KR20170123114A - Screening method of agent for treating muscle disease - Google Patents

Abstract

Binding of fibromodulin to specific amino acid of myostatin prevents coupling of myostatin and activin receptor type IIB (ACVRIIB), thereby promoting differentiation of muscular cells. Based on this, provided are a method for screening treatment agents for muscular disease, a method for screening improvers for meat amount of livestock, a method for screening supplementary agents for myogenesis, or a composition for promoting myodifferentiation.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention [0001]

The present invention relates to a method for inhibiting the binding of myostatin to a 2B type activin receptor (activin receptor type IIB, ACVR IIB) through binding with a specific amino acid of myostatin, And a method for screening a disease therapeutic agent.

Muscle hypofunction, a disease caused by muscle weakness, is defined as the loss of muscle mass associated with aging, which is increasingly recognized as leading to significant consequences for the elderly because muscle hypoxia can be associated with debility, disability and morbidity It is because. The prevalence of myopenia increases from 13-24% among people under 70 years of age to over 50% between those over 80 years of age. Muscle weakness associated with myopenia is known to be associated with increased risk of fatigue, decreased ability to perform the job necessary for independent living, and fractures such as but not limited to femoral neck fractures.

In addition, muscular dystrophy, another disease caused by muscle weakness, is caused by progressive regression of skeletal muscle fibers. A deficiency in one of several proteins located within the plasma membrane or inner membrane increases the likelihood of injury during shrinkage and ultimately leads to fiber regression with severe local inflammation accompanied by immune-competent cell invasion.

Muscular atrophy includes a group of hereditary, progressive muscle disorders that are clinically distinguished by the selective distribution of skeletal muscle weakness. The two most common forms of muscular atrophy are Duchenne and Becker atrophy, resulting from the inheritance of mutations in the dystrophin gene, each located at the Xp21 locus. Other muscular dystrophies include, but are not limited to, limb-girdle muscular dystrophy, fascioscapulohumeral (Landouzy-Dejerine) muscular dystrophy, congenital muscular dystrophy, myotonic dystrophy, (Emery-Dreifuss) < / RTI >

The most serious form is dysmenorrhoea, the regeneration is exhausted, and the skeletal muscle is progressively replaced by fat and fibrous tissue. This symptom is a progressive weakening of the patient, resulting in death due to breathing or heart attack.

Symptoms of dysmenorrhoea are limited to men only, beginning at about 3 to 7 years of age, and most patients are required to move to wheelchairs at age 10 to 12 years and die at 20 years of age due to respiratory complications do.

On the other hand, only corticosteroids such as prednisone, prednisolone, and deflazacort have shown potential for temporary improvement among several medicines that have been tried as potential treatments for muscular atrophy. However, this is not a fundamental treatment because it is mainly due to slowing down or stabilizing muscle and muscle function, and corticosteroid therapy also has side effects.

Therefore, there is a need to develop treatments that can treat muscle loss, which is the cause of myopathy and myopathy.

1. Korean Patent No. 10-1122843.

Accordingly, it is an object of the present invention to provide a screening method for treating a muscle disease drug.

It is another object of the present invention to provide a method for screening animal growth promoters.

In addition, the present invention has another object to provide a method for screening muscle building-aid.

In addition, the present invention has another object to provide a composition for promoting muscle differentiation.

In order to achieve the above object, the present invention includes a method of screening a substance binding to one or more amino acids selected from the group consisting of Ile98, Gly68, Trp31, Trp29, Phe27, Tyr42 and Leu20 of myostatin (MSTN) And a method for screening a therapeutic agent for muscle diseases.

The present invention also provides a method for screening a substance binding to one or more amino acids selected from the group consisting of Ile98, Gly68, Trp31, Trp29, Phe27, Tyr42 and Leu20 of myostatin (MSTN) And a method for screening the animal growth promoting agent.

The present invention also provides a method for screening a substance binding to one or more amino acids selected from the group consisting of Ile98, Gly68, Trp31, Trp29, Phe27, Tyr42 and Leu20 of myostatin (MSTN) And a method for screening a muscle forming auxiliary agent.

In order to achieve the above-mentioned further object, the present invention provides a pharmaceutical composition comprising a substance which binds to at least one amino acid selected from the group consisting of Ile98, Gly68, Trp31, Trp29, Phe27, Tyr42 and Leu20 of myostatin (MSTN) And a composition for promoting muscle differentiation.

The amino acid may further include one or more amino acids selected from the group consisting of Ala70, His57, Met101, Val22 and Pro99 of myostatin (MSTN).

The present invention relates to a method for inhibiting the binding of myostatin to a specific type of amino acid of Myostatin by inhibiting the binding of myostatin to the 2B type activin receptor (Activin receptor type IIB, ACVR IIB) Based on this, a method for screening a therapeutic agent for muscle diseases, a method for screening a meat growth promoting agent for livestock, a method for screening a muscle forming auxiliary agent, or a composition for promoting muscle differentiation are provided.

FIG. 1 shows the result of observing the expression of fibromodulin (FMOD) according to differentiation, wherein A is an observation of FMOD mRNA expression, B is an observation of FMOD protein expression, C is FMOD protein expression and expression site .
FIG. 2 is a result of observing formation of myotube according to knock-down of the FMOD gene, wherein A is the observation of the cell shape according to the FMOD knockdown, B is the normal cell and FMOD gene (FOD) mRNA expression, and D and E were observed for FMOD protein expression and location, respectively.
FIG. 3 shows the results of observing the expression of a marker gene related to muscle cell differentiation according to the knockdown of the FMOD gene. A shows the mRNA expression of MYOD, MYOG (Myogenin) and MYL2 (Myosin light chain 2) MYOD, MYOG, and MYL2, and C is the expression and location of MYOD, MYOG, and MYL2 proteins.
FIG. 4 shows the results of observing the expression of myostatin (MSTN) according to knockdown of the FMOD gene, wherein A is an observation of MSTN mRNA expression, B is an observation of MSTN protein expression, C is MSTN And the expression and expression positions of the protein were observed.
FIG. 5 shows the result of analysis of mutual binding of FMOD, MSTN, 2B type activin receptor type IIB (ACVR IIB) protein through in silico analysis, A shows FMOD and MSTN binding, Is the observation of MSTN and ACVR IIB binding with FMOD and MSTN binding.
FIG. 6 shows FMOD and MSTN binding according to knockdown of FMOD gene. A shows FMOD and MSTN protein binding after 2 days of differentiation, and B shows FMOD and MSTN protein in cells knocked down by FMOD gene .

Hereinafter, the present invention will be described in detail.

The present invention relates to a method for screening a substance binding to one or more amino acids selected from the group consisting of Ile98, Gly68, Trp31, Trp29, Phe27, Tyr42 and Leu20 in the amino acid sequence represented by SEQ ID NO: 1 of myostatin (MSTN) And a method for screening a therapeutic agent for muscle diseases.

The amino acid may further comprise one or more amino acids selected from the group consisting of Ala70, His57, Met101, Val22 and Pro99 in the amino acid sequence of SEQ ID NO: 1 of myostatin (MSTN).

The MSTN is a protein belonging to the TGF-beta (Transforming Growth Factor-beta) family, and is a regulatory factor that inhibits muscle differentiation and growth during muscle differentiation. It is also known as growth differentiation factor 8 (GDF-8) have. Myostatin is mainly produced in bone cells and circulates through the blood to act on muscle tissue. Therefore, it is expected that the muscle will increase if the animal does not have the myostatin gene or inhibits myostatin activity.

The "MSTN" of the present invention may be any eukaryotic protein having MSTN including mammals such as human, bovine, goat, sheep, pig, mouse, rabbit and the like. It is preferable to have an amino acid sequence: DFGLDCDEHSTESRCCRYPLTVDFEAFGWDWIIAPKRYKANYCSGECEFVFLQKYPHTHLVHQANPRGSAGPCCTPTKMSPINMLYFNGKEQIIYGKIPAMVVDRCGCS.

The substance may bind to any one or more amino acids of the amino acid sequence of SEQ ID NO: 1 of myostatin (MSTN) to promote muscle cell differentiation. The substance may be a protein, a compound, a peptide, a peptide mimetic , Substrate analogs, aptamers, and antibodies.

According to an embodiment of the present invention, the material may be fibromodulin (FMOD).

The FMOD is a member of the small proteoglycan (SLRP) family that is rich in leucine. FMOD is a cytoplasmic secretion protein that has a pattern expressed mainly in tissues rich in cartilage, bone, connective tissue collagen, and is involved in fibrillogenesis, cell adhesion, and cytokine activity regulation. In addition, FMOD has been reported to modulate extracellular matrix by binding to the transforming growth factor (TGF) -β isoforms and collagen.

The "FMOD" of the present invention may be any eukaryotic protein having an FMOD including human, bovine, goat, sheep, pig, mouse, rabbit and other mammals, preferably mouse AAH52673.1.

The above-mentioned muscle diseases may be selected from the group consisting of sarcopenia, muscular dystrophy, rigid spinesyndrome, muscle-eye-brain disease, amyotrophic lateral sclerosis (amyotrophic lateral sclerosis, Charcot-Marie-Tooth disease, chronic inflammatory neuropathy, and distal myopathy. The present invention is not limited thereto.

The present invention also provides a method for screening a substance that binds to one or more amino acids selected from the group consisting of Ile98, Gly68, Trp31, Trp29, Phe27, Tyr42 and Leu20 in the amino acid sequence represented by SEQ ID NO: 1 of myostatin (MSTN) And a method for screening a meat growth enhancer of a livestock.

The amino acid may further comprise one or more amino acids selected from the group consisting of Ala70, His57, Met101, Val22 and Pro99 in the amino acid sequence of SEQ ID NO: 1 of myostatin (MSTN).

The substance may bind to any one or more amino acids of the amino acid sequence shown in SEQ ID NO: 1 of myostatin (MSTN) to promote the differentiation of muscle cells of livestock, and is preferably used as a feed additive. But is not limited thereto.

The livestock is any one selected from the group consisting of cattle, goats, sheep, pigs, horses and rabbits, more preferably soda.

The present invention also provides a method for screening a substance that binds to one or more amino acids selected from the group consisting of Ile98, Gly68, Trp31, Trp29, Phe27, Tyr42 and Leu20 in the amino acid sequence represented by SEQ ID NO: 1 of myostatin (MSTN) And a method for screening muscle building-up supplements, which comprises a method for screening muscle building-up supplements.

The amino acid may further comprise one or more amino acids selected from the group consisting of Ala70, His57, Met101, Val22 and Pro99 in the amino acid sequence of SEQ ID NO: 1 of myostatin (MSTN).

The substance may bind to one or more amino acids of the amino acid sequence of SEQ ID NO: 1 of myostatin (MSTN) to promote muscle cell differentiation.

The present invention also relates to a composition comprising a substance which binds to one or more amino acids selected from the group consisting of Ile98, Gly68, Trp31, Trp29, Phe27, Tyr42 and Leu20 in the amino acid sequence represented by SEQ ID NO: 1 of myostatin (MSTN) And a composition for promoting muscle differentiation.

The amino acid may further comprise one or more amino acids selected from the group consisting of Ala70, His57, Met101, Val22 and Pro99 in the amino acid sequence of SEQ ID NO: 1 of myostatin (MSTN).

The substance may bind to any one or more amino acids of the amino acid sequence of SEQ ID NO: 1 of myostatin (MSTN) to promote muscle cell differentiation. The substance may be a protein, a compound, a peptide, a peptide mimetic , A substrate analog, an aptamer, and an antibody, but are not limited thereto.

The composition for promoting muscle differentiation may be used for improving or treating obesity by reducing fat by changing the fat to muscle.

When the composition for promoting muscle differentiation is used as a pharmaceutical composition for treating obesity, it may further include an appropriate carrier, excipient or diluent commonly used in the production of a pharmaceutical composition.

Examples of the carrier, excipient or diluent which can be used in the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil.

The pharmaceutical composition may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols or the like, oral preparations (ointments), suppositories and sterilized injection solutions according to a conventional method .

In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose sucrose), lactose, gelatin, and the like.

In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included .

Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of suppository bases include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like.

The pharmaceutical composition may be administered to mammals such as rats, mice, livestock, humans, and the like in a variety of routes. All modes of administration may be expected, for example, by intramuscular, oral, rectal or intravenous, subcutaneous, intrapulmonary, intrauterine, or intracerebroventricular injection.

In addition, when the composition for promoting muscle differentiation is used as a health functional food for improving obesity, it may be provided in the form of powder, granule, tablet, capsule, syrup or beverage. In addition to the composition for promoting muscle differentiation, It is used together with other food or food additives and can be suitably used according to conventional methods. The amount of the active ingredient to be mixed can be suitably determined according to its use purpose, for example, prevention, health or therapeutic treatment.

There is no particular limitation on the kind of the above health functional food and examples thereof include dairy products including meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gums, ice cream, , Drinks, alcoholic beverages and vitamin complexes.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended to illustrate the contents of the present invention, but the scope of the present invention is not limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

< Example  1> Experimental method

One. C2C12  Cell culture

Rat myofibroblast C2C12 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS, HyClone Laboratories) and 1% penicillin / streptomycin (P / S, Invitrogen) , HyClone Laboratories). For induction of differentiation, cells were cultured for 2 days, 4 days, and 6 days after replacing DMEM (differentiation medium) supplemented with 2% FBS and 1% P / S when the cells grew by about 70% or more. The medium was replaced once every two days and the cells were incubated at 37 ° C.

2. FMOD  gene Knockdown (Knock-down)

When C2C12 cells grew by about 30%, shRNA constructs (1 ng) of FMOD gene were injected into transfection reagent (Santa Cruz Biotechnology) and transfection media (Santa Cruz Biotechnology). After injection of shRNA constructs, puromycin (Santa Cruz Biotechnology) was treated with 2 μg / ml of puromycin, and only the injected cells were selected. The selected cells were grown up to about 70%, and then the cells were changed with the differentiation medium.

The shRNA sequence of the FOMD gene is shown in Table 1, purchased from Santacruz, and the mRNA of the FMOD gene was blocked by the following three sequence combinations.

shRNA Sequence SEQ ID NO:


FMOD





FMOD shRNA Plasmid (m) is a pool of 3 different shRNA plasmids
sc-44823-SHA
Hairpin sequence GATCCCTACATGGCAACCAGATTATTCAAGAGATAATCTGGTTGCCATGTAGTTTTT 2 Sense CUACAUGGCAACCAGAUUAtt 3 AntiSense UAAUCUGGUUGCCAUGUAGTT 4
sc-44823-SHB
Hairpin sequence GATCCCTACTAGACCTGAGTTATATTCAAGAGATATAACTCAGGTCTAGTAGTTTTT 5 Sense CUACUAGACCUGAGUUAUAtt 6 AntiSense UAUAACUCAGGUCUAGUAGtt 7 sc-44823-SHC
Hairpin sequence GATCCGCAACAGGATCAATGAGTTTTCAAGAGAAACTCATTGATCCTGTTGCTTTTT 8 Sense GCAACAGGAUCAAUGAGUUtt 9 AntiSense AACUCAUUGAUCCUGUUGCtt 10

3. Kimja ( Giemsa ) dyeing

Cell culture medium was removed and the cells were washed with phosphate buffered saline (PBS). After washing, 1: 1 volume ratio of methanol: PBS reagent was applied and fixed for 2 minutes. An additional 2: 1 volume ratio of methanol: PBS reagent was added followed by additional fixation for 2 minutes. After 2 minutes, 0.04% Kimja reagent was added and left for 30 minutes. After 30 minutes, the cells were washed with PBS and the appearance of the cells was observed with a microscope.

4. Fusion index

To observe the formation of myotubes in the cells, the cells were photographed three times (300x). In the photographs, the number of fused nuclei in the canaliculus cells was counted, and the number of nuclei in the total cells was counted, and the percentage of nucleated cells was divided by the number of nuclei in the total cells.

5. RNA Extraction and cDNA Synthesis

1 ml of TRIzol ™ reagent was added and the cells were pulverized using a microwave sonicator. The pulverized sample was centrifuged (12,000 rpm, 10 minutes, 4 ° C), and the supernatant was transferred to a new tube. 200 μl of chloroform was added and the mixture was left at room temperature for 10 minutes. Then, a supernatant of a transparent color was obtained by centrifugation (12,000 rpm, 10 minutes, 4 캜). Next, 500 μl of isopropanol was added, and the mixture was allowed to stand for 10 minutes and centrifuged to obtain RNA pellets. The RNA pellet was washed with 70% ethanol (distilled water treated with ethanol + diethylpyrocarbonate (DEPC)), and then completely removed and dried. DEPC treated distilled water was added to the dried transparent RNA and stored at -80 ° C. The total RNA amount was measured by Nanodrop and the 18s, 28s band was confirmed in 1.2% agarose gel. cDNA was synthesized with 1 μg of total RNA, oligo-dT primer, SuperScript ™ II RNase H reverse transcriptase (42 ° C for 50 minutes, 72 ° C for 15 minutes).

6. Confirm gene expression

Real-time PCR was performed to confirm gene expression. For real-time gene expression analysis, gene expression was analyzed using a Power SYBR ^® Green PCR Master Mix containing SYBR green fluorescent material (7500 real-time PCR system). PCR primers were designed with Primer 3 software (http://frodo.wi.mit.edu) according to the nucleotide sequence obtained from NCBI GenBank. The PCR was carried out 40 times at 95 ° C for 10 minutes, at 95 ° C for 33 seconds, at 33 ° C for 33 seconds and at 33 ° C for 33 seconds depending on the gene primer temperature (tm). The PCR products were confirmed by electrophoresis (1.2% agarose gel, 100 v 60 min), and analyzed the gene expression values from the analysis of c (t) values derived through a real-time PCR analysis (fold change 2- ^{△△ Ct} formula). The expression level of the gene in the untreated cell was set to 1 and the gene expression value of the treated cell was calculated. For the analysis of the gene c (t) value, GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) gene was used for normalization.

The sequences of the PCR primers are shown in Table 2 (MYOG (Myogenin), MYL2 (Myosin light chain2), COL1? 1 (Collagen Type I Alpha 1)).

Species Gene Product size (bp) Tm (° C) Sequence direction SEQ ID NO: Mouse



GAPDH 155 55 5'-tgctggtgctgagtatgtcg-3 ' Forward 11 5'-caagcagttggtggtacagg-3 ' Reverse 12 FMOD
155
59 5'-tgcagaagatccctcctgtc-3 ' Forward 13 5'-cttgatctcgttcccatcca-3 ' Reverse 14 MSTN
163
59
5'-acgctaccacggaaacaatc-3 ' Forward 15 5'-ggagtcttgacgggtctgag-3 ' Reverse 16 MYOG
185
59
5'-tccagtacattgagcgccta-3 ' Forward 17 5'-caaatgatctcctgggttgg-3 ' Reverse 18 MYOD
213
59
5'-aggagcacgcacacttctct-3 ' Forward 19 5'-tctcgaaggcctcattcact-3 ' Reverse 20 MYL2
177
59
5'-aaagaggctccaggtccaat-3 ' Forward 21 5'-cctctctgcttgtgtggtca-3 ' Reverse 22

7. Immunostaining ( immunocytochemistry )

The culture medium of the cultured C2C12 cells was removed and washed once with PBS. The washed cells were fixed with 4% formaldehyde (Sigma) for 15 minutes, washed with PBS, and incubated with 0.2% tipton X-100 (Sigma) for 5 minutes Respectively. The cells were washed with PBS, and incubated with enhancer sol. For 30 minutes. Primary antibodies (FMOD, MSTN: 1:50, Santa Cruz Biotechnology) were added and reacted at 4 ° C for 14 hours. Antibodies were removed, washed three times for 10 minutes with PBS, and incubated with a secondary antibody (Alexa Fluor 488 goat anti-rabbit SFX kit) for 1 hour. After incubation, the antibody was removed, washed with PBS for 10 minutes, stained with DAPI (4 ', 6-diamidino-2-phenylindol) and observed for protein expression using a fluorescence microscope.

8. Mutual immunoprecipitation (Co- immunoprecipitation )

Mutual immunoprecipitation was carried out using FMOD and MSTN antibody. Cells with and without FMOD gene expression were cultured in differentiation medium for 2 days. After removing the medium, cells were washed with PBS and incubated with non-denaturing buffer (20 mM Tris HCl (pH 8), 137 mM NaCl, 1% Nonidet P-40, 2 mM EDTA) and protease inhibitor Thermo) was added to dissolve the cells and then the proteins were extracted. 200 μg of the extracted protein was reacted with 3 μg of FMOD or MSTN antibody at 4 ° C. for 16 hours or more. After 16 hours, protein A agarose (Santa Cruz Biotechnology) was added and reacted at 4 ° C for 2 hours. After 2 hours, the cells were washed twice with non-denatured buffer and then subjected to Western blotting such as FMOD or MSTN antibody.

9. Western Blot

The culture medium of the cultured C2C12 cells was removed and washed with PBS. The washed cells were loaded with RIPA buffer (Radioimmunoprecipitation assay buffer, Thermo) and proteolytic enzyme inhibitor (Thermo), and the cells were lysed to extract the proteins. 40 μg of the extracted protein was electrophoresed on an 8 to 10% acrylamide gel and transferred to a PVDF membrane (Polyvinylidene fluoride membrane, Milipore). The cells were blocked with 3% skim milk or bovine serum albumin (BSA) at room temperature for 1 hour. Then, the primary antibodies (FMOD: 1: 400, MSTN: 1: 400, ß-actin: 1: 1000, MYOD: 1: 400, MYOG: 1: 1000, MYL2: 1) diluted in 1% : 1000) was added and reacted at 4 캜 for 16 hours or more. After 16 hours, the cells were washed three times with TBST (Tween 20-containing Tris-Buffered Saline) and the secondary antibody with HRP (horseradish peroxidase) was reacted at room temperature for 1 hour. This was washed three times with TBST and then developed with Thermo (Super Signal West Pico Chemiluminescent Substrate).

10. Protein structure prediction

The crystal structure of MSTN was retrieved from the protein data bank (PDB) (pdb id: 3HH2). In PDB, the structure of FMOD and 2B type of Akt receptor (Activin receptor type IIB, ACVR IIB) was not detected, but the corresponding amino acid sequence was confirmed in UniProt database (Q7TPZ1, P27040). The human 2-type ACV receptor phosphorylated domain crystal structure (pdb id: 2QLU) was used as a template for modeling ACVR IIB using Modeller 9v14. When five different models were generated, the best model was selected based on the dope score (Discrete optimized protein energy). Since there is no suitable template to utilize, the structure of the FMOD is a program that generates a 3D atomic model using ab initio folding and I-TASSER (multi-threading alignment and repetitive structural combination simulation) ) Were used in combination. Homologues can not be seen as a powerful model for FMOD because they show a low ratio of sequence identity in the PDB structure.

Thus, the I-TASSER server (http: //www.national.edu/) provides the best protein model recognized by the "criticality assessment of structural predictions (CASP7, CASP8, a global experiment designed to provide an objective assessment of the latest structural prediction models) /www.zhanglab.ccmb.med.umich.edu/I-TASSER/).

11. Protein-Protein Binding Interaction Analysis

Protein-protein interaction studies were conducted to investigate the binding between MSTN and the receptor of MSTN, ACVR IIB, and to determine whether FMOD inhibits this binding. Protein-protein interactions of MSTN and ACVR IIB were made using the default settings of the PatchDock server (http://bioinfo3d.cs.tau.ac.il/PatchDock). This server is widely used in the study of protein-protein interactions, its algorithm is based on rigid docking, and surface flexibility is handled using intermolecular permeability. PatchDock calculations were scored based on protein-protein interaction energies using the Fire Dock algorithm, resulting in three-dimensional deformations of Cartesian coordinates of a much more improved protein. The structure of the complex with the lowest binding energy was chosen as the final model for later analysis.

< Example  2> Results

One. Muscle stem cells  During differentiation FMOD  Confirmation of increased expression of the gene

After differentiation for 2 days, 2 days, 4 days, and 6 days in C2C12 cells cultured in myoblasts, RNA and protein were extracted and the changes of FMOD gene and protein expression were observed by differentiation. As a result, it was observed that FMOD gene expression was increased about 15 times in the cells at the second day of differentiation compared to pre-differentiation cells (day 0), and the protein showed similar results (FIG. 1).

2. FMOD  Gene knockdown Root canal cell  Formation and muscle differentiation Marker  Confirm gene expression

When the FMOD was knocked down, the formation of canaliculus cells and the expression of the muscle differentiation marker gene were decreased.

To observe the function of FMOD gene by muscle differentiation, the FMOD gene knockdown was performed, and then root canal cell formation and expression of muscle differentiation marker gene were observed. After differentiation of normal and FMOD knockdown cells, the degree of canaliculus formation was observed. As a result, it was observed that the formation of canaliculus cells was decreased in the cells knocked down by FMOD gene compared to normal cells (Fig. 2). In addition, the expression of MYOD, MYOG (Myogenin), and MYL2 (Myosin light chain2) genes of muscle differentiation marker genes in FMOD gene knockdown cells was also decreased compared to normal cells (FIG. 3).

3. FMOD  Gene knockdown MSTN  Confirm gene expression

The expression of MSTN gene and protein in the FMOD gene knockdown cells was examined, and the expression of MSTN gene and protein was increased compared with normal cells (FIG. 4). It was confirmed that FMOD gene suppresses MSTN expression during muscle cell differentiation and regulates muscle differentiation.

4. To FMOD  by MSTN  Confirming promotion of muscle differentiation through inhibition of expression

It was confirmed how the FMOD gene regulates the expression of MSTN and the results are shown in Fig.

In silico analysis, binding of FMOD protein to MSTN protein was observed during differentiation of muscle cells, resulting in decreased binding capacity between MSTN and ACVR IIB. MSTN binds to the ACVRIIB receptor and sends signals into the cell. The global energy of binding of MSTN is -56.99. When two proteins are bound, the amino acid hydrogen bond is formed in 6 sites in ACVRIIB receptor site and 6 sites in MSTN site. Hydrophobic interaction also results in 20 sites on the ACVRIIB receptor site and 17 sites on the MSTN site (Table 3). However, when FMOD binds to MSTN in the binding of MSTN and ACVRIIB receptors, the binding energy is decreased to -29.79 and the binding efficiency is decreased. In particular, when the FMOD protein binds to the MSTN protein, the hydrogen bonding site between the amino acids of the ACVRIIB receptor is reduced to two sites and the hydrogen bonding site of MSTN is reduced to two sites. In addition, the hydrophobic action of the ACVRIIB receptor is reduced to two sites, and the hydrophobic action of MSTN also reduces to two sites (Table 4). This means that when the protein of FMOD binds to MSTN, the MSTN will not bind well to the ACVRIIB receptor.

Complex
Related Residue Hydrogen bond
(hydrogen bonding) Hydrophobic interaction
(hydrophobic interaction) ACVR2B MSTN ACVR2B MSTN ACVRⅡB-MSTN G57, E122, D261, E285, S289, R296 W31, S44, R67, G68, C76, T75 K59, H60, Y119, E122, K253, M255, R256, T258, I259, D261, L264, V274, E277, E278, D281, D283, E285, A286, S289, C292 L20, A26, F27, G28, W29, W31, Y42, R67, G68, S69, C73, T75, P76, M79, Y86, I98, A100

Complex Related Residue

ACVRⅡB-MSTN-FMOD


ACVRⅡB-MSTN
Hydrogen bond Hydrophobic interaction ACVRⅡB MSTN ACVRⅡB MSTN K43, Q44 D30, E91 K71, S74 K90, E91
ACVRⅡB-FMOD
Hydrogen bond Hydrophobic interaction ACVRⅡB FMOD ACVRⅡB FMOD D173, E190 S321, S359 R19, F20, Q41, H175, Q177, A192, N194 R293, G294, F318, V364, V365

In order to prove this in vivo, proteins were extracted from knockdown cells and normal cells of FMOD gene, and immunodomic precipitation was performed to confirm binding of FMOD protein to MSTN protein. As a result, it was confirmed that FMOD protein and MSTN protein were combined and MSTN binding was decreased in the FMOD gene knockdown cells compared to normal cells (FIG. 6). In silico analysis and in vitro analysis, it was confirmed that FMOD regulates MSTN, thereby inhibiting the binding to ACVR IIB and promoting differentiation of muscle cells.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments. It is to be understood that various modifications and changes may be made without departing from the scope of the appended claims.

<110> Research Cooperation Foundation of Yeungnam University <120> Screening method of agent for treating muscle disease <130> ADP-2016-0134 <160> 22 <170> KoPatentin 3.0 <210> 1 <211> 109 <212> PRT <213> Artificial Sequence <220> <223> Myostatin <400> 1 Asp Phe Gly Leu Asp Cys Asp Glu His Ser Thr Glu Ser Arg Cys Cys   1 5 10 15 Arg Tyr Pro Leu Thr Val Asp Phe Glu Ala Phe Gly Trp Asp Trp Ile              20 25 30 Ile Ala Pro Lys Arg Tyr Lys Ala Asn Tyr Cys Ser Gly Glu Cys Glu          35 40 45 Phe Val Phe Leu Gln Lys Tyr Pro His Thr His Leu Val His Gln Ala      50 55 60 Asn Pro Arg Gly Ser Ala Gly Pro Cys Cys Thr Pro Thr Lys Met Ser  65 70 75 80 Pro Ile Asn Met Leu Tyr Phe Asn Gly Lys Glu Gln Ile Ile Tyr Gly                  85 90 95 Lys Ile Pro Ala Met Val Val Asp Arg Cys Gly Cys Ser             100 105 <210> 2 <211> 57 <212> RNA <213> Artificial Sequence <220> <223> FMOD_sc-44823-SHA (Hairpin) <400> 2 gatccctaca tggcaaccag attattcaag agataatctg gttgccatgt agttttt 57 <210> 3 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> FMOD_sc-44823-SHA (Sense) <400> 3 cuacauggca accagauuat t 21 <210> 4 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> FMOD_sc-44823-SHA (Antisense) <400> 4 uaaucugguu gccauguagt t 21 <210> 5 <211> 57 <212> RNA <213> Artificial Sequence <220> <223> FMOD_sc-44823-SHB (Hairpin) <400> 5 gatccctact agacctgagt tatattcaag agatataact caggtctagt agttttt 57 <210> 6 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> FMOD_sc-44823-SHB (Sense) <400> 6 cuacuagacc ugaguuauat t 21 <210> 7 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> FMOD_sc-44823-SHB (Antisense) <400> 7 uauaacucag gucuaguagt t 21 <210> 8 <211> 57 <212> RNA <213> Artificial Sequence <220> <223> FMOD_sc-44823-SHC (Hairpin) <400> 8 gatccgcaac aggatcaatg agttttcaag agaaactcat tgatcctgtt gcttttt 57 <210> 9 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> FMOD_sc-44823-SHC (Sense) <400> 9 gcaacaggau caaugaguut t 21 <210> 10 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> FMOD_sc-44823-SHC (Antisense) <400> 10 aacucauuga uccuguugct t 21 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH primer_sense <400> 11 tgctggtgct gagtatgtcg 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH primer_antisense <400> 12 caagcagttg gtggtacagg 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> FMOD primer_sense <400> 13 tgcagaagat ccctcctgtc 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> FMOD primer_Antisense <400> 14 cttgatctcg ttcccatcca 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> MSTN primer_sense <400> 15 acgctaccac ggaaacaatc 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> MSTN primer_Antisense <400> 16 ggagtcttga cgggtctgag 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> MYOG primer_sense <400> 17 tccagtacat tgagcgccta 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> MYOG primer_Antisense <400> 18 caaatgatct cctgggttgg 20 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> MYOD primer_sense <400> 19 aggagcacgc acacttctct 20 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> MYOD primer_Antisense <400> 20 tctcgaaggc ctcattcact 20 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> MYL2 primer_sense <400> 21 aaagaggctc caggtccaat 20 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> MYL2 primer_Antisense <400> 22 cctctctgct tgtgtggtca 20

Claims (16)

A method for screening a substance which binds to at least one amino acid selected from the group consisting of Ile98, Gly68, Trp31, Trp29, Phe27, Tyr42 and Leu20 in the amino acid sequence represented by SEQ ID NO: 1 of myostatin (MSTN) Wherein the method comprises the steps of: [Claim 2] The method according to claim 1, wherein the amino acid further comprises at least one amino acid selected from the group consisting of Ala70, His57, Met101, Val22 and Pro99 in the amino acid sequence represented by SEQ ID NO: 1 of myostatin (MSTN) &Lt; / RTI &gt; The method according to claim 1, wherein the substance binds to any one or more amino acids of the amino acid sequence of SEQ ID NO: 1 of myostatin (MSTN) to promote muscle cell differentiation. The method according to claim 1, wherein the substance is any one selected from the group consisting of a protein, a compound, a peptide, a peptide mimetic, a substrate analog, an aptamer, and an antibody. The method according to claim 1, wherein the substance is fibromodulin (FMOD). The method of claim 1, wherein the muscle disorder is selected from the group consisting of sarcopenia, muscular dystrophy, rigid spinesyndrome, muscle-eye-brain disease, amyotrophy, amyotrophic lateral sclerosis Amyotrophic lateral sclerosis, Charcot-Marie-Tooth disease, chronic inflammatory neuropathy, and distal myopathy. In the present invention, &Lt; / RTI &gt; A method for screening a substance which binds to at least one amino acid selected from the group consisting of Ile98, Gly68, Trp31, Trp29, Phe27, Tyr42 and Leu20 in the amino acid sequence represented by SEQ ID NO: 1 of myostatin (MSTN) &Lt; / RTI &gt; [Claim 7] The method according to claim 7, wherein the amino acid further comprises at least one amino acid selected from the group consisting of Ala70, His57, Met101, Val22 and Pro99 in the amino acid sequence of SEQ ID NO: 1 of myostatin (MSTN) The method of screening the animal growth promoting agent for livestock. The animal growth promoting agent screening method according to claim 7, wherein the substance binds to any one or more amino acids of the amino acid sequence of SEQ ID NO: 1 of myostatin (MSTN) to promote muscle cell differentiation of livestock Way. A method for screening a substance which binds to at least one amino acid selected from the group consisting of Ile98, Gly68, Trp31, Trp29, Phe27, Tyr42 and Leu20 in the amino acid sequence represented by SEQ ID NO: 1 of myostatin (MSTN) Wherein said muscle-building-aid-screening method comprises the steps of: [Claim 11] The method according to claim 10, wherein the amino acid further comprises at least one amino acid selected from the group consisting of Ala70, His57, Met101, Val22 and Pro99 in the amino acid sequence of SEQ ID NO: 1 of myostatin (MSTN) A muscle-forming auxiliaries. [Claim 11] The method according to claim 10, wherein the substance binds to any one or more amino acids of the amino acid sequence of SEQ ID NO: 1 of myostatin (MSTN) to promote muscle cell differentiation. Characterized by comprising a substance which binds to at least one amino acid selected from the group consisting of Ile98, Gly68, Trp31, Trp29, Phe27, Tyr42 and Leu20 in the amino acid sequence represented by SEQ ID NO: 1 of myostatin (MSTN) A composition for promoting muscle differentiation. [Claim 14] The method according to claim 13, wherein the amino acid further comprises at least one amino acid selected from the group consisting of Ala70, His57, Met101, Val22 and Pro99 in the amino acid sequence of SEQ ID NO: 1 of myostatin (MSTN) Wherein the composition for accelerating muscle differentiation comprises [Claim 13] The composition for promoting muscle differentiation according to claim 13, wherein the substance binds to any one or more amino acids of the amino acid sequence of SEQ ID NO: 1 of myostatin (MSTN) to promote muscle cell differentiation. 14. The composition for promoting muscle differentiation according to claim 13, wherein the substance is any one selected from the group consisting of a protein, a compound, a peptide, a peptide mimetic, a substrate analog, an aptamer, and an antibody.

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