KR101499312B1 - A system for detecting Tau aggregation - Google Patents

Abstract

The present invention relates to a tau protein aggregation detection system, and more particularly, to a fusion protein structure for detecting tau protein aggregation phenomenon, a composition for detecting aggregation phenomenon containing the same, and a tau protein aggregation inhibitor screening composition utilizing the same . Through the present invention, it is possible to provide a more sensitive tau protein aggregation detection system, and moreover, to effectively screen a therapeutic agent for tau protein aggregation inhibitor and Alzheimer's dementia

Description

[0001] The present invention relates to a tau protein aggregation detection system,

The present invention relates to a tau protein aggregation detection system, and more particularly, to a fusion protein structure for detecting tau protein aggregation phenomenon, a composition for detecting aggregation phenomenon containing the same, and a tau protein aggregation inhibitor screening composition utilizing the same .

Dementia is a clinical syndrome that refers to a condition in which a high level of cerebral dysfunction, including memory disorders, language disorders, and personality or emotional changes, is caused by acquired brain lesions.

Dementia can be classified into primary dementia, including Alzheimer's dementia, whose underlying cause is unknown and can not be expected to improve, and vascular dementia caused by a stroke caused by cerebrovascular or cardiovascular disorders. The current treatment for Alzheimer's dementia While cholinesterase inhibitors such as Aricept, Reminyl, and Exelon are used, vascular dementia uses vasodilators and thrombotic inhibitors, and various therapeutic agents are used depending on the type of dementia.

One of the major pathologic features of Alzheimer's disease is neurofibrillary tangles (NFT), which consists of paired helical filaments (PHFs) resulting from hyperphosphorylation of the tau protein. Thus, hyperphosphorylation of the tau protein is understood to be an important step in the formation of NFT.

Tau exists as an isomer that alternates and contains three or four copies of the repeat sequence corresponding to the microtubule-binding domain. The tau in the PHF is proteolytically processed into the core domain, consisting of a phase shifted version of the repeat domain; Only three iterations are involved in stable Ta-tau interaction. Agglutination of hyperphosphorylated tau protein causes destabilization of the microtubule and causes inhibition of various signal transduction through the axon.

In Alzheimer ' s dementia, as a prior patent relating to tau protein, Patent Document 1 discloses a patent for beta-tau aggregation analysis, in which a tau protein-derived polypeptide and a co-deposited beaamyloid are reacted in the presence of a candidate substance, The present invention relates to a technique for measuring the amount of a tau-beta amyloid complex formed, and Patent Document 2 relates to a transgenic animal model expressing Alzheimer's-derived tau protein. In Alzheimer's dementia, Model.

On the other hand, there is not yet a cure for primary dementia such as Alzheimer's dementia, and there is only a slight symptom relief effect of the drugs already released, and there is no drug to cure the cause.

Patent Document 1: US 20020164657 Patent Document 2: US20060112437

In order to solve the problems of the prior art, the present inventors have recognized that the coagulation phenomenon of tau protein is a main disease of Alzheimer's dementia, and that these coagulation inhibitors are targets of a therapeutic agent for dementia and that tau protein And a structure in which a fluorescent protein fragment is bound through a linker peptide, thereby completing the present invention.

It is an object of the present invention to provide a tau protein aggregation detection system.

Another object of the present invention is to provide a structure in which a tau protein and a fluorescent protein fragment are linked through a linker peptide.

Another object of the present invention is to provide a composition for screening a tau protein flocculant inhibitor comprising a plurality of the structures.

It is another object of the present invention to provide a composition for detecting the coagulation of tau protein, which comprises a plurality of the structures.

It is another object of the present invention to provide a base sequence encoding the construct.

It is another object of the present invention to provide a recombinant vector comprising the nucleotide sequence encoding the construct.

It is another object of the present invention to provide a cell transformed with the recombinant vector.

Another object of the present invention is to provide a method for detecting a cancerous cell, comprising the steps of: (a) preparing a culture for the detection composition or the transformed cell; (b) treating the composition or culture of step (a) with a tau protein aggregation inhibiting candidate; And (c) detecting whether or not the tau protein is coagulated after the treatment of the candidate substance in the step (b). The present invention also provides a method for screening a tau protein aggregation inhibitor.

In order to accomplish the above object, the present invention provides a tau protein aggregation detection system (tau-BIFC). This system includes a plurality of structures in which a tau protein and a fluorescent protein fragment are linked by a linker peptide. For example, as shown in Fig. 1, the fluorescent protein fragment is composed of venus 1 and venus 2 fragments in each structure, , When the tau protein (or fragment thereof) is aggregated, the linker peptide can detect the aggregation phenomenon by expressing fluorescence through binding of fragments of each fluorescent protein linked to the tau protein. Such a system can be obtained by treating the present system with a candidate substance for inhibiting tau protein aggregation inhibition or a candidate for treating Alzheimer's disease, and determining whether or not the tau protein aggregates through the treatment, So that the tau protein aggregation inhibitor or the therapeutic agent for Alzheimer's disease can be screened.

In this regard, the present invention provides a construct in which tau protein and fluorescent protein fragments are linked through a linker peptide.

In one embodiment of the present invention, in the structure, the tau protein is a structure comprising the amino acid sequence of SEQ ID NO: 1 or a fragment thereof.

In one embodiment of the present invention, the tau protein is characterized by being composed of the nucleotide sequence of SEQ ID NO: 2 or a fragment thereof.

In one embodiment of the present invention, the fluorescent protein fragment may be a structure having venus, Cerulean, Citrine, mKate, fluorescent proteins having different colors, or a part thereof.

In one embodiment of the present invention, the linker peptide may be any structure, for example, a structure consisting of the amino acid sequence of SEQ ID NO: 3, 4 or 5, or a fragment thereof.

The present invention also provides a composition for screening a tau protein flocculant inhibitor comprising a plurality of said structures.

In one embodiment of the present invention, the structures may be characterized in that their sequences are different from each other, and the sequence of the fluorescent protein fragments in the construct may be different from each other.

In one embodiment of the present invention, the composition may further include a substance that promotes aggregation of tau protein. Such a substance may be beta amyloid, okadaic acid, or HDAC inhibitor.

In addition, the present invention provides a composition for detecting the coagulation of tau protein comprising a plurality of the structures.

The present invention also provides a nucleotide sequence encoding the construct.

In addition, the present invention provides a recombinant vector comprising a nucleotide sequence encoding said construct.

The present invention also provides a cell transformed with the recombinant vector.

The present invention also provides a method for detecting a cancerous cell, comprising the steps of: (a) preparing a culture for the detection composition or the transformed cell; (b) treating the composition or culture of step (a) with a tau protein aggregation inhibiting candidate; And (c) detecting whether the tau protein is coagulated after the candidate material treatment in the step (b).

In one embodiment of the present invention, step (b) of the method may be a step of treating the tau protein aggregation inhibiting candidate substance with a cell lysate obtained by disrupting the cells.

In one embodiment of the present invention, step (c) of the method is not particularly limited as long as it is a method for confirming whether protein aggregation is known in the prior art. Particularly, tau protein aggregation is detected by fluorescence or luciferase Can be measured.

In addition, the present invention provides a composition for screening for the treatment of Alzheimer's dementia comprising a plurality of such structures.

In one embodiment of the present invention, the tau protein in the construct is a tau protein variant consisting of the amino acid sequence of SEQ ID NO: 9.

In the detection system according to the present invention, the cell biology and biochemical analysis (immunostaining, immunoblotting) using the fluorescent protein will provide a better understanding of the pathological phenomenon of the existing tau protein, And can be effectively used for screening tau protein aggregation inhibitor, and furthermore, a therapeutic agent for dementia diseases including Alzheimer's disease.

1 is a block diagram of a tau protein aggregation detection system according to the present invention.
FIG. 2 is a graph showing the results of an increase in fluorescence expression by tau protein aggregation and an aggregation enhancement effect by amyloid.
3 is a graph showing the effect of inhibiting tau aggregation by daunorubicin.
4 is a fluorescence microscope photograph of cells in which tau protein aggregates.
FIG. 5 is a diagram illustrating a structure of a structure in which a plurality of different sequence sets according to the present invention are aggregated. FIG.
FIG. 6 shows the result of FACS measurement of binding between normal and abnormal tau proteins.
FIG. 7 shows the result of western blotting and normalization of normal or abnormal tau protein expressed in the cells, and the result of measuring the phosphorylation status.
Fig. 8 shows the result of fluorescence measurement of binding between normal and abnormal tau proteins.
FIG. 9 is a view showing a process for producing a structure in which a tau protein and a fluorescent protein fragment according to the present invention are linked by a linker.

In order to accomplish the object of the present invention, the present invention relates to a structure in which a tau protein and a fluorescent protein fragment are bound through a linker peptide.

In the present invention, the tau protein of the structure may be a protein consisting of the amino acid sequence of SEQ ID NO: 1 or a fragment thereof. The nucleotide sequence encoding such a tau protein may comprise the nucleotide sequence of SEQ ID NO: 2 or a fragment thereof.

In the present invention, the fluorescent protein fragment may be venus, Cerulean, Citrine, mKate, fluorescent proteins having different colors, or a part thereof.

In one embodiment of the present invention, the fluorescent protein may be a fragment of the venus protein, and may have, for example, the nucleotide sequence of SEQ ID NO: 6 or 7.

In one embodiment of the present invention, the linker peptide can be any structure as long as it does not interfere with the binding between the tau proteins, or conversely, the binding of the fluorescent protein is not disturbed thereby. For example, GGGGSGGGGS), SEQ ID NO: 4 (RPACKIPNDLKQKVMNH), or SEQ ID NO: 5 (AAANSSIDLISVPVDSR) or fragments thereof.

In addition, the present invention relates to a composition for screening a tau protein aggregation inhibitor comprising a plurality of the structures, wherein the fluorescent proteins in the structure are different from each other in one embodiment of the present invention. For example, as shown in FIG. 5, a plurality of structures are combined as a set, and a venus protein, in particular, a fluorescent protein, is attached to one of the structures with amino acids 1-158 (MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPT

LVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTL

VNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQ, SEQ ID NO: 10)

, And 159-240 amino acids (MKNGIKANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRD

HMVLLEFVTAAGITLGMDELYK, SEQ ID NO: 11)

To thereby exhibit fluorescence, thereby detecting tau protein aggregation. This can also be used for screening for coagulation inhibitors.

In one embodiment of the present invention, the composition for screening the coagulation inhibitor may further include a substance that promotes aggregation of tau protein. Examples of the substance include beta amyloid, okadaic acid, alpha-synuclein synuclein < / RTI > protein or an HDAC inhibitor.

It is another object of the present invention to provide a composition for detecting the coagulation of tau protein, which comprises a plurality of the structures.

It is another object of the present invention to provide a base sequence encoding the construct.

It is another object of the present invention to provide a recombinant vector comprising the nucleotide sequence encoding the construct.

It is another object of the present invention to provide a cell transformed with the recombinant vector.

Another object of the present invention is to provide a method for detecting a cancerous cell, comprising the steps of: (a) preparing a culture for the detection composition or the transformed cell; (b) treating the composition or culture of step (a) with a tau protein aggregation inhibiting candidate; And (c) detecting whether or not the tau protein is coagulated after the treatment of the candidate substance in the step (b). The present invention also provides a method for screening a tau protein aggregation inhibitor.

In one embodiment of the present invention, step (b) of the method may be a step of treating the tau protein aggregation inhibiting candidate substance with a cell lysate obtained by disrupting the cells.

In one embodiment of the present invention, step (c) of the method is not particularly limited as long as it is a method for confirming whether or not protein aggregation is known in the prior art, and in particular, whether or not tau protein aggregation is measured using fluorescence or luciferase can do.

Particularly, in the present invention, when the fluorescence measurement method is used as the above detection method, detection is possible in a much shorter time than in the case of confirming the formation of aggregates by biochemical method (immunoblotting) by directly breaking cells, And there is an advantage in the cost part.

Another object of the present invention is to provide a composition for screening Alzheimer's dementia comprising a plurality of the above structures.

 The mutants of tau protein found in Alzheimer's patients with dementia include K257T, I260V, G272V in exon 9, N279K, K280, L284L, P301L, P301S, S305N in exon 10, V337M in exon 12 and G389R in exon 13. Among them, in one embodiment of the present invention, P301L, that is, a typical mutant found in Alzheimer's disease patients in which the 301th amino acid in the amino acid sequence of the normal tau protein is substituted with leucine in proline, , It has been confirmed that the structure of the present invention can be used for the screening of flocculant inhibitors and further for the screening of therapeutic agents for Alzheimer's disease. Such a P301L mutant is composed of the amino acid sequence of SEQ ID NO: 9, and when these mutants are used in the detection method of the present invention, they may be composed of some fragments.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not construed as being limited by these embodiments.

Production of tau protein aggregation detection system according to the present invention

Linker consisting of Venus1 (the amino acid sequence of SEQ ID NO: 10, the nucleotide sequence of SEQ ID NO: 6) and 10 amino acids (SEQ ID NO: 3), the first fragment of the YFP protein VENUS, and the Tau wt (2N4R, No. 8) were amplified by PCR and cloned into mammalian expression vector pcDNA3.1 (Invitrogen, V790-20) (5'BamHI, 3'EcoRI). Construction of a sequence in which linker and tau wt (2N4R) sequences having the same sequence as Venus2 (amino acid of SEQ ID NO: 11 consisting of amino acid sequence of 159-240 amino acid sequence, SEQ ID NO: 7) and sequence of second fragment, were sequentially prepared in the same manner.

Specifically, Fig. 9 briefly shows the production process of the present protein structure,

Venus1-zipper and venus2-zipper constructs prepared according to the method described in Ingrid Remy & Stephen W Michnick, A highly sensitive protein-protein interaction assay based on Gaussia luciferase, Nature Methods 3, 977-979 (2006) As shown in FIG. 9, a linker region is connected after vinus1 / 2, followed by a zipper domain. This zipper domain has a characteristic of attaching to each other, and when these two genes are injected into the cells, the proteins are expressed in the cells to form a zipper bond and thereby the venus protein is completed and fluorescence is exhibited. , We tried to construct a PCR product of venus1 / 2-linker, tau wt / P301L using pcr in the first step because cloning with restriction enzyme was not easy. In the next step, the linked venus1 / 2-linker-tau wt / P301L (final PCR product of FIG. 9) was constructed by hybrid PCR between the products thus constructed and this PCR product was cloned into a pcDNA3.1 vector using a restriction enzyme .

First, venus1-linker (GGT GGC GGT GGC TCT GGG GGT GGT GGG TCC: SEQ ID NO: 18) Only the venus1-linker portion of the zipper plasmid was amplified with pcr (and venus2 also). Four primers were used in this step (primers 1 to 4). tau also produces linker-tau using primers (primers 5-6, linker, linker sequence in primer5 to link to tau gene).

After preparing three PCR products (products 1 to 3), we mixed the PCR fragments in two combinations to make a hybrid. (product 1 + product 3, product 2 + product 3). 9, only 1 + 3 is expressed, but 2 + 3 is the same). When PCR is performed with primer 1 and primer 6, two pcr fragments become single strand at the high temperature, and the linker part is identical to each other. Therefore, when the temperature is lowered, a double strand is formed at the linker part, hybrid PCR fragments were constructed and PCR amplification by primer1,6 was possible using this template. The resulting final fragment was cloned into pcDNA3.1.

The primers used are as follows (the underline is the linker, and the underscores in numbers 2, 5 and 4-5 are complementary).

Primer 1 (SEQ ID NO: 12) AGCTAGGATCCACCATGGTGAGCAAGGGCGAG

Primer 2 (SEQ ID NO: 13) GGACCCACCACCTCCAGAGCCACCGCCACC CTGCTTGTCGGCGGTGATA

Primer 3 (SEQ ID NO: 14) AGCTAGGATCCACCATGAAGAACGGCATCAAGGC

Primer 4 (SEQ ID NO: 15) GGACCCACCACCTCCAGAGCCACCGCCACC CTTGTACAGCTCGTCCATGC

Primer 5 (SEQ ID NO: 16) GGTGGCGGTGGCTCTGGAGGTGGTGGGTCC ATGGCTGAGCCCCGCCAG

Primer 6 (SEQ ID NO: 17) AGCTAGAATTCTCACAAACCCTGCTTGGCCA

Increased fluorescence expression by tau protein aggregation and confirmation of aggregation enhancement effect by amyloid

The venus1-tau wt and venus2-tau wt prepared in Example 1 were transfected into Hela cells (10 ⁴ cells / well) seeded on a 96-well plate to express two constructs in the cells (amyloid-b 0, 0.1, 1 μM treatment conditions). Mock was transfected with venus1-tau wt only, and no fluorescence was observed because no venus complex was formed. Twenty-four hours after transfection, amyloid-beta (bachem, H1368) was treated (final concentration 0, 0.1, 1 μM) and washed twice with PBS buffer for 24 hours. Cells were fixed with 4% paraformaldehyde, and the fluorescence of the cells was measured in the immobilized solution. (Miltilabel reader Victor X4, Perkin-Elmer)

Amyloid-beta oligomers were formed by incubation at 4 ° C for 24 hours. As a result, fluorescence was observed more than 2 times in the condition that two constructs were expressed together (amyloid-beta untreated) compared to a mock in which no fluorescence was observed, which means that tau aggregation occurred in the cells. This coagulation phenomenon is further increased by treatment with amyloid-beta (especially 1 μM) (FIG. 2).

Experiment to confirm tau protein aggregation inhibition effect by Daunorubicin

The venus1-tau wt and venus2-tau wt prepared in Example 1 were transfected into Hela cells (10 ⁴ cells / well) seeded on a 96-well plate to express two constructs in the cells (amyloid-beta 0, 0.1, 1 μM treatment conditions). Mock was transfected with venus1-tau wt only, and no fluorescence was observed because no venus complex was formed. After 24 hours of transfection, Daunorubicin was dissolved in DMEM and treated with the final concentrations (0, 1, 10 μM) indicated in FIG. Twenty-four hours after treatment, the cells were washed twice with PBS buffer. Cells were fixed with 4% paraformaldehyde, and the fluorescence of the cells was measured in the immobilized solution.

As a result, fluorescence was observed more than 2 times in the condition that two constructs were expressed together (amyloid-beta untreated) compared to a mock in which no fluorescence was observed, which means that tau aggregation occurred in the cells. This aggregation phenomenon was observed to be reduced by treatment with daunorubicin, a tau coagulation inhibitor. Thus, in addition to the results in Example 2, this result indicates that the tau-BIFC system can sensitively observe the coagulation phenomenon of tau (Fig. 3).

Fluorescence microscopy of tau protein-aggregated cells

The venus1-tau wt and venus2-tau wt prepared in Example 1 were transfected into Hela cells (10 ⁵ cells / well) seeded on a 12-well plate to express both constructs in the cells. Mock was transfected with venus1-tau wt only, and no fluorescence was observed because no venus complex was formed. 24 hours after transfection, Aβ oligomer (1 mM) was cultured at 4 ℃ for 24 hours and fibrill (1 mM) at 37 ℃ for 24 hours. Cells grown on Coverslip were fixed with 4% paraformaldehyde and observed by fluorescence microscopy. As a result, it was observed that the amount of fluorescence-indicating cells was increased during treatment with amyloid-beta. It was also observed that the oligomer state of the amyloid-beta (monomer <oligomer <fibril) tau increased the fluorescence due to the increase of aggregation phenomenon.

Taken together, the system according to the present invention was found to have excellent sensitivity indicating that the aggregation state of tau which changes under the condition of enhancing or inhibiting tau aggregation is easily observable.

Quantification of binding between normal and abnormal tau proteins

5-1. The binding between normal Tau protein and abnormal Tau protein was quantified using the detection system according to the present invention. SH-SY5Y cells (ATCC (CRL-2266TM), a neuroblastomacellline seeded on a ^6- well plate (5x10 ⁵ cells / well) were subcultured in DMEM (10% FBS, 1% antibiotics) ) In the same manner as described in the previous examples to measure the amount of fluorescence by venous complex formation and tau aggregates produced in the cells, respectively, by flow cytometry And quantified. As a result, fluorescence could not be observed in independent expression of each of the four genes of v1-tau wt / P301L or v2-tau wt / P301L, but v1-tau wt / v2-tau wt or v1-tau P301L / In the combination of v2-tau P301L, formation of venus complex due to binding of tau protein and resulting fluorescence could be observed. In particular, it was observed that fluorescence amount due to binding between abnormal tau (P301L) protein was higher than that between normal tau protein. (Fig. 6)

5-2. In order to confirm whether the fluorescence observed in the above 5-1 was due to aggregate formation, proteins expressed in the cells were observed with western blot under the same expression conditions. The cells were transfected into SH-SY5Y cells ( ⁵ × 10 ⁵ cells / well) seeded on a ^6- well plate with the respective combination conditions (v1-tau wt / v2-tau wt and v1-tau P301L / v2-tau P301L). After 48 hours, the cells expressing the respective proteins were disrupted with hypotonic buffer, and the pellet (P) portion including the supernatant (S) and remaining cell tissue and protein aggregates was separated by SDS-PAGE, (YFP-venus) and tau protein phosphorylation (Thr231) were investigated by western blot. As a result, it was observed that most of the normal tau were present in the supernatant part (S), and in the abnormal tau (P301L), the abnormal tau (P301L) protein was observed in the pellet, (FIG. 7, FIG. A). Figures B and C are graphs comparing these results quantitatively. It can be seen that both the amount of aggregated tau protein and the amount of phosphorylated tau are increased in abnormal tau (P301L).

5-3. Fluorescence of the expressed fluorescent protein complex (v1-tau wt / v2-tau wt, v1-tau P301L / v2-tau P301L) was observed under a microscope. Transfection was performed with two combinations (v1-tau wt / v2-tau wt, v1-tau P301L / v2-tau P301L) in SH-SY5Y cells, which were seeded on a ^6- well plate (5x10 ⁵ cells / well) and Neuroblastomacellline. After 48 hours, the cells grown in Coverslip were fixed with 4% paraformaldehyde and treated with thiazine red and DAPI, and their expression levels were observed by fluorescence microscopy. As a result, we could observe the green of the tau protein complex formed in the cell, and compared the staining pattern of thiazine red (red), a reagent that specifically reacts with the aggregate protein and emits a specific fluorescence, P301L) protein was observed to form aggregates. This indicates that the tau complex observed in the detection system of the present invention forms an aggregate in the case of abnormal tau (P301L) protein, which is consistent with the results obtained in the foregoing 5-1 and 5-2. Taken together, the system of the present invention for detecting the coagulation phenomenon of tau protein shows that aggregation of tau protein can be observed by various methods such as microscopy, flurometer, and FACS using fluorescence, which is suitable for large-scale drug discovery .

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

<110> Nanomol <120> A system for detecting Tau aggregation <130> 2012-01 <160> 18 <170> Kopatentin 2.0 <210> 1 <211> 441 <212> PRT <213> Tau protein <400> 1 Met Ala Glu Pro Arg Glu Glu Phe Glu Val Met Glu Asp His Ala Gly   1 5 10 15 Thr Tyr Gly Leu Gly Asp Arg Lys Asp Gln Gly Gly Tyr Thr Met His              20 25 30 Gln Asp Gln Glu Gly Asp Thr Asp Ala Gly Leu Lys Glu Ser Pro Leu          35 40 45 Gln Thr Pro Thr Glu Asp Gly Ser Glu Glu Pro Gly Ser Glu Thr Ser      50 55 60 Asp Ala Lys Ser Thr Pro Thr Ala Glu Asp Val Thr Ala Pro Leu Val  65 70 75 80 Asp Glu Gly Ala Pro Gly Lys Gln Ala Ala Gln Pro His Thr Glu                  85 90 95 Ile Pro Glu Gly Thr Thr Ala Glu Glu Ala Gly Ile Gly Asp Thr Pro             100 105 110 Ser Leu Glu Asp Glu Ala Ala Gly His Val Thr Gln Ala Arg Met Val         115 120 125 Ser Lys Ser Lys Asp Gly Thr Gly Ser Asp Asp Lys Lys Ala Lys Gly     130 135 140 Ala Asp Gly Lys Thr Lys Ile Ala Thr Pro Arg Gly Ala Ala Pro Pro 145 150 155 160 Gly Gln Lys Gly Gln Ala Asn Ala Thr Arg Ile Pro Ala Lys Thr Pro                 165 170 175 Pro Ala Pro Lys Thr Pro Pro Ser Ser Gly Glu Pro Pro Lys Ser Gly             180 185 190 Asp Arg Ser Gly Tyr Ser Ser Pro Gly Ser Pro Gly Thr Pro Gly Ser         195 200 205 Arg Ser Thr Pro Ser Leu Pro Thr Pro Pro Thr Arg Glu Pro Lys     210 215 220 Lys Val Ala Val Val Arg Thr Pro Pro Lys Ser Pro Ser Ser Ala Lys 225 230 235 240 Ser Arg Leu Gln Thr Ala Pro Val Met Pro Asp Leu Lys Asn Val                 245 250 255 Lys Ser Lys Ile Gly Ser Thr Glu Asn Leu Lys His Gln Pro Gly Gly             260 265 270 Gly Lys Val Gln Ile Ile Asn Lys Lys Leu Asp Leu Ser Asn Val Gln         275 280 285 Ser Lys Cys Gly Ser Lys Asp Asn Ile Lys His Val Pro Gly Gly Gly     290 295 300 Ser Val Gln Ile Val Tyr Lys Pro Val Asp Leu Ser Lys Val Thr Ser 305 310 315 320 Lys Cys Gly Ser Leu Gly Asn Ile His His Lys Pro Gly Gly Gly Gln                 325 330 335 Val Glu Val Lys Ser Glu Lys Leu Asp Phe Lys Asp Arg Val Gln Ser             340 345 350 Lys Ile Gly Ser Leu Asp Asn Ile Thr His Val Pro Gly Gly Gly Asn         355 360 365 Lys Lys Ile Glu Thr His Lys Leu Thr Phe Arg Glu Asn Ala Lys Ala     370 375 380 Lys Thr Asp His Gly Ala Glu Ile Val Tyr Lys Ser Pro Val Val Ser 385 390 395 400 Gly Asp Thr Ser Pro Arg His Leu Ser Asn Val Ser Ser Thr Gly Ser                 405 410 415 Ile Asp Met Val Asp Ser Pro Gln Leu Ala Thr Leu Ala Asp Glu Val             420 425 430 Ser Ala Ser Leu Ala Lys Gln Gly Leu         435 440 <210> 2 <211> 1233 <212> DNA <213> Tau protein <400> 2 atggctgagc cccgccagga gttcgaagtg atggaagatc acgctgggac gtacgggttg 60 ggggacagga aagatcaggg gggctacacc atgcaccaag accaagaggg tgacacggac 120 gctggcctga aagaatctcc cctgcagacc cccactgagg acggatctga ggaaccgggc 180 tctgaaacct ctgatgctaa gagcactcca acagcggaag atgtgacagc acccttagtg 240 gatgagggag ctcccggcaa gcaggctgcc gcgcagcccc acacggagat cccagaagga 300 accagactg aagaagcagg cattggagac acccccagcc tggaagacga agctgctggt 360 cacgtgaccc aagctcgcat ggtcagtaaa agcaaagacg ggactggaag cgatgacaaa 420 aaagccaagg gggctgatgg taaaacgaag atcgccacac cgcggggagc agcccctcca 480 ggccagaagg gccaggccaa cgccaccagg attccagcaa aaaccccgcc cgctccaaag 540 acaccaccca gctctggtga acctccaaaa tcaggggatc gcagcggcta cagcagcccc 600 ggctccccag gcactcccgg cagccgctcc cgcaccccgt cccttccaac cccacccacc 660 cgggagccca agaaggtggc agtggtccgt actccaccca agtcgccgtc ttccgccaag 720 agccgcctgc agacagcccc cgtgcccatg ccagacctga agaatgtcaa gtccaagatc 780 ggctccactg agaacctgaa gcaccagccg ggaggcggga aggtgcaaat agtctacaaa 840 ccagttgacc tgagcaaggt gacctccaag tgtggctcat taggcaacat ccatcataaa 900 ccaggaggtg gccaggtgga agtaaaatct gagaagcttg acttcaagga cagagtccag 960 tcgaagattg ggtccctgga caatatcacc cacgtccctg gcggaggaaa taaaaagatt 1020 gaaacccaca agctgacctt ccgcgagaac gccaaagcca agacagacca cggggcggag 1080 atcgtgtaca agtcgccagt ggtgtctggg gacacgtctc cacggcatct cagcaatgtc 1140 tcctccaccg gcagcatcga catggtagac tcgccccagc tcgccacgct agctgacgag 1200 gtgtctgcct ccctggccaa gcagggtttg tga 1233 <210> 3 <211> 10 <212> PRT <213> linker peptide <400> 3 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser   1 5 10 <210> 4 <211> 17 <212> PRT <213> linker peptide <400> 4 Arg Pro Ala Cys Lys Ile Pro Asn Asp Leu Lys Gln Lys Val Met Asn   1 5 10 15 His     <210> 5 <211> 17 <212> PRT <213> linker peptide <400> 5 Ala Ala Asn Ser Ser Ile Asp Leu Ile Ser Val   1 5 10 15 Arg     <210> 6 <211> 474 <212> DNA <213> Venus1 fragment <400> 6 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120 ggcaagctga ccctgaagct gatctgcacc accggcaagc tgcccgtgcc ctggcccacc 180 ctcgtgacca ccctgggcta cggcctgcag tgcttcgccc gctaccccga ccacatgaag 240 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420 aagctggagt acaactacaa cagccacaac gtctatatca ccgccgacaa gcag 474 <210> 7 <211> 246 <212> DNA <213> Venus2 fragment <400> 7 atgaagaacg gcatcaaggc caacttcaag atccgccaca acatcgagga cggcggcgtg 60 cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc 120 gacaaccact acctgagcta ccagtccgcc ctgagcaaag accccaacga gaagcgcgat 180 cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg 240 tacaag 246 <210> 8 <211> 1233 <212> DNA <213> Tau wt 2N4R <400> 8 atggctgagc cccgccagga gttcgaagtg atggaagatc acgctgggac gtacgggttg 60 ggggacagga aagatcaggg gggctacacc atgcaccaag accaagaggg tgacacggac 120 gctggcctga aagaatctcc cctgcagacc cccactgagg acggatctga ggaaccgggc 180 tctgaaacct ctgatgctaa gagcactcca acagcggaag atgtgacagc acccttagtg 240 gatgagggag ctcccggcaa gcaggctgcc gcgcagcccc acacggagat cccagaagga 300 accagactg aagaagcagg cattggagac acccccagcc tggaagacga agctgctggt 360 cacgtgaccc aagctcgcat ggtcagtaaa agcaaagacg ggactggaag cgatgacaaa 420 aaagccaagg gggctgatgg taaaacgaag atcgccacac cgcggggagc agcccctcca 480 ggccagaagg gccaggccaa cgccaccagg attccagcaa aaaccccgcc cgctccaaag 540 acaccaccca gctctggtga acctccaaaa tcaggggatc gcagcggcta cagcagcccc 600 ggctccccag gcactcccgg cagccgctcc cgcaccccgt cccttccaac cccacccacc 660 cgggagccca agaaggtggc agtggtccgt actccaccca agtcgccgtc ttccgccaag 720 agccgcctgc agacagcccc cgtgcccatg ccagacctga agaatgtcaa gtccaagatc 780 ggctccactg agaacctgaa gcaccagccg ggaggcggga aggtgcaaat agtctacaaa 840 ccagttgacc tgagcaaggt gacctccaag tgtggctcat taggcaacat ccatcataaa 900 ccaggaggtg gccaggtgga agtaaaatct gagaagcttg acttcaagga cagagtccag 960 tcgaagattg ggtccctgga caatatcacc cacgtccctg gcggaggaaa taaaaagatt 1020 gaaacccaca agctgacctt ccgcgagaac gccaaagcca agacagacca cggggcggag 1080 atcgtgtaca agtcgccagt ggtgtctggg gacacgtctc cacggcatct cagcaatgtc 1140 tcctccaccg gcagcatcga catggtagac tcgccccagc tcgccacgct agctgacgag 1200 gtgtctgcct ccctggccaa gcagggtttg tga 1233 <210> 9 <211> 441 <212> PRT <213> Tau P301L 2N4R <400> 9 Met Ala Glu Pro Arg Glu Glu Phe Glu Val Met Glu Asp His Ala Gly   1 5 10 15 Thr Tyr Gly Leu Gly Asp Arg Lys Asp Gln Gly Gly Tyr Thr Met His              20 25 30 Gln Asp Gln Glu Gly Asp Thr Asp Ala Gly Leu Lys Glu Ser Pro Leu          35 40 45 Gln Thr Pro Thr Glu Asp Gly Ser Glu Glu Pro Gly Ser Glu Thr Ser      50 55 60 Asp Ala Lys Ser Thr Pro Thr Ala Glu Asp Val Thr Ala Pro Leu Val  65 70 75 80 Asp Glu Gly Ala Pro Gly Lys Gln Ala Ala Gln Pro His Thr Glu                  85 90 95 Ile Pro Glu Gly Thr Thr Ala Glu Glu Ala Gly Ile Gly Asp Thr Pro             100 105 110 Ser Leu Glu Asp Glu Ala Ala Gly His Val Thr Gln Ala Arg Met Val         115 120 125 Ser Lys Ser Lys Asp Gly Thr Gly Ser Asp Asp Lys Lys Ala Lys Gly     130 135 140 Ala Asp Gly Lys Thr Lys Ile Ala Thr Pro Arg Gly Ala Ala Pro Pro 145 150 155 160 Gly Gln Lys Gly Gln Ala Asn Ala Thr Arg Ile Pro Ala Lys Thr Pro                 165 170 175 Pro Ala Pro Lys Thr Pro Pro Ser Ser Gly Glu Pro Pro Lys Ser Gly             180 185 190 Asp Arg Ser Gly Tyr Ser Ser Pro Gly Ser Pro Gly Thr Pro Gly Ser         195 200 205 Arg Ser Thr Pro Ser Leu Pro Thr Pro Pro Thr Arg Glu Pro Lys     210 215 220 Lys Val Ala Val Val Arg Thr Pro Pro Lys Ser Pro Ser Ser Ala Lys 225 230 235 240 Ser Arg Leu Gln Thr Ala Pro Val Met Pro Asp Leu Lys Asn Val                 245 250 255 Lys Ser Lys Ile Gly Ser Thr Glu Asn Leu Lys His Gln Pro Gly Gly             260 265 270 Gly Lys Val Gln Ile Ile Asn Lys Lys Leu Asp Leu Ser Asn Val Gln         275 280 285 Ser Lys Cys Gly Ser Lys Asp Asn Ile Lys His Val Leu Gly Gly Gly     290 295 300 Ser Val Gln Ile Val Tyr Lys Pro Val Asp Leu Ser Lys Val Thr Ser 305 310 315 320 Lys Cys Gly Ser Leu Gly Asn Ile His His Lys Pro Gly Gly Gly Gln                 325 330 335 Val Glu Val Lys Ser Glu Lys Leu Asp Phe Lys Asp Arg Val Gln Ser             340 345 350 Lys Ile Gly Ser Leu Asp Asn Ile Thr His Val Pro Gly Gly Gly Asn         355 360 365 Lys Lys Ile Glu Thr His Lys Leu Thr Phe Arg Glu Asn Ala Lys Ala     370 375 380 Lys Thr Asp His Gly Ala Glu Ile Val Tyr Lys Ser Pro Val Val Ser 385 390 395 400 Gly Asp Thr Ser Pro Arg His Leu Ser Asn Val Ser Ser Thr Gly Ser                 405 410 415 Ile Asp Met Val Asp Ser Pro Gln Leu Ala Thr Leu Ala Asp Glu Val             420 425 430 Ser Ala Ser Leu Ala Lys Gln Gly Leu         435 440 <210> 10 <211> 158 <212> PRT <213> Venus 1 fragment <400> 10 Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu   1 5 10 15 Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly              20 25 30 Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile          35 40 45 Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr      50 55 60 Leu Gly Tyr Gly Leu Gln Cys Phe Ala Arg Tyr Pro Asp His Met Lys  65 70 75 80 Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu                  85 90 95 Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu             100 105 110 Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly         115 120 125 Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr     130 135 140 Asn Tyr Asn Ser His Asn Val Tyr Ile Thr Ala Asp Lys Gln 145 150 155 <210> 11 <211> 82 <212> PRT <213> Venus 2 fragment <400> 11 Met Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Ile Glu   1 5 10 15 Asp Gly Gly Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile              20 25 30 Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Tyr Gln          35 40 45 Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu      50 55 60 Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu  65 70 75 80 Tyr Lys         <210> 12 <211> 32 <212> DNA <213> primer 1 <400> 12 agctaggatc caccatggtg agcaagggcg ag 32 <210> 13 <211> 49 <212> DNA <213> primer 2 <400> 13 ggacccacca cctccagagc caccgccacc ctgcttgtcg gcggtgata 49 <210> 14 <211> 34 <212> DNA <213> primer 3 <400> 14 agctaggatc caccatgaag aacggcatca aggc 34 <210> 15 <211> 50 <212> DNA <213> primer 4 <400> 15 ggacccacca cctccagagc caccgccacc cttgtacagc tcgtccatgc 50 <210> 16 <211> 48 <212> DNA <213> primer 5 <400> 16 ggtggcggtg gctctggagg tggtgggtcc atggctgagc cccgccag 48 <210> 17 <211> 31 <212> DNA <213> primer 6 <400> 17 agctagaatt ctcacaaacc ctgcttggcc a 31 <210> 18 <211> 30 <212> DNA <213> linker peptide <400> 18 ggtggcggtg gctctggagg tggtgggtcc 30

Claims (16)

(i) a tau protein consisting of the amino acid sequence of SEQ ID NO: 1 or 9 and a fusion protein comprising a fluorescent protein fragment consisting of the amino acid sequence of SEQ ID NO: 10 linked by a linker peptide comprising the amino acid sequence of SEQ ID NO: 3; And
(ii) a tau protein comprising the amino acid sequence of SEQ ID NO: 1 or 9, and a fusion protein fragment comprising the amino acid sequence of SEQ ID NO: 11 linked by a linker peptide comprising the amino acid sequence of SEQ ID NO:
Wherein the tau protein is present in an amount of from about 0.1% to about 10% by weight.
delete delete A method according to claim 1,
(i) a tau protein consisting of the amino acid sequence of SEQ ID NO: 1 or 9 and a fusion protein comprising a fluorescent protein fragment consisting of the amino acid sequence of SEQ ID NO: 10 linked by a linker peptide comprising the amino acid sequence of SEQ ID NO: 3; And
(ii) a tau protein comprising the amino acid sequence of SEQ ID NO: 1 or 9, and a fusion protein fragment comprising the amino acid sequence of SEQ ID NO: 11 linked by a linker peptide comprising the amino acid sequence of SEQ ID NO:
Wherein the tau protein aggregation inhibitor is selected from the group consisting of:
delete 5. The composition of claim 4, wherein the composition further comprises a substance that enhances aggregation of tau protein selected from the group consisting of beta amyloid, okadaic acid, and an HDAC inhibitor.
delete delete delete delete delete delete A tau protein aggregation inhibitor screening method comprising the steps of:
(a) preparing a culture of cells transformed with the nucleotide sequence encoding the composition of claim 4 or the fusion proteins of claim 4;
(b) treating the composition or culture of step (a) with a tau protein aggregation inhibiting candidate; And
(c) detecting the presence or absence of tau protein aggregation by measuring fluorescence after the candidate material treatment in the step (b).
delete A method according to claim 1,
(i) a tau protein consisting of the amino acid sequence of SEQ ID NO: 1 or 9 and a fusion protein comprising a fluorescent protein fragment consisting of the amino acid sequence of SEQ ID NO: 10 linked by a linker peptide comprising the amino acid sequence of SEQ ID NO: 3; And
(ii) a tau protein comprising the amino acid sequence of SEQ ID NO: 1 or 9, and a fusion protein fragment comprising the amino acid sequence of SEQ ID NO: 11 linked by a linker peptide comprising the amino acid sequence of SEQ ID NO:
Or a pharmaceutically acceptable salt thereof.
delete

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