WO2003048202A2 - Gène activant le facteur nucléaire kappa b - Google Patents

Gène activant le facteur nucléaire kappa b Download PDF

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WO2003048202A2
WO2003048202A2 PCT/JP2002/012644 JP0212644W WO03048202A2 WO 2003048202 A2 WO2003048202 A2 WO 2003048202A2 JP 0212644 W JP0212644 W JP 0212644W WO 03048202 A2 WO03048202 A2 WO 03048202A2
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Prior art keywords
protein
activation
seq
gene
polynucleotide
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WO2003048202A3 (fr
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Akio Matsuda
Shuji Muramatsu
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Asahi Kasei Corp
Asahi Kasei Pharma Corp
Asahi Chemical Industry Co Ltd
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Asahi Kasei Corp
Asahi Kasei Pharma Corp
Asahi Chemical Industry Co Ltd
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Priority claimed from JP2001368692A external-priority patent/JP2006166701A/ja
Priority claimed from JP2002291302A external-priority patent/JP2006166705A/ja
Application filed by Asahi Kasei Corp, Asahi Kasei Pharma Corp, Asahi Chemical Industry Co Ltd filed Critical Asahi Kasei Corp
Priority to AU2002349784A priority Critical patent/AU2002349784A1/en
Publication of WO2003048202A2 publication Critical patent/WO2003048202A2/fr
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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
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4705—Regulators; Modulating activity stimulating, promoting or activating activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the present invention relates to a protein capable of activating NF- ⁇ B, a DNA sequence encoding the protein, a method for obtaining the DNA, a recombinant vector containing the DNA, a transformant containing the recombinant vector, and an antibody which specifically reacts with the protein.
  • the present invention also relates to use of the protein, DNA or antibody of the invention in the diagnosis, treatment or prevention of diseases associated with the excessive activation or inhibition of NF- K B.
  • the present invention also relates to a method for screening a substance capable of inhibiting or promoting NF- K B activation by using the protein, DNA, recombinant vector and transformant.
  • NF- K B (nuclear factor kappa B) plays an important role in the transcriptional regulation of various genes involved in inflammation and immunological reactions.
  • NF- K B is a homo- or heterodimer protein which belongs to the Rel family. In unstimulated conditions, NF- K B normally resides in the cytoplasm as an inactive form by forming a complex with an I K B (inhibitory protein of NF- K B) to mask the nuclear transport signal of NF- K B.
  • I K B When cytokines such as interleukin (IL)-1 and tumor necrosis factor (TNF)- stimulate cells, I K B is phosphorylated by IKK (I B kinase) and degraded by the 26S proteasome through ubiquitination. The released NF- K B moves to the nucleus, where it binds to the DNA sequence called the NF- K B binding sequence and induces the transcription of the gene under control of NF- K B.
  • NF- K B is believed to regulate the expression of genes such as those for immunoglobulins, inflammatory cytokines (e.g., IL-1 and TNF- ), interferons and cell adhesion factors.
  • NF- K B is involved in inflammation and immune responses through the expression induction of these genes.
  • NF- K B The inhibition of the function or activation of NF- K B may inhibit the expression of many factors (proteins) involved in inflammatory or immunological diseases or other diseases such as tumor proliferation.
  • NF- / B is a promising target for medicaments against diseases caused or characterized by autoimmunity or inflammation [see e.g., Clinical Chemistry 45, 7-17 (1999); J Clin. Pharmacol. 38, 981-993 (1998); Gut 43, 856-860 (1998); The New England Journal of Medicine 366, 1066-1071 (1997); TiPS 46-50 (1997); The FASEB Journal 9, 899-909 (1995); Nature 395, 225-226 (1998); Science 278, 818-819 (1997); Cell 91, 299-302 (1997)].
  • Extracellular information is converted into a certain signal, which passes through the cell membrane and goes through the cytoplasm to the nucleus, where it regulates the expression of the target gene and causes cell responses. Therefore the elucidation of the mechanism of intracellular signal transduction from extracellular stimuli to NF- K B activation is of very important significance, because it provides very important means of developing new medicaments or therapies against autoimmune diseases and diseases exhibiting inflammatory symptoms.
  • NF- K B activation includes many steps mediated by various transmitters such as protein kinases. Therefore it is desirable for more efficient drug discovery to identify the transmitters which play a key role in the pathway, and to focus research on the transmitters to establish a new drug-screening method.
  • Some signaling molecules involved in NF- K B activation have been identified [e.g., IKK, ubiquitination enzymes and the 26S proteasome described above, as well as TNF receptor associated factor 2 (TRAF2) and NF- K B inducing kinase (NIK)].
  • TNF receptor associated factor 2 TNF receptor associated factor 2
  • NIK NF- K B inducing kinase
  • the object of the present invention is to identify a new gene and protein capable of directly, or indirectly, activating NF- K B, and to provide a method of use of them in medicaments, diagnostics and therapy. That is, an object of the present invention is to provide a new protein capable of activating NF- K B, a DNA sequence encoding the protein, a recombinant vector containing the DNA, a transformant containing the recombinant vector, a process for producing the protein, an antibody directed against the protein or a peptide fragment thereof, and a process for producing the antibody.
  • Another object of the present invention is to provide a method for screening a substance capable of inhibiting or promoting NF- K B activation using the protein, the DNA, the recombinant vector or the transformant, a kit for the screening, a substance capable of inhibiting or promoting NF- K B activation obtainable by the screening method or the screening kit, a process for producing the substance, a pharmaceutical composition containing a substance capable of inhibiting or promoting NF- K B activation, etc.
  • the present inventors have intensively studied to solve the above problems. As a result, the present inventors have succeeded in constructing a full-length cDNA library by using the oligo-capping method; establishing a gene function assay system by expression cloning using 293-EBNA cells; and isolating a new DNA (cDNA) encoding a protein having a function of activating NF- K B by using the assay system.
  • This new DNA molecule induced NF- K B activation by its expression in 293-EBNA cells.
  • This result shows that this new DNA is a signal transduction molecule involved in NF- K B activation.
  • the present invention has been completed.
  • a purified protein selected from the group consisting of: (a) a protein which comprises an amino acid sequence represented by any one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176,
  • a protein that activates NF- K B (Nuclear factor kappa B) and comprises an amino acid sequence having at least one amino acid deletion, substitution or addition in an amino acid sequence represented by any one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170,
  • a protein which comprises an amino acid sequence represented by any one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 200, 202, 204, 206,
  • a protein that activates NF- K B comprises an amino acid sequence having at least one amino acid deletion, substitution or addition in an amino acid sequence represented by any one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82,
  • a polynucleotide sequence which encodes a protein that activates NF- K B and consists of a polynucleotide sequence having at least one nucleotide deletion, substitution or addition in a polynucleotide sequence represented by any one of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151,
  • a polynucleotide sequence which encodes a protein that activates NF- K B and consists of a polynucleotide sequence having at least one nucleotide deletion, substitution or addition in a polynucleotide sequence represented by a coding region of any one of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147,
  • An isolated polynucleotide comprising a nucleotide sequence which encodes a protein that activates NF- K B and has at least 95% identity to the polynucleotide sequence according to (3) over the entire length thereof.
  • An isolated polynucleotide comprising a nucleotide sequence which encodes a protein that activates NF- K B and has at least 95% identity to the polynucleotide sequence according to (4) or (5) over the entire length thereof.
  • a recombinant vector which comprises a polynucleotide according to any one of (3) to (7).
  • a process for diagnosing a disease or susceptibility to a disease related to expression or activity of the protein of (1), (2) or (8) in a subject comprising the steps of:
  • a method for screening compounds capable of inhibiting or promoting NF- K B activation which comprises the steps of:
  • a method for screening compounds capable of inhibiting or promoting NF- K. B activation which comprises the steps of:
  • a compound capable of inhibiting or promoting NF- K B activation which is selected by the method for screening according to (15) or (16).
  • a process for producing a pharmaceutical composition which comprises the steps of:
  • a process for producing a pharmaceutical composition which comprises the steps of:
  • a kit for screening a compound capable of inhibiting or promoting NF- K B activation which comprises:
  • (21) A monoclonal or polyclonal antibody or a fragment thereof, which recognizes the protein according to (1), (2) or (8).
  • a ribozyme or deoxyribozyme capable of inhibiting NF- K B activation which has an action of cleavage of RNA that encodes the protein according to (1), (2) or (8) or an action of cleavage of RNA that encodes a protein involved in a pathway leading to NF- K B activation.
  • a method for treating a disease associated with NF- K B activation which comprises administering to a subject a compound screened by the process according to (15) or (16), and/or a monoclonal or polyclonal antibody or a fragment thereof according to (21) or (22), and/or an antisense oligonucleotide according to (24), and/or a ribozyme or deoxyribozyme according to (25) in an effective amount to treat a disease selected from the group consisting of inflammation, autoimmune diseases, cancers, infectious disease, bone diseases, AIDS, neurodegenerative diseases and ischemic disorders.
  • the pharmaceutical composition according to (27) for the treatment of inflammation, autoimmune diseases, cancers, infectious disease, bone diseases, AIDS, neurodegenerative diseases and/or ischemic disorders is provided.
  • a method of treating inflammation, autoimmune diseases, cancers, infectious diseases, bone diseases, AIDS, neurodegenerative diseases or ischemic disorders which comprises administering a pharmaceutical composition produced by the process according to (18) or (19) to a patient suffering from a disease associated with NF- K B activation.
  • a pharmaceutical composition which comprises a monoclonal or polyclonal antibody or a fragment thereof according to (21) or (22) as an active ingredient.
  • a pharmaceutical composition which comprises an antisense oligonucleotide according to (24) as an active ingredient.
  • a pharmaceutical composition which comprises a ribozyme or deoxyribozyme according to (25) as an active ingredient.
  • composition according to any one of (30) to (32) for the treatment and/or prevention of a disease which is selected from the group consisting of inflammation, autoimmune diseases, infectious diseases, cancers, bone diseases, AIDS, neurodegenerative diseases and ischemic disorders.
  • a computer-readable medium on which a sequence data set has been stored comprising at least one of nucleotide sequence selected from the group consisting of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175,
  • a method for calculating identity to other nucleotide sequences and/or amino acid sequences which comprises comparing data on a medium according to (34) with data of said other nucleotide sequences and/or amino acid sequences.
  • polypeptides comprising all or a part of the amino acid sequences selected from the group consisting of SEQ D NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58,
  • Fig. 1 is a graph showing NF- K B reporter activity inhibition by the proteasome inhibitor MG-132 in Example 3.
  • the axis of abscissa is MG-132 concentration and the transversal axis is relative luciferase activity.
  • a full-length cDNA was produced from mRNA prepared from normal human lung fibroblasts (purchased from Sanko Junyaku Co., Ltd.) and the like, and a full-length cDNA library was constructed in which the cDNA was inserted into the vector pME18S-FL3 (GenBank Accession AB009864).
  • the cDNA library was introduced into E. coli cells, and plasmid preparation was carried out per clone.
  • the pNK K B-LUC reporter plasmid (STRATAGENE) containing a DNA encoding luciferase and the above full-length cDNA plasmid were cotransfected into 293-EBNA cells (Invitrogen).
  • luciferase activity was measured, and the plasmid with significantly increased luciferase activity compared to that of a control experiment (vector pME18S-FL3 is introduced into a cell in place of a full-length cDNA) was selected (the selected plasmid showed a 5-fold or more increase in luciferase activity compared to that of the control experiment), and the entire nucleotide sequence of the cDNA cloned into the plasmid was determined.
  • the protein encoded by the cDNA thus obtained shows that this protein is a signal transduction molecule involved in NF- K B activation.
  • activation of NF- K B refers to activation of NF- K B (including induction of NF- K B activation) when a gene is introduced into a suitable cell and the protein encoded by the gene is excessively expressed.
  • Activation of NF- K B can be measured, for example, by an assay using an NF- K B dependant reporter gene.
  • Activation of NF- / B is reflected by increasing the reporter activity compared to control cells (cells into which the vector only was introduced).
  • Increase in reporter activity is preferably by a factor of 1.5 or more, more preferably by a factor of 2 or more, and still more preferably by a factor of 5 or more.
  • Reporter activity can be measured by cloning a polynucleotide (e.g. cDNA) encoding the protein to be expressed into a suitable expression vector, co-transfecting the expression vector and an NF- K B dependant reporter plasmid into a suitable cell, and after culturing for a certain period, then measuring reporter activity.
  • a polynucleotide e.g. cDNA
  • Suitable expression vectors are well known to those skilled in the art, examples of which include pME18S-FL3, pcDNA3.1 (Invitrogen).
  • the reporter gene can be one which enables a person skilled in the art to easily detect the expression thereof, and examples include a gene encoding luciferase, chloramphenicol acetyl transferase, or ⁇ -galactosidase.
  • Use of a gene encoding luciferase is most preferable, and examples of an NF- K B dependent reporter plasmid include pNF- K B-LUC (STRATAGENE).
  • Suitable cells include cells which exhibit an NF- K B activation response to stimulation by IL-1, TNF- and the like. Examples include 293-EBNA cells. Cell culture and introduction of genes into cells (transfection) can be performed and optimized by a person skilled in the art by known techniques.
  • 293-EBNA cells are inoculated on DMEM medium (Dulbecco's Modified Eagle Medium) containing 5% FBS (Fetal Bovine Serum) in a 96-well cell culture plate to a final cell density of 1 x IO 4 cells/well, and cultured for 24 hours at 37°C in the presence of 5% CO 2 .
  • reporter plasmid pNF- K B-Luc (STRATAGENE) and the expression vector are cotransfected into the cells in a well using FuGENE 6 (Roche).
  • NF- rc B activation is then measured by measuring luciferase activity using a long term luciferase assay system, Picagene LT2.0 (Toyo Ink Mfg).
  • Picagene LT2.0 Toyo Ink Mfg
  • luciferase activity can be measured using PerkinElmer's Wallac ARVOTMST 1420 MUL ⁇ LABEL COUNTER.
  • the method for gene introduction by FuGENE ⁇ , and measurement of luciferase activity by Picagene LT2.0 can be performed respectively according to the attached protocols.
  • the amount of FuGENE ⁇ per 1 well is suitably 0.3 to 0.5 ⁇ 1, preferably 0.3 ⁇ 1; the amount of pNF- K B-Luc plasmid is suitably 50 to lOOng, preferably 50ng; the amount of expression vector is suitably 50-100ng, and preferably lOOng.
  • An ability to activate NF- rc B can be confirmed by an ability to increase the reporter activity (luciferase activity) relative to the control experiment (cells into which only a null vector was introduced).
  • Increase in reporter activity is preferably by a factor of 1.5 or more, more preferably by a factor of 2 or more, and still more preferably by a factor of 5 or more.
  • a protein which comprises an amino acid sequence, which has at least 95% identity, preferably at least 97-99% identity, to one of the above amino acid sequences over the entire length thereof.
  • identity used herein is a relationship between two or more protein sequence or two or more polynucleotide sequences, as determined by comparing the sequences.
  • identity also means the degree of sequence relatedness between protein or polynucleotide sequences, as determined by the match between protein or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences.
  • Identity and similarity can be readily calculated by known methods. Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs.
  • BLAST Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ., J. Mol. Biol., 215: p403-410 (1990), Altschul SF, Madden TL, Schaffer AA, Zhang Z, MiUer W, Lipman DJ,. Nucleic Acids Res. 25: p3389-3402 (1997)).
  • BLAST Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ., J. Mol. Biol., 215: p403-410 (1990)
  • Altschul SF Madden TL
  • Schaffer AA Zhang Z, MiUer W, Lipman DJ,. Nucleic Acids Res. 25: p3389-3402 (1997).
  • the main initial conditions generally used in a BLAST search are as follows, but are not limited to these.
  • An amino acid substitution matrix is a matrix numerically representing the degree of analogy of each pairing of each of the 20 types of amino acid, and normally the default matrix of BLOSUM62 is used.
  • the theory of this amino acid substitution matrix is shown in Altschul S.F., J. Mol. Biol. 219: 555-565 (1991), and apphcability to DNA sequence comparison is shown on States D. J., Gish W., Altschul S.F., Methods, 3: 66-70 (1991).
  • optimal gap cost is determined by experience and in the case of BLOSUM62 preferably parameters of Existence 11, Extension 1 are used.
  • the expected value (EXPECT) is the threshold value concerning statistical significance for a match with a database sequence, and the default value is 10.
  • a protein having, for example, 95% or more identity to the amino acid sequence of SEQ ID NO: 2 may contain in the amino acid sequence up to 5 amino acid changes per 100 amino acids of the amino acid sequence of SEQ ID NO: 2.
  • a protein having 95% or more amino acid sequence identity to a subject amino acid sequence may have amino acids up to 5% of the total number of amino acids within the subject sequence, deleted or substituted by other amino acids, or amino acids up to 5% of the total number of amino acids within the subject sequence may be inserted within the subject sequence.
  • These changes within the subject sequence may exist at the amino terminus or the carboxy te ⁇ ninus of the subject sequence, or may exist at any position between these termini, or may form one or more groups of changes.
  • the Examples described below demonstrate that the protein consisting of an amino acid sequence of any one of the above SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110.
  • a polynucleotide which encodes a protein that activates NF- r B, and comprises a nucleotide sequence which has at least 95% identity, preferably at least 97-99% identity to any one of the above sequences;
  • Polynucleotides which are identical or almost identical to nucleotide sequences contained in the above nucleotide sequences may be used as hybridization probes to isolate full-length cDNA and genomic clones encoding the protein of the present invention, or cDNA or genomic clones of other genes that have a high sequence similarity to the above sequences, or genomic clones, or may be used as primers for nucleic acid amplification reactions.
  • these nucleotide sequences are 70% identical, preferably 80% identical, more preferably 90% identical, most preferably 95% identical to the above sequences.
  • the probes or primers will generally comprises at least 15 nucleotides, preferably 30 nucleotides and may have 50 nucleotides. Particularly preferred probes will have between 30 and 50 nucleotides. Particularly preferred primers have between 20 and 25 nucleotides.
  • the polynucleotide of the present invention may be either in the form of a DNA such as cDNA ,a genomic DNA obtained by cloning or synthetically produced, or may be in the form of RNA such as mRNA.
  • the polynucleotide may be single-stranded or double-stranded.
  • the double-stranded polynucleotides may be double-stranded DNA, double- stranded RNA or DNA:RNA hybrid.
  • the single-stranded polynucleotide may be sense strand also known as coding strand or antisense strand also known as non-coding strand.
  • a protein having the same activity that activates NF- K B as the protein having an amino acid sequence of any one of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168 170, 172, 174, 176, 178, 180, 182, 184, 186
  • One such method involves using conventional mutagenesis procedures for the DNA encoding the protein.
  • Another method is, for example, site-directed mutagenesis (e.g., Mutan-Super Express Km Kit from Takara Shuzo Co., Ltd.). Mutations of amino acids in proteins may also occur in nature.
  • the present invention also includes a mutated protein which is capable of activating NF- K B and which has at least one amino acid deletion, substitution or addition relative to the protein of any one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176
  • substitutions of amino acids are preferably conservative substitutions, specific examples of which are substitutions within the following groups: (glycine, alanine), (valine, isoleucine, leucine), (aspartic acid, glutamic acid), (asparagine, glutamine), (serine, threonine), (lysine, arginine) and (phenylalanine, tyrosine).
  • the present invention also includes a protein that activates NF- K B and comprises an amino acid sequence having a high identity to the amino acid sequence of any one of the above SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180
  • High identity refers to an amino acid sequence having an identity of at least 90%, preferably at least 97 to 99% over the entire length of an amino acid sequence expressed by any one of the above SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176,
  • the proteins of the present invention may be natural proteins derived from any human or animal cells or tissues, chemically synthesized proteins, or proteins obtained by genetic recombination techniques.
  • the protein may or may not be subjected to post-translational modifications such as sugar chain addition or phosphorylation.
  • proteins which are encoded by the genes of the present invention include secretion proteins (growth factors, cytokines, hormones, and the like), protein modification enzymes (protein kinase, protein phosphatase, protease, and the like), signal transduction molecules (protein-protein interaction moleculaes and the like), nuclear proteins (nuclear receptor, transcription factors and the like), and membrane proteins .
  • the membrane proteins include receptors, cell adhesion molecules, ion channels, and transporters.
  • the present invention also includes a polynucleotide encoding the above protein of the present invention.
  • the DNA includes cDNA, genomic DNA, and chemically synthesized DNA.
  • the DNA of the present invention includes a DNA which encodes a protein capable of activating NF- K B and hybridizes under stringent conditions with the DNA sequence of the above nucleotide sequence of any one of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167
  • stringent conditions refer to overnight incubation at 37 °C in a hybridization solution containing 30% formamide, 5 x SSC (0.75 M NaCl, 75mM trisodium citrate), 5 x Denhardt's solution, 0.5% SDS, 100 ⁇ g/ml denatured, sheared salmon sperm DNA) followed by washing (three times) in 2 x SSC, 0.1% SDS for 10 rninutes at room temperature, then followed by washing (two times) in 0.2 x SSC, 0.1% SDS for 10 minutes at 37 °C (low stringency).
  • Preferred stringent conditions are overnight incubation at 42°C in a hybridization solution containing 40% formamide, followed by washing (three times) in 2 x SSC, 0.1% SDS for 10 minutes at room temperature, then followed by washing (two times) in 0.2 x SSC, 0.1% SDS for 10 minutes at 42 °C (moderate stringency). More preferred stringent conditions are overnight incubation at 42T ⁇ in a hybridization solution containing 50% formamide, followed by washing (three times) in 2 x SSC, 0.1% SDS for 10 minutes at room temperature, followed by washing (two times) in 0.2 x SSC, 0.1% SDS for 10 minutes at 50°C (high stringency).
  • the DNA sequence thus obtained must encode a protein capable of activating NF- K B.
  • the present invention also includes a polynucleotide comprising a nucleotide sequence which encodes a protein capable of activating NF- K B and has a high sequence similarity to the nucleotide sequence of the polynucleotide according to above item (3), (4) or (5).
  • these nucleotide sequence are 95% identical, preferably 97% identical, more preferably 98-99% identical, most preferably at least 99% identical to the nucleotide sequence of the polynucleotide according to above item (3), (4) or (5) over the entire length thereof.
  • the above DNA of the present invention can be used to produce the above protein using recombinant DNA techniques.
  • the DNA and peptide of the present invention can be obtained by:
  • Techniques for cloning the DNA encoding the protein of the present invention in the above step (A) include, in addition to the methods described in the specification of the present appHcation, PCR amplification using a synthetic DNA having a part of the nucleotide sequence of the present invention (e.g., any one of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145,
  • a DNA inserted into a suitable vector for example, a commercially available library (e.g., from CLONTECH and STRATAGENE) can be used. Techniques for hybridization are normally used in the art, and can be easily carried out in accordance with various laboratory manuals such as T Maniatis et al., supra. Depending on the intended purpose, the cloned DNA encoding the protein of the present invention can be used as such or if desired after digestion with a restriction enzyme or addition of a linker.
  • the DNA thus obtained may have a nucleotide sequence of any one of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191,
  • the present invention also provides a recombinant vector, which comprises the above DNA sequence.
  • the expression vector for the protein of the present invention can be produced, for example, by excising the desired DNA fragment from the DNA encoding the protein of the present invention, and ligating the DNA fragment downstream of a promoter in a suitable expression vector.
  • Expression vectors for use in the present invention may be any vectors derived from prokaryotes (e.g., E. coli), yeast, fungi, insect viruses and vertebrate viruses so long as such vectors are replicable.
  • the vectors should be selected to be compatible with microorganisms or cells used as hosts. Suitable combinations of host cell - expression vector systems are selected depending on the desired expression product.
  • Plasmid vectors compatible with these microorganisms are generally used as replicable expression vectors for recombinant DNA molecules.
  • the plasmids pBR322 and pBR327 can be used to transform E. coli.
  • Plasmid vectors normally contain an origin of replication, a promoter, and a marker gene conferring upon a recombinant DNA a phenotype useful for selecting the cells transformed with the recombinant DNA.
  • promoters include a j3 -lactamase promoter, lactose promoter and tryptophan promoter.
  • marker genes include an ampicillin resistance gene, and a tetracycline resistance gene.
  • suitable expression vectors include the plasmids pUC18 and pUC19 in addition to ⁇ BR322, pBR327.
  • YEp24 can be used as a replicable vector.
  • the plasmid YEp24 contains the URA3 gene, which can be employed as a marker gene.
  • promoters in expression vectors for yeast cells include promoters derived from genes for 3- ⁇ hosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase and alcohol dehydrogenase.
  • promoters and terminators for use in expression vectors to express the DNA of the present invention in fungal cells include promoters and terminators derived from genes for phosphoglycerate kinase (PGK), glyceraldehyde-3-phosphate dehydrogenase (GAPD) and actin.
  • suitable expression vectors include the plasmids pPGACY2 and pBSFAHY83.
  • promoters for use in expression vectors to express the DNA of the present invention in insect cells include a polyhedrin promoter and P10 promoter.
  • expression vectors which are suitable for insect cells include baculo virus vector.
  • Recombinant vectors used to express the DNA of the present invention in animal cells normally contain functional sequences to regulate genes, such as an origin of replication, a promoter to be placed upstream of the DNA of the present invention, a ribosome-binding site, a polyadenylation site and a transcription termination sequence.
  • Such functional sequences which can be used to express the DNA of the present invention in eukaryotic cells, can be obtained from viruses and viral substances.
  • Examples of such functional sequences include an SR ct promoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter and HSV-TK promoter. Among them, a CMV promoter and SR ⁇ promoter can be preferably used.
  • any promoters can be used so long as they are suitable for use in the above host- vector systems.
  • origins of replication include foreign origins of replication, for example, those derived from viruses such as adenovirus, polyoma virus and SV40 virus. When vectors capable of integration into host chromosomes are used as expression vectors, origins of replication of the host chromosomes may be employed.
  • Suitable expression vectors include the plasmids pSV-dhfr (ATCC 37146), pBPV-l(9-l) (ATCC 37111), pcDNA3.1 (INVITROGEN) and pME18S-FL3.
  • the present invention also provides a transformed cell, which comprises the above recombinant vector.
  • Microorganisms or cells transformed with the replicable recombinant vector of the present invention can be selected from remaining untransformed parent cells based on at least one phenotype conferred by the recombinant vector as memtioned above. Phenotypes can be conferred by inserting at least one marker gene into the recombinant vector. Marker genes naturally contained in replicable vectors can be employed. Examples of marker genes include drug resistance genes such as neomycin resistance genes, and genes encoding dihydrofolate reductase.
  • any of prokaryotes e.g., E. coli
  • microorganisms e.g., yeast and fungi
  • insect and animal cells can be used so long as such hosts are compatible with the expression vectors used.
  • microorganisms include Escherichia coli strains such as E. coli K12 strain 294 (ATCC 31446), E. coli X1776 (ATCC 31537), E. coli C600, E. coh JM109 and E. coli B strain; bacterial strains belonging to the genus Bacillus such as Bacillus subtilis; intestinal bacteria other than E.
  • yeast such as Salmonella typhimurium or Serratia marcescens
  • various strains belonging to the genus Pseudomonas include Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Pichia pastoris.
  • yeast examples include Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Pichia pastoris.
  • fungi examples include Aspergillus nidulans, and Acremonium chrysogenum (ATCC 11550).
  • insect cells for example, Spodoptera frugiperda (Sf cells), High FiveTM cells derived from eggs of Trichoplusiani, etc.
  • insect cells for example, Spodoptera frugiperda (Sf cells), High FiveTM cells derived from eggs of Trichoplusiani, etc.
  • animal cells include HEK 293 cells, COS-1 cells, COS-7 cells, Hela cells, and Chinese hamster ovary (CHO) cells.
  • CHO cells and HEK 293 cells are preferred.
  • combinations of expression vectors and host cells to be used vary with experimental objects. According to such combinations, two types of expression (i.e. transient expression and constitutive expression) can be included.
  • Transformation of microorganisms and cells in the above step (C) refers to introducing DNA into microorganisms or cells by forcible methods or phagocytosis of cells and then transiently or constitutively expressing the trait of the DNA in a plasmid or an intra-chromosome integrated form.
  • Those skilled in the art can carry out transformation by known methods [see e.g., "Idenshi Kougaku Handbook (Genetic Engineering Handbook)", an extra issue of "Jikken Igaku (Experimental Medicine)", YODOSHA CO., LTD.].
  • DNA can be introduced into cells by known methods such as DEAE-dextran method, calcium-phosphate-mediated transfection, electroporation, hpofection, etc.
  • selection can be carried out by clonal selection of the animal cells containing the chromosomes into which the introduced expression vectors have been integrated.
  • transformants can be selected using the above selectable marker as an indication of successful transformation, hi addition, the animal cells thus obtained using the selectable marker can be subjected to repeated clonal selection to obtain stable animal cell strains highly capable of expressing the protein of the present invention.
  • DHFR dihydrofolate reductase
  • MTX methotrexate
  • the above transformed cells can be cultured under conditions which permit the expression of the DNA encoding the protein of the present invention to produce and accumulate the protein of the present invention. In this manner, the protein of the present invention can be produced.
  • the present invention also provides a process for producing a protein, which comprises culturing a transformed cell comprising the isolated polynucleotide according to above item (3) to (7) under conditions providing expression of the encoded protein, and recovering the protein from the culture (that is, cells or culture medium).
  • animal cells can be cultured by methods known to those skilled in the art (see e.g., "Bio Manual Series 4", YODOSHA CO., LTD.).
  • animal cells can be cultured by various known animal cell culture methods including attachment culture such as Petri dish culture, multitray type culture and module culture, attachment culture in which cells are attached to cell culture carriers (microcarriers), suspension culture in which productive cells themselves are suspended.
  • attachment culture such as Petri dish culture, multitray type culture and module culture
  • suspension culture in which productive cells themselves are suspended.
  • media for use in the culture include media commonly used for animal cell culture, such as D-MEM and RPMI 1640.
  • suitable combinations of per se known separation and purification methods can be used.
  • methods include methods based on solubility, such as salting-out and solvent precipitation; methods based on the difference in charges, such as ion-exchange chromatography; methods mainly based on the difference in molecular weights, such as dialysis, ultrafiltration, gel filtration and SDS-polyacrylamide gel electrophoresis; methods based on specific affinity, such as affinity chromatography; methods based on the difference in hydrophobicity, such as reverse phase high performance liquid chromatography; and methods based on the difference in isoelectric points, such as isoelectric focusing.
  • a protein of the present invention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography is employed for purification. Well known techniques for refolding proteins may be employed to regenerate active conformation when the polypeptide is denatured during intracellular synthesis, isolation or purification.
  • the protein of the present invention can also be produced as a fusion protein with another protein. These fusion proteins are also included within the present invention.
  • any vectors can be used so long as the DNA encoding the protein can be inserted into the vectors and the vectors can express the fusion protein.
  • proteins to which a polypeptide of the present invention can be fused include glutathione S-transferase (GST) and a hexa-histidine sequence (6 x His).
  • GST glutathione S-transferase
  • 6 x His hexa-histidine sequence
  • membrane protein When the protein of the present invention is a membrane protein, a transformed cell into which DNA encoding the protein of the present invention has been introduced can express the protein on its membrane.
  • the membrane which is prepared from such transformed cells and contains the protein of the present invention is also included within the present invention.
  • membrane of a cell includes cell membrane, and membrane of cell organelle.
  • the membrane of a cell can be prepared by a method known to those skilled in the art. For example, cells are collected from the culture where transformed cells are cultured, and suspended in a suitable buffer. Then, the cells are lysed by a homogenizer or by vortex after addition of glassbeads.
  • the obtained solution is centrifuged to remove uncrushed cells and the like, and the supernatant is ultracentrifuged under a sutable condition, and the obtained precipitate is suspended in a buffer to prepare a membrabe fraction.
  • the condition for ultracentrifugation can be suitably selected depending on the type of membrane and the like.
  • the present invention also includes a protein capable of inhibiting the activity of the protein of the present invention.
  • proteins include antibodies, or other proteins that bind to active sites of the protein of the present invention, thereby inhibiting the expression of their activity.
  • the present invention also relates to an antibody that reacts with the protein of the present invention or a fragment thereof, and to production of such an antibody. More preferably, the present invention relates to an antibody that specifically react with the protein of the present invention or a fragment thereof, and to production of such an antibody.
  • “specifically” means that closs-reactivity is low, more preferably closs-reactivity is not present.
  • the antibody of the present invention is not specifically limited so long as it can recognize the protein of the present invention.
  • examples of such antibodies include polyclonal antibodies, monoclonal antibodies and their fragments, single chain antibodies and humanized antibodies.
  • Antibody fragments can be produced by known techniques. Examples of such antibody fragments include, but not limited to, F(ab') 2 fragments, Fab' fragments, Fab fragments and Fv fragments.
  • a monoclonal or polyclonal antibody can be produced by administering the protein according to above item (1) or (2) as an antigen or epitope-bearing fragments to a non-human animal.
  • the antibody against the protein of the present invention can be produced by using the protein of the present invention or a peptide thereof as an immunogen according to per se known process for producing antibodies or antisera.
  • Such methods are described, for example, in "Shin Idenshi Kougaku Handbook (New Genetic Engineering Handbook)", the third edition, an extra issue of "Jikken Igaku (Experimental Medicine)", YODOSHA CO., LTD.
  • polyclonal antibodies for example, the protein of the present invention or a peptide thereof can be injected to animals such as rabbits to produce antibodies directed against the protein or peptide, and then their blood can be collected.
  • the polyclonal antibodies can be purified from the blood, for example, by ammonium sulfate precipitation or ion-exchange chromatography, or by using the affinity column on which the protein has been immobilized.
  • mice In the case of monoclonal antibodies, for example, animals such as mice are immunized with the protein of the present invention, their spleen is removed and homogenized to obtain spleen cells, which are then fused with mouse myeloma cells by using a reagent such as polyethylene glycol. From the resulting hybrid cells (i.e. hybridoma cells), the clone producing the antibody directed against the protein of the present invention can be selected. Then, the resulting clonal hybridoma cells can be implanted intraperitoneally into mice, the ascitic fluid recovered from the mice. The resulting monoclonal antibody can be purified, for example, by ammonium sulfate precipitation or ion-exchange chromatography, or by using the affinity column on which the protein has been immobilized.
  • a reagent such as polyethylene glycol.
  • the resulting antibody When the resulting antibody is used to administer it to humans, it is preferably used as a humanized antibody or human antibody in order to reduce its immunogenicity.
  • the humanized antibody can be produced using transgenic mice or other mammals.
  • Humanized chimeric antibodies can be produced by linking a V region of a mouse antibody to a C region of a human antibody. Humanized antibodies can be produced by substituting a sequence derived from a human antibody for a region other than a complementarity-determining region (CDR) from a mouse monoclonal antibody.
  • CDR complementarity-determining region
  • human antibodies can be directly produced in the same manner as the production of conventional monoclonal antibodies by immunizing the mice whose immune systems have been replaced with human immune systems. These antibodies can be used to isolate or to identify clones expressing the protein.
  • these antibodies can be used to purify the protein of the present invention from a cell extract or transformed cells producing the protein of the present invention.
  • These proteins can also be used to construct ELISA, RIA (radioimmunoassay) and western blotting systems.
  • ELISA radioimmunoassay
  • RIA radioimmunoassay
  • western blotting systems can be used for diagnostic purposes for detecting an amount of the protein of the present invention present in a body sample in a tissue or a fluid in the blood of an animal, preferably human.
  • they can be used for diagnosis of a disease characterized by undesirable activation of HF- K B resulting from (expression) abnormality of the protein of the present invention, such as inflammation, autoimmune diseases, infectious diseases, cancers, bone diseases, AIDS, neurodegenerative diseases and ischemic disorders.
  • a standard value (that is, a normal value for the expression of the protein of the present invention) must be established.
  • a method of calculating the standard value comprises binding a body fluid or a cell extract of normal individual of a human or an animal to an antibody against the protein of the present invention under a suitable condition for the complex formation, detecting the amount of the antibody-protein complex by chemical or physical means and then calculating the standard value for the normal sample using a standard curve prepared from a standard solution containing a known amount of an antigen (the protein of the present invention).
  • the presence of a disease can be confirmed by deviation from the standard value obtained by comparison of the standard value with the value obtained from a sample of an individual latently suffering from a disease associated with the protein of the present invention.
  • These antibodies can also be used as reagents for studying functions of the protein of the present invention.
  • the antibody of the present invention can be used as a medicament as mentioned below.
  • an antibody capable of inhibiting the activity of activating NF- K B of the protein of the present invention that is, neutralizing antibody.
  • the present invention is a pharmaceutical composition which comprises the above antibody as an active ingredient, and a method for therapy and/or prevention using the antibody of the present invention.
  • the active ingredient may be combined with other therapeutically or preventively active ingredients or inactive ingredients (e.g., conventional pharmaceutically acceptable carriers or diluents such as immunogenic adjuvants) and physiologically non-toxic stabilizers and excipients.
  • the resulting combinations can be sterilized by filtration, and formulated into vials after lyophilization or into various dosage forms in stabilized and preservable aqueous preparations.
  • Administration to a patient can be intra-arterial aclministration, intravenous administration and subcutaneous administration, which are well known to those skilled in the art.
  • the dosage range depends upon the weight and age of the patient, route of administration and the like. Suitable dosages can be determined by those skilled in the art.
  • These antibodies exhibit therapeutic activity by inhibiting the NF- K B activation mediated by the protein of the present invention. More specifically, the antibody of the present invention is useful as a medicament for treating or preventing a disease associated with abnormality of NF- K B activity such as inflammation, autoimmune diseases, cancers, infectious diseases, bone diseases, AIDS, neurodegenerative diseases and ischemic disorders.
  • the DNA of the present invention can also be used to isolate, identify and clone other proteins involved in intracellular signal transduction processes.
  • the DNA sequence encoding the protein of the present invention can be used as a "bait" in yeast two-hybrid systems (see e.g., Nature 340:245-246 (1989)) to isolate and clone the sequence encoding a protein ("prey") which can associate with the protein of the present invention.
  • prey a protein which can associate with the protein of the present invention.
  • proteins which can associate with the protein of the present invention can be isolated from cell extracts by immunoprecipitation [see e.g., "Shin Idenshi Kougaku Handbook (New Genetic Engineering Handbook)", an extra issue of "Jikken Igaku (Experimental Medicine)", YODOSHA CO., LTD.] using antibodies directed against the protein of the present invention.
  • the protein of the present invention can be expressed as a fusion protein with another protein as described above, and immunoprecipitated with an antibody directed against the fusion protein in order to isolate a protein which can associate with the protein of the present invention.
  • the present invention provides a process for diagnosing a disease or susceptibility to a disease related to expression or activity of the protein of present invention in a subject comprising the steps of:
  • the diagnostic assays offer a process for diagnosing diseases or determining a susceptibility to the diseases through detection of mutation in a gene for the protein of the present invention which has a function of activating NF- K B, by the methods described.
  • diseases may be diagnosised by methods comprising determining from a sample derived from a subject an abonormally decreased or increased level of protein or mRNA.
  • the determination of the presence or absence of a mutation in the nucleotide seqeunce of a the gene encoding the protein of the present invention which has a function of activating NF- K B may involve RT-PCR using a part of the nucleotide sequences of genes encoding these proteins as a primer, followed by conventional DNA sequencing to detect the presence or absence of the mutation.
  • PCR-SSCP Geneomics 5:874-879 (1989); "Shin Idenshi Kougaku Handbook (New Genetic Engineering Handbook)", an extra issue of "Jikken Igaku (Experimental Medicine)", YODOSHA CO., LTD.] can also be used to determine the presence or absence of the mutation.
  • Decreased or increased expression of a gene in a sample can be measured at the RNA level using any of the methods well known in the art for the quantitation of polynucleotides, for example, nucleic acid amplification methods such as RT-PCR, and methods such as RNase protection assay, Northern blotting and other hybridization methods.
  • Assay techniques that can be used to dete ⁇ nine levels of a protein in a sample derived from a host are well-known to those skilled in the art. Such assay methods include radioimmunoassays, competitive-binding assays, Western blot analysis and ELISA assays.
  • the antibody of the present invention mentioned above can be used.
  • the degree of abnormality of expression level of gene in a sample is not particularly Umited.
  • the level of the expressed protein is 2 or more times, or 1/2 or less, as compared with normal case, the subject may be disgnosed to be a disease.
  • the level of the expressed protein is 3 or more times, or 1/3 or less, as compared with normal case, the subject may be disgnosed to be a disease.
  • the DNA of the present invention can be used to detect abnormality in the DNA or mRNA encoding the protein of the present invention or a peptide fragment thereof. Therefore, the DNA is useful for gene diagnosis such as detection of damage, mutation, decreased, increased or excessively increased expression of said DNA or mRNA.
  • the mutation may cause a disease associated with the expression and/or activity of NF- /c B.
  • the abnormal expression of the novel protein of the present invention which acts to activate NF- K B may be responsible for diseases associated with the expression and/or activity of NF- K B.
  • the present invention also relates to a method for screening compounds which inhibit or promote NF- /c B activation using the proteins of the invention.
  • a compound which inhibits NF- K B activation has an activity as an inhibitor of NF- K B in vivo or in vitro as a result of this function, while a compound which promotes NF- K B activation has an activity as an activator of NF- K B in vivo or in vitro as a result of this function. Therefore, an activity as an inhibitor or activator of NF- K B is screened in the method for screening of the present invention.
  • the above compounds have an activity as an inhibitor or activator of NF- K B.
  • the method for screening comprises the steps of: (a) preparing a transformed cell by introducing a gene encoding a protein that promotes activation of NF- K B according to the present invention and a gene encoding a signal which can detect activation of NF- K B into a cell;
  • an activator compound a compound that increases said detectable signal 2-fold or higher than normal
  • an inhibitor compound a compound that decreases said detectable signal half or less than normal
  • genes encoding a signal which can detect activation of NF- K B include reporter genes. Reporter genes are used instead of directly detecting the activation of transcription factors of interest. The transcriptional activity of a promoter of a gene is analyzed by linking the promoter to a reporter gene and measuring the activity of the product of the reporter gene ("Bio Manual Series 4" (1994), YODOSHA CO., LTD.).
  • Any peptide or protein can be used so long as those skilled in the art can measure the activity or amount of the expression product (including the amount of the produced mRNA) of the reporter genes. For example, enzymatic activity of chloramphenicol acetyltransferase, ⁇ -galactosidase, luciferase, etc., can be measured.
  • Any reporter plasmids can be used to evaluate NF- K B activation so long as the reporter plasmids have an NF- K B recognition sequence inserted upstream of the reporter gene.
  • pNF- r B-Luc STRATAGEGE
  • Other examples include NF- K B dependent reporter plasmids described in Tanaka S. et al., J. Vet. Med. Sci. Vol.59 (7); Rothe M. et al., Science Vol.269, p.1424-1427 (1995).
  • Any host cells may be used so long as NF- K B activation can be detected in the host cells.
  • Preferred host cells are mammalian cells such as 293-EBNA cells. Transformation and culture of the cells can be carried out as described above.
  • the method for screening a compound which inhibits or promotes NF- K B activation comprises culturing the transformed cell for a certain period of time, adding a certain amount of a test compound, measuring the reporter activity expressed by the cell after a certain period of time, and comparing the activity with that of a cell to which the test compound has not been added.
  • the reporter activity can be measured by methods known in the art (see e.g., "Bio Manual Series 4" (1994), YODOSHA CO., LTD.).
  • test compounds include, but not limited to, low molecular weight compounds, haigh molecular compounds, and peptides.
  • Test compounds may be artificially synthesized compounds or naturally occurring compounds.
  • Test compounds may be a single compound or mixtures. Usable examples includes a library of low molecular weight compounds, a compound library which was synthesized by combinatorial chemistry, a narurally occurring product containing cells, plants, animals or a part thereof, or an extracred product of such narurally occurring product.
  • the test substance which shows an activity of inhibiting or promoting NF- K B activation can be further screened to isolate a single substance having the activity. Isolate and purification of a desired compound from a mixture can be carried out by using any knonw method such as filteration, extraction, washing, drying, concentration, crystallization or various chromatography in combination.
  • the method for screening according to the present invention can be carried out by the following steps:
  • Methods for measuring the activation of NF- K B in the above method include a method of analyzing the binding of NF- /c B to its binding sequence using cell extraction solution by gel shift (for example, Hayashi T. etal. J.Biol.Chem.268, p.26790-26795 (1993), Nauman M. et al. EMBO J. 13, p4597-4607 (1994)).
  • the amount of mRNA or proteins for genes whose expression is known to be induced by NF- K B activation e.g., genes for IL-1 and TNF- a
  • the amount of mRNA can be measured, for example, by northern hybridization, RT-PCR, etc.
  • the amount of proteins can be measured, for example, by using antibodies.
  • the antibodies may be produced by known methods. Commercially available antibodies(from, e.g., Wako Pure Chemical Industries, Ltd.) can also be used.
  • the present invention further provided a method of producing a pharmaceutical composition, which comprises the following steps (a) to (f):
  • a pharmaceutical composition can be produced by the following steps: (a) preparing a transformed cell by introducing a gene encoding a protein that promotes activation of NF- K B according to the present invention into a cell;
  • step (d) of the method of producing a pharmaceutical composition it is preferable to isolate or identify as an activator compound, a compound that increases said detectable signal 2-fold or higher than normal, and to isolate or identify as an inhibitor compound, a compound that decreases said detectable signal half or less than normal.
  • the protein of the present invention may also be used in a method for the structure-based design of an agonist, antagonist or inhibitor of the protein, by:
  • the present invention also provides a compound which is selected by the above screening method.
  • This compound has a function of inhibiting or promoting NF- /c B activation. More specifically, this compound has a function of inhibiting or promoting NF- K B activation by the protein of the present invention.
  • the compounds obtained by the above screening methods have a function of inhibiting or promoting NF- K B activation, they are useful as therapeutic or preventive pharmaceuticals for the treament of diseases resulting from unfavorable activation or inactivation of NF- K B.
  • a compound which is obtained in the form of a salt can be purified as it is.
  • a compound which is obtained in the free form can be converted into a salt by isolating and purifying a salt obtained by dispersing or dissolving the compound into a suitable solvent and then adding a desired acid or base.
  • Examples of a step to optimize the compounds or salts thereof obtained by the method of the present invention as a pharmceutical composition include methods of formulating according to ordinary processes such as the following.
  • the above compounds or their pharmaceutically acceptable salts in an amount effective as an active ingredient, and pharmaceutically acceptable carriers can be mixed.
  • a form of formulation suitable for the mode of administration is selected.
  • a composition suitable for oral administration includes a solid form such as tablet, granule, capsule, pill and powder, and solution form such as solution, syrup, elixir and dispersion.
  • a form useful for parenteral administration includes sterile solution, dispersion, emulsion and suspension.
  • the above carriers include, for example, sugars such as gelatin, lactose and glucose, starches such as corn, wheat, rice and maize, fatty acids such as stearic acid, salts of fatty acids such as calcium stearate, magnesium stearate, talc, vegetable oil,alcohol such as stearyl alcohol and benzyl alcohol, gum, and polyalkylene glycol.
  • examples of such liquid carriers include generally water, saline, sugar solution of dextrose and the like, glycols such as ethylene glycol, propylene glycol and polyethylene glycol.
  • the present invention also provides a kit for screening compounds which inhibit or promote NF- K B activation.
  • the kit comprises reagents and the like necessary for screening compounds which inhibit or promote NF- K B activation, including:
  • the present invention relates to a diagnostic kit which comprises:
  • a polynucleotide of the present invention having a nucleotide sequence expressed by SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165,
  • a protein of the present invention having an amino acid seqeunce expressed by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166,
  • a kit comprising at least one of (a) to (d) is useful for diagnosing a disease or susceptibility to a disease such as inflammation, autoimmune diseases, cancers, infectious diseases, bone diseases, AIDS, neurodegenerative diseases and ischemic disorders.
  • a disease or susceptibility to a disease such as inflammation, autoimmune diseases, cancers, infectious diseases, bone diseases, AIDS, neurodegenerative diseases and ischemic disorders.
  • NF- K B is involved in a wide variety of pathological conditions such as inflammation, autoimmune diseases, cancers, infectious diseases, bone diseases, AIDS, neurodegenerative diseases and ischemic disorders.
  • NF- K B activity may have significant physiological effects [see e.g., Ann. Rheum. Ds. 57:738-741 (1998); American Journal of Pathology 152:793-803 (1998); ARTHRITIS & RHEUMA ⁇ SM 40:226-236 (1997); Am. J. Respir. Crit. Care Med. 158:1585-1592 (1998); J. Exp. Med. 188:1739-1750 (1998); Gut 42:477-484 (1998); The Journal of Immunology 161:4572-4582 (1998); Nature Medicine 3:894-899 (1997)].
  • the present invention also relates to a method of use of a compound which inhibits the function of the protein capable of activating NF- /c B described above, for inhibiting NF- K B activation. Further, the present invention relates to a method of using a compound which activates the function of the protein capable of activating NF- K B described above, for promoting NF- K B activation.
  • the compound obtained by the above screening method which inhibits NF- K B activation, is useful as a medicament to treat or prevent diseases characterized by undesirable activation of NF- K B, such as inflammation, autoimmune diseases (such as rheumatoid arthritis, systemic lupus erythematosus, asthma, etc), infectious diseases, bone diseases, and graft rejection.
  • diseases characterized by undesirable activation of NF- K B such as inflammation, autoimmune diseases (such as rheumatoid arthritis, systemic lupus erythematosus, asthma, etc), infectious diseases, bone diseases, and graft rejection.
  • NF- K B activation controls apoptosis of cells.
  • the compound obtained by the above screening method which inhibits NF- /c B activation, may be capable of stimulating apoptosis.
  • Diseases which may be treated by the induction of apoptosis include tumors.
  • diseases related to abnormality in NF- K B activation include AIDS (acquired immunodeficiency syndrome), neurodegenerative diseases (Alzheimer's disease, Parkinson's disease, amyoxrophic lateral sclerosis, etc.), ischemic disorders (i.e. those caused by cardiac infarction, reperfusion injury, etc), myelogenesis incompetency syndrome (aplastic anemia, etc), skin diseases (Toxic epidermal necrolysis, etc), proliferative nephritis (IgA nephritis, purpuric nephritis, lupus nephritis, etc) and fulminant hepatitis.
  • a compound obtained by the above screening method which inhibits or promotes NF- K B activation, is useful as a medicament to treat or prevent these diseases.
  • the gene encoding the protein of the present invention is useful for gene therapy to treat various diseases such as inflammation, autoimmune diseases, cancers, infectious diseases, bone diseases, AIDS, neurodegenerative diseases and ischemic disorders.
  • Gene therapy refers to adniinistering into the human body a gene or a cell into which a gene has been introduced for the purpose of therapy of diseases.
  • the protein of the present invention and the DNA encoding the protein can also be used for diagnostic purposes.
  • the present invention provides a agent for gene therapy which comprises a gene encoding the protein of the present invention.
  • the form of the agent for gene therapy is not particularly limited, but includes a pharmaceutical composition which comprises a expression vector containing a gene of the present invention in a pharmaceutical carrier of physiological buffer.
  • the pharmaceutical carrier may contain suitable stabilizer (for example, nuclease inhibitor), chelate agent (for example, EDTA), and/or other auxiliary agent.
  • the agent for gene therapy of the present invention may be provided as a complex of an expression vector containing a gene of the present invention and a liposome.
  • the agent for gene therapy may be applied using a catheter.
  • the agent for gene therapy of the present invention can be directly injected into a blood vessel of patient and the like.
  • the dosage of the agent for gene therapy of the present invention should be selected depending on the conditions such as age, sex, body weight and symptom of patient, and administration route, and is generally about 1 ⁇ g/kg to about 1000 mg/kg, more preferably about 10 ⁇ g/kg to about 100 mg/kg, as an amount of DNA (which is an effective ingredient) per one administration for adult.
  • the number of administration is not particularly hmited.
  • the compound obtained by the screening method of the present invention or a salt thereof can be formulated into the above pharmaceutical compositions (e.g., tablets, capsules, elixirs, microcapsules, sterile solutions and suspensions) according to conventional procedures.
  • the formulations thus obtained are safe and of low toxicity, and can be administered, for example, to humans and mammals (e.g., rats, rabbits, sheep, pigs, cattle, cats, dogs and monkeys).
  • Administration to patients can be carried out by methods known in the art, such as intra-arterial injection, intravenous injection and subcutaneous injection.
  • the dosage and administration mode may vary with the weight and age of the patient, but those skilled in the art can appropriately select suitable administration mode and can appropriately select suitable dosage depending on the administration mode.
  • the DNA can be inserted into a vector for gene therapy, and gene therapy can be carried out.
  • the present invention relates to a medicament which comprises a compound capable of inhibiting or promoting NF- K B activation as an active ingredient.
  • the above compound is useful as a medicament to treat or prevent diseases characterized by abnormal NF- K B activity, such as inflammation, autoimmune diseases, cancers, infectious diseases, bone diseases, AIDS, neurodegenerative diseases and ischemic disorders.
  • diseases characterized by abnormal NF- K B activity such as inflammation, autoimmune diseases, cancers, infectious diseases, bone diseases, AIDS, neurodegenerative diseases and ischemic disorders.
  • the present invention also relates to a medicament to treat or prevent inflammation, autoimmune diseases, cancers, infectious diseases, bone diseases, AIDS, neurodegenerative diseases, ischemic disorders and the like, which comprises a compound capable of inhibiting or promoting NF- K B activation.
  • the compound is useful as a therapeutic and/or prophylactic drug against, for example, rheumatoid arthritis, osteoarthritis, systemic lupus erythematosus, diabetes, sepsis, asthma, allergic rhinitis, ischemic heart diseases, inflammatory intestinal diseases, subarachnoid hemonhage, viral hepatitis, AIDS, atherosclerosis, atopic dermatitis, viral infections, Crohn's disease, gout, hepatitis, multiple sclerosis, cardiac infarction, nephritis, osteoporosis, Alzheimer's, Parkinson's disease, Huntington's chorea, psoriasis, amyo trophic lateral sclerosis, or aplastic anemia.
  • rheumatoid arthritis arthritis
  • osteoarthritis systemic lupus erythematosus
  • diabetes sepsis
  • asthma allergic rhinitis
  • ischemic heart diseases inflammatory intestinal diseases,
  • the present invention also relates to the use of the above compound for manufacturing a medicament for the therapy and/or prevention of inflammation, autoimmune diseases, cancers, infectious diseases, bone diseases, AIDS, neurodegenerative diseases, ischemic disorders and the like.
  • the present invention also provides an antisense ohgonucleotide against the polynucleotide of any one of above items (3) to (7).
  • An antisense ohgonucleotide refers to an oligonucleotide complementary to the target gene sequence.
  • the antisense oligonucleotide can inhibit the expression of the target gene by inhibiting RNA functions such as translation to proteins, transport to the cytoplasm and other activity necessary for overall biological functions.
  • the antisense ohgonucleotide may be RNA or DNA.
  • the DNA sequence of the present invention can be used to produce an antisense oligonucleotide capable of hybridizing with the mRNA transcribed from the gene encoding the protein of the present invention.
  • an antisense oligonucleotide generally has an inhibitory effect on the expression of the corresponding gene (see e.g., Saibou Kougaku Vol.13, No.4 (1994)).
  • the oligonucleotide containing an antisense coding sequence against a gene encoding the protein of the present invention can be introduced into a cell by standard methods.
  • the oligonucleotide effectively blocks the translation of mRNA of the gene encoding the protein of the present invention, thereby blocking its expression and inhibiting undesirable activity.
  • the antisense oligonucleotide of the present invention may be a naturally occurring oligonucleotide or its modified form [see e.g., Murakami & Makino, Saibou Kougaku Vol.13, No.4, p.259-266 (1994); Akira Murakami, Tanpakushitsu Kakusan Kouso (PROTEIN, NUCLEIC ACID AND ENZYME) Vol.40, No.10, p.1364-1370 (1995),Tunenari Takeuchi et al., Jikken Igaku (Experimental Medicien) Vol. 14, No. 4 p85-95(1996)].
  • the oligonucleotide may have modified sugar moieties or inter-sugar moieties.
  • modified forms include phosphothioates and other sulfur-containing species used in the art.
  • at least one phosphodiester bond in the oligonucleotide is substituted with the structure which can enhance the ability of the composition to permeate cellular regions where RNA with the activity to be regulated is located.
  • Such substitution preferably involves a phosphorothioate bond, a phosphoramidate bond, methylphosphonate bond, or a short-chain alkyl or cycloalkyl structure.
  • the antisense ohgonucleotide may also contain at least some modified base forms. Thus, it may contain purine and pyrimidine derivatives other than naturaUy occurring purine and pyrimidine.
  • the furanosyl moieties of the nucleotide subunits can be modified so long as the essential purpose of the present invention is attained. Examples of such modifications include 2'-O-alkyl and 2'-halogen substituted nucleotides.
  • sugar moieties at their 2-position examples include OH, SH, SCH 3 , OCH 3 , OCN or O(CH 2 ) n CH 3 , wherein n is 1 to about 10, and other substituents having similar properties.
  • the analogues are included in the scope of the present invention so long as they can hybridize with the mRNA of the gene of the present invention to inhibit functions of the mRNA.
  • the antisense oligonucleotide of the present invention contains about 3 to about 50 nucleotides, preferably about 8 to about 30 nucleotides, more preferably about 12 to about 25 nucleotides.
  • the oligonucleotide of the present invention can be produced by the well-known sohd phase synthesis technique. Devices for such synthesis are commercially available from some manufactures including Applied Biosystems. Other ohgonucleotides such as phosphothioates can also be produced by methods known in the art.
  • the antisense oligonucleotide of the present invention is designed to hybridize with the mRNA transcribed from the gene of the present invention.
  • Those skilled in the art can easily design an antisense ohgonucleotides based on a given gene sequence (For example, Murakami and Makino: Saibou Kougaku Vol. 13 No.4 p259-266 (1994), Akira Murakami: Tanpakushitsu Kakusan Kouso (PROTEIN, NUCLEIC ACID AND ENZYME) Vol. 40 No.10 pl364-1370 (1995), Tunenari Takeuchi et al., Jikken Igaku (Experimental Medicine) Vol. 14 No. 4 p85-95 (1996)).
  • antisense oligonucleotides which are designed in a region containing 5' region of mRNA, preferably, the translation initaiation site, are most effective for the inhibition of the expression of a gene.
  • the length of the antisense oligonucleotides is preferably 15 to 30 nucleotides and more preferably 20 to 25 nucleotides. It is important to confirm no interaction with other mRNA and no formation of secondary structure in the oligonucleotide sequence by homology search.
  • the evaluation of whether the designed antisense oligonucleotide is functional or not can be determined by introducing the antisence oligonucleotide into a suitable cell and measuring the amount of the target mRNA, for example by northern blotting or RT-PCR, or the amount of the target protein, for example by western blotting or fluorescent antibody technique, to confirm the effect of expression inhibition.
  • Another method includes the triple helix technique.
  • This technique involves forming a triple helix on the targeted intra-nuclear DNA sequence, thereby regulating its gene expression, mainly at the transcription stage.
  • the oligonucleotide is designed mainly in the gene region involved in the transcription and inhibits the transcription and the production of the protein of the present invention.
  • Such RNA, DNA and oligonucleotide can be produced using known synthesizers.
  • the antisense oligonucleotide may be introduced into the ceUs containing the target nucleic acid sequence by any of DNA transfection methods such as calcium phosphate method, electroporation,lipofection, microinjection, or gene transfer methods including the use of gene transfer vectors such as viruses.
  • An antisense oligonucleotide expression vector can be prepared using a suitable rexrovirus vector, then the expression vector can be introduced into the cells containing the target nucleic acid sequence by contacting the vector with the cells in vivo or ex vivo.
  • the DNA of the present invention can be used in the antisense RNA/DNA technique or the triple helix technique to inhibit NF- K B activation mediated by the protein of the present invention.
  • the antisense ohgonucleotide against the gene encoding the protein of the present invention is useful as a medicament to treat or prevent diseases characterized by undesirable activation of NF- K B, such as inflammation, autoimmune diseases, cancers, infectious diseases, bone diseases, AIDS, neurodegenerative diseases and ischemic disorders.
  • diseases characterized by undesirable activation of NF- K B such as inflammation, autoimmune diseases, cancers, infectious diseases, bone diseases, AIDS, neurodegenerative diseases and ischemic disorders.
  • the present invention also provides a medicament which comprises the above antisense ohgonucleotide as an active ingredient.
  • the antisense oligonucleotide can also be used to detect such diseases using northern hybridization or PCR.
  • the present invention also provides a ribozyme or deoxyribozyme which inhibits NF- n B activation.
  • a ribozyme and deoxyribozyme is an RNA capable of recognizing a nucleotide sequence of a nucleic acid and cleaving the nucleic acid (see e.g., Hiroshi Yanagawa, "Jikken Igaku (Experimental Medicine) Bioscience 12: New Age of RNA).
  • the ribozyme or deoxyribozyme can be produced so that it cleaves the selected target RNA (e.g., mRNA encoding the protein of the present invention).
  • the ribozyme or deoxyribozyme specifically cleaving the mRNA of the protein of the present invention can be designed.
  • Such ribozyme has a complementary sequence to the mRNA for the protein of the present invention, complementarily associates with the mRNA and then cleaves the mRNA, which results in reduction or entire loss of the expression of the protein of the present invention.
  • the level of the reduction of the expression is dependent on the level of the ribozyme or deoxyribozyme expression in the target cells.
  • ribozyme or deoxyribozyme commonly used: a hammerhead ribozyme and a hairpin ribozyme.
  • hammerhead ribozymes or deoxyribozymes have been well studied regarding their primary and secondary structure necessary for their cleavage activity, and those skilled in the art can easily design the ribozymes nucleotided solely on the nucleotide sequence information for the DNA encoding the protein of the present invention [see e.g., Iida et al., Saibou Kougaku Vol.16, No.3, p.438-445 (1997); Ohkawa & Taira, Jikken Igaku (Experimental Medicine) Vol.12, No.12, p.83-88 (1994)].
  • the hammerhead ribozymes or deoxyribozymes have a structure consisting of two recognition sites (recognition site I and recognition site II forming a chain complementary to target RNA) and an active site, and cleave the target RNA at the 3 'end of its sequence NUX (wherein N is A or G or C or U, and X is A or C or U) after the formation of a complementary pair with the target RNA in the recognition sites.
  • the sequence GUC (or GUA) has been found to have the highest activity [see e.g., Koizumi, M. et al., Nucl. Acids Res.
  • a ribozyme is designed to form several,up to 10 to 20 complementary base pairs around that sequence.
  • the suitability of the designed ribozyme can be evaluated by checking whether the prepared ribozyme can cleave the target mRNA in vitro according to the method described for example in Ohkawa & Taira, Jikken Igaku (Experimental Medicine) Vol.12, No.12, p.83-88 (1994).
  • the ribozyme can be prepared by methods known in the art to synthesize RNA molecules.
  • the sequence of the ribozyme can be synthesized on a DNA synthesizer and inserted into various vectors containing a suitable RNA polymerase promoter (e.g., T7 or SP6) to enzymatically synthesize an RNA molecule in vitro.
  • a suitable RNA polymerase promoter e.g., T7 or SP6
  • Such ribozymes can be introduced into cells by gene transfer methods such as microinjection.
  • Another method involves inserting a ribozyme DNA into a suitable expression vector and introducing the vector into ceU strains, cells or tissues. Suitable vectors can be used to introduce the ribozyme into a selected cell.
  • vectors commonly used for such purpose include plasmid vectors and animal virus vectors (e.g., retrovirus, adenovirus, herpes or vaccinia virus vectors).
  • animal virus vectors e.g., retrovirus, adenovirus, herpes or vaccinia virus vectors.
  • ribozymes are capable of inhibiting the NF- K B activation mediated by the protein of the present invention.
  • the present invention further provides a process for obtaining a new gene having a function, which comprises using the oligo-capping method to construct a full-length cDNA library, and detecting the presence of a protein having the function by using a signal factor.
  • a signal factor is a reporter gene.
  • the cDNA libraries produced using the oligo-capping method contain full-length cDNA clones in a ratio of 50 to 80%, namely, a 5 to 10-fold increase in full-length cDNA clones compared to the cDNA libraries produced by prior art methods (Sumio Sugano, the monthly magazine BIO INDUSTRY Vol.16, No.ll, p.19-26).
  • Full-length cDNA clones are essential for protein expression in functional analyses of genes, and full-length cDNA clones themselves are very important materials for activity measurement. Thus, cloning of full-length cDNA is necessary for functional analyses of genes.
  • Sequencing of the cDNA not only provides important information for establishing the primary sequence of the protein encoded by the cDNA, but also reveals the entire exon sequence. Thus, the full-length cDNA provides valuable information for identifying a gene, such as information for determining the primary sequence of a protein, exon-intron structure, the transcription initiation site of mRNA, the location of a promoter, etc.
  • the construction of full-length cDNA libraries by the oligo-capping method can be carried out, for example, according to the method described in "Shin Idenshi Kougaku Handbook (New Genetic Engineering Handbook)", the third edition (1999), an extra issue of "Jikken Igaku (Experimental Medicine)", YODOSHA CO., LTD.
  • the oligo-capping method used herein involves substituting a cap structure with a synthetic oligo sequence by using BAP, TAP and an RNA ligase, as described in Suzuki & Sugano, "Shin Idenshi Kougaku Handbook (New Genetic Engineering Handbook)", the third edition (1999), an extra issue of "Jikken Igaku (Experimental Medicine)", YODOSHA CO., LTD.
  • the reporter gene which can be used as a signal factor which indicates the presence of a protein having a function contains one or more suitable expression regulation sequence portion to which a protein factor such as a transcriptional factor can bind, and a structural gene portion which allows the measurement of the activation of the proteins factor.
  • the structural gene portion may encode any peptide or protein so long as those skiUed in the art can measure the activity or amount of its expression product (including the amount of the mRNA produced). For example, chloramphenicol acetyltransferase, ⁇ -galactosidase, luciferase, etc., can be used and their enzymatic activity measured.
  • CREB cAMP responsive element binding protein
  • AP-1 activator protein- 1
  • a CREB-dependent reporter plasmid and an expression vector comprising full-length cDNA produced by the oligo-capping method can be cotransfected into cells, and an expression vector having increased reporter activity can be selected from the cells to attain the purpose.
  • a gene capable of inhibiting CREB is to be obtained, a CREB-dependent reporter plasmid and an expression vector comprising full-length cDNA produced by the oligo-capping method can be cotransfected into cells, and an expression vector having decreased reporter activity can be selected from the cells to attain the purpose.
  • the cDNA clone (expression vector) to be transfected into the cells may be a single clone or multiple clones which may be transfected simultaneously.
  • One embodiment of the process of the present invention is detaUed in Examples herein.
  • a screening system for obtaining a gene capable of inhibiting NF- K B activation can also be constructed by cotransfecting an expression vector comprising full-length cDNA and a reporter gene into cells, stimulating the cells with IL-1 or TNF- and the like, and selecting a clone having subnormally increased reporter activity.
  • a gene encoding a protein capable of activating various factors for example, MAP kinase, transcription factor
  • a gene encoding the protein capable of activating NF- K B can be obtained in addition to a gene encoding the protein capable of activating NF- K B.
  • the process of the present invention uses an in vitro system or a cell-based system, preferably a cell-based system.
  • a cell-based system examples include cells of prokaryotes such as E. coli, microorganisms such as yeast and fungi, as well as insects and animals.
  • Preferred examples include animal cells, in particular, 293-EBNA cells and NIH3T3 cells.
  • the cDNA of the present invention is fuU-length, its 5' end sequence is the transcription initiation site of the corresponding mRNA. Therefore the cDNA sequence can be used to identify the promoter region of the gene by comparing the cDNA with the genomic nucleotide sequence. Genomic nucleotide sequences are available from various databases when the sequences have been deposited in the databases. Alternatively, the cDNA can also be used to clone the desired sequence from a genomic library, for example, by hybridization, and determine its nucleotide sequence. Thus, by comparing the nucleotide sequence of the cDNA of the present invention with a genomic sequence, the promoter region of the gene located upstream the cDNA can be identified.
  • the promoter fragment thus identified can be used to construct a reporter plasmid for evaluating the expression of the gene.
  • the DNA fragment spanning 2kb (preferably lkb) upstream from the transcription initiation site can be inserted upstream of the reporter gene to produce the reporter plasmid.
  • the reporter plasmid can be used to screen for a compound which enhances or reduces the expression of the gene.
  • such screening can be carried out by transforming a suitable cell with the reporter plasmid, culturing the transfonned ceU for a certain period of time, adding a certain amount of a test compound, measuring the reporter activity expressed by the cell after a certain period of time, and comparing the activity with that of a cell to which the test compound has not been added.
  • the present invention also relates to a computer-readable medium on which a sequence data set has been stored, said sequence data set comprising at least one of nucleotide sequence selected from the group consisting of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169
  • the present invention relates to a method for calculating a homology, which comprises comparing data on the above medium with data of other nucleotide sequences.
  • the gene and amino acid sequence of the present invention provide valuable information for determining their secondary and tertiary structure, e.g., information for identifying other sequence having a similar function and high homology.
  • These sequences are stored on the computer-readable medium, then a database is searched using data stored in a known macromolecule structure program and a known search tool such as GCG In this manner, a sequence in a database having a certain homology can be easily found.
  • the computer-readable medium may be any composition of materials used to store information or data. Examples of such media include commercially available floppy disks, tapes, chips, hard drives, compact disks and video disks.
  • the data on the medium allows a method for calculating a homology by comparing the data with other nucleotide sequence data. This method comprises the steps of providing a first polynucleotide sequence containing the polynucleotide sequence of the present invention for the computer-readable medium, and then comparing the first polynucleotide sequence with at least one-second polynucleotide or polypeptide sequence to identify the homology.
  • the present invention also relates to an insoluble substrate to which polynucleotides comprising all or part of the nucleotide sequences selected from the group consisting of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 17
  • a plurality of the various polynucleotides which are DNA probes are fixed on a specifically processed solid substrate such as slide glass to form a DNA microanay and then a labeled target polynucleotide is hybridized with the fixed polynucleotides to detect a signal from each of the probes.
  • the data obtained is analyzed and the gene expression is dete ⁇ nined.
  • the present invention further relates to an insoluble substrate to which polypeptides comprising all or a part of the amino acid sequences selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 18
  • Example 1 Construction of a fuU-length cDNA library using the oligo-capping method
  • Human lung fibroblasts (Cryo NHLF: purchased from Sanko Junyaku Co., Ltd.) were cultured according to the manufacture's protocol. After repeating subculturing the cells to obtain fifty 10cm dishes containing the resulting culture, the cells were recovered with a cell scraper. Then, total RNA was obtained from the recovered cells by using the RNA extraction reagent ISOGEN (purchased from NIPPON GENE) according to the manufacturer's protocol. Then, poly A + RNA was obtained from the total RNA by using an oligo-dT cellulose column according to Maniatis et al., supra.
  • RNA extraction reagent ISOGEN purchased from NIPPON GENE
  • ATDC5 a cell strain cloned from mouse EC (embryonal carcinoma) (Atsumi, T. et al.: CeU Diff. Dev, 30: ⁇ l09-116)(1990) was repeatedly subcultured to obtain fifty 10cm dishes containing the resultant culture. Thereafter, poly A + RNA was obtained by a method similar to that of (1) above. Culture of ATDC5 cells was performed according to the method described in Atsumi, T. et al.: Cell Diff. Dev., 30: pl09-116 (1990).
  • a human T ceU strain purchased from DAINIPPON PHARMACEUTICAL CO.,LTD
  • poly A + RNA was obtained in the same way as in the above (1).
  • Jurkat ceUs were cultured in PRMI 1640 medium (GLBCO) containing 10%FBS (Fetal Bovine Serum: GIBCO) and lOmM HEPES (GIBCO) in the presence of 5% CO 2 at 37T .
  • RAW264.7 cells a mause macrophage-like ceU starain (ATCC Number TTB-71), were cultured to obtain poly A + RNA in the same way as in the above (1).
  • a fuU-length cDNA library was constructed from poly A + RNA of the above human lung fibroblasts, ATDC5 cells, Jurkat cells and RAW264.7 cells by the oligo-capping method according to the method of Sugano S. et al. [e.g., Maruyama, K. & Sugano, S., Gene, 138:171-174 (1994); Suzuki, Y. et al., Gene, 200:149-156 (1997); Suzuki, Y. & Sugano, S. "Shin Idenshi Kougaku Handbook (New Genetic Engineering Handbook)", the third edition (1999), an extra issue of "Jikken Igaku (Experimental Medicine)", YODOSHA CO., LTD.].
  • Sugano S. et al. e.g., Maruyama, K. & Sugano, S., Gene, 138:171-174 (1994); Suzuki, Y. et al., Gene, 200:149-156 (1997); Suzuki,
  • the full-length cDNA library constructed as above was transformed into E. coli strain TOP 10 by electroporation, then spread on LB agar medium containing 100 ⁇ g/ml of ampicillin, and incubated overnight at 37°C. Then, using QIAwell 96 Ultra Plasmid Kit (QIAGEN) according to the manufacturer's protocol, the plasmids were recovered from the colonies grown on ampicillin-containing LB agar medium.
  • the reporter activity of NF- K B was measured using long-term luciferase assay system,PIKKA GENE LT2.0 (TOYO INK) according to the attached manufacturer's instructions.
  • the luciferase activity was measured using WaUac ARVOTMST 1420 MULTILABEL COUNTER (Perkin Elmer).
  • the sequencing was carried out using the reagent Thermo Sequenase II Dye Terminator Cycle Sequencing Kit (Amersham Pharmacia Biotech) or BigDye Terminator Cycle Sequencing FS Ready Reaction Kit (Applied Biosystems) and the device ABI PRISM 377 sequencer or ABI PRISM 3100 sequencer according to the manufacturer's instructions.
  • the fuU-length DNA sequences for the 144 new clones which were obtained by the above screening were determined (SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185,
  • amino acid sequences of the protein coding regions were deduced (SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 200, 202, 204, 206
  • NF- /c B reporter activity The results of measurement of NF- /c B reporter activity (luciferase activity) of 41 clones among the above obtained clones are shown in Table 1 below.
  • the value of activity shown in Table 1 is a relative value to the value of control experiment (luciferase activity of a cell into which pME183-FL3 vector containing no insert has been introduced in place of full length cDNA).
  • Example 3 Screening compounds inhibiting NF- K B activation
  • 293-EBNA cells were seeded on DMEM medium containing 5% FBS in a 96-well cell culture plate to a final cell density of 1 x IO 4 cells/100 ⁇ 1/well, and cultured for 24 hours at 37°C in the presence of 5% CO 2 .
  • 50ng of the plasmid comprising the gene encoding NF- K B activating protein of SEQ ID NO: 12 which was obtained in Example 2 above, and 50ng of the reporter plasmid pNF K B-Luc were cotransfected into the cells in a weU using FuGENE 6.
  • the proteasome inhibitor MG-132 purchased form CALBIOCHEM
  • the present invention provides industrially highly useful proteins capable of activating NF- K B and genes encoding the proteins.
  • the proteins of the present invention and the genes encoding the proteins allow not only screening for compounds useful for treating and preventing diseases associated with the excessive activation or inhibition of NF- /c B, but also production of diagnostics for such diseases.
  • the genes of the present invention are also useful as a gene source used for gene therapy. AU publications, patents and patent applications cited herein are incorporated herein in their entirety.

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Abstract

Cette invention a trait à des protéines capables d'activer le NF-λ B, utilisées pour le diagnostic, le traitement ou la prévention d'états pathologiques associés à une activation excessive ou à l'inhibition du NF-λ B. Grâce à l'utilisation d'un NF-λ B-Luc plasmidique, un ADNc capable d'activer le NF-λ B a été cloné à partir d'une banque d'ADNc issue de fibroblastes pulmonaires et analogue, puis la séquence d'ADN et la séquence aminoacide dérivée déterminées. La protéine, l'ADN codant cette protéine, un vecteur de recombinaison contenant cet ADN ainsi qu'un transformant contenant ce vecteur, se révèlent des plus utiles pour le criblage d'une substance inhibant le NF-λ B ou stimulant son activation.
PCT/JP2002/012644 2001-12-03 2002-12-03 Gène activant le facteur nucléaire kappa b Ceased WO2003048202A2 (fr)

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JP2001368692A JP2006166701A (ja) 2001-12-03 2001-12-03 ＮＦ−κＢ活性化遺伝子
JP2001-368692 2001-12-03
US33582901P 2001-12-05 2001-12-05
US60/335,829 2001-12-05
JP2002-291302 2002-10-03
JP2002291302A JP2006166705A (ja) 2002-10-03 2002-10-03 ＮＦ−κＢ活性化遺伝子
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US60/415,769 2002-10-04

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