KR102279295B1 - Method of screening a material regulating autophagy targeting O-GlcNACylation of ULK1 protein - Google Patents

Abstract

O-GlcNAcylated ULK1 according to the present application binds to ATG14L and plays a pivotal role in inducing its phosphorylation, which is followed by activation of VPS34. Accordingly, phagophore formation and initiation of autophagy due to increase in PI3P The method of the present application based on the mechanism leading to autophagy can be usefully used as a target for the development of new drugs for the treatment of diseases related to autophagy. In particular, it was found that ULK1 O-GlcNAcylation contributes to the initial autophagosome progression rather than the fusion of autophagosome and lysosome, and thus can be used in the development of new drugs useful for regulating early autophagy.

Description

Autophagy modulator screening method targeting O-GlcNAc of ULK1 protein {Method of screening a material regulating autophagy targeting O-GlcNACylation of ULK1 protein}

The present application relates to a method for screening autophagy modulators targeting O-GlcNAcylation of ULK1 protein.

In autophagy, in an environment where intracellular nutrients are scarce, cells surround their unnecessary proteins and aging organelles with lipid bilayers (membrane vacuole, autophagosome), which combine with lysosomes to decompose the enclosed internal material. It is a process of altering and modulating intracellular components in a lysosome-dependent manner (Levine and Klionsky, (2004) Dev Cell 6, 463-377) by removing defective large protein complexes and intracellular organelles. , plays an important role in cell growth, survival and homeostasis.

Therefore, it is known that when there is an abnormality in autophagy regulation, the accumulation of misfolded protein is caused, thereby causing neurodegenerative diseases (Komatsu et al., (2006), Nature, 441, 880-884). .

In addition, autophagy is activated in cancer cells (Ding et al., (2009), Mol. Cancer Ther., 8(7), 2036-2045), and autophagy inhibitors are known to act as anticancer therapeutics (Maiuri et al. ., (2007) Nat. Rev. Cell Biol. 8, 741-752). Moreover, in addition to cancer and neurodegenerative diseases, autophagy is known to be associated with liver disease, heart disease, muscle disease, and pancreatic disease (Levine and Kroemer, (2008), Cell, 132, 27-42; Fortunato and Kroemer, ( 2009), Autophagy, 5(6)).

Therefore, research on the development of various therapeutic agents through the regulation of autophagy of cells is in progress.

U.S. Patent Publication No. 2010-0233730 relates to autophagy control for treatment, and after treatment with a test substance in cells, metabolic stress is applied to observe intracellular changes in autophagosomes, A method of screening is disclosed.

US Patent Application Laid-Open No. 2014-0134661 relates to autophagy regulation for treatment, and discloses a screening method for selecting a substance affecting the p62 protein as a marker as an autophagy modulator.

Korean Patent Registration No. 1645359 discloses a method for screening an autophagy modulator that targets the Beclin1 protein.

Autophagy is a complex biological phenomenon involving various proteins, and it is necessary to develop new targets that can control autophagy at an early stage.

The present application aims to provide a screening method for an autophagy modulator that targets O-GlcNAcylation of ULK1 protein, which can regulate autophagy at an early stage.

In one aspect, the present application provides a method comprising: culturing a cell expressing a ULK protein in a low concentration of glucose; contacting the cell with a test substance expected to regulate O-GlcNAcylation at the 755th threonine residue of ULK1 based on the sequence of SEQ ID NO: 1; and after the contact, measuring the degree of O-GlcNAcylation at the 755th threonine residue of ULK1; and in the measuring step, when there is a change in the O-GlcNAcylation compared to the case treated with the test substance compared to the case not treated with the test substance, the test substance is selected as an autophagy modulator candidate It provides a method for screening autophagy modulators, including.

In one embodiment, human proteins are used herein, but the use of other types of proteins is not excluded. For example, based on the sequence of SEQ ID NO: 1, the residue corresponding to the 755th threonine residue in ULK1 corresponds to the 754th residue in the sequence of another species, for example, a mouse, and a person skilled in the art can easily identify the corresponding residue in other positions will be able to specify

In one embodiment, in the selecting step, when the degree of O-GlcNAcylation at the 755th threonine residue of ULK1 is increased compared to the case where the test substance is not treated with the test substance, the test substance is treated as an autophagy activator, or the When there is no change or a decrease in the degree of O-GlcNAcylation compared to the case where the test substance is not treated, the test substance is selected as an autophagy inhibitor candidate.

In another embodiment, the cell of the present application further expresses AMPK and PPI, and the measuring comprises phosphorylation at serine residues 317 and 556 of ULK1 and dephosphorylation at serine residue 758 of ULK1 and measuring. In this case, when the dephosphorylation and phosphorylation are increased compared to the case where the test substance is not treated as a result of the measurement, the test substance is used as an autophagy activator, and as a result of the measurement, the test substance is not treated with the test substance. When the dephosphorylation and phosphorylation do not change or decrease compared to the case, the test substance is selected as an autophagy inhibitor.

In another embodiment, the cell of the present disclosure further expresses ATG14L and VPS34 lipid kinase, and in this case, the measuring step further comprises measuring the phosphorylation of ATG14 and the activity of VPS34 lipid kinase, wherein the measuring Result When the phosphorylation of ATG14L and the activity of VPS34 lipid kinase were increased compared to the case not treated with the test substance, the test substance was an autophagy activator, and the measurement result compared with the case not treated with the test substance. When the phosphorylation of ATG14L and the activity of VPS34 lipid kinase do not change or decrease, the test substance is selected as an autophagy inhibitor candidate.

In one embodiment, the ULK1 protein according to the present disclosure may be a protein in which the 758th serine residue is dephosphorylated, or the ULK1 protein may be a 317th or 556th serine residue is phosphorylated, in which case the cells may be cultured at a normal glucose concentration. .

Cells used in the method according to the present application are not limited as long as they can achieve the object according to the present application, but for example, the cells include H1299, HEK293, or SW620 cell lines.

The autophagy modulator selected by the method according to the present application can be effectively used for preventing or treating cancer, neurodegenerative disease, autoimmune disease, cardiovascular disease, metabolic disease, or hereditary muscle disease.

O-GlcNAcylated ULK1 binds to ATG14L and plays a pivotal role in inducing its phosphorylation, which in turn consists of activation of VPS34, which in turn leads to phagophore formation and initiation of autophagy due to increase in PI3P. The method of the present application based on this method can be usefully used as a target for the development of new drugs for the treatment of diseases related to autophagy. In particular, it was found that ULK1 O-GlcNAcylation contributes to the initial autophagosome progression rather than the fusion of autophagosome and lysosome, and thus can be used in the development of new drugs useful for regulating early autophagy.

1 shows that O-GlcNAcylation of ULK1 by OGT occurs at residue 754 of threonine (A) co-immunoprecipitation experiment of endogenous ULK1 and OGT in H1299 cells (B) wild-type or ULK1-/- with endogenous ULK1 in HEK293 cells OGT co-immunoprecipitation experiment (C) ULK1 O-GlcNAcylation detection after in vitro O-GlcNAcylation experiment (D) ULK1 O-GlcNAcylation detection through SWGA or immunoprecipitation experiment after treatment with OGA inhibitor Thiamet G (1 μM) in H1299 cells (E) O-GlcNAcylation detection of wild-type or deletion mutant △279-833, 1-600, 279-833 aa ULK1 (F) Wild-type or point mutant T635V, T754Y, T635V/T754 ULK1 after purification using Flag-specific antibody Detection of individual O-GlcNAcylation (G) ETD mass spectrometry revealed that ULK1 O-GlcNAcylation occurs at the T754 site (H) ULK1 O-GlcNAcylation site is evolutionarily conserved (I) Threonine 754 is tyrosine (Y), Asparagine (N), mutated to alanine (A). S757 was mutated to alanine (A) to compare and test T754 and S757 at a different position as a control.
Figure 2 shows that ULK1 O-GlcNAcylation is increased in a glucose deprivation situation (A and B) H1299, HEK293, SW620 cells depleted in glucose for the indicated time. Detect endogenous OGT or O-GlcNAcylation by co-immunoprecipitation of samples with ULK1-specific antibodies. The ratio of OGT/ULK1 or LC3-II/β-actin is shown (C) Harvested by depriving H1299 cells and replacing them with normal media for 4 hours (D) Comparing wild-type HEK293 cells with ULK1-/- HEK293 cells for ULK1 This indicates that O-GlcNAcylation is ULK1-specific. Comparison of O-GlcNAcylation in the presence or absence of NADG similar to free UDP-GlcNAc using SWGA beads (E) Non-specific (siNS) or OGT-specific (siOGT) knockdown followed by glucose starvation at the indicated times (F) H1299 cells LC3 puncta quantification after non-specific or OGT-specific knockdown and GFP-LC3 (green) expression via immunostaining in Nuclei are stained with DAPI (blue). Three independent experiments in the same manner yielded similar results, representing a symbolic image for each sample. Scale bar 10 μm. P value was obtained through t-test (***p<0.0001). Expressed as mean ± SEM. (G) Immunoprecipitation experiment and immunostaining technique were performed simultaneously. Reduction of ULK1 O-GlcNAcylation by OGT knockdown (H) After knockdown of endogenous ULK1 (siULK1) by immunostaining technique, wild-type or mutant ULK1 resistant to siRNA was restored to obtain a symbolic image of the GFP-LC3 puncta pattern. indicate. Autophagy was induced through glucose deprivation in all samples. Similar results were obtained through three independent experiments in the same manner. Scale bar 10 μm. P value was obtained through t-test (***p<0.0001). Expressed as mean ± SEM. Immunoblotting shows the knockdown efficiency and restoration efficiency of wild-type ULK1 and mutant ULK1 shown in the immunostaining technique.
Figure 3 shows that ULK1 dephosphorylation and O-GlcNAcylation occur sequentially (A) ULK1 T754 O-GlcNAcylation and S757 phosphorylation detection after glucose deprivation in H1299 cells according to the indicated time (B) mTOR inhibitor in H1299 cells Torin 1 was treated according to the indicated time and ULK1 O-GlcNAcylation and S757 phosphorylation were detected (C) ULK1 O-GlcNAcylation was detected after overexpressing an activating mutation of mTOR (mTOR CA). ULK1 O-GlcNAcylation was induced in cells through glucose deprivation for 6 hours. (D) HA-labeled wild-type or mutant (T754N, S757A, S757D) ULK1 was overexpressed in HEK293 cells and HA-specific antibody was used after glucose deficiency Immunoprecipitation was performed to show immunoblotting for the indicated antibody (E) Binding of ULK1 to dephosphorylation enzymes PP1, PP2A, and PP5 was compared according to the indicated glucose depletion time (F) Marked glucose depletion time in H1299 cells Co-immunoprecipitation for endogenous ULK1 and PP1 according to the method (G) Using shRNA to detect ULK1 O-GlcNAcylation by performing an immunoprecipitation experiment after glucose deficiency for the indicated time in H1299 cells in which PP1 was knocked down using shRNA (H) H1299 cells ULK1 O-GlcNAcylation detection after Tautomycetin (TMC) treatment (10 nM) according to the time indicated in (I) Co-immunoprecipitation experiment on ULK1 and OGT after PP1 overexpression in HEK293 cells (J) ULK1 O- after PP1 overexpression GlcNAcylation and S757 phosphorylation detection (K) ULK1 O-GlcNAcylation and S757 phosphorylation detection according to the presence or absence of PP1 overexpression after mTOR CA overexpression in H1299 cells (L) Symbolic GFP-LC3 (green) expression pattern according to the presence or absence of endogenous PP1 knockdown in H1299 cells were compared through immunostaining technique. Nuclei are stained with DAPI (blue). Three independent experiments in the same manner yielded similar results, representing a symbolic image for each sample. Scale bar 10 μm. P value was obtained through t-test (***p<0.0001). Expressed as mean ± SEM. The knockdown efficiency of PP1 was shown by immunoblotting in parallel with the immunostaining technique.
Figure 4 shows that ULK1 O-GlcNAcylation activates VPS34 through ATG14L at the stage of initiation of autophagy (A) Between wild-type or T754N/T754Q mutants and endogenous ATG14L, Beclin1, and VPS34 after glucose deprivation in HEK293 cells for 3 hours Binding is shown by performing co-immunoprecipitation experiments. The sample was divided in half and an immunoprecipitation experiment was performed (B) After the endogenous OGT was knocked down and glucose was depleted for 3 hours, a coimmunoprecipitation experiment was performed using a ULK1-specific antibody. Detection with ATG14L, O-GlcNAc, and ULK1-specific antibodies (C) Co-immunoprecipitation experiments of endogenous ULK1 and ATG14L after overexpression of wild-type or mutant OGT in HEK293 cells (D) After overexpression of mTOR CA mutation in HEK293 cells 3 Glucose deficiency for hours. Co-immunoprecipitation experiment was performed using a ULK1-specific antibody and detection with the indicated antibody (E) After overexpressing PP1 in H1299 cells, OGT was knocked down. Immunoprecipitation experiment was performed using a ULK1-specific antibody, followed by immunoblotting using the indicated antibody. (F) Wild-type, T754N, T754Q, S757A, T754N/S757A mutant ULK1 in ULK1-/- HEK293 cells, respectively. After restoration, glucose starvation was observed for 3 hours.
5 shows that phosphorylation by AMPK precedes ULK1 O-GlcNAcylation (A and B) with or without Compound C (10 μM) treatment in H1299 cells (A), or after AMPKα knockdown via shRNA (B) After time-dependent glucose deprivation, immunoprecipitation experiments were performed using ULK1-specific antibodies and blots were observed using the indicated antibodies (C and D). In HEK293 cells, HA-labeled wild-type, S317A, S467A, S555A, T574A, S637A, S777A mutant ULK1 (C) or HA-labeled wild-type, S317D, S467D, S555D, T574D, S637D, S777D mutant ULK1 (D) was overexpressed with OGT and given glucose deprivation for 3 hours. After performing an immunoprecipitation experiment using an HA-specific antibody, the blot was observed using the indicated antibody. The O-GlcNAc/HA ratio is shown.
6 shows that ULK1 dephosphorylation and O-GlcNAcylation regulate the initial stage of autophagy. (A) After overexpressing PP1 in H1299 cells, glucose deprivation was performed for 3 hours and co-immunoprecipitation experiment using ULK1-specific antibody. Performed and observed blots using the indicated antibodies (B) In vitro phosphorylation experiments. Cells overexpressing Flag-ULK1 were deprived of sugar for 3 hours and harvested, followed by immunoprecipitation experiment using Flag-specific M2 beads. Purified protein used for in vitro phosphorylation experiments. The pQE-His-ATG14L protein was amplified and purified using M13pRep, a type of E. coli. ^{Phosphorylation of ATG14L was measured through 32} P (C and D). Flag-VPS34 from cell lysates was purified for in vitro lipid phosphorylation using Flag-specific M2 beads. ^{Synthesis of 32} P-PI(3)P by VPS34 in cells overexpressing either wild-type or mutant ULK1 ^{(C) Synthesis of 32} P-PI(3)P by VPS34 in cells overexpressing PP1 (D) by TLC technique and radiographs Confirmed by shooting experiment. Similar results were obtained through three independent experiments in the same manner. Scale bar 10 μm. P value was obtained through t-test (*p<0.01) or one-way ANOVA (**p<0.001). Expressed as mean ± SEM (E) In a glucose-deficient situation, OGT-dependent ULK1 O-GlcNAcylation occurs through PP1 dephosphorylation and AMPK phosphorylation, and as a result, it induces activation of VPS34 through ATG14L to promote phagopore formation. This is a model drawing that shows
7 is a schematic summary of the mechanisms elucidated herein.

ULK1 (Unc-51 like autophagy activating kinase) is regulated by mTOR (mechanistic target of rapamycin) and AMPK (AMP-activated protein kinase) under energy depletion and regulates the initiation process of autophagy, but the specific mechanism is unknown. didn't In the situation of glucose deficiency, ULK1 is dissociated from mTOR and phosphorylated by AMPK, increasing the activity of ULK1. Herein, ULK1 is O-GlcNAcylated at the 754 residue of threonine by OGT (UDP-N-acetyl-D-glucosamine:protein-O-beta-N-acetyl-D-glucosaminyl transferase) in a sugar-deficient situation, followed by O-GlcNAcylation by mTOR It was confirmed that ULK1 phosphorylated at serine 757 residue was dephosphorylated by PP1 (Protein Phosphatase 1), a dephosphorylation enzyme, which occurred after ULK1 was phosphorylated by AMPK. In addition, O-GlcNAcylation of ULK1 induces the binding of ULK1 and ATG14L and plays a pivotal role in inducing phosphorylation of ATG14L (Beclin 1-associated autophagy-related key regulator), which in turn leads to activation of VPS34, thus increasing PI3P. This is based on the fact that it leads to the formation of phagophores and the initiation of autophagy.

Specifically, in a state in which sugars are not depleted, ULK1 exists in a state in which serine 637 and 757 residues are phosphorylated by mTOR. In a sugar-deficient situation, the phosphorylated serine residue, particularly residue 757, is dephosphorylated by PP1, a dephosphorylation enzyme, and residues 317 and 555 of serine are specifically dephosphorylated by AMPK (5'-AMP-activated protein kinase catalytic subunit alpha-2). It is phosphorylated and activated, followed by O-GlcNAcylation at the 754 residue of threonine by OGT (UDP-N-acetyl-D-glucosamine:protein-O-beta-N-acetyl-D-glucosaminyl transferase), and via ATG14 , It was identified that the activation of VPS34 induces autophagy.

Accordingly, in one aspect, it relates to a screening method for an autophagy modulator that targets the above mechanism.

When referring to the position of the residues herein, the description is based on the mouse sequence, and the residues in the human sequence corresponding to the residues in the mouse sequence are disclosed herein, and those skilled in the art will find the corresponding corresponding residues based on the experimental results and disclosure herein. residues may be specified.

In one embodiment, the method comprises the steps of providing a ULK1 protein or providing a cell (over)expressing ULK1 at a low concentration of glucose; contacting a test substance expected to regulate O-GlcNAcylation at the 754th threonine residue of ULK1; and after the contact, determining a change in O-GlcNAcylation at residue 754 of ULK1; And in the determining step, if there is a change in the O-GlcNAcylation compared to the case treated with the test substance compared to the case not treated with the test substance, the test substance is selected as an autophagy modulator candidate include

As used herein, autophagy (autophagy or autophagocytosis) refers to a catabolism that uses lysosomes to remove various cellular components including unnecessary or denatured proteins in the cell. It is known that when there is an abnormality in autophagy regulation, the accumulation of misfolded protein is caused, thereby causing neurodegenerative disease (Komatsu et al., (2006), Nature, 441, 880-884). In addition, autophagy is activated in cancer cells (Ding et al., (2009), Mol. Cancer Ther., 8(7), 2036-2045), and autophagy inhibitors are known to act as anticancer therapeutics (Maiuri et al. ., (2007) Nat. Rev. Cell Biol. 8, 741-752). Moreover, in addition to cancer and neurodegenerative diseases, autophagy is known to be associated with liver disease, heart disease, muscle disease, and pancreatic disease (Levine and Kroemer, (2008), Cell, 132, 27-42; Fortunato and Kroemer, ( 2009), Autophagy, 5(6)). Therefore, the autophagy modulator and method using the technology disclosed herein can be effectively used for the treatment or prevention of various diseases caused by abnormal autophagy control as described above.

As used herein, modulation includes activation, stimulation or up-regulation, or reduction or down-regulation of a specific biological function, or both, and includes modulation in an in vitro state, modulation in an in vivo state, and in an ex vivo state. It includes all of the control of In this respect, the modulation in the method according to the invention comprises both an autophagy activator or enhancer, or an autophagy inhibitor. In one embodiment, in the step of selecting in the method according to the present application, when the degree of O-GlcNAcylation at residue 754 of ULK1 is increased compared to the case where the test substance is not treated with the test substance, the test substance is used as an autophagy activator , or when the degree of O-GlcNAcylation is decreased compared to the case where the test substance is not treated with the test substance, the test substance may be selected as an autophagy inhibitor candidate.

ULK1 used in the method according to the present application regulates the intracellular localization of VPS34, which is essential for the production of PI(3)P (Phosphatidylinositol 3-phosphate), and PI(3)P is an important molecule for intracellular transport and autophagosome formation It is also an enemy marker (Backer, 2008; Hara et al., 2008; Itakura and Mizushima, 2010). The gene and protein sequences of ULK1 in mammals are known, for example, human proteins and gene sequences are known to GenBank as NP_003556.1, NM_003565.3, and NP_033495.2, NM_009469.3, respectively, for mice, respectively. Human and mouse protein sequences are disclosed herein as SEQ ID NO: 1 and SEQ ID NO: 2, respectively, which have residues corresponding to each other with respect to the residues mentioned herein, Each of the mentioned residues will be readily identifiable in human mouse protein sequences. In one embodiment, human ULK1 protein is used, the residue of human ULK1 corresponding to the 754th threonine of the mouse is the 755th threonine residue, the human ULK1 residue corresponding to the 757th serine residue of the mouse is the 758th serine residue, and the mouse The residue in human ULK1 corresponding to residue 317 is residue 317, and the residue in human ULK1 corresponding to residue 555 in mouse is residue 556. The use of ULK1 derived from other species is not excluded, and even when ULK1 derived from another species is used, those skilled in the art can easily identify and specify the position of the corresponding residue in each species based on the sequences and results disclosed herein. There will be.

In the method according to the present application, a protein sequence of various origins and each origin and a full length or fragment having a sequence substantially identical thereto can be used as long as it has the function according to the present application.

ULK1 that can be used in the method of the present disclosure may be provided in an isolated form or in the form of a cell expressing the same or an animal comprising the cell. In the case of cells, it may be a cell line expressing or overexpressing the protein employed in the present method endogenously, or by introducing a plasmid expressing the same (transient or stable transfection).

Proteins used herein can be prepared using methods known in the art. In particular, the use of genetic recombination technology. For example, a plasmid containing a corresponding gene encoding the protein can be transferred to a prokaryotic or eukaryotic cell such as an insect cell or a mammalian cell, overexpressed, and purified before use. The plasmid may be used, for example, after transfection into an animal cell line as used in the exemplary embodiment of the present application, and then the expressed protein may be purified and used, but is not limited thereto. In this case, the protein may be labeled using a known method or a commercially available protein labeling kit such as various labeling materials, for example, biotin, fluorescent material, acetylation, radioisotope, for convenience of detection, and the labeled material It can be detected using a detection device suitable for

Alternatively, the DNA or RNA sequence encoding the screening protein is expressed in a suitable host cell to use the cell in the method herein, or a cell lysate thereof or in vitro translation of the mRNA of the screening protein is known in the art. The screening protein can be purified by a protein isolation method. Usually, after centrifugation of the cell lysate or the in vitro translated product to remove cell debris, etc., precipitation, dialysis, various column chromatography, etc. are applied. Ion exchange chromatography, gel-permeation chromatography, HPLC, reverse-phase-HPLC, preparative SDS-PAGE, affinity column, etc. are examples of column chromatography. Affinity columns can be made using, for example, anti-screening protein antibodies.

In one embodiment, established cell lines expressing ULK1, for example, cell lines including H1299, HEK293, and SW620 cell lines may be used. However, the present invention is not limited thereto, and those skilled in the art will be able to select an appropriate one with reference to the Examples of the present application.

When cells (over)expressing ULK1 are used in the method according to the invention, the cells are cultured at a low concentration of glucose. Normal glucose concentration is 2000.00 mg/L ~ 4500.00 mg/L, high concentration is 4500.00 mg/L or more, and low concentration is 0.00 mg/L ~ 700 mg/L. As used herein, the low glucose concentration is a concept including depletion or deficiency of glucose.

O-GlcNAc modification (O-GlcNAcylation) refers to a form in which a GlcNAc sugar group is covalently bonded to serine or threonine of a protein. OGT (UDP-N-acetyl-D-glucosamine:protein-O-beta-N-acetyl-D-glucosaminyl transferase) is a biological phenomenon that causes changes in the amount and function of the target mediated by the enzyme. O-GlcNAcylation occurs only by OGT and transfers N-acetylglucosamine from UDP-GlcNAc to the protein substrate. On the other hand, OGA rapidly removes O-GlcNAcylation from protein substrates (Slawson and Hart, 2011; Vocadlo, 2012). These two enzymes with opposing actions induce post-translational modification of proteins and regulate their functions in response to various intracellular signals or processes. In the present application, in a sugar-deficient situation, the phosphorylated serine residue, particularly the 757 residue, is dephosphorylated by the dephosphorylation enzyme PP1, and in particular, the S555 and S317 residues are phosphorylated by AMPK and activated, followed by OGT to the threonine 754 residue. It was identified that O-GlcNAcylation occurred and, via ATG14, activated VPS34, a phosphatidylinositol 3-kinase, to induce autophagy.

The VPS34 forms a stable complex with the VPS15. VPS15 binds to Beclin1, which through VPS34 binds to factors that promote autophagy (ATG14L, UVRAG, Bif1, AMBRA-1) and factors that inhibit autophagy (Bcl2, BclxL, Rubicon) ( Itakura et al., 2008; Liang et al., 2006; Matsunaga et al., 2009; Pattingre et al., 2008; Sun et al., 2008; Takahashi et al., 2008; Zalckvar et al., 2009; Zhong et al., 2009). In particular, ATG14L functions to induce autophagosome formation, because ATG14L regulates the intracellular localization of VPS34, and binding of ATG14L and VPS34 complex is important for AMPK-mediated activation of VPS34 lipid phosphorylation (Kim et al. al., 2013; Matsunaga et al., 2009). However, the mechanism of how ATG14L regulates the VSP34-Beclin1 complex has not been elucidated. Herein, O-GlcNAcylation of ULK1 by OGT occurs at residue 754 of threonine in a sugar-deficient situation, and O-GlcNAcylation of ULK1 is important for inducing normal VPS34 activation through ATG14L, which is important for PI(3)P synthesis and diastole. It was identified that it leads to the initiation of autophagy through pore formation.

Accordingly, in the method according to the present application, the method according to the present application further comprises AMPK and PPI, and the measuring according to the present application comprises the steps of measuring phosphorylation at serine 317 and serine 555 residues of ULK1 and the ULK1 It further comprises the step of measuring dephosphorylation at residue 757, and as a result of the measurement, when the dephosphorylation and phosphorylation are increased compared to the control not treated with the test substance, the test substance is used as an autophagy activator, the measurement As a result, when the dephosphorylation and phosphorylation do not change or decrease, the test substance is selected as an autophagy inhibitor.

The mTOR and AMPK protein and nucleic acid sequences used in the method according to the present application are known: The protein and nucleic acid sequence DB numbers of mTOR are NP_0049491, NM_004958.4, respectively; The protein and nucleic acid sequence DB numbers of AMPK are NP_006242.5 and NM_006251.5, respectively. The protein and nucleic acid sequence DB numbers of PPI (Protein Phosphatase 1) are known as NP_002699.1 and NM_002708.4, respectively.

Also in the method according to the present application the method according to the present invention further comprises ATG14L and VPS34 lipid kinase, these protein and nucleic acid sequences are known as follows: Protein and nucleic acid sequence DB number of ATG14L is NP_055739.2 respectively , NM_014924.4; The protein and nucleic acid sequence DB numbers of VPS34 are known as NP_002638.2 and NM_002647.4, respectively.

In the case of including the protein, the method further comprises measuring the phosphorylation of ATG14L and the activity of VPS34 lipid kinase in the selecting step of the method according to the present application, as a result of which phosphorylation of ATG14L and the activity of VPS34 lipid kinase are measured. A substance that increases the activity is an autophagy activator, and a substance that reduces the phosphorylation of ATG14L and the activity of VPS34 lipid kinase can be selected as an autophagy inhibitor candidate.

Methods for measuring O-GlcNAcylation, phosphorylation level and kinase activity in the method according to the present application are known in the art, and an appropriate one may be selected with reference to the method described in the Examples herein. In addition, since O-GlcNAcylated ULK1 promotes phosphorylation of ATG14L through binding to ATG14L in the method according to the present disclosure, the binding between the proteins can also be measured. Binding or interaction between proteins can be measured using a variety of methods known in the art. Examples include, but are not limited to, yeast-to-hybrid method, confocal microscopy, co-immunoprecipitation method, surface plasma resonance (SPR) and spectroscopy method for confirming intracellular protein binding/interaction. For further references to comparisons and detailed experimental methods, see Berggard et al., (2007) "Methods for the detection and analysis of protein-protein interactions", PROTEOMICS Vol7: pp 2833-2842.

Upstream regulators of ULK1 include mTORC1 and AMPK. ULK1 is phosphorylated at residues 637 and 757 of serine by mTORC1, which inhibits the activity of ULK1 by interfering with binding to AMPK (Hosokawa et al., 2011; Mizushima et al., 2010). Conversely, AMPK regulates cell metabolism to maintain energy homeostasis and induce autophagy. Previous studies have demonstrated that ULK1 is activated through AMPK-dependent phosphorylation. Serine 555 site is the main target site phosphorylated by AMPK. In addition, several AMPK-dependent ULK1 phosphorylation sites such as serine 317, serine 467, threonine 574, serine 637, and serine 777 have been identified (Egan et al., 2011; Kim et al., 2011). Among them, serine 317 and 777 regulate phosphorylation activity of ULK1. Serine 467, serine 555, and threonine 574 are involved in maintaining mitochondrial homeostasis under nutritional deficiencies. Serine 637 is a common site for phosphorylation of mTORC1 and AMPK. Herein, it was identified that O-GlcNAcylation of ULK1 occurs after serine 757 residue phosphorylated by mTOR is dephosphorylated by PP1, a dephosphorylation enzyme, and ULK1 is phosphorylated by AMPK, especially at serine 317 and serine 555 residues. did. In addition, as mentioned above, the residues phosphorylated by mTOR in a glucose-deficient state are dephosphorylated by PP1.

Accordingly, in the method according to the present application, the cell further expresses AMPK and PPI, and further comprising the steps of phosphorylation at serine residues 317 and 555 of ULK1 and dephosphorylation at residue 757 of ULK1 and measuring. and, as a result of the measurement, if the dephosphorylation and phosphorylation were increased compared to the case where the test substance was not treated, the test substance was used as an autophagy activator, and as a result of the measurement, compared with the case where the test substance was not treated, the dephosphorylation When phosphorylation and phosphorylation do not change or decrease, the test substance can be selected as an autophagy inhibitor.

The test substance used in the method of the present application is a substance expected to cause regulation or change of O-GlcNAcylation by acting on ULK1 or the autophagy mechanism of ULK1 identified herein, for example, low molecular weight compounds, high molecular weight compounds, mixtures of compounds (e.g., natural extracts or cell or tissue cultures), or biopharmaceuticals (e.g., proteins, antibodies, peptides, DNA, RNA, antisense oligonucleotides, RNAi, aptamers, RNAzyme and DNAzyme) or sugars and lipids, etc. It is not limited to this.

In one embodiment according to the present application, a low molecular weight compound may be used as a test substance. The test substance may be obtained from a library of synthetic or natural compounds, and methods for obtaining a library of such compounds are known in the art. Synthetic compound libraries are available from Maybridge Chemical Co. (UK), Comgenex (USA), Brandon Associates (USA), Microsource (USA) and Sigma-Aldrich (USA), and natural compound libraries are available from Pan Laboratories (USA) and It can be purchased from MycoSearch (USA). Test substances can be obtained by various combinatorial library methods known in the art, for example, biological libraries, spatially addressable parallel solid phase or solution phase libraries, deconvolution It can be obtained by the required synthetic library method, the "1-bead 1-compound" library method, and the synthetic library method using affinity chromatography selection. Methods for synthesizing molecular libraries are described in DeWitt et al., Proc. Natl. Acad. Sci. U.S.A. 90, 6909, 1993; Erb et al. Proc. Natl. Acad. Sci. U.S.A. 91, 11422, 1994; Zuckermann et al., J. Med. Chem. 37, 2678, 1994; Cho et al., Science 261, 1303, 1993; Carell et al., Angew. Chem. Int. Ed. Engl. 33, 2059, 1994; Carell et al., Angew. Chem. Int. Ed. Engl. 33, 2061; Gallop et al., J. Med. Chem. 37, 1233, 1994, et al. For example, for drug screening purposes, compounds having a low molecular weight therapeutic effect may be used. For example, a compound having a weight of about 1000 Da, such as 400 Da, 600 Da, or 800 Da, may be used. Depending on the purpose, these compounds may form a part of a compound library, and the number of compounds constituting the library varies from tens to millions. Such compound libraries include peptides, peptoids and other cyclic or linear oligomeric compounds, and template-based small molecule compounds such as benzodiazepines, hydantoins, biaryls, carbocycles and polycyclic compounds such as naphthalene, phenothi azine, acridine, steroid, etc.), carbohydrates and amino acid derivatives, dihydropyridine, benzhydryl and heterocycles (such as triazine, indole, thiazolidine, etc.), but these are merely exemplary. It is not limited to this.

Also, for example, biologics can be used for screening. Biologics refers to cells or biomolecules, and biomolecules refer to proteins, nucleic acids, carbohydrates, lipids, or substances produced using cell systems in vivo and in vitro. The biomolecule may be provided alone or in combination with other biomolecules or cells. Biomolecules include, for example, polynucleotides, peptides, antibodies, or other proteins or biological organisms found in plasma.

In one embodiment, the increase or decrease that can select a candidate substance in the method according to the present application can be appropriately selected by those skilled in the art in consideration of the results disclosed herein and common knowledge of those skilled in the art, for example, but not limited thereto, with the test substance about 10% increase or decrease, about 20% increase or decrease, about 30% increase or decrease, about 40% increase or decrease, about 50% increase or decrease, about 60% in the presence of the test substance as compared to an uncontacted control increase or decrease, increase or decrease by about 70%, increase or decrease by about 80%, increase or decrease by about 90%, increase or decrease by about 100%, or more and ranges therebetween.

As mentioned above, the candidate material selected by the method according to the present application may be developed/used as a therapeutic agent for various diseases such as cancer, autoimmune disease, and heart disease.

As used herein, the term “treatment” is a concept including the inhibition, elimination, alleviation, alleviation, amelioration, and/or prevention of a disease, or a symptom or condition caused by the disease.

Hereinafter, examples are presented to help the understanding of the present invention. However, the following examples are provided for easier understanding of the present invention, and the present invention is not limited thereto.

Example

Experimental methods and materials

cell culture

HEK293, H1299, SW620 cells were cultured in DMEM medium (Welgene, LM001-05) supplemented with Zell Shield (Minerva Biolabs GmbH) 1%, 10% FBS, or RPMI-1640 medium (Welgene, LM011-01). To ensure mycoplasma-free conditions, all cells were routinely tested for mycoplasma. For overexpression experiments, transfection was induced by using Lipofectamine 3000 (Life Technologies, 100022052) and Gene In (Global Stem, catalog# 73802, 73012) according to the experimental method recommended by the manufacturer. Cells were harvested 48-72 hours after culture or 24-36 hours after transfection. If not indicated, cultured in normal medium. In the case of glucose deprivation experiment, cells were washed twice with PBS (Welgene, LB001-02), and FBS (TCB, 101DI) dialyzed in DMEM (Welgene, LM001-56) or RPMI1640 (Welgene, LM011-60) from which sugar was removed. ) was mixed and treated.

Construction of ULK1-/- cells using CRISPR/Cas9

To generate KO cells, the cells were transfected with px330 plasmid with the following sequences: ULK1 Fw-5’-AGCAGATCGCGGGCGCCATG-3’, Rv-5’-CATGGCGCCCGCGATCTGCT-3’. After transfection, each cell was isolated and cultured until colonies were formed, and then divided into KOs by protein expression and genomic DNA sequencing.

LC-ETD-MS/MS analysis

Glycosylation was induced by overexpressing Flag-labeled ULK1 together with OGT. ULK1 was isolated by purification with Flag-labeled M2 beads. The protein was modified by adding 8M urea to 50 mM ammonium bicarbonate, reduced, and then alkylated with cysteine thiol. Then, it was digested using trypsin and LC-ETD-MS/MS was performed. ETD-MS/MS data were obtained using an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific), nanoACQUITY UPLC (Waters), in-house packed capillary analytical column and trap column.

plasmid

The px330 sgRNA cloning vector (Addgene, #42230) was used to construct ULK1-/- cells using CRISPR/Cas9. The nPfu-Forte mutagenesis kit (Enzynomics, P410) was used as the taq polymerase required for plasmid mutation.

Antibodies and Reagents

Beclin1-specific antibody (NB110-87318) and LC3B-specific antibody (NB100-2220) were purchased from Novus Biologicals. ULK1-specific antibody (A7481), Flag-specific antibody (F3165), ATG13-specific antibody (SAB4200100), p-serine-specific antibody (P5747), β-actin-specific antibody (A1978), L-α-Phosphatidylinositol sodium Salt (P0639) was purchased from Sigma Aldrich. PP1-specific antibody (sc-7482) and mTOR-specific antibody (sc-1549-R) were purchased from Santa Cruz Biotechnology. ATG14L-specific antibody (Ab173943), ATG101-specific antibody (Ab105387), O-linked N-acetylglucosamine RL II antibody (Ab2739), and OGT-specific antibody (Ab96718) were purchased from Abcam. p-S757 UKL1 specific antibody (#6888), p-S555 ULK1 specific antibody (#5869), p-T172 AMPK specific antibody (#2531), p-S2448 mTOR specific antibody (#2971S), VPS34 specific Red antibody (#3811S) was purchased from Cell Signaling. HA specific antibody (#MMS-101R) was purchased from Covance. PP5 specific antibody (A300-909A) was purchased from Bethyl Laboratories. SWGA beads (AL-1023S) were purchased from Vector Laboratories. Torin 1 (4247) and tautomycetin (2305) were purchased from Tocris.

Co-immunoprecipitation experiment

All cells were harvested after rinsing with PBS at 4°C. Cells were lysed with EBC200 buffer (50 mM Tris-HCl [pH 8.0], 0.2 M NaCl, 0.5% NP-40, protease inhibitor) and stored on ice for 30 minutes. The cell lysate was centrifuged at 14,000 × g at 4°C for 10 minutes to remove the pellet, and quantified and standardized using Bio-Rad Protein Assay Dye Reagent Concentrate (Bio-Rad, #500-0006). In the co-immunoprecipitation experiment, the labeled antibody was first treated with the cell lysate for 2-4 hours, and then stored in a rotator with protein A- and protein G-sepharose beads (GE Healthcare) for 1 hour. The beads were rinsed 5 times with EBC200 buffer and boiled with SDS PAGE-loading buffer. Then, the blot was analyzed by SDS-PAGE.

Immunofluorescence assay

Cells were grown on coated coverslips. For immunofluorescence experiments, cells were washed twice with PBS and fixed with 2% formaldehyde for 30 minutes. The cells were washed twice with PBS containing 0.1% triton-X 100. For the penetration of the antibody, the cells were incubated in PBS with 0.5% triton-X 100 dissolved in them for 5 minutes, and then blocked with a blocking buffer (5% BSA in 0.1% PBS-T) to prevent non-specific binding of the antibody. . The primary antibody was placed in a blocking solution and incubated with the cells. After that, after washing 8 times with 0.1% PBS-T solution, the secondary antibody (donkey anti-mouse IgG; AlexaFluor 488, AlexaFluor 594; donkey anti-rabbit IgG AlexaFluor 488, AlexaFluor 594, Invitrogen, blocking buffer 1:200 dilution) and incubated for 1 hour. After washing again with 0.1% PBS-T solution 8 times, mounting with vectashield (H-1000), and taking a picture with a confocal microscope (Carl Zeiss, LSM700). In the autophagy experiment, cells overexpressed GFP-LC3/mCherry-GFP-LC3 and sub-cultured on coverslips were given glucose deficiency and fixed. In the colocalization experiment, cells were overexpressed with HA-PP1/Flag-ULK1, sub-cultured on coverslips, and fixed with glucose deficiency.

in vitro Phosphorylation experiment

ULK1 protein was immunoprecipitated using Flag-specific M2 agarose affinity gel (Sigma) in HEK293 cells. The resultant was washed three times with BC150 (20 mM Tris-HCL, pH 7.9, 15% glycerol, 1 mM EDTA, 1 mM DTT (dithiothreitol), 0.05% NP40, and 150 mM KCl), BC300 (20 mM Tris-HCL, pH 7.9) , 15% glycerol, 1 mM EDTA, 1 mM DTT (dithiothreitol), 0.05% NP40, and 300 mM KCl) three times, and then again three times with BC150. It was eluted from affinity gel using Flag peptide. His-ATG14L protein was amplified in E. coli strain M15pRep and purified using Ni-NTA resin (QIAGEN). ULK1 and ATG14L proteins were mixed with kinase assay buffer (10 mM HEPES at pH 7.4, 50 mM NaCl, 10 mM MgCl ₂ , 10 mM MnCl ₂ , 1 mM DTT, protease inhibitors, 10 mM cold ATP, 10 μCi per sample [γ- ^32] P] ATP) and reacted at 37°C for 30 minutes, and then SDS sample buffer was added to terminate the reaction. Then, it was analyzed by SDS-PAGE and radiographic measurements.

in vitro VSP34 Lipid Phosphorylation Experiment

HEK293 cells expressing Flag-labeled VSP34 were immunoprecipitated using Flag-specific M2-agarose affinity gel, rinsed with NP-40 buffer, and then rinsed with TBS buffer (150 mM NaCl, 50 mM Tris-HCl [pH 7.4]). 3xFlag peptide was diluted and eluted. Thereafter, the sample was placed in phosphorylation buffer (20 mM HEPES [pH 7.4], 1 mM EGTA, 0.4 mM EDTA, and 5 mM MgCl ₂ ) and stabilized for 10 minutes, followed by 10 mM MnCl ₂ and 2 mg sonicated phosphatidylinositol (Sigma), 10 μCi [ATP- ³² P] and 1 mM cold ATP were mixed and reacted at room temperature for 15 minutes. Add 10 μl 1M HCl to complete the reaction, add 2x volume of methanol/CHCl ₃ (1:1), vortex, and centrifuge to transfer the organic solution to TLC (Analtech) and resolution buffer (CHCl ₃ / methanol / NH ₄ OH (30%) / water (129:100:4.29:24)) was soaked so that only the bottom touched. After the reaction was completed, radiographic measurements were taken.

in vitro OGT experiment

Recombinant Flag-OGT protein was purified from HEK293 cells, and recombinant 6XHis-tagged ULK1 protein was purified from E. coli in reaction buffer (50 mM Tris-HCl pH 7.5, 12.5 mM MgCl ₂ , 2 mM UDP-GlcNAc, 1 mM DTT) to a total volume of 25 μl per sample. The sample was reacted at 37° C. for 24 hours, SDS-PAGE was performed, blotted on PVDF, and O-GlcNAc of ULK1 was detected with an O-GlcNAc specific antibody.

Quantification and Statistical Analysis

All experiments were conducted at least three times in the same manner and in an independent manner. It was expressed as mean±SEM. Significance analysis was performed using GraphPad Prism software in one-tailed unpaired t-test or one-way ANOVA method. P <0.05 was defined as statistically significant. *p < 0.01, **p < 0.001, ***p < 0.0001.

Example 1. Identification of O-GlcNAcylation at ULK1 threonine 754 residue by OGT

Since ULK1 functions as an important regulator in the initiation of autophagy, it is necessary to identify upstream regulators regulating ULK1. Unexpectedly, we observed that ULK1 and OGT bind to each other. It was confirmed that ULK1 and OGT bind endogenously through a co-immunoprecipitation experiment using a ULK1-specific antibody (FIG. 1A). Subsequently, ULK1-/- HEK293 cells were prepared using CRISPR/Cas9, and it was confirmed that ULK1 and OGT bind specifically only to wild-type cells ( FIG. 1B ). Based on this, the possibility of ULK1 being the target of OGT was investigated. In order to confirm whether O-GlcNAcylation occurs in ULK1, an in vitro OGT experiment was performed. It was confirmed that O-GlcNAc of ULK1 increased when OGT was introduced ( FIG. 1C ). In addition, it was confirmed that the O-GlcNAcyation of ULK1 was increased when Thiamet G, an OGA inhibitor, was treated without changing the total protein amount of ULK1 ( FIG. 1D ).

Next, it was decided to find the location where O-GlcNAcyation occurs in ULK1. O-GlcNAcylation was confirmed in several ULK1 deletion mutants. Wild-type, Δ279-833 mutant lacking 279-833 amino acids, 1-600 amino acids, and 279-833 amino acid mutations were overexpressed in HEK293 cells, and an O-GlcNAc band was detected by performing an immunoprecipitation experiment. It was confirmed that ULK1 is O-GlcNAcylation only in the 601-833 amino acid region (FIG. 1E). In a recent study, it was reported that ULK1 is O-GlcNAc at two different sites, threonine 635 and threonine 754 (Ruan et al., 2017), but in this study, threonine 754 residue was overwhelmingly strongly O-GlcNAcylated. was confirmed (FIG. 1F). Electron dissociation (ETD) analysis was performed to confirm the exact O-GlcNAcylation site, and it was confirmed that O-GlcNAcylation was performed at the threonine 754 residue, which is an evolutionarily conserved site of ULK1 (FIGS. 1G, 1H).

In addition, three other threonine 754 mutations (T754A, T754N, T754Y) were to be tested. First, the threonine residue was mutated to alanine. Here, since T754 is very close to mTOR-dependent phosphorylated serine 757 (S757), asparagine (N) and tyrosine (Y) corresponding to the known target motif sequence of mTOR were also mutated (Hsu et al., 2011). The reason for considering mTOR-dependent phosphorylation is that, according to previous studies, mTOR phosphorylates ULK1 under normal conditions, but when nutrient deficiency occurs, mTOR phosphorylation of ULK1 by mTOR disappears and releases the inactive state (Kim et al., 2011). Therefore, a T754 mutant with a conserved residue corresponding to the mTOR target motif was constructed so that phosphorylation by several mTORs occurs normally. Through the co-immunoprecipitation experiment, it was confirmed that O-GlcNAcylation of ULK1 occurred only in wild-type ULK1 and that O-GlcNAcylation did not occur in T754Y, T754N, and T754A, which is also consistent with the ETD mass spectrometry data (FIG. 1I). During the study, it was confirmed that the T754A mutation unexpectedly lost the S757 phosphorylation dynamics, which was judged to be inappropriate for observing the association between O-GlcNAcylation by OGT and phosphorylation by mTOR. Therefore, it was decided to use the T754N mutation to study the O-GlcNAc function of ULK1.

Example 2. Investigation of positive correlation between increased ULK1 O-GlcNAcylation and autophagosome formation in a sugar-deficient situation

The activity of ULK1 largely depends on the effectiveness of nutrients, and nutrient deficiency conditions such as sugar deficiency increase the activity of ULK1 (Kim et al., 2011; Shang and Wang, 2011). Therefore, it was decided to test whether the interaction between ULK1 and OGT is affected by glucose deficiency. As a result of confirming in H1299, HEK293, and SW620 cell lines, it was confirmed that the binding of ULK1 and OGT was increased in a glucose-deficient situation (FIG. 2A). At the same time, it was confirmed that the O-GlcNAc level of ULK1 increased in the glucose-deficient situation ( FIG. 2B ), and it was confirmed that it occurred with the increase of LC3 II conversion. This means that O-GlcNAcylation of ULK1 in cells positively correlates with the progression of autophagy. Furthermore, we predicted that the sugar level would have a negative correlation with the ULK1 O-GlcNAcylation level and that re-addition of sugar would lower the ULK1 O-GlcNAc level again. As expected, restoration of glucose levels following glucose deprivation reversed the modification of ULK1 (Fig. 2C). In addition, it was confirmed using SWGA beads that the ULK1 O-GlcNAcylation band decreased when free N-acetyl D-glucosamine (NADG) mimicking UDP-GlcNAc was treated, and that this phenomenon did not occur in ULK1-/- HEK293 cells ( Figure 2D).

Furthermore, we wanted to see if knockdown of OGT reduces ULK1 O-GlcNAcylation, which leads to inhibition of autophagy. First, it was confirmed that the ULK1 O-GlcNAcylation level was significantly reduced in the OGT knockdown situation using siRNA (FIG. 2E). Next, GFP-LC3 puncta was used as a marker for autophagy progression. It was confirmed that the GFP-LC3 puncta was significantly reduced when the OGT level fell. It is interpreted that OGT knockdown inhibited autophagosome formation (FIGS. 2F, 2G). To more accurately analyze whether O-GlcNAcylation of ULK1 regulates autophagosome formation, siRNA was used to knockdown ULK1 and siRNA-resistant wild-type or T754N mutant ULK1 was restored. It was decided to check the overall LC3 puncta formation and the progress of autophagy by using the mCherry-GFP-LC3 reporter. This reporter decreases the GFP signal in an acidic environment, such as inside the lysosome, but preserves the mCherry signal. Therefore, this reporter is a useful tool to differentiate between autophagosomes (GFP+/mCherry+ yellow puncta) and autolysosomes (GFP/mCherry+ red puncta). As a result of the experiment, it was confirmed that the total number of autophagosomes and the total number of autolysosomes were significantly reduced in T754N than in the wild type (FIG. 2H). Through this, it can be seen that ULK1 O-GlcNAcylation induces autophagosome formation. Using Bafilomycin A1, an inhibitor that inhibits the fusion of autophagosome and lysosome, was used to evaluate the progress of autophagy. It was significantly increased in wild-type and T754N mutant cells when treated with Bafilomycin A _{1 .} As the number of autophagosomes increased due to inability to fuse with lysosomes, it was found that the function of ULK1 O-GlcNAcylation contributes to the initial progression of autophagosomes, not autophagosomes and lysosomes (FIG. 2H).

Example 3. Investigation that dephosphorylation of ULK1 and O-GlcNAcylation occur sequentially

According to one study, when phosphorylation dynamics were quantified with 711 phosphorylated peptides through high-efficiency mass spectrometry, an increase in O-GlcNAcylation led to a decrease in phosphorylation of 280 phosphorylated peptides, while phosphorylation of 148 phosphorylated peptides increased (Wang et al. , 2008). Crosstalk between these two types of modifications may be caused by positional competition according to the spatial arrangement of atoms for the same or close residues, so that one type of modification affects the other type of modification. Since ULK1 is known to be phosphorylated at S757 by mTOR under conditions of sufficient nutrients, we wanted to see if the crosstalk between O-GlcNAcylation and phosphorylation is applied to ULK1 (Hwang et al., 2017; Kim et al., 2011). . S757 is only three amino acids away from the O-GlcNAc site, and it is known that S757 phosphorylation is significantly reduced in nutrient deprivation conditions (Kim et al., 2011). First, the decrease in S757 phosphorylation of ULK1 coincides with the increase in O-GlcNAc of T754, indicating that these two types of modifications are likely to crosstalk with each other (FIG. 3A). In addition, it was confirmed that the binding between ULK1 and mTOR was decreased in the situation of glucose deficiency ( FIG. 3A ).

Based on the above evidence, we wanted to report what kind of correlation there is between O-GlcNAcylation and phosphorylation of ULK1. It is known that phosphorylation of ULK1 by mTOR inhibits the activity of ULK1 in the context of sufficient nutrients. Therefore, when cells were treated with Torin 1, an inhibitor of mTOR, phosphorylation of ULK1 by mTOR was decreased while O-GlcNAcylation of T754 was increased ( FIG. 3B ). Next, in order to establish such a negative correlation, overexpression of an activating mutation of mTOR could reverse O-GlcNAcylation, and the experiment was carried out. The activating mTOR mutant (mTOR CA) was constructed by modifying four residues (I2017T, V2198A, L2216H, and L2260P) (Das et al., 2011). It was confirmed that the O-GlcNAcylation of ULK1 was reversed using the mTOR CA mutation. O-GlcNAcylation of ULK1 decreased corresponding to the expression level of mTOR CA, and lateral phosphorylation increased corresponding to the expression level of mTOR CA ( FIG. 3C ). Through this, it can be seen that T754 O-GlcNAcylation and S757 phosphorylation of ULK1 occur alternately and reversibly. Additionally, we decided to check O-GlcNAcylation and phosphorylation of wild-type and mutant ULK1 under glucose-deficient conditions. As expected, it was observed that O-GlcNAcylation did not occur in the T754N and S757D (S757 phosphorylation mimics) mutants, whereas O-GlcNAcylation occurred in the wild-type and S757A mutants (FIG. 3D). These results support the fact that dephosphorylation and O-GlcNAcylation occur sequentially in the situation of glucose deficiency. Ultimately, O-GlcNAcylation occurs only when phosphorylation of ULK1 S757 is significantly reduced.

Next, we predicted that there would be a dephosphorylation enzyme that promotes the dephosphorylation of ULK1. As a result of comparing binding to ULK1 in a glucose-deficient situation, PP1 showed the strongest binding force than PP2A or PP5 (FIG. 3E). Also, judging from the fact that the level of LC3 II increased when PP5 was knocked down, PP5 did not match the direction of autophagy, whereas PP1 knockdown induced a decrease in LC3 II, suggesting that PP1 was the most likely dephosphorylation enzyme. Although the total protein amount of PP1 did not change, the binding between PP1 and ULK1 was increased in the sugar-deficient situation (FIG. 3F). As the binding between the two molecules increased, the phosphorylation of S757 of ULK1 was also decreased, which may be due to dephosphorylation by PP1. Furthermore, the interaction between ULK1 and PP1 was demonstrated by immunostaining experiments. In the case of sufficient nutrients, ULK1 and PP1 did not bind, but in the case of glucose deficiency, the binding between the two molecules was increased. After confirming the binding of ULK1 and PP1, PP1 was knocked down to see how it affects O-GlcNAcylation of ULK1. In fact, O-GlcNAcylation of ULK1 was significantly reduced due to the absence of PP1 in the glucose-deficient situation (FIG. 3G).

This time, tautomycetin (TMC), a PP1-specific inhibitor, was treated to see if this dephosphorylation was specifically caused by PP1. TMC was treated in the glucose-deficient situation, but as a result, O-GlcNAcylation of ULK1 was significantly reduced (FIG. 3H), indicating that dephosphorylation is essential for O-GlcNAcylation in the glucose-deficient situation. Furthermore, overexpression of PP1 induced binding between ULK1 and OGT ( FIG. 3I ). Accordingly, according to the degree of overexpression of PP1, O-GlcNAcylation of ULK1 was increased correspondingly, and phosphorylation of S757 was significantly decreased ( FIG. 3J ). From this, it can be seen that dephosphorylation of ULK1 by PP1 increases the binding of ULK1 and OGT, thereby promoting O-GlcNAcylation of ULK1. These results provided an additional clue that O-GlcNAcylation of ULK1 cannot be induced only by the separation of ULK1 from mTOR in a glucose-deficient situation, and that binding between ULK1 and OGT is possible only through active dephosphorylation by PP1. In addition, overexpression of PP1 was able to reverse the inhibition of ULK1 O-GlcNAcylation by mTOR ( FIG. 3K ). According to this result, it was predicted that autophagosome formation would be inhibited when PP1 was knocked down, just as autophagosome formation was inhibited when OGT was knocked down as in FIG. 2F, and the experiment was conducted. As expected, when PP1 was knocked down, it was confirmed that the puncta of GFP-LC3 was significantly reduced in the glucose-deficient situation (FIG. 3L). Taken together, these results not only proved the close correlation between ULK1 and PP1, but also maintain the dephosphorylated state of ULK1 while PP1 binds to ULK1 in the sugar-deficient situation, thereby reducing O-GlcNAcylation and O-GlcNAcylation by OGT on ULK1. It was demonstrated that it acts as a gatekeeper between phosphorylation by mTOR.

Example 4. Investigation that ULK1 O-GlcNAcylation plays an important role in the activation of VPS34 through ATG14L in the early stage of autophagy

Since the T754 O-GlcNAcylation site resides in a region that coordinates the function of ULK1, it is possible that this modification affects the function of ULK1 as a kinase. Therefore, it was decided to proceed with an in vitro phosphorylation experiment using ATG14, a known substrate of ULK1. As a result, T754N and T754A mutants showed similar phosphorylation activity to wild-type ULK1. For these mutations, binding between wild-type or mutant ULK1 and ATG13 was compared to rule out the possibility of structural defects. There was no significant difference in the binding between ATG13 and them. Then, we decided to test how O-GlcNAcylation affects functions such as protein-protein interaction of ULK1. Binding affinity with known ULK1 targets, such as ATG14L or VPS34, was checked. In the case of ATG14L, which induces autophagosome formation and acts as an important regulator of VPS34, it was confirmed that binding to T754N and T754Q was significantly lowered when compared with the wild type of ULK1 (FIG. 4A). The T754Q mutation was used in conjunction with T754N, a T754 mutation based on a known target motif of mTOR. Interestingly, it was confirmed that the binding of ULK1 and ATG14L was significantly reduced when OGT was knocked down ( FIG. 4B ). Based on the clue that O-GlcNAcylation of ULK1 is important for binding to ATG14L, we decided to examine how the binding between ULK1 and ATG14L changes depending on the presence or absence of O-GlcNAcylation of ULK1 when wild-type or inactive mutant OGT is overexpressed, respectively. Unlike mutant OGT, binding between ULK1 and ATG14L was increased only when wild-type OGT was overexpressed, suggesting that the interaction between the two molecules is dependent on O-GlcNAcylation (FIG. 4C). In addition, it was predicted that the binding between ULK1 and ATG14L would be reduced when mTOR CA was overexpressed in the same manner based on the clue that O-GlcNAcylation was reduced by overexpression of mTOR CA in FIG. 3C . In fact, it was confirmed that the binding of ULK1 and ATG14L decreased and S757 phosphorylation increased ( FIG. 4D ).

Since binding between ULK1 and ATG14L may be possible only with dephosphorylation, the justification of O-GlcNAcylation may be questioned. To confirm this possibility, ULK1 was dephosphorylated through overexpression of PP1 and then OGT was knocked down to induce dephosphorylation and deO-GlcNAcylation of ULK1. Surprisingly, the binding of ULK1 and ATG14L, which was increased by PP1 overexpression, was abolished in OGT knockdown. Based on this, it can be seen that O-GlcNAcylation has an intrinsic function as well as a function of simply forming a crosstalk with surrounding phosphorylation (Fig. 4E). We decided to confirm the hypothesis using wild-type and O-GlcNAc mutants T754N, T754Q, phosphorylation mutation S757A, and double mutation T754N/S757A ULK1. The T754N/S757A mutant was included in the experiment because it had both dephosphorylated and deO-GlcNAcylated forms. Interestingly, only wild-type and S757A mutants were able to bind ATG14L, but T754N/S757A did not bind, indicating that dephosphorylation alone could not bind ATG14L (FIG. 4F). Taken together, dephosphorylation is essential for O-GlcNAcylation to occur, but O-GlcNAcylation still has its own function, which is important for binding to ATG14L.

Example 5. Investigation that phosphorylation of ULK1 by AMPK occurs before O-GlcNAcylation of ULK1

Based on the fact that AMPK and mTORC1 have opposite functions on ULK1 in previous studies, we tried to find out at what point in this interaction O-GlcNAcylation functions. When the cells were treated with Compound C, an AMPK inhibitor, and sugar deficiency was induced, it was confirmed that AMPK-dependent S555 phosphorylation and O-GlcNAcylation of ULK1 were significantly reduced (FIG. 5A). These results suggest the possibility that phosphorylation by AMPK occurs before O-GlcNAcylation. To provide further clues to this hypothesis, shRNA was used to induce glucose depletion in AMPKα knockdown cells. Similar to the experimental results treated with Compound C, AMPK knockdown significantly reduced ULK1 O-GlcNAcylation as well as AMPK-dependent S555 phosphorylation (FIG. 5B).

However, if AMPK is inhibited or knocked down, overall autophagy is inhibited, and thus OGT may be affected. In order to exclude this question, we decided to check how the AMPK-dependent ULK1 phosphorylation sites S317, S467, S555, T574, S637, and S777 have alanine (SA) mutations, respectively, and how they affect O-GlcNAcylation. Remarkably low O-GlcNAcylation was detected in both SA mutants, which can be interpreted as S317 S555 phosphorylation by AMPK occurs before T754 O-GlcNAcylation (Fig. 5C). Conversely, when the above six sites were replaced with aspartic acid to mimic the phosphorylation state, it was confirmed that O-GlcNAcylation was increased in SD mutants of S317 and S555 ( FIG. 5D ). This suggests the possibility that ULK1 phosphorylation by AMPK occurs before ULK1 O-GlcNAcylation and has a positive effect on ULK1 O-GlcNAcylation. These results suggest that there is a sequential modification process in ULK1 in the situation of glucose deficiency. Following dephosphorylation by PP1 and phosphorylation by AMPK, ULK1 O-GlcNAcylation by OGT occurs. Through this series of processes, binding between ULK1 and ATG14L is made.

Example 6. Investigation that dephosphorylation of ULK1 and O-GlcNAcylation are important for regulating the initiation phase of autophagy

Based on the clue that O-GlcNAcylation of ULK1 is increased by PP1 and the clue that O-GlcNAcylation is important for ULK1-ATG14L binding, we decided to examine whether the binding of ATG14L and ULK1 is affected by S757 dephosphorylation by PP1. When PP1 was overexpressed, it was observed that the binding between ULK1 and ATG14L increased corresponding to the PP1 expression level ( FIG. 6A ). Since ULK1 is a serine/threonine kinase and can phosphorylate VPS34 or Beclin1, elements of the VPS34 complex, it was speculated that the binding of ULK1 and ATG14L could induce phosphorylation of ATG14L, a component of the VPS34 complex. This conjecture is also in the same context as the previous study that ATG14L phosphorylation promotes the kinase activity of VPS34 and induces the formation of phagopores and autophagosomes in the context of nutrient deficiency (Park et al., 2016). In order to confirm whether ULK1 induces phosphorylation of ATG14L, an in vitro phosphorylation experiment was performed. As a result, the phosphorylated form of ATG14L was detected. In addition, phosphorylation of ULK1 due to its own phosphorylation activity was also detected ( FIG. 6B ). Therefore, we found that ULK1 induces phosphorylation of ATG14L.

In the presence of ATG14L, the VPS34 complex is activated by AMPK, whereas in the absence of ATG14L, the VPS34 complex is inhibited by AMPK (Kim et al., 2013). Based on this information, we hypothesized that the lipid kinase activity of VPS34 would be inhibited if the binding of ULK1-ATG14L was not performed, and the experiment was conducted. It was found that only wild-type ULK1 could induce the lipid kinase activity of VPS34 when wild-type or mutant ULK1 was restored through an in vitro VPS34 lipid kinase experiment in ULK1-/- HEK293 cells ( FIG. 6C ). This time, in vitro VPS34 was performed in wild-type HEK293 in a similar manner, but overexpression of PP1 was induced to check the degree of phosphorylation. The increase in VPS34 activity corresponding to the overexpression amount of PP1 ^{was confirmed by detection with 32} P-PI(3)P ( FIG. 6D ).

Since VPS34 can be involved in other cellular processes besides autophagy, we decided to examine whether the O-GlcNAc mutation of ULK1 affects the autophagy-inducing function of VPS34 to confirm this possibility. It is well known that WIPI family proteins recognize PI(3)P in phagophores and act as PI(3)P-binding factors specific for autophagy (Proikas-Cezanne et al., 2015). Since the location of WIPI is dependent on the activity of VPS34, WIPI puncta formation was confirmed in cells in which wild-type or T754N mutant ULK1 was restored. It was confirmed that WIPI puncta was significantly reduced in cells in which T754N mutant ULK1 was restored, unlike wild-type ULK1 in a glucose-deficient situation. These results demonstrate that WIPI puncta formation, which indicates the activity of VPS34, is dependent on O-GlcNAcylation of ULK1. Collectively, these results show that ULK1 O-GlcNAcylation induces the promotion of lipid kinase function of VPS34 by ATG14L, leading to PI(3)P formation and phagopore formation.

This study demonstrates that the initiation of autophagy is regulated by sequential post-translational modifications of ULK1, i.e., dephosphorylation by PP1, phosphorylation by AMPK, and O-GlcNAcylation by OGT in the context of glucose deficiency. In addition, we identified the O-GlcNAcylation site of ULK1 caused by sugar deficiency and revealed that O-GlcNAcylation of ULK1 regulates autophagosome formation by increasing the lipid kinase function of VPS34 through ATG14L (Fig. 6E).

Previous studies have revealed that OGT is recruited to the PI(3)P formation site during growth factor action, suggesting that the signaling cascade triggered by PI3K may be regulated by O-GlcNAcylation (Yang). et al., 2008). In addition to the study result that OGT is recruited to PI(3)P in the cell membrane, it was also predicted that OGT would be recruited to newly formed autophagosomes (Hanover et al., 2010).

In the present application, a specific target of OGT was found in the structure of the preautophagosome, and it was found that ATG14L activates VPS34, a type III PI3K, to induce the formation of PI(3)P. PI(3)P conversion in phagopores by VSP34 is very important for preautophagosome structure formation. According to the results of two other studies, ATG14L promotes the formation of a bilayer structure and induces autophagy, and the absence of ATG14L leads to a defect in autophagosome formation (Matsunaga et al., 2009; Zhong et al., 2009). In addition, it was found that the activity of VPS34, which forms a complex with ATG14L, is highly dependent on ULK1, but detailed mechanisms have not been studied. In this study, we presented evidence that O-GlcNAcylation of ULK1 induces the binding of ULK1 and ATG14L, thereby promoting the lipid kinase function of VPS34.

Furthermore, by suggesting a clue that PP1 promotes O-GlcNAcylation by dephosphorylating S757 close to the glycosylation site of ULK1, it was revealed that O-GlcNAcylation works by mediating dephosphorylation enzymes. This phenomenon has a catalytic function in the situation of glucose deficiency and is considered to be an intermediate process between phosphorylation by mTOR and O-GlcNAcylation by OGT. It is also speculated that the common targets of PP1 and OGT exist in addition to ULK1. This is because it has been reported that global O-GlcNAcylation increases during glucose deficiency in H1299 cells (Yi et al., 2012). This study demonstrated that O-GlcNAcylation of ULK1 is important for activation of VPS34 and phagopore formation through ATG14L.

Interestingly, S317 S555 phosphorylation of ULK1 by AMPK occurs prior to O-GlcNAcylation of ULK1 in the context of glucose deprivation. The phosphorylation status of other phosphorylation sites, S467, T574, and S777, seems to have a minimal effect on O-GlcNAcylation of ULK1, and phosphorylation of S637 does not appear to affect O-GlcNAcylation of ULK1. In a glucose deprivation situation, mTOR is inhibited but AMPK is activated, so the phosphorylation state of S637 in the early stage of autophagy is very unclear (Shang and Wang, 2011). In addition, although S757 is phosphorylated only by mTOR, AMPK inhibits mTOR in a glucose-deficient situation, so knockdown of AMPK or inhibition of its function using an inhibitor may affect phosphorylation of S757. In this regard, further research on the exact mechanism is urgently needed.

This study demonstrates that sequential post-translational modifications on ULK1 in the context of glucose deprivation act as a gatekeeper between pro-autophagy and anti-autophagy, which is an essential determinant of the progression of autophagy. . Since autophagy is a process that consumes a lot of energy and is a critical process that prepares short-term and long-term countermeasures for cell survival, we think that cells use these multiple steps as a checkpoint to determine whether the rest of the reaction will proceed. This study discovered a new target for future drug development by revealing that the initial stage of autophagy is regulated through the crosstalk between dephosphorylation and O-GlcNAcylation.

Although the exemplary embodiments of the present application have been described in detail above, the scope of the present application is not limited thereto, and various modifications and improvements by those skilled in the art using the basic concept of the present application as defined in the following claims are also included in the scope of the present application. will belong to

All technical terms used in the present invention, unless otherwise defined, have the meaning as commonly understood by one of ordinary skill in the art of the present invention. The contents of all publications herein incorporated by reference are incorporated herein by reference.

<110> Seoul National University R&DB Foundation Sookmyung Women's university industry-academic cooperation foundation <120> Method of screening a material regulating autophagy targeting O-GlcNACylation of ULK1 protein <130> DP20190306P <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 1050 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <222> (1)..(1050) <223> ULK1 <400> 1 Met Glu Pro Gly Arg Gly Gly Thr Glu Thr Val Gly Lys Phe Glu Phe 1 5 10 15 Ser Arg Lys Asp Leu Ile Gly His Gly Ala Phe Ala Val Val Phe Lys 20 25 30 Gly Arg His Arg Glu Lys His Asp Leu Glu Val Ala Val Lys Cys Ile 35 40 45 Asn Lys Lys Asn Leu Ala Lys Ser Gln Thr Leu Leu Gly Lys Glu Ile 50 55 60 Lys Ile Leu Lys Glu Leu Lys His Glu Asn Ile Val Ala Leu Tyr Asp 65 70 75 80 Phe Gln Glu Met Ala Asn Ser Val Tyr Leu Val Met Glu Tyr Cys Asn 85 90 95 Gly Gly Asp Leu Ala Asp Tyr Leu His Ala Met Arg Thr Leu Ser Glu 100 105 110 Asp Thr Ile Arg Leu Phe Leu Gln Gln Ile Ala Gly Ala Met Arg Leu 115 120 125 Leu His Ser Lys Gly Ile Ile His Arg Asp Leu Lys Pro Gln Asn Ile 130 135 140 Leu Leu Ser Asn Pro Ala Gly Arg Arg Ala Asn Pro Asn Ser Ile Arg 145 150 155 160 Val Lys Ile Ala Asp Phe Gly Phe Ala Arg Tyr Leu Gln Ser Asn Met 165 170 175 Met Ala Ala Thr Leu Cys Gly Ser Pro Met Tyr Met Ala Pro Glu Val 180 185 190 Ile Met Ser Gln His Tyr Asp Gly Lys Ala Asp Leu Trp Ser Ile Gly 195 200 205 Thr Ile Val Tyr Gln Cys Leu Thr Gly Lys Ala Pro Phe Gln Ala Ser 210 215 220 Ser Pro Gln Asp Leu Arg Leu Phe Tyr Glu Lys Asn Lys Thr Leu Val 225 230 235 240 Pro Thr Ile Pro Arg Glu Thr Ser Ala Pro Leu Arg Gln Leu Leu Leu 245 250 255 Ala Leu Leu Gln Arg Asn His Lys Asp Arg Met Asp Phe Asp Glu Phe 260 265 270 Phe His His Pro Phe Leu Asp Ala Ser Pro Ser Val Arg Lys Ser Pro 275 280 285 Pro Val Pro Val Pro Ser Tyr Pro Ser Ser Gly Ser Gly Ser Ser Ser 290 295 300 Ser Ser Ser Ser Thr Ser His Leu Ala Ser Pro Ser Leu Gly Glu 305 310 315 320 Met Gln Gln Leu Gln Lys Thr Leu Ala Ser Pro Ala Asp Thr Ala Gly 325 330 335 Phe Leu His Ser Ser Arg Asp Ser Gly Gly Ser Lys Asp Ser Ser Cys 340 345 350 Asp Thr Asp Asp Phe Val Met Val Pro Ala Gln Phe Pro Gly Asp Leu 355 360 365 Val Ala Glu Ala Pro Ser Ala Lys Pro Pro Pro Asp Ser Leu Met Cys 370 375 380 Ser Gly Ser Ser Leu Val Ala Ser Ala Gly Leu Glu Ser His Gly Arg 385 390 395 400 Thr Pro Ser Pro Ser Pro Pro Cys Ser Ser Ser Pro Ser Pro Ser Gly 405 410 415 Arg Ala Gly Pro Phe Ser Ser Ser Arg Cys Gly Ala Ser Val Pro Ile 420 425 430 Pro Val Pro Thr Gln Val Gln Asn Tyr Gln Arg Ile Glu Arg Asn Leu 435 440 445 Gln Ser Pro Thr Gln Phe Gln Thr Pro Arg Ser Ser Ala Ile Arg Arg 450 455 460 Ser Gly Ser Thr Ser Pro Leu Gly Phe Ala Arg Ala Ser Pro Ser Pro 465 470 475 480 Pro Ala His Ala Glu His Gly Gly Val Leu Ala Arg Lys Met Ser Leu 485 490 495 Gly Gly Gly Arg Pro Tyr Thr Pro Ser Pro Gln Val Gly Thr Ile Pro 500 505 510 Glu Arg Pro Gly Trp Ser Gly Thr Pro Ser Pro Gln Gly Ala Glu Met 515 520 525 Arg Gly Gly Arg Ser Pro Arg Pro Gly Ser Ser Ala Pro Glu His Ser 530 535 540 Pro Arg Thr Ser Gly Leu Gly Cys Arg Leu His Ser Ala Pro Asn Leu 545 550 555 560 Ser Asp Leu His Val Val Arg Pro Lys Leu Pro Lys Pro Pro Thr Asp 565 570 575 Pro Leu Gly Ala Val Phe Ser Pro Pro Gln Ala Ser Pro Pro Gln Pro 580 585 590 Ser His Gly Leu Gln Ser Cys Arg Asn Leu Arg Gly Ser Pro Lys Leu 595 600 605 Pro Asp Phe Leu Gln Arg Asn Pro Leu Pro Pro Ile Leu Gly Ser Pro 610 615 620 Thr Lys Ala Val Pro Ser Phe Asp Phe Pro Lys Thr Pro Ser Ser Gln 625 630 635 640 Asn Leu Leu Ala Leu Leu Ala Arg Gln Gly Val Val Met Thr Pro Pro 645 650 655 Arg Asn Arg Thr Leu Pro Asp Leu Ser Glu Val Gly Pro Phe His Gly 660 665 670 Gln Pro Leu Gly Pro Gly Leu Arg Pro Gly Glu Asp Pro Lys Gly Pro 675 680 685 Phe Gly Arg Ser Phe Ser Thr Ser Arg Leu Thr Asp Leu Leu Leu Lys 690 695 700 Ala Ala Phe Gly Thr Gln Ala Pro Asp Pro Gly Ser Thr Glu Ser Leu 705 710 715 720 Gln Glu Lys Pro Met Glu Ile Ala Pro Ser Ala Gly Phe Gly Gly Ser 725 730 735 Leu His Pro Gly Ala Arg Ala Gly Gly Thr Ser Ser Pro Ser Pro Val 740 745 750 Val Phe Thr Val Gly Ser Pro Pro Ser Gly Ser Thr Pro Pro Gln Gly 755 760 765 Pro Arg Thr Arg Met Phe Ser Ala Gly Pro Thr Gly Ser Ala Ser Ser 770 775 780 Ser Ala Arg His Leu Val Pro Gly Pro Cys Ser Glu Ala Pro Ala Pro 785 790 795 800 Glu Leu Pro Ala Pro Gly His Gly Cys Ser Phe Ala Asp Pro Ile Ala 805 810 815 Ala Asn Leu Glu Gly Ala Val Thr Phe Glu Ala Pro Asp Leu Pro Glu 820 825 830 Glu Thr Leu Met Glu Gin Glu His Thr Glu Ile Leu Arg Gly Leu Arg 835 840 845 Phe Thr Leu Leu Phe Val Gln His Val Leu Glu Ile Ala Ala Leu Lys 850 855 860 Gly Ser Ala Ser Glu Ala Ala Gly Gly Pro Glu Tyr Gln Leu Gln Glu 865 870 875 880 Ser Val Val Ala Asp Gln Ile Ser Leu Leu Ser Arg Glu Trp Gly Phe 885 890 895 Ala Glu Gln Leu Val Leu Tyr Leu Lys Val Ala Glu Leu Leu Ser Ser Ser 900 905 910 Gly Leu Gln Ser Ala Ile Asp Gln Ile Arg Ala Gly Lys Leu Cys Leu 915 920 925 Ser Ser Thr Val Lys Gln Val Val Arg Arg Leu Asn Glu Leu Tyr Lys 930 935 940 Ala Ser Val Val Ser Cys Gln Gly Leu Ser Leu Arg Leu Gln Arg Phe 945 950 955 960 Phe Leu Asp Lys Gln Arg Leu Leu Asp Arg Ile His Ser Ile Thr Ala 965 970 975 Glu Arg Leu Ile Phe Ser His Ala Val Gln Met Val Gln Ser Ala Ala 980 985 990 Leu Asp Glu Met Phe Gln His Arg Glu Gly Cys Val Pro Arg Tyr His 995 1000 1005 Lys Ala Leu Leu Leu Leu Glu Gly Leu Gln His Met Leu Ser Asp Gln 1010 1015 1020 Ala Asp Ile Glu Asn Val Thr Lys Cys Lys Leu Cys Ile Glu Arg Arg 1025 1030 1035 1040 Leu Ser Ala Leu Leu Thr Gly Ile Cys Ala 1045 1050 <210> 2 <211> 1051 <212> PRT <213> Mus musculus <220> <221> MISC_FEATURE <222> (1)..(1051) <223> ULK1 <400> 2 Met Glu Pro Gly Arg Gly Gly Val Glu Thr Val Gly Lys Phe Glu Phe 1 5 10 15 Ser Arg Lys Asp Leu Ile Gly His Gly Ala Phe Ala Val Val Phe Lys 20 25 30 Gly Arg His Arg Glu Lys His Asp Leu Glu Val Ala Val Lys Cys Ile 35 40 45 Asn Lys Lys Asn Leu Ala Lys Ser Gln Thr Leu Leu Gly Lys Glu Ile 50 55 60 Lys Ile Leu Lys Glu Leu Lys His Glu Asn Ile Val Ala Leu Tyr Asp 65 70 75 80 Phe Gln Glu Met Ala Asn Ser Val Tyr Leu Val Met Glu Tyr Cys Asn 85 90 95 Gly Gly Asp Leu Ala Asp Tyr Leu His Thr Met Arg Thr Leu Ser Glu 100 105 110 Asp Thr Val Arg Leu Phe Leu Gln Gln Ile Ala Gly Ala Met Arg Leu 115 120 125 Leu His Ser Lys Gly Ile Ile His Arg Asp Leu Lys Pro Gln Asn Ile 130 135 140 Leu Leu Ser Asn Pro Gly Gly Arg Arg Ala Asn Pro Ser Asn Ile Arg 145 150 155 160 Val Lys Ile Ala Asp Phe Gly Phe Ala Arg Tyr Leu Gln Ser Asn Met 165 170 175 Met Ala Ala Thr Leu Cys Gly Ser Pro Met Tyr Met Ala Pro Glu Val 180 185 190 Ile Met Ser Gln His Tyr Asp Gly Lys Ala Asp Leu Trp Ser Ile Gly 195 200 205 Thr Ile Val Tyr Gln Cys Leu Thr Gly Lys Ala Pro Phe Gln Ala Ser 210 215 220 Ser Pro Gln Asp Leu Arg Leu Phe Tyr Glu Lys Asn Lys Thr Leu Val 225 230 235 240 Pro Ala Ile Pro Arg Glu Thr Ser Ala Pro Leu Arg Gln Leu Leu Leu 245 250 255 Ala Leu Leu Gln Arg Asn His Lys Asp Arg Met Asp Phe Asp Glu Phe 260 265 270 Phe His His Pro Phe Leu Asp Ala Ser Thr Pro Ile Lys Lys Ser Pro 275 280 285 Pro Val Pro Val Pro Ser Tyr Pro Ser Ser Gly Ser Gly Ser Ser Ser 290 295 300 Ser Ser Ser Ser Ala Ser His Leu Ala Ser Pro Ser Leu Gly Glu 305 310 315 320 Met Pro Gln Leu Gln Lys Thr Leu Thr Ser Pro Ala Asp Ala Ala Gly 325 330 335 Phe Leu Gln Gly Ser Arg Asp Ser Gly Gly Ser Ser Lys Asp Ser Cys 340 345 350 Asp Thr Asp Asp Phe Val Met Val Pro Ala Gln Phe Pro Gly Asp Leu 355 360 365 Val Ala Glu Ala Ala Ser Ala Lys Pro Pro Pro Asp Ser Leu Leu Cys 370 375 380 Ser Gly Ser Ser Leu Val Ala Ser Ala Gly Leu Glu Ser His Gly Arg 385 390 395 400 Thr Pro Ser Pro Ser Pro Thr Cys Ser Ser Ser Pro Ser Pro Ser Gly 405 410 415 Arg Pro Gly Pro Phe Ser Ser Asn Arg Tyr Gly Ala Ser Val Pro Ile 420 425 430 Pro Val Pro Thr Gln Val His Asn Tyr Gln Arg Ile Glu Gln Asn Leu 435 440 445 Gln Ser Pro Thr Gln Gln Gln Thr Ala Arg Ser Ser Ala Ile Arg Arg 450 455 460 Ser Gly Ser Thr Ser Pro Leu Gly Phe Gly Arg Ala Ser Pro Ser Pro 465 470 475 480 Pro Ser His Thr Asp Gly Ala Met Leu Ala Arg Lys Leu Ser Leu Gly 485 490 495 Gly Gly Arg Pro Tyr Thr Pro Ser Pro Gln Val Gly Thr Ile Pro Glu 500 505 510 Arg Pro Ser Trp Ser Arg Val Pro Ser Pro Gln Gly Ala Asp Val Arg 515 520 525 Val Gly Arg Ser Pro Arg Pro Gly Ser Ser Val Pro Glu His Ser Pro 530 535 540 Arg Thr Thr Gly Leu Gly Cys Arg Leu His Ser Ala Pro Asn Leu Ser 545 550 555 560 Asp Phe His Val Val Arg Pro Lys Leu Pro Lys Pro Pro Thr Asp Pro 565 570 575 Leu Gly Ala Thr Phe Ser Pro Gln Thr Ser Ala Pro Gln Pro Cys 580 585 590 Pro Gly Leu Gln Ser Cys Arg Pro Leu Arg Gly Ser Pro Lys Leu Pro 595 600 605 Asp Phe Leu Gln Arg Ser Pro Leu Pro Pro Ile Leu Gly Ser Pro Thr 610 615 620 Lys Ala Gly Pro Ser Phe Asp Phe Pro Lys Thr Pro Ser Ser Gln Asn 625 630 635 640 Leu Leu Thr Leu Leu Ala Arg Gln Gly Val Val Met Thr Pro Pro Arg 645 650 655 Asn Arg Thr Leu Pro Asp Leu Ser Glu Ala Ser Pro Phe His Gly Gln 660 665 670 Gln Leu Gly Ser Gly Leu Arg Pro Ala Glu Asp Thr Arg Gly Pro Phe 675 680 685 Gly Arg Ser Phe Ser Thr Ser Arg Ile Thr Asp Leu Leu Leu Lys Ala 690 695 700 Ala Phe Gly Thr Gln Ala Ser Asp Ser Gly Ser Thr Asp Ser Leu Gln 705 710 715 720 Glu Lys Pro Met Glu Ile Ala Pro Ser Ala Gly Phe Gly Gly Thr Leu 725 730 735 His Pro Gly Ala Arg Gly Gly Gly Ala Ser Ser Pro Ala Pro Val Val 740 745 750 Phe Thr Val Gly Ser Pro Pro Ser Gly Ala Thr Pro Pro Gln Ser Thr 755 760 765 Arg Thr Arg Met Phe Ser Val Gly Ser Ser Ser Ser Leu Gly Ser Thr 770 775 780 Gly Ser Ser Ser Ala Arg His Leu Val Pro Gly Ala Cys Gly Glu Ala 785 790 795 800 Pro Glu Leu Ser Ala Pro Gly His Cys Cys Ser Leu Ala Asp Pro Leu 805 810 815 Ala Ala Asn Leu Glu Gly Ala Val Thr Phe Glu Ala Pro Asp Leu Pro 820 825 830 Glu Glu Thr Leu Met Glu Gin Glu His Thr Glu Thr Leu His Ser Leu 835 840 845 Arg Phe Thr Leu Ala Phe Ala Gln Gln Val Leu Glu Ile Ala Ala Leu 850 855 860 Lys Gly Ser Ala Ser Glu Ala Ala Gly Gly Pro Glu Tyr Gln Leu Gln 865 870 875 880 Glu Ser Val Val Ala Asp Gln Ile Ser Gln Leu Ser Arg Glu Trp Gly 885 890 895 Phe Ala Glu Gln Leu Val Leu Tyr Leu Lys Val Ala Glu Leu Leu Ser 900 905 910 Ser Gly Leu Gln Thr Ala Ile Asp Gln Ile Arg Ala Gly Lys Leu Cys 915 920 925 Leu Ser Ser Thr Val Lys Gln Val Val Arg Arg Leu Asn Glu Leu Tyr 930 935 940 Lys Ala Ser Val Val Ser Cys Gln Gly Leu Ser Leu Arg Leu Gln Arg 945 950 955 960 Phe Phe Leu Asp Lys Gln Arg Leu Leu Asp Gly Ile His Gly Val Thr 965 970 975 Ala Glu Arg Leu Ile Leu Ser His Ala Val Gln Met Val Gln Ser Ala 980 985 990 Ala Leu Asp Glu Met Phe Gln His Arg Glu Gly Cys Val Pro Arg Tyr 995 1000 1005 His Lys Ala Leu Leu Leu Leu Glu Gly Leu Gln His Thr Leu Thr Asp 1010 1015 1020 Gln Ala Asp Ile Glu Asn Ile Ala Lys Cys Lys Leu Cys Ile Glu Arg 1025 1030 1035 1040 Arg Leu Ser Ala Leu Leu Ser Gly Val Tyr Ala 1045 1050

Claims (7)

culturing cells expressing AMPK, PPI and ULK1 proteins in low glucose concentration;
contacting the cell with a test substance expected to regulate O-GlcNAcylation at the 755th threonine residue of ULK1 based on the sequence of SEQ ID NO: 1; and
After the contact, measuring the degree of O-GlcNAcylation at the 755th threonine residue of ULK1, phosphorylation at 317 and 556 serine residues of ULK1, and dephosphorylation at the 758th serine residue of ULK1,
In the measuring step, when the degree of O-GlcNAcylation at the 755th threonine residue of ULK1 is increased and the dephosphorylation and phosphorylation are increased compared to the case where the test substance is not treated, the test substance is activated by autophagy When the degree of O-GlcNAcylation does not change or decreases, and when the dephosphorylation and phosphorylation do not change or decrease compared to the case where the test substance is not treated with the first or the test substance, the test substance is selected as an autophagy inhibitor candidate A method for screening an autophagy modulator.
delete delete The method of claim 1,
The cells further express ATG14L and VPS34 lipid kinase,
The measuring step further comprises measuring the phosphorylation of ATG14L and the activity of VPS34 lipid kinase,
As a result of the measurement, when the phosphorylation of ATG14L and the activity of VPS34 lipid kinase increased compared to the case where the test substance was not treated, the test substance was treated with an autophagy activator, and
As a result of the measurement, when the phosphorylation of ATG14L and the activity of VPS34 lipid kinase do not change or decrease compared to the case where the test substance is not treated with the test substance, the test substance is selected as an autophagy inhibitor candidate. Way.
The method of claim 1,
The ULK1 protein is dephosphorylated at position 758 or
The ULK1 protein is a protein in which the 317th or 556th serine residue is phosphorylated, and the cell is cultured at a normal glucose concentration, autophagy modulator screening method.
6. The method of any one of claims 1, 4 or 5,
The cell line is H1299, HEK293, or SW620 cell line, autophagy modulator screening method.
6. The method of any one of claims 1, 4 or 5,
The autophagy modulator is for the prevention or treatment of cancer, neurodegenerative disease, autoimmune disease, cardiovascular disease, metabolic disease, or hereditary muscle disease, autophagy modulator screening method.

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