KR20230008296A - A pharmaceutical composition containing cell-transducing phosphoglycerate kinase 1 fusion protein for preventing or treating motor neuronal disorder - Google Patents

Abstract

Provided is a medicine for treating neurological diseases, which has little or no side effects and are highly effective. The present invention relates to a pharmaceutical composition for treating neurological diseases, containing a cell-permeable phosphoglycerate kinase 1 fusion protein, wherein the phosphoglycerate kinase 1 fusion protein permeated into cells increased a cell survival rate against hydrogen peroxide toxicity and reduced a level of intracellular reactive oxygen species and hydrogen peroxide-induced DNA fragmentation. The results show that the phosphoglycerate kinase 1 fusion protein has a protective effect against apoptosis, and has a therapeutic effect in protecting nerve cells from damage in neurological diseases.

Description

A pharmaceutical composition containing cell-transducing phosphoglycerate kinase 1 fusion protein for preventing or treating motor neuronal disorder}

The present invention relates to a pharmaceutical composition for preventing or treating neurological diseases comprising a cell-permeable phosphoglycerate kinase 1 fusion protein.

Motor neuron disease is a neurodegenerative disease that affects the motor nerves, the cells that control voluntary muscles in the body. Various patterns of muscle weakness are observed, and muscle spasms and spasms may occur. Walking and talking may become difficult, shortness of breath and respiratory failure may occur, difficulty swallowing saliva or excessive saliva production may also occur. Motor neuron diseases include amyotrophic lateral sclerosis (ALS), also called Lou Gehrig's disease, progressive bulbar palsy (PBP), progressive muscular atrophy (PMA), primary lateral sclerosis (PLS), pseudobulbar palsy, unimodal muscular atrophy (MMA), etc. there is

The causes of amyotrophic lateral sclerosis are not fully understood in most cases, and the mechanisms involved in motor neuron degeneration are complex. There is substantial evidence supporting the hypothesis that oxidative stress is a mechanism leading to motor neuron death. This theory was further supported by the discovery that mutations in the antioxidant enzyme superoxide dismutase 1 (SOD1) cause disease. However, the exact mechanism by which mutant SOD1 causes motor neuron degeneration is not known with certainty.

One of the most widely accepted hypotheses regarding neuronal cell death is that high levels of unsaturated fatty acids and high oxygen consumption by neurons result in increased production of reactive oxygen species (ROS), followed by damage to DNA and cell membranes. (Lin et al, 2003; Song et al, 2013). Free radicals generated during ischemia/reperfusion cause protein destruction, lipid peroxidation and DNA damage in neurons (Olmez and Ozyurt, 2012). Endogenous defense mechanisms such as antioxidants are activated to suppress excess free radicals and attenuate cell damage. Several studies have shown that antioxidant treatment significantly reduces neuronal damage induced by spinal cord ischemia (Hwang et al, 2015; Kim et al, 2012; Zhu et al, 2012).

Phosphoglycerate kinase 1 (hereinafter used interchangeably with "PGK1") is an essential enzyme of aerobic glycolysis. It is a glycolytic enzyme that catalyzes the conversion of 1,3-diphosphoglycerate to 3-phosphoglycerate to produce an ATP molecule.

PGK1 protein is secreted by tumor cells and reduces disulfide bonds in blood vessels, which is a tumor vascular inhibitor, leading to the release of angiostatin, a tumor vascular inhibitor. Enzyme deficiency is associated with a wide range of clinical phenotypes hemolytic anemia and neurological disorders.

The PGK1 protein has a rate-limiting role in coordinating energy production with biosynthesis and redox balance by controlling ATP and 3-PG levels.

In addition to regulating glycolytic metabolism, the PGK1 protein has other functions such as mediating the initiation of herpes zoster in the mammalian cell nucleus and DNA replication and repair.

However, the function or effect of PGK1 protein on neuronal apoptosis caused by reactive oxygen species has not yet been clarified.

An object of the present invention is to provide an effective preventive and therapeutic agent for neurological diseases caused by nerve cell damage and death.

In order to solve the above problems, the present inventors confirmed that protein transport domains such as PEP-1 and HIV Tat peptide were recombined with phosphoglycerate kinase 1 to penetrate NSC34 cells, and cell permeable phosphoglycerate against oxidative stress The protective effect of the kinase 1 fusion protein was studied.

The present inventors attempted to find out whether a PGK1 fusion protein bound to a protein transport domain has a protective effect on neuronal cell death induced by oxidative stress in vitro. In order to study the potential effect of PGK1 fusion protein on ischemic neuronal cell death, the present inventors constructed a PGK1 fusion protein capable of penetrating into cells. The PGK1 fusion protein penetrated into neurons effectively in a concentration-dependent and time-dependent manner. The PGK1 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 PGK1 fusion protein exhibits a protective effect against apoptosis in vitro and can be used as a therapeutic agent for neurological diseases related to oxidative stress.

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

"PGK1 fusion protein" refers to a covalent complex formed by genetic fusion or chemical bonding of a protein transport domain and a target protein (ie, PGK1 protein in the present invention), including a protein transport domain and a PGK1 protein. In this specification, since the PEP-1 protein transport domain is used as a specific example, "PGK1 fusion protein" is used interchangeably with "PEP-1-PGK1" and "PEP-1-PGK1 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 in the present invention, PGK1 is meant.

"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.

Describing the configuration of the present invention in more detail, in one embodiment of the present invention, the present inventors first developed a PGK1 fusion protein expression vector that overexpresses the PGK1 fusion protein and can be easily purified. This expression vector contains cDNA capable of expressing the human PGK1 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 PGK1 fusion protein is characterized by SEQ ID NO: 1 in the sequence listing.

Using this expression vector, the PGK1 fusion protein was overexpressed in E. coli and purified using Ni-affinity chromatography. It was confirmed by Western blot that the PGK1 fusion protein was transported to the cultured cells in a time- and concentration-dependent manner. The PGK1 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 PGK1 fusion protein is well penetrated into the cell, and the function of the PGK1 protein in the cell is well represented. Therefore, these PGK1 fusion proteins suggest the possibility of application to neurological diseases, such as protecting neurons from symptoms related to reactive oxygen species.

A pharmaceutical composition containing a cell-permeable PGK1 fusion protein as an active ingredient can be formulated in an oral or injection form by a conventional method by combining it 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 polyvinylpyrrolidone). ), 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 and treating neurological diseases, comprising the PGK1 fusion protein as an active ingredient and a pharmaceutically acceptable carrier.

The present invention also provides methods for efficiently delivering PGK1 protein into cells. Intracellular delivery of the PGK1 protein molecule according to the present invention is performed by constructing a fusion protein in which a protein transport domain such as the PEP-1 peptide is covalently linked. An example of the protein transport domain of the present invention may include a PEP-1 peptide. However, the protein transport domain of the present invention is not limited to only 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 substituting, adding, or lacking a part of the amino acid sequence of the PEP-1 peptide. Since it is easy for those skilled in the art with ordinary knowledge in the related field, the protein transport domain composed of 7 to 21 amino acids and containing 4 or more lysine or arginine and partial substitution of amino acids therefrom performs the same or similar protein transport function. It will be apparent that fusion proteins using protein transport domains also fall within the scope of the present invention.

Specifically, the present invention relates to a PGK1 fusion protein, an oligonucleotide encoding the PGK1 fusion protein, a vector comprising the oligonucleotide encoding the PGK1 fusion protein, and treatment or prevention of neurological diseases comprising the fusion protein, the oligonucleotide or the vector. It relates to a pharmaceutical composition of interest and a health functional food composition for use in preventing or improving neurological diseases including the fusion protein.

The polynucleotide encoding the PGK1 fusion protein is specifically characterized by SEQ ID NO: 1.

Examples of foods to which the cell-permeable PGK1 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. , available in capsule or beverage form. At this time, the amount of the PGK1 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 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 PGK1 fusion protein as an essential component in the indicated ratio, and may contain various flavoring agents or natural carbohydrates as additional components like conventional beverages. can 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.

The PGK1 fusion protein of the present invention is composed of 7 to 21 amino acid residues and a transport domain containing 3/4 or more arginine or lysine residues is covalently linked to at least one end of PGK1, thereby improving 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 PGK1 fusion protein of the present invention is SEQ ID NO: 2 or SEQ ID NO: 4 in the sequence listing. In preparing the PGK1 fusion protein, fusion proteins of various sequences can be obtained depending on the selection of restriction site sequences, etc., which is obvious to those skilled in the art to which the present invention belongs. It is obvious that the above amino acid sequence is only exemplary and the amino acid sequence of the PGK1 fusion protein is not limited to the sequence listed above.

In addition, the present invention provides a pharmaceutical composition for preventing and treating neurological diseases, comprising the PGK1 fusion protein as an active ingredient and a pharmaceutically acceptable carrier.

In addition, the present invention provides a health functional food composition for preventing or improving neurological diseases, using the PGK1 fusion protein as an active ingredient.

The present invention relates to a cell-transducing PGK1 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 PGK1 protein. In addition, according to the silent change of the PGK1 fusion protein, one or more amino acids in the sequence may be substituted with other amino acid(s) of similar polarity that function equally functionally. 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.

The present invention relates to a PGK1 fusion protein with improved cell penetration efficiency in which a protein transport domain is covalently linked to at least one end of a PGK1 protein.

In addition, the present invention relates to a PGK1 fusion protein characterized in that the amino acid sequence of the PGK1 fusion protein is SEQ ID NO: 1 or SEQ ID NO: 3.

In addition, the present invention relates to a pharmaceutical composition for preventing or treating motor neuron disease containing the PGK1 fusion protein.

In addition, the present invention relates to a recombinant polynucleotide encoding a PGK1 fusion protein in which an oligonucleotide sequence encoding a protein transport domain is linked to at least one end of a cDNA encoding PGK1.

In addition, the present invention relates to a recombinant polynucleotide encoding a PGK1 fusion protein characterized in that the nucleotide sequence of the recombinant polynucleotide is SEQ ID NO: 1 or SEQ ID NO: 3.

In addition, the present invention relates to a pharmaceutical composition for preventing or treating motor neuron disease containing the recombinant polynucleotide.

In addition, the present invention relates to a PGK1 fusion protein expression vector for preventing or treating motor neuron disease, which contains a recombinant polynucleotide encoding a PGK1 fusion protein in which a protein transport domain-encoding oligonucleotide sequence is linked to at least one end of a PGK1-encoding cDNA. .

In addition, the present invention is for preventing or treating motor neuron disease comprising a PGK1 fusion protein expression vector containing a recombinant polynucleotide encoding a PGK1 fusion protein in which a protein transport domain encoding oligonucleotide sequence is linked to at least one end of a PGK1-encoding cDNA. It relates to pharmaceutical compositions.

In addition, the motor neuron disease is amyotrophic lateral sclerosis (ALS), progressive bulbar palsy (PBP), progressive muscular atrophy (PMA), primary lateral sclerosis (PLS), pseudo bulbar palsy or unimodal muscular atrophy (MMA), characterized in that do.

In addition, the present invention relates to a health functional food composition for preventing or improving motor neuron disease, comprising a PGK1 fusion protein as an active ingredient.

In the present invention, the PGK1 fusion protein penetrated into cells increased cell viability against hydrogen peroxide toxicity. These results indicate that the PGK1 fusion protein has a protective effect against neuronal cell death caused by reactive oxygen species, and that the cell-permeable PGK1 fusion protein is useful as a pharmaceutical composition for preventing and treating neurological diseases.

Figure 1 shows the construction of the PEP-1-PGK1 expression vector on the pET-15b vector. Synthetic PEP-1 oligomers were cloned into the NdeI and XhoI sites, and human PGK1 (Phosphoglycerate kinase 1) cDNA was cloned into the XhoI and BamHI sites of pET-15b. (A) shows the construction of expression vectors for PGK1 and PEP-1-PGK1 fusion proteins. The PEP-1-PGK1 fusion protein contains six histidine residues. (B) is the result of expressing each protein by adding IPTG, then purifying the purified PGK1 and PEP-1-PGK1 fusion proteins using 15% SDS-PAGE, and Western blotting with an anti-rabbit polyhistidine antibody.
Figure 2 shows the permeation of the PEP-1-PGK1 fusion protein into NSC34 cells. (A) treated the culture medium with PEP-1-PGK1 fusion protein (0.5-5 μM) and PGK1 protein (0.5-5 μM) for 1 hour, and (B) treated PEP-1-PGK1 fusion protein and PGK1 protein with 15 This is the result of treatment with the culture medium for ~60 minutes and analyzing by Western blotting. (C) The intracellular distribution of the PEP-1-PGK1 fusion protein was visualized by confocal fluorescence microscopy. (D) The stability of the PEP-1-PGK1 fusion protein in NSC34 cells was analyzed by culturing the PEP-1-PGK1 fusion protein (5 μM) in NSC34 cells for 1 to 60 hours and analyzing by Western blotting.
Figure 3a shows the result of measuring cell viability by colorimetric assay using the MTT assay after adding hydrogen peroxide (100 μM) to NSC34 cells pretreated with PEP-1-PGK1 fusion protein or PGK1 protein for 1 hour, and incubating for 1 hour.
Figure 3b shows that hydrogen peroxide (100 μM) was added to NSC34 cells pretreated with PEP-1-PGK1 fusion protein or PGK1 protein for 1 hour, left for 1 hour, and cell viability was measured by colorimetric assay using 5-CFDA and AM assays. is a result
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 PGK1 fusion protein, but those skilled in the art to which the present invention pertains 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 PGK1 by applying the PGK1 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. Mouse motor neuron NSC34 cells were provided by the Korea Cell Line Research Foundation (KCLF). DCF-DA (2',7'-Dichlorofluorescein diacetate) and Bcl-2, Bax, β-actin, P-p53, p53, 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-PGK1 fusion protein

Expression vectors for the PGK1 protein and the PEP-1-PGK1 fusion protein were constructed. The human PGK1 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 BamHI exists on the 5' side. The product obtained through the polymerase chain reaction was linked to a TA vector, cut using XhoI and BamHI, and then linked to an expression vector to prepare a PEP-1-PGK1 fusion protein. Likewise, the control PGK1 protein was prepared using a vector lacking the PEP-1 peptide. After transforming the recombinant PEP-1-PGK1 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 ultrasonically disrupted and purified using a Ni2+-Nitrilostriacetate Sepharose Superflow column to obtain a PEP-1-PGK1 fusion protein. Protein concentration was determined by the Bradford method using bovine serum albumin as a standard.

Introduction of PEP-1-PGK1 fusion protein into NSC34 cells

NSC34 cells were maintained at 37°C, 95% air and 5% CO2, 10% fetal bovine serum (FBS) and 5 mM NaHCO3, antibiotics (100 μg/ml streptomycin, 100 U/ml penicillin), 20 mM HEPES/ It was cultured in humid conditions using DMEM (Dulbecco's Modified Eagle's Medium) containing NaOH (pH 7.4).

The time dependence and concentration dependence of the PEP-1-PGK1 fusion protein and control PGK1 protein transduction 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-PGK1 fusion protein and PGK1 protein in NSC34 cells, the cells were seeded on glass coverslips and treated with the PEP-1-PGK1 fusion protein and PGK1 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. NSC34 cells were blocked with PBS containing 3% bovine serum albumin and 0.1% 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 NSC34 cells were observed under an Fv-300 confocal fluorescence microscope (Olympus, Tokyo, Japan).

Cell viability assay

The viability of NSC34 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-PGK1 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 NSC34 cells treated with and without the PEP-1-PGK1 fusion protein were compared. The PEP-1-PGK1 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 group not treated with the PEP-1-PGK1 fusion protein and the group treated for 1 hour at a concentration of 5 μM to NSC34 cells 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-PGK1 fusion protein

The inventors recombined the PEP-1-vector containing PEP-1, a type of protein transduction domain (PTD), with the PGK1 gene to prepare a permeable PEP-1-PGK1 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-PGK1 fusion protein was confirmed. It was confirmed that it was purified (Fig. 1 A, B).

Result 2: Penetration of PEP-1-PGK1 fusion protein into NSC34 cells

The degree of permeation of the PEP-1-PGK1 fusion protein into NSC34 cells was measured for each time and concentration. As a result, intracellular permeation occurred effectively in a time- and concentration-dependent manner, and PGK1 protein did not permeate (Fig. 2A, B). In addition, cell nuclei were stained with DAPI using a confocal microscope, and the PEP-1-PGK1 fusion protein was stained with green fluorescence, confirming that the protein was effectively penetrated into cells (FIG. 2C). The stability of the PEP-1-PGK1 fusion protein in NSC34 cells was confirmed (Fig. 2D).

Result 3: Cell protective effect of PEP-1-PGK1 fusion protein on cell viability, generation of reactive oxygen species and DNA fragmentation due to oxidative stress

Cells were pretreated with PEP-1-PGK1 fusion protein at different concentrations, and then oxidative stress was induced with hydrogen peroxide. MTT assay was performed to confirm cell viability. When NSC34 cells were treated with 100 μM hydrogen peroxide alone, the number of surviving cells decreased to about 40%, but when PEP-1-PGK1 was pretreated, cell viability increased in a concentration-dependent manner (Fig. 3 A, B)

In addition, in order to confirm that the PEP-1-PGK1 fusion protein effectively inhibits reactive oxygen species induced when hydrogen peroxide is treated, the degree of intracellular oxidation was analyzed using 8-OHdG fluorescent dye. When NSC34 cells were exposed to 100 μM hydrogen peroxide for 1 hour, hydrogen peroxide markedly increased the 8-OHdG signal. However, reactive oxygen species induced by hydrogen peroxide were reduced by the PEP-1-PGK1 fusion protein (Fig. 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 NSC34 cells were exposed to 100 μM hydrogen peroxide for 1 hour, it was confirmed that the PEP-1-PGK1 fusion protein had a protective effect against DNA fragmentation (Fig. 3D).

Therefore, the present invention suggests the possibility that the PEP-1-PGK1 fusion protein can be effectively applied as a therapeutic agent for various diseases and neurodegenerative diseases such as neurological diseases caused by oxidative stress.

<110> Industry Academic Cooperation Foundation, Hallym University <120> A pharmaceutical composition containing cell-transducing phosphoglycerate kinase 1 fusion protein for preventing or treating motor neuronal disorder <130> HallymU-SYCHOI-PGK1-NSC34 <160> 4 <170> KoPatentIn 3.0 <210> 1 <211> 1317 <212> DNA <213> artificial sequence <220> <223> polynucleotide coding Pep-1-Phosphoglycerate kinase 1 fusion protein <400> 1 aaagaaacct ggtgggaaac ctggtggacc gaatggtctc agccgaaaaa aaaacgtaaa 60 gtgatgtcgc tttctaacaa gctgacgctg gacaagctgg acgttaaagg gaagcgggtc 120 gttatgagag tcgacttcaa tgttcctatg aagaacaacc agataacaaa caaccagagg 180 attaaggctg ctgtcccaag catcaaattc tgcttggaca atggagccaa gtcggtagtc 240 cttatgagcc acctaggccg gcctgatggt gtgcccatgc ctgacaagta ctccttagag 300 ccagttgctg tagaactcaa atctctgctg ggcaaggatg ttctgttctt gaaggactgt 360 gtaggcccag aagtggagaa agcctgtgcc aacccagctg ctgggtctgt catcctgctg 420 gagaacctcc gctttcatgt ggaggaagaa gggaagggaa aagatgcttc tgggaacaag 480 gttaaagccg agccagccaa aatagaagct ttccgagctt cactttccaa gctaggggat 540 gtctatgtca atgatgcttt tggcactgct cacagagccc acagctccat ggtaggagtc 600 aatctgccac agaaggctgg tgggtttttg atgaagaagg agctgaacta ctttgcaaag 660 gccttggaga gcccagagcg acccttcctg gccatcctgg gcggagctaa agttgcagac 720 aagatccagc tcatcaataa tatgctggac aaagtcaatg agatgattat tggtggtgga 780 atggctttta ccttccttaa ggtgctcaac aacatggaga ttggcacttc tctgtttgat 840 gaagaggggag ccaagattgt caaagaccta atgtccaaag ctgagaagaa tggtgtgaag 900 attaccttgc ctgttgactt tgtcactgct gacaagtttg atgagaatgc caagactggc 960 caagccactg tggcttctgg catacctgct ggctggatgg gcttggactg tggtcctgaa 1020 agcagcaaga agtatgctga ggctgtcact cgggctaagc agattgtgtg gaatggtcct 1080 gtgggggtat ttgaatggga agcttttgcc cggggaacca aagctctcat ggatgaggtg 1140 gtgaaagcca cttctagggg ctgcatcacc atcataggtg gtggagacac tgccacttgc 1200 tgtgccaaat ggaacacgga ggataaagtc agccatgtga gcactggggg tggtgccagt 1260 ttggagctcc tggaaggtaa agtccttcct ggggtggatg ctctcagcaa tatttag 1317 <210> 2 <211> 438 <212> PRT <213> artificial sequence <220> <223> Pep-1-Phosphoglycerate kinase 1 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 Leu Ser Asn Lys Leu Thr Leu Asp Lys 20 25 30 Leu Asp Val Lys Gly Lys Arg Val Val Met Arg Val Asp Phe Asn Val 35 40 45 Pro Met Lys Asn Asn Gln Ile Thr Asn Asn Gln Arg Ile Lys Ala Ala 50 55 60 Val Pro Ser Ile Lys Phe Cys Leu Asp Asn Gly Ala Lys Ser Val Val 65 70 75 80 Leu Met Ser His Leu Gly Arg Pro Asp Gly Val Pro Met Pro Asp Lys 85 90 95 Tyr Ser Leu Glu Pro Val Ala Val Glu Leu Lys Ser Leu Leu Gly Lys 100 105 110 Asp Val Leu Phe Leu Lys Asp Cys Val Gly Pro Glu Val Glu Lys Ala 115 120 125 Cys Ala Asn Pro Ala Ala Gly Ser Val Ile Leu Leu Glu Asn Leu Arg 130 135 140 Phe His Val Glu Glu Glu Gly Lys Gly Lys Asp Ala Ser Gly Asn Lys 145 150 155 160 Val Lys Ala Glu Pro Ala Lys Ile Glu Ala Phe Arg Ala Ser Leu Ser 165 170 175 Lys Leu Gly Asp Val Tyr Val Asn Asp Ala Phe Gly Thr Ala His Arg 180 185 190 Ala His Ser Ser Met Val Gly Val Asn Leu Pro Gln Lys Ala Gly Gly 195 200 205 Phe Leu Met Lys Lys Glu Leu Asn Tyr Phe Ala Lys Ala Leu Glu Ser 210 215 220 Pro Glu Arg Pro Phe Leu Ala Ile Leu Gly Gly Ala Lys Val Ala Asp 225 230 235 240 Lys Ile Gln Leu Ile Asn Asn Met Leu Asp Lys Val Asn Glu Met Ile 245 250 255 Ile Gly Gly Gly Met Ala Phe Thr Phe Leu Lys Val Leu Asn Asn Met 260 265 270 Glu Ile Gly Thr Ser Leu Phe Asp Glu Glu Gly Ala Lys Ile Val Lys 275 280 285 Asp Leu Met Ser Lys Ala Glu Lys Asn Gly Val Lys Ile Thr Leu Pro 290 295 300 Val Asp Phe Val Thr Ala Asp Lys Phe Asp Glu Asn Ala Lys Thr Gly 305 310 315 320 Gln Ala Thr Val Ala Ser Gly Ile Pro Ala Gly Trp Met Gly Leu Asp 325 330 335 Cys Gly Pro Glu Ser Ser Lys Lys Tyr Ala Glu Ala Val Thr Arg Ala 340 345 350 Lys Gln Ile Val Trp Asn Gly Pro Val Gly Val Phe Glu Trp Glu Ala 355 360 365 Phe Ala Arg Gly Thr Lys Ala Leu Met Asp Glu Val Val Lys Ala Thr 370 375 380 Ser Arg Gly Cys Ile Thr Ile Ile Gly Gly Gly Asp Thr Ala Thr Cys 385 390 395 400 Cys Ala Lys Trp Asn Thr Glu Asp Lys Val Ser His Val Ser Thr Gly 405 410 415 Gly Gly Ala Ser Leu Glu Leu Leu Glu Gly Lys Val Leu Pro Gly Val 420 425 430 Asp Ala Leu Ser Asn Ile 435 <210> 3 <211> 1281 <212> DNA <213> artificial sequence <220> <223> polynucleotide coding Tat-Phosphoglycerate kinase 1 fusion protein <400> 3 aggaagaagc ggagacagcg acgaagaatg tcgctttcta acaagctgac gctggacaag 60 ctggacgtta aagggaagcg ggtcgttatg agagtcgact tcaatgttcc tatgaagaac 120 aaccagataa caaacaacca gaggattaag gctgctgtcc caagcatcaa attctgcttg 180 gacaatggag ccaagtcggt agtccttatg agccacctag gccggcctga tggtgtgccc 240 atgcctgaca agtactcctt agagccagtt gctgtagaac tcaaatctct gctgggcaag 300 gatgttctgt tcttgaagga ctgtgtaggc ccagaagtgg agaaagcctg tgccaaccca 360 gctgctgggt ctgtcatcct gctggagaac ctccgctttc atgtggagga agaagggaag 420 ggaaaagatg cttctgggaa caaggttaaa gccgagccag ccaaaataga agctttccga 480 gcttcacttt ccaagctagg ggatgtctat gtcaatgatg cttttggcac tgctcacaga 540 gcccacagct ccatggtagg agtcaatctg ccacagaagg ctggtgggtt tttgatgaag 600 aaggagctga actactttgc aaaggccttg gagagcccag agcgaccctt cctggccatc 660 ctgggcggag ctaaagttgc agacaagatc cagctcatca ataatatgct ggacaaagtc 720 aatgagatga ttattggtgg tggaatggct tttaccttcc ttaaggtgct caacaacatg 780 gagattggca cttctctgtt tgatgaagag ggagccaaga ttgtcaaaga cctaatgtcc 840 aaagctgaga agaatggtgt gaagattacc ttgcctgttg actttgtcac tgctgacaag 900 tttgatgaga atgccaagac tggccaagcc actgtggctt ctggcatacc tgctggctgg 960 atgggcttgg actgtggtcc tgaaagcagc aagaagtatg ctgaggctgt cactcgggct 1020 aagcagattg tgtggaatgg tcctgtgggg gtatttgaat gggaagcttt tgcccgggga 1080 accaaagctc tcatggatga ggtggtgaaa gccacttcta ggggctgcat caccatcata 1140 ggtggtggag acactgccac ttgctgtgcc aaatggaaca cggaggataa agtcagccat 1200 gtgagcactg ggggtggtgc cagtttggag ctcctggaag gtaaagtcct tcctggggtg 1260 gatgctctca gcaatattta g 1281 <210> 4 <211> 426 <212> PRT <213> artificial sequence <220> <223> Tat-Phosphoglycerate kinase 1 fusion protein <400> 4 Arg Lys Lys Arg Arg Gln Arg Arg Arg Met Ser Leu Ser Asn Lys Leu 1 5 10 15 Thr Leu Asp Lys Leu Asp Val Lys Gly Lys Arg Val Val Met Arg Val 20 25 30 Asp Phe Asn Val Pro Met Lys Asn Asn Gln Ile Thr Asn Asn Gln Arg 35 40 45 Ile Lys Ala Ala Val Pro Ser Ile Lys Phe Cys Leu Asp Asn Gly Ala 50 55 60 Lys Ser Val Val Leu Met Ser His Leu Gly Arg Pro Asp Gly Val Pro 65 70 75 80 Met Pro Asp Lys Tyr Ser Leu Glu Pro Val Ala Val Glu Leu Lys Ser 85 90 95 Leu Leu Gly Lys Asp Val Leu Phe Leu Lys Asp Cys Val Gly Pro Glu 100 105 110 Val Glu Lys Ala Cys Ala Asn Pro Ala Ala Gly Ser Val Ile Leu Leu 115 120 125 Glu Asn Leu Arg Phe His Val Glu Glu Glu Gly Lys Gly Lys Asp Ala 130 135 140 Ser Gly Asn Lys Val Lys Ala Glu Pro Ala Lys Ile Glu Ala Phe Arg 145 150 155 160 Ala Ser Leu Ser Lys Leu Gly Asp Val Tyr Val Asn Asp Ala Phe Gly 165 170 175 Thr Ala His Arg Ala His Ser Ser Met Val Gly Val Asn Leu Pro Gln 180 185 190 Lys Ala Gly Gly Phe Leu Met Lys Lys Glu Leu Asn Tyr Phe Ala Lys 195 200 205 Ala Leu Glu Ser Pro Glu Arg Pro Phe Leu Ala Ile Leu Gly Gly Ala 210 215 220 Lys Val Ala Asp Lys Ile Gln Leu Ile Asn Asn Met Leu Asp Lys Val 225 230 235 240 Asn Glu Met Ile Ile Gly Gly Gly Met Ala Phe Thr Phe Leu Lys Val 245 250 255 Leu Asn Asn Met Glu Ile Gly Thr Ser Leu Phe Asp Glu Glu Gly Ala 260 265 270 Lys Ile Val Lys Asp Leu Met Ser Lys Ala Glu Lys Asn Gly Val Lys 275 280 285 Ile Thr Leu Pro Val Asp Phe Val Thr Ala Asp Lys Phe Asp Glu Asn 290 295 300 Ala Lys Thr Gly Gln Ala Thr Val Ala Ser Gly Ile Pro Ala Gly Trp 305 310 315 320 Met Gly Leu Asp Cys Gly Pro Glu Ser Ser Lys Lys Tyr Ala Glu Ala 325 330 335 Val Thr Arg Ala Lys Gln Ile Val Trp Asn Gly Pro Val Gly Val Phe 340 345 350 Glu Trp Glu Ala Phe Ala Arg Gly Thr Lys Ala Leu Met Asp Glu Val 355 360 365 Val Lys Ala Thr Ser Arg Gly Cys Ile Thr Ile Ile Gly Gly Gly Asp 370 375 380 Thr Ala Thr Cys Cys Ala Lys Trp Asn Thr Glu Asp Lys Val Ser His 385 390 395 400 Val Ser Thr Gly Gly Gly Ala Ser Leu Glu Leu Leu Glu Gly Lys Val 405 410 415 Leu Pro Gly Val Asp Ala Leu Ser Asn Ile 420 425

Claims (10)

A phosphoglycerate kinase 1 fusion protein with improved cell penetration efficiency in which a protein transport domain is covalently linked to at least one end of a phosphoglycerate kinase 1 protein.
The method of claim 1,
The phosphoglycerate kinase 1 fusion protein is a phosphoglycerate kinase 1 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 motor neuron disease containing the phosphoglycerate kinase 1 fusion protein of claim 1 or claim 2.
A recombinant polynucleotide encoding a phosphoglycerate kinase 1 fusion protein in which an oligonucleotide sequence encoding a protein transport domain is linked to at least one end of a cDNA encoding phosphoglycerate kinase 1.
The method of claim 4,
The recombinant polynucleotide encoding a phosphoglycerate kinase 1 fusion protein, characterized in that the base sequence of the recombinant polynucleotide is SEQ ID NO: 1 or SEQ ID NO: 3.
A pharmaceutical composition for preventing or treating motor neuron disease containing the recombinant polynucleotide of claim 4 or claim 5.
Phosphoglycerate for prevention or treatment of motor neuron disease containing a recombinant polynucleotide encoding a phosphoglycerate kinase 1 fusion protein in which a protein transport domain-encoding oligonucleotide sequence is linked to at least one end of a phosphoglycerate kinase 1-encoding cDNA. Late kinase 1 fusion protein expression vector.
A phosphoglycerate kinase 1 fusion protein expression vector containing a recombinant polynucleotide encoding a phosphoglycerate kinase 1 fusion protein in which a protein transport domain-encoding oligonucleotide sequence is linked to at least one end of a phosphoglycerate kinase 1-encoding cDNA. A pharmaceutical composition for preventing or treating motor neuron disease comprising a.
According to any one selected from claims 3, 6, and 8,
The motor neuron disease is amyotrophic lateral sclerosis (ALS), progressive bulbar palsy (PBP), progressive muscular atrophy (PMA), primary lateral sclerosis (PLS), pseudobulbar palsy or unimodal muscular atrophy (MMA), motor neuron disease prevention or a pharmaceutical composition for treatment.
A health functional food composition for preventing or improving motor neuron disease, comprising the phosphoglycerate kinase 1 fusion protein of claim 1 or claim 2 as an active ingredient.

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