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WO2021145743A1 - Mitochondria-targeting protein and use thereof - Google Patents

Mitochondria-targeting protein and use thereof Download PDF

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Publication number
WO2021145743A1
WO2021145743A1 PCT/KR2021/000651 KR2021000651W WO2021145743A1 WO 2021145743 A1 WO2021145743 A1 WO 2021145743A1 KR 2021000651 W KR2021000651 W KR 2021000651W WO 2021145743 A1 WO2021145743 A1 WO 2021145743A1
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Prior art keywords
mitochondrial
stat6
protein
disease
preventing
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PCT/KR2021/000651
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French (fr)
Korean (ko)
Inventor
김현미
주일로
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Ajou University Industry Academic Cooperation Foundation
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Ajou University Industry Academic Cooperation Foundation
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Priority claimed from KR1020200005961A external-priority patent/KR102410556B1/en
Priority claimed from KR1020210005717A external-priority patent/KR102527406B1/en
Application filed by Ajou University Industry Academic Cooperation Foundation filed Critical Ajou University Industry Academic Cooperation Foundation
Publication of WO2021145743A1 publication Critical patent/WO2021145743A1/en
Anticipated expiration legal-status Critical
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/66—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid the modifying agent being a pre-targeting system involving a peptide or protein for targeting specific cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/18—Kallidins; Bradykinins; Related peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression

Definitions

  • the present invention relates to STAT6 protein targeting mitochondria and uses thereof.
  • Mitochondria are important intracellular organelles in cells where the process of oxygen respiration in eukaryotes proceeds. It converts energy stored in various organic substances into the form of adenosine triphosphate (ATP) necessary for life activities through oxidative phosphorylation. Because it metabolizes energy, it acts as a power plant for cells. In addition, it regulates apoptosis by regulating the release of cytochrome c and other proapoptosis factors, and in addition, it is related to signal transduction, cell differentiation, calcium concentration control, reactive oxygen species (ROS) formation and utilization, and the like.
  • ROS reactive oxygen species
  • Neurodegenerative diseases such as Alzheimer's, Huntington's disease, Parkinson's disease, diabetes, which are recently known to be caused by dysfunction of mitochondria, for example, disruption of glycolysis, decrease of mitochondrial enzymes, increase of reactive oxygen species, change in number or size of mitochondria, etc.
  • dysfunction of mitochondria for example, disruption of glycolysis, decrease of mitochondrial enzymes, increase of reactive oxygen species, change in number or size of mitochondria, etc.
  • various diseases such as , cancer and aging-related diseases have been reported, interest in mitochondria and research on them are increasing.
  • Another object of the present invention is to provide a method for moving or distributing a target protein to the mitochondria, and a method for confirming the migration or distribution of the target protein to the mitochondria.
  • Another object of the present invention is to provide a carrier for mitochondrial drug delivery containing the peptide.
  • Another object of the present invention is to provide a composition for preventing or treating diseases related to mitochondrial dysfunction, including a complex to which the peptide and a pharmacologically active substance are bound.
  • Another object of the present invention is to provide a composition for preventing or treating diseases related to mitochondrial dysfunction, comprising the mitochondrial-targeting STAT6 protein as an active ingredient.
  • the present invention provides a mitochondrial targeting peptide consisting of the amino acid sequence of SEQ ID NO: 4.
  • the present invention provides a recombinant vector comprising a gene encoding the amino acid sequence of SEQ ID NO: 4.
  • the present invention provides a method for moving or distributing a target protein to mitochondria, comprising the step of introducing the recombinant vector into a cell.
  • the present invention comprises the steps of introducing a recombinant vector comprising a promoter, a gene encoding the amino acid sequence of SEQ ID NO: 4, a gene encoding a target protein, and a gene encoding a fluorescent protein into a cell; and confirming the movement or distribution of the expressed target protein through fluorescence imaging by expressing the gene; provides a method of confirming the movement or distribution of the target protein in the cell into the mitochondria, including.
  • the present invention provides a carrier for mitochondrial drug delivery containing the mitochondrial targeting peptide as an active ingredient.
  • the present invention provides a pharmaceutical composition for preventing or treating mitochondrial dysfunction-related diseases, comprising as an active ingredient a complex to which the mitochondrial targeting peptide and a pharmacologically active substance are bound.
  • the present invention provides a pharmaceutical composition for preventing or treating diseases related to mitochondrial dysfunction, comprising a promoter, a gene encoding the amino acid sequence of SEQ ID NO: 4, and a recombinant vector to which a gene encoding a target protein is operably linked as an active ingredient.
  • the present invention provides a pharmaceutical composition for preventing or treating diseases related to mitochondrial dysfunction, containing as an active ingredient a STAT6 (Signal transducer and activator of transcription 6) protein comprising a mitochondrial internal target domain and a mitochondrial membrane permeation domain.
  • STAT6 Signal transducer and activator of transcription 6
  • the present invention provides a health functional food composition for preventing or improving mitochondrial dysfunction-related diseases containing STAT6 (Signal transducer and activator of transcription 6) protein comprising a mitochondrial internal target domain and a mitochondrial membrane permeation domain as an active ingredient.
  • STAT6 Synignal transducer and activator of transcription 6
  • the mitochondrial targeting peptide according to the present invention can effectively move or distribute a target protein or drug to the mitochondria by accurately targeting the mitochondria, and using it as a pharmaceutical composition for the prevention or treatment of diseases related to mitochondrial dysfunction can be utilized
  • mitochondrial targeting peptide Through the mitochondrial targeting peptide according to the present invention, while effectively delivering various substances targeting mitochondria, its movement or distribution can be easily and clearly observed or confirmed.
  • the mitochondrial-targeting peptide according to the present invention is also confirmed in mitochondria of other species than humans, and in this regard, it has the advantage of being able to conduct various studies through animal experiments, etc.
  • the STAT6 protein according to the present invention includes a mitochondrial internal target domain and a mitochondrial membrane permeation domain, and when the STAT6 protein is located in the mitochondrial outer membrane by these domains, it induces fragmentation of mitochondria and induces tumorigenesis.
  • the STAT6 protein can be used as a pharmaceutical composition for preventing or treating mitochondrial dysfunction-related diseases, or a health functional food composition to more effectively prevent, treat or improve related diseases.
  • Example 1 shows the human STAT6 protein according to Example 1 of the present invention.
  • FIG. 2 is a microscopic image of the position of the human STAT6 N-terminal sequence of FIG. 1 in a human U373-MG (malignant glioma) cell line.
  • FIG. 3 is a microscopic image of the position of the human STAT6 N-terminal sequence of FIG. 1 in the mouse B16F10 cell line.
  • Figure 4 confirms the transmembrane sequence of the STAT6 protein to be located in the outer mitochondrial membrane according to Example 3 of the present invention.
  • FIG 5 shows the human STAT6 protein (MTS D) from which the human STAT6 protein and the mitochondrial target sequence have been removed according to Example 4 of the present invention.
  • FIG. 6 is a result of observing the effect on mitochondria when the STAT6 proteins of FIG. 5 were overexpressed in the 293T cell line.
  • Example 7 shows a stable cell line in which STAT6 expression is suppressed in the 293T cell line was prepared in order to confirm the effect of STAT6 on tumor formation according to Example 5 of the present invention, and this was performed as a Western blotting experiment. it has been confirmed
  • FIG. 9 is an observation by removing the tumor tissue after cell transplantation in FIG. 7 , the upper figure is a view of a mouse one month after cell transplantation, and the lower figure is the tumor tissue extracted from each numbered mouse in the upper figure. It is a photographed image.
  • FIG. 11 shows human STAT6 protein (STAT6 WT) and STAT6 protein (MTS D) from which the mitochondrial target sequence has been deleted, and each of these STAT6 proteins was overexpressed in the STAT6 KO stable cell line prepared according to FIG. 7 to reconstruct a stable cell line. Therefore, this was confirmed by Western blotting experiments.
  • FIG. 12 is a view of the STAT6 KO and STAT6 WT stable cell lines according to FIG. 11 were transplanted into mice, and the tumor tissue was excised and observed. Tumors are listed in order of size.
  • FIG. 13 is an observation of the tumor tissue excised after transplanting the STAT6 KO and MTS D stable cell lines according to FIG. 11 into mice.
  • the upper figure is a view of a mouse just before tumor extraction one month after transplantation, and the lower figure is the excised figure. Tumors are listed in order of size.
  • the present inventors have completed the present invention by confirming that the specific amino acid sequence of the STAT6 protein N-terminal sequence is a mitochondrial targeting sequence, and that the STAT6 protein is located in the mitochondria, particularly its outer membrane.
  • the present invention provides a mitochondrial targeting peptide consisting of the amino acid sequence of SEQ ID NO: 4.
  • the human STAT6 N-terminal 1-30 sequence among the human STAT6 internal sequences has a mitochondrial targeting (targeting) effect, which is indicated by SEQ ID NO: 4.
  • SEQ ID NO: 4 MSLWGLVSKMPPEKVQRLYVDFPQHLRHLL
  • the mitochondrial targeting peptide or functional equivalent thereof consisting of the amino acid sequence of SEQ ID NO: 4 according to the present invention may be derived from nature, chemically synthesized using a chemical synthesis method or genetic engineering method known in the art, or genetically recombination It may be obtained using technology, but is not limited thereto.
  • the genetic engineering method may produce a DNA sequence encoding the peptide or a functional equivalent thereof according to a conventional method.
  • the DNA sequence can be prepared by PCR amplification using appropriate primers.
  • the DNA sequence can be synthesized by standard methods known in the art, for example, using an automatic DNA synthesizer (such as those sold by Biosearch or Applied Biosystems).
  • the synthesized DNA sequence is inserted into a vector containing one or more expression control sequences that are operably linked thereto to control the expression of the DNA sequence, and transform the host cell with the recombinant expression vector formed therefrom to transform create a transform
  • the "transformant” can be used interchangeably with “host cell”, etc., and is introduced into the cell by any means, for example, electroshock method, calcium phosphatase precipitation method, microinjection method, transformation method, virus infection, etc. It refers to a prokaryotic or eukaryotic cell containing heterologous DNA. Since the expression level and modification of the protein appear differently depending on the host cell, a person skilled in the art can select and use the most suitable host cell for the intended purpose.
  • a substantially pure peptide encoded by the DNA sequence can be recovered from the culture.
  • the recovery may be performed using a method known in the art (eg, chromatography).
  • substantially pure peptide means that the peptide according to the present invention does not substantially contain any other proteins derived from host cells.
  • the peptide according to the present invention may include a peptide having a native amino acid sequence as well as an amino acid sequence variant thereof.
  • amino acid sequence variant refers to a peptide having a different sequence by deletion, insertion, non-conservative or conservative substitution of one or more amino acid residues in the amino acid sequence according to the present invention, substitution of an amino acid analog, or a combination thereof, By the above mutations, the activity of the molecule is not entirely changed.
  • the peptide according to the present invention may be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, or the like.
  • the present invention provides a recombinant vector comprising a gene encoding the amino acid sequence of SEQ ID NO: 4.
  • the gene encoding the amino acid sequence of SEQ ID NO: 4 is a polynucleotide encoding a peptide consisting of the amino acid sequence of SEQ ID NO: 4.
  • the recombinant vector is a means for expressing the gene, and may include a known expression vector such as a plasmid vector, a cosmid vector, or a bacteriophage vector, but is not limited thereto.
  • the vector can be easily prepared by those skilled in the art according to any known method using DNA recombination technology.
  • the recombinant vector according to the present invention may be one in which a promoter, a gene encoding the amino acid sequence of SEQ ID NO: 4, and a gene encoding a target protein are operably linked.
  • the "operably linked” means that an expression control sequence is linked to control transcription and translation of a gene encoding the amino acid sequence of SEQ ID NO: 4, and the gene under the control of an expression control sequence (eg, promoter, enhancer, etc.) and maintaining the correct reading frame so that the peptide according to the invention is expressed and encoded thereby.
  • an expression control sequence eg, promoter, enhancer, etc.
  • the gene encoding the amino acid sequence of SEQ ID NO: 4 is effective for mitochondrial targeting, the gene encoding the amino acid sequence of SEQ ID NO: 4 of the present invention is operably linked to the gene encoding the target protein to be targeted to the mitochondria. In the case of inserting and expressing it, the target protein can be targeted to the mitochondria.
  • FP fluorescent protein
  • the fluorescent protein may be a green fluorescent probe (GFP), a yellow fluorescent protein (YFP), or a red fluorescent protein (RFP), but is not limited thereto.
  • GFP green fluorescent probe
  • YFP yellow fluorescent protein
  • RFP red fluorescent protein
  • the present invention provides a method for moving or distributing a target protein to mitochondria, comprising the step of introducing the recombinant vector into a cell.
  • the introduction into the cell means transforming the cell using a recombinant vector, and the transformation method according to this may use a well-known method well known to those skilled in the art.
  • the present invention comprises the steps of introducing a recombinant vector comprising a promoter, a gene encoding the amino acid sequence of SEQ ID NO: 4, a gene encoding a target protein, and a gene encoding a fluorescent protein into a cell; and confirming the movement or distribution of the expressed target protein through fluorescence imaging by expressing the gene; provides a method of confirming the movement or distribution of the target protein in the cell into the mitochondria, including.
  • the fluorescent protein may be a green fluorescent probe (GFP), a yellow fluorescent protein (YFP), or a red fluorescent protein (RFP), but is not limited thereto.
  • GFP green fluorescent probe
  • YFP yellow fluorescent protein
  • RFP red fluorescent protein
  • the cell By using a fluorescence microscope to continuously image the fluorescence of a specific wavelength emitted from a protein expressed in a cell into which the recombinant vector is introduced, that is, a protein expressed in the transformed cell, the cell is in a living state, and thus the target protein in the cell can be visualized in detail step by step in the process of expression and mitochondrial migration.
  • the 31-50th sequence and the 1-20th sequence, respectively (GFP) was bound to prepare 1-30-GFP, 31-50-GFP, and 1-20-GFP plasmids, and by transfecting them into cells and observing them under a microscope, migration to mitochondria could be confirmed.
  • the present invention provides a mitochondrial drug delivery carrier containing a mitochondrial targeting peptide consisting of the amino acid sequence of SEQ ID NO: 4 as an active ingredient.
  • the peptide is effective at mitochondrial targeting, drugs related to various diseases targeting mitochondria can be delivered directly to the mitochondria, thereby improving drug absorption in the mitochondria.
  • the present invention provides a pharmaceutical composition for preventing or treating mitochondrial dysfunction-related diseases, comprising as an active ingredient a complex to which a mitochondrial targeting peptide and a pharmacologically active substance are bound, which consists of the amino acid sequence of SEQ ID NO: 4.
  • the pharmacologically active substance is a substance capable of inducing a desired pharmacological effect by promoting or inhibiting a physiological function in an animal or human body, and refers to a chemical or biological substance or compound suitable for administration to an animal or human.
  • the pharmacologically active substance has a preventive effect of preventing unwanted biological effects such as preventing infection, alleviating pain or infection resulting from a disease, and can play a role of completely eliminating a disease, so that the pharmacologically active substance is a “drug ” or “therapeutic agent” may be used interchangeably.
  • the complex includes a peptide capable of directing a target to the mitochondria, so that a substance capable of acting on the mitochondria and exhibiting direct pharmacological activity can be effectively delivered to the mitochondria, moved and stayed there. Therefore, it can be usefully used for the prevention or treatment of diseases related to mitochondrial dysfunction.
  • the pharmacologically active substance may be a natural product, compound, protein, or gene (DNA, RNA or PNA) capable of acting on mitochondria to exhibit specific activity, but is not limited thereto.
  • the compound is doxorubicin (doxorubicin), epirubicin (epirubicin), idarubicin (idarubicin), docetaxel (docetaxel), paclitaxel (paclitaxel), cabazitaxel (cabazitaxel), vinblastine (vinblastine), vincristine (vincristine) And it may be one or more anticancer agents selected from the group consisting of cisplatin (cisplain), but is not limited thereto.
  • the compound is selected from the group consisting of flavonoids, polyphenols, resveratrol, carotenoids, alpha-lipoic acid, tocopherol and ascorbic acid. It may be one or more antioxidants, but is not limited thereto.
  • the compound may be an antibiotic, an anti-inflammatory agent, etc., but is not limited thereto.
  • the present invention provides a pharmaceutical composition for preventing or treating diseases related to mitochondrial dysfunction, comprising a promoter, a gene encoding the amino acid sequence of SEQ ID NO: 4, and a recombinant vector to which a gene encoding a target protein is operably linked as an active ingredient.
  • the mitochondrial dysfunction-related disease is ischemic brain disease or ischemic heart disease, stroke, migraine, anemia, Parkinson's disease, Alzheimer's, amyotrophic lateral sclerosis (ALS), mitochondrial encephalomyopathy, cirrhosis, Fanconi's syndrome, atypical phenylketonuria, asthma, diabetes , arteriosclerosis, hypertension, hypercholesterolemia, optic nerve disease, obesity, depression, and may be any one selected from the group consisting of cancer diseases.
  • ALS amyotrophic lateral sclerosis
  • the present invention provides a STAT6 (Signal transducer and activator of transcription 6) protein, specifically, a mitochondrial dysfunction-related disease prevention or treatment pharmaceutical containing STAT6 protein comprising a mitochondrial internal target domain and a mitochondrial membrane permeation domain as an active ingredient A composition is provided.
  • STAT6 Signal transducer and activator of transcription 6
  • STAT6 Signal transducer and activator of transcription 6
  • STAT6 Signal transducer and activator of transcription 6
  • the STAT6 protein may consist of an amino acid sequence represented by SEQ ID NO: 1.
  • the mitochondrial internal target domain may consist of the amino acid sequence represented by SEQ ID NO: 4.
  • the human STAT6 N-terminal 1-30 amino acid sequence among the human STAT6 internal sequences has a mitochondrial targeting effect.
  • the mitochondrial transmembrane domain may consist of the amino acid sequence shown in SEQ ID NO: 11.
  • the mitochondrial membrane permeation domain may localize the STAT6 protein targeting the inside of the mitochondria to the outer mitochondrial membrane.
  • the 624-698 amino acid sequence among the human STAT6 internal sequence serves as a transmembrane domain that allows the protein to be located in the outer mitochondrial membrane, which is SEQ ID NO: 11 indicated as
  • the STAT6 protein can be located in the outer mitochondrial membrane, and when the protein is accumulated in the outer mitochondrial membrane, it induces fragmentation of mitochondria, which is the starting point of apoptosis, and tumor formation can be inhibited, the STAT6 protein can be utilized as a pharmaceutical composition for preventing or treating diseases related to mitochondrial dysfunction.
  • the mitochondrial dysfunction-related disease is ischemic brain disease or ischemic heart disease, stroke, migraine, anemia, Parkinson's disease, Alzheimer's, amyotrophic lateral sclerosis (ALS), mitochondrial encephalomyopathy, cirrhosis, Fanconi's syndrome, atypical phenylketonuria, asthma, diabetes , arteriosclerosis, hypertension, hypercholesterolemia, optic nerve disease, obesity, depression, and may be any one selected from the group consisting of cancer diseases, but is not limited thereto.
  • composition may further include an activator or expression promoter of STAT6 protein, but is not limited thereto.
  • the carrier or pharmaceutical composition for drug delivery according to the present invention may be prepared according to a conventional method in the pharmaceutical field.
  • the drug delivery carrier or pharmaceutical composition may be combined with an appropriate pharmaceutically acceptable carrier according to the formulation, and if necessary, excipients, diluents, dispersants, emulsifiers, buffers, stabilizers, binders, disintegrants, solvents, etc. It can be prepared by including more.
  • the "pharmaceutically acceptable” means that it is not toxic to cells or humans exposed to the pharmaceutical composition, and the appropriate carrier does not inhibit the activity and properties of the mitochondrial targeting peptide or STAT6 protein according to the present invention. It may be selected differently depending on the dosage form and dosage form.
  • the carrier or pharmaceutical composition for drug delivery may be applied in any formulation, and more specifically, it may be formulated into oral formulations, topical formulations, suppositories, and parenteral formulations of sterile injection solutions according to conventional methods.
  • the solid dosage form among the oral dosage forms is in the form of tablets, pills, powders, granules, capsules, etc., and at least one or more excipients, for example, starch, calcium carbonate, sucrose, lactose, sorbitol, mannitol, cellulose, gelatin, etc.
  • excipients for example, starch, calcium carbonate, sucrose, lactose, sorbitol, mannitol, cellulose, gelatin, etc.
  • lubricants such as magnesium stearate and talc may be included.
  • the capsule formulation may further include a liquid carrier such as fatty oil in addition to the above-mentioned substances.
  • liquid formulations include suspensions, solutions, emulsions, syrups, etc.
  • various excipients for example, wetting agents, sweeteners, fragrances, preservatives, etc. may be included. there is.
  • the parenteral formulation may include a sterile aqueous solution, a non-aqueous solution, a suspension, an emulsion, a freeze-dried formulation, and a suppository.
  • Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.
  • As the base of the suppository witepsol, macrogol, Tween 61, cacao butter, laurin fat, glycerogelatin, and the like can be used. Without being limited thereto, any suitable agent known in the art may be used.
  • composition according to the present invention may further add calcium, vitamin D3, or the like to enhance therapeutic efficacy.
  • the pharmaceutical composition according to the present invention may be administered in a pharmaceutically effective amount.
  • pharmaceutically effective amount means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment and not cause side effects.
  • the effective dose level of the pharmaceutical composition is determined by the purpose of use, the age, sex, weight and health status of the patient, the type of disease, severity, drug activity, sensitivity to drug, administration method, administration time, administration route and excretion rate, treatment It may be determined differently depending on factors including the duration, formulation or concurrent use of drugs and other factors well known in the medical field. For example, although not constant, generally 0.001 to 100 mg/kg, preferably 0.01 to 10 mg/kg, may be administered once to several times a day. The above dosage does not limit the scope of the present invention in any way.
  • the carrier or pharmaceutical composition for drug delivery according to the present invention may be administered to any animal capable of developing a disease related to mitochondrial dysfunction, and the animal may be, for example, not only humans and primates, but also cattle, pigs, horses, dogs, etc. of livestock and the like.
  • the carrier or pharmaceutical composition for drug delivery according to the present invention may be administered by an appropriate administration route depending on the form of the formulation, and may be administered via oral or parenteral various routes as long as it can reach the target tissue.
  • the administration method is not particularly limited, and for example, oral, rectal or intravenous, intramuscular, subcutaneous, endobronchial inhalation, intrauterine dural or intracerebroventricular injection, etc. can be administered in a conventional manner. .
  • the pharmaceutical composition according to the present invention may be used alone for the prevention or treatment of diseases related to mitochondrial dysfunction, or may be used in combination with surgery or other drug treatment.
  • the present invention provides a health functional food composition for preventing or improving mitochondrial dysfunction-related diseases, which contains STAT6 (Signal transducer and activator of transcription 6) protein including a mitochondrial internal target domain and a mitochondrial membrane permeation domain as an active ingredient.
  • STAT6 Signal transducer and activator of transcription 6
  • the mitochondrial internal target domain may consist of the amino acid sequence represented by SEQ ID NO: 4.
  • the mitochondrial transmembrane domain may consist of the amino acid sequence shown in SEQ ID NO: 11, and the STAT6 protein targeting the inside of the mitochondria may be located in the outer mitochondrial membrane.
  • the STAT6 protein can be located in the outer mitochondrial membrane, and when the protein is accumulated in the mitochondrial outer membrane, it induces fragmentation of mitochondria, which is the starting point of apoptosis, and inhibits tumor formation. As a result, the STAT6 protein can be used as a health functional food composition for preventing or improving mitochondrial dysfunction-related diseases.
  • the mitochondrial dysfunction-related disease is ischemic brain disease or ischemic heart disease, stroke, migraine, anemia, Parkinson's disease, Alzheimer's, amyotrophic lateral sclerosis (ALS), mitochondrial encephalomyopathy, cirrhosis, Fanconi's syndrome, atypical phenylketonuria, asthma, diabetes , arteriosclerosis, hypertension, hypercholesterolemia, optic nerve disease, obesity, depression, and may be any one selected from the group consisting of cancer diseases, but is not limited thereto.
  • composition may further include an activator or expression promoter of STAT6 protein, but is not limited thereto.
  • the health functional food may be prepared in powder, granule, tablet, capsule, syrup or beverage, etc., and there is no limitation in the form that the health functional food can take, It can include any food with meaning.
  • beverages and various drinks, fruits and their processed foods (canned fruit, jam, etc.), fish, meat and their processed foods (ham, bacon, etc.), breads and noodles, cookies and snacks, dairy products (butter, cheese, etc.) ), etc. are possible, and may include all functional foods in a conventional sense. It may also include food used as feed for animals.
  • the health functional food composition according to the present invention may be prepared by further including pharmaceutically acceptable food additives and other suitable auxiliary ingredients commonly used in the art. Whether or not it is suitable as a food additive can be judged according to the standards and standards for the relevant item in accordance with the general rules and general test methods of the Food Additives Code approved by the Ministry of Food and Drug Safety, unless otherwise specified.
  • the items listed in the 'Food Additives Code' include, for example, chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; natural additives such as persimmon pigment, licorice extract, crystalline cellulose, high pigment, and guar gum; Mixed preparations, such as a sodium L-glutamate preparation, a noodle-added alkali agent, a preservative preparation, and a tar color preparation, etc. are mentioned.
  • chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid
  • natural additives such as persimmon pigment, licorice extract, crystalline cellulose, high pigment, and guar gum
  • Mixed preparations such as a sodium L-glutamate preparation, a noodle-added alkali agent, a preservative preparation, and a tar color preparation, etc. are mentioned.
  • the other auxiliary ingredients include, for example, flavoring agents, natural carbohydrates, sweeteners, vitamins, electrolytes, colorants, pectic acid, alginic acid, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents, etc. may further contain.
  • natural carbohydrate monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol can be used.
  • natural sweeteners such as taumatine and stevia extract or synthetic sweeteners such as saccharin and aspartame may be used.
  • the health functional food composition according to the present invention has the advantage that there are no side effects that may occur during long-term administration of the drug using food as a raw material, unlike general drugs, and has excellent portability, preventing or improving mitochondrial dysfunction-related diseases It can be taken as a supplement for
  • Example 1 is a human STAT6 protein according to Example 1 of the present invention, in detail, the approximate domain constituting the human STAT6 protein (STAT6 wild type, STAT6 WT), STAT6 in which the amino acid sequence at positions 624-847 has been deleted ( TAD-deleted STAT6, TAD D), STAT6 with the 699-847 amino acid sequence deleted (699-847 aa-deleted STAT6, 699-847 D), and human STAT6 N-terminal sequence (1-50 amino acid sequence) it has been shown
  • sequences predicted to act as mitochondrial target sequences are green at the C-terminus, respectively.
  • sequences 1-30, sequences 31-50, sequences 1-20 are green at the C-terminus, respectively.
  • fluorescent protein Green fluorescent protein; GFP
  • 1-30-GFP, 31-50-GFP, and 1-20-GFP plasmids were prepared.
  • the specific preparation method of each plasmid is as follows.
  • the STAT6 wild type plasmid (Origene) was amplified by PCR using SEQ ID NOs: 2 and 3 in Table 1 to obtain human STAT6 N-terminal 1-30 insertion sequences (SEQ ID NO: 4).
  • SEQ ID NO: 2 AGATCTGCCGCCGCGATCGCCATGTCTCTGTGGGGTCTGGTCT (Forward)
  • SEQ ID NO: 3 GCGGCCGCGTACGCGTCAGAAGATGCCGCAGGTGT (Reverse)
  • pCMV6-AC-GFP Origene was also digested with restriction enzymes Sgf1 and Mlu1, and then the inserted sequences 1-30 were subcloned between the cut sequences.
  • the completed 1-30-GFP plasmid was transformed into DH5 ⁇ (RBC) host cells, plated on kanamycin medium, and cultured at 37° C. for 17 hours. Plasmid DNA was extracted from colonies grown after culture.
  • STAT6 wild type plasmid (Origene) was amplified by PCR using SEQ ID NOs: 5 and 6 in Table 2 to obtain human STAT6 N-terminal 31-50 insertion sequences (SEQ ID NO: 7).
  • SEQ ID NO: 5 AGATCTGCCGCCGCGATCGCCGGTGACTGGCTGGAGAGC (Forward)
  • SEQ ID NO: 6 GCGGCCGCGTACGCGTGCAGCAGAAGGCGTC (Reverse)
  • pCMV6-AC-GFP (Origene) was also digested with restriction enzymes Sgf1 and Mlu1, and the 31-50th insertion sequence was subcloned between the cut sequences.
  • the completed 31-50-GFP plasmid was transformed into DH5 ⁇ (RBC) host cells, plated on kanamycin medium, and cultured at 37° C. for 17 hours. Plasmid DNA was extracted from colonies grown after culture.
  • the completed 1-20-GFP plasmid was transformed into DH5 ⁇ (RBC) host cells, plated on kanamycin medium, and cultured at 37° C. for 17 hours. Plasmid DNA was extracted from colonies grown after culture.
  • the three types of plasmids and one type of control GFP plasmid (Origene) prepared by the method 1 above were transfected into human U373-MG malignant glioma cell line and mouse B16F10 melanoma cell line. Each GFP-attached amino acid was overexpressed.
  • the transfection method is as follows.
  • the culture medium of human U373-malignant glioma cell line and mouse B16F10 melanoma cell line distributed from Korea Cell Line Bank was exchanged with OPTI-MEM, treated with the above mixture, and cultured for 48 hours.
  • STAT6 WT TAD D, 699-847 D
  • FIG. 1 stable cell lines expressing each of the STAT6 proteins were prepared.
  • STAT6 TAD-deleted STAT6
  • STAT6 TAD-deleted STAT6
  • mCherry fluorescence was first attached to the C-terminus of human STAT6 protein (STAT6 WT) and STAT6 (MTS D) in which the mitochondrial target sequence was deleted, as shown in FIG. Mitochondrial labeling was stained with TOM20 protein present in the outer mitochondrial membrane.
  • tumor tissues were extracted from mice and the size of the tumor was compared.
  • the size of the tumor was larger and the weight of the tumor was significantly increased than in the case of the 293T parental cells. That is, it can be confirmed that inhibition of STAT6 expression induces tumorigenesis promotion.
  • STAT6 KO stable cell line prepared above
  • human STAT6 protein (STAT6 WT) and STAT6 (MTS D) from which the mitochondrial target sequence was deleted were overexpressed, respectively, to construct a stable cell line as shown in FIG. 11 .
  • the prepared STAT6 KO stable cell line was transplanted into the left leg of a mouse, and the stable cell line overexpressing human STAT6 protein (STAT6 WT) was transplanted into the right leg, and tumor growth was observed for 1 month. As shown in Fig. 12, it was confirmed that the size of the tumor was significantly smaller in the case of transplanting the cells overexpressing STAT6 WT again than in the case of transplanting the STAT6 KO cells.
  • the STAT6 KO stable cell line prepared above was transplanted into the left leg of a mouse, and a stable cell line re-expressing STAT6 (MTS D) with the mitochondrial target sequence deleted was transplanted into the right leg, and the tumor was grown for 1 month.
  • MTS D stable cell line re-expressing STAT6
  • the size of the tumor was measured once every 3 days, and after 1 month, the tumor was excised from the mouse and weighed, and the graph was shown.

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Abstract

The present invention relates to STAT6 protein, which targets mitochondria, and a use thereof. More specifically, the present invention provides a mitochondria-targeting peptide, a pharmaceutical composition comprising a complex in which the mitochondria-targeting peptide and a pharmaceutically active substance are coupled, as an active ingredient for prevention or treatment of a disease associated with mitochondrial dysfunction, a pharmaceutical composition comprising STAT6 protein as an active ingredient for prevention or treatment of a disease associated with mitochondrial dysfunction, and a health functional food composition. The STAT6 protein accurately targets mitochondria and as such, can effectively migrate or disperse a protein or drug of interest into mitochondria, whereby a disease associated with mitochondrial dysfunction can be prevented or treated.

Description

미토콘드리아 표적 단백질 및 이의 용도Mitochondrial target proteins and uses thereof

본 발명은 미토콘드리아를 표적하는 STAT6 단백질 및 이의 용도에 관한 것이다.The present invention relates to STAT6 protein targeting mitochondria and uses thereof.

미토콘드리아(mitochondria)는 진핵 생물에서 산소 호흡의 과정이 진행되는 세포 속의 중요한 세포 내 소기관으로, 여러 유기물질에 저장된 에너지를 산화적 인산화 과정을 통해 생명활동에 필요한 아데노신삼인산(ATP)의 형태로 전환시키는 에너지 대사를 하기 때문에 세포의 발전소 역할을 한다. 또한, 시토크롬c와 다른 세포사멸 유도(proapoptosis) 요소의 방출을 조절함으로써 세포사멸을 조절하며, 이 외에도 신호전달, 세포분화, 칼슘의 농도조절, 활성 산소종(ROS) 형성 및 이용 등과 관련된다. Mitochondria are important intracellular organelles in cells where the process of oxygen respiration in eukaryotes proceeds. It converts energy stored in various organic substances into the form of adenosine triphosphate (ATP) necessary for life activities through oxidative phosphorylation. Because it metabolizes energy, it acts as a power plant for cells. In addition, it regulates apoptosis by regulating the release of cytochrome c and other proapoptosis factors, and in addition, it is related to signal transduction, cell differentiation, calcium concentration control, reactive oxygen species (ROS) formation and utilization, and the like.

최근 미토콘드리아의 기능 장애, 예를 들어, 해당 과정의 붕괴, 미토콘드리아 효소의 감소, 활성 산소종 증가, 미토콘드리아 수 또는 크기의 변화 등에 의해 발병되는 것으로 알려진 알츠하이머와 같은 신경 퇴행성 질환, 헌팅턴병, 파킨슨병, 당뇨병, 암 및 노화관련 질환 등의 다양한 질병들이 보고되어짐에 따라, 미토콘드리아에 대한 관심과 이에 대한 연구가 증가하고 있다.Neurodegenerative diseases such as Alzheimer's, Huntington's disease, Parkinson's disease, diabetes, which are recently known to be caused by dysfunction of mitochondria, for example, disruption of glycolysis, decrease of mitochondrial enzymes, increase of reactive oxygen species, change in number or size of mitochondria, etc. As various diseases such as , cancer and aging-related diseases have been reported, interest in mitochondria and research on them are increasing.

이와 관련하여 약물 또는 유전자와 같은 치료 생리활성 물질을 미토콘드리아로 전달하는 방법들이 연구되고 있으며, 보다 선택적이며 효율적으로 미토콘드리아를 표적함으로써, 관련 질환의 치료 효과를 증진시킬 수 있는 기술의 필요성이 증가하고 있다.In this regard, methods for delivering therapeutically active substances such as drugs or genes to mitochondria are being studied, and by targeting mitochondria more selectively and efficiently, the need for technology that can enhance the therapeutic effect of related diseases is increasing. .

본 발명의 목적은 미토콘드리아를 표적하는 펩타이드 또는 재조합 벡터를 제공하는 데에 있다.It is an object of the present invention to provide a peptide or recombinant vector targeting mitochondria.

본 발명의 다른 목적은 목적 단백질을 미토콘드리아로 이동 또는 분포시키는 방법, 상기 목적 단백질의 미토콘드리아로 이동 또는 분포를 확인하는 방법을 제공하는 데에 있다.Another object of the present invention is to provide a method for moving or distributing a target protein to the mitochondria, and a method for confirming the migration or distribution of the target protein to the mitochondria.

본 발명의 다른 목적은 상기 펩타이드를 함유하는 미토콘드리아 약물 전달용 담체를 제공하는 데에 있다.Another object of the present invention is to provide a carrier for mitochondrial drug delivery containing the peptide.

본 발명의 다른 목적은 상기 펩타이드 및 약리학적 활성 물질이 결합된 복합체를 포함하는 미토콘드리아 기능 장애 관련 질환 예방 또는 치료용 조성물을 제공하는 데에 있다.Another object of the present invention is to provide a composition for preventing or treating diseases related to mitochondrial dysfunction, including a complex to which the peptide and a pharmacologically active substance are bound.

본 발명의 또 다른 목적은 미토콘드리아를 표적하는 STAT6 단백질을 유효성분으로 포함하는 미토콘드리아 기능 장애 관련 질환 예방 또는 치료용 조성물을 제공하는 데에 있다.Another object of the present invention is to provide a composition for preventing or treating diseases related to mitochondrial dysfunction, comprising the mitochondrial-targeting STAT6 protein as an active ingredient.

상기의 목적을 달성하기 위해, 본 발명은 서열번호 4의 아미노산 서열로 이루어진 미토콘드리아 표적용 펩타이드를 제공한다.In order to achieve the above object, the present invention provides a mitochondrial targeting peptide consisting of the amino acid sequence of SEQ ID NO: 4.

본 발명은 서열번호 4의 아미노산 서열을 코딩하는 유전자를 포함하는 재조합 벡터를 제공한다.The present invention provides a recombinant vector comprising a gene encoding the amino acid sequence of SEQ ID NO: 4.

본 발명은 상기 재조합 벡터를 세포 내 도입하는 단계를 포함하는 목적 단백질을 미토콘드리아로 이동 또는 분포시키는 방법을 제공한다.The present invention provides a method for moving or distributing a target protein to mitochondria, comprising the step of introducing the recombinant vector into a cell.

본 발명은 프로모터, 서열번호 4의 아미노산 서열을 코딩하는 유전자, 목적 단백질을 코딩하는 유전자 및 형광 단백질을 코딩하는 유전자를 포함하는 재조합 벡터를 세포 내 도입하는 단계; 및 상기 유전자를 발현시켜 형광 영상을 통해 상기 발현되는 목적 단백질의 이동 또는 분포를 확인하는 단계;를 포함하는 세포 내 목적 단백질의 미토콘드리아로 이동 또는 분포를 확인하는 방법을 제공한다.The present invention comprises the steps of introducing a recombinant vector comprising a promoter, a gene encoding the amino acid sequence of SEQ ID NO: 4, a gene encoding a target protein, and a gene encoding a fluorescent protein into a cell; and confirming the movement or distribution of the expressed target protein through fluorescence imaging by expressing the gene; provides a method of confirming the movement or distribution of the target protein in the cell into the mitochondria, including.

본 발명은 상기 미토콘드리아 표적용 펩타이드를 유효성분으로 함유하는 미토콘드리아 약물 전달용 담체를 제공한다.The present invention provides a carrier for mitochondrial drug delivery containing the mitochondrial targeting peptide as an active ingredient.

본 발명은 상기 미토콘드리아 표적용 펩타이드 및 약리학적 활성 물질이 결합된 복합체를 유효성분으로 포함하는 미토콘드리아 기능 장애 관련 질환 예방 또는 치료용 약학 조성물을 제공한다.The present invention provides a pharmaceutical composition for preventing or treating mitochondrial dysfunction-related diseases, comprising as an active ingredient a complex to which the mitochondrial targeting peptide and a pharmacologically active substance are bound.

본 발명은 프로모터, 서열번호 4의 아미노산 서열을 코딩하는 유전자 및 목적 단백질을 코딩하는 유전자가 작동 가능하게 연결된 재조합 벡터를 유효성분으로 포함하는 미토콘드리아 기능 장애 관련 질환 예방 또는 치료용 약학 조성물을 제공한다.The present invention provides a pharmaceutical composition for preventing or treating diseases related to mitochondrial dysfunction, comprising a promoter, a gene encoding the amino acid sequence of SEQ ID NO: 4, and a recombinant vector to which a gene encoding a target protein is operably linked as an active ingredient.

본 발명은 미토콘드리아 내부 표적 도메인 및 미토콘드리아 막 투과 도메인을 포함하는 STAT6 (Signal transducer and activator of transcription 6) 단백질을 유효성분으로 함유하는 미토콘드리아 기능 장애 관련 질환 예방 또는 치료용 약학 조성물을 제공한다.The present invention provides a pharmaceutical composition for preventing or treating diseases related to mitochondrial dysfunction, containing as an active ingredient a STAT6 (Signal transducer and activator of transcription 6) protein comprising a mitochondrial internal target domain and a mitochondrial membrane permeation domain.

또한, 본 발명은 미토콘드리아 내부 표적 도메인 및 미토콘드리아 막 투과 도메인을 포함하는 STAT6 (Signal transducer and activator of transcription 6) 단백질을 유효성분으로 함유하는 미토콘드리아 기능 장애 관련 질환 예방 또는 개선용 건강기능식품 조성물을 제공한다.In addition, the present invention provides a health functional food composition for preventing or improving mitochondrial dysfunction-related diseases containing STAT6 (Signal transducer and activator of transcription 6) protein comprising a mitochondrial internal target domain and a mitochondrial membrane permeation domain as an active ingredient. .

본 발명에 따른 미토콘드리아 표적용 펩타이드는 미토콘드리아를 정확하게 타겟함으로써, 목적 단백질이나 약물 등을 미토콘드리아로의 효과적으로 이동 또는 분포시킬 수 있고, 이를 이용하여 미토콘드리아 기능 장애와 관련된 질환의 예방 또는 치료를 위한 약학 조성물로 활용할 수 있다. The mitochondrial targeting peptide according to the present invention can effectively move or distribute a target protein or drug to the mitochondria by accurately targeting the mitochondria, and using it as a pharmaceutical composition for the prevention or treatment of diseases related to mitochondrial dysfunction can be utilized

본 발명에 따른 미토콘드리아 표적용 펩타이드를 통해 미토콘드리아를 표적으로 하는 여러 물질을 효과적으로 전달하면서 그의 이동 또는 분포를 쉽고 명확하게 관찰 또는 확인할 수 있다. Through the mitochondrial targeting peptide according to the present invention, while effectively delivering various substances targeting mitochondria, its movement or distribution can be easily and clearly observed or confirmed.

본 발명에 따른 미토콘드리아 표적용 펩타이드는 인간 외 다른 종(species)의 미토콘드리아에서도 확인되는 바, 이와 관련하여 동물 실험 등을 통해 다양한 연구를 할 수 있는 장점이 있다.The mitochondrial-targeting peptide according to the present invention is also confirmed in mitochondria of other species than humans, and in this regard, it has the advantage of being able to conduct various studies through animal experiments, etc.

또한, 본 발명에 따른 STAT6 단백질은 미토콘드리아 내부 표적 도메인 및 미토콘드리아 막 투과 도메인을 포함하고, 상기 도메인들에 의해 STAT6 단백질이 미토콘드리아 외막에 위치할 때, 미토콘드리아의 절편화(fragmentation)를 유도하고 종양 형성을 억제함을 확인한 바, 상기 STAT6 단백질은 미토콘드리아 기능 장애 관련 질환 예방 또는 치료용 약학 조성물, 건강기능식품 조성물로 활용하여 보다 효과적으로 관련 질환을 예방, 치료 또는 개선할 수 있다.In addition, the STAT6 protein according to the present invention includes a mitochondrial internal target domain and a mitochondrial membrane permeation domain, and when the STAT6 protein is located in the mitochondrial outer membrane by these domains, it induces fragmentation of mitochondria and induces tumorigenesis. As it was confirmed that the inhibition, the STAT6 protein can be used as a pharmaceutical composition for preventing or treating mitochondrial dysfunction-related diseases, or a health functional food composition to more effectively prevent, treat or improve related diseases.

도 1은 본 발명의 실시예 1에 따른 human STAT6 단백질을 나타낸 것이다.1 shows the human STAT6 protein according to Example 1 of the present invention.

도 2는 human U373-MG(malignant glioma) 세포주에서 도 1의 human STAT6 N-말단 서열의 위치를 관찰한 현미경 이미지이다.FIG. 2 is a microscopic image of the position of the human STAT6 N-terminal sequence of FIG. 1 in a human U373-MG (malignant glioma) cell line.

도 3은 mouse B16F10 세포주에서 도 1의 human STAT6 N-말단 서열의 위치를 관찰한 현미경 이미지이다.3 is a microscopic image of the position of the human STAT6 N-terminal sequence of FIG. 1 in the mouse B16F10 cell line.

도 4는 본 발명의 실시예 3에 따라 미토콘드리아 외막에 자리잡기 위한 STAT6 단백질의 막 투과(transmembrane) 서열을 확인한 것이다.Figure 4 confirms the transmembrane sequence of the STAT6 protein to be located in the outer mitochondrial membrane according to Example 3 of the present invention.

도 5는 본 발명의 실시예 4에 따라 human STAT6 단백질과 미토콘드리아 표적 서열을 제거한 human STAT6 단백질 (MTS D)을 나타낸 것이다.5 shows the human STAT6 protein (MTS D) from which the human STAT6 protein and the mitochondrial target sequence have been removed according to Example 4 of the present invention.

도 6은 도 5의 STAT6 단백질들을 293T 세포주에 과발현시켰을 때의 미토콘드리아에 미치는 영향을 관찰한 결과이다.6 is a result of observing the effect on mitochondria when the STAT6 proteins of FIG. 5 were overexpressed in the 293T cell line.

도 7은 본 발명의 실시예 5에 따라 STAT6가 종양 형성에 미치는 영향을 확인하기 위해 293T 세포주에서 STAT6 발현이 억제된 안정적 세포주(stable cell line)를 제작하여 이를 웨스턴 블로팅(Western bloting) 실험으로 확인한 것이다.7 shows a stable cell line in which STAT6 expression is suppressed in the 293T cell line was prepared in order to confirm the effect of STAT6 on tumor formation according to Example 5 of the present invention, and this was performed as a Western blotting experiment. it has been confirmed

도 8은 293T Parental 세포와 도 7에 따라 제조된 STAT6 발현이 억제된 세포 (STAT6 KO)를 각각 쥐에 이식한 후, 시간에 따른 종양 부피 변화를 나타낸 그래프이다 (****, P<0.0001; n= 18).8 is a graph showing the change in tumor volume over time after transplanting 293T Parental cells and cells (STAT6 KO) with STAT6 expression suppressed prepared according to FIG. 7 into mice, respectively (****, P<0.0001) ; n = 18).

도 9는 도 7의 세포 이식 후, 종양 조직을 적출하여 관찰한 것으로, 상단 도면은 세포 이식 한 달 뒤의 쥐의 모습이고, 하단 도면은 상단 도면의 각 번호가 매겨진 쥐에서 적출한 종양 조직을 촬영한 이미지이다.9 is an observation by removing the tumor tissue after cell transplantation in FIG. 7 , the upper figure is a view of a mouse one month after cell transplantation, and the lower figure is the tumor tissue extracted from each numbered mouse in the upper figure. It is a photographed image.

도 10은 도 9에 따라 적출한 종양 조직의 무게를 측정한 결과 그래프이다 (**, P< 0.01; n= 18).10 is a graph showing the result of measuring the weight of the tumor tissue extracted according to FIG. 9 (**, P<0.01; n=18).

도 11은 human STAT6 단백질 (STAT6 WT)과 미토콘드리아 표적 서열이 삭제된 STAT6 단백질 (MTS D)로, 도 7에 따라 제조된 STAT6 KO stable cell line에 이들 STAT6 단백질들을 각각 과발현시켜 stable cell line을 다시 제작하여, 이를 Western bloting 실험으로 확인한 것이다.FIG. 11 shows human STAT6 protein (STAT6 WT) and STAT6 protein (MTS D) from which the mitochondrial target sequence has been deleted, and each of these STAT6 proteins was overexpressed in the STAT6 KO stable cell line prepared according to FIG. 7 to reconstruct a stable cell line. Therefore, this was confirmed by Western blotting experiments.

도 12는 도 11에 따른 STAT6 KO와 STAT6 WT stable cell line을 쥐에 이식 후, 종양 조직을 적출하여 관찰한 것으로, 상단 도면은 이식 한 달 뒤 종양 적출 직전의 쥐 모습이고, 하단 도면은 적출된 종양을 크기 순서대로 나열한 것이다.12 is a view of the STAT6 KO and STAT6 WT stable cell lines according to FIG. 11 were transplanted into mice, and the tumor tissue was excised and observed. Tumors are listed in order of size.

도 13은 도 11에 따른 STAT6 KO와 MTS D stable cell line을 쥐에 이식 후, 종양 조직을 적출하여 관찰한 것으로, 상단 도면은 이식 한 달 뒤 종양 적출 직전의 쥐 모습이고, 하단 도면은 적출된 종양을 크기 순서대로 나열한 것이다.13 is an observation of the tumor tissue excised after transplanting the STAT6 KO and MTS D stable cell lines according to FIG. 11 into mice. The upper figure is a view of a mouse just before tumor extraction one month after transplantation, and the lower figure is the excised figure. Tumors are listed in order of size.

도 14는 도 11에 따라 제조된 stable cell line 이식 후, 시간에 다른 종양의 부피 변화를 측정한 그래프이다 (****, P<0.0001; STAT6 KO, n=25; STAT6 WT-myc, n=12; MTS D-myc, n=13).14 is a graph measuring the volume change of different tumors with time after transplantation of the stable cell line prepared according to FIG. 11 (****, P<0.0001; STAT6 KO, n=25; STAT6 WT-myc, n =12; MTS D-myc, n=13).

도 15는 도 11에 따라 제조된 stable cell line 이식 후 한 달 뒤, 쥐에서 적출된 종양의 무게를 측정한 그래프이다 (**, P< 0.01; STAT6 KO, n=25; STAT6 WT-myc, n=12; MTS D-myc, n=13).15 is a graph measuring the weight of tumors excised from mice one month after transplantation of the stable cell line prepared according to FIG. 11 (**, P<0.01; STAT6 KO, n=25; STAT6 WT-myc, n=12; MTS D-myc, n=13).

이하, 본 발명을 상세하게 설명하기로 한다.Hereinafter, the present invention will be described in detail.

본 발명자는 STAT6 단백질 N-말단 서열의 특정 아미노산 서열이 미토콘드리아 타겟팅(targeting) 시퀀스이며, STAT6 단백질이 미토콘드리아, 특히 이의 외막에 위치함을 확인함으로써, 본 발명을 완성하였다.The present inventors have completed the present invention by confirming that the specific amino acid sequence of the STAT6 protein N-terminal sequence is a mitochondrial targeting sequence, and that the STAT6 protein is located in the mitochondria, particularly its outer membrane.

본 발명은 서열번호 4의 아미노산 서열로 이루어진 미토콘드리아 표적용 펩타이드를 제공한다.The present invention provides a mitochondrial targeting peptide consisting of the amino acid sequence of SEQ ID NO: 4.

본 발명의 일 실시예에 따르면, human STAT6 내부 서열 중, human STAT6 N-말단 1-30번째 서열이 미토콘드리아 표적(타겟팅) 효과를 가짐을 확인할 수 있었고, 이를 서열번호 4로 표시하였다.According to an embodiment of the present invention, it was confirmed that the human STAT6 N-terminal 1-30 sequence among the human STAT6 internal sequences has a mitochondrial targeting (targeting) effect, which is indicated by SEQ ID NO: 4.

서열번호 4 : MSLWGLVSKMPPEKVQRLYVDFPQHLRHLLSEQ ID NO: 4: MSLWGLVSKMPPEKVQRLYVDFPQHLRHLL

본 발명에 따른 서열번호 4의 아미노산 서열로 이루어진 미토콘드리아 표적용 펩타이드 또는 이의 기능적 동등물은 천연으로부터 유래될 수 있고, 당 분야에 공지된 화학적 합성 방법 또는 유전 공학적 방법을 이용하여 화학적으로 합성하거나 유전자 재조합 기술을 이용하여 획득될 수 있으나, 이에 제한되는 것은 아니다.The mitochondrial targeting peptide or functional equivalent thereof consisting of the amino acid sequence of SEQ ID NO: 4 according to the present invention may be derived from nature, chemically synthesized using a chemical synthesis method or genetic engineering method known in the art, or genetically recombination It may be obtained using technology, but is not limited thereto.

상기 공지된 화학적 합성(Creighton, Proteins; Structures and Molecular Principles, W. H. Freeman and Co., NY, 1983)의 대표적인 방법으로는 액체 또는 고체상 합성, 단편 응축, F-MOC 또는 T-BOC 화학법이 포함되나(Chemical Approaches to the Synthesis of Peptides and Proteins, Williams et al., Eds., CRC Press, Boca Raton Florida, 1997; A Practical Approach, Athert on ＆ Sheppard, Eds., IRL Press, Oxford, England, 1989), 이에 제한되는 것은 아니다.Representative methods of the known chemical synthesis (Creighton, Proteins; Structures and Molecular Principles, WH Freeman and Co., NY, 1983) include liquid or solid phase synthesis, fragment condensation, F-MOC or T-BOC chemistry, but (Chemical Approaches to the Synthesis of Peptides and Proteins, Williams et al., Eds., CRC Press, Boca Raton Florida, 1997; A Practical Approach, Athert on & Sheppard, Eds., IRL Press, Oxford, England, 1989), However, the present invention is not limited thereto.

상기 유전공학적 방법은 통상적인 방법에 따라 상기 펩타이드 또는 이의 기능적 동등물을 암호화하는 DNA 서열을 제작할 수 있다. 상기 DNA 서열은 적절한 프라이머를 사용하여 PCR 증폭함으로써 제작할 수 있다. 다른 방법으로 당업계에 공지된 표준 방법에 의해, 예컨대, 자동 DNA 합성기(Biosearch 또는 Applied Biosystems 사에서 판매하는 것)을 사용하여 DNA 서열을 합성할 수 있다. 합성된 DNA 서열은 이에 작동가능하게 연결되어 DNA 서열의 발현을 조절하는 하나 이상의 발현 조절 서열(expression control sequence)을 포함하는 벡터에 삽입시키고, 이로부터 형성된 재조합 발현 벡터로 숙주세포를 형질전환시켜 형질전환체를 생성한다.The genetic engineering method may produce a DNA sequence encoding the peptide or a functional equivalent thereof according to a conventional method. The DNA sequence can be prepared by PCR amplification using appropriate primers. Alternatively, the DNA sequence can be synthesized by standard methods known in the art, for example, using an automatic DNA synthesizer (such as those sold by Biosearch or Applied Biosystems). The synthesized DNA sequence is inserted into a vector containing one or more expression control sequences that are operably linked thereto to control the expression of the DNA sequence, and transform the host cell with the recombinant expression vector formed therefrom to transform create a transform

상기 "형질전환체"는 "숙주세포" 등과 호환성 있게 사용될 수 있으며, 임의의 수단, 예를 들어, 전기충격법, 칼슘 포스파타제 침전법, 미세주입법, 형질전환법, 바이러스 감염 등에 의해 세포 내로 도입된 이종성 DNA를 포함하는 원핵 또는 진핵 세포를 의미한다. 숙주세포에 따라서 단백질의 발현량과 수식 등이 다르게 나타나므로, 당업자가 목적하는 바에 가장 적합한 숙주세포를 선택하여 사용할 수 있다. The "transformant" can be used interchangeably with "host cell", etc., and is introduced into the cell by any means, for example, electroshock method, calcium phosphatase precipitation method, microinjection method, transformation method, virus infection, etc. It refers to a prokaryotic or eukaryotic cell containing heterologous DNA. Since the expression level and modification of the protein appear differently depending on the host cell, a person skilled in the art can select and use the most suitable host cell for the intended purpose.

생성된 형질전환체를 상기 DNA 서열이 발현되기에 적절한 배지 및 조건 하에서 배양하여, 배양물로부터 상기 DNA 서열에 의해 코딩된, 실질적으로 순수한 펩타이드를 회수할 수 있다. 상기 회수는 당업계에 공지된 방법(예컨대, 크로마토그래피)을 이용하여 수행할 수 있다. By culturing the resulting transformant under a medium and conditions suitable for expression of the DNA sequence, a substantially pure peptide encoded by the DNA sequence can be recovered from the culture. The recovery may be performed using a method known in the art (eg, chromatography).

상기에서 "실질적으로 순수한 펩타이드"라 함은 본 발명에 따른 펩타이드가 숙주세포로부터 유래된 어떠한 다른 단백질도 실질적으로 포함하지 않는 것을 의미한다. As used herein, the term "substantially pure peptide" means that the peptide according to the present invention does not substantially contain any other proteins derived from host cells.

본 발명에 따른 펩타이드는 천연형 아미노산 서열을 갖는 펩타이드 뿐만 아니라 이의 아미노산 서열 변이체를 포함할 수 있다. The peptide according to the present invention may include a peptide having a native amino acid sequence as well as an amino acid sequence variant thereof.

상기 "아미노산 서열 변이체"란, 본 발명에 따른 아미노산 서열에서 하나 이상의 아미노산 잔기가 결실, 삽입, 비보전적 또는 보전적 치환, 아미노산 유사체의 치환 또는 이들의 조합에 의하여 상이한 서열을 가지는 펩타이드를 의미하나, 상기의 변이에 의해 분자의 활성은 전체적으로 변경되지 않는다.The "amino acid sequence variant" refers to a peptide having a different sequence by deletion, insertion, non-conservative or conservative substitution of one or more amino acid residues in the amino acid sequence according to the present invention, substitution of an amino acid analog, or a combination thereof, By the above mutations, the activity of the molecule is not entirely changed.

또한, 본 발명에 따른 펩타이드는 인산화(phosphorylation), 황화(sulfation), 아크릴화(acrylation), 당화(glycosylation), 메틸화(methylation), 파네실화(farnesylation) 등으로 수식(modification)될 수 있다.In addition, the peptide according to the present invention may be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, or the like.

본 발명은 서열번호 4의 아미노산 서열을 코딩하는 유전자를 포함하는 재조합 벡터를 제공한다.The present invention provides a recombinant vector comprising a gene encoding the amino acid sequence of SEQ ID NO: 4.

상기 서열번호 4의 아미노산 서열을 코딩하는 유전자는 서열번호 4의 아미노선 서열로 이루어진 펩타이드를 코딩하는 폴리뉴클레오티드이다.The gene encoding the amino acid sequence of SEQ ID NO: 4 is a polynucleotide encoding a peptide consisting of the amino acid sequence of SEQ ID NO: 4.

상기 재조합 벡터는 상기 유전자를 발현시키기 위한 수단으로서, 플라스미드 벡터(plasmid vector), 코스미드 벡터(cosmid vector), 박테리오파지 벡터(bacteriophage vector) 등 공지의 발현 벡터를 포함할 수 있으나, 이에 제한되는 것은 아니며, 상기 벡터는 DNA 재조합 기술을 이용한 임의의 공지된 방법에 따라 당업자가 용이하게 제조할 수 있다.The recombinant vector is a means for expressing the gene, and may include a known expression vector such as a plasmid vector, a cosmid vector, or a bacteriophage vector, but is not limited thereto. , the vector can be easily prepared by those skilled in the art according to any known method using DNA recombination technology.

본 발명에 따른 재조합 벡터는 프로모터, 상기 서열번호 4의 아미노산 서열을 코딩하는 유전자 및 목적 단백질을 코딩하는 유전자가 작동 가능하게 연결된 것일 수 있다.The recombinant vector according to the present invention may be one in which a promoter, a gene encoding the amino acid sequence of SEQ ID NO: 4, and a gene encoding a target protein are operably linked.

상기 "작동 가능하게 연결된"이란, 발현 조절 서열이 서열번호 4의 아미노산 서열을 코딩하는 유전자를 전사 및 해독을 조절하도록 연결된 것을 말하며, 발현 조절 서열(예: 프로모터, 인핸서 등)의 조절 하에 상기 유전자가 발현되어 이에 의해 코딩되는 본 발명에 따른 펩타이드가 생성되도록 정확한 해독 프레임을 유지시키는 것을 포함한다.The "operably linked" means that an expression control sequence is linked to control transcription and translation of a gene encoding the amino acid sequence of SEQ ID NO: 4, and the gene under the control of an expression control sequence (eg, promoter, enhancer, etc.) and maintaining the correct reading frame so that the peptide according to the invention is expressed and encoded thereby.

상기 서열번호 4의 아미노산 서열을 코딩하는 유전자는 미토콘드리아 표적에 효과적이므로, 미토콘드리아로 표적하고자 하는 목적 단백질을 코딩하는 유전자에 본 발명의 서열번호 4의 아미노산 서열을 코딩하는 유전자를 작동 가능하게 연결하여 벡터에 삽입하고 이를 발현시키는 경우, 목적 단백질을 미토콘드리아로 표적할 수 있다.Since the gene encoding the amino acid sequence of SEQ ID NO: 4 is effective for mitochondrial targeting, the gene encoding the amino acid sequence of SEQ ID NO: 4 of the present invention is operably linked to the gene encoding the target protein to be targeted to the mitochondria. In the case of inserting and expressing it, the target protein can be targeted to the mitochondria.

상기 재조합 벡터는 형광 단백질(fluorescent protein, FP) 유전자를 더 연결하여 상기 형광 단백질을 발현시킴으로써, 상기 목적 단백질의 이동 또는 분포 여부를 관찰할 수 있다.In the recombinant vector, by further linking a fluorescent protein (FP) gene to express the fluorescent protein, it is possible to observe whether the target protein is moved or distributed.

상기 형광 단백질은 녹색 형광 단백질(green fluorescent probe, GFP), 황색 형광 단백질(yellow fluorescent protein, YFP) 또는 적색 형광 단백질(red fluorescent protein, RFP) 등 일 수 있으나, 이에 제한되는 것은 아니다.The fluorescent protein may be a green fluorescent probe (GFP), a yellow fluorescent protein (YFP), or a red fluorescent protein (RFP), but is not limited thereto.

본 발명은 상기 재조합 벡터를 세포 내 도입하는 단계를 포함하는, 목적 단백질을 미토콘드리아로 이동 또는 분포시키는 방법을 제공한다.The present invention provides a method for moving or distributing a target protein to mitochondria, comprising the step of introducing the recombinant vector into a cell.

상기 세포 내 도입은 재조합 벡터를 이용하여 세포를 형질전환시키는 것을 의미하며, 이에 따른 형질전환 방법은 당업자에게 잘 알려진 공지된 방법을 이용할 수 있다.The introduction into the cell means transforming the cell using a recombinant vector, and the transformation method according to this may use a well-known method well known to those skilled in the art.

본 발명은 프로모터, 상기 서열번호 4의 아미노산 서열을 코딩하는 유전자, 목적 단백질을 코딩하는 유전자 및 형광 단백질을 코딩하는 유전자를 포함하는 재조합 벡터를 세포 내 도입하는 단계; 및 상기 유전자를 발현시켜 형광 영상을 통해 상기 발현되는 목적 단백질의 이동 또는 분포를 확인하는 단계;를 포함하는 세포 내 목적 단백질의 미토콘드리아로 이동 또는 분포를 확인하는 방법을 제공한다.The present invention comprises the steps of introducing a recombinant vector comprising a promoter, a gene encoding the amino acid sequence of SEQ ID NO: 4, a gene encoding a target protein, and a gene encoding a fluorescent protein into a cell; and confirming the movement or distribution of the expressed target protein through fluorescence imaging by expressing the gene; provides a method of confirming the movement or distribution of the target protein in the cell into the mitochondria, including.

상기 형광 단백질은 녹색 형광 단백질(green fluorescent probe, GFP), 황색 형광 단백질(yellow fluorescent protein, YFP) 또는 적색 형광 단백질(red fluorescent protein, RFP) 등 일 수 있으나, 이에 제한되는 것은 아니다.The fluorescent protein may be a green fluorescent probe (GFP), a yellow fluorescent protein (YFP), or a red fluorescent protein (RFP), but is not limited thereto.

상기 재조합 벡터가 도입된 세포, 즉 형질전환된 세포에서 발현되는 단백질로부터 방출되는 특정 파장의 형광을 형광 현미경을 사용하여 상기 세포가 살아있는 상태에서 계속하여 영상화할 수 있게 함으로써, 세포 내에서의 목적 단백질의 발현 과정 및 미토콘드리아로의 이동 과정을 단계별로 세밀하게 가시화할 수 있다.By using a fluorescence microscope to continuously image the fluorescence of a specific wavelength emitted from a protein expressed in a cell into which the recombinant vector is introduced, that is, a protein expressed in the transformed cell, the cell is in a living state, and thus the target protein in the cell can be visualized in detail step by step in the process of expression and mitochondrial migration.

본 발명의 일 실시예에 따르면, STAT6 N-말단서열 (1-50번째 아미노산 서열) 중에서, 1-30번째 서열, 31-50번째 서열 및 1-20번째 서열의 C-말단에 각각 녹색 형광 단백질(GFP)을 결합시켜 1-30-GFP, 31-50-GFP, 1-20-GFP 플라스미드를 제조하였고, 이를 세포 내로 형질주입한 뒤 현미경으로 관찰함으로써, 미토콘드리아로의 이동을 확인할 수 있었다.According to an embodiment of the present invention, among the STAT6 N-terminal sequence (1-50 amino acid sequence), green fluorescent protein at the C-terminus of the 1-30th sequence, the 31-50th sequence and the 1-20th sequence, respectively (GFP) was bound to prepare 1-30-GFP, 31-50-GFP, and 1-20-GFP plasmids, and by transfecting them into cells and observing them under a microscope, migration to mitochondria could be confirmed.

본 발명은 서열번호 4의 아미노산 서열로 이루어진 미토콘드리아 표적용 펩타이드를 유효성분으로 함유하는 미토콘드리아 약물 전달용 담체를 제공한다.The present invention provides a mitochondrial drug delivery carrier containing a mitochondrial targeting peptide consisting of the amino acid sequence of SEQ ID NO: 4 as an active ingredient.

상기 펩타이드는 미토콘드리아 표적에 효과적이므로, 미토콘드리아를 표적으로 하는 여러 가지 질환과 관련된 약물을 미토콘드리아로 직접 전달할 수 있고, 이를 통해 미토콘드리아 내 약물 흡수율을 향상시킬 수 있다.Since the peptide is effective at mitochondrial targeting, drugs related to various diseases targeting mitochondria can be delivered directly to the mitochondria, thereby improving drug absorption in the mitochondria.

본 발명은 서열번호 4의 아미노산 서열로 이루어진 미토콘드리아 표적용 펩타이드 및 약리학적 활성 물질이 결합된 복합체를 유효성분으로 포함하는 미토콘드리아 기능 장애 관련 질환 예방 또는 치료용 약학 조성물을 제공한다.The present invention provides a pharmaceutical composition for preventing or treating mitochondrial dysfunction-related diseases, comprising as an active ingredient a complex to which a mitochondrial targeting peptide and a pharmacologically active substance are bound, which consists of the amino acid sequence of SEQ ID NO: 4.

상기 약리학적 활성 물질은 동물 또는 사람의 체내에서 생리적인 기능을 촉진 또는 억제하여 목적하는 약리학적 효과를 유도할 수 있는 물질로서, 동물 또는 사람에게 투여하기 적합한 화학적 또는 생물학적 물질 또는 화합물을 의미한다. 감염 예방과 같은 원하지 않은 생물학적 효과를 예방하는 예방 효과를 가지고, 질병의 결과로 생기는 고통 또는 감염을 완화시키며, 질병을 완전히 제거할 수 있는 역할을 수행할 수 있기에, 상기 약리학적 활성 물질은 “약물” 또는 “치료제”의 용어와도 혼용될 수 있다.The pharmacologically active substance is a substance capable of inducing a desired pharmacological effect by promoting or inhibiting a physiological function in an animal or human body, and refers to a chemical or biological substance or compound suitable for administration to an animal or human. The pharmacologically active substance has a preventive effect of preventing unwanted biological effects such as preventing infection, alleviating pain or infection resulting from a disease, and can play a role of completely eliminating a disease, so that the pharmacologically active substance is a “drug ” or “therapeutic agent” may be used interchangeably.

상기 복합체는 미토콘드리아로의 표적을 이끌 수 있는 펩타이드를 포함함으로써, 미토콘드리아에 작용하여 직접적인 약리학적 활성을 나타낼 수 있는 물질을 미토콘드리아로 효과적으로 전달하고 이동시켜 머물게 할 수 있다. 따라서 미토콘드리아의 기능 장애와 관련된 질환의 예방 또는 치료에 유용하게 사용될 수 있다.The complex includes a peptide capable of directing a target to the mitochondria, so that a substance capable of acting on the mitochondria and exhibiting direct pharmacological activity can be effectively delivered to the mitochondria, moved and stayed there. Therefore, it can be usefully used for the prevention or treatment of diseases related to mitochondrial dysfunction.

상기 약리학적 활성 물질은 미토콘드리아에 작용하여 특정 활성을 나타낼 수 있는 천연물, 화합물, 단백질 또는 유전자(DNA, RNA 또는 PNA)일 수 있으나, 이에 제한되지 않는다.The pharmacologically active substance may be a natural product, compound, protein, or gene (DNA, RNA or PNA) capable of acting on mitochondria to exhibit specific activity, but is not limited thereto.

상기 화합물은 독소루비신(doxorubicin), 에피루비신(epirubicin), 이다루비신(idarubicin), 도세탁셀(docetaxel), 파클리탁셀(paclitaxel), 카바지탁셀(cabazitaxel), 빈블라스틴(vinblastine), 빈크리스틴(vincristine) 및 시스플라틴(cisplain)으로 이루어진 군에서 선택되는 하나 이상의 항암제일 수 있으나, 이에 제한되는 것은 아니다.The compound is doxorubicin (doxorubicin), epirubicin (epirubicin), idarubicin (idarubicin), docetaxel (docetaxel), paclitaxel (paclitaxel), cabazitaxel (cabazitaxel), vinblastine (vinblastine), vincristine (vincristine) And it may be one or more anticancer agents selected from the group consisting of cisplatin (cisplain), but is not limited thereto.

상기 화합물은 플라보노이드(flavonoid), 폴리페놀(polyphenol), 레스베라트롤(resveratrol), 카로티노이드(carotenoid), 알파리포산(alpha-lipoic acid), 토코페롤(tocopherol) 및 아스코르브산(ascorbic acid)으로 이루어진 군에서 선택되는 하나 이상의 항산화제일 수 있으나, 이에 제한되는 것은 아니다.The compound is selected from the group consisting of flavonoids, polyphenols, resveratrol, carotenoids, alpha-lipoic acid, tocopherol and ascorbic acid. It may be one or more antioxidants, but is not limited thereto.

그 밖에 상기 화합물은 항생제, 항염증제 등 일 수 있으나, 이에 제한되는 것은 아니다.In addition, the compound may be an antibiotic, an anti-inflammatory agent, etc., but is not limited thereto.

본 발명은 프로모터, 서열번호 4의 아미노산 서열을 코딩하는 유전자 및 목적 단백질을 코딩하는 유전자가 작동 가능하게 연결된 재조합 벡터를 유효성분으로 포함하는 미토콘드리아 기능 장애 관련 질환 예방 또는 치료용 약학 조성물을 제공한다.The present invention provides a pharmaceutical composition for preventing or treating diseases related to mitochondrial dysfunction, comprising a promoter, a gene encoding the amino acid sequence of SEQ ID NO: 4, and a recombinant vector to which a gene encoding a target protein is operably linked as an active ingredient.

상기 미토콘드리아 기능 장애 관련 질환은 허혈성 뇌질환 또는 허혈성 심질환, 뇌졸증, 편두통, 빈혈증, 파킨슨병, 알츠하이머, 근위축성측색경화증(ALS), 미토콘드리아뇌근증, 간경변, 판코니 증후군, 비정형 페닐케톤뇨증, 천식, 당뇨병, 동맥경화, 고혈압, 고콜레스테롤증, 시신경 질환, 비만, 우울증 및 암 질환으로 이루어진 군에서 선택되는 어느 하나일 수 있다.The mitochondrial dysfunction-related disease is ischemic brain disease or ischemic heart disease, stroke, migraine, anemia, Parkinson's disease, Alzheimer's, amyotrophic lateral sclerosis (ALS), mitochondrial encephalomyopathy, cirrhosis, Fanconi's syndrome, atypical phenylketonuria, asthma, diabetes , arteriosclerosis, hypertension, hypercholesterolemia, optic nerve disease, obesity, depression, and may be any one selected from the group consisting of cancer diseases.

또한, 본 발명은 STAT6 (Signal transducer and activator of transcription 6) 단백질, 상세하게는 미토콘드리아 내부 표적 도메인 및 미토콘드리아 막 투과 도메인을 포함하는 STAT6 단백질을 유효성분으로 함유하는 미토콘드리아 기능 장애 관련 질환 예방 또는 치료용 약학 조성물을 제공한다.In addition, the present invention provides a STAT6 (Signal transducer and activator of transcription 6) protein, specifically, a mitochondrial dysfunction-related disease prevention or treatment pharmaceutical containing STAT6 protein comprising a mitochondrial internal target domain and a mitochondrial membrane permeation domain as an active ingredient A composition is provided.

본 명세서에서, "STAT6 (Signal transducer and activator of transcription 6)" 단백질은 STAT6 유전자에 의해 인코딩된 단백질로, 사이토카인, 호르몬, 성장인자 등 세포내 신호 전달에 관여하는 전사인자 STAT 패밀리 계열에 속한다.As used herein, the "STAT6 (Signal transducer and activator of transcription 6)" protein is a protein encoded by the STAT6 gene, and belongs to the STAT family of transcription factors involved in intracellular signal transduction such as cytokines, hormones, and growth factors.

상기 STAT6 단백질은 서열번호 1로 표시되는 아미노산 서열로 이루어질 수 있다.The STAT6 protein may consist of an amino acid sequence represented by SEQ ID NO: 1.

바람직하게는, 상기 미토콘드리아 내부 표적 도메인은 서열번호 4로 표시되는 아미노산 서열로 이루어질 수 있다.Preferably, the mitochondrial internal target domain may consist of the amino acid sequence represented by SEQ ID NO: 4.

본 발명의 일 실시예에 따르면, 상기 human STAT6 내부 서열 중, human STAT6 N-말단 1-30번째 아미노산 서열이 미토콘드리아 타겟팅 효과를 가짐을 확인할 수 있었다.According to an embodiment of the present invention, it was confirmed that the human STAT6 N-terminal 1-30 amino acid sequence among the human STAT6 internal sequences has a mitochondrial targeting effect.

바람직하게는, 상기 미토콘드리아 막 투과(transmembrane) 도메인은 서열번호 11로 표시되는 아미노산 서열로 이루어질 수 있다.Preferably, the mitochondrial transmembrane domain may consist of the amino acid sequence shown in SEQ ID NO: 11.

상기 미토콘드리아 막 투과 도메인은 상기 미토콘드리아 내부를 표적하는 STAT6 단백질을 미토콘드리아 외막에 위치시킬 수 있다.The mitochondrial membrane permeation domain may localize the STAT6 protein targeting the inside of the mitochondria to the outer mitochondrial membrane.

본 발명의 일 실시예에 따르면, 상기 human STAT6 내부 서열 중, 624-698번째 아미노산 서열이 상기 단백질이 미토콘드리아 외막에 위치하게 하는 막전위 도메인(transmembrane domain) 역할을 함을 확인할 수 있었고, 이를 서열번호 11로 표시하였다.According to an embodiment of the present invention, it was confirmed that the 624-698 amino acid sequence among the human STAT6 internal sequence serves as a transmembrane domain that allows the protein to be located in the outer mitochondrial membrane, which is SEQ ID NO: 11 indicated as

서열번호 11 :SEQ ID NO: 11:

AFRSHYKPEQMGKDGRGYVPATIKMTVERDQPLPTPELQMPTMVPSYDLGMAPDSSMSMQLGPDMVPQVYPPHSHAFRSHYKPEQMGKDGRGYVPATIKMTVERDQPLPTPELQMPTMVPSYDLGMAPDSSMSMQLGPDMVPQVYPPHSH

상기의 도메인들에 의해 상기 STAT6 단백질은 미토콘드리아 외막에 위치할 수 있고, 상기 단백질이 미토콘드리아 외막에 축적(accumulation) 될 때, 세포사멸의 시발점이 되는 미토콘드리아의 절편화(fragmentation)를 유도하며, 종양 형성을 억제할 수 있는 바, 상기 STAT6 단백질은 미토콘드리아 기능 장애 관련 질환 예방 또는 치료용 약학 조성물로 활용할 수 있다.Due to the above domains, the STAT6 protein can be located in the outer mitochondrial membrane, and when the protein is accumulated in the outer mitochondrial membrane, it induces fragmentation of mitochondria, which is the starting point of apoptosis, and tumor formation can be inhibited, the STAT6 protein can be utilized as a pharmaceutical composition for preventing or treating diseases related to mitochondrial dysfunction.

상기 미토콘드리아 기능 장애 관련 질환은 허혈성 뇌질환 또는 허혈성 심질환, 뇌졸증, 편두통, 빈혈증, 파킨슨병, 알츠하이머, 근위축성측색경화증(ALS), 미토콘드리아뇌근증, 간경변, 판코니 증후군, 비정형 페닐케톤뇨증, 천식, 당뇨병, 동맥경화, 고혈압, 고콜레스테롤증, 시신경 질환, 비만, 우울증 및 암 질환으로 이루어진 군에서 선택되는 어느 하나일 수 있으나, 이에 제한되는 것은 아니다.The mitochondrial dysfunction-related disease is ischemic brain disease or ischemic heart disease, stroke, migraine, anemia, Parkinson's disease, Alzheimer's, amyotrophic lateral sclerosis (ALS), mitochondrial encephalomyopathy, cirrhosis, Fanconi's syndrome, atypical phenylketonuria, asthma, diabetes , arteriosclerosis, hypertension, hypercholesterolemia, optic nerve disease, obesity, depression, and may be any one selected from the group consisting of cancer diseases, but is not limited thereto.

또한, 상기 조성물은 STAT6 단백질의 활성화제 또는 발현촉진제를 더 포함할 수 있으나, 이에 제한되는 것은 아니다.In addition, the composition may further include an activator or expression promoter of STAT6 protein, but is not limited thereto.

본 발명에 따른 약물 전달용 담체 또는 약학 조성물은 약학적 분야의 통상적인 방법에 따라 제조될 수 있다.The carrier or pharmaceutical composition for drug delivery according to the present invention may be prepared according to a conventional method in the pharmaceutical field.

상기 약물 전달용 담체 또는 약학 조성물은 상기 제형에 따라 약학적으로 허용가능한 적절한 담체와 배합될 수 있고, 필요에 따라, 부형제, 희석제, 분산제, 유화제, 완충제, 안정제, 결합제, 붕해제, 용제 등을 더 포함하여 제조할 수 있다. 상기 "약학적으로 허용 가능한"이란, 상기 약학 조성물에 노출되는 세포나 인간에게 독성이 없는 것을 의미하고, 상기 적절한 담체 등은 본 발명에 따른 미토콘드리아 표적용 펩타이드 또는 STAT6 단백질의 활성 및 특성을 저해하지 않는 것으로, 투여 형태 및 제형에 따라 달리 선택될 수 있다.The drug delivery carrier or pharmaceutical composition may be combined with an appropriate pharmaceutically acceptable carrier according to the formulation, and if necessary, excipients, diluents, dispersants, emulsifiers, buffers, stabilizers, binders, disintegrants, solvents, etc. It can be prepared by including more. The "pharmaceutically acceptable" means that it is not toxic to cells or humans exposed to the pharmaceutical composition, and the appropriate carrier does not inhibit the activity and properties of the mitochondrial targeting peptide or STAT6 protein according to the present invention. It may be selected differently depending on the dosage form and dosage form.

상기 약물 전달용 담체 또는 약학 조성물은 어떠한 제형으로도 적용될 수 있고, 보다 상세하게는 통상의 방법에 따라 경구형 제형, 외용제, 좌제 및 멸균 주사용액의 비경구형 제형로 제형화하여 사용될 수 있다.The carrier or pharmaceutical composition for drug delivery may be applied in any formulation, and more specifically, it may be formulated into oral formulations, topical formulations, suppositories, and parenteral formulations of sterile injection solutions according to conventional methods.

상기 경구형 제형 중 고형 제형은 정제, 환제, 산제, 과립제, 캡슐제 등의 형태로, 적어도 하나 이상의 부형제, 예를 들면, 전분, 칼슘카보네이트, 수크로스, 락토오스, 솔비톨, 만니톨, 셀룰로오스, 젤라틴 등을 섞어 조제할 수 있고, 단순한 부형제 이외에 마그네슘 스테아레이트, 탈크 같은 윤활제들도 포함될 수 있다. 또한, 캡술제형의 경우 상기 언급한 물질 외에도 지방유와 같은 액체 담체를 더 포함할 수 있다.The solid dosage form among the oral dosage forms is in the form of tablets, pills, powders, granules, capsules, etc., and at least one or more excipients, for example, starch, calcium carbonate, sucrose, lactose, sorbitol, mannitol, cellulose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc may be included. In addition, the capsule formulation may further include a liquid carrier such as fatty oil in addition to the above-mentioned substances.

상기 경구형 제형 중 액상 제형은 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데 흔히 사용되는 단순 희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다.Among the oral dosage forms, liquid formulations include suspensions, solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients, for example, wetting agents, sweeteners, fragrances, preservatives, etc. may be included. there is.

상기 비경구 제형은 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조 제제, 좌제가 포함될 수 있다. 비수성용제, 현탁제로는 프로필렌글리콜, 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔(witepsol), 마크로골, 트윈 61, 카카오지, 라우린지, 글리세로제라틴 등이 사용될 수 있다. 이에 제한되지 않고, 당해 기술 분야에 알려진 적합한 제제를 모두 사용 가능하다.The parenteral formulation may include a sterile aqueous solution, a non-aqueous solution, a suspension, an emulsion, a freeze-dried formulation, and a suppository. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, witepsol, macrogol, Tween 61, cacao butter, laurin fat, glycerogelatin, and the like can be used. Without being limited thereto, any suitable agent known in the art may be used.

또한, 본 발명에 따른 약학 조성물은 치료 효능의 증진을 위해 칼슘이나 비타민 D3 등을 더 첨가할 수 있다. In addition, the pharmaceutical composition according to the present invention may further add calcium, vitamin D3, or the like to enhance therapeutic efficacy.

본 발명에 따른 약학 조성물은 약학적으로 유효한 양으로 투여될 수 있다. 상기 "약학적으로 유효한 양"이란, 의학적 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분하며 부작용을 일으키지 않을 정도의 양을 의미한다.The pharmaceutical composition according to the present invention may be administered in a pharmaceutically effective amount. The "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment and not cause side effects.

상기 약학 조성물의 유효 용량 수준은 사용 목적, 환자의 연령, 성별, 체중 및 건강 상태, 질환의 종류, 중증도, 약물의 활성, 약물에 대한 민감도, 투여 방법, 투여 시간, 투여 경로 및 배출 비율, 치료기간, 배합 또는 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 달리 결정될 수 있다. 예를 들어, 일정하지는 않지만 일반적으로 0.001 내지 100mg/kg으로, 바람직하게는 0.01 내지 10mg/kg을 일일 1회 내지 수회 투여될 수 있다. 상기 투여량은 어떠한 면으로든 본 발명의 범위를 한정하는 것은 아니다.The effective dose level of the pharmaceutical composition is determined by the purpose of use, the age, sex, weight and health status of the patient, the type of disease, severity, drug activity, sensitivity to drug, administration method, administration time, administration route and excretion rate, treatment It may be determined differently depending on factors including the duration, formulation or concurrent use of drugs and other factors well known in the medical field. For example, although not constant, generally 0.001 to 100 mg/kg, preferably 0.01 to 10 mg/kg, may be administered once to several times a day. The above dosage does not limit the scope of the present invention in any way.

본 발명에 따른 약물 전달용 담체 또는 약학 조성물은 미토콘드리아 기능 장애 관련 질환이 발생할 수 있는 임의의 동물에 투여할 수 있고, 상기 동물은 예를 들어, 인간 및 영장류뿐만 아니라 소, 돼지, 말, 개 등의 가축 등을 포함할 수 있다.The carrier or pharmaceutical composition for drug delivery according to the present invention may be administered to any animal capable of developing a disease related to mitochondrial dysfunction, and the animal may be, for example, not only humans and primates, but also cattle, pigs, horses, dogs, etc. of livestock and the like.

본 발명에 따른 약물 전달용 담체 또는 약학 조성물은 제제 형태에 따른 적당한 투여 경로로 투여될 수 있고, 목적 조직에 도달할 수 있는 한 경구 또는 비경구의 다양한 경로를 통하여 투여될 수 있다. 투여 방법은 특히 한정할 필요 없이, 예를 들면, 경구, 직장 또는 정맥, 근육, 피하, 기관지내 흡입, 자궁내 경막 또는 뇌혈관내(intracere-broventricular) 주사 등의 통상적인 방법으로 투여될 수 있다.The carrier or pharmaceutical composition for drug delivery according to the present invention may be administered by an appropriate administration route depending on the form of the formulation, and may be administered via oral or parenteral various routes as long as it can reach the target tissue. The administration method is not particularly limited, and for example, oral, rectal or intravenous, intramuscular, subcutaneous, endobronchial inhalation, intrauterine dural or intracerebroventricular injection, etc. can be administered in a conventional manner. .

본 발명에 따른 약학 조성물은 미토콘드리아 기능 장애 관련 질환의 예방 또는 치료를 위하여 단독으로 사용될 수 있고, 수술 또는 다른 약물 치료 등과 병용하여 사용될 수 있다.The pharmaceutical composition according to the present invention may be used alone for the prevention or treatment of diseases related to mitochondrial dysfunction, or may be used in combination with surgery or other drug treatment.

본 발명은 미토콘드리아 내부 표적 도메인 및 미토콘드리아 막 투과 도메인을 포함하는 STAT6 (Signal transducer and activator of transcription 6) 단백질을 유효성분으로 함유하는 미토콘드리아 기능 장애 관련 질환 예방 또는 개선용 건강기능식품 조성물을 제공한다.The present invention provides a health functional food composition for preventing or improving mitochondrial dysfunction-related diseases, which contains STAT6 (Signal transducer and activator of transcription 6) protein including a mitochondrial internal target domain and a mitochondrial membrane permeation domain as an active ingredient.

바람직하게는, 상기 미토콘드리아 내부 표적 도메인은 서열번호 4로 표시되는 아미노산 서열로 이루어질 수 있다.Preferably, the mitochondrial internal target domain may consist of the amino acid sequence represented by SEQ ID NO: 4.

바람직하게는, 상기 미토콘드리아 막 투과(transmembrane) 도메인은 서열번호 11로 표시되는 아미노선 서열로 이루어질 수 있으며, 상기 미토콘드리아 내부를 표적하는 STAT6 단백질을 미토콘드리아 외막에 위치시킬 수 있다.Preferably, the mitochondrial transmembrane domain may consist of the amino acid sequence shown in SEQ ID NO: 11, and the STAT6 protein targeting the inside of the mitochondria may be located in the outer mitochondrial membrane.

상기의 도메인들에 의해 상기 STAT6 단백질은 미토콘드리아 외막에 위치할 수 있고, 상기 단백질이 미토콘드리아 외막에 축적될 때, 세포사멸의 시발점이 되는 미토콘드리아의 절편화(fragmentation)를 유도하며, 종양 형성을 억제할 수 있는 바, 상기 STAT6 단백질은 미토콘드리아 기능 장애 관련 질환 예방 또는 개선용 건강기능식품 조성물로 활용할 수 있다.Due to the above domains, the STAT6 protein can be located in the outer mitochondrial membrane, and when the protein is accumulated in the mitochondrial outer membrane, it induces fragmentation of mitochondria, which is the starting point of apoptosis, and inhibits tumor formation. As a result, the STAT6 protein can be used as a health functional food composition for preventing or improving mitochondrial dysfunction-related diseases.

상기 미토콘드리아 기능 장애 관련 질환은 허혈성 뇌질환 또는 허혈성 심질환, 뇌졸증, 편두통, 빈혈증, 파킨슨병, 알츠하이머, 근위축성측색경화증(ALS), 미토콘드리아뇌근증, 간경변, 판코니 증후군, 비정형 페닐케톤뇨증, 천식, 당뇨병, 동맥경화, 고혈압, 고콜레스테롤증, 시신경 질환, 비만, 우울증 및 암 질환으로 이루어진 군에서 선택되는 어느 하나일 수 있으나, 이에 제한되는 것은 아니다.The mitochondrial dysfunction-related disease is ischemic brain disease or ischemic heart disease, stroke, migraine, anemia, Parkinson's disease, Alzheimer's, amyotrophic lateral sclerosis (ALS), mitochondrial encephalomyopathy, cirrhosis, Fanconi's syndrome, atypical phenylketonuria, asthma, diabetes , arteriosclerosis, hypertension, hypercholesterolemia, optic nerve disease, obesity, depression, and may be any one selected from the group consisting of cancer diseases, but is not limited thereto.

이에 상응하는 특징들은 상술된 부분에서 대신할 수 있다.Corresponding features may be substituted for the above-mentioned parts.

또한, 상기 조성물은 STAT6 단백질의 활성화제 또는 발현촉진제를 더 포함할 수 있으나, 이에 제한되는 것은 아니다.In addition, the composition may further include an activator or expression promoter of STAT6 protein, but is not limited thereto.

본 발명에 따른 건강기능식품 조성물에 있어서, 상기 건강기능식품은 분말, 과립, 정제, 캡슐, 시럽 또는 음료 등으로 제조될 수 있고, 상기 건강기능식품이 취할 수 있는 형태에는 제한이 없으며, 통상적인 의미의 식품을 모두 포함할 수 있다. 예를 들어, 음료 및 각종 드링크, 과실 및 그의 가공식품(과일통조림, 잼 등), 어류, 육류 및 그 가공식품(햄, 베이컨 등), 빵류 및 면류, 쿠키 및 스낵류, 유제품(버터, 치즈 등) 등이 가능하며, 통상적인 의미에서의 기능성 식품을 모두 포함할 수 있다. 또한 동물을 위한 사료로 이용되는 식품도 포함할 수 있다.In the health functional food composition according to the present invention, the health functional food may be prepared in powder, granule, tablet, capsule, syrup or beverage, etc., and there is no limitation in the form that the health functional food can take, It can include any food with meaning. For example, beverages and various drinks, fruits and their processed foods (canned fruit, jam, etc.), fish, meat and their processed foods (ham, bacon, etc.), breads and noodles, cookies and snacks, dairy products (butter, cheese, etc.) ), etc. are possible, and may include all functional foods in a conventional sense. It may also include food used as feed for animals.

본 발명에 따른 건강기능식품 조성물은 당업계에서 통상적으로 사용되는 식품학적으로 허용가능한 식품 첨가제 및 적절한 기타 보조 성분을 더 포함하여 제조될 수 있다. 식품 첨가물로서의 적합 여부는 다른 규정이 없는 한, 식품의약품안전처에 승인된 식품첨가물공전의 총칙 및 일반시험법 등에 따라 해당 품목에 관한 규격 및 기준에 의하여 판정할 수 있다. 상기 '식품첨가물공전'에 수재된 품목으로는 예를 들어, 케톤류, 글리신, 구연산칼슘, 니코틴산, 계피산 등의 화학적 합성물; 감색소, 감초추출물, 결정셀룰로오스, 고량색소, 구아검 등의 천연첨가물; L-글루타민산나트륨 제제, 면류첨가알칼리제, 보존료 제제, 타르색소제제 등의 혼합 제제류 등을 들 수 있다.The health functional food composition according to the present invention may be prepared by further including pharmaceutically acceptable food additives and other suitable auxiliary ingredients commonly used in the art. Whether or not it is suitable as a food additive can be judged according to the standards and standards for the relevant item in accordance with the general rules and general test methods of the Food Additives Code approved by the Ministry of Food and Drug Safety, unless otherwise specified. The items listed in the 'Food Additives Code' include, for example, chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; natural additives such as persimmon pigment, licorice extract, crystalline cellulose, high pigment, and guar gum; Mixed preparations, such as a sodium L-glutamate preparation, a noodle-added alkali agent, a preservative preparation, and a tar color preparation, etc. are mentioned.

상기 기타 보조 성분은 예를 들어, 향미제, 천연 탄수화물, 감미제, 비타민, 전해질, 착색제, 펙트산, 알긴산, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알콜, 탄산화제 등을 추가로 함유할 수 있다. 특히, 상기 천연 탄수화물로는 포도당, 과당과 같은 모노사카라이드, 말토스, 수크로오스와 같은 디사카라이드, 및 덱스트린, 사이클로덱스트린과 같은 폴리사카라이드, 자일리톨, 소르비톨, 에리트리톨 등의 당알콜을 사용할 수 있으며, 감미제로서는 타우마틴, 스테비아 추출물과 같은 천연 감미제나 사카린, 아스파르탐과 같은 합성 감미제 등을 사용할 수 있다.The other auxiliary ingredients include, for example, flavoring agents, natural carbohydrates, sweeteners, vitamins, electrolytes, colorants, pectic acid, alginic acid, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents, etc. may further contain. In particular, as the natural carbohydrate, monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol can be used. , as the sweetener, natural sweeteners such as taumatine and stevia extract or synthetic sweeteners such as saccharin and aspartame may be used.

본 발명에 따른 건강기능식품 조성물은 일반 약품과는 달리 식품을 원료로 하여 약품의 장기 복용 시 발생할 수 있는 부작용 등이 없는 장점이 있고, 휴대성이 뛰어나, 미토콘드리아 기능 장애 관련 질환의 예방 또는 개선을 위한 보조제로 섭취될 수 있다.The health functional food composition according to the present invention has the advantage that there are no side effects that may occur during long-term administration of the drug using food as a raw material, unlike general drugs, and has excellent portability, preventing or improving mitochondrial dysfunction-related diseases It can be taken as a supplement for

이하, 본 발명의 이해를 돕기 위하여 실시예를 들어 상세하게 설명하기로 한다. 다만 하기의 실시예는 본 발명의 내용을 예시하는 것일 뿐 본 발명의 범위가 하기 실시예에 한정되는 것은 아니다. 본 발명의 실시예는 당업계에서 평균적인 지식을 가진 자에게 본 발명을 보다 완전하게 설명하기 위해 제공되는 것이다.Hereinafter, to help the understanding of the present invention, examples will be described in detail. However, the following examples are merely illustrative of the contents of the present invention, and the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those of ordinary skill in the art.

<실시예 1> 미토콘드리아 표적 STAT6 단백질<Example 1> Mitochondrial target STAT6 protein

도 1은 본 발명의 실시예 1에 따른 human STAT6 단백질에 관한 것으로, 상세하게는 human STAT6 단백질 (STAT6 wild type, STAT6 WT)을 구성하는 대략적인 도메인, 624-847번째 아미노산 서열이 삭제된 STAT6 (TAD-deleted STAT6, TAD D), 699-847번째 아미노산 서열이 삭제된 STAT6 (699-847 a.a-deleted STAT6, 699-847 D), 그리고 human STAT6 N-말단 서열(1-50번째 아미노산 서열)을 나타낸 것이다.1 is a human STAT6 protein according to Example 1 of the present invention, in detail, the approximate domain constituting the human STAT6 protein (STAT6 wild type, STAT6 WT), STAT6 in which the amino acid sequence at positions 624-847 has been deleted ( TAD-deleted STAT6, TAD D), STAT6 with the 699-847 amino acid sequence deleted (699-847 aa-deleted STAT6, 699-847 D), and human STAT6 N-terminal sequence (1-50 amino acid sequence) it has been shown

* STAT6 단백질의 서열:* Sequence of STAT6 protein:

MSLWGLVSKMPPEKVQRLYVDFPQHLRHLLGDWLESQPWEFLVGSDAFCCNLTSALLSDTVQHLQASVGEQGEGSTILQHISTLESIYQRDPLKLVATFRQILQGEKKAVMEQFRHLPMPFHWKQEELKFKTGLRRLQHRVGEIHLLREALQKGAEAGQVSLHSLIETPANGTGPSEALAMLLQETTGELEAAKALVLKRIQIWKRQQQLAGNGAPFEESLAPLQERCESLVDIYSQLQQEVGAAGGELEPKTRASLTGRLDEVLRTLVTSCFLVEKQPPQVLKTQTKFQAGVRFLLGLRFLGAPAKPPLVRADMVTEKQARELSVPQGPGAGAESTGEIINNTVPLENSIPGNCCSALFKNLLLKKIKRCERKGTESVTEEKCAVLFSASFTLGPGKLPIQLQALSLPLVVIVHGNQDNNAKATILWDNAFSEMDRVPFVVAERVPWEKMCETLNLKFMAEVGTNRGLLPEHFLFLAQKIFNDNSLSMEAFQHRSVSWSQFNKEILLGRGFTFWQWFDGVLDLTKRCLRSYWSDRLIIGFISKQYVTSLLLNEPDGTFLLRFSDSEIGGITIAHVIRGQDGSPQIENIQPFSAKDLSIRSLGDRIRDLAQLKNLYPKKPKDEAFRSHYKPEQMGKDGRGYVPATIKMTVERDQPLPTPELQMPTMVPSYDLGMAPDSSMSMQLGPDMVPQVYPPHSHSIPPYQGLSPEESVNVLSAFQEPHLQMPPSLGQMSLPFDQPHPQGLLPCQPQEHAVSSPDPLLCSDVTMVEDSCLSQPVTAFPQGTWIGEDIFPPLLPPTEQDLTKLLLEGQGESGGGSLGAQPLLQPSHYGQSGISMSHMDLRANLSW (서열번호 1)MSLWGLVSKMPPEKVQRLYVDFPQHLRHLLGDWLESQPWEFLVGSDAFCCNLTSALLSDTVQHLQASVGEQGEGSTILQHISTLESIYQRDPLKLVATFRQILQGEKKAVMEQFRHLPMPFHWKQEELKFKTGLRRLQHRVGEIHLLREALQKGAEAGQVSLHSLIETPANGTGPSEALAMLLQETTGELEAAKALVLKRIQIWKRQQQLAGNGAPFEESLAPLQERCESLVDIYSQLQQEVGAAGGELEPKTRASLTGRLDEVLRTLVTSCFLVEKQPPQVLKTQTKFQAGVRFLLGLRFLGAPAKPPLVRADMVTEKQARELSVPQGPGAGAESTGEIINNTVPLENSIPGNCCSALFKNLLLKKIKRCERKGTESVTEEKCAVLFSASFTLGPGKLPIQLQALSLPLVVIVHGNQDNNAKATILWDNAFSEMDRVPFVVAERVPWEKMCETLNLKFMAEVGTNRGLLPEHFLFLAQKIFNDNSLSMEAFQHRSVSWSQFNKEILLGRGFTFWQWFDGVLDLTKRCLRSYWSDRLIIGFISKQYVTSLLLNEPDGTFLLRFSDSEIGGITIAHVIRGQDGSPQIENIQPFSAKDLSIRSLGDRIRDLAQLKNLYPKKPKDEAFRSHYKPEQMGKDGRGYVPATIKMTVERDQPLPTPELQMPTMVPSYDLGMAPDSSMSMQLGPDMVPQVYPPHSHSIPPYQGLSPEESVNVLSAFQEPHLQMPPSLGQMSLPFDQPHPQGLLPCQPQEHAVSSPDPLLCSDVTMVEDSCLSQPVTAFPQGTWIGEDIFPPLLPPTEQDLTKLLLEGQGESGGGSLGAQPLLQPSHYGQSGISMSHMDLRANLSW (SEQ ID NO: 1)

<실시예 2> human STAT6 내부 서열 중, 미토콘드리아 표적서열의 위치 확인<Example 2> Confirmation of the location of the mitochondrial target sequence among human STAT6 internal sequences

1. 플라스미드(plasmid) 제작1. Plasmid construction

STAT6 N-말단서열 (1-50번째 아미노산 서열) 중에서, 미토콘드리아 표적서열로 작용할 것이라고 예측되는 서열들 (1-30번째 서열, 31-50번째 서열, 1-20번째 서열) C-말단에 각각 녹색 형광 단백질(Green fluorescent protein; GFP)을 결합시켜, 1-30-GFP, 31-50-GFP, 1-20-GFP 플라스미드를 제조하였다. 각 플라스미드의 구체적인 제조방법은 다음과 같다.Among the STAT6 N-terminal sequences (amino acid sequences 1-50), sequences predicted to act as mitochondrial target sequences (sequences 1-30, sequences 31-50, sequences 1-20) are green at the C-terminus, respectively. By binding to a fluorescent protein (Green fluorescent protein; GFP), 1-30-GFP, 31-50-GFP, and 1-20-GFP plasmids were prepared. The specific preparation method of each plasmid is as follows.

1) human STAT6 1-30-GFP 플라스미드 제작1) Construction of human STAT6 1-30-GFP plasmid

먼저, STAT6 wild type 플라스미드 (Origene)에서 하기 표 1의 서열번호 2, 3을 이용하여 PCR에 의해 증폭하여 human STAT6 N-말단 1-30번째 삽입서열 (서열번호 4)을 얻었다.First, the STAT6 wild type plasmid (Origene) was amplified by PCR using SEQ ID NOs: 2 and 3 in Table 1 to obtain human STAT6 N-terminal 1-30 insertion sequences (SEQ ID NO: 4).

서열번호 2SEQ ID NO: 2 AGATCTGCCGCCGCGATCGCCATGTCTCTGTGGGGTCTGGTCT (Forward) AGATCTGCCGCCGCGATCGCCATGTCTCTGTGGGGTCTGGTCT (Forward) 서열번호 3 SEQ ID NO: 3 GCGGCCGCGTACGCGTCAGAAGATGCCGCAGGTGT (Reverse) GCGGCCGCGTACGCGTCAGAAGATGCCGCAGGTGT (Reverse)

pCMV6-AC-GFP (Origene) 또한 제한효소 Sgf1 과 Mlu1으로 절단한 뒤, 절단된 서열 사이에 상기 1-30번째 삽입서열을 서브클로닝(subcloning) 하였다.pCMV6-AC-GFP (Origene) was also digested with restriction enzymes Sgf1 and Mlu1, and then the inserted sequences 1-30 were subcloned between the cut sequences.

완성된 1-30-GFP 플라스미드를 DH5α(RBC) 숙주세포로 형질전환시킨 뒤, 카나마이신(Kanamycin) 배지에 도말하여 37℃에서 17시간 배양하였다. 배양 후 자란 콜로니(colony)로부터 플라스미드 DNA를 추출하였다.The completed 1-30-GFP plasmid was transformed into DH5α (RBC) host cells, plated on kanamycin medium, and cultured at 37° C. for 17 hours. Plasmid DNA was extracted from colonies grown after culture.

* * human STAT6 N-말단 1-30번째 삽입서열:human STAT6 N-terminal insertions 1-30: MSLWGLVSKMPPEKVQRLYVDFPQHLRHLL (서열번호 4) MSLWGLVSKMPPEKVQRLYVDFPQHLRHLL (SEQ ID NO: 4)

2) human STAT6 31-50-GFP 플라스미드 제작2) Construction of human STAT6 31-50-GFP plasmid

STAT6 wild type 플라스미드 (Origene)에서 하기 표 2의 서열번호 5, 6을 이용하여 PCR에 의해 증폭하여 human STAT6 N-말단 31-50번째 삽입서열 (서열번호 7)을 얻었다. STAT6 wild type plasmid (Origene) was amplified by PCR using SEQ ID NOs: 5 and 6 in Table 2 to obtain human STAT6 N-terminal 31-50 insertion sequences (SEQ ID NO: 7).

서열번호 5SEQ ID NO: 5 AGATCTGCCGCCGCGATCGCCGGTGACTGGCTGGAGAGC (Forward) AGATCTGCCGCCGCGATCGCCGGTGACTGGCTGGAGAGC (Forward) 서열번호 6SEQ ID NO: 6 GCGGCCGCGTACGCGTGCAGCAGAAGGCGTC (Reverse) GCGGCCGCGTACGCGTGCAGCAGAAGGCGTC (Reverse)

pCMV6-AC-GFP (Origene) 또한 제한효소 Sgf1 과 Mlu1으로 절단한 뒤, 절단된 서열 사이에 상기 31-50번째 삽입서열을 서브클로닝 하였다.pCMV6-AC-GFP (Origene) was also digested with restriction enzymes Sgf1 and Mlu1, and the 31-50th insertion sequence was subcloned between the cut sequences.

완성된 31-50-GFP 플라스미드를 DH5α (RBC) 숙주세포로 형질전환시킨 뒤, 카나마이신(Kanamycin) 배지에 도말하여 37℃에서 17시간 배양하였다. 배양 후 자란 콜로니로부터 플라스미드 DNA를 추출하였다.The completed 31-50-GFP plasmid was transformed into DH5α (RBC) host cells, plated on kanamycin medium, and cultured at 37° C. for 17 hours. Plasmid DNA was extracted from colonies grown after culture.

* * human STAT6 N-말단 31-50번째 삽입서열:human STAT6 N-terminal 31-50 insertions: GDWLESQPWEFLVGSDAFCC (서열번호 7) GDWLESQPWEFLVGSDAFCC (SEQ ID NO: 7)

3) human STAT6 1-20-GFP 플라스미드 제작3) Construction of human STAT6 1-20-GFP plasmid

상기 1)의 human STAT6 1-30-GFP 플라스미드 제작 방법을 통해 얻은 1-30-GFP 플라스미드에서 하기 표 3의 서열번호 8, 9를 이용하여 human STAT6 N-말단 21-30번째 삽입서열 (서열번호 10)을 잘라내었다.In the 1-30-GFP plasmid obtained through the human STAT6 1-30-GFP plasmid construction method of 1) above, using SEQ ID NOs: 8 and 9 of Table 3 below, the human STAT6 N-terminal 21-30 insertion sequence (SEQ ID NO: 10) was cut out.

서열변호 8SEQ ID NO: 8 ACGCGTACGCGGCCGCTCGAGATGGAGAGCGACGA (Forward) ACGCGTACGCGGCCGCTCGAGATGGAGAGCGACGA (Forward) 서열번호 9SEQ ID NO: 9 GACATAGAGCCGCTGCACTTTTTCTGGGGGCATCTTGG (Reverse) GACATAGAGCCGCTGCACTTTTTCTGGGGGCATCTTGG (Reverse)

완성된 1-20-GFP 플라스미드를 DH5α (RBC) 숙주세포로 형질전환시킨 뒤, 카나마이신(Kanamycin) 배지에 도말하여 37℃에서 17시간 배양하였다. 배양 후 자란 콜로니로부터 플라스미드 DNA를 추출하였다.The completed 1-20-GFP plasmid was transformed into DH5α (RBC) host cells, plated on kanamycin medium, and cultured at 37° C. for 17 hours. Plasmid DNA was extracted from colonies grown after culture.

* * human STAT6 N-말단 1-20번째 삽입서열:human STAT6 N-terminal 1-20 insertions: MSLWGLVSKMPPEKVQRLYV (서열번호 10) MSLWGLVSKMPPEKVQRLYV (SEQ ID NO: 10)

2. human STAT6 1-30-GFP, 31-50-GFP, 1-20-GFP의 세포 내 분포 확인2. Confirmation of intracellular distribution of human STAT6 1-30-GFP, 31-50-GFP, and 1-20-GFP

상기 1의 방법으로 제작된 3종류의 플라스미드와 1종류의 대조군 GFP 플라스미드 (Origene)를 human U373-MG 악성 신경교종 (malignant glioma) 세포주와 mouse B16F10 흑색종 (melanoma) 세포주에 형질주입(transfection) 시켜서 각 GFP가 부착된 아미노산을 과발현 시켰다. The three types of plasmids and one type of control GFP plasmid (Origene) prepared by the method 1 above were transfected into human U373-MG malignant glioma cell line and mouse B16F10 melanoma cell line. Each GFP-attached amino acid was overexpressed.

형질주입 방법은 하기와 같다.The transfection method is as follows.

OPTI-MEM에 리포펙타민 3000 (lipofectamine 3000)을 섞은 후, 5분간 배양(incubation) 시켰다(A). 대조군 GFP 플라스미드, 1-30-GFP, 31-50-GFP, 1-20-GFP 플라스미드를 각각 따로 OPTI-MEM에 섞은 후, P3000 reagent를 추가로 섞고, 여기에 리포펙타민 3000과 섞어 배양한 (A)와 다시 혼합하여 15분간 배양시켰다.After mixing lipofectamine 3000 with OPTI-MEM, it was incubated for 5 minutes (A). Control GFP plasmid, 1-30-GFP, 31-50-GFP, and 1-20-GFP plasmid were separately mixed with OPTI-MEM, then P3000 reagent was additionally mixed, and cultured with Lipofectamine 3000 ( A) was mixed again and incubated for 15 minutes.

한국세포주은행으로부터 분양받은 human U373-malignant glioma 세포주 및 mouse B16F10 melanoma 세포주의 배양액을 OPTI-MEM으로 교환하고 상기의 혼합액으로 처리하여 48시간 배양하였다.The culture medium of human U373-malignant glioma cell line and mouse B16F10 melanoma cell line distributed from Korea Cell Line Bank was exchanged with OPTI-MEM, treated with the above mixture, and cultured for 48 hours.

48시간 후, PBS로 세척하고 4% 포름알데히드(formaldehyde)로 고정한 후, TOM20 항체로 염색하여 밤새 배양하였다. 그 뒤, PBS로 3번 세척하고, Alexa 546-Rb 2차 항체 (invitrogen)로 추가 염색하여, 2시간 상온에서 배양하였다. 끝나면 다시 PBS로 세척하여, 마운팅(mounting) 한 후 공초점 현미경으로 관찰하였다.After 48 hours, washed with PBS, fixed with 4% formaldehyde, stained with TOM20 antibody, and incubated overnight. Then, it was washed 3 times with PBS, and further stained with Alexa 546-Rb secondary antibody (invitrogen), and incubated at room temperature for 2 hours. When finished, it was washed again with PBS, mounted, and observed with a confocal microscope.

그 결과, 도 2에 나타난 바와 같이, human U373-MG 세포주의 미토콘드리아에서 human STAT6 1-30 아미노산 서열이 미토콘드리아에 존재함을 확인할 수 있다. 반면, human STAT6 31-50 아미노산 서열 또는 1-20 아미노산 서열은 미토콘드리아에 존재하지 못함을 확인할 수 있다.As a result, as shown in FIG. 2 , it can be confirmed that the human STAT6 1-30 amino acid sequence is present in the mitochondria of the human U373-MG cell line. On the other hand, it can be confirmed that the human STAT6 31-50 amino acid sequence or 1-20 amino acid sequence does not exist in the mitochondria.

또한, 도 3에 나타난 바와 같이, mouse B16F10 세포주의 미토콘드리아에서 human STAT6 1-30 아미노산 서열이 미토콘드리아에 존재함을 확인할 수 있는 반면, human STAT6 31-50 아미노산 서열 또는 1-20 아미노산 서열은 미토콘드리아에 존재하지 못함을 확인할 수 있다.In addition, as shown in Figure 3, it can be confirmed that the human STAT6 1-30 amino acid sequence is present in the mitochondria in the mouse B16F10 cell line, whereas the human STAT6 31-50 amino acid sequence or the 1-20 amino acid sequence is present in the mitochondria. You can check that you can't.

human STAT6 1-30 아미노산 서열이 인간 외 다른 종(species)의 미토콘드리아에도 위치한다는 사실은 마우스 등을 이용한 동물 실험에도 충분히 사용될 수 있다는 장점을 가진다.The fact that the human STAT6 1-30 amino acid sequence is also located in the mitochondria of species other than humans has the advantage that it can be sufficiently used for animal experiments using mice.

<실시예 3> 미토콘드리아 외막에 자리잡기 위한 STAT6 단백질의 막전위(transmembrane) 서열 확인<Example 3> Confirmation of the transmembrane sequence of the STAT6 protein for positioning in the outer mitochondrial membrane

도 1에 표기된 세 종류의 STAT6 단백질(STAT6 WT, TAD D, 699-847 D)이 미토콘드리아 외막에 위치하는 지를 알아보기 위하여, STAT6 단백질들을 각각 발현하는 안정적인 세포주(stable cell line)를 제작하였다. In order to determine whether the three types of STAT6 proteins (STAT6 WT, TAD D, 699-847 D) shown in FIG. 1 are located in the outer mitochondrial membrane, stable cell lines expressing each of the STAT6 proteins were prepared.

상기 제작된 세포주에서 미토콘드리아를 추출한 후, NaCl을 처리한 결과, 도 4와 같이, STAT6 WT 및 대표적인 미토콘드리아 외막 단백질인 TOM20, TOM40은 막에 존재하기 때문에 펠렛(Pellet, P)에서 검출이 되고, 대표적인 미토콘드리아 내부에 존재하는 soluble 단백질인 아코니타제 2(Aconitase 2) 및 시토크롬 c(Cytochrome c)는 상층액(Supernatant, S)에서 검출됨을 확인할 수 있다.After extracting mitochondria from the prepared cell line, as a result of treatment with NaCl, as shown in FIG. 4, STAT6 WT and TOM20 and TOM40, which are representative mitochondrial outer membrane proteins, are present in the membrane, so they are detected in the pellet (Pellet, P) and representative It can be confirmed that aconitase 2 and cytochrome c, which are soluble proteins present in the mitochondria, are detected in the supernatant (Supernatant, S).

그러나 624-847번째 아미노산 서열이 삭제된 STAT6 (TAD-deleted STAT6)는 S에서 검출이 되었고, 699-847번째 아미노산 서열이 삭제된 STAT6 (699-847-deleted STAT6, TAD-deleted STAT6로부터 일부 서열이 회복됨)는 다시 P에서 검출되는 것으로 나타났다. 이는 STAT6의 624-698번째 아미노산 서열이 미토콘드리아 외막에 존재하기 위한 transmembrane 역할을 하는 것으로 사료된다.However, STAT6 (TAD-deleted STAT6) with the 624-847 amino acid sequence deleted was detected in S, and some sequences from STAT6 (699-847-deleted STAT6, TAD-deleted STAT6) with the 699-847 amino acid sequence deleted. recovered) was again detected in P. It is thought that the 624-698 amino acid sequence of STAT6 acts as a transmembrane to exist in the outer mitochondrial membrane.

* 624-698번째 아미노산 서열:* amino acid sequence at positions 624-698:

AFRSHYKPEQMGKDGRGYVPATIKMTVERDQPLPTPELQMPTMVPSYDLGMAPDSSMSMQLGPDMVPQVYPPHSH (서열번호 11)AFRSHYKPEQMGKDGRGYVPATIKMTVERDQPLPTPELQMPTMVPSYDLGMAPDSSMSMQLGPDMVPQVYPPHSH (SEQ ID NO: 11)

<실시예 4> 미토콘드리아 외막에 존재하는 STAT6 단백질의 영향 확인<Example 4> Confirmation of the effect of STAT6 protein present in the outer mitochondrial membrane

미토콘드리아 외막에 존재하는 STAT6 단백질의 영향을 확인하기 위하여, 먼저 도 5와 같이, human STAT6 단백질 (STAT6 WT)과 미토콘드리아 표적 서열이 삭제된 STAT6 (MTS D)의 C-말단에 mCherry 형광을 부착시켰다. 미토콘드리아 표지는 미토콘드리아 외막에 존재하는 TOM20 단백질로 염색시켰다.In order to confirm the effect of STAT6 protein present on the outer mitochondrial membrane, mCherry fluorescence was first attached to the C-terminus of human STAT6 protein (STAT6 WT) and STAT6 (MTS D) in which the mitochondrial target sequence was deleted, as shown in FIG. Mitochondrial labeling was stained with TOM20 protein present in the outer mitochondrial membrane.

상기 두 종류의 STAT6 단백질을 각각 293T 세포에 과발현시킨 결과, 도 6에 나타난 바와 같이, STAT6 WT의 과발현은 미토콘드리아의 절편화(fragmentation)를 유발하지만, 미토콘드리아 표적 서열이 삭제 (MTS D)되면 미토콘드리아에 타겟하지 못할 뿐만 아니라, 미토콘드리아 형태가 원래대로 회복되는 것을 확인할 수 있었다.As a result of overexpression of the two types of STAT6 proteins in 293T cells, respectively, as shown in FIG. 6 , overexpression of STAT6 WT induces mitochondrial fragmentation, but mitochondrial target sequence deletion (MTS D) results in mitochondrial overexpression. Not only could it not be targeted, but it was also confirmed that the mitochondrial shape was restored to its original state.

<실시예 5> 미토콘드리아 외막 단백질로서의 STAT6가 종양 형성에 미치는 영향 확인<Example 5> Confirmation of the effect of STAT6 as a mitochondrial outer membrane protein on tumor formation

미토콘드리아 외막 단백질로서의 STAT6에 의한 미토콘드리아 절편화 효과가 실제로 어떠한 영향을 미칠 수 있는지에 대해 알아보기 위하여, 미토콘드리아 기능이상으로부터 유래하는 다양한 질병 중, 우선 대표적으로 암-종양 형성에 대해 영향을 주는지 확인하였다.In order to investigate how the mitochondrial fragmentation effect by STAT6 as a mitochondrial outer membrane protein can actually affect, among various diseases derived from mitochondrial dysfunction, it was first confirmed whether it typically affects cancer-tumor formation.

293T 세포주에 CRISPR Cas9을 이용하여 STAT6 발현을 억제시킨 stable cell line (STAT6 KO)을 제조한 후 (도 7), 293T Parental 세포와 STAT6 KO stable cell line을 각각 쥐의 왼쪽다리, 오른쪽 다리에 동시에 이식하여, 종양이 자라는 것을 관찰하였다. 이식 후, 3일에 한 번씩 종양이 자라는 크기를 측정한 결과, 도 8에 나타난 바와 같이, STAT6 발현이 억제된 세포에서 종양 성장이 촉진됨을 확인할 수 있었다 (****, P<0.0001; n= 18).After preparing a stable cell line (STAT6 KO) in which STAT6 expression was suppressed using CRISPR Cas9 in the 293T cell line (FIG. 7), 293T parental cells and STAT6 KO stable cell line were simultaneously transplanted into the left and right legs of mice, respectively. Thus, the growth of the tumor was observed. As a result of measuring the size of tumor growth once every 3 days after transplantation, as shown in FIG. 8 , it was confirmed that tumor growth was promoted in cells in which STAT6 expression was suppressed (****, P<0.0001; n = 18).

또한, 상기 세포주 이식 후 1달 뒤, 쥐에서 종양 조직을 적출하여 종양의 크기를 비교하였다. 그 결과, 도 9 및 도 10에 나타난 바와 같이, STAT6 발현이 억제된 세포의 경우, 293T Parental 세포의 경우보다 종양의 크기가 크고 종양의 무게가 많이 증가한 것으로 나타났다. 즉, STAT6 발현 억제는 종양 형성 촉진을 유도함을 확인할 수 있다.In addition, one month after transplantation of the cell line, tumor tissues were extracted from mice and the size of the tumor was compared. As a result, as shown in FIGS. 9 and 10 , in the case of the cells in which STAT6 expression was suppressed, the size of the tumor was larger and the weight of the tumor was significantly increased than in the case of the 293T parental cells. That is, it can be confirmed that inhibition of STAT6 expression induces tumorigenesis promotion.

상기 제조된 STAT6 KO stable cell line에 다시 human STAT6 단백질 (STAT6 WT)과 미토콘드리아 표적 서열이 삭제된 STAT6 (MTS D)를 각각 과발현시켜서 도 11과 같이 stable cell line을 제작하였다.In the STAT6 KO stable cell line prepared above, human STAT6 protein (STAT6 WT) and STAT6 (MTS D) from which the mitochondrial target sequence was deleted were overexpressed, respectively, to construct a stable cell line as shown in FIG. 11 .

상기 제조된 STAT6 KO stable cell line을 쥐의 왼쪽 다리에 이식하고, human STAT6 단백질 (STAT6 WT)을 다시 과발현시킨 stable cell line을 오른쪽 다리에 이식하여, 1달 동안 종양이 자라는 것을 관찰한 결과, 도 12에 나타난 바와 같이, STAT6 KO 세포를 이식한 경우보다, STAT6 WT를 다시 과발현 시킨 세포를 이식한 경우에서 종양의 크기가 현저히 작아진 것을 확인할 수 있었다.The prepared STAT6 KO stable cell line was transplanted into the left leg of a mouse, and the stable cell line overexpressing human STAT6 protein (STAT6 WT) was transplanted into the right leg, and tumor growth was observed for 1 month. As shown in Fig. 12, it was confirmed that the size of the tumor was significantly smaller in the case of transplanting the cells overexpressing STAT6 WT again than in the case of transplanting the STAT6 KO cells.

또한, 상기 제조된 STAT6 KO stable cell line을 쥐의 왼쪽 다리에 이식하고, 미토콘드리아 표적 서열이 삭제된 STAT6 (MTS D)를 다시 과발현시킨 stable cell line을 오른쪽 다리에 이식하여, 1달 동안 종양이 자라는 것을 관찰한 결과, 도 13에 나타난 바와 같이, 두 경우 모두 비슷한 크기로 종양이 형성되는 것으로 나타났다.In addition, the STAT6 KO stable cell line prepared above was transplanted into the left leg of a mouse, and a stable cell line re-expressing STAT6 (MTS D) with the mitochondrial target sequence deleted was transplanted into the right leg, and the tumor was grown for 1 month. As a result of observing that, as shown in FIG. 13 , in both cases, it was found that tumors of similar size were formed.

상기 세 가지 stable cell line을 이식한 후 3일에 한 번씩 종양이 자라는 크기를 측정하고, 1달 뒤에는 쥐에서 종양을 적출하여 무게 측정하여 그래프로 나타내었다. After transplanting the three stable cell lines, the size of the tumor was measured once every 3 days, and after 1 month, the tumor was excised from the mouse and weighed, and the graph was shown.

그 결과, 도 14 및 도 15에 나타난 바와 같이, STAT6 KO와 MTS D의 경우에는 종양 형성이 촉진되었고, STAT6 WT의 경우에는 종양 형성이 현저히 억제됨을 확인할 수 있었다.As a result, as shown in FIGS. 14 and 15 , in the case of STAT6 KO and MTS D, tumor formation was promoted, and in the case of STAT6 WT, it was confirmed that tumor formation was significantly inhibited.

즉, STAT6 발현을 knock out 시킨 후, 다시 STAT6 WT을 과발현시키면 종양 형성이 억제되고, 반대로 STAT6 발현을 knock out 시킨 후, 다시 미토콘드리아 표적 서열이 삭제된 STAT6를 과발현시키면 종양 형성이 다시 촉진되는 바, STAT6의 종양 형성 억제 효과는 미토콘드리아 외막에 존재하는 STAT6 때문임을 확인할 수 있다. That is, after knocking out STAT6 expression and overexpressing STAT6 WT again, tumor formation was inhibited. Conversely, when STAT6 expression was knocked out and STAT6 was overexpressed again with the mitochondrial target sequence deleted, tumor formation was promoted again. It can be confirmed that the tumor formation inhibitory effect of STAT6 is due to STAT6 present in the outer mitochondrial membrane.

이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적 기술은 단지 바람직한 실시양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백하다. 즉, 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다.As described above in detail a specific part of the content of the present invention, for those of ordinary skill in the art, it is clear that this specific description is only a preferred embodiment, and the scope of the present invention is not limited thereby. do. That is, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims (21)

서열번호 4의 아미노산 서열로 이루어진 미토콘드리아 표적용 펩타이드.A mitochondrial targeting peptide consisting of the amino acid sequence of SEQ ID NO: 4. 서열번호 4의 아미노산 서열을 코딩하는 유전자를 포함하는 재조합 벡터.A recombinant vector comprising a gene encoding the amino acid sequence of SEQ ID NO: 4. 제 2 항에 있어서,3. The method of claim 2, 상기 재조합 벡터는,The recombinant vector is 프로모터, 상기 유전자 및 목적 단백질을 코딩하는 유전자가 작동 가능하게 연결된 것을 특징으로 하는 재조합 벡터.A recombinant vector, characterized in that the promoter, the gene and the gene encoding the target protein are operably linked. 제 2 항 또는 제 3 항에 따른 재조합 벡터를 세포 내 도입하는 단계를 포함하는 목적 단백질을 미토콘드리아로 이동 또는 분포시키는 방법.A method for moving or distributing a target protein to mitochondria comprising the step of introducing the recombinant vector according to claim 2 or 3 into a cell. 프로모터, 서열번호 4의 아미노산 서열을 코딩하는 유전자, 목적 단백질을 코딩하는 유전자 및 형광 단백질을 코딩하는 유전자를 포함하는 재조합 벡터를 세포 내 도입하는 단계; 및 Introducing a recombinant vector comprising a promoter, a gene encoding the amino acid sequence of SEQ ID NO: 4, a gene encoding a target protein, and a gene encoding a fluorescent protein into a cell; and 상기 유전자를 발현시켜 형광 영상을 통해 상기 발현되는 목적 단백질의 이동 또는 분포를 확인하는 단계;를 포함하는 세포 내 목적 단백질의 미토콘드리아로 이동 또는 분포를 확인하는 방법.A method of confirming the movement or distribution of a target protein in a cell to the mitochondria, comprising the step of expressing the gene and confirming the movement or distribution of the expressed target protein through a fluorescence image. 제 1 항에 따른 미토콘드리아 표적용 펩타이드를 유효성분으로 함유하는 미토콘드리아 약물 전달용 담체.A carrier for mitochondrial drug delivery containing the mitochondrial targeting peptide according to claim 1 as an active ingredient. 제 1 항에 따른 미토콘드리아 표적용 펩타이드 및 약리학적 활성 물질이 결합된 복합체를 유효성분으로 포함하는 미토콘드리아 기능 장애 관련 질환 예방 또는 치료용 약학 조성물.A pharmaceutical composition for preventing or treating mitochondrial dysfunction-related diseases, comprising the complex to which the mitochondrial targeting peptide according to claim 1 and a pharmacologically active substance are bound as an active ingredient. 제 7 항에 있어서,8. The method of claim 7, 상기 약리학적 활성 물질은,The pharmacologically active substance is 천연물, 화합물, 단백질 또는 유전자인 것을 특징으로 하는 미토콘드리아 기능 장애 관련 질환 예방 또는 치료용 약학 조성물.A pharmaceutical composition for preventing or treating mitochondrial dysfunction-related diseases, characterized in that it is a natural product, compound, protein or gene. 제 8 항에 있어서,9. The method of claim 8, 상기 화합물은,The compound is 독소루비신(doxorubicin), 에피루비신(epirubicin), 이다루비신(idarubicin), 도세탁셀(docetaxel), 파클리탁셀(paclitaxel), 카바지탁셀(cabazitaxel), 빈블라스틴(vinblastine), 빈크리스틴(vincristine) 및 시스플라틴(cisplain)으로 이루어진 군에서 선택되는 하나 이상인 것을 특징으로 하는 미토콘드리아 기능 장애 관련 질환 예방 또는 치료용 약학 조성물.doxorubicin, epirubicin, idarubicin, docetaxel, paclitaxel, cabazitaxel, vinblastine, vincristine and cisplatin cisplain) a pharmaceutical composition for preventing or treating mitochondrial dysfunction-related diseases, characterized in that at least one selected from the group consisting of. 프로모터, 서열번호 4의 아미노산 서열을 코딩하는 유전자 및 목적 단백질을 코딩하는 유전자가 작동 가능하게 연결된 재조합 벡터를 유효성분으로 포함하는 미토콘드리아 기능 장애 관련 질환 예방 또는 치료용 약학 조성물.A pharmaceutical composition for preventing or treating diseases related to mitochondrial dysfunction comprising a promoter, a gene encoding the amino acid sequence of SEQ ID NO: 4, and a recombinant vector to which a gene encoding a target protein is operably linked as an active ingredient. 제 7 항 내지 제 10 항 중 어느 한 항에 있어서,11. The method according to any one of claims 7 to 10, 상기 미토콘드리아 기능 장애 관련 질환은,The mitochondrial dysfunction-related disease is, 허혈성 뇌질환 또는 허혈성 심질환, 뇌졸증, 편두통, 빈혈증, 파킨슨병, 알츠하이머, 근위축성측색경화증(ALS), 미토콘드리아뇌근증, 간경변, 판코니 증후군, 비정형 페닐케톤뇨증, 천식, 당뇨병, 동맥경화, 고혈압, 고콜레스테롤증, 시신경 질환, 비만, 우울증 및 암 질환으로 이루어진 군에서 선택되는 어느 하나인 것을 특징으로 하는 미토콘드리아 기능 장애 관련 질환 예방 또는 치료용 약학 조성물.Ischemic encephalopathy or ischemic heart disease, stroke, migraine, anemia, Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), mitochondrial encephalomyopathy, liver cirrhosis, Fanconi syndrome, atypical phenylketonuria, asthma, diabetes, arteriosclerosis, hypertension, high blood pressure A pharmaceutical composition for preventing or treating mitochondrial dysfunction-related diseases, characterized in that any one selected from the group consisting of cholesterol, optic nerve disease, obesity, depression and cancer disease. 미토콘드리아 내부 표적 도메인 및 미토콘드리아 막 투과 도메인을 포함하는 STAT6 (Signal transducer and activator of transcription 6) 단백질을 유효성분으로 함유하는 미토콘드리아 기능 장애 관련 질환 예방 또는 치료용 약학 조성물.A pharmaceutical composition for preventing or treating diseases related to mitochondrial dysfunction, comprising as an active ingredient a STAT6 (Signal transducer and activator of transcription 6) protein comprising a mitochondrial internal target domain and a mitochondrial membrane permeation domain. 제 12 항에 있어서,13. The method of claim 12, 상기 미토콘드리아 내부 표적 도메인은,The mitochondrial internal target domain, 서열번호 4로 표시되는 아미노산 서열로 이루어진 것을 특징으로 하는 미토콘드리아 기능 장애 관련 질환 예방 또는 치료용 약학 조성물.A pharmaceutical composition for preventing or treating mitochondrial dysfunction-related diseases, characterized in that it consists of the amino acid sequence represented by SEQ ID NO: 4. 제 12 항에 있어서,13. The method of claim 12, 상기 미토콘드리아 막 투과 도메인은,The mitochondrial membrane permeation domain, 서열번호 11로 표시되는 아미노산 서열로 이루어진 것을 특징으로 하는 미토콘드리아 기능 장애 관련 질환 예방 또는 치료용 약학 조성물.A pharmaceutical composition for preventing or treating mitochondrial dysfunction-related diseases, characterized in that it consists of the amino acid sequence represented by SEQ ID NO: 11. 제 12 항에 있어서,13. The method of claim 12, 상기 미토콘드리아 막 투과 도메인은,The mitochondrial membrane permeation domain, 상기 미토콘드리아 내부를 표적하는 STAT6 단백질을 미토콘드리아 외막에 위치시키는 것을 특징으로 하는 미토콘드리아 기능 장애 관련 질환 예방 또는 치료용 약학 조성물.A pharmaceutical composition for preventing or treating mitochondrial dysfunction-related diseases, characterized in that the STAT6 protein targeting the inside of the mitochondria is located in the outer mitochondrial membrane. 제 12 항에 있어서13. The method of claim 12 상기 STAT6 단백질은,The STAT6 protein is 미토콘드리아 외막에 축적될 때, 미토콘드리아의 절편화(fragmentation)를 유도하는 것을 특징으로 하는 미토콘드리아 기능 장애 관련 질환 예방 또는 치료용 약학 조성물.When it accumulates in the outer mitochondrial membrane, a pharmaceutical composition for preventing or treating mitochondrial dysfunction-related diseases, characterized in that it induces fragmentation of mitochondria. 제 12 항에 있어서13. The method of claim 12 상기 STAT6 단백질은,The STAT6 protein is 미토콘드리아 외막에 축적될 때, 종양 형성을 억제시키는 것을 특징으로 하는 미토콘드리아 기능 장애 관련 질환 예방 또는 치료용 약학 조성물.A pharmaceutical composition for preventing or treating mitochondrial dysfunction-related diseases, characterized in that it inhibits tumor formation when it accumulates in the outer mitochondrial membrane. 제 12 항에 있어서,13. The method of claim 12, 상기 STAT6 단백질은,The STAT6 protein is 서열번호 1로 표시되는 아미노산 서열로 이루어진 것을 특징으로 하는 미토콘드리아 기능 장애 관련 질환 예방 또는 치료용 약학 조성물.A pharmaceutical composition for preventing or treating mitochondrial dysfunction-related diseases, characterized in that it consists of the amino acid sequence represented by SEQ ID NO: 1. 제 12 항에 있어서,13. The method of claim 12, 상기 미토콘드리아 기능 장애 관련 질환은,The mitochondrial dysfunction-related disease is, 허혈성 뇌질환 또는 허혈성 심질환, 뇌졸증, 편두통, 빈혈증, 파킨슨병, 알츠하이머, 근위축성측색경화증(ALS), 미토콘드리아뇌근증, 간경변, 판코니 증후군, 비정형 페닐케톤뇨증, 천식, 당뇨병, 동맥경화, 고혈압, 고콜레스테롤증, 시신경 질환, 비만, 우울증 및 암 질환으로 이루어진 군에서 선택되는 어느 하나인 것을 특징으로 하는 미토콘드리아 기능 장애 관련 질환 예방 또는 치료용 약학 조성물.Ischemic encephalopathy or ischemic heart disease, stroke, migraine, anemia, Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), mitochondrial encephalomyopathy, liver cirrhosis, Fanconi syndrome, atypical phenylketonuria, asthma, diabetes, arteriosclerosis, hypertension, high blood pressure A pharmaceutical composition for preventing or treating mitochondrial dysfunction-related diseases, characterized in that any one selected from the group consisting of cholesterol, optic nerve disease, obesity, depression and cancer disease. 미토콘드리아 내부 표적 도메인 및 미토콘드리아 막 투과 도메인을 포함하는 STAT6 (Signal transducer and activator of transcription 6) 단백질을 유효성분으로 함유하는 미토콘드리아 기능 장애 관련 질환 예방 또는 개선용 건강기능식품 조성물.A health functional food composition for preventing or improving mitochondrial dysfunction-related diseases, comprising, as an active ingredient, a STAT6 (Signal transducer and activator of transcription 6) protein comprising a mitochondrial internal target domain and a mitochondrial membrane permeation domain. 제 20 항에 있어서,21. The method of claim 20, 상기 미토콘드리아 기능 장애 관련 질환은,The mitochondrial dysfunction-related disease is, 허혈성 뇌질환 또는 허혈성 심질환, 뇌졸증, 편두통, 빈혈증, 파킨슨병, 알츠하이머, 근위축성측색경화증(ALS), 미토콘드리아뇌근증, 간경변, 판코니 증후군, 비정형 페닐케톤뇨증, 천식, 당뇨병, 동맥경화, 고혈압, 고콜레스테롤증, 시신경 질환, 비만, 우울증 및 암 질환으로 이루어진 군에서 선택되는 어느 하나인 것을 특징으로 하는 미토콘드리아 기능 장애 관련 질환 예방 또는 개선용 건강기능식품 조성물.Ischemic encephalopathy or ischemic heart disease, stroke, migraine, anemia, Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), mitochondrial encephalomyopathy, liver cirrhosis, Fanconi syndrome, atypical phenylketonuria, asthma, diabetes, arteriosclerosis, hypertension, high blood pressure A health functional food composition for preventing or improving mitochondrial dysfunction-related diseases, characterized in that any one selected from the group consisting of cholesterol, optic nerve disease, obesity, depression and cancer disease.
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