KR20140130655A - Method of screening a substance capable of inhibiting interaction between prp19 and clic proteins and a composition comprising the screened substance - Google Patents

Abstract

The present invention relates to a method for searching for a substance which inhibits an interaction between precursor RNA processing (PRP) 19 proteins and intracellular chloride channel (CLIC) proteins and to a cosmetic composition or a pharmaceutical composition which contains an Elaeocarpus extract searched through the method as an active ingredient. According to the present invention, the substance which inhibits an interaction between PRP19 proteins and CLIC proteins can be rapidly searched on a large scale. The substances searched for through the method of the present invention can inhibit the generation of fat so as to be usefully applied in treating and preventing obesity and diseases which are mediated by an interaction between PRP19 proteins and CLIC proteins.

Description

A method for searching for a substance that inhibits the interaction between PRP19 and CLIC protein, and a composition containing the searched substance TECHNICAL FIELD [METHOD OF SCREENING A SUBSTANCE CAPABLE OF INHIBITING INTERACTION BETWEEN PRP19 AND CLIC PROTEINS AND A COMPOSITION COMPRISING THE SCREENED SUBSTANCE}

The present invention relates to a method for screening a candidate substance that inhibits the interaction of a precursor RNA processing (PRP) 19 protein and an intracellular chloride channel (CLI) protein, and a composition comprising the searched substance.

With the prevalence of Western-style eating habits, obesity is a major threat to modern humans, with the obese population now reaching 1 billion people worldwide. Obesity accumulates fat in each part of the body and causes various diseases. Representative examples include diabetes, hyperlipidemia, fatty liver, low back pain, and gouty arthritis.

Fat is synthesized in fat cells and accumulated in the fat body. Fat cells store excess energy in the form of fat among the calories obtained from food intake, and when necessary, such as exercising or starving, fat cells are decomposed to produce energy, and the center of it is the fat body. The fat body consists of a single layer of phosphorylated lipids, and its periphery is perilipin, adipophilin, TIP47 (tail interacting protein 47), caveolin, cgi58 (comparative gene identification 58), and It is coated with many proteins such as vimentin and myocardial lipid droplet protein (MLDP), and these proteins are involved in the synthesis or decomposition of fat according to various signals. For example, when PKA (protein kinase A) is activated in response to beta-adrenergic stimulation, perilipine present in the fat body is phosphorylated, and hormone sensitive lipase adheres to it to break down fat (Gary M. Clifford , J. Biol. Chem , 275(7): 5011-5015 (2000)).

On the other hand, PRP (precursor RNA processing) 19 protein is significantly increased in adipocytes with differentiation of adipocytes, and when the expression of PRP19 protein is suppressed through RNA interference, the differentiation of adipose is inhibited and SCD1 (stearyl CoA desaturase-1). , S3-12, perilipine, glycerol-3-phosphate acyltransferase, etc. expression levels decrease, and as a result, the amount of fat production is drastically reduced ( J Biol Chem . Jan 26;282 (4): 2456-65 (2007)).

As a result of continuing research to develop a substance that regulates the intracellular action of the PRP19 protein, the present inventors have found for the first time that the U-box domain of the PRP19 protein interacts with the intracellular chloride channel (CLIC) protein to promote adipogenesis. Thus, a method to easily search for substances that inhibit the interaction of the PRP19 protein and the CLIC protein was developed.

Gary M. Clifford, J. Biol. Chem, 275(7): 5011-5015 (2000) J Biol Chem. Jan 26;282(4): 2456-65 (2007)

Accordingly, an object of the present invention is to provide a method for effectively screening a large amount of candidate substances capable of inhibiting the interaction between the PRP19 protein and the CLIC protein.

Another object of the present invention is to provide a cosmetic composition or pharmaceutical composition containing the dampal water extract as an active ingredient.

According to the above object, the present invention is a step of (1) reacting a PRP19 protein or its U-box domain with a CLIC protein or a part thereof in the presence or absence of a candidate substance, wherein any one of the proteins is labeled with a detectable labeling substance. Characterized in that the step; (2) measuring the amount of the labeling substance after the reaction; And (3) selecting a candidate substance that reduces the amount of the labeling substance measured in the presence of the candidate substance compared to the amount of the labeling substance measured in the absence of the candidate substance. It provides a method of searching for a substance that inhibits.

According to the other object, the present invention is dampalsu ( Elaeocarpus sylvestris var. ellipticus (Thunb.) Hara) provides a cosmetic composition or pharmaceutical composition containing the extract as an active ingredient.

According to the search method of the present invention, it is possible to quickly and easily search for a substance that inhibits the interaction between the PRP19 protein and the CLIC protein. In addition, the dampal water extract found by searching by the method of the present invention can inhibit the formation of fat, so it can be usefully used for the treatment and prevention of diseases mediated by the interaction between the PRP19 protein and the CLIC protein, including obesity.

1 is a schematic diagram showing a U-box domain of a precursor RNA processing (PRP) 19 protein that interacts with an intracellular chloride channel (CLIC) protein.
Figure 2 shows the results of electrophoresis of the U-box domain of the PRP19 protein fused with glutathion-s-transferase and the CLIC protein fused with green fluorescence protein.
3 shows the analysis results according to the type of blocking agent used in the enzyme-linked immunosorbent assay (ELISA) (bsa: bovine serum albumin, skim: skim milk, cas: casein).
Figure 4 shows the detection signal according to the type of 96-well plate immobilizing the U-box domain of the PRP19 protein in ELISA analysis.
5 shows the results according to the conditions for immobilizing the U-box domain of the PRP19 protein in ELISA analysis.
6 shows the detection signal according to the concentration of the U-box domain of the PRP19 protein in ELISA analysis.
7 shows the effect of inhibiting the interaction between the U-box domain of the PRP19 protein and the CLIC protein according to the concentration of the dampal water extract in ELISA analysis.
8 is a photomicrograph confirming the degree of fat body formation in a cell line treated with Dampal water extract
9 is an absorbance analysis result of quantifying triglycerides for cell lines treated with dampal water extract.
10 is a result showing the interaction of PRP19 and CLIC protein by immunoprecipitation reaction and Western blot analysis.

The CLIC protein has the amino acid sequence of SEQ ID NO: 8, and acts to increase the amount of fat produced in adipocytes by interacting with the U-box domain of the PRP19 protein.

Accordingly, the present invention is a step of (1) reacting a PRP19 protein or its U-box domain with a CLIC protein or a part thereof in the presence or absence of a candidate material, characterized in that any one of the proteins is labeled with a detectable labeling material. The step of doing; (2) measuring the amount of the labeling substance after the reaction; And (3) selecting a candidate substance that reduces the amount of the labeling substance measured in the presence of the candidate substance compared to the amount of the labeling substance measured in the absence of the candidate substance. It provides a method of searching for a substance that inhibits.

Preferably, the candidate substance may be a substance that directly interferes with the binding between the PRP19 protein and the CLIC protein or inactivates the CLIC protein.

The PRP19 protein or CLIC protein used in the screening method of the present invention may be labeled with a detectable substance. As such a detectable labeling substance, any labeling substance known in the art can be used as long as the interaction between the PRP19 protein and the CLIC protein can be maintained. Specifically, a fluorescent protein (e.g., green fluorescent protein (GFP)), a fluorescent substance, horseradish peroxidase (HRP), a label such as alkaline phosphatase (AP), or such a label A bound antibody (eg, an antibody against the CLIC protein) can be exemplified.

As a method of measuring the amount of a labeling substance in the search method of the present invention, general techniques used to quantitatively analyze proteins in the art may be used. For example, methods for determining the amount of labeling substance bound to PRP19 protein or CLIC protein include enzyme-linked immunosorbent assay (ELISA), western blot on polyacrylamide gel, immunoblot analysis, and immune tissue. There are immunohistochemical staining and the like, and an ELISA method using an antibody against the CLIC protein (or an antibody against the labeling substance) labeled with a labeling substance is preferred. Preferably, ELISA is performed by immobilizing proteins at 4° C. for about 14 hours using a polystyrene well plate such as skim milk as a blocking agent and a Greiner well plate as a protein fixation plate. Can be.

The PRP19 protein and CLIC protein used in the method of the present invention can be prepared, for example, by extracting the mRNA of a mouse gene and then reverse transcription to obtain cDNA, which is cloned into an expression vector and expressed in an appropriate host cell. However, the PRP19 gene and the CLIC gene are not necessarily limited to those derived from mouse genes. In addition, as an expression system, even any cell such as bacteria such as E. coli, yeast, insect cells, mammalian cells, and any expression vector suitable therefor, can express PRP19 and CLIC proteins in a form that maintains their respective functions and structures. Anything can be used.

Dampal water found by the above search method (Elaeocarpus sylvestris var. ellipticus (Thunb.) Hara) extract inhibits adipocyte differentiation and adipogenesis. Accordingly, the present invention provides a cosmetic composition or a pharmaceutical composition for the prevention or treatment of obesity containing the dampal water extract as an active ingredient.

Freshwater extract can be prepared from outposts, stems, roots, leaves, flowers, or mixtures thereof. Preferably, the freshwater extract can be prepared from the stem.

The solvent used to obtain the freshwater extract includes polar solvents, specifically alcohols such as methanol, ethanol, butylene glycol, ethers such as ethyl ether, ketones such as acetone, and polar organics such as esters such as ethyl acetate. A solvent or water may be used, and a mixture thereof may also be used.

Dampal water extract may be obtained by extracting the extract extracted with the polar solvent again with a non-polar solvent. As a usable non-polar solvent, ethyl acetate, hexane, dichloromethane or chloroform may be used, and a mixture thereof may be used.

The freshwater extract may be an extract or extract itself, a concentrate or a dried product of the extract or extract, or a mixture thereof.

The freshwater extract can be obtained, for example, in the following manner. First, about 1 to 15 times of the polar solvent is added to the fresh water, and then extracted 1 to 5 times while heating at about 20 to 100° C. for 1 to 48 hours. The obtained extract is filtered under reduced pressure to distill the extract from which the solid phase has been removed under reduced pressure to obtain a fresh dry matter extract in the form of a solvent from which the solvent has been completely removed. In order to increase the extraction efficiency, as described above, after extraction using a polar solvent, a certain amount of the solvent was removed, and 1 to 4 times the non-polar solvent of the extract was added, and then left at room temperature for 1 to 48 hours to completely separate the layers. , The supernatant is collected and distilled again under reduced pressure to obtain a desired freshwater extract.

In order to examine the effect of the extract obtained by the above method for treating obesity, it was found that the degree of adipogenesis decreased with increasing the concentration of the extract as a result of differentiating 3T3-L1, an undifferentiated adipocyte line, and then treating the dampal water extract. It was confirmed that the obesity suppression effect was excellent.

Thus, the present invention provides a cosmetic composition for a slimming effect, containing the dampal water extract as an active ingredient. Preferably, the freshwater extract may be contained in an amount of 0.0001 to 20% by weight based on the total weight (dry weight) of the cosmetic composition. When the content of the dampal water extract is less than 0.0001% by weight, the effect cannot be expected, and when it exceeds 20% by weight, it is difficult to maintain the formulation.

According to the present invention, it is suitable to prepare the extract or extract itself, the concentrate or dry powder of the extract or extract in a cosmetic formulation and apply it as it is, but may be prepared as a formulation for ordinary internal or external use. .

The cosmetic composition of the present invention can be variously used in cosmetics for slimming effect. For example, the cosmetic composition of the present invention may be formulated into cosmetics such as various creams, lotions, and skins, cleansing products, face wash, soap, beauty liquids, and the like.

The cosmetic composition containing the freshwater extract of the present invention may take the shape of a solution, an emulsion, a viscous mixture, or the like. That is, the cosmetic composition of the present invention is not particularly limited in its formulation, for example, emulsion, cream, lotion, essence, pack, gel, powder, foundation, lotion, ointment, ger patch, essence, cleansing foam, cleansing cream , Cleansing water, soap, spray, etc. can be prepared in a formulation.

In each of the formulations for the cosmetic composition of the present invention, ingredients other than the dampal water extract may be easily selected by a person skilled in the art according to the formulation or purpose of use of the other cosmetic composition and appropriately blended.

The cosmetic composition according to the present invention, in addition to the above-described dampal water extract, other ingredients that are usually added to the cosmetic composition, for example, within the scope of not impairing the object and effect of the present invention, preferably to the object and effect of the present invention. It may contain other ingredients and the like that may have a synergistic effect.

For example, the cosmetic composition of the present invention may further contain a skin absorption promoting substance to enhance the slimming effect.

The cosmetic composition of the present invention may further include components selected from the group consisting of water-soluble vitamins, oil-soluble vitamins, polymer peptides, polymer polysaccharides, sphingo lipids, and seaweed extract.

In addition, the cosmetic composition of the present invention includes fats and oils, moisturizers, emollients, surfactants, organic or inorganic pigments, organic powders, ultraviolet absorbers, preservatives, fungicides, antioxidants, plant extracts, pH adjusters, alcohols, pigments, fragrances. , Blood circulation accelerators, cooling agents, purified water, etc. may additionally be included.

The present invention also provides a pharmaceutical composition for the prevention or treatment of obesity, containing the dampal water extract as an active ingredient. Preferably, the freshwater extract may be contained in an amount of 0.0001 to 100% by weight based on the total weight (dry weight) of the pharmaceutical composition.

If the content of the dampal water extract is less than 0.0001% by weight, the effect cannot be expected, but it is safe to use 100% by weight of the undiluted solution as it is.

The pharmaceutical composition according to the present invention may further include a pharmaceutically acceptable carrier, excipient, diluent, or other additives as the case may be, in addition to the freshwater extract.

Pharmaceutical compositions according to the present invention include oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations such as ointments and creams, suppositories, and sterile injectable solutions according to a conventional method. It can be formulated and used in any form suitable for pharmaceutical preparations.

The pharmaceutical composition according to the present invention may be administered to mammals such as rats, mice, livestock, and humans by various routes such as parenteral and oral. For example, it can be administered by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dura mater or by intracerebroventricular injection. At this time, as a parenteral route, transdermal administration is preferable, and among them, topical application is most preferable.

The dosage of the pharmaceutical composition formulation according to the present invention may vary depending on the age, sex, weight, symptoms, and administration method of the subject in need of inhibition of adipogenesis, but in the case of an external preparation, an appropriate amount is applied several times a day depending on the affected area. It is desirable to continue for more than a month.

In the case of the oral dosage form, it may vary depending on the age, sex, and weight of the patient, but the dampal water extract may be administered once to several times a day in an amount of 0.1 mg to 200 mg/kg. In addition, the dosage may be increased or decreased depending on the route of administration, the severity of symptoms, the patient's age, sex, weight, age, and the like. Therefore, the above dosage does not limit the scope of the present invention in any way.

[Example]

Hereinafter, the present invention will be described in more detail by the following examples. However, the following examples are for illustrative purposes only, and the scope of the present invention is not limited thereto.

Example 1: induction of adipocyte differentiation, and PRP19 Genes and CLIC Genetic Cloning

<Step 1> Adipocyte differentiation induction

Rat ( Mus musculus ) undifferentiated adipocyte line 3T3-L1 (ATCC CL173) was dispensed into a 250 ml tissue culture flask (falcon) and 90% in DMEM (Dulbecco's modified Eagle's Medium, Gibco 1210-0038) medium containing 10% bovine serum. It was incubated in a _{10% CO 2} incubator for 48 hours until it grew longer. Thereafter, the cells were cultured in DMEM medium containing 10% fetal bovine serum, 0.5mM 3-isobutyl-1-methylxanthine (Sigma), 1 μM dexamethasone (Sigma), and 167 nM insulin (Novo-Nordisk) for 48 hours. Then, the cells were cultured again in DMEM medium containing 10% fetal calf serum and 167 nM insulin for 48 hours, and further cultured in DMEM medium containing only 10% fetal calf serum for 48 hours to obtain differentiated adipocytes.

<Step 2> Cloning of PRP19 gene and CLIC gene

After RNA was extracted from the adipocytes obtained in step 1 using the Rneasy mini kit (Qiagen 74104), cDNA was synthesized using a onestep RT-PCR kit (Qiagen 210210), and this was amplified using PCR. At this time, a primer pair having the sequence of SEQ ID NO: 1 to 4 listed in Table 1 was used.

PCR was performed using La taq (TAKARA RR002A) according to the following conditions: reaction at 94° C. for 5 minutes; 30 repetitions of 94°C for 30 seconds, 49°C for 30 seconds, and 69°C for 50 seconds; And final extension at 69° C. for 10 minutes.

The gene encoding the U-box domain of PRP19 obtained in the above PCR process was cloned into the BamHI/EcoRI site of pGEX2T vector (GE Healthcare), and the CLIC gene was a green fluorescence protein (GFP) at the C-terminus of the CLIC protein. ) Was cloned into the BamHI/BamHI site of the pET21b vector (Merk) into which the GFP gene was introduced. The U-box domain of PRP19 is shown in FIG. 1. The nucleotide sequence and amino acid sequence of the U-box domain of PRP19 and CLIC are described as SEQ ID NOs: 5 to 8, respectively.

Example 2: PRP19 Protein and CLIC Confirmation of protein expression

Each vector obtained in step 2 of Example 1 was mixed with E. coli BL21 and then subjected to thermal shock at 42° C. for 92 seconds to transform E. coli. Thereafter, SOC medium (Merk) was added, incubated at 37° C. for 45 minutes, and then uniformly coated on an LB plate containing 100 ppm of ampicillin. The transformed E. coli was selected and cultured in 300 ml of LB medium containing 100 ppm ampicillin, and when the OD (optical density) value reached 1, 300 uM of IPTG was added to induce expression for 4 hours. After obtaining E. coli by centrifugation (6000xg, 10 minutes), the medium was discarded, and 30 ml bugbuster protein extraction solution (Novagen) was added to the obtained E. coli, and allowed to stand for 15 minutes so that the protein was extracted at room temperature. Thereafter, the resultant was centrifuged at 4° C. at 12,000 rpm for 20 minutes with an Eppendorf 5810R centrifuge to take only the supernatant. The extracted protein was subjected to electrophoresis on a 4-12% NuPAGE gel (Invitrogen) and then observed by developing color using a briliant blue R (Sigma). The results are shown in Figure 2. As shown in FIG. 2, expression of the U-box domain in which glutathione-s-transferase is fused and the CLIC protein in which GFP is fused can be confirmed.

Example 3: PRP19 U- box Domain purification

To 30 ml of the extract containing the U-box domain of PRP19 obtained in Example 2, 5 ml of glutathione sepharose resin 4 fast flow (GE healthcare) was added to allow binding between the protein and the resin at 4°C. It was reacted for 5 hours at. Thereafter, centrifugation was performed for 5 minutes at 6000 rpm at 4° C. with an Eppendorf 5810R centrifuge, and then the supernatant was removed, and 10 ml of phosphate buffered saline (pH 7) buffer was added to the remaining precipitate to resuspend. The same method was repeated twice more to wash the precipitate and then the supernatant was removed. Finally, 10 ml of a PBS buffer in which 15 mM of glutathione was dissolved was added to the obtained precipitate to separate the U-box domain of PRP19. This protein separation process was repeated two more times. The obtained protein was subjected to electrophoresis at 200V on a 4-12% NuPAGE gel (invitrogen), and then observed by developing color using a briliant blue R (Sigma).

Example 4: Enzyme-linked immunosorbent assay ( ELISA The optimal conditions for)

(1) blocking agent

The U-box domain of PRP19 obtained in Example 3 was diluted 100 times using 0.1 M sodium carbonate buffer (pH 9.4), and 0 to 100 μl in each well of a Greiner 96-well plate After dispensing at the concentration of, it was reacted at 4°C for 14 hours. After washing the wells 4 times with 200 µl of PBS buffer, 200 µl of PBS buffer in which 0.5% of bovine serum albumin (BSA), skim milk, and casein were used as blocking agents were dissolved in each well. Added. After standing at room temperature for 40 minutes, 200 μl of PBS buffer was added to wash the wells 4 times, and the GFP-fused CLIC protein obtained in Example 2 was added to a blocking buffer (0.5% PBS, pH 7). It was diluted 200 times and added to each well by 100 µl. After standing at room temperature for 1 hour, 200 μl of PBS buffer was added to wash the wells 4 times. Horseradish peroxidase-attached GFP antibody (ab chem) was diluted 6000 times with PBS buffer, and 100 µl was added to each well. After standing at room temperature for 1 hour, 200 µl of PBS buffer was added to wash the wells four times, and 100 µl of TMB substrate solution (Biorad) was added to each well, followed by color development at room temperature for 10 minutes. The results are shown in FIG. 3. As shown in FIG. 3, it can be seen that the signal-to-noise ratio is best when the skim milk is used.

(2) 96-well plate

The U-box domain of PRP19 obtained in Example 3 was diluted 100 times using 0.1M sodium carbonate buffer (pH 9.4), and then 100 μl in a Greiner or nunc 96-well plate. Each was added and reacted at 4° C. for 14 hours. 200 µl of PBS buffer was added to wash the wells four times, and then 200 µl of PBS buffer in which 0.5% skim milk was dissolved was added to each well. After standing at room temperature for 40 minutes, 200 µl of PBS buffer was added to wash 4 times, and then the GFP-fused CLIC protein obtained in Example 2 was 100 with a blocking buffer (0.5% skim milk, PBS). After dilution, 0, 20, 50, and 100 µl were added at a time. After standing at room temperature for 1 hour, 200 μl of PBS buffer was added to wash the wells 4 times. Horseradish peroxidase-attached GFP antibody (ab chem) was diluted 6000 times with PBS buffer, and 100 µl was added to each well. After standing at room temperature for 1 hour, 200 μl of PBS buffer was added to wash the wells 4 times. 100 µl of TMB substrate solution (Biorad) was added to each well, followed by color development at room temperature for 10 minutes. After the reaction was terminated by adding 70 µl of a 20% phosphoric acid solution, the absorbance was measured at 450 nm. The results are shown in Figure 4. As shown in FIG. 4, it can be seen that the Grayner well plate has a better signal-to-noise ratio than the Nunc well plate.

(3) Immobilization conditions

The U-box domain of PRP19 obtained in Example 3 was diluted 100 times using 0.1M sodium carbonate buffer (pH 9.4), and then 100 μl in a Greiner or nunc 96-well plate. Each was added and reacted at 4° C. for 14 hours or at room temperature for 2 hours. 200 µl of PBS buffer was added to wash the wells four times, and then 200 µl of PBS buffer in which 0.5% of skim milk was dissolved was added to each well. After standing at room temperature for 40 minutes, 200 µl of PBS buffer was added again to wash 4 times, and then the GFP-fused CLIC protein obtained in Example 2 was diluted 100 times with a blocking buffer (0.5% skim milk, PBS) to 0 , 20, 50, and 100 µl were added each. After standing at room temperature for 1 hour, 200 μl of PBS buffer was added to wash the wells 4 times. Horseradish peroxidase-attached GFP antibody (ab chem) was diluted 6000 times with PBS buffer, and 100 µl was added to each well. After standing at room temperature for 1 hour, 200 µl of PBS buffer was added and washed 4 times. 100 µl of TMB substrate solution (Biorad) was added to each well, followed by color development at room temperature for 20 minutes. After the reaction was terminated by adding 70 µl of a 20% phosphoric acid solution, the absorbance was measured at 450 nm. The results are shown in FIG. 5. As shown in FIG. 5, it can be seen that when immobilized at 4° C. for 14 hours, better results are obtained than when immobilized at room temperature for 2 hours.

Example 5: PRP19 According to the concentration of protein PRP19 Protein and CLIC Protein interaction analysis

The U-box domain of PRP19 obtained in Example 3 was diluted 100-fold using 0.1M sodium carbonate buffer (pH 9.4), and then 0, 2, 5, 10 in a Greiner 96-well plate. , 20, 50, and 100 μl were added each and reacted at 4° C. for 14 hours. After washing 4 times with 200 µl of PBS buffer, 200 µl of PBS buffer in which 0.5% skim milk was dissolved was added to each well. After standing at room temperature for 40 minutes, 200 µl of PBS buffer was added again to wash the wells four times, and then the GFP-fused CLIC protein obtained in Example 2 was diluted 200 times with blocking buffer, and the resulting solution was added 100 µl each. I did. After standing at room temperature for 1 hour, 200 μl of PBS buffer was added to wash the wells 4 times. Horseradish peroxidase-attached GFP antibody (ab chem) was diluted 6000 times with PBS buffer, and 100 µl was added to each well. After standing at room temperature for 1 hour, 200 μl of PBS buffer was added to wash the wells 4 times. 100 µl of TMB substrate solution (Biorad) was added to each well, and color was developed at room temperature for 10 minutes. After the reaction was terminated by adding 70 µl of a 20% phosphoric acid solution each, the absorbance was measured at 450 nm. The results are shown in Figure 6. As shown in Fig. 6, it can be seen that the interaction with CLIC can be confirmed even with only 20 µl of the solution obtained by diluting the U-box 100 times.

Example 6: ELISA Analysis Freshwater By extract PRP19 Protein and CLIC Confirmation of protein interaction inhibitory effect

The U-box domain of PRP19 obtained in Example 3 was diluted 500 times using 0.1M sodium carbonate buffer (pH 9.4), and then 100 µl was added to a Greiner 96-well plate at 4°C. It was reacted at for 14 hours. 200 µl of PBS buffer was added and washed four times, and then 200 µl of PBS buffer in which 0.5% skim milk was dissolved was added to each well. After leaving for 40 minutes at room temperature, 200 µl of PBS buffer was added to wash 4 times, and then the GFP-fused CLIC protein obtained in Example 2 was diluted 200 times in blocking buffer (0.5% skim milk PBS). Was added by 100 μl. Add 1 µl of Dampal water extract (002-025) purchased from Korea Plant Extract Bank to a concentration of 0, 1 ppm, 2 ppm, 5 ppm, 10 ppm, and 20 ppm, and then stand at room temperature for 1 hour, and then PBS buffer solution 200 µl was added to wash the wells 4 times. Horseradish peroxidase-attached GFP antibody (ab chem) was diluted 6000 times with PBS buffer, and 100 µl was added to each well. Thereafter, it was allowed to stand at room temperature for 1 hour, and 200 µl of PBS buffer was added and washed 4 times. 100 µl of TMB substrate solution (Biorad) was added to each well, and color was developed at room temperature for 20 minutes. After the reaction was terminated by adding 70 µl of a 20% phosphoric acid solution each, the absorbance was measured at 450 nm. The results are shown in FIG. 7. As shown in FIG. 7, it can be seen that the degree of inhibition of the interaction between the PRP19 protein and the CLIC protein increases as the concentration of the dampal water extract increases.

Example 7: Freshwater Confirmation of the effect of extract inhibiting differentiation of adipocytes

<Step 1> Adipocyte differentiation induction and treatment with Dampal water extract

Rat ( Mus musculus ) undifferentiated adipocyte line 3T3-L1 (ATCC CL173) was dispensed into a 12-well plate, and the cells were placed in DMEM (Dulbecco's modified Eagle's Medium, Gibco 1210-0038) medium containing 10% bovine serum. It was incubated in a _{10% CO 2} incubator for 48 hours until it grew more than %. Thereafter, the cells were cultured in DMEM medium containing 10% fetal bovine serum, 0.5mM 3-isobutyl-1-methylxanthine (Sigma), 1 μM dexamethasone (Sigma), and 167 nM insulin (Novo-Nordisk) for 48 hours. Then, it was cultured again in DMEM medium containing 10% fetal calf serum and 167 nM insulin for 48 hours. Thereafter, the dampal water extract used in Example 6 was treated by 3, 10, and 30 ppm so as to be 1/1000 of the medium. Thereafter, the culture was further cultured for 48 hours and cultured for 48 hours in DMEM medium containing only 10% fetal calf serum.

<Step 2> Confirmation of inhibition of fat body formation

The cells obtained in step 1 were fixed with 3.7% formaldehyde, and then washed three times with PBS buffer. In order to stain the fat body, the cells were reacted for 30 minutes in 0.2% oil red solution, and then washed 4 times for 15 minutes each with PBS buffer. The stained cells were observed with a microscope imaging device (Axioplan-2, Carl Zeiss). The results are shown in Figure 8. In Fig. 8, those stained red indicate the triglycerides of the fat body. It can be seen that fat body formation was suppressed in cells (DP 30) treated with Dampal Water extract compared to the control group.

Meanwhile, the cells obtained in step 1 were fixed with 3.7% formaldehyde, and then washed three times with PBS buffer. After staining for 1 hour in 0.5% Sudan II solution, it was washed 4 times for 15 minutes each with PBS buffer. 500 µl of a nonionic surfactant (Nonidet-P40, 4% NP40) and 60% isopropanol solution were added thereto, followed by extraction for 20 minutes. The extracted solution was diluted 1/10 and absorbance was measured at 490 nm. The results are shown in FIG. 9. In FIG. 9, RA was used as a positive control as retinoic acid known to inhibit the differentiation of fat. As shown in FIG. 9, cells treated with dampal water extract (DP3, DP10, DP30) decreased the amount of triglycerides compared to the untreated control cells, and showed similar effects as when using ^{10 -9 M RA.} Are giving.

Example 8: PRP19 And CLIC Between proteins Interaction check

<Step 1> Preparation of PRP19 and CLIC protein fragments

To obtain the entire RPR19 protein, its N-terminal (1-166 amino acids) and C-terminal (167-504 amino acids) fragment, and CLIC protein fragment, murine ( Mus musculus ) After extracting RNA from 3T3-L1 (ATCC CL173) cells, an undifferentiated adipocyte line of musculus, cDNA was synthesized using a onestep RT-PCR kit (Qiagen 210210), and PCR was performed using the primer pairs listed in Table 2 below. I did. A hemagglutinin (HA) sequence was attached to the end of the primer used for the synthesis of the PRP19 protein fragments.

PCR was carried out using a Tpersonal (Whatman, GmbH) instrument for PCR reactions (38 µl of secondary distilled water, 5 µl of 10× buffer, 5 µl of dNTP (2.5 mM each), 0.5 µl (100 pmol) of each primer, and a template. 0.5 [mu]l, PFU Taq polymerase 0.5 [mu]l (5 U/[mu]l)) was performed according to the following conditions: initial denaturation (95[deg.] C. for 5 minutes); 25 repetitions of denaturation (95° C. for 30 seconds), annealing (55° C. for 30 seconds), and extension (2 minutes at 68° C.); And last extension (72° C. for 10 minutes). The obtained PCR product was purified using a MiniEluteTM PCR purification kit (QIAGEN, USA).

The PCR product was cloned according to the manufacturer's instructions with the pCR 2.1-TOPO vector provided in the TOPO TA cloning kit (Invitrogen). Specifically, 4 µl of PCR product, 1 µl of salt solution provided in the TOPO TA cloning kit (Invitrogen), and 1 µl of pCR 2.1-TOPO vector were mixed and reacted at room temperature for 5 minutes, and then TOP10 Escherichia coli ( E. coli ) was cloned by transformation.

PCR 2.1-TOPO-PRP19 and pCR 2.1-TOPO-CLIC vectors from which PCR products were cloned were obtained from the transformed host cells, digested with EcoRI/XbaI and XhoI/BamHI restriction enzymes, respectively, and then PCR products PRP19 and CLIC DNA. Fragments were isolated and cloned into pCDNA3.1 vector treated with the same restriction enzyme. The obtained vectors were named pcDNA3.1 PRP19-HA and pcDNA3.1/Myc-His-CLIC. Sequence analysis was requested to Macrogen Inc. to determine the nucleotide sequence of each clone to confirm the presence of PRP19 and CLIC DNA.

<Step 2> Preparation of human embryonic kidney (Hek) 293 cells transfected with a vector containing PRP19 and CLIC DNA

HeK 293 cells (ATCC CRL-1573) were cultured in a DMEM medium containing 10% bovine serum at 37°C in a 5% CO ₂ incubator. The cultured Hek 293 cells were dispensed into a 6-well plate, and the cells were prepared in step 1 in the presence of the transfection agent Lipofectamine 2000 (Invitrogen) the next day, pcDNA3.1 PRP19-HA and/or pcDNA3.1/Myc-His -CLIC expression vector was mixed with 4g and reacted for 20 minutes. After 8 hours, the solution was removed, washed 3 times with PBS buffer (WelGENE Inc.), exchanged with DMEM medium containing only 10% fetal calf serum, and cultured for 48 hours in _{a 37°C, 5% CO 2 incubator for transfection.} The cells were obtained.

<Step 3> Immunoprecipitation of PRP19 and CLIC protein

The cells obtained in step 2 were washed three times with cold PBS buffer, and then shaken for 5 minutes using 250 μl of M-PER mammalian protein extraction kit (PIERCE. Rockoford, IL). Cell lysate was transferred to a tube and centrifuged at 16,000xg for 20 minutes. After obtaining the supernatant, immunoprecipitation was performed using the ProFound Mammalian HA tag Co-IP kit (PIERCE. Rockoford, IL) according to the method provided in the kit.

Specifically, 200 µl of the cell supernatant obtained previously was placed on a Handee Mini-Spin column, and 6 µl of anti-HA agarose slurry was added thereto, followed by mixing at 4° C. for 16 hours. Thereafter, washed three times with a TBS buffer containing 0.05% Tween-20 (25 mM Tris, 0.15 M NaCl, pH 7.2/ 500 ml), and 25 μl 2X non-reducing sample buffer (0.12) M Tris-HCl, pH 6.8, 2% SDS, 20% glycerol) was added, then boiled at 95° C. for 5 minutes and centrifuged at 10,000xg for 10 seconds to take a supernatant.

On the other hand, the immunoprecipitation reaction was performed in the same manner as above using the ProFound Mammalian Myc Tag Co-IP kit (PIERCE. Rockoford, IL).

<Step 4> Western blot analysis

10 µl of each supernatant obtained in step 3 was separated by size by electrophoresis on 8% SDS-PAGE, and then transferred to a PVDF membrane (Bio-Rad) for 12 hours under 50V conditions. Thereafter, the protein-transferred membrane was blocked with a 5% skim milk solution for 1 hour, and then treated with anti-HA or anti-Myc, a polyclonal antibody as the primary antibody, and horseradish as a secondary antibody. Anti-mouse IgG (Amersham) antibody conjugated with peroxidase (horseradish peroxidase) was treated, and an antibody reaction was performed using an ECL kit (enhanced chemiluminescence kit, Amersham). The reacted PVDF film was sensitized on an X-ray film (Fuji) and developed to confirm the protein expression level. The results are shown in FIG. 10.

Figure 10 (a) is a total cell lysate (lysate) analysis, and (b) is to Myc fused to CLIC after performing an immunoprecipitation reaction using an antibody against HA fused to PRP19 on the same sample. This is the result of Western blotting using an antibody against. When PRP19 or CLIC was expressed alone, nothing was detected in the immunoprecipitation reaction with anti-HA. On the other hand, when both proteins were expressed together, it was confirmed that CLIC was detected by anti-Myc. This shows that the two proteins PRP19 and CLIC specifically interact within cells.

Figure 10 (c) and (d) show the results of performing an immunoprecipitation reaction using an antibody against Myc fused to CLIC and then performing a Western blot using an antibody against HA fused to PRP19. As a result, it can be seen that CLIC interacts with the entire and N-terminus of PRP19.

<110> AMOREPACIFIC CORPORATION <120> METHOD OF SCREENING A SUBSTANCE CAPABLE OF INHIBITING INTERACTION BETWEEN PRP19 AND CLIC PROTEINS AND A COMPOSITION COMPRISING THE SCREENED SUBSTANCE <130> FPD/201410-0025 <160> 16 <170> KopatentIn 1.71 <210> 1 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer for Gene Encoding U-box Domain of PRP19 Protein <400> 1 acttgaggat ccatgtccct gatctgc 27 <210> 2 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Reverse Primer for Gene Encoding U-box Domain of PRP19 Protein <400> 2 acctgaattc ctcaggtggc ggagggagg 29 <210> 3 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer for Gene Encoding CLIC Protein <400> 3 ggatccgatg gctgaagaac aacctcagg 29 <210> 4 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Reverse Primer for Gene Encoding CLIC Protein <400> 4 ggatcccgtt tgagagccct ggccactt 28 <210> 5 <211> 207 <212> DNA <213> U-box Domain of PRP19 Protein of Mus musculus <220> <221> CDS <222> (1)..(207) <400> 5 atg tcc ctg atc tgc tcg atc tcc aat gaa gtg cca gag cac ccg tgc 48 Met Ser Leu Ile Cys Ser Ile Ser Asn Glu Val Pro Glu His Pro Cys 1 5 10 15 gtg tcc cct gtc tct aat cat gtg tat gag cgg cga ctc att gag aag 96 Val Ser Pro Val Ser Asn His Val Tyr Glu Arg Arg Leu Ile Glu Lys 20 25 30 tac att gca gag aat ggc aca gat cct atc aac aac cag cct ctc tca 144 Tyr Ile Ala Glu Asn Gly Thr Asp Pro Ile Asn Asn Gln Pro Leu Ser 35 40 45 gag gag cag ctc atc gac atc aaa gtt gct cac cca atc cga ccc aag 192 Glu Glu Gln Leu Ile Asp Ile Lys Val Ala His Pro Ile Arg Pro Lys 50 55 60 cct ccc tcc gcc acc 207 Pro Pro Ser Ala Thr 65 <210> 6 <211> 69 <212> PRT <213> U-box Domain of PRP19 Protein of Mus musculus <400> 6 Met Ser Leu Ile Cys Ser Ile Ser Asn Glu Val Pro Glu His Pro Cys 1 5 10 15 Val Ser Pro Val Ser Asn His Val Tyr Glu Arg Arg Leu Ile Glu Lys 20 25 30 Tyr Ile Ala Glu Asn Gly Thr Asp Pro Ile Asn Asn Gln Pro Leu Ser 35 40 45 Glu Glu Gln Leu Ile Asp Ile Lys Val Ala His Pro Ile Arg Pro Lys 50 55 60 Pro Pro Ser Ala Thr 65 <210> 7 <211> 726 <212> DNA <213> CLIC of Mus musculus <220> <221> CDS <222> (1)..(723) <400> 7 atg gct gaa gaa caa cct cag gtc gaa ctg ttc gtg aag gct ggc agt 48 Met Ala Glu Glu Gln Pro Gln Val Glu Leu Phe Val Lys Ala Gly Ser 1 5 10 15 gat ggt gcc aag att ggg aac tgc ccc ttc tcc cag aga ctg ttc atg 96 Asp Gly Ala Lys Ile Gly Asn Cys Pro Phe Ser Gln Arg Leu Phe Met 20 25 30 gtg ctc tgg ctc aag gga gtc acc ttc aac gtt acc act gtg gac acc 144 Val Leu Trp Leu Lys Gly Val Thr Phe Asn Val Thr Thr Val Asp Thr 35 40 45 aag aga cgg aca gag act gta cag aag ctc tgc ccc ggt ggg cag ctg 192 Lys Arg Arg Thr Glu Thr Val Gln Lys Leu Cys Pro Gly Gly Gln Leu 50 55 60 ccg ttc ttg ctc tat ggc acc gaa gtg cac aca gac acc aac aag atc 240 Pro Phe Leu Leu Tyr Gly Thr Glu Val His Thr Asp Thr Asn Lys Ile 65 70 75 80 gag gaa ttc ctg gag gcc atg ctg tgc cct ccc agg tac cca aag ctg 288 Glu Glu Phe Leu Glu Ala Met Leu Cys Pro Pro Arg Tyr Pro Lys Leu 85 90 95 gct gcc ctg aac cct gag tcc aac acc tcg gga ctg gac ata ttt gca 336 Ala Ala Leu Asn Pro Glu Ser Asn Thr Ser Gly Leu Asp Ile Phe Ala 100 105 110 aag ttt tct gcc tac atc aag aac tca aac cca gcc ctc aat gac aac 384 Lys Phe Ser Ala Tyr Ile Lys Asn Ser Asn Pro Ala Leu Asn Asp Asn 115 120 125 cta gag aag gga ctc ctg aaa gcc ctg aag gtt cta gac aat tac ctg 432 Leu Glu Lys Gly Leu Leu Lys Ala Leu Lys Val Leu Asp Asn Tyr Leu 130 135 140 aca tcc ccc ctc cca gaa gaa gtg gat gaa acc agc gcc gaa gat gag 480 Thr Ser Pro Leu Pro Glu Glu Val Asp Glu Thr Ser Ala Glu Asp Glu 145 150 155 160 ggc atc tct cag agg aag ttt ctg gac ggc aat gag ctc acc ctg gct 528 Gly Ile Ser Gln Arg Lys Phe Leu Asp Gly Asn Glu Leu Thr Leu Ala 165 170 175 gac tgc aac ctg ctg cca aag ctt cac ata gta cag gtg gtg tgc aaa 576 Asp Cys Asn Leu Leu Pro Lys Leu His Ile Val Gln Val Val Cys Lys 180 185 190 aag tac aga ggc ttc acc atc cca gag gca ttc cgt gga gtg cat cgg 624 Lys Tyr Arg Gly Phe Thr Ile Pro Glu Ala Phe Arg Gly Val His Arg 195 200 205 tac ttg agc aac gct tat gcc cgg gaa gaa ttt gcc tcc acc tgt cca 672 Tyr Leu Ser Asn Ala Tyr Ala Arg Glu Glu Phe Ala Ser Thr Cys Pro 210 215 220 gat gat gaa gag ata gag cta gcc tat gag caa gtg gcc agg gct ctc 720 Asp Asp Glu Glu Ile Glu Leu Ala Tyr Glu Gln Val Ala Arg Ala Leu 225 230 235 240 aaa tga 726 Lys <210> 8 <211> 241 <212> PRT <213> CLIC of Mus musculus <400> 8 Met Ala Glu Glu Gln Pro Gln Val Glu Leu Phe Val Lys Ala Gly Ser 1 5 10 15 Asp Gly Ala Lys Ile Gly Asn Cys Pro Phe Ser Gln Arg Leu Phe Met 20 25 30 Val Leu Trp Leu Lys Gly Val Thr Phe Asn Val Thr Thr Val Asp Thr 35 40 45 Lys Arg Arg Thr Glu Thr Val Gln Lys Leu Cys Pro Gly Gly Gln Leu 50 55 60 Pro Phe Leu Leu Tyr Gly Thr Glu Val His Thr Asp Thr Asn Lys Ile 65 70 75 80 Glu Glu Phe Leu Glu Ala Met Leu Cys Pro Pro Arg Tyr Pro Lys Leu 85 90 95 Ala Ala Leu Asn Pro Glu Ser Asn Thr Ser Gly Leu Asp Ile Phe Ala 100 105 110 Lys Phe Ser Ala Tyr Ile Lys Asn Ser Asn Pro Ala Leu Asn Asp Asn 115 120 125 Leu Glu Lys Gly Leu Leu Lys Ala Leu Lys Val Leu Asp Asn Tyr Leu 130 135 140 Thr Ser Pro Leu Pro Glu Glu Val Asp Glu Thr Ser Ala Glu Asp Glu 145 150 155 160 Gly Ile Ser Gln Arg Lys Phe Leu Asp Gly Asn Glu Leu Thr Leu Ala 165 170 175 Asp Cys Asn Leu Leu Pro Lys Leu His Ile Val Gln Val Val Cys Lys 180 185 190 Lys Tyr Arg Gly Phe Thr Ile Pro Glu Ala Phe Arg Gly Val His Arg 195 200 205 Tyr Leu Ser Asn Ala Tyr Ala Arg Glu Glu Phe Ala Ser Thr Cys Pro 210 215 220 Asp Asp Glu Glu Ile Glu Leu Ala Tyr Glu Gln Val Ala Arg Ala Leu 225 230 235 240 Lys <210> 9 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer for Gene Encoding Whole PRP19 Protein <400> 9 gaattctatg tccctgatct gctcg 25 <210> 10 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> Reverse Primer for Gene Encoding Whole PRP19 Protein <400> 10 tctagactaa agagcgtaat ctggaacatc gtatgggtac agactgtaga atttgag 57 <210> 11 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer for Gene Encoding N-terminal of PRP19 Protein <400> 11 gaattctatg tccctgatct gctcg 25 <210> 12 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> Reverse Primer for Gene Encoding N-terminal of PRP19 Protein <400> 12 tctagactaa agagcgtaat ctggaacatc gtatgggtac accagctcac ccaaatc 57 <210> 13 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer for Gene Encoding C-terminal of PRP19 Protein <400> 13 gaattctatg ggaatgaccc ctgagatt 28 <210> 14 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> Reverse Primer for Gene Encoding C-terminal of PRP19 Protein <400> 14 tctagactaa agagcgtaat ctggaacatc gtatgggtac agactgtaga atttgag 57 <210> 15 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer for Gene Encoding CLIC Protein Fragment <400> 15 ctcgagcggc atggctgaag aacaacctca ggtc 34 <210> 16 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Reverse Primer for Gene Encoding CLIC Protein Fragment <400> 16 ggatccgttc ttgatgtagg cagaaaactt 30

Claims (2)

Elaeocarpus sylvestris there is. ellipticus (Thunb.) Hara) extract as an active ingredient. The method according to claim 1,
Wherein said descendant extract is contained in an amount of 0.0001 to 20% by weight based on the total weight (dry weight) of the cosmetic composition.

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