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WO2002004625A1 - Proteine de liaison de l'$g(a)-synucleine - Google Patents

Proteine de liaison de l'$g(a)-synucleine Download PDF

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Publication number
WO2002004625A1
WO2002004625A1 PCT/JP2001/005890 JP0105890W WO0204625A1 WO 2002004625 A1 WO2002004625 A1 WO 2002004625A1 JP 0105890 W JP0105890 W JP 0105890W WO 0204625 A1 WO0204625 A1 WO 0204625A1
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synuclein
protein
human
binding protein
human synuclein
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Japanese (ja)
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Kenji Ueda
Abdul Alim Muhammad
Kunimasa Arima
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KH Neochem Co Ltd
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Kyowa Hakko Kogyo Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A61P21/04—Drugs for disorders of the muscular or neuromuscular system for myasthenia gravis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to a novel protein that binds to a precursor of a non-amyloid component in amyloid protein [hi-synuclein (also referred to as NACP)], an antibody recognizing the protein, and a protein that binds to thynuclein (his-nuclein binding protein)
  • the present invention relates to a method for screening a substance that inhibits the action of synuclein association or fibril formation promotion by the above method, an agent for preventing the onset of neurodegenerative disease obtained by the method, or an agent for delaying the progress.
  • EGTA Ethylene Glyco-lubis (? -Aminoethyl ether)-N, N, N ,, N,-
  • IPTG isopropyl thiogalactoside
  • PBS phosphate buffered saline
  • RT-P CR reverse transcription-PCR
  • TBS Tris buffered saline
  • T F A trifluoroacetic acid Tris: Tris (hydroxymethyl) aminoniethane
  • NAC non-amyloid beta component
  • human synuclein is a major fibrous form of inclusions called Lewy bodies found in the nerves of Parkinson's disease and certain types of dementia, and intracellular inclusions found in multiple system atrophy. It was reported to be a constituent [Spillantini MG et al., Nature,, 839 (1997)]. Human sph-synuclein also associates in vitro to form fibers.
  • synuclein found in families with familial Parkinson's disease, are more likely to associate or fibrillate in vitro than wild-type human synuclein [ Conway KA et al., Nat.
  • spike synuclein The association or fibril formation of spike synuclein is thought to be involved in the development or progression of these diseases, but the mechanism has not been elucidated yet.
  • most neurodegenerative diseases in which ⁇ -synuclein fibril formation is observed such as Parkinson's disease, Lewy body dementia, multiple system atrophy, and Alzheimer's disease, are mostly familial rather than familial. Because of their onset, the brains of patients with these diseases have factors that promote the initiation and progression of sph-synuclein association and fiber formation, or degrade the sph-synuclein fibers formed. It is considered that the mechanism for performing the operation is reduced.
  • Amyloid /? [Yoshimoto M. et al., Proc. Natl. Acad. Sci., USA, 92, 9141 (1995), Jensen PH et al., Biochem. J., 539 (1997)], 14-3-3 protein [Ostrerova N. et al., J. Neurosci., 12., 5782 (1999)], tau protein [Jensen PH et al., J. Biol. Chem., 21A, 25481 (1999)] and microtubule-associated protein 1B, Jensen PH et al., J. Biol.
  • Tubulin is a major constituent protein of microtubules, and there are two types, tubulin and tubulin.
  • Tubulin is present in Lewy bodies [Galloway PG et al., J. Neuropathol. Exp. Neurol., 41, 654 (1988)], and inhibits the binding of protein to human synuclein. [Jensen PH et al., J. Biol. Chem., M, 25481 (1999)], but reports that it binds to hy-synuclein and reports on its association with association and fibril formation. There is no.
  • the present inventors isolated a 42 kDa and 5 OkDa protein that specifically binds to human synuclein from human brain extract by human synuclein-immobilized affinity chromatography. By analyzing the partial amino acid sequences of these proteins, it was found that the 42 kDa protein is a human synuclein binding protein having a novel amino acid sequence, and the 50 kDa protein is a single tubulin. Identified. In addition, it was found that human synuclein binds not only to single tubulin but also to 5-tubulin (in the present invention, “tubulin” refers to single tubulin or /?-Thubulin).
  • tubulin greatly promotes the association and fibril formation of spike-synuclein in the mouth of the intestine, and that the fiber morphology is very similar to the spike-synuclein fiber in the Lewy bodies of pathological tissues. Completed the invention.
  • [ii] Observe and compare the association or fibril formation of human synuclein when human synuclein is contacted with human synuclein binding protein in the presence of the test substance.
  • a method for screening a substance that inhibits the association or fibril-forming action of human synuclein possessed by a human synuclein-binding protein comprising selecting a substance that inhibits the association or fibril formation of human synuclein from test substances.
  • (8) a protein selected from the group consisting of the protein of (1) or (2) above, tubulin, amyloid / ?, 14_3-3 protein, tau protein and microtubule-associated protein (8).
  • a neutralizing antibody against a human synuclein-binding protein which inhibits the activity of human synuclein-binding protein to promote association or fibril formation of human synuclein.
  • the human synuclein binding protein is selected from the group consisting of the protein of (1) or (2) above, tubulin, amyloid 5, 14-3-3 protein, tau protein, and microtubule-associated protein 1B The method according to (10) above.
  • a medicament comprising the compound of (9) or a pharmaceutically acceptable salt thereof, or the neutralizing antibody of (11), (12) or (14) as an active ingredient.
  • the medicament according to the above (16), wherein the neurodegenerative disease is selected from the group consisting of Parkinson's disease, Lewy body dementia, multiple system atrophy and Alzheimer's disease.
  • (22) Culture the transformant of (20) or (21) above in a culture medium and place it in the culture.
  • a medicament comprising the DNA of (18) or the oligonucleotide or oligonucleotide derivative of (23) as an active ingredient.
  • An agent for preventing the onset of neurodegenerative disease or an agent for delaying the progress comprising as an active ingredient DNA encoding a synuclein-binding protein or a partial DNA thereof.
  • the medicament according to the above (25) or (26), wherein the neurodegenerative disease is selected from the group consisting of Parkinson's disease, Lewy body dementia, multiple system atrophy and Alzheimer's disease.
  • the human synuclein binding protein can be isolated from a biological tissue or cell extract by affinity chromatography using a carrier on which human synuclein is immobilized.
  • the human-synuclein-immobilized carrier is brought into contact with the cell extract to bind the human-synuclein-binding protein to the human-synuclein-immobilized carrier, and then the carrier is washed to remove unbound proteins.
  • the protein can be eluted and isolated from the carrier. it can.
  • the source of the isolation is preferably a living tissue or cells rich in human synuclein, such as the brain. Tissues and cells are disrupted in a buffer containing an appropriate protease inhibitor, and the supernatant obtained by centrifugation can be used as an extract.
  • Examples of the carrier for immobilizing hy-synuclein include agarose-based carriers such as Sepharose-14B.
  • agarose-based carriers such as Sepharose-14B.
  • the sugar such as agarose contained in the carrier or the group of the spacer molecule for immobilization, and reacting with the human synuclein, the human synuclein can be immobilized.
  • Sepharose-14B manufactured by Amersham 'Pharmacia' Biotech
  • cyanogen bromide is reacted with para-synuclein based on the manufacturer's manual to immobilize para-synuclein on sepharose.
  • Hi-synuclein to be bound can be easily obtained with high purity by using a gene recombination method. Specifically, first, a cDNA clone pHBS6-1 of spike-synuclein
  • CDNA was cut out using restriction enzymes, and the base sequence of c-DNA of human synuclein.
  • pET-15b manufactured by Novazidin
  • pET-3d manufactured by Novazidin
  • the transformed E. coli was prepared by introducing this human synuclein expression vector into an appropriate host for E. coli.
  • the cells are recovered, and the synuclein is isolated from the solution obtained by disrupting the cells by a conventional protein purification method such as ammonium sulfate precipitation ion exchange chromatography, hydrophobic chromatography, or reverse phase chromatography. It can be purified.
  • the spun-synuclein-binding protein eluted from the spun-synuclein-immobilized carrier can be separated by SDS polyacrylamide gel electrophoresis (SDS-PAGE) or two-dimensional gel electrophoresis. It can also be isolated and purified by chromatography such as ion exchange chromatography, hydrophobic chromatography, and reverse phase chromatography, or by excising the gel after SDS-PAGE and eluting it from the gel.
  • SDS-PAGE SDS polyacrylamide gel electrophoresis
  • chromatography such as ion exchange chromatography, hydrophobic chromatography, and reverse phase chromatography
  • the molecular weight of the isolated protein can be analyzed by SDS-PAGE or MALD I / TOF mass spectrometry.
  • This isolated protein is used as it is, or after cleavage into peptides by a protease such as lysylendopeptidase or trypsin or chemical cleavage with cyanogen bromide, and the peptide separated by reverse-phase HP LC as a sample.
  • a protease such as lysylendopeptidase or trypsin or chemical cleavage with cyanogen bromide
  • the peptide separated by reverse-phase HP LC as a sample.
  • An isolated amino acid sequence was analyzed by analyzing a known amino acid sequence that matches the obtained amino acid sequence by using a homology analysis program against an amino acid sequence database such as SwissProt or PIR.
  • the novelty of the synuclein binding protein can be determined and identified.
  • Examples of the homology analysis program include BLASTX developed based on the algorithm BLAST [J. Mol.
  • the isolated human synuclein bond The protein is considered to be the protein in the database containing its amino acid sequence.
  • the molecular weight of the protein in the database whose amino acid sequence was consistent was the same as that of the isolated human synuclein binding protein, and the N-terminal amino acid sequence of the isolated human synuclein binding protein was If the amino acid sequence at the N-terminus of the protein in the protein matches, the isolated human synuclein binding protein is almost identified as a protein in the database.
  • the thus isolated human synuclein-binding protein had a molecular weight of 4188.1 (M + H) + by MALD I / T OF mass spectrometry and an amino acid sequence represented by SEQ ID NO: 1.
  • a protein or a tubulin containing an amino acid sequence represented by SEQ ID NO: 2 at the N-terminus can be mentioned.
  • human synuclein binding protein examples include, in addition to the above, —-tubulin, amyloid / ?, 14-13-3 protein, tau protein, microtubule-associated protein 1B, and the like.
  • sph-synuclein-binding protein isolated in (1) is bound to sph-synuclein in vivo can be examined by immunoprecipitation. That is, using a brain extract obtained by the method described in (1) as a sample, an anti-synuclein antibody [EQ
  • the immunoprecipitate containing crane is subjected to immunoblotting using, as a primary antibody, an antibody against the human synuclein binding protein described later in Section 2.
  • an antibody against the human synuclein binding protein described later in Section 2.
  • the human-synuclein binding protein is human tubulin or /?-Tubulin
  • a commercially available anti-human tubulin antibody or anti-tubulin antibody [DM1A etc .: Sigma-Aldrich (Sigma) -Aldrich) can also be used.
  • spike-synuclein-binding protein can be detected in the immunoprecipitate, it can be confirmed that spike-synuclein-binding protein is bound to spike-synuclein even under physiological conditions in vivo.
  • immunoprecipitation was performed using an antibody against the human synuclein binding protein, and the immunoprecipitate containing the obtained human synuclein binding protein was subjected to immunoblotting using an anti-human synuclein antibody as the primary antibody.
  • the sph-synuclein binding protein binds to the sph-synuclein even under physiological conditions in vivo.
  • 5-tubulin, single tubulin and the like can be mentioned as examples of proteins which have been confirmed to bind to sphine-synuclein under physiological conditions in vivo.
  • a synuclein solution using a buffer such as PBS (1.83 g of disodium phosphate, 0.21 g of monopotassium phosphate, 7.65 g of sodium chloride, 1 liter of distilled water, pH 7.2).
  • a buffer such as PBS (1.83 g of disodium phosphate, 0.21 g of monopotassium phosphate, 7.65 g of sodium chloride, 1 liter of distilled water, pH 7.2).
  • PBS 1.83 g of disodium phosphate, 0.21 g of monopotassium phosphate, 7.65 g of sodium chloride, 1 liter of distilled water, pH 7.2.
  • An appropriate concentration of hy-synuclein is 50-700 jumo 1/1.
  • the degree of fibrillation of human synuclein can be quantified as the degree of association of human synuclein by measuring light scattering at 400 nm.
  • a peptide consisting of five or more contiguous sequences in the amino acid sequence of the human synuclein binding protein or its fragment or the human synuclein binding protein to an animal as an antigen allows a polyclonal protein to bind to the human synuclein binding protein.
  • One null antibody can be made.
  • the human synuclein-binding protein include the protein obtained above, tubulin, amyloid, 14-3-3 protein, tau protein, and microtubule-associated protein 1B.
  • the peptide serving as the antigen can be chemically synthesized using a peptide synthesizer.
  • Non-human mammals such as rabbits, goats, rats, mice, and hamsters can be used as animals to which the antigen is administered.
  • the dose of the antigen is preferably 50 to 100 // g per animal.
  • a partial peptide it is desirable that the partial peptide be covalently bound to a carrier protein such as KLH or bovine thyroglobulin as the antigen.
  • the administration of the antigen is performed 3 to 10 times every 1 to 2 weeks after the first administration. After each dose
  • a polyclonal antibody can be obtained by obtaining serum from a non-human mammal whose serum has a sufficient antibody titer against the antigen used for immunization, and separating and purifying the serum.
  • Methods for separation and purification include centrifugation, salting out with ammonium sulfate 50% saturated, and prillic acid precipitation [Harlow E. & LaneD. Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory (1988)], or DEAE- There is a method in which chromatography using a Sepharose column, an anion exchange column, a protein A or protein G column, a gel filtration column, or the like is performed alone or in combination.
  • an animal whose serum shows a sufficient antibody titer against the antigen used for immunization is used as a source of antibody-producing cells.
  • an animal it is preferable to use a rat.
  • the spleen is removed.
  • the spleen is minced in MEM, loosened with forceps, centrifuged at 1,200 rpm for 5 minutes, and the supernatant is discarded.
  • the spleen cells of the obtained precipitate fraction are treated with Tris-ammonium chloride buffer (PH7.65) for 12 minutes to remove red blood cells, washed three times with MEM, and the obtained spleen cells are used as antibody-producing cells. Used.
  • myeloma cells cell lines obtained from mice or rats are used.
  • 8-azaguanine-resistant mouse derived from BALB / c
  • myeloma cell line P3—X63Ag8-Ul Yelton DE et al., Curr. Topics Microbiol. Immunol., SI, 1 (1978), Kohler G. & Milstein C., Eur. J. Immunol., 6, 511 (1976)], SP2 / OA14 [Shulman. Et al., Nature, 269 (1978)], P3-X63-8653 [Seeger RC J. Immunol., M, 1548 (1979)], P3-X63 Ag
  • a normal medium (Hereinafter referred to as a normal medium) and a medium supplemented with 15-g / ml 8-azaguanine], and cultured 3 to 4 days before cell fusion with a normal medium.
  • a normal medium a medium supplemented with 15-g / ml 8-azaguanine
  • a normal medium a medium supplemented with 15-g / ml 8-azaguanine
  • the cell group of the obtained precipitate fraction is loosened well, and the cell group is stirred at 37 ° C while stirring.
  • HAT medium [1 O ⁇ mrno 1/1 hypoxanthine, 1.5 x 10 " 5 mmo 1 in normal medium. / 1 thymidine and 4 x 10- 7 mm o 1/ 1 Aminobuteri down the added medium] is suspended in 100 ml.
  • the full-length or partial fragment of the human synuclein-binding protein used as the antigen was coated on a suitable plate, and the hybridoma culture supernatant or the purified antibody obtained in (2-4) described later was used as the primary antibody.
  • the antibody is reacted with an anti-rat immunoglobulin antibody labeled with piotin, an enzyme, a chemiluminescent substance, a radioactive compound, or the like as a secondary antibody, followed by a reaction according to the labeling substance.
  • Those that specifically react with the combined protein are selected as hybridomas that produce monoclonal antibodies against the sph-synuclein binding protein.
  • Cloning is repeated twice by the limiting dilution method using the hybridoma [the first time uses HT medium (medium in which aminopterin is removed from HA ⁇ medium), and the second time uses normal medium], Those with a stable and strong antibody titer are selected as hybridoma strains producing a monoclonal antibody against the sph-synuclein binding protein. (2-4) Preparation of monoclonal antibody
  • Pristane Pristane; 2, 6, 10, 14-tetramethylpen-decane
  • 2-3 Hyprideoma cells producing the monoclonal antibody to the obtained human synuclein binding protein are injected intraperitoneally with 5 to 20 ⁇ 10 6 cells / animal. In 10 to 21 days, Hypridoma becomes ascites cancer.
  • Ascites is collected from the mouse with ascites tumor and centrifuged at 3,000 rpm for 5 minutes to remove solids.
  • a monoclonal antibody can be purified and obtained in the same manner as the method used for the polyclonal antibody.
  • the antibody subclass is determined using a mouse monoclonal antibody typing kit or a rat monoclonal antibody typing kit.
  • the protein content is calculated by the Lowry method or from the absorbance at 280 nm.
  • Antibodies to the human synuclein binding protein recognize and specifically bind to the human synuclein binding protein, and thus can be used for detection, quantification, and purification of the human synuclein binding protein.
  • Methods for immunologically detecting human synuclein-binding protein using an antibody against human synuclein-binding protein include immunohistochemical staining such as immunohistochemistry and immunocytostaining, immunoblot, and sandwich ELISA.
  • immunohistochemical staining such as immunohistochemistry and immunocytostaining, immunoblot, and sandwich ELISA.
  • Immunohistochemical staining involves fixing tissue sections and cells, reacting an antibody against human synuclein-binding protein as a primary antibody, and labeling the antibody with fluorescent substances, enzymes, biotin, gold colloid, radioactive substances, etc. After reacting the immunoglobulin antibody or antibody fragment as a secondary antibody, if necessary, the labeled antibody is visualized and observed using a microscope to detect human synuclein-binding protein in tissues and cells. It is a way to do it.
  • Fluorescein'isothiocyanate (FITC), tetramethylrhodamine / isothiocyanate, etc., are used for fluorescent substance labeling, and are detected by observation with a fluorescence microscope.
  • enzyme labeling peroxidase, alkaline phosphatase or the like is used, and a color-developing reaction is performed by adding a substrate that develops a color by the enzyme, and the enzyme can be detected by observing with an optical microscope.
  • biotin labeling after reacting avidin labeled with an enzyme such as peroxidase, the same operation as for the enzyme-labeled antibody is performed.
  • a gold colloid label it is detected by observation with an electron microscope.
  • the radioactive materials labeled used is 125 1 or the like, can be detected by observing the silver particles precipitated by radiation by coating the emulsion in an optical microscope.
  • Imnobroth is obtained by fractionating tissues or cells or their crushed solutions by SDS-PAGE, and then printing the gel on a PVDF membrane or a nitrocellulose membrane, and coating the membrane with an antibody against a human synuclein binding protein or an antibody fragment thereof.
  • the reaction is performed as a primary antibody, and enzymes such as peroxidase and alkaline phosphatase
  • a type of enzyme immunoassay monoclonal antibodies to two types of human synuclein binding proteins having different antigen recognition sites are prepared, and one of the monoclonal antibodies or antibody fragments is adsorbed on a plate in advance.
  • the other monoclonal antibody or antibody fragment should be labeled with an enzyme such as peroxidase or alkaline phosphatase.
  • Radioimmunoassay using antibodies labeled with radioactive material 125 1 or the like instead of the enzyme, the same operation as enzyme immunoassay, in a sample by measuring radiation at scintillation counter evening one
  • This is a method for detecting or quantifying a synuclein-binding protein.
  • an antibody against the human synuclein-binding protein and a test sample are mixed in a solution to bind to each other to form a complex of the human synuclein-binding protein and the antibody, and then immobilized on the carrier.
  • This is a method of isolating the complex from a solution using a munoglobulin antibody, the antibody fragment, protein A, or the like.
  • the complex was dissolved by treating the carrier with a sample buffer for SDS-PAGE, and the SDS-PAGE was associated with the sph-synuclein binding protein and the sph-synuclein binding protein in the test sample. Protein can be detected.
  • An antibody against the human synuclein binding protein is immobilized on a carrier as in the case of human synuclein described in 1., and the solution containing the human synuclein binding protein is subjected to affinity mouth chromatography to obtain the human synuclein binding protein. Can be purified. Unlike affinity chromatography immobilized with human-synuclein, it is possible to specifically purify only the human-synuclein-binding protein recognized by the antibody.
  • neutralizing antibodies against the human synuclein binding protein those that inhibit the activity of the human synuclein binding protein to promote the fiber formation of the human synuclein are called neutralizing antibodies against the human synuclein binding protein.
  • the neutralizing antibody against the human synuclein binding protein include an antibody against the human synuclein binding protein that inhibits the binding of the human synuclein binding protein to the human synuclein. Whether an antibody against the human synuclein-binding protein inhibits the activity of promoting human synuclein association or fiber formation depends on the fiber formation system at the mouth of human synuclein described in 1. (3).
  • the human synuclein-binding protein and the antibody against the human synuclein-binding protein are added, compared with the case where only the human synuclein-binding protein is added and the antibody against the human synuclein-binding protein is not added, the human synuclein-binding protein is used. Inhibits the action of sperm-synuclein association or fibril formation by cysteine? You can find out if.
  • human synuclein-binding protein promotes human synuclein association or fiber formation
  • substances that inhibit the binding of human synuclein to human synuclein binding protein are considered to inhibit human synuclein association or fiber formation.
  • the amount of binding of human synuclein or human synuclein-binding protein when the test substance is added is measured.
  • a substance that inhibits the binding of human synuclein to human synuclein binding protein inhibits human synuclein association or fibril formation depends on the in vitro fibril formation of human synuclein described in 1. (3). It is possible to determine whether or not spike-synuclein promotes fibril formation compared to when no test substance is added ( ⁇ -synuclein-binding protein is added) when spike-synuclein binding protein and a test substance are added to the system. it can. Also, even if the test substance does not inhibit the binding of the human synuclein to the human synuclein-binding protein, ⁇ -synuclein may be added to the fibril formation system at the mouth of the human synuclein described in 1. (3).
  • synuclein-binding protein alters the physiological function or properties of the synuclein, such as the degree of phosphorylation or interaction with other proteins
  • the synuclein-binding protein and the synuclein-binding protein By inhibiting the action of this human synuclein-binding protein using a substance that inhibits the binding of human synuclein, neurons involved in the onset and progression of the disease state of human synuclein such as Parkinson's disease and Lewy body dementia. Treatment can prevent the onset of degenerative disease or delay its progress.
  • a drug containing a substance that inhibits the stimulatory action or a neutralizing antibody for human synuclein-binding protein can be administered as a drug alone or as a drug, but is usually pharmacologically acceptable. It is desirable to mix it with one or more carriers as described above and provide it as a pharmaceutical preparation produced by any method well known in the technical field of pharmaceutics.
  • oral administration or parenteral administration such as oral, respiratory, rectal, subcutaneous, intramuscular, and intravenous administration.
  • Administration forms include sprays, capsules, tablets, granules, syrups, emulsions, suppositories, injections, ointments, tapes and the like.
  • Formulations suitable for oral administration include emulsions, syrups, capsules, tablets, powders, granules and the like.
  • liquid preparations such as emulsions and syrups include water, sugars such as sucrose, sorbitol, fructose, glycols such as polyethylene glycol and propylene glycol, oils such as sesame oil, olive oil, soybean oil, p- It can be produced using preservatives such as hydroxybenzoic acid esters and flavors such as strawberry flavor and peppermint as additives.
  • preservatives such as hydroxybenzoic acid esters and flavors such as strawberry flavor and peppermint as additives.
  • excipients such as lactose, glucose, sucrose, mannitol, disintegrants such as starch, sodium alginate, lubricants such as magnesium stearate, talc, polyvinyl alcohol, It can be produced using a binder such as hydroxypropylcellulose and gelatin, a surfactant such as fatty acid ester, and a plasticizer such as glycerin as additives.
  • Formulations suitable for parenteral administration include injections, suppositories, sprays and the like.
  • an injection is prepared using a carrier comprising a salt solution, a glucose solution, or a mixture of both.
  • Suppositories are prepared using carriers such as cocoa butter, hydrogenated fats and carboxylic acids.
  • the propellant does not irritate the substance or the neutralizing antibody itself or the oral and respiratory mucosa of the recipient, and separates the substance or the neutralizing antibody as fine particles. It is prepared using a carrier or the like which is dispersed to facilitate absorption. Specific examples of the carrier include lactose and glycerin. Formulations such as aerosols and dry powders are possible depending on the properties of the compound and the carrier used. Also, in these parenteral preparations, the components exemplified as additives for oral preparations can be added.
  • the dosage or frequency of administration varies depending on the desired therapeutic effect, administration method, treatment period, age, body weight, etc., but is usually 10 mg / kg to 100 mg / kg per day for an adult.
  • the sph-synuclein binding protein isolated in 1. has a novel amino acid sequence
  • the cDNA of the sph-synuclein binding protein which is a polynucleotide encoding the novel sph-synuclein binding protein, is used. It can be isolated as follows.
  • a mixture of multiple nucleotide sequences encoding the partial amino acid sequence obtained in 1. (1) (hereinafter referred to as “degenerate nucleotide sequence”) Can be designed. Since the base sequence of the portion encoding the partial amino acid sequence in the base sequence of the gene for the human synuclein binding protein is one of the above degenerate base sequences, Oligonucleotides are synthesized, and using this as a probe, a cDNA library of the tissue from which the human synuclein-binding protein is isolated is screened by colony hybridization or plaque hybridization. A cDNA clone of the human synuclein binding protein can be isolated. Oligonucleotides having a degenerated base sequence can be chemically synthesized using a DNA synthesizer.
  • the N-terminal amino acid sequence and the internal amino acid sequence are each determined at 5 amino acids or more consecutively. In some cases, or when 20 consecutive amino acids are determined (the first 5 amino acids and the last 5 amino acids yield 2), the degeneracy of the amino acid sequence closer to the N-terminus of both is An oligonucleotide having a base sequence and an oligonucleotide having a base sequence complementary to the degenerate base sequence for the amino acid closer to the C-terminus were synthesized, and the foresight was considered as a first primer and the latter as a reverse primer.
  • RNA of the tissue from which the human synuclein-binding protein was isolated it is possible to amplify and isolate a partial cDNA fragment of the human synuclein binding protein.
  • a cDNA clone encoding the human synuclein binding protein can be isolated.
  • a novel human-synuclein binding protein having an amino acid sequence represented by SEQ ID NO: 1 at the N-terminus and an amino acid sequence represented by SEQ ID NO: 2 therein was isolated.
  • a human brain cDNA library is screened using an oligonucleotide probe having a degenerate nucleotide sequence represented or a nucleotide sequence of 14 or more consecutive nucleotides of these nucleotide sequences, thereby obtaining the human synuclein.
  • the cDNA encoding the binding protein can be isolated.
  • a degenerate nucleotide sequence represented by SEQ ID NO: 5 or a salt of 14 or more consecutive nucleotides of this nucleotide sequence Using a forward primer having a base sequence and a degenerate base sequence represented by SEQ ID NO: 7 or a reverse primer having a continuous base sequence of 14 bases or more of the base sequence, RT-P
  • a forward primer having a base sequence and a degenerate base sequence represented by SEQ ID NO: 7 or a reverse primer having a continuous base sequence of 14 bases or more of the base sequence RT-P
  • EST having a nucleotide sequence identical to the EST nucleotide sequence, and ESTs derived from the same clone as the ES * are collected as ESTs derived from the same gene.
  • the nucleotide sequence of the cDNA of the human synuclein-binding protein can be revealed by connecting the nucleotide sequences considered to be derived from the cDNA of the human synuclein-binding protein. If the nucleotide sequence of cDNA cannot be determined solely from these nucleotide sequences, a sense primer having the most 5 or 5 nucleotides of the nucleotide sequence obtained by splicing or a nucleotide complementary to the 3 or 3 nucleotide sequence is obtained. Using an antisense primer having a sequence, RT—
  • cDNA of the human synuclein binding protein can be obtained.
  • the obtained cDNA is not a full-length cDNA, screening of a cDNA library using this clone as a probe or RACE method [rapid amplification of cDNA ends; Frohman MA et al., Proc. Natl. Acad. Sci. . USA, 8998 (1988)] to obtain a full-length cDNA.
  • the nucleotide sequence of the obtained cDNA clone was determined using a DNA sequencer or the like, and the nucleotide sequence was translated into an amino acid sequence in each frame. Then, the amino acid sequence of the human synuclein binding protein obtained in 1. (1) was determined. Search for an amino acid sequence that matches the partial amino acid sequence.
  • the amino acid sequence of the open reading frame containing the amino acid sequence that matches the partial amino acid sequence can be determined as the amino acid sequence of the whole synuclein-binding protein.
  • polynucleotide encoding the human synuclein binding protein examples include a nucleotide sequence corresponding to the open reading frame and DNAs and RNAs including a nucleotide sequence encoding the amino acid sequence of the entire human synuclein binding protein. I can give it. .
  • the c-DNA or its partial fragment of the c-synuclein binding protein, or the c-DNA base sequence or a nucleotide sequence complementary to the c-DNA sequence of at least 10 consecutive bases is used to form the c-synuclein binding protein.
  • the expression level of the protein at the mRNA level can be measured by a method such as Northern plotting, RT-PCR, or dot hybridization.
  • the above-mentioned cDNA, a partial fragment of the cDNA, an oligonucleotide, or a derivative thereof is used to suppress the expression of the human synuclein-binding protein gene, thereby inhibiting the association of human synuclein or the promotion of fibrosis. It can prevent the onset of neurological diseases related to the onset and progression of the disease state, such as dementia and Lewy body dementia, and treat the disease to delay the progression.
  • Methods for suppressing the expression of the above genes include, for example, antisense RNA / DNA technology [Bioscience and Industry, 322 (1992), Chemistry, 681 (1991).
  • oligonucleotide derivative examples include an oligonucleotide derivative in which a phosphoric ester bond in an oligonucleotide is converted into a phosphorothioate bond, and a phosphoric diester bond in an oligonucleotide in the form of N3,1-P5, phosphoamido.
  • Oligonucleotide derivatives converted to date bonds, ribose and phosphodiester bonds in oligonucleotides are converted to peptide nucleic acid bonds, fe oligonucleotide derivatives and peracyl in oligonucleotides are replaced by C-15 propynyl peracyl
  • Oligonucleotide derivatives, oligonucleotide derivatives in which peracyl in the oligonucleotide is substituted with C-5 thiazole peracyl oligonucleotide derivatives in which cytosine in the oligonucleotide is substituted with C-5 propynylcytosine, oligonucleotides
  • An oligonucleotide derivative in which cytosine in tide is substituted with Fuenokisajin modified cytosine (phenoxazine- modified cytosine), ribonucleic in the oligonucleotide
  • the medicament containing the cDNA, a partial fragment of the cDNA, the oligonucleotide, or the derivative of the oligonucleotide as an active ingredient can be provided in the form and use of the pharmaceutical preparation described in 5. above.
  • the human synuclein binding protein is described in J. Sambrook et al .; Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory (1989), DM Glover and BDHames jDNA Clonin 1: Core Techniques, A Practical Approach, Second. Edition, Oxford University Press (1995) and the like, the polynucleotide encoding the human synuclein binding protein can be expressed in host cells in large quantities and easily.
  • a recombinant vector in which DNA encoding the human synuclein binding protein is inserted downstream of the promoter of an appropriate expression vector is constructed, and the vector is introduced into a host cell to obtain the human synuclein.
  • the human synuclein binding protein By obtaining a transformant expressing the binding protein and culturing the transformant, the human synuclein binding protein can be produced.
  • the expression vector contains a promoter capable of autonomous replication in a host cell or integration into a chromosome and capable of transcribing mRNA from DNA encoding the synuclein binding protein in the host cell. Things are used.
  • any cells that can express the target gene such as bacteria, yeast, animal cells, insect cells, and plant cells, can be used.
  • the method of culturing the above-obtained transformant in a medium can be performed according to a usual method used for culturing a host.
  • the method for producing the human synuclein binding protein includes a method of producing the protein in a host cell, a method of secreting the protein out of the host cell, and a method of producing the protein on the host cell outer membrane. By changing the structure of the protein to be produced, You can choose the method.
  • a conventional method for isolating and purifying an enzyme can be used.
  • the cells are collected by centrifugation after completion of the culture, suspended in an aqueous buffer, and then sonicated with a sonicator, French press, etc.
  • the cells are disrupted with a Manton-Gaurin homogenizer, Dynomill, etc. to obtain a cell-free extract.
  • a normal enzyme isolation / purification method that is, a solvent extraction method, a salting-out method using ammonium sulfate, a desalting method, a precipitation method using an organic solvent, Tylaminoethyl (DEAE)-anion exchange chromatography using a resin such as Sepharose, DIAION HPA-75 (manufactured by Mitsubishi Kasei), or a resin such as S-Sepharose FF (manufactured by Pharmacia) Cation exchange chromatography, hydrophobic chromatography using resins such as petyl sepharose and phenylsepharose, gel filtration using molecular sieves, affinity chromatography, chromatofocusing, A purified sample can be obtained by using techniques such as electrophoresis such as isoelectric focusing alone or in combination.
  • the synuclein-binding protein when expressed by forming an insoluble form in the cells, the cells are similarly collected, crushed, and centrifuged to remove the insoluble form of the protein as a precipitate fraction. to recover.
  • the recovered insoluble form of the protein is solubilized with a protein denaturant.
  • the protein is returned to a normal conformation by diluting or dialyzing the solubilized solution and reducing the concentration of the polypeptide denaturing agent in the solubilized solution. After this operation, a purified sample of the protein can be obtained by the same isolation and purification method as described above.
  • the protein or a derivative of the protein can be recovered in the culture supernatant. That is, the culture is centrifuged in the same manner as described above. The culture supernatant is obtained by further treatment, and a purified sample can be obtained from the culture supernatant by using the same isolation and purification method as described above.
  • the protein thus obtained binds to a single nuclein having an amino acid sequence represented by SEQ ID NO: 1 at the N-terminus and having an amino acid sequence represented by SEQ ID NO: 2 therein.
  • Protein, tubulin, amyloid, 14-3-3 protein, tau protein and microtubule-associated protein 1B binds to a single nuclein having an amino acid sequence represented by SEQ ID NO: 1 at the N-terminus and having an amino acid sequence represented by SEQ ID NO: 2 therein.
  • Protein, tubulin, amyloid, 14-3-3 protein, tau protein and microtubule-associated protein 1B Protein, tubulin, amyloid, 14-3-3 protein, tau protein and microtubule-associated protein 1B.
  • the synuclein-binding protein can also be produced by a chemical synthesis method such as the Fmoc method (fluorenylmethyloxycarbonyl method) and the tBoc method (t-butyloxycarbonyl method). . Chemical synthesis can also be performed using peptide synthesizers such as Advanced ChemTech, Perkin-Elma, Pharmaci, Protein Technology Instrument, Synthecell-Vega, PerSeptive, and Shimadzu. In the drawing
  • FIG. 1 is a view showing the action of tubulin for promoting the fiber formation of spike synuclein.
  • “a” shows the aggregation degree of light-synuclein (light scattering degree of 40 Onm) after 15 days when the concentration of light-synuclein was changed.
  • the horizontal axis is the concentration of spike-synuclein ( ⁇ mo 1 /
  • the vertical axis is the light scattering at 440 nm. If the mouth is only synuclein and nothing is added, ⁇ indicates 1 ⁇ mo 1/1 tubulin is added, Hata is l ⁇ mol
  • b shows the time-course change in the cohesion degree of human synuclein up to 96 hours after addition of 1 mo 1/1 tubulin to 300 / mo 1/1 human synuclein.
  • the horizontal axis is time (unit: time), and the vertical axis is light scattering at 400 nm. Mouth is only for synuclein When nothing was added, ⁇ indicates that 1 ⁇ mo 1/1 tubulin was added, and ⁇ indicates that 1 mo 1/1 tubulin and 1 / mo 1/1 magnesium chloride, GTP and ATP were added. Show the case.
  • c is a bar graph showing the same result as a, showing the specific light scattering at 400 nm for each concentration of sph-synuclein. From the left, C 1: 300 zmo1 / If nothing is added, only C 2: 1 jumo 1/1 tubulin (no spike-synuquine), C 3: 300 ⁇ mo 1/1 spike-synuclein When mo 1/1 BSA is added, C4: 300 ⁇ mol / l BSA, l mo 1/1 tubulin is added (no synuclein is present), 100, 3
  • the figures show the case where 1 zmo 1/1 of tubulin was added to each of 0, 500, and 700-mo 1/1 of human synuclein. + (Black bar graph) indicates the case where l mol / 1 of magnesium chloride, GTP and ATP were further added, and-(gray bar graph) indicates the case where no addition was made. ⁇ ⁇ ⁇ ⁇ ⁇
  • Plasmid pET-hi-synuclein for human sph-synuclein expression was prepared by inserting it between NcoT / RamHT sites of 15b [Novagen]. ⁇
  • Escherichia coli strain transformed with ⁇ —hi-synuclein K s che ri oh i a.
  • IPTG isopropylthiogalactoside
  • the precipitated insoluble fraction was suspended again in the above buffer, centrifuged at 4 ° C. and 18,000 rpm for 60 minutes, and the supernatant was combined with the first supernatant. Add 40 to this combined supernatant.
  • the protein is salted out by adding ammonium sulphate to a saturation of 4%,
  • the mixture was centrifuged at 000 rpm for 60 minutes, and the precipitated protein was recovered. This precipitate is
  • the recombinant dialysate was adsorbed through a solution after dialysis through a DEAE-5PW column (manufactured by Tosoichi Co., Ltd.), washed, and eluted sequentially by increasing the sodium chloride concentration stepwise. Further, the fraction containing the eluted human spleen synuclein was again subjected to DE.
  • Sepharose-4B activated with cyanogen bromide
  • Sepharose-14B was quickly transferred from the column to the ligand immobilization buffer containing 27 mg of the purified recombinant human human synuclein prepared in (1) using an aspirator and mixed. The immobilization reaction was performed by gentle stirring at 4 ° C overnight.
  • Cephalo immobilized with human human synuclein -Synuclein-Sepharose-1B was separated from the solution of human synuclein by centrifugation at 3,000 rpm for 20 minutes, washed with a buffer for immobilizing ligand, and then treated with 50 Ommo
  • the cells were washed with 10 Ommo 1/1 acetate buffer (pH 4.0) containing 1/1 sodium chloride, and then washed again with a ligand immobilization buffer.
  • the washed human synuclein-sepharose-14B was transferred to a ligand immobilization buffer containing 20 Ommo 1/1 glycine (pH 8.0) and reacted at 4 ° C for an additional 16 hours.
  • SDS-PAGE gel concentration: 13.5%
  • a human brain extract before being adsorbed to H-synuclein-Sepharose-14B, an eluate from glycine-Sepharose-14B, a fraction that passed without being adsorbed to H-synuclein-Sepharose-14B, Recombinant human synuclein prepared in (1) was simultaneously subjected to SDS-PAGE.
  • the 42-kDa and 50-kDa human-synuclein-binding proteins eluted from human synuclein-sepharose-14B were further purified by anion exchange HP LC.
  • the eluate containing the protein synthesizing human-synuclein binding from Chichi-Synuclein-Sepharose-14B is passed through a column of £ 5 £ -5 (4.6 x 75 mm, manufactured by Tosoh Corporation).
  • the human synuclein-binding protein was adsorbed, it was washed with 2 Ommo 1/1 Tris-monohydrochloric acid (pH 9.0).
  • Elution was carried out sequentially with 2 Ommo 1/1 tris-hydrochloric acid (pH 9.0) in which the concentration of sodium chloride was increased stepwise by 10 Ommo 1/1 from 10 Ommo 1/1 to 50 Ommo 1/1.
  • MALD I / TOF mass spectrometry was performed on each of the two purified human synuclein-binding proteins obtained in (4). As a result, it was found that the protein of 42 kDa had a molecular weight of 41847. 1 (+ ⁇ ) +, and the protein of 50 kDa had a molecular weight of 501611.1 (M + H) +.
  • the purified human synuclein-binding protein was digested with lysyl endopeptidase at an enzyme: substrate ratio of 1:50 (w / v).
  • the produced peptide was separated by reversed-phase HPLC using Super Sphere RP-100-100 C18 column [4.0 x 250 mm, Merck], ie, 5% acetonitrile. After adsorption of the peptide on the column equilibrated with 0.1% TFA, elution was performed at room temperature, at a flow rate of lml / min for 30 minutes, with 5% -60% linear concentration gradient of acetonitrile—0.1% Made in TF A. The eluted peptide was monitored by absorbance at 210 nm.
  • the amino acid sequences of the obtained peptide and the purified non-enzyme-treated purified human-synuclein binding protein were determined by the Edman degradation method.
  • the 42 kDa protein has the amino acid sequence represented by SEQ ID NO: 1 at the N-terminal. And found to have the amino acid sequence represented by SEQ ID NO: 2 inside.
  • the presence or absence of an amino acid sequence having homology to these amino acid sequences is checked by using the homology search software Frame Search (manufactured by Compugen) with respect to the amino acid sequence databases SwissProt, PIR, and Genpept. As a result, there was no matching sequence, and it was presumed to be a novel protein.
  • the 5 OkDa protein was found to have the amino acid sequence represented by SEQ ID NO: 3 at the N-terminal and the amino acid sequence represented by SEQ ID NO: 4 internally.
  • these amino acid sequences were searched against the amino acid sequence database Swiss PRot using the above-described homology search software, it was found that both of them were included in the amino acid sequence of human human tubulin.
  • the molecular weight of the 50 kDa protein determined by MALD I / T OF mass spectrometry, 50161.1 is in good agreement with the molecular weight, 50158, deduced from the published amino acid sequence of h-tubulin, and the 5-okDa h-synuclein The binding protein was thought to be spike-tubulin.
  • a brain extract was prepared from rat brain in the same manner as in Example 1 (2), and RIPA buffer [
  • Rat brain extracts were prepared using two types of human-synuclein-specific antibodies EQV1 and PQE3 [Arima K. et al., Brain Res.
  • the antigen-antibody conjugate was bound to protein A Sepharose (Sigma-Aldrich). After the reaction, wash the protein A sepharose in PBS containing 1% Triton X-100 and repeat the centrifugation washing operation 5 times, and then in SDS-sample buffer at 100 ° C for 5 minutes. Upon heating, the bound antigen-antibody complex was dissolved in SDS-one sample buffer. The antigen-antibody complex dissolved in the SDS-sample buffer is subjected to SDS-PAGE (gel concentration: 12%), and then electrically transferred to nitrocellulose membrane (manufactured by S & S Portran) and fixed.
  • SDS-PAGE gel concentration: 12%
  • the anti-tubulin antibody TUB 2.1 (Sigma-Aldrich) was used as the primary antibody.
  • TUB 2.1 Sigma-Aldrich
  • the anti-synuclein antibody immunoprecipitated not only with human -synuclein but also with human -tubulin. I understood.
  • physiologically sph-synuclein was bound not only to sph-tubulin but also to ??-tubulin.
  • tubulin and / or tubulin are the major constituent proteins of microtubules, to confirm the binding of microtubules to human synuclein, microtubules were purified from rat brain and examined for the presence of human synuclein. Was.
  • anti-tubulin antibody (1 ': 5000 dilution), anti-tubulin antibody (1: 5000 dilution), anti-tau protein antibody Tau-2 (Neomakers) (1: 500 dilution), anti-MAP5 antibody [Chemicon] (1: 500 dilution) was used as a primary antibody, subjected to the same Imnob analysis as in (1), and purified from brain. It was confirmed that tubulin, tubulin, tau protein, and MAP5 were present in the microtubules.
  • the same purified microtubules were subjected to the same immunoblot analysis using two types of sph-synuclein-specific antibodies EQVI and PQE3 as primary antibodies, and the amount of purified microtubules used was 15, 30, 60.
  • g was increased, gamma-synuclein was detected by both gamma-synuclein-specific antibodies, and the amount of gamma-synuclein was detected in the purified microtubules. confirmed.
  • Rhodamine-labeled anti-magpie Ig G antibody [1:80 dilution "*; made by Biosource International (Biosource International)"
  • FITC-labeled anti-mouse IgG antibody (1:30 dilution, Biosource International) Company).
  • anti-synuclein antibody stained very strongly in all of these tissues for Levi bodies, Pale bodies, which are considered to be the early state of Lewy bodies, and Levi body-associated neurites.
  • the anti-tubulin antibody was weaker than the anti-synuclein antibody, it stained Lewy bodies, Veil bodies, and Lewy body-related neurites in all tissues.
  • tubulin a human synuclein binding protein
  • microtubules isolated from the rat brain in Example 2 (3) were dissociated into tubulin, and subjected to phosphocellulose column chromatography using Whatman P11 [Weingarten M et al., Proc. Natl. Acad. Sci., USA, 11, 1858 (1975)] to purify tubulin.
  • the fibril formation of -synuclein was examined by immuno-microscopy and the aggregation of -synuclein.
  • the cohesion of human synuclein is 400 nm light scattering [MaJ. Etal., Nature, , 92 (1994)] was measured with a spectrophotometer [Gene Spec III manufactured by Hitachi, Ltd.] using the reaction solution 1-1.
  • the formed fibers are isolated, placed on a grid of an electron microscope, and the primary antibody, a sph-synuclein-specific antibody EQV1 (1:40 dilution) or an anti-sph-tubulin antibody (1:40) After the reaction, a secondary antibody bound to gold colloid particles with a diameter of 1 O nm [1:20 dilution, manufactured by British Biocell (British BioCell)] was detected as a probe.
  • magnesium chloride, GTP and ATP which are microtubule formation promoting factors, were added to 1 ⁇ mo 1/1 (normal (Approximately 1/1000 of the concentration used for microtubule formation) was further added, and the same measurement was carried out. As shown in a and b in Fig. 1, compared to the case where only tubulin was added, It was found that aggregation of spike synuclein was promoted.
  • ultrathin sections prepared by fixing the dorsal nucleus tissue of the vagus nerve of a patient with Lewy body dementia to formalin and embedding in LR white resin (manufactured by London Resin) were used for the human synuclein specific antibody PQE 3 ( (1:40 dilution) as the primary antibody.
  • PQE 3 human synuclein specific antibody
  • a gold colloid particle-bound secondary antibody having a diameter of 5 nm was used.
  • the diameter and morphology of the fiber of sph-synuclein formed in the presence of tubulin in vitro were very similar to the fiber of the sph-synuclein in the pathological tissue of patients with dementia with Lewy bodies.
  • a novel human synuclein-binding protein a DNA encoding the protein, and a method for screening a substance that inhibits the association or fibril formation of human synuclein using the human synuclein-binding protein,
  • a prophylactic or retardant drug for neurodegenerative diseases associated with spike synuclein fibril formation such as Parkinson's disease can be developed.
  • SEQ ID NO: 5 synthetic DNA
  • SEQ ID NO: 6 synthetic DNA
  • SEQ ID NO: 7 synthetic DNA

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Abstract

L'invention concerne une nouvelle protéine de liaison de l'α-synucléine ; un ADN codant cette protéine ; un procédé permettant le criblage d'une substance inhibant l'association ou la fibrose de l'α-synucléine au moyen de ladite protéine de liaison de l'α-synucléine ; et des agents préventifs ou retardateurs, qui contiennent la substance susmentionnée et qui sont destinés aux maladies neurodégénératives associées à la fibrose de l'α-synucléine, notamment la maladie de Parkinson.
PCT/JP2001/005890 2000-07-06 2001-07-06 Proteine de liaison de l'$g(a)-synucleine Ceased WO2002004625A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3026057A2 (fr) 2009-03-09 2016-06-01 Ramot at Tel Aviv University Ltd. Compositions pour la prévention et le traitement de maladies neurodégénératives
CN111487414A (zh) * 2019-01-29 2020-08-04 北京现代高达生物技术有限责任公司 一种SNCG/SCC-Ag联检胶体金试纸条及其制备方法和用途

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
[online] UEDA K. ET AL.: "Identification of alpha-tubulin as a binding-partner of NACP(alpha-synuclein) and its involvement in Lewy body formation in Parkinson's disease and in multiple system atrophy", XP002947235, retrieved from 200100075808 accession no. Biosis Database accession no. 12868659 *
GAI W.P. ET AL.: "alpha-Synuclein immunoisolation of glial inclusions from multiple system atrophy brain tissue reveals multiprotein components", J. NEUROCHEM., vol. 73, no. 5, 1999, pages 2093 - 2100, XP002947237 *
ISEKI E. ET AL.: "Accumulation of human alpha-synuclein in different cytoskeletons in Lewy bodies in brains of dementia with Lewy bodies", NEUROSCI. LETT., vol. 290, no. 1, August 2000 (2000-08-01), pages 41 - 44, XP002947236 *
POUL H.J. ET AL.: "alpha-Synuclein binds to tau and stimulates the protein kinase A-catalyzed tau phosphorylation of serine residues 262 and 356", J. BIOL. CHEM., vol. 274, no. 36, 1999, pages 25481 - 25489, XP002947238 *
SOCIETY FOR NEUROSCIENCE ABSTRACTS, vol. 26(1-2), no. 131, November 2000 (2000-11-01) *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3026057A2 (fr) 2009-03-09 2016-06-01 Ramot at Tel Aviv University Ltd. Compositions pour la prévention et le traitement de maladies neurodégénératives
CN111487414A (zh) * 2019-01-29 2020-08-04 北京现代高达生物技术有限责任公司 一种SNCG/SCC-Ag联检胶体金试纸条及其制备方法和用途

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