MG53 mutants described herein retain substantially similar functions as the MG53 mutant, for example, analogs of MG53 mutants have cell repair and/or cardioprotective functions. At the same time, it can avoid or reduce the metabolic side effects caused by wild type MG53, such as insulin resistance, obesity, diabetes, hypertension, dyslipidemia and the like.
  • Conservative substitution of amino acid residues refers to substitutions between amino acids of similar nature, such as substitutions between polar amino acids (such as substitutions between glutamine and asparagine), substitutions between hydrophobic amino acids (such as bright Replacement between amino acid, isoleucine, methionine and proline), and substitution between amino acids with the same charge (such as substitution between arginine, lysine and histidine, or glutamine) Replacement between acid and aspartic acid).
  • the MG53 mutant described herein has only one or more amino acid positions in the non-serine compared to the sequence set forth in SEQ ID NOs: 7-12 and SEQ ID NOs: 147-150. A conservative substitution of amino acids.
  • the MG53 mutants described herein are compared to the sequences set forth in SEQ ID NOs: 7-12 and SEQ ID NOs: 147-150, in two, three, four non-serine, Five, six, seven, eight, nine, ten, fifteen, or twenty amino acid positions have conservative substitutions of amino acids.
  • the MG53 mutant described herein may also contain non-natural amino acids without affecting activity.
  • Non-natural amino acids include, for example, ⁇ -fluoroalanine, 1-methylhistidine, ⁇ -methylene glutamic acid, ⁇ -methyl leucine, 4,5-dehydrolysine, Hydroxyproline, 3-fluorophenylalanine, 3-aminotyrosine, 4-methyltryptophan, and the like.
  • the MG53 mutants of the present application can also be modified using methods well known in the art. For example, but not limited to, PEGylation, glycosylation, amino terminal modification, fatty acylation, carboxy terminal modification, phosphorylation, methylation, and the like.
  • MG53 mutants of the present application retain substantially the same function as the MG53 mutant after modification using methods well known in the art, for example, modified MG53 mutants have cytoremediation functions and / or cardioprotective function, can avoid or reduce the metabolic side effects caused by wild-type MG53, such as insulin resistance, obesity, diabetes, hypertension, dyslipidemia and the like.
  • the present application is directed to a pharmaceutical composition
  • a pharmaceutical composition comprising a MG53 mutant described herein and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier refers to a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering a MG53 mutant to a subject, which does not interfere with MG53 The structure and nature of the mutant.
  • Certain such carriers enable the MG53 mutant to be formulated, for example, as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions and lozenges for oral ingestion by the subject.
  • Certain such vectors enable the MG53 mutant to be formulated for injection, infusion or topical administration.
  • Pharmaceutically acceptable carriers that can be used in the pharmaceutical compositions of the present application include, but are not limited to, for example, pharmaceutically acceptable liquid, gel, or solid carrier, aqueous medium (eg, sodium chloride injection) , Ringer's solution, isotonic glucose injection, sterile water injection, or glucose and lactated Ringer's injection), non-aqueous medium (for example, plant-derived fixed oil, cottonseed oil, corn) Oil, sesame oil, or peanut oil), antimicrobial substances, isotonic substances (such as sodium chloride or glucose), buffers (such as phosphate or citrate buffer), antioxidants (such as sodium bisulfate), anesthetics ( For example, procaine hydrochloride), suspending/dispersing agents (such as sodium carboxymethylcellulose, hydroxypropylmethylcellulose, or polyvinylpyrrolidone), chelating agents (such as EDTA (ethylenediaminetetraacetic acid) or EGTA) (Glycol bis(2-aminoeth
  • the pharmaceutical composition is an oral formulation.
  • Oral formulations include, but are not limited to, capsules, sachets, pills, tablets, lozenges (substrate for taste, typically sucrose and gum arabic or tragacanth), powders, granules, or water or non-aqueous solutions or Suspensions, or water-in-oil or oil-in-water emulsions, or elixirs or syrups, or candy lozenges (for inert matrices such as gelatin and glycerin, or sucrose or gum arabic) and/or mouthwashes and Its analogues.
  • the oral solid preparation (eg, capsules, tablets, pills, dragees, powders, granules, and the like) comprises the MG53 mutant and one or more pharmaceutically acceptable carriers, such as Sodium citrate or dicalcium phosphate, and/or the following: (1) a filler or a supplement such as starch, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) a binder, for example, Carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and/or gum arabic; (3) wetting agents, for example, glycerol; (4) cleavage agents, such as agar, calcium carbonate, Potato or tapioca starch, alginic acid, certain silicates, with sodium carbonate, (5) retarder solutions, such as paraffin wax; (6) accelerated absorbents, such as quaternary ammonium compounds; (7) lubricants, for example, acety
  • oral liquid preparations include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs, and the like.
  • the liquid dosage form may contain conventional inert diluents such as water or other solvents, solubilizers and emulsifiers such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzene.
  • the oral compositions may also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening agents, flavoring agents, pigments, flavors and preservatives.
  • the pharmaceutical composition is an injectable preparation.
  • Formulations for injection include sterile aqueous solutions or dispersions, suspensions or emulsions.
  • the injectable preparation should be sterile and should be liquid to facilitate the injection. It should be stable under the conditions of manufacture and storage and should be contaminated with microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures and/or vegetable oils thereof.
  • the injectable preparation should maintain proper fluidity, and proper fluidity can be maintained by various means, for example, by using a coating such as lecithin, using a surfactant or the like.
  • Antimicrobial contamination can be achieved by the addition of various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc.).
  • the pharmaceutical composition is an oral spray formulation or a nasal spray formulation.
  • spray formulations include, but are not limited to, aqueous aerosols, non-aqueous suspensions, liposomal formulations or solid particulate formulations, and the like.
  • Aqueous aerosols are formulated by combining an aqueous solution or suspension of the agent with a conventional pharmaceutically acceptable carrier with a stabilizer.
  • the carrier and stabilizer vary depending on the needs of the particular compound, but generally include nonionic surfactants (Tween, or polyethylene glycol), oleic acid, lecithin, amino acids such as glycine, buffers, salts, Sugar or sugar alcohol.
  • Aerosols are typically prepared from isotonic solutions and can be delivered by a nebulizer.
  • the pharmaceutical composition can be used in admixture with one or more other drugs.
  • the composition comprises at least one other drug.
  • the other drug is a cardiovascular drug, a drug for treating kidney disease, or a drug for cell repair, and the like.
  • the pharmaceutical composition can be delivered to a subject by an appropriate route including, but not limited to, by an oral route, an injection route (eg, intravenous, intramuscular, subcutaneous, intradermal injection). , intracardiac injection, intrathecal injection, intrapleural injection, intraperitoneal injection, etc.), mucosal route (such as intranasal administration, intraoral administration, etc.), sublingual route, rectal route, transdermal route, intraocular route , the lungs.
  • an injection route eg, intravenous, intramuscular, subcutaneous, intradermal injection.
  • intracardiac injection intrathecal injection
  • intrapleural injection intrapleural injection
  • intraperitoneal injection etc.
  • mucosal route such as intranasal administration, intraoral administration, etc.
  • sublingual route such as intranasal administration, intraoral administration, etc.
  • rectal route transdermal route
  • intraocular route intraocular route
  • the pharmaceutical composition can be administered by an injection route.
  • the present application relates to an isolated nucleic acid comprising a nucleic acid sequence encoding an amino acid sequence of a MG53 mutant described herein.
  • isolated refers to a substance (eg, a polypeptide or nucleic acid) that is separated from its environment in which it normally exists in nature or that exists in an environment different from the environment in which it normally exists in nature.
  • nucleic acid refers to ribonucleic acid (RNA), deoxyribonucleic acid (DNA), or a mixture of ribonucleic acid-deoxyribonucleic acids such as DNA-RNA hybrids.
  • the nucleic acid or polynucleotide may be a single stranded or double stranded DNA or an RNA or DNA-RNA hybrid.
  • the nucleic acid or polynucleotide can be linear or circular.
  • encoding or “encoding to” as used herein, refers to being capable of being transcribed into mRNA and/or translated into a peptide or protein.
  • Coding sequence or “gene” refers to a polynucleotide sequence encoding an mRNA, peptide or protein. These two terms are used interchangeably in this application.
  • the isolated nucleic acid comprises any one of the nucleic acid sequences set forth in SEQ ID NOs: 13-18.
  • SEQ ID NO: 13 is the nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO: 7, the specific sequence of which is shown in FIG.
  • SEQ ID NO: 14 is the nucleic acid sequence encoding the amino acid sequence set forth in SEQ ID NO: 8, the specific sequence of which is shown in FIG.
  • SEQ ID NO: 15 is the nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO: 9, the specific sequence of which is shown in FIG.
  • SEQ ID NO: 16 is the nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO: 10, the specific sequence of which is shown in FIG.
  • SEQ ID NO: 17 is the nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO: 11, the specific sequence of which is shown in FIG.
  • SEQ ID NO: 18 is the nucleic acid sequence encoding the amino acid sequence set forth in SEQ ID NO: 12, the specific sequence of which is shown in FIG.
  • the isolated nucleic acid comprises any of the nucleic acid sequences set forth in SEQ ID NOs: 151-154.
  • SEQ ID NO: 151 is the nucleic acid sequence encoding the amino acid sequence set forth in SEQ ID NO: 147, the specific sequence of which is shown in FIG.
  • SEQ ID NO: 152 is the nucleic acid sequence encoding the amino acid sequence set forth in SEQ ID NO: 148, the specific sequence of which is shown in FIG.
  • SEQ ID NO: 153 is the nucleic acid sequence encoding the amino acid sequence set forth in SEQ ID NO: 149, the specific sequence of which is shown in FIG.
  • SEQ ID NO: 154 is the nucleic acid sequence encoding the amino acid sequence set forth in SEQ ID NO: 150, the specific sequence of which is shown in FIG.
  • the isolated nucleic acids provided herein comprise a nucleic acid sequence having at least 70% homology to any of the nucleic acid sequences set forth in SEQ ID NOs: 13-18, SEQ ID NOs: 151-154, eg, , at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% homologous, and still encoding SEQ ID NOs: 7-12, SEQ ID NOs: 147-150 One of the amino acid sequences shown.
  • the application provides a nucleic acid sequence encoding SEQ ID NOs: 7-12, SEQ ID NOs: 147-150, but the nucleic acid sequence differs from SEQ ID NOs: 13 due to the degeneracy of the genetic code. -18. Any of the nucleic acid sequences set forth in SEQ ID NOs: 151-154.
  • degeneracy of the genetic code refers to the phenomenon that the same amino acid has two or more corresponding codons.
  • proline has four synonymous codons CCU, CCC, CCA, CCG.
  • nucleic acids at certain positions in a known nucleic acid sequence can be replaced without altering the encoded amino acid sequence.
  • One skilled in the art can readily perform a degeneracy replacement of the genetic code, for example, by site directed mutagenesis techniques. Different organisms have different preferences for different codons.
  • the preferred codon of the biological cell can be selected to obtain the corresponding coding sequence and expressed by recombinant expression.
  • the MG53 mutant sequence of the present application e.g., SEQ ID NOs: 7-12, SEQ ID NOs: 147-150 is obtained.
  • the present application relates to an expression vector comprising the coding sequence for the amino acid sequence of the MG53 mutant described herein.
  • the expression vector in the present application may be, for example, a DNA plasmid, a bacterial plasmid, a virus, or the like.
  • Non-limiting examples of expression vectors are, for example, Paul et al, 2002, Nature Biotechnology, 19, 505; Miyagishi and Taira, 2002, Nature Biotechnology, 19, 497; Lee et al, 2002, Nature Biotechnology, 19, 500; and Novina et al, 2002, Nature Medicine, advance Online publication doi: described in 10.1038/nm725.
  • the expression vector may further comprise a promoter, which is related to the amino acid sequence of the MG53 mutant
  • the coding sequences of the sequences are operably linked such that when the expression vector enters a host cell, the promoter is capable of initiating expression of the coding sequence.
  • the expression vector can be introduced into a host cell by an appropriate method, such as, but not limited to, calcium phosphate transfection, lipofection, electroporation transfection, bacterial heat shock, etc., see, for example, Sambrook et al. Molecular Cloning" (Experimental Handbook, Cold Spring Harbor, 1989).
  • an expression vector described herein comprises any one of the nucleic acid sequences set forth in SEQ ID NOs: 13-18.
  • the present application relates to a host cell comprising an expression vector as described herein.
  • the host cell described in the present application may be a eukaryotic cell or a prokaryotic cell.
  • Suitable eukaryotic cells can include, for example, mammalian cells, such as Chinese hamster ovary cells (CHO).
  • Suitable prokaryotic cells can include, for example, bacteria such as E. coli.
  • the present application relates to a method of making the MG53 mutant, and the MG53 mutant provided herein can be prepared by techniques known in the art. For example, it can be prepared by a chemical synthesis method or a genetic engineering method.
  • Solid phase polypeptide synthesis methods mainly include solid phase synthesis and liquid phase synthesis.
  • Solid phase polypeptide synthesis methods include, for example, Merrifield solid phase synthesis, which has been described in detail in the literature "Merrifield, J. Am. Chem. Soc. 85: 2149-2154” and “M. Bodanszky et al., “Peptide Synthesis”. , John Wiley & Sons, Second Edition, 1976” and “J. Meienhofer, "Hormonal Proteins and Peptides", Vol. 2, p. 46, Academic Press (New York), 1983".
  • the Merrifield solid phase synthesis method mainly comprises the steps of: firstly, the protected carboxy terminal amino acid is linked to the resin according to the amino acid sequence of the target polypeptide; after washing, the resin is washed; and the protective group on the amino group of the amino terminal ⁇ of the carboxy terminal is removed (for example, tert-butyl) Oxycarbonyl), when removing this protecting group, it must be ensured that the linkage between the amino acid and the resin is not broken; then the penultimate carboxy-terminal protected amino acid is coupled to the resulting resin for this coupling.
  • the protective group on the amino group of the amino terminal ⁇ of the carboxy terminal is removed (for example, tert-butyl) Oxycarbonyl), when removing this protecting group, it must be ensured that the linkage between the amino acid and the resin is not broken; then the penultimate carboxy-terminal protected amino acid is coupled to the resulting resin for this coupling.
  • polypeptides of the present application can also be prepared by liquid phase synthesis methods, for example, by standard solution peptide synthesis, which has been described in detail in the literature "E. Schroder and K. Kubke, "The Peptides", Vol. 1, Academic Press (New York), 1965".
  • the liquid phase synthesis method mainly includes stepwise coupling of amino acids by chemical or enzymatic methods for forming an amide bond. Or peptide fragments.
  • a genetic engineering method is a method of expressing a corresponding mutant in a suitable host cell using a nucleic acid sequence encoding the corresponding MG53 mutant.
  • a detailed description of this method can be found in "Molecular Cloning" by Sambrook et al. (Experimental Handbook, Cold Spring Harbor, 1989).
  • the method of making the MG53 mutant according to the present application comprises determining one or more positions of a serine that is required to be mutated, and a full length sequence of a plasmid comprising a nucleic acid sequence encoding a wild type MG53 amino acid sequence. Site-directed mutagenesis at the position is performed, the site-directed mutated plasmid is transfected into a host cell, and the host cell is expressed to produce the MG53 mutant.
  • site-directed mutagenesis refers to the introduction of a change of interest into a DNA fragment of interest, including the addition, deletion, substitution, etc. of a base.
  • X One or more serine sites in the coding sequence of the -SPRY domain.
  • SEQ ID NO: 19 is the nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO: 1, the specific sequence of which is shown in Figure 2.
  • SEQ ID NO: 20 is the nucleic acid sequence encoding the amino acid sequence set forth in SEQ ID NO: 2, the specific sequence of which is shown in Figure 3.
  • SEQ ID NO: 21 is the nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO: 3, the specific sequence of which is shown in FIG.
  • SEQ ID NO: 22 is the nucleic acid sequence encoding the amino acid sequence set forth in SEQ ID NO: 4, the specific sequence of which is shown in Figure 5.
  • SEQ ID NO: 23 is the nucleic acid sequence encoding the amino acid sequence set forth in SEQ ID NO: 5, the specific sequence of which is shown in Figure 6.
  • SEQ ID NO: 24 is the nucleic acid sequence encoding the amino acid sequence set forth in SEQ ID NO: 6, the specific sequence of which is shown in Figure 7.
  • SEQ ID NO: 143 is the nucleic acid sequence encoding the amino acid sequence set forth in SEQ ID NO: 139, the specific sequence of which is shown in FIG.
  • SEQ ID NO: 144 is the nucleic acid sequence encoding the amino acid sequence set forth in SEQ ID NO: 140, the specific sequence of which is shown in FIG.
  • SEQ ID NO: 145 is the nucleic acid sequence encoding the amino acid sequence set forth in SEQ ID NO: 141, the specific sequence of which is shown in FIG.
  • SEQ ID NO: 146 is the nucleic acid sequence encoding the amino acid sequence set forth in SEQ ID NO: 142, the specific sequence of which is shown in FIG.
  • the site-directed mutagenesis comprises the steps of:
  • step (2) using the wild type MG53 plasmid as a template, using the primer designed in step (1) for PCR reaction, the PCR product was subjected to agarose gel electrophoresis, and the PCR product was purified;
  • step (3) The purified PCR product in step (2) is digested with a nucleic acid restriction endonuclease, and the digested product is ligated to a suitable plasmid expression vector, and the ligated product is transformed into a bacterial competent cell. to cultivate.
  • the site-directed mutagenesis further comprises the steps of:
  • step (3) Picking the clone obtained in step (3) using the primer designed in the step (1) for colony PCR identification, detecting the PCR product by agarose gel electrophoresis, and then performing DNA sequencing to identify the site-directed mutation. Positive clone.
  • Site-directed mutagenesis of wild-type MG53 can be carried out using various commercially available site-directed mutagenesis kits, for example, with reference to the specification of the Easy Mutagenesis System kit of Beijing Quanjin Company, in which the methods and procedures for site-directed mutagenesis are described in detail.
  • the present application relates to the preparation of the MG53 mutant for the treatment of heart disease, diabetic cerebrovascular disease, diabetic ocular complications, diabetic neuropathy, diabetic foot, kidney disease, and cell and/or tissue damage related diseases.
  • the drug avoids or simultaneously treats a heart disease, a diabetic cerebrovascular disease, a diabetic ocular complication, a diabetic neuropathy, a diabetic foot, a kidney disease, a disease associated with cell and/or tissue damage. Reduces side effects such as insulin resistance, obesity, diabetes, high blood pressure, and dyslipidemia.
  • the heart disease is a disease associated with myocardial damage, including but not limited to, diabetic heart disease, myocardial ischemia, cardiac ischemia/reperfusion injury, myocardial infarction, heart failure, arrhythmia, Heart rupture, angina pectoris, myocarditis, coronary heart disease, pericarditis.
  • the diabetic cerebrovascular disease includes, but is not limited to, cerebral arteriosclerosis, ischemic cerebral blood Tube disease, cerebral hemorrhage, brain atrophy, cerebral infarction.
  • the diabetic ocular complications include, but are not limited to, diabetic retinopathy, diabetic cataract, uveitis associated with diabetes, blindness.
  • the diabetic neuropathy includes, but is not limited to, diabetic peripheral neuropathy.
  • the renal disease includes, but is not limited to, acute glomerulonephritis, chronic glomerulonephritis, nephrotic syndrome, acute kidney injury, diabetic nephropathy, and the like.
  • the disease associated with cell and/or tissue damage includes, but is not limited to, diseases associated with kidney, brain, lung, liver, heart, spleen, cells of the digestive tract, and/or tissue damage, skin Damage-related diseases, such as brain damage, lung injury, spleen injury, spleen rupture, gastric ulcer, gastritis, gastric perforation, gastrointestinal mucosal injury, trauma, burns, ulcers, mucositis, asthma, chronic obstructive pulmonary disease (COPD) ), stroke, skin aging, etc.
  • diseases associated with kidney, brain, lung, liver, heart, spleen, cells of the digestive tract, and/or tissue damage skin Damage-related diseases, such as brain damage, lung injury, spleen injury, spleen rupture, gastric ulcer, gastritis, gastric perforation, gastrointestinal mucosal injury, trauma, burns, ulcers, mucositis, asthma, chronic obstructive pulmonary disease (COPD) ), stroke, skin aging, etc
  • the present application relates to the preparation of the polypeptide of SEQ ID NO: 7 for the treatment of heart disease, diabetic cerebrovascular disease, diabetic ocular complications, diabetic neuropathy, diabetic foot, kidney disease, and cells. And/or use in drugs for tissue damage related diseases.
  • the present application relates to polypeptides set forth in SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, or SEQ ID NO: 150 for use in the preparation of a heart disease, diabetic cerebrovascular disease, Use in diabetic ocular complications, diabetic neuropathy, diabetic foot, kidney disease, drugs associated with diseases associated with cellular and/or tissue damage.
  • the present application relates to a method of treating a heart disease, a diabetic cerebrovascular disease, a diabetic ocular complication, a diabetic neuropathy, a diabetic foot, a kidney disease, a disease associated with cell and/or tissue damage, including administering a desired
  • the subject is therapeutically effective in an amount of the MG53 mutant.
  • the MG53 mutant is in the treatment of heart disease, diabetic cerebrovascular disease, diabetic ocular complications, diabetic neuropathy, diabetic foot, kidney disease, and diseases associated with cellular and/or tissue damage, Metabolic side effects such as insulin resistance, obesity, diabetes, hypertension, dyslipidemia, and the like are avoided or reduced.
  • the heart disease is a disease associated with myocardial damage, including but not limited to, diabetic heart disease, myocardial ischemia, cardiac ischemia/reperfusion injury, myocardial infarction, heart failure, arrhythmia, Heart rupture, angina pectoris, myocarditis, coronary heart disease, pericarditis.
  • the diabetic cerebrovascular disease includes, but is not limited to, cerebral arteriosclerosis, ischemic cerebrovascular disease, cerebral hemorrhage, brain atrophy, cerebral infarction.
  • the diabetic ocular complications include, but are not limited to, diabetic retinopathy, diabetic cataract, uveitis associated with diabetes, blindness.
  • the diabetic neuropathy includes, but is not limited to, diabetic peripheral neuropathy.
  • the renal disease includes, but is not limited to, acute glomerulonephritis, chronic glomerulonephritis, nephrotic syndrome, acute kidney injury, diabetic nephropathy, and the like.
  • the diseases associated with cell and/or tissue damage include, but are not limited to, diseases associated with kidney, brain, lung, liver, heart, spleen, cells of the digestive tract, and/or tissue damage, diseases associated with skin damage, for example, Brain injury, lung injury, spleen injury, spleen rupture, gastric ulcer, gastritis, gastric perforation, gastrointestinal mucosal injury, trauma, burns, ulcers, mucositis, asthma, chronic obstructive pulmonary disease (COPD), stroke, skin aging, etc.
  • diseases associated with kidney, brain, lung, liver, heart, spleen, cells of the digestive tract and/or tissue damage
  • diseases associated with skin damage for example, Brain injury, lung injury, spleen injury, spleen rupture, gastric ulcer, gastritis, gastric perforation, gastrointestinal mucosal injury, trauma, burns, ulcers, mucositis, asthma, chronic obstructive pulmonary disease (COPD), stroke, skin aging, etc.
  • the present application relates to a method of treating heart disease, diabetic cerebrovascular disease, diabetic ocular complications, diabetic neuropathy, diabetic foot, kidney disease, diseases associated with cellular and/or tissue damage, including A therapeutically effective amount of the polypeptide of SEQ ID NO: 7 is administered to a subject in need thereof.
  • the present application relates to a method of treating heart disease, diabetic cerebrovascular disease, diabetic ocular complications, diabetic neuropathy, diabetic foot, kidney disease, diseases associated with cellular and/or tissue damage, including A therapeutically effective amount of a polypeptide of SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149 or SEQ ID NO: 150 is administered to a subject in need thereof.
  • therapeutically effective amount means that the amount of the drug which inhibits or alleviates the disease or symptom of the subject, or which can prevent or prevent the occurrence of the disease or symptom, can be achieved.
  • a therapeutically effective amount can be an amount that relieves one or more diseases or conditions of the subject to a certain extent; one or more physiological or biochemical parameters associated with the cause of the disease or condition can be partially or completely restored to The amount of normal drug; and/or the amount of drug that can reduce the likelihood that a disease or condition will occur.
  • the present application relates to MG53 mutants for use in the treatment of heart disease, diabetic cerebrovascular disease, diabetic ocular complications, diabetic neuropathy, diabetic foot, kidney disease, diseases associated with cellular and/or tissue damage.
  • the MG53 mutant is in the treatment of heart disease, diabetic cerebrovascular disease, diabetic ocular complications, diabetic neuropathy, diabetic foot, kidney disease, and diseases associated with cellular and/or tissue damage, Metabolic side effects such as insulin resistance, obesity, diabetes, high blood pressure, and dyslipidemia are avoided.
  • the heart disease is a disease associated with myocardial damage, including but not limited to, diabetic heart disease, myocardial ischemia, cardiac ischemia/reperfusion injury, myocardial infarction, heart failure, arrhythmia, Heart rupture, angina pectoris, myocarditis, coronary heart disease, pericarditis.
  • the diabetic cerebrovascular disease includes, but is not limited to, cerebral arteriosclerosis, ischemic cerebrovascular disease, cerebral hemorrhage, brain atrophy, cerebral infarction.
  • the diabetic ocular complications include, but are not limited to, diabetic retinopathy, diabetic cataract, uveitis associated with diabetes, blindness.
  • the diabetic neuropathy includes, but is not limited to, diabetic peripheral neuropathy.
  • the renal disease includes, but is not limited to, acute glomerulonephritis, chronic glomerulonephritis, nephrotic syndrome, acute kidney injury, Diabetic nephropathy and the like.
  • the disease associated with cell and/or tissue damage includes, but is not limited to, diseases associated with kidney, brain, lung, liver, heart, spleen, cells of the digestive tract, and/or tissue damage, skin Damage-related diseases, such as brain damage, lung injury, spleen injury, spleen rupture, gastric ulcer, gastritis, gastric perforation, gastrointestinal mucosal injury, trauma, burns, ulcers, mucositis, asthma, chronic obstructive pulmonary disease (COPD) ), stroke, skin aging, etc.
  • diseases associated with kidney, brain, lung, liver, heart, spleen, cells of the digestive tract, and/or tissue damage skin Damage-related diseases, such as brain damage, lung injury, spleen injury, spleen rupture, gastric ulcer, gastritis, gastric perforation, gastrointestinal mucosal injury, trauma, burns, ulcers, mucositis, asthma, chronic obstructive pulmonary disease (COPD) ), stroke, skin aging, etc
  • the present application relates to SEQ ID NO for treating heart disease, diabetic cerebrovascular disease, diabetic ocular complications, diabetic neuropathy, diabetic foot, kidney disease, diseases associated with cellular and/or tissue damage. : The polypeptide shown in 7. In certain embodiments, the present application relates to SEQ ID NO for treating heart disease, diabetic cerebrovascular disease, diabetic ocular complications, diabetic neuropathy, diabetic foot, kidney disease, diseases associated with cellular and/or tissue damage. : 147, SEQ ID NO: 148, SEQ ID NO: 149 or SEQ ID NO: 150.
  • Biological materials involved in all embodiments such as E. coli strains, various cloning and expression plasmids, culture media, kit enzymes, buffers, and various culture methods, protein extraction and purification methods, and other molecular biological methods of operation , are familiar to the skilled person in the field, can refer to "Molecular Cloning” (Lab Handbook, Cold Spring Harbor, 1989) and “Guide to Editing Molecular Biology” edited by Sambrook et al. (US/F. Osbour, etc.) , Yan Ziying and other translations, Beijing, Science Press, 1998).
  • Dpn I digestion The purified PCR product was subjected to a digestion reaction using a nucleic acid restriction endonuclease Dpn I.
  • Transformation of the digested product The Dpn I digested product is subjected to transformation of bacterial competent cells, and is applied to a suitable screening plate for culture.
  • Identification of positive clones Pick the appropriate size clones for colony PCR identification, and perform PCR detection on the agarose gel electrophoresis. The clones with clear and correct size were cultured in small volume and then sequenced. Identification.
  • Human MG53 S255G, S255L, S255V, S255P, S255F, S255W, S255Q, S255C, S255Y, S255D, S255R, S211A, S214A, S246A, S269A, S296A, S297A mutants were prepared in the same manner. The other steps were identical to those of the human MG53 S255A mutant except that the mutant primers were different.
  • the primers for each mutant are as follows:
  • human MG53 S255G represents a MG53 mutant in which the 255th serine of human wild type MG53 (ie, SEQ ID NO: 1) is mutated to glycine.
  • human MG53 S255V represents a MG53 mutant in which the 255th serine of human wild type MG53 (ie, SEQ ID NO: 1) is mutated to proline.
  • human MG53 S255P represents a MG53 mutant in which the 255th serine of human wild type MG53 (ie, SEQ ID NO: 1) is mutated to proline.
  • human MG53 S255F represents a MG53 mutant in which the 255th serine of human wild type MG53 (ie, SEQ ID NO: 1) is mutated to phenylalanine.
  • human MG53 S255W represents a MG53 mutant in which the 255th serine of human wild type MG53 (ie, SEQ ID NO: 1) was mutated to tryptophan.
  • human MG53 S255Q represents the MG53 mutant of the 255th serine of human wild type MG53 (ie SEQ ID NO: 1) mutated to glutamine.
  • human MG53 S255Y represents a MG53 mutant in which the 255th serine of human wild type MG53 (ie, SEQ ID NO: 1) was mutated to tyrosine.
  • human MG53 S255D represents a MG53 mutant in which the 255th serine of human wild type MG53 (ie, SEQ ID NO: 1) is mutated to aspartic acid.
  • human MG53 S255R represents a MG53 mutant in which the 255th serine of human wild type MG53 (ie, SEQ ID NO: 1) is mutated to arginine.
  • human MG53 S211A represents a MG53 mutant in which the 211th serine of human wild type MG53 (ie, SEQ ID NO: 1) is mutated to alanine.
  • Human MG53 S211A forward primer ccttgcgccgggagctgggggccctgaactctt (SEQ ID NO: 49)
  • Human MG53 S211A reverse primer gctgctccaggtaagagttcagggcccccagctcc (SEQ ID NO: 50)
  • human MG53 S214A represents a MG53 mutant in which the serine of position 214 of human wild type MG53 (ie, SEQ ID NO: 1) is mutated to alanine.
  • Human MG53 S214A forward primer gggagctggggagcctgaacgcttacctggagc (SEQ ID NO: 51)
  • human MG53 S246A represents an MG53 mutant in which the serine of position 246 of human wild type MG53 (ie, SEQ ID NO: 1) is mutated to alanine.
  • Human MG53 S246A forward primer tgaaatactgcctggtgaccgccaggctgcaga (SEQ ID NO: 53)
  • Human MG53 S246A reverse primer gccaggatcttctgcagcctggcggtcaccagg (SEQ ID NO: 54)
  • human MG53 S269A represents a MG53 mutant in which the 269th serine of human wild type MG53 (ie, SEQ ID NO: 1) is mutated to alanine.
  • Human MG53 S269A forward primer aggagctgacctttgacccggcctctgcgcacc (SEQ ID NO: 55)
  • Human MG53 S296A reverse primer accaggctcgggtgcgcagaggccgggtcaaag (SEQ ID NO: 56)
  • human MG53 S297A represents a MG53 mutant in which the 297th serine of human wild type MG53 (ie, SEQ ID NO: 1) is mutated to alanine.
  • Human MG53 S297A forward primer agctgacctttgacccgagcgctgcgcaccccga (SEQ ID NO: 57)
  • Human MG53 S297A reverse primer accaccaggctcgggtgcgcagcgctcgggtca (SEQ ID NO: 58)
  • Mouse MG53 S255A, S255G, S255L, S255W, S255Q, S255Y, S255D, S255R mutants were prepared in the same manner as the human MG53 S255A mutant. The other steps were identical to those of the human MG53 S255A mutant except that the mutant primers were different.
  • the primers for each mutant are as follows:
  • mouse MG53 S255A represents an MG53 mutant in which the 255th serine of wild type MG53 (i.e., SEQ ID NO: 2) of the mouse was mutated to alanine.
  • mouse MG53 S255G represents a MG53 mutant in which the 255th serine of wild type MG53 (i.e., SEQ ID NO: 2) of the mouse was mutated to glycine.
  • mouse MG53 S255L represents a MG53 mutant in which the 255th serine of wild type MG53 (i.e., SEQ ID NO: 2) of the mouse was mutated to leucine.
  • mouse MG53 S255W represents a MG53 mutant in which the 255th serine of mouse wild-type MG53 (ie, SEQ ID NO: 2) was mutated to tryptophan.
  • mouse MG53 S255Q represents a MG53 mutant in which the 255th serine of wild type MG53 (i.e., SEQ ID NO: 2) of the mouse was mutated to glutamine.
  • mouse MG53 S255Y represents a MG53 mutant in which the 255th serine of wild type MG53 (i.e., SEQ ID NO: 2) of the mouse was mutated to tyrosine.
  • mouse MG53 S255R represents a MG53 mutant in which the 255th serine of wild type MG53 (i.e., SEQ ID NO: 2) of the mouse was mutated to arginine.
  • Rat MG53 S255A, S255G, S255L, S255W, S255Q, S255Y, S255D, S255R mutants were prepared in the same manner as the human MG53 S255A mutant. The other steps were identical to those of the human MG53 S255A mutant except that the mutant primers were different.
  • the primers for each mutant are as follows:
  • rat MG53 S255A represents a MG53 mutant in which the 255th serine of wild type MG53 (i.e., SEQ ID NO: 3) of the rat was mutated to alanine.
  • Rat MG53 S255A forward primer tgcagaagattctgtcagaggcaccacccccag (SEQ ID NO: 75)
  • Rat MG53 S255A reverse primer tctagccttgctgggggtggtgcctctgacaga (SEQ ID NO: 76)
  • rat MG53 S255G represents a MG53 mutant in which the 255th serine of wild type MG53 (i.e., SEQ ID NO: 3) of the rat was mutated to glycine.
  • Rat MG53 S255G forward primer tgcagaagattctgtcagagggaccacccccag (SEQ ID NO: 77)
  • Rat MG53 S255G reverse primer tctagccttgctgggggtggtccctctgacaga (SEQ ID NO: 78)
  • rat MG53 S255L represents a MG53 mutant in which the 255th serine of wild type MG53 (i.e., SEQ ID NO: 3) of the rat was mutated to leucine.
  • Rat MG53 S255L forward primer tgcagaagattctgtcagagttaccacccccag (SEQ ID NO: 79)
  • Rat MG53 S255L reverse primer tctagccttgctgggggtggtaactctgacaga (SEQ ID NO: 80)
  • rat MG53 S255W represents the MG53 mutant in which the 255th serine of wild type MG53 (i.e., SEQ ID NO: 3) of the rat was mutated to tryptophan.
  • Rat MG53 S255W forward primer tgcagaagattctgtcagagtggccacccccag (SEQ ID NO: 81)
  • Rat MG53 S255W reverse primer tctagccttgctgggggtggccactctgacaga (SEQ ID NO: 82)
  • rat MG53 S255Q represents the MG53 mutant of the 255th serine of wild type MG53 (ie, SEQ ID NO: 3) of the rat which was mutated to glutamine.
  • Rat MG53 S255Q forward primer tgcagaagattctgtcagagcaaccacccccag (SEQ ID NO: 83)
  • Rat MG53 S255Q reverse primer tctagccttgctgggggtggttgctctgacaga (SEQ ID NO: 84)
  • rat MG53 S255Y represents a MG53 mutant in which the 255th serine of wild type MG53 (i.e., SEQ ID NO: 3) of the rat was mutated to tyrosine.
  • Rat MG53 S255Y forward primer tgcagaagattctgtcagagtatccacccccag (SEQ ID NO: 85)
  • Rat MG53 S255D forward primer tgcagaagattctgtcagaggatccacccccag (SEQ ID NO: 87)
  • Rat MG53 S255D reverse primer tctagccttgctgggggtggatcctctgacaga (SEQ ID NO: 88)
  • rat MG53 S255R represents a MG53 mutant in which the 255th serine of wild type MG53 (i.e., SEQ ID NO: 3) of the rat was mutated to arginine.
  • Rat MG53 S255R forward primer tgcagaagattctgtcagagcgaccacccccag (SEQ ID NO: 89)
  • Rat MG53 S255R reverse primer tctagccttgctgggggtggtcgctctgacaga (SEQ ID NO: 90)
  • Monkey MG53 S255A, S255G, S255L, S255W, S255Q, S255Y, S255D, S255R mutants were prepared in the same manner as the human MG53 S255A mutant. The other steps were identical to those of the human MG53 S255A mutant except that the mutant primers were different.
  • the primers for each mutant are as follows:
  • monkey MG53 S255A represents the MG53 mutant in which the 255th serine of wild type MG53 (ie, SEQ ID NO: 4) of monkey is mutated to alanine.
  • monkey MG53 S255G represents the MG53 mutant in which the 255th serine of wild type MG53 (ie, SEQ ID NO: 4) of monkey is mutated to glycine.
  • monkey MG53 S255W represents the MG53 mutant of the 255th serine mutant of wild type MG53 (ie, SEQ ID NO: 4) of monkey to tryptophan.
  • monkey MG53 S255Q represents the MG53 mutant of the 255th serine of wild type MG53 (ie, SEQ ID NO: 4) of monkey to glutamine.
  • monkey MG53 S255Y represents the MG53 mutant in which the 255th serine of wild type MG53 (ie, SEQ ID NO: 4) of monkey is mutated to tyrosine.
  • monkey MG53 S255D represents the MG53 mutant of the 255th serine of wild type MG53 (ie, SEQ ID NO: 4) of monkey to mutated to aspartic acid.
  • Porcine MG53 S255A, S255G, S255L, S255W, S255Q, S255Y, S255D, S255R mutants were prepared in the same manner as the human MG53 S255A mutant. The other steps were identical to those of the human MG53 S255A mutant except that the mutant primers were different.
  • the primers for each mutant are as follows:
  • porcine MG53 S255A represents an MG53 mutant in which the 255th serine of wild type MG53 (i.e., SEQ ID NO: 5) of porcine was mutated to alanine.
  • Pig MG53 S255A forward primer aagatcctggcagaggcgcccccacctgcccgcctg (SEQ ID NO: 107)
  • porcine MG53 S255G represents an MG53 mutant in which the 255th serine of wild type MG53 (i.e., SEQ ID NO: 5) of the pig is mutated to glycine.
  • Pig MG53 S255G forward primer aagatcctggcagaggggcccccacctgcccgcctgg (SEQ ID NO: 109)
  • porcine MG53 S255L represents a MG53 mutant in which the 255th serine of porcine wild-type MG53 (i.e., SEQ ID NO: 5) was mutated to leucine.
  • Pig MG53 S255L forward primer agatcctggcagagttgcccccacctgcccgcctgg (SEQ ID NO: 111)
  • porcine MG53 S255W represents the MG53 mutant of the 255th serine mutated to tryptophan of wild type MG53 (ie, SEQ ID NO: 5) of pig.
  • Pig MG53 S255W forward primer agatcctggcagagtggcccccacctgcccgcctgg (SEQ ID NO: 113)
  • porcine MG53 S255Q represents a MG53 mutant of 255 in which the serine of porcine wild-type MG53 (i.e., SEQ ID NO: 5) was mutated to glutamine.
  • Pig MG53 S255Q forward primer aagatcctggcagagcagcccccacctgcccgcctgg (SEQ ID NO: 115)
  • porcine MG53 S255Y represents the MG53 mutant in which the 255th serine of porcine wild-type MG53 (i.e., SEQ ID NO: 5) was mutated to tyrosine.
  • Pig MG53 S255Y forward primer agatcctggcagagtatcccccacctgcccgcctgga (SEQ ID NO: 117)
  • Pig MG53 S255D forward primer aagatcctggcagaggatcccccacctgcccgcctgga (SEQ ID NO: 119)
  • Pig MG53 S255R forward primer aagatcctggcagagcggcccccacctgcccgcctgg (SEQ ID NO: 121)
  • Dog MG53 S255A, S255G, S255L, S255W, S255Q, S255Y, S255D, S255R mutants were prepared in the same manner as the human MG53 S255A mutant. The other steps were identical to those of the human MG53 S255A mutant except that the mutant primers were different.
  • the primers for each mutant are as follows:
  • dog MG53 S255A represents the MG53 mutant in which the 255th serine of wild type MG53 (ie, SEQ ID NO: 6) of the dog was mutated to alanine.
  • Dog MG53 S255A forward primer aagatcctggcagaagcaccaccgcctgcccgtttg (SEQ ID NO: 123)
  • dog MG53 S255G represents the MG53 mutant in which the 255th serine of wild type MG53 (ie, SEQ ID NO: 6) of the dog was mutated to glycine.
  • Dog MG53 S255G forward primer aagatcctggcagaaggaccaccgcctgcccgtttgg (SEQ ID NO: 125)
  • Dog MG53 S255G reverse primer ccaaacgggcaggcggtggtccttctgccaggatctt (SEQ ID NO: 126)
  • dog MG53 S255L represents a MG53 mutant in which the 255th serine of wild type MG53 (ie, SEQ ID NO: 6) of the dog was mutated to leucine.
  • Dog MG53 S255L forward primer agatcctggcagaattaccaccgcctgcccgtttgg (SEQ ID NO: 127)
  • Dog MG53 S255L reverse primer ccaaacgggcaggcggtggtaattctgccaggatct (SEQ ID NO: 128)
  • dog MG53 S255W represents the MG53 mutant of the 255th serine of the wild type MG53 (ie, SEQ ID NO: 6) of the dog that was mutated to tryptophan.
  • Dog MG53 S255W forward primer agatcctggcagaatggccaccgcctgcccgtttgga (SEQ ID NO: 129)
  • dog MG53 S255Q represents the MG53 mutant of the 255th serine of wild type MG53 (ie, SEQ ID NO: 6) of the dog that was mutated to glutamine.
  • Dog MG53 S255Q forward primer aagatcctggcagaacaaccaccgcctgcccgtttgg (SEQ ID NO: 131)
  • Dog MG53 S255Q reverse primer ccaaacgggcaggcggtggttgttctgccaggatctt (SEQ ID NO: 132)
  • dog MG53 S255Y represents the MG53 mutant in which the 255th serine of wild type MG53 (ie, SEQ ID NO: 6) of the dog was mutated to tyrosine.
  • Dog MG53 S255Y forward primer agatcctggcagaatatccaccgcctgcccgtttgga (SEQ ID NO: 133)
  • dog MG53 S255D represents the MG53 mutant of the 255th serine of wild type MG53 (ie, SEQ ID NO: 6) of the dog that is mutated to aspartic acid.
  • Dog MG53 S255D forward primer aagatcctggcagaagatccaccgcctgcccgtttgga (SEQ ID NO: 135)
  • Dog MG53 S255D reverse primer tccaaacgggcaggcggtggatcttctgccaggatctt (SEQ ID NO: 136)
  • Dog MG53 S255R forward primer aagatcctggcagaacgaccaccgcctgcccgtttgg (SEQ ID NO: 137)
  • Dog MG53 S255R reverse primer ccaaacgggcaggcggtggtcgttctgccaggatctt (SEQ ID NO: 138)
  • Mutants lacking serine deletions and/or mutations of other species MG53 can be prepared in the same manner as the human MG53 S255A mutant. The other steps were identical to those of the human MG53 S255A mutant except that the mutant primers were different. Those skilled in the art can design primers for each mutant using conventional techniques in the art.
  • Example 2 Effect of mouse MG53 S255A mutant on cell repair function and cardioprotective function of mouse wild-type MG53
  • mice MG53 S255A mutant (the amino acid sequence of which is shown in SEQ ID NO: 8) for mice
  • NRVM primary cultured neonatal rat cardiomyocytes
  • the survival of the cells was examined. The results are shown in Figure 14.
  • Hypoxia-reoxygenation of simulated ischemia-reperfusion injury caused massive release of LDVM from lactate dehydrogenase (LDH) and decreased intracellular ATP; overexpression of mouse wild-type MG53 and mouse MG53 S255A Mutants can inhibit the release of LDH and the decrease in ATP to the same extent.
  • LDH lactate dehydrogenase
  • mouse wild-type MG53 has a protective effect on apoptosis and necrosis caused by ischemia-reperfusion, while mouse MG53 S255A mutant does not affect the cell repair function and cardioprotective function of mouse wild-type MG53. That is, phosphorylation of mouse MG53 S255 site does not regulate the cell repair function and cardioprotective function of MG53.
  • Mouse MG53 S255A mutation does not affect MG53 activation of RISK signaling pathway
  • the mechanism of MG53's cardioprotective function is that it is an important molecule in the cardioprotective RISK signaling pathway, which mediates the interaction of caveolin-3 and p85-PI3K proteins, thereby activating RISK signaling pathways, such as downstream important signals.
  • Molecular AKT see Zhang Y. et al, Cardiovascular research 91, 108-115 (2011). Therefore, further, the inventors examined whether the mouse MG53 S255A mutation does not affect the mechanism of MG53 cardioprotection.
  • the mouse MG53 S255A mutant can activate the RISK signaling pathway to the same extent as mouse wild-type MG53. The results of the experiment are shown in Figure 15.
  • mice wild-type MG53 in NRVM can increase the phosphorylation of serine at position 473 of the downstream signal molecule AKT of RISK signaling pathway, ie, activate AKT; whereas mouse MG53S255A mutant can interact with mouse Wild-type MG53 increased the phosphorylation of serine at position 473 of AKT to the same extent, ie, activated AKT.
  • mouse MG53 S255A mutant does not affect the activation of the RISK signaling pathway by wild-type MG53 in mice, ie, the phosphorylation regulation of mouse MG53 S255 does not affect the RISK signaling pathway of cardioprotection.
  • Example 3 Effect of human or mouse MG53 S255A mutant on E3 ubiquitin ligase activity of wild-type MG53
  • the repression of the insulin signaling pathway caused by high expression of MG53 is one of the important mechanisms for the development of insulin resistance and metabolic syndrome, and is also one of the important functions of MG53.
  • the inventors constructed a serine mutant expression plasmid of mouse wild type MG53, mouse MG53 S255A (the amino acid sequence of which is shown in SEQ ID NO: 8). Further, the inventors also constructed an expression plasmid for IRS1, an expression plasmid for ubiquitin, and an expression plasmid for mouse wild-type MG53.
  • MG53 is the E3 ubiquitin ligase of the insulin substrate IRS1, which mediates the proteasomal pathway degradation of its proteins (Song, R.
  • the inventors have expressed the expression plasmid of IRS1, the expression plasmid of ubiquitin, and mouse wild type MG53 or mouse MG53.
  • the plasmid of S255A mutant was co-transfected into HEK293T cell line, and the effect of mouse MG53 S255A mutation on MG53 function was reflected by the change of IRS1 protein content.
  • the results of the experiment are shown in Figure 19. The results showed that wild-type MG53 in mice can greatly reduce the expression of IRS1; while the mouse MG53 S255A mutant can significantly inhibit the MG53-mediated decrease in the amount of IRS1 protein.
  • mouse wild-type MG53 can function as a normal E3 ubiquitin ligase, which mediates the ubiquitination of IRS1.
  • the detected mouse MG53 S255A mutant inhibits E3 of MG53.
  • the ubiquitin ligase function the phosphorylation status of the S255 site of MG53, regulates the E3 ubiquitin ligase function of MG53.
  • MG53 is a RING domain type E3 ubiquitin ligase, and the RING domain of MG53 binds to the E2 ubiquitin cross-linking enzyme to mediate ubiquitination of the substrate and further protease degradation. Therefore, the truncated MG53-D-RING excised by the RING domain lost the E3 ubiquitin ligase function of wild-type MG53.
  • a human MG53 S255A mutant (whose amino acid sequence is shown as SEQ ID NO: 7) or a mouse MG53 S255A mutant (the amino acid sequence thereof is shown in SEQ ID NO: 8) for human or mouse
  • the inventors used human or mouse MG53-D-RING as a positive control to co-express human or mouse wild-type MG53, MG53 S255A mutation in human embryonic kidney epithelial cell line HEK293T.
  • Mouse wild-type MG53 can mediate about 50% degradation of IR ⁇ ; mouse MG53-D-RING truncation and mouse MG53 S255A mutants are almost completely incapable of mediating degradation of IR ⁇ .
  • mouse wild-type MG53 can mediate the degradation of the precursor protein of IR ⁇ protein, while mouse MG53-D-RING truncation and mouse MG53 S255A mutant almost completely inhibit this MG53 mediated degradation.
  • Mouse MG53 S255A mutant inhibits binding of MG53 to substrate IRS1 protein
  • mouse MG53 S255A mutant (whose amino acid sequence is set forth in SEQ ID NO: 8) affects the recognition and binding of MG53 to the substrate IRS1 protein
  • the inventors were in vitro in an isolated HEK293T cell line.
  • the binding ability of the substrate IRS1 to the mouse wild-type MG53 or mouse MG53 S255A mutant was detected by co-immunoprecipitation; on the other hand, the purified protein IRS1 was detected by surface plasmon resonance (SPR) assay. Direct binding ability of mouse wild-type MG53 or mouse MG53 S255A mutant.
  • SPR surface plasmon resonance
  • Figure 18A shows that IRS1 can physically bind to mouse wild-type MG53; while the same amount of IRS1 can only bind a very small amount of mouse MG53 S255A mutant;
  • the mouse MG53 S255A mutant inhibits the recognition and binding of MG53 to the substrate IRS1 protein, thereby inhibiting the E3 ubiquitin ligase activity of MG53.
  • the inventors further analyzed the in vivo activity of the human MG53 S255A mutant by introducing the human MG53 S255A mutant and human wild type MG53 into the rat, respectively.
  • the inventors prepared a human MG53 S255A mutant according to Example 1.
  • 15 male Sprague-Dawley (SD) rats were selected and divided into three groups, experimental group, positive control group and negative control group.
  • Rats in each group were ligated with the left anterior descending coronary artery for 5 minutes after intravenous injection of the above protein for 5 minutes. Before the end of ligation, rats in each group were intravenously injected with the corresponding human MG53 S255A mutant protein, human wild-type MG53 protein, and BSA (6 mg/kg, iv), and then the coronary artery was released. After 24 hours of reperfusion, rats that did not die within 24 hours were defined as survival, and death was defined as death. The results were compared between the experimental group, the positive control group, and the negative control group. Rats in each group were stimulated with insulin, and then the rats were sacrificed.
  • the myocardial infarct size, serum LDH concentration and TUNEL staining of the heart pathological sections were compared between the experimental group, the positive control group and the negative control group. .
  • the area of myocardial infarction in the experimental group was significantly smaller than that in the negative control group, or the rats in the experimental group were able to significantly inhibit the release of LDH compared with the negative control group, or the heart of the experimental group.
  • the pathological section TUNEL staining results were significantly better than the heart pathological section of the negative control group.
  • the changes of p-AKT/t-AKT in the tissues of the experimental group, the positive control group and the negative control group were compared to evaluate the experimental group, the positive control group and the negative control group. Insulin reactivity in various tissues of rats.
  • the ratio of p-AKT/t-AKT in the tissues of the positive control group decreased, indicating that the human wild-type MG53 protein caused a decrease in insulin sensitivity; the ratio of p-AKT/t-AKT in the tissues of the negative control group was normal, indicating BSA There was no effect on insulin sensitivity; the p-AKT/t-AKT ratio in the tissues of the experimental group was higher than that in the positive control group, indicating that the human MG53 S255A mutant can abolish or attenuate the decrease in insulin sensitivity caused by human wild-type MG53 protein.
  • MG53 is produced when at least one serine (especially serine at position 255) in the coiled-coil-SPRY domain of MG53 is deleted or mutated to any other amino acid other than serine or threonine (eg, alanine).
  • the mutant has a cell repair function and/or a cardioprotective function while avoiding or reducing metabolic side effects such as insulin resistance.

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Abstract

一种MG53突变体，在野生型MG53的coiled-coil-SPRY区域内具有至少一个丝氨酸缺失和/或突变为任何其他非丝氨酸或苏氨酸的氨基酸。包括所述MG53突变体的药物组合物；编码所述MG53突变体的核酸；所述MG53突变体的制备方法；所述MG53突变体在制备用于治疗心脏疾病、糖尿病脑血管疾病、糖尿病眼部并发症、糖尿病神经病变、糖尿病足、肾脏疾病、与细胞和/或组织损伤相关疾病的药物中的用途，特别地，所述MG53突变体在治疗心脏疾病、糖尿病脑血管疾病、糖尿病眼部并发症、糖尿病神经病变、糖尿病足、肾脏疾病、与细胞和/或组织损伤相关疾病的同时，能够避免或减少野生型MG53所带来的代谢类副作用，例如，胰岛素抵抗、肥胖、糖尿病、高血压、血脂异常等。

Description

MG53突变体及其制备方法和用途 技术领域
本申请涉及生物医药领域，具体地，本申请涉及一种MG53突变体、包含所述MG53突变体的药物组合物、编码所述MG53突变体的核酸、所述MG53突变体的制备方法、以及MG53突变体在制备用于治疗心脏疾病、糖尿病脑血管疾病、糖尿病眼部并发症、糖尿病神经病变、糖尿病足、肾脏疾病、与细胞和/或组织损伤相关疾病的药物中的用途。
背景技术
Mitsugumin 53(MG53)又称为TRIM72，是三结构域蛋白(Tripartite motif-containing Proteins,TRIM)家族中的一员。MG53由N端的TRIM结构域和C端的SPRY结构域组成，TRIM结构域由依次相连的Ring、B-box、coiled-coil结构域组成(参见Chuanxi Cai et al.,the Journal of Biological Chemistry,Vol.284(5),3314-3322(2009))。MG53主要表达在横纹肌中，对于骨骼肌和心脏甚至整个机体的稳态维持发挥着重要作用。之前发现，MG53具有细胞修复功能和心脏保护功能(参见，例如，Chuanxi Cai et al.,Nature Cell Biology,Vol.11,56-64(2009)；CN101797375B)。另外，进一步的研究发现，在缺血预适应(ischemic preconditioning,IPC)和缺血后适应(ischemic postconditioning,PostC)中，MG53也是起保护作用的重要分子之一，MG53分子的两端可以分别与Caveolin-3以及p85-PI3K激酶相互结合形成复合物，并激活下游的再灌损伤保护激酶(reperfusion injury salvage kinase,RISK)途径，从而实现心肌的保护作用(参见，Chun-Mei Cao et al.,Circulation 121,2565-2574,(2010))。
虽然MG53具有细胞修复功能和心脏保护功能，但是之前的研究还发现，MG53具有E3泛素连接酶活性，参与调控胰岛素抵抗和代谢综合征的发生发展。MG53 N端的TRIM结构域中的Ring结构域具有E3泛素连接酶活性，可以与胰岛素受体(insulin receptor，IR)及胰岛素受体底物(insulin receptor substrate-1，IRS1)结合，并介导它们的泛素化修饰和蛋白酶体途径的降解，进而阻遏胰岛素信号通路，导致以胰岛素抵抗为发病核心环节的肥胖、糖尿病、高血压、血脂异常等代谢类疾病(参见，例如， R.Song et al.,Nature 494,375-379,(2013)；J.S.Yi et al.,Nature communications 4,2354(2013))。由此可见，野生型MG53在修复细胞、保护心脏的同时，会引起胰岛素抵抗、肥胖、糖尿病、高血压、血脂异常等代谢类疾病的副作用，这些副作用是人们所不希望的。
发明概述
本申请涉及一种MG53突变体、包含所述MG53突变体的药物组合物、编码所述MG53突变体的核酸、所述MG53突变体的制备方法以及MG53突变体在制备用于治疗心脏疾病、糖尿病脑血管疾病、糖尿病眼部并发症、糖尿病神经病变、糖尿病足、肾脏疾病、与细胞和/或组织损伤相关疾病的药物中的用途，特别地，所述MG53突变体在具有细胞修复功能和/或心脏保护功能的同时，能够避免或减少野生型MG53所带来的胰岛素抵抗、肥胖、糖尿病、高血压、血脂异常等代谢类副作用。
一方面，本申请涉及一种MG53突变体，其中所述MG53突变体在野生型MG53的氨基酸序列的基础上，在所述野生型MG53的coiled-coil-SPRY区域内具有至少一个丝氨酸缺失和/或突变为任何其他非丝氨酸或苏氨酸的氨基酸。在某些实施方式中，所述coiled-coil-SPRY区域位于野生型MG53的氨基酸序列的第122-477位氨基酸区域。在某些实施方式中，所述野生型MG53来源于动物，优选地，来源于哺乳动物，例如，人、小鼠、大鼠、猴子、猪、狗等。在某些实施方式中，所述野生型MG53的氨基酸序列为SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:139、SEQ ID NO:140、SEQ ID NO:141、SEQ ID NO:142所示的氨基酸序列。
在某些实施方式中，所述MG53突变体在野生型MG53的氨基酸序列的基础上，在所述野生型MG53的coiled-coil-SPRY区域内有至少一个丝氨酸突变为非极性氨基酸。在某些实施方式中，所述非极性氨基酸选自下组：甘氨酸、丙氨酸、亮氨酸、异亮氨酸、缬氨酸、脯氨酸、苯丙氨酸、甲硫氨酸和色氨酸。优选地，在某些实施方式中，所述非极性氨基酸为丙氨酸。在某些实施方式中，所述MG53突变体在野生型MG53的氨基酸序列的基础上，在所述野生型MG53的coiled-coil-SPRY区域内有至少一个丝氨酸突变为除丝氨酸和苏氨酸之外的极性氨基酸。在某些实施方式中，所述极 性氨基酸选自下组：谷氨酰胺、半胱氨酸、天冬酰胺、酪氨酸、天冬氨酸、谷氨酸、赖氨酸、精氨酸和组氨酸。优选地，在某些实施方式中，所述极性氨基酸为半胱氨酸。
在某些实施方式中，发生缺失或突变的丝氨酸位于野生型MG53的氨基酸序列SEQ ID NO:1的第150位、第189位、第211位、第214位、第246位、第255位、第269位、第296位、第297位、第301位、第305位、第306位、第307位、第314位、第341位、第377位、第405位、第418位、第425位或者第430位中的一个或多个位点。
在某些实施方式中，发生缺失或突变的丝氨酸位于野生型MG53的氨基酸序列SEQ ID NO:139的第150位、第189位、第211位、第214位、第246位、第255位、第269位、第296位、第297位、第301位、第305位、第306位、第307位、第314位、第341位、第377位、第405位、第418位、第425位或者第430位中的一个或多个位点。
在某些实施方式中，发生缺失或突变的丝氨酸位于野生型MG53的氨基酸序列SEQ ID NO:140的第150位、第189位、第211位、第214位、第246位、第255位、第269位、第296位、第297位、第301位、第305位、第306位、第307位、第314位、第341位、第377位、第405位、第418位、第425位或者第430位中的一个或多个位点。
在某些实施方式中，发生缺失或突变的丝氨酸位于野生型MG53的氨基酸序列SEQ ID NO:141的第150位、第189位、第211位、第214位、第246位、第255位、第269位、第296位、第297位、第301位、第305位、第306位、第307位、第314位、第341位、第377位、第405位、第418位、第425位或者第430位中的一个或多个位点。
在某些实施方式中，发生缺失或突变的丝氨酸位于野生型MG53的氨基酸序列SEQ ID NO:142的第150位、第189位、第211位、第214位、第246位、第255位、第269位中的一个或多个位点。
在某些实施方式中，发生缺失或突变的丝氨酸位于野生型MG53的氨基酸序列SEQ ID NO:2的第188位、第189位、第210位、第211位、第214位、第246位、 第253位、第255位、第269位、第296位、第297位、第301位、第305位、第306位、第307位、第314位、第341位、第367位、第377位、第418位、第430位或者第440位中的一个或多个位点。
在某些实施方式中，发生缺失或突变的丝氨酸位于野生型MG53的氨基酸序列SEQ ID NO:3的第150位、第188位、第189位、第210位、第211位、第214位、第246位、第253位、第255位、第269位、第296位、第297位、第301位、第305位、第307位、第314位、第341位、第367位、第377位、第418位、第430位、第440位、第464位或者第474位中的一个或多个位点。
在某些实施方式中，发生缺失或突变的丝氨酸位于野生型MG53的氨基酸序列SEQ ID NO:4的第150位、第189位、第211位、第214位、第246位、第255位、第269位、第296位、第297位、第301位、第305位、第306位、第307位、第341位、第377位、第405位、第418位、第425位、第430位或者第464位中的一个或多个位点。
在某些实施方式中，发生缺失或突变的丝氨酸位于野生型MG53的氨基酸序列SEQ ID NO:5的第150位、第189位、第211位、第214位、第246位、第255位、第269位、第296位、第301位、第305位、第307位、第314位、第341位、第377位、第411位、第418位、第425位、第430位或者第474位中的一个或多个位点。
在某些实施方式中，发生缺失或突变的丝氨酸位于野生型MG53的氨基酸序列SEQ ID NO:6的第150位、第189位、第211位、第214位、第246位、第255位、第269位、第296位、第297位、第301位、第305位、第307位、第314位、第341位、第367位、第377位、第418位、第425位或者第430位中的一个或多个位点。
在某些实施方式中，发生缺失或突变的丝氨酸位于野生型MG53的氨基酸序列(例如，SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:139、SEQ ID NO:140、SEQ ID NO:141、或者SEQ ID NO:142)的第150位、第188-189位、第210-211位、第214位、第246位、第253-255位、第269位、第296-297位、第301位、第305-307位、第314位、第341位、第367位、第377位、第405位、第411位、第418位、第425位、第430位、第440位、第464位或者第474位中的一个或多个位点。
在某些实施方式中，发生缺失或突变的丝氨酸位于野生型MG53的氨基酸序列(例如，SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:139、SEQ ID NO:140、SEQ ID NO:141、或者SEQ ID NO:142)的第189位、第211位、第214位、第246位、第253-255位、第269位、第296位、第301位、第305位、第307位、第341位、第377位、第418位或者第430位中的一个或多个位点。
在某些实施方式中，发生缺失或突变的丝氨酸位于野生型MG53的氨基酸序列(例如，SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:139、SEQ ID NO:140、SEQ ID NO:141、或者SEQ ID NO:142)的第211位、第214位、第246位、第253-255位、第269位、第296位或者第297位中的一个或多个位点。
在某些实施方式中，发生缺失或突变的丝氨酸位于野生型MG53的氨基酸序列(例如，SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:139、SEQ ID NO:140、SEQ ID NO:141、或者SEQ ID NO:142)的第211位、第214位、第246位、第255位、第269位、第296位或者第297位中的一个或多个位点。
在某些实施方式中，发生缺失或突变的丝氨酸位于野生型MG53的氨基酸序列(例如，SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:139、SEQ ID NO:140、SEQ ID NO:141、或者SEQ ID NO:142)的第253-255位中的一个或多个位点。在某些实施方式中，发生缺失或突变的丝氨酸位于野生型MG53的氨基酸序列的第253位。在某些实施方式中，发生缺失或突变的丝氨酸位于野生型MG53的氨基酸序列的第255位。在某些实施方式中，发生缺失或突变的丝氨酸位于野生型MG53的氨基酸序列SEQ ID NO:1的第255位。在某些实施方式中，发生缺失或突变的丝氨酸位于野生型MG53的氨基酸序列SEQ ID NO:139、SEQ ID NO:140、SEQ ID NO:141或SEQ ID NO:142的第255位。
在某些实施方式中，所述MG53突变体具有两个或两个以上的丝氨酸突变。在某些实施方式中，所述MG53突变体的两个或两个以上的丝氨酸突变包括在第253-255位中的一个或多个位点上的丝氨酸突变。在某些实施方式中，所述MG53突变体的两 个或两个以上的丝氨酸突变包括在第253位的丝氨酸突变。在某些实施方式中，所述MG53突变体的两个或两个以上的丝氨酸突变包括在第255位的丝氨酸突变。在某些实施方式中，所述MG53突变体的两个或两个以上的丝氨酸突变包括在第253位和第255位的丝氨酸突变。
本领域技术人员可以理解，不同物种的野生型MG53蛋白中丝氨酸所处位点可能有所不同，因此发生缺失或突变的丝氨酸位点也可能有所不同。在某些实施方式中，发生缺失或突变的丝氨酸位于SEQ ID NO:1所示野生型MG53的氨基酸序列中相应丝氨酸位点的上游或下游的1至10个氨基酸之内、1至5个氨基酸之内、或1至3个氨基酸之内。
在某些实施方式中，所述MG53突变体的氨基酸序列为SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:147、SEQ ID NO:148、SEQ ID NO:149或者SEQ ID NO:150所示的氨基酸序列。优选地，在某些实施方式中，所述MG53突变体的氨基酸序列为SEQ ID NO:7所示的氨基酸序列。在某些实施方式中，所述MG53突变体的氨基酸序列与SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:147、SEQ ID NO:148、SEQ ID NO:149、SEQ ID NO:150所示的氨基酸序列之一具有至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少98％、或者至少99％的氨基酸序列同源性，并且所述MG53突变体在具有细胞修复功能和/或心脏保护功能的同时，避免了野生型MG53所带来的代谢类副作用。
另一方面，本申请涉及一种药物组合物，其包括所述MG53突变体和药学上可接受的载体。
另一方面，本申请涉及一种分离的核酸，其包含编码所述MG53突变体的氨基酸序列的核酸序列。在某些实施方式中，所述核酸包含SEQ ID NO:13-18所示的任一核酸序列。在某些实施方式中，所述核酸包含SEQ ID NO:151-154所示的任一核酸序列。
另一方面，本申请涉及一种表达载体，其包含编码所述MG53突变体的氨基酸序列的核酸序列。在某些实施方式中，所述核酸包含SEQ ID NO:13-18所示的任一核酸序列。在某些实施方式中，所述核酸包含SEQ ID NO:151-154所示的任一核酸序列。
另一方面，本申请涉及一种宿主细胞，其包含本申请所述的表达载体。
另一方面，本申请涉及一种所述MG53突变体的制备方法，其包括：确定需要进行突变的丝氨酸的一个或多个位置，对包含编码野生型MG53氨基酸序列的核酸序列的质粒的全长序列进行在所述位置的定点突变，将所述定点突变的质粒转染到宿主细胞，表达所述宿主细胞以产生所述MG53突变体。在某些实施方式中，所述定点突变包括如下步骤：(1)确定需要定点突变的氨基酸在cDNA序列中相应的核苷酸位置，根据突变后的氨基酸修改突变位置的核苷酸序列，并截取包含突变位置的长度为20-40bp的序列设计引物；(2)以野生型MG53质粒为模板，使用步骤(1)中设计的引物进行PCR反应，PCR产物进行琼脂糖凝胶电泳，并对PCR产物进行纯化；(3)步骤(2)中纯化后的PCR产物使用核酸限制性内切酶进行酶切反应，并将酶切产物与合适的质粒表达载体进行连接，将连接产物进行细菌感受态细胞的转化，培养。在某些实施方式中，所述定点突变进一步包括如下步骤：(4)挑取步骤(3)所得克隆使用步骤(1)所设计的引物进行菌落PCR鉴定，对PCR产物进行琼脂糖凝胶电泳检测，然后进行DNA测序鉴定，鉴定出带有所述定点突变的阳性克隆。
另一方面，本申请涉及所述MG53突变体在制备用于治疗心脏疾病、糖尿病脑血管疾病、糖尿病眼部并发症、糖尿病神经病变、糖尿病足、肾脏疾病、与细胞和/或组织损伤相关疾病的药物中的用途。在某些实施方式中，所述药物在治疗心脏疾病、糖尿病脑血管疾病、糖尿病眼部并发症、糖尿病神经病变、糖尿病足、肾脏疾病、与细胞和/或组织损伤相关疾病的同时，避免或减少了代谢类副作用，例如胰岛素抵抗、肥胖、糖尿病、高血压、血脂异常等副作用。在某些实施方式中，所述心脏疾病是与心肌损伤相关的疾病，包括但不限于，糖尿病性心脏病、心肌缺血、心脏缺血/再灌注损伤、心肌梗塞、心力衰竭、心律失常、心脏破裂、心绞痛、心肌炎、冠心病、心包炎。在某些实施方式中，所述糖尿病脑血管疾病包括但不限于，脑动脉硬化、缺血性脑血管病、脑出血、脑萎缩、脑梗塞。在某些实施方式中，所述糖尿病眼部并发症包括但不限于，糖尿病性视网膜病变、糖尿病性白内障、与糖尿病相关的葡萄膜炎、失明。在某些实施方式中，所述糖尿病神经病变包括但不限于，糖尿病周围神经病变。在某些实施方式中，所述肾脏疾病包括但不限于，急性肾小球肾炎、慢性肾小球肾炎、肾病综合征、急性肾损伤、糖尿病肾病等。在某些实施方式中，所述与细胞和/或组织损伤相关疾病包括但不限于与肾脏、脑、肺部、肝脏、心脏、脾脏、消化道的细胞和/ 或组织损伤相关的疾病、皮肤损伤相关的疾病，例如，脑损伤、肺损伤、脾脏损伤、脾脏破裂、胃溃疡、胃炎、胃穿孔、消化道黏膜损伤、外伤、烧伤、溃疡、黏膜炎、哮喘、慢性阻塞性肺疾病(COPD)、中风、皮肤老化等。在某些实施方式中，本申请涉及SEQ ID NO:7所示的多肽在制备用于治疗心脏疾病、糖尿病脑血管疾病、糖尿病眼部并发症、糖尿病神经病变、糖尿病足、肾脏疾病、与细胞和/或组织损伤相关疾病的药物中的用途。在某些实施方式中，本申请涉及SEQ ID NO:147、SEQ ID NO:148、SEQ ID NO:149或者SEQ ID NO:150所示的多肽在制备用于治疗心脏疾病、糖尿病脑血管疾病、糖尿病眼部并发症、糖尿病神经病变、糖尿病足、肾脏疾病、与细胞和/或组织损伤相关疾病的药物中的用途。
另一方面，本申请涉及一种治疗心脏疾病、糖尿病脑血管疾病、糖尿病眼部并发症、糖尿病神经病变、糖尿病足、肾脏疾病、与细胞和/或组织损伤相关疾病的方法，包括给予需要的对象治疗有效量的所述MG53突变体。在某些实施方式中，所述MG53突变体在治疗心脏疾病、糖尿病脑血管疾病、糖尿病眼部并发症、糖尿病神经病变、糖尿病足、肾脏疾病、与细胞和/或组织损伤相关疾病的同时，避免或减少了代谢类副作用，例如胰岛素抵抗、肥胖、糖尿病、高血压、血脂异常等副作用。在某些实施方式中，所述心脏疾病是与心肌损伤相关的疾病，包括但不限于，糖尿病性心脏病、心肌缺血、心脏缺血/再灌注损伤、心肌梗塞、心力衰竭、心律失常、心脏破裂、心绞痛、心肌炎、冠心病、心包炎。在某些实施方式中，所述糖尿病脑血管疾病包括但不限于，脑动脉硬化、缺血性脑血管病、脑出血、脑萎缩、脑梗塞。在某些实施方式中，所述糖尿病眼部并发症包括但不限于，糖尿病性视网膜病变、糖尿病性白内障、与糖尿病相关的葡萄膜炎、失明。在某些实施方式中，所述糖尿病神经病变包括但不限于，糖尿病周围神经病变。在某些实施方式中，所述肾脏疾病包括但不限于，急性肾小球肾炎、慢性肾小球肾炎、肾病综合征、急性肾损伤、糖尿病肾病等。在某些实施方式中，所述与细胞和/或组织损伤相关疾病包括但不限于与肾脏、脑、肺部、肝脏、心脏、脾脏、消化道的细胞和/或组织损伤相关的疾病、皮肤损伤相关的疾病，例如，脑损伤、肺损伤、脾脏损伤、脾脏破裂、胃溃疡、胃炎、胃穿孔、消化道黏膜损伤、外伤、烧伤、溃疡、黏膜炎、哮喘、慢性阻塞性肺疾病(COPD)、中风、皮肤老化等。在某些实施方式中，本申请涉及一种治疗心脏疾病、糖尿病脑血管疾病、糖尿病眼部并发症、糖尿病神经病变、糖尿病足、肾脏疾病、与细胞和/或组织损伤相关疾病的方法， 包括给予需要的对象治疗有效量的SEQ ID NO:7所示的多肽。在某些实施方式中，本申请涉及一种治疗心脏疾病、糖尿病脑血管疾病、糖尿病眼部并发症、糖尿病神经病变、糖尿病足、肾脏疾病、与细胞和/或组织损伤相关疾病的方法，包括给予需要的对象治疗有效量的SEQ ID NO:147、SEQ ID NO:148、SEQ ID NO:149或者SEQ ID NO:150所示的多肽。
另一方面，本申请涉及用于治疗心脏疾病、糖尿病脑血管疾病、糖尿病眼部并发症、糖尿病神经病变、糖尿病足、肾脏疾病、与细胞和/或组织损伤相关疾病的MG53突变体。在某些实施方式中，所述MG53突变体在治疗心脏疾病、糖尿病脑血管疾病、糖尿病眼部并发症、糖尿病神经病变、糖尿病足、肾脏疾病、与细胞和/或组织损伤相关疾病的同时，避免了代谢类副作用，例如胰岛素抵抗、肥胖、糖尿病、高血压、血脂异常等副作用。在某些实施方式中，所述心脏疾病是与心肌损伤相关的疾病，包括但不限于，糖尿病性心脏病、心肌缺血、心脏缺血/再灌注损伤、心肌梗塞、心力衰竭、心律失常、心脏破裂、心绞痛、心肌炎、冠心病、心包炎。在某些实施方式中，所述糖尿病脑血管疾病包括但不限于，脑动脉硬化、缺血性脑血管病、脑出血、脑萎缩、脑梗塞。在某些实施方式中，所述糖尿病眼部并发症包括但不限于，糖尿病性视网膜病变、糖尿病性白内障、与糖尿病相关的葡萄膜炎、失明。在某些实施方式中，所述糖尿病神经病变包括但不限于，糖尿病周围神经病变。在某些实施方式中，所述肾脏疾病包括但不限于，急性肾小球肾炎、慢性肾小球肾炎、肾病综合征、急性肾损伤、糖尿病肾病等。在某些实施方式中，所述与细胞和/或组织损伤相关疾病包括但不限于与肾脏、脑、肺部、肝脏、心脏、脾脏、消化道的细胞和/或组织损伤相关的疾病、皮肤损伤相关的疾病，例如，脑损伤、肺损伤、脾脏损伤、脾脏破裂、胃溃疡、胃炎、胃穿孔、消化道黏膜损伤、外伤、烧伤、溃疡、黏膜炎、哮喘、慢性阻塞性肺疾病(COPD)、中风、皮肤老化等。在某些实施方式中，本申请涉及用于治疗心脏疾病、糖尿病脑血管疾病、糖尿病眼部并发症、糖尿病神经病变、糖尿病足、肾脏疾病、与细胞和/或组织损伤相关疾病的SEQ ID NO:7所示的多肽。在某些实施方式中，本申请涉及用于治疗心脏疾病、糖尿病脑血管疾病、糖尿病眼部并发症、糖尿病神经病变、糖尿病足、肾脏疾病、与细胞和/或组织损伤相关疾病的SEQ ID NO:147、SEQ ID NO:148、SEQ ID NO:149或者SEQ ID NO:150所示的多肽。
附图说明
图1显示野生型MG53的分子结构模式图。
图2显示人野生型MG53的氨基酸序列SEQ ID NO:1及其编码核酸序列SEQ ID NO:19。
图3显示小鼠野生型MG53的氨基酸序列SEQ ID NO:2及其编码核酸序列SEQ ID NO:20。
图4显示大鼠野生型MG53的氨基酸序列SEQ ID NO:3及其编码核酸序列SEQ ID NO:21。
图5显示猴子野生型MG53的氨基酸序列SEQ ID NO:4及其编码核酸序列SEQ ID NO:22。
图6显示猪野生型MG53的氨基酸序列SEQ ID NO:5及其编码核酸序列SEQ ID NO:23。
图7显示狗野生型MG53的氨基酸序列SEQ ID NO:6及其编码核酸序列SEQ ID NO:24。
图8显示人MG53 S255A突变体的氨基酸序列SEQ ID NO:7及其编码核酸序列SEQ ID NO:13。
图9显示小鼠MG53 S255A突变体的氨基酸序列SEQ ID NO:8及其编码核酸序列SEQ ID NO:14。
图10显示大鼠MG53 S255A突变体的氨基酸序列SEQ ID NO:9及其编码核酸序列SEQ ID NO:15。
图11显示猴子MG53 S255A突变体的氨基酸序列SEQ ID NO:10及其编码核酸序列SEQ ID NO:16。
图12显示猪MG53 S255A突变体的氨基酸序列SEQ ID NO:11及其编码核酸序列SEQ ID NO:17。
图13显示狗MG53 S255A突变体的氨基酸序列SEQ ID NO:12及其编码核酸序列SEQ ID NO:18。
图14显示在新生大鼠的原代培养的心肌细胞中使用腺病毒过表达小鼠野生型MG53和小鼠MG53 S255A突变体后，细胞内ATP量和LDH释放量。上图为在新生大鼠的原代培养的心肌细胞中使用腺病毒过表达小鼠野生型MG53和小鼠MG53S255A突变体，经缺氧复氧处理后检测细胞内的ATP的含量，Adv-β-gal表示表达半乳糖苷酶而不表达小鼠野生型MG53及小鼠MG53 S255A突变体的对照病毒， Adv-MG53-myc表示表达小鼠野生型MG53的腺病毒，Adv-MG53-S255A-myc表示表达小鼠MG53 S255A突变体的腺病毒(n＝7,*p＜0.05,**p＜0.01)；下图为在新生大鼠的原代培养的心肌细胞中使用腺病毒过表达小鼠野生型MG53和MG53 S255A突变体，经缺氧复氧处理后检测细胞的LDH的释放量，Adv-β-gal表示表达半乳糖苷酶而不表达小鼠野生型MG53及小鼠MG53 S255A突变体的对照病毒，Adv-MG53-myc表示表达小鼠野生型MG53的腺病毒，Adv-MG53-S255A-myc表示表达小鼠MG53 S255A突变体的腺病毒(n＝7,**p＜0.01,***p＜0.005)。
图15显示小鼠MG53 S255A突变不影响小鼠野生型MG53对AKT的激活。上图为在新生大鼠的原代培养的心肌细胞中使用腺病毒过表达小鼠野生型MG53和小鼠MG53 S255A突变体，蛋白质印迹法(Western Blot)检测细胞内的p-AKT473的量，β-gal表示表达半乳糖苷酶而不表达小鼠野生型MG53及小鼠MG53 S255A突变体的对照腺病毒，MG53表示表达小鼠野生型MG53的腺病毒，S255A表示表达小鼠MG53 S255A突变体的腺病毒；下图为上图的统计图，Advβ-gal表示表达半乳糖苷酶而不表达小鼠野生型MG53及MG53 S255A突变体的对照腺病毒，Adv MG53表示表达小鼠野生型MG53的腺病毒，Adv MG53 S255A表示表达小鼠MG53 S255A突变体的腺病毒(n＝5,*p＜0.05)。
图16显示小鼠MG53 S255A突变抑制了MG53介导的底物降解。图16A的上图是在HEK293T细胞系中过表达IRS1和小鼠野生型MG53、小鼠MG53 S255A突变体、或小鼠MG53-D-RING截短体的质粒，Western Blot检测IRS1的蛋白含量，CON表示不表达小鼠野生型MG53、小鼠MG53 S255A突变体、小鼠MG53-D-RING截短体的空载体质粒对照，MG53表示表达小鼠野生型MG53的载体质粒，S255A表示表达小鼠MG53 S255A突变体的载体质粒，D-RING表示表达小鼠RING结构域切除的MG53截短体MG53-D-RING的载体质粒；图16A的下图是上图的统计图(n＝5，*p＜0.05；**p＜0.01)；图16B的上图是在HEK293T细胞系中过表达IR和小鼠野生型MG53、小鼠MG53 S255A突变体、或小鼠MG53-D-RING截短体的质粒，Western Blot检测IR的蛋白含量，CON表示不表达小鼠野生型MG53、小鼠MG53 S255A突变体、小鼠MG53-D-RING截短体的空载体质粒对照，MG53表示表达小鼠野生型MG53的载体质粒，S255A表示表达小鼠MG53 S255A突变体的载体质粒，D-RING表示表达 小鼠MG53-D-RING截短体的载体质粒；图16B的下图是上图的统计图(n＝5，**p＜0.01)。
图17显示人MG53 S255A突变体抑制了MG53介导的底物降解。上图是在HEK293T细胞系中过表达IRS1和人野生型MG53、人MG53 S255A突变体、或人MG53-D-RING截短体的质粒，Western Blot检测IRS1的蛋白含量，CON表示不表达人野生型MG53、人MG53 S255A突变体、人MG53-D-RING截短体的空载体质粒对照，MG53表示表达人野生型MG53的载体质粒，MG53-dR表示表达人MG53-D-RING截短体的载体质粒；下图是上图的统计图(n＝5，**p<0.01)。
图18显示小鼠MG53 S255A突变体抑制了MG53与底物IRS1的结合。图18A显示HEK293T细胞系中共表达小鼠野生型MG53和MG53的底物IRS1，或者共表达小鼠MG53 S255A突变体和MG53的底物IRS1，然后免疫沉淀IRS1蛋白并进行Western Blot实验检测MG53-IRS1复合物的含量。图18A中的MG53表示小鼠野生型MG53，MG53 S255A表示小鼠MG53 S255A突变体，IRS1表示小鼠胰岛素受体底物；图18B在表面等立体共振(SPR)实验中，固定纯化的蛋白IRS1，检测它对流动相中的纯化蛋白小鼠野生型MG53或小鼠MG53 S255A突变体的结合强度。图18B中的MG53表示小鼠野生型MG53，MG53-S255A表示小鼠MG53 S255A突变体，Rat IRS1表示大鼠胰岛素受体底物。
图19显示小鼠MG53 S255A突变体对IRS1蛋白量的影响。上图是在HEK293T细胞系中过表达IRS1、Ub、MG53、MG53 S255A突变体的质粒，Western Blot检测IRS1的蛋白含量。Ub表示泛素，MG53表示小鼠野生型MG53，MG53-S255A表示小鼠MG53 S255A突变体；下图为上图实验的统计图，CON表示不表达小鼠野生型MG53和小鼠MG53 S255A突变体的空载体质粒对照，MG53表示表达小鼠野生型MG53的载体质粒，MG53-S255A表示表达小鼠MG53 S255A突变体的载体质粒(n＝3,*p<0.05；***p<0.005)。
图20显示人MG53亚型的氨基酸序列SEQ ID NO:139(其对应的NCBI编号为BAD18630.1)及其编码核酸序列SEQ ID NO:143。
图21显示人MG53亚型的氨基酸序列SEQ ID NO:140(其对应的NCBI编号为XP_016878743.1)及其编码核酸序列SEQ ID NO:144。
图22显示人MG53亚型的氨基酸序列SEQ ID NO:141(其对应的NCBI编号为BAC03506.1)及其编码核酸序列SEQ ID NO:145。
图23显示人MG53亚型的氨基酸序列SEQ ID NO:142(其对应的NCBI编号为AAH33211.1)及其编码核酸序列SEQ ID NO:146。
图24显示人MG53亚型S255A突变体的氨基酸序列SEQ ID NO:147及其编码核酸序列SEQ ID NO:151。
图25显示人MG53亚型S255A突变体的氨基酸序列SEQ ID NO:148及其编码核酸序列SEQ ID NO:152。
图26显示人MG53亚型S255A突变体的氨基酸序列SEQ ID NO:149及其编码核酸序列SEQ ID NO:153。
图27显示人MG53亚型S255A突变体的氨基酸序列SEQ ID NO:150及其编码核酸序列SEQ ID NO:154。
图28显示本申请中各物种的全长MG53蛋白及其片段的氨基酸位点的命名示意图。
发明详述
尽管本申请将在以下公开多个方面和实施方式，但是在不违背本申请主题精神和范围的前提下，本领域技术人员显然可以对其进行各种等同改变和修改。本申请公开的多个方面和实施方式仅用于举例说明，其并非旨在限制本申请，本申请的实际保护范围以权利要求为准。除非另外指出，本文中使用的所有技术和科学术语均具有与本发明所属领域中的普通技术人员通常所理解的相同的含义。本申请中引用的所有参考文献、专利、专利申请均通过整体引用并入本文。
一方面，本申请涉及一种MG53突变体，其中所述MG53突变体在野生型MG53的氨基酸序列的基础上，在所述野生型MG53的coiled-coil-SPRY区域内具有至少一个丝氨酸缺失和/或突变为任何其他非丝氨酸或苏氨酸的氨基酸。
本申请中使用的术语“野生型MG53”或“野生型MG53蛋白”是指在对象体内表达的全长MG53蛋白的天然序列或其片段。MG53蛋白是一种多功能蛋白，其结构如图1所示。各物种的全长MG53蛋白长度略有不同，但是通常具有约477个氨基酸，由N端的TRIM结构域和C端的SPRY结构域组成，TRIM结构域由依次相连的Ring、 B-box、coiled-coil结构域(RBCC)组成。MG53蛋白是膜修复的重要组分之一，在缺血再灌注损伤的预适应保护和后适应保护中发挥重要作用，同时MG53蛋白的高表达也会引起胰岛素抵抗和代谢综合征的发生。关于MG53的结构、功能以及与其他蛋白的相互作用，本领域中已有详细报道(参见，例如，Chuanxi Cai et al.,Journal of Biological Chemistry,284(5),3314-3322,(2009)；Xianhua Wang et al.,Circulation Research 107,76-83,(2010)；Eun Young Park et al.,Proteins,790-795(2009))。
本申请中使用的术语“对象”包括人类和非人类的动物。非人类的动物包括所有的脊椎动物，例如哺乳动物和非哺乳动物。“对象”也可以是家畜动物，例如牛、猪、羊、家禽和马；或啮齿类动物，例如大鼠、小鼠；或灵长类动物，例如猿、猴子；或家养动物，例如狗和猫。“对象”可以是雄性或者雌性，可以是老年、成年、青少年、儿童或者婴儿。人类“对象”可以是高加索人、非洲人、亚洲人、闪族人，或其他种族或所述种族背景的混合。
在某些实施方式中，所述野生型MG53优选地来源于哺乳动物，例如，人、猿、猴子、小鼠、大鼠、猪、狗等。本领域技术人员可以通过公开渠道(例如，美国国家生物技术信息中心网站(NCBI))获得各物种的野生型MG53的氨基酸序列，各物种的野生型MG53的氨基酸序列通过引用并入本申请。在某些实施方式中，所述野生型MG53的氨基酸序列如SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5或者SEQ ID NO:6所示，分别对应于人、小鼠、大鼠、猴子、猪、狗等的野生型MG53全长蛋白。
本申请中使用的术语“蛋白”、“多肽”以及“肽”可以互换使用，是指氨基酸的聚合物。本申请所述的蛋白、多肽或肽可以含有天然的氨基酸，也可以含有非天然的氨基酸，或氨基酸的类似物、模拟物。本申请所述的蛋白、多肽或肽可以通过本领域公知的任何方法获得，例如但不限于，通过天然分离、重组表达、化学合成等。
本申请中使用的术语“coiled-coil-SPRY结构域”是野生型MG53的氨基酸序列中对应的一个结构域。coiled-coil结构域(即，卷曲螺旋结构域)存在于大多数TRIM家族蛋白中，该结构域介导TRIM家族成员之间或者TRIM成员与其他蛋白之间的同源性或者异源性结合形成二聚体、多聚体等复合物，从而发挥细胞修复功能(参见，例如，Ozato et al.,Nature Review Immunology,8:849-860(2008)；Sanchez S.et al.,PNAS,111: 2494-2499(2014))。SPRY结构域位于野生型MG53的氨基酸序列的C端，通常位于野生型MG53第288-477位氨基酸区域。在某些实施方式中，本申请中所述的SPRY结构域包括PRY基序和SPRY基序。SPRY结构域在进化上非常保守，从真菌到高等动物中均有表达(参见，例如，Ozato et al.,Nature Review Immunology,8:849-860(2008))。迄今为止，在不同哺乳动物基因组内鉴定到的大约60个TRIM家族成员中，15个成员在TRIM结构域(即Ring-B–box-Coiled-Coil结构域)之后携带一个类似的SPRY结构域，且MG53表现出与这些TRIM亚家族蛋白的保守性一级结构(参见，例如WO2009/073808)。Coiled-coil-SPRY结构域对应的具体氨基酸区域位点在不同物种中可能略有不同，但是本领域技术人员可以通过本领域的现有技术(例如，NCBI中公开的内容)和/或常规实验方法得到不同物种的野生型MG53的coiled-coil-SPRY结构域所对应的具体氨基酸区域位点。在某些实施方式中，本申请中的coiled-coil-SPRY结构域是指野生型MG53的第122-477位氨基酸或者与其结构类似的区域。在某些实施方式中，所述结构类似区域可以是包括第122-477位氨基酸中的70％、80％或者90％的连续的氨基酸序列的区域。在某些实施方式中，所述结构类似区域的N端起始位点可以比第122-477位氨基酸区域的N端增长或者缩短约1个、2个、3个、4个、5个、6个、7个、8个、9个、10个、15个或者20个氨基酸，和/或者其C端终止位点可以比第122-477位氨基酸区域的C端增长或者缩短约1个、2个、3个、4个、5个、6个、7个、8个、9个、10个、15个或者20个氨基酸。例如，在某些实施方式中，本申请中的coiled-coil-SPRY结构域是指人的野生型MG53(即，SEQ ID NO:1)中的coiled-coil-SPRY结构域，其对应于SEQ ID NO:1的第122-477位氨基酸。
本申请中使用的术语“MG53突变体”或者“MG53蛋白突变体”是指野生型MG53蛋白的天然氨基酸序列被修饰的MG53蛋白变体或片段。这类修饰包括但不限于一个或多个氨基酸缺失和/或被取代。在某些实施方式中，本申请中的MG53突变体在野生型MG53的氨基酸序列的基础上，在所述野生型MG53的coiled-coil-SPRY结构域内具有至少一个丝氨酸缺失和/或突变为任何其他非丝氨酸或苏氨酸的氨基酸。
本申请中使用的“至少一个丝氨酸”是指一个或多个丝氨酸。例如，是指野生型MG53的coiled-coil-SPRY结构域内的1个、2个、3个、4个、5个、6个、7个、8个、9个、10个、11个、12个、13个、14个、15个、16个、17个、18个、19个、 20个、21个、22个、23个、24个、25个、26个、27个、28个、29个、30个、31个、32个、33个、34个、35个或更多个丝氨酸。
本申请中使用的“其他非丝氨酸或苏氨酸的氨基酸”是指除了丝氨酸、苏氨酸之外的、任何其他的天然氨基酸、取代的天然氨基酸、非天然氨基酸、取代的非天然氨基酸或其任何组合。天然氨基酸的名称在本申请中表示为标准的单字母或三字母代码。天然氨基酸包括非极性氨基酸和极性氨基酸。除非另有说明，本文所述的任何氨基酸可以是D-或L-构型。
在某些实施方式中，所述野生型MG53的coiled-coil-SPRY结构域内的至少一个丝氨酸(Ser或S)缺失或者突变为非极性氨基酸。所述非极性氨基酸包括甘氨酸(Gly或G)、丙氨酸(Ala或A)、亮氨酸(Leu或L)、异亮氨酸(Ile或I)、缬氨酸(Val或V)、脯氨酸(Pro或P)、苯丙氨酸(Phe或F)、甲硫氨酸(Met或M)、色氨酸(Trp或W)。在某些实施方式中，所述野生型MG53的coiled-coil-SPRY结构域内的至少一个丝氨酸缺失或者突变为甘氨酸或丙氨酸。优选地，在某些实施方式中，所述野生型MG53的coiled-coil-SPRY结构域内的至少一个丝氨酸缺失或者突变为丙氨酸。更优选地，在某些实施方式中，所述野生型MG53的coiled-coil-SPRY结构域内的至少一个丝氨酸突变为丙氨酸。
在某些实施方式中，所述野生型MG53的coiled-coil-SPRY结构域内的至少一个丝氨酸缺失或者突变为极性氨基酸。所述极性氨基酸包括谷氨酰胺(Gln或Q)、半胱氨酸(Cys或C)、天冬酰胺(Asn或N)、酪氨酸(Tyr或Y)、天冬氨酸(Asp或D)、谷氨酸(Glu或E)、赖氨酸(Lys或K)、精氨酸(Arg或R)、组氨酸(His或H)。在某些实施方式中，所述野生型MG53的coiled-coil-SPRY结构域内的至少一个丝氨酸缺失或者突变为半胱氨酸或组氨酸。优选地，在某些实施方式中，所述野生型MG53的coiled-coil-SPRY结构域内的至少一个丝氨酸缺失或者突变为半胱氨酸。更优选地，在某些实施方式中，所述野生型MG53的coiled-coil-SPRY结构域内的至少一个丝氨酸突变为半胱氨酸。
在某些实施方式中，本申请所述的丝氨酸突变包括一个丝氨酸被一个或多个非丝氨酸或苏氨酸的其他氨基酸取代，例如，一个丝氨酸可以被1个、2个、3个、4个、5个、6个、7个、8个、9个或10个非丝氨酸或苏氨酸的其他氨基酸取代。如果有两 个或两个以上丝氨酸被取代，则每个丝氨酸可以分别独立地被一个或多个非丝氨酸或苏氨酸的其他氨基酸取代。
虽然各物种野生型MG53的具体丝氨酸位点随着物种的不同而可能有所不同，但是一旦已知某物种野生型MG53的氨基酸序列及其coiled-coil-SPRY结构域对应的氨基酸区域，本领域技术人员就可以确定coiled-coil-SPRY结构域内的丝氨酸所对应的具体氨基酸位点。例如，本领域技术人员已知人野生型MG53的氨基酸序列如SEQ ID NO:1所示，其coiled-coil-SPRY结构域对应于SEQ ID NO:1的第122-477位氨基酸。在这种情况下，本领域技术人员就可以确定人野生型MG53的coiled-coil-SPRY结构域内的丝氨酸所对应的具体氨基酸位点分别位于SEQ ID NO:1的第150位、第189位、第211位、第214位、第246位、第255位、第269位、第296位、第297位、第301位、第305位、第306位、第307位、第314位、第341位、第377位、第405位、第418位、第425位、第430位。
在某些实施方式中，发生缺失或突变的丝氨酸位于人野生型MG53的氨基酸序列SEQ ID NO:1的第150位、第189位、第211位、第214位、第246位、第255位、第269位、第296位、第297位、第301位、第305位、第306位、第307位、第314位、第341位、第377位、第405位、第418位、第425位、第430位中的一个或多个位点。
在某些实施方式中，本申请所述的MG53蛋白包括全长MG53蛋白，或者全长MG53蛋白的截短体(即全长MG53蛋白的截短的片段)，或者是与全长MG53蛋白或其截短体相比具有一个或多个氨基酸突变或者增加或者删除的突变体。在这种情况下，为了统一不同的MG53蛋白亚型的氨基酸序列中的氨基酸位点数的命名，当本申请中提到某一MG53蛋白亚型的氨基酸位点时，将该MG53蛋白亚型的氨基酸序列与全长MG53蛋白的氨基酸序列(例如，SEQ ID NO:1)进行序列对比，并在必要时在相关氨基酸序列中引入间隔，使相同的氨基酸数目达到最多，将该MG53亚型的氨基酸序列的第1个氨基酸的位点数指定为该氨基酸对应的全长MG53蛋白的氨基酸位点数，从而使得当提及某物种野生型MG53的氨基酸位点数时，不管是全长MG53蛋白还是其截短体，均指的是全长MG53蛋白的氨基酸序列中的位点数。
例如，如图28所示，某物种的全长MG53蛋白具有n个氨基酸，对应的氨基酸位点为第1-n位。MG53亚型1的氨基酸序列与全长MG53蛋白的氨基酸序列相比，含有对应于全长序列的第m-n位氨基酸的截短体，那么就将MG53亚型1的氨基酸序列的第1个氨基酸的位点数指定为第m位，将MG53亚型1的氨基酸序列的第2个氨基酸的位点数指定为第m+1位，以此类推，将MG53亚型1的氨基酸序列的最后一个氨基酸位点指定为第n位。再例如，MG53亚型2的氨基酸序列与全长MG53蛋白的氨基酸序列相比，含有对应于全长序列的第1-s位氨基酸的截短体，那么就将MG53亚型2的氨基酸序列的第1个氨基酸的位点数指定为第1位，将MG53亚型2的氨基酸序列的第2个氨基酸的位点数指定为第2位，以此类推，将MG53亚型2的氨基酸序列的最后一个氨基酸位点指定为第s位。再例如，MG53亚型3的氨基酸序列与全长MG53蛋白的氨基酸序列相比，含有对应于全长序列的第p-q位氨基酸的截短体，那么就将MG53亚型3的氨基酸序列的第1个氨基酸的位点数指定为第p位，将MG53亚型3的氨基酸序列的第2个氨基酸的位点数指定为第p+1位，以此类推，将MG53亚型3的氨基酸序列的最后一个氨基酸位点指定为第q位。
例如，SEQ ID NO:140是人的野生型MG53亚型之一，其具有333个氨基酸，对应于人野生型MG53全长序列SEQ ID NO:1的第145-477位氨基酸片段，在这种情况下，将SEQ ID NO:140的氨基酸序列的第1个氨基酸(蛋氨酸)位点指定为SEQ ID NO:140的第145位，第2个氨基酸位点指定为SEQ ID NO:140的第146位，以此类推，将SEQ ID NO:140的氨基酸序列的最后一个氨基酸位点指定为SEQ ID NO:140的第477位。又例如，SEQ ID NO:141是人的野生型MG53亚型之一，其也具有333个氨基酸，与人野生型MG53全长序列SEQ ID NO:1第145-477位氨基酸相比只有1个氨基酸不同，即SEQ ID NO:1的第315位为谷氨酸，而SEQ ID NO:141的相应位置为甘氨酸，在这种情况下，将SEQ ID NO:141的氨基酸序列的第1个氨基酸(蛋氨酸)位点指定为SEQ ID NO:141的第145位，第2个氨基酸位点指定为SEQ ID NO:141的第146位，以此类推，将SEQ ID NO:141的氨基酸序列的最后一个氨基酸位点指定为SEQ ID NO:141的第477位。再例如，SEQ ID NO:142是人的野生型MG53亚型之一，其具有269个氨基酸，对应于人野生型MG53全长序列SEQ ID NO:1的第1-269位氨基酸片段，在这种情况下，将SEQ ID NO:142的氨基酸序列的第1个氨基酸(蛋氨酸)位点指定为SEQ ID NO:142的第1位，将SEQ ID NO:142的氨基酸序列的第2 个氨基酸位点指定为SEQ ID NO:142的第2位，以此类推，将SEQ ID NO:142的氨基酸序列的最后一个氨基酸位点指定为SEQ ID NO:142的第269位。
在某些实施方式中，发生缺失或突变的丝氨酸位于人野生型MG53亚型的氨基酸序列SEQ ID NO:139的第150位、第189位、第211位、第214位、第246位、第255位、第269位、第296位、第297位、第301位、第305位、第306位、第307位、第314位、第341位、第377位、第405位、第418位、第425位或者第430位中的一个或多个位点。
在某些实施方式中，发生缺失或突变的丝氨酸位于人野生型MG53亚型的氨基酸序列SEQ ID NO:140的第150位、第189位、第211位、第214位、第246位、第255位、第269位、第296位、第297位、第301位、第305位、第306位、第307位、第314位、第341位、第377位、第405位、第418位、第425位或者第430位中的一个或多个位点。
在某些实施方式中，发生缺失或突变的丝氨酸位于人野生型MG53亚型的氨基酸序列SEQ ID NO:141的第150位、第189位、第211位、第214位、第246位、第255位、第269位、第296位、第297位、第301位、第305位、第306位、第307位、第314位、第341位、第377位、第405位、第418位、第425位或者第430位中的一个或多个位点。
在某些实施方式中，发生缺失或突变的丝氨酸位于人野生型MG53亚型的氨基酸序列SEQ ID NO:142的第150位、第189位、第211位、第214位、第246位、第255位、第269位中的一个或多个位点。
在某些实施方式中，发生缺失或突变的丝氨酸位于小鼠野生型MG53的氨基酸序列SEQ ID NO:2的第188位、第189位、第210位、第211位、第214位、第246位、第253位、第255位、第269位、第296位、第297位、第301位、第305位、第306位、第307位、第314位、第341位、第367位、第377位、第418位、第430位、第440位中的一个或多个位点。
在某些实施方式中，发生缺失或突变的丝氨酸位于大鼠野生型MG53的氨基酸序列SEQ ID NO:3的第150位、第188位、第189位、第210位、第211位、第214位、 第246位、第253位、第255位、第269位、第296位、第297位、第301位、第305位、第307位、第314位、第341位、第367位、第377位、第418位、第430位、第440位、第464位、第474位中的一个或多个位点。
在某些实施方式中，发生缺失或突变的丝氨酸位于猴子野生型MG53的氨基酸序列SEQ ID NO:4的第150位、第189位、第211位、第214位、第246位、第255位、第269位、第296位、第297位、第301位、第305位、第306位、第307位、第341位、第377位、第405位、第418位、第425位、第430位、第464位中的一个或多个位点。
在某些实施方式中，发生缺失或突变的丝氨酸位于猪野生型MG53的氨基酸序列SEQ ID NO:5的第150位、第189位、第211位、第214位、第246位、第255位、第269位、第296位、第301位、第305位、第307位、第314位、第341位、第377位、第411位、第418位、第425位、第430位、第474位中的一个或多个位点。
在某些实施方式中，发生缺失或突变的丝氨酸位于狗野生型MG53的氨基酸序列SEQ ID NO:6的第150位、第189位、第211位、第214位、第246位、第255位、第269位、第296位、第297位、第301位、第305位、第307位、第314位、第341位、第367位、第377位、第418位、第425位、第430位中的一个或多个位点。
虽然发生缺失或突变的具体丝氨酸位点随着物种的不同而可能有所不同，但是野生型MG53蛋白的丝氨酸位点在各物种之间具有高度保守性。例如，对于人、小鼠、大鼠、猴子、猪、狗等的野生型MG53蛋白而言，各自野生型MG53蛋白的氨基酸序列的第189位、第211位、第214位、第246位、第255位、第269位、第296位、第301位、第305位、第307位、第341位、第377位、第418位、第430位均是丝氨酸。在某些实施方式中，发生缺失或突变的丝氨酸位于野生型MG53的氨基酸序列(例如，SEQ ID NOs:1-6)的第189位、第211位、第214位、第246位、第255位、第269位、第296位、第301位、第305位、第307位、第341位、第377位、第418位、第430位中的一个或多个位点。
在某些实施方式中，发生缺失或突变的丝氨酸位于野生型MG53的氨基酸序列(例如，SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:139、SEQ ID NO:140、SEQ ID NO:141、或者SEQ ID  NO:142)的第150位、第188-189位、第210-211位、第214位、第246位、第253-255位、第269位、第296-297位、第301位、第305-307位、第314位、第341位、第367位、第377位、第405位、第411位、第418位、第425位、第430位、第440位、第464位或者第474位中的一个或多个位点。
在某些实施方式中，发生缺失或突变的丝氨酸位于野生型MG53的氨基酸序列(例如，SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:139、SEQ ID NO:140、SEQ ID NO:141、或者SEQ ID NO:142)的第189位、第211位、第214位、第246位、第253-255位、第269位、第296位、第301位、第305位、第307位、第341位、第377位、第418位或者第430位中的一个或多个位点。
在某些实施方式中，发生缺失或突变的丝氨酸位于野生型MG53的氨基酸序列(例如，SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:139、SEQ ID NO:140、SEQ ID NO:141、或者SEQ ID NO:142)的第211位、第214位、第246位、第253-255位、第269位、第296位或者第297位中的一个或多个位点。
在某些实施方式中，发生缺失或突变的丝氨酸位于野生型MG53的氨基酸序列(例如，SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:139、SEQ ID NO:140、SEQ ID NO:141、或者SEQ ID NO:142)的第211位、第214位、第246位、第255位、第269位、第296位或者第297位中的一个或多个位点。
在某些实施方式中，发生缺失或突变的丝氨酸位于野生型MG53的氨基酸序列(例如，SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:139、SEQ ID NO:140、SEQ ID NO:141、或者SEQ ID NO:142)的第253-255位中的一个或多个位点。在某些实施方式中，发生缺失或突变的丝氨酸位于野生型MG53的氨基酸序列的第253位。在某些实施方式中，发生缺失或突变的丝氨酸位于野生型MG53的氨基酸序列的第255位。在某些实施方式中，发生缺失或突变的丝氨酸位于野生型MG53的氨基酸序列SEQ ID NO:1的第255位。在 某些实施方式中，发生缺失或突变的丝氨酸位于野生型MG53的氨基酸序列SEQ ID NO:139、SEQ ID NO:140、SEQ ID NO:141或SEQ ID NO:142的第255位。
在某些实施方式中，所述MG53突变体具有两个或两个以上的丝氨酸突变。在某些实施方式中，所述MG53突变体的两个或两个以上的丝氨酸突变包括在第253-255位中的一个或多个位点上的丝氨酸突变。在某些实施方式中，所述MG53突变体的两个或两个以上的丝氨酸突变包括在第253位的丝氨酸突变。在某些实施方式中，所述MG53突变体的两个或两个以上的丝氨酸突变包括在第255位的丝氨酸突变。在某些实施方式中，所述MG53突变体的两个或两个以上的丝氨酸突变包括在第253位和第255位的丝氨酸突变。
本领域技术人员可以理解，不同物种的野生型MG53蛋白中丝氨酸所处位点可能有所不同，因此发生缺失或突变的丝氨酸位点也可能有所不同。在某些实施方式中，发生缺失或突变的丝氨酸位于SEQ ID NO:1所示野生型MG53的氨基酸序列中相应丝氨酸位点的上游或下游的1至10个氨基酸之内、1至5个氨基酸之内、或1至3个氨基酸之内。
在某些实施方式中，所述MG53突变体的氨基酸序列为SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:147、SEQ ID NO:148、SEQ ID NO:149或者SEQ ID NO:150。更优选地，在某些实施方式中，所述MG53突变体的氨基酸序列为SEQ ID NO:7。在某些实施方式中，所述MG53突变体的氨基酸序列与SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:147、SEQ ID NO:148、SEQ ID NO:149、SEQ ID NO:150所示的序列之一具有至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少98％、或至少99％的氨基酸序列同源性，并且所述MG53突变体在具有细胞修复功能和/或心脏保护功能的同时，避免了野生型MG53所带来的代谢类副作用。
“序列同源性”的百分比定义为，针对氨基酸序列而言，将候选氨基酸序列与对比氨基酸序列进行序列对比，并在必要时引入间隔，使相同的氨基酸数目达到最多，并在此基础上计算两条氨基酸序列之间相同氨基酸的百分比；针对核酸序列而言，将候 选核酸序列与对比核酸序列进行序列对比，并在必要时引入间隔，使相同的核苷酸数目达到最多，并在此基础上计算两条核酸序列之间相同核苷酸的百分比。
可以通过本领域所知的多种方式来对比以确定同源性的百分比。例如，可以用公开可用的以下工具进行序列对比，所述工具如BLASTp(美国国家生物技术信息中心网站(NCBI)：http://blast.ncbi.nlm.nih.gov/Blast.cgi，也可参见，Altschul S.F.et al.,J.Mol.Biol.，215:403–410(1990)；Stephen F.et al，Nucleic Acids Res.，25:3389–3402(1997))、ClustalW2(欧洲生物信息研究所网站：http://www.ebi.ac.uk/Tools/msa/clustalw2/，可参见，Higgins D.G.et al.，Methods in Enzymology，266:383-402(1996)；Larkin M.A.et al.，Bioinformatics(Oxford、England)，23(21):2947-8(2007))和TCoffee(瑞士生物信息学研究所网站，可参见，Poirot O.et al.，Nucleic Acids Res.，31(13):3503-6(2003)；Notredame C.et al.，J.Mol.Boil.，302(1):205-17(2000))。使用软件进行序列比对时，可以使用软件提供的默认参数，也可以根据对比的需要适当调整参数，这些都在本领域技术人员的知识范围之内。
在某些实施方式中，SEQ ID NOs:7-12、SEQ ID NOs:147-150以及与SEQ ID NOs:7-12、SEQ ID NOs:147-150具有氨基酸序列同源性的MG53突变体在具有细胞修复功能和/或心脏保护功能的同时，避免或减少了野生型MG53所带来的代谢类副作用。
本申请的发明人惊奇地发现，野生型MG53的coiled-coil-SPRY结构域内至少一个丝氨酸缺失或突变为任何其他非丝氨酸或苏氨酸的氨基酸后，能够避免或减少野生型MG53所带来的代谢类副作用，但是并不影响MG53的细胞修复功能和/或心脏保护功能。不希望受到任何理论的束缚，上述结果可能是由于野生型MG53的coiled-coil-SPRY结构域内的丝氨酸位点的磷酸化能大幅度调控MG53的E3泛素连接酶功能，并通过底物IRβ和IRS1调节MG53对胰岛素信号系统的调控作用，但是该结构域内的丝氨酸位点的磷酸化并不调控MG53的细胞修复功能和/或心脏保护功能。
本申请中使用的“细胞修复功能”是指，在细胞损伤时，特别是在细胞急性损伤时，野生型MG53或MG53突变体能够对损伤细胞的细胞膜进行修补恢复，可选的，通过激活相关信号途径(例如，RISK途径)来减少细胞死亡，促进细胞生存，从而使细胞恢复功能。在某些实施方式中，本申请的野生型MG53或MG53突变体可以修复活的细胞、体外的细胞、或者体内的细胞。本申请的野生型MG53或MG53突变体还可以 修复不同种类的细胞，例如，但不限于，心肌细胞、横纹肌细胞、骨骼肌细胞、肾近端肾小管上皮细胞、肺泡上皮细胞、消化道上皮细胞(例如，口腔上皮细胞、食管上皮细胞、胃上皮细胞、十二指肠上皮细胞、小肠上皮细胞、空肠上皮细胞、回肠上皮细胞、结肠上皮细胞)、粘膜细胞(例如，口腔粘膜细胞、鼻粘膜细胞、胃黏膜细胞、小肠粘膜细胞、结肠黏膜细胞、十二指肠黏膜细胞)、皮肤细胞(例如表皮细胞、上皮细胞、真皮细胞、内皮细胞)、血管细胞(例如血管壁细胞、血管内皮细胞、血管外皮细胞、血管平滑肌细胞)等。在某些实施方式中，本申请提供的野生型MG53或MG53突变体可以修复心肌细胞、骨骼肌细胞、横纹肌细胞、肾近端肾小管上皮细胞、肺泡上皮细胞等。
可以使用本领域公知的方法来测定本申请所述的MG53突变体的细胞修复功能。例如通过在乳鼠心肌细胞(NRVM)中使用腺病毒过表达野生型MG53和本申请所述的MG53突变体，利用缺氧刺激细胞，检测细胞的存活状况(例如，通过MTT法、培养基的ATP和LDH浓度测定法、TUNEL染色法等检测(具体操作步骤参见Zhang.T et al.Nature Medicine,175-184,(2016))，然后比较对照组(例如，不表达野生型MG53、MG53突变体的空载体阴性对照组、过表达野生型MG53的阳性对照组)和实验组(即，过表达本申请所述的MG53突变体的组)的观测指标(例如，细胞内ATP水平、LDH的释放水平等)，来测定本申请所述的MG53突变体的细胞修复功能。
本申请中所述的“心脏保护功能”是指，在心肌损伤时，特别是在心肌急性损伤时，野生型MG53或MG53突变体能够通过对心肌细胞膜损伤的修复，可选的，通过激活心肌细胞的相关信号途径(例如，RISK途径)从而实现对心肌细胞的保护，进而提高心脏保护作用。可以使用本领域公知的方法来测定本申请所述的MG53突变体的心脏保护功能。例如通过在乳鼠心肌细胞(NRVM)中过表达野生型MG53和本申请所述的MG53突变体，利用缺氧刺激细胞，检测细胞的存活状况(例如，通过MTT法、培养基的ATP和LDH浓度测定法、TUNEL染色法等检测(具体操作步骤参见Zhang.T et al.Nature Medicine,175-184,(2016))，然后比较对照组(例如，不表达野生型MG53、MG53突变体的空载体阴性对照组、过表达野生型MG53的阳性对照组)和实验组(即，过表达本申请所述的MG53突变体的组)的观测指标(例如，细胞内ATP水平、LDH的释放水平等)，来考察本申请所述的MG53突变体的心脏保护功能。又例如，分别使用野生型MG53和本申请所述的MG53突变体蛋白孵育心肌细胞，利用各种刺激(例 如，缺氧、H2O2)引导细胞的死亡，通过比较对照组(例如，既不用野生型MG53、又不用MG53突变体孵育的阴性对照组、过表达野生型MG53的阳性对照组)和实验组(即，用本申请所述的MG53突变体孵育的组)的细胞存活率来评价本申请所述的MG53突变体的心脏保护功能。细胞存活率可以通过MTT细胞计数、LDH、ATP或者TUNEL染色的测量来确定。
本申请中所述的“代谢类副作用”是指，在应用了治疗量的药物后所出现的治疗目的之外的、由于代谢紊乱所引起的疾病或不适，包括但不限于，胰岛素抵抗、肥胖、糖尿病、高血压、血脂异常等。不希望受到任何理论的束缚，但是认为可以通过测定MG53的E3泛素连接酶功能来评价代谢类副作用的严重程度。在某些实施方式中，当野生型MG53的coiled-coil-SPRY结构域内的至少一个丝氨酸缺失或突变为任何其他非丝氨酸或苏氨酸的氨基酸后，会抑制野生型MG53至少40％、至少50％、至少60％、至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少98％、至少99％、甚至100％的E3泛素连接酶功能，从而避免或减少野生型MG53产生的代谢类副作用，但是并不影响MG53的细胞修复功能和/或心脏保护功能。“避免或减少野生型MG53产生的代谢类副作用”是指完全没有出现野生型MG53产生的代谢类副作用或者代谢类副作用的严重程度比相应的野生型MG53产生的代谢类副作用降低至少40％、至少50％、至少60％、至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少98％、至少99％、甚至100％。“不影响MG53的细胞修复功能和/或心脏保护功能”是指MG53的细胞修复功能和/或心脏保护功能完全没有受到影响或者MG53的细胞修复功能和/或心脏保护功能降低了至多5％、至多10％、至多15％、至多20％、至多25％、至多30％等。
在某些实施方式中，本申请提供的MG53突变体包含其类似物。所述MG53突变体类似物指的是一种多肽，其具有的功能或结构特征与本申请提供的MG53突变体的全部或部分基本相似，但是所述MG53突变体类似物的氨基酸序列与野生型MG53的氨基酸序列至少在一个氨基酸位点不同。所述MG53突变体类似物可以是MG53突变体的部分片段、衍生物或变体，并可以包含化学或生物的修饰。所述MG53突变体类似物可以具有MG53突变体的一个或多个氨基酸的保守替换、增加、删除、插入、截断、修饰(例如，磷酸化、糖基化、标记等)或其任意组合。MG53突变体类似物可以包括MG53突变体天然存在的变体和人工产生的变体，如通过重组方法或化学合成 获得的人工多肽序列。MG53突变体类似物可以包含非天然存在的氨基酸残基。本领域技术人员可以理解，本申请所述的MG53突变体的类似物仍然保留了与MG53突变体基本上相似的功能，例如，MG53突变体的类似物在具有细胞修复功能和/或心脏保护功能的同时，能够避免或减少野生型MG53所带来的代谢类副作用，例如，胰岛素抵抗、肥胖、糖尿病、高血压、血脂异常等。
氨基酸残基的保守替换是指性质相似的氨基酸之间的替换，例如极性氨基酸之间的替换(如谷氨酰胺和天冬酰胺之间的替换)，疏水性氨基酸之间的替换(如亮氨酸、异亮氨酸、蛋氨酸和缬氨酸之间的替换)，以及带相同电荷的氨基酸之间的替换(如精氨酸、赖氨酸和组氨酸之间的替换，或者谷氨酸和天冬氨酸之间的替换)等。在某些实施方式中，本申请所述的MG53突变体与SEQ ID NOs:7-12、SEQ ID NOs:147-150所示序列相比，仅在非丝氨酸的一个或多个氨基酸位点有氨基酸的保守替换。在某些实施方式中，本申请所述的MG53突变体与SEQ ID NOs:7-12、SEQ ID NOs:147-150所示序列相比，在非丝氨酸的2个、3个、4个、5个、6个、7个、8个、9个、10个、15个、或20个氨基酸位点有氨基酸的保守替换。
在不影响活性的前提下，本申请所述的MG53突变体还可以含有非天然的氨基酸。非天然的氨基酸包括例如，β-氟代丙氨酸、1-甲基组氨酸、γ-亚甲基谷氨酸、α-甲基亮氨酸、4,5-脱氢赖氨酸、羟基脯氨酸、3-氟代苯基丙氨酸、3-氨基酪氨酸、4-甲基色氨酸等。
本申请的MG53突变体也可以使用本领域公知的方法进行修饰。例如，但不限于，PEG化、糖基化、氨基端修饰、脂肪酰化、羧基端修饰、磷酸化、甲基化等。
本领域技术人员可以理解，本申请的MG53突变体使用本领域公知的方法进行修饰之后，仍然保留了与MG53突变体基本上相似的功能，例如，经修饰的MG53突变体在具有细胞修复功能和/或心脏保护功能的同时，能够避免或减少野生型MG53所带来的代谢类副作用，例如，胰岛素抵抗、肥胖、糖尿病、高血压、血脂异常等。
另一方面，本申请涉及一种药物组合物，其包括本申请所述的MG53突变体和药学上可接受的载体。
本申请中使用的术语“药学上可接受的载体”是指，用于给给药对象递送MG53突变体的药学上可接受的溶剂、悬浮剂或任何其它药理上惰性的媒介物，其不干扰MG53 突变体的结构和性质。某些此类载体能使MG53突变体配制成例如片剂、丸剂、胶囊、液体、凝胶剂、糖浆剂、浆液、混悬液和锭剂以给给药对象口服摄入。某些此类载体能使MG53突变体配制成注射、输注或局部给药。
可以用在本申请的药物组合物中的药学上可接受的载体包括，但不限于，例如，药学可接受的液体、凝胶、或固体载剂、水相介质(例如，氯化钠注射液，林格氏液注射液，等渗葡萄糖注射液，无菌水注射液，或葡萄糖和乳酸林格注射液)、非水相介质(例如，植物来源的不挥发性油、棉花子油、玉米油、芝麻油、或者花生油)、抗微生物物质、等渗物质(例如氯化钠或葡萄糖)、缓冲液(例如磷酸盐或枸橼酸盐缓冲液)、抗氧化剂(例如硫酸氢钠)、麻醉剂(例如盐酸普鲁卡因)、悬浮剂/分散剂(例如羧甲基纤维素钠、羟丙基甲基纤维素、或聚乙烯吡咯烷酮)、螯合剂(例如EDTA(乙二胺四乙酸)或EGTA(乙二醇双(2-氨基乙基醚)四乙酸))、乳化剂(例如聚山梨醇酯80(吐温-80))、稀释剂、佐剂、辅料、或无毒辅助物质，其他本领域公知的组分、或以上的多种组合。适用的组分可包括，例如，填充剂、粘合剂、崩解剂、缓冲液、防腐剂、润滑剂、搅味剂、增稠剂、着色剂或乳化剂。
在某些实施方式中，所述的药物组合物为口服制剂。口服制剂包括，但不限于，胶囊、囊剂、药丸、片剂、锭剂(用于味道的基底，通常是蔗糖和阿拉伯胶或黄芪胶)、粉末、颗粒剂、或者是水或非水溶液或悬浮液、或者油包水或水包油的乳剂、或者是酏剂或糖浆、或者是糖果锭剂(适用惰性基质，如白明胶和甘油，或蔗糖或者阿拉伯胶)和/或漱口药及其类似物。
在某些实施方式中，口服的固体制剂(如胶囊、片剂、丸剂、糖衣丸、粉末、颗粒等)中包括所述的MG53突变体与一种或多种药学上可接受的载体，如柠檬酸钠或磷酸二钙，和/或以下物质：(1)填料或者补充剂，例如淀粉、乳糖、蔗糖、葡萄糖、甘露糖醇，和/或硅酸；(2)粘合剂，例如，羧甲基纤维素、藻朊酸盐、明胶、聚乙烯基吡咯烷酮、蔗糖、和/或阿拉伯胶；(3)湿润剂，例如，丙三醇；(4)分裂剂，例如琼脂、碳酸钙、马铃薯或者木薯淀粉、海藻酸、某些硅酸盐、与碳酸钠，(5)阻滞剂溶液，例如石蜡；(6)加速吸收剂，例如季铵化合物；(7)润滑剂，例如，乙酰醇与单硬脂酸甘油酯；(8)吸收剂，例如高岭土与皂土；(9)润滑剂，例如滑石、硬脂酸钙、硬脂酸镁、固体聚乙二醇、十二烷基硫酸钠、及其混合物；与(10)着色剂。
在某些实施方式中，口服的液体制剂包括药学可接受的乳剂、微乳剂、溶液剂、悬浮剂、糖浆剂和酏剂等。除了MG53突变体之外，液体剂型还可以含有常用的惰性稀释剂，例如，水或其他溶剂、增溶剂和乳化剂，如乙醇、异丙醇、碳酸乙酯、乙酸乙酯、苯甲醇、苯(甲)酸苄酯、丙二醇、1,3-丁二醇、油类(特别地，棉籽油、花生油、玉米油、橄榄油、蓖麻油和芝麻油)、甘油、四氢糠醇、聚乙二醇和脂肪酸山梨醇酯、以及混合物。除了惰性稀释液之外，口服组合物也可以添加佐剂例如，润湿剂、乳化剂和悬浮剂、甜味剂、调味剂、颜料、香料和防腐剂。
在某些实施方式中，所述的药物组合物为注射制剂。注射制剂包括无菌水溶液或分散液、混悬剂或乳剂。在所有情况下，所述的注射制剂应当无菌且应当是液体以方便注射。它在生产和贮存条件下应保持稳定，并应当抗微生物(例如细菌和真菌)的污染。载体可以是一种溶剂或分散介质，其包含，例如，水、乙醇、多羟基化合物(例如，甘油、丙二醇，以及液体聚乙二醇等)及其适当的混合物和/或植物油。所述的注射制剂应保持适当的流动性，适当的流动性可通过多种方式维持，例如，通过使用如卵磷脂等涂层，使用表面活性剂等。可以通过加入各种抗菌和抗真菌剂(例如，对羟基苯甲酸酯、氯丁醇、苯酚、山梨酸、硫柳汞等)来实现抗微生物污染。
在某些实施方式中，所述的药物组合物为口腔喷雾制剂或鼻腔喷雾制剂。所述喷雾制剂包括，但不限于，水性气雾剂、非水性悬浮液、脂质体制剂或者固体颗粒制剂等。水性气雾剂是通过将作用剂的水性溶液或者悬浮液和常规的药学上可接受的载体与稳定剂一同配制。该载体与稳定剂根据特定化合物的需要而变化，但是其一般包括非离子型表面活性剂(吐温类、或聚乙二醇)、油酸、卵磷脂、氨基酸如甘氨酸，缓冲液、盐、糖或者糖醇。气雾剂通常由等渗溶液制备，可以通过喷雾器递送。
在某些实施方式中，所述的药物组合物可以和一种或多种其他药物混合使用。在某些实施方式中，所述的组合物包含至少一种其他药物。在某些实施方式中，所述的其他药物为心血管类药物、用于治疗肾脏疾病的药物或者用于细胞修复的药物等。
在某些实施方式中，所述的药物组合物可以通过适当的途径递送到给药对象中，包括但不限于，通过口服途径、注射途径(如静脉注射、肌肉注射、皮下注射、皮内注射、心内注射、鞘内注射、胸膜腔内注射、腹腔内注射等)、粘膜途径(如鼻腔内给药、口腔内给药等)、舌下途径、直肠途径、经皮途径、眼内途径、肺部途径。在某些实施方式中，所述的药物组合物可通过注射途径给药。
另一方面，本申请涉及一种分离的核酸，其包含编码本申请所述MG53突变体的氨基酸序列的核酸序列。
本申请中使用的术语“分离的”是指一种物质(例如多肽或者核酸)与它在自然界中正常存在的环境相分离或存在于与它在自然界中正常存在的环境不同的环境中。
本申请使用的术语“核酸”或“多聚核苷酸”指核糖核酸(RNA)、脱氧核糖核酸(DNA)、或核糖核酸-脱氧核糖核酸的混合物如DNA-RNA杂交物。核酸或多聚核苷酸可以是单链或双链DNA或RNA或DNA-RNA杂交物。核酸或多聚核苷酸可以是线性的或环状的。本申请使用的术语“编码”或“为……编码”指能够转录为mRNA和/或翻译为肽或蛋白。“编码序列”或“基因”指编码mRNA、肽或蛋白的多聚核苷酸序列。在本申请中这两个术语可以互换使用。
在某些实施方式中，所述分离的核酸包含SEQ ID NO:13-18所示的任一核酸序列。
SEQ ID NO:13是编码SEQ ID NO:7所示氨基酸序列的核酸序列，其具体序列参见图8。
SEQ ID NO:14是编码SEQ ID NO:8所示氨基酸序列的核酸序列，其具体序列参见图9。
SEQ ID NO:15是编码SEQ ID NO:9所示氨基酸序列的核酸序列，其具体序列参见图10。
SEQ ID NO:16是编码SEQ ID NO:10所示氨基酸序列的核酸序列，其具体序列参见图11。
SEQ ID NO:17是编码SEQ ID NO:11所示氨基酸序列的核酸序列，其具体序列参见图12。
SEQ ID NO:18是编码SEQ ID NO:12所示氨基酸序列的核酸序列，其具体序列参见图13。
在某些实施方式中，所述分离的核酸包含SEQ ID NO:151-154所示的任一核酸序列。
SEQ ID NO:151是编码SEQ ID NO:147所示氨基酸序列的核酸序列，其具体序列参见图24。
SEQ ID NO:152是编码SEQ ID NO:148所示氨基酸序列的核酸序列，其具体序列参见图25。
SEQ ID NO:153是编码SEQ ID NO:149所示氨基酸序列的核酸序列，其具体序列参见图26。
SEQ ID NO:154是编码SEQ ID NO:150所示氨基酸序列的核酸序列，其具体序列参见图27。
在某些实施方式中，本申请提供的分离的核酸包含与SEQ ID NOs:13-18、SEQ ID NOs:151-154所示的任一核酸序列具有至少70％同源性的核酸序列，例如，至少75％、至少80％、至少85％、至少90％、至少95％、或至少99％的同源性，并且仍能编码SEQ ID NOs:7-12、SEQ ID NOs:147-150所示的氨基酸序列之一。
在某些实施方式中，本申请提供了编码SEQ ID NOs:7-12、SEQ ID NOs:147-150的核酸序列，但其核酸序列由于遗传密码的简并性而不同于SEQ ID NOs:13-18、SEQ ID NOs:151-154所示的任一核酸序列。
本申请中使用的术语“遗传密码的简并性”是指，同一种氨基酸具有两个或更多个对应的密码子的现象。例如，脯氨酸具有4个同义密码子CCU、CCC、CCA、CCG。本领域公知，由于核酸遗传密码的简并性，可以将某已知核酸序列中的某些位置的核酸进行替换，但却不改变编码的氨基酸序列。本领域技术人员可以很容易地进行遗传密码简并性的替换，例如，通过碱基的定点突变技术。不同生物体对不同的密码子的偏好性存在区别，为在某个选定的生物细胞内表达本申请的多肽，可以选择该生物细胞所偏好的密码子，获得相应的编码序列，通过重组表达得到本申请的MG53突变体序列(例如SEQ ID NOs:7-12、SEQ ID NOs:147-150)。
另一方面，本申请涉及一种表达载体，其包含本申请所述的MG53突变体的氨基酸序列的编码序列。
本申请中的表达载体可以是，例如，DNA质粒、细菌质粒、病毒等。表达载体的非限制性例子为如Paul等,2002,Nature Biotechnology,19,505；Miyagishi and Taira,2002,Nature Biotechnology,19,497；Lee等,2002,Nature Biotechnology,19,500；以及Novina等,2002,Nature Medicine,advance online publication doi:10.1038/nm725中所描述的。所述表达载体中可以进一步含有启动子，其与所述MG53突变体的氨基酸序 列的编码序列操作性连接，使得当所述表达载体进入宿主细胞后，所述启动子能够启动编码序列的表达。表达载体可以通过适当的方法导入宿主细胞，例如，但不限于，磷酸钙转染、脂质体转染、电穿孔转染、细菌热休克等，具体的方法请参见，Sambrook等人编著的“分子克隆”(实验手册，冷泉港，1989)。在某些实施方式中，本申请所述的表达载体包含SEQ ID NO:13-18所示的任一核酸序列。
另一方面，本申请涉及一种宿主细胞，其包含本申请所述的表达载体。
本申请中所述的宿主细胞可以是真核细胞，也可以是原核细胞。适当的真核细胞可以包括，例如，哺乳动物细胞，如中国仓鼠卵巢细胞(CHO)。适当的原核细胞可以包括，例如，细菌，如大肠杆菌。
另一方面，本申请涉及所述MG53突变体的制备方法，本申请提供的MG53突变体可以通过本领域的已知技术来制备。例如，可以通过化学合成方法制备，也可以通过基因工程方法制备。
化学合成方法主要包括固相合成和液相合成两种方法。固相多肽合成方法包括，例如Merrifield固相合成法，该方法已详细记载在文献“Merrifield,J.Am.Chem.Soc.85:2149-2154”和“M.Bodanszky等人,"Peptide Synthesis",John Wiley&Sons,Second Edition,1976”以及“J.Meienhofer,"Hormonal Proteins and Peptides",Vol.2,p.46,Academic Press(New York),1983”中。在此通过引用方式将这些文献全文并入到本申请中用作参考。Merrifield固相合成法主要包括以下步骤，根据目标多肽的氨基酸序列，先使被保护的羧基末端氨基酸与树脂连接；连接后洗涤树脂；脱去羧基末端氨基酸α氨基上的保护基(例如，叔丁氧羰基)，脱去这个保护基的时候必须要确保不断裂该氨基酸和树脂间的连接键；然后在所得的树脂上偶联倒数第二个羧基末端被保护的氨基酸，进行这一偶联时，在第二个氨基酸的游离羧基和连在树脂上的第一个氨基酸的氨基之间形成一个酰胺键；根据目标多肽的氨基酸连接顺序，依次重复前述反应过程，直到所有氨基酸都连接到树脂上；最后，从树脂上切落被保护的肽，脱去保护基即可得到目标多肽。本申请的多肽也可以通过液相合成方法制备，例如可以用标准的溶液肽合成法制备，该方法已详细记载在文献“E.Schroder and K.Kubke,"The Peptides",Vol.1,Academic Press(New York),1965”中。在此通过引用方式将该文献全文并入到本申请中用作参考。液相合成法主要包括，利用形成酰胺键的化学或酶方法分步偶联氨基酸 或肽片段。
基因工程方法为利用编码相应MG53突变体的核酸序列在适合的宿主细胞中表达生成相应突变体的方法。有关该方法的详细描述可参考Sambrook等人编著的“分子克隆”(实验手册，冷泉港，1989)。在某些实施方式中，本申请涉及的所述MG53突变体的制备方法包括确定需要进行突变的丝氨酸的一个或多个位置，对包含编码野生型MG53氨基酸序列的核酸序列的质粒的全长序列进行在所述位置的定点突变，将所述定点突变的质粒转染到宿主细胞，表达所述宿主细胞以产生所述MG53突变体。
本申请中使用的术语“定点突变”是指，向目的DNA片段中引入感兴趣的变化，包括碱基的添加、删除、替换等。在某些实施方式中，所述目的DNA片段为野生型MG53的编码序列SEQ ID NOs:19-24、SEQ ID NOs:143-146，引入突变的位点位于所述野生型MG53的coiled-coil-SPRY结构域的编码序列中的一个或多个丝氨酸位点。
SEQ ID NO:19是编码SEQ ID NO:1所示氨基酸序列的核酸序列，其具体序列参见图2。
SEQ ID NO:20是编码SEQ ID NO:2所示氨基酸序列的核酸序列，其具体序列参见图3。
SEQ ID NO:21是编码SEQ ID NO:3所示氨基酸序列的核酸序列，其具体序列参见图4。
SEQ ID NO:22是编码SEQ ID NO:4所示氨基酸序列的核酸序列，其具体序列参见图5。
SEQ ID NO:23是编码SEQ ID NO:5所示氨基酸序列的核酸序列，其具体序列参见图6。
SEQ ID NO:24是编码SEQ ID NO:6所示氨基酸序列的核酸序列，其具体序列参见图7。
SEQ ID NO:143是编码SEQ ID NO:139所示氨基酸序列的核酸序列，其具体序列参见图20。
SEQ ID NO:144是编码SEQ ID NO:140所示氨基酸序列的核酸序列，其具体序列参见图21。
SEQ ID NO:145是编码SEQ ID NO:141所示氨基酸序列的核酸序列，其具体序列参见图22。
SEQ ID NO:146是编码SEQ ID NO:142所示氨基酸序列的核酸序列，其具体序列参见图23。
在某些实施方式中，所述定点突变包括如下步骤：
(1)确定需要定点突变的氨基酸在cDNA序列中相应的核苷酸位置，根据突变后的氨基酸修改突变位置的核苷酸序列，并截取包含突变位置的长度为20-40bp的序列设计引物；
(2)以野生型MG53质粒为模板，使用步骤(1)中设计的引物进行PCR反应，PCR产物进行琼脂糖凝胶电泳，并对PCR产物进行纯化；
(3)步骤(2)中纯化后的PCR产物使用核酸限制性内切酶进行酶切反应，并将酶切产物与合适的质粒表达载体进行连接，将连接产物进行细菌感受态细胞的转化，培养。
在某些实施方式中，所述定点突变进一步包括如下步骤：
(4)挑取步骤(3)所得克隆使用步骤(1)所设计的引物进行菌落PCR鉴定，对PCR产物进行琼脂糖凝胶电泳检测，然后进行DNA测序鉴定，鉴定出带有所述定点突变的阳性克隆。
可以使用各种市售的定点突变试剂盒对野生型MG53进行定点突变，例如参考北京全式金公司的Easy Mutagenesis System试剂盒的说明书，其中详细记载了定点突变的方法和步骤。
另一方面，本申请涉及所述MG53突变体在制备用于治疗心脏疾病、糖尿病脑血管疾病、糖尿病眼部并发症、糖尿病神经病变、糖尿病足、肾脏疾病、与细胞和/或组织损伤相关疾病的药物中的用途。在某些实施方式中，所述药物在治疗心脏疾病、糖尿病脑血管疾病、糖尿病眼部并发症、糖尿病神经病变、糖尿病足、肾脏疾病、与细胞和/或组织损伤相关疾病的同时，避免或减少了代谢类副作用，例如胰岛素抵抗、肥胖、糖尿病、高血压、血脂异常等副作用。在某些实施方式中，所述心脏疾病是与心肌损伤相关的疾病，包括但不限于，糖尿病性心脏病、心肌缺血、心脏缺血/再灌注损伤、心肌梗塞、心力衰竭、心律失常、心脏破裂、心绞痛、心肌炎、冠心病、心包炎。在某些实施方式中，所述糖尿病脑血管疾病包括但不限于，脑动脉硬化、缺血性脑血 管病、脑出血、脑萎缩、脑梗塞。在某些实施方式中，所述糖尿病眼部并发症包括但不限于，糖尿病性视网膜病变、糖尿病性白内障、与糖尿病相关的葡萄膜炎、失明。在某些实施方式中，所述糖尿病神经病变包括但不限于，糖尿病周围神经病变。在某些实施方式中，所述肾脏疾病包括但不限于，急性肾小球肾炎、慢性肾小球肾炎、肾病综合征、急性肾损伤、糖尿病肾病等。在某些实施方式中，所述与细胞和/或组织损伤相关疾病包括但不限于与肾脏、脑、肺部、肝脏、心脏、脾脏、消化道的细胞和/或组织损伤相关的疾病、皮肤损伤相关的疾病，例如，脑损伤、肺损伤、脾脏损伤、脾脏破裂、胃溃疡、胃炎、胃穿孔、消化道黏膜损伤、外伤、烧伤、溃疡、黏膜炎、哮喘、慢性阻塞性肺疾病(COPD)、中风、皮肤老化等。在某些实施方式中，本申请涉及SEQ ID NO:7所示的多肽在制备用于治疗心脏疾病、糖尿病脑血管疾病、糖尿病眼部并发症、糖尿病神经病变、糖尿病足、肾脏疾病、与细胞和/或组织损伤相关疾病的药物中的用途。在某些实施方式中，本申请涉及SEQ ID NO:147、SEQ ID NO:148、SEQ ID NO:149或者SEQ ID NO:150所示的多肽在制备用于治疗心脏疾病、糖尿病脑血管疾病、糖尿病眼部并发症、糖尿病神经病变、糖尿病足、肾脏疾病、与细胞和/或组织损伤相关疾病的药物中的用途。
另一方面，本申请涉及一种治疗心脏疾病、糖尿病脑血管疾病、糖尿病眼部并发症、糖尿病神经病变、糖尿病足、肾脏疾病、与细胞和/或组织损伤相关疾病的方法，包括给予需要的对象治疗有效量的所述MG53突变体。在某些实施方式中，所述MG53突变体在治疗心脏疾病、糖尿病脑血管疾病、糖尿病眼部并发症、糖尿病神经病变、糖尿病足、肾脏疾病、与细胞和/或组织损伤相关疾病的同时，避免或减少了代谢类副作用，例如胰岛素抵抗、肥胖、糖尿病、高血压、血脂异常等副作用。在某些实施方式中，所述心脏疾病是与心肌损伤相关的疾病，包括但不限于，糖尿病性心脏病、心肌缺血、心脏缺血/再灌注损伤、心肌梗塞、心力衰竭、心律失常、心脏破裂、心绞痛、心肌炎、冠心病、心包炎。在某些实施方式中，所述糖尿病脑血管疾病包括但不限于，脑动脉硬化、缺血性脑血管病、脑出血、脑萎缩、脑梗塞。在某些实施方式中，所述糖尿病眼部并发症包括但不限于，糖尿病性视网膜病变、糖尿病性白内障、与糖尿病相关的葡萄膜炎、失明。在某些实施方式中，所述糖尿病神经病变包括但不限于，糖尿病周围神经病变。在某些实施方式中，所述肾脏疾病包括但不限于，急性肾小球肾炎、慢性肾小球肾炎、肾病综合征、急性肾损伤、糖尿病肾病等。在某些实施方式中， 所述与细胞和/或组织损伤相关疾病包括但不限于与肾脏、脑、肺部、肝脏、心脏、脾脏、消化道的细胞和/或组织损伤相关的疾病、皮肤损伤相关的疾病，例如，脑损伤、肺损伤、脾脏损伤、脾脏破裂、胃溃疡、胃炎、胃穿孔、消化道黏膜损伤、外伤、烧伤、溃疡、黏膜炎、哮喘、慢性阻塞性肺疾病(COPD)、中风、皮肤老化等。在某些实施方式中，本申请涉及一种治疗心脏疾病、糖尿病脑血管疾病、糖尿病眼部并发症、糖尿病神经病变、糖尿病足、肾脏疾病、与细胞和/或组织损伤相关疾病的方法，包括给予需要的对象治疗有效量的SEQ ID NO:7所示的多肽。在某些实施方式中，本申请涉及一种治疗心脏疾病、糖尿病脑血管疾病、糖尿病眼部并发症、糖尿病神经病变、糖尿病足、肾脏疾病、与细胞和/或组织损伤相关疾病的方法，包括给予需要的对象治疗有效量的SEQ ID NO:147、SEQ ID NO:148、SEQ ID NO:149或者SEQ ID NO:150所示的多肽。
本申请中使用的术语“治疗有效量”是指，可以实现抑制或缓解对象的疾病或症状，或者可以预防性地抑制或防止疾病或症状发生的药物的量。治疗有效量可以是将对象的一种或多种疾病或症状缓解到一定程度的药物的量；可以将那些跟疾病或症状成因相关的一种或多种生理或生物化学参数部分或完全恢复到正常的药物的量；和/或可以降低疾病或症状发生的可能性的药物的量。
另一方面，本申请涉及用于治疗心脏疾病、糖尿病脑血管疾病、糖尿病眼部并发症、糖尿病神经病变、糖尿病足、肾脏疾病、与细胞和/或组织损伤相关疾病的MG53突变体。在某些实施方式中，所述MG53突变体在治疗心脏疾病、糖尿病脑血管疾病、糖尿病眼部并发症、糖尿病神经病变、糖尿病足、肾脏疾病、与细胞和/或组织损伤相关疾病的同时，避免了代谢类副作用，例如胰岛素抵抗、肥胖、糖尿病、高血压、血脂异常等副作用。在某些实施方式中，所述心脏疾病是与心肌损伤相关的疾病，包括但不限于，糖尿病性心脏病、心肌缺血、心脏缺血/再灌注损伤、心肌梗塞、心力衰竭、心律失常、心脏破裂、心绞痛、心肌炎、冠心病、心包炎。在某些实施方式中，所述糖尿病脑血管疾病包括但不限于，脑动脉硬化、缺血性脑血管病、脑出血、脑萎缩、脑梗塞。在某些实施方式中，所述糖尿病眼部并发症包括但不限于，糖尿病性视网膜病变、糖尿病性白内障、与糖尿病相关的葡萄膜炎、失明。在某些实施方式中，所述糖尿病神经病变包括但不限于，糖尿病周围神经病变。在某些实施方式中，所述肾脏疾病包括但不限于，急性肾小球肾炎、慢性肾小球肾炎、肾病综合征、急性肾损伤、 糖尿病肾病等。在某些实施方式中，所述与细胞和/或组织损伤相关疾病包括但不限于与肾脏、脑、肺部、肝脏、心脏、脾脏、消化道的细胞和/或组织损伤相关的疾病、皮肤损伤相关的疾病，例如，脑损伤、肺损伤、脾脏损伤、脾脏破裂、胃溃疡、胃炎、胃穿孔、消化道黏膜损伤、外伤、烧伤、溃疡、黏膜炎、哮喘、慢性阻塞性肺疾病(COPD)、中风、皮肤老化等。在某些实施方式中，本申请涉及用于治疗心脏疾病、糖尿病脑血管疾病、糖尿病眼部并发症、糖尿病神经病变、糖尿病足、肾脏疾病、与细胞和/或组织损伤相关疾病的SEQ ID NO:7所示的多肽。在某些实施方式中，本申请涉及用于治疗心脏疾病、糖尿病脑血管疾病、糖尿病眼部并发症、糖尿病神经病变、糖尿病足、肾脏疾病、与细胞和/或组织损伤相关疾病的SEQ ID NO:147、SEQ ID NO:148、SEQ ID NO:149或者SEQ ID NO:150所示的多肽。
具体实施方式
所有实施例中的涉及的生物学材料如大肠杆菌菌株、各种克隆与表达质粒、培养基、工具酶、缓冲液，和各种培养方法、蛋白提取和纯化方法、其它的分子生物学操作方法，均为该领域技术人员所熟悉，可以参考Sambrook等人编著的“分子克隆”(实验室手册，冷泉港，1989)及“精编分子生物学实验指南”(美/F.奥斯伯等著，颜子颖等译，北京，科学出版社，1998)。
实施例1：MG53突变体的制备
1.人MG53突变体的制备
人MG53 S255A突变体
(1)设计突变引物：确定需要点突变的氨基酸在cDNA序列中相应核苷酸位置，对照氨基酸密码子表，根据突变后的氨基酸修改突变位置的核苷酸序列，并截取突变位置上游20bp和下游10bp间的序列设计引物。引物序列如下：
人MG53 S255A正向引物：tgcagaagatcctggcagaggctcccccacccg(SEQ ID NO:25)
人MG53 S255A反向引物：tccagacgggcgggtgggggagcctctgccagg(SEQ ID NO:26)
(2)质粒全长克隆的聚合酶链式反应(PCR)：以原基因表达质粒为模板，使用设计的点突变引物，利用高保真Taq酶进行PCR反应，整个基因表达质粒。反应体系和反应条件参照如下：
Figure PCTCN2017093640-appb-000001
(3)PCR产物的琼脂糖凝胶电泳：PCR产物进行琼脂糖凝胶电泳，以鉴定它的纯度和数量。
(4)PCR产物的乙醇沉淀：对于大小正确且条带单一的PCR产物，以乙醇沉淀的方法进行纯化。
(5)Dpn I酶切：纯化后的PCR产物使用核酸限制性内切酶Dpn I进行酶切反应。
(6)酶切产物的转化：对Dpn I酶切产物进行细菌感受态细胞的转化，涂布至合适的筛选平板上进行培养。
(7)阳性克隆的鉴定：挑取大小合适克隆进行菌落PCR鉴定，对PCR产物进行琼脂糖凝胶电泳检测，对于条带清晰并且大小正确的克隆进行小体积摇菌扩增培养，然后进行测序鉴定。
按照相同的方法制备人MG53 S255G、S255L、S255V、S255P、S255F、S255W、S255Q、S255C、S255Y、S255D、S255R、S211A、S214A、S246A、S269A、S296A、S297A突变体。除了突变引物不同之外，其他步骤均与人MG53 S255A突变体的制备步骤相同。各突变体的引物如下所示：
(i)人MG53 S255G的正向和反向引物，人MG53 S255G表示人的野生型MG53(即，SEQ ID NO:1)的第255位丝氨酸突变为甘氨酸的MG53突变体。
人MG53 S255G正向引物：tgcagaagatcctggcagagggtcccccacccg(SEQ ID NO:27)
人MG53 S255G反向引物：tccagacgggcgggtgggggaccctctgccagg(SEQ ID NO:28)
(ii)人MG53 S255L的正向和反向引物，人MG53 S255L表示人的野生型MG53(即，SEQ ID NO:1)的第255位丝氨酸突变为亮氨酸的MG53突变体。
人MG53 S255L正向引物：tgcagaagatcctggcagagcttcccccacccg(SEQ ID NO:29)
人MG53 S255L反向引物：tccagacgggcgggtgggggaagctctgccagg(SEQ ID NO:30)
(iii)人MG53 S255V的正向和反向引物，人MG53 S255V表示人的野生型MG53(即，SEQ ID NO:1)的第255位丝氨酸突变为缬氨酸的MG53突变体。
人MG53 S255V正向引物：tgcagaagatcctggcagaggttcccccacccg(SEQ ID NO:31)
人MG53 S255V反向引物：tccagacgggcgggtgggggaacctctgccagg(SEQ ID NO:32)
(iv)人MG53 S255P的正向和反向引物，人MG53 S255P表示人的野生型MG53(即，SEQ ID NO:1)的第255位丝氨酸突变为脯氨酸的MG53突变体。
人MG53 S255P正向引物：tgcagaagatcctggcagagcctcccccacccg(SEQ ID NO:33)
人MG53 S255P反向引物：tccagacgggcgggtgggggaggctctgccagg(SEQ ID NO:34)
(v)人MG53 S255F的正向和反向引物，人MG53 S255F表示人的野生型MG53(即，SEQ ID NO:1)的第255位丝氨酸突变为苯丙氨酸的MG53突变体。
人MG53 S255F正向引物：tgcagaagatcctggcagagtttcccccacccg(SEQ ID NO:35)
人MG53 S255F反向引物：tccagacgggcgggtgggggaaactctgccagg(SEQ ID NO:36)
(vi)人MG53 S255W的正向和反向引物，人MG53 S255W表示人的野生型MG53(即，SEQ ID NO:1)的第255位丝氨酸突变为色氨酸的MG53突变体。
人MG53 S255W正向引物：tgcagaagatcctggcagagtggcccccacccg(SEQ ID NO:37)
人MG53 S255W反向引物：tccagacgggcgggtgggggccactctgccagg(SEQ ID NO:38)
(vii)人MG53 S255Q的正向和反向引物，人MG53 S255Q表示人的野生型MG53(即，SEQ ID NO:1)的第255位丝氨酸突变为谷氨酰胺的MG53突变体。
人MG53 S255Q正向引物：tgcagaagatcctggcagagcaacccccacccg(SEQ ID NO:39)
人MG53 S255Q反向引物：tccagacgggcgggtgggggttgctctgccagg(SEQ ID NO:40)
(viii)人MG53 S255C的正向和反向引物，人MG53 S255C表示人的野生型MG53(即，SEQ ID NO:1)的第255位丝氨酸突变为半胱氨酸的MG53突变体。
人MG53 S255C正向引物：tgcagaagatcctggcagagtgtcccccacccg(SEQ ID NO:41)
人MG53 S255C反向引物：tccagacgggcgggtgggggacactctgccagg(SEQ ID NO:42)
(ix)人MG53 S255Y的正向和反向引物，人MG53 S255Y表示人的野生型MG53(即，SEQ ID NO:1)的第255位丝氨酸突变为酪氨酸的MG53突变体。
人MG53 S255Y正向引物：tgcagaagatcctggcagagtatcccccacccg(SEQ ID NO:43)
人MG53 S255Y反向引物：tccagacgggcgggtgggggatactctgccagg(SEQ ID NO:44)
(x)人MG53 S255D的正向和反向引物，人MG53 S255D表示人的野生型MG53(即，SEQ ID NO:1)的第255位丝氨酸突变为天冬氨酸的MG53突变体。
人MG53 S255D正向引物：tgcagaagatcctggcagaggatcccccacccg(SEQ ID NO:45)
人MG53 S255D反向引物：tccagacgggcgggtgggggctactctgccagg(SEQ ID NO:46)
(xi)人MG53 S255R的正向和反向引物，人MG53 S255R表示人的野生型MG53(即，SEQ ID NO:1)的第255位丝氨酸突变为精氨酸的MG53突变体。
人MG53 S255R正向引物：tgcagaagatcctggcagagcgtcccccacccg(SEQ ID NO:47)
人MG53 S255R反向引物：tccagacgggcgggtgggggacgctctgccagg(SEQ ID NO:48)
(xii)人MG53 S211A的正向和反向引物，人MG53 S211A表示人的野生型MG53(即，SEQ ID NO:1)的第211位丝氨酸突变为丙氨酸的MG53突变体。
人MG53 S211A正向引物：ccttgcgccgggagctgggggccctgaactctt(SEQ ID NO:49)
人MG53 S211A反向引物：gctgctccaggtaagagttcagggcccccagctcc(SEQ ID NO:50)
(xiii)人MG53 S214A的正向和反向引物，人MG53 S214A表示人的野生型MG53(即，SEQ ID NO:1)的第214位丝氨酸突变为丙氨酸的MG53突变体。
人MG53 S214A正向引物：gggagctggggagcctgaacgcttacctggagc(SEQ ID NO:51)
人MG53 S214A反向引物：tgccgcagctgctccaggtaagcgttcaggctc(SEQ ID NO:52)
(xiv)人MG53 S246A的正向和反向引物，人MG53 S246A表示人的野生型MG53(即，SEQ ID NO:1)的第246位丝氨酸突变为丙氨酸的MG53突变体。
人MG53 S246A正向引物：tgaaatactgcctggtgaccgccaggctgcaga(SEQ ID NO:53)
人MG53 S246A反向引物：gccaggatcttctgcagcctggcggtcaccagg(SEQ ID NO:54)
(xv)人MG53 S269A的正向和反向引物，人MG53 S269A表示人的野生型MG53(即，SEQ ID NO:1)的第269位丝氨酸突变为丙氨酸的MG53突变体。
人MG53 S269A正向引物：aggagctgacctttgacccggcctctgcgcacc(SEQ ID NO:55)
人MG53 S296A反向引物：accaggctcgggtgcgcagaggccgggtcaaag(SEQ ID NO:56)
(xvi)人MG53 S297A的正向和反向引物，人MG53 S297A表示人的野生型MG53(即，SEQ ID NO:1)的第297位丝氨酸突变为丙氨酸的MG53突变体。
人MG53 S297A正向引物：agctgacctttgacccgagcgctgcgcacccga(SEQ ID NO:57)
人MG53 S297A反向引物：accaccaggctcgggtgcgcagcgctcgggtca(SEQ ID NO:58)
2.小鼠MG53突变体的制备
按照人MG53 S255A突变体相同的方法制备小鼠MG53 S255A、S255G、S255L、S255W、S255Q、S255Y、S255D、S255R突变体。除了突变引物不同之外，其他步骤均与人MG53 S255A突变体的制备步骤相同。各突变体的引物如下所示：
(i)小鼠MG53 S255A的正向和反向引物，小鼠MG53 S255A表示小鼠的野生型MG53(即，SEQ ID NO:2)的第255位丝氨酸突变为丙氨酸的MG53突变体。
小鼠MG53 S255A正向引物：tgcagaagatcctgtcagaggcaccaccaccgg(SEQ ID NO:59)
小鼠MG53 S255A反向引物：tctagccttgccggtggtggtgcctctgacagg(SEQ ID NO:60)
(ii)小鼠MG53 S255G的正向和反向引物，小鼠MG53 S255G表示小鼠的野生型MG53(即，SEQ ID NO:2)的第255位丝氨酸突变为甘氨酸的MG53突变体。
小鼠MG53 S255G正向引物：tgcagaagatcctgtcagagggaccaccaccgg(SEQ ID NO:61)
小鼠MG53 S255G反向引物：tctagccttgccggtggtggtccctctgacagg(SEQ ID NO:62)
(iii)小鼠MG53 S255L的正向和反向引物，小鼠MG53 S255L表示小鼠的野生型MG53(即，SEQ ID NO:2)的第255位丝氨酸突变为亮氨酸的MG53突变体。
小鼠MG53 S255L正向引物：tgcagaagatcctgtcagagttaccaccaccgg(SEQ ID NO:63)
小鼠MG53 S255L反向引物：tctagccttgccggtggtggtaactctgacagg(SEQ ID NO:64)
(iv)小鼠MG53 S255W的正向和反向引物，小鼠MG53 S255W表示小鼠的野生型MG53(即，SEQ ID NO:2)的第255位丝氨酸突变为色氨酸的MG53突变体。
小鼠MG53 S255W正向引物：tgcagaagatcctgtcagagtggccaccaccgg(SEQ ID NO:65)
小鼠MG53 S255W反向引物：tctagccttgccggtggtggccactctgacagg(SEQ ID NO:66)
(v)小鼠MG53 S255Q的正向和反向引物，小鼠MG53 S255Q表示小鼠的野生型MG53(即，SEQ ID NO:2)的第255位丝氨酸突变为谷氨酰胺的MG53突变体。
小鼠MG53 S255Q正向引物：tgcagaagatcctgtcagagcaaccaccaccgg(SEQ ID NO:67)
小鼠MG53 S255Q反向引物：tctagccttgccggtggtggttgctctgacagg(SEQ ID NO:68)
(vi)小鼠MG53 S255Y的正向和反向引物，小鼠MG53 S255Y表示小鼠的野生型MG53(即，SEQ ID NO:2)的第255位丝氨酸突变为酪氨酸的MG53突变体。
小鼠MG53 S255Y正向引物：tgcagaagatcctgtcagagtatccaccaccgg(SEQ ID NO:69)
小鼠MG53 S255Y反向引物：tctagccttgccggtggtggatactctgacagg(SEQ ID NO:70)
(vii)小鼠MG53 S255D的正向和反向引物，小鼠MG53 S255D表示小鼠的野生型MG53(即，SEQ ID NO:2)的第255位丝氨酸突变为天冬氨酸的MG53突变体。
小鼠MG53 S255D正向引物：tgcagaagatcctgtcagaggatccaccaccgg(SEQ ID NO:71)
小鼠MG53 S255D反向引物：tctagccttgccggtggtggatcctctgacagg(SEQ ID NO:72)
(viii)小鼠MG53 S255R的正向和反向引物，小鼠MG53 S255R表示小鼠的野生型MG53(即，SEQ ID NO:2)的第255位丝氨酸突变为精氨酸的MG53突变体。
小鼠MG53 S255R正向引物：tgcagaagatcctgtcagagcgaccaccaccgg(SEQ ID NO:73)
小鼠MG53 S255R反向引物：tctagccttgccggtggtggtcgctctgacagg(SEQ ID NO:74)
3.大鼠MG53突变体的制备
按照人MG53 S255A突变体相同的方法制备大鼠MG53 S255A、S255G、S255L、S255W、S255Q、S255Y、S255D、S255R突变体。除了突变引物不同之外，其他步骤均与人MG53 S255A突变体的制备步骤相同。各突变体的引物如下所示：
(i)大鼠MG53 S255A的正向和反向引物，大鼠MG53 S255A表示大鼠的野生型MG53(即，SEQ ID NO:3)的第255位丝氨酸突变为丙氨酸的MG53突变体。
大鼠MG53 S255A正向引物：tgcagaagattctgtcagaggcaccacccccag(SEQ ID NO:75)
大鼠MG53 S255A反向引物：tctagccttgctgggggtggtgcctctgacaga(SEQ ID NO:76)
(ii)大鼠MG53 S255G的正向和反向引物，大鼠MG53 S255G表示大鼠的野生型MG53(即，SEQ ID NO:3)的第255位丝氨酸突变为甘氨酸的MG53突变体。
大鼠MG53 S255G正向引物：tgcagaagattctgtcagagggaccacccccag(SEQ ID NO:77)
大鼠MG53 S255G反向引物：tctagccttgctgggggtggtccctctgacaga(SEQ ID NO:78)
(iii)大鼠MG53 S255L的正向和反向引物，大鼠MG53 S255L表示大鼠的野生型MG53(即，SEQ ID NO:3)的第255位丝氨酸突变为亮氨酸的MG53突变体。
大鼠MG53 S255L正向引物：tgcagaagattctgtcagagttaccacccccag(SEQ ID NO:79)
大鼠MG53 S255L反向引物：tctagccttgctgggggtggtaactctgacaga(SEQ ID NO:80)
(iv)大鼠MG53 S255W的正向和反向引物，大鼠MG53 S255W表示大鼠的野生型MG53(即，SEQ ID NO:3)的第255位丝氨酸突变为色氨酸的MG53突变体。
大鼠MG53 S255W正向引物：tgcagaagattctgtcagagtggccacccccag(SEQ ID NO:81)
大鼠MG53 S255W反向引物：tctagccttgctgggggtggccactctgacaga(SEQ ID NO:82)
(v)大鼠MG53 S255Q的正向和反向引物，大鼠MG53 S255Q表示大鼠的野生型MG53(即，SEQ ID NO:3)的第255位丝氨酸突变为谷氨酰胺的MG53突变体。
大鼠MG53 S255Q正向引物：tgcagaagattctgtcagagcaaccacccccag(SEQ ID NO:83)
大鼠MG53 S255Q反向引物：tctagccttgctgggggtggttgctctgacaga(SEQ ID NO:84)
(vi)大鼠MG53 S255Y的正向和反向引物，大鼠MG53 S255Y表示大鼠的野生型MG53(即，SEQ ID NO:3)的第255位丝氨酸突变为酪氨酸的MG53突变体。
大鼠MG53 S255Y正向引物：tgcagaagattctgtcagagtatccacccccag(SEQ ID NO:85)
大鼠MG53 S255Y反向引物：tctagccttgctgggggtggatactctgacaga(SEQ ID NO:86)
(vii)大鼠MG53 S255D的正向和反向引物，大鼠MG53 S255D表示大鼠的野生型MG53(即，SEQ ID NO:3)的第255位丝氨酸突变为天冬氨酸的MG53突变体。
大鼠MG53 S255D正向引物：tgcagaagattctgtcagaggatccacccccag(SEQ ID NO:87)
大鼠MG53 S255D反向引物：tctagccttgctgggggtggatcctctgacaga(SEQ ID NO:88)
(viii)大鼠MG53 S255R的正向和反向引物，大鼠MG53 S255R表示大鼠的野生型MG53(即，SEQ ID NO:3)的第255位丝氨酸突变为精氨酸的MG53突变体。
大鼠MG53 S255R正向引物：tgcagaagattctgtcagagcgaccacccccag(SEQ ID NO:89)
大鼠MG53 S255R反向引物：tctagccttgctgggggtggtcgctctgacaga(SEQ ID NO:90)
4.猴子MG53突变体的制备
按照人MG53 S255A突变体相同的方法制备猴子MG53 S255A、S255G、S255L、S255W、S255Q、S255Y、S255D、S255R突变体。除了突变引物不同之外，其他步骤均与人MG53 S255A突变体的制备步骤相同。各突变体的引物如下所示：
(i)猴子MG53 S255A的正向和反向引物，猴子MG53 S255A表示猴子的野生型MG53(即，SEQ ID NO:4)的第255位丝氨酸突变为丙氨酸的MG53突变体。
猴子MG53 S255A正向引物：aagatcctggcagaggctcccccacccgcccgtctg(SEQ ID NO:91)
猴子MG53 S255A反向引物：cagacgggcgggtgggggagcctctgccaggatctt(SEQ ID NO:92)
(ii)猴子MG53 S255G的正向和反向引物，猴子MG53 S255G表示猴子的野生型MG53(即，SEQ ID NO:4)的第255位丝氨酸突变为甘氨酸的MG53突变体。
猴子MG53 S255G正向引物：aagatcctggcagagggtcccccacccgcccgtctgg(SEQ ID NO:93)
猴子MG53 S255G反向引物：ccagacgggcgggtgggggaccctctgccaggatctt(SEQ ID NO:94)
(iii)猴子MG53 S255L的正向和反向引物，猴子MG53 S255L表示猴子的野生型MG53(即，SEQ ID NO:4)的第255位丝氨酸突变为亮氨酸的MG53突变体。
猴子MG53 S255L正向引物：agatcctggcagagttacccccacccgcccgtctgga(SEQ ID NO:95)
猴子MG53 S255L反向引物：tccagacgggcgggtgggggtaactctgccaggatct(SEQ ID NO:96)
(iv)猴子MG53 S255W的正向和反向引物，猴子MG53 S255W表示猴子的野生型MG53(即，SEQ ID NO:4)的第255位丝氨酸突变为色氨酸的MG53突变体。
猴子MG53 S255W正向引物：agatcctggcagagtggcccccacccgcccgtctgga(SEQ ID NO:97)
猴子MG53 S255W反向引物：tccagacgggcgggtgggggccactctgccaggatct(SEQ ID NO:98)
(v)猴子MG53 S255Q的正向和反向引物，猴子MG53 S255Q表示猴子的野生型MG53(即，SEQ ID NO:4)的第255位丝氨酸突变为谷氨酰胺的MG53突变体。
猴子MG53 S255Q正向引物：aagatcctggcagagcaacccccacccgcccgtctgga(SEQ ID NO:99)
猴子MG53 S255Q反向引物：tccagacgggcgggtgggggttgctctgccaggatctt(SEQ ID NO:100)
(vi)猴子MG53 S255Y的正向和反向引物，猴子MG53 S255Y表示猴子的野生型MG53(即，SEQ ID NO:4)的第255位丝氨酸突变为酪氨酸的MG53突变体。
猴子MG53 S255Y正向引物：agatcctggcagagtatcccccacccgcccgtctgg(SEQ ID NO:101)
猴子MG53 S255Y反向引物：ccagacgggcgggtgggggatactctgccaggatct(SEQ ID NO:102)
(vii)猴子MG53 S255D的正向和反向引物，猴子MG53 S255D表示猴子的野生型MG53(即，SEQ ID NO:4)的第255位丝氨酸突变为天冬氨酸的MG53突变体。
猴子MG53 S255D正向引物：aagatcctggcagaggatcccccacccgcccgtctgg(SEQ ID NO:103)
猴子MG53 S255D反向引物：ccagacgggcgggtgggggatcctctgccaggatctt(SEQ ID NO:104)
(viii)猴子MG53 S255R的正向和反向引物，猴子MG53 S255R表示猴子的野生型MG53(即，SEQ ID NO:4)的第255位丝氨酸突变为精氨酸的MG53突变体。
猴子MG53 S255R正向引物：aagatcctggcagagcgtcccccacccgcccgtctgg(SEQ ID NO:105)
猴子MG53 S255R反向引物：ccagacgggcgggtgggggacgctctgccaggatctt(SEQ ID NO:106)
5.猪MG53突变体的制备
按照人MG53 S255A突变体相同的方法制备猪MG53 S255A、S255G、S255L、S255W、S255Q、S255Y、S255D、S255R突变体。除了突变引物不同之外，其他步骤均与人MG53 S255A突变体的制备步骤相同。各突变体的引物如下所示：
(i)猪MG53 S255A的正向和反向引物，猪MG53 S255A表示猪的野生型MG53(即，SEQ ID NO:5)的第255位丝氨酸突变为丙氨酸的MG53突变体。
猪MG53 S255A正向引物：aagatcctggcagaggcgcccccacctgcccgcctg(SEQ ID NO:107)
猪MG53 S255A反向引物：caggcgggcaggtgggggcgcctctgccaggatctt(SEQ ID NO:108)
(ii)猪MG53 S255G的正向和反向引物，猪MG53 S255G表示猪的野生型MG53(即，SEQ ID NO:5)的第255位丝氨酸突变为甘氨酸的MG53突变体。
猪MG53 S255G正向引物：aagatcctggcagaggggcccccacctgcccgcctgg(SEQ ID NO:109)
猪MG53 S255G反向引物：ccaggcgggcaggtgggggcccctctgccaggatctt(SEQ ID NO:110)
(iii)猪MG53 S255L的正向和反向引物，猪MG53 S255L表示猪的野生型MG53(即，SEQ ID NO:5)的第255位丝氨酸突变为亮氨酸的MG53突变体。
猪MG53 S255L正向引物：agatcctggcagagttgcccccacctgcccgcctgg(SEQ ID NO:111)
猪MG53 S255L反向引物：ccaggcgggcaggtgggggcaactctgccaggatct(SEQ ID NO:112)
(iv)猪MG53 S255W的正向和反向引物，猪MG53 S255W表示猪的野生型MG53(即，SEQ ID NO:5)的第255位丝氨酸突变为色氨酸的MG53突变体。
猪MG53 S255W正向引物：agatcctggcagagtggcccccacctgcccgcctgg(SEQ ID NO:113)
猪MG53 S255W反向引物：ccaggcgggcaggtgggggccactctgccaggatct(SEQ ID NO:114)
(v)猪MG53 S255Q的正向和反向引物，猪MG53 S255Q表示猪的野生型MG53(即，SEQ ID NO:5)的第255位丝氨酸突变为谷氨酰胺的MG53突变体。
猪MG53 S255Q正向引物：aagatcctggcagagcagcccccacctgcccgcctgg(SEQ ID NO:115)
猪MG53 S255Q反向引物：ccaggcgggcaggtgggggctgctctgccaggatctt(SEQ ID NO:116)
(vi)猪MG53 S255Y的正向和反向引物，猪MG53 S255Y表示猪的野生型MG53(即，SEQ ID NO:5)的第255位丝氨酸突变为酪氨酸的MG53突变体。
猪MG53 S255Y正向引物：agatcctggcagagtatcccccacctgcccgcctgga(SEQ ID NO:117)
猪MG53 S255Y反向引物：tccaggcgggcaggtgggggatactctgccaggatct(SEQ ID NO:118)
(vii)猪MG53 S255D的正向和反向引物，猪MG53 S255D表示猪的野生型MG53(即，SEQ ID NO:5)的第255位丝氨酸突变为天冬氨酸的MG53突变体。
猪MG53 S255D正向引物：aagatcctggcagaggatcccccacctgcccgcctgga(SEQ ID NO:119)
猪MG53 S255D反向引物：tccaggcgggcaggtgggggatcctctgccaggatctt(SEQ ID NO:120)
(viii)猪MG53 S255R的正向和反向引物，猪MG53 S255R表示猪的野生型MG53(即，SEQ ID NO:5)的第255位丝氨酸突变为精氨酸的MG53突变体。
猪MG53 S255R正向引物：aagatcctggcagagcggcccccacctgcccgcctgg(SEQ ID NO:121)
猪MG53 S255R反向引物：ccaggcgggcaggtgggggccgctctgccaggatctt(SEQ ID NO:122)
6.狗MG53突变体的制备
按照人MG53 S255A突变体相同的方法制备狗MG53 S255A、S255G、S255L、S255W、S255Q、S255Y、S255D、S255R突变体。除了突变引物不同之外，其他步骤均与人MG53 S255A突变体的制备步骤相同。各突变体的引物如下所示：
(i)狗MG53 S255A的正向和反向引物，狗MG53 S255A表示狗的野生型MG53(即，SEQ ID NO:6)的第255位丝氨酸突变为丙氨酸的MG53突变体。
狗MG53 S255A正向引物：aagatcctggcagaagcaccaccgcctgcccgtttg(SEQ ID NO:123)
狗MG53 S255A反向引物：caaacgggcaggcggtggtgcttctgccaggatctt(SEQ ID NO:124)
(ii)狗MG53 S255G的正向和反向引物，狗MG53 S255G表示狗的野生型MG53(即，SEQ ID NO:6)的第255位丝氨酸突变为甘氨酸的MG53突变体。
狗MG53 S255G正向引物：aagatcctggcagaaggaccaccgcctgcccgtttgg(SEQ ID NO:125)
狗MG53 S255G反向引物：ccaaacgggcaggcggtggtccttctgccaggatctt(SEQ ID NO:126)
(iii)狗MG53 S255L的正向和反向引物，狗MG53 S255L表示狗的野生型MG53(即，SEQ ID NO:6)的第255位丝氨酸突变为亮氨酸的MG53突变体。
狗MG53 S255L正向引物：agatcctggcagaattaccaccgcctgcccgtttgg(SEQ ID NO:127)
狗MG53 S255L反向引物：ccaaacgggcaggcggtggtaattctgccaggatct(SEQ ID NO:128)
(iv)狗MG53 S255W的正向和反向引物，狗MG53 S255W表示狗的野生型MG53(即，SEQ ID NO:6)的第255位丝氨酸突变为色氨酸的MG53突变体。
狗MG53 S255W正向引物：agatcctggcagaatggccaccgcctgcccgtttgga(SEQ ID NO:129)
狗MG53 S255W反向引物：tccaaacgggcaggcggtggccattctgccaggatct(SEQ ID NO:130)
(v)狗MG53 S255Q的正向和反向引物，狗MG53 S255Q表示狗的野生型MG53(即，SEQ ID NO:6)的第255位丝氨酸突变为谷氨酰胺的MG53突变体。
狗MG53 S255Q正向引物：aagatcctggcagaacaaccaccgcctgcccgtttgg(SEQ ID NO:131)
狗MG53 S255Q反向引物：ccaaacgggcaggcggtggttgttctgccaggatctt(SEQ ID NO:132)
(vi)狗MG53 S255Y的正向和反向引物，狗MG53 S255Y表示狗的野生型MG53(即，SEQ ID NO:6)的第255位丝氨酸突变为酪氨酸的MG53突变体。
狗MG53 S255Y正向引物：agatcctggcagaatatccaccgcctgcccgtttgga(SEQ ID NO:133)
狗MG53 S255Y反向引物：tccaaacgggcaggcggtggatattctgccaggatct(SEQ ID NO:134)
(vii)狗MG53 S255D的正向和反向引物，狗MG53 S255D表示狗的野生型MG53(即，SEQ ID NO:6)的第255位丝氨酸突变为天冬氨酸的MG53突变体。
狗MG53 S255D正向引物：aagatcctggcagaagatccaccgcctgcccgtttgga(SEQ ID NO:135)
狗MG53 S255D反向引物：tccaaacgggcaggcggtggatcttctgccaggatctt(SEQ ID NO:136)
(viii)狗MG53 S255R的正向和反向引物，狗MG53 S255R表示狗的野生型MG53(即，SEQ ID NO:6)的第255位丝氨酸突变为精氨酸的MG53突变体。
狗MG53 S255R正向引物：aagatcctggcagaacgaccaccgcctgcccgtttgg(SEQ ID NO:137)
狗MG53 S255R反向引物：ccaaacgggcaggcggtggtcgttctgccaggatctt(SEQ ID NO:138)
可以按照人MG53 S255A突变体相同的方法制备其他物种MG53各位点丝氨酸缺失和/或突变的突变体。除了突变引物不同之外，其他步骤均与人MG53 S255A突变体的制备步骤相同。本领域技术人员可以使用本领域常规技术手段设计各突变体的引物。
实施例2：小鼠MG53 S255A突变体对小鼠野生型MG53的细胞修复功能和心脏保护功能的影响
为了验证小鼠MG53 S255A突变体(其氨基酸序列如SEQ ID NO:8所示)对小鼠 野生型MG53的细胞修复功能和心脏保护功能的影响，发明人在大鼠的原代培养的乳鼠心肌细胞(NRVM)中使用腺病毒过表达小鼠野生型MG53及小鼠MG53 S255A突变体，检测细胞的存活状况。结果如图14所示，模拟缺血再灌注损伤的缺氧复氧引起NRVM的乳酸脱氢酶(LDH)的大量释放和细胞内ATP的降低；过表达小鼠野生型MG53及小鼠MG53 S255A突变体能差不多同等程度地抑制LDH的释放和ATP的降低。这说明小鼠野生型MG53对缺血再灌注引起的细胞凋亡和坏死等损伤的具有保护作用，而小鼠MG53 S255A突变体并不影响小鼠野生型MG53的细胞修复功能和心脏保护功能，即小鼠MG53 S255位点的磷酸化不调控MG53的细胞修复功能和心脏保护功能。
小鼠MG53 S255A突变不影响MG53对RISK信号通路的激活
MG53的心脏保护功能的机制是：它是心脏保护的RISK信号通路中的重要分子，能介导小窝蛋白caveolin-3和p85-PI3K蛋白相互作用，从而激活RISK信号通路，如下游重要的信号分子AKT(参见Zhang Y.et al,Cardiovascular research 91,108-115(2011))。所以，进一步地，发明人检测小鼠MG53 S255A突变不影响MG53心脏保护的机制是否是小鼠MG53 S255A突变体能和小鼠野生型MG53同等程度地激活RISK信号通路。实验结果如图15所示，在NRVM中过表达小鼠野生型MG53能提高RISK信号通路的下游信号分子AKT的473位丝氨酸的磷酸化，即能激活AKT；而小鼠MG53S255A突变体能与小鼠野生型MG53同等程度地提高AKT的473位丝氨酸的磷酸化，即激活AKT。说明小鼠MG53 S255A突变体不影响小鼠野生型MG53对RISK信号通路的激活，即，小鼠MG53 S255的磷酸化调控不影响心脏保护的RISK信号通路。
实施例3：人或小鼠MG53 S255A突变体对野生型MG53的E3泛素连接酶活性的影响
MG53高表达引起的胰岛素信号通路的阻遏是胰岛素抵抗和代谢综合症发生发展的重要机制之一，也是MG53的重要功能之一。首先，发明人构建了小鼠野生型MG53的丝氨酸突变的表达质粒—小鼠MG53 S255A(其氨基酸序列如SEQ ID NO：8所示)。另外，发明人还构建了IRS1的表达质粒、泛素的表达质粒、小鼠野生型MG53的表达质粒。其次，发明人之前的研究表明，MG53是胰岛素底物IRS1的E3泛素连接酶，可以介导其蛋白质的蛋白酶体途径降解(Song,R.et al.Nature 494,375-379,(2013))。所以发明人将IRS1的表达质粒、泛素的表达质粒、与小鼠野生型MG53或小鼠MG53 S255A突变体的质粒共转染入HEK293T细胞系内，通过IRS1蛋白含量的变化来反应小鼠MG53 S255A突变对MG53功能的影响。实验结果如图19所示，结果表明，小鼠野生型MG53可以极大程度上降低IRS1的表达量；而小鼠MG53 S255A突变体能显著地抑制MG53介导的IRS1的蛋白量降低。说明在HEK293T细胞系的共表达体系中，小鼠野生型MG53可以发挥正常的E3泛素连接酶功能，介导IRS1的泛素化降解；检测到的小鼠MG53 S255A突变体抑制了MG53的E3泛素连接酶功能，即MG53的S255位点的磷酸化状态能调控MG53的E3泛素连接酶功能。
人或小鼠MG53 S255A突变体抑制MG53介导的底物降解
MG53是RING结构域型E3泛素连接酶，MG53的RING结构域与E2泛素交联酶相结合介导底物的泛素化和进一步的蛋白酶降解。所以RING结构域切除的截短体MG53-D-RING就丧失了野生型MG53的E3泛素连接酶功能。进一步地，为了定量地衡量人MG53 S255A突变体(其氨基酸序列如SEQ ID NO:7所示)或小鼠MG53 S255A突变体(其氨基酸序列如SEQ ID NO:8所示)对人或小鼠野生型MG53的E3泛素连接酶功能的影响，发明人以人或小鼠MG53-D-RING为阳性对照，在人胚肾上皮细胞系HEK293T中共表达人或小鼠野生型MG53、MG53 S255A突变体、或MG53-D-RING截短体和MG53的底物IRS1或胰岛素受体IR，检测不同的突变对MG53介导的底物的降解的影响。结果如图16(小鼠)和图17(人)所示，人或小鼠野生型MG53可以极大程度地介导IRS1的降解；人或小鼠MG53-D-RING截短体几乎完全不能介导IRS1的降解；而小鼠MG53 S255A突变体只能介导IRS1约40％的降解，人MG53 S255A突变体只能介导IRS1约10％的降解。小鼠野生型MG53可以介导约50％的IRβ的降解；小鼠MG53-D-RING截短体和小鼠MG53 S255A突变体几乎完全不能介导IRβ的降解。另外，在HEK293T的共表达系统中，小鼠野生型MG53可以介导IRβ蛋白的前体蛋白的降解，同时小鼠MG53-D-RING截短体和小鼠MG53 S255A突变体几乎完全抑制这种MG53介导的降解作用。本实验说明，人或小鼠MG53-D-RING截短体极显著地抑制了MG53的E3泛素连接酶功能；而人或小鼠MG53 S255A突变体大约能抑制MG53 50％以上的E3泛素连接酶功能。
小鼠MG53 S255A突变体抑制MG53与底物IRS1蛋白的结合
为了检测小鼠MG53 S255A突变体(其氨基酸序列如SEQ ID NO:8所示)是否影响MG53与底物IRS1蛋白的识别和结合，发明人一方面在离体的HEK293T细胞系中 通过免疫共沉淀检测活性状态下底物IRS1与小鼠野生型MG53或小鼠MG53 S255A突变体的结合能力；另一方面通过表面等离子体共振(surface plasmon resonance,SPR)实验检测纯化的蛋白IRS1和小鼠野生型MG53或小鼠MG53 S255A突变体的直接结合能力。实验结果如图18所示，图18A显示IRS1可以和小鼠野生型MG53进行物理性结合；而同样量的IRS1只能结合极少量的小鼠MG53 S255A突变体；图18B显示小鼠野生型MG53蛋白可以与IRS1蛋白很好的结合，KD＝64.2nM；而小鼠MG53 S255A突变体蛋白不能和IRS1蛋白结合。由此可见，小鼠MG53 S255A突变体抑制了MG53对底物IRS1蛋白的识别和结合，从而抑制了MG53的E3泛素连接酶活性。
实施例4：人MG53 S255A突变体的体内活性
为了验证人MG53 S255A突变体(其氨基酸序列如SEQ ID NO:7所示)在体内是否仍然具有细胞修复功能和心脏保护功能，并且同时避免或减少了野生型MG53所带来的代谢类副作用，发明人通过将人MG53 S255A突变体和人野生型MG53分别引入大鼠体内，进一步分析人MG53 S255A突变体的体内活性。首先，发明人根据实施例1制备人MG53 S255A突变体。其次，选择15只雄性Sprague-Dawley(SD)大鼠(每只约250g，由北京维通利华实验动物有限公司提供)，分成三组，实验组、阳性对照组、阴性对照组，每组分别5只。30mg/kg戊巴比妥钠腹腔注射麻醉大鼠之后，在左侧第三肋间开胸，暴露出心脏。然后，向实验组大鼠心脏静脉注射人MG53 S255A突变体蛋白(6mg/kg，iv)，向阳性对照组大鼠心脏静脉注射人野生型MG53蛋白(其氨基酸序列如SEQ ID NO:1所示)(6mg/kg，iv)，向阴性对照组大鼠心脏静脉注射牛血清白蛋白(BSA)(6mg/kg，iv)。各组大鼠分别在静脉注射上述蛋白5分钟后结扎心脏冠状动脉左前降支，并持续45分钟。结扎结束之前，各组大鼠再次静脉注射相应的人MG53 S255A突变体蛋白、人野生型MG53蛋白以及BSA(6mg/kg，iv)，然后松开冠状动脉。再灌注24小时之后，24小时内未死亡的大鼠定义为存活，死亡定义为死亡，以此结果比较实验组、阳性对照组和阴性对照组大鼠的生存率。使用胰岛素刺激各组大鼠，随后处死大鼠，比较实验组、阳性对照组和阴性对照组大鼠的心肌梗塞面积、血清LDH浓度以及心脏病理切片TUNEL染色情况，进行各组大鼠心脏损伤评价。实验组大鼠的心肌梗塞面积显著性地小于阴性对照组大鼠的心肌梗塞面积，或者实验组大鼠与阴性对照组大鼠相比能够显著性地抑制LDH释放，或者实验组大鼠的心脏病理切片TUNEL染色结果显著性地优于阴性对照组大鼠的心脏病理切片TUNEL 染色结果，则表明人MG53 S255A突变体并不影响人野生型MG53的细胞修复功能和心脏保护功能，能够用于治疗心脏疾病，例如心肌梗塞、心脏缺血/再灌注损伤等。最后比较实验组、阳性对照组和阴性对照组大鼠各组织(例如，心肌、骨骼肌、肝脏)里p-AKT/t-AKT的变化情况，从而评价实验组、阳性对照组和阴性对照组大鼠各组织的胰岛素反应性。阳性对照组大鼠的组织里p-AKT/t-AKT比值下降，表明人野生型MG53蛋白引起胰岛素敏感性下降；阴性对照组大鼠的组织里p-AKT/t-AKT比值正常，表明BSA对胰岛素敏感性无影响；实验组大鼠的组织里p-AKT/t-AKT比值比阳性对照组高，表明人MG53 S255A突变体可以消除或减弱人野生型MG53蛋白引起的胰岛素敏感性下降。
综上可知，MG53 coiled-coil-SPRY结构域内的至少一个丝氨酸(特别是第255位丝氨酸)缺失或突变为任何其他非丝氨酸或苏氨酸(例如，丙氨酸)的氨基酸时，产生的MG53突变体在具有细胞修复功能和/或心脏保护功能的同时，又避免或减少了胰岛素抵抗等代谢类副作用。
虽然本发明通过引用特定实施例的方式对发明进行特定的展现和描述，但是本领域技术人员都应该理解，在不脱离本发明披露的主旨和保护范围的情况下，上述内容还可以进行各种形式和细节上的变化。

Claims (35)

  1. 一种MG53突变体，其特征在于，所述MG53突变体在野生型MG53的氨基酸序列的基础上，在所述野生型MG53的coiled-coil-SPRY区域内具有至少一个丝氨酸缺失和/或突变为任何其他非丝氨酸或苏氨酸的氨基酸。
  2. 根据权利要求1所述的MG53突变体，其中所述coiled-coil-SPRY区域位于野生型MG53的氨基酸序列的第122-477位氨基酸区域。
  3. 根据权利要求1或2所述的MG53突变体，其特征在于，所述野生型MG53的氨基酸序列为SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5或者SEQ ID NO:6所示的氨基酸序列。
  4. 根据权利要求1-3中任一项所述的MG53突变体，其特征在于，所述丝氨酸突变为非极性氨基酸。
  5. 根据权利要求4所述的MG53突变体，其特征在于，所述非极性氨基酸选自下组：甘氨酸、丙氨酸、亮氨酸、异亮氨酸、缬氨酸、脯氨酸、苯丙氨酸、甲硫氨酸、色氨酸。
  6. 根据权利要求5所述的MG53突变体，其特征在于，所述非极性氨基酸为丙氨酸。
  7. 根据权利要求1-6中任一项所述的MG53突变体，其特征在于，所述丝氨酸突变为除丝氨酸和苏氨酸之外的极性氨基酸。
  8. 根据权利要求7所述的MG53突变体，其特征在于，所述极性氨基酸选自下组：谷氨酰胺、半胱氨酸、天冬酰胺、酪氨酸、天冬氨酸、谷氨酸、赖氨酸、精氨酸、组氨酸。
  9. 根据权利要求8所述的MG53突变体，其特征在于，所述极性氨基酸为半胱氨酸。
  10. 根据权利要求1-9中任一项所述的MG53突变体，其特征在于，发生缺失或突变的丝氨酸位于所述野生型MG53的氨基酸序列的以下一个或多个位点：第189位、第211位、第214位、第246位、第255位、第269位、第296位、第301位、第305位、第307位、第341位、第377位、第418位、第430位。
  11. 根据权利要求1-10中任一项所述的MG53突变体，其特征在于，发生缺失或突变的丝氨酸位于所述野生型MG53的氨基酸序列的以下一个或多个位点：第211位、第214位、第246位、第255位、第269位、第296位、第297位。
  12. 根据权利要求1-11中任一项所述的MG53突变体，其特征在于，发生缺失或突变的丝氨酸位于所述野生型MG53的氨基酸序列的第255位。
  13. 根据权利要求12所述的MG53突变体，其特征在于，所述MG53突变体具有两个或两个以上的丝氨酸突变。
  14. 根据权利要求1-13中任一项所述的MG53突变体，其特征在于，所述MG53突变体的氨基酸序列与SEQ ID NO:7-12所示的任一氨基酸序列具有至少70％的同源性，并且所述MG53突变体在具有细胞修复功能和/或心脏保护功能的同时，避免了野生型MG53所带来的代谢类副作用。
  15. 根据权利要求1-14中任一项所述的MG53突变体，其特征在于，所述MG53突变体的氨基酸序列为SEQ ID NO:7-12所示的任一氨基酸序列。
  16. 根据权利要求1-15中任一项所述的MG53突变体，其特征在于，所述MG53突变体的氨基酸序列为SEQ ID NO:7所示的氨基酸序列。
  17. 一种药物组合物，其特征在于，包括权利要求1-16中任一项所述的MG53突变体和药学上可接受的载体。
  18. 一种分离的核酸，其包含编码权利要求1-16中任一项所述的MG53突变体的氨基酸序列的核酸序列。
  19. 根据权利要求18所述的核酸，其包含SEQ ID NO:13-18所示的任一核酸序列。
  20. 一种表达载体，其包含权利要求18所述的核酸序列。
  21. 根据权利要求20所述的表达载体，其包含SEQ ID NO:13-18所示的任一核酸序列。
  22. 一种宿主细胞，其包含如权利要求20或21所示的表达载体。
  23. 一种权利要求1-16中任一项所述的MG53突变体的制备方法，其特征在于，确定需要进行突变的丝氨酸的一个或多个位置，对包含编码野生型MG53氨基酸序列的核酸序列的质粒的全长序列进行在所述位置的定点突变，将所述定点突变的质粒转染到宿主细胞，表达所述宿主细胞以产生所述MG53突变体。
  24. 根据权利要求23所述的制备方法，其特征在于，所述定点突变包括如下步骤：
    (1)确定需要定点突变的氨基酸在cDNA序列中相应的核苷酸位置，根据突变后的氨基酸修改突变位置的核苷酸序列，并截取包含突变位置的长度为20-40bp的序列设计引物；
    (2)以野生型MG53质粒为模板，使用步骤(1)中设计的引物进行PCR反应，PCR产物进行琼脂糖凝胶电泳，并对PCR产物进行纯化；
    (3)步骤(2)中纯化后的PCR产物使用核酸限制性内切酶进行酶切反应，并将酶切产物与合适的质粒表达载体进行连接，将连接产物进行细菌感受态细胞的转化，培养；
    (4)挑取步骤(3)所得克隆使用步骤(1)所设计的引物进行菌落PCR鉴定，对PCR产物进行琼脂糖凝胶电泳检测，然后进行DNA测序鉴定，鉴定出带有所述定点突变的阳性克隆。
  25. 根据权利要求1-16中任一项所述的MG53突变体在制备用于治疗心脏疾病、糖尿病脑血管疾病、糖尿病眼部并发症、糖尿病神经病变、糖尿病足、肾脏疾病、与细胞和/或组织损伤相关疾病的药物中的用途。
  26. 根据权利要求25所述的用途，其特征在于，所述药物在治疗心脏疾病、糖尿病脑血管疾病、糖尿病眼部并发症、糖尿病神经病变、糖尿病足、肾脏疾病、与细胞和/或组织损伤相关疾病的同时避免了代谢类副作用。
  27. 根据权利要求25或26所述的用途，其特征在于，所述心脏疾病是与心肌损伤相关的疾病。
  28. 根据权利要求25-27中任一项所述的用途，其特征在于，所述心脏疾病包括糖尿病性心脏病、心肌缺血、心脏缺血/再灌注损伤、心肌梗塞、心力衰竭、心律失常、心脏破裂、心绞痛、心肌炎、冠心病、心包炎。
  29. 根据权利要求25或26所述的用途，其特征在于，所述糖尿病脑血管疾病包括脑动脉硬化、缺血性脑血管病、脑出血、脑萎缩、脑梗塞。
  30. 根据权利要求25或26所述的用途，其特征在于，所述糖尿病眼部并发症包括糖尿病性视网膜病变、糖尿病性白内障、与糖尿病相关的葡萄膜炎、失明。
  31. 根据权利要求25或26所述的用途，其特征在于，所述糖尿病神经病变包括糖尿病周围神经病变。
  32. 根据权利要求25或26所述的用途，其特征在于，所述肾脏疾病包括急性肾小球肾炎、慢性肾小球肾炎、肾病综合征、急性肾损伤、糖尿病肾病。
  33. 根据权利要求25或26所述的用途，其特征在于，所述与细胞和/或组织损伤相关疾病包括与肾脏、脑、肺部、肝脏、心脏、脾脏、消化道、皮肤损伤相关的疾病。
  34. 根据权利要求33所述的用途，其特征在于，所述与细胞和/或组织损伤相关疾病包括外伤、烧伤、溃疡、黏膜炎、哮喘、慢性阻塞性肺疾病、中风、皮肤老化。
  35. 根据权利要求25-34中任一项所述的用途，其特征在于，所述MG53突变体的氨基酸序列为SEQ ID NO:7所示的氨基酸序列。
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