METHOD FOR SCREENING FOR AUTOPHAGY ACTIVATOR OR INHIBITOR

Abstract

The present invention provides a method for screening for an autophagy activator or inhibitor comprising the steps of: (a) making a test material to be analyzed come into contact with cells containing Beclin 1 protein; and (b) analyzing the degree of phosphorylation at the 30.sup.th serine amino acid residue of the Beclin 1 protein. The test material is determined to be an autophagy activator when the phosphorylation of the Beclin 1 protein is up-regulated, and the test material is determined to be an autophagy inhibitor when the phosphorylation of the Beclin 1 protein is down-regulated. The present invention first establishes, by ULK1, the mechanism of phosphorylation at the 30.sup.th serine amino acid residue of Beclin 1.

Claims

1-5. (canceled)

6. An antibody specifically binding to Beclin 1 protein with the 30th amino acid residue serine phosphorylated.

7. (canceled)

8. The antibody of claim 6, wherein the antibody is an antibody binding to an epitope composed of the amino acid sequence described by SEQ ID NO: 1.

9. A diagnostic kit for an autophagy-related disease, comprising the antibody of claim 6.

10. The diagnostic kit of claim 9, wherein the antibody is an antibody binding to an epitope composed of the amino acid sequence described by SEQ ID NO: 1.

11. A composition comprising the antibody of claim 6.

12. The composition of claim 11, wherein the antibody is an antibody binding to an epitope composed of the amino acid sequence described by SEQ ID NO: 1.

13. The composition of claim 11, wherein the composition is a pharmaceutical composition comprising a pharmaceutically effective amount of the antibody.

14. The composition of claim 13, further comprising one or more pharmaceutically acceptable carriers.

15. The composition of claim 11, wherein the composition is a diagnostic composition.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0064] FIGS. 1a and 1b shows verification results of phosphorylation of Beclin 1 by ULK1.

[0065] FIG. 2 shows verification results of direct phosphorylation of Beclin 1 by ULK1.

[0066] FIG. 3 shows verification results of Ser30 phosphorylation of Beclin 1 by ULK1.

[0067] FIG. 4 shows verification results of Ser30 phosphorylation of Beclin 1 at starvation state.

[0068] FIG. 5 shows verification results of Ser30 phosphorylation of Beclin 1 by rapamycin treatment.

[0069] FIG. 6 shows verification results of Ser30 phosphorylation of Beclin 1 by torin treatment.

[0070] FIG. 7 shows the importance of Ser30 phosphorylation of Beclin 1 on autophagy activation.

MODE FOR CARRYING OUT THE INVENTION

[0071] Hereinafter, the present invention will be described in detail with reference to examples. These examples are only for illustrating the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

Examples

Materials and Methods

Chemicals and Antibodies

[0072] Anti-ULK1(sc-10900) and Beclin 1 (sc-11427 and sc-10086) antibodies were purchased from Santa Cruz Biotech. Anti-Atg14L antibodies were obtained from MBL (PD026) and Abgent (AP1832a). Anti-myc 9E10 was purchased from EMD Biosciences (OP10), and anti-HA antibody HA.11 was purchased from Covance (AFC-101P).

Plasmid Preparation and Mutagenesis

[0073] Human ULK1 and Atg13 cDNA clones were obtained from Katzusa laboratory. The kinase-dead mutant of ULK1, M92A was made by using the site-directed mutagenesis kit (Stratagene, CA). The cDNA for human Beclin 1 was purchased from Open Biosystems.

Cell Culture and Transfection

[0074] HEK293T, HeLa, and MEF cells were cultured in DMEM (Invitrogen, U.S.)_containing 10% fetal bovine serum (FBS), penicillin, and streptomycin under conditions of 37° C./5% CO.sub.2. For transient expression, the cells were transfected with recombinant DNA using FuGENETM (Rochediagnostics) according to the manufacturer's protocol. Cells were harvested 2 day after transfection, for co-immunoprecipitation.

[0075] Co-Immunoprecipitation and Western Blotting

[0076] For co-immunoprecipitation, cell extracts were prepared in a buffer containing 40 mM HEPES, pH 7.4, 120 mM NaCl, 1 mM EDTA, 50 mM NaF, 1.5 mM Na.sub.3VO.sub.4, 10 mM Mb-glycerophosphate, 1% triton X-100, and protease inhibitor (Roche). Immunoprecipitated proteins were washed four times with a lysis buffer, and loaded onto 8% Tris-glycine gels. The resultant proteins were transferred onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad, USA), and detected with western blotting detection reagents (Perkin Elmer, USA).

Lentiviral Preparation

[0077] Lentiviral vector pLKO.1 coding shRNA targeting Atg14L or scramble sequences was transfected into HEK293T together with lentiviral packaging vectors pHR′8.2ΔR and pCMV-VSV-G using FuGENE 6. Viruses were collected 60 h after transfection, and HeLa cells were infected with the collected viruses in the presence of polybrene. The transfected cells were selected by puromycin treatment. The target sequences for Atg14L shRNA are 5′-CCATAGAACTTGGTCATGTTT-3′ and 5′-CCACTTTCTTTCTATGGGATT-3′, which are positioned at 3′-UTR. This shRNA does not target 3′-UTR-deficient recombinant Atg14L.

Immunostaining

[0078] HeLa or MEF cells were fixed in 4% formaldehyde (in PBS) for 15 min, and permeabilized using 0.3% Triton X-100 at room temperature for 30 min. The permeable cells were subjected to a constant-temperature reaction in PBS containing 1% bovine serum albumin (BSA) for 30 min, and then subjected to an antibody reaction at 4° C. overnight. Endogenous LC3 and Atg14L were stained using anti-LC antibodies (Novus, cat# NB100-2220 and Nanotools, cat#5F10) and anti-Atg14L antibody (MBL, PD026). Recombinant myc and HA-tagged proteins were monitored using Alexa647 fluorescent dye (Cell Signaling, cat#2233)-conjugated anti-myc antibody and anti-HA antibody (Covance, AFC-101P). After primary antibody binding, the cells were subjected to a constant-temperature reaction with Alexa Flour 488-conjugated anti-mouse IgG (Invitrogen, A-11001) and/or Alexa Flour 647-conjugated anti-rabbit IgG (Invitrogen, A-21443). The cells were stained with DAPI (4′-6-Diamidino-2-phenylindole, Invitrogen, D-1306). Images of the stained cells were obtained using Deltavision PersonelDV microscope (Applied Precision). The positioning of Atg14 wild type or S419A mutant of WIPI-1 or LC3 was statistically analyzed by student t-test using SigmaPlot 10.0 software (Systat Software, U.S.).

In Vivo Labeling

[0079] In order to determine the phosphorylation of Beclin 1 in living cells, myc-tagged ULK1 wild type and kinase-dead mutants were temporarily expressed in HEK293T cells. On day 2 after transfection, the reaction was performed in phosphate-free medium containing 10% diluted PBS for 4 h before the reaction for 1 h by adding 0.1 mCi[32P] orthophosphate (MPBiomedicals). For immunoprecipitation, myc-tagged ULK1 was isolated using anti-myc antibody. The isolated proteins were run on SDS-PAGE, and transferred to PVDF membrane, and an autoradiogram was obtained.

Results

Beclin 1 Phosphorylation Induction of ULK1

[0080] In 293 cells, myc-tagged Beclin 1 was co-expressed together with HA-tagged ULK1 wild type or M92A KD (kinase dead) mutant. Myc-Beclin 1 and HA-ULK1 were isolated by immunoprecipitation (IP) using anti-HA antibodies, and the shift aspect on SDS-PAGE was analyzed by western blot. It was confirmed that Beclin 1 and ULK1 or M92A KD mutant were conjugated to each other (FIG. 1a). Beclin 1 co-expressed together with ULK1 showed band shift, but Beclin 1 co-expressed together with ULK1 M92A KD showed no band shift. These results suggest the possibility of the Beclin 1 phosphorylation by the ULK1 wild type. 293T cells co-expressing myc-tagged Beclin 1 together with HA-tagged ULK1 wild type or M92A KD (kinase dead) were treated with/without lambda phosphatase, isolated through immunoprecipitation, and Co-IP was performed using anti-HA antibody, and the shift aspect on SDS-PAGE were analyzed by western blot. It showed that the phosphorylation of Beclin 1 shifted-up by ULK1 was isolated by phosphatase, indicating that ULK1 is a decisive clue in the phosphorylation of Beclin 1 (FIG. 1b).

Beclin 1 Phosphorylation

[0081] The overexpressed myc-tagged ULK1 wild type and KD (M92) mutant were isolated from HEK293T cells by immunoprecipitation (IP) using anti-myc antibody. Recombinant Beclin 1 was isolated from E. coli strain, and used as a substrate. Beclin 1 was subjected to constant-temperature reaction with ULK1 wild type or KD mutant isolated through immunoprecipitation at 37° C. in the presence of 32P-ATP. The incorporation of 32p into Beclin 1 and ULK1 was analyzed by autoradiogram. The levels of Myc-ULK1 and Beclin 1 were analyzed by western blot (FIG. 2). The ULK1 wild type phosphorylates Beclin 1, but KD mutant-added Beclin 1 was not phosphorylated. That is, the results show that the kinase activity of ULK1 is important in the phosphorylation of Beclin 1.

Beclin 1 Ser30 Epitope

[0082] HA-tagged Beclin 1 and myc-tagged ULK1 wild type or KD mutant were expressed and immunoprecipitation was performed using HA antibody. The bands shown on SDS-PAGE were respectively extracted, and the phosphorylation sites of ULK1 were analyzed through LC-MS assay. As an analysis result, Ser30 of Beclin 1 was analyzed as a site with high possibility. In order to develop antibodies specifically binding to Beclin 1 protein with the amino acid residue serine phosphorylated, the epitope of antibody was synthesized as follows. The following synthetic peptide is in a form in which the fifth amino acid residue serine is phosphorylated.

[0083] C-KLDTS FKILDR (26-36, aa) 12-mer

[0084] Cysteine (cys) at the N-terminal was arbitrarily added for conjugation to the carrier protein. The epitopes were synthesized to construct antibodies against Ser30 phosphorylation of Beclin 1. (asked Abfrontier, Korea; http://www.abfrontier.com/cs/antibodyPoly.do).

Beclin 1 Ser30 Phosphorylation

[0085] ULK1+/+ MEF and ULK1−/− MEF were cultured in HBSS media for 2 h. Endogenous Beclin 1 was isolated by immunoprecipitation. The phosphorylation of Ser30 was analyzed using antibodies developed by the present inventors (FIG. 3). The kinase activity of ULK1 was increased in HBSS media, and here, it was estimated that the activated ULK1 would phosphorylate the 30th amino acid residue serine of Beclin 1. As a test result, the self-developed Beclin 1 Ser30 antibody performed a selective reaction on the phosphorylation sites of Beclin 1 phosphorylated by ULK1.

Beclin 1 Ser30 Phosphorylation at Starvation

[0086] MEF cells were cultured in HBSS for predetermined periods of time (0, 2, 4, and 6 h). Endogenous Beclin 1 was isolated by immunoprecipitation. The phosphorylation of Ser30 was analyzed using rabbit antibodies developed in the present invention. It was verified that Ser30 of Beclin 1 was phosphorylated at starvation state (FIG. 4).

Beclin 1 Ser30 Phosphorylation by Rapamycin

[0087] HEK293T cells were treated with rapamycin for predetermined periods of time (0, 15, 30, 60, and 120 min). Endogenous Beclin 1 was isolated by immunoprecipitation. The phosphorylation of Ser30 was analyzed using rabbit antibodies developed in the present invention. It was verified that Ser30 of Beclin 1 was phosphorylated by rapamycin (FIG. 5). Rapamycin increases the activity of ULK1.

Beclin 1 Ser30 Phosphorylation by Torin

[0088] HEK293T cells were treated with torin for predetermined periods of time (0, 15, 30, 60, and 120 min). Endogenous Beclin 1 was isolated by immunoprecipitation. The phosphorylation of Ser30 was analyzed using rabbit antibodies developed in the present invention. It was verified that Ser30 of Beclin 1 was phosphorylated by torin (FIG. 6).

Autophagy Activation

[0089] In Beclin 1-silent HeLa cells, myc-tagged WIPI-1 and HA-tagged Beclin 1 wild type or S30A mutant were temporally expressed. The cells were treated with rapamycin for 1 h, and then immunostained with HA-Beclin 1 (green) and myc-WIPI1 (red). The nucleus (blue) were stained with DAPI. Beclin 1 puncta and WIPI-1 co-location was analyzed (FIG. 7). It was verified that the mutation of the 30th amino acid residue serine of Beclin 1 decreased the autophagy activity.

[0090] Although the present invention has been described in detail with reference to the specific features, it will be apparent to those skilled in the art that this description is only for a preferred embodiment and does not limit the scope of the present invention. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.