WO2022114622A1 - Method for regulating autophagy through arginine methylation of pontin by carm1 and use thereof - Google Patents

Method for regulating autophagy through arginine methylation of pontin by carm1 and use thereof Download PDF

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WO2022114622A1
WO2022114622A1 PCT/KR2021/016485 KR2021016485W WO2022114622A1 WO 2022114622 A1 WO2022114622 A1 WO 2022114622A1 KR 2021016485 W KR2021016485 W KR 2021016485W WO 2022114622 A1 WO2022114622 A1 WO 2022114622A1
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pontin
autophagy
carm1
methylation
protein
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French (fr)
Korean (ko)
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백성희
유영석
신희재
김동하
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서울대학교산학협력단
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

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  • the present application relates to methods for regulating autophagy and uses thereof.
  • an autophagosome (membrane vacuole, autophagosome) composed of a lipid bilayer surrounds its unnecessary proteins and aging organelles in an environment where intracellular nutrients are insufficient, and then merges 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. , play an important role in cell growth, survival and homeostasis.
  • 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).
  • 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)).
  • Korean Patent Registration No. 10-1594168 discloses a method for regulating autophagy mediated by the ZZ region of p62 and its use.
  • U.S. Patent Publication No. 2010-0233730 relates to autophagy control for treatment, and after treating cells with a test substance, metabolic stress is applied to them to observe intracellular changes in autophagosomes, A method of screening is disclosed.
  • autophagy protein-based technologies that act in the cytoplasm
  • autophagy control technologies based on nuclear transcriptional processes or epigenetic mechanisms are unknown.
  • the development of autophagy modulators is necessary.
  • the present application aims to provide a method for regulating autophagy based on a mechanism acting in the nucleus and a screening method using the same.
  • the present application provides a method for screening a substance that regulates autophagy through methylation control of the PONTIN protein by the methyltransferase CARM1 (Coactivator Associated argine methyltransferase 1).
  • the method comprises a first step of culturing cells expressing CARM1 and PONTIN protein as a substrate for the methyltransferase CARM1 in a glucose-deficient state; a second step of treating the cells with a test substance expected to inhibit the methylation activity of the methyltransferase CARM1 for the PONTIN; a third step of isolating the PONTIN protein from the cells: a fourth step of measuring the degree of methylation at residues 333 and 339 based on SEQ ID NO: 1 of the isolated PONTIN protein; and
  • a fifth step of selecting this as a candidate substance for regulating autophagy is included, wherein the increase in methylation is An increase in autophagy, the decrease in methylation, indicates inhibition of autophagy.
  • the first step further expresses the FOXO3a protein, and instead of or in addition to the fourth step, the step comprises measuring the binding of the PONTIN and FOXO3a protein, and the measurement result between the protein An increase in binding indicates an increase in autophagy, and a decrease in binding indicates inhibition of autophagy.
  • the PONTIN according to the present disclosure binds to FOXO3a through residues 624, 627, 628, 640 and 642 of the FOXO3a protein based on SEQ ID NO: 2.
  • the method according to the present disclosure includes, in addition to the fourth step, measuring whether the histone 4 (H4) protein isolated from the cell is acetylated, and as a result of the measurement, the acetylation of the H4 protein An increase indicates an increase in autophagy, and a decrease in binding indicates inhibition of autophagy.
  • H4 histone 4
  • the acetylation of histone 4 occurs at the 6th, 9th, 13th and 17th lysine residues based on the sequence of SEQ ID NO: 3.
  • Autophagy is a mechanism that is activated in various stressful situations such as nutrient deficiency, metabolic stress, viral infection, and aging and plays an important role in maintaining cell survival and homeostasis. is closely related to If the method of regulating autophagy based on the mechanism acting in the nucleus can be known through screening, it will provide theoretical foundational knowledge in the process of discovering new therapeutic targets for diseases caused by autophagy abnormalities (cancer, degenerative brain disease, etc.) can do.
  • the method for regulating autophagy targeting the CARM1-PONTIN-FOXO3a signaling system can be usefully used to develop therapeutic agents for various diseases caused by autophagy regulation problems at various levels.
  • Figure 2 shows that methylation of pontin by CARM1 occurs in the nucleus in a glucose deprivation state.
  • FIG. 1 Measurement of autophagy flux with or without Bafilomycin A1 and Chloroquine treatment.
  • Figures are LC3-II/bactin ratio (h) Representative confocal image after staining using CYTO-ID (i) Representative confocal image using mCherry-GFP-LC3 in Pontin WT, RA MEF. Yellow puncta with mCherry and GFP signals are autophagic spores (phagophores, autophagosomes) that are not bound to lysosomes, and red puncta are acidified autophagic spores (ampisomes, autolysosomes).
  • Figure 4 confirmed through RNA-sequencing that many autophagy and lysosomal genes are regulated by Pontin methylation.
  • (a) RNA-seq analysis method (b) Hierarchical clustering revealed 4131 DEGs (c) Gene ontology and KEGG pathway analysis using DAVID for gene group 1 (d) Representative GSEA results (e) Autophagy process Pontin methylation-dependent genes classified as (f) qRT-PCR results of pontin methylation-dependent autophagy or lysosome-related genes (g) Pontin WT, RK, and RA target protein expression levels in glucose-deficient conditions This is the result confirmed by immunoblot.
  • FIG. 8 shows that the CARM1-Pontin-FOXO3a axis plays a role in enhancer activity of autophagy genes.
  • 3C results performed to see the binding of the promoter and enhancer portion of Map1lc3b. Data confirmed by DNA gel after PCR. The model on the right.
  • 9 is a model figure showing the mechanism identified herein.
  • autophagy 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).
  • autophagy 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 autophagy dysregulation.
  • the present application relates to a method for screening a substance that regulates autophagy through methylation control of PONTIN protein by CARM1 (Coactivator Associated argine methyltransferase 1), a methyltransferase.
  • CARM1 Coactivator Associated argine methyltransferase 1
  • the method comprises a first step of culturing cells expressing CARM1 and PONTIN protein as a substrate for the methyltransferase CARM1 in a glucose-deficient state; a second step of treating the cells with a test substance expected to inhibit the methylation activity of the methyltransferase CARM1 for the PONTIN; a third step of isolating the PONTIN protein from the cells: a fourth step of measuring the degree of methylation at residues 333 and 339 based on SEQ ID NO: 1 of the isolated PONTIN protein; and a fifth step of selecting the methylation as a candidate for regulating autophagy when the methylation increases or decreases when treated with the test substance as compared with the control not treated with the test substance as a result of the measurement, wherein the methylation increases indicates an increase in autophagy, and a decrease in methylation indicates inhibition of autophagy.
  • 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
  • CARM1 coactivator-associated arginine methyltransferase 1 or PRMT4 (protein arginine N-methyltransferase 4) used in the method according to the present application is a nitrogen branch of an arginine residue from S-adenosyl-L-methionine composed of an alpha helix and a beta sheet. It is an enzyme that catalyzes methylation, and the gene and protein sequence of CARM1 in mammals is known, for example, the human gene and protein sequence is known as Gene ID: 10498, Protein ID: NP_954592.1.
  • a protein sequence of various origins and each derived protein sequence and a full-length or fragment having a sequence substantially identical thereto may be used.
  • the substrate of CARM1 is PONTIN.
  • PONTIN herein is a chromatin remodeling factor having ATPase and DNA helicase functions, and for example, human gene and protein sequences are known as Gene ID: 8607 and Protein ID: NP_003698.1, respectively.
  • a protein sequence of various origins and each derived protein sequence and a full-length or fragment having a sequence substantially identical thereto may be used.
  • arginine residues at positions 333 and 339 are methylated based on the sequence of SEQ ID NO: 1 by CARM1 protein.
  • the methylated PONTIN binds to FOXO3a in the next step.
  • the methylated PONTIN binds through contact with residues 624, 627, 628, 640 and 642 of FOXO3a (based on the sequence of SEQ ID NO: 2).
  • FOXO3a is an abbreviation of Forkhead box protein3a, cell growth, proliferation, and differentiation. It is a transcriptional regulator involved in the maintenance of lifespan and homeostasis. Its gene and protein sequence are known as Gene ID: 2309, Protein ID: NP_001446.1, respectively. In the method according to the present application, as long as it has the methylation enzyme function as described above, a protein sequence of various origins and each derived protein sequence and a full-length or fragment having a sequence substantially identical thereto may be used.
  • the method according to the present application may further measure the acetylation of histone 4.
  • Histones are central proteins constituting chromatin, and are known to play an important role in gene expression by acting as a failure to wind up DNA chains and condensing DNA.
  • Histone proteins include H1, H2A, H2B, H3, and H4, and H2A, H2B, H3, and H4 combine two each to form an octameric core histone, and H1 is a linker.
  • the core histone has a very well-conserved sequence, and various variants are also found. For this sequence, reference can be made to the published histone DB2.0.
  • the homology is at least 61% homology, more preferably 70% homology, when the target sequence is aligned to match as much as possible, and the aligned sequence is analyzed using an algorithm commonly used in the art. means a sequence which exhibits homology, even more preferably at least 80% homology, most preferably at least 90% homology, in particular at least 95% homology. Alignment methods for sequence comparison are known in the art.
  • the NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. (1990) 215:403-10) is accessible from NBCI et al. It can be used in conjunction with a sequence analysis program.
  • the BLSAT can be accessed at www.ncbi.nlm.nih.gov/BLAST/, and a method for comparing sequence homology using this program can be found at www.ncbi.nlm.nih.gov/BLAST/blast_help.html.
  • the method according to the present disclosure may be performed using a cell expressing the protein.
  • the protein can be provided to the cell expressing it.
  • the degree of methylation of the PONTIN protein according to the present application may also be performed using a method known in the art, for example, the method described in the Examples herein may be referred to.
  • the test substance used in the method of the present application is a substance expected to regulate PONTIN methylation in a glucose-deficient state by acting on the CARM1-PONTIN-FOXO3a signaling system, and a low molecular weight compound, a high molecular weight compound, a mixture 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, including but not limited to is not doing
  • 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 Required synthetic library methods, "1-bead 1-compound” library methods, and synthetic library methods using affinity chromatography selection can be obtained.
  • 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.
  • compounds having a low molecular weight therapeutic effect may be used.
  • a compound having a weight of about 1000 Da, such as 400 Da, 600 Da, or 800 Da may be used.
  • 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, steroids, etc.), carbohydrates and amino acid derivatives, dihydropyridine, benzhydryl and heterocycles (such as triazine, indole, thiazolidine, etc.), but these are merely exemplary.
  • the present invention is not limited thereto.
  • biologics can be used for screening.
  • Biologics refer to cells or biomolecules
  • 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.
  • 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, for example, an insect cell, a mammalian cell, overexpressed, and then purified and used.
  • 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.
  • 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, and the labeled material. It can be detected using a detection device suitable for
  • the DNA or RNA sequence encoding the screening protein is expressed in an appropriate host cell to prepare a cell lysate, or the mRNA of the screening protein is translated in vitro, and then the screening protein is purified by a protein isolation method known in the art.
  • a protein isolation method known in the art.
  • 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.
  • a substance that modulates autophagy in the presence of the test substance is selected as a candidate substance compared to the control group that is not in contact with the test substance.
  • About 10% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, or about 100% or more, or an increase or decrease of more than 100% may be selected as a candidate substance.
  • the step of detecting the binding of PONTIN to FOXO3a may additionally or instead of methylation of PONTIN.
  • Protein-protein interactions can be measured using a variety of methods known in the art. For example, the yeast-to-hybrid method, confocal microscopy, which confirms the binding/interaction of intracellular proteins. Additional references for comparative and detailed experimental methods regarding these methods include, but are not limited to, coimmunoprecipitation, surface plasma resonance (SPR) and spectroscopy, Berggard et al., (2007) "Methods for The detection and analysis of protein-protein interactions", PROTEOMICS Vol7: pp 2833 - 2842 can be referred to.
  • SPR surface plasma resonance
  • the amount and type of protein, cell type, and test material used in the various screening methods according to the present application vary depending on the specific experimental method and type of the test material used, and those skilled in the art will be able to select an appropriate amount.
  • Autophagy modulators that can be screened by various screening methods according to the present disclosure may be used for the treatment or prevention of various diseases or diseases related to hyperactivity of autophagy due to the accumulation of denatured proteins when there is an abnormality in autophagy. .
  • neurodegenerative diseases including neurodegenerative diseases, liver diseases, autoimmune diseases, cardiovascular diseases, metabolic diseases, hamartoma syndrome, hereditary muscle diseases, muscle diseases or cancer
  • neurodegenerative diseases including neurodegenerative diseases, liver diseases, autoimmune diseases, cardiovascular diseases, metabolic diseases, hamartoma syndrome, hereditary muscle diseases, muscle diseases or cancer
  • Neurodegenerative diseases include, for example, adrenal leukodystrophy, alcoholism, Alexander's disease, Alper's disease, Alzheimer's disease, amyotrophic lateral sclerosis, Ataxiatelangiectasia, Batten disease, bovine spongiform encephalopathy, Canavan disease, cerebral palsy, cockayne syndrome, corticobasal degeneration, Creutzfeldt -Creutzfeldt-Jakob disease, familial fatal insomnia, frontotemporal lobar degeneration, Huntington's disease, HIV-associated dementia, Kennedy's disease, Krabe disease (Krabbe's disease), Lewy body dementia, neuroborreliosis, Machado-Joseph disease, multiple system atrophy, multiple sclerosis , narcolepsy, Niemann Pick disease, Parkinson's disease, Pelizaeus-Merzbacher disease, Pick's disease, primary lateral sclerosis sclerosis, prion disease, progressive supranuclear palsy, Refsum
  • Diseases related to protein degeneration in one embodiment according to the present application include Alzheimer's disease, Parkinson's disease, Lewy body dementia, amyotrophic lateral sclerosis (ALS), Huntington's disease, spinal cerebellar ataxia or spinobulbar musclular atrophy. .
  • the autoimmune disease in which the method according to the present disclosure may be used is alopecia greata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune disease of the adrenal gland, autoimmune hemolytic anemia , autoimmune hepatitis, autoimmune oophoritis and orchitis, autoimmune thrombocytopenia, Behcet's disease, pemphigoid vesicles, cardiomyopathy, celiac sprue-dermatitis, chronic fatigue immune dysfunction syndrome, chronic inflammatory demyelinating multiple.
  • Neuropathy Churg-Strauss syndrome, scar pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, disc lupus, gastrogenetic cold globulinemia, fibromyalgia-fibromyositis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, Hashimoto's thyroiditis , idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura, IgA neuritis, juvenile arthritis, lichen planus, lupus erythematosus, Meniere's disease, mixed connective tissue disease, multiple sclerosis, type I or immune-mediated diabetes mellitus, myasthenia gravis, Pemphigus vulgaris, pernicious anemia, polyarteritis crystallized, polychondritis, autoimmune polyline syndrome, polymyalgia rheumatica, polymyositis and dermatomy
  • the cardiovascular disease for which the method according to the present disclosure can be used is photoarterial heart disease, cardiomyopathy, hypertensive heart disease, heart failure, pulmonary heart disease, cardioversion disorder, endocarditis, inflammatory cardioplasty, myocarditis, valvular heart disease, cerebrovascular disease. It is a cardiovascular disease selected from the group consisting of disorders, lower extremity arterial disease, congenital heart disease, and cardiac rheumatism.
  • the metabolic disease for which the method according to the present application can be used is a metabolic disease selected from the group consisting of obesity, diabetes, dyslipidemia, fatty liver, hypertension, arteriosclerosis, hyperlipidemia and hyperinsulinemia.
  • the cancer for which the method according to the present application can be used is pituitary adenoma, glioma, brain tumor, epipharyngeal cancer, laryngeal cancer, thymoma, mesothelioma, breast cancer, lung cancer, gastric cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, pancreatic endocrine Tumors, gallbladder cancer, penile cancer, ureter cancer, renal cell cancer, prostate cancer, bladder cancer, non-Hodgkin's lymphoma, myelodysplasia.
  • Antibodies recognizing R333 and R339 of Pontin were produced by Peptron using the corresponding peptide.
  • Pontin f/f MEFs were prepared in the 3T3 method using Pontin KO mice.
  • Flag-tagged CARM1 expression in HEK293T After pulling CARM1 with Flag M2 affinity gel (100ul of 50% slurry) (sigma), 20 mM Tris-HCl (pH 7.9), 15% Glycerol, 1 mM EDTA, 1 mM dithiothreitol (DTT), 0.2 mM PMSF, 0.05 % Wash with Nonidet P40, and 150 mM KCl to release non-specific binding. After that, CARM1 and the complex were dropped from the beads using Flag peptide (0.2 mg/ml). After that, the gel was run through SDS-PAGE and protein information was analyzed through LC-MS/MS.
  • GST-tagged construct is used for protein expression after transformation into Rosetta E. coli. It was then pulled out with glutathione beads.
  • M15[pREP4] was grown in E. coli and extracted through Ni-NTA beads.
  • In vitro translated proteins were extracted according to the instructions of the TNT Quick Coupled Transcription/Translation system (Promega). Extracted proteins and in vitro translated proteins were used in GST pulldown experiments in binding buffer (125 mM NaCl, 20 mM Tris [pH 7.5], 10 % glycerol, 0.1 % NP40, and 0.5 mM DTT supplemented with protease inhibitors). After 4 washes, SDS sample buffer treatment and gel run.
  • CARM1 Active Motif, 31347
  • CARM1 Active Motif, 31347 protein in methylation buffer (50 mM Tris-HCl pH 8.5, 20 mM KCl, 10 mM MgCl2, 10 mM -mercaptoethanol, and 250 mM sucrose) with 1 ⁇ Ci of 3H -After putting it in SAM, it was left at 30 °C for one day. After removing the reaction buffer, add 2X sampling buffer, boil for 10 minutes, run on SDS-PAGE, and observe using a film.
  • CARM1 Active Motif, 31347
  • Non-methylated Pontin peptide and methylated Pontin peptide were dotted on the membrane. After drying sufficiently, blocking proceeded with 5% milk dissolved in PBST for 1 hour. Protein-binding buffer (100 mM NaCl, 20 mM Tris-HCl [pH 7.6], 10 % glycerol, 0.1 % Tween-20, 2 % skim milk powder and 1 mM) containing purified GST-FOXO3a proteins (1 ⁇ g/ml) DTT), to bind to the membrane. Afterwards, immunoblot using GST antibody was performed.
  • Protein-binding buffer 100 mM NaCl, 20 mM Tris-HCl [pH 7.6], 10 % glycerol, 0.1 % Tween-20, 2 % skim milk powder and 1 mM
  • 3X Flag-Pontin WT, RK and RA were cloned into pLVX vector, and lentiviral shRNA constructs were cloned into pLKO.1 vector. Lentiviral constructs were transfected into HEK293T cells together with packaging vectors (psPAX2 and VSV-G), and the culture medium was collected through a 0.45- ⁇ m filter after 48 hours.
  • shRNA sequence information Tip60; 5'-GCAACGCCACTTGACCAAATG-3', Pontin; 5'-GTGGCGTCATAGTAGAATTA A-3', FOXO1/3/4; 5'-CTGTTGGCCCTACTTCAAGGA-3'.
  • Autophagic vacuoles were stained using the Cyto-ID autophagy detection kit (Enzo Life Sciences, ENZ-51031). Cells were put on coverslips with 2 x 10 ⁇ 4 pieces of media mixed with Cyto-ID green detection reagent (1:500) and Hoechst 33342 (1:1000). After leaving at 37 °C for 30 min, wash with PBS, add 2% paraformaldehyde/PBS, and fix at room temperature for 10 minutes. Images were then taken with a confocal microscope (Zeiss, LSM700).
  • MEFs were transfected with 6X canonical FOXO response element (6x DBE-luciferase). Luciferase activity was measured 36 hours after transfection and quantified by beta-galactosidase expression.
  • RNA-seq libraries were prepared using the TruSeq RNA sample prep kit v2 (Illumina) method. RNA-seq libraries were sequenced by Illumina HiSeq 4000 (Macrogen) in a paired-end sequence method. RNA-seq data was mapped to the mouse genome (mm9) through the Tophat package. Differential analysis is analyzed through EdgeR package. Differentially regulated genes have a false discovery rate (FDR) cut-off of 1 x 10 ⁇ -5. Hierarchical clustering analysis was performed with gene expression levels in all situations. Ward’s criterion for genes with 1 - (correlation coefficient) is used for distance measure. Clustering heatmap is based on z-scor.
  • ChIP-seq libraries were prepared using the TruSeq DNA Sample prep Kit method. ChIP-seq libraries were sequenced on the Illumina platform (MACROGEN) in a paired-end fashion. ChIP-seq reads were mapped to the mouse reference genome (GRCm38/mm10) using Bowtie2. In the case of methylated Pontin peak, it was produced by Homer (v4.7.2). Pontin methylated antibody prepared by us was used.
  • an antibody capable of recognizing arginine methylation of Pontin was constructed. It was confirmed that the Pontin methylated antibody prepared by us worked well through dot blot (Fig. 1i), and the specificity of the antibody was also confirmed through an in vitro methylation experiment (Fig. 1j). It was confirmed that the antibody works well not only in R333/339A but also in the R333/339K mutant ( FIG. 1k ). When an in vitro methylation experiment was performed using WT of CARM1 and a mutant with impaired enzymatic activity, it was reconfirmed with the homemade antibody that methylation of Pontin was increased only by WT (FIG. 11).
  • Example 2 Identification of the mechanism of methylation of Pontin by CARM1: Occurs in the nucleus in a glucose-deficient state
  • Pontin methylation was observed in Pontin WT MEFs, but not in RK, RA MEFs (Fig. 3c). Pontin methylation was increased not only in glucose deprivation but also in Rapamycin treatment or amino acid depletion conditions (results not shown). We had previously seen that autophagy was disrupted in Pontin KO MEFs through GFP-LC3 puncta assay (results not shown). In order to see the change of autophagy according to pontin methylation, we conducted the GFP-LC3 puncta experiment in our cells.
  • RNA-sequencing data were analyzed with a focus on the regulation of autophagy. Interestingly, it was confirmed that genes related to autophagy initiation, phagopore nucleation and expansion, and cargo recruitment/trafficking were low in Pontin RA MEF (Fig. 4e). Quantitative RT-PCR and immunoblot confirmed that the expression of these genes and proteins was actually Pontin methylation-dependent (Fig. 4f, g). In addition, similar results were obtained in other cells such as HepG2 and HeLa, and similar results were obtained in Rapamycin treatment or amino acid deficiency. Taken together, we found that increased pontin methylation in nutrient-deficient conditions is important for activating autophagy and lysosome-related genes, and that this regulation can affect autophagy from an early stage.
  • FOXO3a was expected to recognize the methylation of pontin at hydrophobic residues M624, I627, and I628.
  • FOXO3a 3A mutant was mutated all three residues above to Pontin using WT.
  • WT hydrophobic residues
  • FOXO3a 3A mutant could not bind to Pontin, and the same result was confirmed through the cell experiment.
  • M624, I627, and I628 of FOXO3a may also be important for methylation recognition of Pontin.
  • methylated Pontin acts as a co-activator of FOXO3a
  • a luciferase experiment was performed using a luciferase reporter having a FOXO-binding site.
  • luciferase activity was increased in the glucose deprivation situation, but not in Pontin RK and RA mutants (Fig. 6a).
  • FOXO3a F640/642L or 3A mutants did not show an additional increase in transcriptional activity by Pontin (FIG. 6b).
  • methylated Pontin can bind not only to the transcriptional initiation periphery but also to H3K4me1 and H3K27Ac-high enhancer regions (Fig. 6c). Interestingly, 36% of the methylated Pontin peaks with the FOXO3a motif were far from the TSS. When the Pontin methylation ChIP-seq peak was observed near the gene of Map1lc3b, a representative autophagy gene, binding was confirmed not only at the transcription start site but also at a distant site (FIG. 6d).
  • Pontin is one of the well-known Tip60 complexes, and in the case of the Tip60 complex, it plays an important role in transcriptional activity through histone acetylation.
  • Tip60 and H4 acetylation change with Pontin at the FOXO3a binding site were confirmed. Through ChIP experiments, we confirmed that H4 acetylation was increased in a glucose deprivation-specific manner with Tip60 (Fig. 6e). Interestingly, it was found that Tip60 and H4 acetylation were not recruited in CARM1 KO MEF, indicating that Pontin methylation status was dependent ( FIG. 6e ).
  • CARM1 Since CARM1 is involved in both the FOXO3a binding site and the TFEB binding site, we checked whether CARM1 can bind to both sites. When viewed through ChIP, CARM1 was bound and H3R17me2 increased in the CLEAR motif, which is the TFEB binding site, but CARM1 binding was not seen in the FOXO3a binding site.
  • Tip60 and H4 acetylation, including methylated Pontin were well increased at the FOXO binding site. However, acetylation of Tip60 and H4 including Pontin did not occur in Pontin RK and RA mutants (FIG. 8a).

Abstract

The present application discloses a method for regulating autophagy or a method for screening an autophagy regulator, each targeting a CARM1-PONTIN-FOXO3a signaling pathway that works in the nucleus. The method according to the present application can be advantageously used at various levels on the basis of the mechanism axis in developing therapeutics for various diseases caused by problems with autophagy regulation.

Description

카름1에 의한 폰틴의 아르기닌 메틸화를 통한 자가포식 조절 방법 및 그 용도 Method for regulating autophagy through arginine methylation of fontin by Karm 1 and its use
본원은 자가포식 조절 방법 및 그 용도에 관한 것이다.The present application relates to methods for regulating autophagy and uses thereof.
자가포식(Autophagy)은 세포내 영양분이 부족한 환경에서 세포가 자신의 불필요한 단백질, 노화된 소기관 등을 지질 이중층으로 구성된 자가포식소체(membrane vacuole, autophagosome)가 둘러싼 후 라이소좀과 합쳐져 둘러싸인 내부 물질을 분해하도록 유도하는 라이소좀-의존 방식으로 세포내 구성물질을 변동, 조정하는 과정 (Levine and Klionsky, (2004) Dev Cell 6, 463-377)으로, 결함이 있는 거대 단백질 복합체 및 세포내 소기관을 제거하여, 세포 성장, 생존 및 항상성에 중요한 역할을 한다.In autophagy, an autophagosome (membrane vacuole, autophagosome) composed of a lipid bilayer surrounds its unnecessary proteins and aging organelles in an environment where intracellular nutrients are insufficient, and then merges 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. , play an important role in cell growth, survival and homeostasis.
따라서 자가포식 조절에 이상이 있는 경우, 변성단백질(misfolded protein)의 축적이 초래되어 이로 인해 신경변성질환이 발생하는 것으로 알려져 있다 (Komatsu et al., (2006), Nature, 441, 880-884). Therefore, it is known that when there is an abnormality in autophagy regulation, the accumulation of misfolded protein is caused, which leads to neurodegenerative disease (Komatsu et al., (2006), Nature, 441, 880-884). .
또한 자가포식은 암세포에서 활성화 (Ding et al., (2009), Mol. Cancer Ther.,8(7), 2036-2045)되며, 자가포식 억제제가 항암 치료제로서 작용하는 것으로 알려져 있다 (Maiuri et al., (2007) Nat. Rev. Cell Biol. 8, 741-752). 아울러 암 및 신경변성 질환에 추가하여 자가포식은 간 질환, 심장 질환, 근육 질환 및 췌장 질환과 연관된 것으로 알려져 있다 (Levine and Kroemer, (2008), Cell, 132, 27-42; Fortunato and Kroemer, (2009), Autophagy, 5(6)).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). 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)).
따라서, 세포의 자가포식의 조절을 통한 다양한 치료제 개발에 대한 연구가 진행되고 있다. Therefore, research on the development of various therapeutic agents through the regulation of autophagy of cells is in progress.
대한민국 등록특허 제10-1594168호에는 p62의 ZZ 영역에 의해 매개되는 자가포식 조절 방법 및 그 용도에 대하여 개시하고 있다.Korean Patent Registration No. 10-1594168 discloses a method for regulating autophagy mediated by the ZZ region of p62 and its use.
미국 공개특허공보 제2010-0233730호는 치료를 위한 자가포식 조절에 관한 것으로, 세포에 시험물질을 처리한 후 대사 스트레스를 가해 자가포식체(autophagosome)의 세포내 변화 관찰을 통해, 자가포식 조절제를 선별하는 방법을 개시하고 있다. U.S. Patent Publication No. 2010-0233730 relates to autophagy control for treatment, and after treating cells with a test substance, metabolic stress is applied to them to observe intracellular changes in autophagosomes, A method of screening is disclosed.
하지만 이들은 세포질에서 작용하는 자가포식 단백질 기반의 기술로서, 핵 내에서의 전사과정이나 후성유전학적 작용 기전에 기반한 자가포식 조절기술에 대하여는 알려지지 않았으며, 보다 근본적인 조절을 위해 핵내에서 작용하는 기전에 기반한 자가포식 조절제의 개발이 필요하다. However, these are autophagy protein-based technologies that act in the cytoplasm, and autophagy control technologies based on nuclear transcriptional processes or epigenetic mechanisms are unknown. The development of autophagy modulators is necessary.
본원은 핵내에서 작용하는 기전에 기반한 자가포식 조절 방법, 이를 이용한 스크리닝 방법을 제공하고자 한다.The present application aims to provide a method for regulating autophagy based on a mechanism acting in the nucleus and a screening method using the same.
한 양태에서 본원은 메틸전달효소인 CARM1 (Coactivator Associated argine methyltransferase 1)에 의한 PONTIN 단백질의 메틸화 조절을 통해 자가포식을 조절하는 물질을 스크리닝하는 방법을 제공한다. In one aspect, the present application provides a method for screening a substance that regulates autophagy through methylation control of the PONTIN protein by the methyltransferase CARM1 (Coactivator Associated argine methyltransferase 1).
일 구현예에서 상기 방법은 포도당이 결핍된 상태에서 CARM1 및 상기 메틸전달효소 CARM1의 기질로서 PONTIN 단백질을 발현하는 세포를 배양하는 제 1 단계; 상기 세포에 메틸전달효소 CARM1의 상기 PONTIN에 대한 메틸화 활성을 억제할 것으로 기대되는 시험물질을 처리하는 제 2 단계; 상기 PONTIN 단백질을 상기 세포로부터 분리하는 제 3 단계: 상기 분리된 PONTIN 단백질의 서열번호 1을 기준으로 333번 및 339번째 잔기에서의 메틸화정도를 측정하는 제 4 단계; 및In one embodiment, the method comprises a first step of culturing cells expressing CARM1 and PONTIN protein as a substrate for the methyltransferase CARM1 in a glucose-deficient state; a second step of treating the cells with a test substance expected to inhibit the methylation activity of the methyltransferase CARM1 for the PONTIN; a third step of isolating the PONTIN protein from the cells: a fourth step of measuring the degree of methylation at residues 333 and 339 based on SEQ ID NO: 1 of the isolated PONTIN protein; and
상기 측정결과 시험물질로 처리되지 않은 대조군과 비교하여, 시험물질로 처리된 경우 상기 메틸화가 증가 또는 감소한 경우, 이를 자가포식을 조절하는 후보물질로 선별하는 제 5 단계를 포함하며, 상기 메틸화 증가는 자가포식 증가, 상기 메틸화 감소는 자가포식 억제를 나타낸다. When the methylation is increased or decreased when treated with the test substance compared to the control not treated with the test substance as a result of the measurement, a fifth step of selecting this as a candidate substance for regulating autophagy is included, wherein the increase in methylation is An increase in autophagy, the decrease in methylation, indicates inhibition of autophagy.
다른 구현예에서는 상기 제 1 단계는 FOXO3a 단백질을 추가로 발현하며, 상기 제 4 단계 대신에, 또는 이에 추가하여, 상기 PONTIN 및 FOXO3a 단백질의 결합을 측정하는 단계를 포함하며, 상기 측정결과 상기 단백질 간의 결합 증가는 자가포식 증가, 결합 감소는 자가포식 억제를 나타낸다. In another embodiment, the first step further expresses the FOXO3a protein, and instead of or in addition to the fourth step, the step comprises measuring the binding of the PONTIN and FOXO3a protein, and the measurement result between the protein An increase in binding indicates an increase in autophagy, and a decrease in binding indicates inhibition of autophagy.
또 다른 구현예에서 본원에 따른 PONTIN은 서열번호 2를 기준으로 상기 FOXO3a 단백질의 624, 627, 628, 640 및 642 잔기를 통해 FOXO3a와 결합한다.In another embodiment, the PONTIN according to the present disclosure binds to FOXO3a through residues 624, 627, 628, 640 and 642 of the FOXO3a protein based on SEQ ID NO: 2.
또 다른 구현예에서 본원에 따른 방법은 상기 제 4 단계에 추가하여, 상기 세포에서 분리된 히스톤 4(H4) 단백질의 아세틸화 여부를 측정하는 단계를 포함하며, 상기 측정결과 상기 H4 단백질의 아세틸화 증가는 자가포식 증가, 결합 감소는 자가포식 억제를 나타낸다. In another embodiment, the method according to the present disclosure includes, in addition to the fourth step, measuring whether the histone 4 (H4) protein isolated from the cell is acetylated, and as a result of the measurement, the acetylation of the H4 protein An increase indicates an increase in autophagy, and a decrease in binding indicates inhibition of autophagy.
또 다른 구현예에서 상기 히스톤 4의 아세틸화는 서열번호 3의 서열을 기준으로 6, 9, 13 및 17번째 라이신 잔기에서 발생한다.In another embodiment, the acetylation of histone 4 occurs at the 6th, 9th, 13th and 17th lysine residues based on the sequence of SEQ ID NO: 3.
자가포식은 영양분 결핍, 대사성 스트레스, 바이러스 감염, 노화 등 다양한 스트레스 상황에서 활성화되어 세포의 생존 및 항상성 유지에 중요한 역할을 하는 기작으로 자가포식 신호 조절에 문제가 생겼을 때 암과 퇴행성 뇌질환 등 여러 질병과 관련이 깊다. 핵 내에서 작용하는 기전에 기반한 자가포식 조절 방법을 스크리닝을 통해 알 수 있다면 자가포식 이상에 기인한 질환들 (암, 퇴행성 뇌 질환 등)의 새로운 치료 표적을 발굴하는 과정에서 이론적인 기반 지식을 제공할 수 있다.Autophagy is a mechanism that is activated in various stressful situations such as nutrient deficiency, metabolic stress, viral infection, and aging and plays an important role in maintaining cell survival and homeostasis. is closely related to If the method of regulating autophagy based on the mechanism acting in the nucleus can be known through screening, it will provide theoretical foundational knowledge in the process of discovering new therapeutic targets for diseases caused by autophagy abnormalities (cancer, degenerative brain disease, etc.) can do.
이에 본원에 따른 CARM1-PONTIN-FOXO3a 신호전달체계를 표적으로 하는 자가포식 조절 방법은 다양한 수준에서 자가포식 조절문제로 인한 다양한 질환의 치료제 개발에 유용하게 사용될 수 있다.Accordingly, the method for regulating autophagy targeting the CARM1-PONTIN-FOXO3a signaling system according to the present application can be usefully used to develop therapeutic agents for various diseases caused by autophagy regulation problems at various levels.
도 1은 CARM1에 의해 Pontin의 R333, R339 잔기가 아르기닌 메틸화 된다. (a) HEK293T에 발현시킨 Flag-CARM1을 Flag-M2 agarose를 이용하여 pulldown함. Mass spectrometric 분석을 통해 포도당 결핍 상황에서 CARM1과 결합하는 단백질인 Pontin을 규명함. (b) affinity chromatography에서 나온 eluate에서 CARM1의 결합 단백질로서 Pontin을 immunoblot으로 보임. (c) CARM1과 Pontin/Reptin의 결합을 GST pulldown 실험을 보임. (d) His-Pontin과 HA-CARM1 WT / R169A 돌연변이를 이용하여 in vitro 메틸화 실험 결과. (e) GST-Pontin WT과 R333/339A 돌연변이를 HA-CARM1을 이용하여 in vitro 메틸화 시킴. 이후 Rme2a 항체로 immunoblot한 결과 (f) Pontin WT, R333K/R339K, R333A/R339A 돌연변이를 이용하여 in vitro 메틸화 실험 결과. RI 이용한 버전 (g) Pontin의 구조와 R333, R339 잔기 (h) 여러 종에서의 Pontin 단백질 서열과 보존된 R333, R339 잔기. (i) 제작한 Pontin 메틸화 항체를 dot blot을 통해 확인. 사용한 펩타이드 Methyl peptide sequence: IVIFASNR(me2)GNCVIR(me2) GTEDITS; Non-methyl peptide sequence: IVIFASNRGNCVIRGTEDITS. (j) GST-Pontin WT, R333A, R339A, R333/339A과 CARM1을 이용하여 in vitro 메틸화 진행. 이후 Rme2a 항체로 immunoblot. (k) GST-Pontin WT, R333K, R339K ,R333/339K로 위와 동일한 실험 결과. Rme2a 항체와 Pontin 메틸화 항체 사용. (l) His-Pontin과 HA-CARM1 WT/R169A를 이용하여 in vitro 메틸화 실험 진행. Pontin 메틸화 항체로 확인한 결과이다. 1 shows that R333 and R339 residues of Pontin are arginine methylated by CARM1. (a) Pulldown of Flag-CARM1 expressed in HEK293T using Flag-M2 agarose. Through mass spectrometric analysis, Pontin, a protein that binds to CARM1 in the situation of glucose deficiency, was identified. (b) Immunoblot showing Pontin as a binding protein of CARM1 in eluate from affinity chromatography. (c) GST pulldown experiment shows the binding of CARM1 and Pontin/Reptin. (d) Results of in vitro methylation experiments using His-Pontin and HA-CARM1 WT / R169A mutants. (e) In vitro methylation of GST-Pontin WT and R333/339A mutants using HA-CARM1. After immunoblot with Rme2a antibody, (f) Results of in vitro methylation experiments using Pontin WT, R333K/R339K, and R333A/R339A mutants. RI version (g) Pontin structure and R333 and R339 residues (h) Pontin protein sequence from several species and conserved R333 and R339 residues. (i) Confirmation of the produced Pontin methylated antibody through dot blot. Peptide used Methyl peptide sequence: IVIFASNR(me2)GNCVIR(me2) GTEDITS; Non-methyl peptide sequence: IVIFASNRGNCVIRGTEDITS. (j) In vitro methylation using GST-Pontin WT, R333A, R339A, R333/339A and CARM1. Then, immunoblot with Rme2a antibody. (k) Same experimental results as above with GST-Pontin WT, R333K, R339K, and R333/339K. Using Rme2a antibody and Pontin methylated antibody. (l) In vitro methylation experiment using His-Pontin and HA-CARM1 WT/R169A. This is the result confirmed by Pontin methylated antibody.
도 2는 CARM1에 의한 Pontin의 메틸화는 포도당 결핍 상태에서 핵 안에서 일어난다. (a) 포도당 결핍 18시간 상황에서 HeLa 세포에서의 CARM1과 Pontin의 결합 (b) CARM1 inhibitor EZM2302 (100 nM), EPZ025654 (100 nM)를 96시간 처리한 뒤 Pontin의 메틸화 측정. (c) CARM1 WT MEF와 knockout MEF에서의 포도당 결핍 상황에서의 Pontin 메틸화 변화 (d) CARM1 KO MEF에 CARM1 WT 혹은 R169A를 발현시킨 뒤 Pontin 메틸화 변화 (e) MEF의 핵과 세포질에서의 Pontin 메틸화 변화 (f) Pontin 메틸화와 CARM1을 찍은 confocal 이미지. Figure 2 shows that methylation of pontin by CARM1 occurs in the nucleus in a glucose deprivation state. (a) Binding of CARM1 and Pontin in HeLa cells under glucose deprivation for 18 hours (b) Measurement of Pontin methylation after treatment with CARM1 inhibitors EZM2302 (100 nM) and EPZ025654 (100 nM) for 96 hours. (c) Changes in pontin methylation under glucose starvation in CARM1 WT MEFs and knockout MEFs (d) Changes in pontin methylation after expression of CARM1 WT or R169A in CARM1 KO MEFs (e) Changes in pontin methylation in the nucleus and cytoplasm of MEFs ( f) Confocal images of Pontin methylation and CARM1.
도 3은 Pontin의 메틸화가 결핍 의존적 자가포식의 증가에 중요하다. (a) Pontin f/f MEF에 Flag-Pontin WT or R333A/R339A (RA) stable하게 발현시키는 방법 모델 (b) Pontin의 protein 발현 레벨 (c) Pontin f/f + Flag Pontin WT/RK,RA MEF에 cre 처리 후 Pontin 메틸화 항체로 IP (d) 대표적인 GFP-LC3 puncta 이미지. Scale bar 20um, 그래프는 LC3-positive 세포수를 정량화 한 것 (e) Pontin WT, RA MEF에서 immuneblot 결과. 숫자는 LC3-II/bactin 비율 (f) bafilomycin A1 처리 유무에 따른 대표적인 GFP-LC3 puncta 이미지. (g) Bafilomycin A1과 Chloroquine 처리 유무에 따른 자가포식 flux 측정. 숫자는 LC3-II/bactin 비율 (h) CYTO-ID를 이용하여 염색 후 대표적인 confocal 이미지 (i) Pontin WT, RA MEF에서 mCherry-GFP-LC3를 이용한 대표적인 confocal 이미지. mCherry와 GFP 시그널이 같이 있는 노란 puncta의 경우 라이소좀과 결합하지 않은 자가포식 포자(파고포어, 오토파고좀)이며, 빨간 puncta는 산성화된 자가포식 포자(앰피좀, 오토라이소좀)임.3 shows that methylation of Pontin is important for the increase in deficiency-dependent autophagy. (a) Stable expression of Flag-Pontin WT or R333A/R339A (RA) in Pontin f/f MEF model (b) Pontin protein expression level (c) Pontin f/f + Flag Pontin WT/RK,RA MEF (d) Representative GFP-LC3 puncta image of IP with Pontin methylated antibody after cre treatment. Scale bar 20um, graph quantifies the number of LC3-positive cells (e) Immuneblot results in Pontin WT, RA MEF. Figures are LC3-II/bactin ratio (f) Representative GFP-LC3 puncta images with and without bafilomycin A1 treatment. (g) Measurement of autophagy flux with or without Bafilomycin A1 and Chloroquine treatment. Figures are LC3-II/bactin ratio (h) Representative confocal image after staining using CYTO-ID (i) Representative confocal image using mCherry-GFP-LC3 in Pontin WT, RA MEF. Yellow puncta with mCherry and GFP signals are autophagic spores (phagophores, autophagosomes) that are not bound to lysosomes, and red puncta are acidified autophagic spores (ampisomes, autolysosomes).
도 4는 많은 자가포식과 라이소좀 유전자들이 Pontin의 메틸화에 의해 조절되는 것을 RNA-sequencing을 통해 확인했다. (a) RNA-seq 분석 방식 (b) Hierarchical clustering을 통해 4131개의 DEG가 나옴 (c) 유전자군 1에대한 DAVID를 이용하여 Gene ontology와 KEGG pathway 분석 (d) 대표적인 GSEA 결과 (e) 자가포식 과정으로 분류한 Pontin 메틸화 의존적인 유전자들 (f) Pontin 메틸화 의존적인 자가포식 혹은 라이소좀 관련 유전자들의 qRT-PCR 결과 (g) 포도당 결핍 상태에서의 Pontin WT,RK,RA에서의 타겟 단백질의 발현 정도를 immunoblot으로 확인한 결과이다. Figure 4 confirmed through RNA-sequencing that many autophagy and lysosomal genes are regulated by Pontin methylation. (a) RNA-seq analysis method (b) Hierarchical clustering revealed 4131 DEGs (c) Gene ontology and KEGG pathway analysis using DAVID for gene group 1 (d) Representative GSEA results (e) Autophagy process Pontin methylation-dependent genes classified as (f) qRT-PCR results of pontin methylation-dependent autophagy or lysosome-related genes (g) Pontin WT, RK, and RA target protein expression levels in glucose-deficient conditions This is the result confirmed by immunoblot.
도 5는 Pontin과 FOXO3a가 메틸화를 통해 결합한다. (a) EnrichR을 이용한 Pontin 메틸화 의존적인 유전자들의 전사인자 분석 (b) Pontin 메틸화 ChIP-seq 결과를 이용한 Binding motif 분석 (c) Pontin과 여러 자가포식 관련 전사인자와의 결합 여부 확인 (d) Pontin WT, RK, RA와 FOXO3a의 결합 여부 확인 (e, f) CARM1 KO에서 Pontin과 FOXO3a의 결합 확인 (g) CARM1 inhibitor인 EZM2302와 EPZ025654 처리 후 Pontin과 FOXO3a의 결합 여부 확인 (h) in vitro 메틸화 실험 이후 진행된 GST pulldown 실험 순서 및 결과 (i) Pontin 메틸화 유무 펩타이드를 이용한 immunodot 실험 결과 (j) Pontin과 FOXO3a의 여러 aromatic 잔기 돌연변이와 결합 여부 확인 (k) FOXO3a WT과 F640/642L 돌연변이를 이용하여 Pontin과의 결합 여부 확인한 결과이다. 5 shows Pontin and FOXO3a bind through methylation. (a) Analysis of transcription factors of Pontin methylation-dependent genes using EnrichR (b) Binding motif analysis using ChIP-seq results of Pontin methylation (c) Confirmation of binding between Pontin and various autophagy-related transcription factors (d) Pontin WT , Confirmation of binding of RK, RA and FOXO3a (e, f) Confirmation of binding of Pontin and FOXO3a in CARM1 KO (g) Confirmation of binding of Pontin and FOXO3a after treatment with CARM1 inhibitor EZM2302 and EPZ025654 (h) After in vitro methylation test GST pulldown test sequence and results (i) Immunodot test results using peptides with or without Pontin methylation (j) Check whether Pontin and FOXO3a combine with various aromatic residue mutations (k) FOXO3a WT and F640/642L mutations It is the result of checking whether or not it is connected.
도 6은 메틸화된 Pontin과 Tip60가 FOXO3a 타겟 유전자들을 H4 acetylation을 통해 활성화 시킨다. (a) Pontin WT/RK를 이용한 루시퍼레이즈 실험 결과 (b) FOXO3a WT, F640/642L을 이용한 루시퍼레이즈 실험 결과 (c) Pontin 메틸화 ChIP-seq 결과를 이용하여 TSS 근처의 peak과 멀리있는 peak들을 Heatmap으로 보여준 결과. (d) Map1lc3b의 프로모터와 인핸서 부근에서의 Pontin 메틸화 ChIP-seq peak 표시 (e) CARM1 WT/KO MEF에서 FOXO3a 결합 부위에서의 CHIP 결과 (f) Pontin 유무에 따른 FOXO3a와 Tip60의 결합 (g) Pontin WT/RK와 Tip60의 결합 확인한 결과이다. 6 shows that methylated Pontin and Tip60 activate FOXO3a target genes through H4 acetylation. (a) Result of luciferase experiment using Pontin WT/RK (b) Results of luciferase experiment using FOXO3a WT, F640/642L (c) Heatmap of peaks near and far from TSS using Pontin methylation ChIP-seq results results shown by . (d) Pontin methylation ChIP-seq peak display near promoter and enhancer of Map1lc3b (e) CHIP result at FOXO3a binding site in CARM1 WT/KO MEF (f) Binding of FOXO3a and Tip60 with and without Pontin (g) Pontin This is the result of confirming the combination of WT/RK and Tip60.
도 7은 Pontin의 메틸화 의존적인 자가포식 유전자들의 활성에 Tip60가 중요하다. (a) Tip60 knockdown 유무에 따른 자가포식 flux 정도. 숫자는 LC3-II/bactin 비율 (b) Tip60 knockdown 유무에 따른 Pontin 메틸화 의존적/비의존적 유전자들의 mRNA 발현 변화 (c) Tip60 knockdown한 세포에서 FOXO3a, Pontin메틸, H4 아세틸화, Tip60에 대한 ChIP 결과 (d) Tip60 knockdown 유무와 Pontin WT/RA에 따른 Pontin 메틸화 의존적 유전자들의 발현 패턴 (e) Tip60 knockdown 유무와 Pontin WT/RA에 따른 LC3 발현 차이. 숫자는 LC3-II/bactin 비율 (f) 모델 그림이다. 7 shows that Tip60 is important for the activity of methylation-dependent autophagy genes of Pontin. (a) Degree of autophagy flux with and without Tip60 knockdown. Numbers indicate the LC3-II/bactin ratio (b) Changes in mRNA expression of Pontin methylation-dependent/independent genes with and without Tip60 knockdown (c) ChIP results for FOXO3a, Pontinmethyl, H4 acetylation, and Tip60 in Tip60 knockdown cells ( d) Expression patterns of Pontin methylation-dependent genes according to the presence or absence of Tip60 knockdown and Pontin WT/RA (e) Differences in LC3 expression according to the presence or absence of Tip60 knockdown and Pontin WT/RA. Figures are the LC3-II/bactin ratio (f) model figure.
도 8은 CARM1-Pontin-FOXO3a 축이 자가포식 유전자들의 인핸서 활성에 역할을 한다. (a, b) FOXO3a, Pontin메틸, H4아세틸, Tip60, H3R17me2를 이용하여 Map1lc3b, Ctns, Atg14에서FOXO3a 결합 부위(a), TFEB 결합 부위(b) 에서 ChIP한 결과. Pontin WT, RK에서 진행함. (c) Map1lc3b의 프로모터와 인핸서 부분의 결합을 보기 위해 진행된 3C 결과. PCR 이후 DNA gel로 확인한 데이터. 오른쪽은 그 모델. (d) Pontin WT/RA MEF에서 자가포식과 라이소좀 관련 유전자들의 eRNA를 qRT-PCR로 확인한 결과이다. FIG. 8 shows that the CARM1-Pontin-FOXO3a axis plays a role in enhancer activity of autophagy genes. (a, b) ChIP at the FOXO3a binding site (a) and TFEB binding site (b) in Map1lc3b, Ctns, and Atg14 using FOXO3a, Pontinmethyl, H4acetyl, Tip60, and H3R17me2. Conducted by Pontin WT, RK. (c) 3C results performed to see the binding of the promoter and enhancer portion of Map1lc3b. Data confirmed by DNA gel after PCR. The model on the right. (d) The result of qRT-PCR confirmation of autophagy and lysosome-related genes in Pontin WT/RA MEF.
도 9는 본원에서 규명된 기전을 나타내는 모델 그림이다.9 is a model figure showing the mechanism identified herein.
본원에서는 CARM1->PONTIN->FOXO3로 이어지는 경로가 포도당 결핍상황에서 세포의 자가포식 조절에 중요한 기전임을 규명하였다. 구체적으로 CARM1은 PONTIN을 메틸화시키고, 메틸화된 PONTIN (Tip60와 결합)은 FOXO3와 결합하여 복합체를 형성하고, 이러한 복합체는 자가포식에 관여하는 유전자에서 발견되는 인핸서에 결합하여 이들 유전자의 전사를 조절하는 방식으로 자가포식 조절에 관여하며, 자가포식이 증가한다. In this paper, it was investigated that the pathway leading to CARM1->PONTIN->FOXO3 is an important mechanism for regulating autophagy in cells in a glucose deprivation situation. Specifically, CARM1 methylates PONTIN, and methylated PONTIN (binding Tip60) binds to FOXO3 to form a complex, and this complex binds to enhancers found in genes involved in autophagy and regulates transcription of these genes. In this way, it is involved in the regulation of autophagy, and autophagy is increased.
세포에게 있어서 영양분 대사의 항상성 유지는 세포의 건강과 기능에 중요하다. 자가포식은 영양분 부족 상태에서 대표적인 방어기작 중 하나로 영양분이 부족할 때 자가포식을 통해 영양분을 스스로 공급받아 생존에 도움을 준다.In cells, maintenance of homeostasis of nutrient metabolism is important for cell health and function. Autophagy is one of the most representative defense mechanisms in nutrient deficient conditions.
본원에서 자가포식(autophagy 또는 autophagocytosis)이란 라이소좀을 이용하여 세포내 불필요하거나 변성된 단백질을 포함하는 다양한 세포구성성분을 제거하는 이화작용을 일컫는 것이다. 자가포식 조절에 이상이 있는 경우, 변성단백질(misfolded protein)의 축적이 초래되어 이로 인해 신경변성질환이 발생하는 것으로 알려져 있다 (Komatsu et al., (2006), Nature, 441, 880-884). 또한 자가포식은 암세포에서 활성화 (Ding et al., (2009), Mol. Cancer Ther.,8(7), 2036-2045)되며, 자가포식 억제제가 항암 치료제로서 작용하는 것으로 알려져 있다 (Maiuri et al., (2007) Nat. Rev. Cell Biol. 8, 741-752). 아울러 암 및 신경변성 질환에 추가하여 자가포식은 간 질환, 심장 질환, 근육 질환 및 췌장 질환과 연관된 것으로 알려져 있다 (Levine and Kroemer, (2008), Cell, 132, 27-42; Fortunato and Kroemer, (2009), Autophagy, 5(6)). 따라서 본원에 개시된 기술을 이용한 자가포식 조절제, 조절방법은 자가포식 조절 이상으로 인한 다양한 질환의 치료 또는 예방에 효과적으로 사용될 수 있다. 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). 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 autophagy dysregulation.
이에 한 양태에서 본원은 메틸전달효소인 CARM1 (Coactivator Associated argine methyltransferase 1)에 의한 PONTIN 단백질의 메틸화 조절을 통해 자가포식을 조절하는 물질을 스크리닝하는 방법에 관한 것이다. Accordingly, in one aspect, the present application relates to a method for screening a substance that regulates autophagy through methylation control of PONTIN protein by CARM1 (Coactivator Associated argine methyltransferase 1), a methyltransferase.
일 구현예에서 상기 방법은 포도당이 결핍된 상태에서 CARM1 및 상기 메틸전달효소 CARM1의 기질로서 PONTIN 단백질을 발현하는 세포를 배양하는 제 1 단계; 상기 세포에 메틸전달효소 CARM1의 상기 PONTIN에 대한 메틸화 활성을 억제할 것으로 기대되는 시험물질을 처리하는 제 2 단계; 상기 PONTIN 단백질을 상기 세포로부터 분리하는 제 3 단계: 상기 분리된 PONTIN 단백질의 서열번호 1을 기준으로 333번 및 339번째 잔기에서의 메틸화정도를 측정하는 제 4 단계; 및 상기 측정결과 시험물질로 처리되지 않은 대조군과 비교하여, 시험물질로 처리된 경우 상기 메틸화가 증가 또는 감소한 경우, 이를 자가포식을 조절하는 후보물질로 선별하는 제 5 단계를 포함하며, 상기 메틸화 증가는 자가포식 증가, 상기 메틸화 감소는 자가포식 억제를 나타낸다. In one embodiment, the method comprises a first step of culturing cells expressing CARM1 and PONTIN protein as a substrate for the methyltransferase CARM1 in a glucose-deficient state; a second step of treating the cells with a test substance expected to inhibit the methylation activity of the methyltransferase CARM1 for the PONTIN; a third step of isolating the PONTIN protein from the cells: a fourth step of measuring the degree of methylation at residues 333 and 339 based on SEQ ID NO: 1 of the isolated PONTIN protein; and a fifth step of selecting the methylation as a candidate for regulating autophagy when the methylation increases or decreases when treated with the test substance as compared with the control not treated with the test substance as a result of the measurement, wherein the methylation increases indicates an increase in autophagy, and a decrease in methylation indicates inhibition of autophagy.
본원에서 조절(modulation)이란, 특정 생물학적 기능의 활성화, 자극 또는 상향 조절, 또는 저하 또는 하향 조절, 또는 양쪽 모두를 포함하며, 인 비트로 상태에서의 조절, 인비보 상태에서의 조절, 엑스비보 상태에서의 조절을 모두 포함하는 것이다.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
본원에 따른 방법에 사용되는 CARM1 (coactivator-associated arginine methyltransferase 1) 또는 PRMT4 (protein arginine N-methyltransferase 4)는 알파 헬릭스 및 베타 시트로 구성된 S-아데노실-L-메치오닌으로부터 알지닌 잔기의 곁가지 질소의 메틸화를 촉매하는 효소이며, 포유동물에서 CARM1의 유전자 및 단백질 서열은 공지된 것으로 예를 들면 인간 유전자 및 단백질 서열은 Gene ID: 10498, Protein ID: NP_954592.1로 공지되어 있다. 본원에 따른 방법에서는 상기와 같은 메틸화효소 기능을 갖는 한 다양한 유래, 그리고 각 유래의 단백질 서열 및 이와 실질적으로 동일한 서열을 갖는 전장 또는 단편이 사용될 수 있다. CARM1 (coactivator-associated arginine methyltransferase 1) or PRMT4 (protein arginine N-methyltransferase 4) used in the method according to the present application is a nitrogen branch of an arginine residue from S-adenosyl-L-methionine composed of an alpha helix and a beta sheet. It is an enzyme that catalyzes methylation, and the gene and protein sequence of CARM1 in mammals is known, for example, the human gene and protein sequence is known as Gene ID: 10498, Protein ID: NP_954592.1. In the method according to the present application, as long as it has the methylation enzyme function as described above, a protein sequence of various origins and each derived protein sequence and a full-length or fragment having a sequence substantially identical thereto may be used.
본원에 따른 상기 방법에서 CARM1의 기질(substrate)은 PONTIN이다. In the method according to the present application, the substrate of CARM1 is PONTIN.
본원에서 PONTIN은 ATPase와 DNA헬리카제 기능을 갖는 염색질 리모델링 인자(chromatin remodeling factor)이며, 예를 들면 인간 유전자 및 단백질 서열은 각각 Gene ID: 8607, Protein ID: NP_003698.1로 공지되어 있다. 본원에 따른 방법에서는 상기와 같은 메틸화효소 기능을 갖는 한 다양한 유래, 그리고 각 유래의 단백질 서열 및 이와 실질적으로 동일한 서열을 갖는 전장 또는 단편이 사용될 수 있다. PONTIN herein is a chromatin remodeling factor having ATPase and DNA helicase functions, and for example, human gene and protein sequences are known as Gene ID: 8607 and Protein ID: NP_003698.1, respectively. In the method according to the present application, as long as it has the methylation enzyme function as described above, a protein sequence of various origins and each derived protein sequence and a full-length or fragment having a sequence substantially identical thereto may be used.
PONTIN은 CARM1 단백질에 의해 서열번호 1의 서열을 기준으로 333번 및 339번째 알지닌 잔기가 매틸화된다. In PONTIN, arginine residues at positions 333 and 339 are methylated based on the sequence of SEQ ID NO: 1 by CARM1 protein.
메틸화된 PONTIN은 그 다음 단계에서 FOXO3a와 결합한다. The methylated PONTIN binds to FOXO3a in the next step.
메틸화된 PONTIN은 FOXO3a의 624, 627, 628, 640 및 642번째 잔기 (서열번호 2의 서열 기준)와 접촉을 통해 결합한다. The methylated PONTIN binds through contact with residues 624, 627, 628, 640 and 642 of FOXO3a (based on the sequence of SEQ ID NO: 2).
본원에서 FOXO3a는 Forkhead box protein3a의 약자로써 세포 성장, 증식, 분화. 수명과 항상성 유지에 관여하는 전사조절인자다. 그 유전자 및 단백질 서열은 각각 Gene ID: 2309, Protein ID:NP_001446.1로 공지되어 있다. 본원에 따른 방법에서는 상기와 같은 메틸화효소 기능을 갖는 한 다양한 유래, 그리고 각 유래의 단백질 서열 및 이와 실질적으로 동일한 서열을 갖는 전장 또는 단편이 사용될 수 있다. Here, FOXO3a is an abbreviation of Forkhead box protein3a, cell growth, proliferation, and differentiation. It is a transcriptional regulator involved in the maintenance of lifespan and homeostasis. Its gene and protein sequence are known as Gene ID: 2309, Protein ID: NP_001446.1, respectively. In the method according to the present application, as long as it has the methylation enzyme function as described above, a protein sequence of various origins and each derived protein sequence and a full-length or fragment having a sequence substantially identical thereto may be used.
본원에서는 특히 CARM1에 의한 PONTIN 메틸화가 FOXO3a의 결합을 유도하고, 결국 CARM1-Pontin-FOXO3a 축이 H4 아세틸화를 통해 인핸서에서 작용하여 자가포식 관련 유전자들의 coactivator로서 작동할 수 있음을 규명하였다. In this paper, in particular, PONTIN methylation by CARM1 induces the binding of FOXO3a, and in the end, the CARM1-Pontin-FOXO3a axis acts as an enhancer through H4 acetylation, and it was identified that it can act as a coactivator of autophagy-related genes.
이에 본원에 따른 방법은 히스톤 4의 아세틸화를 추가로 측정할 수 있다. 히스톤은 염색질(chromatin)을 구성하는 중심단백질로, DNA 사슬을 감는 실패 역할을 해서 DNA를 응축하며, 유전자 발현에 중요한 역할을 하는 것으로 알려져 있다. 히스톤 단백질에는 H1, H2A, H2B, H3 및 H4 등이 존재하며, H2A, H2B, H3 및 H4가 각각 두 개씩 모여 8량체인 코어 히스톤을 형성하고, H1은 연결체이다. 코어 히스톤은 매우 잘 보존된 서열을 갖으며, 다양한 변이체 또한 발견되며, 이러한 서열은 공개된 히스톤 DB2.0을 참조할 수 있다. (https://www.ncbi.nlm.nih.gov/projects/HistoneDB2.0/index.fcgi/browse/). 본원에서 규명된 기전에 관여하는 히스톤 H4의 서열 (Gene ID: 8359, Protein ID: NP_003529.1)에서 H4의 6, 9, 13 및 17번째 라이신 잔기 (서열번호 3의 서열을 기준)를 아세틸화시킨다. Accordingly, the method according to the present application may further measure the acetylation of histone 4. Histones are central proteins constituting chromatin, and are known to play an important role in gene expression by acting as a failure to wind up DNA chains and condensing DNA. Histone proteins include H1, H2A, H2B, H3, and H4, and H2A, H2B, H3, and H4 combine two each to form an octameric core histone, and H1 is a linker. The core histone has a very well-conserved sequence, and various variants are also found. For this sequence, reference can be made to the published histone DB2.0. (https://www.ncbi.nlm.nih.gov/projects/HistoneDB2.0/index.fcgi/browse/). Acetylation of the 6th, 9th, 13th and 17th lysine residues (based on the sequence of SEQ ID NO: 3) of H4 in the sequence of histone H4 (Gene ID: 8359, Protein ID: NP_003529.1) involved in the mechanism identified herein make it
실질적으로 동일한이란, 단백질 및 핵산 서열 수준에 모두 적용되며, 참조 또는 기준이 되는 서열과 비교하여, 염기 또는 아미노산 잔기에 하나 이상의 치환, 결손, 또는 부가가 있을 수 있으나, 전체적으로 볼 때 기능에 차이가 없거나 기능을 나쁘게하지 않는 수준의 기능을 갖는 것을 의미한다. 상동성은 대상이 되는 서열을 최대한 대응되도록 얼라인하고, 당업계에서 통상적으로 이용되는 알고리즘을 이용하여 얼라인된 서열을 분석한 경우에, 최소 61%의 상동성, 보다 바람직하게는 70%의 상동성, 보다 더 바람직하게는 80%의 상동성, 가장 바람직하게는 90% 이상, 특히 95% 이상의 상동성을 나타내는 서열을 의미한다. 서열비교를 위한 얼라인먼트 방법은 당업계에 공지되어 있다. 예를 들면 Smith and Waterman, Adv. Appl. Math. (1981) 2:482; Needleman and Wunsch, J. Mol. Bio. (1970) 48:443; Pearson and Lipman, Methods in Mol. Biol. (1988) 24: 307-31; Higgins and Sharp, Gene (1988) 73:237-44; Higgins and Sharp, CABIOS (1989) 5:151-3; Corpet et al., Nuc. Acids Res. (1988) 16:10881-90; Huang et al., Comp. Appl. BioSci. (1992) 8:155-65 및 Pearson et al., Meth. Mol. Biol. (1994) 24:307-31에 개시되어 있다. NCBI Basic Local Alignment Search Tool(BLAST)(Altschul et al., J. Mol. Biol. (1990) 215:403-10)은 NBCI 등에서 접근 가능하며, blast, blastp, blasm, blastx, tblastn 및 tblastx와 같은 서열 분석 프로그램과 연동되어 이용할 수 있다. BLSAT는 www.ncbi.nlm.nih.gov/BLAST/에서 접속 가능하며, 이 프로그램을 이용한 서열 상동성 비교 방법은 www.ncbi.nlm.nih.gov/BLAST/blast_help.html에서 확인할 수 있다. Substantially the same applies to both protein and nucleic acid sequence levels, and there may be one or more substitutions, deletions, or additions in bases or amino acid residues compared to a reference or reference sequence, but there is no difference in function as a whole It means having a level of functionality that is absent or does not impair functionality. The homology is at least 61% homology, more preferably 70% homology, when the target sequence is aligned to match as much as possible, and the aligned sequence is analyzed using an algorithm commonly used in the art. means a sequence which exhibits homology, even more preferably at least 80% homology, most preferably at least 90% homology, in particular at least 95% homology. Alignment methods for sequence comparison are known in the art. See, for example, Smith and Waterman, Adv. Appl. Math. (1981) 2:482 ; Needleman and Wunsch, J. Mol. Bio. (1970) 48:443; Pearson and Lipman, Methods in Mol. Biol. (1988) 24: 307-31; Higgins and Sharp, Gene (1988) 73:237-44; Higgins and Sharp, CABIOS (1989) 5:151-3; Corpet et al., Nuc. Acids Res. (1988) 16:10881-90; Huang et al., Comp. Appl. BioSci. (1992) 8:155-65 and Pearson et al., Meth. Mol. Biol. (1994) 24:307-31. The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. (1990) 215:403-10) is accessible from NBCI et al. It can be used in conjunction with a sequence analysis program. The BLSAT can be accessed at www.ncbi.nlm.nih.gov/BLAST/, and a method for comparing sequence homology using this program can be found at www.ncbi.nlm.nih.gov/BLAST/blast_help.html.
일 구현예에서 본원에 따른 방법은 상기 단백질을 발현하는 세포를 이용하여 수행될 수 있다. 세포를 이용하여 수행되는 경우 단백질은 이를 발현하는 세포로 제공될 수 있다. In one embodiment, the method according to the present disclosure may be performed using a cell expressing the protein. When carried out using cells, the protein can be provided to the cell expressing it.
본원에 따른 PONTIN 단백질의 메틸화정도 또한 당업계에 공지된 방법을 이용하여 수행될 수 있으며, 예를 들면 본원 실시예에 기재된 방법을 참고할 수 있다. The degree of methylation of the PONTIN protein according to the present application may also be performed using a method known in the art, for example, the method described in the Examples herein may be referred to.
본원의 방법에 사용되는 시험물질은 CARM1-PONTIN-FOXO3a 신호전달시스템에 작용하여 포도당이 결핍된 상태에서 PONTIN 메틸화를 조절할 것으로 기대되는 물질로, 저분자량 화합물, 고분자량 화합물, 화합물들의 혼합물(예컨대, 천연 추출물 또는 세포 또는 조직 배양물), 또는 바이오의약품(예컨대, 단백질, 항체, 펩타이드, DNA, RNA, 안티센스 올리고뉴클레오타이드, RNAi, 앱타머, RNAzyme 및 DNAzyme), 또는 당 및 지질 등을 포함하나 이로 한정하는 것은 아니다.The test substance used in the method of the present application is a substance expected to regulate PONTIN methylation in a glucose-deficient state by acting on the CARM1-PONTIN-FOXO3a signaling system, and a low molecular weight compound, a high molecular weight compound, a mixture 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, including but not limited to is not doing
본원에 따른 일 구현예에서는 저분자화합물이 시험물질로 사용될 수 있다. 상기 시험 물질은 합성 또는 천연 화합물의 라이브러리로부터 얻을 수 있으며 이러한 화합물의 라이브러리를 얻는 방법은 당업계에 공지되어 있다. 합성 화합물 라이브러리는 Maybridge Chemical Co.(UK), Comgenex(USA), Brandon Associates(USA), Microsource(USA) 및 Sigma-Aldrich(USA)에서 구입 가능하며, 천연 화합물의 라이브러리는 Pan Laboratories(USA) 및 MycoSearch(USA)에서 구입 가능하다. 시험 물질은 당업계에 공지된 다양한 조합 라이브러리 방법에 의해 얻을 수 있으며, 예를 들어, 생물학적 라이브러리, 공간 어드레서블 패러럴 고상 또는 액상 라이브러리(spatially addressable parallel solid phase or solution phase libraries), 디컨볼루션이 요구되는 합성 라이브러리 방법, "1-비드 1-화합물" 라이브러리 방법, 그리고 친화성 크로마토그래피 선별을 이용하는 합성 라이브러리 방법에 의해 얻을 수 있다. 분자 라이브러리의 합성 방법은, 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 등에 개시되어 있다. 예를 들면 약물의 스크리닝 목적을 위해서는 화합물은 저분자량의 치료효과를 갖는 것이 사용될 수 있다. 예를 들면 중량이 400 Da, 600 Da 또는 800 Da과 같은 약 1000 Da 내외의 화합물이 사용될 수 있다. 목적에 따라 이러한 화합물은 화합물 라이브러리의 일부를 구성할 수 있으며, 라이브러리를 구성하는 화합물의 숫자도 수십개부터 수백만개까지 다양하다. 이러한 화합물 라이브러리는 펩타이드, 펩토이드 및 기타 환형 또는 선형의 올리고머성 화합물, 및 주형을 기본으로 하는 저분자 화합물, 예컨대 벤조디아제핀, 하이단토인, 바이아릴, 카보사이클 및 폴리사이클 화합물 (예컨대 나프탈렌, 페노티아진, 아크리딘, 스테로이드 등), 카보하이드레이트 및 아미노산 유도체, 디하이드로피리딘, 벤즈하이드릴 및 헤테로사이클 (예컨대 트리아진, 인돌, 티아졸리딘 등)을 포함하는 것일 수 있으나, 이는 단지 예시적인 것으로 이로 한정되는 것은 아니다. 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 Required synthetic library methods, "1-bead 1-compound" library methods, and synthetic library methods using affinity chromatography selection can be obtained. 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 may also vary 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, steroids, etc.), carbohydrates and amino acid derivatives, dihydropyridine, benzhydryl and heterocycles (such as triazine, indole, thiazolidine, etc.), but these are merely exemplary. The present invention is not limited thereto.
또한 예를 들면 바이올로직스가 스크리닝에 사용될 수 있다. 바이올로직스는 세포 또는 바이오분자를 일컫는 것으로, 바이오분자란, 단백질, 핵산, 탄수화물, 지질 또는 생체내 및 생체외에서 세포 시스템 등을 이용하여 생산된 물질을 일컫는 것이다. 바이오분자를 단독으로 또는 다른 바이오분자 또는 세포와 조합으로 제공될 수 있다. 바이오분자는 예를 들면, 폴리뉴클레오타이드, 펩타이드, 항체, 또는 기타 혈장에서 발견되는 단백질 또는 생물학적 유기물질을 포함하는 것이다.Also, for example, biologics can be used for screening. Biologics refer 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.
본원에 사용되는 단백질은 당업계에 공지된 방법을 사용하여 제조될 수 있다. 특히 유전자 재조합 기술을 이용하는 것이다. 예를 들면 상기 단백질을 코딩하는 상응하는 유전자를 포함하는 플라스미드를 원핵 또는 진핵세포 세포 예를 들면 곤충세포, 포유류 세포에 전달하여 과발현 시킨 후 정제하여 사용할 수 있다. 상기 플라스미드는 예를 들면 본원의 예시적 구현예에서 사용한 것과 같은 동물 세포주에 트랜스팩션한 후, 발현된 단백질을 정제하여 사용할 수 있으나, 이로 제한하는 것은 아니다. 이 경우 단백질은 검출의 편이를 위해 다양한 표식물질, 예를 들면 바이오틴, 형광물질, 아세틸화, 방사선 동위원소와 같은 것으로 공지된 방법 또는 시중의 단백질 표지 키트를 사용하여 표지될 수 있으며, 표지된 물질에 적합한 검출기기를 사용하여 검출될 수 있다.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, for example, an insect cell, a mammalian cell, overexpressed, and then purified and used. 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, for the convenience of detection, 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, and the labeled material. It can be detected using a detection device suitable for
또는 스크리닝 단백질을 암호화하는 DNA 또는 RNA 서열을 적당한 숙주 세포에서 발현시켜 그 세포 파쇄물을 만들거나 상기 스크리닝 단백질의 mRNA를 시험관내에서 번역한 후 당업계에 공지된 단백질 분리 방법에 의해 스크리닝 단백질을 정제할 수 있다. 통상, 세포잔여물(cell debris) 등을 제거하기 위해 상기 세포 파쇄물 또는 시험관내 번역한 결과물을 원심분리한 후, 침전, 투석, 각종 컬럼 크로마토그라피 등을 적용한다. 이온교환 크로마토그라피, 겔-퍼미에이션 크로마토그라피, HPLC, 역상-HPLC, 프렙용 SDS-PAGE, 친화성 컬럼 등은 컬럼 크로마토그라피의 예이다. 친화성 컬럼은, 예를 들어, 항-스크리닝 단백질 항체를 이용하여 만들 수 있다.Alternatively, the DNA or RNA sequence encoding the screening protein is expressed in an appropriate host cell to prepare a cell lysate, or the mRNA of the screening protein is translated in vitro, and then the screening protein is purified by a protein isolation method known in the art. can 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.
본원에 따른 방법에서 메틸화 측정결과 시험물질로 처리되지 않은 대조군과 비교하여, 시험물질로 처리된 경우 PONTIN 333 및 339번째 잔기의 메틸화가 증가 또는 감소한 경우, 이를 자가포식을 조절하는 후보물질로 선별하고, 이를 다양한 질환의 치료제로 개발 할 수 있다. As a result of the measurement of methylation in the method according to the present application, when the methylation of residues PONTIN 333 and 339 is increased or decreased when treated with the test substance, compared to the control not treated with the test substance, it is selected as a candidate substance for controlling autophagy, and , it can be developed as a therapeutic agent for various diseases.
실험결과 시험물질과 접촉되지 않은 대조군과 비교하여 시험물질의 존재하에서 자가포식을 조절하는 물질을 후보 물질로 선별한다. 대조군과 비교 약 10% 이상, 약 20% 이상, 약 30% 이상, 약 40% 이상, 약 50% 이상, 약 60% 이상, 약 70% 이상, 약 80% 이상, 약 90% 이상, 또는 약 100% 이상, 또는 이 이상을 증가 또는 감소된 것을 후보물질로 선별할 수 있다.As a result of the experiment, a substance that modulates autophagy in the presence of the test substance is selected as a candidate substance compared to the control group that is not in contact with the test substance. About 10% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, or about 100% or more, or an increase or decrease of more than 100% may be selected as a candidate substance.
본원에 따른 방법에서는 PONTIN과 FOXO3a의 결합을 검출하는 단계를 추가로 또는 PONTIN의 메틸화 대신에 포함할 수 있다. 단백질-단백질 상호작용은 당업계에 공지된 다양한 방법을 이용하여 측정될 수 있다. 예를 들면 세포내 단백질의 결합/상호작용을 확인하는 이스트투하이브리드법, 콘포칼현미경법. 공동면역침전법, 표면플라즈마공명 (SPR) 및 스펙트로스코피법을 포함하나 이로 제한하는 것은 아니며, 이러한 방법에 관한 비교 및 자세한 실험법에 관한 추가의 참고문헌은 Berggard et al., (2007) "Methods for the detection and analysis of protein-protein interactions", PROTEOMICS Vol7: pp 2833 - 2842에 기재된 것을 참고할 수 있다.In the method according to the present application, the step of detecting the binding of PONTIN to FOXO3a may additionally or instead of methylation of PONTIN. Protein-protein interactions can be measured using a variety of methods known in the art. For example, the yeast-to-hybrid method, confocal microscopy, which confirms the binding/interaction of intracellular proteins. Additional references for comparative and detailed experimental methods regarding these methods include, but are not limited to, coimmunoprecipitation, surface plasma resonance (SPR) and spectroscopy, Berggard et al., (2007) "Methods for The detection and analysis of protein-protein interactions", PROTEOMICS Vol7: pp 2833 - 2842 can be referred to.
본원에 따른 다양한 스크리닝 방법에서 사용되는 단백질의 양, 세포의 종류 및 시험물질의 양 및 종류 등은 사용하는 구체적인 실험방법 및 시험물질의 종류에 따라 달라지며, 당업자라면 적절한 양을 선택할 수 있을 것이다. The amount and type of protein, cell type, and test material used in the various screening methods according to the present application vary depending on the specific experimental method and type of the test material used, and those skilled in the art will be able to select an appropriate amount.
본원에 따른 다양한 스크리닝 방법에 의해 스크리닝될 수 있는 자기포식 조절제는, 자가포식에 이상이 있는 경우 변성된 단백질의 축적으로 인해 다양한 질환 또는 자가포식의 과다 활성과 관련된 질환의 치료 또는 예방에 사용될 수 있다.Autophagy modulators that can be screened by various screening methods according to the present disclosure may be used for the treatment or prevention of various diseases or diseases related to hyperactivity of autophagy due to the accumulation of denatured proteins when there is an abnormality in autophagy. .
예를 들면 이로 제한하는 것은 아니나, 신경퇴행성질환을 포함하는 신경변성 질환, 간질환, 자가면역 질환, 심혈관 질환, 대사 질환, 과오종 증후군, 유전성 근육 질환, 근 질환 또는 암(Hara t et al Nature 2006, Mizushima n et al Genes & Dev. 2007, Takamura a et al Genes & Dev. 2011, Ratuo p et al J Hepatology 2010 등 참조) 등의 예방 또는 치료에 사용될 수 있는 물질이다. 예를 들면 신경변성 질환은, 예를 들면 부신성 백색질형성장애(Adrenal Leukodystrophy), 알콜중독, 알렉산더병(Alexander's disease), 알퍼병(Alper's disease), 알츠하이머병(Alzheimer's disease), 근육위축가쪽경화증, 모세관확장실조(ataxiatelangiectasia), 배튼병(Batten disease), 소해면양뇌증(bovine spongiform encephalopathy), 캐너번병(Canavan disease), 뇌성마비, 코케인 증후군(cockayne syndrome), 피질기저퇴화(corticobasal degeneration), 크로이츠펠트-야콥 병(Creutzfeldt-Jakob disease), 치명성 가계 불면증(familial fatal insomnia), 전두엽 측두엽 변성증(frontotemporal lobar degeneration), 헌팅톤병(Huntington's disease), HIV-관련 치매, 케네디병(Kennedy's disease), 크라베병(Krabbe's [0016] disease), 레비소체 치매(Lewy body dementia), 신경보렐리아증(neuroborreliosis), 마카도 조셉 병(Machado-Joseph disease), 다계통 위축증(multiple system atrophy), 다발성경화증(multiple sclerosis), 발작수면 (narcolepsy), 니이만-픽병(Niemann Pick disease), 파킨슨병(Parkinson's disease), 펠리체우스-메르츠바허병(Pelizaeus-Merzbacher disease), 픽병(Pick's disease), 원발가쪽경화증(primary lateral sclerosis), 프리온 질환(prion disease), 진행핵상마비(progressive supranuclear palsy), 레프숨병(Refsum's disease), 잔트호프병(Sandhoff disease), 실더병(Schilder's disease), 유독성 빈혈 속발성의 척수의 아급성연합변성(subacute combined degeneration of spinal cord secondary to pernicious anaemia), 스필마이어-보그트-쇼그렌-배튼 병(Spielmeyer-Vogt-Sjogren-Batten disease), 척수소뇌성 실조증(spinocerebellar ataxia), 척수근위축증(spinal muscular atrophy), 스틸-리차드슨-올스제위스키 병(Steele-Richardson-Olszewski disease), 척수매독(Tabes dorsalis), 독성 뇌병증(toxic encephalopathy)을 포함하나 이로 제한하는 것은 아니다. 본원에 따른 일 구현예에서 단백질변성과 관련된 질환은 알츠하이머병, 파킨슨병, 레비 소체 치매, 근육위축가쪽경화증(ALS), 헌팅톤병, 척수소뇌성 실조증 또는 척수구근 근위축증(spinobulbar musclular atrophy)을 포함한다.For example, but not limited to, neurodegenerative diseases including neurodegenerative diseases, liver diseases, autoimmune diseases, cardiovascular diseases, metabolic diseases, hamartoma syndrome, hereditary muscle diseases, muscle diseases or cancer (Hara t et al Nature 2006) , Mizushima n et al Genes & Dev. 2007, Takamura a et al Genes & Dev. 2011, Ratuo p et al J Hepatology 2010, etc.) It is a substance that can be used for prevention or treatment. Neurodegenerative diseases include, for example, adrenal leukodystrophy, alcoholism, Alexander's disease, Alper's disease, Alzheimer's disease, amyotrophic lateral sclerosis, Ataxiatelangiectasia, Batten disease, bovine spongiform encephalopathy, Canavan disease, cerebral palsy, cockayne syndrome, corticobasal degeneration, Creutzfeldt -Creutzfeldt-Jakob disease, familial fatal insomnia, frontotemporal lobar degeneration, Huntington's disease, HIV-associated dementia, Kennedy's disease, Krabe disease (Krabbe's disease), Lewy body dementia, neuroborreliosis, Machado-Joseph disease, multiple system atrophy, multiple sclerosis , narcolepsy, Niemann Pick disease, Parkinson's disease, Pelizaeus-Merzbacher disease, Pick's disease, primary lateral sclerosis sclerosis, prion disease, progressive supranuclear palsy, Refsum's disease, Sandhoff disease, Schilder's disease, subacute combined degeneration of spinal cord secondary to pernicious anaemia, Spielmeyer-Vogt-Sjogren-Batten disease , including spinocerebellar ataxia, spinal muscular atrophy, Steel-Richardson-Olszewski disease, Tabes dorsalis, and toxic encephalopathy However, it is not limited thereto. Diseases related to protein degeneration in one embodiment according to the present application include Alzheimer's disease, Parkinson's disease, Lewy body dementia, amyotrophic lateral sclerosis (ALS), Huntington's disease, spinal cerebellar ataxia or spinobulbar musclular atrophy. .
또다른 구현예에서, 본원에 따른 방법이 사용될 수 있는 자가면역 질환은 알로페시아 그레아타(alopecia greata), 강직성 척추염, 항인지질 증후군, 자가면역 아디슨 질환, 부신의 자가면역 질환, 자가면역 용혈성 빈혈, 자가면역 간염, 자가면역 난소염 및 고환염, 자가면역 혈소판감소증, 베체트병, 수포성 유천포창, 심근병증, 복강 스프루우-피부염(celiac sprue-dermatitis), 만성 피로 면역이상 증후군, 만성염증성 탈수초 다발성 신경병증, Churg-Strauss 증후군, 반흔성유천포창, CREST 증후군, 한냉 응집소 질환, 크론씨병, 원판성 낭창, 복태성복합한냉글로불린혈증, 섬유근통-섬유근염, 사구체신염, 그레이브스 질환, 귈레인 바레 증후군, 하시모토 갑상선염, 특발성 폐섬유화증, 특발성 혈소판 감소성 자반증, IgA 신경염, 연소자성 관절염, 편평태선, 홍반성 루푸스, 메니에르병, 혼합성 연결 조직 질환, 다발성 경화증, 타입 I 또는 면역-매개 당뇨병, 중증근무력증, 심상성 천포창, 악성 빈혈, 결정성 다발동맥염, 다발연골염, 자가면역성 다선 증후군, 류마티스 다발성근통, 다발성 근염과 피부근염, 일차성 무감마글로불린혈증, 일차성 담증성 간경변, 건선, 건선성 관절염, 레이노 현상, 라이터 증후군, 류마티스 관절염, 사르코이드증, 공피증, 강직인간 증후군, 전신성 홍반성 루푸스, 홍반성 루푸스, 다가야스 동맥염, 일시적 동맥염, 거대세포 동맥염, 궤양성 대장염, 포도막염, 백반증 및 베게너 육아종증로 구성된 군으로부터 선택되는 자가면역질환이다.In another embodiment, the autoimmune disease in which the method according to the present disclosure may be used is alopecia greata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune disease of the adrenal gland, autoimmune hemolytic anemia , autoimmune hepatitis, autoimmune oophoritis and orchitis, autoimmune thrombocytopenia, Behcet's disease, pemphigoid vesicles, cardiomyopathy, celiac sprue-dermatitis, chronic fatigue immune dysfunction syndrome, chronic inflammatory demyelinating multiple. Neuropathy, Churg-Strauss syndrome, scar pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, disc lupus, gastrogenetic cold globulinemia, fibromyalgia-fibromyositis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, Hashimoto's thyroiditis , idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura, IgA neuritis, juvenile arthritis, lichen planus, lupus erythematosus, Meniere's disease, mixed connective tissue disease, multiple sclerosis, type I or immune-mediated diabetes mellitus, myasthenia gravis, Pemphigus vulgaris, pernicious anemia, polyarteritis crystallized, polychondritis, autoimmune polyline syndrome, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynaud's Symptoms, Reiter's syndrome, rheumatoid arthritis, sarcoidosis, scleroderma, ankylosing man syndrome, systemic lupus erythematosus, lupus erythematosus, tagayas arteritis, transient arteritis, giant cell arteritis, ulcerative colitis, uveitis, vitiligo and Wegener's granulomatosis It is an autoimmune disease selected from the group consisting of.
또다른 구현예에서, 본원에 따른 방법이 사용될 수 있는 심혈관질환은 광동맥성심장병, 심근증, 고혈압성심장질환, 심부전, 폐성심, 심율동장애, 심장 내막염, 염증성 심비대증, 심근염, 심장판막증, 뇌혈관장애, 하지동맥질환, 선천성 심질환 및 심장 류머티즘으로 구성된 군으로부터 선택되는 심혈관질환이다.In another embodiment, the cardiovascular disease for which the method according to the present disclosure can be used is photoarterial heart disease, cardiomyopathy, hypertensive heart disease, heart failure, pulmonary heart disease, cardioversion disorder, endocarditis, inflammatory cardioplasty, myocarditis, valvular heart disease, cerebrovascular disease. It is a cardiovascular disease selected from the group consisting of disorders, lower extremity arterial disease, congenital heart disease, and cardiac rheumatism.
또다른 구현예에서, 본원에 따른 방법이 사용될 수 있는 대사질환은 비만, 당뇨, 이상지방혈증, 지방간, 고혈압, 동맥경화, 고지혈증 및 고인슐린혈증으로 구성된 군으로부터 선택되는 대사질환이다.In another embodiment, the metabolic disease for which the method according to the present application can be used is a metabolic disease selected from the group consisting of obesity, diabetes, dyslipidemia, fatty liver, hypertension, arteriosclerosis, hyperlipidemia and hyperinsulinemia.
또다른 구현예에서, 본원에 따른 방법이 사용될 수 있는 암은 뇌하수체선종, 신경교종, 뇌종양, 상인두암, 후두암, 흉선종, 중피종, 유방암, 폐암, 위암, 식도암, 대장암, 간암, 췌장암, 췌내분비종양, 담낭암, 음경암, 요관암, 신세포암, 전립선암, 방광암, 비호지킨성림프종, 골수이형성증.In another embodiment, the cancer for which the method according to the present application can be used is pituitary adenoma, glioma, brain tumor, epipharyngeal cancer, laryngeal cancer, thymoma, mesothelioma, breast cancer, lung cancer, gastric cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, pancreatic endocrine Tumors, gallbladder cancer, penile cancer, ureter cancer, renal cell cancer, prostate cancer, bladder cancer, non-Hodgkin's lymphoma, myelodysplasia.
이하, 본 발명의 이해를 돕기 위해서 실시예를 제시한다. 그러나 하기의 실시예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐 본 발명이 하기의 실시예에 한정되는 것은 아니다.Hereinafter, examples are presented to help the understanding of the present invention. However, the following examples are only provided for easier understanding of the present invention, and the present invention is not limited to the following examples.
실시예Example
실험 방법 및 실험 재료Experimental methods and experimental materials
Pontin의 R333, R339를 인지하는 항체는 해당 펩타이드를 이용하여 Peptron에서 제작하였다. Antibodies recognizing R333 and R339 of Pontin were produced by Peptron using the corresponding peptide.
Generation of PontinGeneration of Pontin f/ff/f MEFs 및 세포배양 MEFs and cell culture
Pontin f/f MEF는 Pontin KO 쥐를 이용하여 3T3 방식으로 제작하였다. Pontin f/f MEFs were prepared in the 3T3 method using Pontin KO mice.
Pontin f/f MEF에 Flag-tagged Pontin WT, RK, RA를 렌티바이러스로 감염 시킨 뒤, hygromycin을 이용하여 1주간 selection하였다. After infecting Pontin f/f MEF with Flag-tagged Pontin WT, RK, and RA with lentivirus, hygromycin was used for selection for 1 week.
친화도 정제를 통한 CARM1 결합 단백질 규명Identification of CARM1 binding protein through affinity purification
HEK293T에 Flag-tagged CARM1 발현시킴. Flag M2 affinity gel (100ul of 50% slurry) (sigma)로 CARM1을 당긴 뒤 20 mM Tris-HCl (pH 7.9), 15 % Glycerol, 1 mM EDTA, 1 mM dithiothreitol (DTT), 0.2 mM PMSF, 0.05 % Nonidet P40, and 150 mM KCl로 wash하여 비특이적 결합 떨어트림. 이후 Flag 펩타이드 (0.2 mg/ml)을 이용하여 bead로부터 CARM1 및 복합체를 떨어트림. 이후 SDS-PAGE를 통해 젤을 달린 뒤 LC-MS/MS를 통해 단백질 정보 분석.Flag-tagged CARM1 expression in HEK293T. After pulling CARM1 with Flag M2 affinity gel (100ul of 50% slurry) (sigma), 20 mM Tris-HCl (pH 7.9), 15% Glycerol, 1 mM EDTA, 1 mM dithiothreitol (DTT), 0.2 mM PMSF, 0.05 % Wash with Nonidet P40, and 150 mM KCl to release non-specific binding. After that, CARM1 and the complex were dropped from the beads using Flag peptide (0.2 mg/ml). After that, the gel was run through SDS-PAGE and protein information was analyzed through LC-MS/MS.
Bacterial expression and GST pull-down assayBacterial expression and GST pull-down assay
GST-tagged construct는 Rosetta E. coli에 transformation된 뒤 단백질 발현에 이용. 이후 glutathione bead로 뽑음. His-tagged Pontin의 경우 M15[pREP4] E. coli에서 키운 뒤 Ni-NTA bead를 통해 뽑음. In vitro translated 단백질은 TNT Quick Coupled Transcription/Translation system (Promega) 설명서대로 뽑음. 뽑은 단백질과 in vitro translated protein은 binding buffer (125 mM NaCl, 20 mM Tris [pH 7.5], 10 % glycerol, 0.1 % NP40, and 0.5 mM DTT supplemented with protease inhibitors)에서 GST pulldown 실험에 이용. 이후 4번의 wash 이후 SDS sample buffer 처리 후 gel 달림.GST-tagged construct is used for protein expression after transformation into Rosetta E. coli. It was then pulled out with glutathione beads. In the case of His-tagged Pontin, M15[pREP4] was grown in E. coli and extracted through Ni-NTA beads. In vitro translated proteins were extracted according to the instructions of the TNT Quick Coupled Transcription/Translation system (Promega). Extracted proteins and in vitro translated proteins were used in GST pulldown experiments in binding buffer (125 mM NaCl, 20 mM Tris [pH 7.5], 10 % glycerol, 0.1 % NP40, and 0.5 mM DTT supplemented with protease inhibitors). After 4 washes, SDS sample buffer treatment and gel run.
In vitro methylation assayIn vitro methylation assay
In vitro methylation 실험은 비드와 결합된 His-Pontin 혹은 GST-Pontin 과 함께 구매한recombinant CARM1 (Active Motif, 31347) 단백질 혹은 HA-CARM1 WT/R169A 돌연변이 단백질과 함께 진행. 반응은 PBS buffer에서 30도 3시간으로 진행되었고 5X sampling buffer를 넣고 5분간 끓여서 종결. SDS-PAGE에 달린 뒤 PVDF membrane에 semi-dry electroblotter를 통해 옮김. Rme2a or Pontin-me 항체를 통해 immunoblot 진행.In vitro methylation experiments were performed with beads-conjugated His-Pontin or GST-Pontin and purchased recombinant CARM1 (Active Motif, 31347) protein or HA-CARM1 WT/R169A mutant protein. The reaction was carried out in PBS buffer at 30 degrees for 3 hours, and 5X sampling buffer was added and boiled for 5 minutes to terminate. After running on SDS-PAGE, transferred to PVDF membrane through semi-dry electroblotter. Immunoblot was performed using Rme2a or Pontin-me antibody.
In vitro methylation assay using 3H-SAMIn vitro methylation assay using 3H-SAM
GST-Pontin 과 구매한 recombinant CARM1 (Active Motif, 31347) 단백질을 methylation buffer (50 mM Tris-HCl pH 8.5, 20 mM KCl, 10 mM MgCl2, 10 mM -mercaptoethanol, and 250 mM sucrose) with 1 μCi of 3H-SAM에 넣은 뒤 30 ℃ 에서 하루 놔둠. Reaction buffer 제거 후 2X sampling buffer를 넣어준 뒤 10분간 끓임 이후 SDS-PAGE에 달린 뒤 필름을 이용하여 관찰.GST-Pontin and purchased recombinant CARM1 (Active Motif, 31347) protein in methylation buffer (50 mM Tris-HCl pH 8.5, 20 mM KCl, 10 mM MgCl2, 10 mM -mercaptoethanol, and 250 mM sucrose) with 1 μCi of 3H -After putting it in SAM, it was left at 30 ℃ for one day. After removing the reaction buffer, add 2X sampling buffer, boil for 10 minutes, run on SDS-PAGE, and observe using a film.
Immunodot blot assayImmunodot blot assay
Non-methylated Pontin peptide와 methylated Pontin peptide를 membrane에 점으로 찍음. 충분히 말린 뒤, PBST에 녹인 5% milk로 1시간 blocking 진행. purified GST-FOXO3a proteins (1 μg/ml)을 넣은 protein-binding buffer (100 mM NaCl, 20 mM Tris-HCl [pH 7.6], 10 % glycerol, 0.1 % Tween-20, 2 % skim milk powder and 1 mM DTT),를 이용하여 membrane에 결합 시킴. 이후 GST antibody를 이용한 immunoblot을 진행.Non-methylated Pontin peptide and methylated Pontin peptide were dotted on the membrane. After drying sufficiently, blocking proceeded with 5% milk dissolved in PBST for 1 hour. Protein-binding buffer (100 mM NaCl, 20 mM Tris-HCl [pH 7.6], 10 % glycerol, 0.1 % Tween-20, 2 % skim milk powder and 1 mM) containing purified GST-FOXO3a proteins (1 μg/ml) DTT), to bind to the membrane. Afterwards, immunoblot using GST antibody was performed.
Lentivirus construction and productionLentivirus construction and production
3X Flag-Pontin WT, RK and RA는 pLVX vector, lentiviral shRNA constructs는 pLKO.1 vector에 클로닝함. Lentiviral constructs는 packaging vectors (psPAX2 and VSV-G)와 함께 HEK293T 세포에 transfection, 48시간 뒤에 0.45-μm filter를 통해 배양액을 모음. shRNA 시퀀스 정보 Tip60; 5’-GCAACGCCACTTGACCAAATG-3’, Pontin; 5’-GTGGCGTCATAGTAGAATTA A-3’, FOXO1/3/4; 5’-CTGTGTGCCCTACTTCAAGGA-3’.3X Flag-Pontin WT, RK and RA were cloned into pLVX vector, and lentiviral shRNA constructs were cloned into pLKO.1 vector. Lentiviral constructs were transfected into HEK293T cells together with packaging vectors (psPAX2 and VSV-G), and the culture medium was collected through a 0.45-μm filter after 48 hours. shRNA sequence information Tip60; 5'-GCAACGCCACTTGACCAAATG-3', Pontin; 5'-GTGGCGTCATAGTAGAATTA A-3', FOXO1/3/4; 5'-CTGTTGGCCCTACTTCAAGGA-3'.
Autophagic vacuole stainingAutophagic vacuole staining
Autophagic vacuoles은 Cyto-ID autophagy detection kit (Enzo Life Sciences, ENZ-51031)를 이용하여 염색함. 세포는 coverslips에 2 x 10^4개와 함께 Cyto-ID green detection reagent (1:500)과 Hoechst 33342 (1:1000)가 섞인 미디어를 넣어줌. 37 ℃에서 30 min 놔둔 뒤 PBS로 wash 후 2% paraformaldehyde/PBS를 넣어준 뒤 상온에서 10분간 고정. 이후 confocal microscope (Zeiss, LSM700)로 이미지 촬영.Autophagic vacuoles were stained using the Cyto-ID autophagy detection kit (Enzo Life Sciences, ENZ-51031). Cells were put on coverslips with 2 x 10^4 pieces of media mixed with Cyto-ID green detection reagent (1:500) and Hoechst 33342 (1:1000). After leaving at 37 °C for 30 min, wash with PBS, add 2% paraformaldehyde/PBS, and fix at room temperature for 10 minutes. Images were then taken with a confocal microscope (Zeiss, LSM700).
Luciferase assayLuciferase assay
MEFs에 6X canonical FOXO response element (6x DBE-luciferase)를 함께 transfection해서 진행. Luciferase activity는 transfection 이후 36시간 뒤에 측정하였으며 beta-galactosidase 발현으로 정량함.MEFs were transfected with 6X canonical FOXO response element (6x DBE-luciferase). Luciferase activity was measured 36 hours after transfection and quantified by beta-galactosidase expression.
RNA-seq analysisRNA-seq analysis
RNA-seq libraries는 TruSeq RNA sample prep kit v2 (Illumina) 방식으로 제작함. RNA-seq libraries는 paired-end sequence 방식으로 Illumina HiSeq 4000 (Macrogen)을 통해 시퀀싱됨. RNA-seq data는 Tophat package를 통해 mouse genome (mm9)에 mapping함. Differential analysis는 EdgeR package를 통해 분석됨. Differentially regulated genes은 false discovery rate (FDR) cut-off 1 x 10^-5. Hierarchical clustering 분석은 모든 상황에서의 유전자 발현 수치로 함. Ward’s criterion for genes with 1 - (correlation coefficient)가 distance measure에 사용됨. Clustering heatmap은 z-scor 기준으로 그림. Functional enrichment analysis of GOBPs and KEGG pathways는 DAVID software를 통해서 하였고 Gene set enrichment analysis는 GSEA (version 3.0) software를 이용. The phenotype label for GSEA was set WT_normal: WT_Glc starv.: RA_normal: RA_Glc starv. = 1: 3: 1: 1.7, then Pearson correlation coefficient was calculated per gene for ranking. Gene sets은 Molecular Signature Database (MSigDB) v6.2에서 얻음.RNA-seq libraries were prepared using the TruSeq RNA sample prep kit v2 (Illumina) method. RNA-seq libraries were sequenced by Illumina HiSeq 4000 (Macrogen) in a paired-end sequence method. RNA-seq data was mapped to the mouse genome (mm9) through the Tophat package. Differential analysis is analyzed through EdgeR package. Differentially regulated genes have a false discovery rate (FDR) cut-off of 1 x 10^-5. Hierarchical clustering analysis was performed with gene expression levels in all situations. Ward’s criterion for genes with 1 - (correlation coefficient) is used for distance measure. Clustering heatmap is based on z-scor. Functional enrichment analysis of GOBPs and KEGG pathways was performed using DAVID software, and gene set enrichment analysis was performed using GSEA (version 3.0) software. The phenotype label for GSEA was set WT_normal: WT_Glc starv.: RA_normal: RA_Glc starv. = 1: 3: 1: 1.7, then Pearson correlation coefficient was calculated per gene for ranking. Gene sets were obtained from Molecular Signature Database (MSigDB) v6.2.
ChIP-seq analysisChIP-seq analysis
ChIP-seq libraries는 TruSeq DNA Sample prep Kit 방식으로 제작함. ChIP-seq libraries는 paired-end 방식으로 Illumina platform (MACROGEN)에서 시퀀싱됨. ChIP-seq reads는 mouse reference genome (GRCm38/mm10)에 Bowtie2를 이용하여 mapping함. methylated Pontin peak의 경우 Homer (v4.7.2)를 통해 제작. 우리가 제작한 Pontin 메틸화 항체 사용하였다. ChIP-seq libraries were prepared using the TruSeq DNA Sample prep Kit method. ChIP-seq libraries were sequenced on the Illumina platform (MACROGEN) in a paired-end fashion. ChIP-seq reads were mapped to the mouse reference genome (GRCm38/mm10) using Bowtie2. In the case of methylated Pontin peak, it was produced by Homer (v4.7.2). Pontin methylated antibody prepared by us was used.
실시예 1. Pontin의 R333, R339 아미노산 잔기가 CARM1에 의해 333과 339 잔기의 아르기닌이 메틸화됨을 규명Example 1. Investigation of methylation of arginine at residues 333 and 339 by CARM1 of R333 and R339 amino acid residues of Pontin
포도당 결핍 상황에서 자가포식 관련 전사 조절을 한다고 알려진 CARM1의 결합 파트너를 찾기 위해 tandem affinity purification을 통해 CARM1과 결합하는 단백질들을 분리했다. 흥미롭게도 LC-MS/MS를 통해 Pontin이라는 크로마틴 재배치 인자가 나왔다 (도 1a). Pontin과 CARM1이 실제로 결합하는지 확인하기 위해 위에서 뽑은 CARM1 복합체 eluate에 Pontin을 인지하는 항체로 확인하였다 (도 1b). Pontin과 CARM1이 직접적으로 결합하는지 확인하기 위해 Gglutathione S-transferase (GST)를 붙은 CARM1과 in vitro로 뽑은 Pontin 단백질과의 결합을 GST pulldown assay를 통해 확인하였다. Pontin과 복합체를 이룬다고 잘 알려진 Reptin도 같이 확인하였다. 실험 결과 CARM1은 Reptin과 결합하지 않고 Pontin과 선택적이게 결합하였다 (도 1c).In order to find a binding partner for CARM1, which is known to regulate autophagy-related transcription in the context of glucose deprivation, proteins that bind to CARM1 were isolated through tandem affinity purification. Interestingly, LC-MS/MS revealed a chromatin rearrangement factor called Pontin (Fig. 1a). In order to check whether Pontin and CARM1 actually bind, it was confirmed as an antibody recognizing Pontin in the CARM1 complex eluate extracted above (FIG. 1b). In order to confirm that Pontin and CARM1 directly bind, the binding of Gglutathione S-transferase (GST)-conjugated CARM1 with Pontin protein extracted in vitro was confirmed by GST pulldown assay. Reptin, which is well known to form a complex with pontin, was also identified. As a result of the experiment, CARM1 was selectively bound to Pontin without binding to Reptin (FIG. 1c).
다음으로 CARM1 WT과 효소 활성이 망가진 R169A 돌연변이를 이용하여 Pontin이 CARM1에 의해 아르기닌 메틸화가 될 수 있는지 in vitro methylation assay를 통해 확인해보았다. 오직 CARM1 WT을 넣어주었을 때만 Pontin이 in vitro 메틸화가 되는 것을 확인하였다 (도 1d). 이를 통해 CARM1의 효소 활성이 Pontin의 아르기닌 메틸화에 중요하다는 것을 확인했다. 다음으로 CARM1에 의해 메틸화되는 Pontin이 아미노산 잔기를 찾기 위해 인터넷에 오픈된 메틸화 예측 프로그램을 사용하였고 예측된 아르기닌 잔기들을 알라닌으로 교체한 돌연변이들을 만들었다. 실험 결과 아르기닌 R333과 R339를 각각 하나씩 알라닌으로 치환한 돌연변이에서 메틸화가 감소되어 있는 것을 보았고 둘을 동시에 돌연변이 시켰을 때 Pontin의 메틸화가 완전히 사라지는 것을 확인하였다 (도 1e). 방사선동위원소 3H-S-Adenosyl methionine (SAM)을 이용하여 Pontin WT과 여러 돌연변이에서 in vitro 메틸화 실험을 진행하였을 때도 Pontin의 WT은 메틸화되는 반면, 333, 및 339번째 아르기닌을 알라닌이나 라이신으로 돌연변이의 경우 메틸화가 되지 않는 것을 확인하였다 (도 1f). 실제로 Pontin의 구조를 확인하였을 때 R333과 R339는 가까운 위치에 존재하였으며 (도 1g), 여러 종에서도 두 아르기닌뿐만 아니라 주변 아미노산 잔기도 종간 잘 보존되어 있는 것을 확인하였다 (도 1h).Next, using CARM1 WT and the R169A mutant with impaired enzyme activity, it was checked whether Pontin could be arginine methylated by CARM1 through an in vitro methylation assay. It was confirmed that Pontin was methylated in vitro only when CARM1 WT was added (Fig. 1d). This confirmed that the enzymatic activity of CARM1 is important for the arginine methylation of Pontin. Next, to find the amino acid residue of Pontin methylated by CARM1, a methylation prediction program opened on the Internet was used, and mutants were made in which the predicted arginine residues were replaced with alanine. As a result of the experiment, it was found that methylation was reduced in the mutants in which arginine R333 and R339 were substituted with alanine, respectively, and it was confirmed that methylation of pontin completely disappeared when both were mutated at the same time (FIG. 1e). When in vitro methylation experiments were performed with Pontin WT and several mutants using the radioisotope 3 HS-Adenosyl methionine (SAM), WT of Pontin was methylated, whereas arginines 333 and 339 were mutated to alanine or lysine. It was confirmed that methylation was not performed (FIG. 1f). In fact, when the structure of Pontin was confirmed, R333 and R339 were located close to each other (FIG. 1g), and it was confirmed that not only the two arginines but also the surrounding amino acid residues were well conserved between species in several species (FIG. 1h).
추가 연구를 위해 Pontin의 아르기닌 메틸화를 인지할 수 있는 항체를 제작하였다. Dot blot을 통해 우리가 제작한 Pontin 메틸화 항체가 잘 작동하는 것을 확인하였으며 (도 1i), in vitro 메틸화 실험을 통해서도 항체의 특이성이 확인되었다 (도 1j). R333/339A뿐만 아니라 R333/339K 돌연변이에서도 동일하게 항체가 잘 작동하는 것을 확인하였다 (도 1k). CARM1의 WT과 효소 활성이 망가진 돌연변이를 이용하여 in vitro 메틸화 실험을 하였을 때 WT에 의해서만 Pontin의 메틸화가 증가하는 것을 직접 만든 항체로 재확인하였다 (도 1l). Pontin의 아르기닌 메틸화가 다른 아르기닌 메틸화 효소에서도 일어나는 것이 보고되었었기에 Pontin 메틸화 항체가 CARM1이 아닌 다른 아르기닌 메틸화 효소에 의해 일어나는 메틸화를 잡는지 확인하였다. 실험 결과 Pontin의 경우 CARM1/PRMT4 뿐만 아니라 PRMT5, PRMT6와도 결합할 수 있었지만 (결과나타내지 않음), 우리가 제작한 Pontin 메틸화 항체는 CARM1에 의한 메틸화만 인지하는 것을 확인하였다. 종합하면 우리는 CARM1에 의해 Pontin의 아르기닌 333, 339번 잔기가 메틸화되는 것을 in vivo, in vitro 실험을 통해 증명하였다.For further study, an antibody capable of recognizing arginine methylation of Pontin was constructed. It was confirmed that the Pontin methylated antibody prepared by us worked well through dot blot (Fig. 1i), and the specificity of the antibody was also confirmed through an in vitro methylation experiment (Fig. 1j). It was confirmed that the antibody works well not only in R333/339A but also in the R333/339K mutant ( FIG. 1k ). When an in vitro methylation experiment was performed using WT of CARM1 and a mutant with impaired enzymatic activity, it was reconfirmed with the homemade antibody that methylation of Pontin was increased only by WT (FIG. 11). Since it has been reported that arginine methylation of pontin occurs in other arginine methylases, it was confirmed whether the pontin methylation antibody captures methylation caused by arginine methylation other than CARM1. As a result of the experiment, Pontin was able to bind not only CARM1/PRMT4 but also PRMT5 and PRMT6 (results not shown), but it was confirmed that the Pontin methylated antibody produced by us only recognized methylation by CARM1. In summary, we have demonstrated through in vivo and in vitro experiments that arginine residues 333 and 339 of pontin are methylated by CARM1.
실시예 2. CARM1에 의한 Pontin의 메틸화의 기전 규명: 포도당 결핍 상태에서 핵 안에서 발생Example 2. Identification of the mechanism of methylation of Pontin by CARM1: Occurs in the nucleus in a glucose-deficient state
CARM1의 단백질이 포도당 결핍 상황에서 증가하는 것이 알려져 있었기에 Pontin과 CARM1의 결합이 결핍 상태에서 증가되는지 확인해보았다. CARM1과 Pontin의 결합이 포도당 결핍 상황에서 증가하는 것을 확인하였다 (도 2a). 우리는 다음으로 이렇게 증가된 결합에 의해 Pontin이 메틸화가 증가되는지 확인해보았다. 우리는 포도당 결핍 상태에서 Pontin의 메틸화가 증가하는 반면, CARM1 특이적인 억제제인 EZM2302 (CAS No. : 1628830-21-6) 와 EPZ025654 (CAS No: 1888328-89-9)를 처리하였을 때 Pontin의 메틸화가 덜 증가하는 것을 확인하였다 (도 2b). CARM1 WT MEF와 CARM1 knockout MEF 세포를 이용하여 보았을 때 CARM1이 존재하는 세포에서만 포도당 결핍 특이적인 Pontin의 메틸화 증가를 관찰하였다 (도 2c). 또한 CARM1 knockout MEF에 CARM1 WT과 효소활성 돌연변이를 발현시켰을 때 CARM1 WT에서만 Pontin의 메틸화가 회복되는 것을 보았다 (도 2d). 이를 통해 Pontin의 메틸화가 CARM1 의존적이라는 것을 다시 한 번 확인하였다. 이전 보고에 따르면 CARM1은 핵 안에서 안정화되기 때문에 우리는 cellular fractionation 실험 (도 2e)과 immunocytochemistry analysis (도 2f)를 이용해 Pontin의 메틸화를 확인해본 결과 포도당 결핍 특이적이게 핵 안에서 증가하는 것을 확인하였다. Since it is known that CARM1 protein is increased in glucose deprivation, it was checked whether the binding of pontin and CARM1 was increased in the deprived condition. It was confirmed that the binding of CARM1 and Pontin was increased in a glucose deprivation situation (FIG. 2a). We next checked whether methylation of Pontin was increased by this increased binding. We found that methylation of Pontin was increased in glucose deprivation condition, whereas methylation of Pontin was increased when CARM1-specific inhibitors EZM2302 (CAS No.: 1628830-21-6) and EPZ025654 (CAS No: 1888328-89-9) were treated. It was confirmed that there was less increase (Fig. 2b). When using CARM1 WT MEF and CARM1 knockout MEF cells, a glucose deprivation-specific increase in Pontin methylation was observed only in CARM1 present cells (FIG. 2c). Also, when CARM1 WT and enzyme-activated mutants were expressed in CARM1 knockout MEFs, methylation of pontin was recovered only in CARM1 WT (Fig. 2d). Through this, it was confirmed once again that methylation of Pontin is CARM1-dependent. According to a previous report, since CARM1 is stabilized in the nucleus, we checked the methylation of Pontin using cellular fractionation experiment (Fig. 2e) and immunocytochemistry analysis (Fig. 2f), and as a result, it was confirmed that glucose depletion-specific increase in the nucleus.
Pontin과 Reptin은 핵 안에서 hetero-dodecamer 복합체를 이루는 것이 알려져 있기에 Pontin의 메틸화가 복합체에 끼치는 영향을 확인해보았다. In vitro 메틸화 실험 이후 in vitro binding assay를 하였을 때 Pontin과 Reptin의 복합체 형성에 Pontin의 메틸화가 영향을 주지 않는 것을 보았다 (결과 나타내지 않음). 또한 아르기닌 돌연변이도 복합체 형성에 영향을 주지 않았다 (결과 나타내지 않음). 종합하면 우리는 Pontin의 메틸화가 CARM1의 Reptin과의 결합에 영향을 주지 않는 것을 확인하였다.Since it is known that Pontin and Reptin form a hetero-dodecamer complex in the nucleus, the effect of Pontin methylation on the complex was checked. After the in vitro methylation experiment, it was found that methylation of Pontin did not affect the formation of a complex between Pontin and Reptin when in vitro binding assay was performed (results not shown). Also, arginine mutation did not affect complex formation (results not shown). Taken together, we confirmed that methylation of Pontin did not affect the binding of CARM1 to Reptin.
실시예 3. Pontin의 메틸화가 포도당 결핍 의존적 자가포식의 증가에 중요함을 규명Example 3. Pontin methylation was found to be important for the increase in glucose deprivation-dependent autophagy
포도당 결핍 상황에서 증가하는 Pontin의 메틸화의 역할을 알아보기 위해 우리는 Flag-tagged Pontin WT과 아르기닌 돌연변이들(R333/339K, R333/339A)을 발현시킨 Pontin f/f MEF를 제작하였다 (도 3a). 이렇게 제작된 세포는 Cre 바이러스를 감염시켜주면 원래 세포의 DNA에서 발현되던 endogenous Pontin이 제거되게 되며, 세포 내에 우리가 발현시킨 Flag-tagged Pontin만 존재하게 된다. 실제로 우리가 제작한 세포를 보면 cre 처리 이후 endogeneous Pontin은 사라지는 것을 확인하였으며, 발현시킨 Pontin WT, RK, RA가 endogenous Pontin의 발현량만큼 발현하는 것을 보았다 (도 3b). 이렇게 제작된 세포를 이용하여 추가 실험들을 진행하였다. Pontin의 메틸화는 Pontin WT MEF에서는 관찰되었지만, RK, RA MEF에서는 관찰되지 않았다 (도 3c). 포도당 결핍 뿐만 아니라 Rapamycin 처리나 아미노산 결핍 상황에서도 Pontin의 메틸화가 증가하였다 (결과 나타내지 않음). 우리는 이전에 Pontin KO MEF에서 자가포식이 망가져 있는 것을 GFP-LC3 puncta assay를 통해 보았었다 (결과 나타내지 않음). Pontin의 메틸화에 따른 자가포식의 변화를 보기 위해 우리가 제작한 세포에서 GFP-LC3 puncta 실험을 진행하였다. 실험 결과 Pontin WT MEF에서는 GFP-LC3 puncta가 포도당 결핍 특이적이게 증가하는 것이 보였으나 Pontin RA MEF에서는 변하지 않는 것을 보았다 (도 3d). Rapamycin 처리나 아미노산 결핍 상황에서도 같은 결과를 얻었다 (결과 나타내지 않음). 또한 western blot을 통해 자가포식의 활성도의 척도 중 하나인 LC3-II의 lipidation 비율을 보았을 때 Pontin WT MEF에서는 LC-3 II가 증가하였으나 Pontin Rk, RA MEF에서는 증가하지 않는 것을 보았다 (도 3e).To investigate the role of increased Pontin methylation in glucose deprivation, we constructed Pontin f/f MEFs expressing Flag-tagged Pontin WT and arginine mutants (R333/339K, R333/339A) (FIG. 3a) . When the cells prepared in this way are infected with Cre virus, the endogenous pontin originally expressed in the DNA of the cell is removed, and only the flag-tagged pontin expressed by us is present in the cell. In fact, looking at the cells produced by us, it was confirmed that endogeneous Pontin disappeared after cre treatment, and the expressed Pontin WT, RK, and RA were found to be expressed as much as the expression level of endogenous Pontin (Fig. 3b). Additional experiments were performed using the cells thus prepared. Pontin methylation was observed in Pontin WT MEFs, but not in RK, RA MEFs (Fig. 3c). Pontin methylation was increased not only in glucose deprivation but also in Rapamycin treatment or amino acid depletion conditions (results not shown). We had previously seen that autophagy was disrupted in Pontin KO MEFs through GFP-LC3 puncta assay (results not shown). In order to see the change of autophagy according to pontin methylation, we conducted the GFP-LC3 puncta experiment in our cells. As a result of the experiment, it was observed that GFP-LC3 puncta increased in a glucose-deficiency-specific manner in Pontin WT MEF, but did not change in Pontin RA MEF (Fig. 3d). Similar results were obtained with rapamycin treatment or amino acid deficiency (results not shown). Also, when looking at the lipidation ratio of LC3-II, which is one of the measures of autophagy activity through western blot, LC-3 II increased in Pontin WT MEF, but not in Pontin Rk and RA MEF (Fig. 3e).
다음으로 우리는 자가포식 flux를 보기 위해 자가포식의 maturation을 방해하는 물질인 Bafilomycin A1이나 Chloroquine을 처리하였다. GFP-LC3 puncta 실험을 통해 Pontin WT MEF의 경우 자가포식의 flux를 막았을 때 GFP-LC3 puncta가 쌓이는 것을 볼 수 있었지만 Pontin RA MEF에서는 쌓이지 않는 것을 확인하였다 (도 3f). Immunoblot 실험을 통해서도 Bafilomycin A1이나 Chloroquine을 처리하였을 때 LC3-II가 WT에서는 증가하지만 RA에서는 많이 증가하지 못하는 것을 보았다 (도 3g). 추가로 자가포식 vacuole을 염색하는 Cyto-ID를 이용하였을 때도 비슷한 결과를 얻었다 (도 3h). 자가포식의 진행 과정 중 어느 단계가 망가져있는지 확인하기 위해 mCherry-GFP-LC3 리포터를 이용한 실험을 진행하였다. 자가포식 과정 중 오토파고좀은 라이소좀과 결합하면서 pH가 낮아지게 되는데 이때 GFP의 초록색 시그널이 약해지게 된다. 반면 mCherry의 빨간색은 pH에 영향을 받지 않기에 오토파고좀의 경우 초록과 빨강이 합쳐진 노란 빛을 띄는 반면, 라이소좀과 결합한 오토라이소좀은 빨간 빛을 띄게 된다. 이를 이용하여 실험하였을 때 Pontin RA MEF에서는 포도당 결핍 상황에서 오토파고좀과 오토라이소좀의 비율이 바뀌지는 않았으며, 전체적인 개수가 WT MEF에 비해 감소하는 것을 보았다 (도 3i). 이 데이터를 통해 우리는 CARM1에 의한 Pontin의 메틸화가 자가포식 자체의 증가에 중요하다는 것을 알 수 있었다.Next, we treated Bafilomycin A1 or Chloroquine, substances that interfere with autophagy maturation, in order to see the autophagy flux. Through the GFP-LC3 puncta experiment, in the case of Pontin WT MEF, it was confirmed that GFP-LC3 puncta was accumulated when the flux of autophagy was blocked, but it was not accumulated in Pontin RA MEF (Fig. 3f). Immunoblot experiments also showed that when Bafilomycin A1 or Chloroquine was treated, LC3-II increased in WT but not significantly increased in RA (FIG. 3g). Additionally, similar results were obtained when Cyto-ID staining autophagic vacuole was used ( FIG. 3h ). An experiment using the mCherry-GFP-LC3 reporter was conducted to determine which stage of the autophagy process is broken. During autophagy, autophagosome binds with lysosome and the pH is lowered. At this time, the green signal of GFP is weakened. On the other hand, since the red color of mCherry is not affected by pH, autophagosomes have a yellow light that is a combination of green and red, whereas autolysosomes combined with lysosomes have red light. When tested using this, in Pontin RA MEF, the ratio of autophagosome and autolysosome did not change in the glucose deprivation situation, and it was seen that the overall number decreased compared to WT MEF (FIG. 3i). From these data, we found that methylation of Pontin by CARM1 is important for the increase of autophagy itself.
Pontin의 메틸화에 의한 자가포식 조절이 보편적인 자가포식 조절기작인지 확인하기 위해 MEF 세포 뿐만 아니라 HepG2, HeLa와 같은 인간 세포에서도 확인해보았다. 포도당 결핍 상황에서 두 세포 모두에서 CARM1의 핵 안에서의 증가와 Pontin의 메틸화의 증가를 볼 수 있었다 (결과는 나타내지 않음). HepG2, HeLa에서도 endogenous Pontin을 knockdown시켜준 뒤 Pontin WT과 RA를 발현시켜주었다. 우리는 이 세포들에서도 Pontin WT 세포에서 Bafilomycin A1 의존적이게 LC3-II가 증가하는 것을 보았으며, Pontin RA 세포에서는 그렇지 못하는 것을 보았다. 우리는 이 데이터를 통해 Pontin의 메틸화에 의한 자가포식 조절이 여러 다양한 다른 세포에서도 공통적이라는 것을 확인했다.To confirm whether autophagy regulation by pontin methylation is a universal autophagy regulation mechanism, we checked not only MEF cells but also human cells such as HepG2 and HeLa. In both cells under glucose deprivation, an increase in the nucleus of CARM1 and an increase in Pontin methylation were observed (results not shown). After knockdown of endogenous Pontin in HepG2 and HeLa, Pontin WT and RA were expressed. We also observed that LC3-II increased in a Bafilomycin A1-dependent manner in Pontin WT cells, but not in Pontin RA cells. From these data, we confirmed that the regulation of autophagy by methylation of pontin is also common in many different cells.
세포에게 있어서 영양분 대사의 항상성 유지는 세포의 건강과 기능에 중요하다. 자가포식은 영양분 부족 상태에서 대표적인 방어기작 중 하나로 영양분이 부족할 때 자가포식을 통해 영양분을 스스로 공급받아 생존에 도움을 준다. Pontin의 메틸화에 따른 세포 생존을 보기 위해 Pontin WT과 RK MEF의 세포 성장율을 보았다. Pontin WT MEF의 경우 포도당 결핍 15-18시간부터 성장이 증가되지 못하며 24시간 이후부터 빠르게 죽기 시작한다. 하지만 Pontin RK MEF의 경우 포도당 결핍 12시간까지는 WT MEF와 큰 차이가 없지만 12시간 이후 빠르게 죽기 시작하였다 (결과 나타내지 않음). 다음으로 우리는 세포의 생존율을 확인해보았다. 성장률과 마찬가지로 Pontin WT MEF의 경우 포도당 결핍 24시간 이후에 세포 생존율이 감소하는 것을 볼 수 있었다. 하지만 Pontin RK MEF의 경우 포도당 결핍 18시간 이후부터 생존율이 감소하였다 (결과 나타내지 않음). 종합하면, 우리의 결과는 Pontin의 메틸화에 따라 포도당 결핍 상황에서 세포의 성장과 생존에 영향을 주며, 지속되는 결핍 상황에서 세포의 생존에 있어서 자가포식의 전사 조절의 중요성을 보여준다.In cells, maintenance of homeostasis of nutrient metabolism is important for cell health and function. Autophagy is one of the most representative defense mechanisms in nutrient deficient conditions. Cell growth rates of Pontin WT and RK MEF were observed to examine cell survival according to Pontin methylation. In the case of Pontin WT MEF, growth does not increase after 15-18 hours of glucose deprivation, and it begins to die rapidly after 24 hours. However, in the case of Pontin RK MEF, there was no significant difference from that of WT MEF until 12 hours of glucose deprivation, but it started to die rapidly after 12 hours (results not shown). Next, we checked the cell viability. Similar to the growth rate, in the case of Pontin WT MEF, cell viability decreased after 24 hours of glucose deprivation. However, in the case of Pontin RK MEF, the survival rate decreased after 18 hours of glucose deprivation (results not shown). Taken together, our results show that Pontin methylation influences cell growth and survival in glucose deprivation conditions, and demonstrates the importance of transcriptional regulation of autophagy in cell survival under persistent deprivation conditions.
실시예 4. 다수의 자가포식과 라이소좀 유전자들이 Pontin의 메틸화에 의해 조절되는 것을 규명Example 4. Finding out that a number of autophagy and lysosomal genes are regulated by Pontin methylation
Pontin의 메틸화가 포도당 결핍 상황에서 핵 안에서 증가하기 때문에 우리는 Pontin의 메틸화가 자가포식 유전자 조절에 중요할 것이라 가정하였다. 어떤 유전자들이 Pontin 메틸화에 의해 조절되는지 확인하기 위해 우리는 Pontin WT, RA MEF에 포도당 결핍 유무에 따라 RNA-sequencing을 진행하였다 (도 4a). PCA를 통해 우리는 Pontin WT, RA MEF가 정상 상황에서는 비슷한 유전자 발현 패턴을 가지지만 포도당 결핍 상황에서는 전체적인 유전자 발현 패턴이 달라지는 것을 확인하였다 (결과 나타내지 않음). 우리는 DEG들을 6개의 유전자군으로 나뉜 히트맵을 얻었다 (도 4b). 유전자군들을 이용하여 Gene ontology와 pathway를 살펴보았을 때 유전자군 1번에서 자가포식과 라이소좀 관련 유전자들이 매우 많이 나왔다 (도 4c). 실제로 자가포식과 라이소좀에 영향을 주는 유전자군을 찾기 위해 우리는 Gene Set Enrichmnet Analysis (GSEA)를 진행하였다. 우리는 결과를 통해 자가포식 조절과 라이소좀 조절, 그리고 파고포어 형성과 관련된 유전자군과 유전자군 1번이 상관 관계가 높은 것을 찾았다 (도 4d).Since pontin methylation is increased in the nucleus under glucose deprivation, we hypothesized that pontin methylation might be important for autophagy gene regulation. To determine which genes are regulated by Pontin methylation, we performed RNA-sequencing in Pontin WT and RA MEFs according to the presence or absence of glucose deficiency (Fig. 4a). Through PCA, we confirmed that Pontin WT and RA MEF had similar gene expression patterns under normal conditions, but changed the overall gene expression pattern under glucose deprivation conditions (results not shown). We obtained a heat map in which DEGs were divided into six gene groups (Fig. 4b). When gene ontology and pathways were examined using gene groups, many genes related to autophagy and lysosome were found in gene group 1 (FIG. 4c). In fact, to find the gene group that affects autophagy and lysosome, we performed Gene Set Enrichmnet Analysis (GSEA). Through the results, we found that the gene group and gene group 1 related to autophagy regulation, lysosomal regulation, and phagopore formation had a high correlation (Fig. 4d).
다음으로 우리는 메틸화된 Pontin이 자가포식 과정에서 어떻게 중요한 역할을 하는지 살펴보았다. 자가포식의 조절을 중심으로 RNA-sequencing 데이터를 분석하였다. 흥미롭게도 자가포식 시작과 파고포어 nucleation과 팽창, 그리고 cargo recruitment/trafficking과 관련된 유전자들이 Pontin RA MEF에서 낮은 것을 확인하였다 (도 4e). Quantitative RT-PCR과 immunoblot을 통해 실제로 이러한 유전자들과 단백질들의 발현이 Pontin 메틸화 의존적이라는 것을 확인했다 (도 4f, g). 또한 HepG2와 HeLa와 같은 다른 세포에서도 같은 결과를 얻었으며, Rapamycin 처리나 아미노산 결핍 상황에서도 비슷한 결과를 얻을 수 있었다. 종합하면 우리는 영양소 결핍 상태에서 증가되는 Pontin의 메틸화가 자가포식과 라이소좀 관련 유전자들을 활성화 시키는데 중요하며, 이런 조절이 자가포식의 초기 단계부터 영향을 줄 수 있다는 사실을 밝혔다.Next, we examined how methylated Pontin plays an important role in the autophagy process. RNA-sequencing data were analyzed with a focus on the regulation of autophagy. Interestingly, it was confirmed that genes related to autophagy initiation, phagopore nucleation and expansion, and cargo recruitment/trafficking were low in Pontin RA MEF (Fig. 4e). Quantitative RT-PCR and immunoblot confirmed that the expression of these genes and proteins was actually Pontin methylation-dependent (Fig. 4f, g). In addition, similar results were obtained in other cells such as HepG2 and HeLa, and similar results were obtained in Rapamycin treatment or amino acid deficiency. Taken together, we found that increased pontin methylation in nutrient-deficient conditions is important for activating autophagy and lysosome-related genes, and that this regulation can affect autophagy from an early stage.
실시예 5. 메틸환된 Pontin과 FOXO3a의 결합 Example 5. Binding of Methyl-Cylated Pontin to FOXO3a
Pontin의 경우 여러 전사인자의 공활성자로 알려져 있기 때문에 우리는 Pontin의 메틸화가 특정 전사인자와의 결합에 영향을 주지 않을까 생각했다. Pontin 메틸화 의존적이게 결합하는 전사인자를 찾기 위해 우리는 RNA-seq에서 얻은 타겟 유전자를 이용하여 전사인자 스크리닝을 하였다. 이를 통해 우리는 자가포식과 관련 깊은 몇 개의 후보 전사인자를 얻을 수 있었다 (도 5a). 추가로 우리는 Pontin 메틸화 항체를 이용하여 ChIP-seq을 진행하였다. Pontin의 메틸화가 결합하는 크로마틴들의 모티프 분석을 통해 우리는 FOXO3a 결합 부분이 높게 나오는 것을 확인하였다 (도 5b). 우리는 후보 전사인자들이 실제로 Pontin과 결합하는지 확인하기 위해 포도당 결핍 상황에서 결합 여부를 확인해보았다. 실험 결과 Pontin이 FOXO3a와는 결합하지만 다른 후보 전사인자들과는 결합하지 않는 것을 확인했다 (도 5c). Pontin과 FOXO3a의 결합은 메틸화 의존적이었으며, Pontin WT은 결합할 수 있지만 Pontin RA, RK는 결합하지 못하는 것을 확인했다 (도 5d). Pontin과 FOXO3a의 결합에 메틸화가 중요한지 더 확실히 하기 위해 우리는 CARM1에 따른 두 단백질의 결합을 보았다. CARM1 KO MEF에서 Pontin과 FOXO3a가 결합하지 못하는 것을 보았으며 (도 5e, f), CARM1 특이적인 inhibitor를 처리하였을 때 Pontin과 FOXO3a의 결합이 감소하는 것을 확인하였다 (도 5g). 이를 통해 Pontin과 FOXO3a의 결합에 있어서 CARM1에 의한 메틸화가 중요하다는 것을 알았다.Since Pontin is known as a coactivator of several transcription factors, we thought that methylation of Pontin might affect the binding to specific transcription factors. To find transcription factors that bind pontin methylation-dependently, we conducted transcription factor screening using target genes obtained from RNA-seq. Through this, we were able to obtain several candidate transcription factors closely related to autophagy (Fig. 5a). In addition, we performed ChIP-seq using Pontin methylated antibody. Through motif analysis of chromatin to which pontin methylation binds, we confirmed that the FOXO3a binding portion was high (Fig. 5b). We checked whether candidate transcription factors actually bind to pontin under glucose deprivation. As a result of the experiment, it was confirmed that Pontin binds to FOXO3a but does not bind to other candidate transcription factors (FIG. 5c). The binding between Pontin and FOXO3a was methylation-dependent, and it was confirmed that Pontin WT could bind but Pontin RA and RK did not ( FIG. 5d ). To further clarify whether methylation is important for the binding of Pontin to FOXO3a, we looked at the binding of the two proteins following CARM1. It was observed that Pontin and FOXO3a could not bind in CARM1 KO MEF (FIG. 5e, f), and it was confirmed that the binding of Pontin and FOXO3a was decreased when CARM1-specific inhibitor was treated (FIG. 5g). Through this, it was found that methylation by CARM1 is important in the binding of Pontin and FOXO3a.
다음으로 우리는 Pontin과 FOXO3a의 직접적인 결합을 보기 위해 in vitro GST pulldown 실험을 진행하였다. In vitro 메틸화를 통해 Pontin을 메틸화 시킨 뒤, 우리는 GST-FOXO3a와 결합을 보았다. 실험 결과 FOXO3a와 메틸화된 Pontin은 결합하는 반면, 메틸화 시키지 않은 Pontin은 결합하지 못하는 것을 보았다 (도 5h). 추가로 Immunodot blot 실험을 통해 FOXO3a가 메틸화된 Pontin 펩타이드와는 결합하는 반면, 메틸화되지 않은 Pontin 펩타이드에는 결합하지 못하는 것을 확인했다 (도 5i). FOXO3a의 어느 위치에 Pontin이 결합하는지 알아보기 위해 우리는 FOXO3a의 여러 돌연변이를 이용하여 Pontin과의 결합을 보았다. 우리는 FOXO3a의 418-673 아미노산 부분에 Pontin이 결합하는 것을 확인하였으며, 추가 실험을 통해 FOXO3a의 CR3 domain인 610-650 아미노산 부분에서 Pontin과의 결합이 일어나는 것을 찾았다.Next, we performed an in vitro GST pulldown experiment to examine the direct binding of Pontin to FOXO3a. After methylation of Pontin via in vitro methylation, we observed binding to GST-FOXO3a. As a result of the experiment, it was found that FOXO3a and methylated pontin were bound, whereas unmethylated pontin did not (Fig. 5h). In addition, it was confirmed through Immunodot blot experiment that FOXO3a bound to methylated Pontin peptide, but failed to bind to unmethylated Pontin peptide (FIG. 5i). To find out where Pontin binds to FOXO3a, we used several mutations of FOXO3a to see the binding to Pontin. We confirmed that Pontin binds to amino acids 418-673 of FOXO3a, and through additional experiments, we found that binding to Pontin occurs in amino acids 610-650, the CR3 domain of FOXO3a.
아르기닌 메틸화를 인지할 수 있는 아르기닌 메틸화 인지 도메인의 경우 aromatic 잔기에 의해 메틸화가 인지된다는 것이 보고되어있다. 우리는 FOXO3a의 메틸화 인지 잔기를 찾기 위해 첫 번째로 CR3 domain에 존재하는 aromatic 잔기들을 하나씩 돌연변이 시켰다. 실험 결과 FOXO3a의 F640과 F642를 류신으로 각각 돌연변이 시켰을 때 Pontin과의 결합이 감소하는 것을 확인하였으며, 두 페닐알라닌을 모두 류신으로 치환하였을 때 Pontin과의 결합이 완전히 사라지는 것을 보았다 (도 5j). 또한 in vitro GST pulldown을 통해 FOXO3a F640/642L 돌연변이가 메틸화된 Pontin과 결합하지 못하는 것을 확인했다 (도 5k). 이를 통해 우리는 FOXO3a의 F640과 F642가 Pontin의 메틸화 인지에 중요할 것이라 생각했다.It has been reported that methylation is recognized by aromatic residues in the case of an arginine methylation recognition domain capable of recognizing arginine methylation. To find the methylation recognition residues of FOXO3a, we first mutated aromatic residues in the CR3 domain one by one. As a result of the experiment, it was confirmed that the binding to Pontin was reduced when F640 and F642 of FOXO3a were mutated with leucine, respectively, and when both phenylalanine were substituted with leucine, the binding to Pontin completely disappeared (Fig. 5j). In addition, it was confirmed that the FOXO3a F640/642L mutant could not bind to methylated Pontin through in vitro GST pulldown (FIG. 5k). From this, we thought that F640 and F642 of FOXO3a might be important for methylation recognition of Pontin.
다른 한편으로 우리는 FOXO3a와 Pontin의 구조를 통해 메틸화 인지 구조를 모델링을 통해 예측해보았다. FOXO3a는 hydrophobic 잔기인 M624, I627, I628이 Pontin의 메틸화를 인지할 수 있을 것으로 예상되었다. 이를 확인해보기 위해 우리는 위의 세 잔기를 모두 돌연변이 시킨 FOXO3a 3A 돌연변이와 WT을 이용하여 Pontin과의 결합을 보았다. GST pulldown 실험을 통해 우리는 FOXO3a 3A 돌연변이의 경우 Pontin과 결합하지 못하는 것을 보았으며, 세포 실험을 통해서도 같은 결과를 확인하였다. 이를 통해 우리는 FOXO3a의 M624, I627, I628 또한 Pontin의 메틸화 인지에 중요할 것을 생각했다.On the other hand, we predicted the methylation recognition structure through modeling through the structures of FOXO3a and Pontin. FOXO3a was expected to recognize the methylation of pontin at hydrophobic residues M624, I627, and I628. To confirm this, we observed the binding of FOXO3a 3A mutant with mutated all three residues above to Pontin using WT. Through the GST pulldown experiment, we saw that the FOXO3a 3A mutant could not bind to Pontin, and the same result was confirmed through the cell experiment. Through this, we thought that M624, I627, and I628 of FOXO3a may also be important for methylation recognition of Pontin.
실시예 6. 메틸화된 Pontin과 Tip60가 FOXO3a 타겟 유전자들을 H4 acetylation을 통해 활성화 시킴을 규명Example 6. Investigation that methylated Pontin and Tip60 activate FOXO3a target genes through H4 acetylation
메틸화된 Pontin이 FOXO3a의 공활성체로 작동하는지 확인해보기 위해 FOXO 결합 위치를 가지는 luciferase reporter를 이용하여 luciferase 실험을 진행하였다. Pontin WT에서는 포도당 결핍 상황에서 luciferase 활성이 증가하였지만 Pontin RK, RA 돌연변이에서는 증가하지 않았다 (도 6a). 또한 FOXO3a F640/642L이나 3A 돌연변이에서는 Pontin에 의한 추가적인 전사 활성의 증가가 안보였다 (도 6b). 메틸화된 Pontin이 어떻게 FOXO3a 타겟 유전자들을 조절하는지 확인하기 위해서 Pontin 메틸화 ChIP-seq 데이터를 살펴보았다. 우리는 메틸화된 Pontin이 전사시작 주변부 뿐만 아니라 H3K4me1와 H3K27Ac이 높은 인핸서 부분에도 결합 할 수 있다는 것을 보았다 (도 6c). 흥미롭게도 메틸화 Pontin peak 중 FOXO3a 모티프를 가지고 있는 것 중 36%가 TSS로부터 멀리 떨어져 있었다. 대표적인 자가포식 유전자인 Map1lc3b의 유전자 근처에서 Pontin 메틸화 ChIP-seq peak을 살펴보았을 때 전사시작부위 뿐만 아니라 먼 부분에서도 결합하는 것을 확인하였다 (도 6d). 우리는 ChIP을 통해 포도당 결핍 상황에서 Pontin이 유전자 전사 시작부분 근처(-1.2kb) 뿐만 아니라 멀리있는 부분(-11.3kb, -5kb)까지 결합 할 수 있다는 것을 확인하였다 (결과 나타내지 않음). 하지만 Pontin RA MEF에서는 Pontin의 recruitment가 망가지는 것을 보았다 (결과 나타내지 않음). 이런 데이터를 통해 우리는 Pontin의 메틸화를 통한 FOXO3a와의 결합을 통해 FOXO3a의 공활성체로서 작동할 수 있음을 보여주었다.To check whether methylated Pontin acts as a co-activator of FOXO3a, a luciferase experiment was performed using a luciferase reporter having a FOXO-binding site. In Pontin WT, luciferase activity was increased in the glucose deprivation situation, but not in Pontin RK and RA mutants (Fig. 6a). In addition, FOXO3a F640/642L or 3A mutants did not show an additional increase in transcriptional activity by Pontin (FIG. 6b). To confirm how methylated Pontin regulates FOXO3a target genes, we looked at Pontin methylated ChIP-seq data. We have seen that methylated Pontin can bind not only to the transcriptional initiation periphery but also to H3K4me1 and H3K27Ac-high enhancer regions (Fig. 6c). Interestingly, 36% of the methylated Pontin peaks with the FOXO3a motif were far from the TSS. When the Pontin methylation ChIP-seq peak was observed near the gene of Map1lc3b, a representative autophagy gene, binding was confirmed not only at the transcription start site but also at a distant site (FIG. 6d). We confirmed through ChIP that Pontin was able to bind not only near the beginning of gene transcription (-1.2 kb) but also far (-11.3 kb, -5 kb) from the beginning of gene transcription in the context of glucose deprivation (results not shown). However, in Pontin RA MEF, Pontin recruitment was impaired (results not shown). From these data, we showed that Pontin can act as a co-activator of FOXO3a through binding to FOXO3a through methylation.
우리는 다음으로 Pontin의 결합이 어떻게 FOXO3a 타겟 유전자들의 전사 활성을 올려줄 수 있는지 살펴보았다. Pontin은 잘 알려진 Tip60의 복합체 중 하나의 요소로 Tip60 복합체의 경우 히스톤 아세틸화를 통해 전사 활성에 중요한 역할을 한다. 이를 확인해보기 위해서 우리는 FOXO3a 결합 위치에 Pontin과 함께 Tip60와 H4 아세틸화가 변하는지 확인해보았다. ChIP 실험을 통해 우리는 Tip60와 함께 H4 아세틸화가 포도당 결핍 특이적이게 증가하는 것을 확인했다 (도 6e). 흥미롭게도 Tip60와 H4 아세틸화가 CARM1 KO MEF에서는 recruit되지 않는 것을 통해 Pontin의 메틸화 상태 의존적이라는 것을 알 수 있었다 (도 6e). FOXO3a와 Tip60의 결합이 Pontin이 존재할 때 증가하는 것을 확인할 수 있었지만 (도 6f), Pontin과 Tip60의 결합은 메틸화 여부와 관련이 없었다 (도 6g). 종합하면 우리는 메틸화된 Pontin이 Tip60를 FOXO3a 위치에 데려와서 자가포식 관련 유전자들의 coactivator로서 작동할 수 있음을 밝혔다. Next, we examined how the binding of Pontin could enhance the transcriptional activity of FOXO3a target genes. Pontin is one of the well-known Tip60 complexes, and in the case of the Tip60 complex, it plays an important role in transcriptional activity through histone acetylation. To confirm this, we checked whether Tip60 and H4 acetylation change with Pontin at the FOXO3a binding site. Through ChIP experiments, we confirmed that H4 acetylation was increased in a glucose deprivation-specific manner with Tip60 (Fig. 6e). Interestingly, it was found that Tip60 and H4 acetylation were not recruited in CARM1 KO MEF, indicating that Pontin methylation status was dependent ( FIG. 6e ). Although it was confirmed that the binding of FOXO3a and Tip60 was increased in the presence of Pontin (Fig. 6f), the binding of Pontin and Tip60 was not related to methylation (Fig. 6g). Taken together, we found that methylated Pontin can act as a coactivator of autophagy-related genes by bringing Tip60 to the FOXO3a site.
실시예 7. Pontin의 메틸화 의존적인 자가포식 유전자들의 활성에 Tip60가 중요함을 규명Example 7. Investigation of the importance of Tip60 in the activity of methylation-dependent autophagy genes of Pontin
자가포식 유전자들의 조절에서 Tip60의 중요성을 보기 위해 shRNA를 이용한 Tip60 knockdown MEF를 제작하였다. Bafilomycin A1을 처리하여 자가포식 flux를 살펴보았을 때 Tip60가 없으면 LC3-II의 증가가 잘 관찰되지 않았다 (도 7a). 이를 통해 Tip60가 자가포식의 증가에도 관여되어 있음을 확인했다. 다음으로 qRT-PCR을 통해 앞에서 살펴본 Pontin 메틸화 의존적인 타겟 유전자들이 Tip60가 없을 때 어떻게 변하는지 보았다. 포도당 결핍 특이적이게 전사가 증가되는 Map1lc3b, Sirt1, Bnip3, Ctns는 WT에서 잘 올라가지만 Tip60가 없을 때 증가하지 못하는 것을 보았다 (도 7b). 반면, Pontin의 메틸화에 의존적이지 않는 자가포식 유전자의 경우 Tip60와도 관련이 없었다 (도 7b). 이를 통해 우리는 Pontin의 메틸화와 Tip60가 연관되어 있음을 다시 한번 확인하였다. 다음으로 우리는 ChIP실험을 통해 Tip60가 없을 때 FOXO3a와 Pontin, 그리고 H4 아세틸화가 어떻게 변하는지 확인하였다. Tip60가 없을 때 FOXO3a와 메틸화된 Pontin은 잘 recruit 되지만 H4 아세틸의 증가가 망가지는 것을 ChIP 실험을 통해 확인하였다 (도 7c).To examine the importance of Tip60 in the regulation of autophagy genes, a Tip60 knockdown MEF using shRNA was constructed. When the autophagy flux was examined by treatment with Bafilomycin A1, no increase in LC3-II was observed without Tip60 (Fig. 7a). Through this, it was confirmed that Tip60 is also involved in the increase of autophagy. Next, through qRT-PCR, we looked at how the Pontin methylation-dependent target genes examined above change in the absence of Tip60. Map1lc3b, Sirt1, Bnip3, and Ctns, which are glucose-deficient-specifically increased transcription, were well elevated in WT, but did not increase in the absence of Tip60 (Fig. 7b). On the other hand, autophagy genes that were not dependent on Pontin methylation were not related to Tip60 (Fig. 7b). Through this, we confirmed once again that the methylation of Pontin and Tip60 are related. Next, we confirmed how FOXO3a, Pontin, and H4 acetylation were changed in the absence of Tip60 through ChIP experiments. In the absence of Tip60, FOXO3a and methylated Pontin were recruited well, but it was confirmed through ChIP experiment that the increase in H4 acetyl was disrupted (FIG. 7c).
Tip60를 knockdown하거나 Pontin의 메틸화를 망가트렸을 때 FOXO 타겟 유전자의 증가가 비슷하게 망가지는 것을 볼 수 있었으며, 둘을 동시에 망가트렸을 때 추가적인 감소가 보이지는 않았다 (도 7d). 이를 통해 우리는 Tip60와 동반하는 H4 아세틸화를 통한 FOXO 타겟 유전자 조절이 Pontin의 메틸화를 통해 일어나는 것을 알 수 있었다. 또한 immunoblot을 통한 LC3-II의 증가 또한 비슷한 결과를 보였다 (도 7e). 종합하면, Pontin의 메틸화가 Tip60의 FOXO3a response element 결합에 중요하며 이에 따른 H4 아세틸화가 자가포식 유전자 활성에 중요하다는 것을 밝혔다 (도 7f).When Tip60 knockdown or methylation of Pontin was disrupted, the increase in the FOXO target gene was similarly disrupted, and when both were disrupted at the same time, no further decrease was seen ( FIG. 7d ). Through this, we found that FOXO target gene regulation through Tip60 and accompanying H4 acetylation occurs through methylation of Pontin. In addition, the increase of LC3-II through immunoblot also showed similar results (Fig. 7e). Taken together, it was revealed that Pontin methylation is important for the binding of Tip60 to the FOXO3a response element, and thus H4 acetylation is important for autophagy gene activity (FIG. 7f).
실시예 8. CARM1-Pontin-FOXO3a 축이 자가포식 유전자들의 인핸서 활성에 역할을 함을 규명Example 8. Clarification of the role of CARM1-Pontin-FOXO3a axis in enhancer activity of autophagy genes
이전 연구에서 우리는 AMPK에 의한 FOXO3a 인산화가 Skp2 억제에 중요하다는 것을 밝혔다. Pontin의 메틸화에 의해 혹시 Skp2가 조절될 수 있는지 확인해보았지만 Skp2 mRNA의 감소에 영향이 없었으며, Skp2 promoter에도 Pontin과 H4 아세틸화가 recruit되지 않았다. 또한 Pontin의 메틸화가 망가져도 CARM1의 안정화에는 영향이 없음을 보았다. 우리는 이전 연구에서 증가된 CARM1이 TFEB과 결합하여 H3R17 메틸화를 증가시키는 것을 밝혔다. Map1lc3b의 경우 두 전사인자 TFEB과 FOXO3a에 의해 조절되기 때문에 CARM1-Pontin-FOXO3a 축과 TFEB-CARM1 축이 어떻게 작동하는지를 살펴보았다.In a previous study, we revealed that FOXO3a phosphorylation by AMPK is important for Skp2 inhibition. It was checked whether Skp2 could be regulated by Pontin methylation, but it had no effect on the decrease in Skp2 mRNA, and Pontin and H4 acetylation were not recruited to the Skp2 promoter either. In addition, it was observed that even if Pontin methylation was disrupted, the stabilization of CARM1 was not affected. We found in a previous study that increased CARM1 binds to TFEB and increases H3R17 methylation. Since Map1lc3b is regulated by two transcription factors, TFEB and FOXO3a, we examined how the CARM1-Pontin-FOXO3a axis and the TFEB-CARM1 axis work.
CARM1은 FOXO3a 결합 부분과 TFEB 결합 부분 모두에 관여하기 때문에 우리는 CARM1이 두 부분에 모두 결합할 수 있는지 확인해보았다. ChIP을 통해 보았을 때 TFEB 결합 부위인 CLEAR 모티프에서는 CARM1이 결합하고 H3R17me2의 증가가 보였지만, FOXO3a 결합 부위에서는 CARM1의 결합이 보이지 않았다. 우리는 FOXO 결합 부위에서는 메틸화된 Pontin을 비롯한 Tip60와 H4 아세틸화가 잘 증가하는 것을 확인하였다. 하지만 Pontin RK, RA 돌연변이에서는 Pontin을 비롯한 Tip60, H4 아세틸화가 오지 않았다 (도 8a). 반면, TFEB 결합 부위에서는 FOXO3a, Pontin, Tip60 모두 recruit되지 않았으며 Pontin의 메틸화 유무와 상관없이 H3R17me2이 증가되어 있는 것을 확인하였다 (도 8b). 종합하면 CARM1-Pontin-FOXO3a 축과 TFEB-CARM1 축이 동일한 유전자를 조절하지만 각각의 결합부위에서 독립적이게 작동함을 확인하였다.Since CARM1 is involved in both the FOXO3a binding site and the TFEB binding site, we checked whether CARM1 can bind to both sites. When viewed through ChIP, CARM1 was bound and H3R17me2 increased in the CLEAR motif, which is the TFEB binding site, but CARM1 binding was not seen in the FOXO3a binding site. We confirmed that Tip60 and H4 acetylation, including methylated Pontin, were well increased at the FOXO binding site. However, acetylation of Tip60 and H4 including Pontin did not occur in Pontin RK and RA mutants (FIG. 8a). On the other hand, it was confirmed that FOXO3a, Pontin, and Tip60 were not recruited at the TFEB binding site, and H3R17me2 was increased regardless of the presence or absence of methylation of Pontin (Fig. 8b). In summary, it was confirmed that the CARM1-Pontin-FOXO3a axis and the TFEB-CARM1 axis regulate the same gene, but operate independently at each binding site.
FOXO3a의 경우 인핸서에서도 작동할 수 있는 것이 알려져 있었기에 Pontin-Tip60가 결합하는 부분이 인핸서로서 작동할 수 있는지 확인해보았다. Map1lc3b의 -11.3kb와 -5kb에 결합하는 Pontin-Tip60가 Map1lc3b의 인핸서로 작동할 수 있는지 확인해보기 위해 3C 실험을 진행하였다. 3C 실험을 통해 Map1lc3b의 -11.3kb와 -5kb 부분이 프로모터 부분과 근접한다는 것을 확인하였다 (도 8c). 우리는 다음으로 인핸서의 활성 여부를 확인하기 위해 eRNA를 qRT-PCR을 통해 확인해보았다 (도 8d). Pontin WT MEF에서는 포도당 결핍 특이적이게 eRNA가 증가하는 것을 확인하였지만 Pontin RA MEF에서는 증가하지 못하는 것을 관찰하였다. 종합하면 CARM1-Pontin-FOXO3a 축이 H4 아세틸화를 통해 인핸서에서 작동할 수 있음을 밝혔다.In the case of FOXO3a, it was known that it can also act as an enhancer, so we checked whether the part where Pontin-Tip60 binds can act as an enhancer. A 3C experiment was conducted to check whether Pontin-Tip60, which binds to -11.3kb and -5kb of Map1lc3b, can act as an enhancer for Map1lc3b. Through the 3C experiment, it was confirmed that the -11.3kb and -5kb regions of Map1lc3b were close to the promoter region ( FIG. 8c ). Next, we checked the eRNA through qRT-PCR to confirm the activity of the enhancer (Fig. 8d). In Pontin WT MEF, it was observed that eRNA was increased in a glucose deprivation-specific manner, but not increased in Pontin RA MEF. Taken together, we revealed that the CARM1-Pontin-FOXO3a axis could act in enhancers via H4 acetylation.
이상에서 본원의 예시적인 실시예에 대하여 상세하게 설명하였지만 본원의 권리범위는 이에 한정되는 것은 아니고 다음의 청구범위에서 정의하고 있는 본원의 기본 개념을 이용한 당업자의 여러 변형 및 개량 형태 또한 본원의 권리범위에 속하는 것이다.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
본 발명에서 사용되는 모든 기술용어는, 달리 정의되지 않는 이상, 본 발명의 관련 분야에서 통상의 당업자가 일반적으로 이해하는 바와 같은 의미로 사용된다. 본 명세서에 참고문헌으로 기재되는 모든 간행물의 내용은 본 발명에 도입된다. 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.

Claims (5)

  1. 메틸전달효소인 CARM1 (Coactivator Associated argine methyltransferase 1)에 의한 PONTIN 단백질의 메틸화 조절을 통해 자가포식을 조절하는 물질을 스크리닝하는 방법으로, 상기 방법은A method for screening a substance that regulates autophagy through methylation control of PONTIN protein by CARM1 (Coactivator Associated argine methyltransferase 1), a methyltransferase, the method comprising:
    포도당이 결핍된 상태에서 CARM1 및 상기 메틸전달효소 CARM1의 기질로서 PONTIN 단백질을 발현하는 세포를 배양하는 제 1 단계; A first step of culturing cells expressing CARM1 and PONTIN protein as a substrate for the methyltransferase CARM1 in a glucose-deficient state;
    상기 세포에 메틸전달효소 CARM1의 상기 PONTIN에 대한 메틸화 활성을 억제할 것으로 기대되는 시험물질을 처리하는 제 2 단계; a second step of treating the cells with a test substance expected to inhibit the methylation activity of the methyltransferase CARM1 for the PONTIN;
    상기 PONTIN 단백질을 상기 세포로부터 분리하는 제 3 단계: A third step of isolating the PONTIN protein from the cells:
    상기 분리된 PONTIN 단백질의 서열번호 1을 기준으로 333번 및 339번째 잔기에서의 메틸화정도를 측정하는 제 4 단계; 및a fourth step of measuring the degree of methylation at residues 333 and 339 based on SEQ ID NO: 1 of the isolated PONTIN protein; and
    상기 측정결과 시험물질로 처리되지 않은 대조군과 비교하여, 시험물질로 처리된 경우 상기 메틸화가 증가 또는 감소한 경우, 이를 자가포식을 조절하는 후보물질로 선별하는 제 5 단계를 포함하며, 상기 메틸화 증가는 자가포식 증가, 상기 메틸화 감소는 자가포식 억제를 나타내는 것인, CARM1 및 PONTIN에 의해 조절되는 자가포식 조절제 스크리닝 방법.When the methylation is increased or decreased when treated with the test substance compared to the control not treated with the test substance as a result of the measurement, a fifth step of selecting this as a candidate substance for regulating autophagy is included, wherein the increase in methylation is An autophagy modulator screening method regulated by CARM1 and PONTIN, wherein the increase in autophagy and the decrease in methylation indicate autophagy inhibition.
  2. 제 1 항에 있어서, The method of claim 1,
    상기 제 1 단계는 FOXO3a 단백질을 추가로 발현하며, The first step further expresses the FOXO3a protein,
    상기 제 4 단계 대신에, 또는 이에 추가하여, 상기 PONTIN 및 FOXO3a 단백질의 결합을 측정하는 단계를 포함하며, 상기 측정결과 상기 단백질 간의 결합 증가는 자가포식 증가, 결합 감소는 자가포식 억제를 나타내는 것인, CARM1 및 PONTIN에 의해 조절되는 자가포식 조절제 스크리닝 방법. Instead of or in addition to the fourth step, comprising the step of measuring the binding of the PONTIN and FOXO3a protein, and as a result of the measurement, an increase in binding between the proteins indicates an increase in autophagy, and a decrease in binding indicates autophagy inhibition. , A screening method for modulators of autophagy regulated by CARM1 and PONTIN.
  3. 제 2 항에 있어서, 3. The method of claim 2,
    상기 PONTIN은 서열번호 2를 기준으로 상기 FOXO3a 단백질의 624, 627, 628, 640 및 642 잔기를 통해 결합하는 것인, CARM1 및 PONTIN에 의해 조절되는 자가포식 조절제 스크리닝 방법. The PONTIN will bind through residues 624, 627, 628, 640 and 642 of the FOXO3a protein based on SEQ ID NO: 2, CARM1 and PONTIN-controlled autophagy modulator screening method.
  4. 제 1 항 내지 제 3 항 중 어느 한 항에 있어서, 4. The method according to any one of claims 1 to 3,
    상기 제 4 단계에 추가하여, 상기 세포에서 분리된 히스톤 4(H4) 단백질의 아세틸화 여부를 측정하는 단계를 포함하며, 상기 측정결과 상기 H4 단백질의 아세틸화 증가는 자가포식 증가, 결합 감소는 자가포식 억제를 나타내는 것인, CARM1 및 PONTIN에 의해 조절되는 자가포식 조절제 스크리닝 방법. In addition to the fourth step, it comprises the step of measuring whether the histone 4 (H4) protein isolated from the cell is acetylated, and as a result of the measurement, an increase in the acetylation of the H4 protein is an increase in autophagy, A method for screening an autophagy modulator regulated by CARM1 and PONTIN, which exhibits phagocytosis inhibition.
  5. 제 4 항에 있어서, 5. The method of claim 4,
    상기 히스톤 4의 아세틸화는 서열번호 3의 서열을 기준으로 6, 9, 13 및 17번째 라이신 잔기에서 발생하는 것인, CARM1 및 PONTIN에 의해 조절되는 자가포식 조절제 스크리닝 방법. The autophagy modulator screening method regulated by CARM1 and PONTIN, wherein the acetylation of histone 4 occurs at the 6th, 9th, 13th and 17th lysine residues based on the sequence of SEQ ID NO: 3.
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