KR20230030060A - Pharmaceutical composition for treating cerebral ischemia containing cell-transducing SH3GL3 fusion protein - Google Patents

Abstract

The present invention is to provide a drug for treatment of cerebral ischemia that has little or no side effects and is highly effective. The present invention relates to a pharmaceutical composition for treating cerebral ischemia containing a cell-permeable SH3GL3 fusion protein, wherein SH3GL3 fusion protein permeated into cells increased cell survival rate against hydrogen peroxide toxicity, and reduced intracellular reactive oxygen species levels and hydrogen peroxide-induced DNA fragmentation. In animal models, the SH3GL3 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 SH3GL3 fusion protein has a protective effect against apoptosis and has a therapeutic effect in protecting neurons from cerebral ischemic damage.

Description

Pharmaceutical composition for treating cerebral ischemia containing cell-transducing SH3GL3 fusion protein {Pharmaceutical composition for treating cerebral ischemia containing cell-transducing SH3GL3 fusion protein}

The present invention relates to a pharmaceutical composition for treating cerebral ischemia comprising a cell-permeable SH3GL3 fusion protein.

"Free radicals" called reactive oxygen compounds are responsible for oxidative stress, and reactive oxygen species (ROS) are unavoidable by-products of a variety of cellular processes involving interaction with oxygen. . If cells are exposed to relatively more active compounds, they are subjected to oxidative stress, resulting in oxidative 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 such as inflammation, ischemia, diabetes and Parkinson's disease. Reactive oxygen species have many effects, such as damaging polymers such as proteins and lipids as well as DNA in cells during the metabolic process of cells. Among them, cerebral ischemia is a disease in which brain cells do not produce enough energy when the artery of the brain is blocked, and cell death occurs as a result. Hypoxic conditions, such as cerebral ischemia, induce the translocation of cytosolic Bax to outer mitochondria, which then promotes the 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 hypoxic conditions, the expression level of Bcl-2 is greatly reduced, which is a major cause of apoptosis. Therefore, if a substance capable of regulating reactive oxygen species is identified, it is expected to have a protective effect against cerebral ischemic damage.

SH3GL3 (SH3-domain GRB2-like 3) is known as endophilin A3 or extra 119 (EEN)-B2 and belongs to SH3 domain containing proteins.

The SH3 domain GRB2-like gene family consists of three types: SH3GL1, SH3GL2, and SH3GL3.

SH3GL3 was first identified in 1998 and identified as a Huntington's disease risk gene.

SH3GL3 is preferentially expressed in mammalian brain and testis and can interact with huntingtinexon 1 protein, dynamin, and synaptojanin.

It has been reported to promote myeloma cell migration, stemness and chemical resistance and can be used as a prognostic marker for melanoma.

However, the function or effect of SH3GL3 protein on cerebral ischemia caused by reactive oxygen species has not yet been clarified.

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

In order to solve the above problems, the present inventors confirmed that protein transport domains such as PEP-1 and HIV Tat peptide recombine with SH3GL3 to penetrate into HT22 cells, and studied the protective effect of cell-penetrating SH3GL3 fusion proteins against oxidative stress did

The present inventors attempted to find out whether the SH3GL3 fusion protein bound to the protein transport domain has a protective effect against ischemic neuronal cell death induced by oxidative stress in vitro. In order to study the potential effect of the SH3GL3 fusion protein on ischemic neuronal cell death, the present inventors constructed a SH3GL3 fusion protein capable of penetrating into cells. The SH3GL3 fusion protein penetrated into neurons effectively in a concentration-dependent and time-dependent manner. The SH3GL3 fusion protein penetrated into cells increased the cell survival rate against hydrogen peroxide toxicity and lowered the level of intracellular reactive oxygen species. These results indicate that the SH3GL3 fusion protein exhibits a protective effect against apoptosis occurring in vitro and can be used as a therapeutic agent for cerebral ischemic damage associated with oxidative stress.

Describing the configuration of the present invention in more detail, in one embodiment of the present invention, the present inventors first overexpressed the SH3GL3 fusion protein and developed a SH3GL3 fusion protein expression vector that can be easily purified. This expression vector contains cDNA capable of expressing the human SH3GL3 protein, one or more protein transport domains of 7 to 21 amino acids, and six histidine residues at the N-terminus. As an example, the amino acid sequence of the SH3GL3 fusion protein is SEQ ID NO: 2 or SEQ ID NO: 4 in the sequence listing.

Using this expression vector, the SH3GL3 fusion protein was overexpressed in E. coli and purified using Ni-affinity chromatography. It was confirmed by Western blot that the SH3GL3 fusion protein was transported to the cultured cells in a time- and concentration-dependent manner. The SH3GL3 fusion protein penetrated into cells was continuously maintained in cells for up to 12 hours and inhibited apoptosis caused by oxidative stress.

These results indicate that the SH3GL3 fusion protein is well penetrated into the cell, and the function of the SH3GL3 protein in the cell is well represented. Therefore, these SH3GL3 fusion proteins suggest the possibility of application to neurological diseases, such as protecting neurons from symptoms related to reactive oxygen species or cerebral ischemic damage.

A pharmaceutical composition containing the cell-permeable SH3GL3 fusion protein as an active ingredient can be formulated in an oral or injection form by a conventional method in combination with a carrier commonly accepted in the pharmaceutical field. Oral compositions include, for example, tablets and gelatin capsules, which contain, in addition to the active ingredient, 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 the tablets may also contain a binder (eg magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and/or polyvinyl pyrrolidone). ), optionally containing disintegrants (eg starch, agar, alginic acid or its sodium salt) or boiling mixtures and/or absorbents, colorants, flavors and sweeteners. Injectable compositions are preferably isotonic aqueous solutions or suspensions, the compositions mentioned being sterile and/or containing adjuvants (eg preservatives, stabilizers, wetting or emulsifying agents, solution accelerators, salts and/or buffers for regulating osmotic pressure). In addition, they may contain other therapeutically useful substances.

The pharmaceutical preparation prepared in this way may be administered orally or parenterally, that is, intravenous, subcutaneous, intraperitoneal or topically applied, as desired. The dose may be administered by dividing the daily dose of 0.0001 to 100 mg/kg into 1 to several times. The dosage level for a specific patient may vary depending on the patient's weight, age, sex, health condition, 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 SH3GL3 fusion protein as an active ingredient and a pharmaceutically acceptable carrier.

The present invention also provides methods for efficiently delivering SH3GL3 proteins into cells. Intracellular delivery of the SH3GL3 protein molecule according to the present invention is performed by constructing a fusion protein in which protein transport domains such as PEP-1 peptide and HIV-Tat peptide are covalently linked. An example of the protein transport domain of the present invention is a PEP-1 peptide or an HIV-Tat peptide. However, the protein transport domain of the present invention is not limited to only the PEP-1 peptide or the HIV-Tat peptide, and has a function similar to those of these protein transport domain peptides due to partial substitution, addition or lack of amino acid sequence of the PEP-1 peptide or HIV-Tat. Since it is easy for those skilled in the art to prepare a peptide to which the present invention belongs, a protein transport domain consisting of 7 to 21 amino acids and containing 4 or more lysine or arginine and a portion of amino acids therefrom It will be apparent that fusion proteins using protein transport domains that perform identical or similar protein transport functions by substitution also fall within the scope of the present invention.

Specifically, the present invention provides a SH3GL3 fusion protein, an oligonucleotide encoding the SH3GL3 fusion protein, a vector containing the oligonucleotide encoding the SH3GL3 fusion protein, a pharmaceutical composition containing the fusion protein for the purpose of treating or preventing cerebral ischemia, and the fusion protein It relates to a health functional food composition for preventing or improving cerebral ischemia comprising a.

The polynucleotide encoding the SH3GL3 fusion protein is specifically characterized by SEQ ID NO: 1 or SEQ ID NO: 3, but is not limited thereto.

Examples of foods to which the cell-permeable SH3GL3 fusion protein of the present invention can be added include various foods, such as beverages, gum, tea, vitamin complexes, and health supplements, including pills, powders, granules, precipitates, and tablets. , can be used in capsule or beverage form. At this time, the amount of the SH3GL3 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 food weight in the case of the health food composition of the present invention, and the health drink composition of the In this case, it may be added at a rate of 0.1 to 30 g, preferably 0.2 to 5 g, based on 100 ml. The health beverage composition of the present invention of the present invention has no particular limitations on the liquid component, except for containing the SH3GL3 fusion protein as an essential component in the indicated ratio, and contains various flavoring agents or natural carbohydrates as additional components like conventional beverages. can do. Examples of the aforementioned natural carbohydrates include monosaccharides such as glucose, fructose, etc. disaccharides such as maltose, sucrose, etc. polysaccharides such as dextrins, cyclodextrins, etc., and xylitol. , sorbitol, erythritol, and the like. As flavoring agents other than those mentioned above, natural flavoring agents (thaumatin, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can advantageously be 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 includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and enhancers (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its It may contain salts, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, and the like. In addition, the compositions of the present invention may contain fruit flesh for preparing natural fruit juice, 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 the range of 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.

"SH3GL3 fusion protein" includes a protein transport domain and a SH3GL3 protein, and refers to a covalent complex formed by genetic fusion or chemical bonding of a protein transport domain and a target protein (ie, SH3GL3 protein in the present invention). In this specification, since the PEP-1 protein transport domain was used as a specific example, "SH3GL3 fusion protein" was used interchangeably with "PEP-1-SH3GL3" and "PEP-1-SH3GL3 fusion protein".

"Target protein" is a molecule that is not a transport domain or a fragment thereof that cannot enter a target cell by itself or enter a target cell at a useful rate, either as such or as the target of a transport domain-target protein complex before being fused with the transport domain. the protein part. Target proteins include polypeptides, proteins, and peptides, and SH3GL3 is meant in the present invention.

"Fusion protein" refers to a complex formed by genetic fusion or chemical bonding of a transport domain and a target protein, including a transport domain and at least one target protein portion.

In addition, 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 into 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 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 in vitro cells, that is, cultured animal cells, human cells, or microorganisms.

The "protein transport domain" in the present invention forms a covalent bond with a high-molecular organic compound, such as an oligonucleotide, peptide, protein, oligosaccharide or polysaccharide, etc. to introduce the organic compound into the cell without requiring a separate receptor, transporter or energy. say what you can

Also, in the present specification, expressions such as “transportation”, “penetration”, “transportation”, “delivery”, “permeation”, and “passing” are used interchangeably with respect to “introduction” of proteins, peptides, and organic compounds into cells.

The SH3GL3 fusion protein of the present invention consists of 7 to 21 amino acid residues and a transport domain containing 3/4 or more arginine or lysine residues is covalently bonded to at least one end of SH3GL3 to improve 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 SH3GL3 fusion protein of the present invention includes SEQ ID NO: 2 or 4 and the like. In preparing the SH3GL3 fusion protein, fusion proteins of various sequences can be obtained depending on the selection of restriction site sequences, etc., which is apparent to those skilled in the art to which the present invention belongs. The above amino acid sequence is only exemplary, and it is obvious that the amino acid sequence of the SH3GL3 fusion protein is not limited to the sequence listed above.

In addition, the present invention provides a pharmaceutical composition for preventing and treating cerebral ischemia, comprising the SH3GL3 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 SH3GL3 fusion protein as an active ingredient.

The present invention relates to a cell-transducing SH3GL3 fusion protein in which a protein transport domain consisting of 7 to 21 amino acids and containing 4 or more lysines or arginines is covalently linked to at least one end of the SH3GL3 protein. In addition, SH3GL3 fusion proteins may be substituted with other amino acid(s) of similar polarity that function equivalently to one or more amino acids in the sequence according to silent change. Amino acid substitutions in the sequence may be selected from other members of the class to which the amino acid belongs. For example, classes of hydrophobic amino acids include alanine, valine, leucine, isoleucine, phenylalanine, valine, tryptophan, proline and methionine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. Positively 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, for example, within the range of 85-100%, are also included in the scope of the present invention.

In the present invention, the SH3GL3 fusion protein penetrated into cells increased cell viability against hydrogen peroxide toxicity. These results indicate that the SH3GL3 fusion protein has a protective effect against apoptosis caused by cerebral ischemia caused by reactive oxygen species and has a therapeutic effect of protecting neurons from cerebral ischemic damage, and that the cell-permeable SH3GL3 fusion protein prevents and treats cerebral ischemia. It tells us that it is useful as a pharmaceutical composition for use.

Figure 1 shows the construction of the PEP-1-SH3GL3 expression vector on the pET-15b vector. Synthetic PEP-1 oligomers were cloned into the NdeI and XhoI sites, and human SH3GL3 (Phosphoglycerate mutase 5) cDNA was cloned into the XhoI and BamHI sites of pET-15b. Figure 1a shows the construction of expression vectors for SH3GL3 and PEP-1-SH3GL3 fusion proteins. The PEP-1-SH3GL3 fusion protein contains six histidine residues. Figure 1b shows the results of expressing each protein by adding IPTG, then purified SH3GL3 and PEP-1-SH3GL3 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-SH3GL3 fusion protein into HT22 cells. 2a shows that PEP-1-SH3GL3 fusion protein (0.5-5 μM) and SH3GL3 protein (0.5-5 μM) were treated in the culture medium for 1 hour, and FIG. 2b shows PEP-1-SH3GL3 fusion protein and SH3GL3 protein at 15-60 This is the result of treatment with the culture medium for 10 minutes and analysis by Western blotting. Figure 2c is a visualization of the intracellular distribution of the PEP-1-SH3GL3 fusion protein by confocal fluorescence microscopy. Figure 2d is the result of analyzing the stability of the PEP-1-SH3GL3 fusion protein in HT22 cells. After transduction with the PEP-1-SH3GL3 fusion protein (5 μM), the cells were cultured for 1-60 hours and analyzed by Western blotting.
Figure 3a shows the result of measuring cell viability using the MTT assay after adding hydrogen peroxide (100 μM) to HT22 cells pretreated with PEP-1-SH3GL3 fusion protein or SH3GL3 protein for 1 hour, and then incubating for 1 hour.
Figure 3b is the result of measuring the intracellular reactive oxygen species level by 5-CFDA staining.
Figure 3c is a result of measuring the level of reactive oxygen species in cells by DCF-DA staining.
Figure 3d is a result of measuring DNA fragmentation by TUNEL staining.

In the following, the configuration of the present invention will be described in more detail with reference to specific embodiments. 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, the PEP-1 peptide was used as a protein transport domain constituting the SH3GL3 fusion protein, but those of ordinary skill in the art to which the present invention belongs can combine the PEP-1 peptide with 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 one or more of the N-terminus or C-terminus of SH3GL3 by applying the SH3GL3 fusion protein. can be easily formed, and there may be slight differences between these fusion proteins, but it can be easily seen that they exhibit similar activities.

ingredient

Ni ^{2+ -} Sepharose nitrilotriacetate Superflow was purchased from Qiagen, and PD-10 column chromatography (Amersham, Brauncschweig, Germany) was purchased. HT22 cells, a mouse motor neuron, 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, 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 of special quality.

Expression and purification of PEP-1-SH3GL3 fusion protein

Expression vectors for the SH3GL3 protein and the PEP-1-SH3GL3 fusion protein were constructed. The human SH3GL3 gene was amplified by polymerase chain reaction using two primers together with cDNA. The sense primer is 5'-CTCGAGGGCAACGCGCAG-3', and a restriction enzyme action site called XhoI is present at the 5' side. The antisense primer is 5'-GGATCCTCAGGAATCTTCGGACTC-3', and a restriction enzyme action site called BamH I is present on the 5' side. The product obtained through the polymerase chain reaction was linked to a TA vector, cut using Xho I and BamH I, and then linked to an expression vector to prepare a PEP-1-SH3GL3 fusion protein. Likewise, a control SH3GL3 protein was prepared using a vector lacking the PEP-1 peptide. The recombinant PEP-1-SH3GL3 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 ultrasonically disrupted and purified using Ni ^{2+ -} Sepharose Nitrilostriacetate Superflow column to obtain PEP-1-SH3GL3 fusion protein. Protein concentration was determined by the Bradford method using bovine serum albumin as a standard.

Introduction of PEP-1-SH3GL3 fusion protein into HT22 cells

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

The time dependence and concentration dependence of the transduction of the PEP-1-SH3GL3 fusion protein and the control SH3GL3 protein into cells were evaluated. Cells were treated in 60 mm dishes at 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 proteins permeated into cells were quantified by the Bradford method and analyzed by Western blotting.

Western blot analysis

For Western blot analysis, proteins in the cell homogenate were separated on a 12% SDS polyacrylamide gel, and the proteins in the gel were electrotransferred to a nitrocellulose membrane (Amersham, UK). The membrane was blocked with TBS-T buffer (25 mM Tris-HCl, 140 mM NaCl, 0.1% Tween 20, pH 7.5). 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 the PEP-1-SH3GL3 fusion protein and SH3GL3 protein in HT22 cells, the cells were seeded on glass coverslips and treated with the PEP-1-SH3GL3 fusion protein and SH3GL3 protein at a concentration of 5 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 containing 3% bovine serum albumin and 01% Triton X-100 (PBS-BT) for 40 minutes at room temperature, and then 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 a colorimetric assay 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 pretreatment with 0.5 to 5 μM of PEP-1-SH3GL3 fusion protein. Absorbance was measured at 570 nm with an ELISA microplate reader (Labsystems Multiskan MCC/340). Cell viability was expressed as a percentage of the untreated control.

Measurement of 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. The levels of reactive oxygen species in HT22 cells treated with and without the PEP-1-SH3GL3 fusion protein were compared. The PEP-1-SH3GL3 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. Then, they were 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 wavelength and 538 nm emission wavelength using a Fluoroskan ELISA plate reader (Labsystems, Helsinki, Finland).

TUNEL assay

Hydrogen peroxide (100 μM) was added to the culture medium of the HT22 cells of the group not treated with the PEP-1-SH3GL3 fusion protein and the group treated for 1 hour at a concentration of 5 μM and treated 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). Results were analyzed using a fluorescence microscope (Nikon eclipse 80i, Japan).

Result 1: Purification and cell penetration of PEP-1-SH3GL3 fusion protein

The present inventors recombined the PEP-1-vector containing PEP-1, a type of protein transduction domain (PTD), with the SH3GL3 gene to prepare a permeable PEP-1-SH3GL3 fusion protein (Fig. 1a). It was overexpressed in Escherichia coli and purified using Ni ^{2+ -} Sepharose nitrilotriacetic acid affinity chromatography column and PD-10 column chromatography, and using SDS-PAGE and Western blot analysis, the PEP-1-SH3GL3 fusion protein was confirmed. It was confirmed that it was purified (Fig. 1a, 1b).

Result 2: Penetration of PEP-1-SH3GL3 fusion protein into HT22 cells

The degree of penetration into HT22 cells was measured according to the time and concentration of the PEP-1-SH3GL3 fusion protein. As a result, intracellular permeation occurred effectively in a time- and concentration-dependent manner, and SH3GL3 protein did not permeate (Figs. 2a and 2b). In addition, cell nuclei were stained with DAPI using a confocal microscope, and the PEP-1-SH3GL3 fusion protein was stained with green fluorescence, confirming that the protein was effectively penetrated into cells (FIG. 2c). In addition, the PEP-1-SH3GL3 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-SH3GL3 fusion protein to HT22 cells (FIG. 2d).

Result 3: Cell protective effect of PEP-1-SH3GL3 fusion protein on cell viability, generation of reactive oxygen species and DNA fragmentation due to oxidative stress

Cells were pretreated with PEP-1-SH3GL3 fusion protein at different 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 viable cells decreased to about 40%, but when pretreated with PEP-1-SH3GL3, cell viability increased in a concentration-dependent manner (FIG. 3a).

In addition, in order to confirm that the PEP-1-SH3GL3 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, hydrogen peroxide markedly increased the 8-OHdG signal. However, reactive oxygen species induced by hydrogen peroxide were reduced by the PEP-1-SH3GL3 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 specifically attached to the DNA fragmentation site. It was performed under the same conditions as in the previous experiment, and when HT22 cells were exposed to 100 μM hydrogen peroxide for 1 hour, it was confirmed that the PEP-1-SH3GL3 fusion protein had a protective effect against DNA fragmentation (FIG. 3d).

Therefore, the present invention suggests the possibility that the PEP-1-SH3GL3 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 SH3GL3 fusion protein <130> HallymU-SYCHOI-SH3GL3-Brain-Ischemia <160> 6 <170> KoPatentIn 3.0 <210> 1 <211> 1107 <212> DNA <213> artificial sequence <220> <223> polynucleotide coding Pep-SH3GL3 fusion protein <400> 1 aaagaaacct ggtgggaaac ctggtggacc gaatggtctc agccgaaaaa aaaacgtaaa 60 gtgatgtcgg tggccgggct gaagaagcag ttccacaaag ccagccagct atttagtgaa 120 aaaataagtg gtgctgaagg aactaaacta gacgatgaat ttcttgacat ggaaaggaaa 180 atagatgtta ccaataaagt tgttgcagaa attctttcaa aaaccactga atatcttcag 240 ccaaatccag catacagagc taagctagga atgctgaaca ctgtgtcgaa gatccgaggg 300 caggtgaaga ccacaggata cccgcagacg gaaggcttgc tgggggactg tatgctgaaa 360 tacgggaagg agctcgggga agactccacc tttggcaatg cattgataga agttggtgaa 420 tccatgaagc taatggctga ggtgaaagac tctcttgata ttaatgtaaa gcaaactttt 480 attgatccac ttcagttact acaagataaa gatttaaaag agatcgggca tcacctgaaa 540 aagctggaag gccgccgcct ggattacgat tataaaaaga aacgagtagg taagatacca 600 gacgaagaag tcagacaagc ggtagaaaaa tttgaagagt caaaggagtt ggctgaaaga 660 agcatgttta actttttaga aaatgatgta gaacaagtca gccagttggc tgtgttcata 720 gaggcagcat tagactatca cagacagtcc acagagattc tgcaggagct gcagagcaag 780 ctacagatgc gaatatcagc tgcatccagt gtccccagac gagaatacaa gccaaggcct 840 gtgaaaagga gttctagtga gctcaatgga gtttccacca cctctgtagt gaagacgaca 900 ggttctaaca ttcccatgga ccagccctgc tgtcgtggtc tctatgactt tgagccagaa 960 aaccaaggag aattaggatt taaagaaggg gacatcatta cattaaccaa tcaaatagat 1020 gaaaactggt atgaaggaat gatacacgga gaatcgggat tcttccccat taattacgtg 1080 gaagtgatcg tgcctttacc tcagtaa 1107 <210> 2 <211> 368 <212> PRT <213> artificial sequence <220> <223> Pep1-SH3GL3 fusion protein <400> 2 Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu Trp Ser Gln Pro Lys 1 5 10 15 Lys Lys Arg Lys Val Met Ser Val Ala Gly Leu Lys Lys Gln Phe His 20 25 30 Lys Ala Ser Gln Leu Phe Ser Glu Lys Ile Ser Gly Ala Glu Gly Thr 35 40 45 Lys Leu Asp Asp Glu Phe Leu Asp Met Glu Arg Lys Ile Asp Val Thr 50 55 60 Asn Lys Val Val Ala Glu Ile Leu Ser Lys Thr Thr Glu Tyr Leu Gln 65 70 75 80 Pro Asn Pro Ala Tyr Arg Ala Lys Leu Gly Met Leu Asn Thr Val Ser 85 90 95 Lys Ile Arg Gly Gln Val Lys Thr Thr Gly Tyr Pro Gln Thr Glu Gly 100 105 110 Leu Leu Gly Asp Cys Met Leu Lys Tyr Gly Lys Glu Leu Gly Glu Asp 115 120 125 Ser Thr Phe Gly Asn Ala Leu Ile Glu Val Gly Glu Ser Met Lys Leu 130 135 140 Met Ala Glu Val Lys Asp Ser Leu Asp Ile Asn Val Lys Gln Thr Phe 145 150 155 160 Ile Asp Pro Leu Gln Leu Leu Gln Asp Lys Asp Leu Lys Glu Ile Gly 165 170 175 His His Leu Lys Lys Leu Glu Gly Arg Arg Leu Asp Tyr Asp Tyr Lys 180 185 190 Lys Lys Arg Val Gly Lys Ile Pro Asp Glu Glu Val Arg Gln Ala Val 195 200 205 Glu Lys Phe Glu Glu Ser Lys Glu Leu Ala Glu Arg Ser Met Phe Asn 210 215 220 Phe Leu Glu Asn Asp Val Glu Gln Val Ser Gln Leu Ala Val Phe Ile 225 230 235 240 Glu Ala Ala Leu Asp Tyr His Arg Gln Ser Thr Glu Ile Leu Gln Glu 245 250 255 Leu Gln Ser Lys Leu Gln Met Arg Ile Ser Ala Ala Ser Ser Val Pro 260 265 270 Arg Arg Glu Tyr Lys Pro Arg Pro Val Lys Arg Ser Ser Ser Glu Leu 275 280 285 Asn Gly Val Ser Thr Thr Ser Val Val Lys Thr Thr Gly Ser Asn Ile 290 295 300 Pro Met Asp Gln Pro Cys Cys Arg Gly Leu Tyr Asp Phe Glu Pro Glu 305 310 315 320 Asn Gln Gly Glu Leu Gly Phe Lys Glu Gly Asp Ile Ile Thr Leu Thr 325 330 335 Asn Gln Ile Asp Glu Asn Trp Tyr Glu Gly Met Ile His Gly Glu Ser 340 345 350 Gly Phe Phe Pro Ile Asn Tyr Val Glu Val Ile Val Pro Leu Pro Gln 355 360 365 <210> 3 <211> 1071 <212> DNA <213> artificial sequence <220> <223> polynucleotide coding Tat-SH3GL3 fusion protein <400> 3 aggaagaagc ggagacagcg acgaagaatg tcggtggccg ggctgaagaa gcagttccac 60 aaagccagcc agctatttag tgaaaaaata agtggtgctg aaggaactaa actagacgat 120 gaatttcttg acatggaaag gaaaatagat gttaccaata aagttgttgc agaaattctt 180 tcaaaaacca ctgaatatct tcagccaaat ccagcataca gagctaagct aggaatgctg 240 aacactgtgt cgaagatccg agggcaggtg aagaccacag gatacccgca gacggaaggc 300 ttgctggggg actgtatgct gaaatacggg aaggagctcg gggaagactc cacctttggc 360 aatgcattga tagaagttgg tgaatccatg aagctaatgg ctgaggtgaa agactctctt 420 gatattaatg taaagcaaac tttattgat ccacttcagt tactacaaga taaagattta 480 aaagagatcg ggcatcacct gaaaaagctg gaaggccgcc gcctggatta cgattataaa 540 aagaaacgag taggtaagat accagacgaa gaagtcagac aagcggtaga aaaatttgaa 600 gagtcaaagg agttggctga aagaagcatg tttaactttt tagaaaatga tgtagaacaa 660 gtcagccagt tggctgtgtt catagaggca gcattagact atcacagaca gtccacagag 720 attctgcagg agctgcagag caagctacag atgcgaatat cagctgcatc cagtgtcccc 780 agacgagaat acaagccaag gcctgtgaaa aggagttcta gtgagctcaa tggagtttcc 840 accacctctg tagtgaagac gacaggttct aacattccca tggaccagcc ctgctgtcgt 900 ggtctctatg actttgagcc agaaaaccaa ggagaattag gatttaaaga agggggacatc 960 attacattaa ccaatcaaat agatgaaaac tggtatgaag gaatgataca cggagaatcg 1020 ggattcttcc ccattaatta cgtggaagtg atcgtgcctt tacctcagta a 1071 <210> 4 <211> 356 <212> PRT <213> artificial sequence <220> <223> Tat-SH3GL3 fusion protein <400> 4 Arg Lys Lys Arg Arg Gln Arg Arg Arg Met Ser Val Ala Gly Leu Lys 1 5 10 15 Lys Gln Phe His Lys Ala Ser Gln Leu Phe Ser Glu Lys Ile Ser Gly 20 25 30 Ala Glu Gly Thr Lys Leu Asp Asp Glu Phe Leu Asp Met Glu Arg Lys 35 40 45 Ile Asp Val Thr Asn Lys Val Val Ala Glu Ile Leu Ser Lys Thr Thr 50 55 60 Glu Tyr Leu Gln Pro Asn Pro Ala Tyr Arg Ala Lys Leu Gly Met Leu 65 70 75 80 Asn Thr Val Ser Lys Ile Arg Gly Gln Val Lys Thr Thr Gly Tyr Pro 85 90 95 Gln Thr Glu Gly Leu Leu Gly Asp Cys Met Leu Lys Tyr Gly Lys Glu 100 105 110 Leu Gly Glu Asp Ser Thr Phe Gly Asn Ala Leu Ile Glu Val Gly Glu 115 120 125 Ser Met Lys Leu Met Ala Glu Val Lys Asp Ser Leu Asp Ile Asn Val 130 135 140 Lys Gln Thr Phe Ile Asp Pro Leu Gln Leu Leu Gln Asp Lys Asp Leu 145 150 155 160 Lys Glu Ile Gly His His Leu Lys Lys Leu Glu Gly Arg Arg Leu Asp 165 170 175 Tyr Asp Tyr Lys Lys Lys Arg Val Gly Lys Ile Pro Asp Glu Glu Val 180 185 190 Arg Gln Ala Val Glu Lys Phe Glu Glu Ser Lys Glu Leu Ala Glu Arg 195 200 205 Ser Met Phe Asn Phe Leu Glu Asn Asp Val Glu Gln Val Ser Gln Leu 210 215 220 Ala Val Phe Ile Glu Ala Ala Leu Asp Tyr His Arg Gln Ser Thr Glu 225 230 235 240 Ile Leu Gln Glu Leu Gln Ser Lys Leu Gln Met Arg Ile Ser Ala Ala 245 250 255 Ser Ser Val Pro Arg Arg Glu Tyr Lys Pro Arg Pro Val Lys Arg Ser 260 265 270 Ser Ser Glu Leu Asn Gly Val Ser Thr Thr Ser Val Val Lys Thr Thr 275 280 285 Gly Ser Asn Ile Pro Met Asp Gln Pro Cys Cys Arg Gly Leu Tyr Asp 290 295 300 Phe Glu Pro Glu Asn Gln Gly Glu Leu Gly Phe Lys Glu Gly Asp Ile 305 310 315 320 Ile Thr Leu Thr Asn Gln Ile Asp Glu Asn Trp Tyr Glu Gly Met Ile 325 330 335 His Gly Glu Ser Gly Phe Phe Pro Ile Asn Tyr Val Glu Val Ile Val 340 345 350 Pro Leu Pro Gln 355 <210> 5 <211> 17 <212> DNA <213> artificial sequence <220> <223> primer <400> 5 ctcgagggca acgcgca 17 <210> 6 <211> 24 <212> DNA <213> artificial sequence <220> <223> primer <400> 6 ggatcctcag gaatcttcgg actc 24

Claims (7)

A pharmaceutical composition for preventing or treating cerebral ischemia comprising a SH3GL3 fusion protein in which a protein transport domain is covalently bonded to at least one end of a SH3GL3 protein to improve cell penetration efficiency.
The method of claim 1,
The SH3GL3 fusion protein is a pharmaceutical composition for preventing or treating cerebral ischemia containing a SH3GL3 fusion protein, characterized in that the amino acid sequence is SEQ ID NO: 2 or SEQ ID NO: 4.
A pharmaceutical composition for preventing or treating cerebral ischemia comprising a recombinant polynucleotide encoding a SH3GL3 fusion protein in which an oligonucleotide sequence encoding a protein transport domain is linked to at least one end of a cDNA encoding SH3GL3.
The method of claim 3,
The recombinant polynucleotide is a pharmaceutical composition for preventing or treating cerebral ischemia, characterized in that the nucleotide sequence is SEQ ID NO: 1 or SEQ ID NO: 3.
A SH3GL3 fusion protein expression vector for preventing or treating cerebral ischemia, comprising a recombinant polynucleotide encoding a SH3GL3 fusion protein in which an oligonucleotide sequence encoding a protein transport domain is linked to at least one end of a SH3GL3-encoding cDNA.
A pharmaceutical composition for preventing or treating cerebral ischemia comprising a SH3GL3 fusion protein expression vector containing a recombinant polynucleotide encoding a SH3GL3 fusion protein in which a protein transport domain encoding oligonucleotide sequence is linked to at least one end of a SH3GL3-encoding cDNA.
A health functional food composition for preventing or improving cerebral ischemia comprising the SH3GL3 fusion protein of claim 1 or claim 2 as an active ingredient.

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