KR101524426B1 - Assay method for autophagy-specific inhibitor screening - Google Patents

Abstract

The present invention relates to a screening method for discovering an autophagy-specific inhibitor, a kit used for the method, and a method for analyzing coherence of a target protein combined to Vps34 or a complex including the same, wherein the method enables to process a candidate substance expected to inhibit autophagy and a library such as various compounds, peptide, aptamer, etc., and to properly determine a high processing amount examination of hindering a formation of a Vps34-Beclin1-ATG14L compound, which plays an important role in an early stage of a phenomenon of the autophagy thereby, in vitro. By using the screening method of the autophagic inhibitor provided in the present invention, the present invention enables to be widely used for the development of a composition for treating various diseases through a restriction of the autophagy as being able to discover the inhibitor for specifically inhibiting the phenomenon of the autophagy.

Description

[0002] Assay method for autophagy-specific inhibitor screening [

The present invention relates to screening methods for identifying autophagic specific inhibitors. More specifically, the present invention relates to a method for screening candidate substances and various compounds, peptides, Thus, a screening method of an autophagy-specific inhibitor capable of discriminating in vitro the inhibition of the formation of the Vps34-Beclin1-ATG14L complex, which plays an important role in the early stage of the self-feeding phenomenon, And a method for analyzing the binding force of a target protein capable of binding to Vps34 or a complex comprising Vps34.

The autophagy phenomenon plays an important role in regulating cell function, such as starvation, protection from infectious bacteria, and regulation of neuronal decay, which is an evolutionarily conserved process in which all eukaryotic cells from yeast to mammals Lt; / RTI > Initially, the cytoplasm and intracellular organs degenerate into a double-membrane-associated structure called autophagosome and the degenerated autophagosome fuses with a lysosome to form an autolysosome, The material degraded by the autolysome is hydrolyzed and reused. However, since the molecular functional groups inherent in this process have not yet been revealed, studies are being actively carried out to identify them.

To date, various genetic / biochemical studies on yeast, fruit fly, etc. have identified the proteins necessary for autophagy in the cell, and studies to analyze their correlation . It is known that autophagy is largely divided into three stages: induction / nucleation, membrane elongation / closure, and vesicle trafficking / maturation / fusion / degradation. In addition, the autophagic pathway contains two unique ubiquitin-like junction systems via Atg8PE (phosphatidylethanolamine) or Atg12Atg5, which pathway is associated with the yeast Vps34 homolog class III PI3K (phosphatidylinositol 3-kinase) and Beclin1 Autogrug) with the complex composed of autopragos.

In many types of cells, stimulation of the class I PI3K / AKT signaling pathway by growth factors and cytokines has the effect of inhibiting autophagy. The inhibitory effect is presumably due to the class I PI3K / AKT-dependent activation of the target of rapamycin (TOR) kinase, which downregulates autophagy. For example, in cells treated with PI 3-kinase inhibitors wortmannin and 3-methyladenine (3-MA), autopagosomal precursors do not develop, suggesting that PI3K- Suggesting the importance of inhibiting the activity of the AKT-mTORC1 signaling system. Inhibition or activation of this self-feeding phenomenon has a great influence on not only simple cell activity, but also physiological or pathological changes in vivo including the cells. Recently, various animal models and genetic analysis of diseases have revealed that autopoiesis process plays an important role in diverse diseases such as diabetes, hypertension, metabolic diseases such as diabetes, degenerative brain disease, infectious diseases, and cancer. . Therefore, the discovery of substances that control autophagy is expected to play a very important role in the development of disease treatments targeted at new action points.

Particularly, studies on substances capable of regulating the self-predation phenomenon related to the treatment of cancer diseases are actively conducted, because a considerable number of cancer cells inhibit the self-predation phenomenon at the early stage of cancer cell growth and induce the growth of cancer cells , And it has also been reported that cancer cells are activated after formation of cancer cells, and show resistance to nutrition, oxygen deficiency and anticancer drugs. Therefore, it can be said that the substance capable of controlling the self-predation phenomenon exhibits an anticancer effect in cancer cells, and researches to develop a method of treating cancer by using such self-predation phenomenon are being actively carried out. For example, Korean Patent Laid-Open Publication No. 2011-0100435 discloses a pharmaceutical composition for preventing or treating auto-predation related diseases and a screening method thereof.

 In addition, the substance capable of inhibiting the self-feeding phenomenon can be used for the treatment of diseases such as inflammatory bowel disease, type 1 diabetes, etc. accompanied by self-feeding phenomenon. For example, Korean Unexamined Patent Publication No. 2008-0088059 discloses an autophosphorylation inhibitor of macrophages containing interleukin-10 as an active ingredient and a binding inhibitor of interleukin-10 and IL-10 receptor or an inhibitor of PI3 kinase I activation, , Which is an autophagy promoter of macrophages.

However, the PI3K-AKT-mTORC1 signaling pathway inhibits autophagy by the absence of a substance that specifically regulates only the autopoiesis process. Thus, activation of the autopoiesis process or activation of the lysosome A method of inhibiting self-feeding by using a substance that interferes with the function is used. However, they are expected to have many adverse effects in the development of drugs targeting autophagic processes by regulating various cellular intracellular physiological activities as well as autophagy.

Under these circumstances, the inventors of the present invention have made extensive efforts to develop a method for effectively identifying a substance that specifically regulates only an autopoiesis process. As a result, it has been found that the formation of Vps34-Beclin1-ATG14L complex specifically involved in self- A high throughput format validation system was designed and used to screen a variety of substances that inhibit the formation of the Vps34-Beclin1-ATG14L complex to identify autophagy-specific inhibitors Thereby completing the present invention.

It is an object of the present invention to provide a method for screening autophagic inhibitors using the Vps34-Beclin1-ATG14L complex.

Another object of the present invention is to provide a kit for screening autopoiesis inhibitors comprising the Vps34-Beclinl complex, ATG14L and agents capable of measuring the levels thereof.

It is another object of the present invention to provide a method for analyzing the binding ability of a target protein capable of binding to Vps34 or a complex comprising Vps34.

The inventors of the present invention have proposed that, in order to develop a method for more effectively providing a substance capable of specifically suppressing the self-predation phenomenon, it is possible to suppress the self-predation by targeting an intracellular factor inducing an autophagy phenomenon It is assumed that the candidate substance can be regarded as a substance capable of specifically suppressing the self-predation phenomenon when the expected candidate substance inhibits the action of the target. While performing various studies based on the assumption, (Induction / nucleation) which induces the phenomenon. The self-predation phenomenon can be divided into induction / nucleation, membrane elongation / closure, and vesicle trafficking / maturation / fusion / degradation. Among them, In addition to self-expression, the various factors involved are also involved in a variety of intracellular transport processes (endosome trafficking, phagocytosis, etc.), so it is appropriate as a target to identify substances that can specifically inhibit the self- It is not.

The ULK1 (ATG1) protein phosphorylation enzyme complex and the PIK3C3 / VPS34 lipid-phosphorylase complex are known to be involved in the induction step. Among them, the ULK1 (ATG1) , The PIK3C3 / VPS34 lipolytic enzyme complex, which has a relatively small number of intracellular interactions to be considered, should be used as a target to specifically identify substances capable of inhibiting the self-propagation phenomenon . However, it is known that the lipid-phosphorylation activity of the complex is involved in various phagocytosis (endosome trafficking / maturation, etc.) other than autophagic phenomenon. Therefore, rather than using the lipid-phosphorylation activity of the complex as a target, As a target.

The PIK3C3 / VPS34 lipolytic enzyme complex can exhibit various forms depending on the constituent elements of the complex. Among these various types of PIK3C3 / VPS34 lipoprotein complexes, the complex (Vps34-Beclin1-ATG14L) containing the ATG14L protein as a component is known as an autophagic specific PIK3C3 / VPS34 complex (pro-autophagy complex) , The suppression of ATG14L expression is specifically suppressed only by the self-predilation phenomenon among the various functions of the PIK3C3 / VPS34 complex. Therefore, the substance that inhibits the formation of the complex can be used as a substance capable of specifically suppressing the self-predation phenomenon Can be expected.

Also, among the complexes (Vps34-Beclin1-ATG14L) containing the ATG14L protein as a constituent, Vps34-Beclin1 is a core region commonly found in various PIK3C3 / VPS34 lipoprotein complexes, so that the self- (Vps34-Beclin1-ATG14L), the binding of ATG14L was used as a practical target. That is, in the formation of the PIK3C3 / VPS34 lipolytic enzyme complex (Vps34-Beclin1-ATG14L), a substance which inhibits the binding of ATG14L can be used as a substance capable of specifically suppressing the self-propagation phenomenon. By confirming whether or not the complex (Vps34-Beclin1-ATG14L) is formed by simultaneously or sequentially adding ATG14L and the candidate substance expected to specifically suppress the self-predation to the constructed complex, Can be screened.

Meanwhile, the method of screening a substance capable of specifically inhibiting the self-propagation phenomenon described above may be modified to analyze the binding force of a target protein capable of binding to Vps34 or a complex containing Vps34. The target protein capable of binding to the complex is treated at various concentrations, the concentration of the conjugate to which the desired protein is bound is determined in the Vps34 or the complex containing the same, and the concentration of the treated target protein and the concentration of the quantified complex Can be used to analyze the binding strength of a target protein that can bind to Vps34 or a complex containing Vps34.

In order to achieve the above-mentioned object, the present invention provides a method for screening an autophagy inhibitor using Vps34, Beclin1 and ATG14L as one embodiment.

As a first method of screening an autophagy inhibitor provided by the present invention, there is provided a method of screening a candidate substance which is expected to specifically inhibit autophagy in a Vps34-Beclin1-ATG14L complex; And (b) identifying the level of Beclinl and ATG14L via said Vps34. At this time, the candidate substance may be a compound, an extract of a natural product, a peptide, an aptamer, etc., though not limited thereto.

The term "autophagy or autophagocytosis" of the present invention means a basic mechanism of action involved in the degradation of intracellular organelles that exhibits an unnecessary or abnormal function using lysosomes. In special circumstances such as malnutrition It is possible to exhibit an effect of improving the cell viability by destroying the intracellular organelles and maintaining the intracellular energy level. Substantial autophagy is characterized by the depletion of cytoplasm and intracellular organs into a double-membrane-bound structure, called autophagosome, which fuses with the lysosomes to form an autolysosome, It is known that the material degraded by lysosomes is hydrolyzed and reused.

In the present invention, the autophoresis can be interpreted as an intracellular action that can be suppressed by a substance that is induced by the formation of Vps34-Beclin1-ATG14L complex and inhibits binding of ATG14L contained in the complex.

The term "Vps34 (vacuolar protein sorting 34)" of the present invention is also referred to as "PIK3C3 (Phosphatidylinositol 3-kinase catalytic subunit type 3) ", and refers to a protein involved in intracellular autophagy. The Vps34 is classified as a class III PI 3-kinase expressed in eukaryotic cells, and exhibits an enzyme activity that phosphorylates phosphatidylinositol to form phosphatidylinositol (3) -phosphate. The specific base sequence and protein information of the gene encoding the protein is known from NCBI (GenBank: NM_002647, NP_002638, etc.). As an example, in the present invention, a protein consisting of the amino acid sequence of SEQ ID NO: 1 was used as Vps34.

The term "Beclin1" of the present invention means a protein expressed by the BECN1 gene and an ortholog protein of Atg6 (autophagy-related gene 6) expressed in yeast. Beclin1 plays an important role in development, cancer development, and neurodegeneration as well as participates in self-predation. The specific base sequence and protein information of the gene encoding the protein are known from NCBI (GenBank: NM_003766, NP_003757, etc.). As one example, in the present invention, a protein consisting of the amino acid sequence of SEQ ID NO: 2 was used as Beclin1.

The term "ATG14L (autophagy related 14)" of the present invention is also referred to as "Barkor (beclin 1-associated autophagy-related key regulator) " The specific base sequence and protein information of the gene encoding the protein are known from NCBI (GenBank: NM_014924.4, NP_055739.2, etc.). As an example, in the present invention, a protein consisting of the amino acid sequence of SEQ ID NO: 3 was used as ATG14L.

The method can be performed to screen for agents capable of specifically removing ATG14L from the Vps34-Beclin1-ATG14L complex that is specifically formed in the autophoresis process, wherein the agent is selected from the group consisting of ATG14L alone in the Vps34-Beclin1-ATG14L complex Only when specifically removed, the formulation can be judged as an autophagy inhibitor. For example, the level of Beclin1 measured in step (b) is not lower than the level of Beclin1 before the reaction with the candidate substance, and the level of ATG14L measured in step (b) Only when the level of ATG14L is lower than that of ATG14L, it can be determined that the candidate substance is an autophagy inhibitor.

In this case, the method of measuring the level of Beclin1 or ATG14L is not particularly limited. For example, the level of Beclin1 or ATG14L may be measured by FRET (Fluorescence Resonance Energy Transfer), western blotting, ELISA (enzyme linked immunosorbent assay) RIA: radioimmunoassay, radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, immunoprecipitation assay, and the like.

The term "antibody" of the present invention means a proteinaceous molecule capable of specifically binding to an antigenic site of a protein or peptide molecule. Such an antibody can be obtained by cloning a gene into an expression vector according to a conventional method, A protein encoded by the gene can be obtained and can be produced from the obtained protein by a conventional method. The form of the antibody is not particularly limited, and any polyclonal antibody, monoclonal antibody or antigen-binding antibody thereof may be included in the antibody of the present invention, and any immunoglobulin antibody may be included, Specific antibodies of the invention. In addition, the antibody comprises a functional fragment of an antibody molecule as well as a complete form having two full-length light chains and two full-length heavy chains. A functional fragment of an antibody molecule means a fragment having at least an antigen-binding function, and can be Fab, F (ab ') 2, F (ab') 2, Fv and the like.

In the present invention, the antibody may be an antibody capable of specifically binding to Vps34, Beclin1, or ATG14L to be measured in the reactant, preferably, a polyclonal antibody capable of specifically binding to each protein An antibody, a monoclonal antibody or a part thereof.

In addition, the Vps34-Beclin1-ATG14L complex may be used either in a non-fixed state or in a fixed state to a carrier. When the Vps34-Beclin1-ATG14L complex is used in a non-fixed state, (b) may be carried out. When used in a fixed state on the carrier, step (b) may be carried out by an enzyme linked immunosorbent assay (ELISA) or the like.

When the complex is used in a form fixed to a carrier, the carrier is not particularly limited. For example, the carrier may be a carrier made of a material such as nitrocellulose, polyvinyl resin, polystyrene resin, or glass. A 96-well plate, a bead or a slide glass, or a carrier having a shape such as a membrane, a 96-well plate, a bead or a slide glass, and as another example, a nitrocellulose, a polyvinyl resin, a polystyrene resin, And the like. In particular, when the carrier has the form of a 96-well plate, a screening method can be simultaneously performed on a plurality of candidate substances.

In order to accurately measure the levels of Beclin1 and ATG14L contained in the Vps34-Beclin1-ATG14L complex immobilized on the carrier in the step (b) when the Vps34-Beclin1-ATG14L complex is immobilized on the carrier, The carrier may be washed to remove Beclinl and ATG14L that are not fixed to the carrier.

As a second method of the screening method of an autophagy inhibitor provided by the present invention, there is provided a method for screening a self-sustaining inhibitor, comprising the steps of: (a) treating ATG14L with a candidate substance which is expected to specifically inhibit autopatching of Vps34-Beclinl complex; And (b) identifying the level of ATG14L bound to the Vps34-Beclinl complex. At this time, the Vps34-Beclin1 complex may also be fixed to the carrier. In this case, the carrier is the same as described above. In step (b), ATG14L contained in the Vps34-Beclin1- ATG14L complex immobilized on the carrier After step (a) is completed, washing the carrier to remove the ATG14L that is not fixed to the carrier, in order to accurately measure the level of the ATG14L. In addition, the method of confirming the level of the candidate substance disclosed in the above method, Vps34, Beclin1, ATG14L, ATG14L and the like are the same as described above.

The second method can screen an autophagic inhibitor having different characteristics from the first provided method. Specifically, the first method provided is a method of screening a substance capable of specifically removing ATG14L from the Vps34-Beclin1-ATG14L complex as an autopoiesis inhibitor, while the second method provides a Vps34-Beclin1 complex A method for screening a substance capable of inhibiting the binding of ATG14L to an ATG14L as an autophagy inhibitor. As a result, an autophagy inhibitor having different characteristics can be individually screened.

Thus, the second method can be performed to screen for agents capable of specifically inhibiting the binding of ATG14L to the Vps34-Beclinl complex specifically formed in the autophoresis process, wherein the agent is a Vps34-Beclinl complex In the case of inhibiting the binding of ATG14L to ATG14L, the agent can be judged as an autophagy inhibitor. For example, if the level of ATG14L measured in step (b) is lower than the level of ATG14L identified by the reaction with only ATG14L without treating the candidate substance, the candidate substance is determined to be an autopoiesis inhibitor Can be used.

The screening method of the self-suppressing agent provided by the present invention will be described with reference to the drawings.

As an example, the autophagic specific inhibitor discovery method disclosed in Fig. 1 can be used.

The GST-Vps34 protein is immobilized in a GSH-coated microwell plate in a fixed direction. Beclin1 and ATG14L proteins were sequentially added to the plate to reconstitute the essential VPS34-Beclin1-ATG14L complex, a PIK3C3 / VPS34 complex for promoting self-stimulation. Binding of each protein was confirmed by ELISA reaction for each protein. To screen autophagic specific inhibitors, a library of the desired substance such as a compound, a peptide, and an aptamer library was added to the prepared assay plate, and the levels of Beclin1 and ATG14L remaining on the plate were measured, It was confirmed whether or not the ginseng substance showed an inhibitory effect. For example, if the material does not affect the Vps34-Beclin1-ATG14L complex, the ELISA detection results for the Beclin1 and ATG14L proteins will not change (A). If the substance causes release of the GST-Vps34 protein from the plate (B) or inhibits the Vps34-Beclin1 response (C), the ELISA detection signal for the Beclin1 and ATG14L proteins will be reduced. Substance (C) inhibiting the Vps34-Beclin1 response can not be an autophagy-specific inhibitor because the Vps34-Beclin1 response is also observed in the visa-predominant PIK3C3 / VPS34 complex. In contrast, if the substance inhibits the binding of ATG14L to the Vps34-Beclinl complex, the ELISA detection signal for ATG14L, which is not Beclin1, will be reduced

In another aspect, the present invention provides a kit for screening an autophagic inhibitor, which comprises a Vps34-Beclin1 complex, ATG14L, a preparation capable of measuring the level of Beclin1, and a preparation capable of measuring the level of ATG14L.

As described above, the Vps34-Beclinl complex may be treated with ATG14L to form a Vps34-Beclin1-ATG14L complex and then treated with the candidate compound expected to be capable of specifically suppressing the self-predation in the formed complex, -Beclin1 complexes with ATG14L and the candidate substance simultaneously can confirm whether the candidate substance can be judged to be an autophagy inhibitor, so that the Vps34-Beclin1 complex can inhibit the level of the immobilized carrier, ATG14L, Beclin1 Kits comprising a measurable agent and an agent capable of measuring the level of ATG14L can be used for screening autophagic inhibitors. At this time, the Vps34-Beclinl complex may be contained in a non-fixed state or may be contained in a fixed state on the carrier, which is the same as described above.

The term " preparation capable of measuring the level of Beclin1 " of the present invention means a means for measuring the level of Beclin1 from a complex bound to a carrier in performing the screening method provided in the present invention, , Which may be an antibody capable of specifically binding to Beclin1, and the antibody is the same as defined above.

The term " agent capable of measuring the level of ATG14L " of the present invention means a means capable of measuring the level of ATG14L from the complex bound to the carrier in performing the screening method provided by the present invention, , Which may be an antibody capable of specifically binding to ATG14L, and the antibody is the same as defined above.

On the other hand, the kit provided by the present invention can be applied to the screening method described above in addition to the above-mentioned components (carrier Vps34-Beclin1 fixed thereto, ATG14L, a preparation capable of measuring the level of Beclin1, and a preparation capable of measuring the level of ATG14L) May comprise one or more other suitable component compositions, solutions or devices as appropriate.

As an example, the kit of the present invention may comprise a kit wherein the Vps34-Beclinl complex is immobilized on a carrier, ATG14L, an antibody capable of specifically binding to Beclinl, an antibody capable of specifically binding to ATG14L, a chromogenic enzyme or a fluorescent substance A secondary antibody, and a chromogenic substrate. The coloring enzyme may be a peroxidase or an alkaline phosphatase. Examples of the fluorescent material include, but are not limited to, FITC (Fluorescein isothiocyanate), RITC (Rhodamine B- (3-ethylbenzothiazoline-6-sulphonic acid), OPD (o-phenylenediamine), TMB (3, 3 ', 5,5'-Tetramethylbenzidine), and the like.

According to another aspect of the present invention, there is provided a method for assaying the binding ability of a complex comprising Vps34 or Vps34 to Vps34 or a complex, comprising the steps of: (a) And (b) measuring the concentration of the conjugate according to the treatment concentration of the target protein, and measuring the binding strength of the target protein to the Vps34 or the complex, wherein the target protein capable of binding to Vps34 or a complex comprising the same The method comprising: At this time, the complex comprising Vps34 or Vps34 may be fixed to the carrier, and the carrier used here is the same as described above. In the step (b), the level of the binding substance immobilized on the carrier is accurately measured The method may further comprise washing the carrier after the step (a) is completed to remove the target protein not immobilized on the carrier.

In the method for analyzing the binding force of a target protein capable of binding to Vps34 or a complex comprising Vps34 provided by the present invention, the complex of step (a) may be a complex containing Vps34 (Vps34-Beclin1 complex) ; The target protein of step (a) may be Beclin1 or ATG14L.

In addition, the binding force measurement in step (b) may be performed using a binding curve prepared using the concentration of the target protein treated in step (a) and the concentration of the conjugate measured in step (b) The concentration of the conjugate may be measured by, for example, fluorescence resonance energy transfer (FRET), western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA) ), Radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, immunoprecipitation assays, and the like.

The use of the self-suppressing agent screening method provided in the present invention can be used to develop a composition for treating various diseases through inhibition of autopoiesis, since a preparation capable of specifically suppressing the self-predation phenomenon can be identified There will be.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic diagram showing an overview of a method for screening autophagic specific inhibitors using the Vps34-Beclin1-ATG14L complex.
FIG. 2 is a photograph showing the results of electrophoresis of proteins expressing and purifying each protein capable of forming Vps34-Beclin1-ATG14L complex.
FIG. 3A is a graph showing the results of verifying the effect of GST and GSH on the binding of the Vps34 protein to the microwell plate. FIG.
Figure 3b shows the binding saturation curve of GST-Vps34 fusion protein to GSH-conjugated microwell plate.
FIG. 3c is a photograph showing Western blot analysis of various concentrations of GST-Vps34 fusion protein bound to GSH-conjugated microwell plate.
FIG. 4A is a graph showing the results of verifying the effect of Vps34 on the binding of Beclin1 protein to Vps34-conjugated microwell plate. FIG.
FIG. 4B shows the binding saturation curve of the Flag-Beclin1 fusion protein to the Vps34-conjugated microwell plate.
FIG. 4C is a photograph showing Western blot analysis of Flag-Beclin 1 fusion proteins bound to Vps34-conjugated microwell plates at various concentrations.
FIG. 5A is a graph showing the results of verifying the effect of Beclin 1 in binding of ATG14L protein to a microwell plate to which Vps34-Beclin 1 complex is bound.
5B shows the binding saturation curve of the Flag-ATG14L fusion protein to the microwell plate to which the Vps34-Beclinl complex is bound.
5C is a photograph showing Western blot analysis of various concentrations of Flag-ATG14L fusion protein bound to a Vps34-Beclin complex conjugated microwell plate.
FIG. 6 is a graph showing the results of verifying the competitive inhibition effect of UVRAG in the binding of ATG14L protein to a microwell plate coupled with Vps34-Beclinl complex.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example  One: Self-predation  Preparation of Proteins Constituting Complex for Acceleration

First, a polynucleotide encoding a fusion protein in which a GST residue is bonded to the N-terminus of Vps34, a polynucleotide encoding a fusion protein in which a Flag residue is bonded to the N-terminus of Beclin1, Polynucleotide encoding a fusion protein in which the Flag residues are combined is prepared and introduced into a mammalian cell expression vector to prepare an expression vector capable of expressing each fusion protein.

Then, each of the above expression vectors was introduced into HEK293 cells cultured in DMEM medium containing glucose (25 mM), 10% FBS and 50 / penicillin / streptomycin, respectively, to obtain transformants capable of expressing the respective fusion proteins Respectively.

Then, each transformant was cultured, and then each of the transformants was washed with cold cell lysis buffer (10 mM Tris, 2 mM EDTA, 100 mM NaCl, 1% NP-40, 50 mM NaF, 1 mM Na3VO4 and protease inhibitor , pH 7.5) to obtain a cell lysate, and the cell lysate was centrifuged (14,000 rpm and 10 minutes) to obtain a supernatant. The supernatant was added to GSH-Sepharose beads (GST-Vps34) or Flag-M2 agarose beads (Flag-Beclin1 or Flag-ATG14L) equilibrated with the cell lysis buffer and incubated for 4 to 2 hours Lt; / RTI > The reaction was centrifuged (1,000 rpm and 2 minutes) to recover the precipitated beads, followed by washing with a cell lysis buffer containing a high concentration of NaCl (0.5 to 1 M). The washed beads were finally washed with cell lysis buffer and then lysed using GSH elution buffer (20 mM GSH and 50 mM Tris, pH 8.0) or 3XFlag elution buffer (cell lysis buffer containing 200 ng / 3XFlag peptide) Proteins were recovered from the beads. The recovered protein was dialyzed overnight at 4 with a dialysis solution (10% glycerol and 20 mM Tris, pH 8.0) to prepare each of the desired fusion proteins (FIG. 2).

FIG. 2 is a photograph showing the results of electrophoresis of proteins expressing and purifying each protein capable of forming Vps34-Beclin1-ATG14L complex. As shown in FIG. 2, it was confirmed that each protein constituting the Vps34-Beclin1-ATG14L complex capable of promoting autophagy was produced in the form of a fusion protein (GST-Vps34, Flag-Beclin1 or Flag-ATG14L).

Example  2: GSH end Combined Microwell  Plate making

A microwell plate capable of binding GST-Vps34 among the fusion proteins prepared in Example 1 was prepared.

Spherically, PBS containing 100 mM GSH (pH 8.0) was added to each well of a 96-well microwell plate and reacted overnight at 4 or completely dried at 60 overnight. Subsequently, the microwell plate was washed with TBST buffer (150 mM NaCl, 0.05% Tween 20 and 10 mM Tris, pH 8.0) to remove unbound GSH, and treated with PBS containing 5% defatted milk to block , And a GSH-coupled microwell plate was produced without causing non-specific binding on the surface.

Example  3: GST - Vps34 Fusion protein GSH end Combined Microwell  Analysis of binding activity of plates

Example  3-1: ELISA analysis

A solution obtained by dissolving the GST-Vps34 fusion protein prepared in Example 1 in TBST buffer was obtained. The solution was dispensed into each well of the GSH-conjugated microwell plate prepared in Example 2, and shaken slowly 4, and for comparison, GSH-conjugated microwell plates were used instead of GSH-conjugated microwell plates, or GST-Vps34 fusion protein was used instead of Vps34 protein . After completion of the reaction, each well of each of the microwell plates was washed with TBST buffer (TBST-P5 buffer) containing 0.5% NP-40, and the primary antibody (1: 500) diluted in TBST buffer Were dispensed in 50 wells into each well, and reacted at room temperature for 1 hour. After completion of the reaction, each of the wells was washed with TBST buffer, and HRP-conjugated secondary antibody was added thereto, followed by reaction at room temperature for 1 hour. Then, the level of the Vps34 protein bound to each microwell plate was quantitated by measuring the color development level at 450 nm by using a TMB solution as a substrate to induce a color development reaction (Fig. 3A).

FIG. 3A is a graph showing the results of verifying the effect of GST and GSH on the binding of the Vps34 protein to the microwell plate. FIG. As shown in FIG. 3A, it was confirmed that when the GST-Vps34 fusion protein was bound to the GSH-conjugated microwell plate, the most effectively Vps34-conjugated microwell plate could be produced.

Example  3-2: Analysis of bond saturation curves

To prepare a binding saturation curve of GST-Vps34 fusion protein to GSH-conjugated microwell plate, various concentrations of GST-Vps34 fusion protein were added to a GSH-conjugated microwell plate and the same as in Example 3-1 , And then the binding saturation curve was prepared using the same (FIG. 3B).

Figure 3b shows the binding saturation curve of GST-Vps34 fusion protein to GSH-conjugated microwell plate. As shown in FIG. 3B, when the GST-Vps34 fusion protein was added to a GSH-conjugated microwell plate at a level of 150 to 200 nM, it was confirmed that their binding level was saturated and no longer increased.

Example  3-3: Immune blot  analysis

Immunoblot analysis was performed to compare the binding activity of GST-Vps34 fusion protein to GSH-conjugated microwell plates at different treatment concentrations. That is, the GST-Vps34 fusion protein of various concentrations (42.5, 85, 170, or 340 nM) was added to each well of the GSH-conjugated microwell plate and the wells were washed and then the SDS-PAGE sample buffer was added , And the GST-Vps34 fusion protein bound to each well was extracted. The extracted GST-Vps34 fusion protein was applied to 10% SDS-PAGE and Western blot analysis using an anti-Vps34 antibody was performed (FIG. 3C).

FIG. 3c is a photograph showing Western blot analysis of various concentrations of GST-Vps34 fusion protein bound to GSH-conjugated microwell plate. As shown in FIG. 3C, when the GST-Vps34 fusion protein is added at a concentration of 170 nM or more, the level of the GST-Vps34 fusion protein bound to the microwell plate is not increased any more, Could know.

Example  4: Flag- Beclin1 Fusion protein Vps34 end Combined Microwell  Analysis of binding activity of plates

Example  4-1: ELISA analysis

A solution obtained by dissolving the Flag-Beclin 1 fusion protein prepared in Example 1 in TBST buffer was obtained. The solution was dispensed into each well of the Vps34-conjugated microwell plate prepared in Example 3-1, For 2 hours, and for comparison, Vps34 was reacted using a microwell plate that was not bound in place of the coupled microwell plate. After the reaction was terminated, the method of Example 3-1 was carried out except that the primary antibody for Beclinl diluted in TBST buffer (1: 100) was used to detect the binding of Vps34 bound to Vps34 The level of Beclin1 protein was quantitated (Figure 4a).

FIG. 4A is a graph showing the results of verifying the effect of Vps34 on the binding of Beclin1 protein to Vps34-conjugated microwell plate. FIG. As shown in FIG. 4A, it was confirmed that when the Flag-Beclin1 fusion protein was bound to the Vps34-conjugated microwell plate, the microwell plate with Vps34-Beclin1 binding most effectively could be produced.

Example  4-2: Binding saturation curve analysis

In order to prepare a binding saturation curve of the Flag-Beclin1 fusion protein to the Vps34-conjugated microwell plate, various concentrations of the Flag-Beclin1 fusion protein were added to the Vps34-conjugated microwell plate, , And then a binding saturation curve was prepared using this (FIG. 4B).

FIG. 4B shows the binding saturation curve of the Flag-Beclin1 fusion protein to the Vps34-conjugated microwell plate. As shown in FIG. 4B, when the Flag-Beclin1 fusion protein was added to a Vps34-conjugated microwell plate at a level of 1.5 M or more, it was confirmed that their binding level was saturated and no longer increased.

Example  4-3: Immune blot  analysis

Immunoblot analysis was performed to compare the binding activity of the Flag-Beclin1 fusion protein to the Vps34-conjugated microwell plate by treatment concentration. That is, a Flag-Beclin 1 fusion protein of various concentrations (40.18, 0.36, 0.73, or 1.46 M) was added to each well of a microwell plate to which Vps34 was conjugated and the wells were washed and then a buffer for SDS- And the Flag-Beclin1 fusion protein bound to each well was extracted. The extracted Flag-Beclinl fusion protein was applied to 10% SDS-PAGE, and Western blot analysis using an anti-Bechlin1 antibody was performed (FIG. 4C).

FIG. 4C is a photograph showing Western blot analysis of Flag-Beclin 1 fusion proteins bound to Vps34-conjugated microwell plates at various concentrations. As shown in FIG. 4C, when the Flag-Beclin 1 fusion protein was added at a concentration of 1.46 M or more, the level of the Vps34-Beclin 1 complex bound to the microwell plate was not increased any more, Could know.

Example  5: Flag- ATG14L Fusion protein Vps34 - Beclin1  Complex Combined Microwell  Analysis of binding activity of plates

Example  5-1: ELISA analysis

A solution obtained by dissolving the Flag-ATG14L fusion protein prepared in Example 1 in TBST buffer was obtained, and the solution was added to each well of the Vps34-Beclin complex conjugated microwell plate prepared in Example 4-1 The reaction was carried out at room temperature for 2 hours. For comparison, a Vps34-Beclin complex conjugated microwell plate was used instead of a microwell plate coupled with Vps34. After the reaction was terminated, the method of Example 3-1 was performed except that the primary antibody for ATG14L (1: 500) diluted in TBST buffer was used to prepare a Vps34-Beclinl complex conjugated microwell plate (Fig. 5A). ≪ tb > < TABLE >

FIG. 5A is a graph showing the results of verifying the effect of Beclin 1 in binding of ATG14L protein to a microwell plate to which Vps34-Beclin 1 complex is bound. As shown in FIG. 5A, it was confirmed that when the Flag-ATG14L fusion protein was bound to the Vps34-Beclin1 complex-bound microwell plate, the microwell plate with the Vps34-Beclin1-ATG14L complex was most effectively produced.

Example  5-2: Analysis of bond saturation curves

In order to prepare a binding saturation curve of the Flag-ATG14L fusion protein to the Vps34-Beclin1 complex-conjugated microwell plate, various concentrations of Flag-ATG14L fusion protein were added to the Vps34-Beclinl complex-bound microwell plate, The color development level was measured in the same manner as in Example 3-1, and then the binding saturation curve was prepared using the same (FIG. 5B).

5B shows the binding saturation curve of the Flag-ATG14L fusion protein to the microwell plate to which the Vps34-Beclinl complex is bound. As shown in FIG. 5B, when the Flag-ATG14L fusion protein was added to a microwell plate conjugated with Vps34-Beclinl complex at a level of 12 nM or more, it was confirmed that their binding level was saturated and no longer increased.

Example  5-3: Immune blot  analysis

Immunoblot analysis was performed to compare the binding activity of the Flag-ATG14L fusion protein to the Viss34-Beclin complex conjugated microwell plate by treatment concentration. That is, a Flag-ATG14L fusion protein of various concentrations (0.78, 1.56, 3.12, 6.25, or 12.5 nM) was added to each well of a microwell plate to which a Vps34- Beclin1 complex was bound and the wells were washed, PAGE sample buffer was added to extract the Flag-ATG14L fusion protein bound to each of the wells. The extracted Flag-ATG14L fusion protein was applied to 10% SDS-PAGE and subjected to Western blot analysis using anti-ATG14L antibody (FIG. 5C).

5C is a photograph showing Western blot analysis of various concentrations of Flag-ATG14L fusion protein bound to a Vps34-Beclin complex conjugated microwell plate. As shown in FIG. 5C, when the Flag-ATG14L fusion protein was added at a concentration of 12.5 nM or more, the level of the Vps34-Beclin1-ATG14L complex bound to the microwell plate was not increased any more, .

Example  6: Vps34 - Beclin1 - ATG14L  Complex Self-predation  Validation of specific inhibitor screening method

Since a substance that inhibits the binding of Beclin1 to ATG14L in the Vps34-Beclin1-ATG14L complex prepared in Example 5-1 can be regarded as an autopoietic-specific inhibitor, a protein capable of competing with ATG14L in binding to Beclin1 UVRAG < / RTI > known as < RTI ID = 0.0 >

First, a Flag-UVRAG fusion protein was prepared by performing the method of Example 1, except that a polynucleotide encoding a fusion protein in which Flag residue was bound to the N-terminus of UVRAG was used.

Next, each of the solutions obtained by dissolving the Flag-ATG14L fusion protein prepared in Example 1 or the Flag-UVRAG fusion protein prepared above in various concentrations in TBST buffer was simultaneously added to the Vps34-Beclin1 The complex was dispensed into each well of a microwell plate coupled, reacted for 2 hours at room temperature, and then diluted in TBST buffer (1: 500), using the respective primary antibody for ATG14L or UVRAG. The method of Example 3-1 was performed to quantitate the level of ATG14L protein bound to a Vps34-Beclinl complex-conjugated microwell plate (Fig. 6).

FIG. 6 is a graph showing the results of verifying the competitive inhibition effect of UVRAG in the binding of ATG14L protein to a microwell plate coupled with Vps34-Beclinl complex. As shown in FIG. 6, when UVRAG was not treated, a high level of ATG14L protein bound to the microwell plate was detected. However, as the level of UVRAG treatment increased, the level of ATG14L protein bound to the microwell plate Respectively.

Therefore, it was found that a substance that inhibits the binding of Beclin1 and ATG14L, which is expected to specifically inhibit autophagy, can be screened through the Vps34-Beclin1-ATG14L complex.

<110> University-Industry Cooperation Group of Kyung Hee University <120> Assay method for autophagy-specific inhibitor screening <130> KPA141262-KR <160> 3 <170> Kopatentin 2.0 <210> 1 <211> 887 <212> PRT <213> Artificial Sequence <220> <223> recombinant Vps34 <400> 1 Met Gly Glu Ala Glu Lys Phe His Tyr Ile Tyr Ser Cys Asp Leu Asp   1 5 10 15 Ile Asn Val Gln Leu Lys Ile Gly Ser Leu Glu Gly Lys Arg Glu Gln              20 25 30 Lys Ser Tyr Lys Ala Val Leu Glu Asp Pro Met Leu Lys Phe Ser Gly          35 40 45 Leu Tyr Gln Glu Thr Cys Ser Asp Leu Tyr Val Thr Cys Gln Val Phe      50 55 60 Ala Glu Gly Lys Pro Leu Ala Leu Pro Val Arg Thr Ser Tyr Lys Ala  65 70 75 80 Phe Ser Thr Arg Trp Asn Trp Asn Glu Trp Leu Lys Leu Pro Val Lys                  85 90 95 Tyr Pro Asp Leu Pro Arg Asn Ala Gln Val Ala Leu Thr Ile Trp Asp             100 105 110 Val Tyr Gly Pro Gly Lys Ala Val Pro Val Gly Gly Thr Thr Val Ser         115 120 125 Leu Phe Gly Lys Tyr Gly Met Phe Arg Gln Gly Met His Asp Leu Lys     130 135 140 Val Trp Pro Asn Val Glu Ala Asp Gly Ser Glu Pro Thr Lys Thr Pro 145 150 155 160 Gly Arg Thr Ser Ser Thr Leu Ser Glu Asp Gln Met Ser Arg Leu Ala                 165 170 175 Lys Leu Thr Lys Ala His Arg Gln Gly His Met Val Lys Val Asp Trp             180 185 190 Leu Asp Arg Leu Thr Phe Arg Glu Ile Glu Met Ile Asn Glu Ser Glu         195 200 205 Lys Arg Ser Ser Asn Phe Met Tyr Leu Met Val Glu Phe Arg Cys Val     210 215 220 Lys Cys Asp Asp Lys Glu Tyr Gly Ile Val Tyr Tyr Glu Lys Asp Gly 225 230 235 240 Asp Glu Ser Ser Ile Leu Thr Ser Phe Glu Leu Val Lys Val Pro                 245 250 255 Asp Pro Gln Met Ser Met Glu Asn Leu Val Glu Ser Lys His His Lys             260 265 270 Leu Ala Arg Ser Leu Arg Ser Gly Pro Ser Asp His Asp Leu Lys Pro         275 280 285 Asn Ala Ala Thr Arg Asp Gln Leu Asn Ile Ile Val Ser Tyr Pro Pro     290 295 300 Thr Lys Gln Leu Thr Tyr Glu Glu Gln Asp Leu Val Trp Lys Phe Arg 305 310 315 320 Tyr Tyr Leu Thr Asn Gln Glu Lys Ala Leu Thr Lys Phe Leu Lys Cys                 325 330 335 Val Asn Trp Asp Leu Pro Gln Glu Ala Lys Gln Ala Leu Glu Leu Leu             340 345 350 Gly Lys Trp Lys Pro Met Asp Val Glu Asp Ser Leu Glu Leu Leu Ser         355 360 365 Ser His Tyr Thr Asn Pro Thr Val Arg Arg Tyr Ala Val Ala Arg Leu     370 375 380 Arg Gln Ala Asp Asp Glu Asp Leu Leu Met Tyr Leu Leu Gln Leu Val 385 390 395 400 Gln Ala Leu Lys Tyr Glu Asn Phe Asp Asp Ile Lys Asn Gly Leu Glu                 405 410 415 Pro Thr Lys Lys Asp Ser Gln Ser Ser Val Ser Glu Asn Val Ser Asn             420 425 430 Ser Gly Ile Asn Ser Ala Glu Ile Asp Ser Ser Gln Ile Ile Thr Ser         435 440 445 Pro Leu Pro Ser Val Ser Ser Pro Pro Pro Ala Ser Lys Thr Lys Glu     450 455 460 Val Pro Asp Gly Glu Asn Leu Glu Gln Asp Leu Cys Thr Phe Leu Ile 465 470 475 480 Ser Arg Ala Cys Lys Asn Ser Thr Leu Ala Asn Tyr Leu Tyr Trp Tyr                 485 490 495 Val Ile Val Glu Cys Glu Asp Gln Asp Thr Gln Gln Arg Asp Pro Lys             500 505 510 Thr His Glu Met Tyr Leu Asn Val Met Arg Arg Phe Ser Gln Ala Leu         515 520 525 Leu Lys Gly Asp Lys Ser Val Arg Val Val Met Arg Ser Leu Leu Ala Ala     530 535 540 Gln Gln Thr Phe Val Asp Arg Leu Val His Leu Met Lys Ala Val Gln 545 550 555 560 Arg Glu Ser Gly Asn Arg Lys Lys Lys Asn Glu Arg Leu Gln Ala Leu                 565 570 575 Leu Gly Asp Asn Glu Lys Met Asn Leu Ser Asp Val Glu Leu Ile Pro             580 585 590 Leu Pro Leu Glu Pro Gln Val Lys Ile Arg Gly Ile Ile Pro Glu Thr         595 600 605 Ala Thr Leu Phe Lys Ser Ala Leu Met Pro Ala Gln Leu Phe Phe Lys     610 615 620 Thr Glu Asp Gly Gly Lys Tyr Pro Val Ile Phe Lys His Gly Asp Asp 625 630 635 640 Leu Arg Gln Asp Gln Leu Ile Leu Gln Ile Ile Ser Leu Met Asp Lys                 645 650 655 Leu Leu Arg Lys Glu Asn Leu Asp Leu Lys Leu Thr Pro Tyr Lys Val             660 665 670 Leu Ala Thr Ser Thr Lys His Gly Phe Met Gln Phe Ile Gln Ser Val         675 680 685 Pro Val Ala Glu Val Leu Asp Thr Glu Gly Ser Ile Gln Asn Phe Phe     690 695 700 Arg Lys Tyr Ala Pro Ser Glu Asn Gly Pro Asn Gly Ile Ser Ala Glu 705 710 715 720 Val Met Asp Thr Tyr Val Lys Ser Cys Ala Gly Tyr Cys Val Ile Thr                 725 730 735 Tyr Ile Leu Gly Val Gly Asp Arg His Leu Asp Asn Leu Leu Leu Thr             740 745 750 Lys Thr Gly Lys Leu Phe His Ile Asp Phe Gly Tyr Ile Leu Gly Arg         755 760 765 Asp Pro Lys Pro Leu Pro Pro Pro Met Lys Leu Asn Lys Glu Met Val     770 775 780 Glu Gly Met Gly Gly Thr Gln Ser Glu Gln Tyr Gln Glu Phe Arg Lys 785 790 795 800 Gln Cys Tyr Thr Ala Phe Leu His Leu Arg Arg Tyr Ser Asn Leu Ile                 805 810 815 Leu Asn Leu Phe Ser Leu Met Val Asp Ala Asn Ile Pro Asp Ile Ala             820 825 830 Leu Glu Pro Asp Lys Thr Val Lys Lys Val Gln Asp Lys Phe Arg Leu         835 840 845 Asp Leu Ser Asp Glu Glu Ala Val His Tyr Met Gln Ser Leu Ile Asp     850 855 860 Glu Ser Val His Ala Leu Phe Ala Ala Val Val Glu Gln Ile His Lys 865 870 875 880 Phe Ala Gln Tyr Trp Arg Lys                 885 <210> 2 <211> 450 <212> PRT <213> Artificial Sequence <220> <223> recombinant Beclin1 <400> 2 Met Glu Gly Ser Lys Thr Ser Asn Asn Ser Thr Met Gln Val Ser Phe   1 5 10 15 Val Cys Gln Arg Cys Ser Gln Pro Leu Lys Leu Asp Thr Ser Phe Lys              20 25 30 Ile Leu Asp Arg Val Thr Ile Gln Glu Leu Thr Ala Pro Leu Leu Thr          35 40 45 Thr Ala Gln Ala Lys Pro Gly Glu Thr Gln Glu Glu Glu Thr Asn Ser      50 55 60 Gly Glu Glu Pro Phe Ile Glu Thr Pro Arg Gln Asp Gly Val Ser Arg  65 70 75 80 Arg Phe Ile Pro Pro Ala Arg Met Met Ser Thr Glu Ser Ala Asn Ser                  85 90 95 Phe Thr Leu Ile Gly Glu Ala Ser Asp Gly Gly Thr Met Glu Asn Leu             100 105 110 Ser Arg Leu Lys Val Thr Gly Asp Leu Phe Asp Ile Met Ser Gly         115 120 125 Gln Thr Asp Val Asp His Pro Leu Cys Glu Glu Cys Thr Asp Thr Leu     130 135 140 Leu Asp Gln Leu Asp Thr Gln Leu Asn Val Thr Glu Asn Glu Cys Gln 145 150 155 160 Asn Tyr Lys Arg Cys Leu Glu Ile Leu Glu Gln Met Asn Glu Asp Asp                 165 170 175 Ser Glu Glu Leu Glu Met Glu Leu Lys Glu Leu Ala Leu Glu Glu Glu             180 185 190 Arg Leu Ile Gln Glu Leu Glu Asp Val Glu Lys Asn Arg Lys Ile Val         195 200 205 Ala Glu Asn Leu Glu Lys Val Gln Ala Glu Ala Glu Arg Leu Asp Gln     210 215 220 Glu Glu Ala Gln Tyr Gln Arg Glu Tyr Ser Glu Phe Lys Arg Gln Gln 225 230 235 240 Leu Glu Leu Asp Asp Glu Leu Lys Ser Val Glu Asn Gln Met Arg Tyr                 245 250 255 Ala Gln Thr Gln Leu Asp Lys Leu Lys Lys Thr Asn Val Phe Asn Ala             260 265 270 Thr Phe His Ile Trp His Ser Gly Gln Phe Gly Thr Ile Asn Asn Phe         275 280 285 Arg Leu Gly Arg Leu Pro Ser Val Pro Val Glu Trp Asn Glu Ile Asn     290 295 300 Ala Ala Trp Gly Gln Thr Val Leu Leu Leu His Ala Leu Ala Asn Lys 305 310 315 320 Met Gly Leu Lys Phe Gln Arg Tyr Arg Leu Val Pro Tyr Gly Asn His                 325 330 335 Ser Tyr Leu Glu Ser Leu Thr Asp Lys Ser Lys Glu Leu Pro Leu Tyr             340 345 350 Cys Ser Gly Gly Leu Arg Phe Phe Trp Asp Asn Lys Phe Asp His Ala         355 360 365 Met Val Ala Phe Leu Asp Cys Val Gln Gln Phe Lys Glu Glu Val Glu     370 375 380 Lys Gly Glu Thr Arg Phe Cys Leu Pro Tyr Arg Met Asp Val Glu Lys 385 390 395 400 Gly Lys Ile Glu Asp Thr Gly Gly Ser Gly Gly Gly Ser Tyr Ser Ile Lys                 405 410 415 Thr Gln Phe Asn Ser Glu Glu Gln Trp Thr Lys Ala Leu Lys Phe Met             420 425 430 Leu Thr Asn Leu Lys Trp Gly Leu Ala Trp Val Ser Ser Gln Phe Tyr         435 440 445 Asn Lys     450 <210> 3 <211> 492 <212> PRT <213> Artificial Sequence <220> <223> recombinant ATG14L <400> 3 Met Ala Ser Pro Ser Gly Lys Gly Ala Arg Ala Leu Glu Ala Pro Gly   1 5 10 15 Cys Gly Pro Arg Pro Leu Ala Arg Asp Leu Val Asp Ser Val Asp Asp              20 25 30 Ala Glu Gly Leu Tyr Val Ala Val Glu Arg Cys Pro Leu Cys Asn Thr          35 40 45 Thr Arg Arg Leu Thr Cys Ala Lys Cys Val Gln Ser Gly Asp Phe      50 55 60 Val Tyr Phe Asp Gly Arg Asp Arg Glu Arg Phe Ile Asp Lys Lys Glu  65 70 75 80 Arg Leu Ser Arg Leu Lys Ser Lys Gln Glu Glu Phe Gln Lys Glu Val                  85 90 95 Leu Lys Ala Met Glu Gly Lys Trp Ile Thr Asp Gln Leu Arg Trp Lys             100 105 110 Ile Met Ser Cys Lys Met Arg Ile Glu Gln Leu Lys Gln Thr Ile Cys         115 120 125 Lys Gly Asn Glu Glu Met Glu Lys Asn Ser Glu Gly Leu Leu Lys Thr     130 135 140 Lys Glu Lys Asn Gln Lys Leu Tyr Ser Arg Ala Gln Arg His Gln Glu 145 150 155 160 Lys Lys Glu Lys Ile Gln Arg His His Arg Lys Leu Gly Asp Leu Val                 165 170 175 Glu Lys Lys Thr Ile Asp Leu Arg Ser His Tyr Glu Arg Leu Ala Asn             180 185 190 Leu Arg Arg Ser Ile Leu Glu Leu Thr Ser Val Ile Phe Pro Ile         195 200 205 Glu Glu Val Lys Thr Gly Val Arg Asp Pro Ala Asp Val Ser Ser Glu     210 215 220 Ser Asp Ser Ala Met Thr Ser Ser Thr Val Ser Lys Leu Ala Glu Ala 225 230 235 240 Arg Arg Thr Thr Tyr Leu Ser Gly Arg Trp Val Cys Asp Asp His Asn                 245 250 255 Gly Asp Thr Ser Ile Ser Ile Thr Gly Pro Trp Ile Ser Leu Pro Asn             260 265 270 Asn Gly Asp Tyr Ser Ala Tyr Tyr Ser Trp Val Glu Glu Lys Lys Thr         275 280 285 Thr Gln Gly Pro Asp Met Glu Gln Ser Asn Pro Ala Tyr Thr Ile Ser     290 295 300 Ala Ala Leu Cys Tyr Ala Thr Gln Leu Val Asn Ile Leu Ser His Ile 305 310 315 320 Leu Asp Val Asn Leu Pro Lys Lys Leu Cys Asn Ser Glu Phe Cys Gly                 325 330 335 Glu Asn Leu Ser Lys Gln Lys Phe Thr Arg Ala Val Lys Lys Leu Asn             340 345 350 Ala Asn Ile Leu Tyr Leu Cys Phe Ser Gln His Val Asn Leu Asp Gln         355 360 365 Leu Gln Pro Leu His Thr Leu Arg Asn Leu Met Tyr Leu Val Ser Pro     370 375 380 Ser Ser Glu His Leu Gly Arg Ser Gly Pro Phe Glu Val Arg Ala Asp 385 390 395 400 Leu Glu Glu Ser Met Glu Phe Val Asp Pro Gly Val Ala Gly Glu Ser                 405 410 415 Asp Glu Ser Gly Asp Glu Arg Val Ser Asp Glu Glu Thr Asp Leu Gly             420 425 430 Thr Asp Trp Glu Asn Leu Pro Ser Pro Arg Phe Cys Asp Ile Pro Ser         435 440 445 Gln Ser Val Glu Val Ser Gln Ser Gln Ser Thr Gln Ala Ser Pro Pro     450 455 460 Ile Ala Ser Ser Ala Gly Gly Met Ile Ser Ser Ala Ala Ser Ser 465 470 475 480 Val Thr Ser Trp Phe Lys Ala Tyr Thr Gly His Arg                 485 490

Claims (16)

(a) treating a candidate substance that is expected to specifically inhibit autopatching in a Vps34 (vacuolar protein sorting 34) -Beclin1-ATG14L (autophagy related 14) complex; And
(b) identifying the level of Beclinl and ATG14L through said Vps34.
The method according to claim 1,
Wherein the Vps34 (vacuolar protein sorting 34) -Beclin1-ATG14L (autophagy related 14) complex is immobilized on a carrier.

The method according to claim 1,
The level of Beclin1 measured in the step (b) is not lower than the level of Beclin1 before the reaction with the candidate substance, and the level of ATG14L measured in the step (b) is lower than the level of ATG14L before the reaction with the candidate substance The candidate substance is determined to be an autophagic inhibitor if the candidate substance is reduced.
(a) treating a candidate substance, ATG14L (autophagy related 14), which is expected to specifically inhibit autopatching in a vacuolar protein sorting 34 (Vps34) -Beclin1 complex; And
(b) identifying the level of ATG14L bound to the Vps34-Beclinl complex.
5. The method of claim 4,
Wherein the Vps34 (vacuolar protein sorting 34) -Beclinl complex is immobilized on a carrier.
5. The method of claim 4,
Wherein the level of ATG14L measured in step (b) is judged to be an autopoiesis inhibitor when the level of ATG14L is reduced compared to the level of ATG14L identified by only reacting ATG14L without treatment of the candidate substance .
A kit for screening autopoiesis inhibitors, comprising an agent capable of measuring the level of Beclin1 and an agent capable of measuring the level of ATG14L, a Vps34 (vacuolar protein sorting 34) -Beclin1 complex, ATG14L (autophagy related 14)
8. The method of claim 7,
Wherein the Vps34-Beclinl complex is immobilized on a carrier.
8. The method of claim 7,
Wherein the agent capable of measuring the level of Beclin1 is an antibody capable of specifically binding to Beclin1.
8. The method of claim 7,
Wherein the agent capable of measuring the level of ATG14L is an antibody capable of specifically binding to ATG14L.
8. The method of claim 7,
Wherein the Vps34-Beclin1-ATG14L complex comprises a fixed 96-well plate, a preparation capable of measuring the level of Beclin1, and an agent capable of measuring the level of ATG14L.
(a) subjecting the complex containing Vps34 (vacuolar protein sorting 34) or Vps34 to treatment with the target protein to measure the binding force with Vps34 or the complex to obtain each conjugate; And
(b) measuring the concentration of the conjugate according to the treatment concentration of the target protein, and measuring the binding strength of the target protein to the Vps34 or the complex, wherein the binding strength of the target protein capable of binding to Vps34 or the complex comprising the same .
13. The method of claim 12,
Wherein the complex comprising Vps34 or Vps34 is immobilized on a carrier.
13. The method of claim 12,
Wherein the complex of step (a) is a Vps34-Beclinl complex.
13. The method of claim 12,
Wherein the target protein of step (a) is Beclin1 or ATG14L (autophagy related 14).
13. The method of claim 12,
Wherein the binding force measurement in step (b) is performed using a binding curve prepared using the concentration of the target protein treated in step (a) and the concentration of the binding substance measured in step (b) .

Images