WO2015050383A1 - Procédé pour la régulation de l'autophagie médiée par le domaine de p62 zz et son utilisation - Google Patents

Procédé pour la régulation de l'autophagie médiée par le domaine de p62 zz et son utilisation Download PDF

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WO2015050383A1
WO2015050383A1 PCT/KR2014/009279 KR2014009279W WO2015050383A1 WO 2015050383 A1 WO2015050383 A1 WO 2015050383A1 KR 2014009279 W KR2014009279 W KR 2014009279W WO 2015050383 A1 WO2015050383 A1 WO 2015050383A1
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disease
end rule
protein
autophagy
region
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Korean (ko)
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권용태
장준민
한동훈
김보연
차현주
유지은
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서울대학교산학협력단
한국생명공학연구원
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/30—Drugs for disorders of the nervous system for treating abuse or dependence
    • A61P25/32—Alcohol-abuse

Definitions

  • the present application is in the field of disease therapeutics due to abnormal accumulation of denatured proteins through autophagy control.
  • autophagy is activated in cancer cells (Ding et al., (2009), Mol. Cancer Ther., 8 (7), 2036-2045), and autophagy inhibitors may act as anticancer agents (Maiuri et al. , (2007) Nat. Rev. Cell Biol. 8, 741-752).
  • autophagy is important for the maintenance of cellular homeostasis through intracellular fluctuations of damaged proteins and organelles.
  • autophagy is degraded, accumulation of denatured protein is induced. This results in neurodegenerative diseases ( Komatsu et al., (2006), Nature, 441, 880-884).
  • substances that enhance cell autophagy can act as therapeutics for the prevention or treatment of neurodegenerative diseases.
  • 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)).
  • US Patent Publication No. 2010-0233730 relates to the control of autophagy for the treatment, by applying a metabolite stress to the cells after treatment of the test substance through the observation of intracellular changes of autophagosome, It relates to a method of screening.
  • European Patent Publication No. 2446886 relates to the treatment of collagen VI deficiency muscle atrophy with autophagy modulators and discloses compositions using Beclin-1, BH3 mimetics and the like.
  • the present application seeks to provide a method and use of autophagy control to target new substances involved in autophagy via the N-end rule pathway.
  • the present disclosure includes contacting a cell with an N-end rule ligand that interacts with a p62 protein, a ZZ region of the p62 protein or a residue from 128 to 163 based on the sequence of SEQ ID NO: 1 of the ZZ region. It provides a method of autophagy control.
  • the method according to the present disclosure may be performed in vitro in one embodiment, and relates to a method for controlling autophagy of cells in vitro using technical features according to the present disclosure.
  • N-end rule ligands used in the methods herein have N-end rule N-terminus of type 1 or type 2, and such N-end rule ligands are Arg-Ala or Xaa-Ile-Phe-Ser-Thr.
  • Tyr-Ile-Phe-Ser-Thr in this sequence is an unstabilized N-terminal sequence derived from sndvis virus nsP4 RNA polymerase.
  • the interaction of the ZZ region with the ligand leads to oligomerization of p62.
  • the present invention also provides a pharmaceutical composition for treating neurodegenerative disease, comprising an N-end rule ligand that interacts with the ZZ region of the p62 protein.
  • N-end rule ligands included in the compositions herein have an N-end rule N-terminus of type 1 or type 2, and such N-end rule ligands are Arg-Ala or Xaa-Ile-Phe-Ser-Thr.
  • the pharmaceutical composition according to the present invention can be used for the treatment of diseases related to protein degeneration (proteinopahty) neurodegenerative diseases, for example, Adrenal Leukodystrophy, alcoholism, Alexander's disease, Alper's disease ( Alper's disease, Alzheimer's disease, muscular dystrophy, ataxia telangiectasia, Batten disease, bovine spongiform encephalopathy, Canervan disease, cerebral palsy, Cockayne syndrome, corticobasal degeneration, Creutzfeldt-Jakob disease, familial fatal insomnia, frontotemporal lobar degeneration, Huntington's disease
  • proteins degeneration proteinopahty neurodegenerative diseases
  • HIV-related dementia HIV-related dementia
  • Kennedy's disease Krabbe's disease
  • Lewy body dementia neuroborreliosis
  • Macadodo-Joseph disease Multiple system atrophy, multiple sclerosis, seizure sleep
  • Neuropsy Niemann Pick disease
  • Parkinson's disease Parkinson's disease
  • Pelizaeus-Merzbacher disease Pick's disease
  • primary lateral sclerosis Subacute union degeneration of the spinal cord of prion disease, progressive supranuclear palsy, Refsum's disease, Sandhoff disease, Schilder's disease, and toxic anemia secondary combined degeneration of spinal cord secondary to pernicious anaemia, Spielmeyer-Vogt-Sjogren-Batten disease, spinocerebellar ataxia, spinal muscular atrophy, steel -Including but not limited to, Richard-Richardson-Olszewski disease, Tabes dorsalis, and toxic encephalopathy.
  • Diseases associated with protein denaturation in one embodiment according to the present invention include Alzheimer's disease, Parkinson's disease, Lewy body dementia, muscular dystrophy (ALS), Huntington's disease, spinal cerebellar ataxia or spinobulbar musclular atrophy .
  • the invention in another aspect relates to a method for screening autophagy modulators that modulate autophagy mediated by p62, the method comprising: a) poly of a p62 protein, a ZZ region of the p62 protein or a 128 to 163 residue of the ZZ region Contacting a sample comprising a peptide with a test substance; And b) detecting an interaction in the ZZ region of the test substance with the protein or peptide, wherein when contacted with the test substance, autophagy fluctuates in comparison with a control group not in contact with the test substance, For example, when increased or decreased, selecting the test substance as an autophagy control candidate.
  • Test substances that can be used for screening according to the present application are those having N-end rule ligands such as N-end rule N-terminus of type 1 or type 2, for example Arg-Ala or Xaa-Ile-Phe-Ser A peptide having the sequence -Thr-Ile-Glu-Gly-Arg-Thr-Tyr-Lys, wherein X has a peptide sequence which is any of Arg, His, Lys, Phe, Trp or Tyr.
  • N-end rule ligands such as N-end rule N-terminus of type 1 or type 2, for example Arg-Ala or Xaa-Ile-Phe-Ser A peptide having the sequence -Thr-Ile-Glu-Gly-Arg-Thr-Tyr-Lys, wherein X has a peptide sequence which is any of Arg, His, Lys, Phe, Trp or Tyr.
  • the sample is provided in the form of a cell, for example, a cell derived from a mammal, for example, HEK21, Mouse embryonic fibroblast, HeLa, Hep3B, or PC3 cells. It is not limited to this.
  • the method according to the present invention also provides a method for screening autophagy modulators, wherein the interaction of the ZZ region with the ligand in place of or in addition to step b) is detected by oligomerization of p62.
  • Autophagy control method, autophagy regulators and autophagy agents comprising the step of contacting the p62 protein according to the present application, the N-end rule ligand interacts with the ZZ region or 128 to 163 residues of the ZZ region of the p62 protein
  • the screening method may be useful for developing a therapeutic method for the treatment or prevention of various diseases caused by abnormal accumulation of denatured proteins through the protein removal mechanism through the N-end rule pathway.
  • FIG. 1A is a result showing that p62 protein binds to type 1 and 2 destabilizing N-terminal residues.
  • Figure 1a schematically shows the ITRAQ analysis of the pulled down protein using each X-peptide according to an embodiment of the present application.
  • Figure 1b is the result confirmed by silver staining and Western blot protein pulled down by the X-peptide.
  • Figure 1c is the result of confirming the p62 protein of the precipitated protein by electrophoresis / silver staining after X-peptide pulldown analysis.
  • Figure 1d is a Western blot confirmed whether binding to the R11 peptide, F11 peptide and V11-peptide of p62 transcribed and translated in vitro.
  • Figure 1E is a characterization of the binding specificity of the N-end rule N-terminus of p62 / sqstm1, pull-down assay using exogenous p62 deletion mutants containing PB1, ZZ and TB regions named endogenous p62 and D3 to be.
  • Figure 1f shows that after binding the biotin-labeled X-peptide to the streptavidin-coated chip, injecting the p62-D3-GST protein into the immobilized peptide and confirmed the binding to the R11 peptide, F11 peptide and V11-peptide to be.
  • Figure 2a schematically shows a series of deletion mutations D1 to D8 at p62 / sqstm1, analyzed by pulldown and Western blot binding of R11 and W11 of each mutation.
  • Figure 2b is the result of comparing the primary sequence of the ZZ region of p62 with the sequence of the UBR region of UBR E3 ligase of various organisms using the ClustalW program.
  • 2C shows Type 1 (R) of six point mutants in which D129, D142_145, D147_149, D151_154, H160_163 and E177 of human p62 (1-440) are substituted with alanine to determine the functional significance of the conserved ZZ-residue -11) and type 2 (W-11) N-end rule N-terminal residue binding was analyzed by X-pull down analysis.
  • FIG. 2D shows deletion mutants generated at the D3 C-terminus to specifically specify sites within the ZZ region important for binding to the Type 1 (R-11) and Type 2 (W-11) N-end rule N-terminus And X-peptide pulldown analysis of binding thereof to the N-end rule N-terminus.
  • FIG. 2E shows the construction of a histidine-deleted mutant and confirms the importance of binding two histidine histidine residues to the N-end rule N-terminus, a construct of a typical zinc finger motif in the ZZ region. , H-11, and K-11) and type 2 (F-11, W-11, Y-11, and L-11) N-end rule results confirmed by in vitro binding analysis with N-terminal residues.
  • 2F is a pulldown analysis showing that deletion of PB1 negatively affects the W-11 (Type-2) peptide of p62.
  • FIG. 3A shows that p62 was removed in the presence or absence of thapsigargin, an ER stress inducer in HEK293, using RNA interference analysis (RNAi) to determine the role of p62 in the regulation of GRP78. This is the result of comparing and analyzing the concentration of GRP78.
  • RNAi RNA interference analysis
  • FIG. 3B shows cyclohexamide follow-up assay using HEK293 cells with p62 or p62 depleted MEF cells with RNAi to determine whether p62 is important for stabilization of GRP78, and GRP78 circulation with p62 control cells and p62 deletion The result is a comparison in the cells.
  • FIG. 3c shows that GRP78 circulation was accelerated by thapsigargin stimulation in contrast to the same experiment as FIG. 3b, and the half-life of GRP78 in MEF and HEK293 control cells was 2 hours and 3 hours below, respectively.
  • Figure 3d shows the results of the pulse tracking analysis, the deletion of p62 resulted in the accumulation of GRP78 and increased half-life in response to thapsigargin.
  • 4A is a Western blot showing that argination of GRP78 was induced in HeLa cells upon stimulation with thapsigargin in the presence of MG132, a proteosome inhibitor.
  • Figure 4b is a result of immunofluorescence showing that the thapsigargin treatment time is longer 0.2-2 micron sized spots containing algination-GRP78 (R-GRP78) was formed.
  • 4C shows HEK293 cell extracts and affinity ligands X-peptide R11 (primary destabilizing residues), E11 (secondary destabilizing residues), and V11 to confirm whether p62 can actually bind to R-GRP78.
  • Peptide pulldown assay results in vitro using (stabilization control).
  • FIG. 5A shows that synthetic binding and full-length p62 are overexpressed with RA, FA or to investigate whether binding of R-GRP78 and other N-end rule proteins to p62 can induce such oligomerization and lead to autophagy.
  • 5B shows Western blot and ELISA that p62 oligomerization by N-end rule interactions is specific for the algination pathway of the N-end rule pathway, and the effect of RA on p62 oligomerization is a dose and time dependent manner.
  • the present application is based on the discovery that p62 is involved in autophagy via the N-end rule pathway, and aims to provide a method for regulating autophagy targeting p62.
  • the present disclosure relates to a method for regulating autophagy comprising contacting a cell with an N-end rule ligand that interacts with a p62 protein, a ZZ region of the p62 protein or 128 to 163 residues of the ZZ region. .
  • autophagy or autophagocytosis refers to catabolism that removes various cellular components, including unnecessary or denatured proteins in cells, using lysosomes.
  • the regulation of autophagy is defined by the normal synthesis of intracellular components, Essential for decomposition and recycling. In this process, autophagosomes are formed, which fuse with the lysosomes, causing cellular components to be degraded or reused.
  • Autophagy includes macro, micro, and sepharon mediated autophagy, all of which may be included herein, and in particular, macromediated autophagy.
  • the N-end rule pathway refers to a proteolytic pathway that degrades a protein having a specific destabilizing moiety, or N-degron, at the N-terminus (which refers to the amino terminus present in the protein or polypeptide).
  • the substrate of the N-end rule pathway is recognized by a protein called N-recognin with an N-degron binding region called a UBR box. This UBR box recognizes the substrate through interaction with the first two residues of the substrate, which is called the N-end rule.
  • Modulation herein includes activation, stimulation or upregulation, or degradation or downregulation, or both, of a biological function. In one embodiment it refers to the activation of autophagy function. Further, the adjustment includes all adjustments in the invitro state, adjustments in the in vivo state, and adjustments in the ex vivo state.
  • P62 (sequestosome-1) according to the present invention is a scaffold protein comprising a plurality of regions present in the cytoplasm, known to bind PKC, p38, RIP1 and TRAF6, and the N-terminal PB1 region that interacts with PKC, ZZ-type zinc finger (ZZ) regions that interact with RIP1, and TRAF6 regions (Sanz L et al., (1999) EMNO J. 18, 3044-3053).
  • p62 protein sequence NP_003891 SEQ ID NO: 1
  • nucleic acid sequence is known as NM_003900.4.
  • the specific mechanism of its N-end rule path is unknown.
  • p62 was identified as a novel N-recognin involved in the N-end rule pathway, and as described in the Examples herein, p62 binds to N-degron through a ZZ region with a higher binding force than previously known N-recognins. do.
  • the combination according to the N-end rule described above causes a conformational change from a closed structure to a closed structure at p62, which leads to activation of autophagy. Subsequently, the denatured protein is removed.
  • Such structural changes include, but are not limited to, oligomerization of p62.
  • the oligomerization of p62 is at least four.
  • autophagy can be activated through contact of a p62 protein, a ZZ region of the p62 protein, or a cell expressing 128 to 163 residues of the ZZ region with an N-end rule ligand.
  • Cells expressing all or part of the above-described p62 according to the present invention are primary cells isolated from mammalian tissue, for example, but not limited to cells derived from brain, liver, pancreas, muscle, established cells derived from mammals. Examples include, but are not limited to, HEK21, Mouse embryonic fibroblast, HeLa, Hep3B, and PC3, and any cell that requires the effects of the present application can be used.
  • the liggen is a natural or synthetic polypeptide or peptide or derivative thereof comprising the N-end rule N-terminus of type 1 or type 2 and includes two or more amino acids in length.
  • a polypeptide consisting of two or eleven amino acids or derivatives thereof is used. These include natural or unnatural amino acid residues (eg D- or L-amino acids), or peptide mimetics.
  • N-end rule ends or destabilizing ends of type 1 and type 2 herein include Arg, Lys, and His (type 1), or Phe, Leu, Trp, Tyr, and Ile (type 2), respectively.
  • the N-end rule ligands may include Arg-Ala (RA) or Arg-Ile-Phe-Ser-Thr-Ile-Glu-Arg-Thr-Tyr (RIFSTIEGRTY).
  • p62 binds to the N-end rule ligand via the ZZ region, which causes structural changes including oligomerization of p62 to activate autophagy.
  • Autophagy is the process of removing intracellular denatured proteins, and when autophagy is abnormal, neurodegenerative diseases, including but not limited to neurodegenerative diseases, liver, due to the accumulation of denatured proteins, such as, but not limited to, liver It can be used to treat diseases and chemotherapy (see Harat 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.)
  • diseases related to protein degeneration such as, but not limited to, Alzheimer's disease, Parkinson's disease, Lewy body dementia, muscular dystrophy (ALS), Huntington's disease, spinal cerebellar ataxia And spinobulbar musclular atrophy.
  • diseases related to protein degeneration such as, but not limited to, Alzheimer's disease, Parkinson's disease, Lewy body dementia, muscular dystrophy (ALS), Huntington's disease, spinal cerebellar ataxia And spinobulbar musclular atrophy.
  • neurodegenerative diseases in which the method according to the invention may be used include adrenal Leukodystrophy, alcoholism, Alexander's disease, Alper's disease, Alzheimer's disease , Atrophic lateral sclerosis, ataxia telangiectasia, Batten disease, bovine spongiform encephalopathy, Canavan disease, cerebral palsy, cockayne syndrome, cortical depression (corticobasal degeneration), Creutzfeldt-Jakob disease, fatal fatal insomnia, frontotemporal lobar degeneration, Huntington's disease, HIV-related dementia, Kennedy's disease (Kennedy's disease), Krabbe's disease, Lewy body dementia, neuroborreliosis, Macadodo-Joseph disease, multiple systems Multiple system atrophy, multiple sclerosis, narcolepsy, Niemann Pick disease, Parkinson's disease, Pelizaeus-Merzbacher disease, Pick's disease, primary lateral sclerosis, prion disease, progressive
  • the invention in another aspect relates to a autophagy regulator or pharmaceutical composition for autophagy control comprising an N-end rule ligand that interacts with the ZZ region of the p62 protein.
  • the ligand included in the autophagy modulator or pharmaceutical composition according to the present application and use thereof are as described above, and may be used, for example, in the treatment or prevention of neurodegenerative diseases.
  • treatment means any action that ameliorates or beneficially alters the associated symptoms by administration of a composition according to the present application.
  • Those skilled in the art to which the present application belongs, will be able to determine the extent to which the composition of the present invention is correct, improved, improved and treated with reference to the data presented by the Korean Medical Association and the like. .
  • prevention means any action that inhibits or delays the development of a related disease by administration of a composition according to the present application. It will be apparent to those skilled in the art that the composition of the present invention, which is effective in removing denatured proteins due to autophagy abnormalities, can prevent these diseases if taken before the onset of symptoms, or symptoms of denatured protein accumulation.
  • the autophagy control agent of the present application may be prepared in a pharmaceutical composition.
  • the pharmaceutical composition may be administered simultaneously or sequentially and may be administered in parallel with other pharmaceutically active ingredients for treating the disease.
  • compositions according to the invention may be formulated in a suitable form with the pharmaceutically acceptable carriers generally used.
  • 'Pharmaceutically acceptable refers to a composition that is physiologically acceptable and does not cause an allergic or similar reaction, such as gastrointestinal disorders, dizziness or the like, when administered to a human.
  • pharmaceutically acceptable carriers include, for example, water, suitable oils, saline, carriers for parenteral administration such as aqueous glucose and glycols, and the like, and may further include stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid.
  • Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol.
  • the composition according to the present invention if necessary according to the administration method or dosage form, suspensions, dissolution aids, stabilizers, isotonic agents, preservatives, adsorption agents, surfactants, diluents, excipients, pH adjusters, analgesics, buffers, Antioxidant etc. can be contained suitably.
  • Pharmaceutically acceptable carriers and formulations suitable for the present invention including those exemplified above, are described in detail in Remington's Pharmaceutical Sciences, latest edition.
  • compositions herein may be prepared in unit dose form by formulating with a pharmaceutically acceptable carrier and / or excipient, according to methods readily available to those of ordinary skill in the art. It can be prepared by incorporation into a dose container. The formulations can then be in the form of solutions, suspensions or emulsions in oil or aqueous media or in the form of powders, granules, tablets or capsules.
  • the method of administering the pharmaceutical composition of the present application may be easily selected according to the formulation, and may be administered to mammals such as domestic animals and humans by various routes.
  • it may be formulated in the form of powders, tablets, pills, granules, dragees, hard or soft capsules, liquids, emulsions, suspensions, syrups, elixirs, external preparations, suppositories, sterile injectable solutions, and so on.
  • Oral or parenteral administration, in particular parenteral administration may be preferred.
  • Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories.
  • non-aqueous solvent and the suspension solvent propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used.
  • base of the suppository witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerol, gelatin and the like can be used.
  • the dosage of the pharmaceutical composition of the present application may vary depending on the weight, age, sex, health condition, diet, time of administration, administration method, excretion rate and severity of the disease, etc. 60 kg), about 1 ng to 10 mg / day, in particular about 1 ⁇ g to 1 mg / day. Since the dosage may vary depending on various conditions, it will be apparent to those skilled in the art that the dosage may be added or subtracted, and thus the dosage does not limit the scope of the present invention in any aspect.
  • the frequency of administration can be administered once a day or divided into several times within the desired range, the administration period is not particularly limited.
  • the disclosure is directed to a method for screening autophagy modulators that regulate autophagy mediated by p62.
  • the method according to the present invention comprises contacting a sample comprising a p62 protein, a ZZ region of the p62 protein or a polypeptide of 128 to 163 residues of the ZZ region with a test substance; And b) detecting an interaction in the ZZ region of the test substance with the protein or peptide, when autophagy is increased in comparison with a control group not contacted with the test substance when contacted with the test substance, Selecting the test substance as an autophagy control candidate.
  • the p62 protein including the ZZ region may be used in the present method depending on the specific method of screening, and may be used in the method of the full length or the N- or C-terminus or a part deleted therebetween. It is all included.
  • In one embodiment comprises 128 to 163 residues based on the human sequence of p62 comprising ZZ that may be used in the methods. Even though they are derived from the same host, for example, humans, there may be sequence variations depending on the specific individual, region, environment, etc., and of course, some sequences have been modified (deleted, substituted, added), but all functionally equivalent variants are present. It can be used in the invention.
  • the gene of the p62 protein includes, but is not limited to, GenBank Accession No NM_003900, and the protein sequence encoded by it is NP_003891.
  • the p62 protein full length, or portions thereof, used herein can be prepared using methods known in the art.
  • the method for producing a protein used for screening is to use a genetic recombination technique.
  • the plasmid containing the corresponding gene encoding the protein can be delivered to prokaryotic or eukaryotic cells such as insect cells and mammalian cells, overexpressed and purified.
  • the plasmid can be used by cloning the gene of interest in a eukaryotic expression vector such as pET28b (Novagen) as used in the exemplary embodiments of the present invention, transferring the cell to a cell line, and then purifying the expressed protein. It is not limiting.
  • a DNA or RNA sequence encoding a protein used for screening in a suitable host cell to make the cell lysate or translating the mRNA of the screening protein in vitro and then screening the protein by a protein isolation method known in the art.
  • the cell lysate or the result of in vitro translation is centrifuged, followed by precipitation, dialysis, and various column chromatography. Ion exchange chromatography, gel-permeation chromatography, HPLC, reverse phase-HPLC, preparative SDS-PAGE, affinity columns and the like are examples of column chromatography. Affinity columns can be made, for example, using anti-screening protein antibodies.
  • the contact between the purified protein and the test substance can be used directly in vitro to confirm binding in the absence or presence of the test substance.
  • the protein may be labeled using a variety of markers such as protein tags, biotin, fluorescent materials, acetylation, radioisotopes or commercially available protein labeling kits for ease of detection, It can be detected using a detector suitable for the labeled material. Protein-protein interactions can be measured using various methods known in the art. For example, the East to Hybrid method and the Confocal Microscopy method for confirming the binding / interaction of intracellular proteins.
  • Co-immunoprecipitation, pull-down analysis, surface plasma resonance (SPR) and spectroscopy methods include, but are not limited to, further references on comparisons and detailed experimental methods for these methods.
  • all or part of the above-described p62 protein according to the present application may be provided in the form of cells expressing it.
  • mammalian cells expressing all or part of the p62 protein Transient or Stable transduction or endogenous expression
  • HEK21 Human embryonic fibroblast
  • HeLa HeLa
  • Hep3B Hep3B
  • PC3 PC3
  • the test substance was added thereto, and after a predetermined time, the total protein was extracted from the cells, for example, by co-immunoprecipitation and immunofluorescence. You can check the interaction.
  • inhibiting autophagy or, alternatively or in combination with, inducing oligomerization of p62 can be selected as a therapeutic candidate.
  • the amount of protein used in the present method, the type of cells and the amount and type of test substance vary depending on the specific test method used and the type of test substance, and those skilled in the art will be able to select an appropriate amount.
  • the "test substance” includes the above-mentioned ligand, and means a substance which is expected to activate autophagy through an interaction including a bond with the ZZ region of p62.
  • a low molecular weight compound may be used as a test substance.
  • the test substance can be obtained from a library of synthetic or natural compounds and methods of obtaining libraries 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 libraries of natural compounds are available from Pan Laboratories (USA) and Available from MycoSearch (USA).
  • Test materials can be obtained by a variety of combinatorial library methods known in the art, for example, biological libraries, spatially addressable parallel solid phase or solution phase libraries, deconvolution Required by the synthetic library method, the "1-bead 1-compound” library method, and the synthetic library method using affinity chromatography screening.
  • Methods of 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.
  • the test substance is a polypeptide comprising an N-end rule residue of type 1 or type 2, for example 2 or more amino acid residues, such as 6, 10, 12, 20 or less Or a polypeptide having more than 20 such as 50 amino acid residues.
  • Biologics can also be used for screening, for example.
  • Biologics refers to cells or biomolecules
  • biomolecules refer to proteins, nucleic acids, carbohydrates, lipids or substances produced using cellular systems in vivo and ex vivo.
  • Biomolecules may be provided alone or in combination with other biomolecules or cells.
  • Biomolecules include, for example, proteins or biological organics found in polynucleotides, peptides, antibodies, or other plasma.
  • the method herein includes detecting an interaction in a ZZ region of a test substance with the p62 protein and associating it with autophagy activation, compared to a control that is not in contact with the test substance when in contact with the test substance.
  • the test substance is selected as a candidate for autophagy control.
  • step a) may be provided in the form of a p62 protein, a ZZ region of the p62 protein or a protein having 128 to 163 residues of the ZZ region. .
  • the increase or decrease of autophagy or oligomerization can be detected by known methods.
  • the former can be detected, for example, by autophagy flux assays using Western blot and confocal microscopy, while the latter can be detected by Western blot, filtertrap assays and / or mass spectrometry.
  • This is not limiting.
  • a substance which induces autophagy activation in the presence of the test substance is selected as compared with the control group which is not in contact with the test substance.
  • At least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about Candidates can be screened for at least 100%, or increased above.
  • Candidates selected by the screening method according to the present application can be developed into new drugs through subsequent studies.
  • HEK293 cell lines were used by transient transfection into plasmids expressing the following.
  • P62 / sqstm1 a series of domain-defective mutants amplified by PCR, was cloned into the pcDNA.3.1 / myc- his plasmids of the EcoRI and XhoI sites, and then transiently transferred to HEK293 cell lines using lipofectamine 2000 (Invitrogen) according to the manufacturer's method. It was.
  • Arg, His, or Lys (Type-1) residues at the N-terminus; Phe, Trp, Tyr, or Leu (Type-2); Or 12-mer peptide (X-I-F-S-T-I-E-G-R-T-Y-K-biotin) comprising Val, Asp, or Gly (stabilization control) was crosslinked to streptavidin agarose resin (Thermo) via C-terminal biotin. The ratio of biotinylated peptide and streptavidin agarose used 0.5 mg of soluble peptide per 1 ml of settling resin. The mixture was diluted in 5 ⁇ volume of PBS and then left at 4 ° C. overnight.
  • Sensor chip SA (GE Healthcare, Piscataway, NJ, USA) with pre-fixed streptavidin in one flow cell was converted to biotinylated biotinylated-R11 (RIFSTIEGRTY) / V11 (VIFSTIEGRTY) / F11 (FIFSTIEGRTY) peptides. Saturated.
  • the Biacore X-100 control software was used to measure the change in RU and create a join curve. Curves obtained using SPR experiments were analyzed and p62-D3-GST dissociation equilibrium constants (KD) for immobilized biotinylated peptides were calculated using kinetic evaluation software.
  • KD dissociation equilibrium constant
  • M dissociation equilibrium constant
  • Plasmids expressing p62 / sqstm1-myc / his were transiently transfected into HEK293 cells using lipofectamine 2000 according to the manufacturer's method. After 24 hours, cells were lysed with lysis buffer (50 mM HEPES, pH 7.4, 0.15 M KCl, 0.1% Nonidet P-40, 10% glycerol, protease inhibitor and phosphatase inhibitor). After freeze-thaw, the cell suspension was allowed to stand on ice for 1 hour and then centrifuged at 4 ° C., 13,000 ⁇ g for 20 minutes. Protein concentration was determined by Bradford assay.
  • oligomerization assays 1 ⁇ g of protein was incubated in the presence / absence of dipeptides dissolved in water at a final concentration of 0.5 or 1 M in the presence of 100 ⁇ M beststatin for 2 hours at room temperature. Samples were mixed in non-reducing loading buffer containing 4% Lithium Dodecyl Sulfate (LDS), heated at 95 minutes for 10 minutes, loaded onto 3% stacking and 12% isolated SDS PAGE, followed by electrophoresis. Monomers, oligomers and aggregates of p62 were detected using anti-p62 and anti-myc antibody mixtures.
  • LDS Lithium Dodecyl Sulfate
  • iTARQ immunobaric tags for relative and absolute quantitation
  • rat testis extracts were used as N-end rule interacting proteins, and X-peptides labeled with biotin immobilized on streptabin beads (R11 (type 1), F11 (type 2), and V11 (stabilization control)).
  • the 10mer linker linked to the X-peptide is derived from Sindbis virus polymerase nsP4, the substrate of the N-end rule, and the proteins pulled down using each X-peptide are of different color using the ITRAQ labeling technique. Labeled with fluorescent material (FIG.
  • Endogenous p62 is capable of binding three types of all type-1 N-end rule N-terminal residues R11, H11 and K11 (FIG. 1E). p62 binds strongly to R11-peptide, moderate to K11 and weakly to H11. Endogenous p62 also bound to three of four N-end rule N-terminal residues, namely F11, W11 and Y11, but not to L11.
  • Arg (R), His (H) and Lys (K) have positively charged side chain groups
  • Phe (F), Typ (W) and Tyr (Y) have aromatic side chains. It can be seen from the characterization of the amino acids to bind p62 that p62 is either positively charged or prefers aromatic side chains. P62, on the other hand, did not favor amino acids with hydrophobic side chains such as Val (V), Leu (L) and Gly (G) and amino acids such as Asp (D) with negatively charged side chains. Exogenous p62 D3 mutations could bind type 1 and type 2 destabilizing N-terminal residues and were less effective than endogenous p62 (FIG. 1E).
  • the D3 mutant did not bind to the stable types of peptides V11, D11 and G11, indicating that the interaction is N-end rule specific as well as p62. These results indicate that p62 can bind to a wide range of Type 1 and Type 2 N-end rule N-terminus, indicating that p62 is a new constituent of the N-end rule path.
  • p62-D3-GST The binding affinity and kinetics of p62-D3 and various N-terminal amino acids were then investigated using surface plasmon resonance biosensors (Biacore, USA). X-peptide pulldown experiments showed that p62 was strongly bound to the R11 peptide, weakly bound to F11, and not bound to V11. In order to determine the binding constant to destabilization and stabilization N-terminus of p62, p62-D3-GST was expressed in E. coli and purified to 50% purity. After biotin labeled X-peptides were immobilized on streptavidin coated chips, p62-D3-GST protein was injected into immobilized peptides.
  • D1 to D8 a series of deletion mutations (D1 to D8) were constructed at p62 / sqstm1 and analyzed for their binding to R11 and W11. It was confirmed.
  • the vector containing the p62 deletion mutant was expressed in HEK293 cells, the lysate of the cells was used for analysis.
  • D1 to D4 are mutants that are continuously deleted at the C-terminus
  • D5 to D8 are mutants that are continuously deleted at the C-terminus (FIG. 2A). Expression in HEK293 cells except D8 was detected by Western blot using anti-histidine antibody.
  • the UBR box is the basic substrate recognition region of 70 residues present in the well-conserved N-end rule E3 family designated UBR1 through UBR7.
  • the primary sequence of the ZZ region of p62 was compared with the sequence of the UBR region of UBR E3 ligase from various organisms using the ClustalW program.
  • the typical C2H2 zinc finger fold of the UBR box was well preserved in p62-ZZ, whereas the binucleate Zn-finger fold of the atypical UBR box was found only partially in p62-ZZ (FIG. 2B).
  • the UBR region contains three very well conserved aspartic acids (D118, D150 and D153), which are essential for binding the substrate's N-terminus to destabilizing residues.
  • the p62-ZZ region also contained three aspartic acid residues, including D129, D147 and D149. Although only D147 of the ZZ region was conserved in UBR box (D118), the three aspartic acid residues on the negatively charged (acidic) stomach appear to be essential for binding to the N-terminal destabilizing residues as in the case of UBR boxes.
  • Type 1 (R-11) and Type 2 (W-11) N-end rule In order to specifically specify the site in the ZZ region that is important for binding to the N-terminus, a deletion mutant is made at the D3 C-terminus, and its N -End rule N-terminal binding was confirmed by X-peptide pulldown analysis. A total of nine p62 deletion mutants (FIG. 2D) were constructed and their expression in HEK293 cells was confirmed by Western blot using anti-myc antibodies (FIG. 2D top panel). CD1 to CD6 bound to both type 1 (R-11) and type 2 (W-11) peptides (FIG. 2D middle and bottom panels).
  • CD-6 binds to the N-end rule N-terminal glass like the CD-5 variant except that it does not bind K-11.
  • the minimum ZZ region at the N-terminus was then determined by X-peptide pulldown analysis.
  • Six N-terminal deletion mutants were created at sites starting from the PB1 region of full length p62 and up to the middle of the ZZ region. Expression of all mutants was confirmed by Western blot using anti-Myc antibody (Fig. 2e top panel).
  • ND-1, ND-2, ND-3 and ND-4 bound to R-11 (Type-1) peptide, but ND-5 and ND-6 failed to bind.
  • ND-5 is free of cysteine residues (C128) and aspartic acid (D129), which are elements of the atypical binucleate Zn-finger conserved in UBR boxes.
  • PB1 has been shown to negatively affect the W-11 (Type-2) peptide of p62, indicating that the PB1 region is important for maintaining the structure of the ZZ region (panel below FIG. 2F). In addition, it may indicate that the PB1 region is required for optimal binding to the N-end rule N-terminus of p62. Taken together, C128 and / or D129 in the ZZ region are important for coupling to the type-1 and type-2 N-end rule N-terminus.
  • N-end rule substrates There are few known substrates in mammals, and these include the proteins belonging to the RGS family, RGS4, RGS5 and RGS16 and BRCA1 and CDC6. Possible N-end rule substrates are GRP78 and PDI, ER Sepharon. Since GRP78, like p62, is involved in the protein quality management, it was first investigated as a substrate for p62.
  • RNA interference analysis was used to remove p62 in the presence or absence of thapsigargin, an ER stress inducer in HEK293, and the effect of p62 removal was The concentrations were evaluated by comparison. Western blot analysis showed that GRP78 concentration was upregulated compared to the stress-free RNAi control due to the removal of p62. As a result, thapsigargin induced expression of GRP78 in both the control siRNA and the p62 siRNA, and the GRP78 concentration was higher in the p62 removal group compared to the RNAi control (FIG. 3A top panel). This indicates that p62 is involved in the regulation of GRP78.
  • RNAi RNA interference analysis
  • p62 clearance does not induce GRP78 mRNA expression, indicating that the p62 clearance effect on GRP78 upregulation is at post-transcriptional levels (FIG. 3A). These results indicate that p62 plays a regulatory role in the onset or stability of GRP78.
  • cyclohexamide follow-up assays were performed using HEK293 cells or p62-depleted MEF cells depleted with RNAi, and GRP78 circulation was compared in p62 control cells and p62 deletion cells. It was.
  • the GRP78 cycle showed a very slow half-life of more than 6 hours for p62-WT MEF cells and 8 hours for control HEK 293 cells.
  • p62-depleted HEK293 cells nor p62-removed MEF cells had a significant effect on GRP78 circulation (FIG. 3B).
  • thapsigargin stimulation accelerated GRP78 circulation, and the half-life of GRP78 in MEF and HEK293 control cells was below 2 and 3 hours, respectively (FIG. 3C).
  • GRP78 accumulated in p62-removed cells, and its half-life was longer than 6 hours compared to the control.
  • GRP78 has been shown to autophagy remove protein complexes comprising GRP78 and misfolded ER clients that have been arginated at E19 in an ATE1-dependent manner.
  • GRP78 is a sepharon that stays in the ER, which plays an important role in the response to a misfolded protein.
  • the GRP78 is truncated after the ER signal sequence has been transferred to the ER, exposing the N-terminal residue, conserved pro-N-degron, E19 and It is arginated by (FIG. 3A).
  • Western blot results showed that argination of GRP78 was induced in HeLa cells upon stimulation with thapsigargin in the presence of the proteosome inhibitor MG132 (FIG.
  • R-GRP78 algination-GRP78
  • R-GRP78 spots were identical to the location of the spots containing p62 induced by thapsigargin. Spot formation was effectively blocked by removal of LC3, indicating that LC3 is important for spot formation of R-GRP78 and p62 induced by thapsigargin. P62 removal blocked the spot formation of R-GRP78, indicating that p62 is important for spot formation of R-GRP78 (FIG. 4B).
  • R-GRP78 The dependence of R-GRP78 on spot formation by p62 and LC3 indicates that R-GRP78 is associated with autophagy to ER stress.
  • peptide pulldown analysis was performed in vitro.
  • HEK293 cell extract as p62 and X-peptide R11 (primary destabilizing residue), E11 (secondary destabilizing residue), and V11 (stabilizing control) were used as affinity ligends.
  • the 10mer linker following the X-peptide was derived from the GRP78 N-terminal residue starting at E19.
  • NHK null of Hong Kong
  • NHK null of Hong Kong
  • ELISA assay using p62 KO MEF cells transfected with p62 full-length or p62-D3 coding plasmid lacking LIR and UBA regions examined whether RA peptides enhanced the interaction of LC3 with p62.
  • the cell lysate was confirmed by Western blot whether the two types of p62 protein expression (FIG. 5B).
  • ELISA assays were performed by incubating cell lysates with LC3 tagged with GST immobilized on glutathione coated plates. The experiment was performed using purified mouse monoclonal antibody against p62 and the absorbance (OD) at 450 nm was measured. Experimental results show that full-length p62 showed significant binding to LC3, while D3 did not bind.

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Abstract

La présente invention concerne un procédé pour la régulation d'autophagie comprenant une étape de mise en contact de cellules avec la protéine p62, le domaine ZZ de la protéine 62, ou un ligand de règle d'extrémité N-terminale avec les résidus 128-163 dans le domaine ZZ ainsi qu'une utilisation de celui-ci. Une élimination de protéine à travers une voie de règle d'extrémité N-terminale au moyen de p62, selon la présente invention, peut être utile pour le développement d'un agent thérapeutique pour le traitement ou la prévention de diverses maladies provoquées par une accumulation anormale de protéines ayant subi au mauvais repliement.
PCT/KR2014/009279 2013-10-02 2014-10-01 Procédé pour la régulation de l'autophagie médiée par le domaine de p62 zz et son utilisation WO2015050383A1 (fr)

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