JP2007121309A - Method for screening a substance that inhibits interaction between amyloid β and amyloid β-binding protein - Google Patents

Abstract

It is an object of the present invention to provide a technique that contributes to the development of a new treatment or prevention method for Alzheimer's disease that is safe and highly effective.
According to the present invention, a protein group that binds to amyloid β protein is comprehensively provided using proteomics technology. The knowledge of these protein groups opens up the possibility of developing therapeutic drugs targeting the protein groups and is considered to contribute greatly to elucidating the causes of Alzheimer's disease.
[Selection figure] None

Description

  The present invention relates to a method for comprehensively identifying a protein (amyloid β-binding protein) having a property of binding to amyloid β that causes Alzheimer's disease using proteomics technology. Furthermore, the present invention relates to a method for using a protein identified by such a method.

  Alzheimer's disease is a disease that develops from the late 40s to 50s in the early age, and develops after the late 70s in the elderly, and includes memory impairment, motivation disorder, judgment disorder, aphasia, apraxia, agnosia, personality Symptoms such as disability, emotional disorder, mirror disorder, and Kluber-Beauty syndrome appear, indicating symptoms of severe dementia. The pathological changes in the brain include geriatric plaques, Alzheimer's neurofibrillary tangles, neuronal loss, etc., and as the symptoms progress, the lesion becomes more severe, leading to prominent brain atrophy.

  As the pathological condition of Alzheimer's disease, abnormal and early deposition of amyloid proteins such as amyloid β40 and amyloid β42, and accumulation of phosphorylated tau protein in neurons are observed. It has also been clarified that there is an abnormal decrease in neurotransmitters such as acetylcholine. Among the Alzheimer-type dementias, familial Alzheimer's disease is also reported. Gene abnormalities in each of chromosomes 14, 19, and 21 have been reported as the cause of the disease, and point mutations in the amino acid sequence of the precursor protein of amyloid β The existence of is proven.

  As described above, in the brain affected by Alzheimer's disease, neuronal cells of the cerebral neocortex and hippocampus are dropped, and fibrous accumulations such as senile plaques appear. The main component of the senile amyloid is the amyloid β protein (Aβ), which is cut out from the extracellular region of the precursor amyloid precursor protein (APP) to the middle of the transmembrane region by a two-step protease action. Secreted. In the early stage of Alzheimer's disease, amyloid protein accumulation is the earliest phenomenon that occurs faster than tau protein phosphorylation. Thus, amyloid protein is considered to play an important role in the onset of Alzheimer's disease, and such a concept is called “amyloid hypothesis”. The latest knowledge about the onset mechanism of Alzheimer's disease is described in detail, for example, in the general remarks by Iwatsubo et al. (Clinical Neuroscience (2002) vol.20 no.6 p626-629: Non-Patent Document 1).

  In the first stage of amyloid protein production, β-cleavage by β-secretase occurs, resulting in the formation of the N-terminus of Aβ. The C-terminal fragment of APP generated by β-cleavage is subsequently cleaved by γ-secretase, and there are diversity in this cleavage site. The main molecular species is Aβ40 consisting of 40 amino acid residues, but it is said that Aβ42 consisting of 42 amino acid residues is also present in about 10%. There are some differences in the properties of Aβ40 and Aβ42, and Aβ42 begins to accumulate from the beginning in the pathological changes of Alzheimer's disease, while Aβ40 accumulates later. Aβ42 is said to exhibit higher aggregation than Aβ40.

  Thus, although the detailed pathology of Alzheimer's disease has been clarified in recent years, an absolute prevention method or treatment method has not yet been found. At the present stage, pharmacotherapy and rehabilitation mainly using brain function improving drugs such as cerebral metabolic activators, cerebral circulation improving drugs, neurotransmitter function adjusting drugs and the like are performed.

  By the way, Aβ vaccine therapy targeting Aβ has attracted attention as a new treatment method for Alzheimer's disease. As an example, Schenk et al. Reported that when an Alzheimer's disease model animal such as a PDAPP mouse was immunized with Aβ, antibodies against Aβ were produced and amyloid deposition was suppressed (Nature (1999) vol. 400 p173-177). Such vaccine therapy is effective because the produced anti-Aβ antibody reacts with the senile plaque in the brain microglia through the blood-brain barrier, and the microglia senile plaque is phagocytosed via the Fc receptor and disappears. It is estimated that. Although this vaccine therapy has gone to the clinical experimental stage, it has not yet been put into practical use due to the disadvantage that an inflammatory reaction is observed. The vaccine therapy is described in detail, for example, in a review by Hariya et al. (Clinical Neuroscience (2002) vol. 20 no. 6 p706-707: Non-patent document 3).

  The blood-brain barrier also plays a role in regulating the equilibrium of Aβ concentration in the blood and in the brain. By administering the Aβ vaccine, the antibody against Aβ and Aβ bind in the blood, It is thought that the balance of excretion at the blood brain barrier is greatly inclined toward the blood side. Such a change in equilibrium promotes the release of Aβ from the brain, and the amount of Aβ in the brain decreases and the amount of Aβ in the blood increases. Thus, the sink hypothesis that drainage through the blood-brain barrier is promoted in vaccine therapy (DeMattos RB et al., Proc Natl Acad Sci USA (2001) vol.98 no.15 p8850-8855: Non Patent document 4) has also been proposed, which is a more powerful idea than the hypothesis of phagocytosis of microglia.

Reiko Wakabayashi, Takeshi Iwatsubo “How far has Alzheimer's disease been elucidated?” Clinical Neuroscience (2002) vol.20 no.6 p626-629 Schenk D. et al., Immunization with amyloid-β attenuates Alzheimer disease like pathology in the PDAPP mouse.Nature (1999) vol.400 p173-177 Yasuo Hariya “Vaccine Therapy” Clinical Neuroscience (2002) vol.20 no.6 p706-707 DeMattos RB et al., Proc Natl Acad Sci USA (2001) vol.98 no.15 p8850-8855

  Therefore, an object of the present invention is to provide a technique that contributes to the development of a new method for treating and preventing Alzheimer's disease that is more effective and safer than vaccine therapy.

  In order to solve the above-mentioned problems, the present inventor has focused on a proteomic technique that has been remarkably advanced in recent years, and comprehensively determined a group of proteins that bind to Aβ using the technique. Knowledge of these protein groups greatly contributes to elucidation of the cause of Alzheimer's disease, and opens up the possibility of developing therapeutic drugs targeting the protein group.

  According to the present invention, a protein group that binds to Aβ is comprehensively provided using proteomics technology. The knowledge of these protein groups opens up the possibility of developing therapeutic drugs targeting the protein groups and is considered to contribute greatly to elucidating the causes of Alzheimer's disease.

  In recent years, a mechanism has been proposed in which Aβ is discharged from the brain into the circulating blood by transport through the blood-brain barrier (BBB). Considering the above amyloid hypothesis, it is considered important to prevent the deposition of Aβ in order to treat and prevent Alzheimer's disease. Some proteins contribute to the mechanism by which Aβ is excreted out of the brain by binding to Aβ, and thus Aβ-binding proteins are thought to be related to the regulation of Aβ levels in the brain. In view of this, it is possible that the deposition of Aβ may be prevented by an approach that targets a protein capable of binding to Aβ and inhibits the interaction with the Aβ-Aβ binding protein.

  In Alzheimer's disease, neurodegeneration is observed, but the relationship between Aβ accumulation and neurodegeneration is still unclear. Also. The mechanism by which Aβ deposition occurs has not yet been clarified. However, it is highly likely that Aβ and binding proteins play some role in these phenomena. From this point of view, it is considered significant to inhibit the interaction between Aβ and Aβ-binding protein in the treatment and prevention of Alzheimer's disease.

  In recent years, a new proteomic technology has been developed that automatically connects a liquid chromatography (LC), a mass spectrometer (MS), and a data analysis system to automatically measure a large amount of protein. Proteomics means that the entire protein (proteome), which is the final product of genome information, is analyzed on a large scale. In the conventional technique, it is considered that it is indispensable to first isolate the target protein in order to identify the protein. For this reason, it is necessary to separate the protein by, for example, electrophoresis.

  Such a method is still an important method for identifying proteins, but it is based on LC-MS / MS and other high-performance protein identification technologies based on the accumulation of genome information and MS innovation. Was created. LC-MS is a device in which LC and MS are connected in series. When a tandem mass spectrometer (MS / MS) is connected to LC, sample fracture analysis in MS (LC-MS / MS) Is possible.

  In proteomics technology using LC-MS / MS, components of protein mixtures and complex complexes consisting of multiple proteins can be directly identified without digestion with a highly specific protease such as trypsin and without separation. . The basis of identification here is the exact mass value of the digested peptide fragment and its cleavage fragment, the terminal amino acid information depending on the protease used, and the PC and algorithm analysis that process it at high speed. This identification method is called the sequence tag method, and it is possible to identify a specific protein based on peptide information. Existing base sequence information including fragmentary information such as EST (expression sequence tag) Are all available. In proteosome research, LC-MS / MS is widely used for protein identification by the sequence tag method. Such proteomics technology is characterized by high throughput compared to conventional methods. For the technology of a large-scale protein analysis system using proteomics, see, for example, a review by Taoka et al. (Experimental Medicine "Proteomics Large-scale Analysis System for Intracellular Functional Complex Analysis" Vol.20 No1. (2002) p8-12 ) And so on.

  Such proteomic technology has many possibilities, but until now, Aβ-binding proteins have not been comprehensively identified using proteomic technology. In the present invention, a number of Aβ-binding proteins have been identified using such a new technique, but the findings of the present invention are useful for the development of new therapeutic agents for Alzheimer's disease and for elucidating the mechanism of Alzheimer's disease. I think that the. Therefore, in the present invention, the Aβ binding protein comprehensively means a group of proteins having the ability to bind Aβ. In the present specification, “generic identification” refers to the concept of identifying a complex consisting of a large number of proteins using proteomics technology without separating them. It is intended to be distinguished from the conventional technique of performing.

  In the following examples, Aβ-binding proteins were identified in proteins derived from mouse brain capillary endothelial cells and cerebral membrane. Crude membrane protein was prepared from the above sample, and Aβ40 was bound to Aβ-binding protein in the crude membrane protein using biotinylated Aβ40, followed by affinity cross-linking. After solubilizing the cross-linked sample, streptavidin beads were added to form a biotinylated Aβ40 and avidin complex. Proteins were separated by electrophoresis, and proteins contained in each obtained band were identified by LC-MS / MS. As a result, in the following Examples, a protein group that binds to Aβ40 was identified. These proteins interact with Aβ40 and are thought to be candidates for factors involved in Aβ transport at the blood-brain barrier.

  The proteins listed in the list below are suitable as the Aβ binding protein of the present invention. However, proteins that bind to Aβ may exist in addition to the proteins described in this list, and the mode of using these other proteins is not excluded in the present invention. In the following list, the Aβ binding proteins identified in the examples are shown together with the registered NCBI numbers.

(1) Plasma membrane Ca ²⁺ -ATPase: NCBI No. 6753140
(2) ATPase, Na ⁺ / K ⁺ transporter alpha (ATPase, Na ⁺ / K ⁺ transporting, alpha): NCBI number 16307541, NCBI number 15488862
(3) Neural cell adhesion molecule 1, 180 kDa isoform precursor (N-CAM 180): NCBI No. 400402
(4) Contactin associated protein: NCBI number 7949100, NCBI number 20347955
(5) Contactin: NCBI No. 6880954
(6) N-ethylmaleimide sensitive fusion protein: NCBI number 20347659, NCBI number 20913355
(7) Neurochondrin: NCBI number 16877778, NCBI number 4512261
(8) Synapsin: NCBI number 7305533, NCBI number 18606446
(9) Heat shock 70KD protein: NCBI number 11612489, NCBI number 20826946
(10) dermcidin: NCBI number 16751921
(11) Alpha-S1 casein precursor: NCBI number 115646
(12) RTN4: NCBI number 23379817, NCBI number 21898577
(13) Ankyrin binding cell adhesion molecule neurofascin [Rattus norvegicus]: NCBI number 1842429
(14) 150-kDa oxygen regulated protein: NCBI number 12831229
(15) Ionotropic glutamate receptor (glutamate receptor, ionotropic): NCBI number 8393313, NCBI number 20872260
(16) Actinin alpha: NCBI number 11230802, NCBI number 21307732
(17) ATPase, H ⁺ / K ⁺ transporter alpha ATPase, H ⁺ / K ⁺ transporting, alpha: NCBI number 9055170, NCBI number 20149728
(18) Dynamin: NCBI number 487855, NCBI number 21961254
(19) Dipeptidyl aminopeptidase-like protein: NCBI number 4038348, NCBI number 22537715
(20) Adapter-related protein complex: NCBI number 20347571, NCBI number 6667563
(21) alpha glucosidase 2, alpha neutral: NCBI number 6679891
(22) pyruvate kinase: NCBI number 20890302, NCBI number 16716633
(23) Atlastin: NCBI number 20909780, NCBI number 19923445
(24) Calcium / calmodulin-dependent protein kinase: NCBI number 6671660, NCBI number 18158420
(25) Tubulin alpha: NCBI number 20893179, NCBI number 6575901
(26) Synaptotagmin: NCBI number 6678197
(27) Catalase: NCBI No. 20857278, NCBI No. 6675272
(28) Tweety (Drosophila) homolog 2: NCBI number 20847934
(29) Protein disulfide-isomerase: NCBI No. 20913929
(30) Aldolase: NCBI number 7548322
(31) Guanine nucleotide binding protein: NCBI number 6680035, NCBI number 6675408
(32) Actin (actin): NCBI number 6675295, NCBI number 20107732, NCBI number 20818844
(33) cAMP-dependent protein kinase catalytic subunit: NCBI number 200367, NCBI number 7110693
(34) Tropomodulin: NCBI number 20890948, NCBI number 7710102
(35) Vacuolar ATP synthase: NCBI number 12585525
(36) Septin: NCBI number 6768563, NCBI number 6675816
(37) Acetyl-Coenzyme A acyltransferase: NCBI number 20896269, NCBI number 20342350
(38) glutamate oxaloacetate transaminase: NCBI number 20889846, NCBI number 90313
(39) Glutamine synthetase: NCBI number 483918, NCBI number 20825772
(40) Glyceraldehyde-3-phosphate dehydrogenase: NCBI number 20897061, NCBI number 20829889
(41) Ribosomal protein L6: NCBI No. 14210106, NCBI No. 6753554
(42) Synaptophysin: NCBI number 20830010, NCBI number 6678195
(43) Capping protein alpha: NCBI number 6667172, NCBI number 12842248
(44) Lactate dehydrogenase: NCBI No.6678674
(45) ATP-ase, H ⁺ transporter ^{(ATPase, H + transporting):} NCBI number 20886367, NCBI No. 15029719
(46) RIKEN cDNA 1010001N11; NADH-ubiquinone oxidoreductase 39KDA subunit precursor (RIKEN cDNA 1010001N11; NADH-UBIQUINONE OXIDOREDUCTASE 39 KDA SUBUNIT PRECURSOR)): NCBI number 20832556, NCBI number 13384720
(47) Acidic ribosomal phosphoprotein PO (NCBI number 6671569, NCBI number 13277927)
(48) RIKEN cDNA 2610020H15 (RIKEN cDNA 2610020H15): NCBI number 20883070
(49) Dimethylarginine dimethylaminohydrolase: NCBI No. 20878745
(50) ATP synthase, H ⁺ transporter mitochondrial F1 complex ^{(ATP synthase, H + transporting mitochondrial} F1 complex): NCBI Number 7,949,003, NCBI No. 6,680,748
(51) Excitatory amino acid transporter: NCBI number 20908477
(52) tubulin, beta: NCBI number 12963615, NCBI number 13324679
(53) 3-oxoacid CoA transferase: NCBI number 18266680
(54) Heterogeneous nuclear ribonucleoprotein G: NCBI number 23274033, NCBI number 20885374
(55) Creatine kinase: NCBI number 10946574
(56) Interleukin enhancer binding factor: NCBI number 13385872
(57) N-myc downstream: NCBI number 7305305
(58) 2 ', 3'-cyclic-nucleotide 3'-phosphodiesterase: NCBI number 309178
(59) Glyceraldehyde 3-phosphate dehydrogenase: NCBI number 20888929, NCBI number 20828351
(60) Maleate dehydrogenase: NCBI No. 6679916
(61) ATP synthase, H ⁺ transporter ^{(ATP synthase, H + transporting)} : NCBI Number 6,680,748
(62) ATP-ase, H ⁺ transporter ^{(ATPase, H + transporting):} NCBI number 19527064, NCBI No. 1,184,661
(63) dermcidin precursor
(64) Na, K-ATPase alpha-1 subunit: NCBI number 205632
(65) ATPase alpha 2, Na / K: NCBI number 358960
(66) (NM_022030) Synaptic vehicle glycoprotein 2a (NCBI number 358960)
(67) (XM_125679) similar to hexokinase 1, isoform HKI-R; brain form hexokinase (NCM number 20860670)
(68) Vascular adenosine triphosphatase subunit B (NCBI number 1184661)
(69) (NM_007505) ATP synthase, H ⁺ transporter, mitochondrial F1 complex, alpha subunit isoform 1 ((NM_007505) ATP synthase, H + transporting, mitochondrial F1 complex, alpha subunit, isoform 1): NCBI Number 6680748
(70) glutamate / aspartate transporter: NCBI number 913796
(71) 2 ', 3'-cyclic-nucleotide 3'-phosphodiesterase, gi | 279547 (2', 3'-cyclic-nucleotide 3'-phosphodiesterase, gi | 279547): NCBI number 279547
(72) actin, gi | 20894101 (actin, gi | 20894101): NCBI number 2089410
(73) Similar to GLUTAMINE SYNTHETASE: NCBI number 20825772
(74) cAMP-dependent protein kinase catalytic subunit: NCBI number 8568079, NCBI number 6755076
(75) Guanine nucleotide binding protein: NCBI No. 6754012
(76) Contactin 1: NCBI number 6880954
(77) ATP synthase, H ⁺ transporter mitochondrial F1 complex ^{(ATP synthase, H + transporting mitochondrial} F1 complex): NCBI Number 7,949,003
(78) Glucose phosphate isomerase 1 complex: NCBI number 20825753
(79) protein kinase, cAMP dependent regulatory type II beta (NCBI number 20847768)
(80) Similar to NEUROTRIMIN PRECURSOR (GP65): NCBI number 20884317
(81) Hypothetical protein XP_166357: NCBI number 20556253
(82) actin: NCBI number 20894101
(83) 2 ', 3'-cyclic-nucleotide 3'-phosphodiesterase: NCBI number 279547
(84) Similar to GLUTAMINE SYNTHETASE: NCBI number 20825772
^{(85) Na + / K +} - exchanged ATP-ase (EC 3.6.1.37) beta-2 chain ^{(Na + / K + -exchanging ATPase} (EC 3.6.1.37) beta-2 chain): NCBI Number 91127
(86) SRC substrate cortactin-like protein (similar to SRC SUBSTRATE CORTACTIN): NCBI No. 20895255
(87) Glyceraldehyde 3-phosphate dehydrogenase-like protein (GAPDH): similar to Glyceraldehyde 3-phosphate dehydrogenase (GAPDH): NCBI number 20343993
(88) capping protein alpha 2 (NCBI number 12842248)
(89) ATP synthase, H ⁺ transporter mitochondrial complex ^{(ATP synthase, H + transporting mitochondrial} complex): NCBI Number 11602916
(90) Ribosomal protein L6 (NCBI number 11602916)
(91) RIKEN cDNA 1010001N11 (RIKEN cDNA 1010001N11): NCBI number 13384720
(92) N-ethylmaleimide sensitive fusion protein attachment protein alpha: NCBI No. 13385392
(93) Voltage-dependent anion channel 1 (NCBI number 6675596)
(94) Syntaxin: NCBI number 6981600
(95) Similar to TRANSCRIPTION FACTOR E2F2 (E2F-2): NCBI number 20848841
(96) Guanine nucleotide-binding protein: NCBI No. 13937391

In addition, although the method employ | adopted in the Example is a suitable method for the objective of extract | collecting A (beta) binding protein, another A (beta) binding protein can also be identified by changing suitably according to the objective. For example, the following reagents can be used as a cross-linking reagent for performing crosslinking, but the cross-linking reagent is not limited thereto.
(1) disuccinimidyl suberate (DSS)
(2) 1,6-Bismaleimidohexane (BMH)
(3) 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)
(4) N- [g-Maleimidobutyryloxy] succinimide ester (GMBS)
(5) Succinimidyl 4- [p-maleimidophenyl] butyrate (SMPB)
(6) beta-maleimidopropionic acid (BMPA)
(7) 1,5-difluoro-2,4-dinitrobenzene (DFDNB)

  In the present invention, affinity cross-linking is used for the purpose of fixing the three-dimensional structure generated between Aβ40 and Aβ-binding protein. The distance between them (spacer) is different. Given that, it is possible to identify other Aβ binding proteins by varying the various cross-linking reagents.

  In the Examples, proteins that bind to Aβ40 were identified, but many of them are considered to bind to Aβ42. However, as described in [0005], there are some differences between Aβ40 and Aβ42. Therefore, when Aβ42 is used, other Aβ-binding proteins can be obtained, and Aβ42-binding proteins also provide useful knowledge regarding the prevention and treatment of Alzheimer's disease. Therefore, a protein that binds to Aβ42 is not excluded from the scope of the present invention.

  The present invention provides a method for screening a substance that inhibits the binding between Aβ and Aβ-binding protein using Aβ-binding protein. As mentioned above, in Alzheimer's disease, there are senile plaques where Aβ accumulates locally in the brain. Since the core protein is required for Aβ aggregation to occur, the obtained binding inhibitor may exert the effect of inhibiting senile plaque formation. Thus, since the binding between Aβ and Aβ-binding protein is strongly correlated with the pathological condition, the activity of inhibiting the formation of senile plaques can be determined by assaying the binding between Aβ and Aβ-binding protein by the usual method. A therapeutic drug for Alzheimer's disease can be screened.

  As already mentioned, nerve loss occurs in Alzheimer's disease, which is the biggest cause of dementia. There is still debate about how Aβ degenerates the nerve, but by using a binding inhibitor to suppress the binding between Aβ and Aβ binding protein to prevent Aβ deposition, There is also the possibility of suppressing neurotoxicity.

  Screening for a substance that inhibits the binding between Aβ and Aβ-binding protein can be performed, for example, using a binding assay method widely used in this technical field. Specifically, when the Aβ-binding protein is a membrane protein, a gene encoding the protein is introduced into the cell and expressed on the cell surface, and the cell expressing Aβ-binding protein and Aβ By reacting, the bond between the two can be measured. In this case, if the compound to be screened is added to the reaction system and Aβ is labeled with radioactivity or the like, the ability of the compound to inhibit binding can be evaluated.

  When the Aβ binding protein is a soluble protein, the tag is obtained by synthesizing the binding protein in an in vitro cell-free system, for example, with the tag attached, and purifying the binding protein with an anti-tag antibody. An added binding protein can be obtained. Thereafter, the compound to be screened is reacted with the purified binding protein and Aβ. By performing immunoprecipitation with an anti-tag antibody, the amount of Aβ co-precipitated with the binding protein can be measured, for example, by Western blot.

  The interaction between the Aβ-binding protein immobilized on the gold thin film and other molecules can also be measured using a machine. Specifically, an Aβ binding protein is immobilized on a gold thin film, then Aβ is added to bind to the binding protein, and a compound to be screened is added. By measuring the dissociation from the Aβ binding protein at this time, the binding inhibition ability of the compound can be evaluated. An apparatus for measuring the interaction between molecules immobilized on a gold thin film and other molecules using surface plasmo resonance is commercially available, and can be used for this purpose. The method using such a machine has an advantage that a large number of specimens can be easily processed.

  An apparatus for measuring the interaction between molecules by vibration of quartz is also commercially available. By fixing the Aβ binding protein on the crystal resonator using the apparatus, the binding inhibition ability of the compound to be screened can be evaluated using the same method as described above.

    Furthermore, a compound that promotes or suppresses the expression of the Aβ binding protein of the present invention can be screened. In vaccine therapy, the presence of antibodies to Aβ in the blood is thought to decrease the level of Aβ in the brain because Aβ is excreted through the blood-brain barrier. In this excretion mechanism, Aβ binding protein and Aβ bind to each other and Aβ is excreted to the blood side by transcytosis, which may promote Aβ excretion. Therefore, by inducing the expression of Aβ binding protein or by promoting the transport function of Aβ binding protein, Aβ elimination ability equivalent to or higher than Aβ vaccine therapy using Aβ antibody can be imparted. there is a possibility. Since this method does not use an immune reaction, unlike vaccine therapy, there is little risk of an inflammatory reaction or the like. Therefore, the Aβ-binding protein of the present invention may provide a new treatment method for Alzheimer's disease that can replace vaccine therapy.

  To screen for compounds that change the expression of Aβ-binding protein, the genome sequence of the binding protein gene is searched and identified from the genome database, and reporter genes such as luciferase and galactosidase are connected to the transcription start site around 5 kbp. Introduce into cultured cells. Since there is a transcriptional control site around the transcription initiation site, the target compound can be screened effectively by measuring the influence of the compound on the expression of the intracellular reporter gene.

  It is also possible to prepare a kit containing the Aβ binding protein of the present invention. The kit is a kit containing one or a plurality of Aβ-binding proteins and labeled Aβ, and preferably also contains a buffer whose salt strength and pH are appropriately adjusted. In this kit, Aβ is labeled in order to detect binding to the binding protein. The labeling method is not particularly limited, and includes various techniques known in the art such as radiolabeling, fluorescent labeling, enzyme labeling, biotin labeling, 2,4-dinitrophenol labeling, and the like. The kit can be used for ordinary binding assays and the like for screening substances that inhibit the binding between Aβ and binding protein. In this kit, it is a preferred embodiment of the present invention to include not only one but also a large number of Aβ binding proteins, and it is particularly preferable to include a plurality of binding proteins having different properties such as membrane proteins and soluble proteins.

  The following examples further illustrate the invention, but are not intended to limit the scope of the invention in any way.

The amyloid β and the biotinylation reagent used in this example are as follows.
Aβ40: DAEFRHDSGY EVHHQKLVFF AEDVGSNKGA IIGLMVGGVV
MW = 4312
Biotinylation reagent: Sulfo-NHS-LC-biotinylation kit (PIERCE)

(Biotinylation of Aβ40)
An aqueous solution of Aβ and an aqueous solution of biotinylation reagent were prepared, and an excess amount of biotinylation reagent was added to Aβ. Thereafter, the biotinylation was carried out by leaving it on an ice bath for 2 hours or at room temperature for 30 minutes. Furthermore, the unreacted biotinylation reagent was removed by the column, and the amount of biotin introduced by the HABA assay was measured.

(Preparation of mouse brain capillary rich membrane protein sample)
Mice were sacrificed by cervical dislocation, the brain was removed, and the cerebrum was isolated (one mouse = 0.3 g). Phosphate buffer was added and homogenized 10 times on ice, dextran solution was added and further homogenized. The homogenate was centrifuged at 4500 × g for 16 minutes (4 ° C.) to remove the upper fat layer and the supernatant. Buffer was added to the pellet for suspension, and the cells were disrupted by the nitrogen cavitation method (800 psi, 30 minutes, 4 ° C.). The suspension was centrifuged at 10,000 × g for 16 minutes (4 ° C.), and the supernatant was further centrifuged at 100,000 × g (40,200 rpm) for 65 minutes (4 ° C.). The suspension buffer was added to the pellet and pipetted, and a protease inhibitor was added to prepare a sample.

(Preparation of mouse cerebral membrane protein sample)
Mice were sacrificed by cervical dislocation, the brain was removed, and the cerebrum was isolated (one mouse = 0.3 g). Density gradient buffer was added and homogenized 10 times on ice in the presence of protease inhibitors. Cells were disrupted by the nitrogen cavitation method (800 psi, 30 minutes, 4 ° C.), and the suspension was placed in a tube and centrifuged (4 ° C.) at 10,000 × g for 16 minutes. Further, the supernatant was centrifuged at 100,000 × g (40,200 rpm) for 65 minutes (4 ° C.). A sample was prepared by adding 600 uL of suspension buffer to the pellet and pipetting, and adding a protease inhibitor.

(Binding to Aβ40)
Biotinylated Aβ was added to the sample so that the final concentration of Aβ was approximately 10 uM. The binding buffer was added so that the total volume was 1.2 mL, and the mixture was stirred and inverted, and left at 25 ° C. for 20 to 30 minutes.

(Cross-crosslinking reaction and protein solubilization)
A cross-linking reagent such as DSS or BMH was dissolved in dimethyl sulfoxide and allowed to stand at 25 ° C. for 30 minutes to carry out Aβ40 cross-linking reaction. Centrifugation was performed at 15,000 rpm for 5 minutes (4 ° C), solubilization buffer and protease inhibitor were added to the centrifuged pellet, pipetting, and then solubilized by shaking overnight (4 ° C) at 400 rpm.

(Purification of Aβ-protein complex)
Streptavidin beads were added to the cross-crosslinked sample. Shake (400C) for 1 hour at 400 rpm to bind biotin and avidin. The resulting complex was obtained by centrifuging at 15,000 rpm for 3 minutes (4 ° C) and removing the supernatant. A sample for electrophoresis was prepared by adding SDS-PAGE sample buffer to the centrifuged pellet and heating at 95 ° C. for 2 min.

(Electrophoresis)
Preparation of electrophoresis (sodium lauryl sulfate polyacrylamide gel electrophoresis: SDS-PAGE) was performed by setting acrylamide gel in the electrophoresis tank and filling the electrophoresis tank with the electrophoresis buffer. Samples were denatured as needed (95 ° C., 3 minutes) and applied to the gel. Samples were electrophoresed under a constant current condition of 0.04 A (voltage was 100 V to 200 V). The gel after electrophoresis was subjected to silver staining by a usual method.

(Mass spectrometry)
The band obtained by the above electrophoresis was cut out, and each band was analyzed by LC-MS / MS to identify a molecule binding to Aβ. As a specific process, Aβ-binding protein was identified by performing a database search based on data obtained by MS / MS measurement. In many cases, a plurality of protein names can be obtained from one MS / MS data, and one of the plurality of proteins having the largest molecular weight was selected. Table 1 shows the peptide fragments detected from each band and the proteins identified from the sequence of the fragments.

  Among the above-mentioned Aβ binding proteins, Guanine nucleotide binding protein (G-protein) is a factor involved in intracellular signal transduction by coupling with a receptor. Given that G-protein plays such a role, it plays an important role in the uptake mechanism of substances bound to receptors. Therefore, G-protein may be involved in the cellular uptake of Ab.

  Synaptic vesicle glycoprotein 2a (SV2a) has a 12-transmembrane transporter-like structure, which may be involved in the transport of Ab. In addition, SV2a knockout mice cause severe convulsions and die immediately after birth, and are thought to play an important role in central nervous function.

  Neural cell adhesion molecule 1 (N-CAM180) has been reported to decrease the expression level of mRNA in dementia diseases such as Alzheimer's disease, suggesting an association with Alzheimer's disease. N-CAM180 is a factor necessary for synaptic binding, and the function of N-CAM180 may be inhibited by interaction with Ab.

  N-ethylmaleimid sensitive fusion protein (NSF), N-ethylmaleimide sensitive fusion protein attachment protein alpha (SNAP, NAPA), and synaptophysin, syntaxin are molecules involved in the regulation of vesicular membrane transport. Therefore, there is a possibility that it is involved in the intracellular transport of Ab or the intracellular transport is inhibited by binding of Ab.

  According to the present invention, a protein group that binds to Aβ is comprehensively provided using proteomics technology. The knowledge of these protein groups opens up the possibility of developing therapeutic drugs targeting the protein groups and is considered to contribute greatly to elucidating the causes of Alzheimer's disease.

Claims (2)

A method of screening for a substance that inhibits the interaction between amyloid β and amyloid β-binding protein using amyloid β-binding protein. The method according to claim 1, wherein the amyloid β-binding protein is a protein selected from the following protein group to which NCBI numbers are assigned.
(1) Plasma membrane Ca ²⁺ -ATPase: NCBI No. 6753140
(2) ATPase, Na ⁺ / K ⁺ transporter alpha (ATPase, Na ⁺ / K ⁺ transporting, alpha): NCBI number 16307541, NCBI number 15488862
(3) Neural cell adhesion molecule 1, 180 kDa isoform precursor (N-CAM 180): NCBI No. 400402
(4) Contactin associated protein: NCBI number 7949100, NCBI number 20347955
(5) Contactin: NCBI No. 6880954
(6) N-ethylmaleimide sensitive fusion protein: NCBI number 20347659, NCBI number 20913355
(7) Neurochondrin: NCBI number 16877778, NCBI number 4512261
(8) Synapsin: NCBI number 7305533, NCBI number 18606446
(9) Heat shock 70KD protein: NCBI number 11612489, NCBI number 20826946
(10) dermcidin: NCBI number 16751921
(11) Alpha-S1 casein precursor: NCBI number 115646
(12) RTN4: NCBI number 23379817, NCBI number 21898577
(13) Ankyrin binding cell adhesion molecule neurofascin [Rattus norvegicus]: NCBI number 1842429
(14) 150-kDa oxygen regulated protein: NCBI number 12831229
(15) Ionotropic glutamate receptor (glutamate receptor, ionotropic): NCBI number 8393313, NCBI number 20872260
(16) Actinin alpha: NCBI number 11230802, NCBI number 21307732
(17) ATPase, H ⁺ / K ⁺ transporter alpha ATPase, H ⁺ / K ⁺ transporting, alpha: NCBI number 9055170, NCBI number 20149728
(18) Dynamin: NCBI number 487855, NCBI number 21961254
(19) Dipeptidyl aminopeptidase-like protein: NCBI number 4038348, NCBI number 22537715
(20) Adapter-related protein complex: NCBI number 20347571, NCBI number 6667563
(21) alpha glucosidase 2, alpha neutral: NCBI number 6679891
(22) pyruvate kinase: NCBI number 20890302, NCBI number 16716633
(23) Atlastin: NCBI number 20909780, NCBI number 19923445
(24) Calcium / calmodulin-dependent protein kinase: NCBI number 6671660, NCBI number 18158420
(25) Tubulin alpha: NCBI number 20893179, NCBI number 6575901
(26) Synaptotagmin: NCBI number 6678197
(27) Catalase: NCBI No. 20857278, NCBI No. 6675272
(28) Tweety (Drosophila) homolog 2: NCBI number 20847934
(29) Protein disulfide-isomerase: NCBI No. 20913929
(30) Aldolase: NCBI number 7548322
(31) Guanine nucleotide binding protein: NCBI number 6680035, NCBI number 6675408
(32) Actin (actin): NCBI number 6675295, NCBI number 20107732, NCBI number 20818844
(33) cAMP-dependent protein kinase catalytic subunit: NCBI number 200367, NCBI number 7110693
(34) Tropomodulin: NCBI number 20890948, NCBI number 7710102
(35) Vacuolar ATP synthase: NCBI number 12585525
(36) Septin: NCBI number 6768563, NCBI number 6675816
(37) Acetyl-Coenzyme A acyltransferase: NCBI number 20896269, NCBI number 20342350
(38) glutamate oxaloacetate transaminase: NCBI number 20889846, NCBI number 90313
(39) Glutamine synthetase: NCBI number 483918, NCBI number 20825772
(40) Glyceraldehyde-3-phosphate dehydrogenase: NCBI number 20897061, NCBI number 20829889
(41) Ribosomal protein L6: NCBI No. 14210106, NCBI No. 6753554
(42) Synaptophysin: NCBI number 20830010, NCBI number 6678195
(43) Capping protein alpha: NCBI number 6667172, NCBI number 12842248
(44) Lactate dehydrogenase: NCBI No.6678674
(45) ATP-ase, H ⁺ transporter ^{(ATPase, H + transporting):} NCBI number 20886367, NCBI No. 15029719
(46) RIKEN cDNA 1010001N11; NADH-ubiquinone oxidoreductase 39KDA subunit precursor (RIKEN cDNA 1010001N11; NADH-UBIQUINONE OXIDOREDUCTASE 39 KDA SUBUNIT PRECURSOR)): NCBI number 20832556, NCBI number 13384720
(47) Acidic ribosomal phosphoprotein PO (NCBI number 6671569, NCBI number 13277927)
(48) RIKEN cDNA 2610020H15 (RIKEN cDNA 2610020H15): NCBI number 20883070
(49) Dimethylarginine dimethylaminohydrolase: NCBI No. 20878745
(50) ATP synthase, H ⁺ transporter mitochondrial F1 complex ^{(ATP synthase, H + transporting mitochondrial} F1 complex): NCBI Number 7,949,003, NCBI No. 6,680,748
(51) Excitatory amino acid transporter: NCBI number 20908477
(52) tubulin, beta: NCBI number 12963615, NCBI number 13324679
(53) 3-oxoacid CoA transferase: NCBI number 18266680
(54) Heterogeneous nuclear ribonucleoprotein G: NCBI number 23274033, NCBI number 20885374
(55) Creatine kinase: NCBI number 10946574
(56) Interleukin enhancer binding factor: NCBI number 13385872
(57) N-myc downstream: NCBI number 7305305
(58) 2 ', 3'-cyclic-nucleotide 3'-phosphodiesterase: NCBI number 309178
(59) Glyceraldehyde 3-phosphate dehydrogenase: NCBI number 20888929, NCBI number 20828351
(60) Maleate dehydrogenase: NCBI No. 6679916
(61) ATP synthase, H ⁺ transporter ^{(ATP synthase, H + transporting)} : NCBI Number 6,680,748
(62) ATP-ase, H ⁺ transporter ^{(ATPase, H + transporting):} NCBI number 19527064, NCBI No. 1,184,661
(63) dermcidin precursor
(64) Na, K-ATPase alpha-1 subunit: NCBI number 205632
(65) ATPase alpha 2, Na / K: NCBI number 358960
(66) (NM_022030) Synaptic vehicle glycoprotein 2a (NCBI number 358960)
(67) (XM_125679) similar to hexokinase 1, isoform HKI-R; brain form hexokinase (NCM number 20860670)
(68) Vascular adenosine triphosphatase subunit B (NCBI number 1184661)
(69) (NM_007505) ATP synthase, H ⁺ transporter, mitochondrial F1 complex, alpha subunit isoform 1 ((NM_007505) ATP synthase, H + transporting, mitochondrial F1 complex, alpha subunit, isoform 1): NCBI Number 6680748
(70) glutamate / aspartate transporter: NCBI number 913796
(71) 2 ', 3'-cyclic-nucleotide 3'-phosphodiesterase, gi | 279547 (2', 3'-cyclic-nucleotide 3'-phosphodiesterase, gi | 279547): NCBI number 279547
(72) actin, gi | 20894101 (actin, gi | 20894101): NCBI number 2089410
(73) Similar to GLUTAMINE SYNTHETASE: NCBI number 20825772
(74) cAMP-dependent protein kinase catalytic subunit: NCBI number 8568079, NCBI number 6755076
(75) Guanine nucleotide binding protein: NCBI No. 6754012
(76) Contactin 1: NCBI number 6880954
(77) ATP synthase, H ⁺ transporter mitochondrial F1 complex ^{(ATP synthase, H + transporting mitochondrial} F1 complex): NCBI Number 7,949,003
(78) Glucose phosphate isomerase 1 complex: NCBI number 20825753
(79) protein kinase, cAMP dependent regulatory type II beta (NCBI number 20847768)
(80) Similar to NEUROTRIMIN PRECURSOR (GP65): NCBI number 20884317
(81) Hypothetical protein XP_166357: NCBI number 20556253
(82) actin: NCBI number 20894101
(83) 2 ', 3'-cyclic-nucleotide 3'-phosphodiesterase: NCBI number 279547
(84) Similar to GLUTAMINE SYNTHETASE: NCBI number 20825772
^{(85) Na + / K +} - exchanged ATP-ase (EC 3.6.1.37) beta-2 chain ^{(Na + / K + -exchanging ATPase} (EC 3.6.1.37) beta-2 chain): NCBI Number 91127
(86) SRC substrate cortactin-like protein (similar to SRC SUBSTRATE CORTACTIN): NCBI No. 20895255
(87) Glyceraldehyde 3-phosphate dehydrogenase-like protein (GAPDH): similar to Glyceraldehyde 3-phosphate dehydrogenase (GAPDH): NCBI number 20343993
(88) capping protein alpha 2 (NCBI number 12842248)
(89) ATP synthase, H ⁺ transporter mitochondrial complex ^{(ATP synthase, H + transporting mitochondrial} complex): NCBI Number 11602916
(90) Ribosomal protein L6 (NCBI number 11602916)
(91) RIKEN cDNA 1010001N11 (RIKEN cDNA 1010001N11): NCBI number 13384720
(92) N-ethylmaleimide sensitive fusion protein attachment protein alpha: NCBI No. 13385392
(93) Voltage-dependent anion channel 1 (NCBI number 6675596)
(94) Syntaxin: NCBI number 6981600
(95) Similar to TRANSCRIPTION FACTOR E2F2 (E2F-2): NCBI number 20848841
(96) Guanine nucleotide-binding protein: NCBI No. 13937391