KR20210158516A - A pharmaceutical composition containing cell-transducing CRYM fusion protein for preventing or treating motor neuronal disorder - Google Patents

Abstract

Provided is a drug for treating neurological diseases having few or no side effects and excellent effects. The present invention relates to a pharmaceutical composition for treating neurological diseases, including a cell-permeable CRYM fusion protein, wherein CRYM fusion protein permeated into cells increases cell viability against hydrogen peroxide toxicity, and reduces intracellular reactive oxygen species levels and hydrogen peroxide-induced DNA fragmentation. These results show that the CRYM fusion protein has a protective effect against apoptosis and shows a therapeutic effect that protects nerve cells from damage to nerve diseases.

Description

A pharmaceutical composition containing cell-transducing CRYM fusion protein for preventing or treating motor neuronal disorder

The present invention relates to a pharmaceutical composition for preventing or treating motor neuron disease comprising a cell-permeable CRYM fusion protein.

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

The causes of amyotrophic lateral sclerosis are not fully understood in most cases, and the mechanisms involved in motor degeneration are complex. There is substantial evidence to support the hypothesis that oxidative stress is one mechanism responsible for 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 clearly known.

One of the most widely accepted hypotheses regarding neuronal death is that high levels of unsaturated fatty acids and high oxygen consumption by neurons increase the 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 breakdown, 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 cellular damage. Several studies have revealed that antioxidant treatment significantly reduces nerve damage induced by spinal cord ischemia (Hwang et al, 2015; Kim et al, 2012; Zhu et al, 2012).

CRYM (Mu-crystallin homolog) was identified as a 35 kDa NADPH-dependent p38 cytoplasmic T3-binding protein (p38CTBP, also known as thyroid hormone (TH) binding protein, i.e. THBP), which was 3,5,3'-triiodo-L-thyronine) is likely to be a major regulatory factor.

Mutation of the CRYM gene leads to non-syndromic hearing loss because the mutation does not participate in the potassium ion recycling system together with Na and K-ATPase.

Many species-specific determinants with NADPH-binding properties are intraocular antioxidants (2), and CRYM, which is upregulated within microglia at late ALS stages, is neurodegenerative, like other NADPHs that require enzymes such as thioredoxin. may have a protective effect on

Mutations in CRYM are potentially associated with amyotrophic lateral sclerosis (ALS), promote reduction of a sulfur-containing substrate known as cyclic ketimine, derived from a sulfur-containing amino acid, and may have a potential role as a neurotransmitter; It may play an important role in the interface between metabolism, neurotransmission and cell survival.

However, the function or effect of CRYM on neuronal apoptosis caused by reactive oxygen species has not yet been clearly elucidated.

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 problem, the present inventors recombined protein transport domains such as PEP-1 and HIV Tat peptide with CRYM protein and confirmed that they penetrate into NSC34 cells, and the protective effect of cell-penetrating CRYM fusion protein against oxidative stress studied.

The present inventors tried to determine whether a CRYM fusion protein bound to a protein transport domain has a protective effect on apoptosis induced by oxidative stress in vitro. In order to study the potential effect of the CRYM fusion protein on ischemic neuronal apoptosis, the present inventors prepared a CRYM fusion protein that can penetrate into cells. The CRYM fusion protein effectively penetrated into neurons in a concentration-dependent and time-dependent manner. The CRYM 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 indicate that the CRYM 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.

To describe the configuration of the present invention in more detail, in an embodiment of the present invention, the present inventors first developed a CRYM fusion protein expression vector capable of overexpressing the CRYM fusion protein and easily purifying it. This expression vector contains cDNA capable of expressing human CRYM 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 CRYM fusion protein is characterized as SEQ ID NO: 1 in the sequence listing.

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

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

The pharmaceutical composition containing the cell-permeable CRYM 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. Compositions for oral use include, for example, tablets and gelatin capsules, which, in addition to the active ingredient, contain 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 (eg 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 in this way can be administered orally, or parenterally, that is, intravenously, subcutaneously, intraperitoneally, or topically administered as desired. The dose may 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 and treating motor neuron diseases, characterized in that it contains the CRYM fusion protein as an active ingredient and a pharmaceutically acceptable carrier.

The motor neuron disease is amyotrophic lateral sclerosis (ALS), progressive bulbar palsy (PBP), progressive muscle atrophy (PMA), primary lateral sclerosis (PLS), pseudobulbular palsy or unilateral muscular atrophy (MMA).

The present invention also provides a method for efficiently delivering a CRYM protein into a cell. The intracellular delivery of the CRYM 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 an HIV Tat peptide is covalently bound. An example of the protein transport domain of the present invention may be an HIV Tat peptide. However, the protein transport domain of the present invention is not limited only to the HIV Tat peptide, and it is in the field of the present invention to prepare a peptide having a function similar to the HIV Tat peptide by partial substitution, addition, or lack of amino acid sequence of the HIV Tat peptide. Since it is easy for those of ordinary skill in the art, it is a protein transport domain composed of 7 to 21 amino acids and containing four or more lysine or arginine and a protein transport that performs the same/similar protein transport function by substituting some amino acids therefrom It will be apparent that fusion proteins using domains also fall within the scope of the present invention.

Specifically, the present invention relates to a CRYM fusion protein, an oligonucleotide encoding the CRYM fusion protein, a vector comprising an oligonucleotide encoding the CRYM fusion protein, the fusion protein, the oligonucleotide or a motor neuron disease treatment comprising the vector or It relates to a pharmaceutical composition for the purpose of prevention and a health functional food composition for preventing or improving motor neuron disease comprising the fusion protein.

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

Foods to which the cell-permeable CRYM fusion protein of the present invention can be added include various foods, for example, beverages, gums, tea, vitamin complexes, health supplements, and the like, and include pills, powders, granules, tablets, and tablets. , can be used in the form of capsules or beverages. At this time, the amount of the CRYM 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 CRYM 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 , a sugar alcohol such as sorbitol and 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 includes various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, coloring agents and thickeners (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its 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.

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

"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 CRYM.

The term "fusion protein" includes a transport domain and one or more target protein portions, and refers to a complex 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, "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.

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

In addition, in this 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 CRYM 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 CRYM, so cell penetration efficiency is improved. It refers to a CRYM fusion protein. In addition, the transport domain refers to one or more of HIV Tat 49-57 residues, Pep-1 peptide, oligolysine, oligoarginine, and oligo (lysine, arginine). In addition, the CRYM fusion protein amino acid sequence of the present invention is SEQ ID NO:2 of the listing. In CRYM fusion protein production, fusion proteins of various sequences can be obtained according to the selection of restriction site sequences, 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 CRYM fusion protein amino acid sequence is not limited to the sequences listed above.

In addition, the present invention provides a pharmaceutical composition for the prevention and treatment of neurological diseases, comprising the CRYM fusion protein as an active ingredient and a pharmaceutically acceptable carrier.

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

The present invention relates to a cell-transducing CRYM fusion protein in which a protein transport domain consisting of 7 to 21 amino acids and including 4 or more lysine or arginine is covalently linked to at least one end of a CRYM protein. In addition, in the CRYM 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 according to silent change. 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 CRYM fusion protein permeated into the cell increased the cell viability against hydrogen peroxide toxicity. These results indicate that the CRYM fusion protein has a protective effect against neuronal cell death caused by reactive oxygen species, and that the cell-permeable CRYM fusion protein is useful as a pharmaceutical composition for the prevention and treatment of neurological diseases.

1 shows the construction of a Tat-CRYM expression vector on a pET-15b vector. Synthetic Tat oligomers were cloned into the Nde I and Xho I sites, and human CRYM cDNA was cloned into the Xho I and BamHI sites of pET-15b. (A) shows the construction of an expression vector of CRYM and Tat-CRYM fusion protein. The Tat-CRYM fusion protein contains six histidine residues. (B) shows the results of expressing each protein by adding IPTG, then purifying the purified CRYM and Tat-CRYM fusion proteins using 15% SDS-PAGE and Western blotting with anti-rabbit polyhistidine antibody.
Figure 2 shows the permeation of Tat-CRYM fusion protein into NSC34 cells. (A) is Tat-CRYM fusion protein (0.5~5μM) and CRYM protein (0.5~5μM) treated in culture medium for 1 hour, (B) is Tat-CRYM fusion protein and CRYM protein for 15~60 minutes It is the result of treatment with a culture medium and analyzed by Western blotting. (C) The intracellular distribution of the Tat-CRYM fusion protein was visualized by confocal fluorescence microscopy. (D) The stability of the Tat-CRYM fusion protein in NSC34 cells was analyzed. After transduction with Tat-CRYM 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 NSC34 cells pretreated with Tat-CRYM fusion protein or CRYM protein for 1 hour, respectively, for 1 hour.
Figure 3b is a result of measuring the level of reactive oxygen species in cells by DCF-DA staining.
3c 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, HIV Tat peptide was used as the protein transport domain constituting the CRYM fusion protein, but those of ordinary skill in the art to which the present invention belongs CRYM fusion in which the Tat peptide is bound to the N-terminus By applying a protein, a fusion protein in which various protein transport domains such as PEP-1 peptide, oligoarginine, oligolysine, and oligo (arginine + lysine) are coupled to one or more of the N-terminus or C-terminus of CRYM can be easily prepared It can be formed, and there may be slight differences between these fusion proteins, but it can be easily seen that they exhibit similar activities.

material

Ni ^2+- Nitrilotriacetate Sepharose Superflow was purchased from Qiagen, and PD-10 column chromatography (Amersham, Brauncschweig, Germany) was purchased. NSC34 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, Casphase 3, Casphase 9, p-p38, p38, p-Erk, Erk, p-JNK and primary antibodies to 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 Tat-CRYM fusion protein

Expression vectors of CRYM protein and Tat-CRYM fusion protein were constructed. The human CRYM 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 Tat-CRYM fusion protein. Similarly, the control CRYM protein was prepared using a vector lacking the Tat peptide. After transforming the recombinant Tat-CRYM plasmid into E. coli BL21, it was induced with 0.5 mM IPTG (isopropyl-β-D-thiogalactoside) and cultured overnight at 18°C. In order to obtain Tat-CRYM fusion protein by pulverizing the cultured cells by ultrasonication, Ni ^2+- Nitrilotriacetate Sepharose Superflow column was used for purification. Protein concentration was determined using the Bradford method using bovine serum albumin as a standard material.

Introduction of Tat-CRYM fusion protein into NSC34 cells

NSC34 cells maintained the conditions of 37° C., 95% air and 5% CO ₂ , 10% fetal calf serum (FBS) and 5 mM NaHCO ₃ , antibiotics (100 μg/ml streptomycin, 100 U/ml penicillin), 20 mM It was cultured under humid conditions using DMEM (Dulbecco's Modified Eagle's Medium) containing HEPES/NaOH (pH 7.4).

The time dependence and concentration dependence of the Tat-CRYM fusion protein and the control CRYM protein 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, 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 Tat-CRYM fusion protein and CRYM protein in NSC34 cells, cells were seeded on glass coverslips and treated with Tat-CRYM fusion protein and CRYM protein at a concentration of 5 μM for 1 hour. Cells were washed twice with PBS and then fixed with 4% paraformaldehyde at room temperature for 5 minutes. NSC34 cells were blocked with PBS (PBS-BT) containing 3% bovine serum albumin and 0.1% Triton X-100 for 40 minutes at room temperature, and washed with PBSBT. The primary antibody (His-probe, Santa Cruz Biotechnology) was diluted at a ratio of 1:10000, 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 colorimetric analysis using MTT {3-(4,5-dimethylthiazol -2-yl)-2,5-dipheyltetrazolium bromide}. Apoptosis was induced by exposing the cells to 100 μM hydrogen peroxide for 1 hour, with or without pre-treating the cells with 0.5-5 μM of Tat-CRYM 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 NSC34 cells were treated with Tat-CRYM fusion protein and when not treated was compared. Tat-CRYM fusion protein was treated with 5 μM for 1 hour and then hydrogen peroxide was treated with 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

NSC34 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 Tat-CRYM 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). Results were analyzed using a fluorescence microscope (Nikon eclipse 80i, Japan).

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

The inventors recombined the CRYM gene with the Tat-vector containing Tat, which is a type of protein transduction domain (PTD), to prepare a permeable Tat-CRYM fusion protein (FIG. 1A). It was overexpressed in Escherichia coli and ^{purified using Ni 2+-} nitrilosamic acetate sepharose affinity chromatography column and PD-10 column chromatography, and Tat-CRYM fusion protein was purified using SDS-PAGE and Western blot analysis. was confirmed (A, B in FIG. 1).

Result 2: Permeation of Tat-CRYM fusion protein into NSC34 cells

The degree of permeation into NSC34 cells was measured by time and concentration of the Tat-CRYM fusion protein. As a result, intracellular permeation occurred effectively in a time- and concentration-dependent manner, and CRYM protein did not permeate (FIG. 2A, B). In addition, cell nuclei were stained with DAPI using a confocal microscope, and the Tat-CRYM fusion protein was stained with green fluorescence to confirm effective penetration into cells (FIG. 2C). The stability of the Tat-CRYM fusion protein in NSC34 cells was confirmed (FIG. 2D).

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

Cells were pretreated with Tat-CRYM fusion protein by concentration, 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, the number of viable cells decreased to about 40%, but when Tat-CRYM was pretreated, the cell viability increased in a concentration-dependent manner (FIG. 3A).

In addition, in order to confirm that the Tat-CRYM 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, the 8-OHdG signal was significantly increased by hydrogen peroxide. However, reactive oxygen species induced by hydrogen peroxide was reduced by the Tat-CRYM fusion protein (FIG. 3B).

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 NSC34 cells were exposed to 100 μM hydrogen peroxide for 1 hour, it was confirmed that the Tat-CRYM fusion protein had a protective effect against DNA fragmentation (FIG. 3C).

Therefore, the present invention suggests the possibility that the Tat-CRYM 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 CRYM fusion protein for preventing or treating motor neuronal disorder <130> HallymU-SYCHOI-CRYM-M_neuronal_disorder <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 972 <212> DNA <213> Artificial Sequence <220> <223> HIV Tat-CRYM fusion protein coding polynucleotide <400> 1 aggaagaagc ggagacagcg acgaagaatg agccgggtac cagcgttcct gagcgcggcc 60 gaggtggagg aacacctccg cagctccagc ctcctcatcc cgcctctaga gacggccctg 120 gccaacttct ccagcggtcc cgaaggaggg gtcatgcagc ccgtgcgcac cgtggtgccg 180 gtgaccaagc acaggggcta cctgggggtc atgcccgcct acagtgctgc agaggatgca 240 ctgaccacca agttggtcac cttctacgag gaccgcggca tcacctcggt cgtcccttcc 300 caccaggcta ctgtgctact ctttgagccc agcaatggca ccctgctggc ggtcatggat 360 ggaaatgtca taactgcaaa gagaacagct gcagtttctg ccattgccac caagtttctg 420 aaacctccca gcagtgaagt gctgtgcatc cttggggctg gggtccaggc ctacagccat 480 tatgagatct tcacagagca gttctccttt aaggaggtga ggatatggaa ccgcaccaaa 540 gaaaatgcag agaagtttgc agacacagtg caaggagagg tacgggtctg ttcttcggtc 600 caggaggctg tggcaggtgc agatgtgatc atcacagtca ccctggcaac agagcccatt 660 ttgtttggtg aatgggtgaa gccaggggct cacatcaatg ctgttggagc cagcagacct 720 gactggagag aactggatga tgagctcatg aaagaagctg tgctgtacgt ggattcccag 780 gaggctgccc tgaaggagtc tggagatgtc ctgctgtcag gggccgagat ctttgctgag 840 ctgggagaag tgattaaggg agtgaaacca gcccactgtg agaagaccac cgtgttcaag 900 tctttgggaa tggcagtgga agacacagtt gcagccaaac tcatctatga ttcctggtca 960 tctggtaaat aa 972 <210> 2 <211> 295 <212> PRT <213> Artificial Sequence <220> <223> HIV Tat-CRYM fusion protein <400> 2 Arg Lys Lys Arg Arg Gln Arg Arg Arg Leu Glu Met Ser Arg Val Pro 1 5 10 15 Ala Phe Leu Ser Ala Ala Glu Val Glu Glu His Leu Arg Ser Ser Ser 20 25 30 Leu Leu Ile Pro Leu Glu Thr Ala Leu Ala Asn Phe Ser Ser Gly 35 40 45 Pro Glu Gly Gly Val Met Gln Pro Val Arg Thr Val Val Pro Val Thr 50 55 60 Lys His Arg Gly Tyr Leu Gly Val Met Pro Ala Tyr Ser Ala Ala Glu 65 70 75 80 Asp Ala Leu Thr Thr Lys Leu Val Thr Phe Tyr Glu Asp Arg Gly Ile 85 90 95 Thr Ser Val Val Pro Ser His Gln Ala Thr Val Leu Leu Phe Glu Pro 100 105 110 Ser Asn Gly Thr Leu Leu Ala Val Met Asp Gly Asn Val Ile Thr Ala 115 120 125 Lys Arg Thr Ala Ala Val Ser Ala Ile Ala Thr Lys Phe Leu Lys Pro 130 135 140 Pro Ser Ser Glu Val Leu Cys Ile Leu Gly Ala Gly Val Gln Ala Tyr 145 150 155 160 Ser His Tyr Glu Ile Phe Thr Glu Gln Phe Ser Phe Lys Glu Val Arg 165 170 175 Ile Trp Asn Arg Thr Lys Glu Asn Ala Glu Lys Phe Ala Asp Thr Val 180 185 190 Gln Gly Glu Val Arg Val Cys Ser Ser Val Gln Glu Ala Val Ala Gly 195 200 205 Ala Asp Val Ile Ile Thr Val Thr Leu Ala Thr Glu Pro Ile Leu Phe 210 215 220 Gly Glu Trp Val Lys Pro Gly Ala His Ile Asn Ala Val Gly Ala Ser 225 230 235 240 Arg Pro Asp Trp Arg Glu Leu Asp Asp Glu Leu Met Lys Glu Ala Val 245 250 255 Leu Tyr Val Asp Ser Gln Glu Ala Ala Leu Xaa Glu Ser Gly Asp Val 260 265 270 Leu Leu Ser Gly Ala Glu Ile Phe Ala Glu Leu Gly Glu Val Ile Lys 275 280 285 Gly Val Lys Thr Ser Pro Leu 290 295

Claims (11)

A CRYM fusion protein with improved cell penetration efficiency by covalently bonding the HIV-Tat protein transport domain to at least one end of the CRYM (Mu-crystallin homolog) protein.
The method according to claim 1,
The CRYM fusion protein is a CRYM fusion protein, characterized in that its amino acid sequence is SEQ ID NO: 2.
A pharmaceutical composition for preventing or treating motor neuron disease containing the CRYM fusion protein of claim 1 or 2.
A recombinant polynucleotide encoding a CRYM fusion protein in which an oligonucleotide sequence encoding an HIV-Tat protein transport domain is bound to at least one end of a CRYM (Mu-crystallin homolog)-encoding cDNA.
5. The method of claim 4,
The recombinant polynucleotide is a recombinant polynucleotide encoding a CRYM fusion protein, characterized in that its base sequence is SEQ ID NO: 1.
A pharmaceutical composition for preventing or treating motor neuron disease containing the recombinant polynucleotide of claim 4 or 5.
CRYM fusion protein expression for preventing or treating motor neuron disease containing a recombinant polynucleotide encoding a CRYM fusion protein in which an oligonucleotide sequence encoding an HIV-Tat protein transport domain is bound to at least one end of a CRYM (Mu-crystallin homolog)-encoding cDNA vector.
A CRYM fusion protein expression vector containing a recombinant polynucleotide encoding a CRYM fusion protein in which an oligonucleotide sequence encoding an HIV-Tat protein transport domain is bound to at least one end of the CRYM (Mu-crystallin homolog) coding cDNA of claim 7. A pharmaceutical composition for preventing or treating motor neuron disease.
The method according to any one of claims 3, 6, 8,
The motor neuron disease is amyotrophic lateral sclerosis (ALS), progressive bulbar palsy (PBP), progressive muscle atrophy (PMA), primary lateral sclerosis (PLS), pseudobulbar palsy or unilateral muscular atrophy (MMA), prevention of motor neuron disease or a therapeutic pharmaceutical composition.
A health functional food composition for preventing or improving motor neuron disease comprising the CRYM (Mu-crystallin homolog) fusion protein of claim 1 or 2 as an active ingredient.
A health functional food composition for preventing or improving motor neuron disease comprising a recombinant polynucleotide encoding the CRYM fusion protein of claim 3 or 4.

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