KR20220147167A - Pharmaceutical composition for treating cerebral ischemia containing cell-transducing PGAM5 fusion protein - Google Patents

Abstract

Provided is a pharmaceutical composition having few side effects or excellent effects of treating cerebral ischemia. The present invention relates to a pharmaceutical composition comprising a cell-penetrating phosphoglycerate mutase 5 (PGAM5) fusion protein for treating cerebral ischemia. The PGAM5 fusion protein penetrated into the cells increased cell survival in toxicity of hydrogen peroxide and reduced the level of reactive oxygen species within cells and DNA fragmentation induced by hydrogen peroxide. The PGAM5 fusion protein showed a protective effect against the death of nerve cells occurring when temporary ischemia was induced in a hippocampal CA1 region of the forebrain in an animal model. Such results suggest that the PGAM5 fusion protein has a protective effect against death of cells and therapeutic effects of protecting nerve cells from damage caused by cerebral ischemia.

Description

Pharmaceutical composition for treating cerebral ischemia comprising cell-permeable PGAM5 fusion protein {Pharmaceutical composition for treating cerebral ischemia containing cell-transducing PGAM5 fusion protein}

The present invention relates to a pharmaceutical composition for treating cerebral ischemia comprising a cell-permeable PGAM5 fusion protein.

"Free radicals" called reactive oxygen compounds are responsible for oxidative stress, and reactive oxygen species (ROS) are unavoidable by-products of various cellular processes, including interactions with oxygen. . If cells are exposed to relatively more active compounds, they are subjected to oxidative stress, resulting in oxidative 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 associated with aging.

Excessive reactive oxygen species generated 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 macromolecules such as proteins and lipids. Among them, cerebral ischemia is a disease in which brain cells do not produce enough energy when an artery in the brain is blocked, resulting in apoptosis. Hypoxia conditions, such as cerebral ischemia, induce cytoplasmic Bax to translocate to outer mitochondria and then promote release of anti-apoptotic factors from mitochondria with a decrease in mitochondrial membrane potential. In contrast, Bcl-2 functions to block cytochrome c release from mitochondria and prevent apoptosis. Under hypoxia, the expression level of Bcl-2 is greatly reduced, which is a major cause of apoptosis. Therefore, it is expected that if a substance with the ability to regulate reactive oxygen species is found, it will be able to show a protective effect against cerebral ischemia damage.

Phosphoglycerate mutase 5 (phosphoglycerate mutase 1; hereinafter referred to as “PGAM5”) displays phosphate activity on serine/threonine residues and activates MAP3K5 kinase by dephosphorylation. It is a substrate of the KEAP1-dependent ubiquitin ligase complex, contributes to the inhibition of NFE2L2-dependent gene expression, and serves as a central mediator for programmed necrosis induced by TNF, reactive oxygen species, and calcium transporters.

Furthermore, the N-terminus of the PGAM5 protein contains a conserved NXESGE motif that binds to the substrate binding pocket in the Kelch domain of Keap1, while the C-terminal PGAM domain binds to Bcl-XL.

In addition, in Kelch ECH, it is used as an antioxidant modulator implicating the KEAP1-NRF2 (protein 1-nuclear factor-E2-related factor 2) signaling pathway and as a serine/threonine phosphatase regulating ASK1 kinase activity. The PGAM5 protein is known to form oligomers on the plasma membrane as well as intracellular and organelle membranes that disrupt their integrity and cause necrosis.

However, the function or effect of PGAM5 protein on cerebral ischemia caused 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 cerebral ischemic injury and nerve cell damage and death caused by cerebral ischemic injury.

In order to solve the above problem, the present inventors confirmed that the protein transport domains such as PEP-1 and HIV Tat peptide were recombined with phosphoglycerate mutase 5 to penetrate into HT22 cells, and cell permeability against oxidative stress. The protective effect of the late mutase 5 fusion protein was studied.

The present inventors tried to determine whether a PGAM5 fusion protein bound to a protein transport domain has a protective effect on ischemic neuronal cell death induced by oxidative stress in vitro. To study the potential effect of the PGAM5 fusion protein on ischemic neuronal apoptosis, the present inventors constructed a PGAM5 fusion protein that can penetrate into cells. The PGAM5 fusion protein effectively penetrated into neurons in a concentration-dependent and time-dependent manner. The PGAM5 fusion protein permeated into the cell increased the cell viability against hydrogen peroxide toxicity and lowered the level of reactive oxygen species in the cell. These results suggest that the PGAM5 fusion protein exhibits a protective effect against apoptosis in vitro, and can be used as a therapeutic agent for oxidative stress-related cerebral ischemia damage.

To describe the configuration of the present invention in more detail, in an embodiment of the present invention, the present inventors first developed a PGAM5 fusion protein expression vector capable of overexpressing and easily purifying a PGAM5 fusion protein. This expression vector contains cDNA capable of expressing human PGAM5 protein, at least one protein transport domain consisting of 7 to 21 amino acids, and 6 histidine residues at the N-terminus. As an example, the amino acid sequence of the PGAM5 fusion protein is characterized as SEQ ID NO: 1 in the sequence listing.

Using this expression vector, the PGAM5 fusion protein was overexpressed in E. coli and purified by Ni-affinity chromatography. It was confirmed by Western blot that the PGAM5 fusion protein was transported to the cells in a time- and concentration-dependent manner in the cultured cells. The PGAM5 fusion protein permeated into the cell was maintained continuously for up to 12 hours in the cell, and apoptosis caused by oxidative stress was suppressed.

These results indicate that the PGAM5 fusion protein is well permeated into the cell and that the function of the PGAM5 protein is well expressed in the cell. Therefore, this PGAM5 fusion protein suggests the possibility of application to cranial nerve diseases, such as protecting nerve cells from reactive oxygen species-related symptoms or cerebral ischemia damage.

The pharmaceutical composition containing the cell-permeable PGAM5 fusion protein as an active ingredient may be formulated in oral or injection form by a conventional method by mixing with a carrier commonly acceptable in the pharmaceutical field. Oral compositions include, for example, tablets and gelatin capsules, which in addition to the active ingredient include diluents (eg lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycine), glidants (eg silica, talc). , stearic acid and its magnesium or calcium salts and/or polyethylene glycol), and tablets also contain binders (e.g. magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and/or polyvinyl pyrrolidone) ), preferably containing a disintegrant (eg, starch, agar, alginic acid or sodium salt thereof) or a boiling mixture and/or absorbent, colorant, flavoring agent and sweetening agent as the case may be. Compositions for injection are preferably isotonic aqueous solutions or suspensions, the compositions mentioned are sterile and/or contain adjuvants (eg, preservatives, stabilizers, wetting or emulsifying agents, solution promoters, salts/or buffers for regulating the osmotic pressure). In addition, they may contain other therapeutically useful substances.

The pharmaceutical preparation prepared as described above may be administered orally, or parenterally, ie, intravenously, subcutaneously, intraperitoneally, or topically, as desired. The dose can be administered in one to several divided doses of 0.0001 to 100 mg/kg of the daily dose. 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, severity of disease, and the like.

Furthermore, the present invention provides a pharmaceutical composition useful for preventing or treating cerebral ischemia, comprising the PGAM5 fusion protein as an active ingredient and a pharmaceutically acceptable carrier.

The present invention also provides a method for efficiently delivering a PGAM5 protein into a cell. The intracellular delivery of the PGAM5 protein molecule according to the present invention is performed by constructing a fusion protein in a form in which a protein transport domain such as a PEP-1 peptide is covalently bound. An example of the protein transport domain of the present invention may be a PEP-1 peptide. However, the protein transport domain of the present invention is not limited only to the PEP-1 peptide, and the present invention is to prepare a peptide having a function similar to that of the PEP-1 peptide by partial substitution, addition, or lack of amino acid sequence of the PEP-1 peptide. Since it is easy for those of ordinary skill in the art to which it belongs, the protein transport domain consisting of 7 to 21 amino acids and containing four or more lysine or arginine and a partial amino acid substitution therefrom performs the same/similar protein transport function It will be apparent that a fusion protein using a protein transport domain is also within the scope of the present invention.

Specifically, the present invention relates to a PGAM5 fusion protein, a vector comprising an oligonucleotide encoding a PGAM5 fusion protein, an oligonucleotide encoding a PGAM5 fusion protein, a pharmaceutical composition for treatment or prevention of cerebral ischemia comprising the fusion protein, and the fusion protein It relates to a functional health food composition for preventing or improving cerebral ischemia comprising a.

The polynucleotide encoding the PGAM5 fusion protein is specifically characterized in that it is SEQ ID NO: 1.

Foods to which the cell-permeable PGAM5 fusion protein of the present invention can be added include various foods, for example, beverages, gum, tea, vitamin complexes, health supplements, and the like, and include pills, powders, granules, needles, and tablets. , can be used in the form of capsules or beverages. In this case, the amount of the PGAM5 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, In this case, it can be added in a ratio of 0.1 to 30 g, preferably 0.2 to 5 g, based on 100 ml. The health beverage composition of the present invention has no particular limitation on the liquid component except that it contains the PGAM5 fusion protein as an essential component in the indicated ratio, and contains various flavoring agents or natural carbohydrates as additional components like a conventional beverage. can do. Examples of the above-mentioned natural carbohydrates include monosaccharides, for example, disaccharides such as glucose and fructose, for example, polysaccharides such as maltose and sucrose, for example, conventional sugars such as dextrin, cyclodextrin, etc., and xylitol , sorbitol, and sugar alcohols such as erythritol. As flavoring agents other than those described above, natural flavoring agents (taumatin, stevia extract (eg, 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 flavoring agents and natural flavoring agents, coloring agents and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts. salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages, and the like. In addition, the compositions of the present invention may contain natural fruit juice and pulp 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 key terms used in the detailed description of the present invention are as follows.

"PGAM5 fusion protein" includes a protein transport domain and a PGAM5 protein, and refers to a covalent complex formed by genetic fusion or chemical bonding of a protein transport domain and a target protein (ie, PGAM5 protein in the present invention). In the present specification, since the PEP-1 protein transport domain was used as a specific example, "PGAM5 fusion protein" was used interchangeably with "PEP-1-PGAM5", "PEP-1-PGAM5 fusion protein", and the like.

"Target protein" refers to a molecule that is not a transport domain or fragment thereof that cannot originally enter the target cell or that cannot enter the target cell at a useful rate, the molecule itself or the target of the transport domain-target protein complex before fusion with the transport domain. protein part. Target proteins include polypeptides, proteins, and peptides, and in the present invention means PGAM5.

The term "fusion protein" refers to a complex comprising a transport domain and one or more target protein portions, and formed by genetic fusion or chemical bonding of the transport domain and target protein.

In addition, the "genetic fusion" refers to a linkage consisting of a linear, covalent bond formed through the genetic expression of a DNA sequence encoding a protein. In addition, the term "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 in or outside the body. That is, the target cell is meant to include a cell in the body, that is, a cell constituting an organ or tissue of a living animal or human, or a microorganism found in a living animal or human. In addition, the target cell is meant to include cells in vitro, that is, cultured animal cells, human cells or microorganisms.

"Protein transport domain" in the present invention is a high molecular weight organic compound, such as oligonucleotide, peptide, protein, oligosaccharide or polysaccharide, etc. covalently formed to introduce the organic compounds into the cell without the need for a separate receptor, carrier, or energy. say what you can

In addition, in the present specification, the expressions "transport", "penetration", "transport", "transfer", "permeation", and "pass" are used interchangeably with respect to "introduction" of a protein, peptide, or organic compound into a cell.

The PGAM5 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 bound to at least one end of PGAM5. It refers to a PGAM5 fusion protein with improved cell penetration efficiency. 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 PGAM5 fusion protein of the present invention includes SEQ ID NO: 4, 6 or 8, and the like. In the preparation of the PGAM5 fusion protein, fusion proteins of various sequences can be obtained depending on the selection of the restriction site sequence, etc., which is apparent to those of ordinary skill in the art to which the present invention pertains. It is apparent that the above amino acid sequence is merely exemplary and the amino acid sequence of the PGAM5 fusion protein is not limited to the sequences listed above.

In addition, the present invention provides a pharmaceutical composition for preventing and treating cerebral ischemia, comprising the PGAM5 fusion protein as an active ingredient and a pharmaceutically acceptable carrier.

In addition, the present invention provides a health functional food composition using the PGAM5 fusion protein as an active ingredient and for preventing or improving brain ischemia.

The present invention relates to a cell-transducing PGAM5 fusion protein in which a protein transport domain consisting of 7 to 21 amino acids and containing 4 or more lysine or arginine is covalently linked to at least one end of the PGAM5 protein. In addition, according to the silent change of the PGAM5 fusion protein, one or more amino acids in the sequence may be substituted with other amino acid(s) of similar polarity that function equally and functionally. An amino acid substitution in a 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 between the fusion protein of the present invention and the amino acid sequence, such as 85-100%, are also included in the scope of the present invention.

In the present invention, the PGAM5 fusion protein permeated into the cell increased the cell viability against hydrogen peroxide toxicity. These results indicate that the PGAM5 fusion protein has a protective effect against apoptosis caused by cerebral ischemia due to reactive oxygen species, and has a therapeutic effect to protect nerve cells from cerebral ischemia damage. It is said to be useful as a pharmaceutical composition for

1 shows the construction of a PEP-1-PGAM5 expression vector on a pET-15b vector. Synthetic PEP-1 oligomers were cloned into the NdeI and XhoI sites, and human PGAM5 (Phosphoglycerate mutase 5) cDNA was cloned into the XhoI and BamHI sites of pET-15b. 1a shows the construction of expression vectors of PGAM5 and PEP-1-PGAM5 fusion proteins. The PEP-1-PGAM5 fusion protein contains six histidine residues. Figure 1b shows the results of expressing each protein by adding IPTG, then purifying the purified PGAM5 and PEP-1-PGAM5 fusion proteins using 15% SDS-PAGE and Western blotting with an anti-rabbit polyhistidine antibody.
2A to 2D show the permeation of the PEP-1-PGAM5 fusion protein into HT22 cells. Figure 2a shows PEP-1-PGAM5 fusion protein (0.5-5 μM) and PGAM5 protein (0.5-5 μM) in culture medium for 1 hour, Figure 2b shows PEP-1-PGAM5 fusion protein and PGAM5 protein 15-60 It is the result of treatment by culture medium for minutes and analyzed by Western blotting. Figure 2c is a visualization of the intracellular distribution of the PEP-1-PGAM5 fusion protein with a confocal fluorescence microscope. Figure 2d is a result of analyzing the stability of the PEP-1-PGAM5 fusion protein in HT22 cells. After transduction with PEP-1-PGAM5 fusion protein (5 μM), cells were cultured for 1 to 60 hours and analyzed by Western blotting.
Figure 3a is the result of measuring the cell viability using the MTT assay after adding hydrogen peroxide (100 μM) to HT22 cells pretreated with PEP-1-PGAM5 fusion protein or PGAM5 protein for 1 hour, respectively, for 1 hour.
Figure 3b is a result of measuring the level of reactive oxygen species in the cell by 5-CFDA staining.
Figure 3c is a result of measuring the level of reactive oxygen species in cells by DCF-DA staining.
3d 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 apparent 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 embodiment of the present invention, PEP-1 peptide was used as the protein transport domain constituting the PGAM5 fusion protein, but those of ordinary skill in the art to which the present invention pertains, the PEP-1 peptide binds to the N-terminus. A fusion protein in which various protein transport domains such as PEP-1 peptide, oligoarginine, oligolysine and oligo (arginine + lysine) are linked to at least one of the N-terminus or C-terminus of PGAM5 by applying the PGAM5 fusion protein 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.

ingredient

Ni ^2+- nitrilotriacetic acid sepharose superflow was purchased from Qiagen, and PD-10 column chromatography (Amersham, Brauncschweig, Germany) was purchased. Mouse motor neurons, HT22 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, p-JNK and primary antibodies of JNK were purchased from Cell signaling Technology (Beverly, MA, USA) and Santa Cruz Biotechnology (Santa Cruz, CA, USA). For all other reagents, premium products were used.

Expression and purification of PEP-1-PGAM5 fusion protein

Expression vectors of PGAM5 protein and PEP-1-PGAM5 fusion protein were constructed. The human PGAM5 gene was amplified by polymerase chain reaction using two primers together with cDNA. The sense primer is 5'-CTCGAGGGCAACGCGCAG-3', and a restriction enzyme site called Xho I exists on the 5' side. The antisense primer is 5'-GGATCCTCAGGAATCTTCGGACTC-3', and a restriction enzyme action site called BamHI exists on the 5' side. The result obtained through the polymerase chain reaction was ligated to a TA vector, cut using Xho I and BamHI , and then ligated to an expression vector to prepare a PEP-1-PGAM5 fusion protein. Similarly, the control PGAM5 protein was prepared using a vector lacking the PEP-1 peptide. After transforming the recombinant PEP-1-PGAM5 plasmid into E. coli BL21, it was induced with 0.5 mM IPTG (isopropyl-β-D-thiogalactoside) and cultured overnight at 18°C. The cultured cells were pulverized by ultrasonication to obtain a PEP-1-PGAM5 fusion protein and purified using a Ni ^2+- Nitrilotriacetate Sepharose Superflow column. Protein concentration was determined using the Bradford method using bovine serum albumin as a standard material.

Introduction of PEP-1-PGAM5 fusion protein into HT22 cells

Mouse hippocampal neurons, HT22 cells, maintain the conditions of 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) containing DMEM (Dulbecco's Modified Eagle's Medium) and cultured under humid conditions.

The time dependence and concentration dependence of the PEP-1-PGAM5 fusion protein and the control PGAM5 protein for transduction into cells were evaluated. Cells were treated in 60 mm dishes for different times (15 to 60 minutes) and different doses (0.5 to 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 proteins in the cell disrupted solution were separated using a 12% SDS polyacrylamide gel, and then the proteins in the gel were electrophoresed on 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). Membranes were analyzed by western blot using the primary antibody 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

To detect PEP-1-PGAM5 fusion protein and PGAM5 protein in HT22 cells, cells were seeded on glass coverslips and treated with PEP-1-PGAM5 fusion protein and PGAM5 protein at a concentration of 5 μM for 1 hour. Cells were washed twice with PBS and fixed with 4% paraformaldehyde for 5 minutes at room temperature. HT22 cells were blocked 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 the dark. 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 assay

The viability of HT22 cells treated with hydrogen peroxide was confirmed by colorimetric analysis using MTT {3-(4,5-dimethylthiazol-2-yl)-2,5-dipheyltetrazolium bromide}. Cell death was induced by exposing the cells to 100 μM hydrogen peroxide for 1 hour, with or without pre-treating the cells with 0.5-5 μM of the PEP-1-PGAM5 fusion protein. 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 level

Intracellular reactive oxygen species levels were measured using DCF-DA (2',7'-dichlorodihydrofluorescein diacetate). DCF-DA is converted to DCF in cells by reactive oxygen species and fluoresces. The level of reactive oxygen species when HT22 cells were treated with PEP-1-PGAM5 fusion protein and when not treated was compared. The PEP-1-PGAM5 fusion protein was treated with 5 μM for 1 hour and then treated with hydrogen peroxide at a concentration of 700 μM for 30 minutes. And washed twice with PBS and treated with DCF-DA at a concentration of 20 μM for 30 minutes. Fluorescence intensity was measured using a Fluoroskan ELISA plate reader (Labsystems, Helsinki, Finland) at 485 nm excitation wavelength and 538 nm emission wavelength.

TUNEL analysis

HT22 cells were treated with hydrogen peroxide (100 μM) by adding hydrogen peroxide (100 μM) to the culture medium of the group not treated with the PEP-1-PGAM5 fusion protein and the group treated at a concentration of 5 μM for 1 hour. 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 of PEP-1-PGAM5 fusion protein and cell permeation

The present inventors recombined the PGAM5 gene with a PEP-1- vector containing PEP-1, a type of protein transduction domain (PTD), to prepare a penetrating PEP-1-PGAM5 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 PEP-1-PGAM5 fusion protein was obtained by SDS-PAGE and Western blot analysis. It was confirmed that it was purified (FIGS. 1a, 1b).

Result 2: Penetration of PEP-1-PGAM5 fusion protein into HT22 cells

The degree of permeation into HT22 cells was measured by time and concentration of the PEP-1-PGAM5 fusion protein. As a result, intracellular permeation occurred effectively in a time- and concentration-dependent manner, and PGAM5 protein did not permeate ( FIGS. 2a and 2b ). In addition, cell nuclei were stained with DAPI using a confocal microscope, and the PEP-1-PGAM5 fusion protein was stained with green fluorescence to confirm effective penetration into cells (FIG. 2c). In addition, the PEP-1-PGAM5 fusion protein (5 μM) was cultured in HT22 cells for 1 to 60 hours and analyzed by Western blotting to confirm the stability of the PEP-1-PGAM5 fusion protein into HT22 cells ( FIG. 2D ).

Result 3: Cell protective effect of PEP-1-PGAM5 fusion protein on cell viability, reactive oxygen species generation and DNA fragmentation due to oxidative stress

Cells were pretreated with PEP-1-PGAM5 fusion protein by concentration, and then oxidative stress was induced with hydrogen peroxide. MTT assay was performed to confirm cell viability. When HT22 cells were treated with 100 μM hydrogen peroxide, the number of viable cells decreased to about 40%, but when PEP-1-PGAM5 was pretreated, cell viability increased in a concentration-dependent manner ( FIG. 3a ).

In addition, in order to confirm whether the PEP-1-PGAM5 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 HT22 cells were exposed to 100 μM hydrogen peroxide for 1 hour, the 8-OHdG signal was significantly increased by hydrogen peroxide. However, the reactive oxygen species induced by hydrogen peroxide was reduced by the PEP-1-PGAM5 fusion protein ( FIGS. 3b and 3c ).

The protective effect of the fusion protein against DNA fragmentation caused by oxidative stress was investigated by the TUNEL staining method using a fluorescent dye that specifically attaches to the DNA fragmentation site. It was performed under the same conditions as the previous experiment, and when HT22 cells were exposed to 100 μM hydrogen peroxide for 1 hour, it was confirmed that the PEP-1-PGAM5 fusion protein had a protective effect against DNA fragmentation ( FIG. 3D ).

Therefore, the present invention suggests the possibility that the PEP-1-PGAM5 fusion protein can be effectively applied as a therapeutic agent for various diseases and neurodegenerative diseases such as cerebral ischemia due to oxidative stress.

<110> Industry Academic Cooperation Foundation, Hallym University <120> Pharmaceutical composition for treating cerebral ischemia containing cell-transducing PGAM5 fusion protein <130> PEP-PGAM5-HT22 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 940 <212> DNA <213> Artificial Sequence <220> <223> PEP-PGAM5 fusion protein coding polyucleotide <400> 1 aaagaaacct ggtgggaaac ctggtggacc gaatggtctc agccgaaaaa aaaacgtaaa 60 gtgtatatgc tcgatggcgt tccggcaggc gctgcagctg gcggcctgcg ggctggccgg 120 gggctcggcc gccgtgctct tctcggccgt ggcggtaggg aagccgcgcg caggcgggga 180 cgcggagcca cgcccggctg agccgccggc ctgggcgggg ggcgcgcggc cgggccccgg 240 tgtctgggac cccaactggg acaggcgaga accactgtct ctgatcaacg tgcggaagag 300 gaacgtggaa tctggggaag aagagctggc gtccaagctg gaccactaca aagccaaggc 360 cacgcggcac atcttcctca tcaggcattc ccagtaccac gtggatggct ccctggagaa 420 ggaccgcact ctgaccccgc tgggtcggga gcaggctgaa ctcactgggc tccgcctggc 480 aagcttgggg ttgaagttta ataaaatcgt ccattcgtct atgacgcgcg ccatagagac 540 caccgatatc atcagccggc acctgccagg cgtctgcaaa gtcagcacag atctgctgcg 600 ggaaggcgcc cccatcgagc cagacccgcc cgtgtctcat tggaagccgg aagctgtgta 660 ttacgaagac ggagcccgga tcgaggccgc cttccggaac tacatccacc gcgcagatgc 720 caggcaggag gaggacagtt acgagatctt catctgtcac gccaacgtca tccgctacat 780 cgtgtgcaga gcactgcagt ttcctcctga aggctggctc cggctctccc tcaataatgg 840 cagcatcacc cacctggtga tccgacccaa cggccgagtt gcgctcagga ccctcgggga 900 cacggggttc atgcctcccg acaagatcac tcgatcctga 940 <210> 2 <211> 334 <212> PRT <213> Artificial Sequence <220> <223> PEP-PGAM5 fusion protein <400> 2 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Ile Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu 20 25 30 Trp Ser Gln Pro Lys Lys Lys Arg Lys Val Tyr Met Leu Glu Met Ala 35 40 45 Phe Arg Gln Ala Leu Gln Leu Ala Ala Cys Gly Leu Ala Gly Gly Ser 50 55 60 Ala Ala Val Leu Phe Ser Ala Val Ala Val Gly Lys Pro Arg Ala Gly 65 70 75 80 Gly Asp Ala Glu Pro Arg Pro Ala Glu Pro Pro Ala Trp Ala Gly Gly 85 90 95 Ala Arg Pro Gly Pro Gly Val Trp Asp Pro Asn Trp Asp Arg Arg Glu 100 105 110 Pro Leu Ser Leu Ile Asn Val Arg Lys Arg Asn Val Glu Ser Gly Glu 115 120 125 Glu Glu Leu Ala Ser Lys Leu Asp His Tyr Lys Ala Lys Ala Thr Arg 130 135 140 His Ile Phe Leu Ile Arg His Ser Gln Tyr His Val Asp Gly Ser Leu 145 150 155 160 Glu Lys Asp Arg Thr Leu Thr Pro Leu Gly Arg Glu Xaa Ala Glu Leu 165 170 175 Thr Gly Leu Arg Leu Ala Ser Leu Gly Leu Lys Phe Asn Lys Ile Val 180 185 190 His Ser Ser Met Thr Arg Ala Ile Glu Thr Thr Asp Ile Ile Ser Arg 195 200 205 His Leu Pro Gly Val Cys Lys Val Ser Thr Asp Leu Leu Arg Glu Gly 210 215 220 Ala Pro Ile Glu Pro Asp Pro Pro Val Ser His Trp Lys Pro Glu Ala 225 230 235 240 Val Tyr Tyr Glu Asp Gly Ala Arg Ile Glu Ala Ala Phe Arg Asn Tyr 245 250 255 Ile His Arg Ala Asp Ala Arg Gln Glu Glu Asp Ser Tyr Glu Ile Phe 260 265 270 Ile Cys His Ala Asn Val Ile Arg Tyr Ile Val Cys Arg Ala Leu Gln 275 280 285 Phe Pro Pro Glu Xaa Trp Leu Arg Leu Ser Leu Asn Asn Gly Ser Ile 290 295 300 Thr Xaa Leu Val Ile Arg Pro Asn Gly Arg Val Ala Leu Arg Thr Leu 305 310 315 320 Gly Asp Thr Gly Phe Met Pro Asp Lys Ile Thr Arg Ser 325 330

Claims (7)

A pharmaceutical composition for preventing or treating cerebral ischemia, comprising a phosphoglycerate mutase 5 fusion protein, wherein the PEP-1 protein transport domain is covalently bound to at least one end of the phosphoglycerate mutase 5 protein to improve cell penetration efficiency.
The method according to claim 1,
The phosphoglycerate mutase 5 fusion protein is a pharmaceutical composition for preventing or treating cerebral ischemia containing a phosphoglycerate mutase 5 fusion protein, characterized in that the amino acid sequence is SEQ ID NO: 2.
For preventing or treating cerebral ischemia containing a recombinant polynucleotide encoding a phosphoglycerate mutase 5 fusion protein in which a PEP-1 protein transport domain-encoding oligonucleotide sequence is linked to at least one end of a phosphoglycerate mutase 5 coding cDNA pharmaceutical composition.
4. The method of claim 3,
The recombinant polynucleotide is a pharmaceutical composition for preventing or treating cerebral ischemia, characterized in that the base sequence is SEQ ID NO: 1.
For preventing or treating cerebral ischemia containing a recombinant polynucleotide encoding a phosphoglycerate mutase 5 fusion protein in which a PEP-1 protein transport domain coding oligonucleotide sequence is linked to at least one end of a phosphoglycerate mutase 5 coding cDNA Phosphoglycerate mutase 5 fusion protein expression vector.
A phosphoglycerate mute containing a recombinant polynucleotide encoding a phosphoglycerate mutase 5 fusion protein in which a PEP-1 protein transport domain-encoding oligonucleotide sequence is linked to at least one end of a phosphoglycerate mutase 5 coding cDNA. A pharmaceutical composition for preventing or treating cerebral ischemia comprising an Izu 5 fusion protein expression vector.
A health functional food composition for preventing or improving cerebral ischemia, comprising the phosphoglycerate mutase 5 fusion protein of claim 1 or 2 as an active ingredient.

Images