KR20120136909A - Method for inducing apoptosis and the composition - Google Patents

Abstract

PURPOSE: A method and a pharmaceutical composition for inducing cell apoptosis are provided to overexpress proteins containing SET domains and to lower cell viability. CONSTITUTION: A method for inducing cell apoptosis comprises a step of overexpressing proteins containing SET domains of amino acid sequence number 1. The proteins are setd3 of sequence number 2. A pharmaceutical composition for inducing cell apoptosis contains the proteins or a gene encoding the same as an active ingredient. The pharmaceutical composition is used for preventing and treating cancer, diabetes, artheriosclerosis, inflammatory bowel diseases, autoimmune diseases, transplantation rejection, post-traumatic immune response, infectious diseases, lymphatic tumor, multiple sclerosis, SLE, pachydermia, ischemia, stroke, uveitis or diabetic retinopathy.

Description

Method for inducing apoptosis and its composition {Method for inducing apoptosis and the Composition}

The present invention is to provide a novel method for inducing apoptosis and a pharmaceutical composition for inducing apoptosis using the same.

Apoptosis is apoptosis caused by the cells themselves under physiological conditions, and is clearly distinguished from necrosis caused by environmental degradation. This apoptosis is characterized by chromosomal aggregation of the cell nucleus, fragmentation of the cell nucleus, loss of cell surface microvillus, aggregation of the cytoplasm, and the like. Cells on which apoptosis occurs are atrophy, and because cell contents are not released to the outside and are quickly incorporated into macrophages or surrounding cells, inflammation does not occur and does not affect surrounding cells. Therefore, many attempts have been made to treat diseases by inducing apoptosis in cells whose presence is harmful to a host organism (for example, cancer cells).

To date, as a means or factor for inducing apoptosis, for example, glucocorticoid treatment, cytotoxicity caused by cytotoxin-T cells, atrophy of hormone-dependent tissues, irradiation, natural killer (NK) cells, killing Cytokines such as killer cells, tumor necrosis factor (TNF) and lymphotoxin (LT) have been reported (Wyllie, AH, Nature, 284: 555-556, 1986; Wyllie, AH et al., Int. Rev. Cytol., 69: 251, 1980; Duvall, E. and Wyllie, AH, Immunology Today, 7: 115-119, 1986; Sellins, KS et al., J. Immunol., 139: 3199, 1987; Yamada, T. et al., Int. J. Radiat. Biol., 53:65, 1988; Schmid, DS et al., Proc. Natl. Acad. Sci., USA, 83: 1881 -1885, 1986; John, C. et al., J. Immunol., 129 (4): 1782-1787, 1982; Howell, DM et al., J. Immunol., 140: 689-692, 1988; Gillian , B. et al., Eur J. Immunol., 17: 689-693, 1987). It is also known to induce apoptosis in antibodies (eg, anti-CD3 antibodies, anti-APO-I antibodies, etc.) (Trauth, BC et al., Science, 245: 301-305, 1989; Smith; , CA et al., Nature, 337: 181-184, 1989; Tadakuma, T. et al., Eur. J, Immunol., 20: 779, 1990). In addition, cycloheximide, a protein inhibitor, is used for acute leukemia cells, Actinomycin D, a RNA inhibitor, for small intestine crypt cells, and both HL-60 cells. Has also been reported to induce apoptosis, respectively (Martin, SJ et al., J. Immunol., 145: 1859-1867, 1990).

However, most of the prior art regarding the induction of such apoptosis are not suitable for clinical use to treat diseases and symptoms characterized by undesirable cell proliferation, such as autoimmune diseases, lymphocyte proliferative diseases, inflammation and cancer. Has not been proven. Many of these treatments are ineffective or toxic, cause side effects, lead to the development of drug resistance or immunosensitivity, and debilitate the subject.

Thus, there is a need for new compositions and methods that can inhibit cell proliferation, induce cell cycle arrest and apoptosis in cells, particularly cancer cells, and treat these diseases and symptoms.

The present invention seeks to provide novel compositions and methods of inducing undesired cell proliferation and inducing cell cycle arrest and apoptosis.

The present invention is to provide a method of inducing apoptosis by overexpressing a protein containing a SET domain represented by the amino acid sequence of SEQ ID NO: 1.

The protein containing the SET domain may be setd3 represented by the amino acid sequence of SEQ ID NO: 2.

The present invention is to provide a pharmaceutical composition for inducing apoptosis, comprising a protein containing a SET domain represented by the amino acid sequence of SEQ ID NO: 1 or a gene encoding the same as an active ingredient.

The protein containing the SET domain may be setd3 represented by the amino acid sequence of SEQ ID NO: 2.

The composition induces cell death by cancer, diabetes, arthritis, inflammatory bowel disease, atherosclerosis, autoimmune disease, transplant rejection, post-traumatic immune response, infectious disease, lymphoid tumor, lymphoma, multiple sclerosis, systemic lupus erythematosus, Scleroderma, ischemia, stroke, uveitis or diabetic retinopathy can be prevented and treated.

According to the present invention, by overexpressing a protein containing a SET domain in the cell, caspase-dependent apoptosis is increased, thereby suppressing aberrant proliferation of cells, ie, autoimmune diseases, lymphocyte proliferative diseases, inflammation, and cancer cells. It can be usefully used for the prevention and treatment of various diseases.

Figure 1 shows that zebrafish setd3 has a conserved SET domain. FIG. 1A shows a schematic of the full-length setd3 and SET domains, FIG. 1B shows the secondary structural elements for the amino acid sequence, the red bars show α-helices in setd3 and the blue arrows show β-sheets , SET domains are indicated by gray boxes. Figure 1C is a three-dimensional model of setd3 (77-490 aa), the secondary structure is visualized in a different color, and the dotted red line shows the gap in setd3. Figures 1D and 1E are three-dimensional modeling of the SET domain. The ssDNA binding motif of setd3 is shown. The structure of the SET domain is generated from 195 aa to 314 aa (FIG. 1D), and this DNA binding motif is represented from 269 aa to 314 aa (FIG. 1E). Arrows indicate flip-loop chains in DNA binding motifs, pink ribbons represent α-helices, and β-sheets are yellow and green ribbons (FIGS. 1D and 1E).
2 shows zebrafish setd3 as a novel HMTase. 2A shows that core histones are used as substrates in HMTase assays. Figure 2B is HMTase analysis with GST-setd3 and core histones, α-H3K4-me2, α-H3K9-me2, α-H3K27-me2, α-H3K36-me1, α-H3K36-me2, α-H3K36-me3 , And Western blotting for the α-H4K20-me2 antibody. 2C shows that cDNA was extracted from HEK293 cells transfected with pcDNA6 or pcDNA6-setd3 and transfected. PCR products were amplified using primers specific for setd3 (upper panel). HEK293 cells were transfected with pcDNA6 or myc-tagged setd3. Total protein was extracted from transfected cells using lysis buffer. Western blot analysis consisted of α-myc or α-β-actin antibodies (lower panel). Figure 2D shows that pcDNA6-setd3-transfected cell extracts were used for Western blot analysis, and changes in histone methylation levels were observed for α-H3K4-me2, α-H3K9-me2, α-H3K27-me2, and α-H3K36-me1. , α-H3K36-me2, α-H3K36-me3, and α-H4K20-me2 antibodies were detected.
Figure 3 shows that zebrafish setd3 induces transcriptional activity. 3A shows a schematic of the domain structure of the setd3 wild type and deletion mutants. 3B and 3C show HEK293 cells with pcDNA6-setd3 (50, 100, or 150 ng), pcDNA6-setd3-N (50, 100, or 150 ng), pcDNA6-setd3-C (50, 100, or 150 ng) , Transiently transfected with MH100-SV40-Luc vector (100 ng) (FIG. 3B), and MH100-TK-Luc vector (100 ng) (FIG. 3C).
4 shows that zebrafish setd3 inhibits cell viability. The vesicle viability was confirmed by MTT assay when setd3 was overexpressed in HEK293 (FIG. 4A), HeLa (FIG. 4B), and NIH-3T3 (FIG. 4C) cells. These cells were transfected with pcDNA6-setd3. Formazan was detected at each time point (24, 36, or 48 h) using an ELISA reader (570 nm), using the pcDNA6 empty vector as a control. It was. FIG. 4D shows total protein extracted from pcDNA6-setd3-transfected HeLa cells, which was applied to Western blot using specific antibodies. β-Actin was used as loading control.

The present invention relates to a method of inducing apoptosis by overexpressing a protein containing a SET domain, and a pharmaceutical composition for inducing apoptosis, comprising a protein containing a SET domain or a gene encoding the same as an active ingredient.

Hereinafter, the present invention will be described in detail.

Many of the prior arts related to the induction of conventional cell death are ineffective or toxic in clinical practice, cause side effects, develop drug resistance or immune sensitization, and debilitate the subject, such as autoimmune diseases, Since it was not suitable for treating diseases and symptoms characterized by undesirable cell proliferation, such as lymphocyte proliferative diseases, inflammation and cancer, etc., the present invention inhibits undesirable cell proliferation, cell cycle arrest and apoptosis. To provide novel compositions and methods for treating these diseases and symptoms.

The present invention provides a method of inducing apoptosis by overexpressing a protein containing a SET domain.

SET domain in the present invention is characterized by being represented by the amino acid sequence of SEQ ID NO: 1, but not limited to, at least 80% homology, preferably at least 90%, more preferably at least 99% homology with the amino acid sequence A protein having may be included in the protein of the present invention.

In the present invention, the kind of the protein containing the SET domain is not particularly limited, but may be setd3.

As shown in FIG. 1, the setd3 may have a SET domain at the N-terminus and a Rubis-subs-bind domain at the C-terminus, and the setd3 protein is represented by the amino acid sequence of SEQ ID NO. Not limited to this, a protein having at least 85% homology with the amino acid sequence, preferably at least 90%, more preferably at least 99%, may be included in the protein of the present invention.

Proteins containing the SET domain of the invention, such as setd3, methylate histone H3K36 as a novel HMTase, activate transcription through this H3K36 HMTase activity, and are associated with apoptosis such as caspase-9 and caspase-3 Regulate genes to lower cell viability.

Proteins containing SET domains used in the present invention can be obtained by chemical synthesis or by using genetic recombination techniques. When prepared by chemical synthesis, it can be obtained using a polypeptide synthesis method well known in the art. When using a recombinant technique, a nucleic acid encoding a protein containing a SET domain is inserted into an appropriate expression vector, the vector is transformed into a host cell, and the host cell is cultured so that the protein containing the SET domain is expressed. It can be obtained by recovering a protein containing a SET domain from the cell. Proteins are expressed in selected host cells and then subjected to conventional biochemical separation techniques, such as treatment with protein precipitants (salting), centrifugation, sonication, ultrafiltration, dialysis, molecular sieve chromatography for isolation and purification. (Gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, and the like can be used. In general, these are used in combination to separate proteins of high purity.

The present invention provides a pharmaceutical composition for inducing apoptosis, comprising a protein containing a SET domain or a gene encoding the same as an active ingredient.

Proteins containing SET domains used in the present invention can be obtained by chemical synthesis or by using genetic recombination techniques. Genomic DNA encoding each of the above proteins, mRNA that is a transcription product of genomic DNA, cDNA synthesized as a template, and the like can be used, but cDNA is particularly preferred. This cDNA can be obtained according to a known method. For example, cDNA using known methods (Mol. Cell Biol., 2: 161-170, 1982; J. Gene, 25: 263-269, 1983; Gene, 150: 243-250, 1994) A cDNA library can be synthesized and the target cDNA can be separated using the produced probe DNA. The obtained cDNA is a conventionally performed gene amplification method such as PCR (Polymerase Chain Reaction), NASBN (Nucleic acid sequence based amplification), TMA (Transcription-mediated amplification) and SDA (Strand displacement amplification) Can be amplified by

SET domain in the present invention is characterized by being represented by the amino acid sequence of SEQ ID NO: 1, but not limited to, at least 80% homology, preferably at least 90%, more preferably at least 99% homology with the amino acid sequence A protein having may be included in the protein of the present invention.

In the present invention, the kind of the protein containing the SET domain is not particularly limited, but may be preferably setd3.

As shown in FIG. 1, the setd3 may have a SET domain at the N-terminus and a Rubis-subs-bind domain at the C-terminus, and the setd3 protein is represented by the amino acid sequence of SEQ ID NO. Not limited to this, a protein having at least 85% homology with the amino acid sequence, preferably at least 90%, more preferably at least 99%, may be included in the protein of the present invention.

Proteins containing the SET domain of the invention, such as setd3, methylate histone H3K36 as a novel HMTase, activate transcription through this H3K36 HMTase activity, and are associated with apoptosis such as caspase-9 and caspase-3 By regulating genes to lower cell viability, the compositions in the present invention are diseases and symptoms caused by abnormal cell growth or abnormal growth regulation, such as cancer, diabetes, arthritis, inflammatory bowel disease, atherosclerosis, autoimmunity Disease, transplant rejection, post-traumatic immune response, infectious disease, lymphoid tumor, lymphoma, multiple sclerosis, systemic lupus erythematosus, scleroderma, ischemia, stroke, uveitis or diabetic retinopathy, preferably cancer It can be used for prevention or treatment.

The cancer is not particularly limited, but the cancer is melanoma, non-small cell lung cancer, small cell lung cancer, lung cancer, liver cancer, retinoblastoma, astrocytoma, glioblastoma, gingival cancer, tongue cancer, leukemia, neuroblastoma, head cancer, neck cancer, breast cancer , Pancreatic cancer, prostate cancer, kidney cancer, bone cancer, testicular cancer, ovarian cancer, mesothelioma, cervical cancer, gastrointestinal cancer, lymphoma, brain cancer, colon cancer, sarcoma or bladder cancer, the composition of the present invention kills cancer cells and cancer cells Induces apoptosis and reduces the growth rate of cancer cells, reduces the incidence or number of metastases, reduces tumor size, inhibits tumor growth, reduces blood supply to tumors or cancer cells, or Promoting an immune response or preventing or inhibiting the progression of cancer can extend the lifespan of patients with cancer.

The pharmaceutical composition comprising the protein containing the SET domain of the present invention or a gene encoding the same as an active ingredient, the active ingredient in an amount of 0.1 to 90% by weight based on the total weight of the composition.

The pharmaceutical composition comprising a protein containing the SET domain of the present invention or a gene encoding the same as an active ingredient may further include appropriate carriers, excipients and diluents commonly used in the preparation of pharmaceutical compositions. The pharmaceutical compositions of the present invention may be prepared in any formulation commonly prepared in the art (eg, Remington's Pharmaceutical Science, latest edition; Mack Publishing Company, Easton PA).

In order to make the protein containing the said SET domain into the form which can be introduce | transduced into a cell, the following means is used, for example.

A protein containing a SET domain can be formulated into a form that can be introduced into a cell by, for example, mixing the protein molecule with a pharmacologically acceptable carrier solution without changing its structure or function.

Such agents can be introduced into the cells by, for example, microinjection methods for in vitro cells. Alternatively, a lipid transduction method (BioPORTER (Gene Therapy Systems, USA), Chariot (Active Motif, USA), etc.) may be employed.

As another example, a fusion protein may be prepared in which a cell-membrane-penetrating peptide is linked to the N-terminal side of the protein to make the protein into a cell. With this cell-membrane-penetrating peptide, proteins pass through the cell membrane and are taken into the cell. As the cell membrane-penetrating peptide, PTD (protein transduction domain) of HIV-T or TAT or PTD of Drosophila homeobox protein antennapedia (Drosophila homeobox protein antennapedia) can be used. For example, in the case of HIV-1 · TAT, the amino acid sequence and the base sequence of the cDNA thereof are known (see Science, 285: 1569-1572, 1999; Genbank Accession NO.U39362 M96155), which corresponds to the PTD. The DNA fragment encoding the region (amino acid sequence 47 to 57 of HIV / TAT) was linked with the cDNA to obtain a fusion DNA fragment, which was expressed in a host cell such as Escherichia coli and the PTD peptide at the N-terminal side. The fusion protein can be prepared by linkage. In addition, since PTNA of antinapedia is also known (for example, GenBank Accession No. AE001573), a fusion protein linked to PTD can be easily produced. In addition, a fusion protein may be prepared by linking a polypeptide and a PTD peptide to a cell membrane-penetrating peptide through a divalent crosslinking agent (for example, EDC or β-alanine or the like).

The gene encoding the protein containing the SET domain of the present invention can be administered directly by injection. Optionally, a vector comprising a gene encoding a protein containing a SET domain of the invention can be administered.

For in vivo cells (i.e., cells in animal subjects), for the purpose of facilitating intracellular growth or enhancing the directivity of a target cell, for example, the cell may be obtained by a viral or nonviral gene introduction vector or the like. It can be introduced inside. A drug in this form can be introduced into a living body for gene therapy (for example, Japanese Patent Laid-Open No. 2003-24092, Japanese Patent Laid-Open No. 2003-501445, etc.). In the above, as a viral vector, for example, an adenovirus vector, a retrovirus vector, a lentivirus vector, an ano-associated virus vector, a vaccinia virus vector , Human immunodeficiency virus (HIV) vectors, herpes virus vectors, and the like. Examples of the non-viral vector include high molecular compounds such as liposomes, artificial lipid vehicles, hollow nanoparticles, and dendrimers. In this case, commercially available reagents for introduction (for example, lipofectin, lipofectamine, DMRIE-C (manufactured by Invitrogen), metafectene, DOTAP (manufactured by BioTex), and Tfx reagent (Promega) can be used.

Apoptosis inducing agents having a protein containing a SET domain or a nucleic acid encoding the same as an active ingredient, and a pharmaceutical composition for treating apoptosis-related diseases may be administered in a suitable formulation together with carriers and diluents known in the art, Depending on the method used, it may have a formulation for parenteral administration, for example, intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, suppository and the like.

For the preparation of pharmaceutical compositions, additives for the preparation of solids or liquids may be used. The additive for preparation may be either organic or inorganic. In other words, when preparing oral solid preparations, excipients, binders, disintegrating agents, lubricants, coloring agents and the like are added to the main medicine, and then tablets, coating tablets, granules, powders, Preparations in the form of capsules and the like can be prepared. Examples of excipients to be used include lactose, sucrose, white sugar, glucose, corn starch, starch, talc, sorbet, crystalline cellulose, dextrin, kaolin, calcium carbonate, silicon dioxide and the like. Examples of the binder include polyvinyl alcohol, polyvinyl ether, ethyl cellulose, methyl cellulose, gum arabic, tragacanth, gelatin, shellac, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, calcium citrate, Dextrin, pectin and the like. Examples of the lubricant include magnesium stearate, talc, polyethylene glycol, silica, and hydrogenated vegetable oil. As a coloring agent, if it is normally permitted to add to a pharmaceutical, all can be used. These tablets and granules can be appropriately coated according to sugar, gelatin coating, and other needs. Moreover, preservatives, antioxidants, etc. can be added as needed.

Inert diluents commonly used in the preparation of liquid preparations for oral administration, for example emulsions, syrups, suspensions, solutions, may be used, for example water or vegetable oils. In addition to the inert diluent, this preparation may be formulated with an adjuvant such as a wetting agent, suspending aid, sweetener, fragrance, colorant or preservative. After the liquid formulation is prepared, it may be filled into a capsule of absorbable material such as gelatin. As a solvent or suspending agent used for preparation of a parenteral administration agent, for example, an injection or a suppository, water, propylene glycol, polyethylene glycol, benzyl alcohol, ethyl oleate, lecithin are mentioned, for example. As a base used for manufacture of a suppository, a cacao butter, an emulsified cacao butter, a laurin butter, and a witezol are mentioned, for example. The preparation method of the formulation is not particularly limited, and any method commonly used in the art can be used.

In the case of injectable preparations, for example, diluents such as water, ethyl alcohol, macrogol, propylene glycol, citric acid, acetic acid, phosphoric acid, lactic acid, sodium lactate, sulfuric acid and sodium hydroxide; PH adjusters and buffers such as sodium citrate, sodium acetate and sodium phosphate; Stabilizers, such as sodium pyrosulfite, ethylenediamine tetraacetic acid, thioglycolic acid, and thio lactic acid, etc. can be used. In this case, in order to prepare an isotonic solution, a sufficient amount of salt, glucose, mannitol, or glycerin may be blended in the formulation, or a conventional dissolution aid, analgesic agent, or local anesthetic may be used.

The dosage of the pharmaceutical of the present invention is not particularly limited, but may be administered once to several times in an amount of 1 ng to 10 mg per kilogram as a weight of the substance which is an active ingredient per adult. It is preferable to appropriately increase or decrease this dosage depending on the age, condition and symptoms of the patient. The daily dose may be administered once a day or divided into two to three times a day at appropriate intervals, or may be administered intermittently at intervals of several days. However, since the dosage of the pharmaceutical composition of the present invention is determined in view of various related factors such as the route of administration, the age, sex, weight of the patient, the severity of the patient, the dosage limits the scope of the present invention in any aspect. It should not be understood to be.

In addition, the pharmaceutical compositions of the present invention can be used in combination with conventional anti-inflammatory compositions, chemotherapy, surgery, radiation, hormone therapy or gene therapy.

Hereinafter, the present invention will be described in more detail by way of examples. However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

< Example >

Example  One: setd3  Protein structure analysis

1-1. Plasmid construct ( Plasmid constructs )

The sequences of all constructs were confirmed by DNA sequencing. A full-length open reading frame of zebrafish setd3 cDNA was purchased from Openbiosystems, which was used in PCR amplification as a template.

pGEX4T1-setd3 was made by PCR amplification using a special primer set for zebrafish setd3 (GeneBank accession no. BC055261). PCR primer sequences are shown in parentheses below. ( Sal I site-introduced primer, forward primer is 5'-GTCGACGGATGGGCAAAAAGAGCAGAGTG-3 ', Not I-site introduced primer, 5'-GCGGCGGCCCCTATTTGCCAGCATCTTTTGG-3' as reverse primer).

For eukaryotic expression, PCR products were subcloned with HA / myc / His-tagged pcDNA6 (Invitrogen, Carlsbad, Calif.). The coding sequence of setd3 was amplified with specific primers. PCR primer sequences are shown in parentheses below ( Hind III-site introduced primer, forward primer is 5'-GCGAAGCTTATGGGCAAAAAGAGCAGAGTG-3 'and Not I-site introduced primer, reverse primer is 5'- GCGCTCGAGTTTGCCAGCATCTTTTGGTTC-3 ').

1-2. Secondary structure modelling  ( Modeling of secondary structures )

The molecular model of the setd3 protein was created using an automated similarity modeling server SWISS-Model ( http://swissmodel.expasy.org/ ). The resulting setd3 protein structure, including full length (77-490 aa), SET domain, and ssDNA binding motif, was visualized with SWISS-Pdb Viewer, which chains in different colors, α-helix, and β-sheet Indicates.

1-3. setd3  Protein structural analysis and results

EnsMart, a data mining tool on the Sanger web site, lists proteins containing SET domains. Among these proteins, we chose setd3, which contains the SET domain. Setd3 is expected to be present on the zebrafish 14th chromosome.

As shown in FIG. 1A below, the data from blasting setd3 for the Conserved Domain Database presented the setd3 structure. SET and Rubis-subs-bind domains are located at the N-terminus (SET domain, 195-314 aa) and C-terminus (the Rubis-subs-bind domain, 343-475 aa) (FIG. 1A). Rubis-subs-bind domains allow these proteins to bind to substrates such as the N-terminal tails of histones H3 and H4.

setd3 protein consists of 17 α-helices and 8 β-sheets in secondary structure (FIG. 1B). In the secondary structure of setd3, the SET domain has three α-helices and three β-sheets (FIG. 1B, gray box).

Three-dimensional prediction of the setd3 protein was performed using the SWISS-Model server. The setd3 protein secondary structure was produced from full length 77 aa to 490 aa (FIG. 1C). In 3D protein modeling, setd3 has one concave gap in the center (FIG. 1C, red dotted line). The SET domain is in the gap and this region will contain the binding site for the substrate (FIG. 1D). The three-dimensional structure of the ssDNA-binding motif in setd3 consists of two antiparallel β-sheets (yellow ribbons) and α-helix (pink ribbon) (FIG. 1E).

Example  2: setd3 of HMTase  activation

2-1. Cell culture ( Cell culture )

Cells were grown in Dulbecco's Modified Eagle's medium (DMEM, Welgene, Daegu, Korea) containing 10% fetal bovine serum (FBS, Welgene) and 0.5% antibiotics (37%, 5% CO ₂ atmosphere). I was.

2-2. Protein Purification ( Protein purification )

For HMTase activity analysis, a full-length open reading frame of setd3 cDNA was inserted into the pGEX4T1 vector. Wild-type protein was expressed in E. coli BL21 (DE3) (Invitrogen) at 37 ° C. for 4 hours and purified by Glutathione-Sepharose 4B. The concentration of this wild type was determined by Coomassie Brilliant Blue R-250 staining of SDS-PAGE 12% gel. Fusion proteins bound to beads were used for HMTase activity assays in vitro.

2-3. HMTase  analysis( HMTase assay )

In vitro 50 mM Tris (pH 8.5), 20 mM KCl, 10 mM MgCl ₂ , 10 mM beta mercaptoethanol, 1.25 M sucrose, 100 nCi S -adenosyl- [methyl- ¹⁴ C] _-L -methionine ( S- adenosyl- [methyl- ¹⁴ C] _-L- methionine, Amersham Bioscience), 1-μg / μL core histone (Roche, Basel, Switzerland) from calf thymus, and 0.5-2.5 μg GST-setd3 or GST HMTase analysis was performed at 30 ° C. for 2 hours in a reaction buffer containing 50 μl volumes.

Proteins were filtered using p81 filter paper (filter paper, Millipore, Bedford, Mass., USA) and washed three times with 10% TCA and 95% ethanol pre-cold at room temperature for 5 minutes. The filter was air dried using 2 mL Gold Ultra (Perkin Elmer, Waltham, Mass., USA), and ¹⁴ C-SAM was measured using a scintillation counter (Perkin Elmer).

Total RNA samples were extracted from each of HEK293 cells transfected with pcDNA6-setd3 using Trizol reagent (Invitrogen). Total RNA (2 μg) was used to synthesize cDNA. Oligo-dT primers (Fermentas, Vilnius, Lithuania) were used for cDNA synthesis, and quantified cDNA was used to analyze setd3 mRNA expression patterns. This primer sequence forward is 5'-GACGATATCAAAAGAGAAGATTACTTCCCTG-3 'and the reverse is 5'-GGATTCCACAAACTCTGCATTTGACCG-3'.

2-4. Western Blot

HEK293 cells were transfected with pcDNA6 vector or pcDNA6-setd3. Transfected cells were lysed, loaded into 14% SDS-PAGE and transferred to nitrocellulose membranes. Anti-cleaved caspase-3 (Millipore), anti-procaspase-9 (Santa Cruz Biotechnology), anti-H3K4-me2, anti-H3K9-me2, anti-H3K27-me2, anti-H3K36-me1, The membrane was probed with anti-H3K36-me2, anti-H3K36-me3, and anti-H4K20-me2 (Millipore).

This blot was incubated with HRP - conjugated goat anti-rabbit antibodies (Santa Cruz Biotechnology) and detected using an ECL system (Santa Cruz Biotechnology).

2-5. setd3 of HMTase  Activity analysis and results

The SET domain is evolutionarily well conserved from humans to lower progressive organisms, suggesting the importance of HMTase activity in the SET domain. To characterize the HMTase activity of setd3, we performed HMTase analysis with GST-setd3 fusion protein purified in scintillation predictor in vitro.

Core histones and S-adenosyl [methyl- ¹⁴ C] _-L- methionine (SAM) were used as substrate and methyl donors, respectively. In vitro assays showed HMTase activity of setd3 in the core histone in a dose-dependent manner (FIG. 2A), but no HMTase activity was observed in GST protein (FIG. 2A).

Western blot analysis shows that GST-setd3 methylates H3K36-me1 / -me2 / -me3 (FIG. 2B). The present inventors then attempt to confirm the HMTase activity of setd3 in vivo. Setd3 was cloned into eukaryotic expression vectors. We transfected HEK293 cells with pcDNA6-setd3, isolated total RNA and amplified setd3 mRNA using specific primers (FIG. 2C, upper panel). PCDNA6-setd3 transiently transfected with eukaryotic cells was detected by Western blot using anti-myc antibody (FIG. 2C, lower panel).

To investigate if setd3 can methylate histones, HEK293 cells were transfected with pcDNA6-setd3 and anti-H3K4-me2, anti-H3K9-me2, anti-H3K27-me2, anti-H3K36-me1 / -me2 Western blot was performed using / -me3, and anti-H4K20-me2 antibodies. Compared with the other sites investigated, it was found that the histone H3K36-mono, di-, and -trimethylating activities of setd3 were increased. (FIG. 2D). Overall, these results indicate that setd3 specifically has HMTase activity against mono-, di-, trimethylation of H3K36 residues both in vivo and ex vivo.

Example  3: Setd3 Induction of transcriptional activity

3-1. temporary Transfection  Reporter analysis ( Transient transfection reporter assay )

Transfection analysis was performed using pcDNA6-setd3, MH100-SV40-Luc, and MH100-TK-luc as internal controls in the indicated sections. In 48-well plates, HEK293 cells were tested for each reporter plasmid (100) in the presence and absence of pcDNA6-setd3 (50, 100, or 150 ng) and pCMV-b-gal plasmid (50 ng) to standardize transfection efficiency. ng) and lipofectamine 2000 (Invitrogen).

Cells were collected 48 hours after transfection and lysed with cell culture lysis reagent (Promega, Madison, Wis.). Luciferase activity was measured using Glomax 20/20 luminometer (Promega). Luciferase activity was normalized to β-galactosidase.

3-2. Setd3 Transcriptional activity and its consequences

To determine whether H3K36 methylation of setd3 could contribute to the activity of general transcription, we performed transient transfection assays using the MH100-SV40-Luc and MH100-Tk-Luc reporter systems.

Transfection of pcDNA6-setd3 to HEK293 resulted in activation of luciferase activity in both reporter systems in a dose-dependent manner. This indicates that relative luciferase activity finally increased by 2.2 and 5 times the basal transcription in both systems (FIGS. 3B and C).

To investigate the role of the SET domain in the regulation of setd3 transcription, reporter analysis was performed with setd3 deletion mutants (FIG. 3A). Similar to the setd3 wild type, transcriptional activity was increased only in cells that were transfected with setd3-N mutants without SET domain intact (SET domain intact setd3-N mutant transfected cells). However, setd3-C lacking the SET domain did not activate transcription in any system (FIGS. 3B and C). These results indicate that setd3 activates transcription through H3K36 HMTase activity.

Example  4: Setd3 Apoptosis by

4-1. MTT  (3- (4,5- dimethylthiazol -2- yl ) -2,5- diphenyltetrazolium  bromide analysis

HEK293, HeLa, and NIH-3T3 cells were seeded in 48-well plates (2 × 10 ³ cells / well) and transiently transfected with pcDNA6-setd3. From 0 to 48 hours after transfection, MTT was added to the cells (20 μl, final concentration 0.5 mg / mL), which was incubated at 37 ° C. for 4 hours, aspiration of medium and DMSO (150 μl) added. . OD was determined with an ELISA reader (Biochrom, Cambridge, England) at 570 nm wavelength. The control group (blank) added with only DMSO was measured and its value was subtracted.

4-2. Setd3 Apoptosis and its consequences

Several HMTases play an important role in development and cell growth. For example, to investigate the effect of setd3 on cell growth, an MTT assay was performed and cell viability was monitored every 12 hours after transfection.

Cell lines from different human tissues (embryonic kidney and cervical cancer) and other species (mouse fibroblast) were used to see the general effect of setd3. The formation of a marker of MTT formazan exocytosis, a needle-like crystal, was significantly reduced in setd3 overexpressed cells as compared to in untreated cells 48 hours after transfection.

When the relative viability in the setd3 transfected cells was compared to the control cells, approximately 40% were gradually reduced in all of the cell lines used (FIGS. 4A, B and C).

These results demonstrate that setd3 inhibits cell proliferation. In order to determine whether zebrafish setd3 overexpressed cells have a change in the expression level of proteins associated with apoptosis, the inventors have determined by Western blot analysis the expression of important modulators in apoptosis such as caspase-9 and caspase-3. Was investigated. The level of inactive procaspase-9 was untreated but decreased in zebrafish setd3 transfected cells when compared to mock.

In contrast to procaspase-9 levels, cleaved caspase-3, an active form of caspase-3 (p17), was significantly increased in zebrafish setd3 overexpressing cells (FIG. 4D). These results indicate that zebrafish setd3 overexpression induces caspase-dependent apoptosis.

<110> CHUNG ANG University Industry Academic Cooperation <120> Method for inducing apoptosis and the Composition <130> DP-2011-0218 <160> 2 <170> Kopatentin 1.71 <210> 1 <211> 120 <212> PRT <213> Set domain # 1 aa in Zebrafish <400> 1 Arg His Leu Leu Ala Thr Gln Ala Ile Gln Asp Val Leu Ser Gln Tyr   1 5 10 15 Lys Asn Thr Ala Arg Gln Tyr Ala Tyr Phe Tyr Lys Val Ile His Thr              20 25 30 His Pro Asn Ala Ser Lys Leu Pro Leu Lys Asp Ala Phe Thr Phe Asp          35 40 45 Asp Tyr Arg Trp Ala Val Ser Ser Val Met Thr Arg Gln Asn Gln Ile      50 55 60 Pro Thr Ala Asp Gly Ser Arg Val Thr Leu Ala Leu Ile Pro Leu Trp  65 70 75 80 Asp Met Cys Asn His Thr Asn Gly Leu Ile Thr Thr Gly Tyr Asn Leu                  85 90 95 Glu Asp Asp Arg Cys Glu Cys Val Ala Leu Lys Asp Tyr Lys Glu Gly             100 105 110 Glu Gln Ile Tyr Ile Phe Tyr Gly         115 120 <210> 2 <211> 596 <212> PRT <213> Setd3 # 2 aa in Zebrafish <400> 2 Met Gly Lys Lys Ser Arg Val Lys Thr Gln Lys Ser Gly Thr Gly Ala   1 5 10 15 Thr Ala Ala Val Ser Pro Lys Glu Met Met Asn Leu Ile Ser Glu Leu              20 25 30 Leu Gln Lys Cys Ser Ser Ala Ala Pro Ser Pro Gly Lys Glu Trp Glu          35 40 45 Glu Tyr Val Gln Ile Arg Ala Leu Val Glu Lys Ile Arg Lys Lys Gln      50 55 60 Lys Gly Met Ser Val Ser Phe Glu Gly Ile Arg Glu Asp Phe Phe Ser  65 70 75 80 Glu Leu Met Ala Trp Ala Ala Glu Cys Arg Ala Ser Cys Asp Gly Phe                  85 90 95 Glu Ile Ser Asn Phe Ala Asp Glu Gly Tyr Gly Leu Lys Ala Thr Lys             100 105 110 Asp Ile Lys Ala Glu Glu Leu Phe Leu Trp Ile Pro Arg Lys Met Leu         115 120 125 Met Thr Val Glu Ser Ala Lys Asn Ser Val Leu Gly Pro Leu Tyr Ser     130 135 140 Gln Asp Arg Ile Leu Gln Ala Met Gly Asn Val Thr Leu Ala Leu His 145 150 155 160 Leu Leu Cys Glu Arg Ala Asn Pro Ser Ser Pro Trp Leu Pro Tyr Ile                 165 170 175 Lys Thr Leu Pro Ser Glu Tyr Asp Thr Pro Leu Tyr Phe Glu Glu Glu             180 185 190 Glu Val Arg His Leu Leu Ala Thr Gln Ala Ile Gln Asp Val Leu Ser         195 200 205 Gln Tyr Lys Asn Thr Ala Arg Gln Tyr Ala Tyr Phe Tyr Lys Val Ile     210 215 220 His Thr His Pro Asn Ala Ser Lys Leu Pro Leu Lys Asp Ala Phe Thr 225 230 235 240 Phe Asp Asp Tyr Arg Trp Ala Val Ser Ser Val Met Thr Arg Gln Asn                 245 250 255 Gln Ile Pro Thr Ala Asp Gly Ser Arg Val Thr Leu Ala Leu Ile Pro             260 265 270 Leu Trp Asp Met Cys Asn His Thr Asn Gly Leu Ile Thr Thr Gly Tyr         275 280 285 Asn Leu Glu Asp Asp Arg Cys Glu Cys Val Ala Leu Lys Asp Tyr Lys     290 295 300 Glu Gly Glu Gln Ile Tyr Ile Phe Tyr Gly Thr Arg Ser Asn Ala Glu 305 310 315 320 Phe Val Ile His Asn Gly Phe Phe Phe Glu Asp Asn Ala His Asp Arg                 325 330 335 Val Lys Ile Lys Leu Gly Val Ser Lys Gly Glu Arg Leu Tyr Ala Met             340 345 350 Lys Ala Glu Val Leu Ala Arg Ala Gly Ile Pro Ala Ser Ser Ile Phe         355 360 365 Ala Leu His Cys Ser Glu Pro Pro Ile Ser Ala Gln Leu Leu Ala Phe     370 375 380 Leu Arg Val Phe Cys Met Thr Glu Glu Glu Leu Arg Asp Tyr Leu Val 385 390 395 400 Gly Asp His Ala Ile Asn Lys Ile Phe Thr Leu Gly Asn Thr Glu Phe                 405 410 415 Pro Val Ser Trp Glu Asn Glu Ile Lys Leu Trp Thr Phe Leu Glu Thr             420 425 430 Arg Ala Ala Leu Leu Leu Lys Thr Tyr Lys Thr Ala Ser Glu Glu Asp         435 440 445 Arg Ser Met Leu Glu Lys Pro Asp Leu Ser Leu His Ser Arg Ile Ala     450 455 460 Ile Lys Leu Arg Leu Ala Glu Lys Glu Ile Leu Glu His Ala Val Ser 465 470 475 480 Cys Gly Arg Ala Lys Arg Leu His Phe Gln Lys Gln Leu Asp Glu Gly                 485 490 495 Ala Pro Leu Pro Leu Tyr Glu Glu Ser Asp Ile Ala Leu Leu Glu Asn             500 505 510 Ala Asp Ala Lys Leu Pro Ile Ile Leu Arg Lys Leu Glu Glu Glu Glu         515 520 525 Glu Glu His Val Glu Glu Met Asn Gln Leu Thr Pro Asp Ala Val Cys     530 535 540 Met Asn Ser Ser Lys Ile Pro Val Leu Asn Gly Gln Cys Lys Asp Leu 545 550 555 560 Asn Gly Thr Gln Glu Asp Pro Pro Gly Gly Gly Ala Val Val Lys Glu                 565 570 575 Ile Glu Lys His Asp Pro Ser Ala Lys Arg Thr Glu Gly Glu Pro Lys             580 585 590 Asp Ala Gly Lys         595

Claims (5)

A method of inducing apoptosis by overexpressing a protein containing a SET domain represented by the amino acid sequence of SEQ ID NO: 1. The method of claim 1, wherein the protein containing the SET domain is setd3 represented by the amino acid sequence of SEQ ID NO: 2. 6. A pharmaceutical composition for inducing apoptosis, comprising a protein containing a SET domain represented by the amino acid sequence of SEQ ID NO: 1, or a gene encoding the same as an active ingredient. 4. The pharmaceutical composition of claim 3, wherein the protein containing the SET domain is setd3 represented by the amino acid sequence of SEQ ID NO: 2. The method of claim 3, wherein the composition induces apoptosis, thereby causing cancer, diabetes, arthritis, inflammatory bowel disease, atherosclerosis, autoimmune disease, transplant rejection, post-traumatic immune response, infectious disease, lymphoid tumor, lymphoma, multiple sclerosis A pharmaceutical composition for preventing and treating systemic lupus erythematosus, scleroderma, ischemia, stroke, uveitis or diabetic retinopathy.

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