KR20200067047A - Composition comprising a peptide derived from DRAK1 protein - Google Patents

Abstract

The present invention relates to: a composition for treating or preventing tumor or inflammation which contains a DRAK1 protein or a fragment thereof, a polynucleotide encoding the DRAK1 protein or the fragment thereof, or a combination thereof; and uses thereof. The present invention can effectively kill cancer cells or suppress metastasis of cancer cells, and also can treat or prevent inflammation.

Description

Composition comprising a peptide derived from DRAK1 protein

The present invention relates to a composition for tumor treatment or anti-inflammatory comprising a peptide derived from the DRAK1 protein as an active ingredient.

DRAK1 (death-associated protein kinase-related apoptosis-inducing kinase 1) is a serine/threonine phosphorylation protein, also known as a serine/threonine kinase 17a protein encoded by the STK17A gene. DRAK1 belongs to DAPK (Death associated protein kinase) and is known to be involved in cell death, but the specific role in cell death of cancer cells is still controversial. Although the role of DRAK1 in cancer development has not been studied, it has been reported that in testicular cancer cells, treatment with cisplatin increases DRAK1 expression and regulates the expression of the tumor suppressing gene p53 gene.

TRAF6 (TNF receptor associated factor 6) protein is an E3 ligase, which is known to induce ubiquitination, which is involved in activation or inhibition of various proteins. In addition, activation of TRAF6 protein is activated by causing auto-ubiquitination due to external stimulation such as various inflammatory cytokines or LPS, or self-expression, and induces various intracellular signaling activation, leading to cancer occurrence and inflammatory reaction. It is known to cause.

One aspect provides a composition for the treatment or prevention of a tumor or inflammation, comprising a DRAK1 protein or fragment thereof, a polynucleotide encoding the DRAK1 protein or fragment thereof, or a combination thereof.

Another aspect provides a method of treating a tumor or inflammation of an individual in need thereof, comprising administering the composition to the individual.

Another aspect comprises the steps of contacting a test substance to a DRAK1 protein or fragment thereof; And detecting a dimer, ubiquitination, or a combination thereof of the TRAF6 protein or a fragment thereof, a method for screening a substance for treating or preventing a tumor or inflammation.

Another aspect is a DRAK1 protein or fragment thereof; TRAF6 protein or fragment thereof; And an antibody, an antigen-binding fragment, a polypeptide, or a combination thereof that specifically binds to the DRAK1 protein or a fragment thereof, and the TRAF6 protein or a fragment thereof, for screening a composition for the treatment or prevention of a tumor or inflammation. to provide.

Hereinafter, the present invention will be described in more detail.

All technical terms used in the present invention, unless defined otherwise, are used in a sense as commonly understood by those skilled in the art in the relevant field of the present invention. In addition, although a preferred method or sample is described herein, similar or equivalent ones are included in the scope of the present invention. In addition, the numerical values described in this specification are considered to include the meaning of “about” even if not specified. The contents of all publications described by reference herein are incorporated herein by reference in their entirety.

One aspect provides a composition for the treatment or prevention of a tumor or inflammation, comprising a DRAK1 protein or fragment thereof, a polynucleotide encoding the DRAK1 protein or fragment thereof, or a combination thereof.

The DRAK1 protein may be defined as GenBank ID: NP_004751.2 in humans, and the polynucleotide encoding the same may be defined as GenBank ID: NM_004760.2.

In one embodiment, the DRAK1 protein or a fragment thereof may include the amino acid sequence of SEQ ID NO: 1.

In one embodiment, the polynucleotide encoding the DRAK1 protein or fragment thereof may be one comprising the polynucleotide sequence of SEQ ID NO: 2.

In one embodiment, the fragment of the DRAK1 protein is the 1 to 193 amino acid sequence of SEQ ID NO: 1, the 62 to 193 amino acid sequence of SEQ ID NO: 1, the 1 to 120 amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 1 It may be a polypeptide comprising an amino acid sequence selected from the 113th to 120th amino acid sequence of, and the 110th to 124th amino acid sequence of SEQ ID NO: 1.

The composition may include a combination of fragments of the DRAK1 protein as defined above. The term'polypeptide' is used interchangeably with'protein'.

In one embodiment, the fragment of the DRAK1 protein may be a polypeptide comprising the amino acid sequence of SEQ ID NO: 3 or a fragment thereof.

Fragments of the DRAK1 protein according to one embodiment may be modified to further include one or more additional amino acids in each defined amino acid sequence. The one or more additional amino acids may be 1 to 1000, 1 to 500, 1 to 300, 1 to 100, 1 to 50, 1 to 30, or 1 to 20. The one or more additional amino acids facilitate the binding of the DRAK1 protein or fragment thereof to the outer wall of the cell or into the cell, for example, a cell membrane protein binding polypeptide, a signal protein leading to a target position in the cell, a folding protein , Cofactor proteins, and the like. The amino acid sequence that facilitates the introduction of the DRAK1 protein or fragment thereof into the cell is 1 to 30, 1 to 20, 1 to 15, 5 to 30, 5 to 20, 5 to 15, 10 to 10 30, 10 to 20, 10 to 15, or about 11 arginine. In addition, the one or more additional amino acids may further include a linker sequence so that the amino acid sequence for improving the targeting or functionality of the DRAK1 protein or fragment thereof does not interfere with the substantial function of the DRAK1 protein or fragment thereof. The linker sequence may be composed of 1 to 30, 1 to 20, 1 to 15, 1 to 10, 1 to 5, or about 3 glycines.

In one embodiment, the modified, polypeptide comprising the amino acid sequence of SEQ ID NO: 3 or a fragment thereof, may be a polypeptide comprising the amino acid sequence of SEQ ID NO: 4.

In one embodiment, the composition may be for inhibiting the activity of TRAF6 (TNF receptor associated factor 6).

The TRAF6 protein may be defined as GenBank ID: NP_665802.1, and the polynucleotide encoding the same may be defined as GenBank ID: NM_145803.2. The TRAF6 protein may include the amino acid sequence of SEQ ID NO: 5, and may be encoded by the polynucleotide sequence of SEQ ID NO: 6.

In one embodiment, the composition may be to inhibit the TRAF6 monomer to form a dimer.

In one embodiment, the DRAK1 protein or fragment thereof may be one that binds to the TRAF domain of TRAF6.

The TRAF domain may also be defined as MATH-TRAF6 ,TRAF DOAMIN, C-terminal MATH subdomain, and may include the amino acid sequence of SEQ ID NO:7. The TRAF domain may be encoded from a polynucleotide having the nucleic acid sequence of SEQ ID NO: 8.

In one embodiment, the composition may be to inhibit the metastasis of cancer cells. Treatment or prevention of the tumor may include controlling or inhibiting metastasis of cancer cells.

In the following examples, the DRAK1 protein binds to the TRAF domain, which is an important site for the autobiquitination activity of the TRAF6 protein, and inhibits the binding of the TRAF6 to the dimer between monomers, leading to destabilization of the TRAF6 protein, which soon leads to cancer development It has been demonstrated that it inhibits TRAF6-mediated signaling, which is important for metastasis and inflammatory responses. In addition, by using a composition containing a polypeptide containing a DRAK1 fragment according to an embodiment that similarly expresses the effect of the DRAK1 protein, it is possible to inhibit cancer development, metastasis, and inflammatory response by inhibiting TRAF6 mediated signaling. Proved.

The tumor can be used interchangeably with cancer. The tumor may be a concept including solid cancer, blood cancer, primary cancer, and metastatic cancer. The tumor may be breast cancer, colorectal cancer, lung cancer, sarcoma, melanoma, head and neck cancer, cervical cancer, uterine cancer, liver cancer, kidney cancer, pancreatic cancer, neuroblastoma, but is not limited thereto.

The composition may include a conventional pharmaceutically acceptable carrier, excipient or additive. The pharmaceutical composition of the present invention can be formulated according to a conventional method, and various oral dosage forms such as tablets, pills, powders, capsules, syrups, emulsions, microemulsions or parenteral such as intramuscular, intravenous or subcutaneous administration. It can be prepared in dosage form. When the composition according to one embodiment is prepared in the form of an oral dosage form, examples of additives or carriers used include cellulose, calcium silicate, corn starch, lactose, sucrose, dextrose, calcium phosphate, stearic acid, magnesium stearate, And calcium stearate, gelatin, talc, surfactant, suspending agent, emulsifying agent, and diluent. When the pharmaceutical composition of the present invention is prepared in the form of an injection, the additive or carrier includes water, saline, an aqueous glucose solution, a similar sugar solution, alcohol, glycol, ether (eg, polyethylene glycol 400), oil, fatty acid, and fatty acid ester , Glycerides, surfactants, suspending agents, emulsifiers, and the like. The composition may further include a pharmaceutically acceptable salt.

The polypeptide or polynucleotide may be composed of each defined amino acid sequence or nucleic acid sequence. The polypeptide or polynucleotide has a sequence identity of 60% or more, for example, 70% or more, 80% or more, 90% or more, 95% or more, 99% or more, or 100% of sequence identity with any one of the defined sequences. It may have an amino acid sequence or a nucleic acid sequence. The polypeptide or polynucleotide may be 1 or more amino acids, 2 or more amino acids, 3 or more amino acids, 4 or more amino acids, 5 or more amino acids, 6 or more amino acids, or 7 or more amino acid residues in any one sequence of each sequence Is a polypeptide having a different amino acid sequence, or one or more bases, two or more bases, three or more bases, four or more bases, five or more bases, six or more bases, or seven or more bases having a different sequence of polynucleotides Can be

Another aspect provides a method of treating a tumor or inflammation of an individual in need thereof, comprising administering the composition according to one embodiment to the individual.

The dosage of the composition is an amount effective for treatment or prevention of an individual or a patient, and may be administered orally or parenterally as desired, and when administered orally, 0.01 to 1000 mg per kg of body weight per day based on the active ingredient , More specifically, to be administered in an amount of 0.1 to 300 mg, when parenterally administered, to an amount of 0.01 to 100 mg per kg of body weight per day based on the active ingredient, and more specifically, to be administered in an amount of 0.1 to 50 mg 1 It can be administered in several times. The dosage for a particular individual or patient should be determined in light of a number of related factors such as the patient's weight, age, sex, health status, diet, time of administration, method of administration, and severity of the disease, and can be appropriately adjusted by a specialist. It should be understood that the above dosage is not intended to limit the scope of the invention in any aspect. Doctors or veterinarians having ordinary skill in the relevant art can readily determine and prescribe an effective amount of the desired composition. For example, a doctor or veterinarian starts at a level lower than that required to achieve the desired therapeutic effect for the dose of the active ingredient of the present invention used in the composition, and gradually doses until the desired effect is achieved. Can be increased.

The term “treating” or “treatment” refers to inhibiting the disease, eg, inhibiting the disease, condition or disorder in an individual who experiences or indicates the pathology or manifestation of the disease, condition or disorder, ie, pathology and/or Or preventing further development of symptoms, or improving the disease, e.g., improving the disease, condition or disorder in an individual experiencing or indicating the pathology or manifestation of the disease, condition or disorder, i.e. pathology and/or Or reversing the signs, such as reducing the severity of the disease.

The term “preventing” or “preventing” herein refers to preventing a disease, for example a disease in an individual who may be prone to a disease, condition or disorder, but has not yet experienced or exhibited the pathology or signs of the disease, Refers to preventing a condition or disorder.

The term "individual" or "patient" as used herein refers to any animal, including mammals, eg, mice, rats, other rodents, rabbits, dogs, cats, pigs, cows, sheep, horses or primates, and humans .

The composition according to one embodiment may be used in combination with one or more other therapeutic agents, for example anti-cancer agents, anti-inflammatory agents or other pharmaceutically active substances.

It is understood that any of the terms or elements mentioned in the composition as mentioned in the description of the claimed treatment method is as mentioned in the description of the composition.

Another aspect comprises the steps of contacting a test substance to a DRAK1 protein or fragment thereof; And detecting a dimer, ubiquitination, or a combination thereof of the TRAF6 protein or a fragment thereof, a method for screening a substance for treating or preventing a tumor or inflammation.

The DRAK1 protein or fragment thereof may be an artificially synthesized polypeptide, or a recombinant microorganism or a polypeptide purified from cells. The DRAK1 protein or a fragment thereof may be in the form of a single isolated polypeptide, or in a mixed form with other proteins derived from cells.

The term “contact” can mean that the test agent binds in a manner that can directly or indirectly affect the DRAK1 protein or fragment thereof. That is, the step of contacting the test substance may be contacted by adding a test substance to a sample containing the extracellularly purified DRAK1 protein or fragment thereof, and the specimen containing the cell expressing the DRAK1 protein or fragment thereof is tested. It may be contact by adding a substance. The sample may be a cell culture. The cells may mean cells that make up the tissue of an individual, or may mean cells isolated from the individual. Further, the cells may be normal cells, or tumor cells or cancer cells. When the DRAK1 protein or fragment thereof and/or TRAF6 protein or fragment thereof is present in a cell, it may be endogenous, exogenous or a combination thereof. When the DRAK1 protein or fragment thereof and/or TRAF6 protein or fragment thereof is in a form present in a cell, polynucleotides encoding each of them may be injected to express in the cell. To express the protein or fragment thereof in a cell, a method known in the art can be used, and it can be stably or temporarily expressed in the cell.

In one embodiment, the DRAK1 protein or fragment thereof may be in contact with the test agent in combination with a TRAF6 protein or fragment thereof. The presence form of the TRAF6 protein or fragment thereof is as described for the DRAK1 protein or fragment thereof.

In one embodiment, the screening method selects a test agent that promotes binding of the DRAK1 protein or fragment thereof to the TRAF6 protein or fragment thereof, or reduces dimerization or ubiquitination of the TRAF6 protein or fragment thereof. It may further include a step. Reduction of dimerization or ubiquitination of the TRAF6 protein or fragments thereof has common knowledge such as immunoblotting (IB or western blotting; WB), immunoprecipitation (IP), immunostaining, fluorescence, FACS, etc. It may be suitably performed using any technique known to the person.

In one embodiment, the screening method comprises comparing the control sample; And comparing the control sample with the test sample to reduce the dimer, ubiquitination, or a combination thereof of the TRAF6 protein or fragment thereof.

The control sample may mean a sample that has not been contacted with the test substance, or a sample that has been contacted with a solvent used to dissolve the test substance. The control sample is for the purpose of screening a substance having a similar or superior effect than a conventional anticancer agent, it is known to be available as an existing anticancer agent, it is known to promote cell death among existing anticancer agents, or to inhibit cancer cell metastasis Or a sample in contact with any material known to be used as a control in a similar screening method in the art.

The screening method includes TRAF6-related factors such as TAK (or Mitogen-activated protein kinase kinase kinase 7; MAP3K7), p38, extracellular signal-regulated kinase (ERK), and JNK (c-Jun N-terminal kinase) or phosphorylation thereof. , Detecting the cancer or inflammation-related factors such as p65, IL-1B and IL-8 in the cytoplasm and nucleus.

It is understood that any of the terms or elements mentioned in the compositions and methods of treatment, such as those mentioned in the description of the claimed screening method, is as mentioned in the description of the composition and method of treatment.

Another aspect is a DRAK1 protein or fragment thereof; TRAF6 protein or fragment thereof; And an antibody, an antigen-binding fragment, a polypeptide, or a combination thereof that specifically binds to the DRAK1 protein or a fragment thereof, and a TRAF6 protein or a fragment thereof, a kit for screening a substance for the treatment or prevention of a tumor or inflammation. to provide.

In one embodiment, the kit for screening may further include an antibody, an antigen-binding fragment, a polypeptide, or a combination thereof specifically binding to ubiquitination enzyme, ubiquitin, and ubiquitinated protein.

The kit comprises one or more other components, solutions, or devices required to measure the binding of a DRAK1 protein or fragment thereof to a TRAF6 protein or fragment thereof, or to measure the expression level of a TRAF6 protein or fragment thereof, dimerization or ubiquitination It may further include. The kit is labeled with, for example, an antibody, polypeptide, or combination thereof specifically binding to DRAK1 protein or fragment thereof or TRAF6 protein or fragment, and a substrate for detecting it, a suitable buffer solution, a chromogenic enzyme, or a fluorescent substance Secondary antibodies, chromogenic substrates, and the like. The coloring substrate may be a nitrocellulose membrane, a 96-well plate synthesized with polyvinyl resin, a 96-well plate synthesized with polystyrene resin, glass slide glass, or the like, and the coloring enzymes may be peroxidase, alkaline phosphatase. (alkaline phosphatase) can be used, FITC, RITC, etc. can be used for the fluorescent material, and the chromogenic substrate is 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) or o -Phenylenediamine (OPD), tetramethyl benzidine (TMB), etc. may be used, but is not limited thereto.

It is understood that any of the terms or elements mentioned in the composition, treatment method or screening method, such as those mentioned in the description of the claimed screening kit, is as mentioned in the description of the composition, treatment method or screening method.

Using a composition comprising a DRAK1 protein or fragment thereof according to an aspect, a polynucleotide encoding the DRAK1 protein or fragment thereof, or a combination thereof, effectively kill cancer cells in an individual in need thereof or inhibit metastasis of cancer cells You can, and you can treat or prevent inflammation.

According to another aspect, contacting the test substance to the DRAK1 protein or a fragment thereof; And detecting a dimer, ubiquitination, or a combination of TRAF6 proteins or fragments thereof, to effectively screen for a tumor or inflammation treatment or prevention agent.

1A shows the interaction of DRAK1 and TRAF6 by the yeast protein hybridization method by immunoprecipitation (IP), and FIG. 1B shows a proteasome-dependent proteolytic inhibitor (MG132) and an autophagy-inducing inhibitor (3MA). When treated, the expression level of TRAF6 was confirmed. Figure 1C confirms the change in expression level of TRAF6 according to the expression level of DRAK1.
FIG. 2A confirms ubiquitination activation of TRAF6 by DRAK1 by pull-down, FIG. 2B confirms inhibition of dimerization of TRAF6 by DRAK1 by IP, and FIG. 2C shows DRAK1 for TRAF domain-mediated dimerization The impact was confirmed by IP.
3A confirms the binding of each domain of DRAK1 to TRAF6, and FIG. 3B confirms the binding of TRAK6 to each detailed site of DRAK1.
Figure 4A is a diagram of each domain and selected amino acid region of DRAK1 protein and its sequence, Figure 4B is confirmed by pull-down of ubiquitination of TRAF6 by selected domain of DRAK1 (11R-DRAK1), Figure 4C is uterine cancer When DRAK1 is knocked down in cells, the expression pattern of the TRAF6 mediated signaling protein is confirmed by Western blot, and FIG. 4D shows whether the NK-kB promoter is activated by luciferase activity analysis.
Figure 5A is a metastasis activity of cervical cancer cells knocked down DRAK1 confirmed by immunostaining method, Figure 5B is a metastasis activity of cervical cancer cells by a selected domain of DRAK1 (11R-DRAK1) confirmed by immunostaining method, Figure 5C Whether the NK-kB promoter was activated was confirmed by luciferase activity analysis.
FIG. 6A confirms the TRAF6 mediated signaling mechanism in DRAK1 knockdown cells by Western blot, FIG. 6B confirms the presence of p65 in the cytoplasm and nucleus by Western blot, and FIG. 6C shows luciferase activity whether the NK-kB promoter is activated It was confirmed by analysis.
FIG. 7A confirms the TRAF6 mediated signaling mechanism in DRAK1 overexpressing cells by Western blot, FIG. 7B confirms the presence of p65 in the cytoplasm and nucleus by Western blot, and FIG. 7C shows luciferase activity whether NK-kB promoter is activated It is the result of the analysis.
FIG. 8A shows the results of RNA sequencing, FIG. 8B shows the clustering by selecting and clustering factors with sequencing changes in RNA sequencing, and FIG. 8C is a graph confirming the expression level of the selected elements in RNA sequencing with qRT-PCR, FIG. 8D is a graph confirming the TRAF6 mediated signaling mechanism by qRT-PCR again.

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

Example One: DRAK1 And TRAF6 Check interaction

1-1. DRAK1 And Between TRAF6 Confirmation

Before confirming the effect of DRAK1 on TRAF6, it was confirmed that DRAK1 can bind TRAF6 directly. The binding of DRAK1 and TRAF6 was performed using a yeast two-hybrid system.

Specifically, 293T was transfected with Flag-TRAF6 and Myc-DRAK1, and after 4 hours, the medium was changed, and after additional incubation for 24 hours, cells were washed with PBS. Cells were then harvested and phosphatase inhibitors (Roche) and proteases with IP buffer (50mM Tris, pH 7.4, 150mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 2mM EDTA, 10% glycerol) The inhibitor (Roche) was added and the cells were lysed. After adding the designated antibody to the cell lysate and incubating for 16 to 20 hours, 30 µl of Dynabeads Protein G (Invitrogen) was added and reacted for about 1 hour, and the beads were separated to obtain each sample. This was Western blotting.

As a result, it was confirmed that DRAK1 binds to TRAF6, and when DRAK1 is present, it is observed that the expression of TRAF6 decreases (FIG. 1A ).

1-2. DRAK1 TRAF6 Determine the impact on stabilization

The mechanism by which DRAK1 binds to TRAF6 and affects the stability of TRAF6 was confirmed. TRAF6 can be stabilized by self-ubiquitination and exhibits activity by suppressing autophagy. Therefore, in order to confirm whether the decrease in the expression of TRAF6 identified in Example 1-1 was induced by proteasome-dependent proteolysis or autophagy, the proteasome-dependent proteolytic inhibitor "MG132" And autophagy induction inhibitor “3MA(3-Methyladenine)” was treated with the cell line of Example 1-1 to obtain each sample.

As a result, it was confirmed that the expression level of TRAF6 was restored when 3MA was treated (FIG. 1B ). Therefore, it can be seen that DRAK1 interacts with TRAF6 to suppress autobiquitination of TRAF6 and to reduce the expression level of TRAF6 as a result of autophagy. In addition, by confirming the tendency for TRAF6 to decrease when overexpressing DRAK1 in a concentration gradient, the correlation can be more clearly seen (FIG. 1C ).

Example 2: DRAK1 by TRAF6 Ubiquitination Active and TRAF6 Dimerization Inhibition analysis

2-1. DRAK1 by TRAF6 Ubiquitination Confirm activation inhibition

It was confirmed whether DRAK1 actually affects the ubiquitination pattern of TRAF6 due to the interaction between DRAK1 and TRAF6 identified in Example 1.

Specifically, His-Ubi, Myc-DRAK1, or Flag-TRAF6 was co-transfected into cells as in Example 1, and pull-down was performed using a Ni-NTA column. The transfected cells were washed with PBS, harvested, and suspended in a solution in which 5 mM N-ethyl maleimide (NEM) was added to 500 mL of PBS. After taking 400 μl PBS, 6 mL binding buffer (6M guanidine HCl, 0.1M Na ₂ HPO ₄ , 0.1M NaH ₂ PO ₄ , 0.01M Tris (pH 8.0), 10mM β-mercaptoethanol, 5mM NEM, 5mM already Dazol), and after adding 40 μl of Ni-NTA agarose (Qiagen), the mixture was reacted at 4° C. for 12 hours. Ni-NTA agarose is A buffer (6 M guanidine HCl, 0.1 M Na ₂ HPO ₄ , 0.1 M NaH ₂ PO ₄ , 0.01 M Tris (pH 8.0), 10 mM β-mercaptoethanol), B buffer (8 M Urea, 0.1 M Na ₂ HPO ₄ , 0.1 M NaH ₂ PO ₄ , 0.01 M Tris (pH 8.0), 10 mM β-mercaptoethanol), C buffer (8 M Urea, 0.1 M Na ₂ HPO ₄ , 0.1 M NaH ₂ PO ₄ , 0.01 M Tris (pH 6.3), 10 mM β-mercaptoethanol, 0.2% Triton X-100), D buffer (8 M urea, 0.1 M Na ₂ HPO ₄ , 0.1 M NaH ₂ PO ₄ , 0.01 M Tris (pH 6.3), 10 mM β-mercaptoethanol, 0.1% Triton X-100). Thereafter, 400 μl of 2x sample buffer (0.72 M β-mercaptoethanol, 200 mM imidazole) was added, incubated at room temperature for 20 minutes, boiled at 95° C. for 5 minutes, and Western blotting was performed with each antibody to perform TRAF6 It was confirmed whether or not ubiquitination.

As a result, when treated with the autophagy inducer 3MA, the total protein expression of TRAF6 reduced by DRAK1 was increased compared to the control, but ubiquitination of TRAF6 did not recover ubiquitination despite treatment with 3MA (FIG. 2A ). ).

2-2. On RAK1 by TRAF6 Dimer Confirmation of formation inhibition

The ubiquitination of TRAF6 is mediated by the formation of dimers by the inter-monomer binding of its own proteins. Therefore, it was confirmed that the reduced ubiquitination of TRAF6 by DRAK1 was a result of inhibiting the dimer formation of TRAF6. It is also known that monomer binding between TRAF6 is mediated by binding between TRAF domains in the TRAF6 protein. Therefore, it was confirmed that the reduced ubiquitination of TRAF6 by DRAK1 was specifically due to the inhibition of binding between TRAF domains.

Specifically, Myc-DRAK1, Flag-TRAF6, Flag-TRAF, HA-TRAF6, or HA-TRAF were co-transfected into cells as in Example 1, followed by IP using an anti-HA antibody, followed by TRAF6 monomer binding. Analysis.

As a result, it was confirmed that DRAK1 inhibits binding between TRAF6 monomers (FIG. 2B ). In addition, DRAK1 was found to inhibit cross-linking between TRAF domains in the TRAF6 protein (Figure 2C). Therefore, it can be seen that DRAK1 inhibits the self-ubiquitination activity of TRAF6 by binding to the domain of TRAF6 and inhibits the mutual binding between TRAF6 monomers, resulting in destabilization of TRAF6.

Example 3: On TRAF6 Combined DRAK1 Amino acid domain analysis

To find the amino acid region in DRAK1 binding to TRAF6, DRAK1 was cut in detail to perform IP for each site.

Specifically, plasmids were prepared to express DRAK1 to express various cut proteins as shown in FIGS. 3A and 3B, and then transformed into cells as in Example 1 to confirm the binding of each DRAK1 fragment to TRAF6 by IP. Did.

As a result, it was observed that the site corresponding to the kinase domain of DRAK1 binds to TRAF6 (FIG. 3A), and specifically, it was confirmed that TRAF6 binds to the amino acid 113th to 120th amino acid sites (FIG. 3B).

Example 4: DRAK1 Fragmentary On TRAF6 Destabilization of Korea

4-1. DRAK1 Fragmentary TRAF6 Self-ubiquitination control

It was confirmed that the DRAK1 fragment was able to induce destabilization by actually inhibiting autoubiquitination by binding to TRAF6.

Specifically, as shown in FIG. 4A, a peptide consisting of 14 amino acids, which is a minimum site, among domains identified as capable of binding to TRAF6 in DRAK1 was selected. To this, 11 arginine was bound to be stably delivered into the cell, and 3 glycines were bound with a linker (SEQ ID NO: 4). The DRAK1 analogue (mimics) polypeptide thus constructed was designated as 11R-DRAK1. 10R of 11R-DRAK1 was treated with 293T cells co-transfected with Flag-TRAF6 and HA-Ubiqutin for 24 hours, and then self-ubiquitination of TRAF6 was confirmed by IP.

As a result, it was confirmed that the autobiquitin activity of TRAF6 was inhibited depending on the treatment concentration of 11R-DRAK1 (FIG. 4B ).

4-2. DRAK1 Fragmentary TRAF6 Inactivation

It has been found that DRAK1 can block various inflammatory responses mediated by TRAF6 or signaling that is important for the development and metastasis of cancer (see Example 6 below). Therefore, it was confirmed that the DRAK1 fragment inhibits the autobiquitination of TRAF6 and consequently, TRAF6 mediated signaling is inhibited.

Specifically, in order to lower the expression of the DRAK1 protein, DRAK1 10 μM of 11R-DRAK1 was treated with CaSki, a cervical cancer cell line transfected with shRNA and pGL2, the backbone plasmid, and incubated for 24 hours. After harvesting the cells, Western blot was performed for each protein. In addition, luciferase analysis, after transforming the NF-κ Luciferase promoter vector and βgal vector into the cell line using FuGENE HD (promega), lysed the cells, and activated the luciferase activity with Luciferase activity kit (Promega). It was measured.

As a result, it was confirmed that 11R-DRAK1 lowered the expression of TRAF6 and reduced phosphorylation of TRAF6 mediated TAK1 and p38 MAPK (FIG. 4C ). In addition, it was confirmed that 11R-DRAK1 also reduces NF-kB transcriptional activation by TRAF6-mediated signaling (FIG. 4D ). Therefore, it can be seen that 11R-DRAK1 induces destabilization of TRAF6 like the DRAK1 protein, thereby effectively suppressing the inflammatory response or the development and metastasis of cancer cells.

Example 5: DRAK1 Confirmation of cancer cell metastasis suppression effect

It was confirmed by using a transwell invasion assay that DRAK1 can inhibit the metastasis of cancer cells by inhibiting the TRAF6 mediated signaling mechanism according to the activation of self-ubiquitination of TRAF6.

First, DRAK1 knockdown cells to be used for analysis were separately prepared. DRAK1 knockdown cells were transfected with LΔ293 cells with Δ8.9 and VSV-G with a shRNA lentiviral vector targeting DRAK1 (Sigma-Aldrich) or a control sh lentiviral vector to harvest the virus in the media two days later. The virus was infected with Hela and CasKi cells, the medium was changed after 24 hours, and after 24 hours, furomycin (2 μg/mL) was treated to select only the infected cells.

DRAK1 knockdown cells of 1X10 ⁵ and control cells were respectively dispensed into 1X10 ⁵ BioCoat Matrigel invasion chambers (BD Biosciences) containing 500 μl of medium, and the chamber was filled with medium without FBS, and then the outside was filled with normal medium. . After incubation for 24 hours, cells outside the chamber were wiped with a cotton swab, fixed in 70% ethanol, and stained with 0.5% toluene. Stained cells were directly counted using a microscope.

As a result, it was confirmed that the invasive ability was increased in the cervical cancer cell line in which DRAK1 was knocked down compared to the control group, and when the TRAF6 was knocked down, the invasive ability was significantly reduced (FIG. 5A ). Therefore, it can be seen that the invasive ability of the cervical cancer cell line increased by knock-down of DRAK1 is induced by activation of TRAF6. On the other hand, when treated with 10 μM of 11R-DRAK1, it was confirmed that the increased cell invasion ability by DARK1 knockdown as well as the control was significantly suppressed (FIGS. 5B and 5C ). Therefore, it can be seen that 11R-DRAK1 can control primary cancer occurrence and cancer metastasis of cervical cancer.

Example 6: DRAK1 TRAF6 Analysis of the effect on the mediated signaling mechanism

TRAF6 is known to be an important protein in the development and metastasis of inflammatory reactions or cancer by inducing activation of various proteins in cells. TRAF6 is known as an important protein for TAK1-p38 or MAPK-NF-kB signaling activity. Based on this, it was confirmed that the inhibition of TRAF6 activity by DRAK1 modulates TRAF6 mediated signaling.

Specifically, DRAK1 knockdown cervical cancer cells were prepared in the same manner as in Example 5. To construct DRAK1 overexpressing cells, first, VS2-G and LPCX-Myc-DRAK1 or control LPCX vectors were transfected into GP293 cells, and virus in the media was harvested two days later. The harvested virus was infected with a cell line with polybrene (8 μl/mL), and after incubation, 2 μg/mL of puromycin was treated to select only the infected cells. IL-1β was treated to induce the inflammatory response, and the expression of each protein was confirmed by Western blot. If necessary, after harvesting the treated cells, the task of separating the cytoplasm and nucleus was added. Luciferase analysis was performed in the same manner as in Example 4.

As a result, in DRAK1 knockdown cells, when IL-1β was treated, phosphorylation of TAK1 and p38 MAPK was significantly higher than that of the control group (FIG. 6A), and intranuclear movement of NF-kB was confirmed by an increase in the substrate, p65 (FIG. 6B). ), the transcriptional activation of NF-kB was also found to be significantly higher (FIG. 6C ).

In addition, when treated with IL-1β in DRAK1 overexpressing cells, phosphorylation of TAK1 and p38 MAPK was significantly reduced compared to the control group (FIG. 7A), intranuclear movement of NF-kB decreased (FIG. 7B), and transcription of NF-kB Activation was also observed to be inhibited (FIG. 7C ). Therefore, it can be seen that DRAK1 can inhibit the activity of TRAF6, thereby controlling the inflammatory response, cancer development, and metastasis.

Example 7: DRAK1 Gene expression analysis induced by expression

In order to investigate the cancer metastasis-related gene regulated by DRAK1 gene expression, after knocking down the DRAK1 gene in HeLa, a cervical cancer cell line, as in Example 5, RNA sequencing was performed (FIG. 8A).

RNA sequencing extracts RNA using TRIzol Reagent (Invitrogen) from each treated cell, purified using DNase I and miRNeasy Mini Kit (Qiagen), and uses the Illumina Mana platform using a 90-bp paired-end library. Each expression level was analyzed. Library quality and purity were determined using an Agilent 2100 BioAnalyzer (Agilent). Each sample was paired-end sequencing using HiSeq Sequencing Kits and Illumina HiSeq 2000, and analyzed using a base-calling pipeline (Sequencing Control Software (SCS), Illumina) program. All paired-end sequences were analyzed based on the hg19 human reference genome (NCBI build 37). Quantitative analysis of the gene was conducted based on the Cufflinks platform, and gene expression analysis was performed by per kb per million reads (RPKM). In addition, gene expression analysis was also performed through 12 Illumina MouseWG-6 v2.0 expression beadchips (4 repetitions for three cell lines). Each gene expression level was normalized by global median adjustment and interquartile range adjustment. After RNA sequencing, RT-qPCR was performed on NK-κ target genes IL-1β and IL-8, which are factors observed differently depending on whether DRAK1 is expressed.

As a result, a gene that has undergone changes in sensitization due to DRAK1 knockdown was clustered and KEGG pathway analysis was performed. As a result, genes related to NF-kB pathway closely related to cell growth and cancer metastasis were activated by DRAK1 knockdown. It was observed. In addition, it was confirmed that the genes related to anticancer drug resistance, inflammatory cytokines, and cell migration were activated during the Go term analysis (FIG. 8B ). Therefore, it can be seen that expression of DRAK1 can control the mechanism of inflammatory cytokine response and the activity of genes related to anticancer drug resistance and metastasis of primary cancer.

In addition, when the expression of DRAK1 is reduced, by confirming an increase in IL1B , IL8 , CXCL1 , CXCL2 , and ABCB1 mRNA expression compared to control cells, the mechanism of DRAK1's inflammatory cytokine response, activity of genes related to anticancer drug resistance , And may contribute to the control of metastasis of primary cancer, which is associated with the inhibition of TRAF6 activity (FIGS. 8C and 8D ).

So far, the present invention has been focused on preferred embodiments. Those skilled in the art to which the present invention pertains will understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an explanatory point of view rather than a restrictive point of view. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the equivalent range should be interpreted as being included in the present invention.

<110> AICT (ADVANCED INSTITUTES OF CONVERGENCE TECHNOLOGY) <120> Composition comprising a peptide derived from DRAK1 protein <130> PN124972 <160> 8 <170> KopatentIn 2.0 <210> 1 <211> 414 <212> PRT <213> Homo sapiens <400> 1 Met Ile Pro Leu Glu Lys Pro Gly Ser Gly Gly Ser Ser Pro Gly Ala 1 5 10 15 Thr Ser Gly Ser Gly Arg Ala Gly Arg Gly Leu Ser Gly Pro Cys Arg 20 25 30 Pro Pro Pro Pro Pro Gln Ala Arg Gly Leu Leu Thr Glu Ile Arg Ala 35 40 45 Val Val Arg Thr Glu Pro Phe Gln Asp Gly Tyr Ser Leu Cys Pro Gly 50 55 60 Arg Glu Leu Gly Arg Gly Lys Phe Ala Val Val Arg Lys Cys Ile Lys 65 70 75 80 Lys Asp Ser Gly Lys Glu Phe Ala Ala Lys Phe Met Arg Lys Arg Arg 85 90 95 Lys Gly Gln Asp Cys Arg Met Glu Ile Ile His Glu Ile Ala Val Leu 100 105 110 Glu Leu Ala Gln Asp Asn Pro Trp Val Ile Asn Leu His Glu Val Tyr 115 120 125 Glu Thr Ala Ser Glu Met Ile Leu Val Leu Glu Tyr Ala Ala Gly Gly 130 135 140 Glu Ile Phe Asp Gln Cys Val Ala Asp Arg Glu Glu Ala Phe Lys Glu 145 150 155 160 Lys Asp Val Gln Arg Leu Met Arg Gln Ile Leu Glu Gly Val His Phe 165 170 175 Leu His Thr Arg Asp Val Val His Leu Asp Leu Lys Pro Gln Asn Ile 180 185 190 Leu Leu Thr Ser Glu Ser Pro Leu Gly Asp Ile Lys Ile Val Asp Phe 195 200 205 Gly Leu Ser Arg Ile Leu Lys Asn Ser Glu Glu Leu Arg Glu Ile Met 210 215 220 Gly Thr Pro Glu Tyr Val Ala Pro Glu Ile Leu Ser Tyr Asp Pro Ile 225 230 235 240 Ser Met Ala Thr Asp Met Trp Ser Ile Gly Val Leu Thr Tyr Val Met 245 250 255 Leu Thr Gly Ile Ser Pro Phe Leu Gly Asn Asp Lys Gln Glu Thr Phe 260 265 270 Leu Asn Ile Ser Gln Met Asn Leu Ser Tyr Ser Glu Glu Glu Phe Asp 275 280 285 Val Leu Ser Glu Ser Ala Val Asp Phe Ile Arg Thr Leu Leu Val Lys 290 295 300 Lys Pro Glu Asp Arg Ala Thr Ala Glu Glu Cys Leu Lys His Pro Trp 305 310 315 320 Leu Thr Gln Ser Ser Ile Gln Glu Pro Ser Phe Arg Met Glu Lys Ala 325 330 335 Leu Glu Glu Ala Asn Ala Leu Gln Glu Gly His Ser Val Pro Glu Ile 340 345 350 Asn Ser Asp Thr Asp Lys Ser Glu Thr Lys Glu Ser Ile Val Thr Glu 355 360 365 Glu Leu Ile Val Val Thr Ser Tyr Thr Leu Gly Gln Cys Arg Gln Ser 370 375 380 Glu Lys Glu Lys Met Glu Gln Lys Ala Ile Ser Lys Arg Phe Lys Phe 385 390 395 400 Glu Glu Pro Leu Leu Gln Glu Ile Pro Gly Glu Phe Ile Tyr 405 410 <210> 2 <211> 3949 <212> DNA <213> Homo sapiens <400> 2 ggtagtctgc ctgccgcagt ccgagcgccg cgctggggag agcgggtgtt tgaaggctcc 60 gcggaccggc actaggagcc gggggcgggt ccgtgaccct ccggctgctc ggagtgaaca 120 ggcggccagg aaagaagcgg gcctgaacac catgatccct ttggagaagc caggcagcgg 180 cggctcctcc ccaggcgcca cctcaggctc gggccgggca ggccggggtc tgagcgggcc 240 gtgccggccg ccgccgccgc cccaggcccg cgggctgctg acagagatac gcgccgtggt 300 gcgcaccgag cccttccagg acggctacag cctgtgcccg ggccgggagc tgggcagggg 360 gaaatttgca gtggtgagaa aatgtataaa gaaagattct gggaaagaat ttgctgcaaa 420 gttcatgaga aaaagaagaa aaggccaaga ttgtcggatg gaaataattc atgagattgc 480 tgtacttgaa ctagcacaag acaatccttg ggtcattaat ttacatgaag tttatgagac 540 tgcatcagaa atgatcttag ttctggaata tgctgctggg ggtgaaatct ttgaccagtg 600 tgttgcagac agagaagaag cctttaaaga aaaagatgtt caaagactta tgcgacagat 660 tttagaaggt gttcactttt tacacactcg tgatgtagtt catcttgatt tgaagcctca 720 gaatattctg ttgacaagtg aatctccatt gggtgacatt aagattgttg attttggcct 780 ttcaagaata ttgaagaaca gtgaagagct ccgagaaatt atgggtaccc ctgaatatgt 840 ggctcctgaa attcttagtt atgatcctat aagcatggca acagatatgt ggagcattgg 900 agtgttaaca tatgtcatgc ttacaggaat atcacctttc ttaggcaatg ataaacaaga 960 aacattctta aacatctcac agatgaattt aagttattct gaggaagaat ttgatgtttt 1020 gtctgagtcg gctgttgatt tcatcaggac acttttagtt aagaaacctg aagatcgagc 1080 cactgctgaa gaatgtctaa agcacccctg gttgacacag agcagtattc aagagccttc 1140 tttcaggatg gaaaaggcac tagaagaagc aaatgccctc caagaaggtc attctgtgcc 1200 tgaaattaat tcggataccg acaaatcaga aaccaaggaa tccattgtaa ccgaagagtt 1260 aattgtagtt acttcatata ctctaggaca atgcagacag tctgaaaaag agaaaatgga 1320 gcaaaaggcc atttccaaac gatttaaatt tgaggaacct ttgctacaag aaattccagg 1380 agaatttatc tactgagcaa tatttccctt tagaacttca agatttctac attgaaaatg 1440 ttaatattat ttatggacct ctggccaaat ggtacatgta ctggaagtgg ataaccagta 1500 tcacttacac aaacaaaaat aactttgtca aatttgtgga gttaggtgga agccagattt 1560 taaaagttgc caaccaggag atttaacagg tacagttacc cgtttcaatg ttatttttaa 1620 gaagggagat gttggcacct ttgaattcta catcctgttt ctccagaatg agaatttgtg 1680 tacaaagata tttgtattca ctttctttaa aaaatccaag taaaagtgcc aaaactacac 1740 ttctgtaaat ctcttgcatt attcatatgt gtatctatat ctgcataatg tttgttaatg 1800 tctactaaaa ttgctacttt ttcactttgg atttgttttt ggcaaaattt tagtctaaac 1860 agacatctaa attttcgaga cttagaataa cattcactaa agtttgataa tgtctttttt 1920 aaatattttc ttactgcttt atagtgactt gatttggttt ctgttgtgtt tttgcctaaa 1980 tactagtaac atatcagtga aaaaccctaa ttttttttct cttcaatgtc actagtaaga 2040 gaggtggtaa tttttcacct ctgaaattat tctgggttta ggtgtcagct cttgagctgc 2100 ttccctcatc acaccacact cacatgcatc tgttctctct cacactctat acccctgcca 2160 cttcctggca cctcccccca tgcttgtcat ttaattttgg ccacttgtag gtatcagtgt 2220 gatctgatca acactctggt taccttggtc agtgaaaaga tgctactata ttgcttttgt 2280 cccaaagtga gtaaaatccc ctagagcaga gagagagaga gagagagaga gtgtcttcat 2340 gccaaccaca gcctgtttct gctgagtttc ttatcagccc tcaagctatg ctaggaaaag 2400 ttataaaatg ccaaaatatt tataaacatt tactttgtcc ataaaaaatt tacattggat 2460 actctgtaag taggaggtac tttgtcccaa aaagatgtat aagaatgtac taatagtttt 2520 atttgattag gattgaacag ttcagttgta tctatgcccc acagtgacca gtaaagtcca 2580 attaaaatat ggaaatgtaa aagtgtatgc cgaatacctt aaagtaacta attatcctta 2640 cacacaaaag gctcagtgca ttaaatattt gcctatcacc tgtgtgctgt gtaccgtggc 2700 catgcctcgc tctgcactat gaacaactca gacctgtccc ttcacggtct gattagggtg 2760 tcacacaaag ctctctccct aacatggttt ctcaactgtc aaaatcatga aaatgacagc 2820 acctaaccac tattttaaag tatgtacagt aaatacccaa atgtgtgtaa atgtcataga 2880 catatacaaa gtcagtaatc tcacccagtc ctgctgtgtg aacctgtcaa agcacctggg 2940 agtgcccttt acaccattag gtgaaattca tcaggcactt cagacaacca ggttgcactg 3000 ttttctcact aaattggctt tatttataca gtttcctgga acctagcatt cctaaacaag 3060 ctccaaaatg cagaagaaga aattgtgcca cagtgataga gtttttaatg aatatccatg 3120 gccacagttt tgagtctcag gtaagtcttg gaagaagtta aacacagtat taggcaaata 3180 tttatctctc tgaaaaaata gggaggaggt gaccaggaaa aaataaccac tattgaatga 3240 cacgtttgta cttgatggat ctgtcatcaa gttttaaaat cagaatcatt tgggcttttt 3300 aaaaaccatt ttatactcgt aataaatata tttaatataa ttgctgtatt tgtgagaaag 3360 atctgttttc ttttagctgg ccacttcagt ttgctgcgta tctttagctg ctgttgtgct 3420 taaatactga gtcatagggt gcttttttaa gaggcccttc agaatatact tgtaaaggtg 3480 tattggcatt ttttggttta taagataaaa acagggcatg aagagccttc atagtattag 3540 gccaaataaa atctattaat aactgccagc tagctctaca cttatctaaa gctgttaatg 3600 ttcctttttt tctatcacca aatttataaa ggactaaaca gtactattga gtttactcgg 3660 tttataaatc cagttttaac tataattcaa caatattttg gtgtggtgaa acgttgttta 3720 tgaaatgtat aaaatgtata agttttaatc aactgggaaa tgatatttga ttgatcttgt 3780 gttttgttgt tttcaaatga aaactaggag gtaattaata cctttctcta gtagtggtat 3840 agaggaagga cagatgctca ttgttgaatc tcttaaattt ttccagctaa cctcagtttg 3900 gaagaaaaat gtattaaagg gttataaaga tcaaaaaaaa aaaaaaaaa 3949 <210> 3 <211> 15 <212> PRT <213> Homo sapiens <400> 3 Ala Val Leu Glu Leu Ala Gln Asp Asn Pro Trp Val Ile Asn Leu 1 5 10 15 <210> 4 <211> 29 <212> PRT <213> Artificial Sequence <220> <223> 11R-DRAK1 <400> 4 Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Gly Gly Gly Ala Val 1 5 10 15 Leu Glu Leu Ala Gln Asp Asn Pro Trp Val Ile Asn Leu 20 25 <210> 5 <211> 522 <212> PRT <213> Homo sapiens <400> 5 Met Ser Leu Leu Asn Cys Glu Asn Ser Cys Gly Ser Ser Gln Ser Glu 1 5 10 15 Ser Asp Cys Cys Val Ala Met Ala Ser Ser Cys Ser Ala Val Thr Lys 20 25 30 Asp Asp Ser Val Gly Gly Thr Ala Ser Thr Gly Asn Leu Ser Ser Ser 35 40 45 Phe Met Glu Glu Ile Gln Gly Tyr Asp Val Glu Phe Asp Pro Pro Leu 50 55 60 Glu Ser Lys Tyr Glu Cys Pro Ile Cys Leu Met Ala Leu Arg Glu Ala 65 70 75 80 Val Gln Thr Pro Cys Gly His Arg Phe Cys Lys Ala Cys Ile Ile Lys 85 90 95 Ser Ile Arg Asp Ala Gly His Lys Cys Pro Val Asp Asn Glu Ile Leu 100 105 110 Leu Glu Asn Gln Leu Phe Pro Asp Asn Phe Ala Lys Arg Glu Ile Leu 115 120 125 Ser Leu Met Val Lys Cys Pro Asn Glu Gly Cys Leu His Lys Met Glu 130 135 140 Leu Arg His Leu Glu Asp His Gln Ala His Cys Glu Phe Ala Leu Met 145 150 155 160 Asp Cys Pro Gln Cys Gln Arg Pro Phe Gln Lys Phe His Ile Asn Ile 165 170 175 His Ile Leu Lys Asp Cys Pro Arg Arg Gln Val Ser Cys Asp Asn Cys 180 185 190 Ala Ala Ser Met Ala Phe Glu Asp Lys Glu Ile His Asp Gln Asn Cys 195 200 205 Pro Leu Ala Asn Val Ile Cys Glu Tyr Cys Asn Thr Ile Leu Ile Arg 210 215 220 Glu Gln Met Pro Asn His Tyr Asp Leu Asp Cys Pro Thr Ala Pro Ile 225 230 235 240 Pro Cys Thr Phe Ser Thr Phe Gly Cys His Glu Lys Met Gln Arg Asn 245 250 255 His Leu Ala Arg His Leu Gln Glu Asn Thr Gln Ser His Met Arg Met 260 265 270 Leu Ala Gln Ala Val His Ser Leu Ser Val Ile Pro Asp Ser Gly Tyr 275 280 285 Ile Ser Glu Val Arg Asn Phe Gln Glu Thr Ile His Gln Leu Glu Gly 290 295 300 Arg Leu Val Arg Gln Asp His Gln Ile Arg Glu Leu Thr Ala Lys Met 305 310 315 320 Glu Thr Gln Ser Met Tyr Val Ser Glu Leu Lys Arg Thr Ile Arg Thr 325 330 335 Leu Glu Asp Lys Val Ala Glu Ile Glu Ala Gln Gln Cys Asn Gly Ile 340 345 350 Tyr Ile Trp Lys Ile Gly Asn Phe Gly Met His Leu Lys Cys Gln Glu 355 360 365 Glu Glu Lys Pro Val Val Ile His Ser Pro Gly Phe Tyr Thr Gly Lys 370 375 380 Pro Gly Tyr Lys Leu Cys Met Arg Leu His Leu Gln Leu Pro Thr Ala 385 390 395 400 Gln Arg Cys Ala Asn Tyr Ile Ser Leu Phe Val His Thr Met Gln Gly 405 410 415 Glu Tyr Asp Ser His Leu Pro Trp Pro Phe Gln Gly Thr Ile Arg Leu 420 425 430 Thr Ile Leu Asp Gln Ser Glu Ala Pro Val Arg Gln Asn His Glu Glu 435 440 445 Ile Met Asp Ala Lys Pro Glu Leu Leu Ala Phe Gln Arg Pro Thr Ile 450 455 460 Pro Arg Asn Pro Lys Gly Phe Gly Tyr Val Thr Phe Met His Leu Glu 465 470 475 480 Ala Leu Arg Gln Arg Thr Phe Ile Lys Asp Asp Thr Leu Leu Val Arg 485 490 495 Cys Glu Val Ser Thr Arg Phe Asp Met Gly Ser Leu Arg Arg Glu Gly 500 505 510 Phe Gln Pro Arg Ser Thr Asp Ala Gly Val 515 520 <210> 6 <211> 8040 <212> DNA <213> Homo sapiens <400> 6 ctcctccccg gcgcgctccc tgcccctcgc tccccgcagc cagcagagaa ggcggaagca 60 gtggcgtccg cagctggggc ttggcctgcg ggcggccagc gaaggtggcg aaggctccca 120 ctggatccag agtttgccgt ccaagcagcc tcgtctcggc gcgcagtgtc tgtgtccgtc 180 ctctaccagc gccttggctg agcggagtcg tgcggttggt gggggagccc tgccctcctg 240 gttcggcctc cccgcgcact agaacgatca tgaacttctg aagggaccca gctttctttg 300 tgtgctccaa gtgatttgca caaataataa tatatatatt tattgaagga gagaatcaga 360 gcaagtgata atcaagttac tatgagtctg ctaaactgtg aaaacagctg tggatccagc 420 cagtctgaaa gtgactgctg tgtggccatg gccagctcct gtagcgctgt aacaaaagat 480 gatagtgtgg gtggaactgc cagcacgggg aacctctcca gctcatttat ggaggagatc 540 cagggatatg atgtagagtt tgacccaccc ctggaaagca agtatgaatg ccccatctgc 600 ttgatggcat tacgagaagc agtgcaaacg ccatgcggcc ataggttctg caaagcctgc 660 atcataaaat caataaggga tgcaggtcac aaatgtccag ttgacaatga aatactgctg 720 gaaaatcaac tatttccaga caattttgca aaacgtgaga ttctttctct gatggtgaaa 780 tgtccaaatg aaggttgttt gcacaagatg gaactgagac atcttgagga tcatcaagca 840 cattgtgagt ttgctcttat ggattgtccc caatgccagc gtcccttcca aaaattccat 900 attaatattc acattctgaa ggattgtcca aggagacagg tttcttgtga caactgtgct 960 gcatcaatgg catttgaaga taaagagatc catgaccaga actgtccttt ggcaaatgtc 1020 atctgtgaat actgcaatac tatactcatc agagaacaga tgcctaatca ttatgatcta 1080 gactgcccta cagccccaat tccatgcaca ttcagtactt ttggttgcca tgaaaagatg 1140 cagaggaatc acttggcacg ccacctacaa gagaacaccc agtcacacat gagaatgttg 1200 gcccaggctg ttcatagttt gagcgttata cccgactctg ggtatatctc agaggtccgg 1260 aatttccagg aaactattca ccagttagag ggtcgccttg taagacaaga ccatcaaatc 1320 cgggagctga ctgctaaaat ggaaactcag agtatgtatg taagtgagct caaacgaacc 1380 attcgaaccc ttgaggacaa agttgctgaa atcgaagcac agcagtgcaa tggaatttat 1440 atttggaaga ttggcaactt tggaatgcat ttgaaatgtc aagaagagga gaaacctgtt 1500 gtgattcata gccctggatt ctacactggc aaacccgggt acaaactgtg catgcgcttg 1560 caccttcagt taccgactgc tcagcgctgt gcaaactata tatccctttt tgtccacaca 1620 atgcaaggag aatatgacag ccacctccct tggcccttcc agggtacaat acgccttaca 1680 attcttgatc agtctgaagc acctgtaagg caaaaccacg aagagataat ggatgccaaa 1740 ccagagctgc ttgctttcca gcgacccaca atcccacgga acccaaaagg ttttggctat 1800 gtaactttta tgcatctgga agccctaaga caaagaactt tcattaagga tgacacatta 1860 ttagtgcgct gtgaggtctc cacccgcttt gacatgggta gccttcggag ggagggtttt 1920 cagccacgaa gtactgatgc aggggtatag cttgccctca cttgctcaaa aacaactacc 1980 tggagaaaac agtgcctttc cttgccctgt tctcaataac atgcaaacaa acaagccacg 2040 ggaaatatgt aatatctact agtgagtgtt gttagagagg tcacttacta tttcttcctg 2100 ttacaaatga tctgaggcag ttttttcctg ggaatccaca cgttccatgc tttttcagaa 2160 atgttaggcc tgaagtgcct gtggcatgtt gcagcagcta ttttgccagt tagtatacct 2220 ctttgttgta ctttcttggg cttttgctct ggtgtatttt attgtcagaa agtccagact 2280 caagagtact aaacttttaa taataatgga ttttccttaa aacttcagtc tttttgtagt 2340 attatatgta atatattaaa agtgaaaatc actaccgcct tgtgctagtg ccctcgagaa 2400 gagttattgc tctagaaagt tgagttctca tttttttaac ctgttataga tttcagagga 2460 tttgaaccat aatccttgga aaacttaagt tctcattcac cccagttttt cctccaggtt 2520 gttactaagg atattcaggg atgagtttaa accctaaata taaccttaat tatttagtgt 2580 aaacatgtct gttgaataat acttgtttaa gtgttccttc tgccttgctt acttatttcc 2640 ttgaggttac gaagtagcat cttccccaga gtttataatg ctgagaacca cgtggatacc 2700 aactgctcat tgttatgcta tgtaaccctt tttgtctatt cagtgcagag tgaatttcac 2760 agctctgcat atgtcttcat ttgtttaatg cttacaagac aggagatgca cacatacaat 2820 cagcaacata aaaattaaaa gtgacccaag tagtcagcgc atgtggcatc tcattggtgg 2880 tgacagaagc tatgtgagcc agaagttttc agctcttttg aataccctct ggtttatttc 2940 gattaaaaag aacaaaattg atttcctaaa atcagaattt tttaaaactt gggagatgat 3000 tggagatacc taggaggtca ccaaactagg attagaagtc acagtggttg tatcacaact 3060 tagcttgagt atgttgctgt agcctaacaa ctgcaggttc tgagaaggat cctgtagaat 3120 cctggaagta accagatttt cctaataggg agatgatttt tttgtgtgcc atcatgtatt 3180 tgttaaaggc ctatatatag atataaaata tcgtggaatc tagttctcag ggagacccgc 3240 aactagtata agcttataaa ggatctaaag atccatccac catttaaagt tgtctggtaa 3300 tgagagatga cattgtatcc cccagagagg ccaaatcaga gtcgccagcc agcgttctag 3360 atcagcctta atttcaagag aaagccaagg acctcatctg caggggagtg tggttttcag 3420 ccccagcgag tgtcactttg aactttccct ttgctttttt ctctcttctc cctccccacc 3480 cacccttagg ctcctgatct ggtgagtttg ttatggagtg aaaataaaag tcaagcagag 3540 accttgtttc ccgtgccacc attagtacca caagctcatg gctagttacc acattacttc 3600 ctggcagttt gtgtccctca gctgtgcctt ccaaccagcg cctgagaatc actgcatacc 3660 accctctagg tagggaaacc tacactgctg ctgttcctgt gattatttta caatgaataa 3720 ataattgtca agttccattt aaaaactgaa cagtagtatt tttgtatttg cgtagaaaaa 3780 gcctgaagga aatatactaa actttttgtt ggcttatttt cctttgcgct tgcttatatt 3840 ttttacattt tctacaataa atgtgtactt ttatcggaga aaaaaattaa atgttgccac 3900 aaaacattta atctccacgc ccccagctca aaaaaggaaa tgatatttaa aagcttcctg 3960 gtcagatttc tattaaaagc actggctgtg cattagatac aaagaggagt catttcctgc 4020 cttggtgata ctattttttt ctactaactc aagagtcttt attaaaaaaa aaagttgttt 4080 tgcctaattt cagcttttag caagcttccc atctgtaaaa tgatttggac cagatatttc 4140 tagagtcccc tccagccata acattctgtc tcaaattaag ttccaaccag cagaacaatg 4200 acaatactta ggaaagtatt ttgccagtat aaaatgtctt taacttactc tttgctgaca 4260 ctgatacttt cctctaattt agtgtctatc agctgggtca catcttaagt aaaatgagca 4320 attttaaccc ccaacatttg gcattttgtc ataaaccagc cagttatttt atgctggtca 4380 ttcatcttga ctacaaagta gaatagtcaa gctgtcattc caaatagaaa actttttact 4440 tcaatcagaa ttaagcctta acctggaaag ttggtttctt ccttacattt tcccaatctc 4500 ctactctatt cttaaacatg ctagtttcac tcagttgggt atacaagcct ttgggcttta 4560 tgttgtatgt tactaaccac cttttaccat atttatcttt tggcatcatt ctgggacatt 4620 gctaaattaa aaaagaaatt gtttccactt ttttctggag atgttcaact aaaggttgtt 4680 ttgttttgtt ttttgttttg agacagtctc accctgacgc tcaggctgga gtgcagtggt 4740 gcaacctcgg ctcactgcaa cctccacctc ccgggctcaa gccattctcc tgcctcagcc 4800 tcccaagcag ctgggattac aggcacccgc caccacgccc agctaatttt tttgtatttt 4860 gagtagagac cgggtttcac catattggcc agtctcgtct ggaactcctg acctcagatg 4920 atccgcccgc ctcagcctcc caaagtgctg ggattacagg catgagccac cacgcccagc 4980 gtccaaccca ctgttggatg aaacttgctg cacgtcatac attttgctgt tggcaaacaa 5040 gtctgaatgt tgatttgaag tttggtagtt tattactatc tattggcagc aaagactgtt 5100 tattggtata ctacaatatg atttaacttt tattttgggg ataaatagta gaaaaaagtg 5160 aaacagaatg aaggcaggtg ttttttattc taatgatgga ataatacaga gatactggac 5220 gatctctagc agttaattat tgtgacccat ataaaattat acaggtcaca gtataattct 5280 ctattaccgt ttttacacca gtaagtctta gataaactaa gcatgcttat gaattatgta 5340 tacagttaga atgcattatt tttacagagg aacaattgct tgtatgtact aacactgttc 5400 tcttggcttg cctcaagttc tactcattat tttatataaa atactattag gctgggcacg 5460 gtggctcacg cctataatcc cagcactttg ggaggtggag gctggcggat tacttgaagc 5520 caggagttcg agaccagcct ggccaaaatg gtgaaacccc atctctataa aaatacaaaa 5580 attagccagg tgtcatgata catgcctgta atcccagctt cttgggaggc tgaggcacgg 5640 gaatcgcttg aacccgggaa gcacaggttg cagtgagcca agatcatgcc actgcacccc 5700 cagcctgggt gacagagtgc aacactgtct cacaaaacaa aacaaaaaca tcagattctg 5760 tttgtgatgc ctagttgctt acaacctaaa cagtgcaatg ccttaaggaa atgaaaagga 5820 gccataagta gtcatttata tttttatttt gaagtgtgct ttttctaaac tcccagattg 5880 acatgatgga ctgtaagtta gtttctctgt ttctgtcttt gtgcctgtag agtgtacttg 5940 gcacttacaa attcccagta tccagaaaga tgatctgatg aaatcaaatt ggatggatct 6000 tggcagactg tgacactcaa ttacagcctt cactttcagt caaaaacgga cacttggcaa 6060 ggaggtgcct ggttgtttca ctaaatgtca cttgtgtgtg taatatttta aagctttttc 6120 cccacaggaa attcgggtca taaaatcctg aaaaataatt ctaggtggga aaagcatttt 6180 aggaaatgag agatgtggtg ctgcttttct tctctcagag tgctttctca gcaggacact 6240 agccctgcct ttaagatggg gaagttgggg catgtgcctc tgctcttact gtctgcagct 6300 ctgaaggtag gtgctgtccc actcggacaa tcgcccaagc agcagtgacc atagttctct 6360 tctatgcaag tccccaggag aaggtaaact gtgtggaatg gggatgtgtt ctggttgctg 6420 ctgaatcccc tcttcttacc acagtgcctg gcacgttgca cacactcaaa tacgtaataa 6480 tgaacattta ttgaaagcag cagttgaagc tgaccaattt ctggtacctt gtcatgtaaa 6540 ttttagatgg taaggcgcag atgttacttt ttttgctttt tttcttcagc acttgatgaa 6600 atttcccaaa catgcagaaa tgttgaaaga cttgtatagt gaacatctac gacctagaat 6660 ctgcagtaat attatgttac atttgcttta tcacttgata gatgttactt ttaatgagac 6720 ttcaagtttg gtttctctaa acaaaatatt ctaaaataac tgaacaactt taatcaattt 6780 gtcttaagtt ctttggggga acttgggaca tttgctttgt aactggaatt gcagccctca 6840 cgttaagcta attttaaact ttgcaaattt gttatgctga atttcagtct tatttatttt 6900 gcctgaaggg gtattttttg taatggattt atttgaaggt ccttgataaa ttgtgcagaa 6960 tattctcgtg ttctttttgc acttgataaa ttatctaatt tctgtggtga gaatgtaatt 7020 tggggcctat tttgtttata caagcttcca gaattatgtt ctcagaggga tgaaaaggtg 7080 taatttagca tataggtcac taaattagga gctaagacac attttctcct gactgaccat 7140 gggtcaatca gttttgtctt cgtgtccttt tccttgtaaa gtagaaacta gaatttgaaa 7200 tttaaatatt aaataatggg taacattcat taatgtatga ctctattaag aaagacactg 7260 tgaatccagg gaggattctc ataattctgt aaactgtatg acaagctgtg gaatgaaatc 7320 tgacttttga aaattgaaag acatccagtg gtcttatcac aaagcctgct tttcctcaga 7380 acttaactat tgccatggaa tttgtaagca gttatcctaa tccatctgga ctctgaaaat 7440 gcatccttta tgagagggag tgaatgcaaa gataagggtg gggaaacact aatcatgaaa 7500 agaatgaaaa tcagtgttca gttttaagag caggttgtat tgaaggaagg gattaaagga 7560 attatccaga tttgaggtgg cacatcttcc accactccct gcaccatcag catgcacgga 7620 gcgcataaaa caagccctgc tcctaatggc agtgaaacct cggatggcct ccatcaggtc 7680 aatacaactg aattgctggg ctgacttaag attgaaggac tccattttag taagtagaga 7740 agtgtgacct ttctcaaccc aggttgtgaa tgtggattca cacttatctc aaaaaggcac 7800 ctggagtttt aactttatgt catgtctcag tactggttgc aaggtatgac caaaagtgtt 7860 ccttgaatgg cacctttttg aatattaatt tagaagaaaa catgccagac tgacatactt 7920 accccctccg cactgttact acttccttac cagccctatg tactgcatca atgtctacaa 7980 gaaagcactc ttcattaaaa tgaaatatat atattaaaat aaaaaaaaaa aaaaaaaaaa 8040 8040 <210> 7 <211> 150 <212> PRT <213> Homo sapiens <400> 7 Gly Ile Tyr Ile Trp Lys Ile Gly Asn Phe Gly Met His Leu Lys Cys 1 5 10 15 Gln Glu Glu Glu Lys Pro Val Val Ile His Ser Pro Gly Phe Tyr Thr 20 25 30 Gly Lys Pro Gly Tyr Lys Leu Cys Met Arg Leu His Leu Gln Leu Pro 35 40 45 Thr Ala Gln Arg Cys Ala Asn Tyr Ile Ser Leu Phe Val His Thr Met 50 55 60 Gln Gly Glu Tyr Asp Ser His Leu Pro Trp Pro Phe Gln Gly Thr Ile 65 70 75 80 Arg Leu Thr Ile Leu Asp Gln Ser Glu Ala Pro Val Arg Gln Asn His 85 90 95 Glu Glu Ile Met Asp Ala Lys Pro Glu Leu Leu Ala Phe Gln Arg Pro 100 105 110 Thr Ile Pro Arg Asn Pro Lys Gly Phe Gly Tyr Val Thr Phe Met His 115 120 125 Leu Glu Ala Leu Arg Gln Arg Thr Phe Ile Lys Asp Asp Thr Leu Leu 130 135 140 Val Arg Cys Glu Val Ser 145 150 <210> 8 <211> 450 <212> DNA <213> Homo sapiens <400> 8 ggaatttata tttggaagat tggcaacttt ggaatgcatt tgaaatgtca agaagaggag 60 aaacctgttg tgattcatag ccctggattc tacactggca aacccgggta caaactgtgc 120 atgcgcttgc accttcagtt accgactgct cagcgctgtg caaactatat atcccttttt 180 gtccacacaa tgcaaggaga atatgacagc cacctccctt ggcccttcca gggtacaata 240 cgccttacaa ttcttgatca gtctgaagca cctgtaaggc aaaaccacga agagataatg 300 gatgccaaac cagagctgct tgctttccag cgacccacaa tcccacggaa cccaaaaggt 360 tttggctatg taacttttat gcatctggaa gccctaagac aaagaacttt cattaaggat 420 gacacattat tagtgcgctg tgaggtctcc 450

Claims (17)

DRAK1 (death-associated protein kinase-related apoptosis-inducing kinase 1) protein or a fragment thereof, a polynucleotide encoding the DRAK1 protein or a fragment thereof, or a combination thereof, a composition for the treatment or prevention of a tumor or inflammation. The method according to claim 1, wherein the DRAK1 protein or fragment thereof comprises the amino acid sequence of SEQ ID NO: 1, composition The method according to claim 1, wherein the fragment of DRAK1 is 1 to 193 amino acid sequence of SEQ ID NO: 1, 62 to 193 amino acid sequence of SEQ ID NO: 1, 1 to 120 of SEQ ID NO: 1, 113 of SEQ ID NO: 1 It is a polypeptide comprising an amino acid sequence selected from the 120th amino acid sequence, and 110th to 124th amino acid sequence of SEQ ID NO: 1. The method according to claim 1, wherein the fragment of DRAK1 is a polypeptide comprising the amino acid sequence of SEQ ID NO: 3 or a fragment thereof. The method according to claim 1, wherein the composition is to inhibit the activity of TRAF6. The composition of claim 1, wherein the composition inhibits TRAF6 monomer from forming a dimer. The composition of claim 1, wherein the DRAK1 protein or fragment thereof binds to the TRAF domain of TRAF6. The method according to claim 1, wherein the TRAF domain comprises the amino acid sequence of SEQ ID NO: 7. The method according to claim 1, wherein the composition is to inhibit the metastasis of cancer cells. A method of treating a tumor or inflammation of an individual in need thereof comprising administering the composition of claim 1 to the individual. Contacting the test substance to the DRAK1 protein or a fragment thereof; And
A method for screening a substance for the treatment or prevention of a tumor or inflammation, comprising detecting a dimer, ubiquitination, or a combination thereof of a TRAF6 protein or fragment thereof. The method according to claim 11, wherein the DRAK1 protein or fragment thereof is in contact with the test substance in combination with a TRAF6 protein or fragment thereof. The method of claim 11, wherein the screening method selects a test substance that promotes the binding of the DRAK1 protein or fragment thereof to the TRAF6 protein or fragment thereof, or reduces the dimerization or ubiquitination of the TRAF6 protein or fragment thereof. The method further comprising the step of. The method of claim 11, wherein the fragment of TRAF6 comprises the SEQ ID NO: TRAF domain. The method of claim 11, wherein the treatment or prevention of the tumor comprises controlling or inhibiting metastasis of cancer cells. DRAK1 protein or fragment thereof;
TRAF6 protein or fragment thereof; And
A kit for screening a compound for the treatment or prevention of a tumor or inflammation, comprising the DRAK1 protein or a fragment thereof, and an antibody, antigen-binding fragment, polypeptide or a combination thereof specifically binding to each of the TRAF6 protein or fragments thereof. The kit for screening according to claim 16, further comprising an antibody, an antigen-binding fragment, a polypeptide, or a combination thereof, specifically binding to ubiquitination enzyme, ubiquitin, and ubiquitinated protein.

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