KR101594168B1 - Method for modulating autophagy mediated by p62 through ZZ domain and its use - Google Patents

Abstract

The present application discloses an autopoiesis control method comprising the step of contacting a cell with a p62 protein, a ZZ region of the p62 protein, or an N-end rule ligation that interacts with residues 128 to 163 of the ZZ region. Protein removal through the N-end rule pathway using p62 according to the present invention may be useful for the development of therapeutic agents for the treatment or prevention of various diseases due to abnormal accumulation of denatured proteins.

Description

Methods of modulating autophagy mediated by p62 through ZZ domain and its use mediated by ZZ domain of p62

The present invention is in the field of therapeutic agents for diseases due to the abnormal accumulation of denatured proteins through autophagy control.

Autophagy is a process in which intracellular constituents are changed and regulated in a lysosomal-dependent manner (Levine and Klionsky, (2004) Dev Cell 6, 463-377), and the defective large protein complexes and intracellular organelles To play an important role in cell growth, survival and homeostasis.

For example, autophagy is activated in cancer cells (Ding et al., 2009), Mol. Cancer Ther., 8 (7), 2036-2045) and autophagic inhibitors may act as anticancer drugs (Maiuri et al (2007) Nat. Rev. Cell Biol. 8, 741-752).

In addition, as mentioned above, autopoiesis is important for maintenance of cellular homeostasis through intracellular fluctuations of damaged proteins and cell organelles. As a result, if self-predation phenomenon is lowered, accumulation of misfolded protein is caused This results in neurodegenerative diseases (Komatsu et al., (2006), Nature, 441, 880-884). Accordingly, a substance that enhances cellular self-proliferation can act as a therapeutic agent for the prevention or treatment of neurodegenerative diseases.

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, ), Autophagy, 5 (6).

U.S. Published Patent Application No. 2010-0233730 discloses a method for controlling autophagy for treatment, which comprises administering a test substance to a cell and observing changes in autophagosome intracellular pathway, And a method of sorting.

European Patent Publication No. 2446886 discloses a composition using Beclin-1, BH3 mimetic and the like, which is related to the treatment of muscular atrophy of collagen-VI female muscle using an autograft control agent.

As mentioned above, a wide variety of diseases can be treated through the control of the self-predation phenomenon, and thus it is necessary to develop new therapeutic agents and methods targeting a substance involved in autopoiesis.

The present invention seeks to provide a method and an application for an autophagy control targeting a novel substance involved in autopoiesis through the N-end rule pathway.

In one embodiment, the invention includes contacting the cell with an N-end rule ligation that interacts with a p62 protein, a ZZ region of the p62 protein, or a 128 to 163 residue based on the sequence of SEQ ID NO: 1 of the ZZ region Provides a self-control method. The method according to the present invention can be carried out in vitro in one embodiment and relates to a method for controlling autologous cellular apoptosis in Invitro using the technical features according to the invention.

The N-end rule ligation used in the method of the present invention has an N-end rule lengend of type-1 or type-2, which is Arg-Ala or Xaa-Ile-Phe-Ser-Thr -Ile-Glu-Gly-Arg-Thr-Tyr-Lys sequence wherein X is Arg, His, Lys, Phe, Trp or Tyr. Tyr-Ile-Phe-Ser-Thr in the above sequence is a destabilized N-terminal sequence derived from sndvis virus nsP4 RNA polymerase.

In the method according to the present invention, the interaction of the ZZ region and the ligation promotes the oligomerization of p62.

In another aspect, the present invention also provides a pharmaceutical composition for the treatment of neurodegenerative diseases comprising an N-end rule ligation that interacts with the ZZ region of the p62 protein.

The N-end rule lysine contained in the composition of the present invention has an N-end rule ligation product of type-1 or type-2 and the N-end rule ligation product is Arg-Ala or Xaa-Ile-Phe-Ser-Thr -Ile-Glu-Gly-Arg-Thr-Tyr-Lys sequence wherein X is Arg, His, Lys, Phe, Trp or Tyr.

The pharmaceutical compositions according to the present invention can be used for the treatment of proteinopahty neurodegenerative diseases associated with protein degeneration and include, for example, adrenal leukodystrophy, alcohol poisoning, Alexander's disease, Alper's disease, Alzheimer's disease, muscular atrophy sclerosis, ataxia telangiectasia, Batten disease, bovine spongiform encephalopathy, Canavan disease, cerebral palsy, Cocker's syndrome, corticobasal degeneration, Creutzfeldt-Jakob disease, familial fatal insomnia, frontotemporal lobar degeneration, Huntington's disease,

Huntedton's disease, HIV-related dementia, Kennedy's disease, Krabbe's disease, Lewy body dementia, neuroborreliosis, Machado-Joseph disease, Multiple system atrophy, multiple sclerosis, seizure sleep < RTI ID = 0.0 >

for example, narcolepsy, Niemann Pick disease, Parkinson ' s disease, Pelizaeus-Merzbacher disease, Pick's disease, primary lateral sclerosis, Prion disease, progressive supranuclear palsy, Refsum's disease, Sandhoff disease, Schilder's disease, toxic anemia secondary subacute of spinal cord secondary Spiegelmeyer-Vogt-Sjogren-Batten disease, spinocerebellar ataxia, spinal muscular atrophy, stellate myelopathy, spinal cord degeneration, - Steele-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, Levy's somatic dementia, muscular atrophy sclerosis (ALS), Huntington's disease, spinal cord cerebral ataxia or spinobulbar musculular atrophy .

In another embodiment, the invention is directed to a method of screening for an autopoiesis modulating agent mediated by p62, the method comprising: a) contacting a p62 protein, a ZZ region of the p62 protein, or a polyline of 128 to 163 residues of the ZZ region Contacting the sample comprising the peptide with the test substance; And b) detecting the interaction of the test substance with the protein or peptide in the ZZ region, wherein the test substance is in contact with the test substance, For example, increasing or decreasing, the test substance is selected as an autograft control candidate substance.

Test substances that can be used in the screening according to the present invention are those having an N-end rule lengthene, for example N-end rule N-terminal of type-1 or type-2, Arg-Ala or Xaa-Ile- -Thr-Ile-Glu-Gly-Arg-Thr-Tyr-Lys sequence, wherein X has a peptide sequence of Arg, His, Lys, Phe, Trp or Tyr.

Screening method according to the present invention The above sample is provided in the form of a cell, for example, a mammalian cell such as HEK21, mouse embryonic fibroblast, HeLa, Hep3B or PC3 cells, But is not limited thereto.

The method according to the present invention also provides a method for screening an autoregulatory agent, wherein the interaction of the ZZ region and the ligation site in place of or in addition to step b) is detected by oligomerization of p62.

Comprising contacting the p62 protein according to the present application, the ZZ region of the p62 protein or an N-end rule ligation product interacting with the 128 to 163 residues of the ZZ region, an autophoresis regulator and an autoregulatory agent The screening method can be useful for the development of a therapeutic method for the treatment or prevention of various diseases due to the abnormal accumulation of the denatured protein through the protein elimination mechanism through the N-end rule pathway.

Figure 1a shows the results showing that the p62 protein binds to destabilizing N-terminal residues of types 1 and 2. FIG. 1A schematically illustrates the ITRAQ analysis of pulldown proteins using each of the X-peptides according to one embodiment of the invention.
Figure 1B shows the results of silver staining and Western blotting of proteins pulled down by X-peptide.
FIG. 1C shows the result of confirming the p62 protein in the precipitated proteins by electrophoresis / silver staining after X-peptide pulldown analysis.
FIG. 1D shows Western blot analysis of the binding of p62 transcribed and translated in Invitro to the R11, F11 and V11-peptides.
FIG. 1E is a graph showing the results of pulldown analysis using an exogenous p62 deletion mutant comprising the PB1, ZZ and TB regions named endogenous p62 and D3, to characterize the binding specificity of the p62 / sqstm1 to the N-end rule N-terminus to be.
FIG. 1F shows the results of binding of the biotin-labeled X-peptide to the streptavidin-coated chip and then injecting the p62-D3-GST protein into the fixed peptide and confirming binding to the R11 peptide, F11 peptide and V11- to be.
FIG. 2a schematically shows a series of deletion mutants D1 to D8 at p62 / sqstm1, and the result of analysis of the binding of R11 and W11 of each mutation to the pull-down and Western blot.
FIG. 2B shows the result of comparing the primary sequence of the ZZ region of p62 with the sequence of the UBR region of UBR E3 ligase derived from various organisms using the ClustalW program.
Figure 2c is a graph showing the effect of six point mutants of type 1 (R (SEQ ID NO: 2)) in which D129, D142_145, D147_149, D151_154, H160_163 and E177 of human p62 (1-440) were replaced with alanine to determine the functional significance of the conserved ZZ- -11) and type 2 (W-11) N-end rule N-terminal residue binding was analyzed by X-pulldown analysis.
FIG. 2d shows the deletion mutants produced at the D3 C-terminus in order to specifically specify the sites in the ZZ region that are important for binding to the N-terminal end of the N-terminal domain of type 1 (R-11) and type 2 (W-11) And its X-peptide pulldown assay for binding to the N-terminal end of the N-end rule.
Fig. 2e shows the structure of a typical zinc finger motif in the ZZ region. In order to confirm the importance of binding of two histidine histidine residues to the N-end rule N-terminus, a histidine deletion mutant was constructed, , H-11, and K-11) and type-2 (F-11, W-11, Y-11 and L-11) N-end rule.
Figure 2f is a pulldown analysis showing that the deletion of PB1 negatively affects the W-11 (Type-2) peptide of p62.
Figure 3a shows the effect of p62 removal in the presence or absence of the ER stress inducer thapsigargin in HEK293 using RNA interference assay (RNAi) to identify the role of p62 in GRP78 regulation, Of the concentration of GRP78.
Figure 3b shows the results of a cyclohexamide tracing assay using p62-deleted HEK293 cells or p62-depleted MEF cells with RNAi to determine whether p62 is important for stabilization of GRP78, and GRP78 circulation in p62 control cells and p62 deletion Cells.
FIG. 3c shows the results of experiments showing that GRP78 circulation was accelerated by thapsigargin stimulation in contrast to that of FIG. 3b, and that the half-life of GRP78 in MEF and HEK293 control cells was 2 hours and 3 hours, respectively.
Figure 3d shows the results of pulse tracing analysis showing that the deletion of p62 resulted in the accumulation of GRP78 and the increased half-life in response to thapsigargin.
4A is a Western blot result indicating that arginylation of GRP78 was induced in HeLa cells upon stimulation with thapsigargin in the presence of the proteasome inhibitor MG132.
FIG. 4B shows the result of immunofluorescence analysis showing that spots having a size of 0.2 - 2 microns including arginylation-GRP78 (R-GRP78) were formed when the treatment time of thapsigargin was prolonged.
Figure 4c is a graph showing the effect of p62 on HEK293 cell extract and affinity ligation of X-peptide R11 (primary destabilizing moiety), E11 (secondary destabilizing moiety), and V11 (Stabilized control group).
FIG. 4d shows the results of immunofluorescence analysis of whether NHK interacts with p62 and R-GRP78 in cells. As a result, the formation of p62 foci and the induction of GRP78 in HeLa cells were induced by NHK overexpression.
FIG. 5A overexpresses synthetic lysing and full-length p62 to determine whether the binding of R-GRP78 and other N-terminal rule proteins to p62 can induce autolymedication to induce autolymidation, Non-reducing SDS-PAGE analysis using HEK293 cell extract cultured in the presence / absence of various N-end rule peptides such as WA or control peptide AA, R or AR and Western blot results using p62 antibody.
Figure 5b shows that p62 oligomerization by the N-end rule interaction is specific for the aligning pathway of the N-end rule pathway and that the effect of RA on p62 oligomerization is dose and time dependent, by Western blot and ELISA This is a result.

This article is based on the discovery that p62 is involved in autopoiesis through the N-end rule pathway and aims to provide a self-control method that targets p62.

In one embodiment herein, the present invention relates to an autophagy control method comprising contacting the cell with an N-end rule ligation that interacts with the p62 protein, the ZZ region of the p62 protein, or the 128 to 163 residues of the ZZ region .

In the present invention, autophagy or autophagocytosis refers to the catabolic action of removing various cellular constituents including unnecessary or denatured proteins in cells using lysosomes. The control of autopoiesis is the normal synthesis of intracellular components, It is essential for disassembly and recycling. In this process, autophagosomes are formed, which are fused with lysosomes, and cell components are degraded or reused. Self-predation has macro-, micro-, and cephalon-mediated self-predation, all of which may be included herein, particularly macro-mediated self-predation.

The N-end rule pathway herein refers to the proteolytic pathway that degrades a protein having a specific destabilizing moiety, i.e., N-degron, at the N-terminus (which refers to the amino terminus present in the protein or polypeptide) will be. The substrate of the N-end rule pathway is recognized by a protein called N-recognin with an N-degron binding region called the UBR box. This UBR box recognizes the substrate through its interaction with the first two residues of the substrate, which is called the N-end rule.

As used herein, modulation refers to activation, stimulation or upregulation of biological function, or degradation or downregulation, or both. In one embodiment, this means activating the self-predation function. Further, the adjustment includes both the adjustment in the in-vivo state, the adjustment in the in-vivo state, and the adjustment in the x-ray state.

It is known that p62 (sequestosome-1) according to the present invention binds to PKC, p38, RIP1 and TRAF6, which is a scaffold protein containing multiple cytoplasmic domains. It is known that PB1 region of N-terminus interacting with PKC, ZZ-type zinc finger (ZZ) region interacting with RIP1, and TRAF6 region (Sanz L et al., (1999) EMNO J. 18, 3044-3053). p62 protein sequence NP_003891 (SEQ ID NO: 1), and the nucleic acid sequence is known as NM_003900.4. However, its specific mechanism in the N-end rule pathway is unknown

Here, we have identified p62 as a novel N-recognin involved in the N-end rule pathway. As described in the examples of this application, p62 binds to N-degron with a higher binding force than the previously known N-recognin through the ZZ region do. Although not limited to this theory, due to the coupling according to the N-end rule, a conformational change is caused to a closed to open structure in p62, Subsequently, the denatured protein is removed. Such structural changes include, but are not limited to, oligomerization of p62. The oligomerization of p62 is four or more.

In this respect, self-predation can be activated through contact of the p62 protein, the ZZ region of the p62 protein, or the N-end rule ligation product with cells expressing the 128 to 163 residues of the ZZ region.

Cells expressing all or a portion of the above-described p62 according to the present invention may be primary cells isolated from mammalian tissues such as, but not limited to, brain, liver, pancreas, muscle derived cells, established mammalian derived cells But are not limited to, for example, HEK21, mouse embryonic fibroblast, HeLa, Hep3B, and PC3, and any cell can be used that requires the effect of the present invention.

As used herein, natural or synthetic polypeptides or peptides or derivatives thereof comprising the N-terminal end of lengendrone type-1 or type-2 include two or more amino acids in length. In one embodiment according to the present invention, a polypeptide consisting of two or ten amino acids or a derivative thereof is used. These include natural or unnatural amino acid residues (e.g., D- or L-amino acids), or peptide mimetics.

The N-end rule termini or destabilizing termini of type 1 and type 2 herein are Arg, Lys, and His (type 1), respectively, or Phe, Leu, Trp, Tyr, and Ile (type 2). In one embodiment according to the present invention, the N-end rule ligation includes Arg-Ala (RA) or Arg-Ile-Phe-Ser-Thr-Ile-Glu-Arg-Thr-Tyr (RIFSTIEGRTY).

As mentioned above, p62 binds to N-end rule ligation through the ZZ region, which leads to structural changes involving oligomerization of p62 and activates autophagy.

Self-predation is the process of removing intracellular denatured proteins. In the case of abnormality in self-feeding, various diseases due to the accumulation of denatured protein are not limited to, for example, neurodegenerative diseases including neurodegenerative diseases, (Hara et al. 2006, Mizushima et al. Genes & Dev. 2007, Takamura et al. Genes & Dev. 2011, Ratuo p et al J Hepatology 2010)

In one embodiment, it is used in a disease associated with protein degeneration (proteinopathy) including, but not limited to, Alzheimer's disease, Parkinson's disease, Levycomie dementia, muscular atrophy sclerosis (ALS), Huntington's disease, And spinobulbar musclular atrophy.

In another embodiment, the neurodegenerative diseases in which the method according to the present invention can be used are selected from the group consisting of Adrenal Leukodystrophy, Alcoholism, Alexander's disease, Alper's disease, Alzheimer's disease, , Ataxia telangiectasia, Batten disease, bovine spongiform encephalopathy, Canavan disease, cerebral palsy, cockayne syndrome, cortical basal degeneration, corticobasal degeneration, Creutzfeldt-Jakob disease, familial fatal insomnia, frontotemporal lobar degeneration, Huntington's disease, HIV-related dementia, Kennedy disease Kennedy's 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, It has been suggested that the disease may be caused by Pick's disease, primary lateral sclerosis, prion disease, progressive supranuclear palsy, Refsum's disease, Sandhoff disease, Schilder's disease, subacute combined degeneration of spinal cord secondary to pernicious anemia, Spielmeyer-Vogt-Sjogren-Batten disease, spinal cord But are not limited to, spinocerebellar ataxia, spinal muscular atrophy, Steele-Richardson-Olszewski disease, Tabes dorsalis, and toxic encephalopathy, It is not limited.

In another embodiment, the present invention relates to a pharmaceutical composition for controlling autopoiesis or autophagy comprising an N-end rule ligation that interacts with the ZZ region of the p62 protein. The lignans contained in the autotrophic regulator or the pharmaceutical composition according to the present invention and their uses are as mentioned above and can be used, for example, for the treatment or prevention of neurodegenerative diseases and the like.

As used herein, the term "treatment" refers to any action that improves or alters the relevant symptoms by administration of a composition according to the invention. Those skilled in the art will be able to ascertain, by reference to the data provided by the Korean Medical Association, the precise criteria of the disease for which the composition of the present invention is effective, .

The term "prophylactic," as used herein, refers to any act that inhibits or delays the onset of a related disorder upon administration of a composition according to the present disclosure. It will be apparent to those skilled in the art that compositions of the present invention that are effective in the removal of denatured proteins due to autophagy abnormalities can prevent such diseases if they are taken prior to the appearance of the symptoms or symptoms of the disease due to accumulation of denatured proteins.

Thus, the self regulating agent of the present invention may be prepared from a pharmaceutical composition. The pharmaceutical composition may be administered simultaneously or sequentially, and may be administered in combination with other pharmacologically active ingredients for the treatment of the diseases.

The therapeutic agent or pharmaceutical composition according to the present invention may be formulated in a suitable form together with a commonly used pharmaceutically acceptable carrier. &Quot; Pharmaceutically acceptable " refers to compositions which are physiologically acceptable and which, when administered to humans, do not normally cause allergic reactions such as gastrointestinal disorders, dizziness, or the like. Examples of pharmaceutically acceptable carriers include, for example, water, suitable oils, saline, aqueous carriers for parenteral administration such as aqueous glucose and glycols, etc., and may further contain 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. In addition, the composition according to the present invention may contain various additives such as a suspending agent, a solubilizer, a stabilizer, an isotonic agent, a preservative, an adsorption inhibitor, an interface activator, a diluent, an excipient, a pH adjuster, An antioxidant, and the like. Pharmaceutically acceptable carriers and formulations suitable for the present invention, including those exemplified above, are described in detail in Remington ' s Pharmaceutical Sciences, Current Edition.

The composition of the present invention may be prepared in a unit dosage form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Into a capacity container. The formulations may 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 administration of the pharmaceutical composition of the present invention can be easily selected according to the formulation, and can be administered to mammals such as livestock, human, and the like in various routes. For example, 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, sterilized injection solutions, Or parenterally, and parenteral administration may be particularly preferable.

Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the non-aqueous solvent and suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As a base for suppositories, witepsol, macrogol, tween 61, cacao paper, laurin, glycerol, gelatin and the like can be used.

The dosage of the pharmaceutical composition of the present invention may vary depending on the patient's body weight, age, sex, health condition, diet, administration time, administration method, excretion rate and severity of disease, 60 kg), it is about 1 ng to 10 mg / day, especially about 1 μg to 1 mg / day. It will be apparent to those skilled in the art that doses may be additive or subtracted, as the dosage can vary depending on various conditions, and thus the dose is not intended to limit the scope of the invention in any way.

The number of administrations can be administered once or several times a day within a desired range, and the administration period is not particularly limited.

In another embodiment, the present invention is directed to a method of screening for an autophagy modulator that modulates 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 in one embodiment with a test substance; And b) detecting the interaction of the test substance with the protein or peptide in the ZZ region, wherein the self-predation is increased when compared to a control that is not contacted with the test substance when contacted with the test substance, And selecting the test substance as an autoposition control candidate substance.

In the screening method of the present invention, the p62 protein including the ZZ region may be used in the present method in various methods depending on the specific method of screening, and may be used in the whole method or in a form in which part of the N- or C- . In one embodiment, from 128 to 163 residues, based on the human sequence of p62, including ZZ, which can be used in the methods. Even if the same host is derived from a human, for example, there may be a sequence variation depending on a specific individual, region, environment, and the like. Of course, some of the sequences are modified (deletion, substitution, addition), but all of the functionally equivalent variants Can be used in the invention.

In one embodiment, the gene for the p62 protein includes, but is not limited to, GenBank Accession No. NM_003900, and the protein sequence encoded thereby is NP_003891.

The p62 protein full-length or part thereof used in the present invention may be prepared using methods known in the art. In particular, a method for producing a protein used for screening is to use a gene recombination technique. For example, a plasmid containing the corresponding gene encoding the protein may be transferred to prokaryotic or eukaryotic cells such as insect cells and mammalian cells for overexpression, followed by purification. For example, the plasmid can be used to purify the expressed protein after transferring the gene to a cell line and cloning the gene into a vector for eukaryotic expression such as pET28b (Novagen) as used in the exemplary embodiment of the present invention, It is not limited.

Or a DNA or RNA sequence encoding a protein used for screening is expressed in an appropriate host cell to prepare a cell lysate thereof or the mRNA of the screening protein is translated in vitro and then screened by a protein separation method known in the art Can be purified. Usually, the cell lysate or the translation product in vitro is centrifuged to remove cell debris and the like, and then precipitation, dialysis, various column chromatography and the like are applied. Ion exchange chromatography, gel-permeation chromatography, HPLC, reversed phase-HPLC, SDS-PAGE for affinity column, and affinity column 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 directly observed in the test tube to confirm whether the test substance is present or absent in the presence of the test substance. In this case, the protein may be labeled using various known markers such as protein tag, biotin, fluorescent substance, acetylation, radioisotope, or a known protein labeling kit for convenience of detection, Can be detected using a detector group suitable for the labeled substance. Protein-protein interactions can be measured using a variety of methods known in the art. For example, the yeast two-hybrid method, the confocal microscope method, which confirms the binding / interaction of intracellular proteins. But are not limited to, co-immunoprecipitation, pulldown analysis, surface plasmon resonance (SPR), and spectroscopy methods. Further references to comparative and detailed methods of such methods are provided in Berggard et al., (2007) &Quot; Methods for the detection and analysis of protein-protein interactions ", PROTEOMICS Vol 7: pp 2833-2842.

In one embodiment, all or a portion of the above-described p62 protein according to the present disclosure may be provided in the form of a cell expressing it. Examples include mammalian cells expressing all or a portion of the p62 protein (transient or stable transmissible or endogenous expression), including, but not limited to, HEK21, mouse embryonic fibroblast, HeLa, Hep3B, and PC3 But is not limited thereto. After such cells are cultured on a cell culture plate, the test substance is added thereto, and after a certain period of time, the total protein is extracted from the cells, and the p62 protein and the lagenated protein are separated from each other by, for example, You can check whether they interact. Compared to the control (when the test substance is not treated), autopoiesis can be suppressed, or alternatively or in conjunction therewith, induction of oligomerization of p62 can be screened as a therapeutic candidate.

The amount of the protein used, the type of the cell, and the amount and type of the test substance used in the present method will depend on the specific experimental method used and the kind of the test substance, and those skilled in the art will be able to select an appropriate amount.

The term "test substance" in the present invention refers to a substance including the above-mentioned ligation, which is expected to activate autopatch through interaction involving binding with the ZZ region of p62, (E. G., Protein, antibody, peptide, DNA, RNA, antisense oligonucleotides, RNAi, aptamers, RNAzymes, and DNAzymes), or mixtures of compounds (e. G., Natural extracts or cell or tissue cultures) Sugar, lipids, and the like.

In one embodiment according to the present application, a low molecular weight compound can be used as a test substance. The test materials can be obtained from libraries of synthetic or natural compounds, and methods for 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 Available from MycoSearch (USA). The test materials can be obtained by various combinatorial library methods known in the art and include, for example, biological libraries, spatially addressable parallel solid phase or solution phase libraries, deconvolution By the desired synthetic library method, "1-bead 1-compound" library method, and by synthetic library methods using affinity chromatography screening. Methods for synthesis of 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, and the like.

In another embodiment according to the present application, the test substance is a polypeptide comprising an N-end rule residue of type 1 or type 2, for example two or more amino acid residues such as 6, 10, 12, 20 or less Or a polypeptide having more than 20, e.g., 50, amino acid residues.

For example, biologics can also be used for screening. Biologics refers to a cell or a biomolecule, which refers to a substance produced using biomolecules, proteins, nucleic acids, carbohydrates, lipids, or in vivo and in vitro cell systems. Biomolecules may be provided alone or in combination with other biomolecules or cells. Biomolecules include, for example, proteins or biological organic materials found in polynucleotides, peptides, antibodies, or other plasma.

The method of the present invention comprises the step of detecting the interaction of the test substance and the p62 protein in the ZZ region and correlating this with the presence of autophagic activation and comparing the test substance with the control substance which is not in contact with the test substance If the self-predation is increased, the test substance is selected as a candidate for self-control. Alternatively or additionally, oligomerization of p62 is detected, wherein 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 .

In the method according to the present invention, whether or not the autopregis is increased or decreased, or oligomerization can be detected by a known method. The former can be detected, for example, by autophagy flux assays using Western blot and confocal microscopy methods, while the latter can be detected by Western blot, filter tap assay and / or mass spectrometry , But is not limited thereto.

As a result of the test, substances which induce the activation of the autopregnancy in the presence of the test substance are selected as candidates as compared with the control group not in contact with the test substance. About 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% 100% or more, or more can be selected as candidates.

Candidate materials selected by the screening method according to the present invention can be developed as new drugs through subsequent studies.

Hereinafter, embodiments are provided to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited to the following examples.

Example

The experimental methods and materials used herein are as follows.

Experimental Methods and Materials

In mammalian cells P62  Overexpression of defective protein

For X-peptide pulldown analysis using endogenous or p62 deficient mutant proteins, the HEK293 cell line was transiently transfected with a plasmid expressing the following. P62 / sqstm1, a series of domain-deficient mutants amplified by PCR, was cloned into the pcDNA.3.1 / myc -his plasmid at EcoRI and XhoI sites, and then transfected with HEK293 cell line using lipofectamine 2000 (Invitrogen) Respectively. After 24 hours, the cells were collected and suspended in a 5X volume of a storage buffer (10 mM KCl, 1.5 mM MgCl ₂ , 10 mM HEPES pH = 7.9) and left for 30 minutes. After 5 times of freezing and thawing, rpm, 4 ° C for 10 minutes.

X-peptide pulldown assay

For this, an Arg, His, or Lys (Type-1) residue at the N-terminus; Phe, Trp, Tyr, or Leu (Type-2); Or 12-mer peptide (XIFSTIEGRTYK-biotin) containing Val, Asp, or GIy (stabilized control) was cross-linked to streptavidin agarose resin (Thermo) via C-terminal biotin. The ratio of the biotinylated peptide and streptavidin agarose was 0.5 mg of the dissolution peptide per 1 ml of sedimented resin. The mixture was diluted in 5X volume of PBS and allowed to stand overnight at 4 < 0 > C. The beads were then electrophoresed at 2000 rpm for 3 minutes and washed three times with PBS. 30 μl of the extract containing 150-200 μg of total protein was taken and diluted with 300 μl of binding buffer (0.05% Tween 20, 10% Glycerol, 0.2 M KCl, 20 mM HEPES pH = 7.9) (50 [mu] l packed vol). The mixture was then reacted at 4 [deg.] C for 2 hours with slight rotation. The beads were precipitated by centrifugation at 2000 rpm for 30 seconds, washed five times with 1m binding buffer or three times for 20 minutes at 4 < 0 > C. Then, the resultant was resuspended in 20 μl 2 × SDS-PAGE, heated at 100 ° C. for 5 minutes, loaded on a gel, and electrophoresed. The pull-down p62 protein was detected as an anti-myc antibody.

Surface plasmon resonance (Biacore) analysis

(GE Healthcare, Piscataway, NJ, USA) with biotinylated-R11 (RIFSTIEGRTY) / V11 (VIFSTIEGRTY) / F11 (FIFSTIEGRTY) peptides with pre-fixed streptavidin in one flow cell Saturated.

140 μl of biotinylated R11 / V11 / F11 peptide (10 μM) was injected into the flow cell at a rate of 10 μl / min for 10 minutes to allow the streptavidin chip to bind R11 / V11 / F11 through biotin-streptavidin binding - < / RTI > peptide. To analyze the binding kinetic, p62-D3-GST introduced into pGEX4T1 was isolated and purified and then expressed in E. coli . P62-D3-GST was then diluted with HBS-EP buffer (0.01 M HEPES, pH7 .4 / 0.15 M NaCl / 3 mM EDTA / 0.005% surfactant P20) and injected into the sensor chip at a rate of 30 μl / min for 150 seconds. A Response Unit (RU) was obtained to determine the association rate constant (ka). After binding, HBS-EP buffer was injected at a rate of 30 l / min for 420 seconds to separate p62-D3 from the fixed R11 / V11 / F11 peptide and RU was collected to determine the dissociation rate constant (kd). RU obtained by injecting 1% elution buffer (20 mM HEPES pH = 7.0, 0.2 M NaCl, 10% Glycerol, 2 mM DTT) was used as a medium control. The change in RU was measured using a Biacore X-100 control software and the coupling curve was created. The curves obtained using SPR experiments were analyzed and the p62-D3-GST dissociation equilibrium constant (KD) for the fixed biotinylated peptides was calculated using kinetic evaluation software. The dissociation equilibrium constant KD (M) is calculated from the equation KD = kd / ka, where kd and ka denote dissociation and binding rate constants, respectively.

Oligomerization analysis

The plasmid expressing p62 / sqstm1-myc / his was transiently transfected into HEK293 cells using lipofectamine 2000 according to the manufacturer's method. After 24 hours, the cells were thawed in a fusion buffer (50 mM HEPES, pH 7.4, 0.15 M KCl, 0.1% Nonidet P-40, 10% glycerol, protease inhibitor and phosphatase inhibitor). After freezing-thawing, the cell suspension was left on ice for 1 hour and then centrifuged at 13,000 x g for 20 minutes at 4 ° C. Protein concentration was determined by Bradford assay. For oligomerization analysis, 1 μg of the protein was incubated in the presence of 100 μM besatin at room temperature for 2 hours at a final concentration of 0.5 or 1 M in the presence / absence of the digested peptides in water. The samples were mixed in a non-reducing loading buffer containing 4% lithium dodecyl sulfate (LDS), heated at 95 for 10 minutes, loaded on 3% stacking and 12% separable SDS PAGE and then electrophoresed. Monomers, oligomers and aggregates of p62 were detected using anti-p62 and anti-myc antibody mixtures.

Example 1 Confirmation of the binding of p62 type 1 and 2 to the destabilizing N-terminal residue

In order to identify proteins interacting with N-degron, Isobaric tags for relative and absolute quantitation were performed in conjunction with X-peptide pulldown analysis and mass spectrometry. Specifically, the rat testis extract was used as the N-end rule interaction protein and the X-peptide (R11 (type 1), F11 (type 2), and V11 (stabilized control) labeled with biotin immobilized on streptavin beads, The 10-mer linker linked to the X-peptide is derived from the Sindbis virus polymerase nsP4, the substrate of the N-end rule. Using the X-peptide, the pull-down protein can be detected using the ITRAQ labeling technique UB2, UBR2, UBR2, and UF3, which are involved in the N-end rule pathway, were identified by the above protein body analysis based on the Mascot score (Fig. UBR4 and UBR5 These results were confirmed by X-peptide pulldown analysis and as a result UBR1 was found in both R11 and F11 pulldown assays and UBR2 only in F11 pulldown assay UBR4 and UBR5 were found in the R11 assay, confirmed by silver stained gel and Western blot (Fig. 1b). In the pull-down analysis using the stabilized control V11, the UBR protein was not precipitated, Indicating that it worked specifically as desired.

In addition to the UBR E3 ligase, the protein identified by mass spectrometry in the R11 sedimentation sample is the sequestosome 1 (p62) protein. p62 / sqstm1 is an autophagic substrate that induces the degradation of ubiquitinated proteins into autophagosomes. It is an important ubiquitin-targeting agent that transfers substrates for proteosomal degradation. These results were confirmed by electrophoresis / silver staining of the precipitated proteins and Western blot after X-peptide pulldown analysis. In the silver-dyed gel, the R11-pulled sample had a strong band around the molecular weight of 62 kDa, but this band was not observed in the negative control V11-pulldone sample. Mass spectrometric analysis showed that this band was p62 (FIG. 1C). Western blot results showed that p62 transcribed and translated in Invitro showed a specifically strong binding to the R11 peptide, while it was weakly bound to the F11 peptide and not to the V11-peptide (Fig. 1d). Based on the amount used in the experiments, the R11 peptide was able to precipitate p62 with an efficiency of greater than 40%, while the efficiency of the F11 peptide was 5%. These results were confirmed by dipeptide competition analysis, in which RA peptide competed most effectively with the R11 peptide in binding to p62, and HA, another type 1 N-end rule peptide, was the next most effective in competition (FIG. 1D). Of the type 2 peptides, WA was less effective and FA was ineffective. Competition analysis with AR, rather than N-end rule peptides, revealed that RA, HA and WA were specific for the N-end rule. These results indicate that p62 can broadly bind to type I and type 2 N-end rule peptides.

Furthermore, to characterize the binding specificity of the p62 / sqstm1 to the N-terminal end of the N-end rule, pulldown analysis was performed using an exogenous p62 deletion mutant comprising the PB1, ZZ and TB regions named endogenous p62 and D3 . Endogenous p62 can bind to all three type-1 N-end rule N-terminal residues R11, H11 and K11 (FIG. 1e). p62 strongly binds to the R11-peptide, moderately to K11 and weakly to H11. Endogenous p62 also bound to three of the four N-terminal end N-terminal residues, F11, W11 and Y11, but not L11. Arg (R), His (H) and Lys (K) have positively charged side chain groups and Phe (F), Typ (W) and Tyr (Y) have aromatic side chains. From the characteristics of the amino acids that bind p62, it can be seen that p62 is positively charged or aromatic side chain is preferred. P62, on the other hand, did not favor amino acids such as Val (V), Leu (L) and Gly (G) and amino acids with hydrophobic side chains and Asp (D) with negatively charged side chains. The exogenous p62 D3 mutation could bind to the destabilizing N-terminal residue of type 1 and type 2 and was less effective than endogenous p62 (FIG. 1e). D3 mutants did not bind to the stable types of peptides V11, D11 and G11, indicating that the interaction is specific to the N-end rule 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 pathway.

Subsequently, the binding affinity and kinetic of p62-D3 and various N-terminal amino acids were examined using a surface plasmon resonance biosensor (Biacore, USA). In the X-peptide pull-down experiment, p62 did not bind strongly to F11 and V11 to R11 peptide. destabilization and stabilization of p62 To determine the binding constant to the N-terminus, p62-D3-GST was expressed in E. coli and purified at 50% purity. The biotin-labeled X-peptide was immobilized on a streptavidin-coated chip, and p62-D3-GST protein was then injected into the fixed peptide. As with the pulldown results, p62-D3-GST did not bind strongly to F11 and V11 to R11 peptide (Fig. 1F). The KD values for R11 and F11 were found to be 30 nM and 300 nM, respectively, which is very good compared to the low micro-molar level binding capacity of the previously known N-recognin. These results indicate the interaction of p62 with the destabilizing N-end rule N-terminal.

Example 2 N-end rule of p62 ZZ region N-terminal linkage identification

destructive N-end rule at p62 To identify the region recognizing the N-terminal end, a series of deletion mutations (D1 to D8) were constructed at p62 / sqstm1 and their binding to R11 and W11 was analyzed by X-peptide pulldown analysis Respectively. After expressing the vector containing the p62 deficient mutation in HEK293 cells, the cell lysate was used for analysis. D1 to D4 are mutants that are consecutively deleted on the C-terminus, and D5 to D8 are mutants that are consecutively deleted on the C-terminus (Fig. Except for D8, expression in HEK293 cells was detected by Western blot using anti-histidine antibody. As in the results of Example 1, the full length p62 was strongly bound to the R11 and W11 peptides. The binding patterns of the mutants to R11 and W11 were also similar. Among the mutants, D2, D3, D4 and D5 including the ZZ type zinc finger (ZZ) region showed the strongest binding force. D3 and D4, including the TRAF region, were shown to bind more strongly to the R11 and W11 peptides compared to D2 and D5, indicating that the TRAF region surrounding the ZZ region is more potent for p62 to be required for optimal binding of the R11 and W11 peptides will be. D1, D6 and D7 without the ZZ region did not bind to the R11 and W11 peptides. These results indicate that the ZZ region is involved in binding to the N-end rule N-terminus of types 1 and 2.

The UBR box is the basic substrate recognition region of 70 residues present in the well conserved N-end rule E3 family named UBR1 to UBR7. In order to investigate the similarity of p62 to the UBR box of the ZZ region, the primary sequence of the ZZ region of p62 was compared to the sequence of the UBR region of UBR E3 ligase from various organisms using the ClustalW program. As a result, the typical C2H2 zinc finger fold of the UBR box was well preserved in p62-ZZ, while the binucleate Zn-finger fold of the atypical UBR box was only partially found in p62-ZZ (Fig. 2B). The UBR region contains three highly conserved aspartic acids (D118, D150 and D153), which are essential for binding to the N-terminal destabilizing moiety of the substrate. The p62-ZZ region also contained three aspartic acid residues including D129, D147 and D149. Only the D147 of the ZZ region was conserved in the UBR box (D118), but the three aspartic acid residues on the negatively charged (acidic) side seem to be essential for binding to the N-terminal destabilizing moiety, as in the case of the UBR box. These results are consistent with pulldown analysis results suggesting that p62 binds to positively charged (basic) side chains. In addition to the C2H2 zinc finger structure and the three aspartic acid residues, three identical (red) residues (Y140, D147 and L150) in the ZZ region and two identical residues (G173 and W184) outside the ZZ region and one (Red) residue (E177) was found.

In order to determine the functional significance of the identical and conserved ZZ-moieties identified in the sequence comparison analysis, the six point mutants D129, D142_145, D147_149, D151_154, H160_163 and E177 of human p62 (1-440) Were constructed and analyzed by X-pulldown analysis. As a result, the mutants in the ZZ region failed to bind to both the type 1 (R-11) and type 2 (W-11) N-end rule N-terminal residues (FIG. 2C). These results indicate that ZZ and UBR have similar binding properties to N-terminal N-terminal residues of type 1 (R-11) and type 2 (W-11), where Cys and His residues act as structural elements , Indicating that aspartic acid plays an important role in N-decrystalline recognition. Mutations of conserved E177 to alanine present outside the ZZ region did not affect binding to the N-terminally terminal residues of type 1 (R-11) and type 2 (W-11). Taken together, these results indicate that the binding properties of the p62 Z-region to the N-end rule N-terminal are similar to those of the UBR box, indicating that the ZZ region and the UBR box are structurally similar.

Type 1 (R-11) and type 2 (W-11) N-end rule In order to specifically specify a site in the ZZ region that is important for binding at the N-terminus, a deletion mutant at the D3 C- -end rule The binding to the N-terminus was confirmed by X-peptide pulldown analysis. A total of 9 p62 deletion mutants (Figure 2d) were constructed and their expression in HEK293 cells was confirmed by Western blot using anti-myc antibodies (Figure 2d top panel). CD1 to CD6 bound to both Type 1 (R-11) and Type 2 (W-11) peptides (Figure 2d middle and bottom panel). Mutants from CD7 without two histidine residues, a constituent of the typical zinc finger motif in the ZZ region, did not bind. (R11, H-11 and K-11) and type-2 (F-11, K-11) in order to confirm the importance of the binding of the two histidine residues to the N- W-11, Y-11 and L-11) N-end rule N-terminal residues were used for in vitro binding analysis (FIG. The CD-5 mutant bound to all type-1 and type-2 N-end rule N-terminal residues except L-11 to a similar degree to endogenous p62. CD-6 bound to the N-end rule N-terminal group as well as the CD-5 variant except that it did not bind to K-11. These results indicate that the minimum ZZ region at the C-terminus indispensable for the binding of the N-end rule N-terminal residue is up to H163.

The minimum ZZ region of the N-terminus was then determined by X-peptide pulldown analysis. Six N-terminal deletion mutants were made in the region starting from the PB1 region of the full length p62 to the middle of the ZZ region. Expression of all mutants was confirmed by Western blot using anti-Myc antibodies (Fig. 2e top panel). ND-1, ND-2, ND-3 and ND-4 bound to R-11 (Type-1) peptide but not ND-5 and ND-6. ND-5 lacks the cysteine residues (C128) and aspartic acid (D129), which are elements of the atypical binucleate Zn-finger conserved in the UBR box. Surprisingly, it has been shown that deletion of PB1 negatively affects the W-11 (Type-2) peptide of p62, indicating that the PB1 region is important for maintaining the structure of the ZZ region (Fig. It can also indicate that the region of PB1 is required for optimal binding to the N-terminal end of p62. Taken together, this indicates that C128 and / or D129 in the ZZ region is important for binding to the N-terminal end of type-1 and type-2 N-end rules.

Example 3 The turnover of GRep78, the ER cephalon by p62,

From the above results, p62 was found to be a new factor in the N-end rule pathway, so the substrate of p62 was examined. First, a known or predictable N-end rule substrate was assessed. There are few known substrates in mammals, and these include RGS4, RGS5 and RGS16, which belong to the RGS family, and BRCA1 and CDC6. Possible N-end rule substrates are GRP78, PDI, and ER cephalon. Since GRP78 participates in the qualitative management of proteins as in p62, it was first investigated as a substrate for p62.

To identify the role of p62 in GRP78 regulation, p62 was eliminated in the presence or absence of thapsigargin, an ER stress inducer, in HEK293 using RNA interference assay (RNAi). The effect of p62 elimination was assessed using GRP78 The concentrations were compared and evaluated. Western blot analysis showed that GRP78 concentration was upregulated compared to the stress-free RNAi control due to the elimination of p62. As a result, thapsigargin induced expression of GRP78 in both control siRNA and p62 siRNA, and the GRP78 concentration was higher in the p62-removed group than in the RNAi control (Fig. 3a top panel). Indicating that p62 is involved in the regulation of GRP78. p62 elimination does not induce GRP78 mRNA expression, indicating that the p62 elimination effect on GRP78 upregulation is at post-transcriptional level (Fig. 3a). These results indicate that p62 plays a modulatory role in the transitional initiation or stability of GRP78. To determine whether p62 is important for stabilization of GRP78, cyclohexamide tracing analysis was performed using p62-depleted H6293 cells or p62-depleted MEF cells with RNAi and GRP78 circulation was compared between p62 control cells and p62 deficient cells Respectively. Under non-stress conditions, GRP78 circulation appeared to be very slow, with a half-life of 6 hours for p62-WT MEF cells and over 8 hours for control HEK 293 cells. Neither p62-depleted HEK293 cells nor p62-depleted MEF cells had a significant effect on the GRP78 circulation (Fig. 3B). In contrast, thapsigargin stimulation accelerated GRP78 circulation, and the half-life of GRP78 in MEF and HEK293 control cells was 2 hours and 3 hours, respectively (Fig. 3c). On the other hand, in the case of p62-removed cells, GRP78 accumulates and its half-life is longer than that of the control group by 6 hours. Pulse tracing analysis was consistent with this, and deletion of p62 resulted in accumulation of GRP78 and increased half-life in response to thapsigargin (FIG. 3D). These results indicate that p62 is involved in thapsigargin-induced GRP78 circulation.

Example 4 p62-dependent autophagolytic degradation of N-degron and ERAD-substrate NHK recognized by p62 by alginination of GRP78

GRP78 has been shown to autophagically clear the protein complex, including GRP78 and the misfolded ER client, in the ATE1-dependent manner at E19. GRP78 is a sepharone that stays in the ER that plays an important role in the response to misfolded proteins. The ER signal sequence is cleaved after ER transition, and the conserved pro-N-degron, E19, (Fig. 3A). Western blotting revealed that the arginylation of GRP78 was induced in HeLa cells by stimulation with thapsigargin in the presence of the proteasome inhibitor MG132 (Fig. 4a), and when the treatment time of thapsigargin was prolonged, the arginylation -GRP78 (R-GRP78) (Fig. 4B). This R-GRP78 spot was identical to the position of the spot containing p62 induced by thapsigargin. Spot formation was effectively blocked by the elimination of LC3, indicating that LC3 is important for spot formation of R-GRP78 and p62 induced by thapsigargin. Removal of p62 resulted in blocking of spot formation of R-GRP78, indicating that p62 is important for spot formation of R-GRP78 (Fig. 4B). The dependence of R-GRP78 on spot formation by p62 and LC3 indicates that R-GRP78 is associated with an autosome to ER stress. To confirm whether p62 could actually bind to R-GRP78, peptide pull down analysis was performed in Invitro. Peptide R11 (primary destabilizing moiety), E11 (secondary destabilizing moiety), and V11 (stabilized control) were used as HEK293 cell extracts and affinity lignans as p62. The 10-mer linker behind the X-peptide was derived from the GRP78 N-terminal residue starting at E19. As a result, p62 showed a strong binding force to R11 (GRP78) peptide and did not bind E11 (GRP78) and V11 (GRP78) peptides (FIG. 4C). The affinity of p62 for R-11 (GRP78) peptide was much lower compared to its affinity for R-11 (nsP4), indicating that variables other than the N-terminal residue determine affinity. ER stress induces arginylation of GRP78, arginylated R-GRP78 interacts with p62 in an N-end rule-dependent manner, and the result that the R-GRP78 / N-end rule interaction is involved in the elimination of misfolded / cohesive ER clients by selective self-predation. To demonstrate this, we used an ERAD substrate called NHK (null of Hong Kong) containing a mutation resulting in abnormal folding in the ER space as a variant of alpha 1-antitrypsin. Whether NHK interacts with p62 and R-GRP78 in cells was examined by immunofluorescence analysis. As a result, NHK overexpression induced the formation of p62 foci and the induction of GRP78 in HeLa cells (Fig. 4d). In most cells containing both proteins, the foci was found to be located fairly between NHK and p62 (upper panel) and between NHK and R-GRP78 (lower panel). Overexpression of NHK-GFP in p62-WT and p62-KO MEF and HEK293 cells transfected with control siRNA or siGRP78 and in the presence of thapsigargin to characterize the role of p62 and GRP78 in the stability of NHK under ER stress conditions Cyclohexamide tracing analysis showed NHK stabilization due to p62 and GRP78 elimination (Figure 4d, right). This indicates that the N-end rule interaction of p62 with GRP78 induced the degradation of misfolded / aggregated proteins by selective autophagy.

Example 5 Production of R-GRP78 Effect by Alloostery Control of p62 by N-end Rule Peptide

Oligomerization of p62 is known to be necessary for self-feeding. The binding of R-GRP78 and other N-end rule proteins to p62 has been shown to induce oligomerization and lead to autophagy. To this end, synthetic lysing capable of inducing the same effects as R-GRP78 and overexpressing full-length p62 and culturing in the presence / absence of various N-end rule peptides such as RA, FA or WA or control peptide AA, R or AR Non-reducing SDS-PAGE analysis using HEK293 cell extracts to investigate whether these N-end rule peptides can induce oligomerization of p62. Western blotting with p62 antibody revealed that RA effectively induced oligomerization of p62 and that the other N-end rule peptides FA and WA and the control groups AA, R and AR were ineffective (Fig. 5A). These results indicate that p62 oligomerization by N-end rule interaction is specific to the pathway of the N-end rule pathway. The effect of RA on p62 oligomerization was dose and time dependent (Figure 5B). Although the oligomerization of p62 precedes the transfer to the autogranite formation site, the interaction with LC3 facilitates the migration of the p62 agglomerate self-predators. Thus, ELISA analysis using p62 KO MEF cells transfected with p62-D3-coding plasmids lacking the p62 full-length or LIR and UBA regions investigated whether RA peptides enhance the interaction between LC3 and p62. The cell melts were confirmed by western blot to express the two types of p62 protein (Fig. 5B). ELISA assays were performed by incubating cell fusion with a GST-tagged LC3 immobilized on glutathione-coated plates. Experiments were performed using purified mouse monoclonal antibody against p62 and the absorbance (OD) at 450 nm was measured. Overall, p62 showed significant binding to LC3, while D3 did not. These ELISA results indicate that p62-LC3 interacted. In order to investigate whether the N-end rule peptide could enhance the interaction between p62 and LC3, N-end rule peptides RA, FA and WA and control peptide AR were incubated with increasing concentrations of p62 and LC3. As shown in FIG. 5B, the interaction of p62 and LC3 was doubled at about 10 mM of RA only, and other peptides were ineffective. These results are consistent with the results of RA (an aldinalizing pathway of the N-end rule pathway) and FA and WA, which were not effective in the oligomerization experiments of p62. These results also indicate that p62 oligomerization is required for binding to LC3 and migration to autophagy formation sites.

While the present invention has been described in connection with what is presently considered to be the preferred embodiments, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, .

All technical terms used in the present invention are used in the sense that they are generally understood by those of ordinary skill in the relevant field of the present invention unless otherwise defined. The contents of all publications referred to herein are incorporated herein by reference.

<110> SNU R & DB Foundation <120> Method for modulating autophagy mediated by p62 through ZZ domain          and its use <130> DP201211013P <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 440 <212> PRT <213> homo sapiens sequestosome-1 isoform 1 <220> <221> PROTEIN <222> (1) <223> sequestosome-1 isoform 1 <400> 1 Met Ala Ser Leu Thr Val Lys Ala Tyr Leu Leu Gly Lys Glu Asp Ala   1 5 10 15 Ala Arg Glu Ile Arg Arg Phe Ser Phe Cys Cys Ser Pro Glu Pro Glu              20 25 30 Ala Glu Ala Gla Ala Ala Gly          35 40 45 Ser Arg Val Ala Leu Phe Pro Ala Leu Arg Pro Gly Gly Phe Gln      50 55 60 Ala His Tyr Arg Asp Glu Asp Gly Asp Leu Val Ala Phe Ser Ser Asp  65 70 75 80 Glu Glu Leu Thr Met Ala Met Ser Tyr Val Lys Asp Asp Ile Phe Arg                  85 90 95 Ile Tyr Ile Lys Glu Lys Lys Glu Cys Arg Arg Asp His Arg Pro Pro             100 105 110 Cys Ala Gln Glu Ala Pro Arg Asn Met Val His Pro Asn Val Ile Cys         115 120 125 Asp Gly Cys Asn Gly Pro Val Val Gly Thr Arg Tyr Lys Cys Ser Val     130 135 140 Cys Pro Asp Tyr Asp Leu Cys Ser Val Cys Glu Gly Lys Gly Leu His 145 150 155 160 Arg Gly His Thr Lys Leu Ala Phe Pro Ser Pro Phe Gly His Leu Ser                 165 170 175 Glu Gly Phe Ser His Ser Arg Trp Leu Arg Lys Val Lys His Gly His             180 185 190 Phe Gly Trp Pro Gly Trp Glu Met Gly Pro Pro Gly Asn Trp Ser Pro         195 200 205 Arg Pro Pro Arg Ala Gly Glu Ala Arg Pro Gly Pro Thr Ala Glu Ser     210 215 220 Ala Ser Gly Pro Ser Glu Asp Pro Ser Val Asn Phe Leu Lys Asn Val 225 230 235 240 Gly Glu Ser Val Ala Ala Leu Ser Pro Leu Gly Ile Glu Val Asp                 245 250 255 Ile Asp Val Glu His Gly Gly Lys Arg Ser Ser Leu Thr Pro Val Ser             260 265 270 Pro Glu Ser Ser Thr Glu Glu Lys Ser Ser Ser Gln Pro Ser Ser         275 280 285 Cys Cys Ser Asp Pro Ser Lys Pro Gly Gly Asn Val Glu Gly Ala Thr     290 295 300 Gln Ser Leu Ala Glu Gln Met Arg Lys Ile Ala Leu Glu Ser Glu Gly 305 310 315 320 Arg Pro Glu Glu Gln Met Glu Ser Asp Asn Cys Ser Gly Gly Asp Asp                 325 330 335 Asp Trp Thr His Leu Ser Ser Lys Glu Val Asp Pro Ser Thr Gly Glu             340 345 350 Leu Gln Ser Leu Gln Met Pro Glu Ser Glu Gly Pro Ser Ser Leu Asp         355 360 365 Pro Ser Gln Glu Gly Pro Thr Gly Leu Lys Glu Ala Ala Leu Tyr Pro     370 375 380 His Leu Pro Pro Glu Ala Asp Pro Arg Leu Ile Glu Ser Leu Ser Gln 385 390 395 400 Met Leu Ser Met Gly Phe Ser Asp Glu Gly Gly Trp Leu Thr Arg Leu                 405 410 415 Leu Gln Thr Lys Asn Tyr Asp Ile Gly Ala Ala Leu Asp Thr Ile Gln             420 425 430 Tyr Ser Lys His Pro Pro Pro Leu         435 440

Claims (19)

Comprising contacting the cell with an N-end rule ligation that interacts with the p62 protein, the ZZ region of the p62 protein, or the 128 to 163 residues based on the sequence of SEQ ID NO: 1 in the ZZ region, Wherein the N-end rule ligation binds to the ZZ region of the p62 protein to induce oligomerization of the p62 thereby regulating autophagy, wherein the N-end rule ligation comprises Arg, Lys, His, Trp, Phe or Tyr Arg-Ala or Xaa-Ile-Phe-Ser-Thr-Ile-Glu-Gly-Arg-Thr-Tyr-Lys sequence as a peptide capable of binding to a target protein through an N- , Wherein Xaa is any one of Arg, His, Lys, Phe, Trp or Tyr and binds to the p62 ZZ region via the N-terminal residue.
delete The method according to claim 1,
The N-end rule ligation that interacts with the p62 ZZ region is an endoplasmic reticulum GRP78 that is N-terminal arginylated, and the N-terminal arginine residue of the protein binds to p62. Self - control method.
The method according to claim 1,
The p62 oligomerization is due to the exposure of the PB1 region of p62 by binding to p62 through the ZZ region of the N-end rule ligation, and by the oligomerization, the p62 binds to the denatured protein coagulum by self- Lt; RTI ID = 0.0 &gt; lysosomes. &Lt; / RTI &gt;
delete delete delete delete delete delete delete A method for screening an autoregulatory agent that modulates self-predation mediated by p62,
a) contacting a sample comprising the ZZ region of the p62 protein or a polypeptide comprising 128 to 163 residues of the ZZ region with a test substance;
b) detecting the interaction of the test substance with the protein or peptide in the ZZ region, wherein when the test substance is contacted with the test substance, the self-predation is increased or decreased , And the test substance is selected as a candidate for self-regulated control.
13. The method according to claim 12, wherein the test substance is an N-end rule ligation.
14. The method of claim 13, wherein the N-end rule ligation product has an N-end rule N-terminus of type-1 or type-2.
delete delete delete delete delete

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