WO1999064454A1 - Novel nucleolar protein - Google Patents

Novel nucleolar protein Download PDF

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Publication number
WO1999064454A1
WO1999064454A1 PCT/JP1999/003014 JP9903014W WO9964454A1 WO 1999064454 A1 WO1999064454 A1 WO 1999064454A1 JP 9903014 W JP9903014 W JP 9903014W WO 9964454 A1 WO9964454 A1 WO 9964454A1
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protein
peptide
dna
seq
amino acid
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PCT/JP1999/003014
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French (fr)
Japanese (ja)
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Nobuhide Ueki
Kazuhiro Yano
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Helix Research Institute
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to novel nucleolar proteins and their genes, and their production and use.
  • Nuclear proteins play important roles in nuclear phenomena such as transcription, mRNA processing, replication, and chromosome organization. Many nuclear proteins carry unique signals that mediate active transport through the nuclear pore complex (NPC).
  • NPC nuclear pore complex
  • Primary sequences for selective translocation into the nucleus known as nuclear localization signals (NLS), and their corresponding receptors, such as the force lipophilin (Kap) family, have already been identified.
  • NLS nuclear localization signals
  • Kap force lipophilin
  • NLSs are lysine / arginine (K / R) -rich basic residues (potential consensus: single-class [K / R] 2 -X- [K / R]; a classic NLS consisting of short stretches of [K / R] 2 -X 1 (2- [K / R] 3 ), consisting of two classes, one of which is karyopherin or impo
  • the type of NLS and the number of Kaps corresponding to them have increased rapidly, which has led to an increase in the protein nucleus. Different transports It is mediated by different pathways.
  • nucleolus is not only a locus where several viral core proteins temporarily accumulate during cell growth (Dang, CV, and Lee, WM (1989) J. Biol. Chem. 264, 18019-18023, Cochrane, AW, Perkins, A., and Rosen, CA (1990) J. Virol. 64, 88 885, Hatanaka, M. (1991) in Genetic Structure and Regulation of HIV (Haseltine, WA, and Wong-Staal, F., Eds.), Pp. 264-287, Raven Press, New York, Siomi, H., Shida, H., Nam, SH, Nosaka, T., Maki, M., and Hatanaka, M.
  • Ribosomal proteins L31 and L5 (Quaye, I. ⁇ ⁇ , Toku, S., and Tanaka, T. (1996) Eur. J. Cell Biol. 69, 151-155, Michael, WM, and Dreyfuss, G. (1996) ) J. Biol. Chem. 271, 11571-11574), the human immunodeficiency virus viral proteins Tat and Rev (Dang, CV, and Lee, W..
  • the present invention provides a novel nucleolar protein, a peptide region involved in its nucleolar transport activity or nuclear transport activity, a nucleic acid molecule encoding them, and their production and use.
  • the present inventors have used a yeast nuclear transport trap (NTT) system (Ueki et al., Nature Biotechnology in press), which has been independently developed, to obtain a plurality of partial cMAs encoding a peptide having nuclear translocation activity. It has been isolated (Japanese Patent Application No. 10-174065).
  • NTT yeast nuclear transport trap
  • the present inventors attempted to isolate a full-length cDNA corresponding to one of the partial cDNAs (accession number AB015339). Specifically, screening of a SUPERSCRIPT human fetal brain cDNA library (Life Technology) was performed by polymerase chain reaction (PCR) using the partial cDNA sequence information and rapid amplification of cDNA ends (RACE). Thus it succeeded in isolating the desired full length cDNA of (this clone N0LP; Nucleolar Local ized Protein named the a
  • the cDNA was found to be A peptide-encoding short E. coli DNA helicase homology region, an acid-rich domain, three potential basic nuclear localization signals (NLS), a serine-rich domain, and a putative Had a coiled-coiled domain.
  • This protein did not show any significant homology to other proteins on a protein-by-protein basis.
  • the present inventors also performed a tissue distribution analysis of the transcript of the N0LP gene, and found that the gene showed a predominant expression in the brain and testis.
  • the present inventors prepared a set of deletion constructs having a fluorescent tag in order to identify a region required for nuclear translocation in the N0LP protein, and added the fluorescent tag to the N0LP protein (fusion protein).
  • fusion protein N0LP protein
  • the present invention relates to a novel nucleolar protein and a peptide region involved in its intracellular localization, a nucleic acid molecule encoding them, and their production and use.
  • a protein comprising the amino acid sequence of SEQ ID NO: 2, or one or more amino acids in the amino acid sequence of the protein are modified by deletion, addition, insertion, and / or substitution with another amino acid
  • a protein consisting of an amino acid sequence and having a nucleoli transfer activity
  • a DNA which specifically hybridizes with MA consisting of the nucleotide sequence of SEQ ID NO: 1 and has a chain length of at least 15 bases.
  • the present inventors have screened a cDNA encoding a peptide having nuclear transfer activity using a yeast nuclear transport trap (NTT) system, and developed a cDNA obtained by the screening.
  • NTT yeast nuclear transport trap
  • the full-length cDNA encoding a novel nucleolar protein named “N0LP” was isolated by screening the human fetal brain cDNA library using the fragment.
  • the amino acid sequence of the “N0LP” protein contained in the protein of the present invention is shown in SEQ ID NO: 2, and the nucleotide sequence of the cDNA encoding the protein is shown in SEQ ID NO: 1.
  • the N0LP protein consists of 524 amino acids and has a calculated molecular weight of 58 kDa.
  • the N0LP protein has an acidic aspartate / glutamic acid-rich region, three basic NLS, a serine-rich region, and a coiled-coil domain.
  • known RM-binding motivations such as the ribonucleoprotein (MP) motif No reefs were found.
  • MP ribonucleoprotein
  • a search for amino acids in the protein database did not find any significant similarity to other known proteins. Therefore, the N0LP protein is considered to be a new type of nucleolar protein.
  • N0LP gene was specifically expressed in the brain and testis (Fig. 2).
  • the expression of the N0LP gene was not detected in normal lung, but several identical EST clones (accession numbers AA121747, AA129951, AA626487, and AA127451) were derived from lung cancer cell lines.
  • the N0LP gene may be an oncogene, and the N0LP protein and its gene may be applied to the development of cancer therapeutics.
  • the present invention includes proteins that are functionally equivalent to the NOLP protein.
  • “functionally equivalent” means that the target protein has an activity of translocating to a nucleolus.
  • the activity of the protein to translocate to the nucleoli in the cell can be easily detected by, for example, modifying the protein with an appropriate substance, or using an immunological detection method using a specific antibody that recognizes the protein.
  • the protein modification method include, for example, labeling the protein directly with a fluorescent dye—gold colloid or the like, or autofluorescent protein such as green fluorescent protein (GFP) or an appropriate epitope antibody (recognition by an antibody). And a method for producing a fusion protein with a short peptide sequence.
  • Such a modified or unmodified protein can be directly introduced into cells by the microinjection method, or cDNA can be introduced by the transfusion method to produce the protein in the host cell, and the detection thereof After performing the necessary treatment, the transfer of the protein to the nucleolus can be observed using a device having an appropriate detection function such as a fluorescence microscope.
  • a protein functionally equivalent to a certain protein can be isolated using a known technique.
  • One embodiment is that the amino acid sequence of the protein is mutated ( Substitutions, deletions, additions and / or insertions).
  • Methods for introducing a mutation into an amino acid sequence in a protein include, but are not limited to, site-directed mutagenesis (Curretnt Protocols in Molecular Biology edit. Ausubel et al. (1987) Publich. Jhon Wily & Sons Section 8.1-8.5)) and PCR (PCR Primer A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1995, 581-621).
  • the present invention relates to a protein in which one or more amino acids are mutated by substitution, deletion, insertion and / or addition in the amino acid sequence (SEQ ID NO: 2) of the N0LP protein, Functionally equivalent proteins are also included.
  • the number and location of amino acid mutations in proteins are not limited as long as their functions are maintained.
  • the number of mutations is typically within 50 amino acids, preferably within 20 amino acids, and more preferably within 5 amino acids.
  • Amino acid mutations in proteins may occur in nature, and N0LP proteins mutated in nature in this manner are also included in the proteins of the present invention.
  • Another embodiment of a method for isolating a protein functionally equivalent to the N0LP protein includes a method utilizing a hybridization technology. That is, if a person skilled in the art uses the hybridization technique (Curretnt Protocols in Molecular Biology edit. Ausubel et al. (1987) Publich. Jhon Wily & Sons Section 6.3-6.4), the MA sequence encoding the NOLP protein is used. (SEQ ID NO: 1) or a DNA fragment having a high homology with the DNA fragment can be usually obtained from the MA to obtain a protein functionally equivalent to the N0LP protein from the MA. Thus, a protein encoded by a MA that encodes the N0LP protein and a MA that hybridizes with the MA, which is functionally equivalent to the N0LP protein, is also included in the protein of the present invention.
  • Organisms that isolate functionally equivalent proteins include, besides humans, for example, rats, mice, bush, ephedra, chicks, potatoes, zebrafish, drosophila, and the like. Not restricted.
  • Hybridization conditions for isolating DNA encoding a functionally equivalent protein are usually about lxSSC, O.lUDS 37 ° C, and more stringent conditions are 0.5xSSC, 0.1 0 SDS is about 42 ° C ”, and more severe conditions are about“ 0.2xSSC, 0.1% SDS, 65 ° C ”.
  • the washing conditions are usually about 0.1 lxSSC, 0.13 ⁇ 4SDS, 2 times at room temperature (15 to 30 minutes) and 3 times at 50 ° C (30 minutes), and more stringent conditions are 0.2xSSC, 0.13 ⁇ 4SDS. , Room temperature (15-30 minutes) twice, 55 ° C (30 minutes) three times ”, and more severe conditions are“ 0.4xSSC, 0.1% SDS, room temperature (15-30 minutes) twice, 65 ° C (30 minutes) 3 times ”. As the hybridization and washing conditions become more stringent, isolation of DNA having higher homology to the probe sequence can be expected.
  • Proteins isolated using such hybridization techniques usually have high homology in amino acid sequence with the NOLP protein.
  • High homology refers to sequence identity of at least 40% or more, preferably 60% or more, more preferably 80% or more. Sequence homology can be determined using a search algorithm such as BLAST, FASTA, Smith-Waterman.
  • PCR gene amplification technique
  • the protein of the present invention may be prepared as a recombinant protein or as a natural protein. It is possible to The recombinant protein can be prepared, for example, by introducing a vector into which a DNA encoding the protein of the present invention is inserted into an appropriate host cell and purifying the protein expressed in the transformant, as described later. It is. On the other hand, a natural protein can be prepared using, for example, an affinity column to which an antibody against the protein of the present invention described below is bound. The antibody used for affinity purification may be a polyclonal antibody or a monoclonal antibody. Further, the protein of the present invention can be prepared by in vitro translation or the like.
  • the present invention also includes a nucleolus localization activity or a peptide having at least a part of the amino acid sequence of the NOLP protein and having nucleolus localization activity.
  • “At least a part of the amino acid sequence of the NOLP protein” refers to an amino acid sequence of at least 5 amino acids or more, preferably 10 amino acids or more.
  • nucleolar localization could be induced by a peptide consisting of amino acids 342-371 of the N0LP protein alone (FIG. 4B-k). Therefore, one preferred embodiment of the peptide having nucleolar transport activity of the present invention contains amino acids 342 to 371 of the NOLP protein.
  • the peptide consisting of amino acids 372-401 of the N0LP protein alone exhibited a nuclear translocation activity (FIG. 4B-j). Therefore, one of the preferred embodiments of the peptide having nuclear translocation activity of the present invention contains amino acids 372-401 of the NOLP protein.
  • the above-mentioned peptide of the present invention has a nucleolus translocation activity or a nuclear translocation activity, it can be used, for example, as a carrier for transporting a desired substance to the nucleolus or nucleus.
  • the substance for transporting the peptide of the present invention as a carrier is not particularly limited.
  • peptides, nucleic acids, polysaccharides, metals, synthetic compounds and the like can be mentioned.
  • These substances can be, but are not limited to, drugs for treating, preventing, or diagnosing a disease. It may be a substance used for purposes other than clinical application, for example, for biological analysis.
  • the bond may be a chemical bridge using a cross-linking agent (Seikagaku Kagaku Kenkyusho, edited by The Biochemical Society of Japan 2, Protein Chemistry (below), Tokyo Kagaku Dojin, 604-618).
  • the DNA encoding the peptide is ligated to the DNA encoding the peptide of the present invention, and these peptides are expressed as a fusion peptide. In some cases, it is possible to combine both peptides.
  • the binding may be via a spacer region.
  • the bonding form other than the above-mentioned covalent bonding form, a bond utilizing mutual reciprocity generated between the peptide of the present invention and a substance for transport to the nucleus may be used.
  • binding forms include, for example, binding using electrostatic affinity, binding using hydrophobic affinity, binding using a binding motif such as protein-protein or protein-nucleic acid, and parent of enzyme and substrate. Coupling using sum power is exemplified. If the substance for transporting the peptide of the present invention does not essentially have these binding abilities, a functional domain having these binding abilities may be newly added and used.
  • the target cells for transporting the substance using the peptide of the present invention as a carrier are not particularly limited, and various cells can be used according to the purpose of using the peptide of the present invention.
  • the target cell may be, for example, a cell in a living body. Alternatively, it may be a cultured cell in the mouth of the in vitro. In addition, these cells can be, for example, animal cells including humans, plant cells, and microbial cells.
  • the peptide of the present invention can be used as a "nucleolus transport support substance" or “nuclear transport indicator” if it can be visually recognized by modification with an appropriate substance. That is, by introducing such a modified peptide into a cell, it becomes possible to monitor mass transfer from the cytoplasm to the nucleolus or nucleus in real time.
  • the peptide may be directly labeled with a fluorescent dye or the like, or a fusion peptide with a fluorescent protein such as EGFP may be prepared.
  • a “nucleolar transport supporting substance” or “nuclear transport indicator” can be applied to, for example, a screening system for developing a nucleolus transport inhibitor or a nuclear transport inhibitor.
  • the present invention also relates to a DNA encoding the protein or the peptide of the present invention.
  • the DNA of the present invention is not particularly limited as long as it can encode the protein or the peptide of the present invention, and includes, for example, cDNA, genomic DNA, and synthetic DNA.
  • the DNA of the present invention is obtained, for example, by screening a human fetal brain-derived cDNA library or a genomic DNA library using, as a probe, a DNA consisting of all or a part of the nucleotide sequence shown in SEQ ID NO: 1. Can be prepared.
  • an oligonucleotide that specifically hybridizes to MA consisting of the nucleotide sequence shown in SEQ ID NO: 1 is used as a primer, and the mRNA, cDNA, or genomic DNA derived from human fetal brain is used as a type III polymerase. It can also be prepared by a chain reaction
  • the DNA of the present invention can be used, for example, for producing the protein or peptide of the present invention.
  • the production of the protein or peptide of the present invention is generally carried out by inserting the DNA of the present invention into an appropriate vector, introducing this into an appropriate host cell, and culturing the obtained transformed cell.
  • the purification is performed by purifying the recombinant protein or peptide expressed in the transformed cell.
  • Conventional general expression systems can be used for expression of a recombinant protein or peptide.
  • Recombinant peptides expressed in this way can be used, for example, by combining conventional chromatography steps such as ion exchange, gel filtration, distribution, and affinity, or by HPLC using an appropriate packing material. It can be purified to a high degree of purity using, for example. Further, the protein or peptide of the present invention is expressed as a fusion peptide with another peptide, for example, GST, 6 ⁇ His, etc. in order to facilitate purification, and a substance having an affinity for the fused peptide is used. It may be purified by affinity.
  • vectors used for expression of the protein or peptide of the present invention include, for example, PBEST vector (promega) for in vitro expression, pET vector (Invitrogen) for Escherichia coli, and culture. In the case of cells and living organisms, examples include the PME18S vector (Mol Cell Biol. 8: 466-472 (1988)). Insertion of the onset light of the DNA into the vector one can this and force s carried out by a ligase reaction using restriction enzyme site by a conventional method (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wi ley & Sons. Section 11.4-11.11).
  • Transduction of vectors into host cells can be performed, for example, by the calcium phosphate precipitation method and the electropulsing method (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons. Section 9.1- 9.9), ribofectamine method (GIBCO-BRL), microinjection method, etc.
  • the DNA encoding the protein of the present invention or the antisense DNA described below may also be applied to gene therapy for diseases caused by mutations in the N0LP gene.
  • adenovirus vectors When used for gene therapy, for example, retrovirus vectors, adenovirus vectors -The DNA or antisense DNA of the present invention is administered to a patient by an ex vivo method or an in vivo method using a virus vector such as an adeno-associated virus vector or a non-viral vector such as a ribosome.
  • the present invention also relates to an antibody that binds to the protein or peptide of the present invention.
  • the form of the antibody of the present invention is not particularly limited, and includes a polyclonal antibody, a monoclonal antibody, and a part thereof having antigen-binding properties. In addition, antibodies of all classes are included. Furthermore, the antibodies of the present invention also include special antibodies such as human antibodies and humanized antibodies.
  • the antibody of the present invention can be obtained by synthesizing an oligopeptide corresponding to the amino acid sequence of the protein or peptide of the present invention and immunizing a rabbit according to a conventional method.
  • a conventional method Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons. Section 11.12-: 11.13
  • monoclonal antibodies they were expressed and purified in Escherichia coli according to standard methods. Mice can be immunized with a protein and obtained from hybridoma cells obtained by fusing spleen cells and myeloma cells (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons. Section 11.4-11.11).
  • Antibodies that bind to the protein or peptide of the present invention may be used for purposes such as treatment of diseases related to the protein of the present invention, in addition to purification and detection of the protein or peptide of the present invention.
  • human antibodies or humanized antibodies are preferred because of their low immunogenicity.
  • Human antibodies were obtained by replacing the immune system with that of a human (eg, “Functional transplant of megabase human immunoglobulin loci recapitulates human antibody response in mice, Mendez, M. J et al. (1997) Nat. Genet. 15 : 146-156j)
  • the humanized antibody can be prepared by genetic recombination using the hypervariable region of a monoclonal antibody (Methods). in Enzymology 203, 99-121 (1991)).
  • the present invention also relates to a DNA that specifically hybridizes with the DNA of SEQ ID NO: 1 and has a chain length of at least 15 nucleotides.
  • “Specifically hybridizes” with the DNA of the present invention means that it hybridizes with the DNA of the present invention under ordinary hybridization conditions, preferably under severe conditions, and hybridizes with other DNA. Means not. Stringency for specific hybridizations can be determined by one skilled in the art by considering the temperature of the hybridization reaction, reaction time, probe or primer concentration, probe or primer length, salt strength, etc. If so, it can be selected as appropriate.
  • Such DNA is used as a probe for detecting and isolating the DNA of the present invention, and as a c-primer which can be used as a primer for amplifying the DNA of the present invention.
  • SEQ ID NO: DNA specifically described in 1 DNA that specifically hybridizes and has a chain length of at least 15 nucleotides” includes antisense DNA for suppressing the expression of the protein of the present invention.
  • the antisense DNA has a chain length of at least 15 bp or more, preferably 100 bp, more preferably 500 bp or more, and usually has a chain length of 3000 bp or less, preferably 2000 bp or less in order to cause an antisense effect.
  • Such antisense DNA is also considered to be applicable to gene therapy for diseases (for example, cancer can be considered) caused by abnormalities (functional or abnormal expression) of the protein of the present invention.
  • FIG. 1 shows the nucleotide and deduced amino acid sequence of human NOLP.
  • the in-frame stop codon position 468) before the start codon is boxed.
  • Three potential nuclei Migration signals are underlined.
  • FIG. 2 shows a sequence alignment of the Tral homology region. Drosophi la virtual protein translated from E. coli Tral / re laxase (1677-1715), human N0LP (7-54), and genomic DNA (accession number AC004360) is shown. Identical and similar amino acid residues are indicated by “*” and “/”, respectively. Highly conserved residues in the core consensus are boxed.
  • FIG. 3 shows a tissue distribution analysis by RT-PCR. The 12 tissues tested are shown above each lane.
  • FIG. 4 shows the sequence requirements for nucleolar localization of the N0LP protein.
  • A. Deletion Constructs are illustrated.
  • a schematic diagram of the putative domain of the N0LP protein is shown on the panel.
  • NLS2 and NS3 indicate potential nuclear localization signals.
  • S-Litsch indicates a serine-rich residue.
  • the subcellular distribution patterns of each construct determined in B are shown on the right.
  • NU / N0 indicates that some are nuclei and some are nucleoli.
  • N0 indicates that it is constitutively a nucleolus.
  • N / C indicates non-specific (whole cell).
  • NU indicates that it is structurally nuclear.
  • B Intracellular distribution of EGFP-hybrid protein after expression in COS-7 cells. Transformed cells were tested after 24 hours.
  • FIG. 5 shows the construction and analysis of the EGFP-galactosidase hybrid protein.
  • A. The fusion protein construct is illustrated.
  • the subcellular distribution patterns of each construct determined in B are summarized on the right.
  • N / C is non-specific (whole cell)
  • PCR Polymerase chain reaction
  • RACE rapid amplification of cDNA ends
  • PCMV-SP0RT2 Lifetime
  • VSP1 Two specific primers "VSP1” (5, -CAC ACA GGA AAC AGC TAT GAC CAC TAG-3, / SEQ ID NO: 3), "VSP2"(5'-AAG CTA TTT AGG TGA CAC TAT AGA AGG TAC-3 '/ SEQ ID NO: 4) and two primers specific to NOLP cDNA "NU164" (5, -TTT GTC GAC ATC CTG CTG TCT CTC CAG ACG-3' / SEQ ID NO: 5) NU162 "(5, -TTT GTC GAC GGC ACG CTC TTG ATA CTC AGG-3 '/ SEQ ID NO: 6) and the partial sequence cDNA (accession number AB015339) obtained so far by nested-PCR.
  • This ORF encoded a predicted polypeptide of 524 amino acids, with a calculated molecular weight of 58 kDa.
  • the protein consists of an acidic aspartate / glutamic acid-rich region (79%, amino acids 268-281), three putative basic NLSs (NLS1, RARKRIR, amino acids 340-346; NLS2, RR KR) , Amino acids 353-357; NLS3, K position, amino acids 392-395), a serine-rich region (42%, amino acids 458-498), and a potential coiled-coil domain (Lupas, A.
  • RNA binding motif such as the ribonucleoprotein (RNP) motif (Burd, CG, and Dreyfuss, G. (1994) Science 265, 615-621).
  • RNP ribonucleoprotein
  • Highly acidic and base rich residues are often found in some nucleolar proteins, but searching for amino acids in the protein database (NCBI Protein Database and Swiss Protein Database) has No significant similarity to known proteins was found.
  • Local homology (50% sequence identity in a 36 amino acid overlap) is due to the N-terminal short residue of N0LP (amino acids 7-40) and the E. coli DNA helicase, Tral / relaxase.
  • RNA type I was prepared from human tissue poly (A) + RNA (CL0NTECH) using excess Superscript II reverse transcriptase (Life Technology) and random hexama-primer.
  • a PCR product of the expected size (664 bp) was exclusively detected in fetal brain, brain, and testis (Fig. 2).
  • Northern plot analysis found a single transcript of approximately 3.5 kb in the brain and testis (not shown overnight). These results partially match the results of the overnight search.
  • RT-PCR and Northern blot analysis did not detect transcript signals in normal lung, but did show some identical EST clones (accession numbers). Nos. AA121747, AA129951, AA626487, and AA127451) were derived from the SCLC cell line (NCI-H69). This fact suggests that N0LP is involved in neuroendocrine tumorigenesis, and therefore may be a potential oncogene or cancer-associated marker.
  • Example 3 Determination of sequence requirements for nucleolus and nucleus localization To determine the sequences required for nucleolus and nucleus localization, first a series of EGFP A tagged N0LP deletion construct was generated. A series of EGFP fusion expression plasmids were constructed by cloning DNA fragments digested with EcoRI / Notl or EcoRI / Sall into the corresponding sites on EGFP-Cl '.
  • pEGFP-Cl ' is a linker to the Bglll / Pstl site of pEGFP-C CLONTECH [oligonucleotide (5'-GAT CTG GAA TTC ATA TGG CCA TGG CGG CCG CTG CA-3' / SEQ ID NO: 8) / (5'-GCG GCC GCC ATG GCC ATA TGA ATT CCA-3, / SEQ ID NO: 9)].
  • a DNA fragment encoding positions 222 to 524 was extracted from the isolated partial cDNA (accession number AB015339) using EcoM / Notl. Others were amplified from full-length NOLP cDNA using PCR and subsequently digested with EcoRI / SalI.
  • the pair of primers used was NU181 / NU204, NU205 / NU204 at positions 12-524, NU205 / NU204 at positions 28-524, and NU131 / NU204, 282-461 at positions 282-524 for cDNA amplification corresponding to amino acids 1-524 of the N0LP protein.
  • NU134 / NU164 The nucleotide sequences of NU181, stroke 4, NU205, NU131, NU166, and NU163 are shown in SEQ ID NOs: 10 to 15, respectively.
  • Plasmid pEGFP- 'lacZ is a modified /?-Galactosidase fragment (containing synthetic Bglll linkers at the 5'- and 3'-ends) to express in-frame EGFP-' lacZ fusion proteins.
  • lacZ was cut at the EcoRI site) and cloned into the Bglll / EcoRI site of pEGFP-C1 '.
  • the PCR products encoding positions 342-371 and 372-401 described above were ligated to the EGFP-'lacZ in frame fusion construct. In order to express the construct, it was ligated to a site corresponding to pEGFP-'lacZ. Confirmation of all constructs was performed by sequencing.
  • DMEN Dulbecco's modified Eagle's medium
  • penicillin G 100 U / ml
  • streptomycin 100 mg / ml
  • NTT method position 222-524
  • Japanese Patent Application No. 10-174065 Japanese Patent Application No. 10-174065
  • Fig. 4-e Japanese Patent Application No. 10-174065
  • N0LP-524 are those that are excluded from the nucleolus with the deformation of the nucleus, are distributed in a point-like manner, are distributed in a patchy manner, are distributed in the nucleolus, etc.
  • three main distribution patterns are shown (Fig. 4-a, b, c).
  • residues 12-524 lacking the N-terminal 11 residues did not show such a distribution pattern, indicating constitutive nucleolar localization (Figure 4-d).
  • amino acids 372-401 acts as an active NLS ( Figure 4B-j).
  • amino acids 342-371 lead a known reporter protein to the nucleolus, and determine whether amino acids 372-401 lead a known repo overnight protein to the nucleolus
  • EGFP-'lacZ EGFP-'lacZ hybrid protein.
  • Amino acids 372-401 are sufficient for nuclear localization ( Figure 5B-c), whereas amino acids 342-371 transfer the chimeric protein to the nucleus, but are excluded from apparent nucleoli Was not transferred to the nucleolus (Fig. 5B-b).
  • a DNA encoding a novel nucleolar protein has been isolated, and a region involved in nucleolar translocation activity and a region involved in nuclear translocation activity in the protein have been identified.
  • the protein or the peptide of the present invention can be used as a carrier for transporting another substance to the nucleus or nucleolus, or as a nuclear transport indicator or a nucleolus transport indicator.
  • the protein of the present invention and its gene may be used as a carcinogenesis marker, or may be used for the development of new therapeutic agents for cancer.
  • the proteins and peptides of the present invention are expected to contribute significantly to research for elucidation of the nucleolar transport mechanism that is still unknown.

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Abstract

A full-length cDNA encoding a novel nucleolar protein which is successfully isolated by screening a fetal human brain cDNA library with the use of a partial cDNA encoding a peptide having a nuclear transport activity isolated by using a yeast nuclear transport trap system (NTT) developed originally. Also, a region necessary for the nucleolar transport activity and a region for the nuclear transport activity of the above protein are identified.

Description

明細書 新規な核小体蛋白質 技術分野  Description Novel nucleolar protein Technical field
本発明は、 新規な核小体蛋白質およびその遺伝子、 並びにそれらの製造およ び用途に関する。 背景技術  The present invention relates to novel nucleolar proteins and their genes, and their production and use. Background art
核蛋白質は、 転写、 mRNAのプロセッシング、 複製、 及び染色体の構成といつ た核の現象において重要な役割を果たしている。 核蛋白質の多くは、 核孔複合 体（NPC) を通じた能動輸送を媒介する固有のシグナルを保有している。核局在 シグナル（NLS)として知られている核内への選択的な移行のための一次配列及 び力リオフヱリン（Kap)フアミ リーのようなこれらに対応したレセプ夕一はす でに同定されている (Gorlich, D. and Mattaj, I. W. (1996) Science 271, 1513-1518、 Nigg, E. A. (1997) Nature 386， 779-787、 Ullman, K. S.， Powers, M. A.， and Forbes, D. J. (1997) Cell 90， 967-970、 Pemberton, L. F.， Blobel, G.， and Rosenblum, J. S. (1988) Curr. Opin. Cell Biol.10， 392-399、 ozniak, R. W.， Rout, M. P., and Aitchison, J. D. (1988) Trends Cell Biol.8, 184-188、 Ohno, M.， Fornerod, M., and Mattaj, I. W. (1998) Cell 92, 327-336) 。 最 もよく知られている NLSは、 リシン/アルギニン（K/R)リヅチな塩基性残基（潜 在的なコンセンサス ：単一のクラス夕一からなる [K/R]2- X- [K/R]; 二つのクラ ス夕一からなる [K/R]2-X1( 2-[K/R]3)の短いストレツチから構成される古典的 な NLSであり、 カリオフェリンあるいはインポ一チン （importin) のひ//?へテ 口ダイマ一によって認識される。最近になって、 NLSの種類及びそれらに対応し た Kapの数が急速に増加した。このことは蛋白質の核内への輸送が異なるさまざ まな経路によって媒介されることを示している。 Nuclear proteins play important roles in nuclear phenomena such as transcription, mRNA processing, replication, and chromosome organization. Many nuclear proteins carry unique signals that mediate active transport through the nuclear pore complex (NPC). Primary sequences for selective translocation into the nucleus, known as nuclear localization signals (NLS), and their corresponding receptors, such as the force lipophilin (Kap) family, have already been identified. (Gorlich, D. and Mattaj, IW (1996) Science 271, 1513-1518, Nigg, EA (1997) Nature 386, 779-787, Ullman, KS, Powers, MA, and Forbes, DJ (1997) Cell 90, 967-970, Pemberton, LF, Blobel, G., and Rosenblum, JS (1988) Curr. Opin. Cell Biol. 10, 392-399, ozniak, RW, Rout, MP, and Aitchison, JD (1988) Trends Cell Biol. 8, 184-188, Ohno, M., Fornerod, M., and Mattaj, IW (1998) Cell 92, 327-336). The best-known NLSs are lysine / arginine (K / R) -rich basic residues (potential consensus: single-class [K / R] 2 -X- [K / R]; a classic NLS consisting of short stretches of [K / R] 2 -X 1 (2- [K / R] 3 ), consisting of two classes, one of which is karyopherin or impo The type of NLS and the number of Kaps corresponding to them have increased rapidly, which has led to an increase in the protein nucleus. Different transports It is mediated by different pathways.
対照的に、 核内の局在性を決定するメカニズム及びトポジヱニック  In contrast, mechanisms and topologies that determine nuclear localization
(topogenic)配列に関してはほとんど知られていない。核小体は、 数種のウイ ルス性コア蛋白質が細胞の増殖中において一時的に蓄積する座位であるばかり でなく (Dang, C. V.， and Lee, W. M. (1989) J. Biol. Chem.264, 18019-18023、 Cochrane, A. W. , Perkins, A.， and Rosen, C. A. (1990) J. Virol.64， 88卜 885、 Hatanaka, M. (1991) in Genetic Structure and Regulation of HIV (Haseltine, W. A.， and Wong-Staal, F.， Eds. ), pp. 264-287, Raven Press, New York、 Siomi, H. , Shida, H.， Nam, S. H. , Nosaka, T.， Maki, M. , and Hatanaka, M. Little is known about (topogenic) sequences. The nucleolus is not only a locus where several viral core proteins temporarily accumulate during cell growth (Dang, CV, and Lee, WM (1989) J. Biol. Chem. 264, 18019-18023, Cochrane, AW, Perkins, A., and Rosen, CA (1990) J. Virol. 64, 88 885, Hatanaka, M. (1991) in Genetic Structure and Regulation of HIV (Haseltine, WA, and Wong-Staal, F., Eds.), Pp. 264-287, Raven Press, New York, Siomi, H., Shida, H., Nam, SH, Nosaka, T., Maki, M., and Hatanaka, M.
(1988) Cell 55， 197-209、 Mears, W. E.， Lam, V·, and Rice, S. A. (1995) J. Virol.69， 935-947) 、 リボゾーム生合成の主要部位である （Hadjiolov, A. A. (1985) The Nucleolus and Ribosome Biogenesis, Springer Verlag, New York、 Scheer, U. , and Weisenberger, D. (1994) Curr. Opin. Cell Biol. 6, 354- 359、 Pederson, T. (1988) Nucleic Acids Res. 26, 3871-3877) 。 さらに最近 になって、 核小体が細胞の老化に深く関与していることを示唆する報告がなさ れている (Johnson, F. B. , Marciniak, R. A. , and Guarente, L. (1998) Curr. Opin. Cell Biol. 10， 332-338) 。 リボゾーム蛋白質 L31及び L5 (Quaye, I. Κ·, Toku, S.， and Tanaka, T. (1996) Eur. J. Cell Biol. 69， 151-155、 Michael, W. M.， and Dreyfuss, G. (1996) J. Biol. Chem. 271, 11571-11574) 、 ヒト 免疫不全ゥィルスのウィルス性蛋白質 Tat及び Rev (Dang, C. V.， and Lee, W. .(1988) Cell 55, 197-209; Mears, WE, Lam, V., and Rice, SA (1995) J. Virol. 69, 935-947), a major site of ribosome biosynthesis (Hadjiolov, AA ( 1985) The Nucleolus and Ribosome Biogenesis, Springer Verlag, New York, Scheer, U., and Weisenberger, D. (1994) Curr.Opin.Cell Biol. 6, 354-359, Pederson, T. (1988) Nucleic Acids Res. 26, 3871-3877). More recently, there have been reports suggesting that nucleoli are deeply involved in cell senescence (Johnson, FB, Marciniak, RA, and Guarente, L. (1998) Curr. Opin. Cell Biol. 10, 332-338). Ribosomal proteins L31 and L5 (Quaye, I. Κ ·, Toku, S., and Tanaka, T. (1996) Eur. J. Cell Biol. 69, 151-155, Michael, WM, and Dreyfuss, G. (1996) ) J. Biol. Chem. 271, 11571-11574), the human immunodeficiency virus viral proteins Tat and Rev (Dang, CV, and Lee, W..
(1989) J. Biol. Chem.264, 18019-18023、 Cochrane, A. W.， Perkins, A.， and Rosen, C. A. (1990) J. Virol. 64, 881-885、 Hatanaka, M. (1991) in Genetic Structure and Regulation of HIV (Haseltine, W. A. , and Wong-Staal, F. , Eds.), pp. 264-287, Raven Press, New York) 、 非リボゾーム蛋白質 nucleolin/C23 (Lapeyre, B.， Bourbon, H.， and Amalric, F. (1987) Proc. Natl. Acad. Sci. USA 84, 1472-1476、 Busch, H. (1984) in Chromosomal Nonhistone Proteins. Vol . 4. (Hnil ica, L. S. ， Ed. ) , pp. 233-286, CRC Press, Boca Raton, FL) 、 N038/B23/nucleophosmin (Busch, H. ( 1984) in Chromosomal Nonhistone Proteins. Vol . 4. (Hnil ica, L. S. ， Ed. )， pp. 233-286, CRC Press, Boca Raton, FL、 Schmidt-Zachmann, M. S. ， Hugle-Dorr, B. ， and Franke, W. W. ( 1987) EMBO J. 6, 1881-1890、 Chan, W. Y. ， Liu, Q. R. , Borjigin, J. , Busch, H.， Rennert, 0. M. ， Tease, L. Aリ and Chan, P. K. ( 1989 ) Biochemistry 28, 1033-1039) 、 転写因子 mUBF (Maeda, Y.， Hisatake, K. ， Kondo, T. ， Hanada, K. ， Song, C. Ζ· , Nishimura, T. ， and Muramatsu, M. ( 1992 ) EMBO J. 11 , 3695-3704) といった 核小体局在に必要とされる数多くの配列が報告されている。 (1989) J. Biol. Chem. 264, 18019-18023, Cochrane, AW, Perkins, A., and Rosen, CA (1990) J. Virol. 64, 881-885, Hatanaka, M. (1991) in Genetic Structure and Regulation of HIV (Haseltine, WA, and Wong-Staal, F., Eds.), Pp. 264-287, Raven Press, New York), nonribosomal protein nucleolin / C23 (Lapeyre, B., Bourbon, H ., And Amalric, F. (1987) Proc. Natl. Acad. Sci. USA 84, 1472-1476, Busch, H. (1984) in Chromosomal Nonhistone. Proteins. Vol. 4. (Hnilica, LS, Ed.), Pp. 233-286, CRC Press, Boca Raton, FL), N038 / B23 / nucleophosmin (Busch, H. (1984) in Chromosomal Nonhistone Proteins. Vol. 4. (Hnilica, LS, Ed.), Pp. 233-286, CRC Press, Boca Raton, FL, Schmidt-Zachmann, MS, Hugle-Dorr, B., and Franke, WW (1987) EMBO J. 6, 1881-1890, Chan, WY, Liu, QR, Borjigin, J., Busch, H., Rennert, 0.M., Tease, L. Ari and Chan, PK (1989) Biochemistry 28, 1033-1039 ), Transcription factor mUBF (Maeda, Y., Hisatake, K., Kondo, T., Hanada, K., Song, C. Ζ ·, Nishimura, T., and Muramatsu, M. (1992) EMBO J. 11) Numerous sequences required for nucleolar localization have been reported.
しかしながら、 核小体局在に関与するコンセンサスモチーフは全く同定され ておらず、 核小体局在のメ力二ズムは明らかではない。 発明の開示  However, no consensus motifs involved in nucleolar localization have been identified, and the mechanism of nucleolar localization is not clear. Disclosure of the invention
本発明は、 新規な核小体蛋白質およびその核小体移行活性または核移行活性 に関与するペプチド領域、 それらをコードする核酸分子、 並びにそれらの製造 および用途を提供する。  The present invention provides a novel nucleolar protein, a peptide region involved in its nucleolar transport activity or nuclear transport activity, a nucleic acid molecule encoding them, and their production and use.
本発明者らは、 独自に開発した酵母の核輸送トラップ（NTT)系（Ueki et al . , Nature Biotechnology in press )を用いて核移行活性を有するぺプチドをコ一 ドする複数の部分 cMAを単離している （特願平 10-174065号）。本発明者らは、 その中の一つの部分 cDNA (ァクセッション番号 AB015339) に対応する全長 cDNA の単離を試みた。 具体的には、 該部分 cDNAの配列情報を利用したポリメラーゼ 鎖反応 （PCR) 及び cDNA末端の急速的増幅 （RACE) によって、 SUPERSCRIPTヒ 卜 胎児脳 cDNAライブラリー（Life Technologie社）のスクリーニングを行い、 これ により目的の全長 cDNAを単離することに成功した （このクローンを N0LP； Nucleolar Local ized Proteinと命名した a The present inventors have used a yeast nuclear transport trap (NTT) system (Ueki et al., Nature Biotechnology in press), which has been independently developed, to obtain a plurality of partial cMAs encoding a peptide having nuclear translocation activity. It has been isolated (Japanese Patent Application No. 10-174065). The present inventors attempted to isolate a full-length cDNA corresponding to one of the partial cDNAs (accession number AB015339). Specifically, screening of a SUPERSCRIPT human fetal brain cDNA library (Life Technology) was performed by polymerase chain reaction (PCR) using the partial cDNA sequence information and rapid amplification of cDNA ends (RACE). Thus it succeeded in isolating the desired full length cDNA of (this clone N0LP; Nucleolar Local ized Protein named the a
単離した全長 N0LP cDNAの構造解析の結果、 該 cDNAは推定 524アミノ酸のポリ ペプチドをコードし、 短い E . col i DNAヘリカーゼ相同領域、 酸性に富んだドメ ィン、 3つの潜在的な塩基性に富んだ核局在シグナル（NLS)、セリンに富んだド メイン、および推定のコイルド -コイルドメインを有していた。 この蛋白質には、 蛋白質デ一夕一ベースにおける他の蛋白質との顕著な相同性は見られなかった。 また、 本発明者らは、 N0LP遺伝子の転写産物の組織分布解析を行い、 該遺伝 子が脳と精巣において優勢な発現を示すことを見出した。 As a result of structural analysis of the isolated full-length N0LP cDNA, the cDNA was found to be A peptide-encoding short E. coli DNA helicase homology region, an acid-rich domain, three potential basic nuclear localization signals (NLS), a serine-rich domain, and a putative Had a coiled-coiled domain. This protein did not show any significant homology to other proteins on a protein-by-protein basis. The present inventors also performed a tissue distribution analysis of the transcript of the N0LP gene, and found that the gene showed a predominant expression in the brain and testis.
さらに、 本発明者らは、 N0LP蛋白質において核移行に必要な領域を特定する ために、蛍光タグを有する 1セッ 卜の欠失構築物を作製し、該夕グを付した N0LP 蛋白質 （融合蛋白質） を COS- 7細胞内で一過的に発現させた。 その結果、 第一お よび第二 NLSとオーバ一ラップする 30アミノ酸の領域が核小体局在に、 また、 こ れに隣接する第三 NLSを含む 30アミノ酸の領域が核局在にそれそれ重要な役割 を果たしていることを見出した。  Furthermore, the present inventors prepared a set of deletion constructs having a fluorescent tag in order to identify a region required for nuclear translocation in the N0LP protein, and added the fluorescent tag to the N0LP protein (fusion protein). Was transiently expressed in COS-7 cells. As a result, a region of 30 amino acids overlapping with the first and second NLS is likely to be in nucleolar localization, and a region of 30 amino acids including the adjacent NLS is likely to be in nuclear localization. It was found to play an important role.
本発明は、 新規な核小体蛋白質およびその細胞内局在性に関与するぺプチド 領域、 それらをコードする核酸分子、 並びにそれらの製造および用途に関し、 より具体的には、  The present invention relates to a novel nucleolar protein and a peptide region involved in its intracellular localization, a nucleic acid molecule encoding them, and their production and use.
( 1 ) 配列番号： 2に記載のアミノ酸配列からなる蛋白質、 または該蛋白質 中のアミノ酸配列において 1若しくは複数のアミノ酸が欠失、 付加、 挿入およ び/または他のアミノ酸による置換により修飾されたアミノ酸配列からなり、 核小体への移行活性を有する蛋白質、  (1) a protein comprising the amino acid sequence of SEQ ID NO: 2, or one or more amino acids in the amino acid sequence of the protein are modified by deletion, addition, insertion, and / or substitution with another amino acid A protein consisting of an amino acid sequence and having a nucleoli transfer activity,
( 2 ) 配列番号： 1に記載の塩基配列からなる DNAとハイブリダィズする DM がコードする蛋白質であって、 核小体への移行活性を有する蛋白質、  (2) a protein encoded by DM, which hybridizes with the DNA consisting of the nucleotide sequence of SEQ ID NO: 1 and has a nucleolar translocation activity;
( 3 ) 配列番号： 2に記載のァミノ酸配列の少なくとも一部を含み、 核小体 への移行活性を有するぺプチド、  (3) a peptide comprising at least a part of the amino acid sequence of SEQ ID NO: 2 and having an activity of translocating to a nucleolus,
( 4 ) 配列番号： 2に記載のアミノ酸配列の 3 4 2位から 3 7 1位を含む、 ( 3 ) に記載のぺプチド、  (4) the peptide of (3), comprising positions 34 to 37 of the amino acid sequence of SEQ ID NO: 2;
( 5 ) 配列番号 ： 2に記載のアミノ酸配列の少なくとも一部を含み、 核への 移行活性を有するぺプチド、 (5) SEQ ID NO: containing at least a part of the amino acid sequence of SEQ ID NO: 2; A peptide having translocation activity,
( 6 ) 配列番号： 2に記載のァミノ酸配列の 3 72位から 40 1位を含む、 ( 5) に記載のペプチド、  (6) the peptide of (5), which comprises positions 372 to 401 of the amino acid sequence of SEQ ID NO: 2;
(7) ( 1 ) 〜 （6) のいずれかに記載の蛋白質またはペプチドと異種ぺプ チドとの融合ペプチド、  (7) a fusion peptide of the protein or peptide according to any of (1) to (6) and a heterologous peptide,
(8) ( 1 ) 〜 （7) のいずれかに記載の蛋白質またはペプチドをコードす る醒、  (8) an awake which encodes the protein or peptide according to any of (1) to (7);
(9) ( 8) に記載の DNAを含むベクタ一、  (9) A vector containing the DNA of (8),
( 1 0) (9 ) に記載のベクタ一を保持する形質転換体、  (10) A transformant carrying the vector according to (9),
( 1 1 ) ( 1 0) に記載の形質転換体を培養する工程を含む、 （ 1 ) 〜 （7) のいずれかに記載の蛋白質またはべプチドの製造方法、  (11) The method for producing a protein or a peptide according to any one of (1) to (7), comprising a step of culturing the transformant according to (10).
( 12) ( 1 ) 〜 （7) のいずれかに記載の蛋白質またはペプチドに結合す る抗体、  (12) an antibody that binds to the protein or peptide according to any one of (1) to (7);
( 1 3) 配列番号： 1に記載の塩基配列からなる MAと特異的にハイブリダィ ズし、 少なくとも 1 5塩基の鎖長を有する DNA、 に関する。  (13) A DNA which specifically hybridizes with MA consisting of the nucleotide sequence of SEQ ID NO: 1 and has a chain length of at least 15 bases.
本発明者らは、 独自に開発した酵母の核輸送トラップ（NTT) 系を用いた核移 行活性を有するぺプチドをコ一ドする cDNAのスクリーニング、 および該スクリ 一二ングにより得られた cDNA断片を利用したヒト胎児脳 cDNAライブラリーのス クリーニングにより、 「N0LP」 と命名された新規な核小体蛋白質をコードする 全長 cDNAを単離した。 本発明の蛋白質に含まれる 「N0LP」 蛋白質のアミノ酸配 列を配列番号： 2に、 該蛋白質をコードする cDNAの塩基配列を配列番号： 1に 示す。  The present inventors have screened a cDNA encoding a peptide having nuclear transfer activity using a yeast nuclear transport trap (NTT) system, and developed a cDNA obtained by the screening. The full-length cDNA encoding a novel nucleolar protein named “N0LP” was isolated by screening the human fetal brain cDNA library using the fragment. The amino acid sequence of the “N0LP” protein contained in the protein of the present invention is shown in SEQ ID NO: 2, and the nucleotide sequence of the cDNA encoding the protein is shown in SEQ ID NO: 1.
N0LP蛋白質は、 524ァミノ酸からなり、計算された分子量は 58 kDaである。 N0LP 蛋白質は、 酸性のァスパラギン酸/グルタミン酸に富んだ領域、 3つの塩基性 に富んだ NLS、セリンに富んだ領域、およびコイルド-コイルドメインを有する。 しかしながら、 リボヌクレオ蛋白質（MP)モチーフのような公知の RM結合モチ ーフは見出されなかった。 また、 蛋白質データーベースにおけるアミノ酸の検 索によっても、 他の公知の蛋白質との有意な類似性は見出されなかった。 従つ て、 N0LP蛋白質は、 新たなタイプの核小体蛋白質であると考えられる。 The N0LP protein consists of 524 amino acids and has a calculated molecular weight of 58 kDa. The N0LP protein has an acidic aspartate / glutamic acid-rich region, three basic NLS, a serine-rich region, and a coiled-coil domain. However, known RM-binding motivations such as the ribonucleoprotein (MP) motif No reefs were found. Also, a search for amino acids in the protein database did not find any significant similarity to other known proteins. Therefore, the N0LP protein is considered to be a new type of nucleolar protein.
RT― PCRおよびノーザンブロッテイングによる N0LP遺伝子の発現解析の結果、 N0LP遺伝子は、 脳および精巣に特異的に発現していた（図 2 )。 また、 興味深い ことに、 N0LP遺伝子は正常肺においてその発現は検出されなかったが、 いくつ かの同一の ESTクローン（ァクセッション番号 AA121747, AA129951, AA626487, および AA127451 )は肺癌細胞系に由来していた。 この事実は、 N0LP蛋白質および その遺伝子が、 発癌マーカーになり得ることを示唆する。 また、 N0LP遺伝子が 癌遺伝子である可能性があり、 N0LP蛋白質やその遺伝子には癌治療薬の開発へ の応用も考えられる。  Analysis of the expression of the N0LP gene by RT-PCR and Northern blotting revealed that the N0LP gene was specifically expressed in the brain and testis (Fig. 2). Interestingly, the expression of the N0LP gene was not detected in normal lung, but several identical EST clones (accession numbers AA121747, AA129951, AA626487, and AA127451) were derived from lung cancer cell lines. Was. This fact suggests that the NOLP protein and its gene can be a marker for carcinogenesis. Also, the N0LP gene may be an oncogene, and the N0LP protein and its gene may be applied to the development of cancer therapeutics.
本発明は、 N0LP蛋白質と機能的に同等な蛋白質を包含する。 ここで 「機能的 に同等」 とは、 対象となる蛋白質が核小体への移行活性を有することを指す。 蛋白質の細胞内における核小体への移行活性は、 例えば、 該蛋白質を適当な物 質で修飾したり、 該蛋白質を認識する特異的な抗体を利用した免疫学的検出法 などによって容易に検出することが可能である。 蛋白質の修飾方法としては、 例えば、 蛋白質を直接的に蛍光色素ゃ金コロイ ドなどで標識したり、 あるいは 緑色蛍光蛋白質（GFP)などの自己蛍光蛋白質や適当なェピトープ夕グ（抗体によ つて認識される短いぺプチド配列） との融合蛋白質を作製する方法などが挙げ られる。 このような修飾蛋白質または非修飾蛋白質を、 マイクロインジェクシ ヨン法によって直接細胞内に導入したり、 あるいはトランスフエクシヨン法に より cDNAを導入して宿主細胞内で蛋白質を生産させ、 それそれの検出に必要な 処理を施した後に、 蛍光顕微鏡などの適当な検出機能を有した装置を用いて該 蛋白質の核小体への移行を観察することができる。  The present invention includes proteins that are functionally equivalent to the NOLP protein. Here, “functionally equivalent” means that the target protein has an activity of translocating to a nucleolus. The activity of the protein to translocate to the nucleoli in the cell can be easily detected by, for example, modifying the protein with an appropriate substance, or using an immunological detection method using a specific antibody that recognizes the protein. It is possible to Examples of the protein modification method include, for example, labeling the protein directly with a fluorescent dye—gold colloid or the like, or autofluorescent protein such as green fluorescent protein (GFP) or an appropriate epitope antibody (recognition by an antibody). And a method for producing a fusion protein with a short peptide sequence. Such a modified or unmodified protein can be directly introduced into cells by the microinjection method, or cDNA can be introduced by the transfusion method to produce the protein in the host cell, and the detection thereof After performing the necessary treatment, the transfer of the protein to the nucleolus can be observed using a device having an appropriate detection function such as a fluorescence microscope.
ある蛋白質と機能的に同等な蛋白質は、 公知の技術を利用して単離すること が可能である。 その一つの態様は、 蛋白質のアミノ酸配列に変異 （アミノ酸の 置換、 欠失、 付加および/または挿入） を導入する方法である。 蛋白質中のァ ミノ酸配列に変異を導入する方法としては、 これらに制限されないが、例えば、 部位特異的変異誘発法（Curretnt Protocols in Molecular Biology edit. Ausubel et al . ( 1987) Publich. Jhon Wi ly & Sons Section 8.1-8.5 ) ) や PCR 法 (PCR Primer A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1995 , 581 - 621) などが知られている。 A protein functionally equivalent to a certain protein can be isolated using a known technique. One embodiment is that the amino acid sequence of the protein is mutated ( Substitutions, deletions, additions and / or insertions). Methods for introducing a mutation into an amino acid sequence in a protein include, but are not limited to, site-directed mutagenesis (Curretnt Protocols in Molecular Biology edit. Ausubel et al. (1987) Publich. Jhon Wily & Sons Section 8.1-8.5)) and PCR (PCR Primer A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1995, 581-621).
本発明には、 このように N0LP蛋白質のアミノ酸配列 （配列番号： 2 ) におい て 1もしくは複数のアミノ酸が置換、 欠失、 挿入および/もしくは付加などに より変異した蛋白質であって、 N0LP蛋白質と機能的に同等な蛋白質も含まれる。 蛋白質におけるアミノ酸の変異数や変異部位は、 その機能が保持される限り制 限はない。 変異数は、 典型的には、 50アミノ酸以内であり、 好ましくは 20アミ ノ酸以内であり、 さらに好ましくは 5アミノ酸以内である。蛋白質中のアミノ酸 の変異は、 自然界において生じることもあり、 このように自然界において変異 した N0LP蛋白質も本発明の蛋白質に含まれる。  The present invention relates to a protein in which one or more amino acids are mutated by substitution, deletion, insertion and / or addition in the amino acid sequence (SEQ ID NO: 2) of the N0LP protein, Functionally equivalent proteins are also included. The number and location of amino acid mutations in proteins are not limited as long as their functions are maintained. The number of mutations is typically within 50 amino acids, preferably within 20 amino acids, and more preferably within 5 amino acids. Amino acid mutations in proteins may occur in nature, and N0LP proteins mutated in nature in this manner are also included in the proteins of the present invention.
N0LP蛋白質と機能的に同等な蛋白質を単離する方法の他の態様としては、 ハ イブリダィゼーシヨン技術を利用する方法が挙げられる。 即ち、 当業者であれ ば、 ノヽィブリダイゼ一ション技術 (Curretnt Protocols in Molecular Biology edit. Ausubel et al . ( 1987) Publich. Jhon Wi ly & Sons Section 6.3-6.4) を用いて NOLP蛋白質をコードする MA配列 （配列番号： 1 ) またはその一部をも とにこれと相同性の高い DNAを単離して、 該 MAから N0LP蛋白質と機能的に同等 な蛋白質を得ることは、通常行いうる。このように N0LP蛋白質をコ一ドする MA とハイプリダイズする MAにコードされる蛋白質であって、 N0LP蛋白質と機能的 に同等な蛋白質もまた本発明蛋白質に含まれる。  Another embodiment of a method for isolating a protein functionally equivalent to the N0LP protein includes a method utilizing a hybridization technology. That is, if a person skilled in the art uses the hybridization technique (Curretnt Protocols in Molecular Biology edit. Ausubel et al. (1987) Publich. Jhon Wily & Sons Section 6.3-6.4), the MA sequence encoding the NOLP protein is used. (SEQ ID NO: 1) or a DNA fragment having a high homology with the DNA fragment can be usually obtained from the MA to obtain a protein functionally equivalent to the N0LP protein from the MA. Thus, a protein encoded by a MA that encodes the N0LP protein and a MA that hybridizes with the MA, which is functionally equivalent to the N0LP protein, is also included in the protein of the present invention.
機能的に同等な蛋白質を単離する生物としては、 ヒト以外に、 例えば、 ラッ ト、 マウス、 ブ夕、 ゥサギ、 ニヮ トリ、 力エル、 ゼブラフィッシュ、 ショウジ ョゥバエなどが挙げられるが、 これらに制限されない。 機能的に同等な蛋白質をコードする DNAを単離するためのハイプリダイゼ一 シヨンの条件は、 通常 「lxSSC、 O. lUDS 37°C」 程度であり、 より厳しい条件 としては 「0.5xSSC、 0. 1¾ SDS 42°C」 程度であり、 さらに厳しい条件としては 「0.2xSSC、0. 1% SDS、65°C」程度である。また、洗浄条件としては、通常「0. lxSSC、 0.1¾SDS 室温 （15〜30分） 2回、 50°C (30分） 3回」 程度であり、 より厳しい 条件としては 「0.2xSSC、 0.1¾SDS、 室温 （15〜30分） 2回、 55°C (30分） 3回」 程度であり、 さらに厳しい条件としては 「0.4xSSC、 0. 1%SDS、 室温（15〜30分） 2回、 65°C (30分） 3回」 程度である。 上記ハイブリダィゼーシヨンおよび洗浄 の条件が厳しくなるほどプローブ配列と高い相同性を有する DNAの単離を期待 しうる。 但し、 上記 SSC、 SDSおよび温度の条件の組み合わせは例示であり、 当 業者であれば、 ハイプリダイゼ一シヨンの厳しさを決定する上記若しくは他の 要素 （例えば、 プローブ濃度、 プローブの長さ、 ハイブリダィゼーシヨン反応 時間など） を適宜組み合わせることにより、 上記と同様の条件を実現すること が可能である。 Organisms that isolate functionally equivalent proteins include, besides humans, for example, rats, mice, bush, ephedra, chicks, potatoes, zebrafish, drosophila, and the like. Not restricted. Hybridization conditions for isolating DNA encoding a functionally equivalent protein are usually about lxSSC, O.lUDS 37 ° C, and more stringent conditions are 0.5xSSC, 0.1 0 SDS is about 42 ° C ”, and more severe conditions are about“ 0.2xSSC, 0.1% SDS, 65 ° C ”. The washing conditions are usually about 0.1 lxSSC, 0.1¾SDS, 2 times at room temperature (15 to 30 minutes) and 3 times at 50 ° C (30 minutes), and more stringent conditions are 0.2xSSC, 0.1¾SDS. , Room temperature (15-30 minutes) twice, 55 ° C (30 minutes) three times ”, and more severe conditions are“ 0.4xSSC, 0.1% SDS, room temperature (15-30 minutes) twice, 65 ° C (30 minutes) 3 times ”. As the hybridization and washing conditions become more stringent, isolation of DNA having higher homology to the probe sequence can be expected. However, the combination of the SSC, SDS and temperature conditions described above is an example, and those skilled in the art will recognize the above or other factors (eg, probe concentration, probe length, hybridizer) that determine the severity of hybridization. The same conditions as described above can be realized by appropriately combining the reaction times.
このようなハイブリダィゼ一シヨン技術を利用して単離される蛋白質は、 通 常、 N0LP蛋白質とアミノ酸配列において高い相同性を有する。高い相同性とは、 少なくとも 40%以上、 好ましくは 60%以上、 さらに好ましくは 80%以上の配列 の同一性を指す。 配列の相同性は、 BLAST、 FASTA、 Smith-Watermanなどの検索 アルゴリズムを用いて決定することができる。  Proteins isolated using such hybridization techniques usually have high homology in amino acid sequence with the NOLP protein. High homology refers to sequence identity of at least 40% or more, preferably 60% or more, more preferably 80% or more. Sequence homology can be determined using a search algorithm such as BLAST, FASTA, Smith-Waterman.
また、 遺伝子増幅技術 (PCR) (PCR Primer A Laboratory Manual , Cold Spring Harbor Laboratory Press, 1995参照） を用いて NOLP蛋白質をコードする DNA配 列 （配列番号： 1 ) の一部をもとにプライマ一を設計し、 NOLP蛋白質をコード する DNA配列またはその一部と相同性の高い DNA断片を単離して、 これをもとに N0LP蛋白質と機能的に同等な蛋白質を得ることも可能である。 このような遺伝 子増幅技術を利用して単離した蛋白質もまた本発明の蛋白質に含まれる。  In addition, using a gene amplification technique (PCR) (see PCR Primer A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1995), a primer was constructed based on a part of the DNA sequence encoding the NOLP protein (SEQ ID NO: 1). It is also possible to isolate a DNA fragment having high homology to the DNA sequence encoding the NOLP protein or a part thereof, and obtain a protein functionally equivalent to the NOLP protein based on the DNA fragment. A protein isolated using such a gene amplification technique is also included in the protein of the present invention.
本発明の蛋白質は、 組み換え蛋白質として、 また天然の蛋白質として調製す ることが可能である。 組み換え蛋白質は、 例えば、 後述するように本発明の蛋 白質をコードする DNAを挿入したベクターを適当な宿主細胞に導入し、形質転換 体内で発現された蛋白質を精製することにより調製することが可能である。 一 方、 天然の蛋白質は、 例えば、 後述する本発明の蛋白質に対する抗体を結合し たァフィ二ティ一カラムを利用して調製することができる。 ァフィ二ティ一精 製に用いる抗体は、 ポリクローナル抗体であってもモノクローナル抗体であつ てもよい。 また、 インビトロトランスレーションなどにより本発明の蛋白質を 調製することも可能である。 The protein of the present invention may be prepared as a recombinant protein or as a natural protein. It is possible to The recombinant protein can be prepared, for example, by introducing a vector into which a DNA encoding the protein of the present invention is inserted into an appropriate host cell and purifying the protein expressed in the transformant, as described later. It is. On the other hand, a natural protein can be prepared using, for example, an affinity column to which an antibody against the protein of the present invention described below is bound. The antibody used for affinity purification may be a polyclonal antibody or a monoclonal antibody. Further, the protein of the present invention can be prepared by in vitro translation or the like.
また、 本発明は、 N0LP蛋白質のアミノ酸配列の少なくとも一部を含み、 核小 体移行活性または核移行活性を有するぺプチドを包含する。 「N0LP蛋白質のァ ミノ酸配列の少なくとも一部」 とは、 少なくとも 5アミノ酸以上、 好ましくは、 10アミノ酸以上のアミノ酸配列を指す。  The present invention also includes a nucleolus localization activity or a peptide having at least a part of the amino acid sequence of the NOLP protein and having nucleolus localization activity. “At least a part of the amino acid sequence of the NOLP protein” refers to an amino acid sequence of at least 5 amino acids or more, preferably 10 amino acids or more.
本発明において、 N0LP蛋白質のアミノ酸 342-371位からなるペプチド単独で核 小体局在を誘導し得ることが判明した（図 4B-k)。 従って、 本発明の核小体移行 活性を有するベプチドの好ましい態様の一つとしては、 N0LP蛋白質のアミノ酸 342- 371位を含有する。 また、 本発明において、 N0LP蛋白質のアミノ酸 372-401 位からなるぺプチド単独で核への移行活性を示すことが示された （図 4B- j )。従 つて、 本発明の核移行活性を有するぺプチドの好ましい態様の一つとしては、 N0LP蛋白質のアミノ酸 372-401位を含有する。  In the present invention, it was found that nucleolar localization could be induced by a peptide consisting of amino acids 342-371 of the N0LP protein alone (FIG. 4B-k). Therefore, one preferred embodiment of the peptide having nucleolar transport activity of the present invention contains amino acids 342 to 371 of the NOLP protein. In addition, in the present invention, it was shown that the peptide consisting of amino acids 372-401 of the N0LP protein alone exhibited a nuclear translocation activity (FIG. 4B-j). Therefore, one of the preferred embodiments of the peptide having nuclear translocation activity of the present invention contains amino acids 372-401 of the NOLP protein.
上記本発明のベプチドは、 核小体移行活性または核移行活性を有するという 特徴を有するため、 例えば、 所望の物質を核小体または核へ輸送するための担 体として利用することが可能である。 本発明のベプチドを担体として輸送する ための物質としては特に制限はない。 例えば、 ペプチド、 核酸、 多糖、 金属、 合成化合物などが挙げられる。 これら物質は、 疾患の治療用、 予防用、 診断用 の薬物でありうるが、 これらに制限されない。 臨床応用以外の目的、 例えば、 生物学的解析などの目的で用いられる物質であってもよい。 本発明のペプチドを用いて、 物質を核小体または核へ輸送するためには、 該 物質を本発明のペプチドに結合させることが必要である。 結合は、 一定の結合 力が得られる限り、 その形態に制限はない。 結合は、 架橋剤を用いた化学的架 橋 （日本生化学会編 続生化学実験講座 2、 蛋白質の化学 （下） 、 東京化学同人、 604-618) でもよく、 例えば、 従来から頻繁に利用されるジメチルスべ口イミ デート二塩酸塩、 スベリン酸ジ -N-ヒドロキシスクシンィミ ドエステル、酒石酸 ジ -N-ヒドロキシスクシンイミ ドエステル、 P-フエ二レンビスマレイミ ド、 メチ ル 4一メルカプトブチルイミデート塩酸塩、 メチル 4一アジドベンゾィミデート 塩酸塩、 グルタルアルデヒド、 N—スクシンィミジル 3- (2-ピリジルジチォ） プ 口ビオネ一ト、 N— （ァ -マレイミ ドブチリルォキシ） スクシンイミ ド、 N- ( £ - マレイミ ドへキサノィルォキシ） スクシンイミ ド、 1-ェチル -3- (3-ジメチルァ ミノプロビル） カルポジイミ ドなどが挙げられる。 また、 輸送するための物質 がべプチドであれば、該ぺプチドをコ一ドする DNAと本発明のぺプチドをコ一ド する DNAを連結して、これらべプチドを融合べプチドとして発現させることによ り両ぺプチドを結合させることも可能である。 結合はスぺーサー領域を介した 結合であってもよい。 一方、 結合形態としては、 上述の共有結合的な結合形態 以外に、 本発明のぺプチドと核へ輸送するための物質の間に生じる相互的な親 和作用を利用した結合でもよい。 このような結合形態として、 例えば、 静電気 的な親和力を利用した結合、疎水的な親和力を利用した結合、蛋白質-蛋白質、 あるいは、 蛋白質一核酸などの結合モチーフを利用した結合、 酵素と基質の親 和力を利用した結合などが挙げられる。 本発明のぺプチドと輸送するための物 質が本質的にこれらの結合能を有していない場合は、 これらの結合能をもつ機 能ドメインを新たに付加して利用することも考えられる。 Since the above-mentioned peptide of the present invention has a nucleolus translocation activity or a nuclear translocation activity, it can be used, for example, as a carrier for transporting a desired substance to the nucleolus or nucleus. . The substance for transporting the peptide of the present invention as a carrier is not particularly limited. For example, peptides, nucleic acids, polysaccharides, metals, synthetic compounds and the like can be mentioned. These substances can be, but are not limited to, drugs for treating, preventing, or diagnosing a disease. It may be a substance used for purposes other than clinical application, for example, for biological analysis. In order to transport a substance to the nucleolus or nucleus using the peptide of the present invention, it is necessary to bind the substance to the peptide of the present invention. There is no limitation on the form of the bond as long as a certain bonding force can be obtained. The bond may be a chemical bridge using a cross-linking agent (Seikagaku Kagaku Kenkyusho, edited by The Biochemical Society of Japan 2, Protein Chemistry (below), Tokyo Kagaku Dojin, 604-618). Dimethyl sulfimidate dihydrochloride, di-N-hydroxysuccinimidyl suberate, di-N-hydroxysuccinimidyl tartrate, P-phenylene bismaleimide, methyl 4-mercaptobutyrimidate hydrochloride Salt, Methyl 4-Azidobenzimidate hydrochloride, glutaraldehyde, N-succinimidyl 3- (2-pyridyldithio) butionate, N— (a-maleimi dobutyryloxy) succinimid, N- (£ -malemid Hexanoyloxy) succinimid, 1-ethyl-3- (3-dimethylaminoprovir) carbopimid and the like. If the substance to be transported is a peptide, the DNA encoding the peptide is ligated to the DNA encoding the peptide of the present invention, and these peptides are expressed as a fusion peptide. In some cases, it is possible to combine both peptides. The binding may be via a spacer region. On the other hand, as the bonding form, other than the above-mentioned covalent bonding form, a bond utilizing mutual reciprocity generated between the peptide of the present invention and a substance for transport to the nucleus may be used. Such binding forms include, for example, binding using electrostatic affinity, binding using hydrophobic affinity, binding using a binding motif such as protein-protein or protein-nucleic acid, and parent of enzyme and substrate. Coupling using sum power is exemplified. If the substance for transporting the peptide of the present invention does not essentially have these binding abilities, a functional domain having these binding abilities may be newly added and used.
本発明のぺプチドを担体として物質を輸送するための標的細胞としては、 特 に制限はなく、 本発明のぺプチドを利用する目的に応じて種々の細胞を用いる ことが可能である。 標的細胞としては、 例えば、 生体における細胞で有り得る し、 またインビト口における培養細胞で有り得る。 また、 これら細胞は、 例え ば、 ヒトを含む動物細胞、 植物細胞、 微生物細胞で有り得る。 The target cells for transporting the substance using the peptide of the present invention as a carrier are not particularly limited, and various cells can be used according to the purpose of using the peptide of the present invention. The target cell may be, for example, a cell in a living body. Alternatively, it may be a cultured cell in the mouth of the in vitro. In addition, these cells can be, for example, animal cells including humans, plant cells, and microbial cells.
本発明のぺプチドは、適当な物質で修飾して視覚的に認識できるようすれば、 「核小体輸送支持物質」 または 「核輸送指示物質」 として利用することも可能 である。 すなわち、 このような修飾を受けたペプチドを細胞内に導入すること により、 細胞質から核小体または核への物質移動をリアルタイムでモニタリン グすることが可能となる。 ペプチドの修飾方法としては、 ペプチドを直接的に 蛍光色素などで標識したり、 あるいは EGFPなどの蛍光蛋白質との融合べプチド を作製すればよい。 このような 「核小体輸送支持物質」 や 「核輸送指示物質」 は、 例えば、 核小体移行阻害剤や核移行阻害剤などを開発するためのスクリー 二ング系に応用することができる。  The peptide of the present invention can be used as a "nucleolus transport support substance" or "nuclear transport indicator" if it can be visually recognized by modification with an appropriate substance. That is, by introducing such a modified peptide into a cell, it becomes possible to monitor mass transfer from the cytoplasm to the nucleolus or nucleus in real time. As a method for modifying the peptide, the peptide may be directly labeled with a fluorescent dye or the like, or a fusion peptide with a fluorescent protein such as EGFP may be prepared. Such a “nucleolar transport supporting substance” or “nuclear transport indicator” can be applied to, for example, a screening system for developing a nucleolus transport inhibitor or a nuclear transport inhibitor.
また、本発明は、上記本発明の蛋白質またはべプチドをコ一ドする DNAに関す る。本発明の DNAは、本発明の蛋白質またはべプチドをコードしうるものであれ ば、 特に制限はなく、 例えば、 cDNA、 ゲノム DNA、 および合成 DNAが含まれる。 本発明の DNAは、 例えば、 配列番号： 1に記載された塩基配列の全部若しくは一 部からなる DNAをプローブとして、ヒト胎児脳由来の cDNAライブラリ一またはゲ ノム DNAライブラリーをスクリ一ニングすることにより調製することができる。 また、 配列番号： 1に記載された塩基配列からなる MAに特異的にハイブリダィ ズするオリゴヌクレオチドをプライマ一として、 ヒ 卜胎児脳由来の mRNA、 cDNA、 またはゲノム DNAを鍊型としたポリメラ一ゼ連鎖反応により調製することもで The present invention also relates to a DNA encoding the protein or the peptide of the present invention. The DNA of the present invention is not particularly limited as long as it can encode the protein or the peptide of the present invention, and includes, for example, cDNA, genomic DNA, and synthetic DNA. The DNA of the present invention is obtained, for example, by screening a human fetal brain-derived cDNA library or a genomic DNA library using, as a probe, a DNA consisting of all or a part of the nucleotide sequence shown in SEQ ID NO: 1. Can be prepared. In addition, an oligonucleotide that specifically hybridizes to MA consisting of the nucleotide sequence shown in SEQ ID NO: 1 is used as a primer, and the mRNA, cDNA, or genomic DNA derived from human fetal brain is used as a type III polymerase. It can also be prepared by a chain reaction
C?る o C o ru o
本発明の DNAは、例えば、本発明の蛋白質またはべプチドの製造に利用するこ とが可能である。 本発明の蛋白質またはペプチドの製造は、 一般的には、 本発 明の DNAを適当なベクターに挿入して、 これを適当な宿主細胞に導入し、得られ た形質転換細胞を培養して、 形質転換細胞に発現させた組み換え蛋白質または ぺプチドを精製することにより行う。組換え蛋白質またはべプチドの発現には、 従来の一般的な発現系を利用することができる。 例えば、 大腸菌、 枯草菌、 酵 母、 昆虫細胞、 植物細胞、 動物細胞などを用いた生体内発現系はもとより、 大 腸菌、 小麦胚芽、 あるいはラビッ トレティキュロサイ トなどの細胞抽出液を用 いた試験管内発現系も利用することが可能である。 このように発現された組み 換えペプチドは、 例えば、 従来から用いられているイオン交換、 ゲルろ過、 分 配、 ァフィ二ティーなどのクロマトグラフィ一のステップを組み合わせたり、 適当な充填剤を使用した HPLCなどを利用するなどして高純度に精製することが 可能である。 また、 本発明の蛋白質またはペプチドは、 精製を容易にするため に他のペプチド、 例えば、 GST、 6 xHisなどとの融合ペプチドとして発現させて、 該融合したぺプチドと親和性を有する物質を利用してァフィ二ティ一精製して もよい。 The DNA of the present invention can be used, for example, for producing the protein or peptide of the present invention. The production of the protein or peptide of the present invention is generally carried out by inserting the DNA of the present invention into an appropriate vector, introducing this into an appropriate host cell, and culturing the obtained transformed cell. The purification is performed by purifying the recombinant protein or peptide expressed in the transformed cell. For expression of a recombinant protein or peptide, Conventional general expression systems can be used. For example, not only in vivo expression systems using Escherichia coli, Bacillus subtilis, yeast, insect cells, plant cells, animal cells, etc., but also cell extracts of Escherichia coli, wheat germ, or rabbit reticulocyte In vitro expression systems can also be used. Recombinant peptides expressed in this way can be used, for example, by combining conventional chromatography steps such as ion exchange, gel filtration, distribution, and affinity, or by HPLC using an appropriate packing material. It can be purified to a high degree of purity using, for example. Further, the protein or peptide of the present invention is expressed as a fusion peptide with another peptide, for example, GST, 6 × His, etc. in order to facilitate purification, and a substance having an affinity for the fused peptide is used. It may be purified by affinity.
本発明の蛋白質またはべプチドの発現に利用されるベクターの一例を示せば、 例えば、 試験管内発現であれば PBESTベクタ一（プロメガ社製） 、 大腸菌であれ ば pETベクター （Invitrogen社製）、 培養細胞、 生物個体であれば PME18Sベクタ ― (Mol Cell Biol . 8:466〜472( 1988)) などが挙げられる。 ベクタ一への本発 明の DNAの挿入は常法により制限酵素サイ トを用いたリガーゼ反応により行う こと力 sできる (Current protocols in Molecular Biology edit. Ausubel et al . ( 1987) Publish. John Wi ley & Sons. Section 11.4〜11.11 ) 。 Examples of vectors used for expression of the protein or peptide of the present invention include, for example, PBEST vector (promega) for in vitro expression, pET vector (Invitrogen) for Escherichia coli, and culture. In the case of cells and living organisms, examples include the PME18S vector (Mol Cell Biol. 8: 466-472 (1988)). Insertion of the onset light of the DNA into the vector one can this and force s carried out by a ligase reaction using restriction enzyme site by a conventional method (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wi ley & Sons. Section 11.4-11.11).
宿主細胞へのベクター導入は、 例えば、 リン酸カルシウム沈殿法、 電気パル ス穿孑し法 (Current protocols in Molecular Biology edit. Ausubel et al . ( 1987) Publ ish. John Wi ley & Sons. Section 9. 1-9.9) 、 リボフェク夕ミン 法（GIBCO- BRL社製）、 マイクロインジェクション法などの方法で行うことが可 能である。  Transduction of vectors into host cells can be performed, for example, by the calcium phosphate precipitation method and the electropulsing method (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons. Section 9.1- 9.9), ribofectamine method (GIBCO-BRL), microinjection method, etc.
本発明の蛋白質をコードする DNAまたは後述のアンチセンス DNAは、 N0LP遺伝 子の変異などに起因する疾患の遺伝子治療への応用も考えられる。 遺伝子治療 に用いる場合には、 例えば、 レトロウイルスベクター、 アデノウイルスベクタ ―、 アデノ随伴ウィルスベクターなどのウィルスベクタ一やリボソームなどの 非ウィルスベクターなどを利用して、 本発明の DNAまたはアンチセンス DNAを、 ex vivo法や in vivo法などにより患者へ投与を行う。 The DNA encoding the protein of the present invention or the antisense DNA described below may also be applied to gene therapy for diseases caused by mutations in the N0LP gene. When used for gene therapy, for example, retrovirus vectors, adenovirus vectors -The DNA or antisense DNA of the present invention is administered to a patient by an ex vivo method or an in vivo method using a virus vector such as an adeno-associated virus vector or a non-viral vector such as a ribosome.
また、 本発明は、 本発明の蛋白質またはペプチドに結合する抗体に関する。 本発明の抗体の形態には特に制限はなく、 ポリクローナル抗体やモノクロ一ナ ル抗体または抗原結合性を有するそれらの一部も含まれる。 また、 全てのクラ スの抗体が含まれる。 さらに、 本発明の抗体には、 ヒト抗体やヒト化抗体など の特殊抗体も含まれる。  The present invention also relates to an antibody that binds to the protein or peptide of the present invention. The form of the antibody of the present invention is not particularly limited, and includes a polyclonal antibody, a monoclonal antibody, and a part thereof having antigen-binding properties. In addition, antibodies of all classes are included. Furthermore, the antibodies of the present invention also include special antibodies such as human antibodies and humanized antibodies.
本発明の抗体は、 ポリクローナル抗体の場合には、 常法に従い、 本発明の蛋 白質またはべプチドのアミノ酸配列に相当するオリゴぺプチドを合成して家兎 に免疫することにより得ることが可能であり （Current protocols in Molecular Biology edit. Ausubel et al . ( 1987) Publ ish. John Wi ley & Sons. Section 11.12〜： 11.13) 、 一方、 モノクローナル抗体の場合には、 常法に従い大腸菌で 発現し精製した蛋白質を用いてマウスを免疫し、 脾臓細胞と骨髄腫細胞を細胞 融合させたハイブリ ドーマ細胞の中から得ることができる（Current protocols in Molecular Biology edit. Ausubel et al . ( 1987) Publish. John Wi ley & Sons. Section 11.4〜11. 11) 。  In the case of a polyclonal antibody, the antibody of the present invention can be obtained by synthesizing an oligopeptide corresponding to the amino acid sequence of the protein or peptide of the present invention and immunizing a rabbit according to a conventional method. (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons. Section 11.12-: 11.13) On the other hand, in the case of monoclonal antibodies, they were expressed and purified in Escherichia coli according to standard methods. Mice can be immunized with a protein and obtained from hybridoma cells obtained by fusing spleen cells and myeloma cells (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons. Section 11.4-11.11).
本発明の蛋白質またはべプチドに結合する抗体は、 本発明の蛋白質やべプチ ドの精製や検出以外にも、 例えば、 本発明の蛋白質に関連した疾患の治療など の目的に利用することも考えられる。抗体を患者の治療目的で用いる場合には、 ヒト抗体またはヒ ト化抗体が免疫原性の少ない点で好ましい。 ヒト抗体は、 免 疫系をヒトのものと入れ換えたマウス （例えば、 「Functional transplant of megabase human immunoglobul in loci recapitulates human antibody response in mice, Mendez, M. J et al . ( 1997) Nat. Genet. 15 : 146-156j 参照） に免疫する ことにより調製することができる。 また、 ヒ ト化抗体は、 モノクローナル抗体 の超可変領域を用いた遺伝子組み換えによつて調製することができる（Methods in Enzymology 203， 99-121 ( 1991 ) )。 Antibodies that bind to the protein or peptide of the present invention may be used for purposes such as treatment of diseases related to the protein of the present invention, in addition to purification and detection of the protein or peptide of the present invention. Can be When antibodies are used for the purpose of treating patients, human antibodies or humanized antibodies are preferred because of their low immunogenicity. Human antibodies were obtained by replacing the immune system with that of a human (eg, “Functional transplant of megabase human immunoglobulin loci recapitulates human antibody response in mice, Mendez, M. J et al. (1997) Nat. Genet. 15 : 146-156j) The humanized antibody can be prepared by genetic recombination using the hypervariable region of a monoclonal antibody (Methods). in Enzymology 203, 99-121 (1991)).
また、 本発明は、 配列番号： 1に記載の DNAと特異的にハイブリダィズし、 少 なくとも 15ヌクレオチドの鎖長を有する DNAに関する。 本発明の DNAと 「特異的 にハイブリダィズする」 とは、 通常のハイブリダィゼーシヨン条件下、 好まし くは厳格な条件下で、 本発明の DNAとハイブリダィズし、 他の DNAとはハイプリ ダイズしないことを意味する。 特異的なハイプリダイゼーシヨンのためのスト リンジエンシーは、 ハイブリダィズ反応の温度、 反応時間、 プローブ又はブラ イマ一濃度、 プローブ又はプライマーの長さ、 塩強度などを考慮することによ り、 当業者であれば適宜選択することができる。  The present invention also relates to a DNA that specifically hybridizes with the DNA of SEQ ID NO: 1 and has a chain length of at least 15 nucleotides. "Specifically hybridizes" with the DNA of the present invention means that it hybridizes with the DNA of the present invention under ordinary hybridization conditions, preferably under severe conditions, and hybridizes with other DNA. Means not. Stringency for specific hybridizations can be determined by one skilled in the art by considering the temperature of the hybridization reaction, reaction time, probe or primer concentration, probe or primer length, salt strength, etc. If so, it can be selected as appropriate.
このような DNAは、 本発明の DNAを検出、 単離するためのプローブとして、 ま た、本発明の DNAを増幅するためのプライマ一として利用することが可能である c プライマ一として用いる場合には、 通常、 15bp〜100bp、 好ましくは 15bp〜35bp の鎖長を有する。 また、 プローブとして用いる場合には、 本発明の DNAの少なく とも一部若しくは全部の配列を有し、少なくとも 15bpの鎖長の DNAが用いられる c また、 「配列番号： 1に記載の DNAと特異的にハイブリダィズし、 少なくとも 15ヌクレオチドの鎖長を有する DNA」には、本発明の蛋白質の発現を抑制するた めのアンチセンス DNAが含まれる。 アンチセンス DNAは、 アンチセンス効果を引 き起こすために、 少なくとも 15bp以上、 好ましくは、 100bp、 さらに好ましくは 500bp以上の鎖長を有し、 通常、 3000bp以内、 好ましくは、 2000bp以内の鎖長を 有する。 このようなアンチセンス DNAには、 本発明の蛋白質の異常（機能異常や 発現異常） などに起因した疾患 （例えば、 癌などが考えられる） の遺伝子治療 への応用も考えられる。 図面の簡単な説明 Such DNA is used as a probe for detecting and isolating the DNA of the present invention, and as a c-primer which can be used as a primer for amplifying the DNA of the present invention. Has a chain length of usually 15 bp to 100 bp, preferably 15 bp to 35 bp. Further, if used as a probe, having a least be of some or all sequences of DNA of the present invention, also c least 15bp chain length DNA is used, "SEQ ID NO: DNA specifically described in 1 "DNA that specifically hybridizes and has a chain length of at least 15 nucleotides" includes antisense DNA for suppressing the expression of the protein of the present invention. The antisense DNA has a chain length of at least 15 bp or more, preferably 100 bp, more preferably 500 bp or more, and usually has a chain length of 3000 bp or less, preferably 2000 bp or less in order to cause an antisense effect. Have. Such antisense DNA is also considered to be applicable to gene therapy for diseases (for example, cancer can be considered) caused by abnormalities (functional or abnormal expression) of the protein of the present invention. BRIEF DESCRIPTION OF THE FIGURES
図 1は、 ヒト N0LPのヌクレオチドおよび推定ァミノ酸配列を示す。 開始コド ンより前のインフレーム終始コ ドン（468位）を四角で囲んだ。 3つの潜在的な核 移行シグナルをアンダーラインで示した。 FIG. 1 shows the nucleotide and deduced amino acid sequence of human NOLP. The in-frame stop codon (position 468) before the start codon is boxed. Three potential nuclei Migration signals are underlined.
図 2は、 Tral相同領域の配列の整列を示す。 E . col i Tral/re laxase ( 1677- 1715 )、 ヒト N0LP ( 7-54)、 およびゲノム DNA (ァクセシヨン番号 AC004360 )から 翻訳したショウジヨウバエ （Drosophi la) 仮想蛋白質を示した。 同一および類 似のアミノ酸残基を 「*」 および 「/」 でそれそれ示した。 コアコンセンサスに おける高度に保存された残基を四角で囲んだ。  FIG. 2 shows a sequence alignment of the Tral homology region. Drosophi la virtual protein translated from E. coli Tral / re laxase (1677-1715), human N0LP (7-54), and genomic DNA (accession number AC004360) is shown. Identical and similar amino acid residues are indicated by “*” and “/”, respectively. Highly conserved residues in the core consensus are boxed.
図 3は、 RT-PCRによる組織分布解析を示す。 試験した 12の組織をそれそれの レーンの上に示した。  FIG. 3 shows a tissue distribution analysis by RT-PCR. The 12 tissues tested are shown above each lane.
図 4は、 N0LP蛋白質の核小体局在のための配列の必要条件を示した。 A.欠失 構築物を図示した。 N0LP蛋白質の推定ドメインの模式図をパネル上に示した。 FIG. 4 shows the sequence requirements for nucleolar localization of the N0LP protein. A. Deletion Constructs are illustrated. A schematic diagram of the putative domain of the N0LP protein is shown on the panel.
「D/E-リツチ」はァスパラギン酸/グル夕ミン酸に富んだ残基を示す。「NLS1」，"D / E-litsch" indicates residues rich in aspartic acid / glutamic acid. "NLS1",
「NLS2」 ， 「NLS3」 は潜在的な核局在シグナルを示す。 「S -リツチ」 はセリンに 富んだ残基を示す。 Bにおいて決定されたそれそれの構築物の細胞内分布パ夕 ーンを右に示した。 「NU/N0」はいくつかは核でいくつかは核小体であることを 示す。 「N0」 は構成的に核小体であることを示す。 「N/C」 は非特異的 （細胞全 体）であることを示す。 「NU」 は構成的に核であることを示す。 B . COS- 7細胞 で発現させた後の EGFP-ハイプリッ ド蛋白質の細胞内分布を示す。形質転換した 細胞は 24時間後に試験した。 “NLS2” and “NLS3” indicate potential nuclear localization signals. "S-Litsch" indicates a serine-rich residue. The subcellular distribution patterns of each construct determined in B are shown on the right. "NU / N0" indicates that some are nuclei and some are nucleoli. "N0" indicates that it is constitutively a nucleolus. “N / C” indicates non-specific (whole cell). "NU" indicates that it is structurally nuclear. B. Intracellular distribution of EGFP-hybrid protein after expression in COS-7 cells. Transformed cells were tested after 24 hours.
図 5は、 EGFP- ガラク トシダーゼハイブリ ヅ ド蛋白質の構築および解析を 示す。 A.融合蛋白質構築物を図示した。 Bで決定したそれそれの構築物の細胞 内分布パターンを右にまとめた。 「N/C」は非特異的 （細胞全体）であることを、 FIG. 5 shows the construction and analysis of the EGFP-galactosidase hybrid protein. A. The fusion protein construct is illustrated. The subcellular distribution patterns of each construct determined in B are summarized on the right. "N / C" is non-specific (whole cell)
「冊」は構成的に核であることを示す。 B . COS- 7細胞での発現後の EGFPハイブ リツ ド蛋白質の細胞内局在を示す。 （a) EGFP- ' lacZ (対照）、 (b) EGFP-"Books" indicate that they are core in composition. B. Intracellular localization of EGFP hybrid protein after expression in COS-7 cells. (A) EGFP- 'lacZ (control), (b) EGFP-
' lacZ- [342-371 ]、 ( c ) EGFP- ' lacZ- [372-401 ] o 形質転換細胞は 24時間後に試 験した。 発明を実施するための最良の形態 'lacZ- [342-371], (c) EGFP-' LacZ- [372-401] o transformed cells were tested 24 hours later. BEST MODE FOR CARRYING OUT THE INVENTION
以下、 本発明を実施例によりさらに詳細に説明するが、 本発明は以下の実施 例に制限されるものではない。  Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to the following Examples.
[実施例 1 ] 全長 NOLP cDNAの単離  [Example 1] Isolation of full-length NOLP cDNA
SUPERSCRIPTヒ ト胎児脳 cDNAライブラリ一（Life Technologie 社）から全長 NOLP cDNAをクローニングするためにポリメラーゼ鎖反応 （PCR) 及び cDNA末端 の急速的増幅 （RACE) を行った。  Polymerase chain reaction (PCR) and rapid amplification of cDNA ends (RACE) were performed to clone full-length NOLP cDNA from SUPERSCRIPT human fetal brain cDNA library (Life Technology).
具体的には、 cDNAライブラリ一のべクタ一である PCMV-SP0RT2 (Life  Specifically, PCMV-SP0RT2 (Lifetime
Technology) 特異的な 2種類のプライマー 「VSP1」 （5， -CAC ACA GGA AAC AGC TAT GAC CAC TAG-3，/配列番号： 3 ) 、 「VSP2」 (5' -AAG CTA TTT AGG TGA CAC TAT AGA AGG TAC-3' /配列番号： 4 ) と NOLP cDNA特異的な 2種類の プライマ一 「NU164」 （5， -TTT GTC GAC ATC CTG CTG TCT CTC CAG ACG-3' /配列番号： 5 ) 、 「NU162」 （5， -TTT GTC GAC GGC ACG CTC TTG ATA CTC AGG-3' /配列番号： 6 ) とを用いた nested-PCRにより、 これまでに得られてい た部分配列 cDNA (ァクセッション番号 AB015339、 特願平 10-174065号） の 5'側上 流の配列取得を試みた。まず、 VSP1/NU162のプライマー対を用いて SUPEERSCRIPT ヒト胎児脳 cDNAライブラリーを鎵型にし、 遺伝子断片の増幅を行った。 次に、 VSP2/NU164のプライマー対を用いて一回目に増幅された DNA断片を鍊型にし、二 回目の増幅を行った。 得られた PCR産物を、 ァガロースゲル電気泳動によって 1.5- 4. Okbのサイズに分画し、 クローニングベクター pT7Blue(R) (Novagen社） に挿入後、 E. coliDH5ひに導入した。 目的の DNA断片を保持しているクローンを 選択するために VSP1/NU162のプライマ一対を用いてコロニー PCRを行い、最も長 いインサート配列 （1652bp) を持つクローンを含むいくつかの陽性クローンを 選択することができた。 これまでに得られている 131 lbpと今回取得した 1652bp は互いに 339bpの 100%同一なオーバ一ラップ領域を共有していた。これら 2つの DNA断片を 1つの cDNAとして統合した結果、 2624bpの鎖長を有する cDNAクローン が得られた。 Technology) Two specific primers "VSP1" (5, -CAC ACA GGA AAC AGC TAT GAC CAC TAG-3, / SEQ ID NO: 3), "VSP2"(5'-AAG CTA TTT AGG TGA CAC TAT AGA AGG TAC-3 '/ SEQ ID NO: 4) and two primers specific to NOLP cDNA "NU164" (5, -TTT GTC GAC ATC CTG CTG TCT CTC CAG ACG-3' / SEQ ID NO: 5) NU162 "(5, -TTT GTC GAC GGC ACG CTC TTG ATA CTC AGG-3 '/ SEQ ID NO: 6) and the partial sequence cDNA (accession number AB015339) obtained so far by nested-PCR. An attempt was made to obtain an array upstream of the 5 'side of Japanese Patent Application No. 10-174065). First, using a primer pair of VSP1 / NU162, the SUPEERSCRIPT human fetal brain cDNA library was transformed into type III, and gene fragments were amplified. Next, the DNA fragment first amplified using the primer pair of VSP2 / NU164 was converted into a 鍊 type, and the second amplification was performed. The resulting PCR product was fractionated to a size of 1.5-4 Okb by agarose gel electrophoresis, inserted into a cloning vector pT7Blue (R) (Novagen), and then introduced into E. coli DH5. Perform a colony PCR using the VSP1 / NU162 primer pair to select clones containing the target DNA fragment, and select several positive clones including the clone with the longest insert sequence (1652 bp) I was able to. The 131 lbp obtained so far and the 1652 bp obtained this time share a 339 bp 100% identical overlapping region with each other. As a result of integrating these two DNA fragments into one cDNA, a cDNA clone with a chain length of 2624 bp was gotten.
このクローンの構造を解析した結果、 推定の開始メチォニンの前にインフレ Analysis of the structure of this clone showed that inflation occurred before
—ム終始コ ドンが存在することが明らかとなり、 こうして 1572 bpの単一の 0RF が推定された （図 1 )。この 0RFは 524アミノ酸からなる推定ポリペプチドをコ —ドし、 計算された分子量は 58 kDaであった。 蛋白質は、 酸性のァスパラギン 酸/グルタミン酸に富んだ領域 （79%， アミノ酸 268- 281位）， 3つの推定された 塩基性に富んだ NLS (NLS1 , RARKRIR, アミノ酸 340- 346位； NLS2, RR KR, アミ ノ酸 353- 357位； NLS3, K職, アミノ酸 392-395位）, セリンに富んだ領域（42%， アミノ酸 458-498位）， および潜在的なコイルド-コイルドメイン （ Lupas, A. ( 1996 ) Methods Enzymol . 266， 513-525 ) (アミノ酸 492-524位）を有していた。 リボヌクレオ蛋白質（RNP)モチーフ（ Burd， C. G. , and Dreyfuss, G. ( 1994) Science 265， 615-621 )のような公知の RNA結合モチーフは見出されなかった。 高度に酸リツチな残基および塩基リツチな残基がいくつかの核小体蛋白質にし ばしば見受けられるが、 蛋白質データ一ベースにおけるアミノ酸の検索（NCBI Protein Databaseおよび Swiss Protein Database ) から、 他の公知の蛋白質と の有意な類似性は見出されなかった。 局所的な相同性（36アミノ酸のオーバ一 ラヅプにおける 50%の配列同一性） が N0LPの N末端の短い残基（アミノ酸 7- 40位） および E. coli DNAヘリ力一ゼ， Tral/relaxaseの C末端部分（アミノ酸 1677- 1715 位）から見出された（図 2 )。 Tral/relaxaseは、接合による DM輸送の開始および 終結に必須である（Byrd， D. R.， and Matson, S. W. ( 1997) Mol . Microbiol . 25, 1011- 1022 )。その触媒活性に重要な領域は N末端に存在すると考えられているが (Balzer, D . , Pansegrau, W. , and Lanka, E. ( 1994) J. Bacteriol . 176, 4285-4295 )、 C末端領域の役割については不明である。 さらに、 高度に類似し た （41アミノ酸のオーバーラップにおける 71%の同一性）仮想翻訳配列がショウ ジヨウバエ （Drosophila) ゲノム DNA (ァクセッション番号 AC004360)から見出 され、 このモチーフが進化の過程でよく保存されていることが示唆された（図 2 )。 It was revealed that a complete stop codon was present, and a single 0RF of 1572 bp was deduced (Fig. 1). This ORF encoded a predicted polypeptide of 524 amino acids, with a calculated molecular weight of 58 kDa. The protein consists of an acidic aspartate / glutamic acid-rich region (79%, amino acids 268-281), three putative basic NLSs (NLS1, RARKRIR, amino acids 340-346; NLS2, RR KR) , Amino acids 353-357; NLS3, K position, amino acids 392-395), a serine-rich region (42%, amino acids 458-498), and a potential coiled-coil domain (Lupas, A. (1996) Methods Enzymol. 266, 513-525) (amino acids 492-524). No known RNA binding motif was found, such as the ribonucleoprotein (RNP) motif (Burd, CG, and Dreyfuss, G. (1994) Science 265, 615-621). Highly acidic and base rich residues are often found in some nucleolar proteins, but searching for amino acids in the protein database (NCBI Protein Database and Swiss Protein Database) has No significant similarity to known proteins was found. Local homology (50% sequence identity in a 36 amino acid overlap) is due to the N-terminal short residue of N0LP (amino acids 7-40) and the E. coli DNA helicase, Tral / relaxase. It was found from the C-terminal part (amino acids 1677-1715) (Figure 2). Tral / relaxase is essential for initiation and termination of DM transport by conjugation (Byrd, DR, and Matson, SW (1997) Mol. Microbiol. 25, 1011-1022). The region important for its catalytic activity is thought to be at the N-terminus (Balzer, D., Pansegrau, W., and Lanka, E. (1994) J. Bacteriol. 176, 4285-4295), but the C-terminus The role of the domain is unknown. In addition, a highly similar virtual translation sequence (71% identity in 41 amino acid overlaps) was found in Drosophila genomic DNA (accession number AC004360), and this motif was evolved during evolution. It is suggested that it is well preserved (Figure 2).
BLASTプログラム（Altschul , S. F .， Gish, W.， Mi l ler, W.， Myers, E. W.， and Lipman, D . J. ( 1990 ) J. Mol . B iol . 215 , 403- 410 )を用いた GenBank/EMBL/DDBJ データ—ベースの検索により、 公知の遺伝子との全体に渡る類似性は見出され なかったが、 ヒト UniGeneデ一夕一ベースにおいて、 ユニークな遺伝子（Hs. 6414) とそのほどんどが同一である多くのヒト EST配列との高度の類似性が認 められた。 このデータ一ベースによれば、 この遺伝子は染色体 18ql2 (マ一力一 D18S457と D18S1124の間）に位置し、 それと同一の ESTクローンは脳、 眼、 精巣、 および小細胞肺癌（ SCLC )細胞系に由来している。  BLAST program (Altschul, S.F., Gish, W., Miller, W., Myers, EW, and Lipman, D.J. (1990) J. Mol. Biol. 215, 403-410) A search of the GenBank / EMBL / DDBJ database used did not reveal any overall similarity to known genes, but a unique gene (Hs. A high degree of similarity with many human EST sequences, most of which were identical, was observed. According to this database, this gene is located on chromosome 18ql2 (between D18S457 and D18S1124), and identical EST clones are found in brain, eye, testis, and small cell lung cancer (SCLC) cell lines. It is derived.
[実施例 2 ] N0LPの組織分布  [Example 2] T0LP tissue distribution
N0LPの発現プロファイルを調べるために、 12の組織における mRNAの発現レべ ルを RT- PCRにより検討した。 プライマ一「NU174」 （5， -TTT GAA TTC CAA TCT TGA AGA AAG AAT GCA AAG TCC-35 /配列番号： 7 ) および 「NU162」 ( 5 ' -TTT GTC GAC GGC ACG CTC TTG ATA CTC AGG-3 ' /配列番号： 6 )を RT— PCRに用いた。 cDNA の錡型は、 過剰な Superscript I I逆転写酵素（Life Technologie社）及びランダ ムなへキサマ一プライマ一を用いてヒト組織のポリ（A)+ RNA ( CL0NTECH )よりそ れそれ作製した。 30サイクルの PCR増幅（95°Cで 20秒および 62°Cで 1分 /1サイク ル）を 10 1の反応液中で行った。 PCR産物は、 2. 5% Nusive GTGァガロースゲル (FMC, Rockland, ME, USA)上で lkb のラダ一 DNAマーカ一 (Life Technologies 社）とともに分離した。 To examine the expression profile of N0LP, the expression level of mRNA in 12 tissues was examined by RT-PCR. Primer "NU174" (5, -TTT GAA TTC CAA TCT TGA AGA AAG AAT GCA AAG TCC-3 5 / SEQ ID NO: 7) and "NU162"(5'-TTT GTC GAC GGC ACG CTC TTG ATA CTC AGG-3 '/ SEQ ID NO: 6) was used for RT-PCR. cDNA type I was prepared from human tissue poly (A) + RNA (CL0NTECH) using excess Superscript II reverse transcriptase (Life Technology) and random hexama-primer. 30 cycles of PCR amplification (20 seconds at 95 ° C and 1 minute / 1 cycle at 62 ° C) were performed in 101 reactions. The PCR products were separated on a 2.5% Nusive GTG agarose gel (FMC, Rockland, ME, USA) with an lkb ladder-DNA marker (Life Technologies).
その結果、 予想されるサイズ（664 bp )の PCR産物が、 胎児脳、 脳、 および精巣 において排他的に検出された（図 2 )。 ノーザンプロッ ト解析により、 脳および 精巣において約 3. 5kbの単一の転写産物が見出された （デ一夕一では示してい ない）。これらの結果は、 デ一夕一ベース検索の結果と部分的に一致している。 特に、 RT-PCRおよびノーザンブロッ ト解析では正常肺において転写産物のシグ ナルは検出されなかったが、 いくつかの同一の ESTクローン（ァクセッション番 号 AA121747, AA129951 , AA626487, および AA127451 )は SCLC細胞系（NCI-H69 )に 由来した。 この事実は、 N0LPが神経内分泌の腫瘍形成に関与し、 それゆえに潜 在的な癌遺伝子または癌関連マ一カーになる可能性を示唆する。 As a result, a PCR product of the expected size (664 bp) was exclusively detected in fetal brain, brain, and testis (Fig. 2). Northern plot analysis found a single transcript of approximately 3.5 kb in the brain and testis (not shown overnight). These results partially match the results of the overnight search. In particular, RT-PCR and Northern blot analysis did not detect transcript signals in normal lung, but did show some identical EST clones (accession numbers). Nos. AA121747, AA129951, AA626487, and AA127451) were derived from the SCLC cell line (NCI-H69). This fact suggests that N0LP is involved in neuroendocrine tumorigenesis, and therefore may be a potential oncogene or cancer-associated marker.
[実施例 3 ] 核小体および核への局在のための配列の必要条件の決定 核小体および核への局在のために必要な配列を決定するために、 まず、 一連 の EGFPでタグを付した N0LPの欠失構築物を作製した。 一連の EGFP融合発現ブラ スミ ドは、 EcoRI/Notlあるいは EcoRI/Sal lで消化した DNA断片を、 EGFP-Cl ' 上 の対応する部位へクローニングすることにより構築した。  Example 3 Determination of sequence requirements for nucleolus and nucleus localization To determine the sequences required for nucleolus and nucleus localization, first a series of EGFP A tagged N0LP deletion construct was generated. A series of EGFP fusion expression plasmids were constructed by cloning DNA fragments digested with EcoRI / Notl or EcoRI / Sall into the corresponding sites on EGFP-Cl '.
pEGFP-Cl 'は pEGFP- C CLONTECH社）の Bgl l l/Pstl部位へのリンカ一 [オリゴ ヌクレオチド（5 ' -GAT CTG GAA TTC ATA TGG CCA TGG CGG CCG CTG CA-3' /配 列番号： 8 )/( 5 ' -GCG GCC GCC ATG GCC ATA TGA ATT CCA -3， /配列番号： 9 ) ]の挿入によって作製した。 222-524位をコードしている DNA断片は、 EcoM/Notlを用いて、 単離された部分 cDNA (ァクセヅシヨン番号 AB015339) か ら取り出した。 それ以外は、 PCRを用いて全長 NOLP cDNAから増幅し、 続いて EcoRI/Sal Iによって消化した。 使用したプライマ一対は N0LP蛋白質のアミノ酸 1-524位に対応する cDNAの増幅には NU181/NU204, 12- 524位には NU205/NU204， 282-524位には NU131/NU204, 282-461位には NU131/NU166 , 282- 401位には NU131/NU164, 282- 371位には NU131/NU163, 282-341位には NU131/NU162, 342- 401位には NU133/NU164， 342- 371位には NU133/NU163, 372-401位には  pEGFP-Cl 'is a linker to the Bglll / Pstl site of pEGFP-C CLONTECH [oligonucleotide (5'-GAT CTG GAA TTC ATA TGG CCA TGG CGG CCG CTG CA-3' / SEQ ID NO: 8) / (5'-GCG GCC GCC ATG GCC ATA TGA ATT CCA-3, / SEQ ID NO: 9)]. A DNA fragment encoding positions 222 to 524 was extracted from the isolated partial cDNA (accession number AB015339) using EcoM / Notl. Others were amplified from full-length NOLP cDNA using PCR and subsequently digested with EcoRI / SalI. The pair of primers used was NU181 / NU204, NU205 / NU204 at positions 12-524, NU205 / NU204 at positions 28-524, and NU131 / NU204, 282-461 at positions 282-524 for cDNA amplification corresponding to amino acids 1-524 of the N0LP protein. NU131 / NU166, 282-401 ranked NU131 / NU164, 282-371 ranked NU131 / NU163, 282-341 ranked NU131 / NU162, 342-401 ranked NU133 / NU164, 342-371 ranked Is in NU133 / NU163, 372-401
NU134/NU164である。 なお、 NU181 , 画 4, NU205, NU131 , NU166, および NU163 の塩基配列は順に配列番号： 1 0から 1 5に示した。 NU134 / NU164. The nucleotide sequences of NU181, stroke 4, NU205, NU131, NU166, and NU163 are shown in SEQ ID NOs: 10 to 15, respectively.
プラスミ ド pEGFP- ' lacZは、 インフレーム EGFP- ' lacZ融合蛋白質を発現させ るために、 改変した/? -ガラク トシダーゼフラグメント（5 ' -末端及び 3' -末端 に合成 Bgl l lリンカ一を含む lacZは EcoRI部位で切り取った）を pEGFP- C1 ' の Bgl l l/EcoRI部位へクローニングすることによって構築した。 上述の 342- 371位 及び 372-401位をコードしている PCR産物を、 EGFP- ' lacZのィンフレーム融合構 築物を発現させるために、 pEGFP- ' lacZに対応した部位に連結した。 すべての 構築物の確認は塩基配列の決定により行った。 Plasmid pEGFP- 'lacZ is a modified /?-Galactosidase fragment (containing synthetic Bglll linkers at the 5'- and 3'-ends) to express in-frame EGFP-' lacZ fusion proteins. lacZ was cut at the EcoRI site) and cloned into the Bglll / EcoRI site of pEGFP-C1 '. The PCR products encoding positions 342-371 and 372-401 described above were ligated to the EGFP-'lacZ in frame fusion construct. In order to express the construct, it was ligated to a site corresponding to pEGFP-'lacZ. Confirmation of all constructs was performed by sequencing.
次いで、 上記構築物を、 10%牛胎児の血清、 ペニシリン G ( 100 U/ml )及びスト レプトマイシン（100 mg/ml )を含むダルベッコ改変イーグル培地（DMEN)で維持 していた C0S-7細胞に導入した。 導入は、 LIPOFECTAMINE PLUS試薬 （Life Technologies, Inc. )を用い製品の使用説明書に従い行った。 これにより EGFP 融合蛋白質を該細胞中で一過的に発現させ、その細胞内分布を検討した（図 4 )。 蛍光性は Axiovert 135 (Carl Zeiss, Tokyo, Japan)を用いた蛍光顕微鏡法によ つて可視化し、 画像は DIGITAL IMAGE F ILE DF-30M (Fujifi lm, Tokyo, Japan) を通して ZVS-3C75DE (Carl Zeiss )装置から集めた。  The above construct was then added to C0S-7 cells maintained in Dulbecco's modified Eagle's medium (DMEN) containing 10% fetal calf serum, penicillin G (100 U / ml) and streptomycin (100 mg / ml). Introduced. The introduction was performed using LIPOFECTAMINE PLUS reagent (Life Technologies, Inc.) according to the instruction manual for the product. Thus, the EGFP fusion protein was transiently expressed in the cells, and its intracellular distribution was examined (FIG. 4). Fluorescence was visualized by fluorescence microscopy using Axiovert 135 (Carl Zeiss, Tokyo, Japan), and the images were passed through DIGITAL IMAGE FILE DF-30M (Fujifi lm, Tokyo, Japan) to ZVS-3C75DE (Carl Zeiss). Collected from equipment.
その結果、 最初に NTT法により単離した部分（222- 524位） （特願平 10- 174065 号） は構成的な核小体への局在を示したが （図 4-e )、 完全な N0LP -524)は、 核の変形を伴なつて、 核小体から除外されて点状に分布しているもの、 斑状に 分布しているもの、 核小体に分布しているものなどのように 3つの主要な分布 パターンを示した（図 4- a， b， c )。対照的に、 N末端の 11残基を欠いた残基 12-524 は、 このような分布パターンを示さず、構成的な核小体局在を示した （図 4-d)。 これらの結果は、その一部が Tral相同領域とォ一バーラップする N末端の 11残基 が、 N0LPの適確な局在および機能に必要であることを示唆する。  As a result, although the portion first isolated by the NTT method (position 222-524) (Japanese Patent Application No. 10-174065) showed a constitutive localization in the nucleolus (Fig. 4-e), it was completely isolated. N0LP-524) are those that are excluded from the nucleolus with the deformation of the nucleus, are distributed in a point-like manner, are distributed in a patchy manner, are distributed in the nucleolus, etc. Thus, three main distribution patterns are shown (Fig. 4-a, b, c). In contrast, residues 12-524 lacking the N-terminal 11 residues did not show such a distribution pattern, indicating constitutive nucleolar localization (Figure 4-d). These results suggest that the N-terminal 11 residues, some of which overlap with the Tral homology region, are required for proper localization and function of NOLP.
構成的な核小体移行に関しては、酸性に富んだ残基を含む N末端の一部（アミ ノ酸 1-281位）の欠失も、 C末端の一部（アミノ酸 372-524位）の欠失も、 核小体の 局在に影響を与えないことが明らかとなった（図 4B-f， i )。 さらに、 アミノ酸 342-371位の欠失は、 完全に排他的な核小体蓄積を崩壊させ（図 4B-j )、 逆に、 ァ ミノ酸 342-371位単独で核小体局在を誘導できた（図 4B-k)。それゆえに、 NLS1お よび NLS2が存在するこの 30残基 （アミノ酸 342- 371位）が、 核小体への局在に十 分であると結論づけた。さらに、 NLS3が存在する隣接する 30ァミノ酸残基（アミ ノ酸 372- 401位）が活性 NLSとして働くと結論づけた （図 4B-j ) . アミノ酸 342- 371位が公知のレポ一ター蛋白質を核小体へ導くか否かを試験 するため、およびアミノ酸 372- 401位が公知のレポ一夕一蛋白質を核小体へ導く か否かを試験するために、 本発明者らはそれそれのペプチドを EGFP- ? -ガラク トシダーゼ （EGFP- ' lacZ) ハイブリッ ド蛋白質の C末端に融合させた。 ァミノ 酸 372-401位が核への局在に十分である（Figure 5B- c )—方、 アミノ酸 342-371 位はキメラ蛋白質を核へは移行させたが、 明白な核小体からの除外を示し核小 体へは移行させなかった（図 5B-b)。 これは適確な核小体への局在を阻害する分 子折り畳み構造のアーティファク トによるのかもしれない。 その理由はいまだ 不明であるが、 核小体局在シグナルを異種のレポ一夕一蛋白質に転移させるこ とができないことは、 以前に他の核小体蛋白質で報告されている（Michael , W. M. and Dreyfuss, G. ( 1996 ) J. Biol . Chem. 271 , 11571-11574 Peculis, B. A and Gall, J. G. ( 1992) J. Cel l Biol . 116, 1-14 Schmidt-Zachmann, M. S. and Nigg, E. A. ( 1993) J. Cell Sci . 105, 799-806 )。 産業上の利用の可能性 With respect to constitutive nucleolar translocation, the deletion of a portion of the N-terminus (amino acids 1-281), including acidic residues, and a deletion of a portion of the C-terminus (amino acids 372-524) The deletion was found to have no effect on nucleolar localization (Fig. 4B-f, i). Furthermore, deletion of amino acids 342-371 completely disrupted exclusive nucleolar accumulation (Figure 4B-j), and conversely, amino acids 342-371 alone induced nucleolar localization. (Figure 4B-k). Therefore, it was concluded that these 30 residues (amino acids 342-371) where NLS1 and NLS2 were present were sufficient for localization to the nucleolus. We further concluded that the adjacent 30 amino acid residue where NLS3 is present (amino acids 372-401) acts as an active NLS (Figure 4B-j). To test whether amino acids 342-371 lead a known reporter protein to the nucleolus, and determine whether amino acids 372-401 lead a known repo overnight protein to the nucleolus To test, we fused each peptide to the C-terminus of the EGFP-?-Galactosidase (EGFP-'lacZ) hybrid protein. Amino acids 372-401 are sufficient for nuclear localization (Figure 5B-c), whereas amino acids 342-371 transfer the chimeric protein to the nucleus, but are excluded from apparent nucleoli Was not transferred to the nucleolus (Fig. 5B-b). This may be due to an artifact of the molecular fold that prevents proper localization to the nucleolus. The reason is still unknown, but the inability to transfer the nucleolar localization signal to a heterologous repo overnight protein has previously been reported for other nucleolar proteins (Michael, WM and Dreyfuss, G. (1996) J. Biol. Chem. 271, 11571-11574 Peculis, B. A and Gall, JG (1992) J. Celll Biol. 116, 1-14 Schmidt-Zachmann, MS and Nigg, EA (1993) J. Cell Sci. 105, 799-806). Industrial applicability
本発明により新規な核小体蛋白質をコードする DNAが単離され、さらに該蛋白 質において核小体移行活性に関与する領域および核移行活性に関与する領域が 同定された。 本発明の蛋白質またはこれらペプチドは、 核や核小体へ他の物質 を輸送するための担体として、 また、 核移行指示物質や核小体移行指示物質と して利用し得る。 また、 本発明の蛋白質およびその遺伝子は、 発癌マーカーと しての利用や、 新たな癌治療薬開発への利用も考えられる。 さらに、 本発明の 蛋白質やべプチドは、 いまだ不明な核小体輸送機構の解明のための研究に大き く貢献し得ると考えられる。  According to the present invention, a DNA encoding a novel nucleolar protein has been isolated, and a region involved in nucleolar translocation activity and a region involved in nuclear translocation activity in the protein have been identified. The protein or the peptide of the present invention can be used as a carrier for transporting another substance to the nucleus or nucleolus, or as a nuclear transport indicator or a nucleolus transport indicator. In addition, the protein of the present invention and its gene may be used as a carcinogenesis marker, or may be used for the development of new therapeutic agents for cancer. Furthermore, the proteins and peptides of the present invention are expected to contribute significantly to research for elucidation of the nucleolar transport mechanism that is still unknown.

Claims

特許請求の範囲  Claims
I . 配列番号： 2に記載のアミノ酸配列からなる蛋白質、 または該蛋白質中 のアミノ酸配列において 1若しくは複数のアミノ酸が欠失、 付加、 挿入および /または他のァミノ酸による置換により修飾されたァミノ酸配列からなり、 核 小体への移行活性を有する蛋白質。 I. A protein consisting of the amino acid sequence of SEQ ID NO: 2, or an amino acid in which one or more amino acids have been modified by deletion, addition, insertion, and / or substitution by another amino acid in the amino acid sequence in the protein A protein consisting of a sequence and having the activity of translocating to the nucleolus.
2 . 配列番号： 1に記載の塩基配列からなる DNAとハイプリダイズする DNAが コ一ドする蛋白質であって、 核小体への移行活性を有する蛋白質。  2. A protein that encodes a DNA that hybridizes with a DNA consisting of the nucleotide sequence of SEQ ID NO: 1 and that has a nucleolar translocation activity.
3 . 配列番号： 2に記載のアミノ酸配列の少なくとも一部を含み、 核小体へ の移行活性を有するぺプチド。  3. A peptide comprising at least a part of the amino acid sequence of SEQ ID NO: 2, and having a nucleoli transfer activity.
4 . 配列番号： 2に記載のアミノ酸配列の 3 4 2位から 3 7 1位を含む、 請 求項 3に記載のペプチド。  4. The peptide according to claim 3, comprising the amino acid sequence of SEQ ID NO: 2 from position 342 to position 371.
5 . 配列番号： 2に記載のアミノ酸配列の少なくとも一部を含み、 核への移 行活性を有するぺプチド。  5. A peptide comprising at least a part of the amino acid sequence of SEQ ID NO: 2 and having nuclear transfer activity.
6 . 配列番号： 2に記載のアミノ酸配列の 3 7 2位から 4 0 1位を含む、 請 求項 5に記載のぺプチド。  6. The peptide according to claim 5, comprising the amino acid sequence of SEQ ID NO: 2 from position 372 to position 41.
7 . 請求項 1〜 6のいずれかに記載の蛋白質またはべプチドと異種べプチド との融合べプチド。  7. A fusion peptide of the protein or peptide according to any one of claims 1 to 6 and a heterologous peptide.
8 . 請求項 1〜 7のいずれかに記載の蛋白質またはべプチドをコ一ドする DNAC 8. DNA C encoding the protein or peptide according to any one of claims 1 to 7
9 . 請求項 8に記載の DNAを含むベクタ一。 9. A vector comprising the DNA according to claim 8.
1 0 . 請求項 9に記載のベクターを保持する形質転換体。  10. A transformant carrying the vector according to claim 9.
I I . 請求項 1 0に記載の形質転換体を培養する工程を含む、 請求項 1〜7 のいずれかに記載の蛋白質またはべプチドの製造方法。  I I. The method for producing a protein or a peptide according to claim 1, comprising a step of culturing the transformant according to claim 10.
1 2 . 請求項 1〜7のいずれかに記載の蛋白質またはべプチドに結合する抗 体。  12. An antibody that binds to the protein or peptide according to any one of claims 1 to 7.
1 3 . 配列番号： 1に記載の塩基配列からなる DNAと特異的にハイブリダィズ し、 少なくとも 1 5塩基の鎖長を有する DNAC 1 3. Specifically hybridizes with DNA consisting of the nucleotide sequence of SEQ ID NO: 1 DNA C with a chain length of at least 15 bases
PCT/JP1999/003014 1998-06-05 1999-06-04 Novel nucleolar protein WO1999064454A1 (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2811681A1 (en) * 2000-07-17 2002-01-18 Commissariat Energie Atomique Vector for identifying modulators of nuclear-cytoplasmic protein translocation, useful e.g. for targeted delivery of therapeutic proteins

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JPH0196196A (en) * 1987-10-08 1989-04-14 Shoichi Hatanaka Signal peptide nos introducing protein into nucleolus of cell

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Publication number Priority date Publication date Assignee Title
JPH0196196A (en) * 1987-10-08 1989-04-14 Shoichi Hatanaka Signal peptide nos introducing protein into nucleolus of cell

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Title
UEKI N, ET AL.: "NOLP: IDENTIFICATION OF A NOVEL HUMAN NUCLEOLAR PROTEIN AND DETERMINATION OF SEQUENCE REQUIREMENTS FOR ITS NUCLEOLAR LOCALIZATION", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, ACADEMIC PRESS INC. ORLANDO, FL, US, vol. 252, 1 January 1998 (1998-01-01), US, pages 97 - 102, XP002919774, ISSN: 0006-291X, DOI: 10.1006/bbrc.1998.9606 *
UEKI N, ET AL.: "SELECTION SYSTEM FOR GENES ENCODING NUCLEAR-TARGETED PROTEINS", NATURE BIOTECHNOLOGY, NATURE PUBLISHING GROUP, US, vol. 18, 1 December 1998 (1998-12-01), US, pages 1338 - 1342, XP002919775, ISSN: 1087-0156, DOI: 10.1038/4315 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2811681A1 (en) * 2000-07-17 2002-01-18 Commissariat Energie Atomique Vector for identifying modulators of nuclear-cytoplasmic protein translocation, useful e.g. for targeted delivery of therapeutic proteins

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