KR20240007777A - Pharmaceutical composition for treating cerebral ischemia containing cell-transducing PFKFB3 fusion protein - Google Patents

Abstract

Challenge: To provide a drug for the treatment of cerebral ischemia that has little or no side effects and is highly effective.
Solution: The present invention relates to a pharmaceutical composition for the treatment of cerebral ischemia containing a cell-permeable PFKFB3 fusion protein. The PFKFB3 fusion protein penetrated into cells increases cell survival against hydrogen peroxide toxicity, and reduces the level of intracellular reactive oxygen species and hydrogen peroxide. Reduced induced DNA fragmentation. In an animal model, the PFKFB3 fusion protein showed a protective effect against neuronal cell death that occurred when transient ischemia was induced in the CA1 portion of the forebrain and hippocampus. These results show that the PFKFB3 fusion protein has a protective effect against cell death and has a therapeutic effect in protecting nerve cells from cerebral ischemic damage.

Description

Pharmaceutical composition for treating cerebral ischemia containing cell-transducing PFKFB3 fusion protein {Pharmaceutical composition for treating cerebral ischemia containing cell-transducing PFKFB3 fusion protein}

The present invention relates to a pharmaceutical composition for treating cerebral ischemia containing a cell-permeable PFKFB3 fusion protein.

“Free radicals”, also called reactive oxygen compounds, are responsible for oxidative stress, and reactive oxygen species (ROS) are unavoidable by-products of various cellular processes involving interaction with oxygen. . If cells are exposed to relatively more active compounds, they are subject to oxidative stress, resulting in oxidation and damage to important components 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 generated from abnormal cellular processes are known to cause various human diseases, including inflammation, ischemia, diabetes, and Parkinson's disease. Reactive oxygen species have many effects, such as damaging not only DNA but also macromolecules such as proteins and lipids within cells during the metabolic process of cells, and as the damage lasts longer, they cause cell death and have a significant impact on human diseases. Among them, cerebral ischemia is a disease in which brain cells cannot produce enough energy when the arteries of the brain are blocked, resulting in cell death. The amount of reactive oxygen species generated rapidly increases in the CA1 region of the hippocampus, causing death of nerve cells. Hypoxic conditions, such as cerebral ischemia, induce the translocation of cytosolic Bax to outer membrane mitochondria, which then promotes the release of anti-apoptotic factors from mitochondria along with a decrease in mitochondrial membrane potential. In contrast, Bcl-2 functions to block cytochrome c release from mitochondria and prevent apoptosis. In hypoxia, the expression level of Bcl-2 drops significantly, becoming a major cause of cell death. Therefore, if a substance with the ability to control reactive oxygen species is discovered, it is expected that it will have a protective effect against cerebral ischemic damage.

PFKFB3 is a bifunctional protein involved in both synthesis and degradation of fructose-2,6-bisphosphate. Additionally, it is a regulatory molecule that regulates the glycolysis process in eukaryotes.

PFKFB3 has a 6-phosphofructo-2-kinase activity that catalyzes the synthesis of F2,6BP (fructose-2,6-bisphosphate) and a fructose-2,6-biphosphatase activity that catalyzes the decomposition of F2,6BP. has. Additionally, this protein is involved in cell cycle progression and apoptosis prevention and is a regulator of cyclin-dependent kinase 1, which links glucose metabolism to cell proliferation and survival of tumor cells.

However, the function or effect of PFKFB3 on neuronal cell death caused by reactive oxygen species has not yet been clearly identified.

The purpose of the present invention is to provide an effective therapeutic agent for treating cerebral ischemic damage and neuronal damage and death caused by cerebral ischemic damage.

In order to solve the above problem, the present inventors confirmed that protein transport domains such as PEP-1 and HIV Tat peptide can penetrate into HT22 cells by recombining them with PFKFB3, and studied the protective effect of the cell-penetrating PFKFB3 fusion protein against oxidative stress. did.

The present inventors sought to determine whether a PFKFB3 fusion protein combined with a protein transport domain has a protective effect on neuronal cell death induced by oxidative stress in vitro. To study the potential effect of PFKFB3 fusion protein on ischemic neuronal death, the present inventors produced a PFKFB3 fusion protein that can penetrate into cells. The PFKFB3 fusion protein effectively penetrated into nerve cells in a concentration-dependent and time-dependent manner. The PFKFB3 fusion protein permeated into cells increased cell survival against hydrogen peroxide toxicity and lowered intracellular reactive oxygen species levels. These results indicate that the PFKFB3 fusion protein exhibits a protective effect against cell death that occurs in vitro and can be used as a treatment for cerebral ischemic damage related to oxidative stress.

To describe the structure of the present invention in more detail, in one embodiment of the present invention, the present inventors first developed a PFKFB3 fusion protein expression vector that can overexpress and easily purify the PFKFB3 fusion protein. This expression vector according to one embodiment contains a cDNA capable of expressing the human PFKFB3 protein, one or more protein transport domains consisting of 7 to 21 amino acids, and 6 histidine residues at the N-terminal portion. As an example, the amino acid sequence of the PFKFB3 fusion protein is characterized as SEQ ID NO: 4 or SEQ ID NO: 6 in the sequence list.

Using this expression vector, the PFKFB3 fusion protein was overexpressed in E. coli and purified using Ni-affinity chromatography. It was confirmed by Western blot that the PFKFB3 fusion protein was transported to the cultured cells in a time- and concentration-dependent manner. The PFKFB3 fusion protein permeated into cells was maintained within the cells for up to 12 hours and inhibited cell death caused by oxidative stress.

These results mean that the PFKFB3 fusion protein penetrates well into cells and demonstrates the function of the PFKFB3 protein within cells. Therefore, this PFKFB3 fusion protein suggests the possibility of application to brain nerve diseases, such as protecting nerve cells from symptoms related to reactive oxygen species or cerebral ischemic damage.

The present invention is a PFKFB3 fusion protein in which the protein transport domain is bound to one or more of the N-terminus and C-terminus of the PFKFB3 (6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3) protein, thereby improving cell penetration efficiency. It relates to a pharmaceutical composition for preventing or treating cerebral ischemia containing.

The protein transport domain improves cell penetration efficiency by binding to the protein, and there is no particular limitation as long as it does not significantly affect the activity of the protein that has penetrated into the cell, and is preferably HIV Tat peptide, Pep-1 peptide, or amino acid residue. It is characterized in that one or more types selected from oligolysine of 7 to 50 amino acid residues, oligoarginine of 7 to 50 amino acid residues, and oligo (lysine + arginine) of 7 to 50 amino acid residues form a monomer or a multimer of dimer or higher.

In addition, the PFKFB3 protein and the protein transport domain can be combined by various binding methods such as ionic bonding and covalent bonding, and preferably, covalent bonding can be used to form a fusion protein.

Additionally, a linker may exist between the PFKFB3 protein and the protein transport domain. The linker is not particularly limited as long as the activity of the cargo molecule transport domain and the activity of the protein are maintained, but is preferably glycine, alanine, leucine, isoleucine, proline, serine, threonine, asparagine, aspartic acid, cysteine, The transport domain monomers of each cargo molecule can be linked using amino acids such as glutamine, glutamic acid, lysine, and arginic acid. More preferably, they can be linked using a linker in which multiple amino acids are linked, most preferably glycine and valine. , leucine, aspartic acid, etc. can be used as a linker by connecting one to five amino acids. In addition to the above amino acid linkers and peptide linkers, chemical linkers can also be used as long as the activity of the cargo molecule transport domain is maintained.

A pharmaceutical composition containing the cell-permeable PFKFB3 fusion protein as an active ingredient can be formulated in oral or injectable form by mixing it with a carrier commonly accepted in the pharmaceutical field and using conventional methods. Oral compositions include, for example, tablets and gelatin capsules, which, in addition to the active ingredient, may contain diluents (e.g. lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycine) and lubricants (e.g. silica, talc). , stearic acid and its magnesium or calcium salts and/or polyethylene glycol), and the tablets may also contain binders (e.g. magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and/or polyvinyl pyrrolidone). ), and in some cases, it is preferred to contain a disintegrant (e.g. starch, agar, alginic acid or its sodium salt) or boiling mixture and/or an absorbent, a colorant, a flavoring agent and a sweetener. Compositions for injection are preferably isotonic aqueous solutions or suspensions, and the compositions mentioned are sterile and/or contain auxiliaries (e.g. preservatives, stabilizers, wetting or emulsifying agents, solution accelerators, salts/or buffers for adjusting osmotic pressure). Additionally, they may contain other therapeutically useful substances.

The pharmaceutical preparation prepared in this way can be administered orally, parenterally, that is, intravenously, subcutaneously, intraperitoneally, or applied topically, depending on the purpose. The daily dose of 0.0001 to 100 mg/kg can be administered in one to several divided doses. The dosage level for a specific patient may vary depending on the patient's weight, age, gender, 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, comprising the PFKFB3 fusion protein as an active ingredient and a pharmaceutically acceptable carrier.

The present invention also provides a method for efficiently delivering PFKFB3 protein into cells. Intracellular delivery of the PFKFB3 protein molecule according to the present invention is performed 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 the HIV Tat peptide. However, the protein transport domain of the present invention is not limited to the HIV Tat peptide, and the field of the present invention is to prepare a peptide that has a similar function to the HIV Tat peptide by substituting, adding, or omitting part of the amino acid sequence of the HIV Tat peptide. As it is easy for those skilled in the art, it is possible to use a protein transport domain consisting of 7 to 50 amino acids and containing a large number of lysines or arginines, and a protein transport domain that performs the same or similar protein transport function by substituting some amino acids therefrom. It will be obvious that fusion proteins also fall within the scope of the present invention.

Specifically, the present invention relates to a PFKFB3 fusion protein, an oligonucleotide encoding the PFKFB3 fusion protein, a vector containing an oligonucleotide encoding the PFKFB3 fusion protein, a pharmaceutical composition for treating or preventing cerebral ischemia containing the fusion protein, and this fusion protein. It relates to a health functional food composition for preventing or improving cerebral ischemia containing.

Specific examples of the polynucleotide encoding the PFKFB3 fusion protein include SEQ ID NO: 3 or SEQ ID NO: 5. However, it is obvious to those skilled in the art that the sequence is not limited to this.

Foods to which the cell-permeable PFKFB3 fusion protein of the present invention can be added include various foods, such as beverages, gum, tea, vitamin complexes, health supplements, pills, powders, granules, precipitates, and tablets. , can be used in capsule or beverage form. At this time, the amount of the PFKFB3 fusion protein in the food or beverage is generally 0.01 to 15% by weight, preferably 0.2 to 10% by weight, of the total weight of the food in the health food composition of the present invention. In this case, it can be added at a rate of 0.1 to 30 g, preferably 0.2 to 5 g, based on 100 ml. The health drink composition of the present invention has no particular limitations on the liquid ingredients other than containing the PFKFB3 fusion protein as an essential ingredient in the indicated ratio, and contains various flavoring agents or natural carbohydrates as additional ingredients like ordinary drinks. can do. Examples of the above-mentioned natural carbohydrates include monosaccharides, such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin, cyclodextrin, etc., and xylitol. Sugar alcohols such as sorbitol and erythritol. As flavoring agents other than those described above, natural flavoring agents (thaumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion 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 contains various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavors, colorants and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its It may contain salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, the compositions of the present invention may contain pulp for the production of natural fruit juice and fruit juice beverages and vegetable beverages. These ingredients can be used independently or in combination. The proportion of these additives is not critical, but is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

The definitions of key terms used in the detailed description of the present invention are as follows.

“PFKFB3 fusion protein” includes a protein transport domain and a PFKFB3 protein, and refers to a covalent complex formed by genetic fusion or chemical bonding of a protein transport domain and a target protein (i.e., PFKFB3 protein in the present invention). In this specification, since the HIV-Tat protein transport domain was used as a specific example, “PFKFB3 fusion protein” was used interchangeably with “Tat-PFKFB3”, “Tat-PFKFB3 fusion protein”, etc.

“Target protein” is a molecule other than a transport domain or fragment thereof that cannot originally enter the target cell or cannot enter the target cell at a useful rate, and is the molecule itself before fusion with the transport domain or the target of the transport domain-target protein complex. It refers to the protein part. Target proteins include polypeptides, proteins, and peptides, and in the present invention, this refers to PFKFB3.

“Fusion protein” includes a transport domain and one or more target protein parts, and refers to a complex formed by genetic fusion or chemical bonding of the transport domain and the target protein.

Additionally, the term “genetic fusion” refers to 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 a target cell refers to a cell inside or outside the body. In other words, target cells 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 include in vitro cells, that is, cultured animal cells, human cells, or microorganisms.

In the present invention, the "protein transport domain" is a covalent bond to a high molecular weight organic compound, such as an oligonucleotide, peptide, protein, oligosaccharide or polysaccharide, which allows the organic compounds to be introduced into cells without the need for a separate receptor, carrier, or energy. Say what you can.

In addition, in this specification, the expressions "transport", "penetration", "transport", "delivery", "permeate", and "pass" are used interchangeably with respect to "introducing" proteins, peptides, and organic compounds into cells.

The PFKFB3 fusion protein of the present invention is composed of 7 to 50 amino acid residues and refers to a PFKFB3 fusion protein in which a transport domain containing more than 3/4 of arginine or lysine residues is covalently linked to at least one end of PFKFB3, thereby improving cell penetration efficiency. Additionally, 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 PFKFB3 fusion protein of the present invention is, for example, SEQ ID NO: 4 or SEQ ID NO: 6 in the sequence listing. In the production of PFKFB3 fusion protein, fusion proteins of various sequences can be obtained depending on the selection of restriction site sequences, etc., and this is obvious to those skilled in the art to which the present invention pertains. It is obvious that the above amino acid sequence is merely an example and that the PFKFB3 fusion protein amino acid sequence is not limited to the above sequence.

Additionally, the present invention provides a pharmaceutical composition for preventing and treating cerebral ischemia, comprising the PFKFB3 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 PFKFB3 fusion protein as an active ingredient.

The present invention relates to a cell-transducing PFKFB3 fusion protein in which a protein transport domain consisting of 7 to 50 amino acids and containing more than 3/4 of lysine or arginine is covalently linked to at least one end of the PFKFB3 protein. In addition, in the PFKFB3 fusion protein, one or more amino acids in the sequence may be replaced with other amino acid(s) of similar polarity that function equally functionally according to silent change. Amino acid substitutions within the sequence may 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. Negatively charged acidic amino acids include aspartic acid and glutamic acid. In addition, fragments or derivatives thereof having the same or similar biological activity within a certain range of homology, for example, 85-100%, between the fusion protein of the present invention and the amino acid sequence are also included in the scope of the present invention.

In the present invention, the PFKFB3 fusion protein permeated into cells increased cell survival against hydrogen peroxide toxicity. These results indicate that the PFKFB3 fusion protein has a protective effect against cell death caused by cerebral ischemia caused by reactive oxygen species and has a therapeutic effect in protecting nerve cells from cerebral ischemic damage, and that the cell-permeable PFKFB3 fusion protein prevents and treats cerebral ischemia. This indicates that it is useful as a pharmaceutical composition.

Figure 1 shows construction of Tat-PFKFB3 expression vector on pET-15b vector. The synthetic Tat oligomer was cloned into NdeI and XhoI sites, and human PFKFB3 cDNA was cloned into XhoI and BamHI sites of pET-15b. (A) shows construction of expression vectors for PFKFB3 and Tat-PFKFB3 fusion proteins. The Tat-PFKFB3 fusion protein contains six histidine residues. (B) shows the results of expressing each protein by adding IPTG, then purifying the PFKFB3 and Tat-PFKFB3 fusion proteins using 15% SDS-PAGE and Western blotting with an anti-rabbit polyhistidine antibody.
Figures 2a to 2d show the permeation of Tat-PFKFB3 fusion protein into HT22 cells. Figure 2a shows Tat-PFKFB3 fusion protein (2~8μM) and PFKFB3 protein (2~8μM) treated in culture medium for 1 hour, and Figure 2b shows Tat-PFKFB3 fusion protein and PFKFB3 protein treated in culture medium for 15~60 minutes. This is the result of processing and analysis by Western blotting. Figure 2c shows the intracellular distribution of Tat-PFKFB3 fusion protein visualized using confocal fluorescence microscopy. Figure 2d shows the results of analyzing the stability of Tat-PFKFB3 fusion protein in HT22 cells. After transduction with Tat-PFKFB3 fusion protein (8 μM), cells were cultured for 1 to 60 hours and analyzed by Western blotting.
Figure 3a shows the results of adding hydrogen peroxide (100 μM) to HT22 cells pretreated with Tat-PFKFB3 fusion protein or PFKFB3 protein, respectively, for 1 hour, and then measuring cell viability using the MTT assay.
Figure 3b shows the results of measuring intracellular reactive oxygen species levels using 5-CFDA staining.
Figure 3c shows the results of measuring intracellular reactive oxygen species levels using DCF-DA staining.
Figure 3d shows the results of measuring DNA fragmentation using TUNEL staining.

Below, the configuration of the present invention will be described in more detail through specific examples. However, it is obvious to those skilled in the art that the scope of the present invention is not limited by the description of the examples. In particular, in the examples of the present invention, HIV Tat peptide was used as the protein transport domain constituting the PFKFB3 fusion protein, but those skilled in the art will know that the PFKFB3 fusion with Tat peptide bound to the N-terminus By applying protein, it is easy to create a fusion protein in which various protein transport domains such as PEP-1 peptide, oligoarginine, oligolysine, and oligo(arginine + lysine) are linked to one or more of the N-terminus or C-terminus of PFKFB3. Although there may be slight differences between these fusion proteins, it can be easily seen that they exhibit similar activities.

ingredient

Ni ^{2+ -} Nitrilotriacetic acid Sepharose Superflow was purchased from Qiagen and PD-10 column chromatography (Amersham, Brauncschweig, Germany). HT22 cells, mouse motor neurons, 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, Caspase 3, Caspase 9, p-p38, p38, p-Erk, Erk, p-JNK and primary antibodies against JNK were purchased from Cell signaling Technology (Beverly, MA, USA) and Santa Cruz Biotechnology (Santa Cruz, CA, USA). All other reagents were special grade products.

Expression and purification of Tat-PFKFB3 fusion protein

Expression vectors for PFKFB3 protein and Tat-PFKFB3 fusion protein were constructed. The human PFKFB3 gene was amplified by polymerase chain reaction using two primers along with cDNA. The sense primer is 5'-CTCGAGGGCAACGGCAG-3', and a restriction enzyme site called XhoI exists on the 5' side. The antisense primer is 5'-GGATCCTCAGGAATCTTCGGACTC-3', and a restriction enzyme site called BamHI exists on the 5' side. The result obtained through polymerase chain reaction was linked to a TA vector, cut using XhoI and BamHI, and then linked to an expression vector to produce Tat-PFKFB3 fusion protein. Likewise, the control PFKFB3 protein was produced using a vector lacking Tat peptide. The recombinant Tat-PFKFB3 plasmid was transformed into E. coli BL21, induced with 0.5mM IPTG (isopropyl-β-D-thiogalactoside), and cultured overnight at 18°C. The cultured cells were pulverized by ultrasonic waves and purified using a Ni ^{2+ -nitrilotriacetic} acid Sepharose superflow column to obtain Tat-PFKFB3 fusion protein. Protein concentration was determined by the Bradford method using bovine serum albumin as a standard.

Introduction of Tat-PFKFB3 fusion protein into HT22 cells

HT22 cells, mouse hippocampal neurons, are maintained at 37°C, 95% air, and 5% CO ₂ , 10% fetal bovine serum (FBS), 5 mM NaHCO ₃ , and antibiotics (100 μg/ml streptomycin, 100 U/ml). Penicillin) and 20 mM HEPES/NaOH (pH 7.4) were cultured in humid conditions using DMEM (Dulbecco's Modified Eagle's Medium).

The time dependence and concentration dependence of the intracellular uptake of Tat-PFKFB3 fusion protein and control PFKFB3 protein were evaluated. Cells were treated in 60 mm dishes at different times (15 to 60 minutes) and doses of each protein (2 to 8 μM). After treatment with trypsin-EDTA (Gibco) and washing with PBS, the fusion protein permeated into the cells was quantified using the Bradford method and analyzed by Western blot.

Western blot analysis

For Western blot analysis, proteins in the cell lysate were separated using a 12% SDS polyacrylamide gel, and then the proteins in the gel were electrotransferred to a nitrocellulose membrane (Amersham, UK). The membrane was blocked with TBS-T buffer (25mM Tris-HCl, 140mM NaCl, 0.1% Tween 20, pH 7.5). Membranes were subjected to Western blot analysis using primary antibodies recommended by the manufacturer. Bound antibody complexes were detected using an enhanced chemiluminescent agent according to the manufacturer's instructions (Amersham, Franklin Lakes, NJ, USA).

Confocal microscopy observation

To detect Tat-PFKFB3 fusion protein and PFKFB3 protein in HT22 cells, cells were seeded on glass coverslips and treated with Tat-PFKFB3 fusion protein and PFKFB3 protein at a concentration of 8 μM for 1 hour. Cells were washed twice with PBS and then fixed with 4% paraformaldehyde for 5 minutes at room temperature. HT22 cells were blocked 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 1:10,000, and the secondary antibody (Alexa fluor 488, Invitrogen) was diluted at a ratio of 1:15,000 and incubated in the dark at room temperature for 1 hour. 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}. Cells were pre-treated with or without 2-8 μM Tat-PFKFB3 fusion protein, and then exposed to 100 μM hydrogen peroxide for 1 hour to induce apoptosis. Absorbance was measured at 570 nm with an ELISA microplate reader (Labsystems Multiskan MCC/340). Cell viability was expressed as a percentage relative to the untreated control.

Measurement of intracellular reactive oxygen species levels

Intracellular reactive oxygen species levels were measured using DCF-DA (2',7'-dichlorodihydrofluorescein diacetate). DCF-DA is converted to DCF within cells by reactive oxygen species and emits fluorescence. We compared the levels of reactive oxygen species when HT22 cells were treated with Tat-PFKFB3 fusion protein and when they were not. Tat-PFKFB3 fusion protein was treated at 5 μM for 1 hour, and then hydrogen peroxide was treated at a concentration of 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. Fluorescence intensity was measured at 485 nm excitation and 538 nm emission wavelength using a Fluoroskan ELISA plate reader (Labsystems, Helsinki, Finland).

TUNEL analysis

HT22 cells were treated with hydrogen peroxide (100 μM) for 1 hour in the culture medium of the group that was not treated with the Tat-PFKFB3 fusion protein and the group that was treated with the Tat-PFKFB3 fusion protein for 1 hour at a concentration of 8 μM. To measure apoptosis, TUNEL (Terminal deoxynucleotidyl transferase-mediated biotinylated dUTP nick end labeling staining) technique was performed using an apoptosis detection kit (Roche Applied Science). The results were analyzed using a fluorescence microscope (Nikon eclipse 80i, Japan).

Result 1: Purification and cell permeation of Tat-PFKFB3 fusion protein

The present inventors recombined the PFKFB3 gene with a Tat-vector containing Tat, a type of protein transduction domain (PTD), to produce a penetrating Tat-PFKFB3 fusion protein (Figure 1). It was overexpressed in E. coli and purified using Ni ^{2+ -} nitrilotriacetic acid Sepharose affinity chromatography column and PD-10 column chromatography, and the Tat-PFKFB3 fusion protein was purified using SDS-PAGE and Western blot analysis. This was confirmed (Figure 1A, B).

Result 2: Permeation of Tat-PFKFB3 fusion protein into HT22 cells

The degree of penetration into HT22 cells was measured for each time and concentration of the Tat-PFKFB3 fusion protein. As a result, intracellular permeation occurred effectively in a time- and concentration-dependent manner, and PFKFB3 protein did not permeate (Figures 2a and 2b). In addition, using a confocal microscope, cell nuclei were stained with DAPI, and the Tat-PFKFB3 fusion protein was stained with green fluorescence to confirm that it effectively penetrated into cells (Figure 2c). Additionally, the stability of the Tat-PFKFB3 fusion protein in HT22 cells was confirmed over time (Figure 2d).

Result 3: Cell protective effect of Tat-PFKFB3 fusion protein against cell survival rate, reactive oxygen species generation, and DNA fragmentation caused by oxidative stress.

Cells were pretreated with Tat-PFKFB3 fusion protein at various concentrations and then oxidative stress was induced with hydrogen peroxide. MTT assay was performed to confirm cell viability. When HT22 cells were single-treated with 100 μM hydrogen peroxide, the number of surviving cells decreased to about 40%, but when Tat-PFKFB3 was pre-treated, cell viability increased in a concentration-dependent manner (Figure 3a).

In addition, the degree of intracellular oxidation was analyzed using 8-OHdG fluorescent dye to confirm whether the Tat-PFKFB3 fusion protein effectively suppresses reactive oxygen species induced when treating hydrogen peroxide. 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-PFKFB3 fusion protein (Figures 3b, 3c).

The protective effect of the fusion protein against DNA fragmentation caused by oxidative stress was examined using the TUNEL staining method using a fluorescent dye that specifically attaches to the DNA fragmentation site. The experiment was performed under the same conditions as the previous experiment, and the results confirmed that the Tat-PFKFB3 fusion protein had a protective effect against DNA fragmentation when HT22 cells were exposed to 100 μM hydrogen peroxide for 1 hour (Figure 3d).

Therefore, the present invention suggests the possibility that the Tat-PFKFB3 fusion protein can be effectively applied as a treatment for various diseases and neurodegenerative diseases such as cerebral ischemia caused by oxidative stress.

Claims (10)

Containing a PFKFB3 fusion protein with improved cell penetration efficiency by binding the protein transport domain to one or more of the N-terminus and C-terminus of the PFKFB3 (6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3) protein. Pharmaceutical composition for preventing or treating cerebral ischemia.
In claim 1,
The protein transport domain is one or more selected from HIV Tat peptide, Pep-1 peptide, oligolysine of 7 to 50 amino acid residues, oligoarginine of 7 to 50 amino acid residues, and oligo(lysine + arginine) of 7 to 50 amino acid residues. A pharmaceutical composition for preventing or treating cerebral ischemia containing a PFKFB3 fusion protein, which is a monomer or a multimer rather than a dimer.
In claim 1,
A pharmaceutical composition for preventing or treating cerebral ischemia containing a PFKFB3 fusion protein, wherein the PFKFB3 protein and the protein transport domain are covalently linked.
In claim 1,
A pharmaceutical composition for preventing or treating cerebral ischemia containing a PFKFB3 fusion protein, wherein a linker exists between the PFKFB3 protein and the protein transport domain.
In claim 1,
A pharmaceutical composition for preventing or treating cerebral ischemia containing a PFKFB3 fusion protein, wherein the PFKFB3 fusion protein has an amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 6.
Prevention of cerebral ischemia containing a recombinant polynucleotide encoding a PFKFB3 fusion protein in which an oligonucleotide sequence encoding a protein transport domain is attached to at least one end of a cDNA encoding the PFKFB3 (6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3) protein. or therapeutic pharmaceutical compositions.
In claim 6,
The recombinant polynucleotide is a pharmaceutical composition for preventing or treating cerebral ischemia, characterized in that the base sequence is SEQ ID NO: 3 or SEQ ID NO: 5.
Prevention or treatment of cerebral ischemia containing a recombinant polynucleotide encoding a PFKFB3 fusion protein in which an oligonucleotide sequence encoding a protein transport domain is attached to at least one end of a cDNA encoding PFKFB3 (6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3). PFKFB3 fusion protein expression vector.
Expression of a PFKFB3 fusion protein containing a recombinant polynucleotide encoding a PFKFB3 fusion protein in which an oligonucleotide sequence encoding a protein transport domain is attached to at least one end of a cDNA encoding PFKFB3 (6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3). A pharmaceutical composition for preventing or treating cerebral ischemia containing a vector.
A health functional food composition for preventing or improving cerebral ischemia containing the PFKFB3 fusion protein of any one of claims 1 to 4 as an active ingredient.