KR20140107988A - Composition for Treating Heart Disease Containing Polypeptide Involved in the activation of Expression of Bradykinin Receptor Derived from Pseudomonas aeruginosa - Google Patents

Abstract

The present invention relates to a composition to treat heart diseases containing Pseudomonas aeruginosa-derived polypeptide involved in the promotion of expressing a bradykinin receptor and, more specifically, to a composition to treat the side effects for diabetes, hypertension, cardiac hypertrophy, or myocardial infarction, containing the Pseudomonas aeruginosa-derived polypeptide involved in the promotion of expressing the bradykinin receptor; and to a method to screen a treatment agent using the Pseudomonas aeruginosa-derived polypeptide. The Pseudomonas aeruginosa-derived PA3807 (nucleoside diphosphate kinase (Ndk); SEQ ID NO: 1) is involved in the regulation of expressing the bradykinin receptor which performs an important action on the regulation of inflammatory responses in various diseases, thus being useful in screening treatment agents for various diseases.

Description

TECHNICAL FIELD [0001] The present invention relates to a composition for treating cardiac diseases, which comprises a green peptides derived from a green antibiotic and is involved in promoting expression of a bradykinin receptor.

The present invention relates to Pseudomonas spp., Which is involved in promoting expression of bradykinin receptor The present invention relates to a composition for treating a diabetic side effect or a cardiovascular disease comprising an aeruginosa- derived polypeptide as an active ingredient. More particularly, the present invention relates to a composition for treating diabetes, an antidiabetic side effect comprising a Pseudomonas aeruginosa polypeptide derived from Pseudomonas aeruginosa, a composition for treating hypertension, cardiac hypertrophy or myocardial infarction, and a method for screening a therapeutic agent using a Pseudomonas aeruginosa-derived polypeptide.

Pseudomonas aeruginosa is a gram-negative aerobic bacterium belonging to the family Pseudomonadaceae . It is a bacteria commonly found in soil and water. Pseudomonas aeruginosa is becoming increasingly recognized as an emerging opportunistic infectious pathogen with clinical relevance. A number of different epidemiological studies have been tracked as emerging as pathogens in hospitals and antibiotic resistance has increased in clinical isolates.

Pseudomonas aeruginosa is an opportunistic pathogen, which initiates infection by using some destruction of the host defense. Pseudomonas aeruginosa infects almost uninfected tissues, but tissue defenses can almost certainly be infected in any way, especially in severe burn patients and immunosuppressed cancers and AIDS patients, including urinary tract infections, respiratory tract infections, dermatitis, soft tissue infections , Bacteremia, osteoarthritis, gastrointestinal infection, and various systemic infections. Pseudomonas infection is a serious problem in patients hospitalized with cancer, cystic fibrosis, and burns. The mortality rate associated with these patients is almost 50%.

Pseudomonas (Pseudomonas) may convey antibiotic resistance plasmids, both R- factor RTF and holds both, and bacterial mechanisms of horizontal gene transfer, these genes by means of mainly transduction and bonding.

Most Pseudomonas infections are invasive and toxin-producing. The ultimate Pseudomonas infection may appear to consist of three distinct stages: (1) bacterial adhesion and clustering; (2) local invasion; (3) Disseminated systemic disease. However, the disease progression can be stopped at any stage. Certain bacterial determinants of toxicity are involved in the characteristic syndromes that mediate each of these stages and eventually accompany the disease.

Quinine is an endogenous peptide formed in plasma and surrounding tissues in response to tissue damage or infection by catalytic degradation of kininogen by the caricature enzyme. Kinin plays an important role in pathological processes involving pain and inflammation.

Bradykinin (BK) is a vasoactive nonapeptide, H-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-OH, formed by various plasma enzymes such as carin- do. In some embodiments, it has an action similar to histamine, and is released from the venule rather than the arterioles, like histamine.

Two types of BK receptors, Bl and B2, are known in mammals (Leeb-Lundberg, et al ., Pharmacol . Rev., 57: 27-77, 2005). BK receptor mediated by B 2 receptors regulates vascular activity (vasodilation, vasoconstriction) as well as increased vascular permeability, inflammatory response and pain (Heitsch H., Drug News Perspect , 13 (4): 213, 2000). In B2 receptors, these effects are related to the role of BK in a variety of diseases such as inflammation, cardiovascular disease and pain. Thus, substances that prevent BK from binding to B2 receptors can inhibit or at least alleviate pathogenic diseases.

B2 receptors are known to be expressed in various cells such as brain, eye, kidney, liver, lung, prostate, skin, small intestine, uterus, and vascular tissue cells while B2 receptor is known to be expressed only in specific tissue cells.

A variety of peptides and non-peptide antagonists of the B2 receptor of BK are known in the art. For example, in WO 2006/40004, WO 03/103671, WO 03/87090, WO 00/2349, WO 00/50418, WO 99/64039, WO 97/41104, WO 97/28153, WO 97/07115, WO 96/13485, EP 0 795 547, EP 0 796 848, EP 0 867 432, and EP 1 213 289 disclose quinoline compounds which are B2 receptor antagonists of BK.

 In addition, peptides that are antagonistic to bradykinin are described in European Patent No. 370,453 B1. In addition, it is known that peptides that are antagonistic to bradykinin can be used in the treatment of osteoarthritis or rheumatoid arthritis (AU 638, 350).

Their biological actions are mediated by two G-protein coupled membrane receptors designated B1 and B2, and the Bl and B2 receptors are cloned (Hess JF et al ., Biochem . Biophys . Res . Commun., 184: 260-268, 1992; Menke JG et al ., J. Biol . Chem ., 269 (34): 21583-21586, 1994), and mechanisms for controlling their expression, self-sustaining and signaling actions have been actively studied (Phagoo SB et al ., Mol . Pharmacol ., 56 (2): 325-333, 1999, Prado GN et al ., J. Cell . Physiol ., 193 (3) 275-286, 2002).

 Thus, there is a need for the development of highly sensitive receptor modulators with improved properties for bradykinin.

On the other hand, as a result of the research that Pseudomonas aeruginosa promotes the expression of bradykinin receptor, substances which are involved in the promotion of expression have been discovered through a highly selective process (Shin HS et al ., FEMS Immunol . Med ., Microbiol ., 63 (3): 418-426, 2011; Curr Microbiol ., 63 (2): 138-144, 2011).

Thus, the present inventors have made to develop a substance that controls the expression of the bradykinin receptor results sought, Pseudomonas aeruginosa (Pseudomonas aeruginosa- derived polypeptide is transferred from bacteria to animal cells by the Pseudomonas aeruginosa system to promote the expression of bradykinin receptor, and the Pseudomonas aeruginosa-derived polypeptide (Ndk) is involved in the side effects of bradykinin receptor- And can be used as a therapeutic agent for cardiovascular diseases and the like, thereby completing the present invention.

It is an object of the present invention to provide a pharmaceutical composition for the treatment of diabetic side effects or cardiovascular diseases comprising a Pseudomonas aeruginosa-derived polypeptide (Ndk) which promotes expression of a bradykinin receptor.

It is another object of the present invention to provide a screening method for a therapeutic agent using a Pseudomonas aeruginosa-derived polypeptide.

In order to achieve the above object, the present invention provides a pharmaceutical composition for treating a diabetic adverse effect or cardiovascular disease, which comprises a polypeptide (Ndk) having the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

The present invention also provides a screening method for a drug for the treatment of diabetic side effects or cardiovascular diseases comprising the steps of: (a) treating a cell having a bradykinin receptor-expressing ability with a polypeptide having the amino acid sequence of SEQ ID NO: 1, Confirming the level of dicinin receptor expression; (b) treating the bradykinin receptor-expressing cell with a candidate substance for treating diabetic side effects or heart disease to confirm a bradykinin receptor expression level; And (c) when the level of expression of step (b) is higher than the level of expression of the bradykinin receptor in step (a), the step of selecting the candidate substance as a therapeutic agent for diabetes or a cardiac disease.

The present invention also provides a method of screening for an inflammatory therapeutic agent comprising the steps of:

(a) treating a cell having a bradykinin receptor-expressing ability with a polypeptide having an amino acid sequence of SEQ ID NO: 1 to confirm a bradykinin receptor expression level; (b) treating the bradykinin receptor-expressing cell with an inflammatory therapeutic agent to confirm a bradykinin receptor expression level; And (c) when the level of expression in step (b) is lower than the level of expression of the bradykinin receptor in step (a), selecting the candidate agent as an inflammatory drug.

The nucleoside diphosphate kinase (Ndk) (SEQ ID NO: 1) derived from P. aeruginosa according to the present invention is involved in the expression control of the bradykinin receptor which is important for the control of the inflammatory reaction in various diseases, .

FIG. 1 shows the effect of a pscF mutant strain on the expression of bradykinin receptor by wild-type P. aeruginosa (a: BR1, b: BR2).
FIG. 2 shows the effect of Exo, ExoT and ExoY on the expression of bradykinin receptors (a: BR1, b: BR2), which are protein factors transmitted through the type III secretion system.
Fig. 3 shows the expression of bradykinin receptor by the culture supernatant obtained from wild-type P. aeruginosa and pscF mutant strain (a: BR1, b: BR2).
FIG. 4 shows Ndk as a new protein factor dependent on the type III secretion system (line 1: protein marker, line 2: wild type, line 3: STY mutant strain, line 4: STYN mutant strain).
FIG. 5 shows expression of bradykinin receptor by wild-type P. aeruginosa and Ndk mutant strain (a: BR1, b: BR2).

In the present invention, it was confirmed that a polypeptide derived from Pseudomonas aeruginosa is involved in the promotion of bradykinin receptor expression.

The activity of the bradykinin receptor of the present invention is regulated by a kininase enzyme and is known to be involved in various inflammatory reactions. For example, an antagonist to bradykinin receptor 2 may be used in the treatment of asthma, rhinitis, arthritis, colitis, pancreatitis, sepsis, edema, Alzheimer's disease and lung cancer. The agonist for bradykinin receptor 2 is known to be useful for the treatment of diabetes and various heart diseases such as hypertension, It is known to be useful for the treatment of cardiac hypertrophy and myocardial infarction.

The various kininase enzymes act on bradykinin to produce activated peptides that act as a major muscle to the bradykinin receptor, and their interaction with the bradykinin receptor modulates various inflammatory responses.

Accordingly, in one aspect, the present invention relates to a pharmaceutical composition for treating a diabetic adverse effect or cardiovascular disease, which comprises a polypeptide having the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

In the present invention, the cardiovascular disease may be selected from the group consisting of hypertension, cardiac hypertrophy, and myocardial infarction.

The amino acid sequence of Nucleoside diphosphate kinase (Ndk) from Pseudomonas aeruginosa is highly homologous to the Ndk of known bacteria and eukaryotes, and the role of Ndk in cell growth and alginate synthesis has been studied (Sundin GW et al., Mol Microbiol , 20 (5): 965-79, 1996).

On the other hand, the amino acid sequence of the present invention is known in the prior art and discloses the effect of Ndk on cell growth and alginate synthesis, but discloses that the amino acid sequence according to the present invention is involved in the expression of bradykinin receptor It was used for other purposes in point.

In order to confirm the induction of inflammatory reaction caused by P. aeruginosa infection as a pathogenic bacterium causing respiratory diseases, A549 cells were treated with cultured P. aeruginosa in order to examine the expression of bradykinin receptor in A549 cells, a lung epithelial cell Next, the expression of bradykinin receptor in A549 cells was confirmed. As a result, it was confirmed that the expression of bradykinin receptor was significantly decreased in the pscF mutant strain compared to the wild type (Fig. 1).

These results suggest that the Pseudomonas aeruginosa protein factor involved in the regulation of bradykinin receptor expression is associated with secretion by the type III secretion system.

In one embodiment of the present invention, in order to confirm the expression of bradykinin receptor by three types of P. aeruginosa protein elements known as ExoS, ExoT and ExoY, which are known to be secreted by the Type III secretion system, Real-time Q-PCR was performed using the STY mutant strain. As a result, it was confirmed that unlike the pscF mutant strain, the expression of bradykinin receptor was not affected at all (FIG. 2).

As a result, it was confirmed that ExoS, ExoT, and ExoY, which are the Pseudomonas aeruginosa protein factors according to the present invention, are not involved in the expression control of the bradykinin receptor.

In the present invention, the Pseudomonas aeruginosa protein factor is related to the type III secretion system for the expression of bradykinin receptor, but the ExoS, ExoT, ExoY protein factor known to be involved is a therapeutic pharmaceutical composition which does not affect the expression do.

 In the present invention, the term " type III secretion system " refers to a protein attached to several gram-negative bacteria, which is secreted when a pathogenic bacterium contacts a eukaryote to induce bacterial infection.

 In the present invention, in order to examine the effect of the factors secreted in the cultivation process of P. aeruginosa, the pscF mutant strain was cultured, and the culture solution in which the cells were removed was treated with A549 cells and then the expression of the bradykinin receptor was examined. And showed no significant difference from the culture solution obtained from P. aeruginosa (Fig. 3).

This result shows that the effect of Pseudomonas aeruginosa on the expression of bradykinin receptor is not due to the increase of bradykinin receptor expression in A549 cells. Although the factor was not secreted, it was confirmed to be regulated by the type III secretion system.

As used herein, the term " mutant " or "variant" means an amino acid sequence in which one or more amino acids have been altered. The variant may have a "conserved" change, wherein the substituted amino acid has similar structural and chemical properties such as, for example, changing leucine to isoleucine. Very rarely, a variant can have a "non-conserved" modification, such as changing glycine to tryptophan. Similar small mutations may include amino acid deletions or insertions or both. Guidance for determining amino acid residues that can be substituted, inserted or deleted without destroying biological or immunological activity can be found, for example, using a computer program such as the DNASTAR software known in the art.

In the present invention, low calcium and low magnesium conditions for activating the type III secretion system were established to identify the protein factor, and the factors obtained by concentrating the protein factors secreted from the wild type, STY mutant strain and STYN mutant strain were analyzed and compared . ExoN mutant was a mutant strain secreted by the type III secretion system and was able to select new protein factors. As a result, the new protein factor was found to be Ndk (nucleoside diphosphate kinase (Ndk); SEQ ID NO: 1), which confirmed that Ndk is involved in the regulation of bradykinin receptor expression (Fig. 4) .

In another embodiment of the present invention, an Ndk mutant strain was prepared and the effect of the strain on the expression of the bradykinin receptor was compared with the wild type strain. The cultured strain was treated with A549 cells and real-time Q-PCR was performed on bradykinin receptor expression. As a result, the expression of bradykinin receptor by the Ndk mutant strain was significantly reduced (FIG. 5). This indicates that Ndk delivered to animal cells through the type III secretion system is a protein factor responsible for the expression of bradykinin receptor by P. aeruginosa.

 In addition, since Ndk of the present invention is involved in the regulation of expression of bradykinin receptor, it can be used as a reporter for target detection of novel drug development screening or signaling mechanism protein detection, which can be used for treating various diseases.

In another aspect, the present invention relates to a screening method for a therapeutic agent for diabetic side effects or cardiovascular diseases comprising the steps of: (a) administering to a cell having bradykinin receptor-expressing ability a polypeptide having the amino acid sequence of SEQ ID NO: 1 (Ndk) to determine the level of expression of the bradykinin receptor; (b) treating the bradykinin receptor-expressing cell with a candidate substance for treating diabetic side effects or heart disease to confirm a bradykinin receptor expression level; And (c) when the level of expression of step (b) is higher than the level of expression of the bradykinin receptor in step (a), the step of selecting the candidate substance as a therapeutic agent for diabetes or a cardiac disease.

The term "screening" in the present invention means to find something having a specific property such as susceptibility or activity to a specific chemical substance such as an antibiotic substance or an enzyme.

In the present invention, "the level of protein expression level" is used to determine the levels of B1 and B2, B1 and B2 receptors, NF-κB (tumor necrosis factor-α) in a subject after administration of a candidate substance to screen for a preventive and therapeutic agent for heart disease and inflammation, Or the step of confirming the presence and the degree of expression of the protein expressed from the mRNA of the green fungus-derived polypeptide, preferably, the amount of the protein can be confirmed by using an antibody that specifically binds to the protein of the gene . Expression of the protein can be confirmed by immunoassay using a specific antibody against each of the B1 and B2, B1 and B2 receptors, NF-κB (tumor necrosis factor-α) And the analysis methods for this are Western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, ouchterlony immunodiffusion, rocket but are not limited to, immunoassays, rocket immunoelectrophoresis, tissue immuno staining, immunoprecipitation assays, complement fixation assays, FACS and protein chips. When an antibody is used as a method of confirming the amount of the protein, a second antibody linked to a specific and detectable label on the target protein can be added. The detection can also be performed by adding the second antibody, A third antibody having a binding affinity and linked to a detectable label may be added. As the label which can be detected in the second antibody or the third antibody, an enzyme capable of exhibiting color development upon culturing with a suitable chromogenic substrate may be used. Detectable moieties can include compositions detectable by spectroscopic, enzymatic, photochemical, biochemical, biotechnological, immunochemical, electrical, optical or chemical means, including, for example, fluorescent markers and dyes, magnetic labels , Linked enzymes, mass spectroscopic tags, spin markers, electron transfer donors and recipients, but are not limited to the above examples.

In addition, Ndk according to the present invention is likely to interact with the NF-κB mechanism, so that it can be used for controlling inflammatory reaction and controlling the infection of P. aeruginosa and for studying the disease mechanism of protein molecules derived from P. aeruginosa on animal cells.

(A) a polypeptide having the amino acid sequence of SEQ ID NO: 1 (Ndk) in a cell having a bradykinin receptor-expressing ability, and a method for screening the anti-inflammatory drug, Lt; RTI ID = 0.0 > bradykinin < / RTI > receptor expression level; (b) treating the bradykinin receptor-expressing cell with an inflammatory therapeutic agent to confirm a bradykinin receptor expression level; And (c) when the level of expression in step (b) is lower than the level of expression of the bradykinin receptor in step (a), selecting the candidate agent as an inflammatory drug.

The compositions of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of the compositions.

The composition of the present invention may be administered orally or parenterally, and it is preferable to select parenterally when injecting parenterally or intraperitoneally, intramuscularly, subcutaneously, intravenously, intramuscularly or intrasternally , But is not limited thereto.

The composition of the present invention can be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterilized injection solutions according to conventional methods . Examples of carriers, excipients and diluents that can be included in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose ), Lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to the commonly used simple diluent, liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included.

Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of suppository bases include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like.

The preferred dosage of the composition of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, the route of administration and the period of time, but can be appropriately selected by those skilled in the art. However, for the desired effect, the composition is preferably administered at a dose of 0.0001 to 1 g / kg per day, preferably 0.001 to 200 mg / kg per day, but is not limited thereto. The above administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example  1: Pseudomonas aeruginosa Bradykinin  Receptor expression and type III secretion system Effect of

Pseudomonas aeruginosa is a pathogenic bacterium that causes respiratory disease. In order to confirm the induction of inflammatory reaction by P. aeruginosa infection, expression of bradykinin receptor was measured in lung epithelial cell A549 (KCLB 10185) cells. A549 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum at 37 ° C and 5% CO 2. Pseudomonas aeruginosa was cultured in LB medium (0.5%; tryptone, 1% ; all w / v) for 16 hours at 37 ° C., cultured for 4 hours to allow the cultured pseudomonas aeruginosa to reach 5-10 cfu per A549 cell, and A549 cells were cultured in TRIZol RNA was obtained. Total RNA was obtained by reverse transcription kit and real-time Q-PCR was performed using SYBR Green PCR Master Mix. The following primers were used to investigate the expression of the bradykinin receptor.

Bradykinin receptor 1 (BK R1);

BK R1-F: 5'-CAACTGAACGTGGCAGAAATCTAC-3 '(SEQ ID NO: 2)

BK R1-R: 5'-CAAGCCCAAGACAAACACCAG-3 '(SEQ ID NO: 3)

Bradykinin receptor 2 (BK R2);

 BK R2-F: 5'-GGGCACACTGCGGACCT-3 '(SEQ ID NO: 4)

BK R2-R: 5'-GCGTTTGCTCACTGTCTGCTC-3 '(SEQ ID NO: 5)

The type III secretion system, which is one of the secretion mechanisms, has the function of transferring bacterial protein factors into animal cells. Therefore, pscF mutant strain (Marx CJ, BMC Res Notes., 26; 1: 2008) are mutants that induce mutations in the type III secretion system and do not secrete bacterial protein factors or transfer them to animal cells. As shown in FIG. 1 (a) and FIG. 1 (b), the expression of the bradykinin receptor was significantly decreased in the pscF mutant strain compared with the wild type strain.

These results suggest that P. aeruginosa protein factors involved in the regulation of bradykinin receptor expression are associated with secretion by the type III secretion system.

STY mutant strains mutating all three P. aeruginosa protein factors (ExoS, ExoT, and ExoY) known to be secreted by the type III secretion system (Bichsel C et al ., PLoS One ., 6 (1): e16465, 2011) was used to perform real-time Q-PCR. As a result, unlike the pscF mutant strain, as shown in Figs. 2 (a) and 2 (b), the expression of bradykinin receptor was not affected at all. This means that the exo, exo, and exoY proteins of pseudomonas aeruginosa do not participate in the regulation of expression of bradykinin receptors.

Example  2: Pseudomonas aeruginosa culture solution Bradykinin  Effect on receptor expression

Expression of the bradykinin receptor was associated with the type III secretion system, but the previously known ExoS, ExoT and ExoY protein factors did not affect expression. Therefore, pscF mutant cell line was cultured in order to confirm the effect of the factors secreted by the culture of P. aeruginosa. The resulting culture was filtered with 0.2 μm pore size filter to remove P. aeruginosa. Real-time Q-PCR was performed on A549 cells treated with 50-100 μl of the filtered culture. As a result, as shown in Figs. 3 (a) and 3 (b), there was no significant difference from the culture obtained in the wild type. This result means that the effect is not due to secreted factors.

Example  3: type III secretion system New secreted by Protein factor  Excavation and Bradykinin  Effect on receptor expression

From the results of Examples 1 and 2, it was confirmed that the expression of bradykinin receptor was affected by an unknown Pseudomonas aeruginosa protein factor and that the protein factor was not secreted but regulated by the type III secretion system. Therefore, in order to identify the protein factors, the factors of activation of the type III secretion system such as low calcium and low magnesium conditions and the factors obtained by concentrating the protein factors secreted from the wild type, STY mutant strain and STYN mutant strain were analyzed Respectively.

ExoN mutant was a mutant strain secreted by the type III secretion system and was able to select new protein factors. As a result of protein analysis, as shown in Fig. 4, the new protein factor was found to be nucleoside diphosphate kinase (Ndk) (SEQ ID NO: 1), which means that Ndk is likely to be involved in the regulation of bradykinin receptor expression.

Example  4: Ndk ( nucleoside diphosphate kinase ) Mutant strain production

To confirm the results of Example 3, an Ndk mutant strain was constructed and the effect of the strain on the expression of the bradykinin receptor was compared with the wild type strain. Cultured strains were treated for 5-10 cfu per A549 cell (KCLB 10185) for 4 h and bradykinin receptor expression was performed by real-time Q-PCR. As a result, as shown in FIG. 5, it was confirmed that the expression of bradykinin receptor was significantly reduced by the Ndk mutant strain. These results suggest that Ndk delivered to animal cells through the type III secretion system is a protein factor responsible for the expression of bradykinin receptor by P. aeruginosa.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereto will be. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

<110> KOREA UNIVERSITY Research and Business Foundation <120> Composition for Treating Heart Disease and Inflammation          Containing Polypeptide Involved in the Activation of Bradykinin          Receptor Derived from Pseudomonas aeruginosa <130> P13-B024 <160> 5 <170> Kopatentin 2.0 <210> 1 <211> 143 <212> PRT <213> PA3807 (nucleoside diphosphate kinase (Ndk)) <400> 1 Met Ala Leu Gln Arg Thr Leu Ser Ile Ile Lys Pro Asp Ala Val Ser   1 5 10 15 Lys Asn Val Ile Gly Glu Ile Leu Thr Arg Phe Glu Lys Ala Gly Leu              20 25 30 Arg Val Val Ala Lys Met Val Gln Leu Ser Glu Arg Glu Ala Gly          35 40 45 Gly Phe Tyr Ala Glu His Lys Glu Arg Pro Phe Phe Lys Asp Leu Val      50 55 60 Ser Phe Met Thr Ser Gly Pro Val Val Glu Val Leu Glu Gly Glu  65 70 75 80 Asp Ala Ile Ala Lys Asn Arg Glu Leu Met Gly Ala Thr Asp Pro Lys                  85 90 95 Lys Ala Asp Ala Gly Thr Ile Arg Asa Asp Phe Ala Val Ser Ile Asp             100 105 110 Glu Asn Ala Val His Gly Ser Asp Ser Glu Ala Ser Ala Ala Arg Glu         115 120 125 Ile Ala Tyr Phe Phe Ala Ala Thr Glu Val Cys Glu Arg Ile Arg     130 135 140 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> BK R1-F <400> 2 caactgaacg tggcagaaat ctac 24 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> BK R1-R <400> 3 caagcccaag acaaacacca g 21 <210> 4 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> BK R2-F <400> 4 gggcacactg cggacct 17 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> BK R2-R <400> 5 gcgtttgctc actgtctgct c 21

Claims (5)

A pharmaceutical composition for the treatment of diabetic side effects or cardiovascular diseases, which comprises a polypeptide having the amino acid sequence of SEQ ID NO: 1 as an active ingredient.
The therapeutic pharmaceutical composition according to claim 1, wherein the polypeptide is associated with secretion by a type III secretion system.
The therapeutic pharmaceutical composition according to claim 1, wherein the cardiovascular disease is selected from the group consisting of hypertension, cardiac hypertrophy and myocardial infarction.
A screening method of a therapeutic agent for diabetic side effects or cardiovascular diseases comprising the steps of:
(a) treating a cell having a bradykinin receptor-expressing ability with a polypeptide having an amino acid sequence of SEQ ID NO: 1 (Ndk) to confirm a bradykinin receptor expression level;
(b) treating the bradykinin receptor-expressing cell with a candidate substance for treating diabetic side effects or heart disease to confirm a bradykinin receptor expression level; And
(c) when the level of expression of step (b) is higher than the level of expression of the bradykinin receptor in step (a), the candidate substance is selected as a therapeutic agent for diabetic side effects or heart disease.
A screening method of an inflammatory therapeutic agent comprising the steps of:
(a) treating a cell having a bradykinin receptor-expressing ability with a polypeptide having an amino acid sequence of SEQ ID NO: 1 (Ndk) to confirm a bradykinin receptor expression level;
(b) treating the bradykinin receptor-expressing cell with an inflammatory therapeutic agent to confirm a bradykinin receptor expression level; And
(c) when the expression level of step (b) is lower than that of the bradykinin receptor expression level in step (a), selecting the candidate agent as an inflammation therapeutic agent.

Images