WO2021210878A1 - Crbn-binding peptide and composition for preventing or treating alzheimer's disease using same - Google Patents

Abstract

The present invention relates to a peptide that binds to CRBN and inhibits the binding of CRBN and DJ2, and a composition for preventing, treating or ameliorating Alzheimer's disease by using same. The peptide of the present invention mimics a CRBN-binding motif in DJ2, and thus binds to CRBN and interferes with binding of DJ2 and CRBN, thereby inhibiting the degradation of DJ2 by CRBN. As a result, since the effect of DJ2 inhibiting the aggregation of tau protein is maintained and improved, DJ2 can be usefully used for the prevention, amelioration or treatment of Alzheimer's disease.

Description

CRBN-binding peptide and composition for preventing or treating Alzheimer's disease using same

The present invention relates to a peptide that binds to CRBN and inhibits the binding of CRBN and DJ2, and a composition for preventing, treating or improving Alzheimer's disease using the same.

Recently, with the rapid development of medicine, the average lifespan of human beings is increasing, and as the elderly population increases, new social problems are emerging. In particular, geriatric neurological diseases such as stroke, Alzheimer's disease, and Parkinson's disease appear as fatal nervous system dysfunction, and there is currently no effective method for treating them. In particular, Alzheimer's disease (AD) is the most common among geriatric neurological diseases. Alzheimer's is the most common degenerative brain disease that causes dementia and is a brain disease that causes problems with memory, thinking and behavior. Dementia is a general term that refers to loss of memory and other intellectual abilities severe enough to interfere with daily life, and Alzheimer's accounts for 60 to 80% of dementia cases.

Although the pathogenesis and cause of Alzheimer's disease is not precise, it is known that the main causes are damage to nerve cells due to decreased synthesis of acetylcholine, a neurotransmitter, deposition of β-amyloid, and hyperphosphorylation of tau protein. Tau protein is a kind of microtubule binding protein having a molecular weight of 50,000 to 70,000 Da, and is mainly involved in entanglement of nerve fibers. Tau proteins exhibit significant molecular diversity, the most well known of which is due to proline-directed phosphorylation.

CRL4 ^{The substrate receptor of CRBN} , E3-ligase substrate recruiter cereblon (CRBN), is a therapeutic target for thalidomide and its derivatives, and is known as an immunomodulatory drug (IMiD). CRBN mutations are associated with autosomal recessive, non-symptomatic mental retardation. CRBN is also implicated in the regulation of AMP-activated protein kinase, glutamine synthetase, MEIS2 and ion channels.

The DNAJ family is involved in a variety of cellular activities, including protein folding/unfolding/refolding. Among them, DJ2 is known to react with DnaK and numerous Hsp70 and Hsp110, which attract and unfold misfolded proteins within stable aggregates. However, it is not clear whether DJ2 can prevent unfolding or aggregation in addition to the ability to recognize unfolded proteins, and the relationship with tau protein is still unknown, and research on this is required.

[Prior art literature]

[Patent Literature]

Republic of Korea Patent Registration 10-2033776

It is an object of the present invention to provide a peptide comprising a CRBN binding motif amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.

It is an object of the present invention to provide a pharmaceutical composition for preventing or treating Alzheimer's disease comprising the peptide.

It is an object of the present invention to provide a food composition for preventing or improving Alzheimer's disease comprising the peptide.

It is an object of the present invention to provide a method for screening a therapeutic agent for Alzheimer's disease.

In order to achieve the above object of the present invention, the present invention provides a peptide comprising a CRBN binding motif amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.

In addition, the present invention provides a pharmaceutical composition for preventing or treating Alzheimer's disease, comprising the peptide.

In addition, the present invention provides a food composition for preventing or improving Alzheimer's disease, comprising the peptide.

In one embodiment of the present invention, the Alzheimer's disease may be caused by phosphorylation of tau.

In addition, the present invention provides a method for screening a therapeutic agent for Alzheimer's disease, comprising the following steps.

a) synthesizing a peptide comprising a CRBN binding motif represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3;

b) analyzing whether the peptide synthesized in step a) can inhibit the binding of CRBN and DJ2 by binding to the DJ2 binding site of CRBN; and

c) determining as a therapeutic agent for Alzheimer's disease when the peptide synthesized in step b) inhibits the binding of CRBN and DJ2.

In addition, the present invention provides a method for preventing or treating Alzheimer's disease, comprising administering to an individual a peptide comprising a CRBN binding motif amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.

In addition, the present invention provides the use of a peptide comprising the CRBN binding motif amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 for preventing or treating Alzheimer's disease.

The peptide of the present invention is a peptide mimicking the CRBN binding motif in DJ2, and since it binds to CRBN and interferes with the binding of DJ2 and CRBN, the degradation of DJ2 by CRBN is suppressed. As a result, since the effect of DJ2 inhibiting the aggregation of tau protein is maintained and improved, it can be usefully used for preventing, improving or treating Alzheimer's disease.

1 shows the identification of Hsp70, DJ1 and DJ2 as endogenous substrates of ^{CRL4 CRBN.} Specifically, Figure 1a shows the results of lysing SH-SY5Y cells, immunoprecipitation with rabbit IgG control or α-CRBN antibody, and blotting with the indicated antibodies. 1b to 1d show that SH-SY5Y cells _{were transiently transfected with scrambled SiRNA (Scr) or siRNA CRBN} and then transfected with HA-Ub 24 h after IP, respectively, with α-Hsp70, α-DJ1 and α- Results performed with the DJ2 antibody and blotted with the indicated antibodies. Figure 1e shows SH-SY5Y cells transiently transfected with FLAG-CRBN and immunoprecipitated with FLAG M2 agarose beads. In vitro ubiquitination of endogenous, co-precipitated chaperones was performed in the presence of E1 + E2 and Ub; Methylated ubiquitin (Me-Ub) was added at the indicated sites, and the reaction was analyzed by SDS-PAGE and the result of immunoblotting with the indicated antibody.

Fig. 1f shows the results of treating Crbn ^−/- and Crbn ^+/+ MEF cells with 2.5 μg/ml CHX and performing immunoblot analysis (statistical analysis is shown from top to bottom in the order of Hsp70, DJ1 and DJ2). Figure 1g shows the results of Crbn ^−/- and Crbn ^+/+ MEF cells treated with 0.5 μg/ml MG132 and subjected to immunoblot analysis (statistical analysis is shown from top to bottom in the order of Hsp70, DJ1 and DJ2).

2 is an experiment on the effect of CRBN and DJ2 on heparin-mediated aggregation of tau. Specifically, Figure 2a shows the results of incubating full-length hTau-44 (5 μM) with the indicated protein combinations for 48 hours, then adding 5 μM ThT and measuring the fluorescence emission at 480 nm with excitation at 440 nm ( Heparin was included in all reactions except control at a concentration of 2.5 μM, Student's t-test was used to quantify the data at 95% significance level). Figure 2b is the result of confirming that the fluorescence emission is excited at 480 nm and excited at 440 nm by incubating the monomer hTau-K18 (5 μM) with the indicated combination (5 μM) and 5 μM ThT for 4 hours (heparin is 2.5 (included in all reactions except control at a concentration of μM) Figure 2d shows the results of treating SHSY5Y cells with monomer K18, aggregating K18 in the presence or absence of DJ2 and CRBN, pictures were taken 48 hours after treatment, and MTT analysis was performed to evaluate cell viability (errors) Bars represent SEM, Student's t-test used to quantify data at 95% significance level)

3 confirms that the N-terminal Lon domain of CRBN binds to the C-terminal domain of DJ2. Specifically, FIGS. 3A and 3B show schematics of the full-length rCRBN and deletion constructs used in FIGS. 3C and 3D below (Cereblon domain of Unknown activity, binding cellular Ligands and Thalidomide), L (linker) , C-terminal domain (CTD)). Figures 3c and 3e show that SH-SY5Y cells were transiently co-transfected with Myc-DJ2 and the indicated plasmids, the cell extracts were immunoprecipitated with α-Myc antibody, followed by SDS-PAGE sorting, and immunoblotting with Myc and HA antibodies. The plotted results are shown (bands at ~55 kDa represent heavy IgG chain (HC) and bands at ~25 kDa represent light IgG chains (LC). Asterisks represent non-specific bands). 3d and 3f show the results of transiently co-transfecting SH-SY5Y cells with HA-CRBN and the designated prmids, and then performing IP with HA antibody 24 hours later.

4 is an experimental result confirming that K32 and K350 are major ubiquitination sites of DJ2. Specifically, FIG. 4a shows the results of immunoblot analysis by transfecting SH-SY5Y cells with wild-type (WT) DJ2 and lysine mutants, and treating the cells with 2.5 μg/ml CHX. Figure 4b is a graph showing the result of Figure 4a. Figure 4c confirms that ubiquitylation is impaired in the case of the double mutant of K32 and K350 residues.

Figure 5 confirms the effect of CRBN KO on CUMS (chronic ultra-mild stress) and tau pathology. Specifically, Figure 5a shows the results of exposing Crbn ^−/- (KO) and Crbn ^+/+ (WT) mice to the CUMS paradigm, then fractionating WBL by SDS-PAGE and performing immunoblotting with antibodies. Figures 5b and 5c show the results of Western blot analysis of the selected pTau epitope and taukinase in WBL of WT and KO mice. Figure 5d shows the results of statistical processing with n = 3 using 5 mice in each group of KO and WT mice. Figures 5e and 5f show the results of statistical analysis of the results of Figures 5b and 5c by the t-test with P < 0.05.

Figure 6 confirms that CRBN KO inhibits the spread of tau pathology. Specifically, Figure 6a is an anatomical schematic diagram of the mouse brain, divided into anterior contralateral (AC), anterior ipsilateral (AI), posterior contralateral (PC), posterior ipsilateral (PI) and cerebellar (Cb) for analysis of western blotting. and the PI region includes the lateral tonsil injection site. 6B shows the results of three-dimensional injection of OA into the brains of WT and KO mice, preparation of WBL in RIPA buffer, and blotting with antibodies of the pTau array. Figure 5c is the relative band intensity quantification results in each group of KO and WT mice n = 3 (error bars indicate SEM).

7 shows the effect of CRBN KO on phosphorylation-mediated tau dimerization. Specifically, Figure 7a shows that HEK293T cells were subjected to CRISPR/Cas9-mediated knockout (KO) of CRBN or used as a negative control (NC), and Myc-tau was transiently transfected into all cell lines and Western blot was performed. am. FIG. 7B shows the result of FIG. 7A on a color gradant scale. Figure 7c shows SH-SY5Y cells _{were transiently transfected with siRNA or siRNA CRBN} , then the cells were transfected with tau40-VN173 and tau-VC155 constructs and treated with OA (30 nM) to generate BiFCs using confocal microscopy. This is the result of the measurement (Hoechst dye is used as counterstain).

8 schematically shows the role of CRBN and DJ2 in tau pathology.

9 is an experiment on whether DJ2 binds to CRBN and tau and thalidomide-independent degradation of DJ1. Specifically, Fig. 9a shows the results of GST pull-down analysis on His-tagged DJ2 and GST-tagged CRBN and blotting with anti-DJ2 and anti-CRBN antibodies, and Fig. 9b shows SH-SY5Y cells in HA-Ub This is the result of transient transfection with , 30 hours later, cells were treated with thalidomide for 24 hours, and cell lysis and IP were performed with mouse IgG control or α-DJ2 antibody. Figure 9c shows SH-SY5Y cells were transiently co-transfected with Myc-tagged Tau and FLAG-tagged DJ2, and 24 hours later, the cell extracts were fractionated by immunoprecipitation and SDS-PAGE with α-Myc antibody and SDS-PAGE with the indicated antibodies. Results from immunoblotting (SE: short exposure, LE: long exposure).

10 is an experiment for localization of a domain responsible for CRBN-DJ2 interaction. Specifically, Figures 10a and 10b show that SH-SY5Y cells were transiently co-transfected with Myc-DJ2 and HA-tagged CRBN digests and after 24 h, the cell extracts were immunoprecipitated with α-HA antibody, sorted by SDS-PAGE. and the results of immunoblotting with antibodies. 10c and 10d show the results of western blotting by transiently co-transfecting SH-SY5Y cells with HA-CRBN and Myc-tagged DJ2 cleavage and 24 hours later, immunoprecipitating the cell extract with α-HA antibody. Figure 10e shows a structural model of the DJ2-CRBN interaction predicted by ClusPro.

11 is an experimental result for the localization of a specific lysine residue responsible for ubiquitination of DJ2. Specifically, Figures 11a and 11b confirm the experimentally reported lysine ubiquitinated in DJ2 using PhosphositePlus and GGbase database. 11c shows that SH-SY5Y cells were transfected with wild-type (WT) DJ2 and various lysine mutants, confirming that K32 and K350 are major ubiquitination sites of DJ2.

12 shows the results of 2D-polyacrylamide gel electrophoresis on whole brain lysates of ^{Crbn −/−} and Crbn ^−/+ , and antioxidant levels of ^{Crbn −/−} brain lysates compared to Crbn ^{−/+ mice.} increase was confirmed.

Figure 13 confirms the effect of CRBN KO on the tau pathology induced by the injection of Okadaic acid. Densitometric quantification results of relative band intensities in each group (n = 3) of ^{Crbn -/-} and Crbn ^{+/+ mice.} is shown graphically.

14 is a result of analyzing the activity of three tau kinases in the same brain sample.

15 shows the effect of CRBN knock-out on tauopathy and the inhibitory effect of the prepared peptide on CRBN and DJ2 binding. Specifically, Fig. 15a shows the confirmation of APP plaques in a murine neuropathic model. Brains were removed from 8-month-old APP knock-in mice, the hippocampus was immunostained with CRBN and APP antibodies for 16 hours, and Alexa- After incubation with conjugated secondary antibody, imaged under a confocal microscope equipped with a 60X lens. Figures 15b and 15c confirmed high levels of CRBN and low DJ2 in brain samples from APP knock-in and 5XFAD mice. Brains were removed from 8-month-old APP knock-in and 5XFAD mice, and half of the brains were placed in frozen sections. and the other half used for Western blot analysis, the hippocampus was immunostained with CRBN and DJ2 antibodies for 16 h, incubated with Alexa-conjugated secondary antibody for 1 h, and imaged under a confocal microscope equipped with a 10X lens. did it Figure 15d shows the amino acid sequence of the peptide inhibitor of the present invention. Figure 15e confirms that the addition of the peptide inhibitor inhibits the binding of CRBN and DJ2. 24 hours after co-transfection of SH-SY5Y cells with Myc-DJ2 and FLAG-CRBN, the cell extract was treated with the α-Myc antibody. This is the result of immunoprecipitation with Myc and HA antibodies, followed by immunoblotting with Myc and HA antibodies (a band of ~55 kDa indicates an IgG heavy chain).

16 is a graph showing the inhibition of CRBN-DJ2 binding by immunoprecipitation using linear (Linear, SEQ ID NO: 1) and circular (SEQ ID NO: 3) peptide forms.

17a to 17d show the results of immunoprecipitation experiments in which CRBN and DJ2 binding inhibition ability was confirmed by immunoprecipitation based on the peptide of SEQ ID NO: 3, and a randomly mixed (Scrambled) peptide of amino acid sequence was added as an experimental control group. (DJ2-peptide is the peptide of SEQ ID NO: 3 and the structure of each peptide is shown in FIG. 17C).

18 shows the sequence and structure of four peptide sequences (Tetra-Peptide sequence, SEQ ID NO: 2) and a control (scrambled tetra-peptide), which are important residues, among the peptides of SEQ ID NO: 1;

19a to 19c confirm that the peptide represented by SEQ ID NO: 2 significantly inhibits the binding of CRBN and DJ2.

The present invention provides a peptide comprising the CRBN binding motif amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.

In the present invention, CRBN is a substrate receptor of the cillin-ring E3 ubiquitin ligase (CRL) complex, and the CRL complex ubiquitinates the substrate from the ubiquitin-conjugation enzyme (E2) to induce degradation in the proteasome. It has also been reported as a target protein of thalidomide, lenalidomide, and pomalidomide, which are immunomodulators (IMiDs) with anticancer effects on multiple myeloma.

As used herein, the term “peptide” refers to a polymer composed of amino acids linked by amide bonds or peptide bonds. For the purposes of the present invention, "peptide comprising a CRBN binding motif amino acid sequence" refers to a peptide having a sequence capable of specifically binding to CRBN and inhibiting the function of CRBN. Specifically, the peptide is a peptide having an activity capable of binding to the DJ2 binding site on CRBN, and acts as a competitive substrate for DJ2, thereby exhibiting an effect of inhibiting the binding of DJ2 and CRBN.

In the present invention, the peptide is included in the scope of the present invention without limitation as long as it is a peptide capable of inhibiting the function of CRBN by binding to the DJ2 binding site on CRBN. The peptide of the present invention may preferably be a peptide comprising an amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, but is not limited thereto, and at least one amino acid in the amino acid sequence is a different amino acid or other compound Even if substituted with , if the effect of inhibiting the function of CRBN by binding to the DJ2 binding site on CRBN is maintained, it is included in the scope of the present invention.

The amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 are as follows.

PISTLDNRTIV (SEQ ID NO: 1)

DNRT (SEQ ID NO: 2)

CPISTLDNRTIVC (SEQ ID NO: 3)

The present invention provides a pharmaceutical composition for preventing or treating Alzheimer's disease, comprising the peptide.

In the present invention, the Alzheimer's disease may be caused by tau phosphorylation.

In the present invention, Alzheimer's disease is the most common degenerative brain disease causing dementia, and the onset gradually deteriorates cognitive functions including memory. The exact pathogenesis and causes of Alzheimer's disease are not precisely known. Currently, it is known as a key mechanism of pathogenesis that a small protein called beta-amyloid is excessively produced and deposited in the brain and has a detrimental effect on brain cells. It is known that hyperphosphorylation of tau protein) also contributes to brain cell damage and affects the pathogenesis.

Tau protein is a cytoskeleton protein, which is a microtubule-associated protein, and is abundantly present in neurons of the central nervous system compared to other cells, and hyperphosphorylated tau The fibrous aggregation of proteins abnormally accumulates in internal neurons, resulting in tauopathy.

Tau disease is distinguished in that the accumulation of amyloid beta (Aβ, β-amyloid), which is considered the main cause of Alzheimer's dementia, occurs in external nerve cells. In this study, it was confirmed that dementia was induced only by tau hyperphosphorylation and aggregation in internal neurons without accumulation of amyloid beta in external neurons (van Swieten and Spillantini, 2007).

As used herein, the term “prevention” refers to any act of inhibiting or delaying the onset of Alzheimer's disease by administering the pharmaceutical composition according to the present invention.

As used herein, the term “treatment” refers to any action in which symptoms due to Alzheimer's disease are improved or beneficially changed by administration of the pharmaceutical composition according to the present invention.

The pharmaceutical composition according to the present invention includes the peptide of the present invention as an active ingredient, and may include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is commonly used in formulation, and includes, but is not limited to, saline, sterile water, Ringer's solution, buffered saline, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, liposome, and the like. It does not, and may further include other conventional additives such as antioxidants and buffers, if necessary. In addition, diluents, dispersants, surfactants, binders, lubricants, etc. may be additionally added to form an injectable formulation such as an aqueous solution, suspension, emulsion, etc., pills, capsules, granules, or tablets. Regarding suitable pharmaceutically acceptable carriers and formulations, formulations can be preferably made according to each component using the method disclosed in Remington's literature. The pharmaceutical composition of the present invention is not particularly limited in formulation, but may be formulated as an injection or oral ingestion.

The pharmaceutical composition of the present invention may be administered orally or parenterally (eg, intravenously or subcutaneously) according to a desired method, and the dosage may vary depending on the patient's condition and weight, the degree of disease, drug form, Although it depends on the route and time of administration, it may be appropriately selected by those skilled in the art.

The composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is the type, severity, and drug activity of the patient. , sensitivity to drugs, administration time, administration route and excretion rate, duration of treatment, factors including concurrent drugs, and other factors well known in the medical field. The composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered single or multiple. In consideration of all of the above factors, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, which can be easily determined by those skilled in the art.

Specifically, the effective amount of the composition according to the present invention may vary depending on the age, sex, and weight of the patient, and may be increased or decreased depending on the route of administration, the severity of the disease, sex, weight, age, and the like.

As used herein, the term "pharmaceutically acceptable salt thereof" may be prepared by a conventional method in the art, for example, hydrochloric acid, hydrogen bromide, sulfuric acid, sodium hydrogen sulfate, phosphoric acid, carbonic acid Drugs with salts with inorganic acids such as formic acid, acetic acid, oxalic acid, benzoic acid, citric acid, tartaric acid, gluconic acid, gestisic acid, fumaric acid, lactobionic acid, salicylic acid, or acetylsalicylic acid (aspirin) Forms pharmaceutically acceptable salts of these acids, or reacts with alkali metal ions such as sodium or potassium to form metal salts thereof, or reacts with ammonium ions to form another pharmaceutically acceptable salt thereof means to form

The present invention provides a food composition for preventing or improving Alzheimer's disease, comprising the peptide.

The food composition includes a health functional food composition.

As used herein, the term “improvement” refers to any action that at least reduces a parameter related to the condition being treated, for example, the degree of symptoms.

In the food composition of the present invention, the active ingredient may be added to food as it is or used together with other food 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 its use (for prevention or improvement). In general, in the production of food or beverage, the composition of the present invention is added in an amount of 15% by weight or less, preferably 10% by weight or less, based on the raw material. However, in the case of long-term ingestion for health and hygiene or health control, the amount may be less than or equal to the above range.

The health functional food composition of the present invention is not particularly limited in other ingredients other than containing the active ingredient as an essential ingredient in the indicated ratio, and may contain various flavoring agents or natural carbohydrates as additional ingredients like a conventional beverage. have. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; disaccharides such as maltose, sucrose and the like; and polysaccharides such as conventional 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 (taumatine, 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 can be appropriately determined by the selection of a person skilled in the art.

In addition to the above, the health functional food composition of the present invention contains various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, coloring agents and thickeners (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 may be contained. These components may be used independently or in combination. The proportion of these additives may also be appropriately selected by those skilled in the art.

The present invention provides a method for screening a therapeutic agent for Alzheimer's disease, comprising the following steps.

a) synthesizing a peptide comprising a CRBN binding motif represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3;

b) analyzing whether the peptide synthesized in step a) can inhibit the binding of CRBN and DJ2 by binding to the DJ2 binding site of CRBN; and

c) determining as a therapeutic agent for Alzheimer's disease when the peptide synthesized in step b) inhibits the binding of CRBN and DJ2.

Step a) is a step of synthesizing any peptide comprising the sequence using a CRBN binding motif having an amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 according to the present invention. In this step, the peptide can be obtained by a chemical method or a recombinant method using a known peptide synthesizer.

Step b) is a step of confirming whether the synthesized peptide has binding activity to CRBN in the presence of DJ2. In this step, whether the peptide of the present invention binds to CRBN can be confirmed by any method capable of analyzing peptide-peptide bonds known in the art, for example, it can be carried out through chemical shift fluctuation analysis. .

Step c) is a step of determining the peptide as a therapeutic agent for Alzheimer's disease when it is confirmed that the synthesized peptide binds to CRBN as a result of the analysis of step b).

The present invention provides a method for preventing or treating Alzheimer's disease, comprising administering to a subject a peptide comprising a CRBN binding motif amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.

As used herein, the term “individual” refers to an animal, and may be a mammal capable of exhibiting beneficial effects by treatment using the peptide of the present invention. Preferred examples of such subjects may include primates such as humans. In addition, such individuals may include all individuals who have or are at risk of having symptoms of Alzheimer's disease.

The present invention provides the use of a peptide comprising a CRBN binding motif amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 for preventing or treating Alzheimer's disease.

The descriptions related to the composition, treatment method, and therapeutic use may be equally applied as long as they do not contradict each other.

Hereinafter, preferred examples are presented to help the understanding of the present invention. However, the following examples are only provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.

<Example 1: Experimental method, materials and reagents>

Example 1-1. Reagents, DNA and Antibodies

Antibodies were commercially purchased from Sigma, Abnova, Abcam, CST, etc. Thalidomide, cycloheximide, MG132, okadaic acid, heparin and ThT were commercially purchased from Sigma, Lipofectamine 2000 was commercially purchased from Invitrogen, ubiquitin, E1 , E2 and methylated ubiquitin were purchased commercially from Boston Biochem.

Example 1-2. Cell Culture and Materials

Human neuroblastoma cell line SH-SY5Y (Invitrogen), HEK-293T cells, and mouse embryonic fibroblast (MEF) cells were purchased from ATCC, and supplemented with 10% fetal bovine serum (FBS; Invitrogen) and 10% fetal bovine serum (FBS; Invitrogen) at 37 °C with _{5% CO 2 .} It was maintained in DMEM supplemented with 100 U ml ⁻¹ antibiotic-antifungal (Invitrogen).

Examples 1-3. plasmid

For mammalian cell expression, Myc-tagged hDJ2 and its mutants were cloned into the pCS2+ MT vector. And HA-tagged rCRBN and its mutants were cloned into pEBB-3HA vector. Tau40-VN173 and Tau40-VC155 for BiFC analysis were provided by Dr. Yunkyung Kim. For bacterial expression, His-tagged Tau40 and Hsp70 were constructed from pET-28a and His-tagged DJ2-pET30b was provided by Dr. Kiseon Kwon. GST-tagged hCRBN in the pGEX-4T-1 vector was provided by Raymond J. Deshaies' laboratory. All cDNAs cloned into mammalian expression vectors and bacterial expression vectors were confirmed by DNA sequence analysis.

Examples 1-4. laboratory animal

The preparation and screening of CRBN-knockout (KO) mice was done using the prior art (Lee et al., 2013). Wild-type and CRBN-KO mice were given standard chow diet and water in pathogen-free conditions with a light-dark cycle of 12 h. All experiments and paradigms were approved by the Gwangju Science and Technology Animal Care Center. The animal's exposure to various stressors is listed in Table 1 below. Protein extracts were prepared from the brain and quantified by the Bradford method (Bio-Rad Laboratories). Samples containing the same amount of protein were separated by SDS-PAGE and subjected to western immunoblotting.

Examples 1-5. 2D-PAGE and image analysis

Whole brain tissue was flash frozen _{, ground to a fine powder in liquid N 2} , and sonicated in urea lysis buffer (7M urea, 2M thiourea, 2% CHAPS, 1% DTT). The lysate was then removed by centrifugation at 45,000 RPM for 30 min and quantified by the Bradford method (Bio-Rad, Hercules, CA). The same amount of protein (300 μg/strip) was separated by IEF (GE Healthcare, pH 3-10, 7 cm) and then separated by SDS-PAGE. After silver staining, image analysis was performed at the Yonsei Proteome Research Center, ^{and spots with significant differences in Crbn −/-} and Crbn ^+/+ were applied to in-gel degradation and MS/MS analysis.

Example 1-6. In vitro ubiquitination assay

The ubiquitination assay was performed as previously known. Briefly, SHSY5Y cells cultured in 6-well plates were transfected with ^Flag CRBN or empty vector and treated with MG132 (10 μM) for 4 hours after 48 hours. Cells were then harvested, lysed in RIPA buffer, and immunoprecipitated with Flag M2 agarose beads at 4 °C for 2-4 h. After washing twice with RIPA buffer and twice with ubiquitination buffer (50 mM Tris-HCl [pH 8.0], 10 mM MgCl ₂ , 0.2 mM CaCl ₂ , 1 mM DTT and 100 nM MG132), the beads were washed with E1 (0.5 μM), UbcH5a (0.5 μM), UbcH3 (1.67 μM), ubiquitin (60 μM) and ATP (4 mM) in 30 μl of ubiquitination buffer and incubated at 30 °C for 1 hour. Methylated ubiquitin (Me-Ub) was also added where indicated, and SDS sample buffer was added to stop the reaction and immunoblot analysis was performed.

Example 1-7. Cell-based ubiquitination assay

SHSY5Y cells cultured in 6-well plates were transfected with siRNA or siRNA _CRBN . After 24 hours, cells were transfected with 1 μg of pRK5-HA-ubiquitin (HA-Ub). After 24 h, cells were harvested, washed twice with cold PBS and lysed in ubiquitination buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 0.5% Triton X-100) supplemented with PMSF and protease inhibitor cocktail. Lysates were incubated with α-DJ2 antibody overnight at 4 °C and immunoprecipitated with proteinG resin. Beads were thoroughly washed with ubiquitination buffer and PBS, the reaction was stopped by adding SDS sample buffer, and SDS-PAGE was separated and transferred to PVDF membrane for immunoblot analysis.

Examples 1-8. Identification of CRBN binding partners using LC-MS/MS

Whole brain tissue powdered with liquid nitrogen was dissolved in lysis buffer (8 M urea, 75 mM NaCl, 50 mM Tris, pH 8.2, protease inhibitor cocktail, 1 mM NaF, 1 mM β-glycerophosphate, 1 mM sodium orthovanadate, 10 mM sodium pyrophosphate, 1 mM PMSF). The protein was then reduced to a final concentration of 5 mM dithiothreitol (DTT) for 30 min at 37 °C and alkylated using IAA to a final concentration of 25 mM for 30 min in the dark at room temperature. After alkylation, the urea concentration was diluted to <2 μM with 25 mM Tris-HCl (pH 8.0) and digested overnight at 37° C. with 1:50 dilution trypsin. The digested protein was subjected to multidimensional protein identification technology (MudPIT) analysis, which is a modification of the known method.

MudPIT column is an analytical column fused silica capillary column containing 7 cm of 5-μm Aqua C18 material (100-μm inner diameter) and 2 cm of 5 μm Partisphere strong cation exchange and 2 cm of 5-μm Aqua C18 reversed-phase column material. The trapping column consisted of a fused silica capillary column (250-μm inner diameter) packed with After elution of the peptides from the microcapillary column, the peptides were electrosprayed with an LTQ linear ion trap mass spectrometer and applied to the waste of the HPLC split with the 2.3 kV atomization voltage used remotely. Cycles of full-scan mass spectra followed by nine data-dependent tandem MS (MS/MS) spectra (400-1400 m/z (mass-to-charge ratio)) at 35% normalized collision energies span the multidimensional separation repeated successively.

The mouse International Protein Index (IPI) database was searched using MS/MS spectra obtained from LC/LC-ESI-MS/MS analysis. SEQUEST was retrieved with a fragment ion mass tolerance of 1.0 Da and a parental mass tolerance of 3.0 Da.

The iodoacetamide derivative of cysteine was accepted as a fixed modification (cysteine +57) in SEQUEST. Oxidation of methionine and acetylation of lysine are variable modifications (methionine +16) allowing up to 3 modifications per peptide (maximum number of modifications per type is 5) and missed cleavage sites for up to 2 trypsin digestions in SEQUEST searches. has been designated

Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the parsimony principle. BIOWORKS version 3.2 was used to filter the search results and apply the following Xcorr values and delta Cn values of 0.08 to the different charge states of the peptides: 1.8 for single charged peptides, 2.5 for double charged peptides, and triple charged peptides. 3.5. The requirement of both tryptic-digested ends was used in the filtering process.

Examples 1-9. CRBN Knockdown

siRNA _CRBN and scrambled siRNA (control) were purchased from Invitrogen, and cell transfection was performed with minor modifications according to the manufacturer's protocol. Target cells were transfected with lipfectamine2000 and siRNA, and knockdown efficiency was analyzed by immunoblot.

Examples 1-10. Bimolecular Fluorescence Complementation (BiFC) Analysis

SH-SY5Y cells were seeded on poly-L-lysine-coated coverslips in complete medium in 6-well plates (1 x 10 ⁴ _{cells/well) and transfected with siRNA or siRNA CRBN} . After 24 h, cells were co-transfected with Tau40-VN173 and Tau-VC155 constructs and then treated with 30 nM okadaic acid (OA) after 16 h. The tau-BiFC fluorescence intensity in cells was analyzed using a FV1000 confocal laser scanning microscope (Olympus) equipped with a 100X objective.

Examples 1-11. K18 agglutination assay

Aggregation of tau protein K18 (5 μM) was performed at 37 °C in the presence of polyanions (heparin, 2.5 μM) in PBS at pH 7.4. Assembly was performed qualitatively by TEM and quantitatively by fluorescence analysis using ThT. Samples were incubated at 37 °C for 6 hours, and dithiothreitol was added to a final concentration of 1 mM to avoid intramolecular disulfide crosslinking. For each state (pre-aggregates, aggregates, aggregates with DJ2), K18 (5 μM) was transferred to a black 98-well plate with PBS containing 5 μM ThT (Sigma). _{ThT signals were measured at λ ex} = 430 nm and λ _em = 480 nm in Biotek, a Synergy H1 hybrid leader.

Examples 1-12. Transmission electron microscopy (TEM)

A 600-mesh carbon-coated copper grid (supplied by SPI) was used for TEM analysis of negatively stained Tau-K18 aggregates. Samples were incubated for 1 minute on a glow-discharge grid, then the solution was removed using filter paper. After washing with distilled water three times, the dye was stained with 1% (w / v) uranyl acetate for 2 minutes, and the final washing step was performed with distilled water. Grids were analyzed by a Tecnai T12 electron microscope equipped with a CCD camera.

Examples 1-13. Expression and purification of recombinant proteins

CRBN-GST was purified from glutathione resin and digested with TEV protease. Tau-6xHis, DJ2-6xHis and Hsp70-6xHis were purified on Ni-NTA affinity resin (Clontech).

Examples 1-14. Immunoblot analysis

Cells were cultured in RIPA buffer (20 mM Hepes, 150 mM NaCl, 1 mM EDTA-EGTA, 1% Triton X-100, 1% NP40, 1% sodium deoxycholate, 2 mM Na ₄ VO) supplemented with PMSF and protease inhibitor cocktail (Roche). ₃ , 100 mM NaF [pH 7.4]).

The lysates were then removed by centrifugation at 12,500 RPM for 30 min and quantified by the Bradford method. Equal amounts of protein (10 μg/lane) were separated by SDS-PAGE and transferred to PVDF membranes. Membranes were blotted with the indicated antibodies. An α-rabbit or α-mouse antibody conjugated to Horseradish peroxidase was used as a secondary antibody, and the signal was ^{detected using ECL™} Western Blotting Detection Reagent (Amersham).

Examples 1-15. immunoprecipitation

Cells were lysed in RIPA buffer and centrifuged at 12,500 RPM for 30 min. Proteins were immunoprecipitated with the designated antibody at 4 °C for 16 hours, and then incubated with protein G Sepharose 4 Fast Flow (GE Healthcare) at 4 °C for 2.5 hours. The beads were then washed twice in RIPA buffer containing beads and twice in PBS. Immunoprecipitated proteins were analyzed by SDS-PAGE, transferred to PVDF membrane, and analyzed by immunoblotting with specific antibodies.

Examples 1-16. Cycloheximide tracking experiment

Cells were seeded overnight in complete medium in 6-well plates (1 x 10 ⁴ cells/wall), and then 2.5 μg/ml Cycloheximide (CHX) was added. Samples were then harvested at indicated time points for immunoblot analysis.

Examples 1-17. Generation of CRBN Knockout HEK293T Cells by CRISPR/Cas9

CRBN CRISPR/Cas9 KO plasmid and CRBN HDR plasmid (sc-412142) were purchased from Santa Cruz and knocked out CRBN according to the manufacturer's protocol. Briefly, HEK293T cells were co-transfected with CRBN CRISPR/Cas9 KO plasmid and CRBN HDR plasmid or control CRISPR plasmid (sc-418922). Cas-9-induced DSB (DNA-containing double-strand break) using Puromycin selected cells that were successful.

Examples 1-18. stress paradigm

In the present invention, a multimodal short-term stress paradigm was used. One group of Crbn ^+/+ C57BL/6 animals and one group of Crbn ^−/- C57BL/6 animals were exposed to CUMS for 5 weeks, and the experiment was repeated 3 times. Specifically, the CUMS paradigm consisted of three daily exposures to one of the following aversive stressors: stroboscopic illumination (6 times per hour / 6 light per sec.), cage tilting (30° position). , soiled cages (beds with bedding soaked with water), paired houses (in each grouping session paired two unfamiliar rats and switched occupants), loud noises (random noise generators (dB level <80; generated for 1 hour at a frequency of 80-300 Hz), confinement (introducing a corrugated sheet along the width of the cage to limit the movable space), and inverted lighting cycle (general indoor lighting off during the day and on at night). The stressors were presented in an unpredictable random order (see Table 1 below).

Examples 1-19. Stereotaxic Injections of okadaic acid (OA) into the mouse brain

Stereotactic surgery was performed as follows. Mice were anesthetized with 6X ketamine (0.1mL/10 g body weight), hair removed by giving local anesthesia (lidocaine 0.3mL), and the mice were placed firmly on the earbars. At an infusion rate of 60 nl/min, 130 nl of 100 μM OA (Sigma) dissolved in DMSO or DMSO alone was unilaterally injected in the lateral amygdala (bregma coordinates: anterior/posterior -1.94, medial/lateral -3.15, dorsal/ ventral -4.5). Then, the needle was held in the inserted state for 5 minutes, then slowly pulled out and the scalp was sewn tightly. Postoperative analgesics and antibiotics were administered intramuscularly once reflexes were restored (septazol 0.05 mL, ketapro 0.05 mL) and mice were placed in their cages and allowed to recover for 24 h. The brain was removed from the skull, perfused with PBS, dissected to obtain the area shown in Figure 6a and snap frozen in liquid nitrogen.

Examples 1-20. statistical analysis

Data are presented as mean ± standard deviation (SD); P-values were calculated using unpaired two-tailed Student's t-test in Microsoft Excel. Po0.05 was considered significant.

<Example 2: Identification of Hsp70 and its co-chaperone as an endogenous substrate of CRBN>

To identify only CRBN-associated substrates, we hypothesized that the endogenous substrate of CRBN ^{could be overexpressed in Crbn −/−} mice and cell lines. To substantiate this hypothesis, quantitative mass spectrometry of whole brain lysate (WBL) from ^{Crbn −/-} and Crbn ^{+/- and Crbn +/- mice was performed.} 3 Crbn ^--- mouse vs. Through Crbn ^+/+ mice experiments, dozens of proteins with higher spectra (average > 2 fold or more) were found. The most differentially expressed proteins are listed in Table 2 below.

Based on LC-MS/MS analysis of WBL (Crbn ^−/− Vs Crbn ^+/+ ), Hsp70 and co-chaperones DJ1 and DJ2 were expected to be novel endogenous substrates of CRBN. Pull-down analysis confirmed that Hsp70, DJ1 and DJ2 interact with CRBN in both endogenous and exogenous systems (Fig. 1a, Fig. 9a). When CRBN expression was suppressed using CRBN-specific siRNA, the introduction of HA-tagged ubiquitin into endogenous Hsp70, DJ1 and DJ2 was greatly reduced, which means that DJ1 and the like are degraded by CRBN-mediated ubiquitination.

In addition, ^Flag CRBN increased co-precipitated endogenous chaperones in in vitro ubiquitination when enriched with recombinant E1, E2, ubiquitin and ATP (Fig. 1e, line 5), and the addition of methylated ubiquitin inhibited ubiquitination. (Fig. 1e, line 4). Since thalidomide does not interfere with the ubiquitination of chaperones, the association of chaperones with IMiDs was not further studied (Fig. 9b).

Next, it was tested whether CRBN knockout promotes chaperone accumulation in cells. WT-MEF and Crbn ^{- / -} using the MEF cells, cycloheximide (CHX) in the presence has been observed that the Hsp70, DJ1 and especially DJ2 decomposition is delayed (Fig. 1f), these chaperones is when treated with MG132 Crbn ^{- / -} accumulated in MEF cells (Fig. 1g). These results suggest that Hsp70, DJ1 and DJ2 are novel endogenous substrates of ^{CRL4 CRBN.}

<Example 3: Effect of CRBN and DJ2 on heparin-mediated aggregation of tau>

Cooperative action of human Hsp70 with DJ1 (but not DJ2) results in an effective reversal of α-synuclein fibril formation. It exhibits a well-regulated substrate-specific preference among J-proteins in partnership with Hsp70. Since the roles of DJ1 and DJ2 in reducing aggregation of α-synuclein were studied, the present invention focused on their role in lowering the aggregation of the microtubule-associated protein tau (MAPT or tau).

First, in vitro aggregation assays were performed with full-length tau and truncated tau (repeat domains of tau or K18) in the presence of different combinations of chaperones and CRBN. Aggregation was induced by heparin and dithiothreitol (DTT) and evaluated using thioflavin T (ThT)-fluorescence analysis and transmission electron microscopy (TEM) analysis ( FIGS. 2a to 2d ). Incubation of chaperone and tau reduced aggregation, as can be seen through reduced binding of ThT dye, and Hsp70/DJ2 was more effective than Hsp70/DJ1 (Fig. 2a). Interestingly, Hsp70 alone could not remove tau aggregation as efficiently as compared to DJ2, which means that DJ2 stabilizes the native conformation of tau to prevent aggregation.

Therefore, co-immunoprecipitation analysis was performed to confirm the binding of DJ2 to tau (FIG. 9c). The binding of tau to DJ2 not only stabilizes the native conformation of tau, but also restricts the reaction with tau kinase in vivo, thereby reducing phosphorylation and aggregation of tau. On the other hand, the removal of DJ2 by CRBN reduced the activity to prevent tau aggregation (Fig. 2a), and it was confirmed that DJ2 effectively removed the aggregation of full-length tau like K18, which aggregated faster than full-length tau (Fig. 2b).

Previously, it was shown that DJ2 has the ability to stabilize the native conformation of tau. Accordingly, in vivo and in vitro studies were performed to determine the effect of DJ2 on removing tau aggregation and whether it was interfered with by CRBN. Tauopathy is characterized by a marked accumulation of fibrillar tau inclusion bodies in the CNS. However, extracellular tau plays an influential role in trans-cell proliferation. Extracellular monomers and seed-competent tau enter the cell and strongly aggregate. To observe the effect of DJ2 on the toxicity of extracellular tau, heparin-induced K18 aggregates were treated for 48 h in the presence or absence of DJ2 and added outside SH-SY5Y cells. Massive apoptosis was observed in SH-SY5Y cells treated with K18 aggregates. However, K18 co-cultured with DJ2 prior to heparin-induced aggregation confirmed no cytotoxicity as visualized through microscopy and MTT analysis (Fig. 2d). Interestingly, CRBN dissociated DJ2 from tau, disrupting its chaperone activity. Fibrillary tangles of relatively large size play an important role in the progression of neurofibrillary tangles (NFTs) toxic effects because they can accumulate inside neurons and cause direct physical interference with axonal movement. These results suggest that DJ2 inhibits tau aggregation, whereas CRBN reduces the tau aggregation-interfering effect of DJ2 through ubiquitination of DJ2.

<Example 4: C-terminal expansion of DJ2 in CRBN-dependent ubiquitination>

To specify the amino acid sequence of DJ2 recognized by CRBN, deletion mutants for Myc-tagged DJ2 (Myc-DJ2) and HA-tagged CRBN (HA-CRBN) were constructed ( FIGS. 3A and 3B ). Pull-down analysis showed that a stretch of ~80 amino acids (Lon-N) at the N-terminus of CRBN interacted with a range of ~100 amino acids at the C-terminus of DJ2 (Figs. 3c and 3d). To pinpoint the sequences of DJ2 and CRBN important for the interaction, we constructed additional deletion mutants of both proteins, which were ß-turn binding to a span of 40 amino acids at the C-terminus of DJ2 and ß3 and ß4 of CRBN. -ß showed the integrity of the loop (Figs. 10a to 10d).

Several structural models of DJ2 docked to CRBN were then analyzed using the 3D coordinates of all atoms defined using ClusPro26, a fully automated algorithm for protein-protein docking. The crystal structure of CRBN (4TZ4) and the PDB file of the structure model of DJ2 generated by the I-Tasser27 server were used. Based on the computer docking data as well as the in vitro co-IP data, the crystal structure of CRBN (4TZ4) and the PDB file of the structure model of DJ2 generated by the I-Tasser27 server were used. Based on in vitro co-IP data and computational docking data, the present inventors manipulated 6 amino acids of DJ2 (T279-T284) and 2 amino acids of CRBN (E152A, F153A, E152A/F153A) individually or in combination. Mutations were constructed ( FIG. 10E ). In the case of CRBN, when E152 and F153 were mutated to alanine, binding strength was reduced (FIG. 3e). Notably, all mutants of DJ2 retained binding to CRBN with the exception of D281A, N282A, R283A and T284A, indicating that these residues are involved in the binding of DJ2 to CRBN (Fig. 3f).

<Example 5: Confirmation of major ubiquitination positions of DJ2>

As DJ2 is ubiquitinated by transfection of exogenous ubiquitin (Fig. 1d), we sought to identify lysine (Lys or K) residues targeted by ^{CRL4 CRBN for endogenous ubiquitination.} According to PhosphoSitePlus28, a comprehensive database of experimentally observed mammalian post-translational modifications, Lys residues of DJ2 undergoing ubiquitination accumulate mainly in the J-domain and C-terminal domains (Figs. 11a and 11b). These Lys residues were individually substituted with arginine (Arg or R) and ubiquitination analysis was performed. The K32R and K350R mutants showed impaired ubiquitination ( FIG. 11C ) and delayed degradation in CHX follow-up analysis ( FIGS. 4A and 4B ). The double mutant K32R/K350R almost completely inhibited ubiquitination compared with WT-DJ2 (Figs. 4a to 4c). This means that K32 and K350 are the major ubiquitination sites for DJ2.

<Example 6: Confirmation of the effect of CRBN reduction on mild stress and tau pathology>

DJ2 was selected for further analysis as a potential co-chaperone for the prevention of tauopathies, and a multimodal chronic ultra-mild stress (CUMS) paradigm was used. In Crbn ^+/+ mice, CUMS produced marked hyperactivity with reduced feeding-related behavior. However, ^{these behavioral changes were prevented in Crbn −/−} mice during the entire stress process (Table 3). Tau phosphorylation was also measured in the WBL of control and stressed mice at the end of CUMS. Surprisingly Crbn ^{+ / +} mice as compared to Crbn ^{- / -} were markedly reduced to the tau phosphorylation in mouse brain, Crbn ^- the DJ2 levels were increased in mice (Fig. 5a and 5b) ^{- /.}

To verify whether the decrease in tau phosphorylation was the result of the increased chaperonic activity of DJ2 alone or the activity of the kinase targeting the tau protein was also impaired, the active form of the tau kinase was further measured. As a result, it was confirmed that the phosphorylation of glycogen synthase kinase 3 (GSK-3) at Ser9 and Ser21 ^{was significantly increased in Crbn −/-} WBL, thereby inhibiting GSK-3α/β ( FIG. 5c ). Likewise, the kinase activity of ERK1/2 and p38 was found to be reduced by their reduced phosphorylation (Fig. 5c). Thus, ^{improved co-chaperone activity and impaired tau kinase activity in Crbn −/−} mice together may affect tau phosphorylation as well as facilitate coping with stressors. 2D-PAGE analysis of WBL from Crbn ^−/- and Crbn ⁺⁺ mice confirmed that the levels of various antioxidant proteins, including thioredoxin, peroxiredoxin, and superoxide dismutase, were significantly increased (see Table 4 below), which resulted from stress ^{Demonstrate the characteristics of Crbn −/−} mice that are resistant to the factor ( FIG. 12 ).

<Example 7: Confirmation of the effect of CRBN reduction on tau-tau interaction and prion-like diffusion>

Topical administration of okadaic acid (OA) can immediately induce phosphorylation and aggregation of tau at anatomically distal sites by inhibiting protein phosphatase 2A (PP2A). The prion-like diffusion of tau can be confirmed by analyzing the phosphorylation status of tau in various anatomical regions of the brain. In the present invention, the ^{response of Crbn −/-} mice to tau phosphorylation and diffusion of tau phosphorylation to other regions of the brain were further verified using the above strategy. OA was sterically injected into Crbn ^−/- and Crbn ^{+/- and Crbn +/+} brains (M & M) and tau phosphorylation levels in the injected and non-injected hemispheres were analyzed (Fig. 6a). Although ^{substantial increases in tau phosphorylation were observed at multiple sites in the Crbn +/+} brain (e.g., pT181, pT231 and pS403/404), it was confirmed that tau phosphorylation was significantly less advanced or significantly prevented in ^{Crbn −/- brains.} (Fig. 6b and Fig. 13).

In addition, OA-induced inhibition of GSK3α/β was more pronounced in the WBL of Crbn ^{−/− than in Crbn +/+ mice ( FIG. 14 ).} Repressed tau phosphorylation was also observed when the CRBN locus was disrupted by CRISPR/Cas9 in the HEK293 cell line overexpressing Myc-tau ( FIGS. 7A and 7B ). ^{Hsp70 has an inverse relationship with tau in the Crbn +/+} HEK293 cell line overexpressing Myc-tau (the same is true for Hsp70 and co-chaperone). Interestingly, chaperone levels were not decreased in the CRISPR/Cas9 ^{-derived Crbn −/-} cell line (GSK3α/β suppressed by OA treatment).

Tau hyperphosphorylation results in pathological tau aggregation. To investigate the effect of CRBN on tau aggregation, we used the Venus-based BiFC technique. _{SHSY5Y cells were transiently transfected with siRNA CRBN} (or control scrambled siRNA) with subsequent co-transfection of the tau-BiFC construct. Although OA-induced hyperphosphorylation increased tau assembly resulting in activation of Venus fluorescence as expected, this effect was greatly reduced by CRBN inhibition ( FIG. 7c ).

These results suggest that the absence of CRBN induces resistance to the aggregation and diffusion of pathological tau.

<Example 8: Confirmation of inhibited activity of DJ2 in murine neuropathic model>

The role of CRBN and DJ2 in disease development and progression in a mouse model of neuropathy was studied. Amyloid-β(Aβ) plaques are one of the triggers of tauopathy because they promote tauopathy by increasing the spread of pathological tau.

To determine whether CRBN is a tauopathy inducer, brain samples from 8-month-old 5X-FAD and APP ^{NL-GF knock-in mice exhibiting late tauopathy were analyzed.} APP antibody immunostaining confirmed the presence of Aβ plaques in brain samples from 5XFAD and APPNL-GF knock-in mice (Fig. 15a).

In addition, the hippocampus of 8-month-old mice was immunolabeled with CRBN and DJ2 antibodies. As confirmed by immunohistochemistry and Western blot, elevated expression of CRBN was observed in hippocampal and cortical regions of ^{5X-FAD and APP NL-GF knock-in mice ( FIGS. 15b and 15c ).} On the other hand, chaperone levels were low at the same location (FIG. 15c). These results suggest that elevated CRBN is a biomarker for neurodegeneration. Thus, CRBN-mediated attenuation of chaperone activity may be an additional neurotoxic mechanism of Alzheimer's disease.

<Example 9: Confirmation of inhibition of interaction of CRBN and DJ2 of the peptide of the present invention>

Peptide inhibitor designs were attempted based on CRBN-DJ2 interactions and hotspot regions of DJ2 interacting with CRBN. Cysteine residues were added to both ends of the peptide to circularize the peptide (Fig. 15d). This was done to obtain the morphology required for the interaction of the DJ2 loop with CRBN (Fig. 10e). As a result of the treatment by changing the concentration of the designed peptide from 0 to 30 μM, the interaction between CRBN and DJ2 was completely blocked as shown in the CoIP analysis results when the peptide inhibitor was treated with a concentration of 10 μM ( FIG. 15e ).

Chemical knockdown of CRBN may not be a desirable strategy for cells for the recruitment and maintenance of ubiquitylation of endogenous substrates other than DJ2. Therefore, the use of peptides based on hotspot residues can mask CRBN, inhibit the interaction of DJ2 with CRBN, thereby inhibiting ubiquitylation of DJ2 and useful for selective inhibition of pathological tau phosphorylation.

<Example 10: Confirmation of CRBN and DJ2 binding inhibition ability using linear and circular peptides>

Considering the above results of CRBN-DJ2 interaction, linear (SEQ ID NO: 1) and circular (SEQ ID NO: 3) peptides were designed based on the hotspot region of DJ2 interacting with CRBN. In the case of a prototype, cysteine residues were added to both ends of the linear peptide to circularize the peptide. As a result of performing immunoprecipitation using the peptide, both types of peptides showed significant CRBN and DJ2 binding inhibition ability as shown in FIG. 16 .

<Example 11: Confirmation of CRBN and DJ2 binding inhibition ability using circular peptides and scrambled peptides thereof>

In order to prove that the amino acid sequence of the peptide of the present invention has the ability to inhibit CRBN and DJ2 binding, immunoprecipitation was performed by preparing a scrambled peptide in which the peptide of SEQ ID NO: 3 and amino acids constituting the peptide of SEQ ID NO: 3 were mixed in random order.

Specifically, SH-SY5Y cells were transiently co-transfected with Myc-DJ2 and FLAG-CRBN. Cell extracts were treated with scrambled peptides or peptides of SEQ ID NO: 3 for 24 hours. The cell extract was immunoprecipitated with α-FLAG antibody from rabbit (Rb) and immunoblotted with Myc and FLAG antibody from mouse (Ms) to remove the band of IgG heavy chain. Data are presented as mean ± SD. n = 5. Student's t test was used.

As a result, as shown in FIGS. 17a to 17d , it was confirmed that the circular peptide (SEQ ID NO: 3, DJ2 peptide of FIG. 17b) inhibited the binding of CRBN and DJ2 better than the scrambled peptide.

<Example 12: Confirmation of CRBN and DJ2 binding inhibition ability of tetrapeptide>

The most potent inhibitors are generally less than 500 Da, so to reduce the size of the peptide inhibitor, the experiment was repeated with a peptide covering the most important residues of the hotspot, confirming that the DNRT sequence (SEQ ID NO: 2) was key (see Fig. 18). Accordingly, DNRT peptide and its scrambled peptide were prepared, and the ability to inhibit CRBN and DJ2 binding was confirmed.

Specifically, SH-SY5Y cells were transiently co-transfected with Myc-DJ2 and FLAG-CRBN. Cell extracts were treated with the scrambled peptide or the peptide of SEQ ID NO: 2 for 24 hours. Cell extracts were immunoprecipitated with α-Myc antibody from mice (Ms) and immunoblotted with FLAG and Myc antibodies from rabbits (Rb). Data are presented as mean ± SD. n = 5. Student's t test was used.

As a result, it was confirmed that a relatively higher concentration of ~30 μM of tetrapeptide significantly blocked the interaction between CRBN and DJ2 as seen in the CoIP analysis, but not the scrambled peptide (see Figs. 19a to 19c).

The peptide of the present invention is a peptide mimicking the CRBN binding motif in DJ2, and since it binds to CRBN and interferes with the binding of DJ2 and CRBN, the degradation of DJ2 by CRBN is suppressed. As a result, since the effect of DJ2 inhibiting the aggregation of tau protein is maintained and improved, it can be usefully used for preventing, improving or treating Alzheimer's disease.

Claims (8)

1. A peptide comprising the CRBN binding motif amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.
2. A pharmaceutical composition for preventing or treating Alzheimer's disease, comprising the peptide of claim 1 .
3. The pharmaceutical composition of claim 2, wherein the Alzheimer's disease is caused by Tau phosphorylation.
4. A food composition for preventing or improving Alzheimer's disease, comprising the peptide of claim 1 .
5. According to claim 4, wherein the Alzheimer's disease is due to Tau (Tau) phosphorylation, the food composition.
6. A method for screening a therapeutic agent for Alzheimer's disease, comprising the steps of:

   a) synthesizing a peptide comprising a CRBN binding motif represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3;

   b) analyzing whether the peptide synthesized in step a) can inhibit the binding of CRBN and DJ2 by binding to the DJ2 binding site of CRBN; and

   c) determining as a therapeutic agent for Alzheimer's disease when the peptide synthesized in step b) inhibits the binding of CRBN and DJ2.
7. A method for preventing or treating Alzheimer's disease, comprising administering to an individual a peptide comprising a CRBN binding motif amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.
8. Use of a peptide comprising the CRBN binding motif amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 for preventing or treating Alzheimer's disease.

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