KR20120121754A - A Composition for Inducing Cell Autophagy - Google Patents

Abstract

PURPOSE: A composition containing DKK3 protein or a polynucleotide encoding the protein for inducing cell autophagy is provided to treat or relieve diseases caused by abnormal cells or proteins. CONSTITUTION: A composition for inducing cell autophagy contains DKK3 protein or a polynucleotide encoding the protein as an active ingredient. DKK3 includes autophagosome formulation in cells. The protein or polynucleotide is derived from human. The protein has an amino acid sequence of sequence number 1. The polynucleotide encoding DKK3 has a nucleic acid sequence of sequence number 2. A polynucleotide encoding DKK3 contains an expression vector for intracellular transduction. A pharmaceutical composition for treating diseases related to autophagy contains the protein or polynucleotide as an active ingredient.

Description

A composition for inducing cell autophagy

The present invention relates to a composition for inducing self-extinguishing of cells, and more specifically, to the present invention is directed to inducing self-extinguishing of cells containing a Dikopf 3 (Dickkopf 3, DKK3) protein or a polynucleotide encoding the same as an active ingredient. The present invention relates to a composition and a pharmaceutical composition capable of treating diseases related to autophagy disorders comprising a Dickkopf 3 (DKK3) protein or a polynucleotide encoding the same as an active ingredient. The present invention also relates to a method for screening a substance that upregulates or downregulates autophagy of a cell using the interaction of a DKK3 polypeptide and a Becklin-1 polypeptide.

Apoptosis of eukaryotic cells is largely manifested by apoptosis, autophagy and necrosis, depending on three mechanisms: morphological and biochemical characteristics (Cell Death Differ. 12 Suppl 2: 1463). -1467, 2005). Among these, autophagy is defined as a catabolic process of autophagosomic-lysosomal protein degradation that is activated under certain circumstances, which is usually triggered under conditions of nutrient deprivation, but also develops and differentiates. , Neurodegenerative diseases, infections and other physiological processes (Reggiori, F. et al. (2002) Eukaryot. Cell 1, 11-21; Codogno, P. et al. (2005) Cell) Death Differ. 12 Suppl 2, 1509-1518; Levine, B. et al. (2005) J. Clin. Invest. 115, 2679-2688). The apoptosis caused by autophagy is classified as type II programmed cell death through a different pathway from apoptosis classified as type 1 apoptosis, and is classified as nutrient starvation or environment. Increased by stress or various compounds, the process results in a double-membrane structure derived from or newly synthesized endoplasmic reticulum (ER), which isolates intracellular organelles (such as mitochondria) and proteins in the cytoplasm. Autophagosomes, which eventually fuse and break down with lysosomes (Nat. Rev. Cancer., 5: 726-734, 2005). If autophagy is above a certain level, apoptosis is induced, and the development of cancer treatments using apoptosis by autophagy is currently drawing attention (Clinical Science, 116: 697-712, 2009).

Autodigestion consists of three or more intracellular proteolytic pathways found anywhere in eukaryotic cells: macroautophagy, microautophagy and chaperone mediated autodigestion. During the macrophages, cytoplasmic material, including organelles, is sequestered into a double membrane covered autophagosome, which is then fused with endosomes and lysosomes. The isolated autophagosome content is then degraded by lysosomal hydrolase and the degradation product is recycled by the cells.

As proteins involved in such autophagy, beclin-1, LC3, mTOR proteins and the like are known. Beclin-1 (BECN1) is one of the important proteins involved in autophagy and is the mammalian orthologue of the yeast cell destructive protein Apg6 / Vps30 (Kametaka, S. et al. (1998) J. Biol) Chem. 273, 22284-22291). Beclin-1 can compensate for the deficiency of autophagy in yeast resulting from the deletion of Apg6 and can cause autophagy when overexpressed in mammalian cells (Liang, XH et al. (1999) Nature 402, 672-676). Mammalian Beclin-1 was originally identified by yeast-to-hybrid screening for proteins that interact with Bcl-2 and with Bax or Bak, which interacts with Bcl-2 and Bcl-xL but is involved in the apoptosis pathway It has been demonstrated to not work (Liang, XH et al, (1998) J. Virol. 72, 8586-8596).

Beclin-1 is commonly expressed in a variety of cells and is ubiquitous in cytoplasmic structures, including mitochondria, but exhibits some nuclear staining and CRM1-dependent nuclear release upon overexpression of beclin-1 (Liang, XH et al. 2001) Cancer Res. 6t1, 3443-3449). Overexpression of beclin-1 in virally infected neurons in vivo resulted in significant protection against Sindbis virus-induced disease and neuronal apoptosis (Liang, XH et al, (1998) J. Virol. 72 , 8586-8596). Light Chain 3 (LC3) is a protein that increases in autodigestion and was originally identified as a subunit of microtubule-associated proteins 1A and 1B (named MAP1LC3) (Mannm SS and Hammarback, JA (1994) J. Biol. Chem. 269, 11492-11497), followed by the yeast protein Apg8 / Aut7 / Cvt5, which plays an important role in autodigestion (Lang, T et al. (1998) EMBO J. 17, 3597 -3607). In humans, three isoforms of LC3 are known, LC3A, LC3B, and LC3C, which undergo post-transcriptional modifications during the autophagy process. LC3 produces a cytoplasmic form LC3-I through the first cleavage of the carboxy terminus immediately following its synthesis and during autodigestion LC3-I is converted to LC3-II due to the ubiquitin-like system involving Apg7 and Apg3 LC3 is linked to autophagosomes. Thus, the presence of LC3 in autophagosomes and the conversion of LC3 to LC3-II, a lower transfer form, are used as indicators of autophagy. The mammlian target of rapamycin (mTOR) is also known as FRAP or RAFP (Sabers, CJ et al. (1995) J. Biol. Chem. 270, 815-822; Brown, EJ et al. (1994) Nature 369, 756 -758, Sabatini, DM et al. (1994) Cell 78, 35-43), Ser / Thr kinase. mTOR is known to respond to changes in ATP and amino acids and to balance nutrient availability and cell growth (Gingras, AC et al. (2001) Genes Dev. 15, 807-826; Dennis, PB et al. (2001) Science 294, 1102-1105). When sufficient nutrients are available, mTOR responds with a phosphatidic acid-mediated signal (Fang, Y. et al. (2001) Science 294, 1942-1945), delivers a positive signal with p70 S6 kinase and 4E, an eIF4E inhibitor Is involved in inactivation of BP1 resulting in translation of certain mRNA groups. mTOR is phosphorylated to Ser2448 via the PI3 kinase / Akt signaling pathway or spontaneously to Ser2481 to produce p-mTOR (Nave, BT et al. (1999) Biochem. J. 344 Pt2, 427-431; Peterson, RT et al. (2000) J. Biol. Chem. 275, 7416-7423). mTOR plays a key role in cell growth and homeostasis, and cell death is known to be inhibited by mTOR.

On the other hand, key regulators of apoptosis include members of the Bcl-2 protein family (Farrow, SN and Brown, R. Curr. Opin. Gen. Dev., 1996, 6: 45-49). The Bcl-2 protein family consists of pro- and anti-self-killing protein family members that act at various levels of self-killing to control self-killing. The Bcl-2 family members comprise one or more Bcl-2 homology (BH) sites (Farrow, SN and Brown, R. Curr. Opin. Gen. Dev., 1996, 6: 45-49). Overexpression of the bcl-2 (B cell lymphoma / leukemia-2) gene may be associated with tumorigenicity (Tsujimoto et al., 1985, Science 228: 1440-1443). The bcl- 2 gene is thought to induce resistance to pathology and treatment of cancer by prolonging cell survival rather than promoting cell differentiation. The human bcl-2 gene is involved in the pathogenesis of certain leukemias, lymphocytic tumors, lymphomas, neuroblastomas and nasopharynx, prostate, breast, colon carcinoma, so non-Hopkinson's lymphoma, lung cancer, breast cancer, colon cancer, prostate cancer, rectal cancer and It has been shown to be overexpressed in various tumors, including acute and chronic leukemia.

It is known that Bcl-2 also affects autophagy. For example, International Patent Publication No. WO06 / 082303 discloses a motif of a Becklin protein that binds to a Bcl-2 protein, a vector comprising a nucleic acid sequence encoding the motif, a transformant transformed with the vector, produced therefrom. Peptides and methods for inducing apoptosis- and offspring-form apoptosis by administering the peptides to cancer patients are disclosed.

As described above, a cell (BCL-2) related to apoptosis that induces apoptosis of eukaryotic cells and a protein (Beclin-1) that participates in autophagy interact with the cell through autophagy. It is known to be involved in death, but the method of controlling its mechanism of action has not been reported yet. Unlike apoptosis, which requires specific conditions, autodigestion can be sufficiently induced by conditions of depletion, so if you can control the induction of autodigestion, you can treat various diseases caused by abnormal cells and proteins. It is expected to be easier to perform, but the results of the research has not been reported so far.

The present inventors have made diligent research efforts to develop a method for controlling the induction of autophagy, and as a result, the use of Dickkopf 3 (DKK3) can inhibit the interaction of BCL-2 with Becklin-1. Accordingly, it was confirmed that the formation of autophagosomes could be promoted, thereby inducing autophagy, and thus the present invention was completed.

It is a main object of the present invention to provide a composition for inducing self-extinguishing of cells comprising Dickkopf 3 (DKK3) or a polynucleotide encoding the same as an active ingredient.

Another object of the present invention is to provide a pharmaceutical composition capable of treating diseases related to autophagy disorders comprising the DKK3 or a polynucleotide encoding the same as an active ingredient.

Still another object of the present invention is to provide a method for screening a substance that up- or down-regulates autophagy of cells by using the interaction of DKK3 and Becklin-1.

According to one embodiment for achieving the above object, the present invention provides a composition for inducing self-extinguishing of a cell comprising a Dikopkop 3 (DKK3) protein or a polynucleotide encoding the same as an active ingredient. Induction of autophagy of the cells can be in vivo or ex vivo.

The term "Dickkopf 3 (DKK3)", as used herein, refers to one family of genes that encode secreted glycoproteins that regulate cell fate during embryonic development, also known as reduced expression in immortalized cells (REIC).

Dicop-3 protein in the present invention is a concept including a DKK3 protein derived from various mammals, including humans, variants or functional fragments that maintain functional equivalents thereof. As used herein, "functionally equivalent variant" refers to a functional equivalent having a function compared with wild-type protein derived from a specific species or a gene sequence thereof, but having a function capable of inducing cell self-extinguishing by binding to beclin-1 protein. The DKK3 protein that can be used in the present invention is preferably DKK3 derived from humans, more preferably DKK3 protein (GenBank Accesstion No. AAQ88744.1) consisting of the amino acid sequence of SEQ ID NO: 1, but is not limited thereto.

DKK3 proteins of the present invention are also within the scope of the present invention as well as proteins having their native amino acid sequences, as well as amino acid sequence variants thereof. A variant of the DKK3 protein refers to a protein in which the DKK3 native amino acid sequence and one or more amino acid residues have different sequences by deletion, insertion, non-conservative or conservative substitution, or a combination thereof. Amino acid exchanges in proteins and peptides that do not alter the activity of the molecule as a whole are known in the art (H. Neurode, R. L. Hill, The Proteins, Academic Press, New York, 1979). The most commonly occurring exchanges involve amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu and Asp / Gly. In some cases, it may be modified by phosphorylation, sulfation, acetylation, glycosylation, methylation, farnesylation, or the like. Such variants include proteins having functional equivalents with substantially equivalent activity or modifications that increase or decrease physicochemical properties. Variants with altered physicochemical properties include, for example, physical factors such as temperature, moisture, pH, electrolytes, reducing sugars, pressurization, drying, freezing, interfacial tension, light, repetition of freezing and thawing, and high concentration conditions. It is a variant with increased structural stability to the external environment of chemical factors such as neutral salts, organic solvents, metal ions, redox agents and proteases.

The DKK3 protein or variant thereof can be isolated or synthesized in nature (Merrifleld, J. Amer. Chem. Soc .. 85: 2149-2156, 1963) or prepared by genetic recombination methods based on DNA sequences (Sambrook). et al, Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, USA, 2nd edition, 1989).

In addition, the polynucleotide encoding the DKK3 protein in the present invention means a nucleic acid molecule encoding the DKK3 protein, these may be isolated or artificially modified synthetically in nature. The polynucleotide may be a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2 (GenBank Accesstion No. AY358378.1), but is not limited thereto.

Polynucleotides that can be used as polynucleotides encoding the DKK3 protein of the present invention are characterized by deletion, substitution or insertion of functional equivalents thereof, such as some nucleotide sequences of the DKK3 polynucleotides. Although modified, the concept includes variants that can function functionally as DKK3 polynucleotides.

In addition, the polynucleotide encoding the DKK3 protein of the present invention may include genomic DNA, cDNA and chemical synthesis DNA, and genomic DNA and cDNA may be prepared according to methods known in the art.

Genomic DNA extracts genomic DNA from, for example, a cell with the DKK3 gene, constructs a genomic library (vectors can be used, for example, plasmids, phages, cosmids, BACs, PACs, etc.) and looks at the library. Colony or plaque hybridization is performed using a probe made on the basis of a polynucleotide encoding the DKK3 protein of the invention (eg, SEQ ID NO: 2), or a polynucleotide encoding the DKK3 protein of the invention (eg, It can also be prepared by preparing a primer specific for SEQ ID NO: 2), and performing a polymerase chain reaction (PCR) using the same.

For example, cDNA synthesizes a cDNA based on mRNA extracted from a cell having a DKK3 gene, constructs a cDNA library by inserting the synthesized cDNA into a vector such as λZAP, and expands the cDNA library. Can be prepared by colony or plaque hybridization or by PCR.

Such separated polynucleotides preferably have high homology with the amino acid sequence of the DKK3 protein (SEQ ID NO: 1) at the amino acid level they encode, and high homology means at least 50% or more, more preferably, throughout the amino acid sequence. Preferably at least 70%, more preferably at least 90%. Homology of amino acid sequences and nucleotide sequences is a program called BLASTN or BLASTX developed based on BLAST (Karlin, S and Altschul, SF. Proc. Natl. Acad. Sci. USA, 90, 5873-5877, 1993) (Altschul, SF. Et al., J. Mol. Biol., 215, 403-410, 1990) and specific methods are known on the following website (http: //www.ncbi.nlm. nih.gov.).

Meanwhile, the polynucleotide encoding the DKK3 protein of the present invention may be provided in an expression vector for intracellular delivery.

Polynucleotides encoding the DKK3 protein of the present invention can be introduced into cells using a variety of transformation techniques such as complexes of DNA and DEAE-dextran, complexes of DNA and nuclear proteins, complexes of DNA and lipids, and the like. The polynucleotide may be in a form contained within a carrier that allows for efficient introduction into a cell. The carrier is preferably a vector, and both viral and non-viral vectors can be used, for example plasmids, phages, cosmids, viral vectors and the like, which can be self-replicating or integrated into the host DNA. .

By "introduction" herein is meant the introduction of foreign DNA into cells by transformation or transduction. Transformation is a sugar such as calcium phosphate-DNA coprecipitation, DEAE-dextran-mediated transformation, polybrene-mediated transformation, electroshock, microinjection, liposome fusion, lipofectamine and protoplast fusion It can be carried out by various methods known in the art. Transduction refers to the transfer of genes into cells using viral or viral vector particles by means of infection.

In addition, DKK3 polynucleotides may be operatively linked in an expression vector with expression control sequences such as promoter / enhancer sequences and other sequences required for transcription, translation or processing. Regulatory sequences may include tissue-specific regulatory and / or inducible sequences as well as direct constitutive expression of nucleotides, and the design of expression vectors may include the host cells to be transformed, the desired level of expression. It may be determined by such factors as.

In addition, the DKK3 polynucleotide of the present invention is introduced into an appropriate eukaryotic cell in a cell culture system in a state of being included in a vector to directly express DKK3, or transformed into prokaryotic cells to express DKK3 protein, and then purify these proteins. Can be used. Such DKK3 protein of the present invention may include, for example, a purified protein, a water soluble protein, or a form fused with a protein or amino acid residue in a form bound to a carrier for delivery or administration to a target cell.

In addition, the present invention may use a substance that activates endogenous DKK3. By "material activating DKK3" is meant a substance that acts directly or indirectly on DKK3 to improve, induce, stimulate, and increase the biological activity of DKK3. Such materials include single compound peptides such as organic or inorganic compounds, complexes of plural compounds and biopolymers such as proteins, nucleic acids, carbohydrates and lipids. The mechanism by which the substance activates DKK3 is not particularly limited. For example, a substance may act as a mechanism for enhancing gene expression such as transcription, translation, or converting an inactive form into an active form. Those skilled in the art can screen for substances that activate DKK3 using known techniques in the art or through the screening methods of the present invention.

The present inventors conducted various studies to confirm the effect of DKK3 on autophagy, and as a result, the DKK3 induces the formation of autophagosome, and as a result, the autophagy can be performed in cells. The protein binds to the BD domain, a BCL-2 binding domain present in the Bcl-1 protein, inhibits the binding of Bcl-1 and BCL-2, thereby inhibiting BCL-2 activity and autophagy of Bcl-1. Inhibition of the PI3K class ImTOR signaling pathway, which maintains the induction effect, inhibits autophagy, and activates the PI3K class III signaling pathway, which activates autophagy, results in inducing autophagy. there was.

According to another embodiment of the present invention, the present invention provides a pharmaceutical composition capable of treating a disease associated with autophagy disorders comprising the Dickkopf 3 (DKK3) protein or a polynucleotide encoding the same as an active ingredient. .

The term "autodigestion disorder related disease" of the present invention means all diseases caused by abnormal cells or proteins occurring and not eliminated by autophagy. Examples include diseases with maturation defects of autophagosomes required for autophagy.

The term "abnormal cell" as used herein refers to a cell that is morphologically or functionally distinct from normal cells. The abnormal cells include nutrient depleted cells, cells genetically modified to impair cell morphology and function, physically damaged cells, and the like. The term "abnormal protein" as used herein means a protein that is morphologically or functionally distinct from normal proteins. The abnormal protein includes all of aggregate-prone mutant proteins and the like, in which the tertiary structure is modified by amino acid mutations.

Specifically, the auto-digestive disorder-related diseases include various neoplastic diseases and various degenerative diseases.

As used herein, the term "degenerative disease" means a disease in which the structure or function of an affected tissue or organ is gradually degraded over time. The degenerative diseases include spinal diseases, joint diseases, cranial nerve diseases, and the like, but the spinal diseases are not particularly limited thereto, but include spinal hypersensitivity, latent spinal cord spinal stenosis, and the like, and the joint diseases are not particularly limited thereto. , Joint stiffness, aseptic bone necrosis, osteomyelitis, bone tumors, and the like, the brain nerve disease is not particularly limited, including Alzheimer's disease, Parkinson's disease, stroke, tau disease, Amyotrophic lateral sclerosis (ALS), Huntington's disease, etc. do.

More specifically, cancer, neurodegenerative diseases (Alzheimer's disease, Huntington's disease, Parkinson's disease, dementia, etc.), desmin-related cardiomyopathy, infections, inflammatory and aging diseases. In addition, autodigestive disorders include muscle degenerative diseases such as α1-antitrypsin deficient liver disease (Perlmutter, 2006) associated with chronic inflammation and cancer, Danon disease associated with cardiomyopathy and myopathy. Beth Levine et al. (2008) Cell. January 11; 132 (1): 2742)

In neurodegenerative diseases, Huntington's disease and spinocerebellar ataxia are associated with the absence of polyglutamine (polyQ) extension tube proteins, and Parkinson's disease does not eliminate mutant alpha cynuclees (α-synucleins). Prefrontal lobe dementia is associated with the absence of mutant tau protein (Williams et al., 2006).

In relation to infection, pathogens that have invaded into cells are degraded by autophagy-dependent pathways (Nakagawa et al., 2004), so infectious diseases can be caused by autophagy disorders. In addition, autodigestion can lead to the rapid removal of apoptosis bodies, thereby preventing tissue inflammation and preventing autoimmune diseases against autoantigens such as systemic lupus erythematosis (Grossmayer et al., 2005). Inflammation or autoimmune diseases can occur due to autodigestion disorders.

In the case of cancer, apoptosis-related pathways are often deficient in most cancer cells, and thus, conventional treatment strategies that simply induce apoptosis have not effectively treated cancer. The present invention can effectively treat cancers resistant to conventional anticancer drugs by promoting autodigestion dependent pathways, which are different from apoptosis.

The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier, excipient or diluent according to conventional methods.

The term "pharmaceutically acceptable" of the present invention refers to compositions and molecules that are physiologically acceptable and typically do not cause unexpected reactions when administered to humans. Preferably, as used herein, the term "pharmaceutically acceptable" means approved by other generally known pharmacopoeia for use in mammals and in particular humans.

Carriers, excipients and diluents that may be included in the compositions of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

Compositions comprising the DKK3 protein of the present invention or a polynucleotide encoding the same as an active ingredient, oral formulations, external preparations, such as powders, granules, tablets, capsules, suspensions, emulsions, syrups and aerosols, respectively, according to a conventional method, It can be formulated in the form of suppositories or sterile injectable solutions.

Specifically, when formulated, it may be prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc. which are commonly used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations may contain at least one excipient such as starch, calcium carbonate, sucrose, or the like. ), Lactose, gelatin and the like can be mixed. In addition to simple excipients, lubricants such as magnesium stearate, talc can also be used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations and suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, macrogol, Tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

In another embodiment of the present invention, the present invention provides a method of treating a disease related to autophagy disorders comprising administering the composition to an individual in need thereof.

According to various methods commonly known to those skilled in the art in consideration of the type, dosage form, and therapeutic effect of diseases related to autophagy disorders, a composition comprising the DKK3 protein of the present invention or a polynucleotide encoding the same as an active ingredient may be appropriately administered. Could be.

According to another embodiment of the present invention, the present invention utilizes the interaction of the DKK3 polypeptide or fragment thereof and the Becklin-1 polypeptide or fragment thereof to determine a substance that upregulates or downregulates the cell's autophagy. It provides a method of screening. Specifically, the interaction between the BCL-2 binding domain of the Becklin-1 polypeptide and the DKK3 polypeptide may be used. The screened material can be used for the study of cell autophagy control and also can be used as a therapeutic agent for diseases related to autophagy disorders.

The interaction is not particularly limited but may be a direct or indirect linkage of the DKK3 polypeptide and the Becklin-1 polypeptide.

Specifically, the binding of the DKK3 polypeptide or fragment thereof to beclin-1 or a fragment thereof may be upregulated by the addition of a substance that induces or promotes this binding to promote autophagy, and conversely inhibits the binding. Or by the addition of a substance that inhibits autodigestion by inhibition. Modulation of the interaction of the BCL-2 binding domain of the Becklin-1 polypeptide with the DKK3 polypeptide directly leads to the regulation of the interaction of Bcl-1 with the BCL-2 protein, thus up- or down-regulating cell autophagy. The material can be screened.

The screening method is not particularly limited thereto, and the method may include adding a substance whose effect on autodigestion in vivo or in vitro to a system in which DKK3 and beclin-1 are present to detect the effect of the substance. It is desirable to. For example, in the screening method, the substance is added to a reactant in which DKK3 and beclin-1 are present under in vitro conditions, and then reacted, and then the effect of the material on the binding of DKK3 and beclin-1 by electrophoresis. May be implemented to include detecting; Expression of the substance in cells expressing DKK3 and Becklin-1 under in vivo conditions, and may be implemented to include detecting the effect of the substance by detecting whether or not autophagy in the cell.

By using the composition comprising the DKK3 protein of the present invention or a polynucleotide encoding the same as an active ingredient, it is possible to treat or ameliorate diseases caused by abnormal cells or proteins due to autodigestion disorders. It can be widely used for treatment. In addition, since the Dikop-3 protein is present and secreted in the human body, side effects that may occur in artificial synthetic drugs can be minimized.

DKK3 induces autophagy.
A. Induction of autophagy through exogenous expression of DKK3
Total cell disruptions were obtained to confirm the relevance of DKK3 for autophagy. Expression of endogenous autophagy proteins was tested using immunoblotting assays on whole cell lysates of HeLa cells stably expressing DKK3 or expressing controls. Expression of DKK3 was confirmed by immunoblot analysis using anti-DKK3 antibodies.
B. Dose-dependent increase in autophagy protein by DKK3
The Flag-DKK3 expression vector was introduced into HeLa cells, and the expression of endogenous autophagy proteins in their whole cell lysates was confirmed by immunoblot analysis. Expression of DKK3 was confirmed by immunoblot analysis using anti-DKK3 antibodies.
C. Damage to DKK3 inhibits DKK3 induced autophagy.
Random siRNAs or DKK3-specific siRNAs were introduced into MRC5 cells, which are normal human lung fibroblasts, and immunoblot analysis of autodigesting proteins was performed using their whole cell disruptions. Anti-GAPDH antibody was used as an internal control.
Figure 2. Promotion of autophagosome formation by DKK3
A. Obtaining HeLa Cells Expressing DKK3
A transformant was obtained by introducing a Flag vector of a control group or a DKK3 vector of an experimental group into the HeLa cells, followed by an immunofluorescence assay using an anti-DKK3 antibody labeled with Alexa 488 to obtain HeLa cells expressing DKK3. At this time, DNA of HeLa cells was stained with DAPI. Expression of DKK3 was also confirmed by immunofluorescence analysis using an anti-DKK3 antibody labeled with Alexa 488. Anti-GAPDH antibody was used as an internal control.
B. Induction of autophagosome formation through DKK3
GFP-LC3 was introduced into HeLa cells transformed with the control and experimental vectors, and autophagosome formation was tested in the cytoplasm through confocal microscopy. 3-MA, an autophagy inhibitor, inhibited the formation of autophagosomes induced by DKK3.
C. Induction of autophagosome formation was quantified by counting GFP-LC3 positive cells in three independent experiments. Error bars indicate standard errors.
D. TEM Analysis of Autophagosome Induction by DKK3
HeLa cells stably expressing the flag vectors of DKK3 or the control group were subjected to electron microscopy by known methods.
DKK3 reacts with Becklin-1 to interfere with the Becklin-1-BCL-2 reaction.
A. DKK3 reacts with beclin-1.
Flag-Beclin-1 vectors were introduced into MCF-7 cells, immunoprecipitated using Flag-agarose beads, and immunoblot analysis was performed using anti-DKK3 antibodies. The same immunoassay was performed using anti-Beclin-1 antibody to confirm the above results.
B. DKK3 reacts with intrinsic beckin-1 through the BD domain of beckin-1.
Cell lysates of HeLa cells expressing DKK3 were immunoprecipitated using protein A / G agarose beads followed by anti-veclin-1 antibodies. Immunoblot analysis was performed with anti-DKK3 antibodies. The same immunoassay with the appropriate antibodies was used to confirm the expression of Beclin-1 and BCL-2.
C. DKK3 and Becklin-1 are in the same position in the cytoplasm.
The location in the cytoplasm of Becklin-1 and DKK3 was confirmed using confocal microscopy. HeLa cells expressing DKK3 were prepared using anti-DKK3 and anti-Beclin-1 antibodies followed by secondary antibodies for detecting DKK3 and becline bound with Alexa 488 (green)-and Alexa 564 (red). Immunofluorescence staining.
D.MCF-7 cells were transformed with a vector comprising the BD domain of Becklin-1 or V5 labeled Becklin-1.
Anti-V5 antibodies were used to immunoprecipitate in the cell disruptions, and immunoblot analysis was performed using anti-DKK3 antibodies (top panel). Expression of beclin-1 was determined by immunoblot analysis using anti-V5 antibodies (bottom panel).
The yeast-to-hybrid screening method confirmed that E. DKK3 protein binds to the BD domain of Becklin-1 protein.
F. The exogenous expression of DKK3 does not affect the formation of the Becklin-1-hVps34 complex.
V5-labeled Becklin-1 was introduced into MCF-7-DKK3 cells, immunoprecipitated with anti-V5 antibody, and immunoblot analysis was performed with anti-hVps34 antibody. Expression of DKK3 was confirmed by immunoblot analysis using anti-DKK3 antibodies in cell disruptions.
Induction of autophagosome formation by DKK3 in intracellular environment
A. Analysis of serum free DKK3 media
Adjusted medium of control vector or DKK3 HEK 293T transducer was collected and concentrated, followed by immunoblot analysis using anti-DKK3 antibody. The content of recombinant DKK3 protein was used to determine the concentration of DKK3 present in serum-free media.
B. Induction of Autodigestion In Vivo by DKK3
In cell lysates of tumor tissues treated with control or DKK3 293T media, the expression of endogenous autophagy related proteins was analyzed by immunoblot analysis. GAPDH was used as an internal control.
C. Autophagy Formation by Electron Microscopy Analysis
Tumor sites were processed and analyzed by TEM analysis. DKK3 treated tumors showed significant autophagosome formation.
Figure 5. Regulation of PI3K signaling pathway by DKK3
Through immunoblot analysis using antibodies of signaling proteins, PI3K signaling pathways of class I and III were analyzed in MCF-7 (A and B) derived cell disruptions. Anti-GAPDH antibody or anti-β-actin antibody was used as internal control.
6. It was confirmed by immunofluorescence microscopy analysis that DKK3 overexpression increased autophagy formation even in nutrient-rich culture conditions. Autophagosome formation was analyzed by confocal microscopy after HeLa-Flag or HeLa-DKK3 stabilized cells were transformed into in a 10% FBS containing medium.

Hereinafter, the present invention will be described in more detail with reference to Examples. These embodiments are only for illustrating the present invention, and the scope of the present invention is not construed as being limited by these embodiments.

Materials and Methods

Cell culture

Human cervical cancer HeLa cells, human embryonic kidney HEK293 cells, human embryonic lung fibroblast MRC5 and human breast cancer MCF-7 cells were treated at 37 ° C. 5% CO ₂ in DMEM medium containing 10% FBS and antibiotics. Were incubated.

Recombinant Vectors and Transduction

Flag-labeled full-length human DKK3 cDNA and human Becklin-1 fragments were obtained by high accuracy PCR using a human fetal brain cDNA library. The fragment was cloned to be introduced into the EcoR1 and XhoI sites of the flag labeled pcDNA vector, yielding a full-length DKK3 expression vector (Flag-DKK3) and a Becklin-1 expression vector (Flag-Beclin 1), respectively. Flag labeled DKK2 (pcDNA3.1-Flag-DKK2) (Flag-DKK2) and Flag labeled DKK3 (pcDNA3.1-Flag-DKK3) (Flag-DKK2) were obtained from Yonsei University professor Kwon Young-geun. The base sequence of all cloned cDNA was confirmed by DNA sequencing. The obtained vector was introduced into the human cell line using Effectene (Qiagen). The control vector and the transducers expressing DKK3 (HeLa, MCF-7, 293T) were produced by obtaining individual colonies in G418-containing medium for 3 weeks and then passage culture. Purity of the transductor was confirmed using indirect immunofluorescence staining with anti-DKK3 antibody. The expression vector containing the full-length Becklin-1 or the BD of Becklin-1 was labeled with V5, introduced into cells using Effectene (Qiagen), and the reaction between DKK3 and Becklin-1 was confirmed.

siRNA introduction

SiRNA oligonucleotide sequences targeting DKK3 were obtained commercially (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). 100 nM DKK3 siRNA was used to reduce the expression of DKK3 using Oligofectamin reagent (Invitrogen). Inhibition of DKK3 expression was confirmed by immunoblot analysis using anti-DKK3 antibodies.

Immunoblot  Assays and Simultaneous Immunoprecipitation Assays

The introduced cells were digested in the presence of protease inhibitor mixtures (Roche, Mannheim, Germany) and dephosphorylation inhibitors (Sigma Aldrich, St. Louis, MO) in radioimmunoprecipitation assay (RIPA) buffer (Park et al. , 2005). Total cell lysates (500 μg) were immunoprecipitated using antibodies. The precipitated protein was washed five times, subjected to 10% SDS-PAGE, then transferred to an ELC membrane (BioRad Laboratories, Hercules, CA) and immunized with an immunoblot using an ECL detection kit (Amersham Biosciences, Uppsala, Sweden). Applied. Anti-β-actin antibody (Sigma, St. Louis, MO) or anti-GAPDH antibody (Sigma Aldrich) was used as an internal control.

Indirect Immunofluorescence Assay

The desired expression vector was introduced into cells cultured on coverslips in 6-well plates. After 30 hours of introduction, the cells were washed with PBS, fixed with 4% formalin solution buffered in PBS, and then subjected to indirect immunofluorescence analysis. After reacting by adding the desired primary antibody, the reaction was performed by adding IgG bound to Alexa 586 or Alexa 488 as a secondary antibody (Invitrogen). Nuclei were stained using DAPI (Roche). Expression and intracellular distribution of the protein of interest were analyzed using confocal microscopy (BioRad, Hertfordshire, UK).

Quantification of GFP-LC3

GFP labeled microtubule binding protein 1 light chain 3 (LC3) expression vector was obtained from Tamos Yoshimori (Osaka University, Osaka, Japan). Into a control vector or MCF-7 or HeLa cells expressing DKK3, a GFP-LC3 expression vector was introduced using a gene transfer reagent (Qiagen, Valencia, Calif.) And subjected to nutrient rich conditions containing 10% FBS or for 4 hours. The cells were cultured under nutrient depletion conditions by culturing in EBSS, and then evaluated by confocal microscopy (Radiance 2000, Biorad). To quantify the induction of autodigestion, confocal microscopy was used to count 200 GFP positive cells with 10 or more points at which the formation of a pointed autophagosome was determined.

Micro Array Analysis and RT-PCR Analysis

To identify downstream targets activated by DKK3 expression, DKK3 expressing cells and control vector cells using TRIZOL reagent (Invitrogen Life Technologies, Grand Island, NY), according to known methods (Lee et al., 2005). Total cell RNA was extracted from the cDNA and subjected to cDNA microarray analysis using a DNA chip (Genetrack Human 17K cDNA chip; Genomictree Products, Taejon, South Korea). For RT-PCR analysis, Supertranscript II Reverse Transcriptase (Invitrogen) and oligo (dT) primers were used to reverse transcription of total cellular RNA extracted from cells of interest, and the resulting cDNA was used as a template for PCR amplification. . The base sequence of the primer for amplifying DKK3 is as follows:

DKK3 forward primer, 5'-GAG CGA GCA GAT CCA GTC-3 '(SEQ ID NO: 3)

DKK3 reverse primer, 5'-AGC CAT GTA GAA CAA ACG GC-3 '(SEQ ID NO: 4).

PCR conditions were first denaturation 95 ℃ 1 minute; Repeat 30 times at 95 ° C for 30 seconds, 60 ° C for 1 minute and 72 ° C for 1 minute; And final extension 72 ° C. for 7 minutes. The resulting sections were subjected to 2% agarose gel electrophoresis and stained with DNA staining reagents (0.01% SYBR safe DNA gel stain, Invitrogen, Carlsbad, CA).

TEM method

Cultured mammalian cells or tumor tissues were fixed with 0.1 M cacodylate buffer (pH 7.4) containing 2.5% glutaraldehyde for 45 minutes at 40 ° C. and buffered without glutaraldehyde. Washed with, postfixed with cacodylate buffer containing 1% OsO4, dehydrated and embedded in Epon. The microtome (Reichert Ultracut S ultramicrotome, Leica) was cut into ultrathin (70 nm). The flakes were stained with 4% uranyl acetate and photographed with an electron microscope (Hitachi H-7100).

Model animals

Five week old female BALB / c nude (nu / nu) mice were obtained commercially (Charles River Laboratory, Japan) and bred under aseptic conditions. Each group contains 5 animals. Serum-free media (CM) derived from control vectors or 293T cells expressing DKK3 were collected by centrifugation and concentrated using Centricon filter devices (Centricon filter devices, MWCO 30, Millipore, Bedford, Mass.). Purity and concentration of the concentrated adjusted medium were determined using immunoblot analysis after SDS-PAGE. After 10 days of transdermal injection of human HeLa cervical cancer cells (5 × 10 ⁶ cells) into the mice, concentrated and purified serum-free media prepared from control vectors or DKK3 expressing transducers for 1 week for 2 weeks. Mice were injected twice a week (43 μg total). The tumor was cut out and weighed and then subjected to immunoblot analysis, immunohistochemical analysis and electron microscopic analysis.

Statistical analysis

The statistical significance of the difference in the experimental results was evaluated by independent analysis of variance, and the probability value was calculated by independent T test. P <0.05 was considered statistically significant.

Example 1: Induction of autophagy of DKK3

The inventors have found that DKK3 is involved in the degradation of β-catenin protein rather than proteasome-mediated ubiquitin degradation activity. Therefore, we tried to determine whether DKK3 is involved in autophagy.

First, after expressing foreign DKK3 protein in HeLa cells lacking endogenous DKK3, the effect of DKK3 on autophagy was confirmed by measuring the expression level of autophagy protein. The outward expression of DKK3 through the introduction of the DKK3 expression vector increased the expression of the endogenous autophagy marker protein beclin-1 and light chain 3 (MAP1 LC3-II) of membrane-bound microtubule related protein 1 (FIG. 1A).

Post-translational modifications that convert LC3-I into LC3-II into the cytoplasm are known to occur after LC3-II is located on the membrane of a nearby autophagosome. We suggested that increased expression of DKK3 induces intrinsic LC3-II and beclin-1 levels and induces dose-dependent DKK3 mediated autophagy (FIG. 1B).

In addition, we studied the role of DKK3 in autophagy via siRNA mediated experiments in human normal MRC5 cells. DKK3-specific siRNA reduced DKK3 protein expression by 80% and decreased intrinsic LC3-II (FIG. 1C).

In order to identify novel DKK3-mediated autophagy induction and explore molecular mechanisms, we present human breast cancer cells that lack caspase 3, exhibit weak activity of caspase-9 and do not express DKK3. MCF-7 cell line was selected. Adenovirus mediated DKK3 gene transfer demonstrated the activity of DKK3 to inhibit tumors by inducing apoptosis. To distinguish DKK3 activity from nonspecific cytotoxic effects, DKK3 was stably expressed in HeLa cells and MCF-7 cells, and the vector of the control group was expressed and then selected in a medium containing G418.

DKK3 protein expression was confirmed by indirect immunofluorescence staining and immunoblot analysis using anti-DKK3 antibodies (FIG. 2A). DKK3 protein was expressed in the cytoplasm of the transformants (FIG. 2A, left).

In order to confirm the induction of DKK3-mediated autophagy in HeLa DKK3 cells, we introduced GFP-labeled autophagosome binding LC3 vectors into control or DKK3 expressing HeLa cells, followed by confocal microscopy. Initial marker autophagosome formation was tested. The binding of phosphatidylethanolamine (PE) to LC3 results in insolubilization of LC3 bound to the membrane of autophagosome under induction conditions. As a result, GFP-LC3 exists in a cell-dispersed form under normal conditions, whereas the presence of GFP-LC3 in the form of a dot reflecting the form bound to the membrane may provide evidence of induction of autophagy. .

Therefore, the inventors conducted experiments confirming the induction of autochemical digestion by production of LC3-II and observation of the intracellular distribution of fluorescently labeled LC3. As expected, confocal microscopy showed that GFP-LC3 was present in cells in a dispersed form in cells expressing the control vector, whereas GFP-LC3 was present in the cells in the form of dots in cells expressing DKK3. From the nutrient-rich condition, it was found that the formation of autophagosomes was induced by the expression of foreign DKK3 (FIG. 6). An increase in autophagosome formation in cells expressing DKK3 was demonstrated in serum-free conditions (FIG. 2B).

In contrast, treatment with 3-MA (3-methyladenine), an inhibitor of class III PI3K (Vps34), prevented DKK3-induced autophagocytosis formation (FIG. 2B, right). Expression of DKK3 in serum-free conditions increased autophagosome formation more than threefold (FIG. 2C).

Next, the inventors confirmed the DKK3-mediated induction of autophagosome formation by TEM analysis. From the morphological approach through the TEM assay, it was confirmed that a large amount of autophagy vacuoles encapsulated by the double membrane was accumulated in the cells expressing DKK3 under nutrient rich conditions compared to the cells of the control group (FIG. 2D). This suggests that exogenous expression of DKK3 at the physiological level enhances the formation of autophagosomes.

Example 2 Interference Effect of DKK3 on the Binding of Becklin-1 and BCL-2

To explain the mechanism of DKK3-mediated autophagosome induction, we identified whether DKK3 physically reacts with Becklin-1, which plays an important role in autophagy formation in the early stages of autophagy. MCF-7-DKK3 cells incorporating Flag-Beclin-1 were analyzed to show a response between DKK3 and Becklin-1 (FIG. 3A).

Next, endogenous beclin-1 also used autodigestion-competent HeLa cells that expressed endogenously at a controllable level. As a result of immunoprecipitation with anti-veclin-1 in HeLa cells expressing DKK3, it was confirmed that an intrinsic beclin-1 and DKK3 complex was formed (FIG. 3B).

To further confirm their response, the intracellular locations of Becklin-1 and DKK3 were determined by direct immunofluorescence staining and confocal microscopy. DKK3 co-located with Becklin-1 in the cytoplasm was observed under confocal microscopy, indicating that DKK3 reacts with Becklin-1 (FIG. 3C).

Next, the present inventors tried to determine whether DKK3 influences the Becklin-1-BCL-2 reaction. Exogenous expression of DKK3 interfered with the Becklin-1-BCL-2 response, suggesting a competition between DKK3 and BCL-2 binding to Becklin-1 (FIG. 3B).

Because BCL-2 is known to react with beckin through the BCL-2 binding domain (BD) of beckin-1, and downregulates beckin-1 dependent autophagy in the nucleation phase of autophagy, The inventors studied whether DKK3 binds to the BD of beclin-1, thereby inhibiting the beclin-1-BCL-2 response.

Expression vectors expressing BD or all of the flag labeled Becklin-1 were introduced into MCF-7 cells expressing DKK3. Simultaneous immunoprecipitation assays using the V5 labeled VECLIN-1 expression vector showed that DKK3 can bind to the BD of VECLIN-1 (FIG. 3D).

Surprisingly, the reaction of the DKK3 with the BD of Beclin-1 was similar to that of the whole Beclin-1 with DKK3 (FIG. 3D). Thus, DKK3 can induce the formation of autophagosomes by inhibiting the Becklin-1-BCL-2 response, thereby promoting the nucleation required for the formation of successful autophagosomes.

In addition, it was confirmed that the binding between the DKK3 protein and the BD domain of Becklin-1 was directly cross-linked through yeast-to-hybrid analysis. Four types of Becklin-1 constructs were constructed and performed as previously described (Park et al., Cancer Res 65 (3): 749-757, 2005). In summary, DNA fragments encoding the Becklin-1 protein were cloned into the EcoRI and XhoI positions of pJG4-5 (Clontech, USA) by PCR method in a cDNA library derived from HeLa cells (Clontech, USA), and the DKK3 full length was LexA Cloning was performed at the BamH I and Xho I positions of the pGilda vector (Clontech, USA) for expression into fusion proteins. The ability to introduce each pGilda and pJG4-5 fusion construct into the experimental yeast strain EGY 48 (Clontech, USA) and grow cells in leucine-deficient media containing 2% galactose, and X-gal, 2% The interaction between the fused proteins was determined through the formation of blue colonies in synthetic medium containing galactose and 2% raffinose.

As can be seen in FIG. 3E, the VECLIN-1 protein, which includes the BD domain alone (1-150) of the VECLIN-1 protein, comparable to that of DKK3 at a level comparable to that of the full-length Becklin-1 protein (1-450). Although interacting, Becklin-1, including other domains, did not interact with DKK3 at all. Thus, these results indicate the importance of the BD domain of the Becklin-1 protein in the interaction of Bcl-1 and DKK3, suggesting that the BD domain of Bcl-1 is essential and sufficient for interaction with DKK3.

The beclin-1-hVps34 complex is known to be involved in the nucleation or early formation of pro-autophagosome membranes7. Because BCL-2 inhibits the formation of beclin-1-hVps34 complex that promotes autophagy and reduces the activity of class III PI3K associated with beclin-1, we believe that beclin- in MCF-7 cells. The effect of DKK3 expression on the 1-hVps34 complex was studied. A gene of Vclin-1 labeled V5 was introduced into MCF7-DKK3 cells, immunoprecipitated with an anti-V5 antibody, and immunoblot analysis was performed using an anti-hVps34 antibody.

Expression of DKK3 had little effect on the Becklin-1-hVps34 response (FIG. 3F). Thus, the binding of Bcl-2 to Bcl-1 was reduced by DKK3 expression as described above (FIG. 3B), whereas the binding of hVps34 to Bcl-1 was not significantly affected by DKK3.

Next, the present inventors confirmed the binding specificity of DKK3 to beclin-1 using a simultaneous immunoprecipitation assay, and confirmed the induction of autophagy protein expression using an immunoblotting assay to form DKK3 mediated autophagy. The specificity of was studied. While DKK3 reacts strongly with Becklin-1 (FIG. 3A), one of the other DKK families, DKK2, failed to react with Becklin-1 as well as the intrinsic autophagy proteins LC3-II and Becklin-1. It did not induce the expression of.

Example  3: DKK3 of Autophagy  Induction

To confirm the effect of DKK3 on the formation of autophagosomes in the in vivo environment, model mice implanted with human cervical cancer were used. Serum-free media derived from 293T cells (293T-DKK3 and 293T-Con) expressing DKK3 or control were compared to recombinant DKK3 protein commercially available by immunoblot assay to assess the purity and concentration of DKK3 (FIG. 4A). ). The content and purity of DKK3 did not show a significant difference between the adjusted medium and the prepared DKK, thus providing a basis for using the adjusted medium.

As in the laboratory environment, immunoblot assay showed dramatic upregulation of Bcl-1 and LC3-II and downregulation of BCL-2 after treatment with DKK3 control media compared to control control media. (FIG. 4B). Next, the formation of DKK3 media mediated autophagy in vivo was confirmed by electron microscopic analysis of tumor tissues treated with DKK3 media or control media injected twice a week for 2 weeks.

Electron microscopic analysis of the tumor tissue, it was confirmed that the number of autophagosomes dramatically increased in the tumor treated with DKK3 adjusted medium compared to the control treated medium (Fig. 4C). These results indicated that the outward expression of DKK3 in vivo induces the formation of autophagosomes.

Example 4 Regulation of PI3K Signaling Pathway by DKK3

Class I PI3K and mTOR regulatory pathways inhibit autophagy, whereas class III PI3K activates autophagy. To confirm the effect of exogenous expression of DKK3 on the PI3K signaling pathway involved in the regulation of autophagy, the immunoblot analysis of DKK3 on the P13K signaling pathway was performed using an immunoblot assay using antibodies to the signaling protein involved in the pathway. Cells expressing DKK3 were used to assess the effect of physiological expression levels (FIG. 5).

Intact DKK3 expression in human breast MCF-7 tumor cells, as it is, the ability of DKK3 to induce autophagy, reduced the phosphorylation of p70S6K, an mTOR target protein, thereby inhibiting mTOR signaling. Inhibiting the signaling of mTOR, as it is, we have shown that the levels of beclin-1 and LC3-II conversions are increased, the levels of Bcl-2 are decreased, and the PI3K class III signaling pathway is activated (FIG. 5A). And 5B).

In addition, Ras plays an important role as a downregulator of autophagy due to nutrient depletion via the class I PI3-kinase signaling pathway (Furuta et al., 2004). Therefore, the present inventors sought to determine the effect of DKK3 on the class I PI3-kinase signaling pathway involved in the regulation of autophagy. Exogenous expression of DKK3 resulted in downregulation of Ras and Raf protein levels, indicating downregulation of the class I PI3-kinase signaling pathway (FIG. 5B).

As a result, it was found that foreign expression of DKK3 suppresses the PI3K class ImTOR signaling pathway, but induces autodigestion by activating the PI3K class III signaling pathway.

<110> SAMSUNG LIFE PUBLIC WELFARE FOUNDATION <120> A Composition for Inducing Cell Autophagy <130> PA110272 / KR <160> 4 <170> Kopatentin 2.0 <210> 1 <211> 350 <212> PRT <213> Human Dickkopf 3 <400> 1 Met Gln Arg Leu Gly Ala Thr Leu Leu Cys Leu Leu Leu Ala Ala Ala   1 5 10 15 Val Pro Thr Ala Pro Ala Pro Ala Pro Thr Ala Thr Ser Ala Pro Val              20 25 30 Lys Pro Gly Pro Ala Leu Ser Tyr Pro Gln Glu Glu Ala Thr Leu Asn          35 40 45 Glu Met Phe Arg Glu Val Glu Glu Leu Met Glu Asp Thr Gln His Lys      50 55 60 Leu Arg Ser Ala Val Glu Glu Met Glu Ala Glu Glu Ala Ala Ala Lys  65 70 75 80 Ala Ser Ser Glu Val Asn Leu Ala Asn Leu Pro Pro Ser Tyr His Asn                  85 90 95 Glu Thr Asn Thr Asp Thr Lys Val Gly Asn Asn Thr Ile His Val His             100 105 110 Arg Glu Ile His Lys Ile Thr Asn Asn Gln Thr Gly Gln Met Val Phe         115 120 125 Ser Glu Thr Val Ile Thr Ser Val Gly Asp Glu Glu Gly Arg Arg Ser     130 135 140 His Glu Cys Ile Ile Asp Glu Asp Cys Gly Pro Ser Met Tyr Cys Gln 145 150 155 160 Phe Ala Ser Phe Gln Tyr Thr Cys Gln Pro Cys Arg Gly Gln Arg Met                 165 170 175 Leu Cys Thr Arg Asp Ser Glu Cys Cys Gly Asp Gln Leu Cys Val Trp             180 185 190 Gly His Cys Thr Lys Met Ala Thr Arg Gly Ser Asn Gly Thr Ile Cys         195 200 205 Asp Asn Gln Arg Asp Cys Gln Pro Gly Leu Cys Cys Ala Phe Gln Arg     210 215 220 Gly Leu Leu Phe Pro Val Cys Thr Pro Leu Pro Val Glu Gly Glu Leu 225 230 235 240 Cys His Asp Pro Ala Ser Arg Leu Leu Asp Leu Ile Thr Trp Glu Leu                 245 250 255 Glu Pro Asp Gly Ala Leu Asp Arg Cys Pro Cys Ala Ser Gly Leu Leu             260 265 270 Cys Gln Pro His Ser His Ser Leu Val Tyr Val Cys Lys Pro Thr Phe         275 280 285 Val Gly Ser Arg Asp Gln Asp Gly Glu Ile Leu Leu Pro Arg Glu Val     290 295 300 Pro Asp Glu Tyr Glu Val Gly Ser Phe Met Glu Glu Val Arg Gln Glu 305 310 315 320 Leu Glu Asp Leu Glu Arg Ser Leu Thr Glu Glu Met Ala Leu Gly Glu                 325 330 335 Pro Ala Ala Ala Ala Ala Ala Leu Leu Gly Gly Glu Glu Ile             340 345 350 <210> 2 <211> 1053 <212> DNA <213> Human Dickkopf 3 <400> 2 atgcagcggc ttggggccac cctgctgtgc ctgctgctgg cggcggcggt ccccacggcc 60 cccgcgcccg ctccgacggc gacctcggct ccagtcaagc ccggcccggc tctcagctac 120 ccgcaggagg aggccaccct caatgagatg ttccgcgagg ttgaggaact gatggaggac 180 acgcagcaca aattgcgcag cgcggtggaa gagatggagg cagaagaagc tgctgctaaa 240 gcatcatcag aagtgaacct ggcaaactta cctcccagct atcacaatga gaccaacaca 300 gacacgaagg ttggaaataa taccatccat gtgcaccgag aaattcacaa gataaccaac 360 aaccagactg gacaaatggt cttttcagag acagttatca catctgtggg agacgaagaa 420 ggcagaagga gccacgagtg catcatcgac gaggactgtg ggcccagcat gtactgccag 480 tttgccagct tccagtacac ctgccagcca tgccggggcc agaggatgct ctgcacccgg 540 gacagtgagt gctgtggaga ccagctgtgt gtctggggtc actgcaccaa aatggccacc 600 aggggcagca atgggaccat ctgtgacaac cagagggact gccagccggg gctgtgctgt 660 gccttccaga gaggcctgct gttccctgtg tgcacacccc tgcccgtgga gggcgagctt 720 tgccatgacc ccgccagccg gcttctggac ctcatcacct gggagctaga gcctgatgga 780 gccttggacc gatgcccttg tgccagtggc ctcctctgcc agccccacag ccacagcctg 840 gtgtatgtgt gcaagccgac cttcgtgggg agccgtgacc aagatgggga gatcctgctg 900 cccagagagg tccccgatga gtatgaagtt ggcagcttca tggaggaggt gcgccaggag 960 ctggaggacc tggagaggag cctgactgaa gagatggcgc tgggggagcc tgcggctgcc 1020 gccgctgcac tgctgggagg ggaagagatt tag 1053 <210> 3 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 gagcgagcag atccagtc 18 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 agccatgtag aacaaacggc 20

Claims (14)

Dickkopf 3 (DKK3) protein or a composition for inducing autophagy of a cell comprising a polynucleotide encoding the same as an active ingredient.
The method of claim 1,
The DKK3 composition to induce the formation of autophagosomes in the cell (autophagosome).
The method of claim 1,
The DKK3 inhibits the binding of Bcl-1 and BCL-2 to inhibit the activity of BCL-2 and maintains the effect of inducing autophagy of beclin-1.
The method of claim 3,
The DKK3 binds to the BCL-2 binding domain present in the Becklin-1 protein and inhibits the binding of Bcl-1 and BCL-2.
The method of claim 1,
Said DKK3 inhibits the PI3K class ImTOR signaling pathway that inhibits autophagy and activates the PI3K class III signaling pathway that activates autophagy.
The method of claim 1,
The DKK3 protein or a polynucleotide encoding the same is characterized in that the human origin.
The method of claim 1,
Wherein said DKK3 protein has the amino acid sequence of SEQ ID NO: 1.
The method of claim 1,
The polynucleotide encoding the DKK3 has a nucleic acid sequence of SEQ ID NO: 2.
The method of claim 1,
The polynucleotide encoding the DKK3 is included in an expression vector for intracellular delivery.
A pharmaceutical composition capable of treating diseases related to autophagy disorders comprising Dikopkop 3 (DKK3) protein or a polynucleotide encoding the same as an active ingredient.
The method of claim 10,
The autophagy disorder-related disease is a tumorous disease or a degenerative disease.
The method of claim 10,
The diseases include spinal hypersensitivity, latent spinal cord, spinal canal stenosis, joint stiffness, aseptic osteonecrosis, osteomyelitis, Alzheimer's disease, Parkinson's disease, stroke, tauopathy, amyotrophic lateral sclerosis (ALS), Huntington's disease, frontal lobe dementia, dementia, Selected from the group consisting of spinal cerebellar ataxia, cancer, desmin-related cardiomyopathy, infectious disease, inflammatory disease, α1-antitrypsin deficiency liver disease (Perlmutter, 2006) and Danon disease Phosphorus pharmaceutical composition.
A method of screening a substance that upregulates or downregulates autophagy of a cell using the interaction of a Dkk-3 polypeptide and a Becklin-1 polypeptide.
The method of claim 13,
Wherein said interaction is a binding of a BCL-2 binding domain of a Becklin-1 polypeptide to a DKK3 polypeptide.

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