KR20090042105A - A composition for regulating sirt1 activity - Google Patents

Abstract

A composition containing AROS(active regulator of sirt1) proteins or genes thereof is provided to treat or prevent diabetes, obesity, cancer, neurodegenerative diseases or aging-related diseases by regulating SIRT1 activity. A composition for regulating SIRT1 activity comprises AROS(ACTIVE REGULATOR OF SIRT1) proteins or genes coding the AROS proteins as active ingredients. The composition for regulating SIRT1 activity includes AROS protein antibody, AROS SiRNA, AROS antisense DNA or cell strain containing the DNA thereof. The AROS proteins or the genes coding the AROS proteins are administered to mammal to prevent and treat diabetes, obesity, cancer, neurodegenerative diseases or aging-related diseases.

Description

A composition for regulating SIRT1 activity

The present invention relates to a composition for modulating activity of SIRT1. In addition, the present invention relates to methods and uses for prevention or treatment using the same.

Human sirtuin 1 (or SIRT1) belongs to the mammalian SIRT family and is most similar to Sir2 of yeast with NAD ⁺ -dependent class III histone deacetyl activity. Mammalian SIRT1 is involved in chromatin reconstitution, DNA damage response and dietary restriction associated with gene expression inhibition (Chen et al., Science 310 , 1641, 2005). Human SIRT1, like Sir2 in yeast, reconstructs chromatin and inhibits gene expression through histone deacetylation (Vaquero et al., Mol. Cell 16 , 93105, 2004). SIRT1 also induces deacetylation of various transcription factors related to cell growth, stress response, and endocrine regulation, in addition to histone proteins. In addition, according to a recent study, a technique applied to diabetes, obesity, neurodegenerative diseases or aging-related diseases by increasing the deacetylation activity of the SIRT1 is disclosed in US Patent Publication No. US 2006/0025337 A1 or International Patent Publication No. WO 2005/002672 It is disclosed in A2 etc. The most well known substrate of SIRT1 to date is the cancer suppressor protein p53, which is important for cell cycle and cell natural death regulation. p53 is a sequence-specific transcription factor that promotes the expression of p21WAF1, GADD45, Bax genes and the like. DNA damage or histone acetylatransferase (HAT) such as CBP / p300 induces acetylation of p53 to increase p53's DNA binding activity, thereby increasing transcriptional activity (Gu and Roeder, Cell 90 , 595606). , 1997; Liu et al., Mol. Cell. Biol. 19 , 12021209, 1999; Sakaguchi et al., Genes Dev. 12 , 28312841, 1998). Conversely, SIRT1 binds to p53 and induces deacetylation to inhibit the transcriptional activity of p53 (Langley et al., EMBO J. 21, 23832396, 2002; Luo et al., Cell 107 , 137148, 2001; Vaziri et al., Cell 107 , 149159, 2001). Furthermore, deacetylation of p53 inhibits cell aging and apoptosis caused by DNA damage or stress. Other substrates of SIRT1 include Ku70 (Cohen et al., Science 305 , 390392, 2004) involved in DNA repair, FOXO1 (Brunet et al., Science 303 , 20112015, 2004; Motta et al, associated with glucose metabolism and insulin secretion). , Cell 116 , 551563, 2004), NF-kB (Yeung et al., EMBO J. 23 , 23692380, 2004), involved in neuronal proliferation, PGC-1α (Rodgers et al., Nature 434 , important for energy metabolism). 113118, 2004), and PPARγ associated with obesity (Picard et al., Nature 429, 771776, 2004).

Substrates of SIRT1 are well known as described above, but only some indirect SIRT1 activity regulators have been reported for SIRT1 activity regulators, and no direct regulators are known.

In addition, the AROS (Active Regulator of SIRT1) protein discovered through the present invention is a protein of NCBI accession number BC037573, known only as ribosomal S19-binding protein (Maeda et al., Biochem. Biophys. Res. Commun. 339, 41-46, 2006), its function is unknown.

The technical problem to be solved by the present invention is to provide a composition for regulating SIRT1 activity.

In addition, the technical problem to be solved by the present invention is to provide a composition for the prevention or treatment of diabetes, obesity, cancer, neurodegenerative diseases or aging-related diseases and uses thereof.

In addition, the technical problem to be solved by the present invention is to provide a method for preventing or treating diabetes, obesity, cancer, neurodegenerative diseases or aging-related diseases.

In addition, the technical problem to be solved by the present invention is to provide an antibody of the AROS protein and its use.

In addition, the technical problem to be solved by the present invention is to provide a method for detecting AROS protein.

In addition, the technical problem to be solved by the present invention is to provide a search method for AROS expression regulator.

In addition, the technical problem to be solved by the present invention is to provide a search method for AROS protein and SIRT1 binding modulator.

In addition, the technical problem to be solved by the present invention is to provide a composition for inhibiting AROS protein activity and its use.

In addition, the technical problem to be solved by the present invention is to provide a method for inhibiting AROS protein activity.

The present invention provides a composition for enhancing SIRT1 activity, containing an AROS protein or a gene encoding an AROS protein as an active ingredient.

The present invention provides a composition for preventing or treating diabetes, obesity, cancer, neurodegenerative diseases or aging-related diseases, which contains an AROS protein or a gene encoding an AROS protein as an active ingredient and which is effective by SIRT1 activation.

In the present invention, the gene may be any one or more selected from DNA, cDNA, and mRNA. It is possible to produce the AROS protein intracellularly or extracellularly by transcription and translation of the DNA encoding the AROS protein.

The AROS (Active Regulator of SIRT1) protein has an amino acid sequence shown in Figure 1 as a factor that binds to SIRT1, and serves to enhance the activity of SIRT1. SIRT1 is a deacetylase of sirtuin 1 in humans belonging to the mammalian SIRT family.

 The cDNA may be composed of a sequence disclosed in NCBI Accession Number BC037573, and may include a sequence located at 128 to 538 of the sequence (FIG. 1). In addition, the mRNA may have a nucleotide sequence complementary to the sequence disclosed in NCBI accession number BC037573.

The present invention also provides the use of a gene encoding an AROS protein or AROS protein for the preparation of a therapeutic agent for diabetes, obesity, cancer, neurodegenerative diseases or aging-related diseases, which is effected by SIRT1 activation.

In the present invention, the gene may be any one or more selected from DNA, cDNA, and mRNA.

In the present invention, the neurodegenerative disease may be Alzheimer's disease, Amyotrophic axon sclerosis, Parkinson's disease, Huntington's disease, or multiple sclerosis.

In the present invention, the aging-related disease may be heart disease, arthritis, high blood pressure, or Alzheimer's disease.

In addition, the present invention is a method for preventing or treating diabetes, obesity, cancer, neurodegenerative diseases or aging-related diseases, which are effective by SIRT1 activation by administering to a mammal a therapeutically effective amount of an AROS protein or a gene encoding an AROS protein. To provide.

In the present invention, the gene may be any one or more selected from DNA, cDNA, and mRNA.

In the present invention, the neurodegenerative disease may be Alzheimer's disease, Amyotrophic axon sclerosis, Parkinson's disease, Huntington's disease, or multiple sclerosis.

In the present invention, the aging-related disease may be heart disease, arthritis, high blood pressure, or Alzheimer's disease.

A composition comprising an AROS protein or a gene encoding an AROS protein enhances the activity of SIRT1 in vivo, intracellularly, or externally.

In applying the composition of the present invention to a patient it may be administered in a dose of 1ng to 1g per day per 1kg body weight based on the active ingredient.

The present invention provides a composition for inhibiting SIRT1 activity, comprising an AROS protein antibody, AROS SiRNA, AROS antisense DNA, or a cell line comprising the DNA as an active ingredient.

The present invention provides a polyclonal antibody that specifically binds to an AROS protein.

In the present invention, the antibody can recognize 25-33 residues of the AROS protein.

The residue is a site excellent in antigenicity selected using a Protean (DNA Star) program.

The present invention provides a method for detecting AROS protein comprising binding the antibody and AROS protein.

The present invention provides an anticancer composition comprising the polyclonal antibody as an active ingredient and exhibiting an effect by inhibiting SIRT1 activity. The anticancer composition may be due to the anticancer action that is enhanced by the activity of p53 by the inhibition of SIRT1 activity, it may exhibit an effect on colon cancer, lung cancer and the like.

The present invention provides a use of a polyclonal antibody that specifically binds to an AROS protein for the preparation of a cancer therapeutic agent that is effective by inhibiting SIRT1 activity.

In the present invention, the antibody may recognize 25-33 residues of the AROS protein.

The present invention provides a method for preventing or treating cancer, which is effective by inhibiting SIRT1 activity by administering to a mammal a polyclonal antibody that specifically binds to a therapeutically effective amount of AROS protein. The prophylactic or therapeutic method may be due to the anti-cancer effect of enhanced p53 activity by inhibiting SIRT1 activity, and may have an effect on colorectal cancer or lung cancer.

In the present invention, the polyclonal antibody may recognize 25-33 residues of the AROS protein.

In the present invention, by measuring the content of the AROS protein, the polyclonal antibody may provide a composition, a diagnostic method, or a use capable of diagnosing a disease that may be prevented or treated due to the action or inhibition of the AROS protein. .

In the case of intravenous administration, the polyclonal antibody preparation of the present invention can generally be administered at 0.001 to 1000 mg / kg of body weight per adult, based on the active ingredient.

The present invention provides a method for searching for an AROS expression regulator comprising binding an antibody of the AROS protein to the AROS protein.

The present invention provides a method for searching for AROS protein and SIRT1 binding modulator, comprising binding AROS protein and SIRT1.

The search method may be by east-to-hybrid method, GST pull-down analysis method, or immunoprecipitation analysis method.

The present invention provides a composition for inhibiting AROS protein activity, containing a duplex formed by SEQ ID NO: 1 and SEQ ID NO: 2 as an active ingredient.

The present invention provides a duplex formed by SEQ ID NO: 1 and SEQ ID NO: 2 as an active ingredient, and has an effect by inhibiting SIRT1 activity, and provides a composition for preventing or treating cancer.

The present invention provides the use of a duplex formed by SEQ ID NO: 1 and SEQ ID NO: 2 for the preparation of an AROS protein activity inhibitor.

The present invention provides the use of a duplex formed by SEQ ID NO: 1 and SEQ ID NO: 2 for the manufacture of a prophylactic or therapeutic agent for cancer that is effective by inhibiting SIRT1 activity.

The present invention provides a method for inhibiting AROS protein activity by administering an effective amount of a duplex formed by SEQ ID NO: 1 and SEQ ID NO: 2 to a mammal.

The present invention provides a method for preventing or treating cancer, wherein the mammal is administered with a therapeutically effective amount of a duplex formed by SEQ ID NO: 1 and SEQ ID NO: 2, thereby suppressing SIRT1 activity.

The duplex is a siRNA formed by combining the sense 5'-UCUCGUCCUGGUCAGAAACUUCAGG-3 'of SEQ ID NO: 1 and the antisense 5'-CCUGAAGUUUCUGACCACGAGA-3' of SEQ ID NO: 2, and may inhibit intracellular AROS protein expression. The siRNA is for inhibiting intracellular AROS protein expression, and may be used for cancer treatment by increasing p53 activity, which is a cancer suppressor.

The siRNA of the present invention is in its own complete form, that is, a form which is directly synthesized in vitro and then transduced into a cell through transfection, or a single chain oligonucleotide to have such form after administration in vivo. The fragment and its reverse complement are transformed into a form that can be derived from a single chain polynucleotide separated by a spacer, for example, an siRNA expression vector or a PCR-derived siRNA expression cassette prepared to express siRNA in a cell. Alternatively, the cells may be introduced into cells through an infection process.

SiRNAs of the invention include variants having one or more substitutions, insertions, deletions and combinations thereof that have a change that does not degrade their activity, ie a functional equivalent. Such variants may exhibit at least 70% homology with the siRNA sequences described above, preferably at least 80%, more preferably at least 90%, most preferably at least 95%. Such homology is well known in the art for computer algorithms, such as the Align or BLAST algorithms (Altschul, J. Mol. Biol. 219-555-565, 1991, Heniloff and Henikoff, Proc. Natl. Acad. Sci. USA 89 : 10915-10919, 1992) can be readily determined by comparing the sequence of the polynucleotides to the corresponding portions of the target polynucleotides.

The cancer includes all cancers that can be treated by increasing p53 activity, which is a cancer suppressor such as lung cancer and colon cancer.

The siRNA may further be used to include additional substances that inhibit the proliferation of cancer cells in cancer patients for the prevention or treatment of cancer, and agents for promoting the influx of siRNA, for example liposomes (US Pat. No. 4,897,355, 4,394,448, 4,235,871, 4,231,877, 4,224,179, 4,753,788, 4,673,567, 4,247,411, 4,814,270 or one of many sterols, including cholesterol, cholate and deoxycholic acid It can also be combined with the lipophilic carrier.

The range of effective dosage of the siRNA of the present invention may vary depending on various factors such as sex, severity, age, administration method, target cell, expression level, etc., and may be easily determined by those skilled in the art, but about 0.01 ug To about 100 ug / kg / day.

The present invention provides a composition for inhibiting AROS protein activity, comprising AROS antisense DNA of SEQ ID NO: 5 or a cell line comprising the DNA as an active ingredient.

The present invention provides a composition for preventing or treating cancer, having an AROS antisense DNA of SEQ ID NO: 5 or a cell line including the DNA as an active ingredient and exhibiting an effect by inhibiting AROS protein expression.

The present invention provides the use of an AROS antisense DNA of SEQ ID NO: 5 or a cell line comprising said DNA for the preparation of an AROS protein inhibitor.

The present invention provides the use of an AROS antisense DNA of SEQ ID NO: 5 or a cell line comprising said DNA for the manufacture of a prophylactic or therapeutic agent for cancer.

The present invention provides a method of inhibiting AROS protein activity by administering to a mammal an effective amount of AROS antisense DNA of SEQ ID NO: 5 or a cell line comprising the DNA.

The present invention provides a method for preventing or treating cancer, wherein the mammal is administered with a therapeutically effective amount of AROS antisense DNA of SEQ ID NO: 5 or a cell line comprising the DNA to inhibit the AROS protein activity.

The range of effective dosage of the AROS antisense DNA of the present invention or a cell line including the DNA may vary depending on various factors such as sex, severity, age, administration method, expression level, and can be easily determined by those skilled in the art. But may range from about 0.01 ug to about 100 ug / kg / day.

The composition comprising the AROS protein, SiRNA, polyclonal antibody or AROS antisense DNA according to the present invention as an active ingredient can be administered by parenteral routes such as injection, and may be formulated in liquid or lyophilized form, if necessary. It may also optionally contain a pharmaceutically acceptable carrier such as stabilizers, preservatives, isotonic agents and the like. Pharmaceutically acceptable carriers include mannitol, lactose, saccharose, human albumin and the like in the case of lyophilized preparations. In the case of liquid preparations, for example, saline solution, water for injection, phosphate buffer, and the like can be given. However, it is not limited only to these. Dosages vary depending on the age, weight, sex, general state of health, extent of disease, and composition of the composition to be administered.

 The composition comprising the gene encoding the AROS protein, AROS antisense DNA, or SiRNA not only comprises adenovirus, retrovirus, adenovirus-associated virus (AAV) vectors, but also directly injects or contains oligonucleotide-liposomes. can do. When the DNA encoding the protein is inserted into a viral vector, the composition of the present invention prepares a viral particle comprising the recombinant DNA or a transduced cell line using the viral particle, and then the substrate used for gene therapy. It can manufacture by mixing. Cell lines comprising the DNA according to the invention can also be prepared in the same manner.

 The gene therapy substrate can be any substrate commonly used in injections. For example, the substrate may be distilled water, sodium chloride solution, mixtures of sodium chloride and inorganic water salts or the like, solutions of mannitol, lactose, dextran, glucose and the like, amino acid solutions such as glycine, arginine, organic acid solutions or salt solutions and glucose Mixing solutions and similar solutions. Injectables may also be prepared as solutions, suspensions or colloidal solutions by adding osmotic pressure regulators, pH regulators, vegetable oils such as sesame oil or soybean oil, or surfactants such as lecithin, nonionic surfactants, etc., in the usual manner. . Such injectables may be prepared in powder form, lyophilized form and dissolved immediately before use. The composition of the present invention may be dissolved in a pre-sterilized substrate if necessary immediately before gene treatment in the solid phase, or may be used as it is without further treatment in the liquid phase. It may also be administered with a physiologically acceptable carrier.

As the cell line including the vector, all cells which have the same or similar MHC (major histocompatibility complex) antigen of the patient, which does not cause tissue rejection at the time of transplantation, may be used. It is preferable to use it for gene transfer because it can completely exclude the tissue transplant rejection reaction.

According to the present invention, it is possible to modulate SIRT1 activity, thereby preventing or treating diabetes mellitus, obesity, cancer, neurodegenerative diseases, or aging-related diseases, and providing methods and uses for the prevention or treatment using the same. It is also possible to search for substances related to AROS and SIRT1.

Advantages and features of the present invention and methods for achieving them will be apparent with reference to the embodiments described below in detail. However, the present invention is not limited to the embodiments disclosed below, but will be implemented in various forms, and only the embodiments are intended to complete the disclosure of the present invention, and the general knowledge in the technical field to which the present invention pertains. It is provided to fully convey the scope of the invention to those skilled in the art, and the present invention is defined only by the scope of the claims.

[ Production Example  1] Preparation of AROS Protein Expression Plasmid in Various Cells

1) Escherichia coli

AROS cDNA obtained by PCR (ALa cDNA amplified by PCR using HeLa cDNA library as a template (NCBI accession number BC037573)) was used to express E. coli expression vector [PET15b for His-tag protein (Invitrogen, Carlsbad, CA), GST- pGEXT-4T for tag protein (Amersham Pharmacia Biotech, Piscataway, NJ)] was introduced as a conventional cloning method, and induction of E. coli expression with IPTG as necessary, followed by nickel-affinity and glutathione-friendly columns (Amersham Pharmacia Biotech, Piscataway, NJ). His-tag, GST-tagged AROS protein was purified purely.

2) yeast

AROS cDNA of SEQ ID NO: 3 obtained by PCR was used for the yeast expression vector (pBTM116 for LexA DBD-tag and pASV3 for VP16 AD-tag (Kim et al., J. Biol. Chem. 277 , 3202032028, 2002), Gal4 PGAD10 for AD-tag (BD Biosciences)] was introduced by conventional cloning.

3) animal cells

The AROS cDNA of SEQ ID NO: 3 obtained by PCR was introduced into a conventional cloning method in the vector for animal cell expression [pcDNA3 for flag-tag (Invitrogen), pEGFP for GFP-tag, and pHcRed for BD-HcRed-tag (BD Biosciences)]. .

[ Production Example  2] cell line preparation

Cell lines H1299 and HCT116 used in the present invention were cultured in RPMI1640 medium and other cells were cultured in DMEM medium (5% heat inactive FBS and antibiotic-antifungal; Gibco BRL, Gaithersburg, MD). The culture was carried out at 37 ° C., 5% carbon dioxide atmosphere.

AROS cDNA secured in the sense and antisense directions by PCR was introduced into pcDNA3 (G418-resistant), a vector for animal cell expression. The plasmid DNA thus prepared was introduced into colon cancer cell line HCT116 and treated with G-418 (Amersham Pharmacia Biotech) at 1 mg / ml for 24 hours, after 3-5 days, and then cultured in a medium containing G-418 for 2 weeks. Later, colonies having resistance to G-418 were obtained, and Western blot confirmed whether the expression of the AROS protein was increased or decreased.

[ Example  One] AROS Polyclonal  Preparation of Antibodies

The synthesized AROS peptide antigen (amino acids 25-33) (peptron, Korea) was conjugated with KLH by glutaraldehyde conjugation and dissolved in 0.2 mg / ml in saline. 5 ml of the dissolved solution was injected subcutaneously into SPF white rabbits to immunize rabbits. After immunization three times every two weeks, the antibody value was confirmed by ELIZA method, and a large amount of blood was collected from the ear artery. Antisera were obtained from the collected blood. AROS protein antigens were immobilized on CNBr-activated Sepharose 4V (GE Healthcare) to prepare an affinity column, equilibrated with physiological saline, and passed through 5 ml of antiserum. After washing 8 times with physiological saline (+ 1M NaCl), eluted with 100mM glycine (pH 2.5), the elution fraction was collected in a tube containing 1M Tris-Cl (pH 8.0) in advance to obtain a purified antibody.

[ Example 2 ] AROS Polyclonal  By antibody AROS  Confirmation of protein detection effect

Western blot and fluorescence microscope (AX70) after reaction with AROS protein, polyclonal antibody prepared in Example 1 (1: 100 dilution) and Texas Red-conjugated anti-rabbit IgG (1: 200 dilution; Amersham Pharmacia Biotech) ; Olympus Optical Co, Tokyo, Japan). The results are shown in Figure 1 C (Western blot) and Figure 1 D (fluorescence microscope), it was confirmed that the AROS protein is easily detected by the antibody of the present invention.

[ Example  3] AROS  For suppressing expression siRNA  Produce

SiRNAs consisting of duplexes of SEQ ID NO: 1 and SEQ ID NO: 2, ie, sense 5'-UCUCGUCCUGGUCAGAAACUUCAGG-3 'and antisense 5'-CCUGAAGUUUCUGACCACGAGA-3, were provided in duplex form by Invitrogen (USA). After dissolving in water at the final concentration of 100 mM, 50 and 100 nM were introduced into animal cells (HCT116) with Lipofectamine 2000 (Invitrogen), and expression inhibition of AROS was confirmed by Western blot.

[ Example  4] siRNA On by AROS  Expression inhibition effect confirmed

As shown in Preparation Example 2, the AROS cDNA of SEQ ID NO: 2 obtained by PCR was inserted into the animal cell expression vector pcDNA3 (Invitrogen) in the forward direction (AROS cDNA of SEQ ID NO: 3) or the reverse direction (AROS cDNA of SEQ ID NO: 5). To prepare AROS sense and antisense expression vectors, respectively. After the prepared AROS sense and antisense expression vectors were introduced into animal cells (H1299 cells), the expression and inhibition of expression of AROS protein were confirmed by Western blot (FIGS. 2D and 4C, respectively). On the other hand, Western blot with proteins extracted from the AROS sense expression HCT116 cell line (S, 1; S # 1) and antisense expression HCT116 cell line (AS, 2; AS # 1, AS # 2) prepared in Preparation Example 2 As a result, it was confirmed that AROS protein was increased in AROS-S # 1, and AROS protein expression was inhibited in AROS-AS # 1 and AROS-AS # 2 (FIG. 6A, left).

[ Example  5] AROS  Protein SIRT1  Identify the effects on expression regulation

1) Isolation of AROS Protein

AROS protein was isolated from animal cells by the following method.

Intracellular factors that directly regulate SIRT1 activity using Yeast two-hybrid screening method using the cDNA library (BD Biosciences, Palo Alto, CA) of HeLa cells connected to Gal4 AD as bait with SIRT1 linked to LexA DBD Explored. The experimental method was followed by a known method (Kim et al., J. Biol. Chem. 277, 32020-32028, 2002) except that SIRT1 linked with LexA was used as a bait. As a result, AROS (Active Regulator of SIRT1: GenBank number BC037573) was isolated as a factor that binds to SIRT1.

Figure 1 A shows the full-length amino acid sequence of the human AROS protein according to the present invention and the nucleic acid sequence of the cDNA encoding the same, as shown in Figure 1 A is a full-length cDNA encoding the human AROS protein (SEQ ID NO: 3) is 135 It can be seen that the dog amino acid is encoded to make an AROS protein (SEQ ID NO: 4).

2) Confirmation of expression of AROS mRNA

A biotin-labeled DNA probe (AROS and control β-actin) was prepared using a kit from KPL (Gaithersburg, MD), and a human AP was detected by purchasing a detector AP using human multiple tissue issue (MTN) Blot Membrane (# 7780-1; BD Biosciences). Northern blot analysis was performed using a Chemiluminescent Blotting Kit (# 54-30-02 KPL), and the results are shown in FIG. 1B.

Figure 1 B shows the results of the analysis of the Northern blot AROS mRNA expression in human tissue. β-actin was used as an internal control.

As shown in FIG. 1B, AROS mRNA was relatively high in the heart, liver, small intestine, and lung.

3) Confirmation of the expression regulation effect of SIRT1 by AROS protein

In order to analyze the correlation between AROS protein and SIRT1, Western blot analysis was performed using the AROS antibody and SIRT1 antibody (Santa Cruz Biotechnology) prepared in Example 1 using the ECL system (Amersham Pharmacia Biotech). Was performed. The result is shown in FIG. 1C.

As shown in FIG. 1C, it was confirmed that AROS expression and SIRT1 expression were expressed in a similar pattern.

4) Confirm the distribution of AROS protein in cells

Intracellular AROS protein was reacted with polyclonal antibody (1: 100 dilution) and Texas Red-conjugated anti-rabbit IgG (1: 200 dilution; Amersham Pharmacia Biotech), followed by fluorescence microscopy (AX70; Olympus Optical Co, Tokyo, Japan) and the results are shown in FIG. 1D. 1D shows the intracellular AROS and the right shows the overexpressed AROS. In addition, Hoechst staining was used to represent the nucleus. As shown in FIG. 1D, the AROS protein was present in the cell nucleus, and when overexpressed GFP-AROS, it was confirmed that the AROS protein was present in a specific region (nuclear phosphorus or heterochromatin) in the cell nucleus.

From the above results, it is understood that AROS protein and SIRT1 are distributed in the cell nucleus in the same manner, and the pattern expressed in the cells is also the same.

[ Example  6] AROS Wow SIRT1  The coupling between

1) Confirmation of specific binding between AROS protein and SIRT1

The known yeast two-hybrid assay (Kim et al., J. Biol. Chem. 277 , 3202032028, 2002) using LexA DBD-SIRT family and Gal4 AD-AROS. The reaction was confirmed and the results are shown in FIG. 2A.

Interactions were measured by β-gal assay and folding activity represents the relative value of Gal4 AD empty control. Data represent mean ± standard deviation of three experiments.

As shown in FIG. 2A, it was confirmed that the AROS protein specifically binds to SIRT1 of the SIRT family.

2) Check the joint

Various deletions of SIRT1 were prepared in the LexA DBD vector (pBTM116) by PCR and analyzed for two-hybrid yeast by known methods (Kim et al., J Biol Chem., 277, 32020-32028, 2002). Was done.

2B shows a map of the SIRT1 domain interacting with AROS protein. Folding activity represents the relative β-gal value compared to the sum of the Gal4 AD empty control and LexA DBD-SIRT1. Data represent mean ± standard deviation of three experiments.

As shown in FIG. 2B, the AROS protein was found to bind to amino acids 114-217 only present in SIRT1 in the SIRT family.

3) Confirmation of binding between AROS protein and SIRT1 in vitro

In vitro with GST-SIRT1 (amino acids 114-217) and TNT reticulocyte system (Promega, Madison, WI) isolated from E. coli with glutathione-Sepharose beads (Amersham Pharmacia Biotech) Western blot was performed with GST pull-down and AROS antibody using AROS protein synthesized in vitro.

Figure 2 C shows the results, it was confirmed that AROS protein directly binds to SIRT1 in vitro.

4) Confirmation of binding between AROS protein and SIRT1 in cells

Intracellular binding between AROS white matter and SIRT1 was confirmed by immunoprecipitation (IP) under the following two conditions.

4-1) Confirmation with SIRT1 antibody after immunoprecipitation with Flag antibody

First, after introducing Flag-AROS expression vector into lung cancer cell line H1299, cell extracts were obtained and immunoprecipitated with Flag antibody (mouse monoclonal; Sigma, St. Louis, MO) and SIRT1 antibody (rabbit polyclonal; Santa Cruz Biotechnology, Santa Cruz, CA) Western blotting was performed and the experiment was performed in the opposite direction.

The results are shown in FIG. 2D and the binding of AROS protein and SIRT1 was confirmed.

4-2) Confirmation with SIRT1 antibody after immunoprecipitation with AROS antibody

The binding between AROS protein and SIRT1 present in the cells was confirmed by performing Western blot with IP and SIRT1 antibody with AROS antibody, and the results are shown in FIG. 2E.

As shown in FIG. 2E, it was confirmed that binding between AROS protein and SIRT1 had no effect on DNA damage conditions (6 hours treatment with 20 μM Etoposide + 0.5 μM TSA).

5) Fluorescence microscope analysis

In order to reconfirm intracellular binding between AROS protein and SIRT1, each primary antibody (polyclonal antibody produced by AROS, monoclonal antibody purchased from Santa Cruz Biotechnology for SIRT1) and Texas Red for AROS Secondary antibody In the case of (Amersham Pharmacia Biotech) and SIRT1, intracellular SIRT1 and AROS proteins were analyzed by fluorescence microscopy (AX70; Olympus Optical Co, Tokyo, Japan) with a secondary antibody (Santa Cruz Biotechnology) with FITC. To this end, H1299 cells washed with physiological saline were fixed with 4% paraformaldehyde and punched with 0.5% Triton X-100 to react with each antibody.

The results are shown in Figure 2F, confirming that both AROS protein and SIRT1 are present in the nucleus.

[ Example  7] AROS  By protein p53 of Deacetylation ( deacetylation ) Confirmation of promotion effect

In order to analyze the effect of the AROS protein of the present invention on the deacetylation of p53 protein, a representative activity of SIRT1, was carried out as follows.

1) in vitro testing

Isolate GST-AROS and SIRT1 from E. coli, purchase a fluorescent acetyl p53 peptide (amino acids 379-382) ( Biomol, Plymouth Meeting, and PA ), and use the Flour de Lys-SIRT1 Assay Kit (Biomol) (Langley et al., EMBO J. 21, 23832396, 2002).

The experimental results are shown in FIG. 3A. As a result, SIRT1 alone induced deacetylation of p53, but the addition of GST-AROS significantly increased p53 deacetylation compared to GST alone.

2) in vivo testing

2-1) Confirmation of p53 Acetylation Inhibitory Effect Induced by CBP

Acetylation of p53 by CBP was performed according to known methods (Gu and Roeder, Cell 90, 595606, 1997).

In combination with SIRT1, AROS, or SIRT1 and AROS, p53, HA-labeled CBP expression vector was cotransfected into H1229 cells. Cell lysates were analyzed by Western blot with antibodies to HA, Ac-p53 (K-382), and p53.

The results are shown in Figure 3B.

As shown in FIG. 3B, acetylation of p53 induced by CBP with HAT (histone acetyltransferase) activity is reduced when overexpression of SIRT1 or AROS in H1299 cells by Western blot using an antibody that recognizes only p53 (Santa Cruz Biotechnology). It was confirmed that there was no change in the amount of total p53 protein.

2-2) Confirmation of p53 Acetylation Inhibitory Effect Induced by Etoposide and Trichostatin

Acetylation of p53 by etoposide and trichostatin was performed according to known methods (Luo et al., Proc Natl Acad Sci USA. 101 , 2259-2264, 2004).

SIRT1, AROS, or SIRT1 and AROS expression vectors were introduced into HCT116 cells and treated with 20 μM etoposide and 0.5 μM TSA. Cytolysates were analyzed by Western blot using antibodies against Flag, Ac-p53 and p53.

The results are shown in Figure 3C.

As shown in FIG. 3C, the treatment of etoposide, which induces DNA damage, and HDAC (histone deacetylase) inhibitor TSA (tricostatin A), which increases the acetylation of p53, increased the acetylation of p53 in HCT116 cells, overexpressing SIRT1 or AROS. It was found to decrease further, and further decreased by simultaneously expressing SIRT1 and AROS.

These results indicate that p53 acetylation is inhibited by the action of SIRT1 and AROS protein.

3) RNA inhibition experiment

The experiment was performed using AROS siRNA according to the experimental method shown in Example 3.

The HCT116 cells were treated with AROS siRNA (50, 100 nM: Invitrogen) for one day, and treated with etoposide / TSA (20 μM, 0.5 μM for 6 hours, respectively) for acetylation of p53. The expression of AROS and the acetyl level of p53 were confirmed by Western blot. At this time, the expression of β-actin was analyzed by Western blot with β-tin antibody by loading control.

3D shows the results of RNA inhibition experiments.

As a result, when AROS siRNA (50, 100 nM: Invitrogen) was treated for the RNA inhibition μ experiment, the amount of intracellular AROS protein was reduced compared to the control siRNA. Thus, etoposide / TSA treatment (20 μM, 0.5 μM, respectively, 6 hours) The acetylation of p53 induced by treatment) is further increased.

That is, it can be seen that the expression of the AROS protein is inhibited by the AROS siRNA according to the present invention, and as a result, the acetylation of p53 is increased.

These results suggest that AROS protein is a factor that enhances the deacetylation of p53 by helping the intracellular SIRT1.

[ Example  8] AROS  Protein p53  Confirmation of transcriptional inhibitory effect

In order to analyze the effect of the AROS protein on the transcriptional activity of p53 was tested as follows.

1) AROS protein effects on SIRT1 wild type and deacetylase defective type

The effect of deacetylation of p53 induced by AROS protein on the transcriptional activity of p53 was analyzed by transfection assay.

 Introducing Bax-Luciferase, which binds a promoter of p53-induced Bax gene to luciferase reporter gene in H1299 cells (Korea Cell Line Bank, College of Medicine, Seoul National University) without p53, Increasing amounts of AROS, Flag-SIRT1 wild type (WT), or Flag-SIRT1 HY variant (deacetylase deficient) (0.1, 0.3, 0.5 μg) were introduced, and p53 expression vectors were introduced in combination. .

The combination is as shown in the X axis of Fig. 4A. + Means contained, and-means not contained. The right triangle means that the injection amount is increased, and the rectangular shape means that the injection amount is constant.

Luciferase activity was determined using the luciferase assay after normalizing to the observed β-gal activity. Fold-luciferase activity on the Y axis shows the relative ratios obtained with and without the p53 vector. Data represent mean ± standard deviation of three experiments. The variance was shown by calculating the p value.

The results are shown in Figure 4A.

As shown in FIG. 4A, luciferase activity increased by p53 is decreased by AROS and SIRT1. On the other hand, the SIRT1 HY mutant without deacetyl activity did not affect p53 activity and did not respond to AROS. These results confirm that the AROS protein according to the present invention induces deacetylation of p53 together with SIRT1 and further inhibits the transcriptional activity of p53.

2) Confirmation of p53 transcriptional inhibitory effect by SIRT1 and AROS protein

P53, SIRT1, and AROS expression vectors were introduced into H1299 cells. Cells were treated with increasing concentrations of nicotinamide (NAM) (1, 5, 10 mM) for 12 hours. Cell lysates were then analyzed by luciferase assay. Data represent mean ± standard deviation of three experiments. The variance was shown by calculating the p value.

The combination is as shown in the X axis of Fig. 4B. + Means contained, and-means not contained. The right triangle means that the injection amount is increased, and the rectangular shape means that the injection amount is constant.

Luciferase activity was determined using the luciferase assay after normalizing to the observed β-gal activity. Fold-luciferase activity on the Y axis shows the relative ratios obtained with and without the p53 vector. Data represent mean ± standard deviation of three experiments. The variance was shown by calculating the p value.

4B shows the result.

As shown in FIG. 4B, nicotinamide (NAM), which is a well known inhibitor of SIRT1 activity, was inhibited by AROS or SIRT1 for 12 hours of treatment with 1, 5, and 10 mM for 12 hours. These results indicate that SIRT1 is required for inhibition of p53 transcriptional activity by AROS.

3) Influence of intracellular p53 transcriptional activity following introduction of AROS antisense

The experiment was performed using AROS antisense according to the experimental method shown in Preparation Example 2 and Example 4.

P53 and AROS antisense expression vector were introduced into H1299 cells, and cell eluate was measured by luciferase assay. The levels of acetylated p53, AROS, and p53 were measured by Western blot. Luciferase activity was determined using the luciferase assay after normalizing to the observed β-gal activity. Fold-luciferase activity on the Y axis shows the relative ratios obtained with and without the p53 vector. Data represent mean ± standard deviation of three experiments.

The results are shown in Figure 4C.

As shown in Figure 4C, it can be seen that the introduction of AROS antisense in H1299 cells to reduce the amount of intracellular AROS increases the transcriptional activity and acetylation of p53 without affecting the expression of p53. These results indicate that SIRT1 is required for inhibition of p53 transcriptional activity by AROS.

4) Effect on p21WAF1 expression following AROS overexpression

 AROS overexpression vector (AROS-pcDNA3) was prepared according to the experimental method described in Preparation Example 1-3, and the experiment was performed.

On the other hand, the expression of p21WAF1 (another target gene of p53) induced by etoposide / TSA (20 μM, 0.5 μM, respectively) treatment when AROS was overexpressed was tested to determine the degree of acetyl p53 and p21WAF1. The amount of, AROS and p53 was confirmed by Western blot.

The results are shown in FIG. 4D. Overexpression of AROS also inhibits the expression of p21WAF1, another target gene for p53 induced by etoposide / TSA treatment (20 μM, 0.5 μM for 6 hours).

These results suggest that AROS, together with SIRT1, induces deacetylation of p53 and further inhibits p53 transcriptional activity.

[ Example  9] AROS Wow SIRT1 On by p53 Check the inhibitory effect

In order to prove that the inhibitory activity of p53 protein by AROS of the present invention is due to its action with SIRT1, the following experiment was performed.

1) GST pull-down analysis

GST pull-down analysis was performed using GST-p53 protein and Flag-SIRT1 or Flag-AROS synthesized in vitro. Binding protein is shown by SDS-PAGE using anti-Flag antibody and Western blot.

The results are shown in FIG. 5A, and as shown in FIG. 5A, it was confirmed that SIRT1 directly binds to p53 instead of AROS.

This means that AROS protein directly binds to SIRT1 and inhibits p53 activity.

2) In vivo Immunoprecipitation (Immunoprecipitation)

After introducing the Flag-AROS expression vector into the HCT116 cell line, the in vivo immunoassay method confirmed the correlation between SIRT1, AROS and p53. Flag-AROS vectors were introduced into HCT116 cells with and without SIRT1 siRNA treatment. The cell eluate was immunoprecipitated with preimmune serum or p53 antibody, followed by western blot with anti-Flag antibody. Expression values of SIRT1, p53, and Flag-AROS are shown in Western blot.

The results are shown in FIG. 5B.

As shown in FIG. 5B, after immunoprecipitation with p53 antibody, Western blot analysis was performed with Flag antibody to confirm significant binding between p53 and AROS, and SIRT1 siRNA (Invitrogen) was further treated under the same conditions to express SIRT1. When inhibited (identified by WB), the bonds between them were lost. This means that p53 and SIRT1 bind.

3) Identification using SIRT1 non-expressed cell line

Bax-Luciferase reporter analysis was performed using a HeLa cell line (purchased from the Korea Cell Line Bank: see FIG. 1C) with little SIRT1 expression. HeLa cells were transduced with increasing amounts of Flag-AROS (0.1, 0.3, and 0.5 μg) with p53 or / and SIRT1 vectors.

The results are shown in FIG. 5C, and the data show the mean ± standard deviation of three experiments. The variance was shown by calculating the p value.

As a result, only when SIRT1 was introduced from the outside, inhibition of p53 transcriptional activity by increase of AROS (0.1, 0.3, and 0.5 μg) was observed. These results indicate that only SIRT1 is directly involved in the inhibition of p53 transcriptional activity.

4) Identification using SIRT1 non-expressed cell line

Experiments were performed using SIRT1 siRNA according to known methods (Luo et al., Cell 107, 137-148, 2001). SIRT1 siRNA was used in duplex form of sense 5'-ACUUUGCUGUAACCCUGUA (dTdT) -3 'and antisense 5'-UACAGGGUUACAGCAAAGU (dTdT) -3' from Invitrogen.

Inhibition of p53 transcriptional activity inhibition by AROS when SIRT1 siRNA (100 nM) was treated with H1299 cell line (purchased from Korea Cell Line Bank: See FIG. 1C) that overexpressed SIRT1 It was confirmed. The Flag-AROS vector was introduced into HCT116 cells and treated with 100 nM of SIRT1 siRNA or control siRNA. Fold-luciferase activity is indicative of the relative ratios obtained in the presence and absence of Bax-luciferase. SIRT1 and AROS values are shown in Western blot. Data represent mean ± standard deviation of three experiments. The variance is shown by calculating the p value.

The results are shown in FIG. 5D.

As shown in FIG. 5D, when SIRT1 siRNA (100 nM) was treated to H1299 cell line (FIG. 1D) overexpressing SIRT1, SIRT1 expression was inhibited (identified by Western blotting), and AROS inhibited transcription of p53. Was lost, indicating that only SIRT1 is directly involved in the inhibition of transcriptional activity of p53.

5) Effects of SIRT1 Expression Inhibition and AROS Overexpression on Acetylation of p53

Experiments were performed using the AROS sense vector for overexpression according to the experimental method shown in Preparation Example 3) and Example 4, and siRNA according to the experimental method shown in Example 9-4 for SIRT1 expression inhibition.

The effects of SIRT1 inhibition and AROS overexpression on the acetylation of p53 induced by etoposide / TSA (20 μM, 0.5 μM 6 hours) treatment in HCT116 cell lines were analyzed. Flag-AROS, SIRT1 siRNA, or control siRNA (represented by "C") was introduced into HCT116 cells. After 1 day, cells were treated with or without etoposide / TSA (20 μM, 0.5 μM each 6 hours). Protein values were determined by Western blot using antibodies against Flag (AROS), Ac-p53, SIRT1 and β-actin.

The results are shown in FIG. 5E. As shown in FIG. 5E, acetylation of p53 is increased by inhibition of expression of SIRT1, decreased by overexpression of AROS protein, and the effect of AROS protein is considerably lost when both conditions are present. These results indicate that SIRT1 has a significant effect on the inhibition of p53 transcriptional activity.

6) Confirmation of p53 Deacetylation Inhibition of AROS Protein by Splitomycin Treatment

Experiments were performed using splitomicin, an inhibitor of SIRT1, according to a known method (Bedalov et al., Proc Natl Acad Sci USA. 98, 15113-15118, 2001).

Experiments were performed to confirm that deacetylation of p53 by AROS was inhibited when splitomicin, an inhibitor of SIRT1, was treated. P53, CBP, and AROS vectors were introduced into H1299 cells and treated with splitomicin (240 μM, 9 hours; Calbiochem, La Jolla, CA). Cell eluate was analyzed by Western blot using an antibody.

The results are shown in Figure 5F.

As shown in FIG. 5F, when treatment with splitomicin, a well-known inhibitor of SIRT1, treatment of deacetylation of p53 by AROS was confirmed.

As a result, it can be seen that the deacetylase activity of SIRT1 and SIRT1 is required for inactivation of p53 by AROS (deacetylation and transcriptional inhibition).

[ Example  10] p53 of  Effect on Cell Growth Regulatory Activity AROS  Identify the effects of protein

The effect of the AROS protein according to the present invention on the cell growth regulation activity of p53 was analyzed by the following method.

As described above, AROS was shown to induce deacetylation of p53 and further inhibit the transcriptional activity of p53 by increasing the enzyme activity of SIRT1 through binding to SIRT1. On the other hand, it is well known that p53 acts as a cancer suppression transcription factor and is involved in cancer cell proliferation inhibition and cell natural death. Therefore, the effect of AROS on cancer cell growth was analyzed based on the above results.

1) Confirmation of cell proliferation effect by AROS protein

Cell lines overexpressing AROS (sense: 1, S # 1) or inhibiting intracellular expression of AROS (2, AS # 1 and AS # 2) according to the assays described in Preparation Examples 2 and 4 Was prepared and used in the experiment.

Using HCT116 cells, a cell line overexpressing AROS or antisense intracellular expression of AROS was prepared (cell line of Preparation Example 2) to confirm AROS expression by Western blotting and cell proliferation was confirmed by MTT assay. It was. HCT116 cells stably expressing Flag-empty, Flag-AROS sense (S), or AROS antisense (AS) were treated with 1 μM etoposide and 0.5 μM TSA at the time indicated in FIG. 6A (right) graph. AROS protein expression was analyzed by Western blot with anti-AROS antibody (left). Cell viability was confirmed by MTT assay (right). Data represent mean ± standard deviation of three experiments.

The results are shown in FIG. 6A.

As shown in FIG. 6A (left), intracellular expression of AROS can be effectively inhibited by the siRNA of the present invention. As shown in FIG. 6A (right), HCT116 cells inhibit proliferation and inhibit AROS. Overexpressing cells were resistant to proliferation inhibition by etoposide / TSA treatment, and cell lines that inhibited intracellular expression of AROS were more sensitive to etoposide / TSA treatment. That is, when the intracellular expression of the AROS protein according to the present invention is inhibited, the cell viability is reduced, and the viability is increased by the AROS protein.

2) Confirm the effect of AROS protein on the expression of p21WAF1

According to the experimental methods shown in Preparation Example 2 and Example 4, a cell line in which AROS was overexpressed (sense) or inhibited intracellular expression of AROS (antisense) was prepared and used for the experiment.

HCT116 cells were used to prepare antisense cell lines overexpressing AROS or inhibiting intracellular expression of AROS (by Preparation Example 2) to express p21WAF1 expression as a target gene for p53 and important for cell proliferation inhibition. Was increased in cells overexpressing and in cells inhibiting intracellular expression of AROS by Westingblot. HCT116 stable cells were treated as in 1) above. After 2 days of treatment, the cell eluate was confirmed by Western blot.

The results are shown in FIG. 6B.

As shown in FIG. 6B, the expression of p21WAF1, which is a target gene of p53 and is important for cell proliferation, was significantly reduced in cells overexpressing AROS, and increased in cells inhibiting intracellular expression of AROS. That is, it can be seen that the AROS protein according to the present invention inhibits the transcriptional activity of p53.

3) Identify the effect of AROS protein on cell cycle progression

FACS analysis was performed according to known methods (Kim et al., J Biol Chem, 277, 32020-32028, 2002).

Experimental results using a FACS Calibur flowcytometer (Becton-Dickinson Immunocytometry System, San Jose, Calif.) Were used to analyze the cell cycle. The cells were treated in the same manner as 2) above.

The results are shown in Figure 6C.

As shown in FIG. 6C, G0 / G1 cells increased during etoposide / TSA treatment, decreased for AROS overexpressed cells, and G0 / G1 cells increased in cells inhibiting intracellular expression of AROS. That is, it can be seen that the cytostatic effect of p53 is reduced by the intracellular expression of AROS.

4) Confirmation of effect of AROS protein on cell natural death

DAPI staining was performed according to a known method (Kim et al., J Biol Chem, 277, 32020-32028, 2002).

Cells were treated in the same manner as in 2) above, and DAPI staining and quantitative apoptosis analysis (quantification of DNA fragments) were used to analyze the effect of AROS on apoptosis.

The results are shown in Figure 6D.

After etoposide / TSA treatment, the condensation and fragmentation of chromatin in the cell nucleus, which are characteristic of cellular natural death, were confirmed as shown in FIG. 6D by DAPI staining. As a result, in cells overexpressing AROS, phenomena such as condensation and fragmentation of chromatin in the cell nucleus, which are characteristic of cell death, were reduced, and cell death was further increased. From these results, it can be seen that the cellular natural death is reduced by the intracellular expression of the AROS protein.

5) Quantification of DNA Segments

DNA fragmentation experiments were performed according to known methods (Holdenrieder et al., Clin Chem Lab Med. 39, 596-605, 2001).

ELISA analysis (Roche Diagnostics) was performed to quantify DNA fragmentation, an indicator of cellular natural death. Cells were treated overnight with control or SIRT1 siRNA (100 nM) and 2 days with 1 μM etoposide and 0.5 μM TSA. Data represent mean ± standard deviation of three experiments.

The result is shown in FIG. 6E.

As a result, as shown in Fig. 6E, it showed a quantitative pattern similar to the result of 4), and such a pattern was mostly lost when siRNA suppressed the expression of intracellular SIRT1. Proved.

In conclusion, in the present invention, as shown schematically in FIG. 6F, the AROS protein induces inactivation of p53 by increasing the activity of SIRT1 through binding to SIRT1, and furthermore, cell proliferation inhibition, which is a representative activity of intracellular p53, and It can be seen that it inhibits the induction of cellular natural death.

That is, it can be seen that the AROS protein according to the present invention can be used as a therapeutic agent for the treatment and prevention of aging such as cell aging due to the increase of p53 activity by inhibiting the activity of p53. In addition, genes encoding the AROS protein (DNA, cDNA, mRNA, etc.) can also be produced by transcription or / and translation of the AROS protein, it can be used as a gene therapy for the treatment and prevention of aging such as cell aging.

In addition, it can be used for diseases such as diabetes, neuropathy, obesity, acquired immunodeficiency, cancer, etc., which require SIRT1 activation for prevention or treatment.

Figure 1 A shows the amino acid sequence of the human AROS protein and cDNA sequence encoding it.

Figure 1 B is a diagram comparing the expression level of human AROS mRNA by tissue.

1C is a diagram comparing the correlation between AROS protein and SIRT1 in various cancer cell lines.

Figure 1 D is a diagram confirming the intracellular presence of AROS protein.

2A is a diagram showing specific binding of AROS protein and SIRT1.

2B is a diagram showing a map of SIRT1 domain that binds to AROS protein.

Figure 2C is a diagram confirming the binding of AROS protein and GST-SIRT1 isolated from E. coli in vitro.

Figure 2 D is a diagram confirming the binding to SIRT1 after introducing the Flag-AROS expression vector into the lung cancer cell line.

Figure 2E is a diagram confirming the binding of AROS protein and SIRT1 under DNA damage conditions.

FIG. 2F is an immunofluorescence microscope assay confirming that most AROS and SIRT1 coexist in the nucleus.

FIG. 3A is a diagram illustrating the effect of AROS protein on p53 deacetylation by SIRT1 in vitro. FIG.

3B is a diagram illustrating in vitro the effect of AROS protein on the deacetylation of p53 induced by CBP.

Figure 3C confirms that AROS protein, along with SIRT1, reduces the deacetylation of endogenous p53 induced by DNA damage.

Figure 3D is a diagram measuring the acetylation of p53 caused by DNA damage by AROS siRNA.

4A is a diagram confirming that the AROS protein inhibits the transcriptional activity induced by p53 together with SIRT1.

4B is a diagram showing the inhibition of inactivation of p53 induced by AROS and SIRT1 by nicotinamide.

Figure 4C is a diagram confirming the increase in p53 transcriptional activity by the introduction of AROS antisense.

4D is a diagram confirming the expression inhibitory effect of p21WAF1 induced by DNA damage by AROS.

Figure 5A is a diagram confirming the protein binding to p53 by GST pull-down analysis.

FIG. 5B is a diagram illustrating siRNA tSIRT1 involved in the interaction of AROS protein and p53 using siRNA in vivo. FIG.

Figure 5C is a diagram confirming the effect of the AROS protein on the transcriptional activity of p53.

5D is a diagram confirming the effect of SIRT1 siRNA on the transcriptional activity of p53 induced by AROS.

FIG. 5E is a diagram analyzing the effect of SIRT1 expression inhibition and AROS overexpression on acetylation of p53 induced by DNA damage.

5F is a diagram confirming that deacetylation of p53 by AROS is inhibited when splitomicin treatment is an inhibitor of SIRT1.

Figure 6 A is a diagram confirming the effect of AROS on cell proliferation.

Figure 6B is a diagram confirming the effect of AROS on the expression of p21WAF1.

6C is a diagram showing the effect of AROS expression on the cell cycle.

Figure 6 D is a diagram analyzing the effect of AROS protein on natural apoptosis.

6E is a quantitative analysis of DNA fragmentation, which is an indicator of cellular natural death.

6F is a diagram schematically showing the effect of AROS on p53 activity through binding to SIRT1.

<110> CHEBIGEN INC. <120> A composition for regulating SIRT1 activity <130> P07-052-KBG <160> 5 <170> KopatentIn 1.71 <210> 1 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> sense strand of siRNA for AROS <400> 1 ucucguccug gucagaaacu ucagg 25 <210> 2 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> antisense strand of siRNA for AROS <400> 2 ccugaaguuu cugaccacga ga 22 <210> 3 <211> 411 <212> DNA <213> Artificial Sequence <220> <223> sense cDNA <400> 3 atgtccgccg ccctgctgcg gcggggcctg gagctgctgg cggcgtccga ggccccccgg 60 gaccctccag gtcaggccaa gccgagaggg gctccggtga aacggccccg gaagacgaag 120 gcaattcagg cccagaaact gcggaactcg gccaagggaa aggtgcccaa gtcggcactg 180 gacgagtacc ggaagcgaga gtgtcgagac cacctcagag taaacctgaa gtttctgacc 240 aggacgagaa gcaccgtggc tgagtctgtg agccagcaga ttttgcgcca gaaccggggc 300 cgcaaggcct gtgaccggcc tgtggccaag accaagaaga agaaggctga gggcaccgtg 360 ttcaccgagg aagacttcca gaagttccag caggaatact tcggcagcta g 411 <210> 4 <211> 135 <212> PRT <213> Homo sapiens <400> 4 Met Ser Ala Ala Leu Leu Arg Arg Gly Leu Glu Leu Leu Ala Ala Ser   1 5 10 15 Glu Ala Pro Arg Asp Pro Pro Gly Gln Ala Lys Pro Arg Gly Ala Pro              20 25 30 Val Lys Arg Pro Arg Lys Thr Lys Ala Ile Gln Ala Gln Lys Leu Arg          35 40 45 Asn Ser Ala Lys Gly Lys Val Pro Lys Ser Ala Leu Asp Glu Tyr Arg      50 55 60 Lys Arg Glu Cys Arg Asp His Leu Arg Val Asn Leu Lys Phe Leu Thr  65 70 75 80 Arg Thr Arg Ser Thr Val Ala Glu Ser Val Ser Gln Gln Ile Leu Arg                  85 90 95 Gln Asn Arg Gly Arg Lys Ala Cys Asp Arg Pro Val Ala Lys Thr Lys             100 105 110 Lys Lys Lys Ala Glu Gly Thr Val Phe Thr Glu Glu Asp Phe Gln Lys         115 120 125 Phe Gln Glu Tyr Phe Gly Ser     130 135 <210> 5 <211> 411 <212> DNA <213> Artificial Sequence <220> <223> antisense cDNA <400> 5 gatcgacggc ttcataagga cgaccttgaa gaccttcaga aggagccact tgtgccacgg 60 gagtcggaag aagaagaacc agaaccggtg tccggccagt gtccggaacg ccggggccaa 120 gaccgcgttt tagacgaccg agtgtctgag tcggtgccac gaagagcagg accagtcttt 180 gaagtccaaa tgagactcca ccagagctgt gagagcgaag gccatgagca ggtcacggct 240 gaacccgtgg aaagggaacc ggctcaaggc gtcaaagacc cggacttaac ggaagcagaa 300 ggccccggca aagtggcctc ggggagagcc gaaccggact ggacctccca gggccccccg 360 gagcctgcgg cggtcgtcga ggtccggggc ggcgtcgtcc cgccgcctgt a 411  

Claims (37)

A composition for enhancing the activity of SIRT1 containing an AROS protein or a gene encoding an AROS protein as an active ingredient. A composition for preventing or treating diabetes, obesity, cancer, neurodegenerative diseases or aging-related diseases, containing an AROS protein or a gene encoding an AROS protein as an active ingredient and exhibiting effects by SIRT1 activation. The composition of claim 2, wherein the gene is at least one selected from DNA, cDNA, and mRNA. The composition of claim 2, wherein the neurodegenerative disease is Alzheimer's disease, Amyotrophic axon sclerosis, Parkinson's disease, Huntington's disease, or multiple sclerosis. The composition of claim 2, wherein the aging-related disease is heart disease, arthritis, hypertension, or Alzheimer's. Use of an AROS protein or gene encoding an AROS protein for the manufacture of a medicament for the treatment of diabetes, obesity, cancer, neurodegenerative diseases or aging related diseases, which is effected by SIRT1 activation. 7. The use of claim 6, wherein the gene is at least one selected from DNA, cDNA, and mRNA. 7. The use according to claim 6, wherein the neurodegenerative disease is Alzheimer's, muscular dystrophy, Parkinson's disease, Huntington's disease, or multiple sclerosis. Use according to claim 6, wherein the aging-related disease is heart disease, arthritis, hypertension, or Alzheimer's. A method of preventing or treating diabetes, obesity, cancer, neurodegenerative diseases or aging related diseases, which is effected by SIRT1 activation by administering to a mammal a therapeutically effective amount of an AROS protein or a gene encoding an AROS protein. The method of claim 10, wherein the gene is at least one selected from DNA, cDNA, and mRNA. The method of claim 10, wherein the neurodegenerative disease is Alzheimer's disease, muscular dystrophy, Parkinson's disease, Huntington's disease, or multiple sclerosis. The method of claim 10, wherein the aging-related disease is heart disease, arthritis, high blood pressure, or Alzheimer's disease. A composition for inhibiting SIRT1 activity comprising an AROS protein antibody, AROS SiRNA, AROS antisense DNA, or a cell line comprising the DNA as an active ingredient. Polyclonal antibody that specifically binds to AROS protein. The polyclonal antibody of claim 15, wherein the antibody recognizes 25-33 residues of the AROS protein. A method for detecting an AROS protein comprising binding an AROS protein with an antibody of any one of claims 15 or 16. The anti-cancer composition comprising the polyclonal antibody of any one of claims 15 or 16 as an active ingredient and exhibiting effects by inhibiting SIRT1 activity. Use of a polyclonal antibody that specifically binds to an AROS protein for the manufacture of a cancer therapeutic agent that is effective by inhibiting SIRT1 activity. The use of a polyclonal antibody according to claim 19, wherein the antibody recognizes 25-33 residues of the AROS protein. A method of preventing or treating cancer, wherein the mammal is administered by inhibiting SIRT1 activity by administering a polyclonal antibody that specifically binds to a therapeutically effective amount of AROS protein. The method of claim 21, wherein the polyclonal antibody recognizes 25-33 residues of the AROS protein. 17. A method for searching for an AROS expression regulator comprising binding an AROS protein with an antibody of any one of claims 15 or 16. Searching method of the AROS protein and SIRT1 binding force modulator comprising the step of binding the AROS protein and SIRT1. 25. The method according to claim 24, wherein said screening method is based on a yeast two-hybrid method, a GST pull-down assay, or an immunoprecipitation assay. Containing a duplex formed by SEQ ID NO: 1 and SEQ ID NO: 2 as an active ingredient, AROS protein activity inhibitory composition. A duplex formed by SEQ ID NO: 1 and SEQ ID NO: 2 as an active ingredient and exhibits effects by inhibiting SIRT1 activity, cancer prevention or treatment composition. Use of a duplex formed by SEQ ID NO: 1 and SEQ ID NO: 2 for the preparation of an AROS protein inhibitor. Use of a duplex formed by SEQ ID NO: 1 and SEQ ID NO: 2 for the preparation of a cancer prophylactic or therapeutic agent that is effective by inhibiting SIRT1 activity. A method of inhibiting AROS protein activity by administering an effective amount of a duplex formed by SEQ ID NO: 1 and SEQ ID NO: 2 to a mammal. A method of preventing or treating cancer, wherein the mammal is administered with a therapeutically effective amount of a duplex formed by SEQ ID NO: 1 and SEQ ID NO: 2, thereby inhibiting SIRT1 activity. AROS antisense DNA of SEQ ID NO: 5 or a cell line comprising the DNA as an active ingredient, AROS protein activity inhibitory composition. AROS antisense DNA of SEQ ID NO: 5 or a cell line containing the DNA as an active ingredient, and has an effect by inhibiting AROS protein activity, a composition for preventing or treating cancer. Use of the AROS antisense DNA of SEQ ID NO: 5 or a cell line comprising said DNA for the preparation of an AROS protein inhibitor. Use of an AROS antisense DNA of SEQ ID NO: 5 or a cell line comprising said DNA for the manufacture of a prophylactic or therapeutic agent for cancer. A method of inhibiting AROS protein activity by administering to a mammal an effective amount of AROS antisense DNA of SEQ ID NO: 5 or a cell line comprising said DNA. A method of preventing or treating cancer, wherein the mammal is administered with a therapeutically effective amount of AROS antisense DNA of SEQ ID NO: 5 or a cell line comprising the DNA to inhibit AROS protein activity.

Images