KR20210003342A - Pharmaceutical composition for treating cerebral ischemia containing phosphoglycerate mutase 1 fusion protein - Google Patents

Abstract

The present invention is to provide a pharmaceutical composition for treating cerebral ischemia which causes little to no side effects and has an excellent effect. The present invention relates to a pharmaceutical composition for treating cerebral ischemia including cell permeable PGAM1 (phosphoglycerate mutase 1) fusion protein. The PGAM1 fusion protein permeated into cells increases cell viability against hydrogen peroxide toxicity and reduces the level of active oxygen species in cells and DNA fragmentation caused by hydrogen peroxide. In addition, the PGAM1 fusion protein shows a protection effect with respect to neuronal cell death occurring when transient ischemia is induced at the CA1 forebrain hippocampus portion in an animal model. Such results suggest that the PGAM1 fusion protein shows a protection effect with respect to neuronal cell death and a therapeutic effect of protecting neuronal cells from brain ischemia damage.

Description

Pharmaceutical composition for treating cerebral ischemia containing phosphoglycerate mutase 1 fusion protein {Pharmaceutical composition for treating cerebral ischemia containing phosphoglycerate mutase 1 fusion protein}

The present invention relates to a pharmaceutical composition for the treatment of cerebral ischemia comprising a cell-permeable phosphoglycerate mutase 1 fusion protein.

"Free radicals", called reactive oxygen compounds, are responsible for oxidative stress, and reactive oxygen species (ROS) are inevitable by-products in various cellular processes including interactions with oxygen. . If cells are exposed to more active compounds, they are subjected to oxidative stress, resulting in oxidation and damage to important elements such as proteins, DNA and fats. Oxidative stress is the cause of many diseases such as cancer and Alzheimer's, and these diseases are also related to aging.

Excessive reactive oxygen species arising from abnormal cellular processes are known to cause various human diseases such as inflammation, ischemia, diabetes and Parkinson's disease. During the metabolic process of cells, reactive oxygen species have many effects, such as damaging not only DNA in cells, but also polymers such as proteins and lipids, and causing apoptosis as the damage time increases, causing a lot of impact on human diseases. Among them, cerebral ischemia is a disease in which brain cells fail to produce enough energy when the arteries of the brain are blocked, and apoptosis occurs due to this.As the amount of reactive oxygen species increases rapidly in the hippocampus CA1 region, neuronal cell death occurs. Hypoxic conditions, such as cerebral ischemia, induce cytoplasmic Bax to metastasize to the mitochondria of the outer membrane and then promote the release of anti-apoptotic factors from the mitochondria with a decrease in mitochondrial membrane potential. In contrast, Bcl-2 functions to prevent cytochrome c release from mitochondria and prevent cell death. In hypoxia, the level of expression of Bcl-2 drops significantly, which is a major cause of cell death. Therefore, if a substance capable of regulating reactive oxygen species is identified, it is expected that it will have a protective effect against cerebral ischemia.

Phosphoglycerate mutase 1 (phosphoglycerate mutase 1; hereinafter used together with "PGAM1") is an enzyme that catalyzes the glycolysis process, and converts 3-phosphoglycerate into 2-phosphoglycerate. The function or effect of phosphoglycerate mutase 1 on neuronal cell death by reactive oxygen species has not yet been clearly elucidated.

An object of the present invention is to provide an effective therapeutic agent for treating nerve cell damage and death caused by cerebral ischemia injury and cerebral ischemia injury.

In order to solve the above problem, the present inventors confirmed that protein transport domains such as PEP-1 and HIV Tat peptide recombined with phosphoglycerate mutase 1 to penetrate into HT22 cells, and cell-penetrating phosphoglycerides against oxidative stress The protective effect of the late mutase 1 fusion protein was studied.

The present inventors tried to find out whether the PGAM1 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 PGAM1 fusion protein on ischemic neuronal cell death, the present inventors constructed a PGAM1 fusion protein that can penetrate into cells. The PGAM1 fusion protein effectively penetrated into neurons in a concentration-dependent and time-dependent manner. The PGAM1 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 PGAM1 fusion protein exhibits a protective effect against apoptosis occurring in vitro, and can be used as a therapeutic agent against cerebral ischemic injury related to oxidative stress.

To explain the configuration of the present invention in more detail, in an embodiment of the present invention, the present inventors first developed a PGAM1 fusion protein expression vector that can overexpress and easily purify the PGAM1 fusion protein. This expression vector contains a human PGAM1 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 PGAM1 fusion protein is characterized by SEQ ID NO: 1.

Using this expression vector, the PGAM1 fusion protein was overexpressed in E. coli and purified using Ni-affinity chromatography. It was confirmed by Western blot that the PGAM1 fusion protein was transported to the cells in a time and concentration dependent manner. The PGAM1 fusion protein permeated into the cell was continuously maintained for up to 12 hours in the cell and suppressed apoptosis due to oxidative stress.

These results imply that the PGAM1 fusion protein is well permeated into the cell, and the function of the PGAM1 protein is well expressed in the cell. Therefore, this PGAM1 fusion protein presents the possibility of application to neurological diseases such as protecting neurons from symptoms related to reactive oxygen species or brain ischemia damage.

The pharmaceutical composition containing the cell-penetrating PGAM1 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 tablet also contains a binder (e.g. magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidone). ), 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 preparations 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 00001 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 severity of disease.

Furthermore, the present invention provides a pharmaceutical composition useful for preventing or treating cerebral ischemia, characterized in that it comprises the PGAM1 fusion protein as an active ingredient and a pharmaceutically acceptable carrier.

The present invention also provides a method for efficiently delivering the PGAM1 protein into cells. Intracellular delivery of the PGAM1 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 a PGAM1 fusion protein, an oligonucleotide encoding the PGAM1 fusion protein, an oligonucleotide encoding the PGAM1 fusion protein, a pharmaceutical composition for the treatment or prevention of cerebral ischemia comprising the fusion protein, and the fusion protein It relates to a functional food composition for preventing or improving cerebral ischemia comprising a.

The polynucleotide encoding the PGAM1 fusion protein is specifically characterized by SEQ ID NO: 1.

Foods to which the cell-permeable PGAM1 fusion protein of the present invention can be added include various foods, such as beverages, gums, teas, vitamin complexes, health supplement foods, and the like, pills, powders, granules, needles, tablets. , Capsules or beverages can be used. At this time, the amount of the PGAM1 fusion protein in the food or beverage may be generally added in 001 to 15% by weight, preferably 02 to 10% by weight of the total food weight, in the case of the health food composition of the present invention. In the case, it may be added in a ratio of 01 to 30 g, preferably 02 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 PGAM1 fusion protein as an essential component in the indicated ratio, and contains various flavoring agents or natural carbohydrates as an additional component like a normal beverage. can do. 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.

"PGAM1 fusion protein" includes a protein transport domain and a PGAM1 protein, and refers to a covalently bonded complex formed by genetic fusion or chemical bonding of a protein transport domain and a target protein (ie, PGAM1 protein in the present invention). In the present specification, since the HIV-Tat protein transport domain was used as a specific example, "PGAM1 fusion protein" was mixed with "Tat-PGAM1" and "Tat-PGAM1 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 PGAM1.

"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 PGAM1 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 PGAM1, thereby improving cell penetration efficiency.It refers to a PGAM1 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 amino acid sequence of the PGAM1 fusion protein of the present invention includes SEQ ID NO: 4, 6 or 8, and the like. In the production of the PGAM1 fusion protein, fusion proteins of various sequences can be obtained depending on the selection of the restriction site sequence, etc., which is obvious to those of ordinary skill in the art. It is obvious that the above amino acid sequence is exemplary only, and that the amino acid sequence of the PGAM1 fusion protein is not limited to the sequence listed above.

In addition, the present invention provides a pharmaceutical composition for the prevention and treatment of cerebral ischemia, including the PGAM1 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 cerebral ischemia, using the PGAM1 fusion protein as an active ingredient.

The present invention relates to a cell-transducing PGAM1 fusion protein in which a protein transport domain consisting of 7 to 21 amino acids and including 4 or more lysine or arginine is covalently bonded to at least one end of the PGAM1 protein. In addition, the PGAM1 fusion protein may be substituted with other amino acid(s) of similar polarity in which one or more amino acids function equally functionally in the sequence 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 PGAM1 fusion protein permeated into the cell increased the cell viability against hydrogen peroxide toxicity. These results indicate that the PGAM1 fusion protein has a protective effect against apoptosis due to cerebral ischemia caused by reactive oxygen species, and has a therapeutic effect to protect nerve cells from cerebral ischemia damage, and the cell permeable PGAM1 fusion protein prevents and treats cerebral ischemia. It says that it is useful as a pharmaceutical composition for use.

Figure 1 shows the construction of the Tat-PGAM1 expression vector on the pET-15b vector. Synthetic Tat oligomers were cloned into the Nde I and Xho I sites, and the human PGAM1 (Phosphoglycerate mutase 1) cDNA was cloned into the Xho I and BamH I sites of pET-15b. (A) shows the construction of the expression vector of the PGAM1 and Tat-PGAM1 fusion proteins. The Tat-PGAM1 fusion protein contains six histidine residues. (B) was expressed by adding IPTG. Purified PGAM1 and Tat-PGAM1 fusion proteins were purified using 15% SDS-PAGE and western blotted with anti-rabbit polyhistidine antibody.
Figure 2 shows the penetration of Tat-PGAM1 fusion protein into HT22 cells. (A) is the Tat-PGAM1 fusion protein (0.5 ~ 5μM) and PGAM1 protein (0.5 ~ 5μM) treated in the culture medium for 1 hour, (B) the Tat-PGAM1 fusion protein and PGAM1 protein for 15 ~ 60 minutes The culture medium was treated and analyzed by Western blot. (C) The intracellular distribution of the Tat-PGAM1 fusion protein was visualized with a confocal fluorescence microscope.
3A shows hydrogen peroxide (100 μM) was added to HT22 cells pretreated with Tat-PGAM1 fusion protein and PGAM1 protein for 1 hour and left for 1 hour. Cell viability was measured by colorimetric analysis using MTT.
Figure 3b is the level of reactive oxygen species in cells was measured by DCF-DA staining.
3c shows DNA fragmentation was measured 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 a protein transport domain constituting the PGAM1 fusion protein, but those of ordinary skill in the art to which the present invention belongs, the PEP-1 peptide is bound to the N-terminus. By applying the PGAM1 fusion protein, a fusion protein in which various protein transport domains such as PEP-1 peptide, oligoarginine, oligolysine and oligo (arginine + lysine) are bound to one or more of the N-terminus or C-terminus of PGAM1. It can be formed easily, and there may be slight differences between such fusion proteins, but 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 hippocampal nerve cells, HT22 cells, were provided by the Korea Cell Line Research Foundation (KCLF). 2',7'-DCF-DA (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.

Tat- PGAM1 Fusion protein Expression and purification

Expression vectors of the PGAM1 protein and the Tat-PGAM1 fusion protein were constructed. PGAM1, one of the human genes, was amplified by polymerase chain reaction (PCR) using two primers along with cDNA. The sense primer is composed of 5'-CTCGAGGGCAACGCGCAG-3', and a restriction enzyme action site called Xho1 exists on the 5'side. And the antisense primer is composed of 5'-GGATCCTCAGGAATCTTCGGACTC-3', and a restriction enzyme site called BamH1 is present on the 5'side. The result obtained through PCR was ligated to a TA vector, cut using Xho1 and BamH1, and ligated to an expression vector to prepare a Tat-PGAM1 fusion protein. Similarly, the control PGAM1 protein was prepared using a vector lacking the Tat peptide. The recombinant Tat-PGAM1 plasmid was transformed with E. coli BL21, followed by induction with 05mM IPTG (isopropyl-β-D-thiogalactoside), and cultured at 18°C overnight. The cultured cells were pulverized with ultrasonic waves and purified using a Ni ^{2 +-} nitrilotriacetic acid Sepharose Superflow column to obtain a Tat-PGAM1 fusion protein. The protein concentration was determined by the Bradford method using bovine serum albumin as a standard.

Tat- with HT22 cells PGAM1 Fusion protein Introduction

Mouse hippocampal neuron HT22 cells maintain conditions of 37°C, 95% air and 5% CO2, and 10% fetal calf serum (FBS) and 5 mM NaHCO ₃ , antibiotics (100mg/ml streptomycin, 100U/ml penicillin) , DMEM (Dulbecco's Modified Eagle's Medium) consisting of 20 mM HEPES/NaOH (pH 7.4) was used to incubate in humid conditions.

The time dependence and concentration dependence of the Tat-PGAM1 fusion protein and the control PGAM1 protein were evaluated. Cells were treated in a 60mm dish at different times (15-60 minutes) and doses (0.5-5 μM) of each protein. 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, 01% 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 Microscopic observation

To detect the Tat-PGAM1 fusion protein and PGAM1 protein in HT22 cells, the cells were seeded on a glass coverslip, and the Tat-PGAM1 fusion protein and PGAM1 protein were treated at a concentration of 5 μM for 1 hour. The cells were washed twice with PBS and then fixed with 4% paraformaldehyde for 5 minutes at room temperature. HT22 cells were blocked for 40 minutes with PBS (PBS-BT) containing 3% bovine serum albumin and 01% 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 HT22 cells were observed under an Fv-300 confocal fluorescence microscope (Olympus, Tokyo, Japan).

Cell viability analysis

The survival rate of HT22 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 with or without 0.5-5 μM of Tat-PGAM1 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.

Intracellular Reactive oxygen species Level measurement

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. The levels of reactive oxygen species were compared when HT22 cells were treated with Tat-PGAM1 fusion protein and when not treated. Tat-PGAM1 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 the Tat-PGAM1 fusion protein and the group treated with a concentration of 5 μM for 1 hour to HT22 cells, followed by treatment 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).

Outcome 1: Tat-PGAM1 Fusion protein Purification and cell penetration

The present inventors recombined the Tat-vector and the PGAM1 gene containing Tat, which is a kind of protein transport domain PTD (Protein Transduction Domain), to produce an invasive Tat-PGAM1 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-PGAM1 fusion protein was purified using SDS-PAGE and Wespunblot analysis. It was confirmed that (A, B in Fig. 1).

Results 2: Tat-PGAM1 into HT22 cells Fusion protein Transmission

The degree of penetration into HT22 cells by time and concentration of the Tat-PGAM1 fusion protein was measured. As a result, time- and concentration-dependent intracellular permeation occurred effectively, and the PGAM1 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-PGAM1 fusion protein was stained with green fluorescence to effectively permeate the cell (FIG. 2C).

Outcome 3: With oxidative stress Cell viability due to, Reactive oxygen species Tat- for generation and DNA fragmentation PGAM1 Fusion protein Cell protection effect

After pretreatment of the Tat-PGAM1 fusion protein by concentration in the cells, oxidative stress was induced with hydrogen peroxide. MTT analysis was performed to confirm cell viability. When HT22 cells were treated with 100 μM hydrogen peroxide, the number of surviving cells decreased to about 40%, but when pre-treated with Tat-PGAM1, the cell viability increased in a concentration-dependent manner (Fig. 3A).

In addition, the degree of intracellular oxidation was analyzed using 8-OHdG fluorescent dye to confirm whether the Tat-PGAM1 fusion protein effectively inhibited reactive oxygen species induced when hydrogen peroxide was treated. When HT22 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 the Tat-PGAM1 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 HT22 cells were exposed to 100 μM hydrogen peroxide for 1 hour, it was confirmed that the result that the Tat-PGAM1 fusion protein has a protective effect against DNA fragmentation (FIG. 3C).

Accordingly, the present invention proposes the possibility that the Tat-PGAM1 fusion protein can be effectively applied as a therapeutic agent for neurodegenerative diseases such as cerebral ischemia due to oxidative stress and various diseases.

<110> Industry Academic Cooperation Foundation, Hallym University <120> Pharmaceutical composition for treating cerebral ischemia containing phosphoglycerate mutase 1 fusion protein <130> HallymU-SYCHOI_Tat_PGAM1_BrainIschemia <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 792 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide coding Tat-PGAM1 fusion protein <400> 1 aggaagaagc ggagacagcg acgaagaatg gccgcctaca aactggtgct gatccggcac 60 ggcgagagcg catggaacct ggagaaccgc ttcagcggct ggtacgacgc cgacctgagc 120 ccggcgggcc acgaggaggc gaagcgcggc gggcaggcgc tacgagatgc tggctatgag 180 tttgacatct gcttcacctc agtgcagaag agagcgatcc ggaccctctg gacagtgcta 240 gatgccattg atcagatgtg gctgccagtg gtgaggactt ggcgcctcaa tgagcggcac 300 tatgggggtc taaccggtct caataaagca gaaactgctg caaagcatgg tgaggcccag 360 gtgaagatct ggaggcgctc ctatgatgtc ccaccacctc cgatggagcc cgaccatcct 420 ttctacagca acatcagtaa ggatcgcagg tatgcagacc tcacagaaga tcagctaccc 480 tcctgtgaga gtctgaagga tactattgcc agagctctgc ccttctggaa tgaagaaata 540 gttccccaga tcaaggaggg gaaacgtgta ctgattgcag cccatggcaa cagcctccgg 600 ggcattgtca agcatctgga gggtctctct gaagaggcta tcatggagct gaacctgccg 660 actggtattc ccattgtcta tgaattggac aagaacttga agcctatcaa gcccatgcag 720 tttctggggg atgaagagac ggtgcgcaaa gccatggaag ctgtggctgc ccagggcaag 780 gccaagaagt ga 792 <210> 2 <211> 265 <212> PRT <213> Artificial Sequence <220> <223> Tat-PGAM1 fusion protein <400> 2 Arg Lys Lys Arg Arg Gln Arg Arg Arg Leu Glu Met Ala Ala Tyr Lys 1 5 10 15 Leu Val Leu Ile Arg His Gly Glu Ser Ala Trp Asn Leu Glu Asn Arg 20 25 30 Phe Ser Gly Trp Tyr Asp Ala Asp Leu Ser Pro Ala Gly His Glu Glu 35 40 45 Ala Lys Arg Gly Gly Gln Ala Leu Arg Asp Ala Gly Tyr Glu Phe Asp 50 55 60 Ile Cys Phe Thr Ser Val Gln Lys Arg Ala Ile Arg Thr Leu Trp Thr 65 70 75 80 Val Leu Asp Ala Ile Asp Gln Met Trp Leu Pro Val Val Arg Thr Trp 85 90 95 Arg Leu Asn Glu Arg His Tyr Gly Gly Leu Thr Gly Leu Asn Lys Ala 100 105 110 Glu Thr Ala Ala Lys His Gly Glu Ala Gln Val Lys Ile Trp Arg Arg 115 120 125 Ser Tyr Asp Val Pro Pro Pro Pro Met Glu Pro Asp His Pro Phe Tyr 130 135 140 Ser Asn Ile Ser Lys Asp Arg Arg Tyr Ala Asp Leu Thr Glu Asp Gln 145 150 155 160 Leu Pro Ser Cys Glu Ser Leu Lys Asp Thr Ile Ala Arg Ala Leu Pro 165 170 175 Phe Trp Asn Glu Glu Ile Val Pro Gln Ile Lys Glu Gly Lys Arg Val 180 185 190 Leu Ile Ala Ala His Gly Asn Ser Leu Arg Gly Ile Val Lys His Leu 195 200 205 Glu Gly Leu Ser Glu Glu Ala Ile Met Glu Leu Asn Leu Pro Thr Gly 210 215 220 Ile Pro Ile Val Tyr Glu Leu Asp Lys Asn Leu Lys Pro Ile Lys Pro 225 230 235 240 Met Gln Phe Leu Gly Asp Glu Glu Thr Val Arg Lys Ala Met Glu Ala 245 250 255 Val Ala Ala Gln Gly Lys Ala Lys Lys 260 265

Claims (6)

A pharmaceutical composition for the prevention or treatment of cerebral ischemia containing the HIV-Tat protein transport domain is covalently linked to at least one end of the phosphoglycerate mutase 1 protein to improve cell penetration efficiency.
The method according to claim 1,
The phosphoglycerate mutase 1 fusion protein is a pharmaceutical composition for preventing or treating cerebral ischemia containing a phosphoglycerate mutase 1 fusion protein, characterized in that the amino acid sequence is SEQ ID NO: 2.
For the prevention or treatment of cerebral ischemia containing a recombinant polynucleotide encoding a phosphoglycerate mutase 1 fusion protein by conjugating an oligonucleotide sequence encoding an HIV-Tat protein transport domain to at least one end of the phosphoglycerate mutase 1 coding cDNA Pharmaceutical composition.
The method of claim 4,
The recombinant polynucleotide is a pharmaceutical composition for preventing or treating cerebral ischemia, characterized in that the base sequence is SEQ ID NO: 1.
Phosphoglycerate mute comprising a recombinant polynucleotide encoding a phosphoglycerate mutase 1 fusion protein by linking an oligonucleotide sequence encoding an HIV-Tat protein transport domain to at least one end of the phosphoglycerate mutase 1 coding cDNA Izu 1 fusion protein expression vector containing a pharmaceutical composition for preventing or treating cerebral ischemia.
A health functional food composition for preventing or improving cerebral ischemia using the phosphoglycerate mutase 1 fusion protein of claim 1 or 2 as an active ingredient.

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