KR20230110922A - Pharmaceutical Composition comprising USP17L2 for Preventing or Treating of Bone Related Diseases - Google Patents

Abstract

The USP17L2 peptide of the present invention has the effect of promoting the differentiation of osteoblasts by increasing the expression levels of osteoblast differentiation marker genes ALP (alkaline phosphatase), COL1A1 (Collagen Type I Alpha 1 Chain), OC (Osteocalcin), OSX (osterix) and RUNX2 (Runt-related transcription factor 2), which can prevent or treat bone diseases such as osteoporosis. The composition is expected to be used as a therapeutic agent that exhibits excellent therapeutic effects for the above bone diseases for which there is no effective treatment.

Description

Pharmaceutical composition comprising USP17L2 for preventing or treating bone diseases

The present invention relates to a pharmaceutical composition for preventing or treating bone disease containing USP17L2 (ubiquitin crboxyl-terminal hydrolase 17 like family member 2) and a pharmaceutical preparation containing the composition.

Bone is composed of an extracellular matrix, and bone tissue is composed of various types of cells, such as an extracellular matrix, osteoblasts that form new bone tissue, osteoclasts that absorb bone tissue, and osteocytes. Normal adult bone tissue is an active tissue in which the activities of osteoclasts and osteoblasts are balanced to maintain homeostasis, and bone formation and resorption constantly occur.

Bone remodeling is a phenomenon in which old bone is destroyed and removed (bone resorption), and fixation (bone formation) filling in the missing new bone is repeated throughout life even after bone growth is stopped. The interaction between osteoblasts and osteoclasts is balanced so that bone resorption and bone formation are balanced so that bone homeostasis is maintained and the calcium concentration in the blood is kept constant.

BACKGROUND OF THE INVENTION Osteoporosis, a bone metabolic disease, is a complex disease influenced by factors such as genetic factors and dietary habits, and has emerged as a social problem from social and medical interest due to the rapid aging of modern society.

Currently, calcium supplements, bisphosphonates, estrogens, calcitonin, and thiazide diuretics are used to treat osteoporosis. However, estrogen used for the treatment of menopausal osteoporosis has a problem in that long-term use can cause side effects such as weight gain and breast cancer. Therefore, there is a need for the development of a therapeutic agent that promotes bone formation and restores bone loss without such side effects.

In addition, the deubiquitin enzyme USP17L2 (ubiquitin crboxyl-terminal hydrolase 17 like family member 2) has been reported to be involved in the inhibition or induction of inflammation and cancer, but the role of USP17L2 related to bone metabolism has not been reported to date.

Korea Patent No. 1799533

One aspect is to provide a pharmaceutical composition for the prevention or treatment of bone disease comprising the USP17L2 peptide having the amino acid sequence represented by SEQ ID NO: 1 or a polynucleotide encoding the same as an active ingredient.

Another aspect is to provide a health functional food composition for preventing or improving bone disease, comprising the USP17L2 peptide having the amino acid sequence represented by SEQ ID NO: 1 or a polynucleotide encoding the same as an active ingredient.

Another aspect is to provide a pharmaceutical preparation for preventing or treating bone disease, including the pharmaceutical composition.

1. A pharmaceutical composition for the prevention or treatment of bone disease comprising the USP17L2 peptide having the amino acid sequence represented by SEQ ID NO: 1 or a polynucleotide encoding the same as an active ingredient.

2. The method of 1 above, wherein the composition is selected from the group consisting of ALP (alkaline phosphatase), COL1A1 (Collagen Type I Alpha 1 Chain), OC (Osteocalcin), OSX (osterix) and RUNX2 (Runt-related transcription factor 2) Characterized in that it increases the expression of one or more genes, a pharmaceutical composition.

3. The pharmaceutical composition according to 2 above, characterized in that the composition increases the expression of the OSX gene.

4. The pharmaceutical composition according to 1 above, wherein the USP17L2 peptide promotes the differentiation of osteoblasts.

5. The bone disease according to 1 above, wherein the bone disease is at least one selected from the group consisting of osteoporosis, periodontal disease, osteomalacia, osteopenia, bone defect, bone atrophy, osteonecrosis, fracture, osteolysis, osteoarthritis, and bone formation imperfecta. disease, the pharmaceutical composition.

6. The pharmaceutical composition according to 1 above, wherein the USP17L2 peptide is contained in an amount of 0.1 μg to 2.5 μg.

7. The pharmaceutical composition according to 6 above, wherein the USP17L2 peptide is contained in an amount of 0.2 μg to 2.0 μg.

8. A health functional food composition for preventing or improving bone disease, comprising the USP17L2 peptide having the amino acid sequence represented by SEQ ID NO: 1 or a polynucleotide encoding the same as an active ingredient.

9. A pharmaceutical formulation for preventing or treating bone disease, comprising the composition of 1 above.

Compositions and preparations comprising the USP17L2 peptide of the present invention are the bone cell differentiation marker gene ALP (alkaline phosphatase), Col1a1 (Collagen Type I Alpha 1 Chain), OC (OSxin), OSX (OSX) and Runx2 By increasing the expression amount of nt-related transcript factor 2), there is an effect of promoting the differentiation of bone cells, thereby showing the prevention or treatment of bone diseases such as osteoporosis.

Figure 1 (A) is a diagram showing the results of comparing the differentiation of osteoblasts by ALP staining when C2C12 cells were transduced with the USP17L2 gene under conditions inducing osteoblast differentiation using BMP4. 1(B) is a graph comparing ALP activity according to USP17L2 content in a pharmaceutical composition.
Figure 2 is a diagram showing the results of analyzing the expression levels of ALP-Luc (Fig. 2(A)), BSP-Luc (Fig. 2(B)) and OC-Luc (Fig. 2(C)) luciferase reporters, which are osteoblast differentiation reporter plasmids, in C2C12 cells when USP17L2 gene was transduced.
Figure 3 shows the osteoblast differentiation marker genes ALP (Fig. 3(A)), OC (Fig. 3(B)), COL1A1 (Fig. 3(C)), RUNX2 (Fig. 3(D)), OSX (Fig. 3(E)), and USP17le (Fig. 3(F)) when USP17L2 gene was transduced into C2C12 cells under conditions inducing osteoblast differentiation using BMP4. It is a diagram showing the result of comparing the expression level.
Figure 4 shows the result of confirming the expression level of OSX protein, which is an osteoblast differentiation marker, when the USP17L2 content is changed according to whether or not BMP4 is included. It is a diagram showing the result of analysis that the expression of OSX protein is increased by USP17L2 relative to the case of including BMP4 (FIG. 4 (B)) compared to the case of not including BMP4 (FIG. 4 (A)).
5 is a diagram showing the results of analyzing the binding between USP17L2 and OSX proteins when USP17L2 and OSX genes were transduced into C2C12 cells.
FIG. 6 (A) is a diagram showing the results of analysis that USP17L2 and OSX genes are transduced into C2C12 cells, protein synthesis is inhibited with cyclohexamid, and the expression level of OSX protein synthesized before cyclohexamid treatment is compared, and that the stability of OSX protein is increased by USP17L2. Figure 6 (B) is a diagram showing the change in relative density of bands over time when comparing the expression level of the OSX protein.
7 is a diagram showing the results of analyzing the reduction of ubiquitination of OSX protein by USP17L2 when USP17L2, OSX, and ubiquitin (Ub) genes were transduced into C2C12 cells.
8 is a diagram showing the USP17L2 amino acid sequence.

The present inventors have confirmed that the USP17L2 peptide having the amino acid sequence represented by SEQ ID NO: 1 exhibits an excellent prevention or treatment effect of bone disease by osteoblast differentiation through the expression of osteoblast activation-related genes or the stabilization of the peptides by the genes.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for preventing or treating bone disease comprising the USP17L2 peptide or a polynucleotide encoding the same as an active ingredient.

As used herein, "USP17L2" refers to a deubiquitination enzyme that removes conjugated ubiquitin from specific proteins to regulate various cellular processes. In one embodiment of the present invention, the USP17L2 peptide can promote osteoblast differentiation by stabilizing osteoblast activation-related peptides or proteins.

In one embodiment of the present invention, the USP17L2 peptide may be a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1.

As used herein, the term "prevention" refers to all activities of suppressing or delaying the onset of bone diseases by the pharmaceutical composition according to the present invention.

As used herein, the term "treatment" refers to all activities in which symptoms of bone disease are improved or beneficially changed by the pharmaceutical composition according to the present invention.

In the present invention, the peptide is composed of the amino acid sequence of SEQ ID NO: 1, and osteoblast differentiation marker genes ALP (alkaline phosphatase), COL1A1 (Collagen Type I Alpha 1 Chain), OC (Osteocalcin), OSX (osterix) and RUNX2 (Runt-related transcription factor 2) can be promoted by increasing the expression of osteoblast differentiation. At this time, the peptide is at least 70%, preferably at least 80%, more preferably at least 90%, most preferably at least 95%, 96%, 97%, 98%, 99% or more sequence homology with the amino acid sequence of SEQ ID NO: 1 It may also include an amino acid sequence.

In one embodiment of the present invention, the composition may increase the expression of one or more genes selected from the group consisting of alkaline phosphatase (ALP), collagen type I alpha 1 chain (COL1A1), osteocalcin (OC), osterix (OSX), and runt-related transcription factor 2 (RUNX2).

In one embodiment of the present invention, it was confirmed that the composition increased the expression of the OSX gene (see Experimental Example 4), and it was confirmed that the stability of the OSX protein was increased by the USP17L2 peptide. Therefore, the USP17L2 peptide can be usefully used as an active ingredient of a composition for preventing or treating bone disease.

As used herein, the term "alkaline phosphatase (ALP)" refers to an enzyme primarily produced in bone and liver tissue. Alkaline phosphatase blood levels can be elevated in conditions that affect the bone or liver, including tumors, often when cancer has spread to the bone, but can also be elevated in benign conditions such as fractures.

In the present specification, the term "Collagen Type I Alpha 1 Chain (COL1A1)" refers to a gene responsible for the synthesis of collagen and hyaluronic acid. If the expression of the COL1A1 gene is abnormal, there may be a problem with bone formation.

As used herein, the term "OC (Osteocalcin)" refers to a non-collagen protein present in bone, cartilage, dentin, and cementum. The OC protein may be secreted from osteoblasts and may be involved in bone formation.

In the present specification, the term "OSX (osterix)" refers to a protein that regulates cell differentiation in the process of forming bone and dentin (hard tissue constituting most of a tooth).

RUNX2 (Runt-related transcription factor 2)" refers to a protein involved in osteoblast activity.

In one embodiment of the present invention, the USP17L2 peptide can promote the differentiation of osteoblasts, thereby exhibiting an effect of preventing or treating bone diseases.

In the present specification, the bone disease may be a metabolic bone disease or an orthopedic bone disease.

The “metabolic bone disease” refers to a condition or disease caused by excessive production or activity of osteoclasts, and may include a bone mass-lowering disease. The bone mass reduction disease refers to a condition or disease in which a decrease in bone mass is accompanied by symptoms such as a decrease in bone density and softening of bone tissue.

The bone disease may be at least one selected from the group consisting of osteoporosis, periodontal disease, osteomalacia, osteopenia, bone defect, bone atrophy, osteonecrosis, fracture, osteolysis, osteoarthritis and osteogenesis imperfecta, but is not limited thereto. .

The pharmaceutical composition of the present invention may be administered orally or parenterally (for example, intravenously, subcutaneously, intraperitoneally, intranasally, inhaled or applied topically) according to the desired method, and the dosage may be appropriately selected by those skilled in the art, although it varies depending on the condition and weight of the patient, the severity of the disease, the form of the drug, the route of administration and time.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat or diagnose a disease at a reasonable benefit / risk ratio applicable to medical treatment or diagnosis, and the effective dose level may be determined according to the patient's disease type, severity, drug activity, sensitivity to the drug, administration time, administration route and discharge rate, treatment period, factors including concurrently used drugs, and other factors well known in the medical field. The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered single or multiple times. Considering all of the above factors, it is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects, which can be easily determined by those skilled in the art.

Specifically, the effective amount of the pharmaceutical composition of the present invention may vary depending on the patient's age, sex, condition, body weight, absorption rate, inactivity rate and excretion rate of the active ingredient in the body, disease type, and concomitant drugs. In addition, in the cycle, it can be administered daily or every other day, or divided into 1 to 3 times a day. However, since it may increase or decrease depending on the route of administration, severity of obesity, gender, weight, age, etc., the dosage is not limited to the scope of the present invention in any way.

In one embodiment of the present invention, the USP17L2 peptide is added to the pharmaceutical composition in an amount of 0.1 μg to 3.0 μg, 0.1 μg to 2.5 μg, 0.1 μg to 2.0 μg, 0.15 μg to 2.0 μg, 0.2 μg to 2.0 μg, 0.25 μg to 2.0 μg, 0.25 μg to 1.5 μg, or 0.2 μg to 1.5 μg. 5 μg to 1.0 μg.

When the compounds are administered as a composition, they may be formulated with a pharmaceutically acceptable vehicle or carrier in suitable amounts to provide a suitable dosage form.

Meanwhile, the composition may further include a carrier, an excipient, and a diluent used in preparing a pharmaceutical composition.

The carrier is commonly used and includes, but is not limited to, saline, sterile water, Ringer's solution, buffered saline, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, liposome, etc., and may further contain other conventional additives such as antioxidants and buffers, if necessary.

In addition, excipients, diluents, dispersants, surfactants, binders, lubricants, etc. may be additionally added to formulate formulations for injections such as aqueous solutions, suspensions, emulsions, etc., pills, capsules, granules, or tablets.

The excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil, but is not limited thereto.

Regarding a suitable pharmaceutically acceptable carrier and formulation, it can be preferably formulated according to each component using the method disclosed in Remington's literature. The pharmaceutical composition of the present invention is not particularly limited in dosage form, but may be formulated into an injection form, an injection form, a spray form, an inhalant form, or an external skin preparation.

In addition, the composition may be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories and sterile injection solutions.

Solid preparations for oral administration may be tablets, pills, powders, granules, capsules, etc., and the solid preparations may be prepared by mixing tectorgenin and its fractions with at least one excipient, such as starch, calcium carbonate, sucrose, lactose, or gelatin. In addition to the above excipients, lubricants such as magnesium styrate and talc may be used.

Liquid preparations for oral administration may include suspensions, internal solutions, emulsions, syrups, and the like, and various excipients such as wetting agents, sweeteners, fragrances, and preservatives in addition to simple diluents such as water and liquid paraffin.

Preparations for parenteral administration may include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used as the non-aqueous solvent or suspending agent. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin fat, and glycerogeratin may be used.

The composition may be used for at least one material selected from the group consisting of artificial bone, artificial joint, bone fixation material, bone substitute, bone restoration material, bone filling material, and bone graft material.

In addition, the present invention provides a health functional food composition for preventing or improving bone disease, comprising the USP17L2 peptide having the amino acid sequence represented by SEQ ID NO: 1 or a polynucleotide encoding the same as an active ingredient.

As used herein, the term "improvement" refers to any activity that at least reduces the parameters related to the condition being treated, for example, the severity of symptoms.

In this case, the health functional food composition may be used before or after the onset of the disease in order to prevent or improve the disease, simultaneously with or separately from the drug for treatment.

In the health functional food composition of the present invention, the active ingredient may be added to food as it is or used together with other food or food ingredients, and may be appropriately used according to conventional methods. The mixing amount of the active ingredient can be suitably determined depending on its purpose of use (for prevention or improvement). In general, the composition of the present invention can be added in an amount of preferably 15% by weight or less, preferably 10% by weight or less, based on the raw material during production of food or beverage. However, in the case of long-term intake for the purpose of health and hygiene or health control, the amount may be less than the above range.

In addition to containing the active ingredient, the health functional food composition of the present invention may contain other ingredients as essential ingredients without particular limitation. For example, it may contain various flavoring agents or natural carbohydrates as additional ingredients like a normal beverage. Examples of the aforementioned natural carbohydrates include monosaccharides such as glucose, fructose, and the like; disaccharides such as maltose, sucrose and the like; and polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin, and the like, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (thaumatin, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can advantageously be used. The ratio of the natural carbohydrates can be appropriately determined by a person skilled in the art.

In addition to the above, the health functional food composition of the present invention may contain various nutrients, vitamins, minerals (electrolyte), flavoring agents such as synthetic flavoring agents and natural flavoring agents, colorants and enhancers (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, and the like. These components can be used independently or in combination, and the ratio of these additives can also be appropriately selected by those skilled in the art.

"USP17L2", "bone disease", "prevention" and the like may be within the foregoing ranges.

In addition, the present invention provides a pharmaceutical formulation for preventing or treating bone disease, including the pharmaceutical composition.

In addition, the present invention provides a method for preventing or treating bone disease comprising administering the pharmaceutical composition to a subject.

In the present invention, "individual" means a subject in need of treatment of a disease, and more specifically, mammals such as humans or non-human primates, mice, rats, dogs, cats, horses, and cattle. It means.

"USP17L2", "bone disease", "administration", "prevention", "treatment" and the like may be within the foregoing ranges.

In addition, the present invention provides a use of the pharmaceutical composition for preventing or treating bone disease.

In addition, the present invention provides a use of the USP17L2 peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 or a polynucleotide encoding the same for preparing a drug for treating bone disease.

In addition, the present invention provides the use of a composition comprising the USP17L2 peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 or a polynucleotide encoding the same as an active ingredient for preparing a drug for treating bone disease.

Hereinafter, the present invention will be described in more detail through Examples and Experimental Examples. However, these examples are intended to illustrate the present invention by way of example, and the scope of the present invention is not limited to these examples and experimental examples.

Example

Experimental Example 1. Analysis of osteoblast differentiation promotion by USP17L2

USP17L2 gene was transduced into C2C12 cells and cultured in a culture medium containing BMP-4 for 3 days. BMP-4 protein was purchased from R&D Systems (Mc-Kinley Place NE, MN, USA).

The cultured cells were fixed in 4% paraformaldehyde solution for 10 minutes, and then washed several times with PBS at room temperature. Then, after staining with NBT/BCIP solution (Sigma-Aldrich) for 15 minutes at room temperature, 610 nm absorbance was measured to compare and analyze ALP relative activity.

As a result, it was confirmed that the relative activity of ALP increased as the content of USP17L2 increased (see FIG. 1).

Experimental Example 2. Analysis of osteoblast differentiation marker luciferase reporter expression increase by USP17L2

To analyze the expression level of the osteoblast differentiation marker gene by USP17L2, C2C12 cells were transduced with the USP17L2 gene and ALP-Luc, BSP-Luc or OC-Luc reporter plasmids. After 1 day, using the Human Luciferase Reporter Assay Kit (Promega, Madison, WI, USA), the luciferase expression level was comparatively analyzed according to the experimental conditions of the kit.

As a result, it was confirmed that fold induction of ALP-Luc, BSP-Luc or OC-Luc reporters increased as the content of USP17L2 increased (see FIG. 2).

Experimental Example 3 . Analysis of osteoblast differentiation marker gene expression level increase by USP17L2

In order to confirm the increase in osteoblast differentiation marker gene expression by USP17L2, C2C12 cells were transduced with the USP17L2 gene and cultured in a culture medium containing BMP-4 for 3 days.

Thereafter, the RNA of the cultured cells was extracted with TRIzol reagent (Invitrogen), 1 μg of RNA was synthesized using a random hexamer primer (reverse transcription kit, Invitrogen), and SYBR Premix Ex Taq kit (TaKaRa Bio, Japan) and RT-PCR was performed using a MiniOpticon real-time PCR System. Expression levels of ALP, COL1A1, OC, OSX, and RUNX2, which are osteoblast differentiation marker genes, were compared and analyzed through the RT-PCR.

RNA 1 μg was synthesized using random hexamer primers (reverse transcription kit, Invitrogen), SYBR Premix Ex Taq kit (TaKaRa Bio, Japan) and MiniOpticon real-time PCR system were measured.

As a result, it was confirmed that the mRNA relative expression of osteoblast differentiation marker genes increased as the content of USP17L2 increased, compared to the case where USP17L2 was not included (see FIG. 3 ).

This indicates that as the content of USP17L2 increases, the expression of osteoblast differentiation genes is increased, thereby promoting the differentiation of osteoblasts.

Experimental Example 4. Analysis of osteoblast differentiation marker OSX protein expression increase by USP17L2

In order to analyze the increase in the expression of the osteoblast differentiation marker OSX protein by USP17L2, the USP17L2 gene was transduced into C2C12 cells and cultured in a culture medium containing BMP-4 for 3 days.

Proteins from the cultured cells were extracted, and the expression levels of OSX proteins, which are osteoblast differentiation marker proteins, were compared and analyzed using immunoblotting.

Specifically, the immunoblotting was performed by washing the cells in the culture dish twice with PBS, and then using a cell disruption solution (25 mM HEPES (pH 7.5), 150 mM NaCl, 1% NP-40, 0.25% sodium deoxycholate, 10% glycerol, 25 mM NaF, 1 mM EDTA, 1 mM Na3VO4, 250 μM PMSF, 10 μg/ml leupept in, 10 μg/ml peptidase and 10 μg/ml Aprotinin) for 15 minutes, and then the cell disruption solution was centrifuged at 4° C. and 13,000 rpm. The proteins in the centrifuged supernatant were separated by SDS-PAGE electrophoresis, and the separated proteins were attached to a PVDF membrane, reacted with a primary antibody and a secondary antibody, stained with ECL solution, and imaged.

As a result, it was confirmed that the expression of OSX protein, an osteoblast differentiation marker protein, increased as the content of USP17L2 increased, compared to the case where USP17L2 was not included (see FIG. 4 ).

This indicates that as the content of USP17L2 increases, the expression of OSX protein increases, thereby promoting the differentiation of osteoblasts.

Experimental Example 5. Analysis of USP17L2 and OSX protein binding

To analyze the binding between USP17L2 and OSX proteins, C2C12 cells were transduced with USP17L2 and OSX genes and cultured for 2 days. The cultured cell proteins were extracted and the binding between USP17L2 and OSX protein was analyzed using immunoprecipitation and immunoblotting.

Specifically, the cell lysate was reacted with antibody and protein A-agarose beads at 4° C. for 12 hours, washed three times with PBS, separated proteins with SDS-PAGE sample solution, subjected to SDS-PAGE electrophoresis and immunoblotting.

As a result, it was confirmed that the band appeared thick in the cells transduced with both the USP17L2 and OSX proteins (see FIG. 5).

Experimental Example 6. Analysis of OSX protein stability increase by USP17L2

To confirm the increase in OSX protein stability by USP17L2, USP17L2 and OSX genes were transduced into C2C12 cells, protein synthesis was inhibited with cyclohexamid, and the residual amount of OSX protein synthesized before cyclohexamid treatment was compared and analyzed using immunoblotting.

As a result, it was confirmed that the residual amount of the OSX protein was relatively high in the cells transduced with both the USP17L2 and the OSX protein. In addition, it was confirmed that the relative density of the Osterix band was higher when both OSX and USP17L2 were treated than when only OSX was treated (see FIG. 6).

This indicates that USP17L2 may increase the stability of the OSX protein, thereby promoting the differentiation of osteoblasts.

Experimental Example 7. Analysis of OSX protein ubiquitination reduction by USP17L2

To analyze the decrease in ubiquitination of OSX protein by USP17L2, USP17L2, OSX, and ubiquitin (Ub) genes were transduced into C2C12 cells and cultured for 2 days. The cultured cell proteins were extracted and the effect of USP17L2 on the ubiquitination of OSX protein was comparatively analyzed using immunoprecipitation and immunoblotting.

As a result, it was confirmed that the ubiquitination of the OSX protein was reduced when USP17L2 was included compared to when USP17L2 was not included (see FIG. 7 ).

This indicates that USP17L2 may increase the stability of the OSX protein, thereby promoting the differentiation of osteoblasts.

<110> Ewha University - Industry Collaboration Foundation INDUSTRY FOUNDATION OF CHONNAM NATIONAL UNIVERSITY <120> Pharmaceutical Composition comprising USP17L2 for Preventing or Treating Bone Related Diseases <130> PD22-003 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 530 <212> PRT <213> artificial sequence <220> <223> USP17L2 <400> 1 Met Glu Asp Asp Ser Leu Tyr Leu Gly Gly Glu Trp Gln Phe Asn His 1 5 10 15 Phe Ser Lys Leu Thr Ser Ser Arg Pro Asp Ala Ala Phe Ala Glu Ile 20 25 30 Gln Arg Thr Ser Leu Pro Glu Lys Ser Pro Leu Ser Ser Glu Ala Arg 35 40 45 Val Asp Leu Cys Asp Asp Leu Ala Pro Val Ala Arg Gln Leu Ala Pro 50 55 60 Arg Lys Lys Leu Pro Leu Ser Ser Arg Arg Pro Ala Ala Val Gly Ala 65 70 75 80 Gly Leu Gln Asn Met Gly Asn Thr Cys Tyr Glu Asn Ala Ser Leu Gln 85 90 95 Cys Leu Thr Tyr Thr Pro Pro Leu Ala Asn Tyr Met Leu Ser Arg Glu 100 105 110 His Ser Gln Thr Cys Gln Arg Pro Lys Cys Cys Met Leu Cys Thr Met 115 120 125 Gln Ala His Ile Thr Trp Ala Leu His Ser Pro Gly His Val Ile Gln 130 135 140 Pro Ser Gln Ala Leu Ala Ala Gly Phe His Arg Gly Lys Gln Glu Asp 145 150 155 160 Ala His Glu Phe Leu Met Phe Thr Val Asp Ala Met Lys Lys Ala Cys 165 170 175 Leu Pro Gly His Lys Gln Val Asp His His Ser Lys Asp Thr Thr Leu 180 185 190 Ile His Gln Ile Phe Gly Gly Cys Trp Arg Ser Gln Ile Lys Cys Leu 195 200 205 His Cys His Gly Ile Ser Asp Thr Phe Asp Pro Tyr Leu Asp Ile Ala 210 215 220 Leu Asp Ile Gln Ala Ala Gln Ser Val Lys Gln Ala Leu Glu Gln Leu 225 230 235 240 Val Lys Pro Glu Glu Leu Asn Gly Glu Asn Ala Tyr His Cys Gly Leu 245 250 255 Cys Leu Gln Arg Ala Pro Ala Ser Lys Thr Leu Thr Leu His Thr Ser 260 265 270 Ala Lys Val Leu Ile Leu Val Leu Lys Arg Phe Ser Asp Val Thr Gly 275 280 285 Asn Lys Leu Ala Lys Asn Val Gln Tyr Pro Glu Cys Leu Asp Met Gln 290 295 300 Pro Tyr Met Ser Gln Gln Asn Thr Gly Pro Leu Val Tyr Val Leu Tyr 305 310 315 320 Ala Val Leu Val His Ala Gly Trp Ser Cys His Asp Gly His Tyr Phe 325 330 335 Ser Tyr Val Lys Ala Gln Glu Gly Gln Trp Tyr Lys Met Asp Asp Ala 340 345 350 Lys Val Thr Ala Cys Ser Ile Thr Ser Val Leu Ser Gln Gln Ala Tyr 355 360 365 Val Leu Phe Tyr Ile Gln Lys Ser Glu Trp Glu Arg His Ser Glu Ser 370 375 380 Val Ser Arg Gly Arg Glu Pro Arg Ala Leu Gly Ala Glu Asp Thr Asp 385 390 395 400 Arg Arg Ala Thr Gln Gly Glu Leu Lys Arg Asp His Pro Cys Leu Gln 405 410 415 Ala Pro Glu Leu Asp Glu Arg Leu Val Glu Arg Ala Thr Gln Glu Ser 420 425 430 Thr Leu Asp His Trp Lys Phe Pro Gln Glu Gln Asn Lys Thr Lys Pro 435 440 445 Glu Phe Asn Val Arg Lys Val Glu Gly Thr Leu Pro Pro Asn Val Leu 450 455 460 Val Ile His Gln Ser Lys Tyr Lys Cys Gly Met Lys Asn His His Pro 465 470 475 480 Glu Gln Gln Ser Ser Leu Leu Asn Leu Ser Ser Thr Thr Arg Thr Asp 485 490 495 Gln Glu Ser Val Asn Thr Gly Thr Leu Ala Ser Leu Gln Gly Arg Thr 500 505 510 Arg Arg Ser Lys Gly Lys Asn Lys His Ser Lys Arg Ala Leu Leu Val 515 520 525 Cys Gln 530

Claims (9)

A pharmaceutical composition for the prevention or treatment of bone diseases comprising, as an active ingredient, a USP17L2 (ubiquitin crboxyl-terminal hydrolase 17 like family member 2) peptide having the amino acid sequence represented by SEQ ID NO: 1 or a polynucleotide encoding the same.
The method according to claim 1, wherein the composition is selected from the group consisting of ALP (alkaline phosphatase), COL1A1 (Collagen Type I Alpha 1 Chain), OC (Osteocalcin), OSX (osterix) and RUNX2 (Runt-related transcription factor 2) Characterized in that it increases the expression of one or more genes, the pharmaceutical composition.
The pharmaceutical composition according to claim 2, wherein the composition increases the expression of the OSX gene.
The pharmaceutical composition according to claim 1, wherein the USP17L2 peptide promotes the differentiation of osteoblasts.
The method according to claim 1, wherein the bone disease is at least one disease selected from the group consisting of osteoporosis, periodontal disease, osteomalacia, osteopenia, bone defect, bone atrophy, osteonecrosis, fracture, osteolysis, osteoarthritis and osteogenesis imperfecta. pharmaceutical composition.
The pharmaceutical composition according to claim 1, wherein the USP17L2 peptide is contained in an amount of 0.1 μg to 2.5 μg.
The pharmaceutical composition according to claim 6, wherein the USP17L2 peptide is contained in an amount of 0.2 μg to 2.0 μg.
A functional food composition for preventing or improving bone disease, comprising as an active ingredient a USP17L2 (ubiquitin crboxyl-terminal hydrolase 17 like family member 2) peptide having the amino acid sequence represented by SEQ ID NO: 1 or a polynucleotide encoding the same.
A pharmaceutical formulation for preventing or treating bone disease, comprising the composition of claim 1.