KR20170023045A - Autophagy stimulation using p62 ZZ domain binding compounds or arginylated BiP for the prevention or treatment of neurodegenerative disease - Google Patents

Abstract

The present invention relates to treatment and prevention of neurodegenerative diseases through activities of autophage mediated by arginated BiP or ligand binding to p62 ZZ domain.

Description

{Autophagy stimulation using p62 ZZ domain binding compounds or arginylated BiP for the prevention or treatment of neurodegenerative disease}

The present invention relates to the treatment of neurodegenerative diseases through autophagy control by a ligand that binds to the ZZ domain of the p62 protein involved in autophage and an arginated Bip (arginylated Bip; R-Bip) protein.

The N-end rule (N-end rule) pathway is a proteolytic system using a specific protein N-terminus as a decomposition signal (Figs. 1 and 2). N-endrule decomposition signals include base residues having type 1 N-terminal arginine (Nt-Arg), lysine (Nt-Lys) and histidine (Nt-His), and type 2 phenylalanine (Nt-Phe), leucine. There are hydrophobic residues of (Nt-Leu), tryptophan (Nt-Trp), tyrosine (Nt-Tyr) and isoleucine (Nt-Ile) (Fig. 2). These N-terminal residues are bound to ligands (N-ligandra business card) of specific recognition elements (N-recognins; N-lecognin) (Fig. 3). The present inventors first discovered or cloned previously known N-lecognins, that is, UBR1, UBR2, UBR4, and UBR5, and revealed that they have a UBR box as a substrate recognition domain (Tasaki et al. 2005) (Tasaki et al. 2005). Fig. 3). In addition, it was revealed that the UBR box recognizes the substrate by binding type-1 N-endrule ligands (Nt-Arg, Nt-Lys, Nt-His) such as the N-terminal Arg residue and attaches the ubiquitin chain to the substrate. . UBR1 and UBR2 also have an N-domain, and the N-domain plays an important role in binding to type-2 N-end rule ligands (Nt-Trp, Nt-Phe, Nt-Tyr, Nt-Leu, Nt-Ile). It was also revealed that it was done (Sriram et al., 2011). The ubiquitinated substrate produced by the binding of N-lecognin to the N-end rule ligand is transferred to the proteasome and broken down into short peptides. In this process, specific N-terminal residues (Nt-Arg, Nt-His, Nt-Lys, Nt-Trp, Nt-Phe, Nt-Tyr, Nt-Leu, Nt-Leu) are N-recognin N-end It is an essential determinant for binding and an active ingredient of a ligand because it provides most of the hydrogen bonds required when targeting the rule substrate (Figs. 3c and 3d) (Sriram and Kwon, 2010).

Degenerative brain disease refers to a degenerative disease that develops in the brain as we age. Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), prion disease/human mad cow disease (Prion disease), multiple sclerosis, muscle It can be divided into atrophic lateral sclerosis (Lou Gehrig's disease). Most of the known degenerative brain diseases occur because the pathogenic protein metabolism does not occur smoothly and accumulates in the form of a coagulum, and thus belongs to the group of protein misfoldiong disorders. In particular, the mutant Huntington protein of Huntington's disease (Williams et al., 2008; Tsoi et al. 2012), the mutant alpha synuclein of Parkinson's disease (Martin et al. 2011), the mutant prion of human mad cow disease (Griffith, 1967), Lou Gehrig's disease The mutants of SOD1 and TDP-34 (Andersen et al. 2011) are rapidly transformed into coagulants and thus become toxic substances to neurons.

Among them, Parkinson's disease is a representative neurological degenerative disease that shows dopamine neuron loss and is accompanied by symptoms such as resting tremor, stiffness, slow motion, and postural instability. Drug treatment for Parkinson's disease is divided into symptomatic therapy and neuroprotective therapy. Symptomatic treatment drugs include levodopa, dopamine agonists, anticholinergic drugs, COMT inhibitors, and amantadine, and neuroprotective treatments include antioxidants and MAO-B inhibitors. Among them, the drug that exerts the strongest effect is levodopa, which is the most effective because dopamine precursors are metabolized to produce dopamine. Since levodopa is metabolized not only by L-dopa decarboxylase but also by catechol-O-methyltransferase (COMT), the combination of a COMT inhibitor can block metabolism by COMT in the blood, thereby increasing the effect and action time of levodopa. In the case of dopamine replacement therapy using levodopa, it is effective in many Parkinson's disease patients, but most of the patients show side effects such as movement abnormalities and changes in motor response after 5-10 years after administration, or autonomic nervous system dysfunction, posture. Instability, etc. develop, and levodopa treatment no longer improves.

A representative Huntington's disease remedy is tetrabenazine (TBZ). The mechanism of action of TBZ is not well known, but it depletes monoamines such as dopamine, serotonin, and norepinephrine by inhibiting the vesicular monoamine transporter in presynaptic neurons. It is also weak against the D2 dopamine receptor in the post-synaptic neuron, but it also functions as an antagonist. Clozapine is an atypical neuroleptic that effectively reduces movement disorders. Amantadine, which acts as an NMDA receptor antagonist, is also used to treat Huntington's disease. Huntington's disease is caused by excessive excitation of dopamine and glutamic acid receptors, so symptoms can be alleviated by inhibition of glutamic acid receptors by amantadine or rizol.

A common problem with Parkinson's disease and Huntington's disease treatment is that the treatment proceeds with symptomatic therapy and neuroprotective therapy, and since it is far from the elimination of the root cause of the disease, there are many limitations in terms of treatment effect and persistence. Therefore, there is a limitation in the treatment of diseases by supplying and controlling neurotransmitters without fundamentally removing the denatured protein coagulations that cause the disease.

Misfolded proteins that do not fold properly in cells aggregated when left unfolding for a long time and become cytotoxic substances such as blocking the proteasome or reducing other cellular functions, so ubiquitin ligase is used. By ubiquitination, it is delivered to the proteasome and decomposed (Fig. 4). In normal cells, this process is smooth and the damage caused by denatured proteins is minimized, whereas in the elderly neurons, denatured proteins accumulate because this process is slow, and these excess protein debris are converted back to coagulation. In addition, the neurons of patients suffering from degenerative brain diseases such as Huntington's disease, Parkinson's disease, human mad cow disease, and Lou Gehrig's disease have a strong property of transforming specific mutant proteins into coagulants, so they are not decomposed into the above-described proteasome. Because the proteasome has a narrow inner diameter of 13 angstroms, the denatured protein must be unfolded, but when the protein is coagulated, it cannot be resolved. The key technology in the present invention is to effectively remove the denatured protein coagulation that causes degenerative brain disease by simultaneously activating p62 and autophagy.

Autophagy is a major intracellular proteolytic system along with the ubiquitin-proteasome system. Autophagy is an essential proteolytic process to maintain cell homeostasis and genetic stability by degrading aging or malfunctioning organelles and damaged or poorly folded proteins. In particular, when the denatured protein coagulum accumulates in the cytoplasm, it becomes a cytotoxic substance, and thus autophagy must receive and decompose them (FIG. 4). At this time, p62/SQRSM1/Sequestosome-1 binds to the denatured protein and its coagulant and transfers them to the autophagosome (FIG. 4). When delivering a denatured protein to the autophagosome, p62 undergoes self-oligomerization as a key process. At this time, the denatured protein is concentrated together, reducing the volume and facilitating decomposition by autophagy. It is the PB1 domain that p62 mediates self oligomerization, and its regulatory mechanism is not well known. The denatured protein-p62 conjugate delivered to the autophagosome is decomposed by the enzyme of the lysosome as the autophagosome binds to the lysosome (FIG. 4). Through this mechanism, autophagy is important for maintaining cell homeostasis through intracellular fluctuations of damaged proteins and organelles.If autophagy is degraded, the accumulation of misfolded proteins is caused, resulting in neurodegenerative diseases. It may occur. The core technology of the present invention is to provide a method for activating intracellular autophagy.

Research on activating autophagy to treat degenerative brain diseases has been actively conducted. The modulator that normally inhibits autophage is mTOR, and the method of activating autophage using mTOR inhibitors is the most widely used. Specifically, in an AD animal model that uses rapamycin and overexpresses APP, amyloid beta (Ab) and tau (tau) were removed and cognition was improved at the same time (Caccamo et al., 2010), and AD overexpressing tau was improved. Tau was removed from the animal model (Rodriguez-Navarro et al., 2010), and the mutant a-synuclein protein coagulum overexpressed in the PD mouse model was removed (Webb et al., 2003). It was confirmed that huntingtin coagulation was effectively removed from the HD mice using CCI-779, a rapamycin-like substance, and improved animal behavior and cognition (Ravikumar et al., 2002). However, mTOR plays a very important role in a wide variety of intracellular pathways such as NF-kB, and therefore, those known to use mTOR as a drug target despite their excellent activity in removing degenerative protein coagulations of degenerative brain diseases. There are limitations in using autophage activators as therapeutic agents.

Studies using autophage activators as drug targets other than mTOR have also been reported. Specifically, in the HD mouse model, huntingtin coagulum was successfully removed using Rilmenidine, which activates autophage without passing through mTOR (Rose et al., 2010). In prion disease/human mad cow disease model mice, overexpressed mutant prion (PrPSc) coagulum was successfully removed using lithium, which activates autophagy as an inositol monophosphatase inhibitor (Heiseke et al., 2009). . The mutant alpha-synuclein protein coagulum overexpressed in the PD mouse model was removed using lithium (Sarkar et al., 2005), and the mutant SOD1 G93A overexpressed in the brain of the amyotrophic lateral sclerosis (ALS) mouse model. The coagulum was removed (Feng et al., 2008; Pizzasegola et al., 2009). Trehalose, a known substance extracted from plants, is known to activate autophagy without passing through mTOR. Using Trehalose, mutant huntingtin in the brain of the HD mouse model (Sarkar et al., 2007), improved motor performance and longevity in the PD mouse model (Tanaka et al., 2004), and A30P and overexpressed in PD model cells and The A53T mutant alpha synuclein coagulum was removed (Sarkar et al., 2007). Finsetin, a kind of natural extract, flavone, is known to activate autophagy through TORC1 and AMPK. It was confirmed by using finsetin to relieve or improve symptoms in an animal model of degenerative brain disease (Maher et al., 2011). Substances that do not pass through mTOR or activate autophagy through two or more targets including mTOR are also limited in use as therapeutic agents as these targets play various roles in the intracellular signaling system.

As described above, there is no treatment for most degenerative brain diseases such as Huntington's disease, and mTOR inhibitor, a compound most commonly used as an autophagy activator, regulates overall gene expression in cells in response to stimulation from various external environments in addition to controlling autophagy. It is not suitable as a therapeutic agent because it plays a wide range of roles. In the present invention, by activating p62, which directly transfers the denatured protein coagulum to the autophagosome, there is provided a technology for removing the denatured protein coagulum, which is a causative factor of degenerative brain diseases.

The mechanism of action and key technologies of the present invention are summarized in FIG. 1. Specifically, mutant huntingtin or alpha synuclein is transformed into a water-insoluble denatured body (misfolded) and then coagulated together to grow into an oligomer-shaped coagulum (①). These denatured bodies act as cytotoxic substances in nerve cells and grow further and grow into large oligomers (②) or fibrillar coagulations (③), and ultimately, inclusion bodies are formed (step ④). . In the above process, in young neurons, endoplasmic reticulum chaperons such as BiP produce a large amount of Nt-Arg through N-terminal arginization (⑤) by ATE1 R-transferase, and then the arginized BiP (R-BiP) comes out into the cytoplasm. It binds to denatured Huntington or Alpha Synuclein (⑥). Nt-Arg of R-BiP binds to the ZZ domain of p62 as a ligand (⑦). Due to the binding, the normally closed inactive form of p62 is changed to an open form, and structural activation is induced (step ⑧), thereby exposing the PB1 and LC3-binding domains. Based on the oligomerization (⑨) by the PB1 domain, it binds to the Huntington and alpha-synuclein protein coagulants and is concentrated into a coagulant capable of autophagy degradation, that is, a p62 body (body or sequestosome) (⑩). After that, autophagy targeting (⑪) and lysosomal protein degradation are completed through the binding of p62 with LC3 protruding from the autophagosome membrane. Young neurons have strong autophagy proteolysis consisting of steps ⑤-⑪, so cytotoxic protein coagulants (①-⑤) do not accumulate, but aging neurons have weakened autophagy protein degradation in steps ⑤-⑪. As the solidified body (①-⑤) accumulates, it falls into a vicious cycle. In this study, by using a low-mass ligand of the p62 ZZ domain, p62 was artificially activated (⑫, ⑬), and thus Huntington and alpha synuclein protein coagulum were effectively removed. Specifically, p62 bound to the ligand through step ⑫ promotes p62-R-BiP-denatured protein oligomerization (⑨) and autophagy coagulation (⑩), and also through step ⑬, the ligand-p62 conjugate is autophagy. It acts as an activator (⑭) and promotes the synthesis of LC3 and the conversion of LC3-I to LC3-II to promote autophagosome formation (⑮).

As a result of literature investigation, p62 is the first-in-class target of the autophagy activator proposed by the present inventors (Figs. 1 and ⑧). In addition, there are no examples of p62 being studied as a drug target for the development of an autophagy activator or the removal of protein coagulation in degenerative brain diseases. Therefore, as a therapeutic agent for neurodegenerative diseases, there is a need to develop new therapeutic agents and methods targeting substances involved in autophagy.

Accordingly, the present inventors have endeavored to develop a neurodegenerative disease prevention and therapeutic agent using a substance involved in autophagy activity. As a result, the ligand binding to the p62 ZZ domain binds to LC3 and activates the autophagy to Huntington and Alpha Syne. By effectively removing the Klein protein coagulant and the like, the present invention was completed by confirming a pharmaceutical composition for treatment or prevention of neurodegenerative diseases and a control method thereof.

It is an object of the present invention to provide a pharmaceutical composition for preventing and treating neurodegenerative diseases containing a ligand that binds to the p62 ZZ domain as an active ingredient.

Another object of the present invention is to provide a health functional food for preventing and improving neurodegenerative diseases containing a ligand that binds to the ZZ domain of the p62 protein as an active ingredient.

An object of the present invention is to (1) induce p62 oligomerization and structural activation, (2) activate autophage, and (3) denatured protein comprising the step of treating cells with a ligand that binds to the ZZ domain of p62. It is to provide a way to remove the coagulum.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing and treating neurodegenerative diseases containing a ligand that binds to the ZZ domain of the p62 protein as an active ingredient.

In addition, the present invention provides a health functional food for preventing and improving neurodegenerative diseases containing a ligand that binds to the ZZ domain of the p62 protein as an active ingredient.

Further, comprising the step of treating a cell or p62 protein with a ligand binding to the ZZ domain of p62 (1) inducing p62 oligomerization and structural activation, (2) enhancing the binding of p62 to LC3, (3) Provides a method of enhancing the delivery of p62 to the autophagosome, (4) activating autophagy, and (5) removing denatured protein coagulation.

The present invention relates to a p62 activity modulator, an autophagy activator, and a therapeutic agent for degenerative brain diseases, using a ligand that binds to the ZZ domain of p62 as an active ingredient. The p62 ZZ domain ligand of the present invention is an X-11 peptide (X=Nt-Arg, Nt-Phe, Nt-Tyr, Nt-Trp), Arg-Ala(RA), Trp-Ala(WA), ZZ-L1 and ZZ-L2 digests, and N-terminal arginated BiP (Nt-arginylated BiP; R-Bip). In particular, the active ingredients of the X-11 peptide, Arg-Ala, Trp-Ala, and R-BiP include Nt-Arg, Nt-Phe, Nt-Tyr and Nt-Trp attached to the N-terminus. Therefore, the ligand binding to the p62 ZZ domain can be usefully used as an active ingredient of a composition for preventing or treating neurodegenerative diseases through autophagy control.

1 is a diagram showing a drug target and mechanism proposed in the present invention.
2 is a schematic diagram showing an N-terminal proteolysis pathway. In this pathway, N-terminal residues such as Nt-Arg act as decomposition ligands and are recognized by N-lecognin and then bound, and the binding induces decomposition of the substrate. In the existing pathway, Nt-Arg serves to enhance targeting of the substrate to the proteasome by inducing ubiquitination. In the present invention, it was found that Nt-Arg not only activates p62 by binding to p62 of autophage, but also activates the autophagy pathway.
3 is a diagram showing the structure and sequence of N-lecognins in the N-terminal proteolytic pathway. (a) A schematic diagram of the primary structure of the UBR box protein group (UBR1-UBR7). They have a UBR box that can be combined with Nt-Arg, and it was found that UBR1, UBR2, UBR4, and UBR5 can actually combine with Nt-Arg. (b) A diagram comparing the sequences of UBR boxes. (c, d) Crystal structure of UBR box of UBR1.
4 is a schematic diagram showing how ubiquitin-proteasome (UPS), chaperone-mediated autophagy, and macroautophagy cooperate with each other to remove denatured proteins.
5 is a diagram showing that p62 can be selectively combined with N-ligands such as Nt-Arg and Nt-Trp as a novel N-lecognin of N-endrule. (a) In order to discover new N-lecognin that binds to N-degron, a schematic diagram of what proteins are attached in animal tissue extract by synthesizing a peptide with N-degron attached to the N-terminus. (b, c) The results of analyzing the proteins obtained by performing X-peptide pulldown using rat testis extracts by silver staining method. (d) The results of iTRAQ (Isobaric tags for relative and absolute quantitation) linked to mass spectrometry on proteins obtained by the X-peptide pull-down technique. (ei) The results of immunoblot analysis of proteins obtained using the X-peptide pull-down technique.
6 is a diagram showing the results of measuring the binding strength of p62 to Nt-Arg (Kd, 40 nM) and Nt-Phe (Kd, 3.4 mM) using Biacore assay.
7 is a diagram showing the result of comparing p62 with the N-endrule ligand through the ZZ domain, and comparing the primary structure of the ZZ domain with the UBR box. (a, c, e, g, I) Schematic diagram showing various pieces of p62. (b, d, f, h, j) These are the results of X-peptide pull-down analysis with p62 fragments. l, p62 ZZ domain and the result of comparative analysis of the primary structure of the UBR box.
8 is a diagram showing that Arg-Ala induces self-oligomerization of p62 and increases binding of p62 to LC3. (ac) Analysis of self oligomerization of p62. In order to investigate whether the N-ligand Arg-Ala induces self-oligomerization and aggregate formation of p62 when bound to the p62 ZZ domain, a full-length p62-expressing HEK293 cell lysate was used in vitro using non-reducing SDS-PAGE. In vitro oligomerization assays were performed. (d) The results of in vitro p62 oligomerization analysis using a p62 ZZ mutant that cannot bind to the Arg-11 peptide. (e) In order to investigate whether the PB1 domain of p62 is involved in the aggregate of p62, the aggregation ability of the D69A mutant was evaluated. (fh) To confirm the effect of Arg-Ala on the binding of p62 and LC3, ELISA analysis was performed. (i) A schematic diagram of inducing structural activation of p62 by binding of Nt-Arg to the p62 ZZ domain.
9 is a diagram comparing the primary protein sequences of endoplasmic reticulum chaperons. When these proteins enter the endoplasmic reticulum after translation, the signal peptide is cut off, which exposes the N-ligand to the N-terminal residue of the mature protein (marked in a box). When denatured proteins accumulate in the cytoplasm, these chaperons migrate to the cytoplasm through N-terminal arginization.
10 is a diagram showing the discovery of endoplasmic reticulum proteins that obtain Nt-Arg as an N-ligand through N-terminal arginization. (a) Schematic diagram explaining the process of N-terminal arginization by ATE1 R-transferase when the cytoplasm is exposed to stress, such as accumulation of denatured proteins. (b) Results of a peptide binding assay to measure the specificity and avidity of the R-BiP antibody. (c) Dot blot analysis using R-BiP antibody. (d) After overexpressing ATE1 R-transferase isomerase, it was confirmed that R-BiP formation was induced using the R-BiP antibody. (e) When ATE1 was knocked down using siRNA, R-BiP It was confirmed that the formation of is reduced.
11 is a diagram illustrating that BiP, CRT, and PDI mediate N-terminal arginization by ATE1. (a) Arginylation was assayed by expressing Ub-X-BiP-flag (X=Glu or Val). (b) Schematic diagram of generating the recombinant protein Ub-X-Bip-myc/his structure. (c) Using this structure, it was confirmed that X-BiP was arginized to Nt-Glu19. (d) R-BiP was not produced in ATE1 knockout cells. (e) Using thapsigargin, which causes ER stress, R-BiP is not caused by ER stress, but is a product of an enzymatic reaction caused by ATE1 R-tranferase. (f) Using the cell fractionation technique, it was revealed that the R-BiP produced in this way migrates to a large amount of cytoplasm. (g) It was confirmed that R-BiP transferred to the cytoplasm using the cycloheximide proteolysis technique was not relatively degraded compared to the non-arginated BiP in the endoplasmic reticulum. (h) As a result of investigating whether CRT and PDI also undergo N-terminal arginization by ATE1, it was confirmed that R-CRT and R-PDI were generated when ATE1 isoenzyme was overexpressed.
12 is a diagram illustrating that the foreign DNA of the cytoplasm induces R-BiP production. (a, b) The generation of R-BiP is specifically induced when double stranded DNA (dsDNA) is introduced into the cytoplasm. (b, c) Similar to R-BiP, R-CRT was confirmed to be induced when dsDNA was introduced into the cytoplasm. (d) Using the cell fractionation technique, it was observed that R-BiP induced by dsDNA migrates to the cytoplasm. (eg) Using poly(dA:dT) that mimics a pathogen containing DNA (virus or bacteria), R-BiP, R-CRT, P-PDI, etc. are generated as part of the innate immune response against invading DNA. Confirmed. (f) At this time, it was confirmed that the autophagy action was activated together.
13 is a diagram confirming that R-BiP is delivered to the autophagosome of the autophage along with p62. (a) Using immunostaining, it was confirmed that R-BiP migrated to the cytoplasm and then gathered in cell structures such as puncta. (bd) The R-BiP spot was colocalized with the p62 spot (a, c), and it was confirmed that it was also co-located with LC3 (bd). (e) It was confirmed that BiP was transferred to the LC3-positive autophagosome even in the mouse embryonic heart. (fh) When ATE1 or BiP was knocked down using siRNA, R-BiP could not move to the autophagosome. The p62 knockout showed similar results.
14 is a diagram confirming that Nt-Arg is required when R-BiP is delivered to the autophagosome. (a) Schematic diagram of production of X-BiP-GFP by overexpressing Ub-X-BiP-GFP recombinant protein (X = Arg, Glu, or Val) in cells. (b) Using immunostaining techniques, the status of migration by autophagy is investigated. (c) R-BiP was delivered to autophagy autophagosomes. On the other hand, Val-BiP (Nt-Glu19 could not be a substrate for arginization because it was substituted with Val) could not be delivered to autophagy autophagosomes because there was no Nt-Arg. (d) Similar results were obtained when the intracellular location of R-BiP was compared with LC3 spots. (e) After removing most of BiP ^{and overexpressing Ub-X-BiP Δ} (Fig. 14A), ^{leaving only residues 19-124, the intracellular location of X-BiP ΔD} was investigated using an immunostaining technique. (f) Similar results were obtained when the intracellular locations of X-BiP and LC3 were examined by immunostaining.
15 is a diagram confirming that Nt-Arg of R-BiP directly binds to the p62 ZZ domain. (a, b) X-peptide pull-down analysis to investigate whether the Nt-Arg of R-BiP binds to the p62 ZZ domain. (c, d) R-BiP peptide pulled down p62 from the cell extract, but E-BiP or V-BiP peptide did not pull down p62. (e) Schematic diagram of dividing p62 into small pieces to investigate where Nt-Arg of R-BiP binds to p62. (f) As a result of performing a pull-down experiment with the p62 fragments and R-Bip peptide, it was shown that Nt-Arg of R-BiP binds to the p62 ZZ domain. (g) Only the p62 ZZ domain was cut out and named p62-ZZ83-175-GST and p62-ZZ(D129A)83-175. (h) GST pull-down analysis was performed using the p62 fragments. (i) When p62-ZZ83-175-RFP and ubiquitin-X-Bip19-124-GFP were co-expressed in MEF cells, Arg18-Bip19-124 and p62-ZZ83-175-RFP showing puncta formation Although the co-migration form was shown, Val19-Bip19-124-GFP and p62-ZZ83-175-RFP were not in the form of spots and co-migration.
16 is a diagram confirming that R-BiP is decomposed by autophagy depending on p62. (a, b) Ub-X-BiP ^Δ -GST was prepared and overexpressed in +/+ and p62-/- cells, and then the degradation of BiP was analyzed. (c) R-BiP of Ub-X-BiP ^Δ -GST was accumulated without degradation in ATG5-/- cells lacking the autophagy process. (d, e) When a quantitative analysis of cycloheximide proteolysis was performed with Ub-R-BiP-myc/his, it was confirmed that R-BiP was remarkably stabilized in ATG5-/- cells.
FIG. 17 is a diagram confirming that R-BiP is produced by cytoplasmic ubiquitinated proteins and then binds to cytoplasmic denatured proteins and transfers them to autophagy. (a) HeLa cells were treated with poly(dA:dT) and then immunoblot was performed. As a result, when R-BiP production was induced, intracellular protein groups bound to ubiquitin were also accumulated. (b) It was confirmed that some of the ubiquitinated intracellular proteins were transferred to the p62 body in the form of spots, and co-located with R-BiP and p62 in this structure. (c) After treating cells with various chemicals, the formation of R-BiP was investigated using an immunoblot technique. As a result, proteasome inhibitors commonly induced R-BiP formation. (d, e) It is a result that the production of R-BiP, R-CRT, and R-PDI is more strongly induced when simultaneously treatment with a proteasome inhibitor and thapsigargin, which causes stress in the endoplasmic reticulum. (f) As a result of promoting the formation of intracellular denatured proteins by treatment with geldenamycin, an inhibitor of Hsp90, it was confirmed that the formation of R-BiP was induced. (g) When overexpressing YFP-CL1, a model substrate that is denatured by self-folding, it was confirmed that R-BiP was directly bound. (h, i) It was confirmed that YFP-CL1 was delivered to autophage vacuoles using immunofluorescence staining, and co-located with R-BiP and p62 in the structure.
18 is a diagram confirming that ZZ-L1 and ZZ-L2, which are small compounds, increase p62 oligomerization and binding to LC3. (a, b) ZZ-L1 was treated with p62 overexpressing cell extracts, and then the degree of p62 aggregate was measured using an in vitro oligomerization assay. (c) As a result of investigating whether ZZ-L1 activates p62 in a similar way to the experiment that increased the binding of p62 to LC3 using Arg-Ala, ZZ-L1 was concentration dependent (0, 10, 25, 100, 500). , 1000 μM) confirmed to increase the binding with LC3. (df) To confirm whether ZZ-L1 and ZZ-L2 induce p62 p62 formation, it was observed using immunofluorescence confocal microscopy.
19 is a diagram showing that ZZ-L1 and ZZ-L2 are autophagy activators. (a) Using immunofluorescence staining, it was revealed that the ZZ ligand promoted the formation of LC3 autophage spots in addition to p62 autophage spots in HeLa cells. This indicates that the ZZ ligands function as autophagy activators. (b) Western blot was run to investigate the effect of ZZ-L1 and ZZ-L2 on autophagy. (c) The increase of LC3 and the formation of LC3-II were induced by the ZZ compounds ZZ-L1 and ZZ-L2, which transfected si-control with blood, but the effect was inhibited by p62 knock down. . (d) Similar experiments using +/+ and p62-/- cells showed that ZZ-L1 (5 mM) did not act as an autophagy activator in p62-/- cells. (e) ZZ ligand-treated cells were treated with hydroxychloroquine (HCQ), an autophagy inhibitor, and then the amount of LC3 was investigated using an immunoblot. (f) Using the HeLa cells stably transformed with RFP-GFP-LC3 in which acid-sensitive GFP and insensitive RFP were combined, autophagy dynamic analysis was performed in the same manner as used above. (g) It is a schematic diagram showing that p62 activated by the ZZ ligand functions as an autophagy activator.
20 is a diagram showing that p62 bound to ZZ ligand activates autophage regardless of mTOR. (a) HeLa cells were treated with ZZ-L1 and a cycloheximide proteolysis assay was performed, indicating that ZZ-L1 promotes the degradation of p62. (b, c) In order to compare the efficacy and mechanism of ZZ ligand and rapamycin as autophagy activators, the results of comparing the production of LC3 and the conversion of LC3-II after treating HeLa cells by concentration or by time. (d) Rapamycin is an inhibitor of mTOR (mammalian target of rapamycin), and when mTOR is inhibited, key regulators of autophagy (ULK, Beclin, etc.) are activated, through the synthesis of LC3 and conversion to the LC3-II form. Schematic diagram showing that the production of autophagosomes is increased. (e) After treatment with ZZ ligand or rapamycin in HeLa cells, the phosphorylation of p70S6K regulated by mTOR was investigated. (f) Whether the ZZ ligand promotes the migration of ubiquitinated intracellular proteins to autophagy was investigated by immunofluorescence staining. It was confirmed that 5 mM ZZ-L1 induced ubiquitinated proteins into autophagy vacuoles. In addition, when the autophagy inhibitor hydroxychloroquine was treated, it was confirmed that ubiquitinated proteins accumulate in the autophage blisters.
21 is a diagram showing that the ZZ ligand removes the mutant huntingtin protein coagulum. (a) Schematic diagram of wild-type huntingtin (HDQ25-GFP) and mutant huntingtin (HDQ103-GFP; CAG repeats 103 times). (b) The results of investigating the remaining huntingtin proteins in cells using 10 mM ZZ-L1 or 1 mM rapamycin in cells overexpressing the huntingtin proteins, and using a dot blot assay. (c) HeLa cells overexpressing the HDQ103-GFP protein coagulant were treated with 10 mM ZZ ligand, followed by immunofluorescence staining. (d) After dividing the cells into an insoluble fraction (including coagulant) in 0.5% Triton X-100, immunoblot was performed, and as a result of performing an immunoblot, in the coagulant fraction treated with ZZ-L1. It is confirmed that huntingtin denatured protein is effectively removed. (e) HeLa cells were treated with ZZ-L1, ZZ-L2, and rapamycin, followed by immunoblot technique. (f) +/+ and ATG5-/- cells were treated with 10 mM ZZ ligand and 1 mM rapamycin, and then immunoblot was performed.
FIG. 22 shows R-BiP and p62 co-located in the inclusion body formed by the HDQ103-GFP huntingtin protein coagulant. (a, b, c) A diagram showing the results of immunofluorescence staining after treatment with 10 mM ZZ ligand in HeLa cells overexpressing the HDQ103-GFP protein coagulum.
23 is a diagram showing the results of comparing the chemical properties of p62-ZZ1 (ZZ-L1), p62-ZZ2 (ZZ-L2), and rapamycin.
24 is a diagram showing the results of comparing the pharmacokinetic profile of p62-ZZ1 (ZZ-L1) in mice.

Hereinafter, the present invention will be described in detail.

The present invention provides a method of activating the function of p62 by containing a ligand that binds to the ZZ domain of p62 as an active ingredient.

In addition, the present invention provides an autophage activator using a ligand that binds to the p62 ZZ domain as an active ingredient.

In addition, the present invention provides a method of removing a denatured protein coagulum using a ligand that binds to the p62 ZZ domain as an active ingredient.

Furthermore, the present invention provides a pharmaceutical composition for preventing and treating neurodegenerative diseases containing a ligand that binds to the p62 ZZ domain as an active ingredient.

The p62 has an amino acid sequence set forth in SEQ ID NO: 1. The ZZ domain is characterized in that it comprises residues 128 to 163 of the amino acid sequence of the p62 protein represented by SEQ ID NO: 1.

The ligand includes the following peptides. The active ingredient that directly binds to the p62 ZZ domain in the ligands described in SEQ ID NOs: 2 to 9 below is Nt-Arg (Formula 1), Nt-Phe (Formula 2), Nt-Trp (Formula 3), or Nt-Tyr It is the N-terminal residue of (Formula 4):

SEQ ID NO: 2: Arg-Ala;

SEQ ID NO: 3: Phe-Ala;

SEQ ID NO: 4: Trp-Ala;

SEQ ID NO: 5: Tyr-Ala;

SEQ ID NO: 6: Arg-Ile-Phe-Ser-Thr-Ile-Glu-Gly-Arg-Thr-Tyr-Lys (R-11);

SEQ ID NO: 7: Trp-Ile-Phe-Ser-Thr-Ile-Glu-Gly-Arg-Thr-Tyr-Lys (W-11);

SEQ ID NO: 8: BiP protein (BiP); And

SEQ ID NO: 9: Bip protein (R-BiP) in which the N-terminal Glu19 of the BiP protein is arginized.

[Formula 1]

[Formula 2]

[Formula 3]

[Formula 4]

In addition, the ligand is the following Chemical Formula 5 (ZZ-L1; 2-(3,4-bis(benzyloxy)benzylamino)ethanol) or Chemical Formula 6 (ZZ-L2; 1-(3,4-bis(benzyloxy)phenoxy)- 3-(isopropylamino)propanol). In particular, p62-ZZ2 is a material known as NCI314953.

[Formula 5]

[Formula 6]

In addition, the ligand binds to the p62 ZZ domain, activates the PB1 domain and the LIR domain of the p62 protein to induce p62 oligomerization and aggregates, and increases autophagosome formation through p62 aggregate induction. It is done. Through the above process, the denatured protein coagulum is smoothly removed (see FIG. 1).

The neurodegenerative diseases include Lyme borreliosis, Fatal familial insomnia, Creutzfeldt-Jakob Disease (CJD), multiple sclerosis (MS), dementia, Alzheimer's disease, epilepsy, Parkinson's disease, stroke, Huntington's disease, Picks disease and amyotrophic lateral sclerosis (ALS). Is selected from the group.

In a specific embodiment of the present invention, the present inventors have identified that p62 is N-lecognine capable of binding to Nt-Arg, Nt-Phe, Nt-Trp, and Nt-Tyr (see Fig. 5), and in particular p62 is It was confirmed that the binding power to Nt-Arg was excellent (see FIG. 6), and it was confirmed that the ZZ domain of p62 is involved in the binding (see FIG. 7). It was confirmed that the N-ligand Arg-Ala induced auto-oligomerization and aggregate formation of p62 (see FIG. 8D), and increased the binding of p62 and LC3 (see FIG. 8F). As a result of the discovery of endoplasmic reticulum proteins that acquire Nt-Arg through N-terminal arginization, N-terminal arginylated BiP (R-BiP), calreticulin (R-CRT), and protein disulfide isomearase (R-PDI) were obtained. It was confirmed (see FIG. 9), and they confirmed that N-terminal arginization was mediated by ATE1 (see FIG. 10). In addition, it was confirmed that foreign DNA in the cytoplasm induces R-BiP production (see FIG. 12), and it was confirmed that the R-BiP was transferred to the autophagosome like p62 (see FIG. 13). It was confirmed that R-BiP was decomposed by autophagy and became ubiquitinated in a p62-dependent manner (see Fig. 16). Digestive compounds (ZZ-L1 and ZZ-L2) structurally similar to Nt-Trp were discovered (see FIG. 18), and it was confirmed that the ZZ-L1 and ZZ-L2 increased the formation of intracellular autophagosomes (Fig. 19). In addition, it was confirmed that the ZZ compound (ligand) fuses the autophagosomes and lysosomes formed by them (see Fig. 20), and removes the denatured protein including the Huntington coagulant through autophagy activation (Figs. 21 and Figs. 22).

Accordingly, the ligand binding to the p62 ZZ domain of the present invention can be usefully used as an active ingredient in a pharmaceutical composition for preventing or treating neurodegenerative diseases through autophagy control by inducing oligomerization and aggregation of p62.

The therapeutic agent or pharmaceutical composition according to the present invention may be formulated in a suitable form together with a generally used pharmaceutically acceptable carrier. "Pharmacologically acceptable" refers to a composition that is physiologically acceptable and, when administered to a human, does not usually cause allergic reactions such as gastrointestinal disorders, dizziness, or similar reactions. For example, the pharmaceutically acceptable carrier includes, for example, a carrier for parenteral administration such as water, suitable oil, saline, aqueous glucose and glycol, and may further include a stabilizer and a preservative. Suitable stabilizers are antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid. Suitable preservatives are benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. In addition, the composition according to the present invention is a suspension agent, a solubilizer, a stabilizer, an isotonic agent, a preservative, an adsorption inhibitor, a surfactant, a diluent, an excipient, a pH adjuster, a painless agent, a buffer agent, if necessary depending on the administration method or formulation. , Antioxidants, etc. may be appropriately included. Pharmaceutically acceptable carriers and formulations suitable for the present invention, including those exemplified above, are described in detail in Remington's Pharmaceutical Sciences, the latest edition.

The composition of the present invention is prepared in unit dosage form by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily carried out by a person having ordinary knowledge in the technical field to which the present invention belongs, or It can be manufactured by being enclosed in a multi-volume container. In this case, the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or may be in the form of powder, granules, tablets, or capsules.

The compound represented by [Chemical Formula 5] or [Chemical Formula 6] of the present invention can be used in the form of a pharmaceutically acceptable salt, and the salt is an acid addition salt formed by a pharmaceutically acceptable free acid. useful. Acid addition salts include inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid, and aliphatic mono and dicarboxylates, phenyl-substituted alkanoates, hydroxy alkanoates and alkanes. It is obtained from non-toxic organic acids such as dioates, aromatic acids, aliphatic and aromatic sulfonic acids. Such pharmaceutically non-toxic salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide, ioda Id, fluoride, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate , Sebacate, fumarate, maleate, butine-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methyl benzoate, dinitro benzoate, hydroxybenzoate, me Toxibenzoate, phthalate, terephthalate, benzenesulfonate, toluenesulfonate, chlorobenzenesulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, hydroxybutyrate, glycolate, Maleate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate or mandelate.

The acid addition salt according to the present invention is a conventional method, for example, by dissolving a benzofuran-based compound represented by [Chemical Formula 5] or [Chemical Formula 6] in an excessive amount of an aqueous acid solution, and dissolving the salt in a water-miscible organic solvent, eg For example, it can be prepared by precipitation using methanol, ethanol, acetone or acetonitrile. In addition, the mixture may be dried by evaporating a solvent or an excess of acid, or may be prepared by suction filtration of the precipitated salt.

In addition, a pharmaceutically acceptable metal salt can be made using a base. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the undissolved compound salt, and evaporating and drying the filtrate. At this time, it is pharmaceutically suitable to prepare sodium, potassium or calcium salt as the metal salt. In addition, the corresponding silver salt is obtained by reacting an alkali metal or alkaline earth metal salt with a suitable negative salt (eg, silver nitrate).

The method of administering the pharmaceutical composition of the present invention can be easily selected according to the dosage form, and can be administered to mammals such as livestock and humans by various routes. For example, powders, tablets, pills, granules, dragees, hard or soft capsules, liquids, emulsions, suspensions, syrups, elixirs, external preparations, suppositories, sterile injectable solutions, etc. It may be administered orally or parenterally, and parenteral administration may be particularly desirable.

Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, etc. may be used as the non-aqueous solvent and suspension. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin, glycerol, gelatin, and the like can be used.

Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, suppositories, and the like.

Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, etc. may be used as the non-aqueous solvent and suspension. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin, glycerol, gelatin, and the like may be used.

Solid preparations for oral administration include tablets, patients, powders, granules, capsules, troches, and the like, and these solid preparations include at least one or more benzofuran-based compounds represented by Formula 5 or Formula 6 of the present invention. It is prepared by mixing an excipient such as starch, calcium carbonate, sucrose, lactose, or gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, liquid solutions, emulsions, or syrups.In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as humectants, sweeteners, fragrances, and preservatives are included. I can.

The dosage of the pharmaceutical composition of the present invention may vary depending on the patient's weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and severity of disease, but the effective dosage is usually For adults (60 kg), it is about 1 ng to 10 mg/day, especially about 1 g to 1 mg/day. Since the dosage is variable according to various conditions, it is apparent to those skilled in the art that the dosage may be varied, and therefore, the dosage does not limit the scope of the present invention in any way.

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

In addition, the present invention provides a health functional food for preventing and improving neurodegenerative diseases containing a ligand that binds to the ZZ domain of the p62 protein as an active ingredient.

The ligands are Arg-Ala (SEQ ID NO: 2), Phe-Ala (SEQ ID NO: 3), Trp-Ala (SEQ ID NO: 4), Tyr-Ala (SEQ ID NO: 5), R-11 (SEQ ID NO: 6), W- 11 (SEQ ID NO: 7), BiP (SEQ ID NO: 8), and R-BiP (SEQ ID NO: 9) is selected from the group consisting of, the active ingredient of the ligand is an N-terminal residue, the Nt-Arg (Formula 1 ), Nt-Phe (Chemical Formula 2), Nt-Trp (Chemical Formula 3), and Nt-Tyr (Chemical Formula 4).

In addition, the ligand is ZZ-L1 (Chemical Formula 5), or ZZ-L2 (Chemical Formula 6; NCI314953).

There is no particular limitation on the type of food to which the compound of the present invention is added, and includes all health functional foods in the usual sense.

The compound of the present invention may be added to food as it is or may be used together with other foods or food ingredients, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be appropriately determined depending on the purpose of use (for prevention or improvement). In general, the amount of the compound in the health functional food may be added in an amount of 0.1 to 90 parts by weight of the total food weight. However, in the case of long-term intake for the purpose of health and hygiene or for the purpose of health control, the amount may be less than the above range, and there is no problem in terms of safety, so the active ingredient may be used in an amount above the above range.

When the health food composition according to the present invention is a beverage composition, there is no particular limitation on other ingredients other than containing the compound as an essential ingredient in the indicated ratio, and various flavoring agents or natural carbohydrates, etc., as in ordinary beverages, as additional ingredients. It may contain. Examples of the above-described natural carbohydrates include monosaccharides such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose, and the like; And polysaccharides, for example, common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavoring agents (taumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 10 g per 100 of the composition of the present invention.

In addition, the health food composition according to the present invention includes various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and heavy weight agents (cheese, chocolate, etc.), pectic acid and salts thereof, Alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonates used in carbonated beverages, and the like. In addition, it may contain flesh for the manufacture of natural fruit juice and fruit juice beverages and vegetable beverages.

These components may be used independently or in combination. The proportion of these additives is not limited, but is generally selected from 0.1 to about 20 parts by weight per 100 parts by weight of the compound of the present invention.

Therefore, the ligand binding to the p62 ZZ domain of the present invention can be usefully used as an active ingredient in a health functional food for preventing or improving neurodegenerative diseases through autophagy control by inducing oligomerization and aggregation of p62.

In addition, the present invention induces the oligomerization of p62 by binding the ligand to the ZZ domain of the p62 protein, comprising the step of treating a ligand that binds to the ZZ domain of the p62 protein to cells or p62 protein, and binding of p62 to LC3. A method of increasing p62 activity including a process of increasing it, a method of enhancing the delivery of p62 to the autophagosome by binding a ligand to the p62 ZZ domain, and activating autophagy by binding the ligand to the p62 ZZ domain. Method, a method of activating the process of transferring a denatured protein coagulum to an autophagosome by binding a ligand to the p62 ZZ domain, and a denatured protein coagulant by a lysosome by binding the ligand to the p62 ZZ domain to activate autophagy Provides a way to increase decomposition.

Accordingly, the ligand binding to the p62 ZZ domain of the present invention can be usefully used in the prevention or treatment of neurodegenerative diseases through autophagy control by inducing oligomerization and aggregation of p62.

Hereinafter, the present invention will be described in detail by examples and experimental examples.

However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the contents of the present invention are not limited to the following Examples.

< Example 1> p62 end New N- That it is lecognin discovery

In order to discover new N-lecognin that binds to N-degron, a peptide with N-degron attached to the N-terminus was synthesized to investigate what proteins adhered in the animal tissue extract.

Specifically, for this purpose, iTRAQ (Isobaric tags for relative and absolute quantitation) linked to X-peptide pull-down analysis and mass spectrometry was performed. Rat testis extract was used as the N-endrule interacting protein, and biotin-labeled X-peptide (R11 sequence: RIFSTIEGRTY (type 1)) immobilized on streptabine beads, F11 sequence: FIFSTIEGRTY (type 2), and V11 (Stabilization control) (VIFSTIEGRTY) was used (Fig. 5a, d). The 10 mer linker linked to the X-peptide is derived from the Sindbis virus polymerase nsP4, the substrate of the N-endrule. The mixture was diluted with 5 times the volume of PBS and incubated overnight at 4°C. Proteins pulled down using each X-peptide were labeled with fluorescent substances of different colors using the ITRAQ labeling technique. About 200 proteins were identified through the proteomic analysis based on the Mascot score, and the known mammalian UBR box E3 family, UBR1, UBR2, UBR4 and UBR5 involved in the N-endrule pathway were included ( Figure 5b, c).

The results were confirmed by an X-peptide pull-down analysis followed by an immunoblot technique (Fig. 5e-i). UBR1 was found in both R11 and F11 pulldown analysis, UBR2 was found only in F11 pulldown analysis, and UBR4 and UBR5 appeared in R11 analysis. In the pull-down analysis using the stabilization control V11, the UBR protein did not precipitate. In addition to the UBR E3 ligase, the sequestosome 1 (p62) protein was confirmed by mass spectrometry in the R11 sedimentation sample. The results were confirmed by electrophoresis/silver staining and western blot of the precipitated protein after X-peptide pull-down analysis.

As a result, as shown in Fig. 1c, in the silver-stained gel, the R11-pull-down sample had a strong band near the molecular weight of 62 kDa, but this band was not observed in the negative control, V11-pull-down sample. As a result of performing mass spectrometry, the band was found to be a p62/sqstm1 (hereinafter p62) protein (FIG. 5C). As a result of these experiments, the present inventors confirmed that p62 is a novel N-lecognin.

< Example 2> p62 end Nt - Arg , Nt - Phe , Nt -Trp, and Nt - Tyr Identified as N-lecognine that can bind to

In order to more reliably identify the p62 protein identified in Example <1-1>, the following experiment was performed.

Specifically, Western blot was performed using a mouse monoclonal p62 antibody (1:500, ab56416, Abcam, Cambridge, UK) produced by a recombinant full-length protein corresponding to amino acids 1-440 of human p62. . As a result, as shown in Fig. 5D, p62 transcribed and translated in vitro showed a specific strong binding to the R11 peptide, but weakly to the F11 peptide and did not bind to the V11-peptide (Fig. 1D). Based on the amount used in the experiment, the R11 peptide was able to sediment p62 with an efficiency in excess of 40%, whereas the efficiency of the F11 peptide was 5%. The results were confirmed by dipeptide competition assay, and RA peptide (Arg-Ala) competed most effectively with R11 peptide in binding to p62, and another type 1 N-endrule peptide, HA (His- Ala) was next effective in competition (Fig. 5h, i). Among the type 2 peptides, WA (Trp-Ala) had an effect, and FA (Phe-Ala) had no effect. Competition analysis with AR, not N-endrule peptide, revealed that RA, HA and WA were N-endrule specific. The above results indicate that p62 can bind a wide range of type 1 and type 2 N-endrule peptides.

In order to directly determine the binding specificity for the N-terminal residue of p62, the binding of a peptide having various N-terminal residues to p62 was determined using the X-peptide pull-down technique.

As a result, as shown in FIG. 5, p62 was able to bind to all three types of type 1 N-endrule N-terminal residues R11, H11, and K11 (FIG. 5H). p62 binds strongly to R11-peptide, moderately to K11 and weakly to H11. Endogenous p62 also bound to three of the four types of N-endrule N-terminal residues, that is, F11, W11 and Y11, but not L11 (Fig. 5H). Arg(R), His(H) and Lys(K) have positively charged side chain groups, and Phe(F), Trp(W) and Tyr(Y) have aromatic side chains. It can be seen from the nature of the amino acid that binds p62 that p62 is positively charged or favors aromatic side chains. On the other hand, p62 did not prefer 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. The above results indicate that p62 selectively binds to Nt-Arg, Nt-Trp, Nt-Phe, and Nt-Tyr.

< Example 3> p62 of Nt - Arg Wow Nt - Phe Determine the cohesion for

In order to measure the binding constant of p62 to Nt-Arg and Nt-Phe, the following experiment was performed.

Specifically, p62-D3-GST was expressed in E. coli and purified with 50% purity. Biotin-labeled X-peptide was immobilized on a streptavidin-coated chip using surface plasmon resonance biosensors (Biacore), and then p62-D3-GST protein was injected into the immobilized peptide.

As a result, as shown in FIG. 6, p62-D3-GST was strongly bound to the R11 peptide, weakly to F11, and did not bind to V11 (FIG. 6 ). The Kd values of R11 and F11 were found to be 44 nM and 3.4 μM, respectively. In particular, the binding force to Nt-Arg was about 50 times higher than that of N-lecognin having a UBR box (Kd, 3.2 μM). The above results indicate that p62 is a novel drug target.

< Example 4> Nt - Arg Wow Nt - Phe end p62 of ZZ Make direct binding to domain

In order to determine which site Nt-Arg binds to p62, a series of deletion mutants (D1 to D8) were constructed in p62, and then their binding to R11 and W11 was determined in the same manner as in <Example 1>. As a method, it was confirmed by X-peptide pull-down analysis.

Specifically, D1 to D4 are mutants continuously deleted at the C-terminal region, and D5 to D8 are mutants consecutively deleted at the N-terminal region. Nt-Arg and Nt-Trp both bound to D2, D3, D4 and D5 including the ZZ domain, but did not bind to D1, D6, and D7 without the ZZ domain (Figs. 7a-b). In order to more accurately determine the site of p62 binding to Nt-Arg and Nt-Trp, a deletion mutant was made at the C-terminus of p62 D3, and its binding to the N-endrule N-terminus was determined by X-peptide pull-down analysis. It was confirmed (Fig. 7c). Of a total of 9 p62 deletion mutants, CD1 to CD6 bound to both Nt-Arg and Nt-Trp (FIG. 7D). The mutant from CD7 without two histidine residues, which is the structure of the zinc finger motif of the ZZ domain, did not bind. In addition, the minimum ZZ domain at the N-terminus was determined by X-peptide pull-down analysis (Fig. 7e). Six N-terminal deletion mutants were made in the region starting from the PB1 region of full-length p62 to the middle of the ZZ domain. ND-1, ND-2, ND-3, and ND-4 bound to Nt-Arg, but not ND-5 and ND-6 (Fig. 7f). ND-5 is devoid of the cysteine residue (C128) and aspartic acid (D129), elements of the atypical binucleate Zn-finger conserved in the UBR box. The mutant p62 without the ZZ domain was unable to bind to Nt-Arg (Fig. 7g-h). A fragment cut off only the ZZ domain (#83-175) could also bind to Nt-Arg, but the D129A mutation could not (Fig. 7i-j). The above results indicate that Nt-Arg and Nt-Trp bind to the ZZ domain of p62.

Six point mutations in which D129, D142_145, D147_149, D151_154, H160_163, and E177 in p62(1-440) were substituted with alanine to determine if binding of Nt-Arg and Nt-Trp to p62 follows the N-end rule. Sieves (point mutants) were constructed to perform X-peptide pull-down analysis. Mutants in the ZZ domain were unable to bind to Nt-Arg and Nt-Trp (Fig. 7k). The above results indicate that both ZZ and UBR boxes bind to Nt-Arg and Nt-Trp based on the N-endrule, where Cys and His residues act as structural elements, and aspartic acid is responsible for N-degron recognition. It indicates that it is playing an important role. Taken together, the above results show that the binding property of the ZZ domain of p62 to the N-endrule N-terminus is similar to that of the UBR box, indicating that the ZZ domain and the UBR box are structurally similar. In addition, when X-peptide binds to p62, it indicates that N-terminal residues such as Nt-Arg or Nt-Trp are active ingredients of the N-ligand.

< Example 5> p62 of ZZ The domain is UBR N- Lecognin UBR box and structural and functional Homology Existence check

The UBR box consisting of 70 residues is a substrate recognition domain present in the N-endrule E3 family named UBR1 to UBR7. The primary sequence of the p62 ZZ domain was compared with the sequence of the UBR region of UBR E3 ligase derived from various organisms using the ClusterW program.

As a result, the p62 ZZ domain showed structural similarity to the UBR box (Fig. 7l). While the typical C2H2 zinc finger fold of the UBR box was well preserved in p62-ZZ, the binucleate Zn-finger fold of the atypical UBR box was only partially found in p62-ZZ. . The UBR region contains three very well conserved aspartic acids (D118, D150 and D153), which are essential for the binding of the substrate to the N-terminal destabilizing moiety. The p62-ZZ domain also contained three aspartic acid residues including D129, D147 and D149. Only D147 of the ZZ domain was conserved in the UBR box (D118), but the three aspartic acid residues above the negatively charged (acidic) appear to be essential for binding to the N-terminal destabilizing residue as in the case of the UBR box. The above results are consistent with the results of a pull-down analysis in which p62 favors binding to a positively charged (basic) side chain. In addition to the C2H2 zinc finger structure and three aspartic acid residues, three identical (red) residues (Y140, D147 and L150) within the ZZ domain in all sequences and two identical residues (G173 and W184) outside the ZZ domain and one conserved A (red) residue (E177) was found (Fig. 2B).

< Example 6> N- Ligandin Arg -Ala p62 Chair Oligomerization And confirming that it induces the formation of aggregates

In order to investigate whether the N-ligand Arg-Ala induces self-oligomerization and aggregate formation of p62 when bound to the p62 ZZ domain, a full-length p62-expressing HEK293 cell lysate was used in vitro using non-reducing SDS-PAGE. Oligomerization assays (in vitro oligomerization assays) were performed (FIG. 8).

Specifically, HEK293 cells were transfected with plasmid DNA expressing p62-myc/his. After 24 hours, the cells were lysed in a lysis buffer (50 mM Hepes, pH 7.4, 0.15 M KCl, 0.1% Nonidet P-40, 10% glycerol, protease inhibitor and phosphotease inhibitor) and the cooling-thaw cycle was repeated. I did. The cell suspension was incubated on ice for 1 hour and then centrifuged at 4° C. and 13,000 x g for 20 minutes. Protein concentration was determined by Bradford assay. For the p62 oligomerization experiment, 1 μg of protein was cultured with or without dipeptide (dissolved in water to a final concentration of 0.5 or 1 M) in the presence of 100 μM vestatin at room temperature. The sample was mixed with a non-reducing loading buffer containing 4% lithium dodecyl sulfate (LDS), heated at 95° C. for 10 minutes, and subjected to SDS electrophoresis. Monomers, oligomers, and aggregates of p62 were detected with a mixture of p62 and myc antibodies. Various dipeptides such as Arg-Ala, Lys-Ala and His-Ala (type 1), Phe-Ala, Trp-Ala and Tyr-Ala (type 2), and Ala-Arg and Ala After culturing -Phe (stabilized), Western blot was performed using the myc/p62 antibody.

As a result, as shown in Fig. 8a, it was shown that only Arg-Ala having Nt-Arg among various dipeptides specifically induces p62 oligomerization and aggregate formation (Figs. 8a-ca). The above results indicate that the N-ligand, Arg-Ala, induces self-oligomerization and aggregate formation of p62.

In addition, in order to investigate whether the PB1 domain of p62 is involved in the p62 aggregate, as a result of evaluating the aggregation ability of the D69A mutant, the D69A mutant normally binds to Nt-Arg, but Arg-Ala is the D69A p62 mutant. It was shown that the formation of aggregates could not be induced (Fig. 8E).

< Example 7> Arg -Ala p62 of Oligomerization When inducing ZZ Confirm that you need a domain

In order to find out whether binding to the ZZ domain is required when Arg-Ala induces oligomerization of p62, p62 oligomerization analysis was performed using a p62 ZZ mutant that cannot bind to the Arg-11 peptide.

As a result, as shown in Fig. 8d, in the presence of Arg-Ala, wild-type p62 effectively formed an oligomer/aggregate, whereas the ZZ mutant did not form an oligomer/aggregate at all (FIG. 8D). This indicates that the p62 oligomer is induced by binding Arg-Ala to the ZZ domain.

< Example 8> Arg -Ala p62 of LC3 Confirmation of increased binding with

The oligomerization of p62 is initially required for the recruitment of p62 at the autophagosome initiation site, and then introduced into the autophagosome membrane through the interaction of the p62 with the LC3. It is known (Itakura et al., 2011). In order to confirm the effect of Arg-Ala on the binding of p62 and LC3, ELISA analysis was performed (Fig. 8f-h).

Specifically, full-length p62 and p62 mutants were infected with p62 KO mouse embryonic fibroblasts, harvested 24 hours later, dissolved in a lysis buffer, and centrifuged at 4° C. and 1,3000 rpm. Type 1 and Type 2 dipeptide cultured GSH-coated plate (Priece, 15140) with GST-tagged LC3 recombinant protein (Enzo lifescience, BML-UW1155) and 20 μg of cell lysate at room temperature. Incubated for 1.5 hours at. The bound p62 was incubated with an anti-p62 antibody (SC-28359, santa cruz) at room temperature for 1 hour, and a secondary antibody to which HRP was attached was incubated for 45 minutes. After washing three times with PBS, TMB (3,3',5,5'-Tetramethylbenzidine) substrate (Priece, 34021) was added to each well, and the color was changed in the dark for 10 minutes at room temperature. TMB stop solution 2N H2SO4 was added, and absorbance was measured at 450 nm.

As a result, as shown in Fig. 8f, Arg-Ala is the only one among various dipeptides to enhance the binding between p62 and LC3 (Fig. 8f). Furthermore, p62 ZZ mutants, such as D129A, C142/145A, D147/149A, C151/154A, H160/163A, and ZZ deletion (ZZdel) and PB1 mutants, such as D69A, are Did not work (Fig. 8g, h). Therefore, from the above results, it was shown that Arg-Ala binds to the p62-ZZ domain to induce a conformational change, activates the p62 aggregate via the PB1 domain, and induces p62-LC3 interconnection via the LIR domain. 8I is a diagram for easily showing the inactivated form and the activated form of p62.

< Example 9> N-terminal Arginated Through Nt - Arg Discovery of endoplasmic reticulum proteins that acquire

Since Nt-Arg, a physiological N-ligand of the p62 ZZ domain, can be produced through N-terminal arginization by ATE1 R-transferase, the N-terminal sequence of endoplasmic reticulum proteins was investigated using bioinformatics.

As a result, according to the N-terminal degradation pathway, Nt-Asp and Nt-Glu can act as substrates for arginization by ATE1 R-transferase, and the resulting Nt-Arg is likely to function as an N-ligand. Yes (Fig. 10A). Using information biology techniques, many endoplasmic reticulum chaperons, such as BiP, calreticulin (CRT), protein disulfide isomerase (PDI), GRP94, ERdJ5, etc., have Nt-Asp or Nt-Glu, and thus arginization by ATE1 R-transferase is prevented. It was found that the amino acid L-Arg can be obtained through (FIG. 9).

< Example 10> N-terminal Arginized BiP , CRT and PDI Of antibodies specific for a subpopulation of

Among the endoplasmic reticulums, N-terminal arginylated protein for production of antibodies specifically recognizing N-terminal arginated BiP (R-BiP), calreticulin (R-CRT), and protein disulfide isomearase (R-PDI) Peptides corresponding to the N-terminal region of the, ie, REEEDKKEDVG (R-BiP), REPAVYFKEQ (R-CRT), and RDAPEEEDHVL (R-PDI) were synthesized, respectively. The peptide was inoculated into rabbits to obtain antibody serum, and then the antibody was purified using IgG chromatography. The antibody group was subjected to chromatography using EEEDKKEDVGC, EPAVYFKEQ, and DAPEEEDHVL, that is, a peptide that does not undergo arginization as a ligand, to remove unspecified antibodies. Finally, REEEDKKEDVGC (R-BiP), REPAVYFKEQ (R-CRT), and RDAPEEEDHVL (R-PDI), that is, arginylated peptide was passed through chromatography using as a ligand to purify an arginylation-specific antibody (Fig. 10b). .

< Example 11> BiP , CRT and PDI end ATE1 N-terminal arginization by Mediated Identification

After overexpressing the ATE1 R-transferase isomerase, it was confirmed that R-BiP formation was induced using the R-BiP antibody (FIG. 10D). In addition, when ATE1 was knocked down using siRNA, it was confirmed that the formation of R-BiP was reduced (FIG. 10e). Furthermore, arginization was assayed by expressing Ub-X-BiP-flag (X=Glu or Val) (FIG. 11A). Ub-X-BiP-flag is separated into Ub and X-BiP-flag by a deubiquitinating enzyme at the same time as translation. Using the recombinant protein, it was revealed that Nt-Glu19 of X-BiP is an arginylated residue (Fig. 11A).

Another recombinant protein, namely, Ub-X-Bip-myc/his construct was generated (FIG. 11B). Here, X has Arg18, Glu19 or Val19, and since the structure does not contain an ER signal, it remains in the cytoplasm, and the N-terminal portion is exposed after ubiquitin is removed by ubiquitin hydrolases. Using this structure, it was confirmed once again that X-BiP was arginized to Nt-Glu19 (FIG. 11C). R-BiP was not produced in ATE1 knockout cells (Fig. 11D). Using thapsigargin, which causes ER stress, it was revealed that R-BiP was not caused by ER stress, but was a product of an enzymatic reaction caused by ATE1 R-tranferase (FIG. 11E). In addition, it was found that the R-BiP produced in this way migrates to the cytoplasm in a large amount using the cell fractionation technique (FIG. 11F). It was confirmed that R-BiP moved to the cytoplasm using a cycloheximide protein degradation technique was not relatively degraded compared to BiP that was not arginized in the endoplasmic reticulum (FIG. 11g). This is consistent with the experimental results that R-BiP is delivered by autophagy. These results indicate that BiP undergoes arginization at the N-terminal Nt-Glu19 by ATE1, and the product, R-BiP, moves to the cytoplasm.

As a result of investigating whether CRT and PDI also undergo the N-terminal arginization process by ATE1, it was confirmed that R-CRT and R-PDI were generated when the ATE1 isoenzyme was overexpressed (Fig. 11h). These results indicate that in addition to BiP, CRT, and PDI, a number of endoplasmic reticulum chaperons and other proteins can be modified by N-terminal arginization mediated by ATE1.

< Example 12> The outer DNA of the cytoplasm is R- BiP Finding out that it causes the generation

In order to find out the cause of R-BiP production, stress conditions causing N-terminal arginization of endoplasmic reticulum proteins were investigated by various methods.

As a result, as shown in Fig. 12, it was confirmed that the generation of R-BiP is specifically induced when double stranded DNA (dsDNA) is introduced into the cytoplasm (Fig. 12a, b). In addition to R-BiP, it was confirmed that R-CRT is similarly induced when dsDNA is introduced into the cytoplasm (Fig. 12b, c). It was observed that R-BiP induced by dsDNA migrates to the cytoplasm using a cell fractionation technique (Fig. 12D). Using poly(dA:dT) that mimics a pathogen containing DNA (virus or bacteria), it was confirmed that R-BiP, R-CRT, P-PDI, etc. are generated as part of the innate immune response against invading DNA. (Fig. 12e-g). In addition, at this time, it was confirmed that the autophagy action was activated together (FIG. 12b, f).

< Example 13> R- BiP this p62 With Autophagy To autophagosome Confirm delivery

To determine whether R-BiP and p62 were delivered to the autophagosome at the same time, it was observed using immunostaining.

As a result, as shown in FIG. 13, it was confirmed that R-BiP migrated to the cytoplasm and then collected in a cell structure such as a puncta (FIG. 13A). The R-BiP spots were colocalized with the p62 spots (Figs. 13a, c), and it was confirmed that they were also co-located with LC3 (Figs. 13b-d). It was also confirmed that BiP was transferred to the LC3-positive autophagosome in the mouse embryonic heart (Fig. 13e). When ATE1 or BiP was knocked down using siRNA, R-BiP could not migrate to the autophagosome (Fig. 13f-h). The p62 knockout also showed similar results (Figs. 13f-h). These results indicate that R-BiP comes out of the cytoplasm, is transferred to the autophagosome through p62, and is eventually degraded by the lysosome.

< Example 14> R- BiP this To autophagosome When delivered Nt - Arg Make sure you need

Ub-X-BiP-GFP recombinant protein (X = Arg, Glu, or Val) was overexpressed in cells to generate X-BiP-GFP (Fig. 14A), and then the migration situation by autophagy using an immunostaining technique. Investigated. R-BiP was delivered to the autophagy autophagosome (Fig. 14c). On the other hand, Val-BiP (Nt-Glu19 could not be a substrate for arginization because it was substituted with Val) was not delivered to the autophagy autophagosome because there was no Nt-Arg (FIG. 14C). Similar results were obtained when the intracellular location of R-BiP was compared with LC3 spots (FIG. 14D). Since other sites of the BiP protein can affect autophage targeting, Ub-X-BiP ^Δ (Fig. 14A), which removes most of the BiP protein and leaves only residues 19-124, is overexpressed, and then X-BiP ^Δ The intracellular location was investigated using an immunostaining technique (Fig. 14e). R-BiP ^Δ formed cytoplasmic spots in common in +/+ and p62-/- cells, and also moved autophagy autophagosomes (FIG. 14E). On the other hand, since E-BiP ^Δ can be arginated in +/+ cells, it normally migrated to the autophagy opophagosome, whereas it did not show such activity in p62-/- cells (FIG. 14E). In addition, V-BiP ^Δ did not migrate to the autophagosome in both +/+ and p62-/- cells (FIG. 14E). Similar results were obtained when the intracellular locations of X-BiP and LC3 were examined by immunostaining technique (FIG. 14f). The above results indicate that when BiP or other endoplasmic reticulum proteins are arginized and released into the cytoplasm (after binding to denatured proteins or other substrates), Nt-Arg resulting from post-translational modification acts as an N-ligand and binds to the p62 ZZ domain. .

< Example 15> R- BiP of Nt - Arg end p62 ZZ Confirm that it binds directly to the domain

In order to investigate whether Nt-Arg of R-BiP binds to the p62 ZZ domain, an X-peptide pull-down analysis was performed (FIGS. 15a, b).

As a result, as shown in Fig. 15, the R-BiP peptide pulled down p62 from the cell extract, but the E-BiP or V-BiP peptide did not (Fig. 15c, d). In order to investigate where the Nt-Arg of R-BiP binds to p62, p62 was divided into small pieces (FIG. 15E). As a result of performing a pull-down experiment with the p62 fragments and R-BiP peptide, it was shown that Nt-Arg of R-BiP binds to the p62 ZZ domain (FIG. 15F).

To be sure that R-BiP binds to the p62 ZZ domain, only the p62 ZZ domain was cut out and named p62-ZZ83-175-GST and p62-ZZ(D129A)83-175, and GST pull-down analysis was performed (Fig. 15G). ). ZZ83-175 pulled down Arg18-Bip19-654 but failed to pull down Val19-Bip19-654, and ZZ mutant ZZ(D129A)83-175 failed to pull down either Arg-Bip18-654 or Val-Bip19-654. Therefore, the ZZ domain of p62 is required for the interaction of the arginized Bip and p62 in an N-endrule-dependent manner (Fig. 15H).

In addition, when p62-ZZ83-175-RFP and ubiquitin-X-Bip19-124-GFP were co-expressed in MEF cells, Arg18-Bip19-124 was at the same time as p62-ZZ83-175-RFP, which showed puncta formation. Although the migration form was shown, Val19-Bip19-124-GFP and p62-ZZ83-175-RFP did not perform mottling and simultaneous migration (FIG. 15i).

< Example 16> R- BiP this p62 Dependently Autophagy Confirm that it is decomposed by action

In order to determine whether R-BiP is degraded by autophagy through p62, Ub-X-BiP ^Δ -GST was prepared and overexpressed in +/+ and p62-/- cells (FIG. 16A).

As a result, as shown in Fig. 16, R-BiP degraded well in +/+ cells compared to V-BiP, but was not degraded in p62-/- cells and accumulated (Fig. 16a, b). On the other hand, V-BiP was not degraded and accumulated in both +/+ and p62-/- cells (Fig. 16a, b). When the autophagy inhibitor Hydroxychloroquine was treated, R-BiP was not decomposed and accumulated (Figs. 16a and b). In addition, R-BiP was accumulated without being degraded in ATG5-/- cells lacking the autophagy process (FIG. 16C). When a quantitative analysis of cycloheximide proteolysis was performed with Ub-R-BiP-myc/his, it was confirmed that R-BiP was remarkably stabilized in ATG5-/- cells (Fig. 16d, e). The above results indicate that R-BiP is delivered to and degraded by p62 to the lysosome.

< Example 17> R- BiP This cytoplasmic Ubiquitinated Confirm that it is induced by protein

In order to find out the type of stress induced by R-BiP, cells were treated with various chemicals, and then the formation of R-BiP was investigated using an immunoblot technique.

As a result, it was found that the proteasome inhibitor commonly induces R-BiP formation (FIG. 17C). When R-BiP production was induced, it was confirmed that ubiquitin accumulates like the bound intracellular protein groups (FIG. 17C), and this tendency was also commonly observed when poly(dA:dT) was treated (FIG. 17A). Some of the ubiquitinated intracellular proteins were delivered in the form of spots rather than p62 (a structure that temporarily exists in the form of a coagulum before the proteins pulled by p62 are delivered to the autophagosome), where R-BiP and p62 and It was confirmed that it is located at the same time (FIG. 17B). These results indicate that when denatured proteins accumulated in the cytoplasm are ubiquitinated and then transferred to autophagy, signals are given to the endoplasmic reticulum, promoting N-terminal arginization of chaperons present in the endoplasmic reticulum and their migration to the cytoplasm. R-BiP indicates that it migrates to the p62 body like ubiquitinated proteins.

< Example 18> R- BiP Binds to these cytoplasmic denatured proteins and binds them Autophagy Make sure to pass

Geldenamycin, an inhibitor of Hsp90, was treated to promote the formation of intracellular denatured proteins.

As a result, it was confirmed that the formation of R-BiP was induced (FIG. 17F), and when overexpressing YFP-CL1, a model substrate that is denatured by self-folding, was confirmed that R-BiP was directly bound (FIG. 17G). . In addition, it was confirmed that YFP-CL1 was transferred to autophage vacuoles using immunofluorescence staining, and co-located with R-BiP and p62 in this structure (FIG. 17h, i). These results indicate that R-BiP binds to the denatured protein accumulated in the cytoplasm and moves to the autophagy autotagosomes along with p62.

When synthesizing these results, as described in Fig. 1, when the denatured protein and its coagulant accumulate in the cytoplasm, a signal is sent to the endoplasmic reticulum and various chaperons such as BiP, CRT, and PDI in the endoplasmic reticulum are Nt. -Arg ligand is mounted and moves to the cytoplasm. Then, they bind to the denatured protein while binding to the p62 ZZ domain through the Nt-Arg ligand. As a result of the binding, p62 changes from a closed structure to an open structure, thereby exposing the PB1 domain (with oligomerization) and undergoing self-oligomerization to form a p62 body along with the denatured protein, R-BiP. In addition, the LC3 binding domain is also exposed to promote binding to LC3 protruding from the membrane of the autophagosome. As a result, the denatured protein-R-BiP-p62 is delivered to the autophagosome in the form of a complex coagulum, and is then degraded by the lysosome degrading enzyme.

< Experimental example 1> Low mass Compound phosphorus ZZ -L1 of p62 Oligomerization And confirming that the binding with LC3 is increased.

In an effort to further discover the ligand of the p62 ZZ domain in addition to Arg-Ala, 2-((3,4-bis(benzyloxy)benzyl)amino), a digestive compound having structural similarity to the N-endrule ligand, Nt-Trp. ethan-1-ol hydrochloride (named ZZ-L1; Chemical Formula 5) and 1-(3,4-bis(benzyloxy)phenoxy)-3-(isopropylamino)-2-propanol (named ZZ-L2; Chemical Formula 6) ) Was found to increase the activity of p62, especially oligomerization and autophagy. In particular, ZZ-L2 is a material known as NCI314953. These low-mass compounds have structural similarities to Phe-Ala or Trp-Ala rather than Arg-Ala, and are therefore thought to bind to the type-2 binding subdomain of the ZZ domain.

It was investigated whether these low-mass compounds induce oligomerization and increased activity of p62 like Arg-Ala when bound to the p62 ZZ domain.

Specifically, the degree of p62 aggregate was measured in a concentration-dependent manner (0, 10, 100, 1000 μM) using an in vitro oligomerization assay.

As a result, it was confirmed that, as shown in Fig. 18, similarly to Arg-Ala, ZZ-L1 induces p62 aggregates in a concentration-dependent manner (Figs. 18a and b). In addition, as a result of investigating whether ZZ-L1 activates p62 in a similar way to the experiment that increased the binding of p62 to LC3 using Arg-Ala, ZZ-L1 was concentration dependent (0, 10, 25, 100, 500, 1000). μM) was confirmed to increase the binding to LC3 (Fig. 18c). From this, it can be seen that ZZ-L1 induces structural activation of p62 in a pattern similar to Arg-Ala by binding to the p62 ZZ domain.

In addition, to confirm whether ZZ-L1 and ZZ-L2 induce p62 puncta formation, it was observed using immunofluorescence confocal microscopy (Fig. 18d-g). Specifically, HeLa cells on the coverslip were treated with XIE ZZ compounds at different concentrations (0, 1, 2.5, 5, and 10 μM) for 12 hours (Fig. 18f, g) or at different times (0, 1, 3, 6, and 12 hours) was treated with 10 μM of the XIE ZZ compound (Fig. 18e, e). As a result, ZZ-L1 and ZZ-L2 spot p62 concentration-dependently (0, 1, 5, 10 μM) for 12 hours or time-dependent (1, 3, 6, 12 h) at 10 μM in HeLa cells. It was found that it induces the formation (puncta formation) (Fig. 18d-g).

< Experimental example 2> ZZ - L1 and ZZ - L2 end Intracellular Autophagosome Confirming that it increases formation

Western blot was performed to investigate the effect of ZZ-L1 and ZZ-L2 on autophagy.

Specifically, ZZ-L1 and ZZ-L2 increased the amount of LC3 in concentration-dependent (0, 1, 5, 10 μM) and time-dependent (1, 3, 6, 12 h), and LC3-I (inactive form) The conversion of to LC3-II (active form) was also enhanced (FIG. 19B ). Using immunofluorescence staining, it was revealed that the ZZ ligand promoted the formation of LC3 autophage spots in addition to p62 autophage spots in HeLa cells (FIG. 19A). This indicates that the ZZ ligands function as autophagy activators.

It was investigated whether the increase of the compound-induced LC3 protein and the formation of LC3-II were caused by p62. HeLa cells (1x106/well) in a 6-well plate were treated with 40 nM of p62 siRNA using RNAi Max reagent (Invitrogen) to knock down p62, and at this time, siRNA control was also treated in parallel. Then, DMSO or 5 mM ZZ compound was incubated for 3 hours or 6 hours.

As a result, the increase of LC3 and the formation of LC3-II were induced by the ZZ compounds ZZ-L1 and ZZ-L2 transfected with si-control, but the effect was completely by p62 knock down. It was inhibited (Fig. 19c). In addition, in a similar experiment using +/+ and p62-/- cells, it was shown that ZZ-L1 (5 mM) did not act as an autophage activator in p62-/- cells (FIG. 19D). The above results indicate that the ZZ compound mimics the effect induced by Arg-Ala binding to the ZZ domain of p62, inducing aggregates of p62 via the PB1 domain of p62, and further increasing autophagosome formation.

< Experimental example 3> ZZ Ligand is Autophagy The flux Confirm that it increases

The increase in LC3 synthesis and LC3-II form when treated with ZZ ligand may be caused by activation of the upstream of the autophage flux (synthesis of key factors, transcription, etc.), but downstream (fusion of lysosomes with autophagosomes, etc.) It is also possible that the degradation process in the lysosome) is blocked. In order to confirm whether the autophage flow was normal when the ZZ ligand was treated, the cells treated with the ZZ ligand were treated with an autophage inhibitor, and then the amount of LC3 was investigated using an immunoblot. Specifically, using NH4Cl (Sigma, A9434), Bafilomycin A1 (Sigma, B1793) and hydroxychloroquine (HCQ)) (Sigma, H0915), inhibitors that prevent degradation in lysosomes. Autophagy dynamic analysis (autophagy flux assay) was performed (Fig. 19e). After the XIE ZZ compound was first treated for 3 hours, an autophagy inhibitor was added and incubated for 3 more hours. After obtaining a cell extract, Western analysis was performed to measure the LC3-II band intensity with image J. The degree of increase in formation was calculated after normalizing to GAPDH.

As a result, even after treatment with the XIE ZZ compound, inhibition of autophagy by hydroxychloroquine (HCQ) increased the autophage dynamics including the accumulation of LC3-II by more than two times (FIG. 19E). The results show that the ZZ ligand not only promotes the formation of autophagosomes by actually increasing the synthesis of key components of autophagy such as LC3, but also the substrates (p62, R -BiP, denatured protein, etc.) decomposes (Fig. 19g).

< Experimental example 4> ZZ On the ligand Formed by Autophagosome Confirmation of fusion with lysosomes

In order to confirm whether the autophagosome formed by the ZZ ligand is delivered to the lysosome, the same method used above was used using HeLa cells stably transformed with RFP-GFP-LC3 stably transformed with a combination of acid-sensitive GFP and insensitive RFP. Another autophagy dynamic analysis was performed.

As a result, it was confirmed that ZZ-L1 and ZZ-L2 promote the formation of RFP+GFP+ autophagosomes (neutral pH), and at the same time, it was confirmed that the GFP+ signal disappeared in some RFP+ spots (FIG. 19F). These RFP+GFP- spots are because when RFP+GFP+ autophagosomes fuse with lysosomes to form autolysosomes (acid pH), acid-sensitive GFP fluorescence disappears in an acidic environment. These results indicate that the autophagosome formed by the ZZ ligand is normally delivered to the lysosome and decomposed (Fig. 19g).

As another method of measuring the change in autophage flow by the ZZ ligand, the change in the intracellular concentration of p62 was directly measured. HeLa cells were treated with ZZ-L1 and a cycloheximide proteolysis assay was performed. As a result, it was shown that ZZ-L1 promotes the decomposition of p62 (FIG. 20A). These results indicate that p62 bound to the ZZ ligand acts as an autophagy activator.

< Experimental example 5> ZZ Ligand and bound p62 end mTOR Regardless of Autophagy Activation Check sikim

A representative compound known as an autophagy activator is rapamycin. Rapamycin is an inhibitor of mTOR (mammalian target of rapamycin) [ref]. As mTOR is inhibited, key regulators of autophagy (ULK, Beclin, etc.) are activated, through the synthesis of LC3 and conversion to the LC3-II form. The production of autophagosomes is increased (FIG. 20D). Rapamycin's efficacy as an autophagy activator is of high value as a therapeutic agent, but development of an autophage activator that does not undergo mTOR is urgent because of side effects such as a wide range of biological effects through mTOR.

In order to compare the efficacy and mechanism of ZZ ligand and rapamycin as autophagy activators, the production of LC3 and conversion of LC3-II were compared after treating HeLa cells by concentration or by time.

As a result, it was found that the overall efficacy of the ZZ ligand was similar to that of rapamycin, or even better in some cases (FIGS. 20b, c).

To investigate whether the ZZ ligand activates autophage via mTOR, HeLa cells were treated with ZZ ligand or rapamycin, and then phosphorylation of p70S6K regulated by mTOR was investigated (FIG. 20D). As expected, rapamycin inhibited mTOR, thereby inhibiting the phosphorylation of p70S6K, but the ZZ ligand did not inhibit the phosphorylation of 70S6K (FIG. 20E). The above results indicate that ZZ ligand activates autophagy without passing through mTOR, unlike conventional rapamycin, and thus ZZ ligand is a novel autophagy activator that has not been known until now.

< Experimental example 6> ZZ Ligand is Intracellular To ubiquitin By Targeted Proteins Five Confirmation of induction to topage

Intracellular denatured proteins are primarily targeted by ubiquitin and then degraded by the proteasome. Whether the denatured protein is coagulated (e.g., mutant huntingtin protein) or the proteasome activity is reduced, or in a situation where it cannot enter the proteasome, it is collected by autophage and then degraded by lysosomes. Based on the results that the ZZ ligand enhances autophage flow, it was investigated whether the ZZ ligand enhances the migration of ubiquitinated intracellular proteins to the autophage by immunofluorescence staining.

As a result, it was confirmed that 5 mM ZZ-L1 induced ubiquitinated proteins into autophage vacuoles (FIG. 20F). In addition, when the autophagy inhibitor hydroxychloroquine was treated, it was confirmed that ubiquitinated proteins accumulate in the autophage blisters (FIG. 20F). The above results indicate that the ZZ ligand promotes autophagy collection of intracellular denatured proteins.

< Experimental example 7> ZZ Ligand is Mutation Huntingteen protein Coagulate Confirm to be removed

Huntington's disease is a ubiquitin-proteasome system due to the property of being easily denatured (misfolded) due to excessive repetition of the huntingtin protein's CAG codon (~36 times or more), as well as being rapidly converted into aggregates. There is no decomposition (Fig. 21A). A technology has been developed to remove rapamycin by activating autophage, but it is difficult to use it as a therapeutic because rapamycin has an activity that affects various biological pathways through mTOR. Considering that the ZZ ligand activates autophagy without passing through mTOR, it was investigated whether the huntingtin protein coagulum could be removed.

Specifically, when wild-type huntingtin (HDQ25-GFP) and mutant huntingtin (HDQ103-GFP; CAG repeats 103 times) were expressed in HeLa cells and observed by immunofluorescence staining, HDQ25-GFP was spread throughout the cells. On the other hand, HDQ103-GFP was observed to be gathered in the form of a protein coagulum (Fig. 21c). Cells overexpressing the huntingtin proteins were treated with 10 mM ZZ-L1 or 1 mM rapamycin, and the remaining huntingtin proteins in the cells were investigated using a dot blot assay. As a result, ZZ-L1 was more effectively mutated. It was shown that the huntingtin protein coagulum was removed (FIG. 21B). In a similar experimental setting, cells were divided into insoluble fractions (including coagulants) in 0.5% Triton X-100, and immunoblots were performed. As a result, the coagulum fraction treated with ZZ-L1 It was confirmed that huntingtin denatured protein was effectively removed from (FIG. 21D). Rapamycin also showed a similar effect (FIG. 21D). Similar bran was obtained in another experiment in which HeLa cells were compared with ZZ-L1 and ZZ-L2 with rapamycin (FIG. 21E).

To investigate whether the ZZ ligand removes huntingtin coagulation through autophagy activation, +/+ and ATG5-/- (autophagosomes do not form without ATG5) in cells with 10 mM ZZ ligand and 1 mM. After treatment with rapamycin, immunoblot was performed. In the +/+ cells, the ZZ ligand effectively removed the huntingtin coagulum, whereas the ATG5-/- cells did not show an effect (FIG. 21F). This indicates that the ZZ ligand removes the huntingtin protein coagulum by activating autophage.

In order to investigate the mechanism by which the ZZ ligand removes the huntingtin coagulum, HeLa cells overexpressing the HDQ103-GFP protein coagulum were treated with 10 mM ZZ ligand, and then immunofluorescence staining was performed. In cells not treated with ZZ-L1, HDQ103-GFP formed an inclusion body, in which p62 and R-BiP were co-located (FIGS. 22a and b). In addition, in the cells treated with ZZ-L1, it was found that the huntingtin inclusion body was effectively reduced or disappeared (FIGS. 22a-c). When the above results are summarized, it can be seen that the ZZ ligand can effectively remove the denatured protein coagulum by activating autophagy.

<110> Seoul National University Korea Research Institute of Bioscience and Biotechnology <120> Autophagy stimulation using p62 ZZ domain binding compounds or arginylated BiP for the prevention or treatment of neurodegenerative disease <130> 2017P-02-007 <160> 9 <170> KoPatentIn 3.0 <210> 1 <211> 440 <212> PRT <213> Homo sapiens <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 Glu Ala Ala Ala Gly Pro Gly Pro Cys Glu Arg Leu Leu 35 40 45 Ser Arg Val Ala 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 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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 <210> 2 <211> 2 <212> PRT <213> Artificial Sequence <220> <223> Arg Ala peptide <400> 2 Arg Ala One <210> 3 <211> 2 <212> PRT <213> Artificial Sequence <220> <223> Phe Ala peptide <400> 3 Phe Ala One <210> 4 <211> 2 <212> PRT <213> Artificial Sequence <220> <223> Trp Ala peptide <400> 4 Trp Ala One <210> 5 <211> 2 <212> PRT <213> Artificial Sequence <220> <223> Tyr Ala peptide <400> 5 Tyr Ala One <210> 6 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> R-11 peptide <400> 6 Arg Ile Phe Ser Thr Ile Glu Gly Arg Thr Tyr Lys 1 5 10 <210> 7 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> W-11 peptide <400> 7 Trp Ile Phe Ser Thr Ile Glu Gly Arg Thr Tyr Lys 1 5 10 <210> 8 <211> 636 <212> PRT <213> Homo sapiens <400> 8 Glu Glu Glu Asp Lys Lys Glu Asp Val Gly Thr Val Val Gly Ile Asp 1 5 10 15 Leu Gly Thr Thr Tyr Ser Cys Val Gly Val Phe Lys Asn Gly Arg Val 20 25 30 Glu Ile Ile Ala Asn Asp Gln Gly Asn Arg Ile Thr Pro Ser Tyr Val 35 40 45 Ala Phe Thr Pro Glu Gly Glu Arg Leu Ile Gly Asp Ala Ala Lys Asn 50 55 60 Gln Leu Thr Ser Asn Pro Glu Asn Thr Val Phe Asp Ala Lys Arg Leu 65 70 75 80 Ile Gly Arg Thr Trp Asn Asp Pro Ser Val Gln Gln Asp Ile Lys Phe 85 90 95 Leu Pro Phe Lys Val Val Glu Lys Lys Thr Lys Pro Tyr Ile Gln Val 100 105 110 Asp Ile Gly Gly Gly Gln Thr Lys Thr Phe Ala Pro Glu Glu Ile Ser 115 120 125 Ala Met Val Leu Thr Lys Met Lys Glu Thr Ala Glu Ala Tyr Leu Gly 130 135 140 Lys Lys Val Thr His Ala Val Val Thr Val Pro Ala Tyr Phe Asn Asp 145 150 155 160 Ala Gln Arg Gln Ala Thr Lys Asp Ala Gly Thr Ile Ala Gly Leu Asn 165 170 175 Val Met Arg Ile Ile Asn Glu Pro Thr Ala Ala Ala Ile Ala Tyr Gly 180 185 190 Leu Asp Lys Arg Glu Gly Glu Lys Asn Ile Leu Val Phe Asp Leu Gly 195 200 205 Gly Gly Thr Phe Asp Val Ser Leu Leu Thr Ile Asp Asn Gly Val Phe 210 215 220 Glu Val Val Ala Thr Asn Gly Asp Thr His Leu Gly Gly Glu Asp Phe 225 230 235 240 Asp Gln Arg Val Met Glu His Phe Ile Lys Leu Tyr Lys Lys Lys Thr 245 250 255 Gly Lys Asp Val Arg Lys Asp Asn Arg Ala Val Gln Lys Leu Arg Arg 260 265 270 Glu Val Glu Lys Ala Lys Arg Ala Leu Ser Ser Gln His Gln Ala Arg 275 280 285 Ile Glu Ile Glu Ser Phe Tyr Glu Gly Glu Asp Phe Ser Glu Thr Leu 290 295 300 Thr Arg Ala Lys Phe Glu Glu Leu Asn Met Asp Leu Phe Arg Ser Thr 305 310 315 320 Met Lys Pro Val Gln Lys Val Leu Glu Asp Ser Asp Leu Lys Lys Ser 325 330 335 Asp Ile Asp Glu Ile Val Leu Val Gly Gly Ser Thr Arg Ile Pro Lys 340 345 350 Ile Gln Gln Leu Val Lys Glu Phe Phe Asn Gly Lys Glu Pro Ser Arg 355 360 365 Gly Ile Asn Pro Asp Glu Ala Val Ala Tyr Gly Ala Ala Val Gln Ala 370 375 380 Gly Val Leu Ser Gly Asp Gln Asp Thr Gly Asp Leu Val Leu Leu Asp 385 390 395 400 Val Cys Pro Leu Thr Leu Gly Ile Glu Thr Val Gly Gly Val Met Thr 405 410 415 Lys Leu Ile Pro Arg Asn Thr Val Val Pro Thr Lys Lys Ser Gln Ile 420 425 430 Phe Ser Thr Ala Ser Asp Asn Gln Pro Thr Val Thr Ile Lys Val Tyr 435 440 445 Glu Gly Glu Arg Pro Leu Thr Lys Asp Asn His Leu Leu Gly Thr Phe 450 455 460 Asp Leu Thr Gly Ile Pro Pro Ala Pro Arg Gly Val Pro Gln Ile Glu 465 470 475 480 Val Thr Phe Glu Ile Asp Val Asn Gly Ile Leu Arg Val Thr Ala Glu 485 490 495 Asp Lys Gly Thr Gly Asn Lys Asn Lys Ile Thr Ile Thr Asn Asp Gln 500 505 510 Asn Arg Leu Thr Pro Glu Glu Ile Glu Arg Met Val Asn Asp Ala Glu 515 520 525 Lys Phe Ala Glu Glu Asp Lys Lys Leu Lys Glu Arg Ile Asp Thr Arg 530 535 540 Asn Glu Leu Glu Ser Tyr Ala Tyr Ser Leu Lys Asn Gln Ile Gly Asp 545 550 555 560 Lys Glu Lys Leu Gly Gly Lys Leu Ser Ser Glu Asp Lys Glu Thr Met 565 570 575 Glu Lys Ala Val Glu Glu Lys Ile Glu Trp Leu Glu Ser His Gln Asp 580 585 590 Ala Asp Ile Glu Asp Phe Lys Ala Lys Lys Lys Glu Leu Glu Glu Ile 595 600 605 Val Gln Pro Ile Ile Ser Lys Leu Tyr Gly Ser Ala Gly Pro Pro Pro 610 615 620 Thr Gly Glu Glu Asp Thr Ala Glu Lys Asp Glu Leu 625 630 635 <210> 9 <211> 637 <212> PRT <213> Homo sapiens <400> 9 Arg Glu Glu Glu Asp Lys Lys Glu Asp Val Gly Thr Val Val Gly Ile 1 5 10 15 Asp Leu Gly Thr Thr Tyr Ser Cys Val Gly Val Phe Lys Asn Gly Arg 20 25 30 Val Glu Ile Ile Ala Asn Asp Gln Gly Asn Arg Ile Thr Pro Ser Tyr 35 40 45 Val Ala Phe Thr Pro Glu Gly Glu Arg Leu Ile Gly Asp Ala Ala Lys 50 55 60 Asn Gln Leu Thr Ser Asn Pro Glu Asn Thr Val Phe Asp Ala Lys Arg 65 70 75 80 Leu Ile Gly Arg Thr Trp Asn Asp Pro Ser Val Gln Gln Asp Ile Lys 85 90 95 Phe Leu Pro Phe Lys Val Val Glu Lys Lys Thr Lys Pro Tyr Ile Gln 100 105 110 Val Asp Ile Gly Gly Gly Gln Thr Lys Thr Phe Ala Pro Glu Glu Ile 115 120 125 Ser Ala Met Val Leu Thr Lys Met Lys Glu Thr Ala Glu Ala Tyr Leu 130 135 140 Gly Lys Lys Val Thr His Ala Val Val Thr Val Pro Ala Tyr Phe Asn 145 150 155 160 Asp Ala Gln Arg Gln Ala Thr Lys Asp Ala Gly Thr Ile Ala Gly Leu 165 170 175 Asn Val Met Arg Ile Ile Asn Glu Pro Thr Ala Ala Ala Ile Ala Tyr 180 185 190 Gly Leu Asp Lys Arg Glu Gly Glu Lys Asn Ile Leu Val Phe Asp Leu 195 200 205 Gly Gly Gly Thr Phe Asp Val Ser Leu Leu Thr Ile Asp Asn Gly Val 210 215 220 Phe Glu Val Val Ala Thr Asn Gly Asp Thr His Leu Gly Gly Glu Asp 225 230 235 240 Phe Asp Gln Arg Val Met Glu His Phe Ile Lys Leu Tyr Lys Lys Lys 245 250 255 Thr Gly Lys Asp Val Arg Lys Asp Asn Arg Ala Val Gln Lys Leu Arg 260 265 270 Arg Glu Val Glu Lys Ala Lys Arg Ala Leu Ser Ser Gln His Gln Ala 275 280 285 Arg Ile Glu Ile Glu Ser Phe Tyr Glu Gly Glu Asp Phe Ser Glu Thr 290 295 300 Leu Thr Arg Ala Lys Phe Glu Glu Leu Asn Met Asp Leu Phe Arg Ser 305 310 315 320 Thr Met Lys Pro Val Gln Lys Val Leu Glu Asp Ser Asp Leu Lys Lys 325 330 335 Ser Asp Ile Asp Glu Ile Val Leu Val Gly Gly Ser Thr Arg Ile Pro 340 345 350 Lys Ile Gln Gln Leu Val Lys Glu Phe Phe Asn Gly Lys Glu Pro Ser 355 360 365 Arg Gly Ile Asn Pro Asp Glu Ala Val Ala Tyr Gly Ala Ala Val Gln 370 375 380 Ala Gly Val Leu Ser Gly Asp Gln Asp Thr Gly Asp Leu Val Leu Leu 385 390 395 400 Asp Val Cys Pro Leu Thr Leu Gly Ile Glu Thr Val Gly Gly Val Met 405 410 415 Thr Lys Leu Ile Pro Arg Asn Thr Val Val Pro Thr Lys Lys Ser Gln 420 425 430 Ile Phe Ser Thr Ala Ser Asp Asn Gln Pro Thr Val Thr Ile Lys Val 435 440 445 Tyr Glu Gly Glu Arg Pro Leu Thr Lys Asp Asn His Leu Leu Gly Thr 450 455 460 Phe Asp Leu Thr Gly Ile Pro Pro Ala Pro Arg Gly Val Pro Gln Ile 465 470 475 480 Glu Val Thr Phe Glu Ile Asp Val Asn Gly Ile Leu Arg Val Thr Ala 485 490 495 Glu Asp Lys Gly Thr Gly Asn Lys Asn Lys Ile Thr Ile Thr Asn Asp 500 505 510 Gln Asn Arg Leu Thr Pro Glu Glu Ile Glu Arg Met Val Asn Asp Ala 515 520 525 Glu Lys Phe Ala Glu Glu Asp Lys Lys Leu Lys Glu Arg Ile Asp Thr 530 535 540 Arg Asn Glu Leu Glu Ser Tyr Ala Tyr Ser Leu Lys Asn Gln Ile Gly 545 550 555 560 Asp Lys Glu Lys Leu Gly Gly Lys Leu Ser Ser Glu Asp Lys Glu Thr 565 570 575 Met Glu Lys Ala Val Glu Glu Lys Ile Glu Trp Leu Glu Ser His Gln 580 585 590 Asp Ala Asp Ile Glu Asp Phe Lys Ala Lys Lys Lys Glu Leu Glu Glu 595 600 605 Ile Val Gln Pro Ile Ile Ser Lys Leu Tyr Gly Ser Ala Gly Pro Pro 610 615 620 Pro Thr Gly Glu Glu Asp Thr Ala Glu Lys Asp Glu Leu 625 630 635

Claims (16)

A pharmaceutical composition for preventing and treating Huntington's disease comprising the compound (ZZ-L1) represented by the general formula (5) as an active ingredient.
[Chemical Formula 5]

The pharmaceutical composition for preventing and treating Huntington's disease according to claim 1, wherein the compound binds to the ZZ domain of the p62 protein having the amino acid sequence of SEQ ID NO: 1.
The pharmaceutical composition for preventing and treating Huntington's disease according to claim 2, wherein the ZZ domain comprises 128 to 163 residues of the amino acid sequence of p62 protein represented by SEQ ID NO: 1.
The composition of claim 1, wherein said composition is selected from the group consisting of Arg-Ala (SEQ ID NO: 2), Phe-Ala (SEQ ID NO: 3), Trp-Ala (SEQ ID NO: 4), Tyr- 6), W-11 (SEQ ID NO: 7) and R-BiP (SEQ ID NO: 9).
The method according to claim 1, wherein the compound (ZZ-L1) binds to the p62 ZZ domain and activates the PB1 domain and the LIR domain of the p62 protein to induce oligonucleation and aggregation of p62 A pharmaceutical composition.
The pharmaceutical composition for preventing and treating Huntington's disease according to claim 1, wherein the compound (ZZ-L1) forms an autophagosome through induction of p62 agglutination.
A health functional food for improving Huntington's Disease containing the compound (ZZ-L1) represented by the general formula (5) as an active ingredient.
[Chemical Formula 5]

The health functional food for prevention and improvement of Huntington's disease according to claim 7, wherein the compound (ZZ-L1) binds to the ZZ domain of the p62 protein having the amino acid sequence of SEQ ID NO: 1.
9. The pharmaceutical composition according to claim 8, wherein the ZZ domain comprises 128 to 163 residues of the amino acid sequence of p62 protein represented by SEQ ID NO: 1.
8. The composition of claim 7, wherein said composition is selected from the group consisting of Arg-Ala (SEQ ID NO: 2), Phe-Ala (SEQ ID NO: 3), Trp-Ala (SEQ ID NO: 4), Tyr- 6), W-11 (SEQ ID NO: 7) and R-BiP (SEQ ID NO: 9).
The method according to claim 7, wherein the compound (ZZ-L1) binds to the p62 ZZ domain and activates the PB1 and LIR domains of the p62 protein to induce oligonucleotides and aggregates of p62 Health functional foods.
The health functional food for prevention and improvement of Huntington's disease according to claim 7, wherein the compound (ZZ-L1) forms an autophagosome through induction of p62 aggregates.
1) increasing the activity of the p62 protein by treating the compound (ZZ-L1) represented by the general formula (5) with an animal cell or p62 protein other than human;
2) inducing oligomerization of the activated p62 protein;
3) the step of increasing the formation of autopagosomes
4) binding of the p62 protein to the denatured protein; and
5) transferring the p62 protein to autopagosum.
(SEQ ID NO: 2), Phe-Ala (SEQ ID NO: 3), Trp-Ala (SEQ ID NO: 4), Tyr- (SEQ ID NO: 6), W-11 (SEQ ID NO: 7) and R-BiP (SEQ ID NO: 9).
14. The method according to claim 13, wherein the oligomerization of the p62 protein in step 2) is induced by binding of the p62 protein to the PB1 domain.
14. The method according to claim 13, wherein the step (5) of transferring the p62 protein to autophagosus is performed by binding LC3-binding domain of p62 protein with LC3 of autophagosome.

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