KR101071305B1 - NF-κB Inhibitor containing ARH1 protein or gene encoding the same - Google Patents

Abstract

The present invention relates to a novel use of the ARH1 protein or a gene encoding the same, and more particularly, an NF-κB inhibitor comprising an ARH1 protein or a gene encoding the same as an active ingredient, an angiogenesis-related disease containing the inhibitor, It relates to a composition for treating inflammatory diseases, autoimmune diseases or viral diseases.

ARH1, TRADD, NF-κB inhibitor, angiogenesis

Description

NF-κB inhibitor or ARH1 protein or gene encoding the same}

The present invention relates to a novel use of the ARH1 protein or a gene encoding the same, and more particularly, an NF-κB inhibitor comprising an ARH1 protein or a gene encoding the same as an active ingredient, an angiogenesis-related disease containing the inhibitor, It relates to a composition for treating inflammatory diseases, autoimmune diseases or viral diseases.

NF-κB (nuclear factor kappa B) is one of the most actively studied transcription factors since it was discovered in 1986 as a factor that binds to the immunoglobulin kappa light chain in B cells (Baeuerle and Baltimore, Cell, 87). : 13-20, 1996).

NF-κB is a transcription factor that regulates the expression of genes involved in the activation of immune and inflammatory responses and inhibition of apoptosis (Beg and Baltimore, Science, 274: 782-784, 1996), p52 / p100 (NF-κB2 ), c-Rel, RelB and p65 (RelA). These NF-κBs form the homopolymer or heteropolymer of the p50 subunit group (p50, p52) and the p65 subunit group (p65, c-Rel, RelB) in cells, and are most abundant and The active form of NF-κB is a heteropolymer of p50 / p65 (p50 / relA). They are present in the cytoplasm in an inactivated state by binding to their inhibitors, IκB proteins (IκBα, IκBβ, IκBγ, IκBε, Bcl3). However, phosphorylated and degraded IκB proteins by various external stimuli such as cytokines (TNF-α, IL-1, etc.), bacterial / virus infections (LPS, dsRNA, etc.), stress (ROI, UV, adriamycin, radiation, etc.). The free NF-κB is activated and migrates to the nucleus, which then binds to the NF-κB binding site of the target gene to induce the expression of the gene (Rothwarf et al., Nature, 395: 297-300, 1998; Yamaoka et. al., Cell, 93: 1231-1240, 1998).

Although the transcription factor NF-κB is important for normal physiological functions such as development and immune response, abnormal NF-κB activation is known to be closely related to the pathogenesis of various diseases. Therefore, it is degenerative and refractory by regulating abnormal NF-κB activation. It is possible to suppress the onset and progression of the disease, attracting attention as a target substance for drug development.

Tumor suppressor (TNF) can induce gene expression that can alter inflammatory response and immune function by activating the transcriptional regulators NF-κB and AP-1 in response to TNFR1. TNF forms trimers and induces multiplexing of receptors in the process of binding to TNFR1. TNFR1 binds to the death domain of a cytoplasmic protein called TRADD (TNFR-associated death domain) through a death domain in its cytoplasmic region. TRADD acts as an intermediate mediator that allows multiple signaling factors to bind to activated TNFR1. Proteins that can react with TNFR1 through TRADD include TRAF2, RIP, and FADD. TRAF2 (TNF-associated factor-2) and receptor-interacting protein (RIP) activate NF-κB and JNK / AP-1, while FADD induces cell suppression. TRAF2 activates NIK (NF-κB-inducing kinase), and NIK activates IKK (I-κB kinase complex). IKK phosphorylates I-κB (inhibitor of κB) to promote the degradation of I-κB, allowing NF-κB to move into the nucleus and induce transcription of several genes (Hsu, H. et al., Cell , 81 , 495-504, 1995; Van Antwerp, DJ . Et al., Science , 274 , 787-789, 1996; Hsu, H. et al., Cell , 84 , 299-308, 1996b; Locksley, RM. et al., Cell , 104 , 487-501, 2001). Therefore, it can be seen that the binding patterns between intracellular proteins are closely related to apoptosis, inhibition of degradation, and the presence or absence of intracellular nuclei migration. It can be expected to be able to regulate -κB activation.

On the other hand, ARH1 protein is a gene containing a GTP binding domain of about 26 kDa is known to exist in eukaryotic cells of various species, it is known that the expression is decreased in breast and ovarian cancer and can induce cell growth inhibition (Yu, Y. et al., Proc. Natl. Acad. Sci. USA, 96, 214-219, 1999; Luo, RZ. Et al., Oncogene, 22, 2897-2909, 2003).

We searched for genes that could inhibit NF-κB activity by regulating interprotein binding on the TNF signaling pathway, while ARH1 proteins interact with TRADD, which plays an essential role in TNF signaling. The present invention has been completed by inhibiting NF-κB activity by nucleating NF-κB by competitively blocking the binding of and TRAF2.

It is an object of the present invention to provide an NF-κB inhibitor comprising an ARH1 protein or a gene encoding the same as an active ingredient.

Still another object of the present invention is to provide a composition for treating a disease associated with abnormal activation of NF-κB containing the NF-κB inhibitor.

Still another object of the present invention is to provide a method of inhibiting NF-κB activity in a cell by using an ARH1 protein or a gene encoding the same.

Still another object of the present invention is to provide a method of treating a disease associated with abnormal activation of NF-κB using the NF-κB inhibitor.

In one embodiment, the present invention relates to an NF-κB inhibitor comprising an ARH1 protein or a gene encoding the same as an active ingredient.

As used herein, the term "NF-κB inhibitor" refers to an agent that reduces the expression or activity of NF-κB in a cell, and specifically, acts directly on NF-κB or on the signaling pathway of NF-κB. Reducing the expression level of NF-κB or its activity by indirectly acting on a higher regulator to reduce expression of NF-κB at the transcription level, increase degradation of expressed NF-κB, or interfere with its activity It means a formulation to make. When NF-κB is activated in cells, they move inside the nucleus to bind to the target site to induce the expression of other genes. Herein, NF-κB activity means such substantial gene expression. Inhibition of activity is a concept that includes inhibiting gene expression by inhibiting their movement into the nucleus.

The ARH1 protein is a gene containing a GTP binding domain of about 26 kDa and is known to exist in eukaryotic cells of various species. The ARH1 protein of the present invention or the gene encoding the same has a human NF-κB inhibitory activity. It is a concept that includes a variety of mammalian-derived ARH1 protein, genes encoding them or variants functionally equivalent thereto. As used herein, "functionally equivalent variant" refers to a wild type protein derived from a particular species or to a gene having a function of inhibiting NF-κB activity although there is a variation.

ARH1 proteins or genes encoding them that can be used in the present invention are animals including humans, such as monkeys, pigs, horses, cows, sheeps, dogs ), Cats, mice, rabbits, and the like, preferably ARH1 derived from human, more preferably ARH1 protein described in SEQ ID NO: 1 (National Institutes of Health Gene Bank ( NIH GenBank) (AAG35625) or gene encoding the ARH1 protein described in SEQ ID NO: 2 (Permission No. AF202543 of the National Institutes of Health Genetic Bank), but is not limited thereto.

ARH1 proteins of the present invention are also within the scope of the present invention as well as proteins having their native amino acid sequences, as well as amino acid sequence variants thereof. A variant of an ARH1 protein refers to a protein in which the ARH1 native amino acid sequence and one or more amino acid residues have a different sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof. Amino acid exchanges in proteins and peptides that do not alter the activity of the molecule as a whole are known in the art (H. Neurode, R. L. Hill, The Proteins, Academic Press, New York, 1979). The most commonly occurring exchanges are amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Phe, Ala / Exchange between Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, Asp / Gly. In some cases, it may be modified by phosphorylation, sulfation, acetylation, glycosylation, methylation, farnesylation, or the like.

The ARH1 protein or variant thereof can be extracted from nature or synthesized (Merrifleld, J. Amer. Chem. Soc .. 85: 2149-2156, 1963) or by genetic recombination methods based on DNA sequences (Sambrook). et al, Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, USA, 2nd edition, 1989).

In addition, the gene encoding the ARH1 protein in the present invention means a nucleic acid molecule encoding the ARH1 protein, these may be isolated from nature or artificially modified.

An ARH1 nucleic acid molecule that can be used as a gene encoding an ARH1 protein of the present invention is a functional equivalent of a nucleic acid molecule constituting the same, for example, some sequences of the ARH1 nucleic acid molecule are deleted, substituted or inserted. It is a concept that includes variants that have been modified by insertion, but that can function functionally the same as the ARH1 nucleic acid molecule.

In addition, nucleic acid molecules encoding the ARH1 protein of the present invention may include genomic DNA, cDNA and chemical synthesis DNA, genomic DNA and cDNA can be prepared according to methods known in the art.

Genomic DNA, for example, extracts genomic DNA from cells with the ARH1 gene and constructs a genomic library (vectors can be used, for example, plasmids, phages, cosmids, BACs, PACs, etc.) and viewed the library. Colony or plaque hybridization is performed using a probe constructed on the basis of DNA encoding the protein of the invention (eg, SEQ ID NO: 2), or DNA encoding the protein of the present invention (eg, SEQ ID NO: 2). It can also be prepared by preparing a primer specific for the polymerase chain reaction (Polymerase Chain Reaction, PCR) using the same.

For example, cDNA synthesizes cDNA based on mRNA extracted from a cell having an ARH1 gene, inserts the synthesized cDNA into a vector such as λZAP, prepares a cDNA library, and expands the cDNA library. Can be prepared by colony or plaque hybridization or by PCR.

Such separated genes preferably have high homology with the amino acid sequence of the ARH1 protein (SEQ ID NO: 1) at the amino acid level they encode, and high homology means at least 50% or more, more preferably, throughout the amino acid sequence. Indicates at least 70%, more preferably at least 90% sequence identity. Homology of amino acid sequences and nucleotide sequences is a program called BLASTN or BLASTX developed based on BLAST (Karlin, S and Altschul, SF. Proc. Natl. Acad. Sci. USA, 90, 5873-5877, 1993) (Altschul, SF. Et al., J. Mol. Biol., 215, 403-410, 1990) and specific methods are known on the following website (http: //www.ncbi.nlm. nih.gov.).

On the other hand, the gene encoding the ARH1 protein of the present invention may be provided included in the expression vector for intracellular delivery.

Genes encoding the ARH1 protein of the present invention can be introduced into cells using a variety of transformation techniques, such as complexes of DNA and DEAE-dextran, complexes of DNA and nuclear proteins, complexes of DNA and lipids. The ARH1 gene may be in a form contained within a carrier that allows for efficient introduction into a cell. The carrier is preferably a vector, and both viral and non-viral vectors can be used, for example plasmids, phages, cosmids, viral vectors and the like, which can be self-replicating or integrated into the host DNA. .

By "introduction" herein is meant the introduction of foreign DNA into cells by transformation or transduction. Transformation is a sugar such as calcium phosphate-DNA coprecipitation, DEAE-dextran-mediated transformation, polybrene-mediated transformation, electroshock, microinjection, liposome fusion, lipofectamine and protoplast fusion It can be carried out by various methods known in the art. Transduction refers to the transfer of genes into cells using viral or viral vector particles by means of infection.

In addition, the ARH1 gene may be combined with expression control sequences such as promoter / enhancer sequences and other sequences necessary for transcription, translation or processing in the expression vector. Regulatory sequences may include tissue-specific regulatory and / or inducible sequences as well as direct constitutive expression of nucleotides, and the design of expression vectors may include the host cells to be transformed, the desired level of expression. It may be determined by such factors as.

In addition, the ARH1 gene of the present invention is introduced into an appropriate eukaryotic cell in a cell culture system in a state of being included in a vector, or directly expressing ARH1 or transforming into prokaryotic cells to express ARH1 protein, and then purifying these proteins. Can be used. Such ARH1 proteins of the present invention may include, for example, purified proteins, water soluble proteins, or forms fused with protein or amino acid residues in a form bound to a carrier for delivery or administration to a target cell.

On the other hand, the ARH1 protein of the present invention exhibits NF-κB inhibitory activity. Such NF-κB inhibitory activity is preferably achieved by inhibiting the migration of NF-κB into the nucleus of the p50 or p65 subunit of NF-κB.

In addition, such inhibition of NF-κB activity is preferably achieved by the interaction of the ARH1 protein and TRADD (TNF receptor-associated death domain) protein. Such interaction may inactivate NF-κB or result in inhibition of migration of NF-κB into the nucleus.

Specifically, the ARH1 protein of the present invention may bind to the TRADD protein, preferably the N-domain of the TRADD protein. This competitively blocks the binding of TRADD and TRAF2 (TNF receptor-associated factor 2) to inhibit the NF-κB activation delivered by TRAF2.

TRADD protein is a protein that acts as an intermediate mediator to allow multiple signal transduction factors to bind to activated TNFR1. TRADD2 is a protein that can react with TNFR1 through TRADD protein, such as TRAF2, RIP, and FADD. In particular, TRAF2 (TNF-associated factor-2) and RIP (receptor-interacting protein) activate NF-κB and JNK / AP-1. TRAF2 and RIP activate NIF (NF-κB-inducing kinase), NIK activates I-κB kinase complex (IKK), and activated IKK phosphorylates I-κB (Inhibitor of κB) By promoting degradation, NF-κB can move into the nucleus to induce transcription of several genes.

The ARH1 protein of the present invention binds to these TRADD proteins and competitively blocks the binding of TRADD and TRAF2, thereby inhibiting NF-κB activation delivered by TRAF2, which in turn is either p50 or p65, a subunit of NF-κB. Inhibits the migration of the gene into the nucleus, thereby inhibiting the transcription of several genes.

In a specific embodiment of the present invention, the present inventors confirmed that ARH1 protein inhibits NF-κB activity, and this inhibitory mechanism in the cell inhibits the binding of TRAF2 and TRADD by blocking the binding of TRAF2 and TRADD. It was confirmed that this is achieved by inhibiting NF-κB transcriptional activity on the signaling pathway and inhibiting the transfer of the p65 / p50 gene known as a gene into the nucleus.

In addition, experiments have shown that ARH1 protein affects angiogenesis that can be induced by the activation of NF-κB. ARH1 protein can inhibit angiogenesis and expression of factor VEGF, which is known to promote angiogenesis. It was confirmed to inhibit.

In another aspect, the present invention relates to a composition for treating diseases associated with abnormal activation of NF-κB containing an NF-κB inhibitor comprising the ARH1 protein or a gene encoding the same as an active ingredient.

Since the ARH1 protein or the gene encoding the same of the present invention exhibits NF-κB inhibitory activity by interacting with TRADD protein in the cell and inhibiting the migration of subunit p50 or p65 into the nucleus, NF-κB It can be provided as a composition for the treatment and prevention of diseases associated with abnormal activation.

The disease to which the NF-κB inhibitor of the present invention can be applied is not particularly limited as long as the disease is caused by abnormal activation of NF-κB, for example, angiogenesis-related diseases, inflammatory diseases, autoimmune diseases or the like. Viral diseases, and more specific examples include asthma, allergic rhinitis, atopic dermatitis, gallbladder, conjunctivitis, psoriasis, ulcerative colitis, systemic inflammatory syndrome, sepsis, polymyositis, dermatitis, nodular polyarthritis, Mixed connective histosis, Sjogren's syndrome, gout, Alzheimer's dementia, Parkinson's disease, Amyotrophic lateral sclerosis, chronic arthrosis, type I diabetes, type II diabetes, diabetic retinopathy, multiple sclerosis, Crohn's disease, chronic thyroiditis (Hashimoto disease ), Ceriac disease, myasthenia gravis, vulgaris ulcer, systemic erythematodes, viral diseases (HIV, herpes, Sendai rotavirus, phosphorus Rouenza virus, rabies virus, bullous stomatitis virus, rhinovirus, hepatitis A virus, hepatitis B virus, rubella virus, etc.), bacterial disease, radiation disorders, arteriosclerosis, hemangiomas, angiofibromas, reperfusion disorders and heart Hypertrophy, etc.

As used herein, "prevention" means any action that inhibits the formation or delays the onset of the disease by administration of the composition, and "treatment" means any action that improves or advantageously changes the condition of the disease by administration of the composition. It means.

The composition for treating diseases related to NF-κB abnormal activation containing an NH-κB inhibitor comprising an ARH1 protein of the present invention or a gene encoding the same as an active ingredient may further include a pharmaceutically acceptable carrier. Can be formulated together. As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not irritate an organism and does not inhibit the biological activity and properties of the administered compound. Examples of the pharmaceutical carrier which is acceptable for the composition to be formulated into a liquid solution include sterilized and sterile water suitable for the living body such as saline, sterilized water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, One or more of these components may be mixed and used. If necessary, other conventional additives such as an antioxidant, a buffer, and a bacteriostatic agent may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.

The composition for treating diseases associated with abnormal activation of NF-κB of the present invention can be applied to any formulation including the same as an active ingredient, and can be prepared in oral or parenteral formulations. Pharmaceutical formulations of the invention may be oral, rectal, nasal, topical (including the cheek and sublingual), subcutaneous, vaginal or parenteral (intramuscular, subcutaneous). And forms suitable for administration by inhalation or insufflation.

Oral dosage forms containing the composition of the present invention as an active ingredient include, for example, tablets, troches, lozenges, water-soluble or oily suspensions, preparation powders or granules, emulsions, hard or soft capsules, syrups or elixirs. can do. For formulation into tablets and capsules, lactose, saccharose, sorbitol, mannitol, starch, amylopectin, binders such as cellulose or gelatin, excipients such as dicalcium phosphate, disintegrants such as corn starch or sweet potato starch, stearic acid masne It may include a lubricating oil such as calcium, calcium stearate, sodium stearyl fumarate or polyethylene glycol wax, and in the case of a capsule, it may further contain a liquid carrier such as fatty oil in addition to the above-mentioned materials.

Formulations for parenteral administration comprising the composition of the present invention as an active ingredient, for injection, such as subcutaneous injection, intravenous injection or intramuscular injection, a suppository injection method or aerosol for spraying by inhalation through the respiratory system It can be formulated as. To formulate injectable formulations, the compositions of the present invention may be mixed in water with stabilizers or buffers to prepare solutions or suspensions, which may be formulated for unit administration of ampoules or vials. For infusion into suppositories, it may be formulated as a rectal composition such as suppositories or body enema including conventional suppository bases such as cocoa butter or other glycerides. When formulated for spraying such as aerosols, a propellant or the like may be combined with the additives to disperse the dispersed dispersion or wet powder.

In another aspect, the present invention relates to a method of treating a disease associated with NF-κB abnormal activation using said NF-κB inhibitor.

In the present invention, the treatment of the disease may include administering a pharmaceutical composition for treating a disease associated with abnormal activation of NF-κB containing an NF-κB inhibitor. As used herein, the term "administration" means introducing a pharmaceutical composition of the present invention to a patient in any suitable manner, and is carried by viral or nonviral techniques of the ARH1 gene or transplantation of cells expressing the ARH1 gene. It includes.

The route of administration of the composition of the present invention may be administered via various routes orally or parenterally as long as it can reach the target tissue, and specifically, oral, rectal, topical, intravenous, intraperitoneal, intramuscular, intraarterial, It may be administered in a conventional manner via transdermal, nasal, inhaled, intraocular or intradermal routes. Preferably it is administered topically to the tissue showing the disease lesion.

The treatment method of the present invention includes administering a composition of the present invention in a pharmaceutically effective amount. It will be apparent to those skilled in the art that a suitable total daily dose may be determined by the practitioner within the correct medical judgment. The specific therapeutically effective amount for a particular patient may be based on the specific composition, including the type and severity of the reaction to be achieved, whether or not other agents are used in some cases, the age, weight, general health, sex and diet of the patient, time of administration, It is desirable to apply differently depending on the route of administration and the rate of release of the composition, the duration of treatment, and the various factors and similar factors well known in the medical arts, including drugs used with or concurrent with the specific composition. Therefore, the effective amount of a pharmaceutical composition suitable for the purpose of the present invention is preferably determined in consideration of the above matters.

In addition, the treatment method of the present invention is applicable to any animal in which a disease caused by abnormal activation of NF-κB can occur, and the animal is not only human and primate but also domestic animals such as cattle, pigs, sheep, horses, dogs and cats. It includes.

In another aspect, the present invention relates to a method of inhibiting NF-κB activity in a cell by using an ARH1 protein or a gene encoding the same.

Treatment of the ARH1 protein of the present invention directly to the cells, or the gene encoding the ARH1 protein to the cells to express these proteins, it is possible to inhibit NF-κB activity by inhibiting the migration of NF-κB into the nucleus. . Specifically, the ARH1 protein interacts with the TNF receptor-associated death domain (TRADD) protein to competitively block the binding of TRADD and TNF receptor-associated factor 2 (TRAF2) into the nucleus of the p50 or p65 subunit of NF-κB. Because it can inhibit the migration of, it can effectively inhibit abnormal NF-κB activity in the cell.

At this time, as a method of processing the ARH1 gene of the present invention into a cell, a viral or non-viral transport technique capable of introducing a nucleic acid molecule constituting the gene into the cell can be used. Virus delivery mechanisms include, but are not limited to, lentivirus, retrovirus, adenovirus, herpes virus, avidox virus, and the like. Nonviral delivery mechanisms may include lipid mediated transfection, liposomes, immunoliposomes, lipofectin, cationic surface amphiphiles and combinations thereof. As a result, eukaryotic cells in which the ARH1 gene is introduced and express the ARH1 protein can be used as cell therapy for diseases associated with abnormal activation of NF-κB.

Since the ARH1 protein according to the present invention can inhibit NF-κB activity in cells by interacting with the TRADD protein and blocking the migration of NF-κB into the nucleus, it can act as an inhibitor of NF-κB, and the NF of the present invention -κB inhibitors have been shown to substantially inhibit angiogenesis, a type of lesion associated with abnormal activation of NF-κB, at the cellular and tissue levels.

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Example  1. Cell culture

Human cervical cancer-derived HeLa cells, ovarian cancer cell lines 2774, SKOV-3, OVCA-3 and breast cancer cell lines MCF-7 (American Type Culture Collection, USA) are heat inactivated 10% fetal bovine serum and penicillin / streptomycin Incubated at 37 of 5% CO ₂ in DMEM and RPMI medium (Life Technologies, USA) supplemented with (100 unit / ml). HUVEC cell lines (human umbilical vein endothelial cells, Clonetics, San Diego, USA) were incubated at 37 of 5% CO ₂ in EGM medium (Clonetics, San Diego, USA) containing 2% FBS (Fetal Bovine Serum).

Example  2: ARH1 , TRADD , TRAF2  Expression Vector Preparation

A vector containing all genes other than the human ARH1 gene was prepared. PGEX4T-1-ARH1 (FIG. 2C), pGEX4T-1-TRAF2 (FIG. 2C), and pET28-TRADD (N) (FIG. 2C) as prokaryotic expression vectors, and pcDNA3.1 / ARH1 ( 1B, 3C, 3D, 4A), pcDNA4 / HisMax-ARH1 (FIG. 2B), pEGFPN3-ARH1 (FIG. 2D, 3C), pEGFPC1-TRADD (N) (FIGS. 2B and 2D) and pcDNA4 / HisMax-TRADD (FIG. 2b) was produced by cloning the respective expression vectors in the following manner.

In summary, ARH1 gene fragments were obtained by PCR and then Eco RI was expressed in pcDNA3.1 (Invitrogen, USA), pcDNA4 / HisMax-ARH1 (Invtrogen), pGEX4T-1 (Amersham Biosciences, Sweden) and pEGFPN3 (Clonetech, USA) expression vectors. And the Xho I positions, and the Bgl II and Eco RI positions were inserted into the pEGFPC1 and pEGFPN3 vectors. The gene encoding TRAF2 was inserted into the Bam HI and Sal I sites in the expression vector of pGEX4T-1, and the recombinant protein was isolated and purified. Segments of genes encoding TRADD were constructed by inserting into the Eco RI and Xho I positions in pET28 (Novagen, USA) and pcDNA4 / HisMax (Invitrogen) expression vectors, respectively. Sequencing was performed by Applied Biosystems, USA.

When the vectors are prokaryotic cells, the E. coli BL21DE3 transformed in each of the E. coli BL21DE3 cells in a 37 ° C. LB culture containing 100 μg / ml ampicillin and 75 μg / ml kanamycin, has a 595 nm absorbance of 0.5˜. Incubate to 0.6 and induce the expression of recombinant protein for 3-4 hours at 37 ° C. with 0.5 mM IPTG (isopropyl-bD-thiogalactopyranoside) at 37 ° C., respectively, using lysis buffer (50 mM Tris-HCl (pH8.0). ), 100 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1 mM PMSF, 0.5 mM DTT) and triturated by ultrasound. Denatured proteins were purified and purified using GST and Ni ⁺ columns, respectively.

Example 3: Verification of functional inhibition of transcriptional activity of NF-κB by ARH1 protein

Luciferase reporter construct pNFB- of NF-κB purchased from Stratagen after growth in HEK 293 normal cell line culture vessel (24 well, Costar Co.) Luc 250 ng, Renililla luciferase vector pRL-TK (Promega) 25 ng and the concentration of ARH1 0.1 and 0.5 ㎍, respectively, or control was transformed using the electroshock method without the presence of ARH1. After 24 hours the cells were incubated in medium lacking antibiotics temporarily for at least 2 hours and then stimulated by addition of 10 ng / ml human TNF-α. Cells were harvested and treated with cells using lysis buffer according to the dual-luciferase assay manual (Promega), and luciferase activity was measured using a luminometer. As a result, it was confirmed that NF-κB transcriptional activity of the cells transformed with the ARH1 gene was inhibited (FIGS. 1A and 1B).

On the other hand, in order to observe intracellular migration and nuclear localization of NF-κB by ARH1, SKOV-3 ovarian cancer cell line was transfected with an expression vector containing ARH1 protein and then intracellular protein expression level was examined. In summary, lysis buffer [50 mmol / L Tris-HCl (pH 8.0), 150 mmol / L NaCl, 1, transfected SKOV-3 cells transfected with full length ARH1 expression vector or control vector not containing ARH1 gene. % NP40, 0.1% SDS, 10 mmol / L sodium deoxylate]. 20 ug of total cell eluate was isolated from SDS-10% polyacrylamide gel and delivered to nylon membrane, incubated with anti-p65 antibody (Santa Cruz, USA) and ECL Chemiluminescent Kit (Amersham, UK) And visualized by chemiluminescence. HSP 90 (Santa Cruz, USA) protein was used as a cytoplasmic marker for the purpose of quantifying the amount of total protein, and Western blot was performed using Histone H1 (Santa Cruz, USA) antibody as a marker in the nucleus.

As a result, cells expressing ARH1 protein showed higher p65 protein expression level in the cytoplasm and lower p65 protein expression level in the nucleus than the control group (FIG. 1C). This is because overexpression of ARH1 prevents the migration of NF-κB from the cytoplasm into the nucleus, ARH1 promotes proteolytic degradation of NF-κB (p65) in the cytoplasm and at the same time inactivates its NF-κB (p65) protein. It has a new function to block the movement into the nucleus.

Example 4: ARH1  Protein and TRADD  Protein interactions

To understand the NF-κB inhibition mechanism of the ARH1 protein at the molecular level, a yeast two-hybrid assay developed by Gyuris et al. Was performed as previously described (Gyuris et al., Cell, 75: 791-803, 1993; Park et al., Cancer Res., 65: 749-757, 2005). Proteins that interact with ARH1 protein were screened. In summary, the ARH1 fragment was inserted into the Eco RI and Xho I positions in the pGilda vector (Clontech) containing the yeast, E. coli common vector LexA regulatory gene. The construct was then introduced into EGY 48 (Clontech), an experimental yeast strain, to obtain colonies expressed from the culture medium lacking histidine, a primary amino acid selective marker, and then used to cDNA library derived from HeLa cells ( Clontech) was used to select colonies that bind secondarily. Thereafter, to verify the binding protein through various identification methods (the ability of cells to grow in leucine-deficient medium containing 2% galactose, whether blue colonies were formed in synthetic medium containing 2% galactose and 2% raffinose). After the bound genes are selected, genes obtained by sequencing are determined using the BLAST program provided by NCBI. As a result, it was found that ARH1 cross-links with TRADD (FIGS. 2A and 2B), and DNA fragments encoding TRADD for reconfirmation were obtained by PCR and then transferred to the Eco RI and Xho I positions of pJG4-5 (Clontech). It was cloned, transformed into yeast strain EGY 48 and confirmed through various test methods. Direct coupling between TRADD and ARH1 protein was confirmed by co-immunoprecipitation of endogenous TRADD and externally injected GFP-tagged ARH1 (GFP-ARH1) protein. The results confirmed that ARH1 directly binds to TRADD (FIG. 2C). In addition, the intracellular location of the nucleus, ARH1 and TRADD in the cell was confirmed by fluorescence microscopy (FIG. 2D).

These results show the importance of the TRADD N-domain in the interaction of ARH1 with TRADD, indicating that N-domain is an essential and sufficient condition for interaction with ARH1. In addition, it suggests a new function involved in transcriptional regulation through protein-protein interaction rather than N-domain as a motif of the conventional TRAF2 binding domain.

In addition, the synthesis of Examples 3 and 4 shows that when ARH1 is overexpressed, cross-linking with TRAF2 is blocked (FIGS. 2A and 2B), and p65 / p50 gene known as NF-κB gene is inhibited into the nucleus, thereby inhibiting p65. As the / p50 protein accumulates in the cytoplasm, it can be seen that NF-κB activity is inhibited (FIG. 1C). The results indicate that ARH1 competitively blocks TRADD-TRAF2 interactions intracellularly, inhibiting transcriptional regulation mechanisms and inhibiting NF-κB activity by impeding the transfer of p65 / p50 gene, known as NF-κB gene, into the nucleus. You can see that it has new features.

Example 5 Validation of Angiogenesis and Metastasis Inhibitory Activity of ARH1 in Human Ovarian Cancer Cells

Investigations have been reported to inhibit angiogenesis through inhibition of NF-κB activity. An example of many associations with angiogenesis and NF-κB activity in endothelial cells was revealed by integrin-linked kinase (ILE) (Lee, SP. Et al., Circulation, 114 , 150-159, 2006), ILK is an intracellular protein bound to the intracellular ends of integrins and plays an important role in cell structure, survival and proliferation. When the tissue is in ischemia, the cells in the tissue respond to overcome this problem. According to this paper, vascular endothelial cells under hypoxia enhance the amount of intracellular ILK and its kinase function. This enhanced ILK has been shown to regulate nuclear transfer of transcription factors HIF-1 and NF-κB through Akt, Erk, and IB under hypoxia. In this nucleus, both HIF-1 and NF-κB regulate the expression of intercellular adhesion molecule-1 (IMC-1) and stromal cell-derived factor-1 (SDF-1). It has been shown to play an important role in regression to ischemic tissue. Genetic enhancement or inhibition of ILK in the future suggests that ultimately an important way to control angiogenesis. In addition, recently reported that overexpression of angiopoietin-1 (Ang-1), which plays an important role in cancer angiogenesis, mediates the activity of the transcription factor NF-κB (Mitola, S. et al., Blood, 112 , 1154-1157, 2008), the newly discovered cancer and angiogenic protein angiosidin require phosphorylation when NF-κB migrates into the nucleus, where IB, p50, and p65 A gene that causes phosphorylation has been reported (Gaurnier-Hausser, A. et al., Cancer Res., 68 , 5905-5914, 2008).

The present inventors investigated whether ARH1 protein could inhibit angiogenesis. Angiogenesis occurs only when all the various processes including endothelial cell growth, migration and proliferation are completed. In other words, the angiogenic ability is achieved by the proliferation, migration and penetration of endothelial cells, and tube formation. First, in order to investigate the inhibitory activity of angiogenic ability of ARH1 protein in vitro, ARH1 protein was expressed in endothelial cells and its inhibitory ability was examined. The specific experimental method is as follows.

First, in order to accurately perform the experiment on endothelial cells, the expression of ARH1 gene was overexpressed, and the level of ARH1 mRNA was determined by RT-PCR. At the same time, it was confirmed that the protein level was normally expressed by Western blot. In addition, siARH1 (siRNA that inhibits the expression of ARH1 gene) was prepared as a control thereof, and the effect of inhibiting the expression of ARH1 mRNA and protein was confirmed by the same method (FIG. 3A). In order to investigate whether ARH1 protein affects the degree of proliferation of endothelial cells, DNA synthesis was performed as follows by the [ ³ H ] cymidine binding assay. Endothelial cells (4.5 x 10 ³ cells / well) were placed in gelatin-fixed yang containers (96 wells, Nunc) and incubated at 37 ° C. for one day, followed by cell growth medium [(Medium 199/10% FBS / 10 mM HEPES (pH 7.4)], pretreated with these endothelial cells and ARH1 protein, a control without ARH1 protein, and siRNA peptides that prevent ARH1 expression, followed by addition of VEGF (10 ng / ml). Then treated with endothelial cells for 24 hours with [ ³ H ] cymidine (0.5 μCi / well, 76 Ci / mmol, AP Biotech), washed with 0.1% albumin-containing phosphate buffer and 20 at room temperature with 0.4 N NaOH. After lysing the cells, the cells were neutralized with 2N HCl, and the amount of radioactivity used for DNA synthesis was measured using a liguid scintillation counter, which confirmed that the ARH1 protein inhibited the proliferation of endothelial cells (FIG. 3B).

In addition, cell migration experiments were performed to determine whether they are involved in endothelial cell migration and infiltration. Specifically, cell migration experiments were performed using a transwell plate (pore size 8 um, Costar, USA). The bottom surface of the transwell was reacted overnight at 4 ° C. with each ARH1 solution (10 ug / ml) and then blocked for 1 hour 30 with VEGF (25 ng / ml) in M199 containing 1% FBS. . After HUVEC cells were suspended in the culture medium at a concentration of 3 × 10 ⁵ cells / ml, 0.1 ml of cell suspension was pre-cultured at 37 ° C. for 30 minutes, respectively, by adding ARH1 protein and siARH1 peptide. Next, the cells were allowed to move at 37 ° C. for 6-8 hours. Cell migration was terminated by removing the cells from the top of the filter with a cotton swab and the filter was fixed with 8% glutaraldehyde and stained with crystal violet. Cell migration was observed by light microscopy, and cell numbers were measured at eight randomly selected sites under HPF (microscopic high power fields, x 200).

As a result, it was confirmed that endothelial cell migration and infiltration in the Transwell system was promoted by coating of VEGF, and the effect was inhibited by ARH1 protein (FIGS. 3C, 3D).

On the other hand, as a result of testing whether the ARH1 protein can inhibit the expression level of VEGF, which is a factor that induces angiogenesis in the cells, when the ARH1 protein is treated in HUVEC cells and ovarian cancer cells, the expression of both VEGF is inhibited. It could be seen (Fig. 3e). These results indicate that ARH1 protein can prevent the inhibition of angiogenesis by reducing the expression of VEGF.

Example 6: Validation of endothelial tube formation inhibitory activity of human ovarian cancer cells of ARH1

In order to determine whether the ARH1 protein inhibits angiogenesis, we analyzed the effect on endothelial tube formation. Specifically, Matrigel (matrigel, Chemicon, USA) was polymerized by adding 100 ul to each well of 96 well plate. After HUVEC cells were suspended in culture medium at a concentration of 3 × 10 ⁵ cells / ml, 0.1 ml of cell suspension was added to each well coated with Matrigel. After adding the protein and anti-ARH1 and incubating at 37 ° C. for 16-18 hours, the cells were photographed and the tubes were counted to obtain an average value. As a result, endothelial tube formation was selectively inhibited in Matrigel by proteins and antibodies against ARH1, but tube formation, which was inhibited by ARH1, was recovered when siRNA was used as a control (FIG. 4A). . Taken together, the results suggest that ARH1 protein may act as a potent inhibitor of angiogenesis. This possibility was confirmed using an ex vivo system before investigating in an in vivo mouse model. For this purpose, it was confirmed that angiogenesis was rapidly inhibited even in the results of the vessel sprouting assay (FIG. 4B).

Example 7: In vivo angiogenesis inhibition by ARH1 protein

To investigate ARH1 inhibitory ability in vivo, a tumor mouse model was prepared by injecting 2774 ovarian cancer cell lines subcutaneously in mice. When the tumor size reached 70-100 mm ³ , ARH1 protein was injected directly into the tumor three times at three day intervals. As a result, in the case of the control group (mouse treated with PBS), the tumor size increased to an average of 1500 mm ³ , whereas the ARH1 protein expression did not increase the tumor size (FIG. 5). In addition, as a result of examining the angiogenesis of the tumor section by the immunostaining method for CD31, the formation of a large number of blood vessels in the control group, whereas the formation of the blood vessels in the tumor was very low when the ARH1 protein was treated (FIG. 5).

These results indicate that expression of ARH1 causes cancer cell killing by inhibiting angiogenesis. The above results confirm that NF-κB is inhibited through ARH1 expression, thereby inhibiting angiogenesis that may be caused by abnormal activation of NF-κB at the tissue level.

Since the ARH1 protein according to the present invention can effectively inhibit NF-κB activity in a cell, the NF-κB inhibitor including such an ARH1 protein or a gene encoding the same may be used for various diseases caused by NF-κB abnormal activity. For example, it can be effectively used for the development of therapeutic agents for angiogenic diseases, inflammatory diseases, autoimmune diseases and viral diseases.

Figure 1a is a graph of the expression of ARH1 intracellular protein according to the concentration reduces the activity of the NF-κB-reporter gene.

Figure 1b is a graph that inhibits transcriptional activity mediated by NF-κB expression of ARH1 intracellular protein according to the concentration.

1c is a diagram showing a phenomenon in which p65 protein known as NF-κB gene does not move into the nucleus and accumulates in the cytoplasm with or without ARH1 protein expression.

FIG. 2A is a photograph (left) and a graph (right) showing the activity of the TRADD gene obtained by screening using an ovarian cDNA library directly through a two-hybrid analysis.

Figure 2b is a photograph of the reconfirmation of the in vitro interaction using the cell line in the Western blot using the ARH1 antibody and TRADD antibody after constructing the gene using each different expression vector system.

Figure 2c is a photograph showing the intracellular competitive binding capacity that can occur simultaneously in vitro after purification of each protein using different expression vector systems.

Figure 2d is a photograph of the intracellular location of the nucleus (DAPI), ARH1 and TRADD in the human cell line by fluorescence microscope.

Figure 3a is a photograph of observing the mRNA level of the gene expressing the ARH1 gene and RNA inhibiting the expression through RNA interference using a GFP vector containing RT-PCR and fluorescent material fluorescence microscope (siARH1 Expression inhibition upon treatment).

3B is a graph showing inhibition of proliferation of HUVEC cells using the protein for ARH1 (recovered upon siARH1 treatment).

3C and 3D are photographs (top) and graphs (bottom) (recovery upon siARH1 treatment) showing inhibition of migration (C) and infiltration (D) of HUVEC cells using proteins for ARH1.

3E is a photograph showing a phenomenon in which endothelial growth hormone, one of angiogenesis inducers, is inhibited by expression of ARH1 protein.

FIG. 4A is a photograph (top) and graph (bottom) (recovery upon siARH1 treatment) showing inhibition of tube formation of HUVEC cells using the protein for ARH1.

Figure 4b is a photograph (top) and graph (bottom) (recovered again upon treatment with siARH1) showing inhibition of angiogenesis in ex vivo using mouse muscle using the protein for ARH1.

Figure 5 is a photograph and graph that ARH1 protein effectively inhibits angiogenesis of human ovarian cancer cells in vivo.

<110> National Cancer Center <120> NF-kB Inhibitor containing ARH1 protein or gene encoding the same <130> PA080390 <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 229 <212> PRT <213> Homo sapiens <400> 1 Met Gly Asn Ala Ser Phe Gly Ser Lys Glu Gln Lys Leu Leu Lys Arg   1 5 10 15 Leu Arg Leu Leu Pro Ala Leu Leu Ile Leu Arg Ala Phe Lys Pro His              20 25 30 Arg Lys Ile Arg Asp Tyr Arg Val Val Val Val Gly Thr Ala Gly Val          35 40 45 Gly Lys Ser Thr Leu Leu His Lys Trp Ala Ser Gly Asn Phe Arg His      50 55 60 Glu Tyr Leu Pro Thr Ile Glu Asn Thr Tyr Cys Gln Leu Leu Gly Cys  65 70 75 80 Ser His Gly Val Leu Ser Leu His Ile Thr Asp Ser Lys Ser Gly Asp                  85 90 95 Gly Asn Arg Ala Leu Gln Arg His Val Ile Ala Arg Gly His Ala Phe             100 105 110 Val Leu Val Tyr Ser Val Thr Lys Lys Glu Thr Leu Glu Glu Leu Lys         115 120 125 Ala Phe Tyr Glu Leu Ile Cys Lys Ile Lys Gly Asn Asn Leu His Lys     130 135 140 Phe Pro Ile Val Leu Val Gly Asn Lys Ser Asp Asp Thr His Arg Glu 145 150 155 160 Val Ala Leu Asn Asp Gly Ala Thr Cys Ala Met Glu Trp Asn Cys Ala                 165 170 175 Phe Met Glu Ile Ser Ala Lys Thr Asp Val Asn Val Gln Glu Leu Phe             180 185 190 His Met Leu Leu Asn Tyr Lys Lys Lys Pro Thr Thr Gly Leu Gln Glu         195 200 205 Pro Glu Lys Lys Ser Gln Met Pro Asn Thr Thr Glu Lys Leu Leu Asp     210 215 220 Lys Cys Ile Ile Met 225 <210> 2 <211> 690 <212> DNA <213> Homo sapiens <220> <221> gene (222) (1) .. (690) <223> nucleotide sequence of ARH1 gene <400> 2 atgggtaacg ccagctttgg ctccaaggaa cagaagctgc tgaagcggtt gcggcttctg 60 cccgccctgc ttatcctccg cgccttcaag ccccacagga agatcagaga ttaccgcgtc 120 gtggtagtcg gcaccgctgg tgtggggaaa agtacgctgc tgcacaagtg ggcgagcggc 180 aacttccgtc atgagtacct gccgaccatt gaaaatacct actgccagtt gctgggctgc 240 agccacggtg tgctttccct gcacatcacc gacagcaaga gtggcgacgg caaccgcgct 300 ctgcagcgcc acgttatagc ccggggccac gccttcgtcc tggtctactc agtcaccaag 360 aaggaaaccc tggaagagct gaaggccttc tatgagctga tctgcaagat caaaggtaac 420 aacctgcata agttccccat cgtgctggtg ggcaataaaa gtgatgacac ccaccgggag 480 gtggccctga atgatggtgc cacctgtgcg atggagtgga attgcgcctt catggagatt 540 tcagccaaga ccgatgtgaa tgtgcaggag ctgttccaca tgctgctgaa ttacaagaaa 600 aagcccacca ccggcctcca ggagcccgag aagaaatccc agatgcccaa caccactgag 660 aagctgcttg acaagtgcat aatcatgtga 690  

Claims (12)

Asthma, Allergic Rhinitis, Atopic Dermatitis, Galline, Conjunctivitis, Psoriasis, Ulcerative Colitis, comprising as an active ingredient an ARH1 protein having the amino acid sequence set forth in SEQ ID NO: 1 as an NF-κB inhibitor or a gene encoding the same Systemic Inflammatory Syndrome, Sepsis, Multiple Myositis, Dermatitis, Nodular Polyarthritis, Mixed Connective Tissue Syndrome, Scheren's Syndrome, Gout, Alzheimer's Dementia, Parkinson's Disease, Amyotrophic Lateral Sclerosis, Chronic Arthritis, Type I Diabetes, Type II Diabetes mellitus, diabetic retinopathy, multiple sclerosis, Crohn's disease, chronic thyroiditis, ceria disease, myasthenia gravis, vulgaris ulcer, systemic erythematodes, viral diseases, bacterial diseases, radiation disorders, arteriosclerosis, hemangiomas, angiofibromas A composition for treating a disease associated with abnormal activation of NF-κB selected from the group consisting of reperfusion disorders and cardiac hypertrophy. The composition of claim 1, wherein said ARH1 protein inhibits the migration of NF-κB into the nucleus of the p50 or p65 subunit. The composition of claim 1, wherein the NF-κB inhibition is achieved by binding an ARH1 protein and a TNF receptor-associated death domain (TRADD) protein. The composition of claim 3, wherein the ARH1 protein binds to TRADD to competitively block binding of TRADD and TNF receptor-associated factor 2 (TRAF2). The composition of claim 1, wherein the ARH1 protein or a gene encoding the same is derived from a human. The composition of claim 1, wherein the ARH1 gene is included in an expression vector for intracellular delivery. delete delete delete The composition of claim 1 further comprising a pharmaceutically acceptable carrier. A method of inhibiting NF-κB activity in isolated cells in vitro using an ARH1 protein or a gene encoding the same. 12. The p50 or p65 subtype of NF-κB according to claim 11, wherein the ARH1 protein interacts with a TNF receptor-associated death domain (TRADD) protein to competitively block the binding of TRADD and TNF receptor-associated factor 2 (TRAF2). A method of inhibiting NF-κB activity by inhibiting migration of the unit into the nucleus.

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