KR20210018620A - A pharmaceutical composition containing cell-transducing endophilin-A1 fusion protein for preventing and treating neuronal disorder - Google Patents

Abstract

The present invention provides a drug for treating neurological diseases with few or no side effects and excellent effects. The present invention relates to a pharmaceutical composition for treating neurological diseases comprising a cell-permeable endophylline-A1 fusion protein, wherein the intracellular endophylline-A1 fusion protein increases cell viability against hydrogen peroxide toxicity, and decreases a level of intracellular reactive oxygen species and hydrogen peroxide-induced DNA fragmentation. The above results suggest that the endophyllin-A1 fusion protein has a protective effect against apoptosis and has a therapeutic effect to protect nerve cells from damage to neurological diseases.

Description

A pharmaceutical composition containing cell-transducing endophilin-A1 fusion protein for preventing and treating neuronal disorder}

The present invention relates to a pharmaceutical composition for preventing and treating neurological diseases comprising a cell-permeable endophylline-A1 fusion protein.

In the clinic, paraplegia develops into unpredictable complications between 1 and 21 days after surgery, depending on the duration and extent of ischemia after reconstruction of descending and thoracic aortic aneurysms (Crawford and Coseelli, 1991; Etz et al, 2006; Kouchoukos, 1991; Panthee and Ono, 2015). Rabbits are the best animals for spinal cord ischemia studies as they do not have ancillary blood vessels in the thoracic spinal cord (Mazensky et al, 2011). Obstruction of the abdominal aorta below the kidney causes massive but delayed nerve damage in the abdominal processes of the rabbit spinal cord (Zivin and DeGirolami, 1980). One of the most widely accepted hypotheses regarding delayed neuronal death is that high levels of unsaturated fatty acids and high oxygen consumption by neurons lead to increased production of reactive oxygen species (ROS), followed by DNA and cell membrane damage. (Lin et al, 2003; Song et al, 2013). Free radicals generated during ischemia/reperfusion cause protein destruction, lipid peroxidation and DNA damage in neurons (Olmez and Ozyurt, 2012). Endogenous defense mechanisms such as antioxidants are activated to inhibit excess free radicals and weaken cell damage. Several studies have shown that antioxidant treatment significantly reduces nerve damage induced by spinal cord ischemia (Hwang et al, 2015; Kim et al, 2012; Zhu et al, 2012).

Endophilin-A1 is encoded by the SH3GL2 gene, and maintains homeostasis by mediating protein-protein interactions important for intracellular division of epidermal growth factor receptor (EGFR). Downregulation of endophylline-A1 is associated with cancer development. The SH3GL2 gene, which encodes endophylline-A1, is a tumor suppressor gene. Endophylline-A1 is mainly distributed in the central nervous system, and is particularly concentrated in the presynaptic ganglion. Endophylline-A1 plays an important biological role in the proliferation, differentiation and other physiological functions of cells, mainly through the SH3 domain protein and other downstream proteins.

However, the function or effect of endophylline-A1 on neuronal cell death by reactive oxygen species has not yet been clearly identified.

An object of the present invention is to provide a pharmaceutical composition for effective prevention or treatment of neurological diseases caused by nerve cell damage and death.

In order to solve the above problems, the present inventors confirmed that protein transport domains such as PEP-1 and HIV Tat peptides recombined with endophylline-A1 and penetrated into NSC34 cells, and endophyllin-A1 fusion protein, which is cell-penetrating against oxidative stress. The protective effect of was studied.

The present inventors attempted to determine whether the endophylline-A1 fusion protein bound to the protein transport domain has a protective effect on neuronal cell death induced by oxidative stress in vitro. In order to study the potential effect of the endophylline-A1 fusion protein on ischemic neuronal death, the present inventors constructed an endophylline-A1 fusion protein that can penetrate into cells. The endophylline-A1 fusion protein effectively penetrated into neurons in a concentration-dependent and time-dependent manner. The endophylline-A1 fusion protein permeated into the cells increased the cell viability against hydrogen peroxide toxicity and lowered the level of reactive oxygen species in the cells. These results indicate that the endophylline-A1 fusion protein exhibits a protective effect against apoptosis occurring in vitro and can be used as a therapeutic agent for neurological diseases related to oxidative stress. To explain the composition of the present invention in more detail, in one embodiment of the present invention, the present inventors first developed an endophylline-A1 fusion protein expression vector that can overexpress and easily purify the endophyllin-A1 fusion protein. This expression vector contains a human endophylline-A1 protein, at least one protein transport domain consisting of 7 to 21 amino acids, and a cDNA capable of expressing 6 histidine residues at the N-terminus. As an example, the amino acid sequence of the endophylline-A1 fusion protein is characterized by SEQ ID NO: 1.

Using this expression vector, the endophyllin-A1 fusion protein was overexpressed in E. coli and purified using Ni-affinity chromatography. It was confirmed by Western blot that the endophylline-A1 fusion protein was transported to the cells in a time and concentration dependent manner. The endophyllin-A1 fusion protein permeated into the cell was continuously maintained for up to 12 hours in the cell, and apoptosis caused by oxidative stress was suppressed.

These results imply that the endophylline-A1 fusion protein is well permeated into the cell and shows the function of the endophylline-A1 protein well in the cell. Therefore, this endophylline-A1 fusion protein offers a possibility to be applied to neurological diseases such as protecting nerve cells from symptoms related to reactive oxygen species.

The pharmaceutical composition containing the cell-penetrating endophylline-A1 fusion protein as an active ingredient may be formulated in an oral or injectable form by a conventional method by blending with a carrier commonly accepted in the pharmaceutical field. Oral compositions include, for example, tablets and gelatin capsules, which, in addition to the active ingredient, include diluents (e.g. lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycine), lubricants (e.g. silica, talc). , Stearic acid and its magnesium or calcium salt and/or polyethylene glycol), and the tablets also contain a binder (e.g. magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and/or polyvinyl pyrrolidone). ), and in some cases a disintegrant (eg starch, agar, alginic acid or sodium salt thereof) or a boiling mixture and/or an absorbent, colorant, flavoring and sweetening agent. The composition for injection is preferably an isotonic aqueous solution or suspension, and the compositions mentioned are sterilized and/or contain adjuvants (e.g., preservatives, stabilizers, wetting or emulsifying agent solution accelerators, salts/or buffers for controlling the osmotic pressure). In addition, they may contain other therapeutically useful substances.

The pharmaceutical preparation thus prepared may be administered orally, or parenterally, ie, intravenously, subcutaneously, intraperitoneally or topically, as desired. The dose can be administered by dividing the daily dose of 0.0001 to 100 mg/kg in 1 to several times. The dosage level for a specific patient may vary depending on the patient's weight, age, sex, health status, administration time, administration method, excretion rate, and disease severity.

Further, the present invention provides a pharmaceutical composition useful for the prevention and treatment of neurological diseases, characterized in that it comprises the endophylline-A1 fusion protein as an active ingredient and a pharmaceutically acceptable carrier.

The present invention also provides a method for efficiently delivering endophyllin-A1 protein into cells. Intracellular delivery of the endophyllin-A1 protein molecule according to the present invention is carried out by constructing a fusion protein in which a protein transport domain such as an HIV Tat peptide is covalently linked. An example of the protein transport domain of the present invention is an HIV Tat peptide. However, the protein transport domain of the present invention is not limited to the HIV Tat peptide, and it is in the field to which the present invention pertains to producing a peptide that functions similar to the HIV Tat peptide by partial substitution, addition, or absence of the amino acid sequence of the HIV Tat peptide. Because it is easy for those skilled in the art, protein transport consisting of 7 to 21 amino acids and containing 4 or more lysine or arginine, and a protein transport that performs the same or similar protein transport function by partial substitution of amino acids therefrom It will be apparent that fusion proteins using domains are also within the scope of the present invention.

Specifically, the present invention is a vector comprising an oligonucleotide encoding an endophylline-A1 fusion protein, an endophylline-A1 fusion protein, an oligonucleotide encoding an endophylline-A1 fusion protein, and a neurological disease treatment comprising the fusion protein Or it relates to a pharmaceutical composition for the purpose of preventing and a functional food composition for preventing or improving neurological diseases comprising the fusion protein.

The polynucleotide encoding the endophylline-A1 fusion protein is specifically characterized by SEQ ID NO: 1.

Foods to which the cell-permeable endophylline-A1 fusion protein of the present invention can be added include various foods, such as beverages, gums, teas, vitamin complexes, health supplements, and the like, pills, powders, granules, It can be used as a pill, tablet, capsule or beverage. At this time, the amount of the endophylline-A1 fusion protein in food or beverage is generally 0.01 to 15% by weight, preferably 0.2 to 10% by weight of the total food weight in the case of the health food composition of the present invention, and health In the case of a beverage composition, it may be added in an amount of 0.1 to 30 g, preferably 0.2 to 5 g, based on 100 ml. The health beverage composition of the present invention of the present invention has no particular limitation on the liquid component except for containing the endophylline-A1 fusion protein as an essential component in the indicated ratio, and various flavoring agents or natural carbohydrates, etc. are added as in ordinary beverages. It can be contained as an ingredient. Examples of the above-described natural carbohydrates include monosaccharides such as disaccharides such as glucose and fructose, polysaccharides such as maltose and sucrose, for example, conventional sugars such as dextrin and cyclodextrin, and xylitol. , Sugar alcohols such as sorbitol and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (taumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention. In addition to the above, the composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and heavy weight agents (cheese, chocolate, etc.), pectic acid and salts thereof, alginic acid and its Salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonates used in carbonated beverages, and the like may be contained. In addition, the compositions of the present invention may contain natural fruit juice and flesh for the production of fruit juice beverages and vegetable beverages. These components may be used independently or in combination. The proportion of these additives is not critical, but is generally selected from 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

Definitions of main terms used in the detailed description of the present invention are as follows.

The term "endophylline-A1 fusion protein" includes a protein transport domain and an endophylline-A1 protein, and is a genetic fusion or chemical bond between a protein transport domain and a target protein (ie, endophylline-A1 protein in the present invention). It means a covalent bond formed by the complex. In the present specification, since the HIV-Tat protein transport domain was used as a specific example, "endophylline-A1 fusion protein" was mixed with "Tat-SH3GL2" and "Tat-SH3GL2 fusion protein".

"Target protein" refers to a molecule that is not a transport domain or fragment thereof that cannot enter the target cell by nature or cannot enter the target cell at an inherently useful rate, and the molecule itself or the transport domain-target protein complex before fusion with the transport domain. It means the target protein part. The target protein includes a polypeptide, a protein, and a peptide, and in the present invention, it means an endophylline-A1 protein.

"Fusion protein" refers to a transport domain and a portion of one or more target proteins, and refers to a complex formed by genetic fusion or chemical bonding between the transport domain and the target protein.

In addition, the "genetic fusion" means a linear, covalent linkage formed through genetic expression of a DNA sequence encoding a protein. In addition, "target cell" refers to a cell to which a target protein is delivered by a transport domain, and the target cell refers to a cell inside or outside the body. That is, target cells are meant to include cells in the body, that is, cells constituting organs or tissues of living animals or humans, or microorganisms found in living animals or humans. In addition, target cells are meant to include extracorporeal cells, that is, cultured animal cells, human cells, or microorganisms.

The "protein transport domain" in the present invention is a polymer organic compound, such as oligonucleotide, peptide, protein, oligosaccharide or polysaccharide, etc. by covalent bonding to introduce the organic compounds into cells without requiring a separate receptor, carrier, or energy. Say what you can.

In addition, in the present specification, "transport", "penetration", "transport", "transport", "permeation", and "pass through" expressions for "introducing" proteins, peptides, and organic compounds into cells were used interchangeably.

The endophylline-A1 fusion protein of the present invention is composed of 7 to 21 amino acid residues, and a transport domain containing 3/4 or more of arginine or lysine residues is covalently bonded to at least one end of endophylline-A1 to improve cell penetration efficiency. Endophylline-A1 fusion protein. In addition, the transport domain refers to one or more of HIV Tat 49-57 residues, Pep-1 peptide, oligolysine, oligoarginine, or oligo (lysine, arginine). In addition, the endophylline-A1 fusion protein amino acid of the present invention. The sequence includes SEQ ID NO: 2 and the like. In the production of endophylline-A1 fusion protein, fusion proteins of various sequences can be obtained depending on the selection of the restriction site sequence, etc., which is obvious to a person of ordinary skill in the art to which the present invention belongs. It is obvious that the above amino acid sequence is only exemplary, and that the endophylline-A1 fusion protein amino acid sequence is not limited to the sequence listed above.

In addition, the present invention provides a pharmaceutical composition for the prevention and treatment of neurological diseases, including the endophylline-A1 fusion protein as an active ingredient and a pharmaceutically acceptable carrier.

In addition, the present invention provides a health functional food composition for preventing or improving neurological diseases, using the endophylline-A1 fusion protein as an active ingredient.

The present invention is composed of 7 to 21 amino acids, a protein transport domain containing 4 or more lysine or arginine is covalently bonded to at least one end of the endophylline-A1 protein cell-transducing endophylline-A1 fusion It's about protein. In addition, the endophylline-A1 fusion protein may be substituted with other amino acid(s) of similar polarity in which one or more amino acids function in the sequence equally functionally according to silent change. Amino acid substitutions in a sequence can be selected from other members of the class to which the amino acid belongs. For example, the hydrophobic amino acid class includes alanine, valine, leucine, isoleucine, phenylalanine, valine, tryptophan, proline and methionine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. Positive basic amino acids include arginine, lysine and histidine. Acidic amino acids with negative charges include aspartic acid and glutamic acid. In addition, fragments or derivatives thereof having a certain range of homology between the fusion protein of the present invention and the amino acid sequence, such as the same and similar biological activity within the range of 85-100%, are included in the scope of the present invention.

In the present invention, the endophyllin-A1 fusion protein permeated into the cells increased the cell viability against hydrogen peroxide toxicity. These results indicate that the endophylline-A1 fusion protein has a protective effect against neuronal cell death by reactive oxygen species, and that the cell-permeable endophylline-A1 fusion protein is useful as a pharmaceutical composition for preventing and treating neurological diseases.

Figure 1 shows the construction of the Tat-endophylline-A1 expression vector on the pET-15b vector. Synthetic Tat oligomers were cloned into the NdeI and XhoI sites, and human endophylline-A1 cDNA was cloned into the XhoI and BamHI sites of pET-15b. (A) shows the construction of an expression vector of endophylline-A1 and Tat-endophylline-A1 fusion proteins. The Tat-endophylline-A1 fusion protein contains six histidine residues. (B) is the expression of each protein by adding IPTG, and then the purified endophylline-A1 protein and Tat-endophylline-A1 fusion protein were purified using 15% SDS-PAGE, followed by western blotting with anti-rabbit polyhistidine antibody. One result.
Figure 2 shows the penetration of Tat-endophylline-A1 fusion protein into NSC34 cells. (A) is a Tat-endophylline-A1 fusion protein (0.5-5 μM) and an endophylline-A1 protein (0.5-5 μM) treated in a culture medium for 1 hour, (B) is a Tat-endophylline-A1 fusion protein And endophylline-A1 protein was treated in the culture medium for 15 to 60 minutes and analyzed by Western blotting. (C) The intracellular distribution of the Tat-endophylline-A1 fusion protein was visualized with a confocal fluorescence microscope. In (D), in order to investigate the intracellular stability of the Tat-endophylline-A1 fusion protein, the Tat-endophylline-A1 fusion protein (5 μM) was cultured for 1 to 48 hours and analyzed by Western blotting.
3A shows hydrogen peroxide (100 μM) was added to NSC34 cells pretreated with Tat-endophylline-A1 fusion protein or endophylline-A1 protein for 1 hour, respectively, and allowed to stand for 1 hour, and then cell viability was measured by colorimetric analysis using MTT assay. One result.
3B is a result of measuring the level of reactive oxygen species in cells by DCF-DA staining.
3C is a result of measuring DNA fragmentation by TUNEL staining.

Hereinafter, the configuration of the present invention will be described in more detail with reference to specific embodiments. However, it is obvious to those of ordinary skill in the art that the scope of the present invention is not limited by the description of the examples. In particular, in the embodiment of the present invention, the HIV Tat peptide was used as the protein transport domain constituting the endophylline-A1 fusion protein, but the Tat peptide binds to the N-terminus if one of ordinary skill in the art belongs to the present invention. By applying the endophylline-A1 fusion protein, various protein transport domains such as PEP-1 peptide, oligoarginine, oligolysine and oligo (arginine + lysine) are transferred to one of the N-terminus or C-terminus of endophylline-A1. The fusion protein bound to the above can be easily formed, and although there may be some differences between such fusion proteins, it can be easily seen that they exhibit similar activities.

material

Ni ^{2 +-} nitrilotriacetic acid Sepharose Superflow was purchased from Qiagen, PD-10 column chromatography (Amersham, Brauncschweig, Germany). Mouse motor neuron NSC34 cells were provided by the Korea Cell Line Research Foundation (KCLF). DCF-DA (2',7'-Dichlorofluorescein diacetate) and Bcl-2, Bax, β-actin, P-p53, p53, casphase 3, casphase 9, p-p38, p38, p-Erk, Erk, Primary antibodies of p-JNK and JNK were purchased from Cell signaling Technology (Beverly, MA, USA) and Santa Cruz Biotechnology (Santa Cruz, CA, USA). All other reagents were used with special grade products.

Expression and purification of Tat-endophylline-A1 fusion protein

Expression vectors of endophylline-A1 protein and Tat-endophylline-A1 fusion protein were constructed. The SH3GL2 gene encoding the human endophylline-A1 protein was amplified by a polymerase chain reaction using two primers along with cDNA. The sense primer is 5'-CTCGAGGGCAACGCGCAG-3', and a restriction enzyme site called Xho I is present on the 5'side . The antisense primer is 5'-GGATCCTCAGGAATCTTCGGACTC-3', and a restriction enzyme site called BamH I exists on the 5'side. The resultant obtained through the polymerase chain reaction was ligated to a TA vector, cut using Xho I and BamH I, and ligated to an expression vector to prepare a Tat-SH3GL2 fusion protein. Similarly, the control endophylline-A1 protein was prepared using a vector lacking the Tat peptide. The recombinant Tat-endophylline-A1 plasmid was transformed with E. coli BL21, followed by induction with 0.5mM IPTG (isopropyl-β-D-thiogalactoside), and cultured at 18°C overnight. The cultured cells were pulverized by ultrasonic waves and purified using a Ni ^{2 +-} nitrilotriacetic acid Sepharose Superflow column to obtain a Tat-endophylline-A1 fusion protein. The protein concentration was determined by the Bradford method using bovine serum albumin as a standard.

Introduced Tat-endophylline-A1 fusion protein into NSC34 cells

NSC34 cells maintain the conditions of 37°C, 95% air and 5% CO ₂ , 10% fetal calf serum (FBS) and 5 mM NaHCO ₃ , antibiotics (100 μg/ml streptomycin, 100 U/ml penicillin), 20 mM It was cultured under humid conditions using DMEM (Dulbecco's Modified Eagle's Medium) containing HEPES/NaOH (pH 7.4).

The time dependence and the concentration dependence of the Tat-endophylline-A1 fusion protein and the control endophylline-A1 protein were evaluated. The cells were treated in a 60 mm dish at different times (15-60 minutes) and the dosage of each protein (0.5-5 μM). After treatment with trypsin-EDTA (Gibco) and washing with PBS, the fusion protein permeated into the cells was quantified by the Bradford method and analyzed by Western blot.

Western blot analysis

For western blot analysis, the protein in the cell pulverization solution was separated with a 12% SDS polyacrylamide gel, and then the protein in the gel was electrophoresed to a nitrocellulose membrane (Amersham, UK). The membrane was blocked with TBS-T buffer (25 mM Tris-HCl, 140 mM NaCl, 0.1% Tween 20, pH 7.5). The membrane was analyzed by Western blot using the manufacturer's recommended primary antibody. The bound antibody complex was detected according to the manufacturer's instructions (Amersham, Franklin Lakes, NJ, USA) using an enhanced chemiluminescent agent.

Confocal microscopy

In order to detect Tat-endophylline-A1 fusion protein and endophylline-A1 protein in NSC34 cells, cells were seeded on glass coverslips, and Tat-endophylline-A1 fusion protein and endophylline-A1 protein were added at a concentration of 5 μM for 1 hour. Treated during. The cells were washed twice with PBS and then fixed with 4% paraformaldehyde for 5 minutes at room temperature. NSC34 cells were blocked for 40 minutes with PBS (PBS-BT) containing 3% bovine serum albumin and 0.1% Triton X-100 for 40 minutes at room temperature, and washed with PBSBT. The primary antibody (His-probe, Santa Cruz Biotechnology) was diluted at a ratio of 1:10000, and the secondary antibody (Alexa fluor 488, Invitrogen) was diluted at a ratio of 1:15000 and incubated for 1 hour at room temperature in a dark place. Cell nuclei were stained with DAPI (4',6-diamidino-2-phenylindole 1 mg/ml; Roche, Mannheim, Germnay) for 3 minutes. Stained NSC34 cells were observed under an Fv-300 confocal fluorescence microscope (Olympus, Tokyo, Japan).

Cell viability analysis

The survival rate of NSC34 cells treated with hydrogen peroxide was confirmed by colorimetric analysis using MTT {3-(4,5-dimethylthiazol -2-yl)-2,5-dipheyltetrazolium bromide}. Apoptosis was induced by exposing the cells to 100 μM hydrogen peroxide for 1 hour with or without pre-treatment of 0.5-5 μM of Tat-endophylline-A1 fusion protein. Absorbance was measured at 570 nm with an ELISA microplate reader (Labsystems Multiskan MCC/340). Cell viability was expressed as a percentage of the untreated control.

Measurement of the level of reactive oxygen species in cells

The level of reactive oxygen species in cells was measured using DCF-DA (2',7'-dichlorodihydrofluorescein diacetate). DCF-DA is converted to DCF in cells by reactive oxygen species and fluoresces. We compared the levels of reactive oxygen species when treated with Tat-endophylline-A1 fusion protein and without treatment in NSC34 cells. Tat-endophylline-A1 fusion protein was treated with 5 μM for 1 hour, and then hydrogen peroxide was treated with 700 μM for 30 minutes. Then, it was washed twice with PBS and treated with DCF-DA at a concentration of 20 μM for 30 minutes. Fluoroskan ELISA plate reader (Labsystems, Helsinki, Finland) was used to measure the fluorescence intensity of 485 nm excitation wavelength and 538 nm emission wavelength.

TUNEL analysis

Hydrogen peroxide (100 μM) was added to the culture medium of the group not treated with Tat-endophylline-A1 fusion protein and the group treated for 1 hour at a concentration of 5 μM to NSC34 cells and treated for 1 hour. In order to measure apoptosis, the TUNEL (Terminal deoxynucleotidyl transferase-mediated biotinylated dUTP nick end labeling staining) technique was performed using a cell death detection kit (Roche Applied Science). The results were analyzed using a fluorescence microscope (Nikon eclipse 80i, Japan).

Results 1: Purification and cell penetration of Tat-endophylline-A1 fusion protein

The inventors recombined Tat-vector and SH3GL2 gene containing Tat, which is a kind of protein transport domain PTD (Protein Transduction Domain), to produce an invasive Tat-endophylline-A1 fusion protein (Fig. 1A). It was overexpressed in E. coli and purified using Ni ^{2 +-} Nitrilotriacetic acid Sepharose affinity chromatography column and PD-10 column chromatography, and Tat-endophylline-A1 fusion protein using SDS-PAGE and Western blot analysis. It was confirmed that this was purified (Fig. 1A, B).

Results 2: Penetration of Tat-endophylline-A1 fusion protein into NSC34 cells

The degree of permeation into NSC34 cells was measured for each time and concentration of the Tat-endophylline-A1 fusion protein. As a result, time- and concentration-dependent permeation within cells was effectively performed, and endophylline-A1 protein did not permeate (Fig. 2A, B). In addition, it was confirmed that the cell nucleus was stained with DAPI using a confocal microscope, and the Tat-endophylline-A1 fusion protein was stained with green fluorescence to effectively permeate the cell (FIG. 2C). It was confirmed that the Tat-endophylline-A1 fusion protein permeated into the cell was continuously maintained for up to 12 hours in the cell (FIG. 2D).

Outcome 3: With oxidative stress Cell viability due to, Reactive oxygen species Cellular protective effect of Tat-endophylline-A1 fusion protein on production and DNA fragmentation

After pretreatment of Tat-endophylline-A1 fusion protein by concentration in the cells, oxidative stress was induced with hydrogen peroxide. MTT analysis was performed to confirm cell viability. When NSC34 cells were single-treated with 100 μM hydrogen peroxide, the number of surviving cells decreased to about 40%, but when pretreated with Tat-endophylline-A1, the cell viability increased in a concentration-dependent manner (FIG. 3A).

In addition, in order to confirm whether the Tat-endophylline-A1 fusion protein effectively inhibits reactive oxygen species induced when hydrogen peroxide is treated, the degree of intracellular oxidation was analyzed using 8-OHdG fluorescent dye. When NSC34 cells were exposed to 100 μM hydrogen peroxide for 1 hour, the 8-OHdG signal was significantly increased by hydrogen peroxide. However, reactive oxygen species induced by hydrogen peroxide were reduced by Tat-endophylline-A1 fusion protein (Fig. 3b).

The protective effect of the fusion protein on DNA fragmentation caused by oxidative stress was investigated by the TUNEL staining method using a fluorescent dye specifically attached to the DNA fragmentation site. It was performed in the same state as the previous experiment, and when NSC34 cells were exposed to 100 μM hydrogen peroxide for 1 hour, it was confirmed that the Tat-endophylline-A1 fusion protein had a protective effect against DNA fragmentation (FIG. 3C).

Accordingly, the present invention proposes a possibility that the Tat-endophylline-A1 fusion protein can be effectively applied as a therapeutic agent for neurodegenerative diseases such as neurological diseases caused by oxidative stress and various diseases.

<110> Industry Academic Cooperation Foundation, Hallym University <120> A pharmaceutical composition containing cell-transducing endophilin-A1 fusion protein for preventing and treating neuronal disorder <130> HallymU-SYChoi-SH3GL2 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 1092 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding Tat-endophilin-A1 fusion protein <400> 1 aggaagaagc ggagacagcg acgaagactc gagatgtcgg tggccggcct caagaagcag 60 ttccataaag ccactcagaa agtgagtgag aaggttggag gagctgaagg aaccaagcta 120 gatgatgact tcaaagagat ggaaaggaaa gtggatgtca ccagcagggc tgtgatggaa 180 ataatgacta aaacaattga ataccttcaa cccaatccag cttccagagc taagctcagc 240 atgatcaaca ccatgtcaaa aatccgtggc caggagaagg ggccaggcta tcctcaggca 300 gaggcgctgc tggcagaggc catgctcaaa tttggaagag agcttggaga tgattgcaac 360 tttggcccag cacttggtga ggtcggggag gccatgcggg aactgtcgga ggtcaaagac 420 tctttggaca tagaagtgaa gcagaacttc attgaccctc ttcagaatct tcatgacaaa 480 gatcttaggg aaattcaaca tcatctaaag aagttggagg gtcgacgcct ggattttgat 540 tataagaaga aacgacaagg caagattccg gatgaagagc ttcgtcaagc tctagagaaa 600 tttgatgagt ctaaggaaat tgctgagtca agcatgttca atctcttgga gatggatatt 660 gaacaagtga gccagctctc tgcacttgtg caagctcagc tggagtacca caagcaggca 720 gtccagatcc tgcagcaagt cacggtcaga ctggaagaaa gaataagaca ggcttcatct 780 cagcctagaa gggaatatca acctaaacca cgaatgagcc tggagtttcc aactggagac 840 agtactcagc ccaatggggg tctctcccac acaggcactc ccaaaccttc aggtgtccaa 900 atggatcagc cctgctgccg agctctgtac gactttgaac ctgaaaatga aggggagttg 960 ggatttaaag agggcgatat catcacactc actaaccaaa ttgatgagaa ctggtatgag 1020 gggatgctgc atggccattc aggcttcttc cccatcaatt atgtggaaat tctggttgcc 1080 ctgccccatt ag 1092 <210> 2 <211> 271 <212> PRT <213> Artificial Sequence <220> <223> Tat-endophilin-A1 fusion protein <400> 2 Arg Lys Lys Arg Arg Gln Arg Arg Arg Leu Glu Met Ser Val Ala Gly 1 5 10 15 Leu Lys Lys Gln Phe His Lys Ala Thr Gln Lys Val Ser Glu Lys Val 20 25 30 Gly Gly Ala Glu Gly Thr Lys Leu Asp Asp Asp Phe Lys Glu Met Glu 35 40 45 Arg Lys Val Asp Val Thr Ser Arg Ala Val Met Glu Ile Met Thr Lys 50 55 60 Thr Ile Glu Tyr Leu Gln Pro Asn Pro Ala Ser Arg Ala Lys Leu Ser 65 70 75 80 Met Ile Asn Thr Met Ser Lys Ile Arg Gly Gln Glu Lys Gly Pro Gly 85 90 95 Tyr Pro Gln Ala Glu Ala Leu Leu Ala Glu Ala Met Leu Lys Phe Gly 100 105 110 Arg Glu Leu Gly Asp Asp Cys Asn Phe Gly Pro Ala Leu Gly Glu Val 115 120 125 Gly Glu Ala Met Arg Glu Leu Ser Glu Val Lys Asp Ser Leu Asp Ile 130 135 140 Glu Val Lys Gln Asn Phe Ile Asp Pro Leu Gln Asn Leu His Asp Lys 145 150 155 160 Asp Leu Arg Glu Ile Gln His His Leu Lys Lys Leu Glu Gly Arg Arg 165 170 175 Leu Asp Phe Asp Tyr Lys Lys Lys Arg Gln Gly Lys Ile Pro Asp Glu 180 185 190 Glu Leu Arg Gln Ala Leu Glu Lys Phe Asp Glu Ser Lys Glu Ile Ala 195 200 205 Glu Ser Ser Met Phe Asn Leu Leu Glu Met Asp Ile Glu Gln Val Ser 210 215 220 Gln Leu Ser Ala Leu Val Gln Ala Gln Leu Glu Tyr His Lys Gln Ala 225 230 235 240 Val Gln Ile Leu Gln Gln Val Thr Val Arg Leu Glu Glu Arg Ile Arg 245 250 255 Gln Ala Ser Ser Gln Pro Arg Arg Glu Tyr Gln Pro Lys Pro Arg 260 265 270

Claims (6)

A pharmaceutical composition for the prevention or treatment of neurological diseases containing an endophylline-A1 fusion protein, wherein the HIV-Tat protein transport domain is covalently bonded to at least one end of the endophylline-A1 protein.
The method according to claim 1,
The endophylline-A1 fusion protein is a pharmaceutical composition for preventing or treating neurological diseases containing an endophylline-A1 fusion protein, characterized in that the amino acid sequence is SEQ ID NO: 2.
A pharmaceutical composition for preventing or treating neurological diseases containing a recombinant polynucleotide encoding an endophyllin-A1 fusion protein in which an oligonucleotide sequence encoding an HIV-Tat protein transport domain is bound to at least one end of the cDNA encoding the endophyllin-A1 protein .
The method of claim 3,
The recombinant polynucleotide is a pharmaceutical composition for preventing or treating neurological diseases, characterized in that the base sequence is SEQ ID NO: 1.
Neurological diseases containing an endophylline-A1 fusion protein expression vector containing a recombinant polynucleotide encoding an endophylline-A1 fusion protein in which an HIV-Tat protein transport domain-encoding oligonucleotide sequence is bound to at least one end of the endophylline-A1 coding cDNA Pharmaceutical composition for prophylaxis or treatment.
A health functional food composition for preventing or improving neurological diseases using the endophylline-A1 fusion protein of claim 1 or 2 as an active ingredient.

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