JP4948845B2 - Method for screening substances for treatment of neurodegenerative diseases - Google Patents

Description

  The present invention relates to a method for screening a substance for treating a neurodegenerative disease using intracellular proteasome activity as an index.

  In many neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, proteinaceous aggregates are formed in nerve cells. Aggregates called neurofibrillary tangles in Alzheimer's disease and Lewy bodies in Parkinson's disease are all fibrous deposits composed of various proteins. Tau and Lewy bodies are the main components of neurofibrillary tangles. Α-synuclein has been identified as a major component of each. Particularly in Parkinson's disease, a gene encoding α-synuclein has been found as one of the causative genes from genetic analysis of familial disease families. Since there is a correlation between the site where these aggregates appear and the site where neurons drop out, a mechanism is considered that aggregates appearing inside cells become cytopathic, eventually leading to the death and development of neurons. Although not experimentally proven.

  In recent years, there have been reports that molecules such as oligomers and / or protofibrils, which are considered to be pre-stage states of fibers, are cytotoxic bodies, and the fibers are not toxic but rather protective. Therefore, it is important to clarify not only the fiber but also the structure, generation mechanism, and toxicity mechanism of these molecules.

One of the mechanisms of aggregate formation is an abnormality in the intracellular proteolytic system. That is, it is considered that an abnormality occurs in the intracellular proteolytic system for some reason, and the protein that should be decomposed originally accumulates without being decomposed, and such an aggregate is formed. In recent years, intracellular protein quality control mechanisms have attracted attention, and what is thought to play a central role is the degradation mechanism of the ubiquitin-proteasome system. The proteasome is a giant proteolytic complex involved not only in protein quality control but also in various life phenomena. It mainly degrades ubiquitinated proteins, but some proteins are known to undergo ubiquitin-independent degradation.
Gilon, T., Chomsky, O., and Kulka, RG (1998) Degradation signals for ubiquitin system proteolysis in Saccharomyces cerevisiae. The EMBO J. 17, 2759-2766 Bence, NF, Sampat, RM, and Kopit, RR (2001) Impairment of the ubiquitin-proteasome system by protein aggregation.Science 292, 1552-1555

  An object of the present invention is to provide a screening method for a substance for treating a neurodegenerative disease.

  In order to solve the above-mentioned problems, the present inventor considered that the proteasome is involved in the accumulation of intracellular α-synuclein in Parkinson's disease and the like, and conducted intensive research on the relationship between intracellular α-synuclein and proteasome activity. . Then, an assay system was constructed using GFP-CL1 in which a proteasome degradation signal was added to green fluorescent protein (GFP), and it was clarified that α-synuclein expressed in cells suppressed proteasome activity, and the present invention was completed. It came to do.

That is, the present invention is as follows.
(1) A method for screening a substance that suppresses inhibition of proteasome function, wherein a test substance is brought into contact with a cell expressing α-synuclein, a proteasome degradation signal protein, and a labeling substance, and the signal of the labeling substance is detected. When the signal intensity of the detected labeling substance is lower than the signal intensity of the labeling substance in the control cells not contacted with the test substance, the test substance is selected as a substance that suppresses the inhibition of proteasome function. Said method.

(2) A method for screening a substance that promotes the function of proteasome, comprising a protein capable of forming amyloid fibrils, a cell expressing a proteasome degradation signal protein, and a labeling substance, or a proteasome degradation signal protein and a label A test substance is brought into contact with a cell expressing the substance to detect the signal of the labeling substance, and the signal intensity of the detected labeling substance is higher than the signal intensity of the labeling substance in the control cell not contacted with the test substance. When it decreases, the test substance is selected as a substance that promotes the function of the proteasome.

(3) A method for screening a substance that suppresses at least one selected from the group consisting of oligomerization, protofibrillation and fibrosis of a protein capable of forming amyloid fibrils, and a protein capable of forming amyloid fibrils; A test substance is brought into contact with a cell expressing a proteasome degradation signal protein and a labeling substance to detect the signal of the labeling substance, and the signal intensity of the detected labeling substance in the control cell not contacted with the test substance When the signal intensity is lower than the signal intensity of the labeling substance, the test substance is selected as a substance that suppresses at least one selected from the group consisting of protein oligomerization, protofibrillation and fibrosis that can form amyloid fibrils. And said method.

  In the method of the present invention, a substance that suppresses inhibition of proteasome function by a protein capable of forming amyloid fibrils and a substance that promotes the function of proteasome can be used for the treatment of neurodegenerative diseases. In addition, a substance that suppresses at least one selected from the group consisting of oligomerization, protofibrillation, and fibrosis of proteins capable of forming amyloid fibrils can also be used for the treatment of neurodegenerative diseases.

  Here, examples of the protein capable of forming amyloid fibril include at least one selected from the group consisting of α-synuclein, tau, β-amyloid or a precursor thereof, polyglutamine, and prion.

  The present invention provides a method for screening a substance that suppresses inhibition of proteasome function inhibition by α-synuclein, and a method for screening a substance that suppresses oligomerization and / or protofibrillation of α-synuclein. Substances to be screened according to the present invention can be used as therapeutic agents for neurodegenerative diseases. In addition, since the method of the present invention can be assayed using the fluorescence intensity or the like as an index, it is possible to easily increase the throughput.

Hereinafter, the present invention will be described in detail.
1. Overview The relationship between neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease and the intracellular proteolytic system represented by the proteasome has attracted attention. That is, in non-dividing cells such as nerve cells, protein quality control by proteasome is indispensable for maintaining cell homeostasis, and some abnormality occurs in the intracellular proteolytic system, and undegraded protein accumulates in the cell. Therefore, a mechanism is considered that the failure of quality control to exert cytotoxicity causes neurodegenerative diseases. The proteasome is an enzyme that forms a multi-subunit complex, plays a central role in selectively degrading intracellular proteins localized in the nucleus and cytoplasm. This enzyme plays various roles in vivo. For example, in the cell cycle, apoptosis, metabolic regulation, immune response, signal transduction, transcriptional control, quality control, stress response, DNA repair, etc. Perform function control. Proteasome proteolysis is controlled by a ubiquitin-proteasome system linked to ubiquitin, which serves as a landmark for degrading target proteins. Proteasome is an enzyme that is indispensable for the survival of cells, and suppression of intracellular proteasome activity is thought to ultimately lead to cell death.

The present inventor examined the influence of a protein capable of forming amyloid fibrils, which is one of the causative substances of neurodegenerative diseases and cell death, on proteasome activity.
As a system for measuring the proteasome activity in the cell, any experimental method can be used as long as it can confirm the degradation of the desired protein in the cell. For example, the fluorescent fluorescent protein (GFP) Examples include systems (reference documents (1) and (2)) that measure the intensity of the labeling signal using GFP-CL1 in which a sequence that is degraded by the proteasome (referred to as proteasome degradation signal sequence) is bound to the C-terminal side.

  The present inventor constructed an assay system using GFP-CL1 obtained by adding a proteasome degradation signal sequence (CL1) consisting of 16 amino acids to a protein emitting green fluorescence called GFP (FIG. 1). This method is characterized in that the proteasome activity in living cells can be easily monitored as the fluorescence intensity of GFP. When GFP is expressed in a cell, the cell emits green fluorescence and the cell glows green. In addition, when α-synuclein is expressed in cells, intracellular proteasome activity is suppressed.

  When the GFP-CL1 is expressed in cells, the GFP-CL1 is decomposed by intracellular proteasome activity, and green fluorescence is not emitted in the cells. When an agent that suppresses proteasome activity is allowed to coexist in this assay system, proteasome activity is suppressed, so that degradation of GFP-CL1 is suppressed, and the cells glow green. In other words, this assay system has the property that when there is proteasome activity, the cells do not glow, and when the proteasome activity is suppressed, the cells glow green. Therefore, in this assay system, proteasome activity in living cells can be examined by measuring the fluorescence intensity of GFP.

  Using the GFP-CL1 system, co-expression of α-synuclein and GFP-CL1 in the cells results in an increase in the fluorescence intensity of the cells compared to the expression of GFP-CL1 alone. It can be confirmed that the intracellular proteasome activity is suppressed when α-synuclein is expressed. That is, by monitoring the fluorescence intensity of GFP-CL1, the effect of α-synuclein on proteasome activity can be examined.

  In previous reports, polyglutamine protein accumulated and fibrotic in cells inhibited intracellular proteasome activity (Reference (2)), but in the present invention, fibrosis of α-synuclein was not detected in the cells. It was. That is, in the case of α-synuclein, it is thought that soluble molecules rather than fibers inhibit proteasome activity and ultimately damage cells. In particular, molecules such as oligomers and protofibrils that are in a pre-fibrotic state play a role. That is, proteasome activity can be promoted by inhibiting not only α-synuclein fibrillation but also oligomerization and protofibrillation. In this way, the molecule in which α-synuclein acts on the proteasome is a completely different type of molecule from the previous report, and the mechanism by which α-synuclein of the present invention acts on the suppression of proteasome activity is completely different from the previous report. It is.

  As described above, GFP-CL1 is degraded by the proteasome, so cells do not glow green in the absence of α-synuclein, whereas when GFP-CL1 and α-synuclein are co-expressed in cells, green fluorescence An increase in strength is observed. This phenomenon is the result of α-synuclein reducing proteasome activity in the cell.

  By the way, many of the mutants of synuclein have increased fiber formation in vitro compared with the wild type. When such mutant synuclein is co-expressed with GFP-CL1 as described above, the proteasome activity is suppressed more strongly than the wild type. On the other hand, suppression of proteasome activity is not observed when a delta mutant that cannot form fibrils in vitro is co-expressed with GFP-CL1.

  In summary, the following can be said.

(1) α-synuclein suppresses intracellular proteasome activity.
(2) Suppression of proteasome activity by α-synuclein is positively correlated with its ability to form fibrosis.
(3) Mutant α-synuclein that cannot fibrosis cannot suppress proteasome activity.

  Therefore, as a mechanism for suppressing the proteasome activity, the expressed α-synuclein becomes an oligomer or protofibril due to a conformational change in the cell, which is considered to suppress the proteasome activity. It is considered that a mutant having a high fibril forming ability is more likely to be oligomerized and / or protofibrillated in the cell, and thus the degree of suppression of proteasome activity is also strong. On the other hand, mutants that do not form fibrosis cannot suppress proteasome activity because they do not cause such structural changes.

  From the above viewpoint, the present inventor used a GFP-CL1 expression system used as an assay system for proteasome activity as a protein capable of forming amyloid fibrils (for example, α-synuclein, tau, β-amyloid or a precursor thereof, polyglutamine, prion Etc.) was considered to be applied to a system for detecting oligomerization or protofibrillation, or fibrosis. That is, in the present invention, when the protein capable of forming the amyloid fibril and GFP-CL1 are expressed in the cell, the proteasome activity is suppressed when the oligomer and / or protofibril of the protein is present. Fluoresces green, but in the absence of such structures, cells do not fluoresce, taking advantage of not only fibrosis but also oligomerization and / or protofibrillation at the pre-fibrosis stage This is to screen for a compound that suppresses the above.

  By utilizing this method, it becomes possible to screen for compounds that have an action of protecting cells by preventing inhibition of proteasome function by α-synuclein or the like. In fact, in compounds such as polyphenol, the effect of preventing the inhibition of proteasome activity by α-synuclein was confirmed.

  Thus, by using the method of the present invention, high-throughput screening of various compounds becomes possible, and it can be said that the screened substance becomes a therapeutic drug for neurodegenerative diseases having a completely new mechanism of action.

2. Screening Method The screening method of the present invention aims to search for a protein oligomer capable of forming amyloid fibrils and / or protofibrillation, or a substance that suppresses fibrillation, and a protein capable of forming amyloid fibrils, a proteasome degradation signal and A test substance is brought into contact with a cell that expresses a labeled protein, and the test substance is selected by using a decrease in the labeling signal as an index as compared with a control cell. Examples of proteins that can form amyloid fibrils include α-synuclein, tau, polyglutamine, and the like.

  Alpha synuclein is a substance accumulated in the cytoplasm identified as a pathogen of Parkinson's disease, and aggregation of alpha synuclein is considered to be a cause of cell death. The base sequence of the α-synuclein gene is known, and the sequence can be easily obtained through a public database such as GenBank. The base sequence of the α-synuclein gene is shown in SEQ ID NO: 1 (P37804), and the amino acid sequence is shown in SEQ ID NO: 2 (L08850).

Tau is a kind of microtubule-binding protein and accumulates in nerve cells in large quantities as neurofibrillary tangles in a group of diseases called tauopathy typified by Alzheimer's disease. There are three repeats of the microtubule binding site (Tau 3R) and 4 repeats (Tau 4R). The base sequence of the tau gene is known, and its sequence information can be easily obtained through public databases such as GenBank. The nucleotide sequence of the tau gene is SEQ ID NO: 3 (NP005901), and the amino acid sequence is SEQ ID NO: 4 (NM 005910.2).

  β-amyloid (Aβ) is known as a protein that accumulates inside and outside the cell and is involved in the pathogenesis of Alzheimer's disease. In addition, β amyloid precursor protein (Amyloid Precursor Proterin; APP) is a protein before being cleaved by α, β or γ secretase. β-amyloid is thought to interact weakly with tau. Some Alzheimer's disease causative genes are consistent with β-amyloid precursors. The base sequence of β-amyloid gene is known, and its sequence information can be easily obtained through public databases such as GenBank. The base sequence of the β amyloid gene is shown in SEQ ID NO: 5 (P5067), and the amino acid sequence is shown in SEQ ID NO: 6 (Y00264).

Polyglutamine is a protein accumulated in Huntington's disease and triplet repeat disease, and is considered to exert cytotoxicity. The base sequence of the polyglutamine gene is known, and its sequence information can be easily obtained through public databases such as GenBank. The base sequence of the polyglutamine gene is SEQ ID NO: 7 (NP002102), and the amino acid sequence is SEQ ID NO: 8 (NM 002111.5).

A prion is a highly agglutinating protein with abnormal accumulation observed in mad cow disease and Creutzfeldt-Jakob disease, and is known to accumulate and accumulate in nerve cells or outside. It is considered that the higher order structure of the prion protein is changed and the beta sheet structure is increased, whereby the normal prion protein is converted into a pathogenic prion protein and causes neuronal cell death. The base sequence of the prion gene is known, and its sequence information can be easily obtained through public databases such as GenBank. The base sequence of the prion gene is SEQ ID NO: 9 (NP898902), and the amino acid sequence is SEQ ID NO: 10 (NM 183079.1).

Hereinafter, α-synuclein will be exemplified and described as a protein capable of forming amyloid fibrils.
Proteasome is an enzyme that is indispensable for cell survival, and suppression of intracellular proteasome activity eventually leads to cell death. That is, the strong expression of α-synuclein itself is an undesirable situation for cells, and in order for cells to continue to survive, suppression of proteasome activity must be avoided. Therefore, it is considered that the screening method of the present invention makes it possible to search for compounds that avoid inhibition of proteasome activity by α-synuclein, compounds that promote proteasome activity, and the like. That is, various candidate compounds (test substances) are brought into contact with a fusion of a proteasome degradation signal and a labeling substance, or a cell in which α-synuclein, a proteasome degradation signal and a labeling substance are co-expressed, and from an untreated cell Also, a substance that reduces the intensity of the label signal (for example, fluorescence intensity) is selected. The compound has an effect of protecting cells by avoiding inhibition of proteasome activity by α-synuclein, or promotes the function of proteasome, and these compounds can be used as the therapeutic agent for neurodegenerative diseases of the present invention. it can. Here, the “substance that promotes the function of the proteasome” includes substances unrelated to the substance that suppresses the inhibition of the function of the proteasome. Therefore, in the method of the present invention, it is possible to screen a substance that promotes the function of the proteasome by expressing a labeling substance and a proteolytic signal in cells.

As described above, proteasome activity is suppressed when α-synuclein is expressed in cells. For example, the presence of soluble molecules such as oligomers and protofibrils in the pre-fibrosis state among α-synuclein molecules inhibits proteasome activity. In addition, fibrosis occurs in parallel with such oligomerization or protofibrillation, and the proteasome activity may be inhibited.
Therefore, (i) such a substance that inhibits the formation of α-synuclein molecule oligomers or protofibrils, and (ii) a substance that inhibits synuclein fibrosis promotes proteasome activity, and thus is a therapeutic agent for neurodegenerative diseases. A candidate is also preferable, and (iii) a substance that promotes the function of the proteasome of a cell is also preferable as a candidate for a therapeutic agent for neurodegenerative diseases.

  In the screening method of the present invention, an expression vector in which a proteasome degradation signal is operably linked to a labeling substance and an α-synuclein expression vector are prepared, and these are introduced into cells and co-expressed. However, α-synuclein, proteasome degradation signal, and label signal sequence may be incorporated as a cassette in one vector, or may be linked to separate vectors and introduced separately into cells. When screening for a substance that promotes the function of the proteasome, only an expression vector in which a proteasome degradation signal is operably linked to the labeling substance, for example, an expression vector of a labeling substance fused with the degradation signal may be expressed alone. .

  Expression vector construction and vector introduction into cells are well known and can be performed using conventional techniques in the art (eg, Molecular Cloning, A Laboratory Manual 2nd ed. (Cold Spring Harbor Laboratory Press (1989 As a vector for linking each gene encoding a labeling substance, a proteasome degradation signal, or α-synuclein, commercially available products can be used.

  A proteasome degradation signal is a signal that is present in a protein that is degraded by the proteasome, is recognized by the proteasome, and triggers proteolysis. For example, a CL1 sequence consisting of 16 amino acid residues (ACKNWFSSLSHFVIHL) (SEQ ID NO: 11) can be mentioned. However, the proteasome degradation signal is not limited to the above CL1, and CL2 (SLISLPLPTRVKFSSLLLIRIMKIITMTFPKKLRS) (SEQ ID NO: 12), CL6 (FYYPIWFARVLLVHYQ) (SEQ ID NO: 13), etc. can also be used.

  The labeling substance is used as a detection means for detecting a proteasome degradation signal by the proteasome, and any of a fluorescent labeling substance, a radiolabeling substance, and an antibody labeled with these labeling substances can be used. . In view of easy detection operation, green fluorescent protein (GFP) derived from Aequorea victorea is preferable. Examples of the fluorescent labeling substance include GFP, EGFP (Enhanced-humanized GFP) or rsGFP (red-shift GFP), which is a GFP-modified mutant (GFP variant), and the like. It is also possible to use GFP derived from yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), blue fluorescent protein (BFP), Renilla reniformis And the genes encoding them can be used in the present invention. The base sequences of the genes encoding these are known, and commercially available products can be used, or the sequence information can be easily obtained through public databases such as GenBank.

  The cell into which the expression vector has been introduced is divided into a test cell for contacting the test substance and an untreated control cell, and the test signal is contacted by adding the test substance to the test cell to detect the labeled signal. Here, “contact” means that the test substance is treated so as to interact with the cell into which the expression vector has been introduced. The test substance is added to the cell culture system, or the cell is cultured in the presence of the test substance. It means to do both. The signal intensity (signal intensity) of the labeling substance can be determined by quantifying by a method suitable for each labeling protein such as fluorescence intensity, band density in electrophoresis, or by visual inspection. Then, the test substance when the signal intensity is lower than that of the control is selected as a substance that suppresses the inhibition of proteasome function, or a substance that suppresses α-synuclein oligomerization and / or protofibrillation.

  In the control cell, since the proteasome activity is suppressed by α-synuclein, the recombinant protein in which the expressed labeled protein and the proteasome degradation signal are combined is not degraded by the proteasome activity and emits fluorescence. In contrast, when a test substance that can be a candidate substance is added, formation of oligomers and protofibrils of α-synuclein is inhibited, so suppression of proteasome activity by α-synuclein no longer occurs, and proteasome activity is normal. When exerted, the expressed recombinant protein is degraded. Therefore, the label signal emitted from the cell after contacting the test substance is weaker than the intensity of the label signal emitted from the control cell.

  In the present invention, in addition to an immunoblot using an anti-labeled protein antibody such as an anti-GFP antibody, it is possible to detect the labeling substance by changing the fluorescence intensity of GFP-CL1, so that multiple samples can be collected in a short time. Can be processed and high-throughput screening can be performed.

  Candidate substances to be screened according to the present invention include, for example, peptides, proteins, non-peptide compounds, synthetic compounds (polymers or low molecular compounds), fermentation products, cell extracts, cell culture supernatants, and plant extracts. Fluids, tissue extracts of mammals (eg, mice, rats, pigs, cows, sheep, monkeys, humans, etc.), plasma and the like. These compounds may be novel compounds or known compounds. There may be. These candidate substances may form salts, and the salts of candidate substances include salts with physiologically acceptable acids (for example, inorganic acids and organic acids) and bases (for example, metal acids). Is used.

  For example, the candidate substance includes two types of compounds, Purpurogallin (2L) and Myricetin (C7), which are a kind of polyphenol. These compounds all have an action of inhibiting α-synuclein fibrosis in vitro, as shown in Examples described later. In the screening method described above, when the above compound is not added, GFP-CL1 is not decomposed and a band appears because proteasome activity remains inhibited by α-synuclein expression (lane 2 in FIG. 6). When 2L and C7 are added, the amount of GFP-CL1 band decreases dramatically (lanes 4 and 5). It can be said that these compounds have an action of preventing the inhibition of proteasome activity by α-synuclein and protecting the cells so that cell death is not induced.

  According to the screening method of the present invention, it is possible to search for compounds that protect cells by avoiding inhibition of intracellular proteasome activity by α-synuclein. In addition, by administering a substance that inhibits the expression of α-synuclein, preferably an oligomer of α-synuclein or protofibril, to animals such as humans searched by the screening method described above, by promoting proteasome activity, Can be treated and / or prevented. Thus, the screening method of the present invention is expected to contribute to the development of a novel therapeutic agent for neurodegenerative diseases having a new mechanism of action.

3. Use of Screened Substance The substance screened in the present invention can be used as a therapeutic agent for various neurodegenerative diseases. Neurodegenerative disease means a disease called neurodegeneration in which nerve cells die without any obvious cause such as trauma or bacterial infection. Alzheimer's disease, mainly dementia, and movement disorders The main symptoms include Parkinson's disease. In addition to these diseases, Huntington's disease, triplet repeat disease, amyotrophic lateral sclerosis, Lewy body dementia, multiple system atrophy, Creutzfeldt-Jakob disease, Gerstmann-Straussler syndrome, mad cow disease, bulbar spinal cord Muscular atrophy, spinocerebellar ataxia, dentate nucleus erythroleukemia Louisa atrophy, FTDP-17, progressive epithelial paralysis, corticobasal degeneration, Pick disease, etc. In the present invention, Parkinson's disease associated with α-synuclein deposition, Lewy body dementia and multiple system atrophy, Alzheimer's disease associated with tau and β-amyloid or its precursor, Huntington's disease associated with polyglutamine and triplet repeat disease, Also particularly preferred are Creutzfeldt-Jakob disease and mad cow disease, which are associated with prions.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

(1) Preparation of GFP-CL1, α-synuclein and tau expression vectors
In the GFP-CL1 expression vector (pEGFP-CL1), a CL1 sequence consisting of 16 amino acid residues (ACKNWFSSLSHFVIHL) (SEQ ID NO: 11) is added to the Bgl II site of the multicloning site of the pEGFP-C1 vector (Clontech). It was prepared by inserting a base sequence corresponding to the CL1 amino acid sequence.

The α-synuclein expression vector (pcDNA3-αsyn) is obtained by using the pRK172-αsyn vector (MRC Laboratories, UK, Dr. Michel Goedert) as a template and the α-synuclein coding region amplified by the PCR method on the Nhe I site of pcDNA3 vector (Invitrogen) Introduced and produced.
As tau expression vectors, pSG5-tau3R and 4R (distributed by Dr. Michel Goedert, UK MRC Laboratory) were used.

(2) Introduction of plasmid into SH-SY5Y cells Neuroblast SH-SY5Y was cultured in an incubator under conditions of 37 ° C. and 5% CO ₂ using DMEM / F12 medium containing 10% calf serum.

  pEGFP-CL1 (0.3 μg), pcDNA3-αsyn vector (1 μg), pSG5-tau3R or 4R (1 μg) were introduced into SH-SY5Y cells using FuGENE6 transfection reagent (Roche). FuGENE6 of 3 times the total amount of plasmid was mixed with the plasmid, allowed to stand at room temperature for 15 minutes, and then mixed with the cell solution. The cells were cultured for 2 to 3 days and used for cell lysate preparation or immunohistochemical staining.

Observation with confocal laser microscope SH-SY5Y cells cultured on a cover glass are mixed with pFGFP-C1 (0.3 μg) or pEGFP-CL1 (0.3 μg) alone or with pcDNA3-αsyn (1 μg). In the presence of FuGENE6. The culture was continued for 2 days, and the cells were fixed in a 4% paraformaldehyde solution. The fixed cells were treated with 0.2% Triton X-100, blocked with a 5% bovine serum albumin solution, and reacted with an anti-α-synuclein antibody (1000-fold diluted) at 37 degrees for 1 hour. Washed with 50 mM Tris-HCl, pH 7.5 (TBS-T) containing 0.05% Tween 20 and 150 mM NaCl. Thereafter, the mixture was reacted with TRITC-labeled anti-mouse secondary antibody) at 37 ° C. for 1 hour. After washing with TBS-T, the cells were reacted with TO-PRO-3 (Invitrogen, diluted 3000 times) at 37 ° C. for 40 minutes for nuclear staining. This was encapsulated on a slide glass and then analyzed with a confocal laser microscope (Zeiss).

  In order to confirm whether or not the constructed pEGFP-CL1 was degraded by the proteasome in the cells, the expressed cells were observed with a confocal laser microscope and shown in FIG. The green color in the figure indicates fluorescence by GFP, and the blue color indicates a nuclear staining image by TO-PRO-3. As shown in FIG. 2, when only GFP was expressed, many cells emitted green fluorescence due to GFP (left in FIG. 2). When GFP-CL1 with a degradation signal coupled to GFP was expressed, the number of fluorescent cells was significantly reduced. (Figure 2 middle). On the other hand, when MG132, which is a proteasome inhibitor, was treated at this time, the number of cells emitting fluorescence increased compared to the case where only GFP-CL1 was expressed (right side of FIG. 2). From the above, it was shown that when GFP-CL1 is expressed, GFP-CL1 is degraded by intracellular proteasome activity, and thus fluorescence decreases. This was consistent with the results obtained in reference (2). It was also found that GFP, unlike GFP-CL1, is not degraded or difficult to be degraded by intracellular proteasomes.

Analysis by immunoblot
In SH-SY5Y cells, pFGFP-C1 (0.3 μg) or pEGFP-CL1 (0.3 μg) alone or with them and pcDNA3-αsyn (1 μg), pSG5-tau3R or 4R (1 μg) Added in the presence. The culture was continued for 3 days, and the cells were collected by centrifugation (1,800 g, 5 minutes, 4 ° C.). After washing the cells with phosphate buffered saline (PBS), 100 μL of disruption buffer (50 mM Tris-HCl, pH 7.5 / 150 mM NaCl / 5 mM ethylenediaminetetraacetic acid / 5 mM ethylene glycol bis (β-aminoethyl) Ether) -N, N, N, N-tetraacetic acid / protease inhibitor cocktail) followed by sonication. The cell lysate was subjected to ultracentrifugation (290,000 g, 20 minutes, 4 ° C.), and the tris-soluble fraction of the supernatant was recovered. The tris-soluble fraction was quantified using the BCA Protein assay kit (PIERCE), and then added with a sample buffer for sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (SDS-PAGE). A sample was used. On the other hand, the precipitate fraction was sonicated with a disruption buffer containing 100 μL of 1% Triton X-100 (TX), and ultracentrifugated under the same conditions (290,000 g, 20 minutes, 4 ° C.). The obtained supernatant was used as a TX soluble fraction, and an SDS sample buffer was added to prepare a sample for electrophoresis. The TX-treated precipitate fraction was sonicated by adding 100 μL of SDS-PAGE sample buffer, and used as a sample for electrophoresis.

  Tris-Tricine SDS-PAGE using 13.5% gel was performed on all the obtained fractions. After electrophoresis, the gel was transferred to a polyvinylidene difluoride (PVDF) membrane, blocked with a 3% gelatin solution, anti-GFP antibody (MBL, diluted 2,000 times), anti-α synuclein antibody (1,000 times diluted) or Anti-tau antibody (1,000-fold dilution) was reacted overnight at room temperature. After the reaction, the PVDF membrane was washed with Tris-buffered saline (TBS), and then reacted with horse radish peroxidase-labeled mouse Ig G (10,000-fold dilution) at room temperature for 1 hour. After the reaction, the membrane was washed with TBS, treated with an immunostar reagent (Wako Pure Chemical Industries), and exposed to an X-ray film (Fuji Film) to detect a band.

  When only GFP-CL1 is expressed, GFP-CL1 is decomposed by intracellular proteasome activity and the band disappears (lane 1 in FIG. 3). When GFP-CL1 and α-synuclein are co-expressed, degradation of GFP-CL1 is suppressed and a band appears (lane 3). In addition, when GFP-CL1-expressing cells are treated with MG132, a proteasome inhibitor, the proteasome activity is suppressed and the degradation of GFP-CL1 is inhibited, so that a band appears (lane 2). From the above results, it is shown that proteasome activity is inhibited when α-synuclein is expressed in cells.

Inhibition of proteasome activity by intracellular α-synuclein To determine whether α-synuclein expressed in cells affects proteasome activity, co-expression of α-synuclein and GFP-CL1 in SH-SY5Y cells, immunoblotting and confocal laser Microscopic analysis was performed.

  When only GFP-CL1 is expressed (left panel), the cell hardly emits fluorescence because GFP-CL1 is degraded by the proteasome. When GFP-CL1 and α-synuclein are co-expressed (right panel), more cells are shown to fluoresce than when only the left GFP-CL1 is expressed. This shows that proteasome activity is inhibited when α-synuclein is expressed in cells, as in the immunoblot results of FIG.

  That is, when only GFP-CL1 is expressed, GFP-CL1 is degraded by the intracellular proteasome activity, so almost no GFP-CL1 band is detected (lane 1 in FIG. 3), and the cells that emit fluorescence Less (figure 4 left panel). When α-synuclein and GFP-CL1 were co-expressed, a GFP-CL1 band was detected (Fig. 3, lane 3), and more fluorescent than when GFP-CL1 alone was expressed (Fig. 4, left panel). A significant increase in cell number was observed (FIG. 4, right panel). On the other hand, when GFP-CL1-expressing cells were treated with MG132, a proteasome inhibitor, a remarkable band of GFP-CL1 was detected (FIG. 3, lane 2). The above results indicate that the intracellular proteasome activity is inhibited when α-synuclein is expressed in cells.

Effect of mutational effects of α-synuclein on proteasome activity α-synuclein has been reported to have three mutations linked to the onset (references (3), (4), (5)), but these All are known to be more fibrotic in vitro than wild type. Moreover, it is known that a mutant (Δ73-83) lacking residues 73 to 83 in the amino acid sequence constituting α-synuclein does not fibrosis in vitro. The effect of the mutational effects of these α-synucleins on intracellular proteasome activity was examined.

  Of the three types of mutations, A30P in which Ala at residue 30 was mutated to Pro and A53T in which Ala at residue 53 was mutated to Thr were used. These mutants were coexpressed with SH-SY5Y cells together with GFP-CL1 and cultured for 3 days, and then cell lysates were prepared and immunoblotted.

  The results are shown in FIG. Lane 1 in FIG. 5 is a result of expressing only GFP-CL1, and GFP-CL1 is degraded by proteasome activity, and thus the band disappears. When wild-type α-synuclein is co-expressed, proteasome activity is suppressed (lane 2), and thus a GFP-CL1 band can be detected. On the other hand, when synuclein mutants A30P and A53T were expressed (lanes 3 and 4), a GFP-CL1 band was detected more strongly than lane 2 in which the wild type was expressed. In addition, when the delta mutant Δ73-83, which does not fibrosis in vitro, was expressed, almost no GFP-CL1 band was detected (lane 5).

  These results indicate that mutant α-synuclein, which is more likely to fibrosis in vitro than wild-type, strongly inhibits proteasome activity, but mutants that do not fibrosis in vitro cannot suppress proteasome activity. . That is, it was suggested that the suppression of proteasome activity by synuclein is related to its fibrosis.

Inhibition of proteasome activity by tau As in Examples 4 and 5, whether tau expressed in cells affects proteasome activity was analyzed by immunoblotting.

  Even if only GFP-CL1 is expressed, the band is hardly detected (lane 1 in FIG. 6). However, when α-synuclein and GFP-CL1 are co-expressed, the GFP-CL1 band is detected as described above ( In FIG. 6, lane 2) and when Δ73-83 was expressed, almost no GFP-CL1 band was detected (FIG. 6, lane 3). On the other hand, when tau 3R with 3 repeats and tau 4R with 4 repeats were expressed together with GFP-CL1, the GFP-CL1 band was remarkably detected in both cases (lanes 4 and 5 in FIG. 6), and α-synuclein and Similarly, intracellular tau was found to suppress proteasome activity.

Compounds that avoid inhibition of proteasome activity by α-synuclein From the above results, it was revealed that when α-synuclein is expressed in cells, intracellular proteasome activity is inhibited. It was also revealed that the inhibitory effect was stronger for mutants that were more likely to be fibrotic, and that the non-fibrotic mutant had almost no inhibitory effect. Based on the above, as a mechanism for the inhibition of proteasome activity by α-synuclein, some α-synuclein forms fibrils or oligomers or protofibrils that are considered to be the pre-stage state of fibers in cells, and these molecules suppress proteasome activity. Possible possibility. If this hypothesis is correct, proteasome activity is not inhibited if α-synuclein oligomerization is suppressed in the cell. We have found that compounds such as polyphenols inhibit α-synuclein fibrosis in vitro, and examined whether these compounds avoid inhibition of proteasome activity by α-synuclein in cells. As compounds, three types of compounds were used: Ferric-dehydroporphyrin IX (2D), which is a porphyrin compound, and Purpurogallin (2L), Myricetin (C7), which is a kind of polyphenol. All of these compounds have an action of inhibiting α-synuclein fibrosis in vitro.

  Α-synuclein and GFP-CL1 were co-expressed in the cells, and after 2 hours, the above compound was added to the medium to a final concentration of 40 μM, and the culture was continued for 3 days. Three days later, cell lysates were prepared and immunoblotted with an anti-GFP antibody. If the compound suppresses the inhibition of proteasome by α-synuclein, GFP-CL1 is degraded and the GFP-CL1 band disappears, but if the added compound has no effect, the proteasome activity remains inhibited. So GFP-CL1 is not degraded and a GFP-CL1 band appears (Figure 7). Thus, screening for compounds that avoid inhibition of proteasome activity by α-synuclein was performed using the appearance and disappearance of the GFP-CL1 band as an index.

  The result is shown in FIG. When no compound is added, since proteasome activity remains inhibited by α-synuclein expression, GFP-CL1 is not degraded and a GFP-CL1 band is detected (FIG. 7, lane 2). When 40 μM 2D was added, a GFP-CL1 band appeared (lane 3). In other words, even when the compound was added, the proteasome activity remained inhibited, and it can be said that this compound has no protective effect on the cells. From this result, 2D does not have the effect of suppressing the inhibition of proteasome activity due to the expression of α-synuclein. It has been shown. On the other hand, when 2 L and C7 were added, the amount of GFP-CL1 band was drastically reduced compared to the case of untreated lane 2 (same lanes 4 and 5). In other words, it was thought that the addition of the compound eliminated the inhibition of proteasome activity by synuclein, and thus the band disappeared.These compounds prevented the inhibition of proteasome activity by α-synuclein and prevented cell death from being induced. It was shown that there is an effect to protect.

Reference (1) Gilon, T., Chomsky, O., and Kulka, RG (1998) Degradation signals for ubiquitin system proteolysis in Saccharomyces cerevisiae. The EMBO J. 17, 2759-2766
(2) Bence, NF, Sampat, RM, and Kopit, RR (2001) Impairment of the ubiquitin-proteasome system by protein aggregation. Science 292, 1552-1555
(3) Polymeropoulos, MH, Lavedan, C., Leroy, E., Ide, SE, Dehejia, A., Dutra, A., Pike, B., Root, H., Rubenstein, J., Boyer, R. , Stenroos, ES, Chandrasekharappa, S., Athanassiadou, A., Papapetropoulos, T., Johnson, WG, Lazzarini, AM, Duvoisin, RC, Iorio, GD, Golbe, LI, and Nussbaum, RL (1997) Mutation in the alpha-synuclein gene identified in families with Parkinson's disease. Science 276, 2045-2047
(4) Kruger, R., Kuhn, W., Muller, T., Woitalla, D., Graeber, M., Kosel, S., Przuntek, H., Epplen, JT, Schols, L., and Riess. O. (1998) Ala30Pro mutation in the gene encoding alpha-synuclein in Parkinson's disease. Nat. Genet. 18, 106-108
(5) Zarranz, JJ, Alegre, J., Gomez-Esteban, JC, Lezcano, E., Ros, R., Ampuero, I., Vidal, L., Hoenicka, J., Rodriguez, O., Atares, B., Llorens, V., Gomez Tortosa, E., del Ser, T., Munoz, DG, and de Yebenes, JG (2004) The new mutation, E46K, of alpha-synuclein causes Parkinson and Lewy body dementia. Ann Neurol. 55, 164-73

It is a figure which shows the outline | summary of the system which measures the proteasome activity in the cell using the system using Green fluorescent protein. It is the photograph which expressed GFP and GFP-CL1 in the cell, and observed the cell with the confocal laser microscope. It is a figure which shows the result confirmed by immunoblotting with the antibody of GFP about whether intracellular alpha synuclein affects proteasome activity. It is the photograph which observed the cell with the confocal laser microscope about whether the alpha synuclein expressed in the cell inhibits proteasome activity. In order to investigate whether the mutation effect of α-synuclein affects the proteasome activity, it is a figure showing the results of immunoblotting analysis by co-expressing three known mutants with GFP-CL1. It is a figure which shows the result of having performed the immunoblot analysis about whether the tau expressed in the cell suppresses proteasome activity. It is a figure which shows the result of having performed the immunoblot analysis about some compounds which suppressed the fibrosis of synuclein in vitro.

Claims (7)

A method for screening a substance that suppresses the inhibition of proteasome function, wherein a protein capable of forming amyloid fibrils and a protein comprising the amino acid sequence represented by SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 13 are located on the C-terminal side A test substance is brought into contact with a cell expressing any one of the labeling substances selected from the group consisting of GFP derived from GFP, EGFP, rsGFP, YFP, CFP, BFP and Renilla, and the signal of the labeling substance is obtained. When the signal intensity of the detected labeling substance detected is lower than the signal intensity of the labeling substance in the control cells not contacted with the test substance, the test substance is used as a substance that suppresses the functional inhibition of the proteasome. Selecting the method. A method for screening a substance that promotes the function of a proteasome, wherein a protein capable of forming amyloid fibrils and a protein comprising the amino acid sequence represented by SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 13 are added to the C-terminal side The test substance is brought into contact with a cell expressing any one of the labeling substances selected from the group consisting of GFP derived from GFP, EGFP, rsGFP, YFP, CFP, BFP and Renilla, and the signal of the labeling substance is detected. When the signal intensity of the detected labeling substance is lower than the signal intensity of the labeling substance in the control cells not contacted with the test substance, the test substance is selected as a substance that promotes the function of the proteasome. And said method.   The method according to claim 1, wherein the substance that suppresses the inhibition of the function of the proteasome is used for the treatment of a neurodegenerative disease.   The method according to claim 2, wherein the substance that promotes the function of the proteasome is used for treatment of a neurodegenerative disease.   The method according to any one of claims 1 to 4, wherein the protein capable of forming amyloid fibrils is at least one selected from the group consisting of α-synuclein, tau, β-amyloid or a precursor thereof, polyglutamine, and prion. . The method according to any one of claims 1 to 5, wherein the protein comprising the amino acid sequence represented by SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 13 is a protein comprising the amino acid sequence represented by SEQ ID NO: 11. . The method according to any one of claims 1 to 6, wherein the labeling substance is GFP.

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