WO2018034379A1 - Methylated-rack1 protein detecting antibody and use thereof - Google Patents

Methylated-rack1 protein detecting antibody and use thereof Download PDF

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WO2018034379A1
WO2018034379A1 PCT/KR2016/012454 KR2016012454W WO2018034379A1 WO 2018034379 A1 WO2018034379 A1 WO 2018034379A1 KR 2016012454 W KR2016012454 W KR 2016012454W WO 2018034379 A1 WO2018034379 A1 WO 2018034379A1
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cancer
antibody
rack1
protein
lysine
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French (fr)
Korean (ko)
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김정애
박경찬
양석진
염영일
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한국생명공학연구원
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer

Definitions

  • the present invention relates to an antibody that specifically recognizes a RACK1 protein (RACKlK271me2) in which lysine, the second amino acid from the N-terminus, is dimethylated, and its use.
  • Oxygen homeostasis is an integral part of metazoan physiology.
  • hypoxic state cells induce hypoxia responses to adapt to and survive harsh environments.
  • Hypoxic reactions including metabolic adaptation, upregulation of oxygen transporters, maintenance of pH homeostasis, and stimulation of angiogenesis, are mediated by a variety of genes (Harris, AL, Nat. Rev. Cancer. 2002 (2) , 38- 47, Cassavaugh, J. ⁇ & Lounsbury.M., J. Cell.Biochem, 2011 (112), 735-744).
  • Hypoxia-inducible factor-1 (HIF-1) is a protein that regulates the expression of genes that are critical for hypoxic reactions due to high induction expression in hypoxia. There are two proteins, ex and ⁇ .
  • HIF-la protein is hydroxylated using oxygen as substrate and destroyed by ubiquitin system.
  • the lack of oxygen substrate inhibits the hydroxylation of HIF-la protein, which is known to stabilize the protein itself because the destruction by the ubiquitin system does not occur.
  • the stability of the HIF-la protein can be regulated in an oxygen-independent manner.
  • the mechanism of inducing destruction by the ubiquitin system is well known by binding to a protein called RACK1.
  • the binding of RACK1 to HIF-la protein is not dependent on the hydroxylation of HIF-la, and it has been reported that the inhibition of RACK1 increases the stability of HIF-la (Liu et al., Mol Cell).
  • Stabilized HIF-la binds to the nucleus of HIF-1 ⁇ , ARNT, and moves to the nucleus to produce erythropoiesis and angiogenesis. And control the expression of genes involved in glycolysis and the like.
  • HIF-la has been reported as an important molecule that controls the growth and growth of cancer cells in the hypoxic state, especially the amount of HIF-la protein and the prognosis of cancer patients is known to have a close correlation.
  • HIF-1 When HIF-1 is activated by hypoxic conditions induced by the growth of cancer cells into solid cancer, hemokinase 2, glucose transporter Kglucose transporter 1, erythropoietin, IGF-2, and endoglin ), By inducing expression of genes such as VEGFA, MMP-2, uPAR, MDRl, etc., and obtaining traits such as resistance to apoptosis, increased angiogenesis, increased cell proliferation, and invasion Eventually cancer cells become more malignant. As HIF plays an important role in the growth, proliferation, and malignancy of cancer, in particular, solid cancer, researches to develop anticancer drugs by targeting the stability regulation of HIF-la protein have been actively conducted (Cancer Res. 62, 4316).
  • RACKKReceptor for activated protein kinase C is an adapter protein with seven WD motifs of 40 amino acids, a 36kDa protein consisting of 317 amino acids, and is primarily present in the cytosol and cytosolic membranes.
  • HIF-la protein Cell Co ⁇ un Signal. 2011; 9:22.
  • RACK1 protein Through binding to several proteins, including HIF-la protein (Cell Co ⁇ un Signal. 2011; 9:22.), It regulates various cell responses such as cell growth, differentiation, adhesion and immunity (Li JJ, Xie D. Oncogene. 20159; 34 (15): 1890-8.).
  • overexpression of RACK1 protein has been reported to reduce the expression level of intracellular HIF-la protein under hypoxic conditions (Liu et al. Mol Cell. 2007 Jan 26; 25 (2): 207-17.)
  • RACK1 is overexpressed in many cancer tissues such as melanoma, breast cancer, colon cancer, lung cancer, liver cancer, esophageal cancer, and oral cancer, resulting in cancer growth.
  • the present invention provides an antibody or an immunologically active fragment thereof, wherein the second lysine specifically binds to the dimethylated RACK1 protein (RACKlK271me2).
  • the present invention is the second lysine (lysine) is dimethylated (diniethylat ion)
  • kits for diagnosing cancer in a sample comprising an antibody or immunologically active fragment thereof that specifically binds to RACKl protein (RACKlK271nie2).
  • Antibody (antibody) of the present invention was confirmed that the second lysine (lysine) can specifically recognize and bind the dimethyl at ion (RACKlK271me2), the antibody is methylated in the field of cell biology It can be usefully used for RACK1 protein research. [Brief Description of Drawings]
  • Stomach ectopic expression of LSD1 and RACK1 from HEK-293T cells and observed through the interaction of LSD1 and RACK1 with all immunoblotting;
  • FIG. 2 is a diagram illustrating methylation of the second stage lysine residue of RACK1 purified from HEK293T cells by tandem mass spectorometry analysis.
  • FIG. Figure 3 is RACK1 Gibbon 2 lysine residues (1 271 and the protein: 172 times lysine residue (K172) to the use of the methylation of the mutant proteins RACK1 ant i -pan-methyl -lysine antibody replaced with alanine immune blotting ( Figure is observed through i ⁇ unoblotting.
  • FIG. 4 is a diagram illustrating hydrophobici ty and antigenicity analysis in order to obtain an amino acid sequence for use as an antigen:
  • Circle shows the amino acid sequence site used for antigen generation, and has the amino acid sequence of 266 to 276 of SEQ ID NO: 1.
  • 5 is a diagram confirming the immune effect of the prepared anti-RACK1 antibody.
  • Figure 6 is a diagram confirming the specificity of the prepared anti-RACK1 antibody.
  • FIG. 7 is a diagram illustrating the methylation of the second lysine residue of the RACK1 protein by immunoblotting using the antibody of the present invention.
  • Figure 8 shows the second lysine residues of RACK1 protein in hypoxic condition (1% 02 condition)
  • HIF-la protein The interaction of HIF-la protein is observed through immunoblotting using HIF-la antibody.
  • FIG. 9 is a diagram illustrating the interaction between RACK1 and HIF-la protein according to the presence or absence of LSD1 gene inhibition by immunoblotting using HIF-la antibody.
  • the present invention provides an antibody or immunologically active fragment thereof in which lysine 271 specifically binds to dimethylat ionized RACKl protein (RACKlK271me2).
  • the ACK1 protein is preferably composed of the amino acid sequence of SEQ ID NO: 1, but is not limited thereto.
  • the immunologically active fragment is Fab.
  • it is any one selected from the group consisting of Fab ', F (ab') 2, Fv, Fd, single chain Fv (scFv) and disulfide stabilized Fv (dsFv).
  • the antibody provides an antibody or an immunologically active fragment thereof in which the lysine specifically binds to a dimethylat ion ACKl (Rece tor for activated protein kinase C) epitope in the amino acid sequence of SEQ ID NO: 2 do.
  • the antibody is preferably a monoclonal antibody or a polyclonal antibody, but is not limited thereto.
  • the polyclonal antibody may be produced by a conventional method of injecting a protein marker of the present invention into an animal and collecting blood from the animal to obtain a serum containing the antibody.
  • Such polyclonal antibodies can be purified by any method known in the art and can be made from any animal species host, such as goats, rabbits, sheep, monkeys, horses, pigs, cattle, dogs and the like.
  • Such monoclonal antibodies can be prepared using any technique that provides for the production of antibody molecules through the culture of continuous cell lines.
  • Such techniques include, but are not limited to, hybridoma technology, human B-cell hybridoma technology, and EBV-hybridoma technology (Kohler G et al., Nature 256: 495-497, 1975; Kozbor D et al., J Immunol Methods 81: 31—42, 1985; Cote RJ et al., Proc Natl Acad Sci 80: 2026-2030, 1983; Cole SP et al., Mol Cell Biol 62: 109-120, 1984 ).
  • the present invention include, but are not limited to, hybridoma technology, human B-cell hybridoma technology, and EBV-hybridoma technology (Kohler G et al., Nature 256: 495-497, 1975; Kozbor D et al., J Immunol Methods 81: 31—42, 1985; Cot
  • Preparing a transformant by transforming a host cell into a host cell comprising a RACKl gene in which lysine is dimethylated;
  • step 2) Incubate the transformant of step 1) to dimethylate lysine (d imethyl at i on) preparing the RAC 1 protein;
  • the experimental animal is a rabbit. It is preferably any one selected from the group consisting of mice, rats, cattle and monkeys, and more preferably rabbits, but is not limited thereto.
  • the gene encoding the antigen of the present invention may be due to the degeneracy of the codon or in view of the preferred codon in the organism in which the antigen is to be expressed.
  • Various modifications may be made to the coding region within the range of not changing the amino acid sequence of the antigen expressed from the coding region, and various modifications or modifications may be made to the region except for the coding region without affecting the expression of the gene. There is. Those skilled in the art will appreciate that such modified genes are also within the scope of the present invention. That is, the antigen used in the present invention may be mutated by one or more nucleic acid bases encoding proteins having equivalent activity by substitution, deletion, insertion, or a combination thereof, which are also included in the scope of the present invention.
  • Expression vectors used in the present invention include, but are not limited to, plasmid vectors, cosmid vectors, bacteriophage vectors and viral vectors. Suitable expression vectors include signal sequences or leader sequences for membrane targeting or secretion in addition to expression control elements such as promoters, operators, initiation codons, termination codons, polyadenylation signals and enhancers, and can be prepared in various ways depending on the purpose. have.
  • the promoter of the expression vector can be constitutive or inducible.
  • the signal sequence includes a host of the genus Escherichia. (Escherichia sp.) Bacteria, the PhoA signal sequence, OmpA signal sequence, etc., if the host is Bacillus (Baci llus sp.) Bacteria, ⁇ -amylase signal sequence, subtilisin signal sequence, etc., the host is yeast In the case of), MFa signal sequence, SUC2 signal sequence, etc. may be used, but if the host is an animal cell, an insulin signal sequence, an ⁇ -interferon signal sequence, an antibody molecule signal sequence, or the like may be used, but is not limited thereto.
  • the expression vector may also include a selection marker for selecting a host cell containing the vector. If it is a replicable expression vector it includes the origin of replication.
  • Demethylation of the second stage lysine of RACK1 protein provides an antibody or immunologically active fragment, characterized by LSDKLysine-specific demethylase 1).
  • Methylation of the second stage lysine of the RACK1 protein provides an antibody or immunologically active fragment thereof characterized by the degradation of HIF-la.
  • the present inventors have a high possibility of producing a specific antibody (hydrophobicity) through the hydrophobicity and antigenicity analysis (Hydrophobicity and Antigenicity analysis) to secure the amino acid sequence to be used as an antigen ) Is low.
  • a sequence having high antigenicity (ant igenicity) and highly exposed to the outside in the tertiary structure is selected (see FIG. 4).
  • the present invention also provides a kit for diagnosing cancer in a sample comprising the antibody or immunologically active fragment thereof according to the present invention.
  • the cancer is a melanoma, breast cancer, colon cancer, lung cancer, liver cancer.
  • Urethral cancer Penile cancer Prostate cancer, Chronic or acute leukemia, Solid tumor of childhood. Differentiated lymphoma.
  • Bladder cancer kidney cancer Any one selected from the group consisting of renal cell carcinoma, renal pelvic carcinoma, first central nervous system lymphoma spinal contraction tumor, brainstem glioma and pituitary adenoma is not limited thereto.
  • the present invention Any one selected from the group consisting of renal cell carcinoma, renal pelvic carcinoma, first central nervous system lymphoma spinal contraction tumor, brainstem glioma and pituitary adenoma is not limited thereto.
  • the present invention Any one selected from the group consisting of renal cell carcinoma, renal pelvic carcinoma, first central nervous system lymphoma spinal contraction tumor, brainstem glioma and pituitary adenoma is not limited thereto.
  • step 2) when the level of dimethylation of step 1) is reduced compared to a normal control group, the level of dimethylation for providing cancer diagnosis information, including the step of determining a high risk of cancer or an individual having cancer, It provides a measuring method of.
  • the biological sample of step 1) is preferably selected from the group consisting of bone marrow serum, blood and blood, but is not limited thereto.
  • Immunoblotting (I ⁇ unoblot ting) using methyl-lysine 3 ⁇ 4 ⁇ sieve confirmed that methylation of RACK1 is down-regu l at ion when LSD1 is overexpressed (see FIG. 1). ).
  • the present inventors have identified a polyclonal antibody that specifically recognizes RACKl (RACKlK271me2) dimethylated dimethylated lysine residues prepared in the present invention to determine how LSD1 regulates methylation of the 2nd lysine residue of RACK1.
  • This lysis immunoblotting was performed to confirm that when RSD1 was overexpressed, RACK1 dimethylated with lysine residue 271 was significantly reduced (see FIG. 7).
  • the present inventors have examined the effects of demethylation of RACKl (RACKlK271me2) dimethylated on LSD1-mediated 2nd lysine residues on HIF-la protein stability.
  • LSI 1 is associated with RACK1 through the regulation of methylation of RACiaK271me2.
  • HIF-la modulates the physical interactions of HIF-la
  • co-immunoprecipitation assay with HACK-mutant (K271A) protein and wild-type RACK1 and 2nd lysine substituted with alanine After doing. Immunoblotting was performed to confirm that demethylation of RACKlK271me2 mediated by LSD1 inhibited the binding of RACK1 and HIF-la (see FIG. 9).
  • the polyclonal antibody of the present invention specifically recognizes RACK1 protein (RACKlK271me2) in which lysine is dimethylated, and when LSD1 is inactivated using the antibody, RACK1 is activated.
  • RACK1 protein RACKlK271me2
  • methylation of RACK1 can be utilized as a marker of cancer diagnosis and the antibody can be usefully used for methylated RACK1 protein research in the field of cell biology.
  • the present inventors performed the following experiments to confirm the physical interaction of the LSD1 protein and the RACK1 protein.
  • HEK293T cell line was purchased from American Type Culture Collection (ATCC) and Korean Cell Line Bank in the United States, Rosvvell Park with 10% fetal bovine serum (GIBCO BRL) and IX antibiotic-antifungal agent (GIBCO BRL) Incubated with Memorial Institute 1640 medium (RPMI1640; GIBCO BRL).
  • the hypoxic environmental conditions were cultured using 02 / C02 incubator (MC0—5M. Sanyo, Japan) under 1% -3% 02, 5% C02 and 923 ⁇ 4-94% N2 conditions.
  • the inventors performed the following experiment to determine how LSD1 regulates methylation of RACK1.
  • Co-imnmnoprecipitation was performed using HEK293T cells expressing ACK1 ectopically as follows.
  • Vector 3 which expresses flag-labeled RACKKFlag tagged RACK1) and ectopic expression of Myc-posited LSDKMyc tagged LSD1), was mixed and lipofectamin (invitrogen) was added for 20 minutes at room temperature. After reaction, HEK293T cells were transfected. After 48 hours, HEK293T intracellular protein was extracted using RIPA buffer (50 mM Tris-HCl pH7.5, 150 mM NaCl, 100 mM EDTA, 1% Triton-X100, Protease inhibitor) and EzviewTMRed Anti-c- Co-immunoprecipitation reaction was performed at 4 ° C.
  • RIPA buffer 50 mM Tris-HCl pH7.5, 150 mM NaCl, 100 mM EDTA, 1% Triton-X100, Protease inhibitor
  • KCl 100 mM Na2HPI4, 2 mM H2P04, 0.1% Tween20
  • a flag antibody (Sigma) was reacted on a blocked membrane (immunoblot membrane) at a ratio of 1: 1,000 at 4 ° C for 16 hours, washed three times with PBST buffer for 10 minutes, and then HRP conjugated.
  • HRP horse-radish peroxidase
  • the horse-radish peroxidase (HRP) conjugated mouse secondary antibody was reacted for 1 hour at room temperature at a ratio of 1: 2, then washed three times for 10 minutes using PBST buffer.
  • Enhanced Chemi luminescence emission of light using ECU solution
  • LAS- 4 000 Luminescent Image analyzer, FUGIFILM
  • the inventors performed the following experiments to analyze which sites of RACK1 methylated.
  • the gel band of immunoprecipitated RACK1 was cut out in ⁇ Example 1-2> and reduced using high-density DT, and then alkylated with indole-3 acetic acid. Then, the cut gel band was washed with lOmM ammonium bicarbonate and 50% ACN, and then immersed in a dissolution buffer (50mM ammonium bicarbonate, 5 mM CaC12, andl g trypsin) and reacted at 37 ° C for 16 hours to dissolve (Park et al. , 2013). The dissolved peptide was added twice with 50 mM animonuim bicarbonate and 100% acetonitrile (acetonitr i le; ACN).
  • a dissolution buffer 50mM ammonium bicarbonate, 5 mM CaC12, andl g trypsin
  • MS / MS spectra were analyzed following a software analysis protocol using Proteome discover software version 1.3 with the protein sequence of RACK1 (UnitProtKB-63244). Trypsin with two missed cleavages was selected as the peptide cleavage enzyme.
  • the carbam ' idomethylation of cysteine was selected as the static modi fi cat ion, and the mono-, di and tr i-methylat ions in the oxidation and lysine of methionine were selected as vari able and modi fi cat ion. Only high scoring peptides were considered genuine. The high scoring peptide was consistent with the peptide above the threshold in our invention (Xcorr>
  • Flag-labeled wild-type RACKKFlag tagged RACK1 as a template, PCR grade water 35 ⁇ of KOD-Plus-Mut agenesis Kit (TOYOBO), 10x Buffer 5 L 2mM dNTPs 5 seed flag for iPCR (Flag tagged) wild type RACKl (50ng / ul) 1 id, KOD-Plus-DNA Polyineraseinverse Ifd and primers at 10 ⁇ ol / ul concentration
  • Flag- RACK1 K172A Forward primer 5'-TG (X; ACGCGCTGGTCMGGTATGG— 3 '(SEQ ID NO: 3);
  • Flag-RAC l K172A Reverse primer 5'-GCCACAGGAGACGATGATAGGGTTGCT-3 '(SEQ ID NO: 4);
  • Flag-RACKl K271A Forward primer 5'-G / ⁇ CTGGCGCMGAAGTTATCAGT-3 '(SEQ ID NO:
  • Flag-RACKl K271A Reverse primer 5'-ATCTACMTGATCmCCCTCTAAATC-3 '(SEQ ID NO: 6)] was performed inverse PCR using 1.5 / £. At this time, the PCR conditions were performed inverse PCR for 10 minutes at 94 ° C 2 minutes, denatured for 10 seconds at 98'C, annealing for 30 seconds at 60 ° C, elongation at 72 ° C for 7 minutes. After completion of the inverse PCR reaction, treated with Dpnl restriction enzyme of 2 / z. (! For 1 hour at 37 ° C.
  • PCR product 1 PCR grade water 7 ⁇ , ligation high 5 ⁇ and ⁇ 4 polynucleotide kinase treated with Dpnl restriction enzyme
  • the phosphorylation / ligation reaction was carried out for 1 hour at 16 ° C.
  • the phosphorylation / ligation product 1 ⁇ was transformed into E. coli and DNA was extracted.
  • RACKKFlag tagged RACK ⁇ 72 ⁇ in which labeled lysine residues were replaced with alanine and lagine labeled 2nd lysine residues as alanine, thus obtaining a 3 ⁇ 4 RACKl (Flag tagged RAC 1 K271A) vector.
  • the vector was transfected into HEK293T cells using lipofectamin (lipofectamin), and after 48 hours, the protein was extracted using RIPA buffer, and the extracted protein lnig and anti-Flag M2 30 ⁇ £ were mixed. Immunoprecipitation reaction was performed at 4 ° C. for 16 hours. Thereafter, anti-Flag M2 was washed three times for 10 minutes using RIPA buffer, 2x sample buffer was added, and the immunoprecipitated protein was eluted by boiling at 10 C for 5 minutes. Thereafter, immunoblotting was performed using the anti-pan-methyl-lysine antibody in the same manner as in ⁇ Example 1-2>.
  • a polyclonal antibody (RACKlK271me2) that specifically recognizes RACK1 having dimethylated lysine residues dimethylated (dimethylated) was prepared by the following method.
  • an amino acid sequence to be used as an antigen hydrophobicity with low possibility of producing a specific antibody through hydrophobicity and antigenicity analysis is low, and antigen A sequence having high antigenicity and having a high possibility of being exposed to the outside in the tertiary structure is selected (FIG. 4), and based on this, the final amino acid sequence [amino acid sequence having dimethylation at lysine residue: IVDELK (me2 ) QEVIS (SEQ ID NO: 2)]. Based on the sequence, a peptide having dimethylation at a lysine residue was synthesized and used as an antigen.
  • SEQ ID NO: 2 Production of polyclonal antisera is described by SEQ ID NO: 2 from the RACK1 protein (RAC lK271me2) dimethylated with lysine residues 2 times in rabbits in which a preimmune response has been induced (park 0) for 4 weeks.
  • Peptides were used as antigen and subcutaneously injected (50 «g / rabbit) to boost antibody production first (week 4-primary boosting).
  • two weeks after the first antibody-induced immune response reaction the production of the antibody was induced secondarily (500 / rabbit; six weeks—second boosting), and additionally after three weeks, the production of the antibody was induced three times (500 ⁇ g / rabbit). ; Week 8-3rd boosting). A week later (Week 9).
  • Serum was obtained by cardiac puncture to obtain anti-RACK1 polyclonal antiserum and RAC 1 peptide [IVDELK (me2) QEVIS; dimethylation at lysine residue 2; Amino acids 266 to 276, (SEQ ID NO: 2)] were purified by affinity chromatography to obtain a rabbit anti-RACK1 polyclonal antibody.
  • the enzyme immunoassay was performed in the following manner.
  • the antigen is ligated to 2 g / n with a coating buffer and dispensed into each well 50, followed by overnight at 4 ° C. or three hours at 37 ° C. for coating. Put it. Thereafter, the coating solution was discarded and the 2% skim milk / TBS-T solution was dispensed and blocked at 37 ° C. for one hour. After an hour wash with TBS-T solution. Primary antibodies were dispensed into each well and reacted at 37 ° C. for 2 hours. Secondary antibodies washed three times with TBS-T and diluted 1: 5000 were then dispensed 50 ⁇ into each well and reacted at 37 ° C for one hour.
  • the blocking test is a competitive iti on assay.
  • the antibody and antigen are reacted together in the step of attaching the secondary antibody to the antibody, and the protein of the membrane and If the antibody has specificity as a principle of competing antigens, only the target band disappears or disappears relatively clearly from the result of adding the antigen, thereby confirming the specificity of the antibody.
  • Western blotting was performed in the following manner.
  • the synthesized RACK1K271 peptide was prepared without the methylation of K271nie0 peptide, only one K271mel fabtide, two K271me2 peptide and three K271me3 peptide, a total of four peptides, from Ong to 1000ng by nylon membrane (nylon membrane) After aliquoting and drying, the nylon membrane was blocked with 5% BSA. Thereafter, the polyclonal antibody RACKlK271me2 prepared in Example 1 was diluted with 1: 1.000 in 5% BSA (prepared by dissolving BSA in PBST) and reacted at room temperature for 1 hour, and then washed three times with PBST for 10 minutes. .
  • the anti-rabbit antibody IgG-HRP (Mi l lore) was reacted at a ratio of 1: 2,000 for 1 hour in real plants, and then washed three times for 10 minutes using PBST buf fer.
  • Luminescence was enhanced using an Enhanced Chemi luminescence (ECU solution) and analyzed by LAS-4000 (Luminescent Image analyzer, FUGIFILM).
  • polyclonal antibody RACKlK271me2 and K271me2 antigen (2 / zg / ml) were mixed for 30 minutes in a block for the blocking test, and then reacted to the nylon on membrane prepared as described above.
  • the polyclonal antibody RACKlK271me2 could not react with the K271me2 peptide.
  • the polyclonal antibody RACKlK271me2 prepared in Example 3 has an antibody specificity that specifically recognizes RACK1 in which lysine residue 271 is dimethylated (FIG. 6). .
  • Example 4 Confirmation of the Effect of the (RACKlK271me2) Polyclonal Antibody Specifically Recognizing RACK1 Dimethylated by Lysine No. 2 Lysine
  • RACK1 Overexpression of RACK1 is known to reduce the level of intracellular HIF-la protein under hypoxic conditions (Liu, YV et al., MolCell 2007. 25, 207-217.), Resulting in demethylation of RAC l 271me2 by LSD1. In order to confirm the effect of HIF-la protein stability on the stability of the RACK1 mutant was substituted for the second stage lysine of the RACK1 protein with alanine, and the following experiment was performed.
  • HEK293T cells were cultured and immunoblotting was performed using the HIF-la antibody (BD Biosciences. 610959) in the same manner as in ⁇ Example 2-2>.
  • the wild-type RACK1 protein which is the 2nd stage of the RACK1 protein, had higher levels of HIF-la protein than the RACK1 mutation (K271A), which replaced the 2nd stage of the lysine with alanine. It was confirmed to be low (FIG. 8). Accordingly, it was confirmed that the second stage lysine (K271) of the RACK1 protein is an important residue in the degradation of HIF-la mediated by RACK1.
  • the second lysine (K271) of the RACK1 protein is adjacent to the TO6 domain of the RACK1 protein, which is essential for the association of RACK1 with HIF-la, so that LSD1 modulates the physical interaction of RACK1 and HIF-la through the regulation of methylation of RACKlK271me2.
  • interaction ion the interaction ion
  • knockdown HEK293T cells and HEK293T cells, which were not, were overexpressed using lipofectamine (1 ipofect ion) as in ⁇ Example 1-2>, and after 48 hours, ⁇ MG132 (Calbiochem) was 4 Time was processed.
  • Proteins were extracted in the same manner as described above, and 3 mg of protein and ANTI—Flag M2 (Sigma-Aldr ich) 35 ⁇ were mixed, and co-immunoprecipitation reaction was performed at 4 ° C. for 16 hours. Wash precipitated anti-Flag M2 three times with RIPA buffer. 2Xsample buffer was added and the mixture was boiled at 100 ° C. for 5 minutes to elute the precipitated protein. The eluted protein was separated using a 10% SDS-PAGE gel, and the separated protein was transferred to a nylon membrane for 2 hours at 90V electric partial pressure at 4 ° C, and 5) blocking for 30 minutes on BSA. (blocking).
  • Blocking the HA antibody in the film (membrane) (Santa Cruz) for 1: After washing three to 1,000 rate and banung at 4 ° C for 16 hours, 10 minutes in PBST buffer times, HRP is bonded Sat "meal A secondary antibody (horse irradiated peroxidase (HRP) —conjugated rabbit secondary antibody, Millipore) was reacted at room temperature for 1 hour at a ratio of 1: 2, 000. After washing three times using PBST buffer. Emission was performed using an Enhanced Chemi luminescence (ECL) solution and analyzed by LAS-4000 (Luminescent Image Analyzer, FUGIFILM).
  • ECL Enhanced Chemi luminescence
  • RACK1 when LSD1 was inactivated, it was confirmed that RACK1 was methylated and methylated RACK1 could degrade HIF-1 la and such methylation of RACK1 could be used as a marker for cancer treatment.

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Abstract

The present invention relates to: an antibody that specifically recognizes a receptor for activated protein kinase C (RACK1) protein (RACK1K271me2) of which lysine, the 271st amino acid from the N-terminus, is dimethylated; and a use thereof. Specifically, it is confirmed that RACK1 is methylated when lysine-specific demethylase 1 (LSD1) is inactivated by using a polyclonal antibody of the present invention and that the methylated RACK1 can degrade hypoxia-inducible factor-1 (HIF-1a), and thus the methylation of RACK1 can be used as a marker for cancer diagnosis and the antibody can be useful in research on a methylated RACK1 protein in the field of cell biology.

Description

【명세서】  【Specification】
【발명의 명칭】  [Name of invention]
메틸화 -RACK1 단백질 검출 항체 및 그의 용도 【기술분야】  Methylated-RACK1 Protein Detection Antibody and Uses thereof
본 발명은 N-말단으로부터 2기번째 아미노산인 리신 (lysine)이 다이메틸화 (dimethylation) 된 RACK1 단백질 (RACKlK271me2)을 특이적으로 인식하는 항체 (antibody) 및 이의 용도에 관한 것이다. 【배경기술】  The present invention relates to an antibody that specifically recognizes a RACK1 protein (RACKlK271me2) in which lysine, the second amino acid from the N-terminus, is dimethylated, and its use. Background Art
산소 항상성 (Oxygen homeostasis)은 후생동물 (metazoan) 생리학의 필수적인 부분이다. 저산소 상태에서 세포는 혹독한 환경에 적응하고 생존하기 위하여 저산소 반웅 (hypoxia responses)을 유도한다. 대사 적응, 산소전달체의 상향조절， pH 항상성의 유지, 및 혈관신생 (angiogenesis)의 자극을 포함하는 저산소 반웅은, 다양한 유전자에 의해 매개된다 (Harris, A. L. , Nat. Rev. Cancer . 2002(2), 38- 47,Cassavaugh, J. <& Lounsbury. . M. , J. Cell. Biochem, 2011(112), 735-744). 저산소유도인자 (Hypoxia-inducible factor-l; HIF-1)는 저산소 상태에서 다량 유도 발현되어 저산소 반응에 핵심적인 유전자들의 발현을 조절하는 단백질이다. ex와 β 두 가지 단백질이 존재하는데 정상산소 분압에서는 HIF-la 단백질이 산소를 기질로 이용하여 히드록실화 되어 유비퀴틴 시스템에 의해 파괴되나. 저산소 상태에서는 산소 기질의 결핍으로 인해 HIF-la 단백질의 히드록실화가 저해되어 유비퀴틴 시스템에 의한 파괴가 발생하지 않아 단백질 자체가 안정화되는 것으로 알려져 있다. HIF-la 단백질의 안정성은 산소비의존적으로도 조절을 받을 수 있는데, 특히, RACK1이라는 단백질과의 결합을 통해 유비퀴틴 시스템에 의한 파괴가 유도되는 기전이 잘 알려져 있다. RACK1과 HIF-la 단백질의 결합은 HIF— la의 히드록실화에 의존 이지 않으며 , RACK1과의 결합을 저해할 경우, HIF-la의 안정성이 증가하는 것으로 보고되어 있다 (Liu et al. , Mol Cell , 2007(25), 207-217). 안정화된 HIF-la는 HIF-1 β인 ARNT와 결합하여 핵으로 이동하여 적혈구 생성 (erythropoiesis), 신혈관생성 (angiogenesis) 및 해당과정 (glycolysis)등에 관련된 유전자의 발현을 조절한다. 한편 HIF-la는 저산소 상태에 대한 암세포의 적웅 및 성장을 조절하는 중요한 분자로도 보고되어 있는데 특히 HIF-la 단백질의 양과 암 환자의 예후는 밀접한 상관 관계를 갖는 것으로 알려져있다. 암세포가 고형암으로 성장하면서 유도되는 저산소 조건에 의해 HIF-1이 활성화되면 핵소키나아제 2(hexokinase 2), 글루코스 전달체 Kglucose transporter 1), 에리쓰로포이에틴 (erythropoietin), IGF-2, 엔도글린 (endoglin), VEGFA, MMP-2, uPAR, MDRl 등과 같은 유전자의 발현을 유도하여 세포사 (apoptosis)에 대한 내성, 혈관신생능의 증가, 세포증식능의 증가， 침윤 (invasion)능의 증가 등의 형질을 획득하게 되어 결국 암세포는 보다 악성화되게 된다. 이와 같이 HIF는 암, 특히 고형암의 성장, 증식 및 악성화에 중요한 역할을 하기 때문에 이러한 HIF-la 단백질의 안정성 조절을 표적으로 하여 항암제를 개발하려는 연구가 매우 활발히 진행되고 있다 (Cancer Res. 62, 4316, 2002； Nat Rev Drug Discovery 2,1, 2003； Semenza et al . Nature Reviews Cancer 2003. 3, 721-732； Yi Qin et al,. Cancer Letter, 2014, 347, 225-232). 이외에도 HIF-1의 활성은 암 발생과 전이 뿐만 아니라 류마티스성 관절염, 허혈성 뇌졸증， 동맥경화증 등 다양한 만성 대사성 질환의 병리학적 기전과도 밀접한 관계가 있기 때문에 최근 주요 신약 표적으로 부상하고 있다. RACKKReceptor for activated protein kinase C)은 40개의 아미노산으로 구성된 7개의 WD 모티프를 가진 어댑터 단백질로서 317개의 아미노산으로 구성된 36kDa 크기의 단백질이며 , 세포기질 (cytosol)과 세포질막 (cytosol ic membrane)에 주로 존재하여 HIF-la 단백질을 포함한 여러 단백질들과 결합을 통해 (Cell Co隱 un Signal. 2011； 9： 22.) 세포성장, 분화, 부착 및 면역과 같은 다양한 세포반웅을 조절하는 역할을 한다 (Li JJ, Xie D. Oncogene.20159；34(15)： 1890-8. ) . 특히, RACK1 단백질의 과발현은 저산소환경 조건에서 세포내 HIF-la 단백질의 발현 수준을 감소시킨다고 보고된 바 있으며 (Liu et al. Mol Cell . 2007 Jan 26；25(2) :207-17.), 최근에는 흑색종, 유방암, 대장암, 폐암， 간암, 식도암, 구강암과 같은 많은 암조직에서 RACK1이 과발현되어 암의 성장 (growth). 침윤 (invasion) 및 전이 (metastasis)에 관여함이 알려졌으나 그 구체적인 분자 기작은 보고된 바가 없다 (Zhong X. et al . , Ann Surg Oncol . 2013 ;20： 1044-52. ) . 본 발명에서 본 연구자들은 RACK1이 리신-특이적 탈메틸화효소인 LSD1과 상호결합함을 확인, RACK1의 메틸화가능성을 확인하였고， 이에 RACK1의 메틸화부위를 확인하고 이를 검출할 수 있는 항체를 제작, 본 발명의 항체를 활성화된 다이메릴화 (diniethylation)된 RACK1 단백질의 검출 및 이를 이용한 암진단에 유용하게 이용될 수 있음을 확인하여 본 발명을 완성하였다. 【발명의 상세한 설명】 Oxygen homeostasis is an integral part of metazoan physiology. In a hypoxic state, cells induce hypoxia responses to adapt to and survive harsh environments. Hypoxic reactions, including metabolic adaptation, upregulation of oxygen transporters, maintenance of pH homeostasis, and stimulation of angiogenesis, are mediated by a variety of genes (Harris, AL, Nat. Rev. Cancer. 2002 (2) , 38- 47, Cassavaugh, J. <& Lounsbury.M., J. Cell.Biochem, 2011 (112), 735-744). Hypoxia-inducible factor-1 (HIF-1) is a protein that regulates the expression of genes that are critical for hypoxic reactions due to high induction expression in hypoxia. There are two proteins, ex and β. At normal oxygen partial pressure, HIF-la protein is hydroxylated using oxygen as substrate and destroyed by ubiquitin system. In the hypoxic state, the lack of oxygen substrate inhibits the hydroxylation of HIF-la protein, which is known to stabilize the protein itself because the destruction by the ubiquitin system does not occur. The stability of the HIF-la protein can be regulated in an oxygen-independent manner. In particular, the mechanism of inducing destruction by the ubiquitin system is well known by binding to a protein called RACK1. The binding of RACK1 to HIF-la protein is not dependent on the hydroxylation of HIF-la, and it has been reported that the inhibition of RACK1 increases the stability of HIF-la (Liu et al., Mol Cell). , 2007 (25), 207-217). Stabilized HIF-la binds to the nucleus of HIF-1 β, ARNT, and moves to the nucleus to produce erythropoiesis and angiogenesis. And control the expression of genes involved in glycolysis and the like. On the other hand, HIF-la has been reported as an important molecule that controls the growth and growth of cancer cells in the hypoxic state, especially the amount of HIF-la protein and the prognosis of cancer patients is known to have a close correlation. When HIF-1 is activated by hypoxic conditions induced by the growth of cancer cells into solid cancer, hemokinase 2, glucose transporter Kglucose transporter 1, erythropoietin, IGF-2, and endoglin ), By inducing expression of genes such as VEGFA, MMP-2, uPAR, MDRl, etc., and obtaining traits such as resistance to apoptosis, increased angiogenesis, increased cell proliferation, and invasion Eventually cancer cells become more malignant. As HIF plays an important role in the growth, proliferation, and malignancy of cancer, in particular, solid cancer, researches to develop anticancer drugs by targeting the stability regulation of HIF-la protein have been actively conducted (Cancer Res. 62, 4316). , 2002; Nat Rev Drug Discovery 2,1, 2003; Semenza et al. Nature Reviews Cancer 2003. 3, 721-732; Yi Qin et al, Cancer Letter, 2014, 347, 225-232). In addition, HIF-1 activity has recently emerged as a major drug target because it is closely related to the pathogenesis of various chronic metabolic diseases such as rheumatoid arthritis, ischemic stroke, and atherosclerosis as well as cancer development and metastasis. RACKKReceptor for activated protein kinase C) is an adapter protein with seven WD motifs of 40 amino acids, a 36kDa protein consisting of 317 amino acids, and is primarily present in the cytosol and cytosolic membranes. Through binding to several proteins, including HIF-la protein (Cell Co 隱 un Signal. 2011; 9:22.), It regulates various cell responses such as cell growth, differentiation, adhesion and immunity (Li JJ, Xie D. Oncogene. 20159; 34 (15): 1890-8.). In particular, overexpression of RACK1 protein has been reported to reduce the expression level of intracellular HIF-la protein under hypoxic conditions (Liu et al. Mol Cell. 2007 Jan 26; 25 (2): 207-17.), Recently, RACK1 is overexpressed in many cancer tissues such as melanoma, breast cancer, colon cancer, lung cancer, liver cancer, esophageal cancer, and oral cancer, resulting in cancer growth. It is known to be involved in invasion and metastasis, but no specific molecular mechanisms have been reported (Zhong X. et al., Ann Surg Oncol. 2013; 20: 1044-52.). In the present invention, the present inventors confirmed that RACK1 interacts with LSD1, a lysine-specific demethylase, and confirms the methylation potential of RACK1, and thus, an antibody capable of identifying and detecting the methylation site of RACK1 is present. The present invention was completed by confirming that the antibody of the present invention can be usefully used for detecting activated dimerylated RACK1 protein and diagnosing cancer using the same. [Detailed Description of the Invention]
【기술적 과제】  [Technical problem]
본 발명의 목적은 2기번 리신 (lysine)이 다이메틸화 (diinethylat ion) 된 RACKl 단백질 (RACKlK271me2)에 특이적으로 결합하는 항체 또는 이의 면역학적 활성 단편을 제공하는 것이다.  It is an object of the present invention to provide an antibody or immunologically active fragment thereof in which second-order lysine specifically binds to a dimethylated RACKl protein (RACKlK271me2).
【기술적 해결방법】 Technical Solution
상기 목적을 달성하기 위하여, 본 발명은 2기번 리신 (lysine)이 다이메틸화 (diniethylation) 된 RACK1 단백질 (RACKlK271me2)에 특이적으로 결합하는 항체 또는 이의 면역학적 활성 단편을 제공한다.  In order to achieve the above object, the present invention provides an antibody or an immunologically active fragment thereof, wherein the second lysine specifically binds to the dimethylated RACK1 protein (RACKlK271me2).
아울러, 본 발명은 2기번 리신 (lysine)이 다이메틸화 (diniethylat ion) 된 In addition, the present invention is the second lysine (lysine) is dimethylated (diniethylat ion)
RACKl 단백질 (RACKlK271nie2)에 특이적으로 결합하는 항체 또는 이의 면역학적 활성 단편을 포함하는 시료 내 암 진단용 키트를 제공한다. Provided is a kit for diagnosing cancer in a sample comprising an antibody or immunologically active fragment thereof that specifically binds to RACKl protein (RACKlK271nie2).
【유리한 효과】 Advantageous Effects
본 발명의 항체 (antibody)는 2기번 리신 (lysine)이 다이메틸화 (dimethyl at ion) 된 RACKl 단백질 (RACKlK271me2)을 특이적으로 인식하여 결합할 수 있음을 확인, 상기 항체는 세포생물학 분야에서 메틸화된 RACK1 단백질 연구에 유용하게 사용될 수 있다. 【도면의 간단한 설명】 Antibody (antibody) of the present invention was confirmed that the second lysine (lysine) can specifically recognize and bind the dimethyl at ion (RACKlK271me2), the antibody is methylated in the field of cell biology It can be usefully used for RACK1 protein research. [Brief Description of Drawings]
도 1은 LSD1과 RACK1의 상호작용을 확인한 도이다:  1 is a diagram confirming the interaction of LSD1 and RACK1:
위： HEK-293T 세포로부터 LSD1과 RACK1을 ectopic하게 발현시켜 LSD1과 RACK1의 상호작용올 면역블 ¾팅(1隱 unoblotting)을 통해 관찰한 도;  Stomach: ectopic expression of LSD1 and RACK1 from HEK-293T cells and observed through the interaction of LSD1 and RACK1 with all immunoblotting;
아래: ACK1 단백질의 메틸화를 anti-pan-methyl-lysine 항체를 이용하여 면역블럿팅 (immunoblotting)을 통해 관찰한 도.  Below: Figure observed methylation of ACK1 protein via immunoblotting using anti-pan-methyl-lysine antibody.
도 2는 이증질량분석 (tandem mass spectorometry analyse)을 통해 HEK293T세포에서 정제한 RACK1의 2기번째 리신 잔기의 메틸화를 관찰한 도이다. 도 3는 RACK1 단백질의 2기번 리신 잔기 (1(271)와 : 172번 리신 잔기 (K172)를 알라닌으로 바꾼 RACK1 돌연변이 단백질의 메틸화를 ant i -pan-methyl -lysine 항체를 이용하여 면역블럿팅 (i關 unoblotting)을 통해 관찰한 도이다. FIG. 2 is a diagram illustrating methylation of the second stage lysine residue of RACK1 purified from HEK293T cells by tandem mass spectorometry analysis. FIG. Figure 3 is RACK1 Gibbon 2 lysine residues (1 271 and the protein: 172 times lysine residue (K172) to the use of the methylation of the mutant proteins RACK1 ant i -pan-methyl -lysine antibody replaced with alanine immune blotting ( Figure is observed through i 關 unoblotting.
도 4는 항원으로 사용할 아미노산 서열을 확보하기 위하여 소수성과 항원성 분석 (hydrophobici ty and antigenicity analysis)을 수행한 도이다:  FIG. 4 is a diagram illustrating hydrophobici ty and antigenicity analysis in order to obtain an amino acid sequence for use as an antigen:
동그라미: 항원생성에 사용한 아미노산 서열 부위를 나타낸 것으로, 서열번호 1의 266부터 276번째 아미노산 서열을 갖는다.  Circle: shows the amino acid sequence site used for antigen generation, and has the amino acid sequence of 266 to 276 of SEQ ID NO: 1.
도 5는 제조한 항— RACK1 항체의 면역효과를 확인한 도이다.  5 is a diagram confirming the immune effect of the prepared anti-RACK1 antibody.
도 6은 제조한 항 -RACK1 항체의 특이성을 확인한 도이다.  Figure 6 is a diagram confirming the specificity of the prepared anti-RACK1 antibody.
도 7은 RACK1 단백질 2기번째 리신 잔기의 메틸화를 본 발명의 항체를 이용하여 면역블럿팅 (immunoblotting)을 통해 관찰한 도이다.  7 is a diagram illustrating the methylation of the second lysine residue of the RACK1 protein by immunoblotting using the antibody of the present invention.
도 8은 저산소조건 (1% 02 조건)에서 RACK1 단백질 2기번째 리신 잔기와 Figure 8 shows the second lysine residues of RACK1 protein in hypoxic condition (1% 02 condition)
HIF-la 단백질의 상호작용을 HIF-la 항체를 이용하여 면역블럿팅 (immunoblotting)을 통해 관찰한 도이다. The interaction of HIF-la protein is observed through immunoblotting using HIF-la antibody.
도 9는 LSD1 유전자 저해 유무에 따라 RACK1과 HIF-la 단백질의 상호작용을 HIF-la 항체를 이용하여 면역블럿팅 (immunoblotting)을 통해 관찰한 도이다.  9 is a diagram illustrating the interaction between RACK1 and HIF-la protein according to the presence or absence of LSD1 gene inhibition by immunoblotting using HIF-la antibody.
【발명의 실시를 위한 최선의 형태】 [Best form for implementation of the invention]
이하, 본 발명을 상세히 설명한다. 본 발명은 271번 리신 (lysine)이 다이메틸화 (dimethylat ion) 된 RACKl 단백 질 (RACKlK271me2)에 특이적으로 결합하는 항체 또는 이의 면역학적 활성 단편을 제 공한다. Hereinafter, the present invention will be described in detail. The present invention provides an antibody or immunologically active fragment thereof in which lysine 271 specifically binds to dimethylat ionized RACKl protein (RACKlK271me2).
상기 ACK1 단백질은 서열번호 1의 아미노산 서열로 구성된 것이 바람직하 나 이에 한정되지 않는다.  The ACK1 protein is preferably composed of the amino acid sequence of SEQ ID NO: 1, but is not limited thereto.
상기 면역학적 활성 단편은 Fab. Fab', F(ab')2, Fv, Fd, 단일쇄 Fv (scFv) 및 디설파이드 안정화 Fv (dsFv)로 구성된 군으로부터 선택된 어느 하나인 것이 바 람직하나 이에 한정되지 않는다.  The immunologically active fragment is Fab. Preferably, it is any one selected from the group consisting of Fab ', F (ab') 2, Fv, Fd, single chain Fv (scFv) and disulfide stabilized Fv (dsFv).
상기 항체는 서열번호 2의 아미노산 서열에 있어서, 리신이 다이메틸화 (dimethylat ion) 된 RACKl (Rece tor for activated protein kinase C) 에피토프 (epitope)에 특이적으로 결합하는 항체 또는 이의 면역학적 활성 단편을 제공한다. 상기 항체는 단일클론항체 (monoclonal antibody) 또는 다클론항체 (polyclonal antibody)인 것이 바람직하나, 이에 한정되지 않는다.  The antibody provides an antibody or an immunologically active fragment thereof in which the lysine specifically binds to a dimethylat ion ACKl (Rece tor for activated protein kinase C) epitope in the amino acid sequence of SEQ ID NO: 2 do. The antibody is preferably a monoclonal antibody or a polyclonal antibody, but is not limited thereto.
상기 다클론 항체는 본 발명의 단백질 마커를 동물에 주사하고 해당 동물로 부터 채혈하여 항체를 포함하는 혈청을 수득하는 종래의 방법에 의해 생산할 수 있 다. 이러한 다클론 항체는 당업계에 알려진 어떠한 방법 의해서든 정제될 수 있 고, 염소, 토끼, 양, 원숭이, 말, 돼지, 소, 개 등의 임의의 동물 종 숙주로부터 만들어질 수 있다.  The polyclonal antibody may be produced by a conventional method of injecting a protein marker of the present invention into an animal and collecting blood from the animal to obtain a serum containing the antibody. Such polyclonal antibodies can be purified by any method known in the art and can be made from any animal species host, such as goats, rabbits, sheep, monkeys, horses, pigs, cattle, dogs and the like.
상기 단클론 항체는 연속 세포주의 배양을 통한 항체 분자의 생성을 제공하 는 어떠한 기술을 사용하여도 제조할 수 있다. 이러한 기술로는 이들로 한정되는 것은 아니지만 하이브리도마 기술， 사람 B-세포 하이브리도마 기술 및 EBV-하이브 리도마 기술 등이 포함된다 (Kohler G et al. , Nature 256:495-497, 1975； Kozbor D et al. , J Immunol Methods 81:31—42, 1985； Cote RJ et al . , Proc Natl Acad Sci 80:2026-2030, 1983； Cole SP etal. , Mol Cell Biol 62:109-120, 1984). 또한 본 발명은  Such monoclonal antibodies can be prepared using any technique that provides for the production of antibody molecules through the culture of continuous cell lines. Such techniques include, but are not limited to, hybridoma technology, human B-cell hybridoma technology, and EBV-hybridoma technology (Kohler G et al., Nature 256: 495-497, 1975; Kozbor D et al., J Immunol Methods 81: 31—42, 1985; Cote RJ et al., Proc Natl Acad Sci 80: 2026-2030, 1983; Cole SP et al., Mol Cell Biol 62: 109-120, 1984 ). In addition, the present invention
D 271번 리신 (lysine)이 다이메틸화 (dimethylat ion) 된 RACKl 유전자를 포 함하는 백터를 숙주 세포에 형질전환시켜 형질전환체를 제조하는 단계;  Preparing a transformant by transforming a host cell into a host cell comprising a RACKl gene in which lysine is dimethylated;
2) 단계 1)의 형질전환체를 배양하여 2기번 리신 (lysine)이 다이메틸화 (d imethyl at i on) 된 RAC 1 단백질을 제조하는 단계; 2) Incubate the transformant of step 1) to dimethylate lysine (d imethyl at i on) preparing the RAC 1 protein;
3) 단계 2)의 재조합 단백질을 항원으로 실험동물에 주입하여 면역반웅을 유도하는 단계; 및  3) inducing an immune reaction by injecting the recombinant protein of step 2) into an experimental animal as an antigen; And
4) 단계 3)의 실험동물의 혈청을 수득하여 정제하는 단계를 포함하는 제조 방법으로 제조되는 것을 특징으로 하는 항체 또는 이의 면역학적 활성 단편의 제조 방법을 제공한다.  4) It provides a method for producing an antibody or an immunologically active fragment thereof, characterized in that it is prepared by a manufacturing method comprising the step of obtaining and purifying the serum of the experimental animal of step 3).
상기 실험동물은 토끼. 마우스, 래트, 소 및 원숭이로 이루어진 군으로 부 터 선택되는 어느 하나인 것이 바람직하며， 토끼인 것이 더욱 바람직하나, 이에 한 정되지 않는다.  The experimental animal is a rabbit. It is preferably any one selected from the group consisting of mice, rats, cattle and monkeys, and more preferably rabbits, but is not limited thereto.
본 발명의 항원을 코딩하는 유전자는 코돈의 축퇴성 ( degeneracy)으로 인하 여 또는 상기 항원을 발현시키고자 하는 생물에서 선호되는 코돈을 고려하여. 코딩 영역으로부터 발현되는 항원의 아미노산 서열을 변화시키지 않는 범위 내에서 코딩 영역에 다양한 변형이 이루어질 수 있고, 코딩영역을 제외한 부분에서도 유전자의 발현에 영향을 미치지 않는 범위 내에서 다양한 변형 또는 수식이 이루어질 수 있 으며. 그러한 변형 유전자 역시 본 발명의 범위에 포함됨을 당업자는 잘 이해할 수 있을 것이다. 즉, 본 발명에서 사용된 항원은 이와 동등한 활성을 갖는 단백질을 코딩하는 한 하나 이상의 핵산 염기가 치환, 결실， 삽입 또는 이들의 조합에 의해 변이될 수 있으며, 이들 또한 본 발명의 범위에 포함된다.  The gene encoding the antigen of the present invention may be due to the degeneracy of the codon or in view of the preferred codon in the organism in which the antigen is to be expressed. Various modifications may be made to the coding region within the range of not changing the amino acid sequence of the antigen expressed from the coding region, and various modifications or modifications may be made to the region except for the coding region without affecting the expression of the gene. There is. Those skilled in the art will appreciate that such modified genes are also within the scope of the present invention. That is, the antigen used in the present invention may be mutated by one or more nucleic acid bases encoding proteins having equivalent activity by substitution, deletion, insertion, or a combination thereof, which are also included in the scope of the present invention.
상기 발현 백터의 제작 시에는. 상기 항원을 생산하고자 하는 숙주세포의 종류에 따라 프로모터 (promoter ) , 종결자 ( terminator ) , 인헨서 ( i nhancer ) 등과 같 은 발현조절 서열， 막 표적화 또는 분비를 위한 서열 등을 적절히 선택하고 목적에 따라 다양하게 조합할 수 있다. 본 발명에서 사용된 발현 백터는 플라스미드 백터， 코즈미드 백터, 박테리 오 파아지 백터 및 바이러스 백터 등을 포함하나 이에 제한되지 않는다. 적합한 발 현 백터는 프로모터， 오퍼레이터， 개시코돈, 종결코돈, 폴리아데닐화 시그널 및 인 핸서 같은 발현 조절 엘리먼트 외에도 막 표적화 또는 분비를 위한 시그널 서열 또 는 리더 서열을 포함하며 목적에 따라 다양하게 제조될 수 있다. 발현 백터의 프로 모터는 구성적 또는 유도성일 수 있다. 상기 시그널 서열에는 숙주가 에쉐리키아속 (Escherichia sp.)균인 경우에는 PhoA 시그널 서열， OmpA 시그널 서열 등이, 숙주 가 바실러스속 (Baci llus sp.)균인 경우에는 α-아밀라아제 시그널 서열, 서브틸리 신 시그널 서열 등이， 숙주가 효모 (yeast)인 경우에는 MFa 시그널 서열, SUC2 시 그널 서열 등이, 숙주가 동물세포인 경우에는 인슐린 시그널 서열， α-인터페론 시 그널 서열, 항체 분자 시그널 서열 등을 이용할 수 있으나， 이에 제한되지 않는다. 또한 발현 백터는 백터를 함유하는 숙주 세포를 선택하기 위한 선택 마커를 포함할 수 있고. 복제 가능한 발현 백터인 경우 복제 기원을 포함한다. In the production of the expression vector. According to the type of host cell to produce the antigen, appropriately select expression control sequences such as promoters, terminators, enhancers, etc., sequences for membrane targeting or secretion, and the like. It can be combined in various ways. Expression vectors used in the present invention include, but are not limited to, plasmid vectors, cosmid vectors, bacteriophage vectors and viral vectors. Suitable expression vectors include signal sequences or leader sequences for membrane targeting or secretion in addition to expression control elements such as promoters, operators, initiation codons, termination codons, polyadenylation signals and enhancers, and can be prepared in various ways depending on the purpose. have. The promoter of the expression vector can be constitutive or inducible. The signal sequence includes a host of the genus Escherichia. (Escherichia sp.) Bacteria, the PhoA signal sequence, OmpA signal sequence, etc., if the host is Bacillus (Baci llus sp.) Bacteria, α-amylase signal sequence, subtilisin signal sequence, etc., the host is yeast In the case of), MFa signal sequence, SUC2 signal sequence, etc. may be used, but if the host is an animal cell, an insulin signal sequence, an α-interferon signal sequence, an antibody molecule signal sequence, or the like may be used, but is not limited thereto. The expression vector may also include a selection marker for selecting a host cell containing the vector. If it is a replicable expression vector it includes the origin of replication.
상기 RACK1 단백질 2기번째 리신의 탈메틸화 (demethylation)는 LSDKLysine-specific demethylase 1)에 의한 것을 특징으로 하는 항체 또는 면역 학적 활성 단편을 제공한다.  Demethylation of the second stage lysine of RACK1 protein provides an antibody or immunologically active fragment, characterized by LSDKLysine-specific demethylase 1).
상기 RACK1 단백질 2기번째 리신의 메틸화는 HIF-la를 분해시키는 것을 특 징으로 하는 항체 또는 이의 면역학적 활성 단편을 제공한다. 본 발명의 구체적인 실시예에서, 본 발명자들은 항원으로 사용할 아미노산 서열을 확보하기 위하여 소수성과 항원성 분석 (Hydrophobic ity and Antigenicity analysis)을 통해 특이항체 (speci f ic antibody)의 제작 가능성이 높은 소수성 (hydrophobicity)이 낮고. 항원성 (ant igenicity)이 높으며 3차구조에서 외부로 노 출되어 있을 가능성이 높은 서열을 선정하고 (도 4 참조 이를 기반으로 최종 아미 노산 서열 [리신 잔기에 다이메틸화를 가지는 아미노산 서열: IVDELK(me2)QEVIS (서 열번호 2)]을 선정하고, 상기 리신 잔기에 다이메틸화 (dimethylation)를 가지는 아 미노산 서열번호 2의 서열을 이루는 펩타이드를 합성하여 사용하였다. 다클론 항혈 청 (polyclonal ant i sera)의 생산은 상기 항원을 토끼에 주사하여 면역반웅을 유도 하였으며 3차까지 끌어올려 (boosting), 심장 천공법으로 혈청을 수득해 항 -RACK1 다클론 항혈청을 획득하고ᅳ 수득한 혈청 내의 항체의 면역효과를 ELISA 분석하여 3 차로 끌어올린 (boosting) 항체의 면역효과가 있음을 확인하였고, 웨스턴블¾팅 (western blotting)을 수행하여 상기 항체가 2기번 리신 잔기가 다이메틸화 (dimethylation)된 RACK1을 특이적으로 인식함을 확인함으로써, 성공적으로 다클론 항체가 생성되었음을 알 수 있었다 (도 5 및 도 6 참조). 또한, 본 발명은 본 발명에 따른 항체 또는 이의 면역학적 활성 단편을 포 함하는 시료 내 암 진단용 키트를 제공한다. Methylation of the second stage lysine of the RACK1 protein provides an antibody or immunologically active fragment thereof characterized by the degradation of HIF-la. In a specific embodiment of the present invention, the present inventors have a high possibility of producing a specific antibody (hydrophobicity) through the hydrophobicity and antigenicity analysis (Hydrophobicity and Antigenicity analysis) to secure the amino acid sequence to be used as an antigen ) Is low. A sequence having high antigenicity (ant igenicity) and highly exposed to the outside in the tertiary structure is selected (see FIG. 4). Based on this, the final amino acid sequence [amino acid sequence having dimethylation at the lysine residue: IVDELK (me2 ) QEVIS (SEQ ID NO: 2)] and a peptide comprising the amino acid sequence of SEQ ID NO: 2 having dimethylation in the lysine residue was synthesized and used polyclonal ant i sera. Induced immune response by injecting the antigen into rabbits and boosted up to 3rd (boosting) to obtain serum by cardiac puncture to obtain anti-RACK1 polyclonal antiserum ELISA analysis confirmed that there is an immunological effect of the third boosted (boosting) antibody, Western blotting was carried out by the antibody lysine residues 2 times By ensuring that the tilhwa (dimethylation) the RACK1 specific to recognize, it can be seen that it has successfully polyclonal antibody is generated (see FIGS. 5 and 6). The present invention also provides a kit for diagnosing cancer in a sample comprising the antibody or immunologically active fragment thereof according to the present invention.
상기 암은 혹색종, 유방암, 대장암， 폐암, 간암. 식도암, 구강암, 위암, 골 암 . 췌장암 , 피부암 , 두경부암 , 자궁암 , 난소암 , 직장암， 결장암 , 유방암 , 자궁 육 종, 나팔관 암종， 자궁내막 암종, 자궁경부 암종, 질 암종. 외음부 암종， 식도암， 후두암, 소장암, 갑상선암， 부갑상선암. 연조직의 육종. 요도암ᅵ 음경암ᅳ 전립선암, 만성 또는 급성 백혈병 , 유년기의 고상 종양 . 분화 림프종 . 방광암 , 신장암 . 신장 세포 암종, 신장 골반 암종, 제 1중추신경계 림프종 척수축 종양, 뇌간 신경교종 및 뇌하수체 아데노마로 이루어진 군으로부터 선택되는 어느 하나이나 이에 한정 되지 않는다. 아울러， 본 발명은  The cancer is a melanoma, breast cancer, colon cancer, lung cancer, liver cancer. Esophageal Cancer, Oral Cancer, Gastric Cancer, Bone Cancer. Pancreatic cancer, Skin cancer, Head and neck cancer, Uterine cancer, Ovarian cancer, Rectal cancer, Colon cancer, Breast cancer, Uterine sarcoma, Fallopian tube carcinoma, Endometrial carcinoma, Cervical carcinoma, Vaginal carcinoma. Vulvar carcinoma, esophageal cancer, laryngeal cancer, small intestine cancer, thyroid cancer, parathyroid cancer. Sarcoma of soft tissue. Urethral cancer Penile cancer Prostate cancer, Chronic or acute leukemia, Solid tumor of childhood. Differentiated lymphoma. Bladder cancer, kidney cancer Any one selected from the group consisting of renal cell carcinoma, renal pelvic carcinoma, first central nervous system lymphoma spinal contraction tumor, brainstem glioma and pituitary adenoma is not limited thereto. In addition, the present invention
1) 생물학적 시료로부터 RACK1 단백질의 N-말단으로부터 2기번째 리신 ( lysine)의 다이메틸화 (dimethyl at ion) 수준을 측정하는 단계 ;  1) measuring the dimethyl at ion level of the second lysine from the N-terminus of the RACK1 protein from the biological sample;
2) 상기 단계 1)의 다이메틸화 수준이 정상 대조군에 비하여 감소된 경우, 암에 걸릴 위험이 높거나, 암에 걸린 개체로 판정하는 단계를 포함하는, 암 진단의 정보를 제공하기 위한 다이메틸화 수준의 측정방법을 제공한다.  2) when the level of dimethylation of step 1) is reduced compared to a normal control group, the level of dimethylation for providing cancer diagnosis information, including the step of determining a high risk of cancer or an individual having cancer, It provides a measuring method of.
상기 단계 1)의 생물학적 시료는 골수 혈청, 혈 및 혈액으로 구성된 군 으로부터 선택되는 것이 바람직하나, 이에 한정되지 않는다. 본 발명의 구체적인 실시예에서， LSD1과 RACK1의 상호작용을 확인하기 위해 ectopi c하게 RACK1과 LSD1을 발현하는 HEK293T 세포를 사용하여 공면역침전법 (co- imniunoprec ipi tat ion) 및 ant i -pan-methyl - lysine ¾■체를 이용해 면역블럿팅 ( i隱 unoblot t ing)을 수행하여, LSD1이 과발현 (overexpression)되면 RACK1의 메틸화 가 하향 조절 (down-regu l at ion)됨을 확인하였다 (도 1 참조) . The biological sample of step 1) is preferably selected from the group consisting of bone marrow serum, blood and blood, but is not limited thereto. In a specific embodiment of the present invention, co-imniunoprec ipi tat ion and ant i -pan- using HEK293T cells expressing RACK1 and LSD1 ectopi c to confirm the interaction between LSD1 and RACK1. Immunoblotting (I 隱 unoblot ting) using methyl-lysine ¾ ■ sieve confirmed that methylation of RACK1 is down-regu l at ion when LSD1 is overexpressed (see FIG. 1). ).
또한. 본 발명자들은 LSD1이 RACK1의 어느 부위를 메틸화시키는지 분석하기 위해 이중질량분석 ( t andem mass spectoromet ry ana lyse) 및 2기번 리신 잔기와 또 다른 메틸화 가능한 잔기인 172번 리신 잔기를 알라닌으로 바꾸어 준 후, ant i- pan-me thy 1- lysine 항체를 이용하여 면역블럿팅 ( i隱 unoblot t ing)을 수행하여 , LSD1 에 의해 조절받는 RACK1 단백질 메틸화의 부위는 RACK1 단백질의 2기번째 리신 잔 기임을 확인하였다 (도 2 및 도 3 참조). Also. We analyzed t andem mass spectorometry ana lyse and 2nd lysine residue and another methylable residue 172 lysine residue to alanine to analyze which region of RACK1 methylated. LSD1 was immunoblotted using ant i-pan-me thy 1-lysine antibody. It was confirmed that the site of RACK1 protein methylation regulated by the second stage lysine residue of RACK1 protein (see FIGS. 2 and 3).
또한. 본 발명자들은 LSD1이 RACK1의 2기번 리신 잔기의 메틸화를 어떻게 조절하는지 알아보기 위해 본 발명에서 제조한 2기번 리신 잔기가 다이메틸화 (dimethylation)된 RACKl(RACKlK271me2)을 특이적으로 인식하는 다중클론항체를 이 용해 면역블 ¾팅^隱 unoblotting)을 수행하여, LSD1이 과발현되면 271번 리신 잔기 가 다이메틸화 (dimethylation)된 RACK1이 상당히 감소함을 확인하였다 (도 7 참조). 또한, 본 발명자들은 LSD1이 매개하는 2기번째 리신 잔기에 다이메틸화 (dimethylation) 된 RACKl(RACKlK271me2)의 탈메틸화가 HIF-la 단백질 안정성에 미치는 영향에 대해 확인해보기 위해 야생형 RACK1과 2기번 리신을 알라닌으로 치 환한 RAC 1 돌연변이 (K271A)에 대해 HIF-la 항체 (BD Biosciences, 610959)를 이용 해 면역블럿팅 (imnumoblotting)을 수행하여, RACK1에 의해 매개되는 HIF-la의 분 해에는 RACK1 단백질의 2기번째 리신 (K271)이 중요한 잔기 (residue)임을 확인하였 다 (도 8 참조).  Also. The present inventors have identified a polyclonal antibody that specifically recognizes RACKl (RACKlK271me2) dimethylated dimethylated lysine residues prepared in the present invention to determine how LSD1 regulates methylation of the 2nd lysine residue of RACK1. This lysis immunoblotting was performed to confirm that when RSD1 was overexpressed, RACK1 dimethylated with lysine residue 271 was significantly reduced (see FIG. 7). In addition, the present inventors have examined the effects of demethylation of RACKl (RACKlK271me2) dimethylated on LSD1-mediated 2nd lysine residues on HIF-la protein stability. Immunoblotting was performed using the HIF-la antibody (BD Biosciences, 610959) for the RAC 1 mutant (K271A), which was converted to RACK1. It was confirmed that the fourth lysine (K271) is an important residue (see Figure 8).
아울러, 본 발명자들은 LSI)1이 RACiaK271me2의 메틸화 조절을 통해 RACK1과 In addition, the present inventors have found that LSI) 1 is associated with RACK1 through the regulation of methylation of RACiaK271me2.
HIF-la의 물리적인 상호작용 (interact ion)을 조절하는지 알아보고자 야생형 RACK1 과 2기번 리신을 알라닌으로 치환한 RACK1 돌연변이 (K271A) 단백질과 HIF-la 단백 질 공면역침전법 (co-immunoprecipitation assay)을 수행한 후. 면역블럿팅 (inimunoblotting)을 수행하여, LSD1에 의해 매개된 RACKlK271me2의 탈메틸화는 RACK1과 HIF-la의 결합을 저해한다는 것을 확인하였다 (도 9 참조). To determine if HIF-la modulates the physical interactions of HIF-la, co-immunoprecipitation assay with HACK-mutant (K271A) protein and wild-type RACK1 and 2nd lysine substituted with alanine After doing. Immunoblotting was performed to confirm that demethylation of RACKlK271me2 mediated by LSD1 inhibited the binding of RACK1 and HIF-la (see FIG. 9).
따라세 본 발명의 다중클론항체 (polyclonal antibody)는 2기번 리신 (lysine)이 다이메틸화 (dimethylation) 된 RACK1 단백질 (RACKlK271me2)을 특이적으 로 인식하며, 상기 항체를 이용하여 LSD1이 불활성화되면 RACK1이 메틸화되고 메틸 화된 RACK1은 HIF-la를 분해시킬 수 있음을 확인함으로써， RACK1의 메틸화는 암진 단의 마커로 활용될 수 있으며 상기 항체는 세포생물학 분야에서 메틸화된 RACK1 단백질 연구에 유용하게 사용될 수 있다. 이하, 본 발명을 하기 실시예에 의해 상세히 설명한다.  Accordingly, the polyclonal antibody of the present invention specifically recognizes RACK1 protein (RACKlK271me2) in which lysine is dimethylated, and when LSD1 is inactivated using the antibody, RACK1 is activated. By confirming that methylated and methylated RACK1 can degrade HIF-la, methylation of RACK1 can be utilized as a marker of cancer diagnosis and the antibody can be usefully used for methylated RACK1 protein research in the field of cell biology. Hereinafter, the present invention will be described in detail by the following examples.
단, 하기 실시예는 본 발명을 예시하는 것일 뿐， 본 발명의 내용이 하기 실 시예에 의해 한정되는 것은 아니다. However, the following examples are merely to illustrate the present invention, the contents of the present invention It is not limited by the example.
<실시예 1> LSD1과 RACK1의 물리적 상호작용 확인 Example 1 Confirmation of Physical Interaction of LSD1 and RACK1
본 발명자들은 LSD1 단백질과 RACK1 단백질의 물리적 상호작용 (physical interaction)을 확인하기 위하여 하기와 같은 실험을 수행하였다.  The present inventors performed the following experiments to confirm the physical interaction of the LSD1 protein and the RACK1 protein.
<1-1> 세포배양 <1-1> Cell Culture
HEK293T 세포주는 미국의 ATCC(American Type Culture Col lection)와 한국 세포주은행 (Korean Cell Line Bank)에서 구매하여, 10% 소태아혈청 (GIBCO BRL)과 IX항생-항진균제 (GIBCO BRL)를 넣어준 Rosvvell Park Memorial Institute 1640 배지 (RPMI1640; GIBCO BRL)로 배양하였다. 저산소 환경 조건은 02/C02 배양기 (MC0—5M. Sanyo, Japan)를 사용하여 1%-3% 02, 5% C02와 92¾-94% N2 조건으로 만들어서 세포 를 배양하였다. <1-2> LSD1 과발현에 의한 RACK1의 메틸화 확인  HEK293T cell line was purchased from American Type Culture Collection (ATCC) and Korean Cell Line Bank in the United States, Rosvvell Park with 10% fetal bovine serum (GIBCO BRL) and IX antibiotic-antifungal agent (GIBCO BRL) Incubated with Memorial Institute 1640 medium (RPMI1640; GIBCO BRL). The hypoxic environmental conditions were cultured using 02 / C02 incubator (MC0—5M. Sanyo, Japan) under 1% -3% 02, 5% C02 and 92¾-94% N2 conditions. <1-2> Methylation of RACK1 by LSD1 Overexpression
본 발명자들은 LSD1이 RACK1의 메틸화를 어떻게 조절하는지 알아보기 위하 여 하기와 같은 실험을 수행하였다.  The inventors performed the following experiment to determine how LSD1 regulates methylation of RACK1.
구체적으로. ectopic하게 ACK1을 발현하는 HEK293T 세포를 사용하여 공면 역침전법 (co-imnmnoprecipitation)을 하기와 같이 수행하였다.  Specifically. Co-imnmnoprecipitation was performed using HEK293T cells expressing ACK1 ectopically as follows.
Flag이 표지된 RACKKFlag tagged RACK1)을 ectopic 하게 발현하는 백터 3 과 Myc이 포지된 LSDKMyc tagged LSD1)을 ectopic하게 발현시키는 백터 3/ 을 섞고 리포펙타민 (lipofectamin, invitrogen)을 첨가하여 20분 동안 실온에서 반웅 시킨 후, HEK293T 세포에 트랜스펙션 (transfection)하였다. 48시간 후, RIPA buffer (50mM Tris-HCl pH7.5, 150mM NaCl , lOmM EDTA, 1% Triton-X100, Protease inhibitor)를 이용하여 HEK293T 세포내 단백질을 추출하고 추출된 단백질 lmg에 EzviewTMRed Anti-c-Myc Affinity Gel (Si ma-Aldr ich) 30ul를 첨가하여 16시간 동 안 4°C에서 공면역침전법 (co-immunoprecipitation) 반웅을 수행하였다. 침전된 anti-c-Myc affinity gel을 RIPA buffer로 세 번 세척하고, 2XSDS sample buffer (120mM Tris-HCl pH6.8, 20% glycerol , 4% SDS, 5% β -mercaptoethanol )를 넣고 100°C에서 5분간 끓여서 침전된 단백질을 용출 (elution)하였다. 10% SDS-PAGE gel 을 이용하여 용출 (elution)된 단백질을 분리하고， 4°C에서 90V로 2시간 동안 분리 된 단백질을 나일론막 (nylon membrane)으로 이동시킨 후, 5% BSA가 첨가되어 있는 PBST( 137mM NaCl . 2.7niM KCl, lOmM Na2HPI4, 2mM H2P04, 0.1% Tween20) 에 30분간 블로킹 (blocking) 하였다. 블로킹 (Blocking)된 막 (immunoblot membrane)에 Flag 항 체 (Sigma)를 1:1,000 비율로 16시간 동안 4。(：에서 반응시키고, PBST buffer로 10분 간 세 번 세척 한 후, HRP가 접합된 쥐 2차 항체 (horse— radish peroxidase (HRP)- conjugated mouse secondary antibody)를 1:2, 000의 비율로 실온에서 1시간 동안 반웅시켰다. 이 후， PBST buffer를 이용하여 10분씩 세 번 세척한 후, Enhanced Chemi luminescence (ECU 용액을 이용하여 발광시키고, LAS-4000 (Luminescent Image analyzer, FUGIFILM)으로 분석하였다. Vector 3, which expresses flag-labeled RACKKFlag tagged RACK1) and ectopic expression of Myc-posited LSDKMyc tagged LSD1), was mixed and lipofectamin (invitrogen) was added for 20 minutes at room temperature. After reaction, HEK293T cells were transfected. After 48 hours, HEK293T intracellular protein was extracted using RIPA buffer (50 mM Tris-HCl pH7.5, 150 mM NaCl, 100 mM EDTA, 1% Triton-X100, Protease inhibitor) and EzviewTMRed Anti-c- Co-immunoprecipitation reaction was performed at 4 ° C. for 16 hours by adding 30ul of Myc Affinity Gel (Si ma-Aldr ich). Wash the precipitated anti-c-Myc affinity gel three times with RIPA buffer, add 2XSDS sample buffer (120mM Tris-HCl pH6.8, 20% glycerol, 4% SDS, 5% β-mercaptoethanol) The precipitated protein was eluted by boiling at 100 ° C. for 5 minutes. The eluted protein was separated using a 10% SDS-PAGE gel, and the separated protein was transferred to a nylon membrane for 2 hours at 90V at 4 ° C., followed by 5% BSA. PBST (137 mM NaCl. 2.7 niM KCl, 100 mM Na2HPI4, 2 mM H2P04, 0.1% Tween20) was blocked for 30 minutes. A flag antibody (Sigma) was reacted on a blocked membrane (immunoblot membrane) at a ratio of 1: 1,000 at 4 ° C for 16 hours, washed three times with PBST buffer for 10 minutes, and then HRP conjugated. The horse-radish peroxidase (HRP) conjugated mouse secondary antibody was reacted for 1 hour at room temperature at a ratio of 1: 2, then washed three times for 10 minutes using PBST buffer. , Enhanced Chemi luminescence (emission of light using ECU solution), and analyzed by LAS- 4 000 (Luminescent Image analyzer, FUGIFILM).
그 결과. Myc이 표지된 LSD Myc tagged LSD1)과 Flag이 표지된 RACKKFlag tagged RACK1)은 물리적으로 상호작용 ( interact ion) 하고 있음을 확인하였다.  As a result. Myc-labeled LSD Myc tagged LSD1) and Flag-labeled RACKKFlag tagged RACK1) were confirmed to be physically interacting.
또한, 물리적 상호작용에 의한 RACK1의 메틸화 정도를 확인하기 위해. 상기 와 같은 방법으로 준비한 면역블럿 (i画 unoblot)에 ant i— pan-methyl -lysine 항체 (Abeam, ab23366)를 1:500 비율로 반응시켰다.  In addition, to determine the degree of methylation of RACK1 by physical interaction. An ant i-pan-methyl-lysine antibody (Abeam, ab23366) was reacted in an 1: 500 ratio to an immunoblot prepared in the same manner as described above.
그 결과, LS1H은 RACK1과 물리적으로 상호작용 (interact ion)하고 있으며, LSD1이 과발현 (over express ion)되면 RACK1의 메틸화 정도가 하향 조절 (down- regulation)됨을 확인하였다 (도 1).  As a result, it was confirmed that LS1H physically interacts with RACK1, and when LSD1 is overexpressed, methylation of RACK1 is down-regulated (FIG. 1).
<실시예 2> LSD1에 의해 메틸화되는 RACK1의 메틸화 위치 확인 Example 2 Methylation Location of RACK1 Methylated by LSD1
본 발명자들은 LSD1이 RACK1의 어느 부위를 메틸화시키는지 분석하기 위하 여 하기와 같은 실험을 수행하였다.  The inventors performed the following experiments to analyze which sites of RACK1 methylated.
구체적으로, 상기 <실시예 1-2>에서 면역침전한 RACK1의 gel band를 잘라내 고ᅳ DT를 이용하여 환원시킨 후， indole-3 acetic acid로 알킬화 하였다. 이후, 잘라낸 gel band를 lOmM ammonium bicarbonate와 50% ACN으로 세척 후, 용해 버퍼 (50mM ammonium bicarbonate, 5 mM CaC12,andl g trypsin)에 담구어 37°C에서 16 시간 반웅하여 용해 시켜주었다 (Park et al. , 2013). 용해시킨 펩타이드를 50mM animonuim bicarbonate와 100%아세토니트릴 (acetonitr i le; ACN)로 두 번에 거쳐 추 출하여 갑압하에 건조시킨 (lyophilize) 후， 1% 포름산 (formic acid)에 용해하고. 이를 Nano Acquity UPLC system을 사용하여 (Waters, USA) 이중질량분석 (tandem mass spectorometry analyse)을 하기와 같이 수행하였다. 5 ^의 펩타이드 용해액을 주입하여, C18 trap-column (i.d. 300^ηι , length 5mm, particle size 5/ni; Waters) 에서 탈염 (desalting)후, 농축시키고, 6卿의 직경을 가지고 있는 75 - silica tube에 C18 (Aqua; particle size 3ᅳ «ni)을 레진으로 패킹 (packing)하여 제작한 100- 画 microcapillary column을 통해 분리하였다. 이후, 5% 이동상 B를 5분 동안 홀려 주고, 15% 이동상 B를 5분 동안 50% 이동상 B를 75분 동안 95% 이동상 B를 5분 동 안 홀려주고 마지막으로 5% 이동상 B를 5분 동안 홀려주었다. MS/MS 스펙트럼은 RACK1의 단백질 서열 (UnitProtKB-63244)과 함께 Proteome discover software version 1.3을 사용하여 소프트웨어 분석 프로토콜을 따라 분석하였다. 펩타이드 절단 효소로써 2개의 missed cleavage를 가지는 트립신을 선택하였고. 시스테인의 carbam'idomethylation을 static modi fi cat ion으로 선택하였으며， 메티오닌의 산화 와 리신에 있는 mono- , di , tr i—methylat ion을 vari able , modi fi cat ion으로 선택하 였고. 높은 점수를 가진 펩타이드 (high scoring peptide)만이 진짜로 일치하다고 간주하였다. 높은 점수를 가진 펩타이드 (high scoring peptide)는 우리 발명에서 기준값 (threshold)보다 위인 펩타이드와 일치하였다 (Xcorr >Specifically, the gel band of immunoprecipitated RACK1 was cut out in <Example 1-2> and reduced using high-density DT, and then alkylated with indole-3 acetic acid. Then, the cut gel band was washed with lOmM ammonium bicarbonate and 50% ACN, and then immersed in a dissolution buffer (50mM ammonium bicarbonate, 5 mM CaC12, andl g trypsin) and reacted at 37 ° C for 16 hours to dissolve (Park et al. , 2013). The dissolved peptide was added twice with 50 mM animonuim bicarbonate and 100% acetonitrile (acetonitr i le; ACN). The solution was dried and dried under reduced pressure, and then dissolved in 1% formic acid. This was carried out using a Nano Acquity UPLC system (Waters, USA) double mass spectrometry (tandem mass spectorometry analyse) as follows. 5 ^ peptide lysate was injected, concentrated after desalting in C18 trap-column (id 300 ^ ηι, length 5mm, particle size 5 / ni; Waters), concentrated, and C18 (Aqua; particle size 3 ᅳ «ni) in a silica tube was separated by packing a resin with a 100- 画 microcapillary column. Then, 5% mobile phase B is held for 5 minutes, 15% mobile phase B is held for 5 minutes, 50% mobile phase B is held for 75 minutes, 95% mobile phase B is held for 5 minutes, and 5% mobile phase B is held for 5 minutes. I gave it to you. MS / MS spectra were analyzed following a software analysis protocol using Proteome discover software version 1.3 with the protein sequence of RACK1 (UnitProtKB-63244). Trypsin with two missed cleavages was selected as the peptide cleavage enzyme. The carbam ' idomethylation of cysteine was selected as the static modi fi cat ion, and the mono-, di and tr i-methylat ions in the oxidation and lysine of methionine were selected as vari able and modi fi cat ion. Only high scoring peptides were considered genuine. The high scoring peptide was consistent with the peptide above the threshold in our invention (Xcorr>
2.5 f or doub 1 echarged ion,≥3fortr ipl eandmor echarged i on). 2.5 f or doub 1 echarged ion, ≥3 fortr ipl eandmor echarged i on).
그 결과, RACK1 2기번째 잔기에 다이메틸화 (dimethylation)가 되어있음올 확인하였다 (도 2).  As a result, it was confirmed that dimethylation (dimethylation) was performed on the 2nd residue of RACK1 (FIG. 2).
또한, 하기와 같은 방법으로 DNA 돌연변이 유발 (mutagenesis)을 하여. 271 번 리신 잔기와 또 다른 메틸화 가능한 잔기인 172번 리신 잔기를 알라닌으로 바꾸 어 주었다.  In addition, by DNA mutagenesis in the following manner. The 271 lysine residue and another methylable residue 172 lysine residue were replaced with alanine.
구체적으로， Flag이 표지된 야생형 RACKKFlag tagged RACK1)을 주형으로 하여, KOD-Plus-Mut agenesis Kit(TOYOBO)의 PCR grade water 35^, iPCR용 10x Buffer 5 L 2mM dNTPs 5씨 Flag이 표지된 (Flag tagged) 야생형 RACKl(50ng/ul ) 1 id, KOD-Plus-DNA Polyineraseinverse Ifd 및 10隱 ol/ul 농도의 프라이메 Flag- RACK1 K172A Forward 프라이머: 5'-TG(X;ACGCGCTGGTCMGGTATGG— 3' (서열번호 3); Flag-RAC l K172A Reverse 프라이머: 5'-GCCACAGGAGACGATGATAGGGTTGCT-3' (서열번호 4)； Flag-RACKl K271A Forward 프라이머: 5'-G/\CTGGCGCMGAAGTTATCAGT-3' (서열번호Specifically, Flag-labeled wild-type RACKKFlag tagged RACK1) as a template, PCR grade water 35 ^ of KOD-Plus-Mut agenesis Kit (TOYOBO), 10x Buffer 5 L 2mM dNTPs 5 seed flag for iPCR (Flag tagged) wild type RACKl (50ng / ul) 1 id, KOD-Plus-DNA Polyineraseinverse Ifd and primers at 10 隱 ol / ul concentration Flag- RACK1 K172A Forward primer: 5'-TG (X; ACGCGCTGGTCMGGTATGG— 3 '(SEQ ID NO: 3); Flag-RAC l K172A Reverse primer: 5'-GCCACAGGAGACGATGATAGGGTTGCT-3 '(SEQ ID NO: 4); Flag-RACKl K271A Forward primer: 5'-G / \ CTGGCGCMGAAGTTATCAGT-3 '(SEQ ID NO:
5)； Flag-RACKl K271A Reverse 프라이머 : 5'-ATCTACMTGATCmCCCTCTAAATC-3' (서열 번호 6)] 1.5/£를 이용하여 inverse PCR을 수행하였다. 이때, PCR 조건은 94°C에서 2분, 98'C에서 10초간 변성 , 60°C에서 30초간 어닐링， 72°C에서 7분간 신장의 조건 으로 10회 반복하여 inverse PCR올 수행하였다. Inverse PCR 반웅 종료 후, 2/z.(!의 Dpnl 제한효소를 37°C에서 1시간 처리한 후. Dpnl 제한효소가 처리된 PCR 산물 1 , PCR grade water 7 μί , ligation high 5μί 및 Τ4 polynucleotide kinase 를 이용 하여 인산화 /연결 (kination/ligation) 반응을 1시간 동안 16°C에서 실시하였다. 이 후, 인산화 /연결 (kination/ligation) 산물 1 ^를 10 대장균에 형질 전환하고 DNA 를 추출하여 Flag이 표지된 172번 리신 잔기가 알라닌으로 바뀐 RACKKFlag tagged RACK ΚΓ72Α)와 Flag이 표지된 2기번 리신 잔기가 알라닌으로 바¾ RACKl(Flag tagged RAC 1 K271A) 백터를 확보하였다. 상기 백터를 HEK293T 세포에 리포펙타민 (lipofectamin)을 이용하여 트랜스펙션 (transfect ion) 시키고 48시간 후, RIPA buffer를 이용하여 단백질을 추출하고, 추출된 단백질 lnig과 항 -Flag M2 30Λ£를 섞 어 16시간 동안 4°C에서 면역침전반웅을 수행하였다. 이후, RIPA buffer를 이용하 여 항 -Flag M2를 10분씩 세 번 세척하고, 2x sample buffer를 첨가한 후, 10C C에 서 5분간 끓여 면역침전된 단백질을 용출 (elution)하였다. 이 후, anti-pan- methyl-lysine 항체를 이용하여 상기 <실시예 1-2>와 동일한 방법으로 면역블롯팅 (munoblotting)을 수행하였다. 5); Flag-RACKl K271A Reverse primer: 5'-ATCTACMTGATCmCCCTCTAAATC-3 '(SEQ ID NO: 6)] was performed inverse PCR using 1.5 / £. At this time, the PCR conditions were performed inverse PCR for 10 minutes at 94 ° C 2 minutes, denatured for 10 seconds at 98'C, annealing for 30 seconds at 60 ° C, elongation at 72 ° C for 7 minutes. After completion of the inverse PCR reaction, treated with Dpnl restriction enzyme of 2 / z. (! For 1 hour at 37 ° C. PCR product 1, PCR grade water 7 μί, ligation high 5 μί and Τ4 polynucleotide kinase treated with Dpnl restriction enzyme The phosphorylation / ligation reaction was carried out for 1 hour at 16 ° C. Subsequently, the phosphorylation / ligation product 1 ^ was transformed into E. coli and DNA was extracted. RACKKFlag tagged RACK κΓ72Α) in which labeled lysine residues were replaced with alanine and lagine labeled 2nd lysine residues as alanine, thus obtaining a ¾ RACKl (Flag tagged RAC 1 K271A) vector. The vector was transfected into HEK293T cells using lipofectamin (lipofectamin), and after 48 hours, the protein was extracted using RIPA buffer, and the extracted protein lnig and anti-Flag M2 30Λ £ were mixed. Immunoprecipitation reaction was performed at 4 ° C. for 16 hours. Thereafter, anti-Flag M2 was washed three times for 10 minutes using RIPA buffer, 2x sample buffer was added, and the immunoprecipitated protein was eluted by boiling at 10 C for 5 minutes. Thereafter, immunoblotting was performed using the anti-pan-methyl-lysine antibody in the same manner as in <Example 1-2>.
그 결과, LSD1에 의해 조절받는 RACK1 단백질 메틸화의 부위는 RACK1 단백 질의 2기번째 리신 잔기임을 확인하였다 (도 3).  As a result, it was confirmed that the site of RACK1 protein methylation regulated by LSD1 is the second lysine residue of the RACK1 protein (FIG. 3).
<실시예 3> 271번 리신 잔기가 다이메틸화 (dimethylation)된 RACK1을 특이 적으로 인식하는 (RACKlK271me2) 다중클론항체의 제조 Example 3 Preparation of a (RACKlK271me2) Polyclonal Antibody Specific for Recognizing RACK1 Dimethylated with Lysine No. 271
2기번 리신 잔기가 다이메틸화 (dimethylation)된 RACK1을 특이적으로 인식 하는 (RACKlK271me2) 다증클론항체를 하기의 방법으로 제조하였다.  A polyclonal antibody (RACKlK271me2) that specifically recognizes RACK1 having dimethylated lysine residues dimethylated (dimethylated) was prepared by the following method.
구체적으로, 항원으로 사용할 아미노산 서열을 확보하기 위하여 소수성과 항원성 분석 (Hydrophobic ity and Antigenicity analysis)을 통해 특이항체 (specific antibody)의 제작 가능성이 높은 소수성 (hydrophobic ity)이 낮고, 항원 성 (antigenicity)이 높으며 3차구조에서 외부로 노출되어 있을 가능성이 높은 서열 을 선정하고 (도 4), 이를 기반으로 최종 아미노산 서열 [리신 잔기에 다이메틸화 (diniethylation)를 가지는 아미노산 서열: IVDELK(me2)QEVIS (서열번호 2)]을 선정 하였다. 상기 서열을 기반으로 리신 잔기에 다이메틸화 (dimethylation)를 가지는 펩타이드를 합성하여 항원으로 사용하였다 Specifically, in order to secure an amino acid sequence to be used as an antigen, hydrophobicity with low possibility of producing a specific antibody through hydrophobicity and antigenicity analysis is low, and antigen A sequence having high antigenicity and having a high possibility of being exposed to the outside in the tertiary structure is selected (FIG. 4), and based on this, the final amino acid sequence [amino acid sequence having dimethylation at lysine residue: IVDELK (me2 ) QEVIS (SEQ ID NO: 2)]. Based on the sequence, a peptide having dimethylation at a lysine residue was synthesized and used as an antigen.
다클론 항혈청 (polyclonal antisera)의 생산은 4주 동안 예비면역 반응이 (0 주차)유도된 토끼에 2기번 리신 잔기가 다이메틸화 (dimethylation)된 RACK1 단백질 (RAC lK271me2) 유래의 서열번호 2로 기재되는 펩타이드를 항원으로 이용， 피하 주 사하여 (50 «g/rabbit) 1차로 항체 생산을 끌어 올렸다 (4주차- 1차 boosting). 또한, 1차 항체생산 면역 반웅 유도 2주 후에 2차로 항체 생산을 끌어을렸으며 (500 /rabbit; 6주차— 2차 boosting), 추가로 2주 후에 3차로 항체 생산을 끌어을렸다 (500^g/rabbit; 8주차- 3차 boosting). 그로부터 1주 후 (9주차). 심장 천공법으로 혈청을 수득하여 항 -RACK1 다클론 항혈청을 획득하고 리신 2기번 잔기에 다이메틸 화 (dimethylation)가 되어있는 RAC 1 펩타이드 [IVDELK(me2)QEVIS; 아미노산 266 ~ 276, (서열번호 2)]를 이용하여 친화 크로마토그래피를 통해 정제하여 토끼 항- RACK1 다클론항체를 얻었다.  Production of polyclonal antisera is described by SEQ ID NO: 2 from the RACK1 protein (RAC lK271me2) dimethylated with lysine residues 2 times in rabbits in which a preimmune response has been induced (park 0) for 4 weeks. Peptides were used as antigen and subcutaneously injected (50 «g / rabbit) to boost antibody production first (week 4-primary boosting). In addition, two weeks after the first antibody-induced immune response reaction, the production of the antibody was induced secondarily (500 / rabbit; six weeks—second boosting), and additionally after three weeks, the production of the antibody was induced three times (500 ^ g / rabbit). ; Week 8-3rd boosting). A week later (Week 9). Serum was obtained by cardiac puncture to obtain anti-RACK1 polyclonal antiserum and RAC 1 peptide [IVDELK (me2) QEVIS; dimethylation at lysine residue 2; Amino acids 266 to 276, (SEQ ID NO: 2)] were purified by affinity chromatography to obtain a rabbit anti-RACK1 polyclonal antibody.
<3-1> 항 -RACK 1 항체의 면역효과 확인 <3-1> Immune effect of anti-RACK 1 antibody
토끼에서 얻은 혈청 내의 항 -RACK1 항체의 면역효과를 확인하기 위하여 , 효 소면역분석법 (ELISA)을 하기와 같은 방법으로 수행하였다.  In order to confirm the immune effect of the anti-RACK1 antibody in the serum obtained from rabbits, the enzyme immunoassay (ELISA) was performed in the following manner.
구체적으로, 항원을 코팅버퍼 (coating buffer)와 함께 2 g/n 이 되도록 회 석시키고 각 웰에 50 씩 분주한 후, 코팅이 되도록 4°C에서 오버나잇 (overnight) 하거나 37°C에서 세시간 동안 두었다. 그 후, 코팅 용액을 버리고 의 2% skim milk/TBS-T 용액을 분주하여 37°C에서 한시간 동안 블로킹 (blocking) 시켰다. 한시 간 후에 TBS-T 용액으로 세척하고. 의 1차 항체를 각각의 웰에 분주하여 37°C 에서 두시간 동안 반응시켰다. 그 후 TBS-T로 3번 세척하고 1:5000으로 희석시킨 2 차 항체를 각 웰에 50 ^씩 분주하고 37°C에서 한시간 동안 반웅시켰다. 그 후 TBS- T로 5번 세척한 후 0PD 또는 TMB(color reagent)를 50 씩 각 웰에 분주하고 색깔 이 변하면 정지용액 (stop solution; IN H2S04)을 넣어서 반웅을 증단시킨 후, 495nm에서 0D값을 측정하였다. Specifically, the antigen is ligated to 2 g / n with a coating buffer and dispensed into each well 50, followed by overnight at 4 ° C. or three hours at 37 ° C. for coating. Put it. Thereafter, the coating solution was discarded and the 2% skim milk / TBS-T solution was dispensed and blocked at 37 ° C. for one hour. After an hour wash with TBS-T solution. Primary antibodies were dispensed into each well and reacted at 37 ° C. for 2 hours. Secondary antibodies washed three times with TBS-T and diluted 1: 5000 were then dispensed 50 ^ into each well and reacted at 37 ° C for one hour. After washing 5 times with TBS-T, dispense 0PD or TMB (color reagent) into each well by 50 and change the color. Then, add a stop solution (IN H2S04) to increase reaction. The 0D value was measured at 495 nm.
그 결과, 1/5000에서 0D값이 1.0 이상으로 나타났고 이를 통해 항 -RACK1 항 체는 면역효과가 있음을 확인하였다 (도 5) . <3-2> 항 -RACK 1 항체의 특이성 확인  As a result, 0D value was found to be greater than 1.0 at 1/5000, and it was confirmed that the anti-RACK1 antibody had an immune effect (FIG. 5). <3-2> Specificity of anti-RACK 1 antibody
차단검사 (blocking test )는 경쟁적 분석 (compet i t i on assay)의 하나로서 웨 스턴 블럿팅을 할 때 2차 항체를 붙이는 단계에서 항체와 항원을 함께 반웅시켜 항 체에 대하여 막 ( membrane)의 단백질과 항원을 경쟁하는 원리로 항체가 특이성이 있 으면 항원을 함께 넣어 준 결과에서 목적한 밴드 ( target band)만이 사라지거나 상 대적으로 확연히 사라지는 양상을 보여 이를 통해 항체의 특이성을 확인할 수 있다. 상기 항 -RACK1 항체의 특이성을 확인하기 위하여. 웨스턴블럿팅 (western blot t i ng) 을 하기와 같은 방법으로 수행하였다.  The blocking test is a competitive iti on assay. When western blotting, the antibody and antigen are reacted together in the step of attaching the secondary antibody to the antibody, and the protein of the membrane and If the antibody has specificity as a principle of competing antigens, only the target band disappears or disappears relatively clearly from the result of adding the antigen, thereby confirming the specificity of the antibody. To confirm the specificity of the anti-RACK1 antibody. Western blotting was performed in the following manner.
구체적으로, 합성한 RACK1K271 펩타이드에 메틸화가 없는 K271nie0 펩타이드, 하나만 있는 K271mel 팹타이드, 두 개 있는 K271me2 펩타이드 및 세 개 있는 K271me3 펩타이드, 총 4 종의 펩타이드를 제작하여 Ong 내지 1000ng씩 나일론막 (nylon membrane)에 분주시키고 말린 후, 5% BSA로 나일론막 (nylon membrane)을 블 로킹 (blocking)하였다. 이후 상기 <실시예 1>에서 제조한 다중클론 항체 RACKlK271me2를 5% BSA(PBST에 BSA를 녹여서 제조)에 1 : 1.000으로 희석하여 1시간 동안 실온에서 반응한 후, PBST로 10분간 세 번 세척하였다. 이후, 항—토끼 항체 IgG-HRP (Mi l l ipore)를 1 : 2 , 000의 비율로 실은에서 1시간 동안 반응시킨 후, PBST buf fer를 이용하여 10분간 세 번 세척하였다. Enhanced Chemi luminescence (ECU 용액을 이용하여 발광시키고, LAS-4000 (Luminescent Image analyzer , FUGIFILM)으 로 분석하였다.  Specifically, the synthesized RACK1K271 peptide was prepared without the methylation of K271nie0 peptide, only one K271mel fabtide, two K271me2 peptide and three K271me3 peptide, a total of four peptides, from Ong to 1000ng by nylon membrane (nylon membrane) After aliquoting and drying, the nylon membrane was blocked with 5% BSA. Thereafter, the polyclonal antibody RACKlK271me2 prepared in Example 1 was diluted with 1: 1.000 in 5% BSA (prepared by dissolving BSA in PBST) and reacted at room temperature for 1 hour, and then washed three times with PBST for 10 minutes. . Subsequently, the anti-rabbit antibody IgG-HRP (Mi l lore) was reacted at a ratio of 1: 2,000 for 1 hour in real plants, and then washed three times for 10 minutes using PBST buf fer. Luminescence was enhanced using an Enhanced Chemi luminescence (ECU solution) and analyzed by LAS-4000 (Luminescent Image analyzer, FUGIFILM).
그 결과, 제조한 다중클론 항체 RACKlK27 ie2는 다이메틸화 (dimethyl at ion) 가 되어 있는 K271me2 펩타이드에만 특이적으로 반웅하는 것을 확인하였다.  As a result, it was confirmed that the prepared polyclonal antibody RACKlK27 ie2 specifically reacted only with the K271me2 peptide that has been dimethylated (dimethyl at ion).
또한 차단검사 (blocking test )를 위해 다중클론 항체 RACKlK271me2와 K271me2 항원 (2/zg/ml )을 흔합하여 실은에서 30분간 반응시킨 후 상기와 같이 준비 된 나일론막 (nyl on membrane)에 반응시켰다.  In addition, the polyclonal antibody RACKlK271me2 and K271me2 antigen (2 / zg / ml) were mixed for 30 minutes in a block for the blocking test, and then reacted to the nylon on membrane prepared as described above.
그 결과, 다중클론 항체 RACKlK271me2가 K271me2 펩타이드와 반웅하지 못하 는 것을 확인함으로써, <실시예 3>에서 제조한 다중클론 항체 RACKlK271me2는 271 번 리신 잔기가 다이메틸화 (dimethylation)된 RACK1을 특이적으로 인식하는 항체 특이성 (specificity)이 있음을 확인하였다 (도 6) . <실시예 4> 2기번 리신 잔기가 다이메틸화 (dimethylation)된 RACK1을 특이 적으로 인식하는 (RACKlK271me2) 다중클론항체의 효과 확인 As a result, the polyclonal antibody RACKlK271me2 could not react with the K271me2 peptide. By confirming that, the polyclonal antibody RACKlK271me2 prepared in Example 3 has an antibody specificity that specifically recognizes RACK1 in which lysine residue 271 is dimethylated (FIG. 6). . Example 4 Confirmation of the Effect of the (RACKlK271me2) Polyclonal Antibody Specifically Recognizing RACK1 Dimethylated by Lysine No. 2 Lysine
LSI)1이 RACK1의 2기번 리신 잔기의 메틸화를 어떻게 조절하는지 알아보기 위하여 하기와 같은 실험을 수행하였다.  To determine how LSI) 1 regulates methylation of the 2nd lysine residue of RACK1, the following experiment was performed.
구체적으로 , 상기 <실시예 3>에서 제조한 2기번 리신 잔기가 다이메틸화 (dimethylation)된 RACKl(RACKlK271me2)을 특이적으로 인식하는 다중클론항체를 이 용하여 상기 <실시예 1-2>와 동일한 방법으로 면역블럿팅 (i隱 unoblotting)을 수행 하였다.  Specifically, the same method as in <Example 1-2> by using a polyclonal antibody that specifically recognizes RACKl (RACKlK271me2), in which the 2nd lysine residue prepared in <Example 3> is dimethylated (dimethyl) Immunblotting (i 隱 unoblotting) was performed.
그 결과, LSD이 과발현되면 271번 리신 잔기가 다이메틸화 (dimethylation) 된 RACK1이 상당히 감소함을 확인하였다 (도 7).  As a result, it was confirmed that when LSD is overexpressed, RACK1 in which lysine residue 271 is dimethylated is significantly reduced (FIG. 7).
상기 <실시예 3> 및 <실시예 4>를 통해 RACKl K27의 메틸화는 LSD1에 의해 직접적으로 조절됨을 확인하였다.  Through <Example 3> and <Example 4> it was confirmed that the methylation of RACKl K27 is directly controlled by LSD1.
<실시예 5> RACK1 단백질 2기번째 리신의 탈메틸화 효과 확인 Example 5 Demethylation Effect of RACK1 Protein 2nd Lysine
<5-1> HIF-la 단백질 안정성에 미치는 영향 확인  <5-1> Effect of HIF-la Protein Stability
RACK1의 과발현은 저산소환경 조건에서 세포내 HIF-la 단백질의 레벨을 감 소시킨다고 알려져있고 (Liu, Y. V.et al . , MolCell 2007. 25, 207-217.), 이에 LSD1에 의한 RAC l 271me2 탈메틸화가 HIF-la 단백질 안정성에 미치는 영향에 대 해 확인해보기 위하여 RACK1 단백질의 2기번째 리신을 알라닌으로 치환한 RACK1 돌 연변이를 제조한 후, 하기와 같은 실험을 수행하였다.  Overexpression of RACK1 is known to reduce the level of intracellular HIF-la protein under hypoxic conditions (Liu, YV et al., MolCell 2007. 25, 207-217.), Resulting in demethylation of RAC l 271me2 by LSD1. In order to confirm the effect of HIF-la protein stability on the stability of the RACK1 mutant was substituted for the second stage lysine of the RACK1 protein with alanine, and the following experiment was performed.
구체적으로 , 저산소환경 조건에서 상기 <실시예 2-1>과 같은 방법으로 Specifically, in the same manner as in <Example 2-1> under low oxygen environment conditions
HEK293T세포를 배양하여 HIF-la 항체 (BD Biosciences. 610959)를 이용해 상기 <실 시예 2-2>와 동일한 방법으로 면역블럿팅 (immunoblotting)을 수행하였다. HEK293T cells were cultured and immunoblotting was performed using the HIF-la antibody (BD Biosciences. 610959) in the same manner as in <Example 2-2>.
그 결과, RACK1 단백질 2기번째가 리신인 야생형 RACK1 단백질이 2기번 리 신을 알라닌으로 치환한 RACK1 돌연변이 (K271A)보다 HIF-la 단백질의 레벨이 더 낮은 것을 확인하였다 (도 8). 이에 , RACK1에 의해 매개되는 HIF-la의 분해에는 RACK1 단백질의 2기번째 리신 (K271)이 중요한 잔기 (residue)라는 것을 확인하였다. As a result, the wild-type RACK1 protein, which is the 2nd stage of the RACK1 protein, had higher levels of HIF-la protein than the RACK1 mutation (K271A), which replaced the 2nd stage of the lysine with alanine. It was confirmed to be low (FIG. 8). Accordingly, it was confirmed that the second stage lysine (K271) of the RACK1 protein is an important residue in the degradation of HIF-la mediated by RACK1.
<5-2> RAC 1 및 HIF-la의 결합에 미치는 영향 확인 <5-2> Confirmation of the effect on the binding of RAC 1 and HIF-la
RACK1 단백질의 2기번째 리신 (K271)은 RACK1과 HIF-la의 결합 (associat ion) 에 필수적인 RACK1 단백질의 TO6 도메인과 인접해있으므로 LSD1이 RACKlK271me2의 메틸화 조절을 통해 RACK1과 HIF-la의 물리적인 상호작용 ( interact ion)을 조절하 는지 알아보고자 하기와 같은 실험을 수행하였다.  The second lysine (K271) of the RACK1 protein is adjacent to the TO6 domain of the RACK1 protein, which is essential for the association of RACK1 with HIF-la, so that LSD1 modulates the physical interaction of RACK1 and HIF-la through the regulation of methylation of RACKlK271me2. In order to find out whether the interaction (interaction ion) is controlled, the following experiment was performed.
구체적으로, Flag이 표지된 야생형 ACKKFlag tagged RAC 1), 혹은 Flag이 표지된 2기번 리신을 알라닌으로 치환한 돌연변이 RACK1(K271A) 유전자 5 을, HA 가 표지된 야생형 HIF-la 5 과 함께 LSD1이 녹다운 (knockdown) 되어 있는 HEK293T 세포와 그렇지 않은 HEK293T 세포에 리포펙타민 ( 1 ipofect ion)을 이용하여 <실시예 1-2>와 같이 과발현 (overexpression) 시킨 후, 48시간 후에 ΙΟμΜ MG132(Calbiochem)를 4시간 처리하였다. 상기와 같은 방법으로 단백질을 추출하여 각각 3mg의 단백질과 ANTI— Flag M2 (Sigma-Aldr ich) 35^를 섞어 16시간 동안 4°C 에서 공면역침전법 (co-immunoprecipitation) 반웅을 수행하였다. 침전된 항 -Flag M2를 RIPA buffer로 세 번 씻어주고. 2Xsample buffer를 넣고 100°C에서 5분간 끓 여주어 침전된 단백질을 용출 (elution) 하였다. 10% SDS-PAGE gel을 이용하여 용출 된 단백질을 분리하고ᅳ 분리된 단백질을 4°C에서 90V 전기 분압으로 2시간 동안 나 일론막 (nylon membrane)으로 이동시킨 후, 5) BSA에 30분간 블로킹 (blocking)하였 다. 블로킹 (blocking) 된 막 (membrane)에 HA 항체 (Santa Cruz)를 1:1,000 비율로 16시간 동안 4°C에서 반웅시키고， PBST buffer로 10분간 세 번 세척한 후, HRP가 접합된 토'끼 2차 항체 (horse一 radish peroxidase (HRP)— conjugated rabbit secondary antibody, Millipore)를 1:2, 000의 비을로 1시간 동안 실온에서 반웅하 였다. PBST buffer를 이용하여 세 번 씻어 준 후. Enhanced Chemi luminescence (ECL) 용액을 이용하여 발광시키고, LAS-4000 (Luminescent Image analyzer, FUGIFILM)으로 분석 하였다. Specifically, LSD1 knocked down the wild-type ACKKFlag tagged RAC 1), or the mutant RACK1 (K271A) gene 5 in which the flag-labeled wild-type lysine was substituted with alanine. (knockdown) HEK293T cells and HEK293T cells, which were not, were overexpressed using lipofectamine (1 ipofect ion) as in <Example 1-2>, and after 48 hours, ΙΟμΜ MG132 (Calbiochem) was 4 Time was processed. Proteins were extracted in the same manner as described above, and 3 mg of protein and ANTI—Flag M2 (Sigma-Aldr ich) 35 ^ were mixed, and co-immunoprecipitation reaction was performed at 4 ° C. for 16 hours. Wash precipitated anti-Flag M2 three times with RIPA buffer. 2Xsample buffer was added and the mixture was boiled at 100 ° C. for 5 minutes to elute the precipitated protein. The eluted protein was separated using a 10% SDS-PAGE gel, and the separated protein was transferred to a nylon membrane for 2 hours at 90V electric partial pressure at 4 ° C, and 5) blocking for 30 minutes on BSA. (blocking). Blocking (blocking) the HA antibody in the film (membrane) (Santa Cruz) for 1: After washing three to 1,000 rate and banung at 4 ° C for 16 hours, 10 minutes in PBST buffer times, HRP is bonded Sat "meal A secondary antibody (horse irradiated peroxidase (HRP) —conjugated rabbit secondary antibody, Millipore) was reacted at room temperature for 1 hour at a ratio of 1: 2, 000. After washing three times using PBST buffer. Emission was performed using an Enhanced Chemi luminescence (ECL) solution and analyzed by LAS-4000 (Luminescent Image Analyzer, FUGIFILM).
그 결과, LSD1 유전자 발현이 저해되면 HIF-la와 야생형 RACK1 사이에 물 리적인 상호작용 (interact ion)이 크게 증가된 반면 2기번 리신을 알라닌으로 치환 한 RACKl 돌연변이 (K271A)와 HIF-l a는 LSD1 유전자 발현 저해에 영향이 없음을 확 인함으로써, LSI)1에 의해 매개된 RACKlK271nie2의 탈메틸화는 RACK1과 HIF-l a의 결 합을 저해한다는 것을 확인하였다 (도 9) . As a result, the inhibition of LSD1 gene expression significantly increased the physical interaction between HIF-la and wild-type RACK1, while replacing lysine with alanine. We confirmed that one RACKl mutation (K271A) and HIF-l a had no effect on inhibition of LSD1 gene expression, indicating that demethylation of RACKlK271nie2 mediated by LSI) 1 inhibited the binding of RACK1 and HIF-l a. It was confirmed (FIG. 9).
즉, LSD1이 불활성화되면 RACK1이 메틸화되고 메틸화된 RACK1은 HIF-l a를 분해시킬수 있음을 확인하였고 이러한 RACK1의 메틸화는 암치료의 마커로 활용될 수 있음을 확인하였다. That is, when LSD1 was inactivated, it was confirmed that RACK1 was methylated and methylated RACK1 could degrade HIF-1 la and such methylation of RACK1 could be used as a marker for cancer treatment.
Figure imgf000021_0001
Figure imgf000021_0001
psoo swi Hml- psoo sw i Hm l-
Figure imgf000022_0001
Figure imgf000022_0001
10 1 0
<220> <220>
<221> INIT_MET <221> INIT_MET
<222> (6) <222> (6)
<400> 2 <400> 2
lie Val Asp Glu Leu Lys Gin Glu Val lie Ser lie Val Asp Glu Leu Lys Gin Glu Val lie Ser
5 10  5 10
<210> 3  <210> 3
<211> 24 <211> 24
<212> DNA <212> DNA
<213> Artificial Sequence <213> Artificial Sequence
<220> <220>
<223> Flag-RACKl K172A Forward primer <223> Flag-RACKl K172A Forward primer
<400> 3 <400> 3
tgggacgcgc tggtcaaggt atgg 24 <210> 4 tgggacgcgc tggtcaaggt atgg 24 <210> 4
<211> 27  <211> 27
<212> DNA <212> DNA
<213> Artificial Sequence <213> Artificial Sequence
<220> <220>
<223> Flag-RACKl K172A Reverse primer <223> Flag-RACKl K172A Reverse primer
<400> 4 <400> 4
gccacaggag acgatgatag ggttgct gccacaggag acgatgatag ggttgct
<210> 5  <210> 5
<211> 24  <211> 24
<212> DNA  <212> DNA
<213> Artificial Sequence <223> Flag-RACKl K271A Forward primer <213> Artificial Sequence <223> Flag-RACKl K271A Forward primer
<400> 5 <400> 5
gaactggcgc aagaagttat cagt 24 <210> 6 gaactggcgc aagaagttat cagt 24 <210> 6
<211> 27 <211> 27
<212> DNA <212> DNA
<213> Artificial Sequence  <213> Artificial Sequence
<220> <220>
<223> Flag-RACKl K271A Reverse primer  <223> Flag-RACKl K271A Reverse primer
<400> 6 <400> 6
atctacaatg atctttccct ctaaatc atctacaatg atctttccct ctaaatc

Claims

【청구의 범위】 [Range of request]
【청구항 1】  [Claim 1]
N-말단으로부터 2기번째 리신 (lysine)이 다이메틸화 (dimethylat ion) 된 RAC 1 단백질 (RACKlK271me2)에 특이적으로 결합하는 항체 또는 이의 면역학적 활성 단편 .  An antibody or immunologically active fragment thereof in which the second lysine from the N-terminus specifically binds to the RAC 1 protein (RACKlK271me2) dimethylated.
【청구항 2] [Claim 2]
제 1항에 있어서, 상기 RACK1 단백질은 서열번호 1의 아미노산 서열로 구성된 것을 특징으로 하는 항체 또는 이의 면역학적 활성 단편.  The antibody or immunologically active fragment thereof of claim 1, wherein the RACK1 protein consists of an amino acid sequence of SEQ ID NO: 1.
【청구항 3】 [Claim 3]
제 1항에 있어서, 상기 면역학적 활성 단편은 Fab.. Fab', F(ab')2, Fv, Fd, 단일쇄 Fv (scFv) 및 디설파이드 안정화 Fv (dsFv)로 구성된 군으로부터 선텍된 어느 하나인 것을 특징으로 하는 항체 또는 이의 면역학적 활성 단편.  The method of claim 1, wherein the immunologically active fragment is any one selected from the group consisting of Fab .. Fab ', F (ab') 2, Fv, Fd, single chain Fv (scFv) and disulfide stabilized Fv (dsFv). An antibody or immunologically active fragment thereof, characterized in that.
【청구항 4】 [Claim 4]
제 1항에 있어서, 상기 항체는 서열번호 2의 아미노산 서열에 있어서, 리신이 다이메틸화 (dimethylation) 된 RACKU Receptor for activated protein kinase C) 에피토프 (epitope)에 특이적으로 결합하는 항체 또는 이의 면역학적 활성 단편.  The antibody or immunological activity thereof according to claim 1, wherein the antibody specifically binds to RACKU dimethylated RACKU Receptor for activated protein kinase C) epitope in amino acid sequence of SEQ ID NO: 2 snippet.
【청구항 5】 [Claim 5]
제 1항에 있어서, 상기 항체는 단일클론항체 (monoclonal antibody) 또는 다 클론항체 (polyclonal antibody)인 것을 특징으로 하는 항체 또는 이의 면역학적 활성 단편.  The antibody or immunologically active fragment thereof according to claim 1, wherein the antibody is a monoclonal antibody or a polyclonal antibody.
【청구항 6】 [Claim 6]
제 1항에 있어서, 상기 면역학적 활성 단편은  The method of claim 1, wherein said immunologically active fragment is
1) 2기번 리신 (lysine)이 다이메틸화 (dimethylation) 된 RACK1 유전자를 포함하는 백터를 숙주 세포에 형질전환시켜 형질전환체를 제조하는 단계; 1) 2nd lysine is dimethylated RACK1 gene Preparing a transformant by transforming the vector into a host cell;
2) 단계 1)의 형질전환체를 배양하여 2기번 리신 ( lys ine)이 다이메틸화 (diniethyl at ion) 된 RACK1 단백질을 제조하는 단계 ;  2) culturing the transformant of step 1) to prepare a RACK1 protein in which lysine is dimethylated (diniethyl at ion);
3) 단계 2)의 재조합 단백질을 항원으로 실험동물에 주입하여 면역반웅을 유도하는 단계 ; 및  3) inducing an immune response by injecting the recombinant protein of step 2) into the experimental animal as an antigen; And
4) 단계 3)의 실험동물의 혈청을 수득하여 정제하는 단계를 포함하는 제조 방법으로 제조되는 것을 특징으로 하는 항체 또는 이의 면역학적 활성 단편.  4) An antibody or immunologically active fragment thereof, which is prepared by a manufacturing method comprising the step of obtaining and purifying the serum of a laboratory animal of step 3).
【청구항 7】 [Claim 7]
제 1항에 있어서, 상기 RACK1 단백질 2기번째 리신의 탈메틸화는 The method of claim 1, wherein the demethylation of the second stage lysine RACK1 protein is
LSDKLys ine-spec i f i c deniethylase 1)에 의한 것을 특징으로 하는 항체 또는 이의 면역학적 활성 단편. LSDKLys ine-spec i f i c deniethylase 1) or an immunologically active fragment thereof.
【청구항 8】 [Claim 8]
제 1항에 있어서, 상기 RACK1 단백질 2기번째 리신의 메틸화는 HIF- l a (Hypoxi a-induc ible factor- 1 α )를 분해시기는 것을 특징으로 하는 항체 또는 이의 면역학적 활성 단편.  The antibody or immunologically active fragment thereof of claim 1, wherein methylation of the second lysine of the RACK1 protein degrades HIF-1 a (Hypoxi a-inducible factor-1 α).
【청구항 9】 [Claim 9]
제 1항의 항체 또는 이의 면역학적 활성 단편을 포함하는 시료 내 암 진단용 키트.  A kit for diagnosing cancer in a sample comprising the antibody of claim 1 or an immunologically active fragment thereof.
【청구항 10】 [Claim 10]
제 9항에 있어서, 상기 암은 흑색종, 유방암， 대장암, 폐암, 간암， 식도암， 구강암， 위암, 골암,. 췌장암 , 피부암, 두경부암， 자궁암, 난소암, 직장암, 결장암， 유방암. 자궁 육종, 나팔관 암종, 자궁내막 암종, 자궁경부 암종. 질 암종, 외음부 암종, 식도암. 후두암. 소장암, 갑상선암, 부갑상선암, 연조직의 육종, 요도암, 음경암, 전립선암, 만성 또는 급성 백혈병, 유년기의 고상 종양, 분화 림프종， 방광암. 신장암, 신장 세포 암종 신장 골반 암종, 저 U증추신경계 림프종, 척수축 종양, 뇌간 신경교종 및 뇌하수체 아데노마로 이루어진 군으로부터 선텍되는 어느 하나인 것을 특징으로 하는 암 진단용 키트. 10. The method of claim 9, wherein the cancer is melanoma, breast cancer, colon cancer, lung cancer, liver cancer, esophageal cancer, oral cancer, gastric cancer, bone cancer. Pancreatic cancer, skin cancer, head and neck cancer, uterine cancer, ovarian cancer, rectal cancer, colon cancer, breast cancer. Uterine sarcoma, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma. Vaginal carcinoma, vulvar carcinoma, esophageal cancer. Laryngeal cancer. Small bowel cancer, thyroid cancer, parathyroid cancer, sarcoma of soft tissue, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, solid tumors of childhood, differentiated lymphoma, bladder cancer. Kidney cancer, Kidney cell carcinoma Kidney pelvic carcinoma, Hypoproliferative nervous system lymphoma, Spinal contraction Cancer diagnostic kit, characterized in that any one selected from the group consisting of tumor, brain stem glioma and pituitary adenoma.
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Citations (1)

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