KR102220907B1 - Method for screening agent for anti-obesity using phosphorylation of histon - Google Patents

Abstract

In the present invention, a candidate compound expected to inhibit differentiation or accumulation of fat is treated on stem cells, and the expression level of S6K1 (Ribosomal S6 kinase 1) or H2B phosphorylation level is measured in the administered stem cells. It relates to a method for screening an agent capable of preventing or treating obesity, comprising the step of: The method of screening for an agent capable of preventing or treating obesity of the present invention uses the phosphorylation of Serine residue 36 of H2B by S6K1, which exhibits the same action at the cellular level as well as in vivo, so it is widely used in the development of more effective obesity inhibitors. It will be possible.

Description

Obesity inhibitor screening method using histone phosphorylation {Method for screening agent for anti-obesity using phosphorylation of histon}

The present invention relates to a method for screening obesity inhibitors using histone phosphorylation, and more specifically, the present invention treats stem cells with a candidate compound expected to inhibit differentiation or accumulation of fat, and in the administered stem cells It relates to a method for screening an agent capable of preventing or treating obesity, comprising measuring the level of expression of S6K1 (Ribosomal S6 kinase 1) or the level of phosphorylation of H2B.

Obesity generally means that there is an excess of fat tissue in the body, and it is a phenomenon in which excess energy is accumulated as body fat because the energy consumed by food cannot be balanced with the energy consumed by physical activities. Abnormal increase in body fat due to energy imbalance over a long period of time causes various metabolic diseases and adult diseases such as diabetes, hyperlipidemia, heart disease, stroke, arteriosclerosis, fatty liver, etc., which is a serious social problem in Korea as well as in the West. .

Obesity is caused by a variety of causes, such as genetic factors, environmental factors due to westernized diet, psychological factors due to stress, and abnormal energy metabolism. And the type can be divided into simple obesity due to overeating and lack of exercise and symptomatic obesity due to endocrine, hypothalamic, hereditary, and metabolic, depending on the cause.

In the evaluation of obesity, there are a method of measuring weight and a method of measuring the thickness of skin wrinkles. In general, obesity is defined if the obesity index is 25 or higher. Body obesity index (body mass index, BMI) is a value obtained by dividing weight (kg) by the square of height (m). Westerners are over 30, but in Korea, over 25 is evaluated by taking into account differences between races. .

Multi-faceted research is underway for the development of obesity treatments around the world. Drugs for the treatment of obesity can be largely divided into mechanisms of suppression of fat absorption, promotion of fat breakdown and heat generation, regulation of appetite and satiety, inhibition of protein metabolism, and emotional regulation related to food consumption. Representative obesity treatments include Xenical™, which suppresses fat absorption using oristat, and Reductil™, which suppresses appetite by stimulating the sympathetic nervous system with sibutramine as the main ingredients. . However, in the case of Xenical™, side effects such as fatty stool, abdominal pain, vomiting, itching, and liver damage have been reported. In the case of Reductil™, it causes serious cardiovascular side effects as well as side effects such as headache, loss of appetite, insomnia, and constipation. There is a controversy over the recent reinforcement of usage standards.

In addition to drug therapy through these obesity drugs, there are dietary therapy to limit food intake and exercise therapy to increase energy consumption as methods for preventing and treating obesity, and psychotherapy, behavioral therapy, and surgical therapy are also being implemented.

As a preferable treatment method for obesity, it is suggested that the most safe and effective method is to promote energy consumption through exercise and to use drugs for treating obesity with little side effects. However, in the case of drugs for the treatment of obesity, serious side effects have been reported as in the examples of Xenical™ and Reductil™, and clear confidence in safety is not supported. Therefore, there is a need for development of a material that can guarantee safety while exhibiting excellent anti-obesity effects on the human body.

In addition to drug therapy through these obesity drugs, there are dietary therapy to limit food intake and exercise therapy to increase energy consumption as methods for preventing and treating obesity, and psychotherapy, behavioral therapy, and surgical therapy are also being implemented.

As a preferable treatment method for obesity, it is suggested that the most safe and effective method is to promote energy consumption through exercise and to use drugs for treating obesity with little side effects. However, in the case of drugs for the treatment of obesity, serious side effects have been reported as in the examples of Xenical™ and Reductil™, and clear confidence in safety is not supported. Therefore, there is a need for development of a material that can guarantee safety while exhibiting excellent anti-obesity effects on the human body.

On the other hand, in order to develop a material that guarantees such stability, research is being actively conducted to develop a material that exhibits anti-obesity effects for various natural products. For example, sprout barley is known to contain significantly higher amounts of plant fiber, carotene, and vitamin C compared to other vegetables and fruits. In addition, in terms of pharmacological activity, the sprout barley is known to exhibit effects such as cholesterol lowering, constipation prevention, arteriosclerosis prevention, antioxidant activity, aging prevention, osteoporosis prevention, insomnia improvement, anemia prevention, etc. On the nutritional value and anti-oxidation, anti-cancer, anti-diabetic and whitening effect of the food, global food and food culture, 2010; Korean Patent Registration No. 1174497; Korean Patent Publication No. 2013-0051181). In particular, luteorin contained in sprout barley is known to exhibit superior antioxidant activity compared to vitamin C and genistein, which are known as antioxidants, and luteorin is known to exhibit superior antidiabetic activity than acarbose, a diabetes treatment agent.

However, even after the development of an anti-obesity substance that has secured safety in this case, it is never easy to commercialize it. Although it exhibits the effect of inhibiting fat accumulation at the cellular level, the effect of inhibiting fat accumulation at the living body level is sometimes reduced. Because there are many, this is predicted to be due to homeostasis in the body, and as a result, there is a problem that enormous cost and loss of time occur. If, in the initial selection process of anti-obesity substances, the possibility of differentiation or accumulation of fat at the cellular level as well as at the biological level can be predicted in advance, it is expected that such problems can be substantially improved. It is not reported.

Under this background, the present inventors have made extensive research efforts to develop a method to predict the possibility of differentiation or accumulation of fat at the cellular level as well as at the biological level in the initial selection process of anti-obesity substances. It was found that phosphorylation of H2B, a kind of H2B, is involved in the differentiation of fat, and the association between H2B phosphorylation and fat differentiation is maintained not only at the cellular level but also at the living body level, and by confirming the phosphorylation of H2B, screening for anti-obesity substances It was confirmed that it could be done, and the present invention was completed.

One object of the present invention is to provide a method for screening an agent capable of preventing or treating obesity by using a change in the level of intracellular phosphorylation of H2B, a type of histone.

While conducting various studies to develop a method for screening an agent that can more effectively prevent or treat obesity, the present inventors paid attention to S6K1 (Ribosomal S6 kinase 1). The S6K1 is known as a key regulator that affects the growth, size and cell metabolism of cells in the cell, and it was confirmed that the phosphorylation of histones performed in the nucleus of S6K1 is essential for the differentiation or accumulation of fat. Specifically, the S6K1 phosphorylates the 36th serine residue of H2B, a type of histone. When stem cells whose expression is inhibited are differentiated into adipocytes, the level of markers of adipocytes decreases, whereas the expression thereof is increased. In this case, it was confirmed that the marker level of adipocytes increased, and when serine 36 of H2B, a substrate of S6K1, was phosphorylated, when the phosphorylation of the serine was inhibited, it was confirmed that the mRNA level of PPARγ was remarkably decreased.

As such, the present inventors first identified that phosphorylation of H2B is performed in the process of promoting adipocyte differentiation through activation of S6K1, and that the phosphorylation of H2B is essential for adipocyte differentiation.

Therefore, S6K1 phosphorylates the 36th serine residue of H2B and is involved in the differentiation or accumulation of fat in vivo, and it is expected that S6K1 can be utilized in the development of a formulation capable of inhibiting the differentiation or accumulation of fat when using it.

As an embodiment for achieving the above object, the present invention treats stem cells with a candidate compound expected to inhibit differentiation or accumulation of fat, and S6K1 (Ribosomal S6 kinase 1) in the administered stem cells. It provides a method for screening an agent capable of preventing or treating obesity, comprising the step of measuring the expression level of H2B or the level of phosphorylation of H2B.

Specifically, the method of screening for an agent capable of preventing or treating obesity of the present invention is a candidate compound that is expected to inhibit differentiation or accumulation of fat in stem cells capable of differentiating into adipocytes. Compound treatment step; (b) measuring the level of expression of Ribosomal S6 kinase 1 (S6K1) and the level of phosphorylation of histone H2B in cells treated with the candidate compound; And (c) selecting a candidate compound having reduced expression level or phosphorylation level compared to a negative control group not treated with the candidate compound.

In this case, the expression level of S6K1 is preferably measured through its mRNA level or the level of the protein expressed therefrom, and more preferably, an agent for measuring the level of the mRNA of S6K1 or the protein expressed therefrom is used, or It can be measured using a composition or kit containing the agent, and when the level measured in the experimental group is significantly reduced compared to the level measured in the control group, the candidate compound prevents obesity, which inhibits differentiation or accumulation of fat. Alternatively, it can be determined that it can be used as a therapeutic agent.

The term "enjoyment cell" of the present invention refers to an undifferentiated adult stem cell that has excellent self-renewal ability and can differentiate into various cells such as liver, bone, cartilage, fat, blood vessels, heart, nervous system, and the like.

In the present invention, the stem cells may be interpreted as being stem cells capable of differentiating into adipose tissues among various tissues, preferably mesenchymal stem cells (MSCs), and more preferably Adipose tissue that has advantages in terms of accessibility, stability, effectiveness, and economics due to its easy and safe acquisition process in that adipose tissue can be extracted in large quantities, there is no restriction on the supply and demand of tissue, and easy in vitro culture. adipose tissue) present in adipose-derived stem cells (ASCs), and most preferably, 10T1/2 cells in which a method of differentiation into adipocytes is established among mesenchymal stem cells.

The term "S6K1 (Ribosomal S6 kinase 1)" of the present invention, also called Ribosomal protein S6 kinase beta-1, is a protein that is a key regulator affecting the growth, size and cell metabolism of cells in the cell, and the RPS6KB1 gene It means a protein encoded by. The specific amino acid sequence or nucleotide sequence and protein information of the S6K1 or the gene encoding it are known from NCBI (GenBank: NP_001107806, NM_001114334).

The term "agent for measuring the mRNA level of a gene" of the present invention refers to an agent used in a method of measuring the level of mRNA transcribed from the target gene in order to confirm the expression of a target gene included in a sample. , Preferably RT-PCR, competitive RT-PCR, real-time RT-PCR, RNase protection assay (RPA), Northern blotting , It may be a probe or primer capable of specifically binding to a target gene used in a method such as a DNA chip analysis method, but is not particularly limited thereto.

The term "primer" of the present invention is a nucleic acid sequence having a short free 3'hydroxyl group, which can form a complementary template and a base pair, and is a starting point for template strand copying. It refers to a short nucleic acid sequence that functions as. The primers can initiate DNA synthesis in the presence of a reagent for polymerization (ie, DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates at an appropriate buffer and temperature.

In the present invention, the primer may be a primer that can be used for amplification of a gene of a gene encoding S6K1, and as long as it can be amplified by a PCR method by complementary binding to the gene, the nucleotide sequence of the primer is Not limited.

The term "probe" of the present invention refers to a nucleic acid fragment such as RNA or DNA corresponding to a short number of bases to several hundred bases capable of specific binding to a gene or mRNA, and an oligonucleotide probe, It may be produced in the form of a single stranded DNA (single stranded DNA) probe, a double stranded DNA (double stranded DNA) probe, an RNA probe, and the like, and may be labeled for easier detection.

In the present invention, the probe may be a probe capable of complementary binding to the gene encoding the S6K1, and the nucleotide sequence of the probe is not limited as long as it can complementarily bind to the gene.

The term "agent for measuring the level of protein" of the present invention means an agent used in a method of measuring the level of a target protein contained in a sample, preferably western blotting, ELISA (enzyme linked immunosorbent). assay), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immune diffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay (Immunoprecipitation Assay), complement It may be an antibody used in methods such as a Complement Fixation Assay, FACS, and protein chip assay.

The term "antibody" of the present invention refers to a proteinaceous molecule capable of specifically binding to an antigenic site of a protein or peptide molecule, and such an antibody is a gene cloned into an expression vector according to a conventional method, and the marker A protein encoded by a gene can be obtained, and can be prepared from the obtained protein by a conventional method. The form of the antibody is not particularly limited, and if a polyclonal antibody, a monoclonal antibody, or any one having antigen binding properties, a part thereof is also included in the antibody of the present invention, and all immunoglobulin antibodies may be included, as well as humanized antibodies, etc. It may also contain a special antibody of. In addition, the antibody comprises a complete form having two full-length light chains and two full-length heavy chains, as well as functional fragments of antibody molecules. The functional fragment of an antibody molecule refers to a fragment having at least an antigen-binding function, and may be Fab, F(ab'), F(ab') 2, Fv, and the like.

In the present invention, the antibody may be an antibody capable of specifically binding to S6K1 or phosphorylated H2B, and preferably a polyclonal antibody or a monoclonal antibody capable of specifically binding to each of the proteins. Or you could be part of it.

On the other hand, in performing the method of the present invention, the kit comprising an agent for measuring the level of the mRNA of each gene or the level of the protein expressed therefrom that can be used is the level of the mRNA of the gene or the level of the protein expressed therefrom In addition to primers, probes, or antibodies for measuring, one or more other constituent compositions, solutions, or devices suitable for the assay method may be included.

As a specific example, the kit for measuring the mRNA expression level of the gene encoding S6K1 of the present invention may be a kit including essential elements necessary for performing RT-PCR. The RT-PCR kit includes, in addition to each primer pair specific for the gene, a test tube or other suitable container, reaction buffer (varies in pH and magnesium concentration), deoxynucleotides (dNTPs), Taq-polymerase and reverse transcriptase. Such enzymes, DNase, RNAse inhibitors, DEPC-water, sterile water, and the like may be included. In addition, a primer pair specific to a gene used as a quantitative control may be included.

As another example, the kit of the present invention may include essential elements necessary to perform a DNA chip analysis method. The DNA chip analysis kit may include a substrate to which cDNA corresponding to a gene or a fragment thereof is attached as a probe, and reagents, agents, enzymes, etc. for preparing a fluorescently labeled probe. In addition, the substrate may include a cDNA corresponding to a quantitative control gene or a fragment thereof.

As another example, the kit of the present invention may be a protein chip analysis kit for measuring the level of S6K1 or phosphorylated H2B, the kit is not particularly limited thereto, It may contain a buffer solution, a secondary antibody labeled with a color developing enzyme or a fluorescent substance, a color developing substrate, and the like. The substrate is not particularly limited thereto, but a nitrocellulose membrane, a 96-well plate synthesized with polyvinyl resin, a 96-well plate synthesized with polystyrene resin, and a slide glass made of glass may be used, and the color developing enzyme is not particularly limited thereto. However, peroxidase and alkaline phosphatase may be used, and the fluorescent material is not particularly limited thereto, but may be FITC, RITC, etc., and the color developing substrate solution is not particularly limited thereto, but ABTS (2, 2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) or OPD (o-phenylenediamine), TMB (tetramethyl benzidine).

According to an embodiment of the present invention, as a result of confirming whether S6K1 can phosphorylate histones, H2B is specifically phosphorylated (FIG. 1A), and when the S6K1 inhibitor PF-4708671 is treated, concentration-dependently H2B Decreases the phosphorylation of (Fig. 1b), the S6K1 phosphorylates the amino acid site 7 to 26 of H2B (Fig. 1c), and phosphorylates serine 36 of the serine located at amino acids 7 to 26 (Fig. 1d), This was confirmed through immunoblot analysis (FIG. 1E). On the other hand, as a result of confirming the effect of the effect of S6K1 on the differentiation of adipocytes, the mRNA levels of the adipogenic transcription factors PPARγ and C/EBPα decreased in adipocytes differentiated using stem cells that suppressed the expression of S6K1. (Fig. 2a), in adipocytes differentiated using stem cells expressing structurally activated S6K1, the mRNA levels of the adipogenic transcription factors PPARγ and C/EBPα are significantly increased (Fig. 2b), and serine of H2B histones When phosphorylation of is promoted, the mRNA levels of PPARγ and C/EBPα are increased, whereas when the phosphorylation of serine is suppressed, the mRNA level of PPARγ is remarkably decreased (Fig. 2c). In addition, more phosphorylation of H2B histone serine was observed in the white adipose tissue of the rat inducing obesity than the white adipose tissue of the rat fed a normal diet (Fig. 3a), and similarly, it was normal in the abdominal subcutaneous fat tissue of the overweight and obese patients. It was confirmed that phosphorylation of H2B histone serine was increased than that of abdominal subcutaneous fat tissue of patients with BMI (FIG. 3B ).

Therefore, it was found that S6K1 phosphorylates the 36th serine residue of H2B, and thus is involved in the differentiation or accumulation of fat in vivo, thus inhibiting the expression level of S6K1 or inhibiting the phosphorylation of the 36th serine residue of H2B. It was found that a substance capable of inhibiting the differentiation or accumulation of fat.

The method of screening for an agent capable of preventing or treating obesity of the present invention uses the phosphorylation of Serine residue 36 of H2B by S6K1, which exhibits the same action at the cellular level as well as in vivo, so it is widely used in the development of more effective obesity inhibitors. It will be possible.

1A is a photograph showing the results of phosphorylation of various histones by S6K1.
Figure 1b is a photograph showing the change in the phosphorylation level of H2B according to the treatment concentration of the S6K1 inhibitor PF-4708671.
1C is a photograph showing the result of confirming which part of the histone phosphorylation is performed.
1D is a photograph showing the result of confirming phosphorylation by S6K1 for a mutant peptide in which serine residues included in a recombinant peptide containing amino acids 7 to 26 of H2B are substituted with alanine.
1E is an immunoblot analysis picture showing the inhibition of the phosphorylation of Serine residue 36 of H2B by inhibition of S6K1 expression in 10T1/2 cells, where H2BS36Ph represents H2B containing phosphorylated Serine residue 36.
2A is a graph showing the results of comparing the mRNA levels of the adipogenic transcription factors PPARγ and C/EBPα in adipocytes differentiated from stem cells in which S6K1 is normally expressed or its expression is suppressed.
Figure 2b is a wild-type S6K1 gene (S6K1-WT), structurally activated S6K1 gene (S6K1-CA) and dominant negative S6K1 gene (S6K1-DN) PPARγ measured in adipocytes differentiated in each 10T1/2 cells containing And a graph showing the result of comparing the mRNA levels of C/EBPa.
Figure 2c shows adipocytes differentiated from each 10T1/2 cells expressing wild-type H2B (WT), variant H2B (36D) in which serine 36 is substituted with aspartic acid, or H2B (36A) variant H2B (36A) in which serine 36 is substituted with alanine. It is a graph showing the results of comparing the mRNA levels of PPARγ and C/EBPα measured in.
Figure 3a is a photograph showing the result of comparing the degree of H2BS36Ph as an immunoblot in white adipose tissue of a mouse induced obesity (HFD) through a high fat diet and a mouse ingested a normal diet (NCD) as an immunoblot.
3B is a photograph showing the result of comparing the phosphorylation level of S6K1 and the expression level of H2BS36Ph in abdominal subcutaneous fat tissues of obese patients (Overweight) and normal people (Normal).

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

Example One: S6K1 On by Histone Phosphorylation

Example 1-1: various Histone Phosphorylation

Various recombinant histones (H2A, H2B, H3, H4 or H5) were added _{to a kinase buffer containing 5 mM MgCl 2} and 0.3 mM ^γ-32 PATP (0.5 Ci/ml), and under conditions with or without S6K1. And reacted at 30° C. for 1 hour. After the reaction was completed, the protein or peptide was subjected to SDS-PAGE. The electrophoretic gel was stained with Coomassie blue R-150 and visualized, put in an AGFA 100 NIF film, and exposed at -70°C for 24 hours to take a fluorescence picture (Fig. 1a).

1A is a photograph showing the results of phosphorylation of various histones by S6K1. As shown in FIG. 1A, S6K1 preferably phosphorylated H2B, but other histone proteins were not effectively phosphorylated.

In addition, while reacting the H2B and S6K1, the S6K1 inhibitor PF-4708671 was treated at various concentrations (0, 10, 50 or 100 mM) to measure the phosphorylation level of H2B (Fig. 1b).

Figure 1b is a photograph showing the change in the phosphorylation level of H2B according to the treatment concentration of the S6K1 inhibitor PF-4708671. As shown in Figure 1b, the protein level of H2B did not change, but it was confirmed that the phosphorylation level of H2B decreased as the treatment level of PF-4708671 increased, so that the phosphorylation of H2B was performed by S6K1. .

Example 1-2: Histone Identification of phosphorylation sites

In order to confirm at which site of the H2B phosphorylation is performed, a recombinant peptide containing amino acids 7 to 26 of the H2B and a recombinant peptide containing amino acids 27 to 46 were used as substrates to react with S6K1 (Fig. 1c).

1C is a photograph showing the result of confirming which part of the histone phosphorylation is performed. As shown in Figure 1c, it was confirmed that the recombinant peptide containing amino acids 7 to 26 of H2B was phosphorylated.

In addition, it was confirmed that serine that can be phosphorylated in the recombinant peptide containing amino acids 7 to 26 of H2B corresponds to amino acids 32, 36 and 38, and each variant peptide obtained by substituting each serine with alanine It was produced as follows.

27-46: N-KKRKRSRKESYSIYVYKVLK-C (SEQ ID NO: 1)

S32A: N-KKRKRSRKESYSIYVYKVLK-C (SEQ ID NO: 2)

S36A: N-KKRKRSRKESYSIYVYKVLK-C (SEQ ID NO: 3)

S38A: N-KKRKRSRKESYSIYVYKVLK-C (SEQ ID NO: 4)

Each of the mutant peptides prepared above was used as a substrate to react with S6K1 (Fig. 1D).

1D is a photograph showing the result of confirming phosphorylation by S6K1 for a mutant peptide in which serine residues included in a recombinant peptide containing amino acids 7 to 26 of H2B are substituted with alanine. As shown in FIG. 1D, since it was confirmed that the mutant peptide in which serine 36 was substituted with alanine was not phosphorylated, it was found that S6K1 phosphorylated serine 36 of histone H2B.

Example 1-3: Within the cell S6K1 On by Histone Phosphorylation verification

10T1/2 cells were cultured in DMEM medium containing 10% BCS and 1% penicillin/streptomycin (P/S). The cultured 10T1/2 cell cells were treated with rapamycin (Calbiochem, 100 nM) and PF-4708671 (Tocris, 20 μM) to inhibit S6K1 activity. Subsequently, in order to knock down S6K1, siRNA against S6K1 was introduced into cells using a lentiviral vector using Lipofectamine 2000 reagent (Life Technologies), and the cells in which siRNA was expressed by the introduction of the lentivirus was puromycin (2 Μg/ml) was used for selection. The siRNA sequence for S6K1 is as follows:

#03 F: 5'-GGACCAGCCAGAAGAUGCAGGCUCU-3' (SEQ ID NO: 5)

#03 R: 5'-AGAGCCUGCAUCUUCUGGCUGGUCC-3' (SEQ ID NO: 6)

#04 F: 5'-CACCCUUUCAUUGUGGACCUGAUUU-3' (SEQ ID NO: 7)

#04 R: 5'-AAAUCAGGUCCACAAUGAAAGGGUG-3' (SEQ ID NO: 8)

Immunoblot analysis using an anti-p70 S6K1 antibody (Cell Signaling Technology) and an anti-histone H2B Ser36 phosphorylation antibody (ECM biosciences) was performed on 10T1/2 cells expressing the two siRNAs (Fig. 1e). At this time, actin was used as an internal control group.

1E is an immunoblot analysis picture showing the inhibition of the phosphorylation of Serine residue 36 of H2B by inhibition of S6K1 expression in 10T1/2 cells, where H2BS36Ph represents H2B containing phosphorylated Serine residue 36. As shown in Fig. 1e, it was found that phosphorylation of Serine residue No. 36 of H2B was performed by S6K1 in 10T1/2 cells, which are stem cells.

Example 2: Effect on the induction of fat differentiation of stem cells S6K1 Investigation of the effectiveness of

It was intended to confirm how the S6K1 affects the differentiation of adipocytes.

Example 2-1: S6K1 of Whether or not it appears Follow PPAR γ and C/ EBP change in expression level of α

In stem cells, it was attempted to confirm whether the expression levels of the adipogenic transcription factors PPARγ and C/EBPα were changed depending on the expression of S6K1.

First, 10T1/2 cells and 10T1/2 cells (siS6K1 #03 and siS6K1 #04) expressing the two siRNAs produced in Example 1-3 were 10% FBS, 1% P/S, and 10 μg/ml. Cultured in DMEM medium containing BMP4 for 4 days to differentiate into adipocytes, and differentiate the differentiated adipocytes into 10% FBS, 1% P/S, 0.5 mM IBMX (3-isobuyl-1-methylxanthine), 1 μM It was cultured in DMEM medium containing dexamethasone and 1 µg/ml insulin to differentiate into adipocytes. Total RNA was obtained from the differentiated cells, and the obtained total RNA was reverse transcribed using a Reverse Transcription kit (Promega, USA) to obtain cDNA. Subsequently, quantitative real-time PCR (qPCR) using the cDNA as a template was performed using KAPATM SYBR FAST qPCR (KAPABIOSYSTEMS) including CFX96TM and Chromo4TM real-time PCR detector (Bio-Rad). The mRNA levels were measured and compared (FIG. 2A ).

2A is a graph showing the results of comparing the mRNA levels of the adipogenic transcription factors PPARγ and C/EBPα in adipocytes differentiated from stem cells in which S6K1 is normally expressed or its expression is suppressed. As shown in FIG. 2A, it was confirmed that the mRNA levels of the adipogenic transcription factors PPARγ and C/EBPα were decreased in stem cells in which the expression of S6K1 was suppressed.

Example 2-2: activated S6K1 On by PPAR γ and C/ EBP change in expression level of α

From the results of Example 2-1, it was confirmed that the expression levels of PPARγ and C/EBPα were reduced according to the suppression of the expression of S6K1, so to confirm the effect of the activation of S6K1 on the expression levels of PPARγ and C/EBPα. I did.

Specifically, each transformant was obtained by introducing a structurally activated S6K1 gene (S6K1-CA) or a dominant negative S6K1 gene (S6K1-DN) into 10T1/2 cells, and these transformants were subjected to fat After differentiation into cells, the expression levels of PPARγ and C/EBPα expressed in differentiated adipocytes were measured by the method of Example 2-1, and then compared (FIG. 2B). At this time, untransformed 10T1/2 cells having a wild-type S6K1 gene (S6K1-WT) were used as a control.

Figure 2b is a wild-type S6K1 gene (S6K1-WT), structurally activated S6K1 gene (S6K1-CA) and dominant negative S6K1 gene (S6K1-DN) PPARγ measured in adipocytes differentiated in each 10T1/2 cells containing And a graph showing the result of comparing the mRNA levels of C/EBPa. As shown in FIG. 2B, in stem cells into which the structurally activated S6K1 gene (S6K1-CA) was introduced, the mRNA levels of PPARγ and C/EBPα increased, and in particular, it was confirmed that the mRNA level of PPARγ was remarkably increased.

Example 2-3: Histone H2B By phosphorylation of PPAR γ and C/ EBP change in expression level of α

From the results of Example 2-2, it was confirmed that when the activity of S6K1 is increased, the expression levels of PPARγ and C/EBPα are increased, so whether this phenomenon is related to the phosphorylation of serine 36 of histone H2B phosphorylated by S6K1. I wanted to confirm.

Specifically, a vector containing a polynucleotide encoding a variant obtained by substituting serine at No. 36 of H2B with aspartic acid similar to serine (36D) or alanine different from serine (36A) was introduced into 10T1/2 cells, respectively. Was obtained, the transformants were differentiated into adipocytes, and the expression levels of PPARγ and C/EBPα expressed in the differentiated adipocytes were measured by the method of Example 2-1, and then compared. (Fig. 2c). At this time, untransformed 10T1/2 cells having a wild-type H2B gene (WT) were used as a control.

Figure 2c shows adipocytes differentiated from each 10T1/2 cells expressing wild-type H2B (WT), variant H2B (36D) in which serine 36 is substituted with aspartic acid, or H2B (36A) variant H2B (36A) in which serine 36 is substituted with alanine. It is a graph showing the result of comparing the mRNA levels of PPARγ and C/EBPα measured in. As shown in Figure 2c, compared to cells containing wild-type H2B, in cells containing a variant in which serine 36 is substituted with aspartic acid, the mRNA levels of PPARγ and C/EBPα are increased, whereas serine 36 is converted to alanine. In cells containing the substituted variant, it was confirmed that the mRNA level of PPARγ was significantly reduced.

Therefore, it was found that phosphorylation of Serine No. 36 of H2B may affect the expression levels of PPARγ and C/EBPα.

Example 3: Verification of biological adipose tissue

From the results of Examples 1 to 2, it was confirmed that inhibition of the expression of S6K1 can inhibit the differentiation of fat through inhibition of phosphorylation of Serine No. 36 of H2B. I did.

Example 3-1: Analysis of the degree of histone phosphorylation in obese mice

Immunoblot analysis was performed using an adventitious anti-histone H2B Ser36 phosphorylation antibody (ECM biosciences) for obesity-induced mice (HFD) and normal diet-fed mice (NCD) through a high fat diet (Fig. 3A). ). At this time, actin was used as an internal control group.

Figure 3a is a photograph showing the result of comparing the degree of H2BS36Ph as an immunoblot in white adipose tissue of a mouse induced obesity (HFD) through a high fat diet and a mouse ingested a normal diet (NCD). As shown in FIG. 3A, it was confirmed that the level of phosphorylation of Serine residue 36 of H2B was increased in the adipose tissue of the mouse induced obesity.

Example 3-2: obesity In the patient To S6K1 Analysis of the degree of phosphorylation of histones by

From the results of Example 3-1, it was confirmed that phosphorylation of serine 36 of histone H2B was increased in adipose tissue of obese mice, so to confirm whether this phenomenon was observed in human adipose tissue,

The phosphorylation level of S6K1 and the expression level of H2BS36Ph in the abdominal subcutaneous fat tissue of overweight and obese patients with a BMI of 23 or more (Overweight) and a normal patient with a BMI of less than 23 (Normal) were compared through Western blot analysis (Fig. 3b).

3B is a photograph showing the result of comparing the phosphorylation level of S6K1 and the expression level of H2BS36Ph in abdominal subcutaneous fat tissue of obese patients (Overweight) and normal people (Normal). As shown in FIG. 3B, it was confirmed that the amount of activated S6K1 protein (P-S6K1) was increased compared to the total amount of S6K1 protein in the adipose tissue of obese patients. In addition, it was confirmed that the phosphorylation level of serine residue 36 of H2B was also increased.

Therefore, it was confirmed in living tissues that serine phosphorylation at No. 36 of histone H2B by S6K1 can be a target of an anti-obesity agent.

<110> SUNGKYUNKWAN UNIVERSITY Foundation for Corporate Collaboration <120> Method for screening agent for anti-obesity using phosphorylation of histon <130> KPA140984-KR <160> 9 <170> KopatentIn 2.0 <210> 1 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> peptide <400> 1 Lys Lys Arg Lys Arg Ser Arg Lys Glu Ser Tyr Ser Ile Tyr Val Tyr 1 5 10 15 Lys Val Leu Lys 20 <210> 2 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> peptide <400> 2 Lys Lys Arg Lys Arg Ser Arg Lys Glu Ser Tyr Ser Ile Tyr Val Tyr 1 5 10 15 Lys Val Leu Lys 20 <210> 3 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> peptide <400> 3 Lys Lys Arg Lys Arg Ser Arg Lys Glu Ser Tyr Ser Ile Tyr Val Tyr 1 5 10 15 Lys Val Leu Lys 20 <210> 4 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> peptide <400> 4 Lys Lys Arg Lys Arg Ser Arg Lys Glu Ser Tyr Ser Ile Tyr Val Tyr 1 5 10 15 Lys Val Leu Lys 20 <210> 5 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 5 ggaccagcca gaagaugcag gcucu 25 <210> 6 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 6 agagccugca ucuucuggcu ggucc 25 <210> 7 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 7 cacccuuuca uuguggaccu gauuu 25 <210> 8 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 8 aaaucagguc cacaaugaaa gggug 25 <210> 9 <211> 126 <212> PRT <213> Artificial Sequence <220> <223> Histone H2B, P33778 <400> 9 Met Pro Glu Pro Ser Lys Ser Ala Pro Ala Pro Lys Lys Gly Ser Lys 1 5 10 15 Lys Ala Ile Thr Lys Ala Gln Lys Lys Asp Gly Lys Lys Arg Lys Arg 20 25 30 Ser Arg Lys Glu Ser Tyr Ser Ile Tyr Val Tyr Lys Val Leu Lys Gln 35 40 45 Val His Pro Asp Thr Gly Ile Ser Ser Lys Ala Met Gly Ile Met Asn 50 55 60 Ser Phe Val Asn Asp Ile Phe Glu Arg Ile Ala Gly Glu Ala Ser Arg 65 70 75 80 Leu Ala His Tyr Asn Lys Arg Ser Thr Ile Thr Ser Arg Glu Ile Gln 85 90 95 Thr Ala Val Arg Leu Leu Leu Pro Gly Glu Leu Ala Lys His Ala Val 100 105 110 Ser Glu Gly Thr Lys Ala Val Thr Lys Tyr Thr Ser Ser Lys 115 120 125

Claims (11)

(a) a candidate compound treatment step of treating a candidate compound predicted to be capable of inhibiting differentiation or accumulation of fat in the isolated stem cells capable of differentiating into adipocytes;
(b) measuring the expression level of Ribosomal S6 kinase 1 (S6K1) and the phosphorylation level of serine 36 of histone H2B in cells treated with the candidate compound; And
(C) a method for screening an agent capable of preventing or treating obesity, comprising the step of selecting a candidate compound having reduced expression level and phosphorylation level compared to a negative control group not treated with the candidate compound.
The method of claim 1,
The method of the stem cells are mesenchymal stem cells (Mesenchymal stem cells; MSCs).
The method of claim 1,
The method of measuring the expression level of S6K1 is performed using a primer capable of amplifying a gene encoding S6K1 or a probe capable of specifically binding to the gene.
The method of claim 1,
The method of measuring the expression level of S6K1 is performed using an antibody capable of specifically binding to S6K1.
The method of claim 1,
The phosphorylation level of serine 36 of H2B is performed using an antibody capable of specifically binding to phosphorylated H2B.
The method of claim 1,
The candidate compound inhibits the expression of S6K1 and exhibits an activity of inhibiting the phosphorylation of serine 36 of H2B.
The method of claim 1,
The histone H2B is characterized in that the amino acid sequence represented by SEQ ID NO: 9 is translated.
Expression or activity of S6K1; And A composition for inhibiting the differentiation of stem cells into adipocytes, comprising an inhibitor of phosphorylation of serine 36 of histone H2B as an active ingredient.
The method of claim 8,
The inhibitor is a composition for inhibiting the differentiation of stem cells into adipocytes, characterized in that siRNA or a compound.
The method of claim 9,
The siRNA is an siRNA comprising at least one nucleotide sequence selected from the group consisting of SEQ ID NO: 5 to SEQ ID NO: 8, the composition for inhibiting the differentiation of stem cells into adipocytes.
The method of claim 9,
The compound is a composition for inhibiting differentiation of stem cells into adipocytes, characterized in that rapamycin or PF-4708671.

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