KR20120030810A - Composition for preventing and treating amyloid diseases comprising cyclophilin b - Google Patents

Abstract

PURPOSE: A composition for treating or preventng amyloid disease comprising cyclophilin B is provided to form neural plate by being deposited with other substances, thereby effectively treating amyloid disease. CONSTITUTION: A composition for treating or preventng amyloid disease comprising cyclophilin B(CypB) includes protein. The CypB protein has amino acid sequence which is represented by sequence number 1. The composition for treating or preventng amyloid disease comprises nucleic acid having nucleotide sequence which codes the cyclophilin B (CypB) protein. The nucleic acid having the nucleotide sequence which codes the CypB protein has nucleotide sequence which codes the amino acid sequence of the sequence number 1. The nucleic acid having nucleotide sequence which codes the CypB protein has nucleotide sequence of sequence number 2. The nucleic acid having the nucleotide sequence which codes the CypB protein is a CypB protein expression recombinant vector. The CypB protein expression recombinant vector comprises nucleotide sequence which codes the amino acid sequence of the sequence number 1. The CypB protein expression recombinant vector comprises the nucleotide sequence of the sequence number 2.

Description

Composition for preventing and treating amyloid diseases comprising cyclophilin Ｂ

The present invention relates to a composition for treating or preventing amyloid diseases comprising a cyclophilin B (CypB) protein, a nucleic acid having a nucleotide sequence encoding a CypB protein or a CypB protein expressing recombinant vector.

Amyloids are a variety of specific protein deposits (either intracellular or extracellular) that appear in a number of different diseases, and despite their diversity in their occurrence, all amyloid deposits have common morphological properties and specific dyes (e.g. , Congo red), and shows a characteristic red rust birefringence pattern in polarization after staining. In addition, they share a common ultrafine structure and a common X-ray diffraction and infrared spectrum. Resistant to acids or alkalis and similar to glassy or colloidal, which, when deposited, leads to amyloid degeneration resulting in local and systemic amyloidosis.

Amyloid-β peptides (Aβ) are 39-43 amino acid peptides derived by proteolysis on large proteins known as beta amyloid precursor proteins (“βAPP”). KLVFFA, the 16-21 region of Aβ peptides, is responsible for the intermolecular interactions of Aβ during β-sheet formation and fibrogenesis (Tjernberg LO, et al., J. Biol. Chem. 272, 12601-05 (1997). Findeis, et al., Biochemistry 38, 6791-6800 (1999); Findeis, et al., Amyloid, 231-41 (Dec 2001).

Amyloid-β peptides deposit with other substances to form neural plaques, which in turn cause neurotoxic effects, resulting in amyloid diseases such as chronic inflammation, multiple myeloma, II It is known to cause type II diabetes, Creutzfeldt-Jakob, Alzheimer's disease, and the like.

Cyclophilins are one of the immunophillin families and are known for the first time as cell binding proteins for the immunosuppressive Cyclosporin A (CsA). Cyclophylline is classified into CypB, CypB, CypC, CypD, Cyp40 and the like, in particular, it is reported that there is peptidyl-proyl isomer (PPIase) activity. PPIase activity acts as molecular chaperones that promote protein folding, intracellular trafficking, and maintenance of multiprotein complex safety. Among cyclophilins, CypB is distinguished from others by the presence of signal sequences directed to the endoplasmic reticulum. CypB is found not only in the endoplasmic reticulum but also in the extracellular space and the nucleus. CypB, a 21-kDa protein comprising a 25-amino acid sequence directed to the endoplasmic reticulum, plays an important role in the endoplasmic reticulum response and protects cells from ER stress. CypA as well as CypB show higher content in neurons than glial cells.

However, no neuroprotective role of CypB has been found in amyloidogenic diseases caused by neurotoxicity due to amyloid-β peptide deposition.

Under this background, the present invention is one of the immunophilin families, the present invention by identifying cyclophilin B having PPIase activity found mainly in neurons, and confirming that it can be used for treating or preventing amyloidogenic diseases. Was completed.

One object of the present invention is to provide a composition for treating or preventing amyloid diseases comprising cyclophilin B (CypB) protein.

Another object of the present invention is to provide a composition for treating or preventing amyloidogenic diseases comprising a nucleic acid having a nucleotide sequence encoding a CypB protein.

It is another object of the present invention to provide a composition for treating or preventing amyloidogenic diseases comprising a CypB protein expressing recombinant vector.

Another object of the present invention is to administer a pharmacologically effective amount of a CypB protein, a nucleotide sequence encoding a CypB protein, or a CypB protein expressing recombinant vector to a subject in need of treating or preventing amyloidogenic diseases, thereby treating or preventing amyloidogenic diseases. To provide a way.

Still another object of the present invention is to provide a method for screening a drug for treating or preventing amyloidogenic diseases by assaying a substance that promotes expression of CypB protein.

In one embodiment, the present invention provides a composition for treating or preventing amyloid diseases, including cyclophilin B (CypB) protein.

An "amyloidosis" or "amyloidogenic disease" to be treated or prevented in accordance with the present invention refers to a pathological condition characterized by the presence of amyloid fibers. Amyloid is a generic term that refers to a diverse group of specific protein deposits (intracellular or extracellular) that appear in numerous different diseases. In particular, the amyloid-β peptide (Aβ) is a 39-43 amino acid peptide derived by proteolysis in a large protein known as beta amyloid precursor protein ("βAPP"). The "amyloidosis" or "amyloidogenic disease" of the present invention includes "amyloid-β disease" or "amyloid-β related disease". The term "amyloid-β disease" includes diseases, symptoms, pathologies, and other structural or dysfunctions of the brain and its components whose cause is amyloid, thereby including memory loss, facial or jitter symptoms. Specifically includes diseases caused by neurotoxicity due to amyloid-β peptide deposition. The brain region affected by amyloid-β disease may be a matrix comprising parenchyma, including the pulsed or functional or anatomical site, or the neuron itself. Amyloid-β diseases of the present invention may include age-related cognitive decline, mild cognitive impairment (“MCI”), vascular dementia, cerebral amyloid angiopathy (“CAA”), hereditary intracranial hemorrhage, senile dementia, Down syndrome, inclusion body myositis (“IBM "), Age-related macular degeneration (" ARMD "), chronic inflammation, multiple myeloma, type II diabetes, Creutzfeldt-Jakob, Alzheimer's disease disease). Amyloid-β peptides deposit with other substances to form neural plaques and thereby cause neurotoxic effects, leading to amyloid diseases.

Cerebral amyloid angiopathy (“CAA”) refers to the specific deposition of amyloid fibrils in the walls of arterioles, cortical and leptomingeal arteries, and in capillaries and veins. CAA can occur sporadically or be inherited. Clinically related to intracranial hemorrhage. Exemplary CAA disorders include, but are not limited to, hereditary intracranial hemorrhage (HCHWA-I) with Icelandic amyloidosis; Dutch modifications of HCHWA (HCHWA-D; mutations in Aβ); Flemish mutation of Aβ; Arctic mutation of Aβ; Italian mutations of Aβ; Iowa mutation of Aβ; Familial British dementia; And familial Danish dementia.

Age-related macular degeneration (“ARMD”) causes irreversible vision loss in older people.

Aβ _25-35 is an active virulence segment, in particular the levels of Aβ _1-42 are increased in serum or fibroblasts of brains of patients with amyloidogenic diseases, and the deposited Aβ _1-42 and Aβ _25-35 are isolated from the brain. Neurotoxicity was observed in both hippocampus and neurons.

CypB proteins used in the compositions of the present invention include all CypB proteins from yeast, plant or animal. Variants of CypB protein by deletion, insertion, non-conservative or conservative substitution, or combinations thereof, including wild type CypB proteins, which serve to protect cells from neurotoxicity induced by Aβ in the cell It includes. As one specific example, there may be mentioned CypB protein having the amino acid sequence of SEQ ID NO: 1, substitution, insertion, deletion variants of such a sequence can also be used in the composition of the present invention.

A variant of CypB protein refers to a protein having a sequence in which the amino acid sequence of the wild type and one or more amino acid residues are different. Insertion typically consists of a contiguous sequence of about 1 to 20 amino acids, although larger insertions may be possible. Deletion typically consists of about 1 to 30 residues, but in some cases larger deletions may also be possible, such as one of the domains may be deleted. Such variants can be prepared by chemical peptide synthesis methods known in the art or by recombinant methods based on DNA sequences (Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, USA, 2d Ed. , 1989). Amino acid exchanges in proteins and peptides that do not alter the activity of the molecule as a whole are known in the art (H. Neurode, R. L. Hill, The Proteins, Academic Press, New York, 1979). The most commonly occurring exchanges are amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Phe, Ala / Exchange between Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, Asp / Gly.

In addition, the CypB protein may be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation and the like in some cases.

The variant or modification may be a functional equivalent that exhibits the same biological activity as a natural protein, or may be a variant or modification that modifies the properties of the protein as needed. Preferably, the protein has increased structural stability against heat, pH, etc., or increased protein activity due to variation and modification on amino acid sequence.

CypB proteins can be obtained by extraction and purification in nature by methods well known in the art. Alternatively, since the sequence of the CypB protein is known, it can be obtained by chemical synthesis (Merrifleld, J. Amer. Chem. Soc .. 85: 2149-2156, 1963) or by using genetic recombination techniques. When prepared by chemical synthesis, it can be obtained using a polypeptide synthesis method well known in the art. When using a recombinant technique, a nucleic acid encoding CypB is inserted into an appropriate expression vector, the vector is transformed into a host cell, the host cell is cultured to express CypB, and the CypB is recovered from the host cell. Can be.

As the expression vector for expressing the CypB protein, any conventional expression vector can be used. Depending on the host cell, the expression level and expression of the protein are different. Therefore, the host cell may be selected and used according to the purpose. Host cells that can be used include fungi (eg Aspergillus), yeast (eg Pichia pastoris, Saccharomyces cerevisiae, Shh) Cells derived from higher eukaryotes, including lower eukaryotic cells, insect cells, plant cells, mammals, and the like, such as irradiated Carromaces (Schizosaccharomyces) and Neurospora crassa can be used as host cells.

Proteins are expressed in selected host cells and then subjected to conventional biochemical separation techniques, such as treatment with protein precipitants (salting), centrifugation, sonication, ultrafiltration, dialysis, molecular sieve chromatography for isolation and purification. (Gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, and the like can be used. In general, these are used in combination to separate proteins of high purity. (Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982); Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Press (1989) Deutscher, M., Guide to Protein Purification Methods Enzymology, vol. 182. Academic Press. Inc., San Diego, CA (1990)).

Compositions of the invention that exert physiological effects in vivo in the brain may be more useful if they have access to target cells in the brain. Examples of brain cells include, but are not limited to, neurons, glial cells (astrocytes, oligodendrocytes, microglia), cerebrovascular cells (muscle cells, endothelial cells), and cells constituting meninges. The blood brain barrier (BBB) typically limits access to brain cells by acting as a physical and functional obstacle that separates the brain parenchyma from the systemic circulation (see, eg, Pardridge, et al., J. Neurovirol. 5 (6), 556-69 (1999); Rubin, et al., Rev. Neurosci. 22, 11-28 (1999)). Circulating molecules can normally access brain cells by either of two processes: lipid-mediated transport through BBB by free diffusion, or active (or catalyzed) transport.

In order to facilitate the transport of the composition of the invention through the BBB, the composition may be coupled to a BBB transport vector (see Bickel, et al., Adv. Drug Delivery Reviews 46 for a review of BBB transport vectors and mechanisms). , 247-79 (2001). Exemplary transport vectors include OX26 monoclonal antibodies directed against cationized albumin or transferrin receptors; These proteins perform absorption-mediated and receptor-mediated transcytosis through BBB, respectively.

In another aspect, the present invention provides a composition for treating or preventing amyloidogenic diseases comprising a nucleic acid having a nucleotide sequence encoding a CypB protein.

The nucleotide sequence encoding the CypB protein in the composition of the present invention is a nucleotide sequence encoding a CypB protein in the wild-type or variant form as described above, wherein one or more bases may be mutated by substitution, deletion, insertion, or a combination thereof. It can be isolated from nature or prepared using chemical synthesis.

When chemically synthesizing a nucleotide sequence encoding a CypB protein, synthesis methods well known in the art are described, for example as described in Engels and Uhlmann, Angew Chem Int Ed Eng., 37: 73-127, 1988. Methods can be used, such as tryster, phosphite, phosphoramidite and H-phosphate methods, PCR and other autoprimer methods, oligonucleotide synthesis on solid supports, and the like.

One specific example of the nucleotide sequence encoding the CypB protein is the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1, and the sequence of SEQ ID NO: 2 will be exemplified as the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: Can be.

The nucleic acid having the above nucleotide sequence may be single or double stranded, and may be a DNA molecule (genomic, cDNA or synthetic) or an RNA molecule.

In a preferred embodiment, the nucleotide sequence encoding the CypB protein is provided to a recombinant expression vector operably linked to the vector expressing it.

In the present invention, "vector" means a means for introducing a nucleic acid sequence (eg, DNA, RNA, etc.) encoding a gene of interest into a host cell. As used herein, an "expression vector" refers to a gene construct that is capable of expressing a target protein or RNA of interest in a suitable host cell and includes essential regulatory elements operably linked to express the gene insert.

In the present invention, "operably linked" refers to a functional linkage of a nucleic acid expression control sequence and a nucleic acid sequence encoding a protein or RNA of interest to perform a general function. For example, a promoter and a nucleic acid sequence encoding a protein or RNA may be operably linked to affect expression of the coding sequence. Operational linkage with recombinant vectors can be prepared using genetic recombination techniques well known in the art, and site-specific DNA cleavage and ligation uses enzymes commonly known in the art and the like.

Vectors of the invention include plasmid vectors, cosmid vectors, viral vectors and the like. Suitable expression vectors include signal or leader sequences for membrane targeting or secretion in addition to expression control elements such as promoters, operators, initiation codons, termination codons, polyadenylation signals, enhancers and can be prepared in various ways depending on the purpose. Initiation and termination codons are generally considered to be part of the nucleotide sequence encoding the target protein and must be functional in the individual when the gene construct is administered and must be in frame with the coding sequence. The promoter of the vector may be constitutive or inducible. The expression vector also includes a selectable marker for selecting a host cell containing the vector and, in the case of a replicable expression vector, a replication origin. The vector may be self-replicating or integrated into the host DNA.

Nucleic acids having a nucleotide sequence encoding a CypB protein can be directly injected into known methods in the art, such as naked DNA in vector form (Wolff et al., Science, 247: 1465-8, 1990: Wolff et al. , J Cell Sci. 103: 1249-59, 1992), liposomes, cationic polymers and the like can be delivered into patient cells for the purpose of treating or preventing amyloidogenic diseases. Liposomes are phospholipid membranes prepared by mixing cationic phospholipids such as DOTMA or DOTAP for gene transfer. Nucleic acid-liposomal complexes are formed when cationic liposomes and anionic nucleic acids are mixed in a proportion. Cis and stay in endosomes (Schaefer-Ridder M et al., Sceience. 215 (4529): 166-168, 1982; Hodgson et al., Nat Biotechnol., 14 (3): 339-342, 1996) . The degree of gene transfer from the endosome to the nucleus through the cytoplasm determines gene transfer and treatment efficiency. This gene transfer method has the advantage of low immunogenicity, repeated administration and high safety. Cationic polymers used for gene transport include poly-L-lysine, spermine, polyethylenimine (PEI) and chitosan (Hashida, Br J Cancer). , 90 (6): 1252-1258, 2004; Wiseman, Gene Ther., 10 (19): 1654-1662, 2003; Koping-Hoggard, Gene Ther., 8 (14): 1108-1121, 2001). When the gene is administered in the form of a complex with a cationic polymer, the residence time and expression duration of the gene have been found to be significantly increased compared to naked DNA.

In another aspect, the present invention provides a composition for treating or preventing amyloidogenic diseases comprising a CypB protein expressing recombinant vector. The CypB protein expression recombinant vector may include a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1, may comprise a nucleotide sequence of SEQ ID NO: 2.

When transfecting a recombinant vector engineered to express CypB into a patient cell in a cell caused by Aβ toxicity, the expression efficiency can be increased, so the therapeutic effect is excellent.

Vectors used in gene therapy can be viral vectors and include, but are not limited to, replication defective retroviruses, adenoviruses and adeno-associated viruses. Viral vectors must meet the following criteria: (1) the vector must be capable of infecting the cells of interest and thus a viral vector with an appropriate host range should be selected; (2) the delivered gene must be able to be conserved and expressed in cells for an appropriate period of time; (3) The vector must be safe for the host. Other viral vectors that can be used for gene transfer into cells include retroviruses such as the Molone murine leuchemia virus (MMLV); Parvobaviruses such as JC, SV40, polyoma, adenovirus; Epstein-Barr decanter (EBV); Papilloma virus, such as bovine papilloma virus type I (BPV); Vaccinia and poliovirus and other human and animal viruses. Other methods include many methods, including nonviral and nonprotein vectors, viral vectors (eg, adenoviruses, herpes virus, vaccinia, retroviruses), DNA encapsulated in liposomes, lipid delivery vehicles, and naked DNA and Delivery vehicles can be used.

Host expression systems are well known in the art and are used in one aspect. The mammalian promoter can be any DNA sequence that binds to a mammalian RNA polymerase and can initiate downstream (3 ') transcription of the coding sequence of the cyclophilin protein into mRNA.

Promoters usually have a TATA box using a transcription initiation region located proximal to the 5 'end of the coding sequence and 25 to 30 base pairs located upstream of the transcription initiation site. The TATA box is believed to direct RNA polymerase II so that RNA synthesis occurs at the correct position. Mammalian promoters also typically contain an upstream promoter element located 100 to 200 base pairs upstream of the TATA box. The upstream promoter element determines the rate of transcription initiation and can act in either direction. Particularly used as mammalian promoters are promoters derived from mammalian viral genes because viral genes are often highly expressed and have a wide host range. Examples include the SV40 early promoter, mouse breast tumor virus LTR promoter, adenovirus major late promoter and herpes simplex virus promoter.

As one example, the cyclophilin gene can be achieved by operatively linking it to a very tightly regulated promoter such as the T7 expression vector (Rosenberg, et al., Gene, 56: 125-135, 1987). Insertion of a potent vector identifies a convenient restriction target near both ends of the cyclophilin gene and a suitable restriction target on the vector near its promoter and inserts into the vector such that the cyclophilin gene is oriented under the transcriptional and translational control of the potent promoter. Can be achieved. Alternatively, the cyclophylline gene can be overexpressed by establishing a strong ribosomal binding site upstream of the gene to increase expression of the gene (Shine and Dalgarno, Proc. Natl. Acad. Sci. USA, 71: 1342-1346, 1974).

Transcriptional termination and polyadenylation sequences, typically recognized by mammalian cells, are regulatory regions located 3 'of the translational termination codon, thus contiguous with the coding sequence with the promoter element. The 3 'end of the mature mRNA is formed by site-specific posttranslational cleavage and polyadenylation. Examples of transcription terminators and polyadenylation signals include those derived from SV40.

In another embodiment, the present invention is directed to treating a amyloidogenic disease by administering a pharmacologically effective amount of CypB protein, a nucleic acid having a nucleotide sequence encoding the CypB protein, or a CypB protein recombinant vector to a subject in need of treatment or prevention of an amyloidogenic disease. Or provide a method of prevention. CypB protein, nucleic acid having a nucleotide sequence encoding CypB protein or CypB protein expression recombinant vector used in the treatment method of the present invention is as described above.

 The treatment method of the present invention is for the prevention or treatment of amyloidogenic diseases of vertebrates, vertebrates include mammals such as humans, domestic animals and the like.

The composition of the present invention comprising a CypB protein as described above, a nucleic acid having a nucleotide sequence encoding a CypB protein or a CypB protein expression recombinant vector can be used to treat or prevent amyloidogenic diseases.

The composition of the present invention may comprise a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers can be used as oral administration binders, suspending agents, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, pigments, flavorings, etc., in the case of injections, buffers, preservatives, analgesic Topical agents, solubilizers, isotonic agents, stabilizers and the like can be used in combination, and for topical administration, bases, excipients, lubricants, preservatives and the like can be used. The formulation of the pharmaceutical composition of the present invention may be prepared in various ways by mixing with a pharmaceutically acceptable carrier as described above. For example, in the case of oral administration, it may be prepared in the form of tablets, troches, capsules, elesir, suspensions, syrups, wafers, etc., and in the case of injections, may be prepared in unit dosage ampoules or multiple dosage forms.

The composition of the present invention may be administered via any general route as long as it can reach the desired tissue. Thus, the compositions of the present invention can be administered topically, orally, parenterally, intranasally, intravenously, intramuscularly, subcutaneously, intraocularly, transdermally, and formulated into solutions, suspensions, tablets, pills, capsules, sustained release formulations, and the like. can do. Preferred formulations are injections. Injections can be subcutaneous, intramuscular or intravenous.

 The composition of the present invention may be administered in a therapeutically or prophylactically effective amount. Dosage may vary depending on various factors such as disease type and severity, age, sex, administration method, target cell, expression level, etc. of the patient, and can be easily determined by those skilled in the art.

In another embodiment, the present invention provides a method of preparing a CypB protein-expressing cell, (b) contacting the cells of step (a) with a candidate compound, and (c) expressing the CypB protein in step (b). Comparing the amount to the expression level of a control group that is not in contact with the candidate compound, and (d) identifying a compound that exhibits an increase in the CypB protein expression level, the method of screening for a drug that treats or prevents amyloidogenic diseases It is about. In the above method, the difference in the expression level of CypB protein can be confirmed at the protein level or mRNA level. The screened drug can be used to increase intracellular CypB expression, preferably to increase neuronal CypB expression.

The level of protein expression can be confirmed by electrophoresis after loading each protein into the gel, but preferably by immunoassay through the antigen-antibody complex formation using an antibody against CypB protein. Such assays include Western blot, RIA, immunoprecipitation assay, and the like.

In the detection method, the amount of antigen-antibody complex formed can be quantitatively measured through the magnitude of a signal of a detection label. Such a detection label can be selected from the group consisting of enzymes, fluorescent materials, ligands, luminescent materials, microparticles, redox molecules and radioisotopes, but is not necessarily limited thereto. Colorimetric method, electrochemical method, fluorimetric method, luminometry, particle counting method, visual assessment and the like to detect antigen-antibody complex formation. A method selected from the group consisting of scintillation counting methods may be used, but is not limited thereto.

The level of mRNA expression level can be confirmed using primers specific for CypB protein. Specific analysis methods include RT-PCR or Northern blot. RT-PCR, which is a simple assay that can quantitatively measure the transcription of CypB to mRNA by checking the pattern and intensity of the band, is preferable.

In a specific embodiment of the present invention, immunohistochemistry and immunocytochemical methods confirmed that CypB is neuron-specific in the cytoplasm of cerebral cortex, hippocampus, teeth and neurons of normal rat and mouse brain tissues. In addition, it was confirmed that CypB inhibits Aβ-induced neuronal cell death by regulating redox response in neurons. Specifically, it was confirmed that overexpressed CypB protects neurons from neurotoxicity induced by Aβ _25-35 , and neurotoxicity induced by Aβ _25-35 through overexpressing CypB reduces the incidence of intracellular ROS. It was confirmed that the decrease.

The composition for preventing and treating amyloidogenic diseases comprising cyclophilin B of the present invention is amyloid diseases caused by amyloid-β peptides deposited with other substances to form neural plaques and thereby cause neurotoxic effects. ), Eg, age-related cognitive decline, mild cognitive impairment ("MCI"), vascular dementia, cerebral amyloid angiopathy ("CAA"), hereditary intracranial hemorrhage, senile dementia, Down syndrome, inclusion body myositis ("IBM") Age-related macular degeneration ("ARMD"), chronic inflammation, multiple myeloma, type II diabetes, Creutzfeldt-Jakob, Alzheimer's disease Can effectively cure

1 is a result showing that cyclophilin B is specifically found in mainly neurons.
2 shows that overexpressed cyclophilin B is present at Aβ _25-35 . By reducing apoptosis induced.
3 shows that overexpressed cyclophilin B is present at Aβ _25-35 . Results in reducing apoptosis induced and inhibiting ROS generation.
4 shows that overexpressed cyclophilin B is present at Aβ _25-35 . It is a result indicating that it reduces the apoptosis signal induced by.
5 is a result of analyzing the knockdown efficiency of cyclophylline-siRNA by Western blotting.
Figure 6 is the result of analyzing the knockdown efficiency of cyclophylline-siRNA by Western blotting.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Materials and Methods

Example 1: Material

Mongolian gerbil mice (3 weeks old) and Sprague-Dawley (SD; 3 months old) rats were purchased from a central laboratory animal (Seocho, Seoul, KR), and were cortical and dental. The hippocampus was collected from Kyunghee University Medical Center. Fetal bovine serum and RPMI 1640 medium were purchased from Lonza (Walkersvile, MD, USA). Horse serum and Lipofectamine 2000 were purchased from Gibco, Invitrogen (Grand Island, NY, USA). PC12 cell lines were purchased from the American Type Culture Collection, Rockville, MD, USA. Aβ _25-35 and Aβ _35-25 were purchased from Bachem (Torrance, CA, USA). 2 ', 7'-dichlorofluorescein diacetate (DCF-DA), Alexa Fluor 488 anti-goat and rabbit, Alexa Fluor 594 Mouse Fluor 594 anti-mouse antibody, Hochest 33342, and DAPI nucleic acid staining were purchased from Molecular Probes (Eugene, OR, USA). Mouse anti-neuronal nuclei (Neun) monoclonal antibodies are described by Chemicon International, Inc. (Temecula, CA, USA). Normal horse serum and ABC kit are available from Vector Laboratories, Inc. (Burlingame, CA, USA). Antibodies against AKT are from Abcam (Cambridge, UK), Bax, Bcl-2, Bcl-xL, α-actinine, and actin are from Santa cruz Technology (Santa cruz, CA, USA), and caspase-3 and PARP are Calbiochem. (San Diego, CA, USA), ERK1 / 2, JNK and p38 were purchased from Cell Signaling Technology (Boston, Mass., USA). 2.3% crystal violet solution, 3- (4, 5-dimethylthiazol-3-yl) -2, 5-diphenyl tetrazolium bromide (3- (4, 5-dimethylthiazol-2-yl ) -2, 5-diphenyl tetrazolium bromide (MTT), 4% paraformaldehyde, 36.5% formaldehyde, collagen (type I), ethanol, hydrogen peroxide, methanol, phosphate buffered saline, PBS), poly-L-lysine, sodium dodecyl sulfate (SDS), Triton X-100, xylene, and other chemicals Unless purchased from Sigma (St. Louis, MO, USA).

Example 2: Immunohistochemistry

The brains of Mongolian gerbil mice (3 weeks old) and SD rats (3 months old) were taken out immediately. Whole brains were fixed, dehydrated in 0.1 M PBS with 4% (w / v) paraformaldehyde dissolved in paraffin overnight at 4 ° C. Vibratome (Leica VT 1000S; Nussloch, DE) was used to make a 30 μm-thick surface, with free-floating coronal sections containing 1% (including the cerebral cortex, hippocampus, and dentate gyrus). v / v) blocked endogenous peroxidase for 15 minutes with hydrogen peroxide. Then washed three times with 0.1 M PBS for 5 minutes, the primary CypB (1: 200) antibody and the special neuron marker Neun (1: 500) were 5% (v / v) normal goat serum, 2% (w / v) incubated overnight at 4 ° C. with BSA, 0.1% (v / v) Triton X-100. Second, the biotinylated HRP system was used, and immunohistochemical staining was performed using the avidin-biotin compleximmunoperoxidase method (ABC kit, Vector Laboratories Inc .; Burlingame, CA). Used. Color development was performed using a diaminobenzidine (DAB) blush horseradish peroxidase system.

Example 3: Immunocytochemistry

The brain tissues of mice and rats were fixed for 30 minutes at room temperature in 0.1 M PBS containing 4% (w / v) paraformaldehyde. Cells were permeated for 30 minutes at room temperature in PBS containing 3% (w / v) BSA and 0.1% (v / v) Triton X-100, followed by anti-CypB antibody (1: 200) and Alexa Incubated in Fluor 488 rabbit (Alexa Fluor 488 rabbilt), anti-Neun antibody (1: 500), Alexa Fluor 594 mouse, DAPI (1: 1000). Microtubule networks were visualized using a fluorescence confocal laser microscope (META 510; Zeiss) at 100 and 400 magnifications.

Example 4: Beta Amyloid Section _25-35 (beta amyloid fragment _25-35 ) And beta amyloid fragments _35-25 (beta amyloid fragment _35-25 Preparation of

Beta β-amyloid Aβ _25-35 and Aβ _35-25 were purchased from Bachem (Torrance, Calif.), _Dissolved in deionized distilled water at a concentration of 1 mM, and sealed for storage at -80 ° C. . Aβ _25-35 and Aβ _35-25 were aged in a humid container at 37 ° C. before use and added to the culture medium.

Example 5: Cell Culture and Treatment

PC12 cells were treated with 12.5% (v / v) heat-inactivated horse serum, 2.5% (v / v) fetal bovine serum and 1% (v / v) antibiotic: penicillin 37 ° C., 5 in RPMI 1640 medium supplemented with 100 mg / ml G sodium, 0.25 mg / ml streptomycin sulfate (antibiotics: penicillin G sodium 100 mg / ml, streptomycin sulfate 0.25 mg / ml, Gibco; Grand Island, NY, USA) Incubated in% wet carbon dioxide gas, 95% air. When cells reach 70-80% of the confluence before the experiment (Morishima Y., et al. (2002) Beta-amyloid induces neuronal apoptosis via a mechanism that involves the c-Jun N-terminal kinase pathway and The induction of Fas ligand.J. Neurosci. 21, 7551-7560.) Sprinma (Sigma; Saint Louis, MO, USA) was sprayed on 100 mm dish pre-covered. The medium was changed every 2-3 days and trypsinized during exponential growth and then in collagen-covered 60 mm dishes for Western blot analysis and clonality analysis at a density of 5x10 ⁴ cells / cm ² , for cell viability analysis. a 24-well plate ^{(well plates) 1x10 6 cells /} cm 2, ROS and analyzed for flow cytometry (flowcytometry) in 6-well plate ^{(well plates) 1x10 5 cells /} cm 2, 2x10 6 cells for immunocytochemistry / cm ² Resuspended in 12-well plates covered with poly-L-lysine. After 24 hours, cell growth was stopped in pre-culture for one hour at RPMI 1640 given 1% (v / v) fetal bovine serum and then 20 to 100 μM / mL of Aβ _25-35 or reverse peptide for the next 48 hours. _Incubated with Aβ _35-25 . Each experiment was established on three samples and cultures were replaced after two months for passages where fresh portions of frozen cells were thawed.

Example 6: Transfection with Plasmids

Wild-type human cyclophilin B (CypB / WT)

forward primer, 5'-TT AAGCTT ATGCTGCGCCTCTCCGAACG-3 '( HindIII ) (SEQ ID NO: 3); reverse primer 1, 3'-TATGGGTA GATATC CTCCTTGGCGATGGCA-5 '( EcoRV ) (SEQ ID NO: 4); reverse primer 2, 5'-CC CTCGAG AGCGTAGTCTGGGACGTCGTATGGGTAGAT-3 '( XhoI ) (SEQ ID NO: 5); The C-terminal end was amplified from the entire RNA sample by RT-PCR by tagging HA tag 5'-TACCCATACGACGTCCCAGACTACGCT-3 '(SEQ ID NO: 6). PPIase-unsafe mutant CypB / R95A was prepared with the following primers: 5'-CAAATTCCAT GCG GTAATCAAG-3 '(SEQ ID NO: 7); The ariginine residue (R95) was mutated to Alanine (A) using 3'-CTTGATTGC CGC ATGGAATTG-3 '(SEQ ID NO: 8). DNA transfection was performed into PC12 cells with Lipofectamine 2000 drug according to the manufacturer's protocol. Cells were treated with Aβ _25-35, Aβ _35-25 or H ₂ O ₂ for 48 hours after 48 hours from _aliquot .

Example 7: RNAi Interference

Small interfering RNAs (siRNA) specific for cyclophilin B (si-CypB) or scrambled siRNA were purchased from Dharmacon (Chicago, IL, USA). CypB siRNA transfection was performed with the same DNA construct and after 72 hours of culture, Aβ _25-35, Aβ _35-25 or H ₂ O ₂ were treated for 48 hours. The efficiency of siRNA interference in the production of CypB was analyzed by Western blot analysis.

Example 8: Cell Viability Assay (MTT Reduction)

3- (4, 5-dimethylthiazol-3-yl) -2, 5-diphenyl tetrazolium bromide (3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyl tetrazolium bromide, MTT The assay was performed to determine cell viability at Aβ. Cells were seeded in 24 well plates as before and after 24 hours, either Aβ _25-35 or Aβ _35-25 alone or with pcDNA3, CypB / WT, CypB / R95A for 24 hours or si- for 48 hours. Treatment was performed after transfection of RNA with si-CypB / WT. MTT was added to each well at a concentration of 0.5 mg / ml in PBS. Absorbance at 595 nm was measured in a microplate reader (Bio-Rad; Hercules, Calif., USA). The control group used untreated cells and Aβ _35-25 treated cells and was expressed as the percentage of absorbance associated with untreated cells.

Example 9: Clonogenicity Analysis

Clonogenic assays were performed on growing cells based on the following conditions: 1) PC12 cells were each dispensed as described above, Aβ _25-35 , Treatment was for 48 hours with or without Aβ _35-25 . 2) PC12 cells were each dispensed as described above and transfected with pcDNA3, CypB / WT, CypB / R95A for 24 hours and then treated for 48 hours with or without Aβ _25-35 . 3) PC12 cells were each dispensed as described above and transfected with si-control and si-CypB / WT for 48 hours and then treated for 48 hours with or without Aβ _25-35 . Cells were trypsinized and aliquoted 1: 100 diluted in normal growth medium. Within 14 to 17 days, 50 to 60 colonies formed. Colonies were fixed with 100% methanol and stained with 2.3% (v / v) crystal violet solution for 30 minutes followed by several washes with PBS every 10 minutes.

Example 10 Western Blot Analysis

For whole cell lysis samples, cells were treated with LIPA buffer (150 mM NaCl, 50 mM Tris, pH 7.4, 1% Na-deoxycholate, 1% NP-40, 5 mM EDTA 1 mM Na ₃ VO ₄ , 1 mM NaF) Homogenized. The same amount of protein samples (30 μg) were separated using SDS-PAGE on a 9-14% (v / v) polyacrylamide gel and transferred to nitrocellulose membrane. Membranes were then blocked for one hour in TBST containing 5% (w / v) skim milk and primary antibodies were incubated overnight at 4 ° C., followed by secondary antibodies for one hour. For detection, enhanced chemiluminescent agents (Santa cruz Biotechnology; Santa Cruz, CA, USA) were used. Except in particular, α-actinin and actin proteins were immunosampled to standardize the amount of sample protein.

Example 11: Hochest 33342 Staining

PC12 cells were simply dispensed onto poly-L-lysine-covered coverglass in 24-well plates as described above and the cells attached to the coverglass were freshly loaded with poly-L-lysine containing fresh medium. Removed with covered 24-well plates. Aβ _25-35 (50 μΜ / mL) was either treated single or after 48 hours of transfection of pcDNA3, CypB / WT, CypB / R95A or si-CypB for 72 hours. Cells were washed with PBS and then fixed for 10 minutes at 20 ° C. with 4% (v / v) formaldehyde dissolved in PBS. Cells were then stained for 30 minutes with Hochest 33342, 1: 1000 and then visualized using a fluorescence confocal laser microscope (META 510; Zeiss).

Example 12 Measurement of Intracellular Reactive Oxygen Species

In order to measure the intracellular development of ROS, a fluorescent probe, 2 ', 7'-dichlorofluorescein diacetate (2', 7'-dichlorofluorescein diacetate, DCF-DA) was used. PC12 cells were dispensed as above and transfected with pcDNA3, CypB / WT, CypB / R95A for 24 hours or si-control and si-CypB / WT for 48 hours. The following day, cells were treated for 3 hours with or without Aβ _25-35 (50 μM / mL) and then 10 μM DCF-DA was added at 37 ° C. for one hour. Cells were washed twice with PBS and centrifuged at 1500 g for 5 minutes. ROS generation was detected by Flow Cytometry (Beckman Coulter).

Example 13: Detection of Apoptosis PC12 Cells by Flowcytometry

To detect apoptotic cells by flowcytometry, PC12 cells were transfected with pcDNA3, CypB / WT, CypB / R95A for 24 hours or si-control and si-CypB / WT for 48 hours. After 24 hours, cells were exposed to Aβ _25-35 (50 μM / mL) for 48 hours, washed twice and fixed for one hour with 70% ethanol. Cells were then harvested by floating in 0.5 mL PBS and centrifuged at 1500 g for 5 minutes. Cells were resuspended in 0.5 mL PBS with 0.05 mg / mL PI and 2 μl / mL of RNase (stock 10 mg / mL) was added and incubated at 37 ° C. for 30 minutes. Samples were washed twice with PBS and suspended in 0.5-1 mL PBS. DNA content was detected by flowcytometry (Beckman Coulter) to analyze the rate of apoptosis.

Example 14 Statistical Analysis

In the examples, the results were expressed as mean 표준 standard deviation (SD) of data from at least three independent experiments and were statistically analyzed using a Student t-test. P * <0.05 value and P ** <0.001 were considered significant.

result

1. Neuron-specifically located cyclophilin B in neurons

Cytophyllin B (cypB) is present in the cytoplasm of the cerebral cortex, hippocampus, and dentate gyrus neurons in normal rat and mouse brain tissues. Physiological evaluation was performed. The results are shown in Figure 1a.

The number of neurons relative to the brain cell nucleus was expressed by the neuron-specific nuclear protein marker Neun (neuronal nuclei) in the sense that neurons and cypB would be in the same position. To assess the possibility that cypB could be neuron-specific, immunocytochemistry was performed to see if neurons and cypB were directly located in the exact overlap of the nucleus and cypB of neurons in each neuron. The results are shown in Figure 1b. This result also occurred in the brain of mice (FIG. 1B). However, the fact that DAPI stained cells are not associated with Neun or cypB stained cells indicates that approximately 90% of the cells in the cortical, hippocampus, and dentate gyrus are neurons and cypB is a neuron- It can be seen that it is specific.

2. Beta Amyloid in PC12 Cells _25-35 (beta amyloid _25-35 Effect of overexpressed cyclophilin B on neurotoxicity induced by

Neurotoxicity induced by beta amyloid (Aβ _25-35 ) was measured by MMT analysis. The viability of PC12 cells after 48 hours of Aβ _25-35 (20-100 μM / mL) was 81.98% (20 μM / mL), 53.02% (40 μM / mL), 48.80%, respectively, in a dose-dependent manner. (60 μM / mL), 40.35 (80 μM / mL) (FIG. 2A). After treatment with 50 μM / mL of Aβ _25-35 for 48 hours, cell viability in PC12 cells was reduced to 36.81% of the control group (FIG. 2B). In addition, cell death induced by Aβ _25-35 was magnified 400 times as originally observed by microscopy through clonality assay (FIG. 2C) and treated with 50 μM / mL of Aβ _25-35 for 48 hours in MTT reduction assay. The survival rate of the Aβ _35-25 control group, 91%, was reduced to 42%. However, Aβ _35-25 , the reverse peptide of Aβ _25-35 , did not affect the viability of PC12 cells. The percentage of survival was calculated as Aβ-treated sample OD / control OD. To test the effect of cyclophilin B (CypB) on neurotoxicity induced by Aβ _25-35 , overexpressing HA-tagged CypB / WT, or HA-tagged CypB / R95A, which is defective in PPIase activity in PC12 cells. . After both CypB / WT and CypB / R95A established transfections, expression levels of CypB / WT and CypB / R95A were analyzed by Western blot analysis (FIG. 2E). Relevant overexpression levels of the two proteins were quantitatively quantified according to α-actinin and actin. No difference was seen in the overexpression numbers. FIG. 2F shows significantly increased survival of PC12 cells after 50 μM / mL Aβ _25-35 treatment for 48 hours in overexpressed CypB / WT. However, mock overexpression or overexpressed CypB / R95A mutant protein did not yield Aβ _25-35 treated PC12 cells. In addition, no differences were seen in the viability of the Aβ _35-25 treated group. In addition, CypB / WT overexpressed Clonogenicity up to 86%, but virtual (51%) or CypB / R95A (57%) overexpression did not. These results indicate that overexpressed CypB protects PC12 cells from neurotoxicity induced by Aβ _25-35 .

3. Beta-amyloid in PC12 Cells _25-35 (beta amyloid _25-35 Effect of overexpressed cyclophilin B (Cyp B) on apoptosis induced by

Chromatin contraction and fragmentation, which can be detected by Hochest 33342 staining through staining of nuclei of Aβ _25-35 (50 μM / mL) treated cells with fluorescent nucleotides in recognition of DNA fragmentation, are well known features of apoptosis. Treatment with Aβ _25-35 (50 μM / mL) for 48 hours showed characteristics that were recognized as apoptosis by 30%, _{whereas the} control and Aβ _35-25 did not (FIG. 3A). In addition, overexpressed CypB / WT showed no chromatin contraction and fragmentation compared to mock (42%) or CypB / R95A expressed (53%) (FIG. 3B).

Aβ _25-35 can induce H ₂ O ₂ formation through metal ion reduction in the Alzheimer's disease model system (Huang X., et al. (1999) The A beta peptide of Alzheimer's disease directly produces hydrogen peroxide through metal ion Reduction.Biochemistry.38, 7609-7616.), ROS generation in CypB / WT-overexpressed PC12 cells after Aβ _25-35 treatment. Incidence levels of intracellular ROS were measured using DCF-DA. As presented in Figure 3e and 3f, Aβ _25-35 to prove ROS accumulation in the PC12 cells treated for 3 hours Aβ _25-35 (50 μM / mL) untreated given into the DCF-DA in PC12 cells after a treatment for Comparison of cells (35.89%) with Aβ _35-25 treated cells (57.13%) showed a significant increase in DCF positive cells (86.80%). 200 μM / mL H ₂ O ₂ -treated cells (98.72%) were used as positive controls. Surprisingly, CypB / WT-overexpressed PC12 cells significantly reduced ROS accumulation after Aβ _25-35 treatment, indicating that neurotoxicity induced by Aβ _25-35 through overexpressed CypB reduces the incidence of intracellular ROS. It can be seen that it decreases (Figs. 3e and 3f). However, CypB / R95A mutant-overexpressed PC12 cells did not reduce the incidence of intracellular ROS.

FACS analysis was then performed to assess the DNA fragmentation that occurs in each cell as an indicator of apoptosis in pcDNA, CypB / WT, CypB / R95A transfections after exposure to Aβ _25-35 . First, cell division on sub-G1 of the cell cycle was determined after 50 μM / mL of Aβ _25-35 , 50 μM / mL Aβ _35-25 or 200 μM / mL H ₂ O ₂ treatment for 48 hours. 200 μM / mL H ₂ O ₂ was used as a positive control for cell death of apoptosis. As shown in FIG. 3c, the sub-G1 group peak was evident after 50 μM / mL of Aβ _25-35 treatment, whereas untreated or Aβ _35-25 treated cells showed no peak. Aβ _25-35 treated cells (25.04%) showed almost 9.2 and 4.5 times the cell death of apoptosis compared to untreated control (2.72%) and Aβ _35-25 treated cells (5.55%), respectively (FIG. 3C). Thus, we measured apoptosis in pcDNA, CypB / WT, CypB / R95A transfection after exposure to Aβ _25-35 . Less apoptosis cell death was seen in CypB / WT transfection than in pcDNA and CypB / R95A transfection (FIG. 3D). As shown in FIG. 3D, overexpressed CypB / WT reduced apodosis induced by Aβ _25-35 by 12.99%, while reducing pcDNA (24.22%) or overexpressed CypB / R95A mutation (22.06%). I couldn't.

As expected, capspase-3 and PARP cleavage was evident in Aβ _25-35 treated cells when Aβ _25-35 was exposed to 48 hours, while Bax, Bcl-2 and Bcl-xL were reversed (FIGS. 3G and 3H). . However, capspase-3 cleavage was inhibited by overexpressed CypB / WT, but not by overexpressed CypB / R95A mutations (FIG. 3I). PARP cleavage was evident in cells exposed to Aβ _25-35 by the appearance of 89 kDa fragments derived from 116 kDa nucleopoly (ADP-ribose) polymerase enzymes, Bax and Bcl-2, Bcl-xL was reversed (FIG. 3J).

4. Beta Amyloid _25-35 (beta amyloid _25-35 Effect of Overexpressed Cyclophilin B (Cyp B) on Apoptosis Signal Induced by

To understand the effects of overexpressed CypB on the neurotoxic apoptosis signaling pathway induced by Aβ _25-35 , since several studies have pointed out several signaling pathways including the Aβ-induced MAPK and PI-3K / AKT1 pathways We studied stress signal transduction pathways such as, MAPK and PI-3K / AKT1 pathways. Depending on coordination among cell types and states, MAPK and PI-3K / AKT1 regulate cell death and cell survival. Western blot analysis of Aβ _25-35 (50 μM / mL) treated p-JNK, p-p38, ERK1 / 2 and AKT1 in a time-dependent manner (FIGS. 4A and 4B). However, overexpressed CypB / WT blocked p-JNK, p-p38, ERK1 / 2 and AKT1 induced by Aβ _25-35 , whereas the overexpressed CypB / R95A mutations were not (FIGS. 4C and 4D). . Taken together, these results show that overexpressed CypB protects PC12 cells against neurotoxicity induced by Aβ _25-35 .

5. Effect of Overexpressed Cyclophilin B (Cyp B) on siRNA Knock Down

Furthermore, in order to investigate and confirm the biochemical principles responsible for the protective role of CypB against neurotoxicity induced by Aβ _25-35 , RNA interference experiments were performed using siRNA to knock down CypB protein levels in PC12 cells. And then cell survival was assessed after Aβ _25-35 treatment. In this experiment, the efficiency of siRNA was assessed by Western blot analysis. Expression levels of endogenous CypB were inhibited by significantly specific siRNA interference (FIG. 5A). CypB, as is proposed in order to provide necessary to reduce the neurotoxicity induced by Aβ _25-35, MTT reduction assay (Figure 5b) and immunogenic clone analysis (Figure 5c), The neurotoxicity induced by Aβ _25-35 Significantly increased in CypB knock down after Aβ _25-35 treatment compared to control-siRNA transfection. In this example, MTT reduction assay expressing control-siRNA showed 50% of cell viability compared to control, si-CypB was 39% and clonality assay was control-siRNA (56%) and si-CypB (39 %). However, Aβ _35-25 did not affect cell viability.

As predicted in FIG. 5D, chromatin condensation and nuclear fragility, which appear to be affected by apoptosis factors as well as neurotoxicity in cells, was more pronounced in knockdown compared to control siRNA. The cleavage of Caspase-3 and PARP was dramatically activated after knock down of cypB as in Bax, but Bcl-2 was reversed (FIGS. 5E and 5F). This shows the survival pathway between Bcl-2 proteins.

6. Beta Amyloid _25-35 (beta amyloid _25-35 In the apoptotic signal induced by Cyclophylline  B ( Cyp  B) knock down Effect

To determine if MAPK and PI-3K / AKT1 are important in Aβ _25-35 induced cell death, the effects of CypB on MAPK and PI-3K / AKT1 signals by knockdown of CypB were examined. Execution of cypB-siRNA knockdown also increased MAPK and PI-3K / AKT1 levels in PC12 cells (FIGS. 6A and 6B). When treated with Aβ _25-35 (50 μM / mL) for 48 hours, the contents of p-JNK, p-p38, p-ERK1 / 2 and AKT1 showed an increase compared to control-siRNA. The results show that MAPK and PI-3K are associated with Aβ-mediated apoptosis cell death and cypB inhibits Aβ-induced changes by inhibiting antioxidant enzyme activity and other cytokine levels.

<110> University-Industry Cooperation Group of Kyung Hee University <120> Composition for preventing and treating amyloid diseases          comprising cyclophilin B <130> PA100448 / KR <160> 8 <170> KopatentIn 1.71 <210> 1 <211> 216 <212> PRT <213> Homo sapiens <400> 1 Met Leu Arg Leu Ser Glu Arg Asn Met Lys Val Leu Leu Ala Ala Ala   1 5 10 15 Leu Ile Ala Gly Ser Val Phe Phe Leu Leu Leu Pro Gly Pro Ser Ala              20 25 30 Ala Asp Glu Lys Lys Lys Gly Pro Lys Val Thr Val Lys Val Tyr Phe          35 40 45 Asp Leu Arg Ile Gly Asp Glu Asp Val Gly Arg Val Ile Phe Gly Leu      50 55 60 Phe Gly Lys Thr Val Pro Lys Thr Val Asp Asn Phe Val Ala Leu Ala  65 70 75 80 Thr Gly Glu Lys Gly Phe Gly Tyr Lys Asn Ser Lys Phe His Arg Val                  85 90 95 Ile Lys Asp Phe Met Ile Gln Gly Gly Asp Phe Thr Arg Gly Asp Gly             100 105 110 Thr Gly Gly Lys Ser Ile Tyr Gly Glu Arg Phe Pro Asp Glu Asn Phe         115 120 125 Lys Leu Lys His Tyr Gly Pro Gly Trp Val Ser Met Ala Asn Ala Gly     130 135 140 Lys Asp Thr Asn Gly Ser Gln Phe Phe Ile Thr Thr Val Lys Thr Ala 145 150 155 160 Trp Leu Asp Gly Lys His Val Val Phe Gly Lys Val Leu Glu Gly Met                 165 170 175 Glu Val Val Arg Lys Val Glu Ser Thr Lys Thr Asp Ser Arg Asp Lys             180 185 190 Pro Leu Lys Asp Val Ile Ile Ala Asp Cys Gly Lys Ile Glu Val Glu         195 200 205 Lys Pro Phe Ala Ile Ala Lys Glu     210 215 <210> 2 <211> 1045 <212> DNA <213> Homo sapiens <400> 2 actatccggc gccgagccgg aggggggaaa cggcgcccgc cgcccgcccg gagcccgcga 60 gcaaccccag tcccccccac ccgcgcgtgg cggcgccggc tccctagcca ccgcggcccc 120 accctcttcc ggcctcagct gtccgggctg ctttcgcctc cgcctgtgga tgctgcgcct 180 ctccgaacgc aacatgaagg tgctccttgc cgccgccctc atcgcggggt ccgtcttctt 240 cctgctgctg ccgggacctt ctgcggccga tgagaagaag aaggggccca aagtcaccgt 300 caaggtgtat tttgacctac gaattggaga tgaagatgta ggccgggtga tctttggtct 360 cttcggaaag actgttccaa aaacagtgga taattttgtg gccttagcta caggagagaa 420 aggatttggc tacaaaaaca gcaaattcca tcgtgtaatc aaggacttca tgatccaggg 480 cggagacttc accaggggag atggcacagg aggaaagagc atctacggtg agcgcttccc 540 cgatgagaac ttcaaactga agcactacgg gcctggctgg gtgagcatgg ccaacgcagg 600 caaagacacc aacggctccc agttcttcat cacgacagtc aagacagcct ggctagatgg 660 caagcatgtg gtgtttggca aagttctaga gggcatggag gtggtgcgga aggtggagag 720 caccaagaca gacagccggg ataaacccct gaaggatgtg atcatcgcag actgcggcaa 780 gatcgaggtg gagaagccct ttgccatcgc caaggagtag ggcacaggga catctttctt 840 tgagtgaccg tctgtgcagg ccctgtagtc cgccacaggg ctctgagctg cactggcccc 900 ggtgctggca tctggtggag cggacccact cccctcacat tccacaggcc catggactca 960 cttttgtaac aaactcctac caacactgac caataaaaaa aaatgtgggt tttttttttt 1020 ttaatataaa aaaaaaaaaa aaaaa 1045 <210> 3 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 ttaagcttat gctgcgcctc tccgaacg 28 <210> 4 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 tatgggtaga tatcctcctt ggcgatggca 30 <210> 5 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 ccctcgagag cgtagtctgg gacgtcgtat gggtagat 38 <210> 6 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> HA tag <400> 6 tacccatacg acgtcccaga ctacgct 27 <210> 7 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 caaattccat gcggtaatca ag 22 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 cttgattgcc gcatggaatt g 21

Claims (10)

Comprising cyclophylline B (CypB) protein, composition for preventing and treating amyloidogenic diseases. The composition of claim 1, wherein the CypB protein has the amino acid sequence of SEQ ID NO: 1. A composition for preventing and treating amyloidogenic diseases, comprising a nucleic acid having a nucleotide sequence encoding a cyclophylline B (CypB) protein. The composition of claim 3, wherein the nucleic acid having the nucleotide sequence encoding the CypB protein has a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1. 4. The composition of claim 3, wherein the nucleic acid having the nucleotide sequence encoding the CypB protein has the nucleotide sequence of SEQ ID NO: 2. The composition of claim 3, wherein the nucleic acid having the nucleotide sequence encoding the CypB protein is a CypB protein expression recombinant vector. The composition of claim 6, wherein the CypB protein expression recombinant vector comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1. The composition of claim 6, wherein the CypB protein expression recombinant vector comprises the nucleotide sequence of SEQ ID NO: 2. 8. The method of claim 1 or claim 3, wherein the amyloidogenic disease is aging-related cognitive decline, mild cognitive impairment, vascular dementia, cerebral amyloid angiopathy, hereditary intracranial hemorrhage, senile dementia, Down syndrome, inclusion body myositis, aging-related macular degeneration, chronic A composition that is an inflammation, multiple myeloma, type II diabetes, Creutfeldt-Jakob disease or Alzheimer's disease. (a) culturing CypB protein expressing cells,
(b) contacting the cells of step (a) with a candidate compound,
(c) comparing the expression level of the CypB protein in step (b) with the expression level of the control group not contacted with the candidate compound, and
(d) identifying a compound that exhibits an increase in the amount of CypB protein expression, the method for screening a drug for treating or preventing amyloidogenic diseases.

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