WO2004048565A9 - アポトーシス関連蛋白質およびその用途 - Google Patents
アポトーシス関連蛋白質およびその用途Info
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- WO2004048565A9 WO2004048565A9 PCT/JP2003/014794 JP0314794W WO2004048565A9 WO 2004048565 A9 WO2004048565 A9 WO 2004048565A9 JP 0314794 W JP0314794 W JP 0314794W WO 2004048565 A9 WO2004048565 A9 WO 2004048565A9
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4747—Apoptosis related proteins
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P5/00—Drugs for disorders of the endocrine system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
Definitions
- the present invention relates to a functional fragment of a protein that binds to and activates ASK1, and various uses of the protein and the fragment, in particular, pharmaceutical uses.
- Apoptosis plays a role in the elimination of unnecessary and abnormal cells during development, homeostasis, and a host defense function that removes damaged cells, and the mechanism at the molecular level is also increasingly clear. It is becoming. Abnormality of these molecules and disruption of the regulatory mechanism impair the physiological function of apoptosis, and may cause various diseases and exacerbate them. For example, if apoptosis is excessively suppressed, the cells that should be removed will proliferate abnormally and induce neoplastic diseases and autoimmune diseases, etc. Conversely, if apoptosis is abnormally enhanced, the cells that are essentially required It can lead to death and neurodegenerative diseases.
- Mitogen-activated protein (MAP) kinase cascade is activated by inflammatory cytokines such as physiochemical stress, tumor necrosis factor a (TNF-H), and interleukin-1 (IL-1).
- MAPKK is a signaling mechanism that sequentially activates MAP kinase kinase (MAPKK) and MAP kinase (MAPK). In response to these stimuli, cells survive, proliferate, differentiate, and die (apoptosis). Is shown.
- JNK c-Jun N-terminal kinase
- p38 MAP kinase
- MAPKs that are part of the signaling pathway that induces apoptosis (eg, Science, 270, 1326 (1995)). Furthermore, it is involved in inducing the inflammatory response by inducing the production of inflammatory site force-in.
- JNK and p38 are derived from MAPKK MKK4Z7 and MKK3Z6. Are activated respectively. These MAP KKs are activated by one MAP KKK called Apoptosis signal-regulating kinase 1 (ASK 1) (JP 10-93A, Science, 275, 90-94 (1997)). Many other MAP KKKs have been reported besides ASK1, but ASK1 is characterized by its ability to induce apoptosis in cells through signaling through activation of JNK and / or p38. Attached. Recently, it has been suggested that ASK 1 activation is involved in cell differentiation such as keratinocyte differentiation and neurite outgrowth of PC12 cells.ASK 1 is not only apoptotic but also controls cell fate. It is becoming clear that it is playing an important role. Furthermore, it has become clear that induction of the production of inflammatory cytokines is involved in inducing an inflammatory response.
- ASK 1 is an important molecule that determines the future fate of cells, it is thought that various factors are involved in its activation and that it is under complex control. So far, it has been reported that activation of ASK1 requires the formation of homo-oligomers between ASKs and the subsequent phosphorylation of threonine in the activation loop.
- rotein phosphatase 5 (PP 5) is H 2 0 (2 ) It is thought that activated ASK 1 is returned to an inactive state by directly binding to ASK 1 under stimulation and dephosphorylating threonine (EMBO Journal, 20, 6028-6036 (2001)) Furthermore, in the absence of oxidative stress, thioredoxin, a redox regulator, constantly binds to the N-terminal domain of ASK1 and acts as an activating inhibitor of ASK1 to exert oxidative stress.
- TNF receptor-associated factor 2 (TRAF 2) binds to the C-terminal domain of ASK1 during ASK 1 activation by TNF- ⁇ Activation of ASK 1 (Molecular Cell, 2, 389-395 (1998)), and 14-3-3 protein inhibits ASK 1 activation by binding to the C-terminal domain.
- ASK1 is closely related to ER stress-induced apoptosis because treatment of ASK1 knockout mouse cells with ER stress inducers significantly suppresses apoptosis compared to wild-type mouse cells. Therefore, ASK1 inhibitors such as thioredoxin and 14-13-3 protein, ASK1 dominant negative mutant, and ASK1 antisense oligonucleotide described above are used for neurodegenerative diseases (eg, polyglutamine disease). Etc.) have been suggested to be effective in preventing and treating ER stress-related diseases (W02 / 38179).
- ASK1 inhibitors such as thioredoxin and 14-13-3 protein, ASK1 dominant negative mutant, and ASK1 antisense oligonucleotide described above are used for neurodegenerative diseases (eg, polyglutamine disease). Etc.) have been suggested to be effective in preventing and treating ER stress-related diseases (W02 / 38179).
- an object of the present invention is to provide novel knowledge on the activation mechanism of ASK1 and the mechanism of apoptosis-induced Z inflammatory response induction through the mechanism. That is, a first object of the present invention is to identify a novel ASK1-binding protein that has not been known so far, and to clarify the mechanism of ASK1 activation control by the protein. Another object of the present invention is to provide a novel preventive and therapeutic means for various diseases involving ASK1, based on the interaction between the protein and ASK1. Disclosure of the invention
- the present inventors used a human ASK1 full-length cDNA as a bait and prepared an expression library derived from human fetal brain by a yeast two-hybrid method.
- a protein of unknown function consisting of 127 amino acids encoded by a known gene named PGR1 as a new ASK1 binding protein.
- ABP 1 a protein of unknown function consisting of 127 amino acids encoded by a known gene named PGR1 as a new ASK1 binding protein.
- the present inventors have found that the protein not only binds to ASK1, but also activates ASK1 and its downstream JNK and p38, and induces caspase-dependent apoptosis. .
- the present inventors have conducted further studies based on these findings, and have completed the present invention.
- a peptide or a salt thereof which contains the same or substantially the same amino acid sequence as a part of the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4, and is capable of activating ASK 1;
- a method for producing the peptide or the salt thereof which comprises culturing the transformant according to the above [7] and collecting the peptide or the salt thereof according to the above [1] from the obtained culture.
- [9] contains an amino acid sequence identical or substantially identical to a part of the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4, and does not activate ASK 1; Or a peptide or a salt thereof that can be inactivated or
- ASK 1 comprising a protein having the same or substantially the same amino acid sequence as the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4, the peptide according to the above [1], or a salt thereof.
- Activation accelerator
- a medicine comprising a protein containing the same or substantially the same amino acid sequence as the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 or the peptide of the above-mentioned [1], or a salt thereof;
- the disease is selected from the group consisting of cancer, autoimmune disease, viral infection, endocrine disease, blood disease, organ hyperplasia, restenosis after angioplasty, and recurrence after cancer resection
- the disease is selected from the group consisting of cancer, autoimmune disease, viral infection, endocrine disease, blood disease, organ hyperplasia, restenosis after angioplasty, and recurrence after cancer resection
- the disease is selected from the group consisting of cancer, autoimmune disease, viral infection, endocrine disease, blood disease, organ hyperplasia, restenosis after angioplasty, and recurrence after cancer resection [20] ]
- Apoptosis or apoptosis comprising a base sequence encoding a protein having the same or substantially the same amino acid sequence as the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4, or a polynucleotide containing a part thereof; Diagnostics for inflammation-related diseases,
- Diseases include cancer, autoimmune disease, viral infection, endocrine disease, blood disease, organ dysplasia, restenosis after angioplasty, recurrence after cancer resection, transplant rejection, graft-versus-host disease, Immune deficiency, neurodegenerative disease, ischemic heart disease, radiation damage, ultraviolet light disorder, toxic disease, nutritional disorder, inflammatory disease, ischemic neuropathy, diabetic neuropathy, vascular disease, respiratory disease and cartilage disease
- a polynucleotide comprising a nucleotide sequence complementary to a nucleotide sequence encoding a protein having the same or substantially the same amino acid sequence as the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4, or a part thereof
- An ASK 1 activation inhibitor comprising:
- a polynucleotide comprising a nucleotide sequence complementary to a nucleotide sequence encoding a protein having the same or substantially the same amino acid sequence as the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4, or a part thereof. Medicines containing
- Diseases include viral infection, endocrine disease, blood disease, organ dysplasia, transplanted organ rejection, graft-versus-host disease, immunodeficiency, neurodegenerative disease, ischemic heart disease, radiation injury, ultraviolet radiation damage, medium Selected from the group consisting of toxic diseases, nutritional disorders, inflammatory diseases, ischemic neuropathies, diabetic neuropathies, vascular diseases, respiratory diseases and cartilage diseases.
- a diagnostic agent for an apoptosis or inflammation-related disease comprising an antibody against a protein having the same or substantially the same amino acid sequence as the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 or a salt thereof,
- the disease is cancer, autoimmune disease, viral infection, endocrine disease, blood disease, organ dysplasia, restenosis after angioplasty, recurrence after cancer resection, transplant rejection, graft-versus-host disease , Immunodeficiency, neurodegenerative disease, ischemic heart disease, radiation damage, ultraviolet light disorder, toxic disease, nutritional disorder, inflammatory disease, ischemic neuropathy, diabetic neuropathy, vascular disease, respiratory disease and cartilage
- the diagnostic agent of the above-mentioned [30] which is selected from the group consisting of diseases;
- an ASK1 activation inhibitor comprising an antibody against a protein or a salt thereof having the same or substantially the same amino acid sequence as the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4,
- a medicament comprising an antibody against a protein or a salt thereof having the same or substantially the same amino acid sequence as the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4,
- Diseases include viral infection, endocrine disease, blood disease, organ dysplasia, transplanted organ rejection, graft-versus-host disease, immunodeficiency, neurodegenerative disease, ischemic heart disease, radiation damage, ultraviolet light damage, medium Selected from the group consisting of toxic diseases, nutritional disorders, inflammatory diseases, ischemic neuropathies, diabetic neuropathies, vascular diseases, respiratory diseases and cartilage diseases.
- a protein containing the same or substantially the same amino acid sequence as the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4, or the peptide of the above-mentioned [1] or a salt thereof, and ASK1 or N-terminal activation The method according to the above [38], wherein binding to a partial peptide containing a regulatory domain or a salt thereof is measured.
- a protein containing the same or substantially the same amino acid sequence as the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4, the peptide of the above-mentioned [1] or a salt thereof, or a cell producing the same A kit for screening for an ASK-1 activation modulator,
- kit according to [40] further comprising ASK1 or a partial peptide thereof containing an N-terminal activation control domain, a salt thereof, or a cell producing the same.
- Activation of ASK1 or the partial peptide or a salt thereof in a cell producing ASK1 or a partial peptide containing an N-terminal activation control domain and a kinase domain or a salt thereof is performed by: (1) SEQ ID NO: 2 or (2) SEQ ID NO: 2 or SEQ ID NO: 4 in the presence of a protein containing the same or substantially the same amino acid sequence as the amino acid sequence shown in SEQ ID NO: 4, or the peptide of the above [1] or a salt thereof.
- Described method, (43) (1) a protein containing the same or substantially the same amino acid sequence as the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4, or the peptide of the above-mentioned [1] or a salt thereof, and 2) Activation of ASK1 or its partial peptide or its salt in cells producing ASK1 or its partial peptide containing the N-terminal activation regulatory domain and kinase domain or its salt is determined by the presence or absence of the test substance.
- a polynucleotide comprising a nucleotide sequence encoding a protein having the same or substantially the same amino acid sequence as the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 or a part thereof, or SEQ ID NO: 2 or the sequence
- the method according to the above-mentioned [44], wherein an antibody against a protein containing the same or substantially the same amino acid sequence as the amino acid sequence shown in No. 4 or a salt thereof is used.
- a polynucleotide comprising a nucleotide sequence encoding a protein having the same or substantially the same amino acid sequence as the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4, or a polynucleotide containing a part thereof, or SEQ ID NO: 2
- a screening kit for an ASK1 activation modulator comprising an antibody against a protein having the same or substantially the same amino acid sequence as the amino acid sequence shown in SEQ ID NO: 4, or a salt thereof
- an apoptosis-inducing agent comprising a substance having an amino acid sequence identical or substantially identical to the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 or a substance that increases the expression or activity of a salt thereof,
- the disease is selected from the group consisting of cancer, autoimmune disease, viral infection, endocrine disease, blood disease, organ hyperplasia, restenosis after angioplasty, and recurrence after cancer resection [49]
- an apoptotic or inflammatory cytokine comprising a substance having the same or substantially the same amino acid sequence as the SEQ ID NO: 2 or SEQ ID NO: 4 or a substance that reduces the expression or activity of a salt thereof Production inhibitor,
- a medicament comprising a substance that decreases the expression or activity of a protein or a salt thereof having the same or substantially the same amino acid sequence as the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4,
- the medicament according to the above [52] which is an agent for preventing or treating a disease that is therapeutically effective by suppressing apoptosis or inflammation, and
- Diseases include viral infection, endocrine disease, blood disease, organ dysplasia, transplanted organ rejection, graft-versus-host disease, immunodeficiency, neurodegenerative disease, ischemic heart disease, radiation damage, ultraviolet light damage, medium
- the drug according to the above [53] which is selected from the group consisting of a toxic disease, a nutritional disorder, an inflammatory disease, an ischemic neuropathy, a diabetic neuropathy, a vascular disease, a respiratory disease, and a cartilage disease.
- a pharmaceutical comprising an ASK 1 partial peptide or a salt thereof that does not contain
- Diseases include viral infection, endocrine disease, blood disease, organ dysplasia, transplanted organ rejection, graft-versus-host disease, immunodeficiency, neurodegenerative disease, ischemic heart disease, radiation damage, ultraviolet light damage, medium 41.
- the medicament according to claim 40 which is selected from the group consisting of a toxic disease, a nutritional disorder, an inflammatory disease, an ischemic neuropathy, a diabetic neuropathy, a vascular disease, a respiratory disease, and a cartilage disease.
- An ASK1 activation inhibitor comprising the peptide of the above-mentioned [9] or a salt thereof,
- a medicament comprising the peptide of the above-mentioned [9] or a salt thereof,
- Diseases include viral infection, endocrine disease, blood disease, organ dysplasia, transplanted organ rejection, graft-versus-host disease, immunodeficiency, neurodegenerative disease, ischemic heart disease, radiation damage, ultraviolet light damage, medium
- the present invention provides the medicament of the above-mentioned [63], which is selected from the group consisting of toxic diseases, nutritional disorders, inflammatory diseases, ischemic neuropathies, diabetic neuropathies, vascular diseases, respiratory diseases and cartilage diseases.
- FIG. 1 shows the alignment of the AB.P1 amino acid sequence of human (top row) and mouse (bottom row). The portion surrounded by a square indicates the partial amino acid sequence used as an antigen for producing an antibody. Boxes indicate partial sequences used as antigenic peptides for producing the two antibodies (ELA antibody and LVR antibody) of the present invention.
- FIG. 2A shows the tissue distribution of ABP1 mRNA expression in mouse tissues.
- FIG. 2B shows the daily change of ABP1 mRNA expression in mouse fetal tissues.
- FIG. 3 shows the increase in detection intensity in a plasmid amount-dependent manner when the ABP1 protein is overexpressed by transfecting HEK293 cells with a plasmid that expresses ABP1 without a tag. Cells were collected 24 hours after gene transfer, each cell extract was divided into two, SDS-PAGE was performed, and Imno plot (IB) was performed using the ELA antibody and the LVR antibody.
- ABP1-PCMA3 (-) indicates non-transformed HEK293 cells, and the gradient indicates that the amount of plasmid increases from left to right.
- Figures 4A-B show the results of a binding test between ABP1 and ASK1.
- Figure 4A Transfection of F1ag-ABP1 and Myc-ASK1 plasmids into HEK293 cells, harvested cells 24 hours later, immunoprecipitated with anti-F1ag antibody, and immunoblotted with anti-Myc antibody Analysis was performed.
- Figure 4B Transfection of F1ag-ASK1 and CFP-ABP1 plasmids into HEK293 cells, harvested cells 24 hours later, immunoprecipitated with anti-F1ag antibody, and immunoblot analysis with anti-GFP antibody was done. In addition, it is a CFI ⁇ GFP mutant and can be recognized by an anti-GFP antibody.
- FIG. 4A the right two lanes with B, H 2 ⁇ 2 treatment (0. 5 mM, 1 hour) was also performed.
- the lower part shows a part of each cell lysate before immunoprecipitation, which was separately electrophoresed.
- IP stands for immunoprecipitation
- IB stands for immunoblot.
- FIG. 5A shows a schematic diagram of an ASK1-deficient mutant.
- An HA tag was added to the N-terminal of each plasmid containing the ASK1-deficient mutant.
- Amino acid numbers 678-936 (shaded area) indicate the kinase domain.
- FIG. 5B shows the results of a binding test between ABP1 and an ASK1-deficient mutant. The lower panel shows a portion of each cell lysate before immunoprecipitation, which was separately electrophoresed. IP represents immunoprecipitation, and IB represents immunoblot.
- FIGS. 6A-B show the induction of cell death by ABP 1 in HeLa cells.
- Figure 6A Fluorescence microscope image (upper) and differential interference image (lower) of the same field of view 36 hours after transfection of CFP or CFP-ABP1 plasmid into HeLa cells.
- Figure 6B Percentage of cell death (%) 36 hours after gene transfer.
- FIGS. 7A-B show the induction of cell death by ABP1 in PAE- ⁇ cells.
- Figure 7 A PAE-ABP1 cells in the presence (+) and absence (1) of tetracycline (Tet) Shows a phase-contrast microscope image 36 hours after culturing.
- Figure 7B Percentage of cell death (%) after culture in the presence of tetracycline (Oh) and at 24 and 48 hours after tetracycline removal.
- FIG. 8 shows cell death induction (DNA fragmentation) by ABP1 in PAE-E cells. After removing tetracycline from ⁇ - ⁇ cells, DNA fragmentation at each time was examined by agarose gel electrophoresis (upper panel). Both ends indicate molecular weight markers (M), and the right end indicates size. The lower panel shows the expression of My c-ABP 1 protein in each cell confirmed by immunoblot analysis using an anti-My c antibody.
- FIGS. 9A-B show the caspase dependence of cell death by ABP1.
- Figure 9A PAE-ABP1 cells were cultured without tetracycline and caspase 3 activity was measured after 0, 12, 24, and 36 hours.
- FIGS. 10A-B show cell death induction by ABP1-deficient mutants.
- FIG. 10A shows a schematic diagram of an ABP1-deficient mutant. A CFP evening was added to the N-terminal side in the same manner as in the wild type.
- Figure 10B Percentage of cell death (%) 36 hours after gene transfer in cells transfected with ABP1-deficient mutants.
- Figures 11A-B show activation of 31: 1, JNK :, p38 according to 88-1.
- Figure 11A ⁇ - ⁇ cells were cultured without tetracycline, and endogenous JNK and p38 activation was performed by immunoblot analysis with each anti-phosphoprotein antibody.
- Figure 1 IB PAE-ABP1 cells were cultured without tetracycline, and the activation of endogenous ASK1 was examined by immunoblot analysis using an anti-phosphorylation protein antibody. IB stands for immunoblot.
- ABSP 1 of the present invention is a protein containing an amino acid sequence identical or substantially identical to the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4.
- the ABP 1 of the present invention can be prepared from warm-blooded animals (eg, humans, mice, rats, guinea pigs, hamsters, puppies, higgs, goats, pigs, puppies, puppies, birds, cats, dogs, salmon, chimpanzees, etc.).
- warm-blooded animals eg, humans, mice, rats, guinea pigs, hamsters, puppies, higgs, goats, pigs, puppies, puppies, birds, cats, dogs, salmon, chimpanzees, etc.
- Cells eg, hepatocytes, spleen cells, nerve cells, glial cells, splenic i3 cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, goblet cells, endothelial cells, smooth muscle cells, fibers Blast cells, fiber cells, muscle cells, fat cells, immune cells (eg, macrophages, T cells, B cells, natural killer cells, mast cells, neutrophils, basophils, eosinophils, monocytes), Megakaryocytes, synovial cells, chondrocytes, bone cells, osteoblasts, osteoclasts, mammary cells, hepatocytes or stromal cells, or precursors, stem cells or cancer cells of these cells) Or any tissue where these cells are present, such as the brain, parts of the brain (eg, olfactory bulb, amygdala, basal sphere, hippocampus, thalamus, hypothala
- the “substantially identical amino acid sequence” to the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 is about 70% or more, preferably about 8% or more to the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4.
- the “protein containing an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4” includes, for example, a protein substantially identical to the amino acid sequence represented by SEQ ID NO: 2 or 4 shown above. Having the same amino acid sequence, and substantially the same as the protein having the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4. Proteins having activity are preferred.
- Substantially the same activity includes, for example, ASK 1 or a kinase group located downstream thereof (eg, MKK4 / 7, MKK3 / 6, dish, p38, etc.) Apoptosis-inducing activity and the like. Substantially the same means that the properties are qualitatively (eg, physiologically or pharmacologically) the same. Therefore, it is preferable that the activities such as promotion of ASK1 cascade activation are the same, but the quantitative factors such as the degree of these activities and the molecular weight of the protein may be different (for example, About 0.1 to 100 times, preferably about 0.1 to 10 times, and more preferably 0.5 to 2 times).
- ASK1 cascade activation promoting activity can be measured by a known method, for example, by using a labeled phosphate group donor and using a kinase group located at or downstream of ASK1 (eg, MKK4 / 7, MKK3 / 6, JNK, p38), etc., and apoptosis-inducing activity should be measured by measuring cell death induction rate, morphological observation of cells, detection of DNA fragmentation, etc. Can be.
- ABP 1 of the present invention includes, for example, 1) 1 or 2 or more (preferably about 1 to 30, preferably 1 to 1) in the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4; About 0 amino acids, more preferably an amino acid sequence in which the number of amino acids (1 to 5) has been deleted, (2) one or more amino acid sequences in the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 (preferably, An amino acid sequence having about 1 to 30 amino acids, preferably about 1 to 10 amino acids, and more preferably a number of (1 to 5) amino acids; 3) an amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4; Or an amino acid sequence into which two or more (preferably about 1 to 30, preferably about 1 to 10, and more preferably a number (1 to 5)) amino acids have been inserted; One or more of the amino acid sequences shown in No.
- amino acid sequence in which about 1 to 30 amino acids, preferably about 1 to 10 amino acids, and more preferably a number (1 to 5) amino acids are substituted with another amino acid, or a combination thereof.
- muteins such as proteins having the same amino acid sequence.
- ABP1 of the present invention is preferably human ABP1 (hABPl) having the amino acid sequence shown in SEQ ID NO: 2 or mouse ABP1 (mABPl) having the amino acid sequence shown in SEQ ID NO: 4, Alternatively, it is a homolog thereof in other warm-blooded animals (eg, rats, guinea pigs, hamsters, egrets, higgins, goats, pigs, lions, sesame, birds, cats, dogs, monkeys, chimpanzees, etc.).
- warm-blooded animals eg, rats, guinea pigs, hamsters, egrets, higgins, goats, pigs, lions, sesame, birds, cats, dogs, monkeys, chimpanzees, etc.
- hABPl is a protein consisting of 127 amino acids which is encoded by a known gene (GenBank accession number: AF116272) derived from a human T cell named PGR1, but its function has not been reported so far.
- Mouse ABP1 is a protein consisting of 125 amino acids that is encoded in a mouse brain-derived cDNA named clone MNCb-1039 (GenBank accession numberAB041651). Absent.
- ABP 1 of the present invention including a protein containing the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4, has a C-terminal lipoxyl group (one COOH), a carboxylate (_COO one), an amide (-CONH 2 ) or ester (one COOR).
- R in the ester e.g., methyl, Echiru, n- propyl, isopropyl
- alkyl groups such as n- butyl; for example, cyclopentyl, C 3 _ 8 cycloalkyl group such as cyclohexyl; e.g., phenyl, " - C 6 one 12 Ariru group such as naphthyl le; for example, benzyl, phenylene Lou C t _ 2 alkyl group such as phenethyl; single-naphthyl, such as ⁇ - naphthylmethyl - Ci_ 2 (7 _ 14 Ararukiru such as an alkyl group A pivaloyloxymethyl group or the like is used.
- ABP 1 has a lipoxyl group (or carboxylate) at a position other than the C-terminus
- ABP 1 in which the lipoxyl group is amidated or esterified is also included in the ABP 1 of the present invention.
- the ester in this case, for example, the above-mentioned C-terminal ester and the like are used.
- amino acid residues e.g., Mechionin residues
- N-terminal Amino group protecting groups e.g., formyl group, such as ⁇ ⁇ 6 Al force Noiru such Asechiru group ⁇ e Ashiru Group
- N-terminal glutamine residue formed by cleavage in vivo, pyroglutamine oxidation
- Substituent on the side chain of amino acid in the molecule for example, --OH, SH, amino group, imidazo Ichiru group, i Ndoru group, Guanijino group, etc.
- a suitable protecting group e.g., formyl group, C i _ 6 Ashiru groups such as C E _ 6 Al force Noiru group such Asechi Le group
- complex proteins such as so-called glycoproteins to which sugar chains are bound.
- the present invention provides a peptide having the partial amino acid sequence of ABP1 described above, and having substantially the same activity as ABP1.
- substantially the same activity has the same meaning as described above.
- the “substantially the same activity” can be measured in the same manner as described above.
- the partial peptide is hereinafter referred to as “activating peptide of the present invention”.
- the activating peptide of the present invention is not particularly limited as long as it has the above properties.
- the activating peptide of the present invention has about 60 amino acids or more, preferably about 60 to about 1 Peptides having the same or substantially the same amino acid sequence as the partial amino acid sequence consisting of 100 amino acids, more preferably about 60 to about 80 amino acids, and the like.
- the partial amino acid sequence may be an N-terminal sequence of ABP1, a C-terminal sequence, or an internal sequence. Alternatively, a combination of these partial sequences may be used.
- the activating peptide of the present invention is at least about 60 amino acids on the N-terminal side of the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4, more preferably about 60 to about 100 amino acids, particularly preferably It contains a partial amino acid sequence of about 60 to about 80 amino acids.
- the activating peptide of the present invention may exhibit an activity even higher than that of the full-length protein (eg, ASK1 cascade activation promoting activity, apoptosis inducing activity, etc.).
- the partial peptides of ABP 1 include those that can function as (antagonistic) inhibitors of ABP 1 or the “activating peptide of the present invention”. Examples of such partial peptides include those which have binding activity to ASK1, but cannot activate the kinase.
- the partial peptide is hereinafter referred to as “the inhibitory peptide of the present invention”.
- the inhibitory peptide of the present invention contains the same or substantially the same amino acid sequence as a part of the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4, and does not activate or inactivate ASK1. It is a peptide that can be converted.
- the inhibitory peptide include those containing a partial amino acid sequence consisting of about 35 amino acids or less in the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4, and preferably containing a partial amino acid sequence on the N-terminal side.
- the partial peptide of ABP 1 of the present invention (including both the activating peptide of the present invention and the inhibitory peptide of the present invention; hereinafter, it may be simply abbreviated as “partial peptide of the present invention”) has a C-terminal. carboxyl group (one COOH :), Karupokishireto (one COO-), amide (- CONH 2), or may be any of the ester (-COOR).
- examples of R in the ester include the same as described above for ABP1.
- these peptides have a carboxyl group (or propyloxylate) other than the C-terminal, those in which the lipoxyl group is amidated or esterified are also included in the partial peptide of the present invention.
- the ester in this case, for example, the above-mentioned C-terminal ester or the like is used.
- the partial peptides of the present invention include those in which the amino group of the N-terminal methionine residue is protected with a protecting group, and those in which the N-terminal side is cleaved in vivo to form G 1 Also includes those in which n is pyroglutamine-oxidized, those in which the substituent on the side chain of the amino acid in the molecule is protected by an appropriate protecting group, and those which are combined with a sugar chain such as a so-called glycopeptide. .
- Examples of the salt of ABP1 or a partial peptide thereof of the present invention include physiologically acceptable salts with an acid or a base, and a physiologically acceptable acid addition salt is particularly preferable.
- Such salts include, for example, inorganic acids (eg, hydrochloric acid, phosphoric acid, bromide water) Salts with organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzene) And sulfonic acid).
- inorganic acids eg, hydrochloric acid, phosphoric acid, bromide water
- Salts with organic acids eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid
- the ABP1 of the present invention or a salt thereof can be prepared from the cells or tissues of a warm-blooded animal by the protein purification method known per se. Specifically, the tissue or cells of a warm-blooded animal are homogenized, and the soluble fraction is separated and purified by chromatography such as reverse phase chromatography, ion exchange chromatography, affinity chromatography, etc. ABP1 or a salt thereof can be produced.
- ABP1 of the present invention or a partial peptide thereof or a salt thereof can also be produced according to a known peptide synthesis method.
- the peptide synthesis method may be, for example, either a solid phase synthesis method or a liquid phase synthesis method.
- the target protein can be produced by condensing a partial peptide or amino acid capable of constituting ABP1 with the remaining portion and, when the product has a protecting group, removing the protecting group.
- condensation and the elimination of the protecting group are performed according to a method known per se, for example, the methods described in the following 1 to 5.
- the ABPs 1 thus obtained can be isolated and purified by a known purification method.
- purification methods include, for example, solvent extraction, distillation, and column chromatography. Chromatography, liquid chromatography, recrystallization, and combinations thereof.
- the free form can be converted into an appropriate salt by a known method or a method analogous thereto, and conversely, the protein (peptide) is converted into a salt.
- the salt can be converted to a free form or another salt by a known method or a method analogous thereto.
- commercially available resins for protein synthesis can be used for the synthesis of the ABP1s of the present invention.
- Such resins include, for example, chloromethyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, MM resin, 4 I-Hydroxymethylmethylphenylacetamidomethyl resin, polyacrylamide resin, 4- (2 ', 4, dimethoxyphenylhydroxymethyl) phenoxy resin, 4- (2', 4'dimethoxyphenyl-Fmocaminoethyl) phenoxy resin And the like.
- an amino acid having an amino group and a side chain functional group appropriately protected can be replaced with a target protein or peptide (hereinafter sometimes collectively referred to as “white matter, etc.”) according to the sequence.
- the condensation is carried out on the resin according to various condensation methods known per se. At the end of the reaction, proteins and the like are cut out from the resin, and at the same time, various protecting groups are removed. Further, an intramolecular disulfide bond formation reaction is carried out in a highly diluted solution to obtain a target protein or the like or an amide thereof.
- the protected amino acid may be added directly to the resin along with a racemization inhibitor additive (eg, HOBt, ⁇ ⁇ ⁇ ⁇ ⁇ ), or symmetric anhydrides or HOBt esters or HOOBt
- a racemization inhibitor additive eg, HOBt, ⁇ ⁇ ⁇ ⁇ ⁇
- symmetric anhydrides or HOBt esters or HOOBt The ester can be added to the resin after activation of the protected amino acid in advance as an ester.
- Solvents used to activate protected amino acids and condense with resins are used in protein condensation reactions. It can be appropriately selected from solvents known to be usable. For example, acid amides such as N, N-dimethylformamide, N, N-dimethylacetamide, N-methylpyrrolidone, halogenated hydrocarbons such as methylene chloride and chloroform, alcohols such as trifluoroethanol , Sulfoxides such as dimethylsulfoxide, amines such as pyridine, ethers such as dioxane and tetrahydrofuran, nitriles such as acetonitrile and propionitrile, esters such as methyl pentoxide and ethyl acetate, or esters thereof.
- acid amides such as N, N-dimethylformamide, N, N-dimethylacetamide, N-methylpyrrolidone
- halogenated hydrocarbons such as methylene chloride and chloroform
- alcohols such
- the reaction temperature is appropriately selected from the range known to be usable for the protein bond formation reaction, and is usually appropriately selected from the range of about 120 T: to 50.
- the activated amino acid derivative is usually used in a 1.5 to 4-fold excess.
- the protection of the functional group which should not be involved in the reaction of the raw materials, the protecting group, the elimination of the protective group, the activation of the functional group involved in the reaction, and the like can be appropriately selected from known groups or known means.
- Examples of the protecting group for the starting amino group include Z, Boc, tertiary pentoxycarbonyl, isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, C1_Z, Br—Z, a Damantyloxycarbonyl, trifluoroacetyl, phthaloyl, formyl, 212-trophenylsulfenyl, diphenylphosphinothioyl, Fmoc and the like are used.
- the lipoxyl group may be, for example, an alkyl esterified (eg, methyl, ethyl, propyl, butyl, tert-butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl, etc.) Or cyclic alkyl esterification), aralkyl esterification (eg, benzyl ester, 4-nitrobenzyl ester, 4-methoxybenzyl ester, 4-cyclobenzyl ester, benzhydryl esterification), fenash Esterification, ben It can be protected by ziroxycarbonyl hydrazide, tertiary butoxycarbonyl hydrazide, trityl hydrazide, or the like.
- an alkyl esterified eg, methyl, ethyl, propyl, butyl, tert-butyl, cyclopen
- the hydroxyl group of serine can be protected, for example, by esterification or etherification.
- a group suitable for this esterification for example, a lower alkanol group such as an acetyl group, an aroyl group such as a benzoyl group, a group derived from carbonic acid such as a benzyloxycarbonyl group, an ethoxycarponyl group, and the like are used.
- Examples of the group suitable for etherification include a benzyl group, a tetrahydrobiranyl group, and a t-butyl group.
- the protecting group of the phenolic hydroxyl group of tyrosine for example, B z 1, C l 2 - B z 1, 2- two Torobenjiru, B r- Z, such as tertiary butyl is used.
- the protecting group for imidazole of histidine for example, Tos, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum, Boc, Trt, Fmoc and the like are used.
- Methods for removing (eliminating) protecting groups include, for example, catalytic reduction in a hydrogen stream in the presence of a catalyst such as Pd-black or Pd-carbon, or hydrogen fluoride anhydride, methanesulfonic acid, or trifluoromethane.
- a catalyst such as Pd-black or Pd-carbon, or hydrogen fluoride anhydride, methanesulfonic acid, or trifluoromethane.
- Acid treatment with dichloromethane, trifluoroacetic acid or a mixture thereof, base treatment with diisopropylethylamine, triethylamine, piperidine, piperazine, etc., reduction with sodium in liquid ammonia, etc. Is also used.
- the elimination reaction by the above acid treatment is generally carried out at a temperature of about 120 ° C to 40 ° C.
- anisol for example, anisol, phenol, thioanisole, methcresol, paracresol, dimethylsulfide
- a force-thione scavenger such as 1,4-butanedithiol, 1,2-ethanedithiol and the like.
- the 2,4-dinitrophenyl group used as an imidazole protecting group of histidine is removed by thiophenol treatment
- the formyl group used as an indole protecting group of tributofan is 1,2-ethanedithiol, 1,4-
- alkali treatment with dilute sodium hydroxide solution, dilute ammonia and the like.
- activated raw oxypoxyl groups include, for example, corresponding acid anhydrides, azides, and active esters [alcohols (eg, pentachlorophenol, 2,4,5-trichlorophenol, 2,4 —Dinitrophenol, cyanomethyl alcohol, paranitrophenol, HONB, N-hydroxysuccinimide, N-hydroxyphthalimide, ester with HOB t)].
- active esters eg, pentachlorophenol, 2,4,5-trichlorophenol, 2,4 —Dinitrophenol, cyanomethyl alcohol, paranitrophenol, HONB, N-hydroxysuccinimide, N-hydroxyphthalimide, ester with HOB t
- activated amino group of the raw material for example, a corresponding phosphoric amide is used.
- an amide form of a protein or the like for example, firstly, after amidating and protecting the carboxy-terminal amino acid of the amino acid, a peptide (protein) chain is added to the amino group side to a desired length. After the elongation, a protein or the like from which only the protecting group for the N-terminal ⁇ -amino group of the peptide chain has been removed and a protein or the like from which only the protecting group for the C-terminal lipoxyl group has been removed are produced. Are condensed in a mixed solvent as described above. Details of the condensation reaction are the same as described above.
- the crude protein or the like is purified by various known purification means, and the main fraction is freeze-dried to obtain an amide such as a desired protein.
- ester of a protein or the like for example, after condensing a carboxy terminal amino acid lupoxyl group with a desired alcohol to form an amino acid ester, the ester of the desired protein or the like is obtained in the same manner as the amide of a protein or the like. You can get the body.
- the partial peptide of the present invention or a salt thereof can also be produced by cleaving the peptide 1 or a salt thereof with an appropriate peptidase.
- ABPs 1 of the present invention can be obtained by culturing a transformant containing DNA encoding ⁇ 1 or a partial peptide thereof, and isolating and purifying ⁇ ⁇ 1 from the resulting culture. It can also be manufactured.
- Examples of the DNA encoding ABP 1 or a partial peptide thereof of the present invention include genomic DNA, genomic DNA library, human or other warm-blooded animals (eg, human, mouse, rat, guinea pig, hamster, egret, sheep) , Goat, bush, Poma, bird, cat, dog, monkey, chimpanzee, and any other cell (eg, splenocytes, nerve cells, glial cells, spleen cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, Endothelial cells, fibroblasts, fibrocytes, muscle cells, adipocytes, immune cells (eg, macrophages, T cells, B cells, natural killer cells, mast cells, neutrophils, basophils, eosinophils, monocytes ), Megakaryocytes, synovial cells, chondrocytes, osteocytes, osteoblasts, osteoclasts
- the vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like. Alternatively, it can be directly amplified by Reverse Transcriptase Polymerase Chain Reaction (hereinafter abbreviated as lRT-PCR) using a total RNA or mRNA fraction prepared from the cells and tissues described above.
- lRT-PCR Reverse Transcriptase Polymerase Chain Reaction
- Examples of the DNA encoding ABP 1 of the present invention include, for example, DNA containing the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3, or the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 and high stringency Activity that is substantially the same as that of a protein containing a nucleotide sequence that hybridizes under simple conditions and having the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 (eg, ASK1 cascade activation promoting activity) , An apoptosis-inducing activity, etc.).
- DNA encoding ABP 1 of the present invention include, for example, DNA containing the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3, or the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 and high stringency Activity that is substantially the same as that of a protein containing a nucleotide sequence that hybridizes under simple conditions and
- DNA that can hybridize with the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 under high stringency conditions examples include, for example, SEQ ID NO: 1 or SEQ ID NO: 4794
- Hybridization can be performed by a method known per se or a method analogous thereto, for example, the method described in Molecular Cloning, 2nd edition (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). And so on. When a commercially available library is used, hybridization can be performed according to the method described in the attached instruction manual. Hybridization can preferably be performed according to high stringency conditions. '
- High stringency conditions refer to, for example, a sodium concentration of about 19 to 40 mM, preferably about 19 to 20 mM, and a temperature of about 50 to 70 ° C, preferably about 60 to 65 ° C. In particular, a sodium concentration of about 19 mM and a temperature of about 65 are preferred.
- the DNA encoding ABP1 of the present invention is preferably hABP1 DNA containing the nucleotide sequence shown in SEQ ID NO: 1 or mABP1 DNA containing the nucleotide sequence shown in SEQ ID NO: 3, or other warm blood. And homologs thereof in animals (eg, rats, guinea pigs, hamsters, egrets, sheep, goats, goats, bushes, puppies, pomas, birds, cats, dogs, monkeys, chimpanzees, etc.).
- animals eg, rats, guinea pigs, hamsters, egrets, sheep, goats, goats, bushes, puppies, pomas, birds, cats, dogs, monkeys, chimpanzees, etc.
- the DNA encoding the partial peptide of the present invention is not limited as long as it contains a base sequence encoding the same or substantially the same amino acid sequence as a part of the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4. It may be something. Further, any of genomic DNA, genomic DNA library, cDNA derived from the above-described cells and tissues, cDNA library derived from the above-described cells and tissues, and synthetic DNA may be used.
- the vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like. Alternatively, amplification can be performed directly by the RT-PCR method using an mRNA fraction prepared from the above-mentioned cell'tissue.
- the DNA encoding the partial peptide of the present invention includes, for example, (1) DNA having a partial base sequence of DNA having the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3, or.
- DNA encoding a peptide having an activity of inhibiting the activity of a protein containing an amino acid sequence encoded by the DNA eg, ASK1 cascade activation inhibitory activity, apoptosis inhibitory activity, etc.
- the DNA encoding ABP1 of the present invention or a partial peptide thereof can be amplified by PCR using a synthetic DNA primer having a part of the nucleotide sequence encoding the protein or peptide, or can be prepared in an appropriate expression vector.
- the integrated DNA can be cloned by hybridizing with a DNA fragment encoding a part or the entire region of the protein of the present invention or with a labeled synthetic DNA. Hybridization can be carried out, for example, according to the method described in Molecular 'Cloning, 2nd edition (described above). When a commercially available library is used, hybridization can be performed according to the method described in the instruction manual attached to the library.
- the DNA base sequence can be determined using known kits such as Mutan TM -Super Express Km (Takara Shuzo Co., Ltd.), Mutan TM -K (Takara Shuzo Co., Ltd.), and the like.
- the conversion can be carried out according to a method known per se, such as the dup 1 ex method or the Kunke method, or a method analogous thereto.
- the cloned DNA can be used as it is depending on the purpose, or after digestion with a restriction enzyme, if desired, or after adding a linker.
- the DNA may have ATG as a translation initiation codon at its 5 'end and TAA, TGA or TAG as a translation termination codon at its 3' end. These translation initiation codon and translation termination codon can be added using a suitable synthetic DNA adapter.
- the DNA expression vector encoding ABP1 or a partial peptide thereof of the present invention can be obtained, for example, by cutting out a DNA fragment of interest from DNA encoding ABP1, and ligating the DNA fragment downstream of a promoter in an appropriate expression vector. Can be manufactured.
- Plasmids derived from Escherichia coli eg, pBR322, pBR325, pUC12, pUC13
- plasmids derived from Bacillus subtilis eg, pUBll0, pTP5, pC194
- plasmids derived from yeast For example, pSH19, pSHl5
- Pacteriophage such as ⁇ phage
- animal viruses such as retrovirus, vaccinia virus, and baculovirus
- pA1-11, pXTl, pXTl, pRc / CMV, pRc / RSV, pc DNA I ZNeo and the like are used.
- Any promoter may be used as long as it is appropriate for the host used for gene expression.
- SRa promoter when the host is an animal cell, SRa promoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, HSV-TK promoter and the like are used. Among them, a CMV promoter, an SR promoter and the like are preferable.
- the host is a bacterium belonging to the genus Escherichia, a trp promoter, a lac promoter
- re cA promoter primary AP L promoter
- lpp promoter mono- such as the T7 opening motor are preferred.
- the host is a Bacillus genus, SPOL promoter, SP02 promoter First, pen P promoter is preferred.
- PH-5 promoter When the host is yeast, PH-5 promoter, PGK promoter, GAP promoter, ADH promoter and the like are preferable.
- a polyhedrin promoter When the host is an insect cell, a polyhedrin promoter, a P10 promoter and the like are preferable.
- an expression vector in addition to the above, those containing an enhancer, a splicing signal, a polyA addition signal, a selection marker, an SV40 replication origin (hereinafter sometimes abbreviated as SV40 ori), and the like, if desired, may be used. be able to.
- the selection marker one, for example, dihydrofolate reductase (hereinafter sometimes abbreviated as dh fr) gene [methotrexate (MTX) resistance], ampicillin resistant gene (hereinafter sometimes abbreviated as Amp r), neomycin resistance gene (hereinafter sometimes abbreviated as Ne o 1 ", G418 resistant) and the like.
- dh fr gene deficient Chinese hamster cells when using the dh fr gene as a selectable Ma one car, The target gene can also be selected on a thymidine-free medium.
- a signal sequence suitable for the host may be added to the N-terminal side of the protein of the present invention. If the host is Escherichia, the PhoA signal sequence, OmpA signal sequence, etc .; if the host is Bacillus, the polymerase, the subtilisin signal sequence, etc .; In some cases, MF ⁇ signal sequence, SUC2 signal sequence, etc .; when the host is an animal cell, insulin signal sequence, a-interferon signal sequence, antibody molecule, signal sequence, etc. are used, respectively.
- the transformant containing the “DNA encoding ABP 1 of the present invention or a partial peptide thereof” obtained as described above is transformed into a host with an expression vector containing the DNA according to a known method. It can be manufactured by conversion.
- the expression vectors include those described above.
- Escherichia bacteria for example, Escherichia bacteria, Bacillus bacteria, yeast, insect cells, insects, animal cells, and the like are used.
- Escherichia bacteria for example, Escherichia coli K12 ⁇ DH1 [Processing's of the National 'Academy', 'Science', 'The' USA. Acad. Sci. USA), 60, 160 (1968)], JM103 [Nucleic Acids Research, 9th volume, 309 (1981)], JA 221 [Jianalileo ob.
- Bacillus subtilis Bacillus subtilis
- MI 114 Gene, 24, 255 (1983)
- 207—21 Journal of Biochemistry, 95, 87 (1984)].
- yeast examples include, for example, Saccharomyces cerevisiae AH22, AH22R—, ⁇ 87-11 A, DKD-5D, 20 B—12, Schizosaccharomyces pombe NC YC 193 3, NC YC 2036 Pichia pastoris K M71 or the like is used.
- Insect cells include, for example, when the virus is Ac NPV, a cell line derived from a larva of night moth (Spodoptera frugiperda cell; S f cell), an MG1 cell derived from the midgut of Trichoplusia ni, or an egg derived from egg of Trichoplusia ni High Five TM cells, cells derived from Mamestra brassicae, cells derived from Estigmena acrea and the like are used.
- Sf cells include Sf9 cells (ATCC CRL1711) and Sf21 cells (Vaughn, JL et al., In Vivo, 13, 213-217, (1977)) and the like. Is used.
- insects for example, silkworm larvae are used [Maeda et al., Nature, 315, 592 (1985)].
- animal cells include monkey cells COS-7, Vero, Chinese Hams Yuichi cell CHO (hereinafter abbreviated as CHO cells), dh fr gene-deficient Chinese Hamster Yuichi cell CHO (hereinafter CHO (dh fr-1 2)
- CHO cells Chinese Hams Yuichi cell CHO (hereinafter abbreviated as CHO cells)
- CHO (dh fr-1 2) Mouse L cells, mouse AtT-20, mouse myeloma cells, rat GH3, human FL cells, etc. are used.
- Transformation can be performed according to a known method depending on the type of host. Escherichia bacteria are described, for example, in Proc. Natl. Acad. Sci. USA, 69, 21. 10 (1972) and Gene (17), 107 (1 982).
- Bacillus can be transformed, for example, according to the method described in Molecular & General Genetics, Vol. 168, 11 (1979).
- Yeasts are described, for example, in Methods in Enzymology, Vol. 194, 182–187 (1991), Processings of the National Academy, Obs. Transformation can be performed according to the method described in The USA (Proc. Natl. Acad. Sci. USA), 75, 1929 (1978).
- Insect cells and insects can be transformed according to the method described in, for example, Bio / Technology, 6, 47-55 (1988).
- Animal cells are transformed, for example, according to the method described in Cell Engineering Separate Volume 8, New Cell Engineering Experimental Protocol. 263-267 (1995) (published by Shujunsha), Virology, 52, 456 (1973). be able to.
- the transformant can be cultured according to a known method depending on the type of the host.
- a liquid medium is preferably used as the culture medium.
- the medium preferably contains a carbon source, a nitrogen source, inorganic substances, etc. necessary for the growth of the transformant.
- carbon sources include, for example, glucose, dextrin, soluble starch, sucrose, etc .
- nitrogen sources include, for example, ammonium salts, nitrates, corn steep liquor, peptone, casein, meat extract, Inorganic or organic substances such as soybean meal and potato extract
- examples of inorganic substances include calcium chloride, sodium dihydrogen phosphate, and magnesium chloride.
- the medium may be supplemented with yeast extract, vitamins, growth promoting factors and the like.
- the pH of the medium is preferably about 5-8.
- an M9 medium containing glucose and casamino acid As a culture medium for culturing a transformant whose host is a bacterium belonging to the genus Escherichia, for example, an M9 medium containing glucose and casamino acid [Miller, Jauna lab espermentin] Molecular'Genetics (Journal of Experiments in Molecular Genetics), 431-433, Cold Spring Harbor Laboratory, New York 1972] is preferred. If necessary, an agent such as 3) 3-indolylacrylic acid may be added to the medium to make the mouth motor work efficiently.
- Culture of a transformant whose host is a genus Escherichia is usually carried out at about 15 to 43 ° C for about 3 to 24 hours. If necessary, ventilation or stirring may be performed.
- Burkholder's minimal medium As a medium for culturing a transformant in which the host is yeast, for example, Burkholder's minimal medium [Bostian, KL et al., Processings' ob. The National Academy of Cultures, Inc.] Pro atl. Acad. Sci. USA, 77, 4505 (1980)] and 0.
- the culture medium for culturing insect cells or transformants whose insects are insects is, for example, inactivated by Grace's Insect Medium (Grace, TCC, Nature, 195, 788 (1962)). For example, those to which additives such as 10% serum obtained as described above are appropriately added are used.
- the pH of the medium is preferably about 6.2 to 6.4.
- the cultivation is usually performed at about 27 ° C for about 3 to 5 days. Ventilation and stirring may be performed as necessary.
- Examples of a medium for culturing a transformant in which the host is an animal cell include a MEM medium containing about 5 to 20% fetal bovine serum [Science, Vol. 122, 501 (1952)], DMEM medium [Virology, 8, 396 (1959)], RPMI 1640 medium [Journal 'ob' the 'American' Medicafre 'association- The Journal of the American Medical Association, volume 199 Proceding of the Society for the Biological Medicine, Vol. 73, 1 (1950)], 199 Medium [Proceding of the Society for the Biological Medicine] Are used.
- the pH of the medium is preferably about 6-8.
- the cultivation is usually performed at about 30 ° C to 40 ° C for about 15 to 60 hours. Ventilation and stirring may be performed as necessary.
- ABP1 of the present invention or a partial peptide thereof or a salt thereof can be produced inside or outside the cells of the transformant.
- the ABPs of the present invention can be separated and purified from a culture obtained by culturing the transformant according to a method known per se.
- the cells or cells collected from the culture by a known method are suspended in an appropriate buffer, and then subjected to ultrasound, lysozyme and / or freeze-thawing. After the cells or cells are destroyed by, for example, a method of obtaining a crude extract of a soluble protein by centrifugation or filtration is used as appropriate.
- the buffer may contain a protein denaturant such as urea or guanidine hydrochloride, or a surfactant such as Triton X-100 TM .
- the isolation and purification of the ABPs 1 of the present invention contained in the soluble fraction thus obtained can be carried out according to a method known per se.
- Such methods include methods using solubility such as salting-out and solvent precipitation; mainly using differences in molecular weight such as dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis.
- a method utilizing a difference in charge such as ion exchange chromatography; a method utilizing a specific affinity such as affinity chromatography; a method utilizing a difference in hydrophobicity such as reverse phase high performance liquid chromatography.
- the free form can be converted to a salt by a method known per se or a method analogous thereto, and when the ABP 1 is obtained as a salt, The salt can be converted into a free form or another salt by a method known per se or a method analogous thereto.
- ABP1s produced by the transformant can be arbitrarily modified or the polypeptide can be partially removed before or after purification by the action of an appropriate protein modifying enzyme.
- an appropriate protein modifying enzyme for example, trypsin, chymotrypsin, arginyl endopeptidase, protein kinase, glycosidase and the like are used.
- the presence of the ABPs 1 of the present invention thus obtained can be confirmed by, for example, enzymatic immunoassay western blotting using a specific antibody.
- the ABPs 1 of the present invention are characterized in that RNA corresponding to the DNA encoding the above-described ABP 1 or a partial peptide thereof is a type II, and is obtained from a heron reticulocyte lysate, a wheat germ lysate, an Escherichia coli lysate, etc. It can also be synthesized by in vitro translation using a cell-free protein translation system. Alternatively, DNA encoding ABP1 or a partial peptide thereof can also be synthesized as type III using a cell-free transcription / translation system containing RNA polymerase.
- An antibody against ABP 1 or a salt thereof of the present invention may be any of a polyclonal antibody and a monoclonal antibody as long as it can recognize ABP 1 or a salt thereof. It may be.
- ABP 1 or its An antibody against a salt can be produced by using the ABP 1s of the present invention as an antigen according to a method for producing an antibody or antiserum known per se.
- the ABP 1s of the present invention used as an antigen any of the above-mentioned ABP 1s or partial peptides thereof or salts thereof may be used.
- a monoclonal antibody or a polyclonal antibody against ABP1 or a salt thereof can be produced, for example, as follows.
- the ABP 1s of the present invention are administered to a mammal at a site where antibody production is possible upon administration, together with itself or a carrier or diluent.
- Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability upon administration. Administration is usually performed once every 2 to 6 weeks, for a total of about 2 to 10 times. Examples of mammals to be used include monkeys, egrets, dogs, guinea pigs, mice, rats, sheep, and goats, and mice and rats are preferably used.
- an individual with an antibody titer is selected from a mammal immunized with an antigen, such as a mouse, and the spleen or lymph node is collected 2 to 5 days after the final immunization, and the antibody-producing cells contained therein are collected.
- an antigen such as a mouse
- myeloma cells of the same or different species a monoclonal antibody-producing hybridoma can be prepared.
- the measurement of the antibody titer in the antiserum can be performed, for example, by reacting the labeled protein described below with the antiserum, and then measuring the activity of the labeling agent bound to the antibody.
- the fusion operation can be performed according to a known method, for example, the method of Köhler and Milstein [Nature, 256, 495 (1975)].
- the fusion promoter include polyethylene glycol (PEG) and Sendai virus, but PEG is preferably used.
- myeloma cells include mammalian myeloma cells such as NS-1, P3U1, SP2Z0, and AP-1, but P3U1 is preferably used.
- the preferred ratio between the number of antibody-producing cells (spleen cells) and the number of myeloma cells used is 1: 1 to 20: about 1, PEG (preferably PEG 1000 to PEG 600) is added at a concentration of about 10 to 80%, and 20 to 40 ° C, preferably 1 to 30 to 37 ° C. Efficient cell fusion can be achieved by incubating for 10 minutes. .
- Monoclonal antibody-producing hybridomas are prepared, for example, by adding a hybridoma culture supernatant to a solid phase (eg, microplate) on which an antigen is directly or adsorbed together with a carrier, and then labeling with an immunoglobulin labeled with a radioactive substance or an enzyme.
- a solid phase eg, microplate
- Antibody (if mouse used for cell fusion is mouse, anti-mouse immunoglobulin antibody is used) or protein A, and monoclonal antibody bound to solid phase is detected; anti-immunoglobulin antibody or protein A is adsorbed
- a method of adding a hybridoma culture supernatant to the solid phase, adding a protein labeled with a radioactive substance, an enzyme, or the like, and detecting monoclonal antibody bound to the solid phase. can be.
- the selection of the monoclonal antibody can be performed according to a method known per se or a method analogous thereto.
- the selection of monoclonal antibodies can usually be performed in a medium for animal cells supplemented with HAT (hypoxanthine, aminopterin, thymidine).
- HAT hyperxanthine, aminopterin, thymidine
- any medium may be used as long as it can grow eight hybridomas. Examples of such a medium include RPMI 1640 medium containing 1 to 20%, preferably 10 to 20% fetal bovine serum, and GIT medium containing 1 to 10% fetal bovine serum (Wako Pure Chemical Industries, Ltd.
- a serum-free medium for hybridoma culture (SFM-101, Nissui Pharmaceutical Co., Ltd.) can be used.
- the culture temperature is usually 20 to 40 ° C, preferably about 37 ° C.
- the cultivation time is usually 5 days to 3 weeks, preferably 1 week to 2 weeks.
- Culture can be usually performed under 5% carbon dioxide gas.
- the antibody titer of the culture supernatant of the eight hybridomas can be measured in the same manner as the measurement of the antibody titer in the antiserum described above.
- the monoclonal antibody thus obtained can be obtained by a method known per se, for example, an immunoglobulin separation / purification method [eg, salting out method, alcohol precipitation method, isoelectric point precipitation method, electrophoresis, ion exchanger, (Eg, DEAE) adsorption / desorption method, ultracentrifugation method, gel filtration A specific purification method in which only the antibody is collected using an antigen-binding solid phase or an active adsorbent such as protein A or protein G, and the bond is dissociated to obtain the antibody.
- an immunoglobulin separation / purification method eg, salting out method, alcohol precipitation method, isoelectric point precipitation method, electrophoresis, ion exchanger, (Eg, DEAE) adsorption / desorption method, ultracentrifugation method, gel filtration
- an immunoglobulin separation / purification method eg, salting out method, alcohol precipitation method, isoelectric point precipitation method,
- a polyclonal antibody against ABP1 or a salt thereof can be produced according to a method known per se.
- a immunizing antigen (ABPs 1) itself or a complex thereof with a carrier protein is prepared, and a mammal is immunized in the same manner as in the above-described method for producing a monoclonal antibody. It can be produced by collecting the product and separating and purifying the antibody.
- other than mammals, chickens and the like can be used.
- the type of carrier protein and the mixing ratio of the carrier and the hapten are determined by the antibody against the hapten immunized by cross-linking the carrier. As long as it can be efficiently performed, any kind may be cross-linked at any ratio.For example, serum albumin, thyroglobulin, hemocyanin, etc., in a weight ratio of about 0.1 to 2 per hapten per hapten. A method of coupling at a rate of 0, preferably about 1 to 5 is used.
- various condensing agents for example, an active ester reagent containing a glutaldehyde aldehyde, a maleimide active ester, a thiol group, or a dithioviridyl group are used.
- the condensation product is administered to a mammal at a site capable of producing an antibody itself or together with a carrier and a diluent.
- Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability upon administration.
- the administration is usually made once every about 2 to 6 weeks, for a total of about 3 to 10 times.
- the polyclonal antibody can be collected from blood, ascites, etc., preferably from blood, of the mammal immunized by the above method.
- the measurement of the polyclonal antibody titer in the antiserum can be performed in the same manner as the measurement of the antibody titer in the antiserum described above.
- Polyclonal antibodies can be separated and purified according to the same immunoglobulin separation and purification methods as for monoclonal antibodies described above.
- a partial peptide of ABP1 When a partial peptide of ABP1 is used as an antigen, its position on ABP1 is not particularly limited, and examples include an oligopeptide having a partial amino acid sequence in a region well conserved among various warm-blooded animals. Specifically, a peptide having the amino acid sequence shown in SEQ ID NO: 5 or SEQ ID NO: 6 which is completely conserved between human and mouse is preferably exemplified.
- a polynucleotide containing a nucleotide sequence complementary to the nucleotide sequence encoding ABP1 of the present invention or a part thereof may be ABP1.
- nucleotide sequence a nucleotide sequence encoding ABP1 or a partial peptide thereof and a nucleotide sequence capable of hybridizing under physiological conditions of a cell expressing the protein, more specifically About 70% or more, preferably about 80% or more, more preferably about 90% or more, most preferably about 90% or more, with respect to the overlapping portion with the complementary strand of the nucleotide sequence encoding ABP 1 or a partial peptide thereof.
- a nucleotide sequence having about 95% or more homology is exemplified.
- the antisense polynucleotide of the present invention can be designed and synthesized based on the nucleotide sequence information of the cloned or determined polynucleotide of the present invention. Such a polynucleotide can inhibit the replication or expression of the gene encoding ABP1. That is, the antisense polynucleotide of the present invention can hybridize with RNA transcribed from the gene encoding ABP1, and inhibits the synthesis (processing) or function (translation into protein) of mRNA. Can be.
- the length of the target region of the antisense polynucleotide of the present invention is not particularly limited as long as the translation of ABP1 is inhibited by the hybridization of the antisense polynucleotide, resulting in ABP1 May be the entire sequence or a partial sequence of the RNA encoding the gene, a short one of about 15 bases and a long one And the entire sequence of mRNA or early transcript. Considering the ease of synthesis and the problem of antigenicity, oligonucleotides consisting of about 15 to about 30 bases are preferred, but not limited thereto.
- the initiation codon, the 3 ′ end untranslated region, the 3 ′ end palindrome region, and the 3 ′ end hairpin loop can be selected as target regions, but any region within the gene can be selected as a target.
- the antisense polynucleotide of the present invention not only inhibits translation into protein by hybridizing with mRNA encoding ABP1 or an early transcript, but also encodes ABP1, which is double-stranded DNA. It may bind to a gene to form a triplex, thereby inhibiting RNA transcription.
- Antisense polynucleotides are dexoxyliponucleotides containing 21-deoxy D-reports, liponucleotides containing D-ribose, or other types of nucleotides that are N-glycosides of purine or pyrimidine bases.
- polymers having a non-nucleotide backbone eg, commercially available protein nucleic acids and synthetic sequence-specific nucleic acid polymers
- polymers containing special bonds provided that such polymers are found in DNA and RNA
- nucleotides having a configuration that allows base pairing and base attachment can be double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, and even DNA: RNA hybrids, and can be unmodified polynucleotides (or unmodified oligonucleotides), or even known.
- nucleotides capped, methylated, one or more natural nucleotides replaced by analogs, intramolecular nucleotides Modified, eg, having uncharged bonds (eg, methylphosphonate, phosphotriester, phosphoramidate, olebamate, etc.), charged or sulfur-containing bonds (eg, phosphorothioate, Such as proteins (nucleases, nucleases, etc.) Nucleases, which have side-chain groups such as inhibitors, toxins, antibodies, signal peptides, poly-L-lysine, etc., and sugars (eg, monosaccharides); Acridine, psoralen, etc.), contains chelating compounds (eg, metals, radioactive metals, boron, oxidizable metals, etc.), contains alkylating agents, has modified bonds (Eg, a anomeric nucleic acid).
- uncharged bonds eg, methylphosphonate, phosphotriester
- nucleoside may include not only those containing purine and pyrimidine bases but also those having other modified heterocyclic bases. Such modifications can include methylated purines and pyrimidines, acylated purines and pyrimidines, or other heterocycles. Modified nucleotides and modified nucleotides may also be modified at the sugar moiety, e.g., where one or more hydroxyl groups have been replaced by halogens or aliphatic groups, or by functional groups such as ethers, amines, etc. It may be converted.
- Antisense polynucleotides are RNA, DNA, or modified nucleic acids (RNA, DNA).
- modified nucleic acid include, but are not limited to, sulfur derivatives of nucleic acids, thiophosphate derivatives, and polynucleoside amides, which are resistant to degradation of oligonucleoside amides.
- the antisense polynucleotide of the present invention can be preferably designed according to the following policy. I.e., to make the antisense polynucleotide more stable in the cell, to increase the cell permeability of the antisense polynucleotide, to have a greater affinity for the target sense strand, and to be toxic.
- Antisense polynucleotides may contain altered or modified sugars, bases, or linkages, and are provided in specialized forms such as ribosomes and microspheres. Can be given, applied by gene therapy, or given in an added form.
- Such additional forms include polythiones, such as polylysine, which act to neutralize the charge on the phosphate backbone, and lipids, which enhance interaction with cell membranes and increase nucleic acid uptake. (Eg, phospholipid, cholesterol, etc.). Preferred lipids for addition include cholesterol and its derivatives (eg, cholesteryl chromate formate, cholic acid, etc.). Such a substance can be attached to the 3, 5 or 5 'end of a nucleic acid, and can be attached via a base, a sugar, or an intramolecular nucleoside bond.
- polythiones such as polylysine
- lipids which enhance interaction with cell membranes and increase nucleic acid uptake.
- Preferred lipids for addition include cholesterol and its derivatives (eg, cholesteryl chromate formate, cholic acid, etc.).
- Such a substance can be attached to the 3, 5 or 5 'end of a nucleic acid, and can be attached via a base,
- capping groups include, but are not limited to, hydroxyl-protecting groups known in the art, such as glycols such as polyethylene glycol and tetraethylene glycol.
- a liposome capable of specifically cleaving mRNA or gene early transcript encoding ABP 1 within the coding region is also included in the antisense polynucleotide of the present invention. May be included.
- the term "lipozyme” refers to an RNA having an enzymatic activity for cleaving a nucleic acid, but recently it has been revealed that oligo DNA having the nucleotide sequence of the enzyme active site also has a nucleic acid-cleaving activity. However, in the present specification, the term is used as a concept including DNA as long as it has a sequence-specific nucleic acid cleavage activity.
- the most versatile liposomes include self-splicing RNAs found in infectious RNAs such as viroids and willoids, and hammerhead and hairpin types are known.
- the hammerhead type exhibits enzymatic activity with about 40 bases, and the bases at both ends (approximately 10 bases in total) adjacent to the hammerhead structure take the desired cleavage site of mRNA.
- By using a complementary sequence it is possible to specifically cleave only the target mRNA.
- This type of lipozyme has the additional advantage of not attacking genomic DNA since it uses only RNA as substrate.
- RNA encoding ABP 1 has a double-stranded structure by itself, use a hybrid liposome linked to an RNA motif derived from a viral nucleic acid that can specifically bind to RNA helicase. Can make the target sequence single-stranded [Proc. Natl. Acad. Sci. USA, 98 (10): 5572-5577 (2001)].
- a hybrid liposome further linked to a sequence modified with tRNA is used to promote the transfer of the transcript to the cytoplasm. [Nucleic Acids Res., 29 (13): 2780-2788 (2001)].
- RNAi RNA interference
- the antisense oligonucleotide and lipozyme of the present invention determine the target region of mRNA or the initial transcript based on the cDNA sequence or the genomic DNA sequence encoding the protein of the present invention, and can be used to automatically synthesize commercially available DNAZRNA. Using a machine (Applied Biosystems, Beckman, etc.) to synthesize a sequence complementary thereto.
- the sense strand and the antisense strand were synthesized using a DNA / RNA automatic synthesizer, respectively, and denatured in an appropriate annealing buffer, for example, at about 90 to about 95 ° C for about 1 minute. Thereafter, it can be prepared by annealing at about 30 to about 70 for about 1 to about 8 hours.
- a longer double-stranded polynucleotide can be prepared by synthesizing complementary oligonucleotide chains so as to alternately overlap each other, annealing them, and ligating them with a ligase.
- ABP 1 or the activated peptide of the present invention binds and activates ASK 1 Therefore, it can promote the induction of apoptosis through the ASK1 kinase cascade. Therefore, (1) ABP1 or the activation peptide of the present invention, (2) a polynucleotide encoding ABP1 or the activation peptide of the present invention, (3) a polynucleotide containing a base sequence encoding ABP1 or a part thereof, (4) the present invention
- the antibody, the antisense polynucleotide of the present invention, and the inhibitory peptide of the present invention have the following uses.
- a ⁇ 1 binds to and activates A S1 to promote apoptosis induction through the ASK1 kinase cascade. Therefore, ABP1 or the activating peptide of the present invention or a salt thereof is added to the cells, or a polynucleotide encoding ABP1 or the activating peptide of the present invention is introduced into the cells for expression, and the amount of ABP1 in the cells is increased. By increasing the amount of ASK1, intracellular ASK1 activation can be promoted or apoptosis can be induced in the cells. For example, it can be used as a reagent for apoptosis research.
- ABP1 or the activated peptide of the present invention or a salt thereof is used as the above-mentioned ASK1 activation promoter or apoptosis inducer
- water or an appropriate buffer eg, phosphate buffer, PBS, Tris Dissolution so as to obtain an appropriate concentration.
- an appropriate buffer eg, phosphate buffer, PBS, Tris Dissolution so as to obtain an appropriate concentration.
- commonly used preservatives, stabilizers, reducing agents, tonicity agents and the like may be added.
- the polynucleotide when ABP1 or a polynucleotide encoding the activating peptide of the present invention is used as the above-mentioned ASK1 activation promoter or apoptosis inducer, the polynucleotide may be used alone or in a retrovirus vector, adenovirus vector, or the like. 1. After transfection into a suitable vector such as an adenovirus associated virus vector, the cells can be introduced into cells by the above-mentioned transformation method (eg, ribosome method, electo-portion method, etc.).
- transformation method eg, ribosome method, electo-portion method, etc.
- ABP 1 has a function of activating ASK 1 and inducing apo 1 ⁇ cis in cells, and thus ABP 1 or a nucleic acid encoding it in vivo (eg, Genes, mRNAs, etc.), when they are defective or deficient, or when their expression levels are abnormally reduced, and the induction of apoptosis in cells by some other factor is excessive.
- ABP 1 or a nucleic acid encoding it in vivo eg, Genes, mRNAs, etc.
- various diseases such as cancer, autoimmune disease, viral infection, endocrine disease, blood disease, and organ hyperplasia occur.
- a) the ABP1 or the activating peptide of the present invention or a salt thereof is used.
- the cells are transplanted into the patient, etc., so that ABP1 in the body of the patient is obtained.
- ASK1 cascade-mediated apoptosis can be induced in abnormal or unwanted cells.
- ABP1 or the activation peptide of the present invention or a salt thereof, or b) a polynucleotide encoding ABP1 or the activation peptide of the present invention is prevented from inducing apoptosis.
- Therapeutically effective diseases such as cancer (eg, leukemia, esophageal cancer, stomach cancer, colon cancer, rectal cancer, lung cancer, liver cancer, kidney cancer, breast cancer, uterine cancer, ovarian cancer, prostate cancer, melanoma, myeloma, bone Sarcomas, brain tumors, etc.), autoimmune diseases (eg, systemic lupus erythematosus, scleroderma, rheumatoid arthritis, siedalen syndrome group, multiple sclerosis, insulin-dependent diabetes mellitus, psoriasis, ulcerative colitis, idiopathic platelets Decreased purpura, Crohn's disease, glomerulonephritis, etc., viral infections (eg, hemorrhagic fever, T-cell leukemia, positiosarcoma, infectious mononucleosis, lymphoma, nasopharyngeal carcinoma, uterine face cancer, skin Cancer, he
- ABP1 or the activating peptide of the present invention or a salt thereof is combined with the above-mentioned prophylactic and therapeutic agents.
- the polynucleotide encoding ABP1 or the activating peptide of the present invention when used as the above-mentioned prophylactic / therapeutic agent, the polynucleotide may be used alone or after insertion into an appropriate vector such as a Ted virus vector. It can be formulated according to the means.
- the polynucleotide can be administered as it is or together with an auxiliary for promoting uptake by a catheter such as a gene gun or a hydrogel catheter.
- a) ABP 1 or the activating peptide of the present invention or a salt thereof, or b) a polynucleotide encoding ABP 1 or the activating peptide of the present invention may be a sugar-coated tablet or capsule, if necessary. It can be used orally as an elixir, microcapsule or the like, or parenterally in the form of an injection such as a sterile solution with water or another pharmaceutically acceptable liquid, or a suspension.
- ABP1 or the activating peptide of the present invention or a salt thereof, or b) a polynucleotide encoding ABP1 or the activating peptide of the present invention may be prepared by using a known physiologically acceptable carrier, flavoring agent, or excipient. It can be manufactured by mixing with excipients, vehicles, preservatives, stabilizers, binders and the like in the unit dosage form generally required for the practice of formulations. The amount of the active ingredient in these preparations is such that an appropriate dose in the specified range can be obtained.
- Additives that can be incorporated into tablets, capsules, etc. Swelling agents such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, and flavoring agents such as peppermint, cocoa oil or cherry.
- Swelling agents such as magnesium stearate
- sweeteners such as sucrose, lactose or saccharin
- flavoring agents such as peppermint, cocoa oil or cherry.
- the unit dosage form is a capsule
- the above type of material can further contain a liquid carrier such as an oil or fat.
- Sterile compositions for injection are formulated according to standard pharmaceutical practice, such as dissolving or suspending the active substance in vehicles such as water for injection, and naturally occurring vegetable oils such as sesame oil and coconut oil. You can.
- aqueous solution for injection for example, physiological saline, isotonic solution containing glucose and other adjuvants (eg, D-sorbitol, D-mannitol, sodium salt, etc.) and the like are used.
- Solubilizers such as alcohol (eg, ethanol), polyalcohol (eg, propylene glycol, polyethylene glycol), nonionic surfactants (eg, Polysorbate 80 TM , HCO-50)
- oily liquid for example, sesame oil, soybean oil, and the like are used, and may be used in combination with a solubilizing agent such as benzyl benzoate or benzyl alcohol.
- prophylactic / therapeutic agents examples include, for example, buffers (eg, phosphate buffer, sodium acetate buffer), soothing agents (eg, benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (eg, human serum It may be combined with preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants, etc.
- buffers eg, phosphate buffer, sodium acetate buffer
- soothing agents eg, benzalkonium chloride, procaine hydrochloride, etc.
- stabilizers eg, human serum It may be combined with preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants, etc.
- preservatives eg, benzyl alcohol, phenol, etc.
- the preparations obtained in this way are safe and low toxic, such as, for example, humans and other warm-blooded animals (eg, rats, mice, hamsters, egrets, sheep, sheep, goats, bushes, dogs, dogs, Cats, dogs, monkeys, chimpanzees, birds, etc.).
- warm-blooded animals eg, rats, mice, hamsters, egrets, sheep, sheep, goats, bushes, dogs, dogs, Cats, dogs, monkeys, chimpanzees, birds, etc.
- the dose of ABP 1 or the activating peptide of the present invention varies depending on the administration subject, target organ, symptom, administration method, and the like.
- oral administration generally, for example, a cancer patient (60 kg) is used.
- L 00 mg preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg.
- the single dose varies depending on the administration target, target organ, symptoms, administration method, etc.
- it is usually used, for example, in cancer patients (60 kg).
- the dose converted per 60 kg can be administered.
- Administration of ABP 1 or a polynucleotide encoding an activating peptide of the invention The amount varies depending on the administration target, target organ, symptoms, administration method, and the like. However, in the case of oral administration, for example, in a cancer patient (as 60 kg), about O.lmg to I: 00 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg. In the case of parenteral administration, the single dose varies depending on the administration target, target organ, symptoms, administration method, etc. ), It is convenient to administer about 0.01 to 30 mg per day, preferably about 0.1 to 2 Omg, more preferably about 0.1 to 10 mg per day. In the case of other animals, a dose converted per 6 O kg can be administered.
- a polynucleotide containing the nucleotide sequence encoding ABP1 or a part thereof (hereinafter, referred to as “sense polynucleotide of the present invention”) and the antisense polynucleotide of the present invention can be used as a probe to obtain human or other DNA or mRNA encoding ABP 1 in warm-blooded animals (e.g., rats, mice, hamsters, rabbits, sheep, sheep, goats, pigs, pigs, pomeras, cats, dogs, monkeys, chimpanzees, birds, etc.) Since abnormalities (gene abnormalities) can be detected, for example, genetic diagnostic agents such as DNA damage or mutation, mRNA splicing abnormalities or decreased expression, or DNA amplification or mRNA overexpression.
- genetic diagnostic agents such as DNA damage or mutation, mRNA splicing abnormalities or decreased expression, or DNA amplification or mRNA overexpression.
- the polynucleotide containing a part of the nucleotide sequence encoding ABP1 is not particularly limited as long as it has a necessary length (for example, about 15 bases or more) as a probe, and encodes a partial peptide of ABP1. (It is in frame).
- the above-described genetic diagnosis using the sense or antisense polynucleotide of the present invention includes, for example, known Northern hybridization, quantitative RT-PCR, PCR-SSCP method, allele-specific PCR, PCR- It can be performed by the SSOP method, DGGE method, RNase protection method, PCR-RFLP method, and the like.
- diseases associated with apoptosis or suppression of inflammatory site force-in production such as cancer (eg, leukemia, esophageal cancer, stomach cancer, colorectal cancer, rectal cancer, lung cancer, liver cancer, kidney cancer, breast cancer) , Uterine cancer, ovarian cancer, prostate cancer, melanoma, myeloma, osteosarcoma, brain tumor, etc.), autoimmune diseases (eg, systemic lupus erythematosus, scleroderma, rheumatoid arthritis, Scheddaren syndrome, multiple sclerosis) Disease, insulin-dependent diabetes mellitus, psoriasis, ulcerative colitis, idiopathic thrombocytopenic purpura, Crohn's disease, glomerulonephritis, etc., viral infections (eg, hemorrhagic fever, T-cell leukemia, lipopositive meat) , Infectious mononucleosis, lymphom
- ABP1 overexpression is detected by Northern hybridization or quantitative RT-PCR, diseases associated with enhanced apoptosis or inflammation, such as viral infections (eg, AIDS, influenza, Unknown fever), endocrine diseases (eg, hormonal deficiency, site force in deficiency, etc.), blood diseases (eg, cytopenia, renal anemia, etc.), organ dysplasia (eg, thyroid atrophy, cleft palate, etc.), transplantation Organ rejection, graft-versus-host disease, immunodeficiency, neurodegenerative disease (eg, polyglutamine disease, Alfheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, prion disease, cerebellar degeneration, etc.), ischemic Heart disease (eg, angina pectoris, myocardial infarction, etc.), radiation damage, UV damage (eg, sunburn, etc.), toxic disease (eg, renal tubular cell injury due to heavy metals, liver due to alcohol)
- viral infections e
- the antibody of the present invention can specifically recognize ABP1, it can be used for detecting ABP1 in a test solution.
- the two kinds of antibodies recognize different portions of ABP1. For example, if one antibody recognizes the N-terminal portion of ABP 1 (eg, a portion having the amino acid sequence shown in SEQ ID NO: 5), the other antibody will recognize the C-terminal portion of ABP 1 (eg, And the like having a portion having the amino acid sequence shown in SEQ ID NO: 6).
- ABP 1 can be quantified using a monoclonal antibody against ABP 1, and detection by tissue staining or the like can also be performed.
- the antibody molecule itself may be used, or the F (ab ′) 2 , Fab ′, or Fab fraction of the antibody molecule may be used.
- the method for quantifying ABP 1 or a salt thereof using the antibody of the present invention is not particularly limited, and may be an antibody, antigen, or antibody-antigen complex corresponding to the amount of antigen (eg, ABP 1) in a test solution.
- Any measurement method may be used as long as the amount of the body is detected by chemical or physical means, and the amount is calculated from a standard curve prepared using a standard solution containing a known amount of antigen.
- nephelometry, a competition method, an immunometric method and a sandwich method are suitably used, but in terms of sensitivity and specificity, it is particularly preferable to use a sandwich method described later.
- a labeling agent used in a measurement method using a labeling substance for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance and the like are used. Radioisotopes, if example embodiment, [1 2 5 I], [1 3 1 I], [3 H], and [1 4 C] used.
- a stable enzyme having a large specific activity is preferable. For example, ⁇ -galactosidase, ⁇ -dalcosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used.
- fluorescent substance for example, fluorescamine, fluorescein isothiosinate and the like are used.
- luminescent substance for example, luminol, luminol derivative, luciferin, lucigenin and the like are used.
- a biotin- (strept) avidin system can be used for binding the antibody or antigen to the labeling agent.
- the carrier include insoluble polysaccharides such as agarose, dextran, and cellulose; synthetic resins such as polystyrene, polyacrylamide, and silicon; and glass.
- a test solution is reacted with the insolubilized antibody of the present invention (primary reaction), and further reacted with another labeled antibody of the present invention (secondary reaction), followed by labeling on the insolubilized carrier.
- primary reaction the insolubilized antibody of the present invention
- secondary reaction another labeled antibody of the present invention
- the primary reaction and the secondary reaction may be performed in reverse order, may be performed simultaneously, or may be performed with a time delay.
- the labeling agent and the method of insolubilization can be in accordance with those described above.
- the antibody used for the immobilized antibody or the labeled antibody does not necessarily need to be one kind, and two or more kinds of antibodies are used for the purpose of improving measurement sensitivity and the like. A mixture may be used.
- antibodies having different binding sites for ⁇ ⁇ 1 are preferably used.
- the antibody used in the secondary reaction recognizes the C-terminal of ABP 1
- the antibody used in the primary reaction is preferably the C-terminal or less.
- an antibody that recognizes the N-terminal is used.
- the antibody of the present invention can be used in a measurement system other than the sandwich method, for example, a competition method, an immunometric method, a nephrometry method, or the like.
- the competition method after the antigen in the test solution and the labeled antigen are allowed to react competitively with the antibody, the unreacted labeled antigen (F) is separated from the labeled antigen (B) bound to the antibody. (BZF separation), the labeling amount of either B or F is measured, and the amount of antigen in the test solution is quantified.
- This reaction method uses a soluble antibody as the antibody, a liquid phase method in which BZF separation is performed using polyethylene glycol or a secondary antibody against the above-mentioned antibody (primary antibody), or an immobilized antibody as the primary antibody Alternatively, a solid-phase method using a soluble primary antibody and a solid-phased antibody as the secondary antibody is used.
- the antigen in the test solution and the immobilized antigen are subjected to a competitive reaction with a certain amount of labeled antibody, and then the solid phase and the liquid phase are separated. After reacting the antigen with an excess amount of the labeled antibody, the immobilized antigen is added to bind the unreacted labeled antibody to the solid phase, and then the solid phase and the liquid phase are separated. Next, the amount of the label in either phase is measured to determine the amount of the antigen in the test solution.
- the amount of insoluble sediment resulting from the antigen-antibody reaction in a gel or in a solution is measured. Even when the amount of antigen in the test solution is small and only a small amount of sediment is obtained, laser nephrometry utilizing laser scattering is preferably used.
- ABP1 or a salt thereof can be quantified with high sensitivity by using the antibody of the present invention.
- a biological sample eg, blood, plasma, urine, biopsy, etc.
- concentration of ABP 1 or a salt thereof in the test sample is determined using the antibody of the present invention.
- apoptosis suppression is associated with a disease, such as cancer (eg, leukemia, esophageal cancer, stomach cancer, colorectal cancer, rectal cancer, lung cancer, liver cancer, kidney cancer, breast cancer) , Uterine cancer, ovarian cancer, prostate cancer, melanoma, myeloma, osteosarcoma, brain tumor, etc.), autoimmune diseases (eg, systemic lupus erythematosus, scleroderma, rheumatoid arthritis, siedalen syndrome, multiple sclerosis, insulin) Dependent diabetes mellitus, psoriasis, ulcerative colitis, idiopathic thrombocytopenic purpura, Crohn's disease, glomerulonephritis, etc., viral infections (eg, hemorrhagic fever, T-cell leukemia, cystic sarcoma, infectious disease) Disease, lymphoma
- cancer eg, leukemia, es
- diseases associated with apoptosis or inflammation such as viral infections (eg, AIDS, influenza, unknown fever, etc.), endocrine diseases (eg, hormones) Deficiency, site power in deficiency, etc.), blood disease Disease (eg, cytopenia, renal anemia, etc.), organ dysplasia (eg, thyroid atrophy, cleft palate, etc.), transplanted organ rejection, graft-versus-host disease, immunodeficiency, neurodegenerative disease (eg, polyglutamine disease) , Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, prion disease, cerebellar degeneration, etc.), ischemic heart disease (eg, angina pectoris, myocardial infarction, etc.), radiation damage, UV damage (eg, Sunburn, etc.) Toxic diseases (eg, renal tubular cell injury due to heavy metals, liver cell injury due to alcohol,
- the antibody of the present invention inhibits the binding of ABP1 to ASK1 by specifically binding to ABP1, and inactivates ASK1 activation and apoptosis Z inflammatory cytokine production induction promoting action ( That is, neutralization).
- the antisense polynucleotide of the present invention inhibits the expression of ABP1 and reduces the production of the protein, and consequently ASK1 activation by ABP1 and induction of apoptosis Z inflammatory cytokine production. Acceleration can be reduced.
- the antibody of the present invention to cells to inactivate ABP 1, or introducing the antisense polynucleotide of the present invention into cells to reduce the amount of ABP 1 in the cells, It can inhibit ASK1 activation and suppress apoptosis / inflammatory cytokine production of the cells, and can be used, for example, as a reagent for studying apoptosis and inflammatory response.
- water or an appropriate buffer eg, phosphate buffer, PBS, Tris-HCl buffer, etc.
- an appropriate buffer eg, phosphate buffer, PBS, Tris-HCl buffer, etc.
- PBS phosphate buffer
- Tris-HCl buffer Tris-HCl buffer
- the antisense polynucleotide of the present invention is the ASK1 activation inhibitor described above.
- the polynucleotide may be used alone or after being introduced into a suitable vector such as a retrovirus vector, an adenovirus vector, an adenovirus associated virus vector, and the like.
- the cells can be introduced into cells using a conversion method (eg, ribosome method, electoral poration method, etc.).
- ABP 1 since ABP 1 has a function of activating ASK 1 to induce apoptosis in cells or inducing inflammation, ABP 1 or a nucleic acid encoding it in vivo (eg, a gene, mRNA, etc.) ) Is abnormal (expression of a highly active mutant), or its expression level is abnormally increased, and is caused by apo1 ⁇ -cis induction or inflammatory If cytokine production is abnormally elevated, e.g., viral infection, endocrine disease, blood disease, organ dysplasia, transplant rejection, graft-versus-host disease, immunodeficiency, neurodegenerative disease, ischemic heart disease, radiation Disorders, UV damage, toxic diseases, nutritional disorders, inflammation, ischemic neuropathy (cerebral ischemia), diabetic peripheral neuropathy, vascular diseases, respiratory diseases, cartilage diseases, etc. To.
- cytokine production is abnormally elevated, e.g., viral infection, endocrine disease, blood disease, organ dysp
- a) the antibody of the present invention or b) the antisense polynucleotide of the present invention which inhibits apoptosis or inflammation by preventing or treating the disease effectively; for example, a disease caused by overexpression of ABP 1;
- viral infections eg, AIDS, influenza, unknown fever, etc.
- endocrine diseases eg, hormone deficiency, Dysfunction, blood disorders (eg, cytopenia, renal anemia, etc.), dysplasia (eg, thyroid atrophy, cleft palate, etc.), transplanted organ rejection, graft-versus-host disease, immunodeficiency, God Degenerative diseases (eg, polyglutamine disease, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, prion disease, cerebellar degeneration, etc.), ischemic heart disease (eg, angina pectoris, myocardial infarction, etc.) , Radiation damage, ultraviolet light damage (eg, sunburn, en
- the antibody of the present invention When used as the above-mentioned prophylactic / therapeutic agent, it can be formulated in the same manner as the above-mentioned medicament containing ABP1 or the activating peptide of the present invention or a salt thereof.
- the antisense polynucleotide of the present invention When used as the above-mentioned prophylactic / therapeutic agent, it may be formulated in the same manner as the above-mentioned drug containing ABP1 or a polynucleotide encoding the activating peptide of the present invention. Can be.
- the preparations obtained in this way are safe and low toxic, such as, for example, humans and other warm-blooded animals (eg, rats, mice, eight-month-old, egrets, higgins, goats, pigs, pigs, dogs, Cats, dogs, monkeys, chimpanzees, birds, etc.).
- warm-blooded animals eg, rats, mice, eight-month-old, egrets, higgins, goats, pigs, pigs, dogs, Cats, dogs, monkeys, chimpanzees, birds, etc.
- the dose of the antibody of the present invention varies depending on the administration subject, target organ, symptoms, administration method, and the like. It is about 0.1 to 10 Omg per day, preferably about 1.0 to 5 Omg, and more preferably about 1.0 to 2 Omg per day. In the case of parenteral administration, the single dose varies depending on the administration subject, target organ, symptoms, administration method, and the like. )), It is convenient to administer about 0.01 to 30 mg, preferably about 0.1; It is. In the case of other animals, it is possible to administer the amount converted to 60 kg. Giru.
- the dosage of the antisense polynucleotide of the present invention varies depending on the administration subject, target organ, disease state, administration method, and the like.
- oral administration in general, for example, in patients with poly and glutamine diseases (60 kg) Is about 0.1 mg to 100 mg per day, preferably about 1.0 to 5 mg, more preferably about 1.0 to 20 mg per day.
- parenteral administration the single dose varies depending on the administration target, target organ, disease state, administration method, and the like.
- parenteral injection it is usually used in patients with polyglutamine disease (6 Okg ), it is convenient to administer about 0.01 to 3 Omg per day, preferably about 0.1 to 20 mg, more preferably about 0.1 to 1 Omg per day. In the case of other animals, the dose can be administered in terms of 60 kg.
- the inhibitory peptide of the present invention can function as an (antagonist) inhibitor of ABP1, that is, it can bind to ASK1, but does not activate or inactivate it.
- ABP1 an apoptosis / inflammatory cytokine production inhibitor, or a prophylactic / therapeutic agent for a disease associated with enhanced apoptosis or inflammation.
- the inhibitory peptide of the present invention When the inhibitory peptide of the present invention is used as an inhibitor of ASK1 activation-Apoptosis or inflammatory cytokine production inhibitor, ASK1 activation containing the above-mentioned ABP1 or the activating peptide of the present invention or a salt thereof is promoted.
- ⁇ Reagents can be prepared in the same manner as apoptosis inducers.
- the inhibitory peptide of the present invention is used as the above-mentioned prophylactic / therapeutic agent, it can be formulated in the same manner as the above-mentioned drug containing ABP1, the activated peptide of the present invention or a salt thereof.
- the preparations obtained in this way are safe and low toxic, they can be used, for example, in humans and other warm-blooded animals (eg, rats, mice, hamsters, egrets, sheep, sheep, goats, pigs, pigs, dogs, cats). , Dogs, monkeys, chimpanzees, birds, etc.).
- warm-blooded animals eg, rats, mice, hamsters, egrets, sheep, sheep, goats, pigs, pigs, dogs, cats.
- the dosage of the inhibitory peptide of the present invention depends on the administration target, target organ, symptoms, administration method, etc.
- oral administration for example, in a patient with polyglutamine disease (as 60 kg), about 0.1 mg to 100 mg, preferably about 1.0 to 50 mg, More preferably, it is about 1.0 to 20 mg.
- parenteral administration the single dose varies depending on the subject of administration, target organ, symptoms, administration method, etc.
- it is usually used, for example, in patients with polyglutamine disease (60 kg ), It is convenient to administer about 0.01 to 3 Omg per day, preferably about 0.1 to 2 Omg, more preferably about 0.1 to 10 mg per day.
- the dose can be administered in terms of 6 O kg.
- ASK1 partial peptide having no phosphorylation activity or salt thereof As demonstrated in Examples described later, since the site involved in binding of ASK1 to ABP1 is an N-terminal activation control domain, The partial peptide of ASK1, which has the activation control domain but lacks the kinase domain responsible for activating MAPKK located downstream of the cascade, can function as an ASK1 (antagonist) inhibitor. That is, since the peptide can bind to ABP1 but cannot be activated by it, it can competitively inhibit the binding between ASK1 and ABP1 that are endogenous to cells and the activation of ASK1 thereby. Therefore, the partial peptide can be used as an ABP1 inhibitor, an inhibitor of apoptosis or inflammatory cytokine production, or an agent for preventing or treating diseases associated with enhanced apoptosis or inflammation.
- the kinase domain of ASK1 for example, in the case of human ASK1, an amino acid sequence represented by amino acid numbers 678 to 936 in the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing of Patent Document 1 (Japanese Patent Laid-Open No. 10-93). Is mentioned. Therefore, the partial peptide of ASK1 having an activation control domain on the N-terminal side and lacking the kinase domain (hereinafter sometimes abbreviated as “ASK1 partial peptide of the present invention”) is as described above.
- amino acid sequence identical or substantially identical to all or a part of the amino acid sequence represented by amino acid numbers 1 to 677 in the amino acid sequence of human ASK 1 (hereinafter, collectively referred to as “ASK1-N sequence”) And a peptide having the ability to bind to ABP1.
- substantially identical amino acid sequence Is preferably about 70% or more, preferably about 80% or more, more preferably about 90% or more, particularly preferably about 90% or more of the amino acid sequence represented by amino acid numbers 1 to 677 in the amino acid sequence of human ASK 1 described above.
- the ASK1 partial peptide of the present invention includes, for example, 1) 1 or 2 or more in the ASK1-N sequence (preferably about 1 to 30, preferably about 1 to 10, more preferably 1 to 10; Amino acid sequence in which the amino acid sequence has about 5 to 5) amino acids; (2) 1 or more (preferably about 1 to 30, preferably about 1 to 10, more preferably 1 to 10 amino acids in the ASK1-N sequence) (3) ASK 1-N sequence with 1 or more (preferably about 1 to 30, preferably about 1 to 10, more preferably about 1 to 10) amino acids 51) or 2 or more (preferably about 1 to 30, preferably about 1 to 10, and more preferably Amino acid sequence in which 1-5) amino acids have been replaced with other amino acids, or Mutant peptides containing the combined amino acid sequence are also included.
- the A SKI partial peptide of the present invention can be prepared by the same method as the above-described partial peptide of ABP1.
- ASK1 activation containing the above-mentioned ABP1 or the activated peptide of the present invention or a salt thereof is used.
- Acceleration • Reagents can be prepared in the same manner as the apoptosis inducing agent.
- the ASK1 partial peptide of the present invention is used as the above-mentioned prophylactic / therapeutic agent, it can be formulated in the same manner as the above-mentioned drug containing ABP1 or the activated peptide of the present invention or a salt thereof.
- the preparations obtained in this way are safe and low toxic, so that, for example, humans and other warm-blooded animals (for example, rats, mice, hams, egrets, higgins, goats, bushes, squirrels, Poma, cat, dog, monkey, chimpanzee, bird, etc.).
- the dose of the ASK1 partial peptide of the present invention varies depending on the administration subject, target organ, symptoms, administration method, and the like. However, in the case of oral administration, for example, in a patient with polydaltamin disease (as 60 kg), About 0.1 mg to 100 mg per day, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg.
- the single dose varies depending on the subject of administration, target organ, symptoms, administration method, etc.
- it is usually, for example, polydaltamin disease patient (60 kg)
- it is convenient to administer about 0.01 to 30 mg, preferably about 0.1 to 2 Omg, more preferably about 0.1 to 10 mg per day.
- the dose can be administered in terms of 6 Okg.
- the present invention also provides a method of screening for an ASK1 activation modulator by using ABP1 or the activating peptide of the present invention or a salt thereof, or a cell that produces it.
- the screening method is roughly classified into (A) a method using the binding property of ABP1 and ASK1, (B) a method using ASK1 activation as an index, and (C) a method using ABP1 expression level as an index. Is done.
- a partial peptide thereof that retains binding to ASK1 or ABP1 that is, a partial peptide containing at least the N-terminal activation control domain
- a salt thereof or a salt thereof is produced.
- the cells used are further used.
- ABP1 can bind to and activate ASK1
- a binding agent using ABP1 or the activation peptide of the present invention and its partial peptide containing ASK1 or the N-terminal activation control domain is used.
- screening of a compound having the same action as ABP1 or the activation peptide of the present invention can be carried out. Screening can be performed. That is, the present invention provides a method for screening an ASK1 activation regulator using ABP1 or the activation peptide of the present invention and ASK1 or a partial peptide thereof containing an N-terminal activation control domain. More specifically, the present invention provides
- ASK 1 or the partial peptide containing the N-terminal activation control domain and ABP 1 or the activation peptide of the present invention in contact with (2) ASK 1 or N-terminal activity ABP1 or the partial peptide containing the N-terminal activation control domain, which is obtained when the activation peptide of the present invention and a test substance are contacted with the partial peptide containing the activation control domain.
- a method for screening for an ASK 1 activation-regulating substance which comprises comparing the amount of binding between a peptide and ABP 1 or the activated peptide of the present invention
- a method for screening for an ASK1 activation-regulating substance which comprises comparing the binding amount between a partial peptide containing a domain and ABP1 or the activated peptide of the present invention.
- ASK1 or its partial peptide containing the N-terminal activation control domain and ABP1 or the activation peptide of the present invention are produced, and when both bind, the transcription of the reporter gene is activated It is intended to provide a method for screening for an ASK1 activation-regulating substance, characterized by comparing the expression level of the reporter gene in cells in the presence and absence of a test substance.
- ASK1 used in the above-mentioned screening method (a) or (b) is isolated from human or other warm-blooded animal cells using a method known per se (for example, the same method as described above for ABP1). It can be purified.
- the partial peptide of ASK 1 including the N-terminal activation control domain is not particularly limited as long as it has the amino acid sequence of the N-terminal activation control domain described above, and may be a part of the kinase domain or the C-terminal amino acid sequence. May be included.
- the partial peptide can be obtained by digesting ASK1 with an appropriate protease.
- ASK1 and its partial peptide containing the N-terminal activation control domain are genes known per se. After cloning the DNA encoding it according to engineering techniques,
- ABP1 and the activating peptide of the present invention (hereinafter sometimes simply referred to as ABP1) can be prepared according to the above-mentioned method.
- the cell that produces ASK1 is not particularly limited as long as it is a human or other warm-blooded animal cell that expresses ASK1, and examples include HeLa cells and HEK293 cells.
- Examples of cells that produce ASK1 or a partial peptide thereof containing an N-terminal activation control domain include transformants prepared by the above-described genetic techniques. Is done.
- Test substances include, for example, proteins, peptides, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, etc., and these substances are novel Or a known one.
- a compound that binds to ASK1 and inhibits the binding between ABP1 and ASK1 can be selected as an ASK1 activation modulator.
- the reaction between ABP1 and ASK1 can usually be performed at about 37 ° C for about several hours.
- ASK1 standard by suspending ASK1 or cells producing it in a buffer suitable for screening.
- the buffer is a buffer which does not inhibit the binding between ABP1 and ASK1, such as a phosphate buffer having a pH of about 4 to: L0 (preferably, a pH of about 6 to 8) and a tris-hydrochloride buffer. Any may be used.
- CHAPS, T Surfactants such as we en-80 TM (Kao-Atlas), digitonin, and dexcholate can also be added to the buffer.
- PMSF PMSF
- leptin bacitracin
- aprotune protein inhibitor
- E-64 protein inhibitor
- pepsintin protein inhibitor
- the ABP1 and ASK1 cells are left immobilized in the incubator, that is, while the cells are grown, or using cells fixed with daltaraldehyde / paraformaldehyde. Can be combined.
- the buffer a medium / Hank's solution is used as the buffer.
- ABP1 for example, 0.0 lm1 to 10 m1
- [125 1] coexist 1 0 one 4 M ⁇ L 0- 1Q analyte M simultaneously.
- NBS non-specific binding
- the solution is filtered through a glass fiber filter paper (when using ASK 1 producing cells) or BZF separated (when using purified ASK 1), washed with an appropriate amount of the same buffer, and left on the glass fiber filter paper or solid phase.
- radioactive e.g., the amount of [125 1]
- the screening kit of the present invention contains ABP1, preferably further contains ASK1 or a cell that produces it.
- screening kit of the present invention examples include, but are not limited to, the following.
- HeLa cells or HEK293 cells were subcultured into 12-well plates at 5 ⁇ 10 5 wells, and 3 ⁇ l. Flip, 5% C0 2, followed by culturing for 2 days at 95% air.
- ABP 1 A solution containing 0.1% ⁇ serum albumin (manufactured by Sigma) in PBS containing 0.1 mM and stored at 20 ° C.
- test substance solution M After adding 10- 1Q 5 IX 1 a test substance solution M, 5 nM of labeled AB? 1 5 1 pressurized tut, to react at room temperature for one hour. To determine the nonspecific binding A supplementary ABP 1 to 5 1 of 10- 4 M in place of the test substance.
- the cells used in the screening method (c) above include (1) DNA binding of ASK1 or its partial peptide containing the N-terminal activation control domain to a transcription factor (eg, GAL4, VP16, etc.).
- a transcription factor eg, GAL4, VP16, etc.
- a DNA encoding a fusion protein with either the domain or the transcription activation domain (2) a DNA encoding a fusion protein of ABP 1 or the activation peptide of the present invention and the other domain of the transcription factor, And (3) a cell containing a reporter gene under the control of a promoter activated by the transcription factor, preferably a yeast cell, a mammalian cell, or the like.
- Reporter genes include, for example, luciferase gene; 3-galactosidase gene, alkaline phosphatase gene, peroxididase gene, chloramphenico-l-acetyltransferase gene, and Darin fluorescein protein (GFP) gene. Is mentioned.
- a compound that inhibits reporter gene expression can be selected as an ASK1 activation modulator.
- the present invention relates to (1) ABP1 or the present invention, wherein the activation of the protein or the peptide in cells producing ASK1 or a partial peptide thereof including an N-terminal activation control domain and a kinase domain is performed.
- the present invention provides a method for screening for an ASK1 activation-regulating substance by measuring and comparing the presence and absence of the activation peptide of the present invention with (2) the presence of the ABP1 or the activation peptide of the present invention and a test substance. .
- ASK1 or N-terminal activation control domain and Cells that produce the partial peptide containing the ASK1 and kinase domain include cells that endogenously produce ASK1 (eg, HeLa cells, HEK293 cells, etc.) and ASK1 Animal cells into which DNA encoding the 1- or N-terminal activation control domain and its partial peptide containing the kinase domain has been introduced.
- the partial peptide of ASK 1 containing the N-terminal activation control domain and the kinase domain is represented by amino acid numbers 1 to 936 in the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing of Patent Document 1 (JP-A-10-93).
- Contains the same or substantially the same amino acid sequence as all or a part of the amino acid sequence (hereinafter, collectively referred to as “ASK1-NK sequence”), and is activated by binding to ABP1.
- ASK1-NK sequence a part of the amino acid sequence
- the “substantially identical amino acid sequence” refers to at least about 70%, preferably about 80% or more, more preferably about 90% or more, particularly preferably about 95% or more of the human ASK1-NK sequence. Most preferably, the amino acid sequence has about 98% or more homology.
- the partial peptides include, for example, 1) 1 or 2 or more in the ASK1-NK sequence (preferably about 1 to 30, preferably about 1 to 10, more preferably 1 to 10 5) amino acid sequences, and (2) one or more (preferably about 1 to 30, preferably about 1 to 10, more preferably 1 to 5) amino acids in the ASK1-NK sequence ) Amino acid sequence, 3 ASK 1— 1 or more (preferably 1 to 30, preferably 1 to 10 and more preferably 1 to 5) ) Amino acid sequence, or 1 or 2 or more (preferably about 1 to 30, preferably about 1 to 10, more preferably Amino acid sequence in which 1-5) amino acids have been replaced with other amino acids, or Mutations base peptide containing a set only the combined amino acid sequences are also included.
- ABP1 or the activated peptide of the present invention may be added to cells from the outside, or may be produced by ASK1-producing cells themselves. . In the latter case, ABP 1 may be produced endogenously,
- the transformant may be a genetically engineered transformant using ASK1-producing cells as a host in accordance with the above-described ABP1 expression method.
- Test substances include, for example, proteins, peptides, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, etc., and these substances are novel Or a known one.
- ASK 1 activation can be caused by ASK 1 autophosphorylation, kinases located downstream of the ASK 1 cascade (eg, MKK4 / 7, MK 3/6, J, p38, etc.) and other potential ASK 1 substrates It can be evaluated by measuring the phosphorylation of protein or synthetic peptide, the rate of inducing cell death, and the like.
- ASK1-producing cells are cultured on a multiwell plate or the like. Before performing the screening, replace the medium with a fresh medium or an appropriate buffer that is not toxic to the cells in advance, and add the test substance (or ABP 1 if the cells do not produce ABP 1) etc. for a certain period of time. After incubation, cells are extracted or the supernatant is collected, and the products and phenomena generated are quantified according to each method.
- Phosphorylation of ASK 1 and other proteins or peptides can be detected by Western blot analysis using antibodies specific to phosphorylated proteins (peptides) or by incorporation of [ 32 P] -labeled ATP into substrate proteins (peptides). It can be performed by a method such as detection by gel electrophoresis and autoradiography.
- the cell death assay is based on a system that can confirm the expression of ABP1 (for example, a cell into which DNA encoding a fusion protein of ABP1 and a fluorescent protein has been introduced, or an ABP1 expression vector that further contains a selectable marker gene). Introduced cells) are used to calculate the percentage of dead cells in all ABP1-expressing cells by using detachment from the plate or morphological observation (eg, membrane blebbing, fragmentation, etc.) as an index. It can be carried out.
- ABP1 for example, a cell into which DNA encoding a fusion protein of ABP1 and a fluorescent protein has been introduced, or an ABP1 expression vector that further contains a selectable marker gene. Introduced cells) are used to calculate the percentage of dead cells in all ABP1-expressing cells by using detachment from the plate or morphological observation (eg, membrane blebbing, fragmentation, etc.) as an index. It can be carried out.
- the substance when ASK1 is activated when a test substance is added to an ASK1 producing cell, the substance can be selected as an ASK1 activation promoting substance.
- the substance when ASK 1 was inactivated when the test substance was added, In this case, the substance can be selected as an ASK1 activation inhibitor.
- the present invention relates to, for example, ABP contained in (i) a) blood of a non-human mammal, b) a specific organ, c) a tissue or cell isolated from an organ, or (ii) a transformant.
- the present invention provides a method for screening a substance that changes the expression level of ABP1, that is, an ASK1 activation modulator, by measuring the mRNA level of 1 or the amount of ABP1 protein.
- ABP1 mRNA amount or protein amount is specifically performed as follows.
- a drug eg, TNF- a, IL-11, Fas, anticancer drugs, etc.
- physicochemical stress eg, UV, active oxygen, ischemia, etc.
- ABP1 mRNA contained in the obtained cells can be quantified by, for example, extracting mRNA from cells or the like by a conventional method and using a method such as RT-PCR, or It can also be quantified by known Northern blot analysis.
- the amount of ABP1 protein can be quantified by extracting the protein from cells or the like by an ordinary method and, for example, performing Western blot analysis using a labeled anti-ABP1 antibody.
- a transformant expressing ABP 1 or the activating peptide of the present invention is prepared according to the above-described method, and ABP 1 or the activating peptide of the present invention or the mRNA thereof contained in the transformant is prepared. It can be quantified and analyzed in the same way.
- a given time before drug or physicochemical stress is given to a normal or disease model non-human mammal (30 minutes to 24 hours, preferably 30 minutes to 12 hours, more preferably Is from 1 hour to 6 hours before) or after a certain time (30 minutes to 3 days, preferably 1 hour to 2 days, more preferably 1 hour to 24 hours), or drug or physicochemical stress
- the test substance is administered, and after a lapse of a certain time after the administration (30 minutes to 3 days, preferably 1 hour to 2 days, more preferably 1 hour to 24 hours), the ABP contained in the cells It can be performed by quantifying and analyzing the amount of mRNA or the amount of protein in 1.
- test substance When the transformant is cultured according to a conventional method, the test substance is mixed into the medium, and after culturing for a certain period of time (from 1 day to 7 days, preferably from 1 day to 3 days, more preferably after 2 days) (3 days later), by quantifying and analyzing the mRNA amount or protein amount of ABP1 or the activating peptide of the present invention contained in the transformant.
- Test substances include peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, and the like. These substances may be novel substances or known substances.
- a substance that increases the expression level of ABP1 can be selected as an ASK1 activation promoting substance, and a substance that decreases the expression level of ABP1 can be selected as an ASK1 activation inhibitor.
- the ASK1 activation promoter When the ASK1 activation promoter is used as an apoptosis-inducing agent, dissolve it in water or an appropriate buffer (eg, phosphate buffer, PBS, Tris-HCl buffer, etc.) to an appropriate concentration. Can be prepared. If necessary, commonly used preservatives, stabilizers, reducing agents, tonicity agents and the like may be added.
- the ASK-1 activation promoting substance has the function of inducing apoptosis in cells.Therefore, in some patients, it is not possible to expect removal of unnecessary or abnormal cells by apoptosis due to reduction of ABP1 or other factors. By administering an ASK 1 activation promoting substance to the patient to activate ASK 1, apoptosis via the ASK 1 cascade is induced in abnormal or unnecessary cells in the patient's body. Can be led.
- inducing apoptosis of ASK1 activation promoting substance is a prophylactic and therapeutically effective disease, such as cancer (eg, leukemia, esophageal cancer, stomach cancer, colorectal cancer, rectal cancer, lung cancer, liver cancer, kidney) Cancer, breast cancer, uterine cancer, ovarian cancer, prostate cancer, melanoma, bone myeloma, osteosarcoma, brain tumor, etc., autoimmune diseases (eg, systemic lupus erythematosus, scleroderma, rheumatoid arthritis, rheumatoid arthritis, siedalen syndrome) , Multiple sclerosis, insulin-dependent diabetes mellitus, psoriasis, ulcerative colitis, idiopathic thrombocytopenic purpura, Crohn's disease, glomerulonephritis, etc., viral infections (eg, hemorrhagic fever, T-cell leukemia, Sarcoma
- the ASK1 activation inhibitor When used as the above-mentioned prophylactic / therapeutic agent, it can be formulated in the same manner as the above-mentioned drug containing ABP1 or the activating peptide of the present invention or a salt thereof.
- the preparations obtained in this way are safe and low toxic, such as, for example, humans and other warm-blooded animals (eg, rats, mice, hamsters, egrets, sheep, sheep, goats, bushes, dogs, dogs, Cats, dogs, monkeys, chimpanzees, birds, etc.).
- warm-blooded animals eg, rats, mice, hamsters, egrets, sheep, sheep, goats, bushes, dogs, dogs, Cats, dogs, monkeys, chimpanzees, birds, etc.
- the dose of the ASK-1 activation promoting substance varies depending on the administration target, target organ, symptoms, administration method, and the like.
- the single dose varies depending on the administration target, target organ, symptoms, administration method, and the like.
- the dose can be administered in terms of 6 O kg.
- ASK1 activation inhibitor (hereinafter synonymous in the present specification) Can inhibit signal transduction by the ASK1 cascade to suppress cell apoptosis and inflammatory cytokine production. Therefore, by adding an ASK1 activation inhibitor to a cell, apoptosis / inflammatory cytokine production of the cell can be suppressed, and for example, it can be used as a research reagent for apoptosis, inflammatory reaction, etc. .
- an ASK1 activation inhibitor When used as an inhibitor of apoptosis or inflammatory cytokine production, adjust the concentration to the appropriate level in water or an appropriate buffer (eg, phosphate buffer, PBS, Tris-HCl buffer). It can be prepared by dissolving in water. If necessary, commonly used preservatives, stabilizers, reducing agents, tonicity agents and the like may be added.
- an appropriate buffer eg, phosphate buffer, PBS, Tris-HCl buffer.
- ASK1 activation inhibitors have the function of suppressing apoptosis of cells and production of inflammatory cytokines.
- ASK1 activation inhibitor is administered to a patient to inhibit the activation of ASK1 in a patient who has lost or has developed an inflammatory disease. It can suppress apoptosis death and inflammatory response.
- inhibiting ASK1 activation by inhibiting apoptosis or inflammation can prevent or treat diseases that are therapeutically effective, such as viral infections (eg, AIDS, influenza, unknown fever, etc.), endocrine diseases (eg, hormones) Deficiency, site force in deficiency, etc.), blood diseases (eg, cytopenia, renal anemia, etc.), organ dysplasia (eg, thyroid atrophy) Congenital, cleft palate, etc.), transplanted organ rejection, graft-versus-host disease, immunodeficiency, neurodegenerative disease (eg, polyglutamine disease, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, prion disease, cerebellar degeneration) Etc.), ischemic heart disease (eg, angina pectoris, myocardial infarction, etc.), radiation damage, UV damage (eg, sunburn, etc.) Toxic disease (eg, renal tubular cell damage by heavy metals, liver cell damage by
- the ASK1 activation inhibitor When used as the above-mentioned prophylactic / therapeutic agent, it can be formulated in the same manner as the above-mentioned drug containing ABP1 or the activating peptide of the present invention or a salt thereof.
- the preparations obtained in this way are safe and low toxic, they can be used, for example, in humans and other warm-blooded animals (eg, rats, mice, hamsters, egrets, sheep, sheep, goats, pigs, pigs, dogs, cats). , Dogs, monkeys, chimpanzees, birds, etc.).
- warm-blooded animals eg, rats, mice, hamsters, egrets, sheep, sheep, goats, pigs, pigs, dogs, cats.
- the dose of the ASK1 activation inhibitor varies depending on the administration target, target organ, symptoms, administration method, and the like.
- oral administration in general, for example, in polyglutamine disease patients (60 kg) Is about 0.1 mg / day to: 00 mg L, preferably about 1.0 to 5 Omg, more preferably about 1.0 to 20 mg.
- parenteral administration the single dose varies depending on the administration subject, target organ, symptoms, administration method, etc.
- it is usually used, for example, for patients with polyglutamine disease (6 Okg ), It is convenient to administer about 0.01 to 30 mg per day, preferably about 0.1 to 2 Omg, more preferably about 0.1 to 10 mg per day.
- the amount converted per 6 Okg can be administered.
- the present invention relates to an exogenous DNA encoding ABP 1 (hereinafter referred to as the exogenous DN of the present invention).
- A) or a mutant DNA thereof (sometimes abbreviated as the foreign mutant DNA of the present invention).
- Non-human mammal used in the present invention is not limited to,
- Non-human mammals having the exogenous DNA of the present invention or the mutant DNA thereof include unfertilized eggs, fertilized eggs, germ cells including spermatozoa and their progenitor cells, and the like.
- the calcium phosphate method, the electric pulse method, the Lipofection method It can be produced by transferring the target DNA by the agglutination method, microinjection method, particle gun method, DEAE-dextran method and the like.
- the exogenous DNA of the present invention intended for somatic cells, organs of living organisms, tissue cells and the like can be transferred and used for cell culture, tissue culture and the like.
- the DNA transgenic animal of the present invention can also be produced by fusing the cells with the above-mentioned germ cells by a cell fusion method known per se.
- mice for example, porcupines, pigs, higgins, goats, magpies, dogs, cats, guinea pigs, hamsters, mice, rats and the like are used.
- a crossing strain a B6C3F strain, a BDF ⁇ strain, a BeDSFi strain, a BALBZc strain, an ICR strain, etc.
- a rat eg, Wistar, SD, etc.
- mammal in the recombinant vector that can be expressed in mammals, human and the like can be mentioned in addition to the above-mentioned non-human mammals.
- the exogenous DNA of the present invention is not DNA that encodes ABP1 originally possessed by a non-human mammal, but ABP1 that has been once isolated and extracted from a mammal. Refers to the DNA to be loaded.
- mutant DNA of the present invention DNA having a mutation (for example, mutation) in the base sequence of the original ABP1-encoding DNA, specifically, base addition, deletion, substitution with another base
- DNA that is used is used, and also includes abnormal DNA.
- the abnormal DNA means a DNA that expresses abnormal ABP1, and for example, a DNA that expresses abnormal ABP1 that suppresses the function of normal ABP1 is used.
- the exogenous DNA of the present invention may be derived from a mammal that is the same or different from the animal of interest.
- various mammals having ABP1 DNA having high homology thereto for example, egret, dog, cat, guinea pig, hams, rat, mouse
- DNA constructs eg, vector
- DNA encoding human ABP1 is linked downstream of various promoters that can express DNA of interest from fertilized eggs of target mammals, such as mouse fertilized eggs.
- ABP1 expression vectors include plasmids derived from Escherichia coli, plasmids derived from Bacillus subtilis, plasmids derived from yeast, bacteriophages such as ⁇ phage, retroviruses such as Moloney leukemia virus, vaccinia virus or vaccinia virus.
- animal viruses such as Among them, a plasmid derived from Escherichia coli, a plasmid derived from Bacillus subtilis or a plasmid derived from yeast are preferably used.
- promoters that regulate DNA expression include: (1) promoters of DNA derived from viruses (eg, simian virus, cytomegalovirus, Moroni leukemia virus, JC virus, breast cancer virus, poliovirus, etc.); (2) Promoters derived from various mammals (human, egret, dog, cat, guinea pig, hamster, rat, mouse, etc.), for example, albumin, insulin II, perobrakin II, Erasase, erythropoietin, endothelin, muscle creatine Kinase, glial fibrillary acidic protein, daltathione S-transferase, platelet-derived growth factor] 3, keratin Kl, K10 and ⁇ 14, collagen type I and type II, cyclic AMP-dependent protein kinase] 3 I subunit Stoma fin, tartrate-resistant alkaline phosphatase, atrial natriuretic factor, endothelial receptor thymic
- cytomegalovirus promoter capable of high expression throughout the body
- a human peptide chain elongation factor 1 (EF-1a) promoter a human and a chicken / 3-actin promoter, and the like are preferable.
- the above vector preferably has a sequence that terminates transcription of the target mRNA in a DNA transgenic mammal (generally referred to as "tamineta").
- tamineta a DNA transgenic mammal
- the sequence of each of the above DNAs can be used, and preferably, SV40 terminator of Simian virus or the like is used.
- the splicing signal of each DNA, the enhancer region, and a part of the intron of eukaryotic DNA are translated into the 5 'upstream of the promoter region and the promoter region for the purpose of further expressing the target exogenous DNA. Linkage between regions or 3 ′ downstream of the translation region is also possible depending on the purpose.
- the normal ABP 1 translation region is derived from liver B derived from humans or various mammals (eg, egrets, dogs, cats, guinea pigs, hamsters, rats, mice, etc.).
- Thyroid cells Thyroid cells, fibroblast-derived DNA and various commercially available DNA
- an exogenous abnormal DNA can produce a translation region obtained by mutating a normal ABP1 translation region obtained from the above-mentioned cells or tissue by a point mutagenesis method.
- the translation region can be prepared as a DNA construct that can be expressed in a transgenic animal by a conventional DNA engineering technique in which it is ligated downstream of the above-mentioned promoter and, if desired, upstream of the transcription termination site.
- Transfer of the exogenous DNA of the present invention at the fertilized egg cell stage is ensured to be present in all germ cells and somatic cells of the target mammal.
- the presence of the exogenous DNA of the present invention in the germinal cells of the transgenic animal after DNA transfer means that the progeny of the transgenic animal retains the exogenous DNA of the present invention in all of its germ cells and somatic cells Means to do.
- the progeny of this type of animal that has inherited the exogenous DNA of the present invention has the exogenous DNA of the present invention in all of its germ cells and somatic cells.
- the non-human mammal to which the exogenous normal DNA of the present invention has been transferred is confirmed to stably maintain the exogenous DNA by mating, and is subcultured as an animal having the DNA in a normal breeding environment. I can do it.
- Transfer of the exogenous DNA of the present invention at the fertilized egg cell stage is ensured to be present in excess in all germ cells and somatic cells of the target mammal.
- Excessive presence of the exogenous DNA of the present invention in the germinal cells of the animal after the transfer of the DNA indicates that the offspring of the animal of the present invention will have the foreign DNA of the present invention in all of its germinal and somatic cells. It means having excess.
- the progeny of such animals that have inherited the exogenous DNA of the present invention have an excess of the exogenous DNA of the present invention in all of their germinal and somatic cells.
- the non-human mammal having the exogenous normal DNA of the present invention in which the normal DNA of the present invention is highly expressed, promotes the function of endogenous normal DNA, and ultimately ABP1 hyperfunction. May develop and can be used as a model animal for the disease state. For example, using the normal DNA-transferred animal of the present invention, it is possible to elucidate the pathological mechanism of ABP1 hyperfunction and diseases associated with ABP1, and to examine treatment methods for these diseases.
- diseases associated with hyperactivity of ABP1 for example, viral infections (e.g., AIDS, Influenza, unknown fever, etc., endocrine diseases (eg, hormone deficiency, site force deficiency, etc.), blood diseases (eg, cytopenia, renal anemia, etc.), genital dysplasia (eg, thyroid atrophy, cleft palate, etc.) ), Transplant organ rejection, graft-versus-host disease, immunodeficiency, neurodegenerative diseases (eg, polyglutamine disease, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, prion disease, cerebellar degeneration, etc.), Ischemic heart disease (eg, angina pectoris, myocardial infarction, etc.), radiation damage, UV damage (eg, sunburn, etc.) Toxic disease (eg, renal tubular cell damage by
- a non-human mammal having the exogenous abnormal DNA of the present invention can be subcultured in a normal breeding environment as an animal having the DNA after confirming that the exogenous DNA is stably maintained by mating.
- the target foreign DNA can be incorporated into the above-mentioned plasmid and used as a raw material.
- the DNA construct with the promoter can be prepared by ordinary DNA engineering techniques. The transfer of the abnormal DNA of the present invention at the fertilized egg cell stage is ensured to be present in all germ cells and somatic cells of the target mammal.
- the presence of the abnormal DNA of the present invention means that all the offspring of the produced animal have the foreign abnormal DNA of the present invention in all of its germ cells and somatic cells.
- Type refractory disease eg, cancer (eg, leukemia, esophageal cancer, stomach cancer, colorectal cancer, rectal cancer, lung cancer, liver cancer, kidney cancer, breast cancer, uterine cancer, ovarian cancer, prostate cancer, melanoma, myeloma, bone) Sarcomas, brain tumors, etc.
- autoimmune diseases eg, systemic lupus erythematosus, scleroderma, rheumatoid arthritis, Sheddalen syndrome, multiple sclerosis, insulin-dependent diabetes mellitus, psoriasis, ulcerative colitis, idiopathic Thrombocytopenic purpura, Crohn's disease, glomerulonephritis, etc.
- viral infections eg.
- the abnormal DNA-highly expressing animal of the present invention elucidates the inhibition of normal ABP1 function (dominant negative action) by abnormal ABP1 in ABP1 inactive type refractory disease. Model.
- the mammal to which the foreign abnormal DNA of the present invention has metastasized since the mammal to which the foreign abnormal DNA of the present invention has metastasized has abnormal ABP 1 increasing symptoms, it can be used for a therapeutic drug screening test for ABP 1 function inactive refractory disease. .
- a non-human mammalian embryonic stem cell in which the DNA encoding ABP 1 has been inactivated [2] the DNA has introduced a reporter gene (eg, a / 3-galactosidase enzyme gene derived from Escherichia coli)
- a reporter gene eg, a / 3-galactosidase enzyme gene derived from Escherichia coli
- the DNA is inactivated by introducing a reporter gene (eg, a [3-galactosidase gene derived from Enterobacteriaceae), and the control of a promoter for the DNA encoding the reporter gene ABP1 is performed.
- a reporter gene eg, a [3-galactosidase gene derived from Enterobacteriaceae
- the non-human mammal according to (6) which can be expressed under
- a non-human mammalian embryonic stem cell in which the DNA encoding ABP 1 has been inactivated refers to whether the expression of the DNA is suppressed by artificially mutating the ABP 1 DNA of the non-human mammal. Alternatively, by substantially losing the activity of ABP1 encoded by the DNA, the DNA does not substantially have the ability to express ABP1 (hereinafter sometimes referred to as the knockout DNA of the present invention. )
- Non-human mammal embryonic stem cells hereinafter abbreviated as ES cells).
- Examples of a method for artificially mutating the DNA encoding ABP 1 include, for example, deletion of part or all of the DNA sequence by genetic engineering, and insertion or substitution of another DNA. it can.
- the knockout DNA of the present invention may be prepared by, for example, shifting the codon reading frame or disrupting the function of the promoter or exon by these mutations.
- non-human mammalian embryonic stem cells in which the DNA encoding ABP 1 has been inactivated include, for example, ABP1 DNA of the target non-human mammal is isolated, and its exon portion is a drug resistance gene typified by a neomycin resistance gene, a hygromycin resistance gene, or lacZ ( ⁇ -galactosidase enzyme), Inserting a reporter gene such as cat (chloramphenicol acetyltransferase gene) disrupts the function of exons, or terminates transcription of the gene in the intron between exons (eg, And polyA addition signal) to prevent the synthesis of complete mRNA, resulting in gene
- a DNA chain having a DNA sequence constructed so as to be integrated is introduced into the chromosome of the animal by, for example
- ES cells from which the DNA encoding ABP 1 is inactivated by the homologous recombination method or the like for example, those already established may be used as described above, or the known Evans and Kaufman method may be used. It may be newly established according to. For example, in the case of mouse ES cells, currently, 129 ES cells are generally used, but since the immunological background is not clear, an alternative pure immunogenic genetic background is used. For example, to obtain ES cells for which C57BL / 6 BDF mice in which the number of mice and the number of eggs collected by C57BL6 have been improved by crossing with DBAZ2 (such as those established using F of C57BLZ6 with DBA / 2) can also be used successfully.
- DBAZ2 such as those established using F of C57BLZ6 with DBA / 2
- BDFi mice In addition to the advantage of high number of eggs collected and the robustness of the eggs, it has C57BLZ6 mice as a background, and the ES cells obtained using these cells It can be advantageously used in that the genetic background can be replaced by C57BLZ6 mice by backcrossing with C57BLZ6 mice.
- blastocysts 3.5 days after fertilization are generally used, but in addition to this, a large number of cells can be efficiently collected by collecting 8-cell embryos and culturing them up to blastocysts. Early embryos can be obtained.
- male ES cells are generally more convenient for producing a germline chimera. It is also desirable to discriminate between males and females as soon as possible in order to reduce cumbersome culture labor.
- An example of a method for determining the sex of ES cells is a method of amplifying and detecting a gene in the sex-determining region on the Y chromosome by PCR. With this method, the number of ES cells in a colony was about 1 (about 50), compared to about 10 6 cells for karyotype analysis.
- the primary selection of ES cells in the initial stage can be performed by gender discrimination, and the early selection of male cells can greatly reduce the labor required in the initial culture.
- the secondary selection can be performed, for example, by confirming the number of chromosomes by the G-banding method.
- the embryonic stem cell line obtained in this way usually has very good growth potential, but it must be carefully subcultured because it tends to lose its ability to generate individuals.
- LIF (1-1000) on a suitable feeder cell such as STO fibroblasts (OU / ml) in a CO2 incubator (preferably 5% CO2, 95% air or 5% oxygen, 5% CO2, 90% air) at about 37 ° C
- the cells are treated with trypsin ZEDTA solution (usually 0.001-0.5% trypsin / 0.1-5 mM EDTA, preferably about 0.1% trypsin Z ImM EDTA) to obtain single cells.
- trypsin ZEDTA solution usually 0.001-0.5% trypsin / 0.1-5 mM EDTA, preferably about 0.1% trypsin Z ImM EDTA
- Such subculture is usually performed every 1 to 3 days. At this time, it is desirable to observe the cells and, if any morphologically abnormal cells are found, discard the cultured cells.
- ES cells can be transformed into various types of cells, such as parietal, visceral, and cardiac muscle, by monolayer culture up to high density or suspension culture until cell clumps are formed under appropriate conditions. It is possible to differentiate [MJ Evans and MH Kaufman, Nature 292, 154, 1981; GR Martin Proceedings of National Academy of Sciences, USA (Proc. Natl. Acad. Sci. USA) Vol. 78, p. 7634, 1981; TC Doetschman et al., Journal of Obembliology and 'Experimental Morphology, Vol. 87, p. 27, 1985]
- the DNA-deficient cells of the present invention obtained by differentiating the ES cells of the present invention are useful in the cell biological study of ABP1 in in vitro.
- the non-human mammal deficient in DNA expression of the present invention can be distinguished from a normal animal by measuring the mRNA amount of the animal using a known method and indirectly comparing the expression levels.
- non-human mammal the same one as described above is used.
- the targeting vector prepared as described above is introduced into mouse embryonic stem cells or mouse egg cells, and the ABP 1 DNA of the targeting vector is inactivated by the introduction.
- the homologous recombination of the NA sequence by gene homologous recombination replaces the ABP1 DNA on the chromosome of mouse embryonic stem cells or mouse egg cells, thereby knocking out the DNA encoding ABP1. it can.
- Cells in which the DNA encoding ABP 1 has been knocked out can be analyzed by Southern hybridization analysis using the DNA sequence on or near ABP 1 DNA as a probe, or the DNA sequence on the evening getter vector and the evening getter It can be determined by PCR analysis using the DNA sequence of the neighboring region other than the mouse-derived ABP1 DNA used in the study as a primer.
- a cell line in which DNA encoding ABP1 has been inactivated by homologous recombination is cloned, and the cells are cloned at an appropriate time, for example, at the 8-cell stage.
- the chimeric embryo is injected into a non-human mammal embryo or blastocyst, and the resulting chimeric embryo is transplanted into the uterus of the pseudo-pregnant non-human mammal.
- the animals produced are chimeric animals composed of both cells with a normal ABP1 locus and cells with an artificially mutated ABP1 DNA locus.
- all the tissues are artificially mutated from a population obtained by crossing such a chimeric individual with a normal individual. It can be obtained by selecting individuals composed of cells having the added ABP1 DNA locus, for example, by judging coat color. The individuals obtained in this manner are usually individuals with ABP1 heterozygous expression deficiency. Can be obtained.
- a transgenic non-human mammal having a targeting vector introduced into a chromosome can be obtained by injecting a DNA solution into the nucleus of an egg cell by a microinjection method. Compared to non-human mammals, it can be obtained by selecting those having a mutation in the ABP1 DNA locus by homologous recombination.
- the individual in which ABP1 DNA has been knocked out can be reared in a normal breeding environment after confirming that the DNA has been knocked out in the animal obtained by mating. .
- the germline can be obtained and maintained according to a standard method. That is, by crossing male and female animals having the inactivated DNA, the inactivated DNA is A homozygous animal having both homologous chromosomes can be obtained. The obtained homozygous animal can be efficiently obtained by rearing the mother animal in such a manner that there are one normal animal and one homozygote. By mating male and female heterozygous animals, homozygous and heterozygous animals having the inactivated DNA are bred and subcultured.
- a non-human mammalian embryonic stem cell in which ABP1 DNA has been inactivated is extremely useful for producing the non-human mammal deficient in expression of the DNA of the present invention.
- non-human mammal deficient in DNA expression of the present invention lacks various biological activities that can be induced by ABP1, and thus can serve as a model for a disease caused by inactivation of the biological activity of ABP1. Therefore, it is useful for investigating the causes of these diseases and examining treatment methods.
- the non-human mammal deficient in DNA expression of the present invention may be a disease caused by ABP1 DNA deficiency or damage, such as cancer (eg, leukemia, esophageal cancer, stomach cancer, colon cancer, rectum cancer, lung cancer, liver cancer, Kidney cancer, breast cancer, uterine cancer, ovarian cancer, prostate cancer, melanoma, myeloma, osteosarcoma, brain tumor, etc., autoimmune diseases (eg, systemic lupus erythematosus, scleroderma, rheumatoid arthritis, Schedalen) Syndrome, multiple sclerosis, insulin-dependent diabetes mellitus, psoriasis, ulcerative colitis, idiopathic thrombocytopenic purpura, Crohn's disease, glomerulonephritis, etc., inflammation (eg, arthritis, nephritis, etc.), viral infection ( Eg, hemorrhagic fever, T-cell
- the present invention provides a disease caused by ABP1 DNA deficiency or damage, which comprises administering a test compound to a non-human mammal deficient in expressing the DNA of the present invention and observing and measuring changes in the animal. Or a compound that has a therapeutic or preventive effect on And a method for screening for a salt.
- Examples of the non-human mammal deficient in DNA expression of the present invention used in the screening method include those described above.
- Test compounds include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and plasma.These compounds are novel compounds. Or a known compound.
- a non-human mammal deficient in expression of the DNA of the present invention is treated with a test compound and compared with a non-treated control animal, and changes in organs, tissues, disease symptoms and the like of the animal are indicated.
- the therapeutic and prophylactic effects of the test compound can be tested.
- test compound for example, oral administration, intravenous injection and the like are used, and it can be appropriately selected according to the symptoms of the test animal, the properties of the test compound, and the like.
- the dose of the test compound can be appropriately selected according to the administration method, the properties of the test compound, and the like.
- the symptom of the test animal is, for example, about 10% or more, preferably about 30% or more, more preferably
- the test compound can be selected as a compound having a therapeutic / preventive effect on the above-mentioned diseases.
- Compounds obtained by the screening method are compounds selected from the test compounds described above, and are used as pharmaceuticals such as safe and low toxic therapeutic and prophylactic agents for the above-mentioned diseases caused by ABP1 deficiency or damage. Can be used. Furthermore, compounds derived from the compounds obtained by the above screening can be used in the same manner.
- the compound obtained by the screening method may form a salt.
- the salt of the compound include physiologically acceptable acids (eg, inorganic acids, organic acids, etc.) and bases (eg, alkali metals, etc.). And the like, and a physiologically acceptable acid addition salt is particularly preferable.
- Such salts include, for example, salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.) and organic acids (eg, acetic acid, formic acid, Salts with on-acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, etc. are used.
- inorganic acids eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.
- organic acids eg, acetic acid, formic acid, Salts with on-acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, etc.
- a drug containing the compound or a salt thereof obtained by the screening method can be produced and used in the same manner as the drug containing ABP1 or the activating peptide of the present invention.
- the present invention relates to a compound which promotes or inhibits the activity of a promoter for ABP1 DNA, which comprises administering a test compound to a non-human mammal deficient in expressing DNA of the present invention and detecting the expression of a repo overnight gene. Or a method for screening a salt thereof.
- the non-human mammal deficient in expression of DNA of the present invention may be one of the above-mentioned non-human mammals deficient in expression of DNA of the present invention in which ABP1 DNA is introduced by introducing a reporter gene. Those which are activated and which can express the reporter gene under the control of a promoter for ABP1 DNA are used.
- test compound examples include the same compounds as described above.
- the reporter gene the same one as described above is used, and an iS-galactosidase gene (1 acZ), a soluble alkaline phosphatase gene, a luciferase gene and the like are preferable.
- ABP 1 when a portion of the DNA region encoding ABP 1 is replaced with a 3-galactosidase gene (1 ac Z) derived from Escherichia coli, the ABP 1 expressing Instead, / 3_galactosidase is expressed. So, for example, An ABP 1 animal can be easily prepared by staining with a reagent that
- 3-galactosidase is a substrate, such as 5-bromo-4-chloro-3-indolyl iS-galactopyranoside (X-ga 1).
- an ABP1-deficient mouse or a tissue section thereof is fixed with dartalaldehyde, washed with phosphate buffered saline (PBS), and then stained with X-ga1 at room temperature or at 37 ° C. After reacting for about 30 minutes to 1 hour in the vicinity of C, the ⁇ -galactosidase reaction may be stopped by washing the tissue specimen with an lmM EDTAZPBS solution, and the coloration may be observed.
- mRNA encoding 1 ac Z may be detected according to a conventional method.
- the compound or a salt thereof obtained by using the above-mentioned screening method is a compound selected from the test compounds described above, and is a compound that promotes or inhibits the activity of the promoter for ABP1 DNA.
- the compound obtained by the screening method may form a salt.
- the salt of the compound include physiologically acceptable acids (eg, inorganic acids) and bases (eg, organic acids). And the like, and particularly preferred are physiologically acceptable acid addition salts.
- physiologically acceptable acids eg, inorganic acids
- bases eg, organic acids
- physiologically acceptable acid addition salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.), and organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid) Succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, etc.).
- inorganic acids eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.
- organic acids eg, acetic acid, for
- a compound or a salt thereof that promotes ABP 1 DNA promoter activity can promote ABP 1 expression and promote ABP 1 function.
- prevention and treatment of diseases related to ABP 1 dysfunction It can be used as a medicine such as a drug.
- the compound includes, for example, cancer (eg, leukemia, esophageal cancer, stomach cancer, colorectal cancer, rectal cancer, lung cancer, liver cancer, kidney cancer, breast cancer, uterine cancer, ovarian cancer, prostate cancer, melanoma).
- autoimmune diseases eg, systemic lupus erythematosus, scleroderma, rheumatoid arthritis, siedalen syndrome, multiple sclerosis, insulin-dependent diabetes mellitus, psoriasis, ulcerative colon
- Inflammation idiopathic thrombocytopenic purpura, Crohn's disease, glomerulonephritis, etc.
- viral infections eg, hemorrhagic fever, T-cell leukemia, lipopositive meat
- Tumors infectious mononucleosis, lymphoma, nasopharyngeal cancer, uterine face cancer, skin cancer, hepatitis, liver cancer, etc.
- endocrine diseases eg, hyperhormoneism, cytotoxicity, etc.
- blood diseases eg, blood Polycythemia, B-cell lymphoma, polycythemia, etc.
- a compound or a salt thereof that inhibits the promoter activity of ABP 1 DNA can inhibit the expression of ABP 1 and inhibit the function of ABP 1, such as a disease associated with excessive expression of ABP 1 It is useful as a medicament such as a prophylactic or therapeutic agent.
- the compound may be, for example, a viral infection (eg, AIDS, influenza, unknown fever, etc.), an endocrine disease (eg, hormone deficiency, cytotoxic deficiency, etc.), a blood disease (eg, cytopenia, Renal anemia, etc.), organ dysplasia (eg, thyroid atrophy, cleft palate, etc.), transplanted organ rejection, graft-versus-host disease, immunodeficiency, neurodegenerative disease (eg, polyglutamine disease, Alzheimer's disease, Parkinson's disease, muscle) Amyotrophic lateral sclerosis, prion disease, cerebellar degeneration, etc.), ischemic heart disease (eg, angina, myocardial infarction, etc.), radiation damage, UV damage (eg, sunburn, etc.) Toxic disease (eg, heavy metal) Renal tubular cell injury, alcohol-induced hepatocellular injury, etc.), nutritional disorders (eg, thymus atrophy due to vitamin and
- a medicament containing the compound or a salt thereof obtained by the screening method can be produced and used in the same manner as the medicament containing ABP1 or the activated peptide of the present invention.
- the non-human mammal deficient in DNA expression of the present invention has ABP 1 DNA. It is extremely useful in screening for compounds or salts thereof that promote or inhibit the activity of promoters against them, and greatly contribute to the investigation of the causes of various diseases caused by insufficient expression of ABP1 DNA or the development of therapeutic drugs. Can be.
- transgenic animal In addition, using a DNA containing the promoter region of the ABP1 gene, genes encoding various proteins are ligated downstream of the DNA and injected into egg cells of an animal to produce a so-called transgenic animal (transgenic animal). ), It becomes possible to synthesize the protein in a tissue-specific and / or time-specific manner, and to examine its action in living organisms. Furthermore, if a suitable reporter gene is linked to the above promoter, and a cell line that expresses this is established, a low-molecular-weight compound that specifically promotes or suppresses the production of ABP1 itself in the body Can be used as a search system.
- TC thiazolidine-1 (R) monolithic lipoxamide group
- HONB 1-hydroxy-5-norpolene-2,3-dicarpoxyimide
- FIG. 2 shows the nucleotide sequence of the amino acid coding region of human ABP 1 cDNA.
- [SEQ ID NO: 3] 2 shows the nucleotide sequence of the amino acid coding region of mouse ABP1 cDNA.
- the amino acid sequence of a peptide used as an antigen for producing an anti-ABP1 antibody is shown.
- ASK1-KR in which the lysine (K) at the ATP-binding site (position 709) in the kinase domain of human ASK1 was replaced with arginine (R) as a bait.
- Brent's system [Zervos et al., Cell, Vol. 72, p. 223-232, 1993; Gyuris et al., Cell, Vol. 75, p. 791-803, 1993] using the yeast two-hybrid method. As a result, about 1200 positive clones were obtained.
- ABP 1 a database search based on the obtained nucleotide sequence shows that a part of the protein coding region of the gene named "PGR 1" (corresponding to amino acids 35 to 127 in amino acids) Part).
- the protein encoded by this gene consists of 127 amino acids, but the information in the database is only about the structure of the gene, and there was no report on its function. Therefore, the full-length cDNA encoding the protein of this gene was cloned by the PCR method. Since this protein was identified as an ASK1 binding protein, it was named ASK1 binding protein 1 (ABP1). Homologous search revealed that ABP 1 had similar molecules in mammals such as mice, but no molecules with high homology were found in flies and nematodes. It was. In addition, existing motifs and functional domains did not exist when motif search or domain search was performed.
- Figure 1 shows the alignment of the human and mouse ABP 1 amino acid sequences.
- Example 2 ABP 1 niRNA expression distribution
- a polyclonal antibody of Egret against ABP1 was prepared. Based on the amino acid sequence information obtained from the nucleotide sequence of ABP 1 cDNA, two types of peptide antibodies using ABP 1 specific peptides (SEQ ID NO: 5 and SEQ ID NO: 6) as antigens were prepared. It was named LVR antibody. Both antibodies were purified using the affinity of the antigen peptide.
- HeLa cells and HEK293 cells were cultured in Dulbecco's modified Eagle's medium (DMEM; SIGMA) containing high-concentration glucose (4.5 mg / ml) in the presence of 5% C ⁇ 2 .
- DMEM Dulbecco's modified Eagle's medium
- the culture medium was supplemented with 10% fetal calf serum (FBS) and 100 units of Zml / nicillin.
- FBS fetal calf serum
- Porcine aortic endothelial (PAE) cell culture was performed by adding 10% FBS, 10 mM HEPES, and 100 units per ml of nicillin to an F12 medium (Invitrogen).
- Each cell was treated with a lysis buffer [150 mM NaC1, 20 mM Tris-HC1 (pH 7.5), 1 OmM EDTA, 1% Triton X-100, 1% dexcholate, 1.5% aprotinin, ImM PMSF]
- the lysate is centrifuged to remove the supernatant, and SDS sample buffer [lO OmM Tris-HC1 (pH 8.8), 0.01% bromophenol blue, 36% glycerol, 4% SDS] is added.
- SDS polyacrylamide gel electrophoresis SD S-PAGE
- Two fusion proteins, F1ag-ABP1 and Myc—ASK1 were co-expressed in HEK293 cells'.
- the cells are lysed using a lysis buffer (described above), the lysate is centrifuged, the supernatant is collected, reacted with an anti-F1ag antibody (Clone M2; SIGMA), and added with Protein A-sepharose4B (Zymed Laboratories). Incubate for 30 minutes, wash three times with lysis buffer, add SDS sample buffer (described above), and confirm binding of both proteins by the same procedure as in the immunoblotting method. ( Figure 4A).
- Antibodies used were an anti-F1ag antibody (described above) and an anti-Myc antibody (Clone 9E10, Calbiochem). This binding was enhanced by treatment with H 2 O 2 , an activator of ASK1. Next, when CFP-ABP1 and Myc-ASK1 plasmids were co-expressed in HEK293 cells and immunoprecipitated with anti-F1ag antibody, the binding of both was confirmed (Fig. 4B). . Antibodies used were an anti-F1ag antibody (described above) and an anti-GFP antibody (Medical & Biological Laboratories CO.). The binding was also enhanced by H 2 0 2 treatment. This indicates that ABP1 and ASK1 bind in mammalian cells, and that the binding is enhanced by H 2 O 2 treatment. Example 5 Identification of ASK 1 binding site for ABP 1
- Binding of various HA-tagged ASK1-deficient mutants shown in Fig. 5A to ABP1 was carried out by co-expression with F1ag-ABP1 as in Example 3, followed by immunoprecipitation with anti-F1ag antibody. Searched by law.
- Antibodies used were anti-HA antibody (Clone 3F10, Roche Diagnostics K.K) and anti-F1 ag antibody (Clone M2, SIGMA).
- the expression vector of ASKl used was the one described in Saitoh et al. (Supra).
- ABP1 co-precipitated with ASK1-NT and ASK1-AC, but did not show coprecipitation with ASK1- ⁇ SK and ASK1-K (FIG. 5 ⁇ ). This revealed that ABP1 binds to the ⁇ -terminal domain of ASK1.
- Example 6 Cell death induction by ABP 1
- ABP 1 was considered to be a protein having cell death inducing ability.
- PAE-ABP1 cells a cell line that can induce ABP1 expression in PAE cells in a tetracycline-dependent manner.
- the production of PAE-ABP1 cells was carried out by partially modifying the method of Takeda et al. (J. Biol. Chem., 275, 9805-9913, 2000).
- the entire length of the Myc-tagged ⁇ ⁇ ⁇ ⁇ 1 cDNA was subcloned into the pTet-Splice-neo vector, and this and the pTet-tTAk-hyg plasmid were simultaneously transfected into PAE cells, unlike the method of Takeda et al. , 500 ng / m 1 tetracycline (Sigma), 400 units 1111 Hygromycin B (ako), 24 OmgXm 1 Neomycin (Geneticin, Life Technologies, Inc.) Cells that had formed colonies were designated as PAE-ABP1 cells.
- the maintenance culture of the cells was performed by adding 500 ng / m 1 tetracycline, 200 units of 1111 hygromycin B, and 3 Omg / ra 1 neomycin to the culture solution for maintaining the PAE cells described above.
- PAE-ABP1 cells do not express ABP1 when cultured in the presence of tetracycline.However, in the absence of tetracycline, the expression of ABP1 protein with Myc group can be expressed by immunoblotting after about 6 hours. ABP1 expression can be confirmed stably thereafter. Immunostaining with anti-Myc antibody (Clone 9E10, Calbiochem) showed stable expression of Myc—ABP1 in most cells. T / JP2003 / 014794
- PAE-ABP1 cells (2 ⁇ 10 6 cells) were lysed with 200 ml of lysis buffer [20 mM Tris-HC 1 (H7.5), 1 OmM EDTA, 0.5% Triton X 100] . The lysate was centrifuged and the supernatant was collected and treated with 0.2 mg / ml of protein K at 42 ° C for 1 hour. Next, the DNA was purified by phenol-chloroform extraction and ethanol precipitation, and this was added to a TE buffer (1 OmM Tris-HC1, 1 mM EDTA) containing 0.2 mg / ml liponuclease A. After re-dissolving and electrophoresing on a 2% agarose gel, the cells were stained with ethidium bromide and the migration pattern was photographed.
- lysis buffer 20 mM Tris-HC 1 (H7.5), 1 OmM EDTA, 0.5% Triton X 100
- PAE-ABP1 cells formed a DNA ladder with ABP1 protein expression, coincident with the appearance of cell death when observed under a microscope (FIG. 8). This indicated that cell death due to ABP1 was so-called apoptosis with DNA fragmentation. This was also confirmed by TUNEL staining of PAE-ABP1 cells.
- ABP 1 does not have a functional domain of an existing apoptosis-related factor. Therefore, in order to examine domains required for apoptosis by ABP1, plasmids expressing fusion proteins of various ABP1-deficient mutants and CFP shown in FIG. 10A were prepared, and a plasmid was prepared in the same manner as in Example 6 (1). HeLa cells were expressed to examine the presence or absence of cell death.
- An ABP1 expression vector was prepared by subcloning ABP1 cDNA or ABP1-deficient mutant cDNA A obtained by PCR into pECFP-CI (Clontech). Gene transfer of each plasmid into cells was performed using FuGENE6 (Roche Diagnostics K.K) as described in the instruction manual.
- the activation of JNK and ⁇ 38 during the induction of AB 1 expression using ⁇ -ABP1 cells was examined using the respective anti-phosphorylation protein antibodies.
- the antibodies used were anti-phosphorylated J J antibody (Cell Signaling), anti-phosphorylated p38 antibody (Cell Signaling), and anti-phosphorylated AS K1 antibody (Tobiume et al., EMB0 Rep., 2, 222-228, 2001). .
- the cells are Lyse in the same manner as in Example 4, using a lysis buffer (described above), centrifuge the lysate, collect the supernatant, react with anti-Myc antibody (Clone 9E10, Calbiochem), and use Protein A-separose4B (Zymed Laboratories) Was added, incubated for 30 minutes, washed three times with the lysis buffer, and then added with the SDS sample buffer (described above).
- the ABP1 of the present invention has an effect of activating the ASK1 cascade to induce apoptosis in cells and to induce the production of inflammatory site. Therefore, the ABP1, the polynucleotide encoding the same, and the like of the present invention are useful as a prophylactic / therapeutic agent for diseases that can be expected to have a preventive / therapeutic effect by inducing apoptosis in cells.
- the ABP1 inhibitor of the present invention suppresses apoptosis and production of inflammatory site force-in.
- ABP 1 and ASK 1 of the present invention provides a means for screening a novel preventive / therapeutic agent for apoptosis or inflammation-related disease.
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AU2003284597A AU2003284597A1 (en) | 2002-11-22 | 2003-11-20 | Apoptosis-associated protein and use thereof |
US10/535,931 US7390625B2 (en) | 2002-11-22 | 2003-11-20 | Apoptosis-associated protein and use thereof |
EP03774087A EP1582586A4 (en) | 2002-11-22 | 2003-11-20 | APOPTOSIS ASSOCIATED PROTEIN AND ITS USE |
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WO2012068464A2 (en) * | 2010-11-18 | 2012-05-24 | University Of Delaware | Methods of treating and preventing thrombotic diseases using ask1 inhibitors |
US9045485B2 (en) | 2010-12-16 | 2015-06-02 | Convergence Pharmaceuticals Limited | ASK 1 inhibiting pyrrolopyrimidine derivatives |
WO2013128418A2 (en) * | 2012-03-02 | 2013-09-06 | Council Of Scientific & Industrial Research | Peptide inhibitors as novel anti-hiv therapeutics |
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AU3885999A (en) * | 1998-05-05 | 1999-11-23 | Incyte Pharmaceuticals, Inc. | Human transcriptional regulator molecules |
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US7485414B2 (en) * | 2002-08-30 | 2009-02-03 | Rigel Pharmaceuticals, Inc. | Modulators of angiogenesis |
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US7390625B2 (en) | 2008-06-24 |
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WO2004048565A1 (ja) | 2004-06-10 |
AU2003284597A1 (en) | 2004-06-18 |
US20060240023A1 (en) | 2006-10-26 |
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