WO2006057217A1 - Dfrp proteins regulating drg proteins and utilization of the same - Google Patents

Dfrp proteins regulating drg proteins and utilization of the same Download PDF

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Publication number
WO2006057217A1
WO2006057217A1 PCT/JP2005/021361 JP2005021361W WO2006057217A1 WO 2006057217 A1 WO2006057217 A1 WO 2006057217A1 JP 2005021361 W JP2005021361 W JP 2005021361W WO 2006057217 A1 WO2006057217 A1 WO 2006057217A1
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gene
seq
drg1
drg2
protein
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PCT/JP2005/021361
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French (fr)
Japanese (ja)
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Kosuke Ishikawa
Jun-Ichiro Inoue
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The University Of Tokyo
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention relates to a DFRP protein that controls DRG protein and a method for controlling DRG protein.
  • the developmentally regulated GTP-binding protein (DRG) subfamily constitutes a division of the GTPase superfamily (see Non-Patent Document 1).
  • Drg was first identified by the subtractive cDNA cloning method as a gene (NEDD-3) that is expressed at high levels in the developing mouse brain (see Non-Patent Documents 2 to 4).
  • NEDD-3 subtractive cDNA cloning method
  • Genes homologous to mouse drg have been reported in a wide variety of eukaryotic and archaeal species. These DRG proteins show remarkable similarities, suggesting that the role of DRG in cells is very important. Schenker et al.
  • Non-Patent Document 5 conducted a DRG sequence search and reported that the DRG subfamily contains two closely related proteins, DRG1 (original DRG) and DRG2, but these DRG1 proteins And the physiological functions of DRG2 proteins and their regulatory mechanisms have not been elucidated so far.
  • Non-Patent Document 1 Leipe, D. D. et al., (2002) Classification and evolution of P— loop GTP ases and related ATPases. J. Mol. Biol. 317, 41-72.
  • Non-Patent Document 2 Kumar, S. et al., (1992) Identification of a set of genes with develop mentally down-regulated expression in the mouse brain. Biochem. Biophys. Res. Commun. 185, 1155—1161.
  • Non-Patent Document 3 Sazuka, T. et al., (1992) DRG: a novel developmentally regulated G TP— binding protein. Biochem. Biophys. Res. Commun. 189, 363—370.
  • Non-Patent Document 4 Sazuka, T. et al., (1992) Expression of DRG during murine embryonic development. Biochem. Biophys. Res. Commun. 189, 371-377.
  • Non-Special Publication 5 Schenker, T. and Trueb, B. (1997) Assignment of the gene for a dev elopmentally regulated GTP-binding protein (DRG2) to human chromosome bands 17pl3——>> pl2 by in situ hybridization. Cytogenet. Cell Genet. 79, 274-275.
  • DRG2 dev elopmentally regulated GTP-binding protein
  • An object of the present invention is to elucidate the physiological functions and regulatory mechanisms of DRG proteins (DRG1 and DRG2) which are considered to be important regulators of cell proliferation.
  • Another object of the present invention is to provide novel proteins DFRP1 and DFRP2 that specifically bind to DRG1 protein and DRG2 protein and regulate their expression.
  • DRG1 and DRG2 have high homology over the entire length. Regions with particularly strong homology include the characteristic G-motif domain that can be the active center of GTPase and the C-terminal TGS domain associated with RNA binding (Ishikawa, K. et al., (2003) Cloning and char acterization of Xenopus laevis drg2, a member of the developmentally regulated GT P-binding protein subfamily. Gene 322, 105-112.). Based on these similarities, the phylogenetic analysis of DRG1 and DRG2 indicates that these proteins are likely to have similar functions. DRG1 and DRG2 belong to separate evolutionary branches ( Li, B. and Trueb, B.
  • DRG represents a family of two closely related uTP-Dinding proteins. Biochim. Biophys. Acta. 1491, 196-204. And Etheridge, N., et al, (1 999 ) Characterization of ATDRG1, a member of a new class of GTP-binding protein s in plants. Plant Mol. Biol. 39, 1113-1126.). Therefore, we hypothesized that DRG1 and DRG2 have distinct functions.
  • the present inventors have proposed the physiological function of DRG protein for the first time in the present invention.
  • DFRP DRG family regulatory proteins
  • DFRP RG family regula tory nrotein
  • Two new protein families, DFRP1 and DFRP2 were identified ( Figures 1B and 3).
  • DFRP domain One interesting feature of DFRP is the existence of a unique domain that is named DFRP domain by the present inventors and shows high sequence identity among DFRPs (FIG. 1A). It was found that this DFRP domain is essential for binding to the DRG family ( Figure 2). Thus, for evolutionary conservation in eukaryotes, the binding of DFRP to DRG family members and the proper expression of the DRG protein is considered very important.
  • SRG TAL-l
  • TAL-l a transcription factor involved in hematopoiesis that can bind to DRG1
  • over-expresses it also stabilizes the DRG1 protein
  • DRG1 protein a transcription factor involved in hematopoiesis that can bind to DRG1 protein
  • XH XH (199b; Association of a novel GTP binding protein, DRG, with TAL oncogenic proteins. Oncogene 12, 2343-2350.
  • degradation occurs when DRG protein is released from a stable protein complex.
  • Such degradation of free DRG molecules can be attributed to ubiquitin activase (El), conjugase (E2) and ligase. (E3) may be mediated by polyubiquitin by concerted action. Specific domains of DFRP can bind to these ubiquitin-related enzymes.
  • DFRP2 is a protein that contains other domains such as RING fingers, IBR (In Between Ring fingers), UBA ⁇ UBC, and WD repeat domains, which are characteristic of proteins involved in degradation by the ubiquitin proteasome pathway. Has an RWD domain often seen.
  • two consecutive repeats of CCCH-type Zn fingers of DFRP1 are similar to C3HC4-type and C3H2C3-type RING fingers. We believe that these two Zn-finger repeats may be RI NG finger variants. In addition, one Zn finger can bind to ubiquitin.
  • NPL4 novel zinc finger (NZF) domain force ER—associated degradation (ERAD) (Wang, B., Alam, S. L ”Meyer, H H “et al. (2003) Structure and ubiquitin interactions of the conserved zinc finger domain of Npl4. J. Biol. Chem. 278, 20225-20234.) 0 polyubiquitin -associated zinc finger (PAZ)
  • PAZ polyubiquitin -associated zinc finger
  • Histone deacetylase 6 binds polyubiquitin through its zinc finger ( PAZ domain) and copurifies with deubiq uitinating enzymes. Proc. Natl. Acad. Sci. USA 99, 13425-13430.).
  • LEREP04 renamed Faraca et al., was first identified as a gene that is rapidly transcribed in response to erythropoietin (Epo) signaling via the C-terminal truncated Epo receptor in erythroleukemia SKT6 cells.
  • Epo erythropoietin
  • Epo receptors relay key signals for growth (Shikama, Y., Barber, DL, D 'Andrea, AD and Sieff, CA (1996) A constitutively activated chimeric cytokine r eceptor confers factor-independent growth in hematopoietic Blood lines, Blood 88, 45 5-464.) o When stability is required for DRG1 cell function, the cell induces DFRP1 by such growth signals and consequently initiates DRG1 function. It is possible. However, abnormal expression of the DRG family results in cell transformation or cell cycle arrest (Mahajan, M. A "Park, ST and Sun, XH (1996) Association of an ovel GTP binding protein, DRG, with TAL oncogenic proteins.
  • DRG1, DRG2, DFRP1, and DFRP2 are knocked out or knocked down and normal in the same manner as yeast when normal cancer cells that are not cancer cells, normal cells can be obtained by knocking down DRG1, DRG2, DFRP1, and DFRP2. There is a possibility that growth can be attenuated specifically in cancer cells.
  • DRG1 and DFRP1 KD cells showed changes in mRNA of various factors. Involved in the stability of ribosomes on mRNA, directly or indirectly controlled mRNA stabilization. it seems to do.
  • DFRP1 and DFRP2 are specific regulatory proteins of the DRG family (DRG1 and DRG2), respectively, and possibly suppressed the degradation by ubiquitin by binding, whereby DRG1 and DRG2 It was clarified that the expression of this protein is positively controlled. It was clarified that the expression of DRG1 and DRG2 did not exist!
  • DR G1 knockdown cells using the human cancer cell line HeLaS3 have increased cell motility 'invasion' and metastasis capacity, while DRG2 knockdown cells have decreased, and cell adhesion is abnormally enhanced.
  • the present invention specifically binds to DRG protein which is an important regulator of cell proliferation.
  • DRG protein which is an important regulator of cell proliferation.
  • novel proteins DFRP1 and DFRP2 that regulate their expression [1] the polypeptide according to (1) or (2) below,
  • amino acid sequence of SEQ ID NO: 2 or 4 consisting of an amino acid sequence in which one or more amino acids are substituted, deleted, inserted or added in the amino acid sequence from position 234 to position 295, Polypeptide having action ability
  • a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2 (human DFRP1 amino acid sequence) or SEQ ID NO: 4 (mouse DFRP1 amino acid sequence)
  • SEQ ID NO: 9 base sequence of human drgl
  • SEQ ID NO: 10 base sequence of mouse drgl
  • a DRG1 protein stabilizer comprising the polypeptide according to [1] or [2] or the nucleic acid according to [3] as an active ingredient
  • a cell motility inhibitor comprising the polypeptide according to [1] or [2] or the nucleic acid according to [3] as an active ingredient,
  • a cancer cell growth inhibitor comprising the nucleic acid according to [6] or [7] as an active ingredient
  • a screening method for a cell motility inhibitor comprising the following steps (1) to (3).
  • test substance When the binding activity in the presence of the test substance is greater than the binding activity in the absence of the test substance, the test substance is selected as a candidate for cell motility inhibitor [12] And a screening method for a cell motility inhibitor having the step of (2),
  • a screening method for a cell motility inhibitor having the following steps (1) and (2):
  • a screening method for a cancer cell growth inhibitor comprising the following steps (1) to (3):
  • test substance is selected as a candidate for a cancer cell growth inhibitor.
  • a DRG2 protein comprising an amino acid sequence having one or more amino acids substituted, deleted, inserted or added in the amino acid sequence from position 132 to position 187 in the amino acid sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 8. Having the ability to interact with
  • a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 8
  • a gene comprising the base sequence described in SEQ ID NO: 5 (base sequence of human dfrp2) or SEQ ID NO: 7 (base sequence of mouse dfrp2) (2) A gene that hybridizes under stringent conditions with the nucleotide sequence of SEQ ID NO: 5 (base sequence of human dfrp2) or SEQ ID NO: 7 (base sequence of mouse dfrp2)
  • nucleic acid having an activity of annealing to the transcription product of the gene described in (1) or (2) below, having a length of 15 bases or more, preferably 19 bases or more,
  • a gene comprising the base sequence described in SEQ ID NO: 11 (base sequence of human drg2) or SEQ ID NO: 12 (base sequence of mouse drg2)
  • a DRG2 protein stabilizer comprising the polypeptide according to [17] or [18] or the nucleic acid according to [19] as an effective component
  • An intercellular adhesion promoter comprising the nucleic acid according to [22] or [23] as an active ingredient
  • a cell movement inhibitor comprising the nucleic acid according to [22] or [23] as an active ingredient
  • a cancer cell growth inhibitor comprising the nucleic acid according to [22] or [23] as an active ingredient, [28] an intercellular adhesion promoter or cell movement having the following step (1) force (3) Screening method for inhibitors,
  • test substance When the binding activity in the presence of the test substance is lower than the binding activity in the absence of the test substance !, the test substance is selected as a candidate for an intercellular adhesion promoter or cell motility inhibitor.
  • a screening method for an intercellular adhesion promoter or cell movement inhibitor comprising the following steps (1) and (2):
  • a screening method for an intercellular adhesion promoter or cell motility inhibitor comprising the following steps (1) and (2): (1) A step of allowing a test substance to act on a cell having a promoter and a drg2 gene
  • a screening method for a cancer cell growth inhibitor comprising the following steps (1) to (3):
  • a screening method for a cancer cell growth inhibitor comprising the following steps (1) and (2):
  • a method for treating cancer comprising a step of administering the cell motility inhibitor according to [9] or [26] to a patient,
  • a method for treating cancer comprising a step of administering the cancer cell proliferation inhibitor according to [10] or [27] to a patient,
  • a method for treating cancer comprising a step of administering the intercellular adhesion promoter according to [25] to a patient. Law.
  • FIG. 1 is a diagram and a photograph relating to the identification of DFRP.
  • A Domain structure and partial sequence alignment of mouse DFRP2 (GIR2) and DFRP1 (LEREP04).
  • DFRP1 and DFRP2 had a region of high homology that we named “DFRP domain” (hatched box).
  • the peptide sequences of mouse (M. musculus) (Mm), D. melanogaster (Dm) and S. cerevisiae (Sc) DFRP2 and DFRP1 were aligned.
  • NCBI accession numbers are XP-125585 (DFRP2—Mm), NP.651227 (DFRP2—Dm), NP_010436 (DFRP2—Sc), NP—0812 10 (DFRPLMn), NP—610401 (DFRP1—Dm) and NP—014734 (DFRP1—Sc).
  • B shows the interaction between DFRP2 (GIR2) or DFRP1 (LEREP04) and DRG protein. Extracts of 293T cells co-transfected with FLAG-labeled mouse DRG1 or DRG2, or empty control and Myc-labeled mouse DFRP2 or DFRP1 expression vector were immunoprecipitated using anti-FLAG antibody.
  • IP Immunoprecipitation
  • C It is a figure shown about the joint assembly in an in vivo. The protein extract of HeLa S3 cells was immunoprecipitated using antibodies against DFRP1, DRG1 and control IgG. The immunoprecipitation complex was then probed with anti-DRG1 antibody. The membrane was repeatedly stripped and re-searched repeatedly with anti-DFRP1 and anti-DRG2 antibodies.
  • D Photograph showing immunofluorescence analysis of subcellular localization of DFRP1 and DRG1. HeLa S3 cells were stained with polyclonal antibodies against DFRP1 and DRG1.
  • FIG. 2 is a diagram and a photograph showing the necessity of a DFRP domain for binding of DFRP and DRG protein.
  • A Structural map of DFRP2 (upper section) and DFRP1 site deletion mutant (lower section). The hatched box indicates the DFRP domain. The gray filled bar corresponds to the highly conserved region of DFRP1 in eukaryotes shown in Figure 1 (A).
  • B This figure shows the determination of the essential region of DFRP2 to interact with DRG2.
  • GST fusion DFRP2 11 or its site deletion mutant and FLAG label An extract of 293T cells co-transfected with the DRG2 expression vector was incubated with dartathione sepharose beads.
  • FIG. 3 is a photograph showing the regulation of DRG protein expression by DFRP.
  • A It is a photograph showing an increase in the expression of DRG protein by co-expression with DFRP. Extracts of 293T cells co-transfected with Myc-labeled DRG1 or DRG2 and expression vector or FLAG-tagged DFRP1 or DFRP2 expression vector were analyzed by Western blotting using anti-Myc and anti-FLAG antibodies.
  • B A photograph showing inhibition of ubiquitination of DRG protein by DFRP binding.
  • the expression vector encoding HA-ubiquitin (Ub) (3 / ⁇ ) and 1 ⁇ LAG-DRG1 (left panel) or DRG2 (right panel) (3 ⁇ g) is Myc-DFRPl or DFRP2 ( 293T cells transfected with 1 or 5 ⁇ g) were treated for 3 hours in the presence (+) or absence ( ⁇ ) of 10 ⁇ M MG132 and then harvested.
  • the IP panel and cell lysate were immunoprecipitated using an anti-FLAG antibody.
  • the immunoprecipitated complex was searched with an anti-HA antibody to detect a polyubiquitin chain.
  • the same membrane was used after repeated stripping and re-searched repeatedly with anti-FLAG and anti-Myc antibodies to detect DRG and DFRP, respectively.
  • the black and white arrows indicate full-length Myc-DFRPl and Myc-DFRP2, respectively.
  • the amount of protein applied was adjusted so that the amount of FLAG-DRG1 and FLAG-DRG2 was the same in all lanes.
  • the loading of each sample was determined by preliminarily assessing the concentration on a separate plot using the same sample.
  • the lysate panel and the cell lysate used for immunoprecipitation were analyzed by Western blot using anti-Myc antibody to detect DFRP. Same membrane After stripping the len, it was re-searched with anti-FLAG antibody to detect DRG1 and DRG2. For this membrane, we loaded an equal number of cell extracts onto SDS-PAGE.
  • FIG. 4 is a photograph showing the disruption of the dfrpl gene in DT40 cells.
  • A Structure of a partial Gallus gallus dfrpl locus and knockout construct. The hatched box indicates the exon of the duckpl dfrpl gene. The left exon contains the ZnF-1 domain.
  • Bsr blasticidin
  • HisD histidinol
  • FIG. 5 is a photograph showing the regulation of DRG1 expression by DFRP1 in vivo.
  • A Western blot of DT40 wild type, dfrpl —, —, dfrpl "'mil and dfrpl —, — mA Dl cells (mouse DFRP1 fl or ⁇ Dl (cells rescued in FIG. 2 (A)) (top Panel) and Northern blot (bottom panel) analysis, whole cell extracts separated by SDS-PAGE, then DRG1, DRG2, DFRP1 and tubulin (for internal load control) were detected with antibodies against. membrane iterates Sutoritsubingu was used Shi repeatedly.
  • FIG. 6A is a photograph showing the expression analysis of dfrpl and drgl in Xenopus laevis (X. laevis).
  • A Spatial expression of dfrpl and drgl transcripts during Xenopus embryonic development.
  • A, b ventral view, front left;
  • I, j Benzyl alcohol: Embryo clarification with benzyl benzoate (2: 1).
  • ba arch; bcs, branch crest segment; bi, blood island; de, developing eye; e, eye; ft), forebrain; hb, hindbrain; hcs, hyoid crest Segment (hyoid crest segment); le, crystal; mb, middle moon; mcs, mandibular crest segment; nc, notochord; ov, otocyst; pr, pronephros; sc, spinal cord; sm, segment ; tnc, trunk nerve trunk.
  • FIG. 6BC is a photograph showing the expression pattern of dfrpl and drgl in Xenopus laevis (X. laevis).
  • B Photograph showing the tissue-specific expression of dfrpl mRNA in an adult xenopath. Total RNA was isolated from the adult tissue shown and used in the Northern blot method.
  • C Temporal expression of dfrpl transcripts during X. laevis embryo development. Xenopus embryos were isolated at the indicated developmental stage and used in the Northern blot method.
  • Fig. 7 shows the evaluation of DRG1, DRG2, DFRP1, and DFRP2 knockdown cells by Doxycyclin supplementation and the changes in mRNA expression of genes that control cell motility, morphology and cytoskeleton It is a photograph.
  • Fig. 8 is a photograph showing a change in morphology of DRG1 knockdown.
  • the lower photo is an enlarged view of the upper photo.
  • FIG. 9 is a photograph showing changes in the actin skeleton of DRG1 knockdown cells.
  • FIG. 10 is a graph showing cell motility evaluation of DRG1 knockdown cells.
  • FIG. 11 is a photograph showing the morphological change of DFRP1 knockdown cells. HeLaS3 with DFRP1 RNAi vector introduced (right) and control with an empty vector introduced (left).
  • Fig. 12 is a photograph showing a change in morphology of DRG2 knockdown.
  • FIG. 13 is a photograph showing a transient morphological change of DRG2 knockdown cells. HeLas3 ⁇ DRG2 RNAi vector introduced (right) and empty vector as a control (left) are shown.
  • FIG. 14 is a graph showing changes in growth of DRG1 and DRG2 knockdown cells.
  • FIG. 15 is a model diagram of control of the DRG family and DFRPs.
  • FIG. 16 is a diagram and a photograph showing a crude fraction obtained by centrifuging mouse liver homogenates of DRG1 and DRG2. The left is a diagram showing the fractionation procedure, and the right is a photograph showing the results of Western plot analysis.
  • FIG. 17 is a graph and a photograph showing polysome fractionation by sucrose gradient.
  • the present inventors have developed a novel protein capable of regulating the expression of these proteins by specifically binding to DRG1 protein and DRG2 protein, which are considered to act as important regulators of cell proliferation.
  • DFRP1 and DFRP2 were identified.
  • the base sequence of the human dfrpl gene is shown in SEQ ID NO: 1
  • the amino acid sequence of the human DFRP1 protein is shown in SEQ ID NO: 2
  • the base sequence of the mouse dfrpl gene is shown in SEQ ID NO: 3
  • the mouse D The amino acid sequence of the FRP1 protein is SEQ ID NO: 4
  • the nucleotide sequence of the human dfrp2 gene is SEQ ID NO: 5
  • the amino acid sequence of the human DFRP2 protein is SEQ ID NO: 6
  • the base sequence of the mouse dfrp2 gene is SEQ ID NO: 7 shows the amino acid sequence of the mouse DFRP2 protein in SEQ ID NO: 8
  • the base sequence of human drgl in SEQ ID NO: 9
  • the base sequence of the human drg2 gene is shown in SEQ ID NO: 12.
  • domain (DFR P domain) force showing high sequence identity between DFRP1 and DFRP2 is essential for binding to the DRG family.
  • the DFRP domain In the amino acid sequence of the DFRP1 protein (SEQ ID NO: 2 or SEQ ID NO: 4), the DFRP domain has a 234 position and a 295 position.
  • the DFRP domain In the amino acid sequence of the DFRP2 protein (SEQ ID NO: 6 or SEQ ID NO: 8), the DFRP domain is located at positions 132 to 187.
  • the present invention first provides the polypeptide (DFRP1 polypeptide) described in (1) or (2) below.
  • the present invention also provides the polypeptide (DFRP1 polypeptide) described in (1) or (2) below.
  • a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4.
  • amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 one or more amino acids are deleted, inserted, or added, and the amino acid sequence is also a polymorphism capable of interacting with the DRG1 protein.
  • the present invention also provides the polypeptide (DFRP2 polypeptide) described in (1) or (2) below.
  • a DRG2 protein comprising an amino acid sequence having one or more amino acids substituted, deleted, inserted or added in the amino acid sequence from position 132 to position 187 in the amino acid sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 8.
  • the present invention also provides the polypeptide (DFRP2 polypeptide) described in (1) or (2) below.
  • a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 8
  • amino acid sequence shown in SEQ ID NO: 6 or SEQ ID NO: 8 consists of an amino acid sequence in which one or more amino acids are substituted, deleted, inserted or added, and is capable of interacting with DRG2 protein.
  • protein means a polymer having a plurality of amino acid strengths.
  • oligopeptides and polypeptides are also included in the concept of proteins. Proteins are meant to include both those that are not modified from the naturally occurring state and those that are modified. Modifications include acetylation, acylation, ADP-ribosylation, amidation, flavin covalent bond, heme moiety covalent bond, nucleotide or nucleotide derivative covalent bond, lipid or lipid derivative covalent bond, phosphatidylinositol.
  • Examples include white matter degradation, phosphorylation, prenylation, racemization, selenoylation, sulfation, arginyl transfer of amino acids to proteins such as RNA-mediated addition, ubiquitin.
  • Natural proteins include, for example, DRG protein (including DRG1 protein and DRG2 protein) and DFRP protein (including DFRP1 protein and DFRP2 protein) expressed in cells (tissues) that are considered to be expressed.
  • the extract can be prepared by a method using affinity chromatography using an antibody against DRG protein or DFRP protein.
  • the recombinant protein can be prepared by culturing cells transformed with DNA encoding DRG protein or DFRP protein.
  • “expression” includes “transcription” from a gene or “translation” into a polypeptide and “degradation inhibition” of a protein. “Expression of DRG protein or DFRP protein” means that transcription and translation of a gene encoding DRG protein or DFRP protein occurs, or that transcription or translation of these genes produces DRG protein or DFRP protein. .
  • a person skilled in the art will appropriately determine whether or not any polypeptide has the “ability to interact with DRG1 protein” or “ability to interact with DRG2 protein” in (2) above. Can do. For example, it can be determined using the binding activity between the test polypeptide and the DRG1 protein or DRG2 protein as an index. For example, when any polypeptide has binding activity with DRG1 protein or DRG2 protein, the test polypeptide is determined to have “DRG1 protein interaction ability” or “DRG2 protein interaction ability”.
  • the present invention also provides the DFRP1 protein or DFRP2 protein in the human or mouse, and a variant (mutant) of these proteins.
  • polypeptide having the amino acid sequence set forth in SEQ ID NO: 2 or 4 or the polypeptide having the amino acid sequence set forth in SEQ ID NO: 6 or 8, and one or more amino acids in the amino acid sequence
  • “functionally equivalent” means that the target polypeptide is the same as the polypeptide of the present invention. It has the same biological or biochemical activity or ability. Examples of such ability include ability to interact with DRG1 protein or ability to interact with DRG2 protein.
  • the gene of the present invention is encoded using gene amplification technology (PCR) (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons Section 6.1-6.4).
  • a primer is designed based on a part of the DNA sequence (SEQ ID NO: 1, 3, 5, or 7), and a DNA fragment highly homologous to the DNA sequence encoding the polypeptide of the present invention is isolated, and the DNA Based on the above, it is possible to obtain a polypeptide functionally equivalent to the polypeptide of the present inventor.
  • Amino acid mutations may also occur in nature.
  • a polypeptide having an amino acid sequence in which one or more amino acids are mutated in the amino acid sequence of the polypeptide of the present invention and functionally equivalent to the polypeptide is also included in the polypeptide of the present invention.
  • the number of amino acids to be mutated is usually within 50 amino acids, preferably within 30 amino acids, and more preferably within 10 amino acids (for example, within 5 amino acids).
  • amino acid side chain properties include hydrophobic amino acids (A, I, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G , H, K, S, T), amino acids with lunar aliphatic side chains (G, A, V, L, I, P), amino acids with hydroxyl-containing side chains (S, T, Y), amino acids with sulfur atom-containing side chains (C, M), amino acids with carboxylic acid and amide-containing side chains (D, N, E, Q), amino acids with base-containing side chains (R, K, ⁇ ), and amino acids having aromatic side chains (H, F, Y, W) can be mentioned (the parentheses indicate single letter amino acids).
  • hydrophobic amino acids A, I, M, F, P, W, Y, V
  • hydrophilic amino acids R, D, N, C, E, Q, G , H, K, S, T
  • amino acids with lunar aliphatic side chains G, A, V,
  • the present invention also provides a nucleic acid encoding the DFRP1 polypeptide of the present invention or a nucleic acid encoding the DFRP2 polypeptide of the present invention.
  • the present invention also provides a vector carrying a nucleic acid encoding the DFRP1 polypeptide of the present invention or a vector carrying a nucleic acid encoding the DFRP2 polypeptide of the present invention.
  • the vector of the present invention is not particularly limited as long as it can stably hold (carry) the inserted DNA.
  • a pBluescript vector (Stratagene) is used as a cloning vector.
  • An expression vector is particularly useful when a vector is used for the purpose of producing the polypeptide of the present invention.
  • the expression vector is not particularly limited as long as it is a vector that expresses a polypeptide in vitro, in E.
  • coli in cultured cells, or in an organism.
  • pBEST vector manufactured by Promega
  • E. coli for in vitro expression.
  • PET vector manufactured by In vitrogen
  • pME18S-FL3 vector GenBank Accession No. A B009864
  • pME18S vector Mol Cell Biol. 8: 466-472 (1988) for living organisms
  • the insertion of the DNA of the present invention into a vector can be performed by a conventional method, for example, by a ligase reaction using a restriction enzyme site (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley). & Sons. Section 11.4—11.11).
  • the present invention also provides an antibody against (recognizing or binding to) the DFRP1 polypeptide of the present invention or an antibody against the DFRP2 polypeptide.
  • the "antibody” in the present invention is preferably an antibody for which a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a human antibody or a mixture thereof is also selected.
  • humanized antibodies, low molecular weight antibodies (including antibody fragments), multispecific antibodies, and antibody modified products are also available. It may be included.
  • the DFRP1 polypeptide or DFRP2 polypeptide of the present invention is used for immunization to produce an antibody that binds to the DFRP1 polypeptide or DFR P2 polypeptide of the present invention. It can also be used as a source.
  • Antibodies that bind to the DFRP1 polypeptide or DFRP2 polypeptide of the present invention can be prepared by methods known to those skilled in the art.
  • a polyclonal antibody can be obtained as follows. Serum is obtained by immunizing small animals such as rabbits with the DFRP1 polypeptide or DFRP2 polypeptide of the present invention or a fusion protein thereof with GST. This is prepared, for example, by purifying with ammonium sulfate precipitation, protein A, protein G column, DEAE ion exchange chromatography, a DFRP1 polypeptide or DFRP2 polypeptide-coupled affinity column of the present invention, and the like.
  • a monoclonal antibody for example, a small animal such as a mouse is immunized with the DFRP1 polypeptide or DFRP2 polypeptide of the present invention, and the spleen is removed from the mouse, and this is ground to isolate cells.
  • a clone that produces an antibody that binds to the DFRP2 polypeptide is selected from the fused cells (neubridoma) made by fusing myeloma cells with a reagent such as polyethylene glycol. To do.
  • the DFRP1 polypeptide or DFRP2 polypeptide of the invention can be prepared by purification using a coupled column or the like.
  • the antibody against the DFRP1 polypeptide or DFRP2 polypeptide of the present invention may be applied to antibody therapy for diseases involving the DFRP protein of the present invention.
  • antibody therapy In order to reduce epidemiogenicity, it is preferable to use human antibodies or human antibodies.
  • a chimeric antibody is an antibody produced by combining sequences derived from different animals.
  • the antibody comprises a mouse antibody heavy chain and light chain variable region and a human antibody heavy chain and light chain constant region.
  • Such as an antibody is an antibody.
  • a chimeric antibody can be prepared by a known method. For example, DNA encoding an antibody V region and DNA encoding a human antibody C region are ligated, incorporated into an expression vector, and introduced into a host. It is obtained by making it produce.
  • the low molecular weight antibody is not particularly limited as long as it includes an antibody fragment in which a part of a full-length antibody (such as whole IgG) is deleted and has an ability to bind to an antigen.
  • the antibody fragment is not particularly limited as long as it is a part of a full-length antibody.
  • Specific examples of antibody fragments include, for example, Fab, Fab ′, F (ab ′) 2, and Fv.
  • Specific examples of the low molecular weight antibody include, for example, Fab, Fab ′, F (ab ′) 2, Fv, scFv (single chain Fv), diabodies, sc (Fv) 2 (single chain (Fv) 2 ) And the like.
  • Humanized antibodies are modified antibodies, also referred to as reshaped human antibodies. Humanized antibodies are constructed by transplanting CDRs of antibodies derived from immunized animals into complementarity determining regions of human antibodies. The general genetic recombination technique is also known.
  • variable region e.g, FR
  • constant region amino acids in the variable region or constant region may be substituted with other amino acids.
  • modified antibodies include antibodies bound to various molecules such as polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • the substance to be bound is not limited.
  • it can be obtained by chemically modifying the obtained antibody.
  • the antibody against the DFRP1 polypeptide or DFRP2 polypeptide of the present invention is the DFRP1 polypeptide of the present invention! /, which is used for isolation, identification and purification of DFRP2 polypeptide and cells expressing the same. Can do.
  • shRNA short hairpin RNA
  • DRG1, DFRP1, DRG2, and DFRP2 mRNA DRG1, DFRP1, DRG2, DFR
  • RNAi is a double-stranded RNA (hereinafter abbreviated as "dsRNA") consisting of a sense RNA consisting of a sequence homologous to the mRNA of the target gene and an antisense RNA consisting of a complementary sequence. It is a phenomenon that can induce the destruction of the target gene mRNA and suppress the expression of the target gene by introducing it into the cell. RNAi is thus attracting attention as a simple gene knockout method to replace the conventional complicated, low-efficiency gene disruption method by homologous recombination because it can suppress the expression of the target gene. The above RNAi was first discovered in nematodes. 7 (S) (Fire, A. et al.
  • dsRNA In vitro, dsRNA is known to target mRNA for cleavage in lysates of early Drosophila embryos or extracts of cultured Drosophila S2 cells (Tuschl T., et al. Genes Dev. 13: 3191-3197, 1999; Hammond SM, et al., Nature 404: 293-296, 2000; Zamore P., et al., Cell 101: 25-33, 2000). In vitro RNAi reactions require ATP (Zamore P., et al., Cell 101: 25-33, 2000).
  • RNAi provides a way to inactivate target genes and thus provides a powerful tool for studying gene function in C. eleg ans, Drosophila and plants . Specific inhibition of gene expression can also be obtained by stable and inducible expression of dsRNA in animals and plants (Kennerdell and Carthew Nature Biotechnol. 18: 896-898, 2000; Tavernarakis N. et al., Nature Genetics 24: 18 0-183, 2000; Hammond SM et al "Nature Reviews Genetics 2: 110-119, 2001). In mouse embryo cancer EC cells and embryonic stem (ES) cells, gene inactivation by dsRNA was successful. However (Billy E.
  • Rigoadelate (2-5A) synthase which is activated by dsRN A and synthesizes 2-5A, which is required for the activity of a sequence-specific RNase called RNase L (Silverman RH in Ribonucleases: Structures and Functions, eas. D'Alessio, G. and Riordan JF (Academic, New York) pp. 515-551)
  • RNase L a sequence-specific RNase called RNase L (Silverman RH in Ribonucleases: Structures and Functions, eas. D'Alessio, G. and Riordan JF (Academic, New York) pp. 515-551)
  • the other is protein kinase PKR, its activity
  • the type phosphorylates the translation factor eukaryotic cell initiation factor (eIF2), leading to general inhibition of protein synthesis and cell death (Cle mens MJ and Elia A., J. Interferon Cytokine
  • RNAi-related proteins such as rde-l, mut-7 and Dicer have been identified (Tabara H. et al., Cell 99: 123-132, 1999; Ketting RF et al., Cell 99: 133-141, 1999; Bernstein E. et al., Nature 409: 363-366, 2 001).
  • tandem type has two U6 promoters, each of which independently transcribes sense RNA and antisense RNA.
  • the transcribed sense and antisense RNA forms double-stranded RNA in the cell and acts as siRNA.
  • the stem-loop type has a single promoter, and has a structure that is a V connecting the sense strand and the antisense strand in a loop downstream of it.
  • RNA with this stem-loop RNA structure is processed by a cleavage enzyme dicer or the like to produce siRNA.
  • the above “stem loop” is composed of a double-stranded part (stem) caused by hydrogen bonding between inverted repeats existing on single-stranded RNA and a partial force of the loop sandwiched between them. Refers to the structure, also called the hairpin loop.
  • RNA molecules capable of inducing RNAi against DRG1, DFRP1, DRG2, and DF RP2 mRNA that form this hairpin loop are also included in the present invention.
  • the present invention provides a nucleic acid having an activity of annealing to the transcription product of the gene (dfrpl gene) described in (1) or (2) below.
  • the present invention also provides a nucleic acid having an activity of annealing to the transcription product of the gene (drgl gene) described in (1) or (2) below.
  • the present invention also provides a nucleic acid having an activity of annealing to the transcription product of the gene (dfrp2 gene) described in (1) or (2) below.
  • the present invention also provides a nucleic acid having an activity of annealing to the transcription product of the gene (drg2 gene) described in (1) or (2) below.
  • the nucleic acid having an annealing activity for the dfrpl gene or drgl gene, or the transcription product of the dfrp2 gene or drg2 gene in the present invention is preferably a nucleic acid having a length of 15 bases or more, more preferably 19 bases or more ( A nucleic acid having a length of 25 bases or less is preferred.
  • Stringent hybridization conditions can be appropriately selected by those skilled in the art. For example, in a hybridization solution containing 25% formamide, 50% formamide under more severe conditions, 4 X SSC, 50 mM Hepes pH 7.0, 10 X Denhardt's solution, 20 g / ml denatured salmon sperm DNA, After prehybridization at 42 ° C, add a labeled probe and incubate overnight at 42 ° C for hybridization.
  • the combinations of the above SSC, SDS and temperature conditions are exemplary, and those skilled in the art will recognize the above and other factors that determine the stringency of the hybridization, such as probe concentration, By combining the probe length, hybridization reaction time, etc.) as appropriate. Similar stringency can be achieved.
  • probe concentration By combining the probe length, hybridization reaction time, etc.
  • dfrpl gene or drgl gene or a dfrp2 gene or drg2 gene in another organism, based on the base sequence of the dfrpl gene, drgl gene, dfrp 2 gene or drg2 gene. Can be obtained as appropriate.
  • the present inventors have also found that DFRP1 protein strength specifically stabilizes RG1 protein, and DFRP2 protein specifically stabilizes DRG2 protein. That is, the present invention relates to a DRG1 protein stabilizer comprising a DFRP1 polypeptide or a nucleic acid encoding a DFRP1 polypeptide as an active ingredient, or a DRG2 protein stabilizer comprising a nucleic acid encoding a DFRP2 polypeptide or a DFRP2 polypeptide as an active ingredient. I will provide a. The stabilizer is thought to impart stability to the DRG protein and contribute to normal cell growth.
  • containing nucleic acid as an active ingredient means t containing nucleic acid as a main active ingredient, and does not limit the content of nucleic acid.
  • DRG1 has been knocked down by adding doxycycline (Dox) have an increased ability to move and infiltrate compared to cells without doxycycline.
  • Dox doxycycline
  • DRG1 is thought to negatively regulate the expression of mRNA, a factor that promotes cell motility. That is, if the DRG1 force S is stabilized, it is considered that the cell has a higher movement speed, and DFRP1, which can stabilize the function of the DRG1, can be used to suppress the cell movement speed.
  • the present invention provides a cell motility inhibitor (compound having an action of inhibiting cell motility) comprising the DFRP1 polypeptide or a nucleic acid encoding the DFRP1 polypeptide as an active ingredient.
  • the cell motility inhibitor is a disease caused by, for example, abnormal cell motility (increased tl), such as cancer, solid tumor, tumor metastasis, benign tumor (eg hemangioma, acoustic schwannoma, neurofibroma, trachoma and purulent).
  • Granulomas vascular dysfunction, inflammation and immune disorders, Behcet's disease, gout, arthritis, rheumatoid arthritis, psoriasis, diabetic retinopathy and other ocular vascular diseases (e.g., posterior lens fibroproliferation, macular degeneration, Corneal transplant rejection, neovascular glaucoma), osteoporosis, various skin diseases accompanied by changes in the activity of fibrinolytic proteases, especially skin irritation caused by stimulation of drying / cleaning agents, etc.
  • Behcet's disease e.g., arthritis, rheumatoid arthritis, psoriasis, diabetic retinopathy and other ocular vascular diseases (e.g., posterior lens fibroproliferation, macular degeneration, Corneal transplant rejection, neovascular glaucoma), osteoporosis, various skin diseases accompanied by changes in the activity of fibrinolytic proteases, especially skin irritation caused by stimulation of drying / cleaning
  • Skin condition skin condition, cancer cell invasiveness and metastasis, inflammatory bowel disease, precancerous colon adenoma Septic arthritis, osteoarthritis, rheumatoid arthritis (provided direct involvement of excess u-PA production), osteoporosis, cholesterin tumor and excess plasminogen activity
  • Some skin and corneal diseases such as corneal ulcers, keratitis, epidermolysis bullosa, psoriasis and pemphigus, trauma, acute circulatory failure, inflammation, thrombosis, splenitis and cancer metastasis and invasion It can be used for the treatment of diseases such as diseases caused by increased activity of proteases.
  • the present invention includes cancer, solid tumor, tumor metastasis, benign tumor (eg, hemangioma, acoustic schwannoma, neurofibroma, trachoma, and purulent), comprising the step of administering the cell motility inhibitor to a patient.
  • benign tumor eg, hemangioma, acoustic schwannoma, neurofibroma, trachoma, and purulent
  • Granulomas vascular dysfunction, inflammation and immune disorders, Behcet's disease, gout, arthritis, rheumatoid arthritis, psoriasis, diabetic retinopathy and other ocular vascular diseases (e.g., posterior lens fibroproliferation, macular) Degeneration, corneal transplant rejection, neovascular glaucoma), osteoporosis, various skin diseases with changes in the activity of fibrinolytic proteases, particularly skin with proliferative abnormalities of the epidermis such as rough skin caused by stimulation of dry 'cleansing agents' Condition, aggressiveness and metastasis of cancer cells, inflammatory bowel disease, pre-cancerous colon adenoma, septic arthritis, osteoarthritis, rheumatoid arthritis (provided direct involvement of excess u-PA production), osteo Some skin and corneal diseases that have been shown to be pathogenic due to loci, cholesterol, and excessive plasminogen activity, such as corneal ulcers, keratitis,
  • the present invention also provides a cancer, solid tumor, tumor metastasis, benign tumor (eg, hemangioma, acoustic schwannoma, neurofibroma, trachoma, and purulent granulation) of DFRP1 polypeptide or nucleic acid encoding DFRP1 polypeptide.
  • benign tumor eg, hemangioma, acoustic schwannoma, neurofibroma, trachoma, and purulent granulation
  • Vascular dysfunction e.g., Vascular dysfunction, inflammation and immune disorders, Behcet's disease, gout, arthritis, rheumatoid arthritis, psoriasis, diabetic retinopathy and other ocular vascular diseases (e.g., posterior lens fibroproliferation, macular degeneration, Corneal transplant rejection, neovascular glaucoma), osteoporosis, various skin diseases with altered activity of fibrinolytic proteases, especially skin with proliferative abnormalities of the epidermis, such as rough skin caused by stimulation of dry 'cleansing agents' etc.
  • Behcet's disease e.g., arthritis, rheumatoid arthritis, psoriasis, diabetic retinopathy and other ocular vascular diseases (e.g., posterior lens fibroproliferation, macular degeneration, Corneal transplant rejection, neovascular glaucoma), osteoporosis, various skin diseases with altered activity of fibrino
  • the present invention relates to use in the manufacture of a therapeutic agent for diseases such as diseases caused by enhancement.
  • treatment generally means obtaining a pharmacological and Z or physiological effect.
  • An effect may be prophylactic in that it completely or partially interferes with the disease or symptom, or may be therapeutic in that it completely or partially treats the symptom of the disease.
  • treatment includes all treatment of diseases in mammals, particularly humans.
  • the term includes a predisposition to a disease but is still diagnosed and prevents the onset of the patient, suppresses the progression of the disease, or reduces the disease.
  • the present inventors have found that the cells in which DRG2 is knocked down have very high cell-cell adhesion and reduced cell motility. In other words, it stabilizes the function of DRG2, suppresses the function or activity of DFRP2, suppresses the function or activity of dfrp2 gene, or has the activity of annealing to the drg2 gene transcript.
  • the nucleic acid is considered to have an action of promoting cell-cell adhesion or an action of reducing cell motility.
  • the present invention provides an intercellular adhesion promoter (an action that promotes intercellular adhesion), which comprises, as an active ingredient, a nucleic acid that has an activity of annealing to the transcription product of the dfrp2 gene or drg2 gene of the present invention.
  • a cell motility inhibitor (a compound having an action of inhibiting cell motility).
  • Such cell motility inhibitors are cancers, solid tumors, tumor metastases, benign tumors (e.g.
  • skin diseases accompanied by changes in activity, especially dryness Skin conditions that cause proliferative abnormalities of the epidermis such as rough skin, invasive and metastatic cancer cells, inflammatory bowel disease, precancerous colon adenoma, septic arthritis, osteoarthritis, rheumatoid arthritis ( Direct involvement of excess u-PA production has been demonstrated), osteoporosis, cholesterol tumors, and some skin and corneal diseases that have been shown to be caused by excessive plasminogen activity such
  • the present invention includes cancer, solid tumor, tumor metastasis, benign tumor (for example, hemangioma, acoustic schwannoma, neurofibroma, trachoma and suppuration), which comprises the step of administering the cell motility inhibitor to a patient.
  • benign tumor for example, hemangioma, acoustic schwannoma, neurofibroma, trachoma and suppuration
  • Granulomas vascular dysfunction, inflammation and immune disorders, Behcet's disease, gout, arthritis, rheumatoid arthritis, psoriasis, diabetic retinopathy and other ocular vascular diseases (e.g., posterior lens fibroproliferation, macular) (Degeneration, corneal transplant rejection, neovascular glaucoma), osteoporosis, various skin diseases accompanied by changes in the activity of fibrinolytic proteases, especially rough skin caused by stimulation of dry 'cleansing agents', such as acne, proliferative abnormalities of the epidermis Perceived skin condition, cancer cell invasiveness and metastasis, inflammatory bowel disease, precancerous colon adenoma, septic arthritis, osteoarthritis, rheumatoid arthritis (provided direct involvement of excess u-PA production) , Osteopolo Some skin and corneal diseases whose etiology has been shown to be caused by cis, cholesterol tumors and
  • the present invention also relates to cancers, solid tumors, tumor metastases, benign tumors (eg, hemangiomas, acoustic schwannomas, neurofibromas) of nucleic acids having the activity of annealing to dfrp2 gene or drg2 gene transcripts.
  • benign tumors eg, hemangiomas, acoustic schwannomas, neurofibromas
  • vascular dysfunction e.g., vascular dysfunction, inflammation and immune disorders, Behcet's disease, gout, arthritis, rheumatoid arthritis, psoriasis, diabetic retinopathies and other ocular vascular diseases (e.g., posterior lens fiber growth) Disease, macular degeneration, corneal transplant rejection, angiogenic glaucoma), osteoporosis, various changes in fibrinolytic protease activity Skin diseases, especially dry 'rough skin caused by stimulation with detergents', acne, etc.
  • Behcet's disease gout, arthritis, rheumatoid arthritis, psoriasis, diabetic retinopathies and other ocular vascular diseases (e.g., posterior lens fiber growth) Disease, macular degeneration, corneal transplant rejection, angiogenic glaucoma), osteoporosis, various changes in fibrinolytic protease activity
  • Skin diseases especially dry 'rough skin caused by stimulation with detergents', acne,
  • the present invention relates to use in the manufacture of a therapeutic agent for diseases such as diseases caused by enhancement.
  • the intercellular adhesion promoter can be used for the treatment of diseases caused by abnormalities (decrease) in intercellular adhesion, such as cancer. That is, the present invention relates to a method for treating cancer or the like, comprising the step of administering the intercellular adhesion promoter to a patient. The present invention also relates to the use of a nucleic acid having an activity of annealing to a transcription product of the dfrp2 gene or drg2 gene in the manufacture of a therapeutic agent for cancer or the like.
  • the intercellular adhesion promoter of the present invention is considered to have an effect on skin looseness or wrinkles caused by decreased adhesion between cells.
  • the intercellular adhesion promoter may be applied as a cosmetic or cosmetic agent to improve these symptoms.
  • DRG1, DRG2, DFRP1, and DFRP2 knockout cell growth and morphology are almost normal in yeast cells, but in human cancer cell line HeLaS3, DRG1, DRG2, It was found that when DFRP1 and DFRP2 were knocked down, cell growth decreased and the morphology became abnormal. In other words, knockout or knockdown of DRG1, DRG2, DFRP1, and DFRP2 may be able to attenuate the growth of cancer cells specifically without affecting normal cells. There is a possibility that it can function as an agent (a compound having an action of suppressing the growth of cancer cells).
  • the present invention provides a nucleic acid having an activity to bind to a transcription product of the dfrpl gene or drgl gene, or a nucleic acid having an activity to anneal to a transcription product of the dfrp2 gene or drg2 gene.
  • a cancer cell growth inhibitor as an active ingredient.
  • the cancer cell growth inhibitor is used for cancer treatment, for example, as a cancer therapeutic agent. It is thought that it can be done.
  • the present invention also relates to a method for treating cancer comprising the step of administering the cancer cell growth inhibitor to a patient.
  • the present invention also relates to a cancer therapeutic agent comprising a nucleic acid having an activity to anneal to a transcription product of a dfrpl gene or a drgl gene, or a nucleic acid having an activity to anneal to a transcription product of a dfrp2 gene, V or drg2 gene.
  • the target of the cancer cell growth inhibitor of the present invention is not particularly limited as long as it is a cancer cell! ,.
  • patient is usually a human, but is not necessarily limited to only humans, and is a non-human organism (preferably a mammal, more preferably a mouse, rat, monkey, i.e. Vertebrates such as cats and cats).
  • a non-human organism preferably a mammal, more preferably a mouse, rat, monkey, i.e. Vertebrates such as cats and cats.
  • the present invention also provides a screening method for a cell motility inhibitor.
  • a preferred embodiment of the screening method of the present invention is a method using as an index the binding activity of the DRG1 protein and the DFRP1 polypeptide of the present invention, or the binding activity of the DRG2 protein and the DFRP2 polypeptide of the present invention.
  • DRG1 protein and DFRP1 polypeptide are allowed to act in the presence or absence of a test substance.
  • test substance used in the screening method of the present invention there is no particular limitation on the test substance used in the screening method of the present invention.
  • natural compounds, organic compounds, inorganic compounds, proteins, peptides and other single compounds as well as compound libraries, gene library expression products, cell extracts, cell culture supernatants, fermented microorganism products, marine organism extracts Products, plant extracts and the like, but are not limited thereto.
  • these test substances can be appropriately labeled as necessary.
  • the label include a radiolabel and a fluorescent label.
  • the DRG1 protein and the DFRP1 polypeptide, or the DRG2 protein and the DFRP2 polypeptide are, for example, the DRG1 protein and the DFRP1 polypeptide depending on the indicator for detecting the binding to the test substance! /,
  • the DRG2 protein and It can be a purified form of DFRP2 polypeptide, a form expressed intracellularly or extracellularly, or a form bound to an affinity column.
  • DRG1 protein and DFRP1 polypeptide, or DR Measure binding activity to G2 protein and DFRP2 polypeptide The binding between the DRG1 protein and the DFRP 1 polypeptide, or the binding between the DRG2 protein and the DFRP2 polypeptide is performed by, for example, the label attached to the test substance bound to the DRG1 protein and the DFRP1 polypeptide or the DRG2 protein and the DFRP2 polypeptide. Can be detected.
  • the test substance is selected.
  • a substance that increases in the case of binding activity with DRG1 protein and DFRP1 polypeptide and a substance that decreases in the case of binding activity with DRG2 protein and DFRP2 polypeptide is selected.
  • Substances (compounds) selected (isolated) by this method are useful as candidate substances for cell motility inhibitors.
  • test substance that reduces the binding activity to the DRG2 protein and DFRP2 polypeptide selected as described above can also be used as an intercellular adhesion promoter.
  • the method uses expression of dfrpl gene, dfrp2 gene, drgl gene, or drg2 gene as an index. That is, a compound that increases the expression of the dfrpl gene, a compound that decreases the expression of the dfrp2 gene, a compound that increases the expression of the drgl gene, or a compound that decreases the expression of the drg2 gene must have a cell motility inhibitory effect. There is expected.
  • test substance (compound) is allowed to act on a dfrpl gene, dfrp2 gene, drgl gene, or drg2 gene-containing cell having a promoter.
  • the "cell” used in the present method is not particularly limited, but is preferably a human-derived cell.
  • ⁇ Cells holding dfrpl gene '', ⁇ cells holding dfrp2 gene '', ⁇ cells holding drgl gene '', or ⁇ cells holding drg2 gene '' include endogenous dfrpl gene, dfrp2 gene, Cells containing the drgl gene or drg2 gene, or cells into which an exogenous dfrpl gene, dfrp2 gene, drgl gene, or drg2 gene has been introduced can be used.
  • Cells that carry exogenous dfrpl gene, dfrp2 gene, drgl gene, or drg2 gene are usually introduced into host cells with an expression vector inserted with dfrpl gene, dfrp2 gene, drgl gene, or drg2 gene It can produce by doing.
  • the expression vector can be produced by general gene engineering techniques.
  • test substance used in this method can be appropriately labeled as necessary.
  • label include radiolabels and fluorescent labels as described above.
  • “Making a test substance act” can be performed by adding a test substance to a culture medium of a cell retaining dfrpl gene, dfrp2 gene, drgl gene, or drg2 gene, for example. It is not limited to this method.
  • the test substance is a protein or the like, it can be “acted” by introducing a DNA vector expressing the protein into the cell.
  • the expression level can be measured easily by those skilled in the art, and can be appropriately performed by a general method such as the Northern plot method or the Western plot method.
  • the measurement can be performed using as a marker the label attached to the test substance that has acted on the cells that express the dfrpl gene, dfrp2 gene, drgl gene, or drg2 gene.
  • expression means either transcription from a gene encoding a protein (generation of mRNA) or translation from a transcription product of the gene.
  • Test substance with increased expression level test substance with decreased expression level of dfrp2 gene, test substance with increased expression level of drg 1 gene, or test substance with decreased expression level of drg2 gene Select.
  • the compound obtained by the screening method is expected to be usable as a cell motility inhibitor.
  • test substance in which the expression level of the dfrp2 gene is reduced or the test substance in which the expression level of the drg2 gene is reduced is expected to have an intercellular adhesion promoting action. Can also be used.
  • the present invention also provides a screening method for a cancer cell growth inhibitor.
  • a preferred embodiment of the screening method of the present invention is a method using as an index the binding activity of the DRG1 protein and the DFRP1 polypeptide of the present invention, or the binding activity of the DRG2 protein and the DFRP2 polypeptide of the present invention. That is, the compound that decreases the binding activity is expected to have a cancer cell growth inhibitory effect.
  • DRG 1 protein and DFRP1 polypeptide are allowed to act in the presence or absence of a test substance.
  • DRG2 protein and DFRP2 polypeptide are allowed to act in the presence or absence of the test substance.
  • DRG1 protein and DFRP1 polypeptide, or DRG2 protein and DFRP2 polypeptide are purified according to the indicator for detecting binding to the test substance. Morphology, expressed intracellularly or extracellularly, or bound to the affinity column. It can be.
  • the test substance used in this method can be appropriately labeled as necessary. Examples of the label include radiolabels and fluorescent labels as described above.
  • the binding activity between the DRG1 protein and the DFRP1 polypeptide or the binding activity between the DRG2 protein and the DFRP2 polypeptide is measured.
  • the binding between the DRG1 protein and the DF RP1 polypeptide, or the binding between the DRG2 protein and the DFRP2 polypeptide, for example, the label attached to the test substance bound to the DRG1 protein and the DFRP1 polypeptide, or the DRG2 protein and the DFRP2 polypeptide. Can be detected.
  • test substance when the binding activity in the presence of the test substance is lower than the binding activity in the absence of the test substance, the test substance is selected.
  • Substances (compounds) selected (isolated) by this method are useful as candidate substances for cancer cell growth inhibitors.
  • DRG1 protein and DFRP1 polypeptide when the binding activity of DRG1 protein and DFRP1 polypeptide is increased as compared with that of normal cells during cancer cell growth, as an inhibitor against the growth of cancer cells, DRG1 protein and DFRP1 polypeptide Select a test substance that reduces the binding activity to a normal level.
  • the binding activity of DRG2 protein and DFRP2 polypeptide when the binding activity of DRG2 protein and DFRP2 polypeptide is increased compared to that of normal cells during cancer cell growth, the binding activity of DRG2 protein and DFRP2 polypeptide can be used as an inhibitor against that cancer cell growth. Select a test substance that reduces to a normal level.
  • Another embodiment of the screening method of the present invention is a method using the expression of the dfrpl gene or the dfrp2 gene as an index. Substances that reduce the expression level of the dfrpl gene or the dfrp2 gene are expected to be candidate compounds for suppressing cancer cell growth.
  • test substance is allowed to act on cells having a dfrpl gene or a dfrp2 gene having a promoter.
  • a test substance that reduces the expression level of the dfrpl gene or the dfrp2 gene is selected.
  • This method can be appropriately performed following the above-described screening method for cell motility inhibitors. The substance thus selected is considered useful as a cancer cell growth inhibitor.
  • Another embodiment of the screening method of the present invention is a method using the expression of drgl gene or drg2 gene as an index. Substances that reduce the expression level of drgl gene or drg2 gene are expected to be candidate compounds for suppressing cancer cell growth.
  • a test substance is allowed to act on a cell having a drgl gene or drg2 gene having a promoter.
  • a test substance that reduces the expression level of the drgl gene or drg2 gene is selected.
  • This method can be appropriately carried out following the screening method for a cancer cell proliferation inhibitor described above. The substance thus selected is considered useful as a cancer cell growth inhibitor.
  • drgl gene or drg2 gene when the expression level of drgl gene or drg2 gene is increased compared to normal cells during cancer cell growth, the expression of drgl gene or drg2 gene is used as an inhibitor against the cancer cell growth.
  • a test substance that reduces the amount to a normal level is selected.
  • the nucleic acid encoding the polypeptide having the amino acid sequence ability of the protein, the protein, and the polypeptide can be obtained by methods well known to those skilled in the art. It can be realized by introducing it.
  • the expression of the DRG1 protein, DFRP1 protein, DRG2 protein, and DFRP2 protein of the present invention is decreased, the nucleic acid that causes RNAi, the antibody that binds to the protein, and the property of being dominant negative with respect to the protein. It can be realized by introducing a protein having a low molecular weight compound that binds to the protein.
  • nucleic acid that causes RNAi examples include nucleic acids having an activity of annealing to the dfrpl gene, drgl gene, dfrp2 gene or drg2 gene transcription product as described above.
  • antibody that binds to the protein examples include antibodies against the DFRP1 polypeptide or DFRP2 polypeptide as described above, and antibodies against the DRG1 or DRG2 polypeptide.
  • An antibody against DRG1 or DRG2 polypeptide can be obtained in the same manner as the antibody against DFRP1 or DFRP2 polypeptide as described above.
  • the “protein having a dominant negative property” refers to a protein having a function of eliminating or reducing the activity of an endogenous wild-type protein by expressing a gene encoding the protein.
  • the “low molecular weight compound (low molecular weight substance)” may be a natural or artificial compound. Usually a person skilled in the art It is a compound that can be produced or obtained by using methods known to those skilled in the art.
  • the agent of the present invention when used as a pharmaceutical composition, it can be formulated by methods known to those skilled in the art. For example, it can be used parenterally in the form of a sterile solution with water or other pharmaceutically acceptable liquid, or an injection in suspension.
  • a pharmacologically acceptable carrier or medium such as sterilized water or physiological saline, vegetable oil, emulsifier, suspending agent, surfactant, stabilizer, flavoring agent, excipient, vehicle, preservative It is conceivable to formulate the drug by combining it in a unit dosage form that is generally required for pharmaceutical practice, in appropriate combination with drugs, binders, etc. The amount of active ingredient in these preparations is such that an appropriate volume within the indicated range is obtained.
  • Sterile compositions for injection can be formulated according to conventional pharmaceutical practice using a vehicle such as distilled water for injection.
  • aqueous solutions for injection examples include isotonic solutions containing physiological saline, glucose and other adjuvants, such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride.
  • solubilizers such as alcohols, specifically ethanol, polyalcohols such as propylene glycol, polyethylene glycol, nonionic surfactants such as polysorbate 80 (TM), HCO-50 may be used in combination.
  • oily liquid examples include sesame oil and soybean oil, which may be used in combination with benzyl benzoate or benzyl alcohol as a solubilizing agent.
  • a buffering agent such as phosphate buffer, sodium acetate buffer, a soothing agent such as hydrochloric acid pro-in, a stabilizer such as benzyl alcohol, phenol, or an acid proofing agent may be blended.
  • the prepared injection is usually filled in a suitable ampoule.
  • Examples of the dosage form of the drug include tablets, powders, pills, powders, granules, fine granules, soft and hard capsules, film coating agents, pellets, sublingual agents,
  • Examples of parenteral preparations such as pastes include injections, suppositories, transdermal preparations, nasal preparations, pulmonary preparations, ointments, plasters, and liquids for external use.
  • Examples of the injection form can be administered systemically or locally by, for example, intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, and the like. Persons skilled in the art can select the optimal dosage form according to the administration route and administration subject.
  • a viral vector such as retrovirus, adenovirus, or Sendai virus, or a non-viral vector such as liposome can be used.
  • administration methods include in vivo methods and ex vivo methods.
  • the administration method can be appropriately selected depending on the age and symptoms of the patient.
  • the dosage and administration method of the pharmaceutical agent of the present invention vary depending on the patient's weight, age, symptoms, etc., but those skilled in the art can appropriately select them.
  • the present invention also relates to an antibody against a polypeptide encoded by the drgl gene, which has no cross-reactivity with the polypeptide encoded by the drg2 gene and binds to a DRG1 protein antibody (anti-DRG1 Protein antibody). That is, an antibody against the polypeptide encoded by the gene described in SEQ ID NO: 9 or SEQ ID NO: 10, wherein the polypeptide encoded by the gene described in SEQ ID NO: 11 or SEQ ID NO: 12 is cross-reactivity. It does not have an anti-DRG1 protein antibody.
  • the present invention also relates to an antibody against a polypeptide encoded by the drg2 gene, which has no cross-reactivity with the polypeptide encoded by the drgl gene and binds to a DRG2 protein antibody (anti-DRG2 Protein antibody). That is, an antibody against the polypeptide encoded by the gene described in SEQ ID NO: 11 or SEQ ID NO: 12, and the polypeptide encoded by the gene described in SEQ ID NO: 9 or SEQ ID NO: 10 and closliatati
  • the present invention relates to an anti-DRG2 protein antibody that does not have a bite.
  • Blasticidin, histidinol and puromycin were purchased from Sigma.
  • MG 132 was purchased from Peptide Institute.
  • FLAG-tagged, Myc-tagged and GST-fusion expression vectors were constructed by inserting cDNA fragments into modified pME-FLAG, pME-Myc or pME-GST from the SRa promoter-derived expression vector pME18S (Shiio, Y., Y amamoto, T. and Yamaguchi, N. (1992) Negative regulation of Rb expression by the p53 gene product. Proc. Natl. Acad. Sci. USA 89, 5206-5210.).
  • DFRPlfl Mouse DFRPlfl, DFRP1 ⁇ N (aa 231 to 426), DFRP1 ⁇ C (aa 1 to 263), DFRP2, DRG1 and DRG2 cDNA fragments are fused to pAD-GAL4-2.1 (Stratagene) mouse neural tube cDNA library Amplified from scratch by PCR.
  • DFRP2 cDNA was digested with Ncol, blunt-ended with Klenow enzyme, and used to generate AC1 and ⁇ ⁇ 1 site deletion mutants of DFRP2.
  • EcoRV was used to generate the AC2 and ⁇ 2 site deletion mutants of DF RP2.
  • D FRPI A DI (aa 236-260 deletion type) is inserted into the Bglll-extension pME-FLAG-DFRPlfl vector by inserting the Bglll site at the appropriate position between the two Bglll sites of the DFRP1 cDNA by the Kunkel method. It was created by inserting the longer of the Bglll-digested fragments.
  • HA-labeled ubiquitin was prepared by cloning into pcDNA3.1 vector (Invitrogen). Drug resistance gene cassette, pA-puro vector and DT40 genomic library for constructing DT40 stable cell line were provided by Dr. Kurosaki (RIKEN, Japan).
  • CDNA encoding mouse DFRPl (aa 1-270, ⁇ C2), DFRP2, DRG1 and DRG2 was subcloned into pGEX—4T—1 (Amersham Biosciences) and pMAL—c (New England BioLabs) expression vectors .
  • the vector was transformed into Escherichia coli (BL21).
  • the expressed GST and MBP fusion proteins (GST-DFRP1 ⁇ C2, GST-DFRP2, GST-DRG1, GST-DRG2, MBP-DRG1, and MBP-DRG2) are each glutathione sepharose according to the manufacturer's instructions. Purified from bacterial lysates using 4B beads (Amersham Biosciences) and amylose sucrose (New England BioLabs).
  • GST-DFRP1 A C2 GST-DFRP2, GST-DRG1, MBP-DRG1, or GST first emulsified in 50% Freund's complete adjuvant and then emulsified in 50% Freund's incomplete adjuvant (Sigma) to produce antiserum -Immunized with DRG2 subcutaneously at 2-week intervals.
  • DFRP1 AC2 antiserum was affinity purified on a MAbTrap TM GII column (Amersham Biosciences). Antiserum against GST-DFRP2 was roughly purified by ammonium sulfate precipitation. Antisera against MBP-DRG1 and GST-DRG2 were finally adsorbed on GST-DRG1 and MBP-DRG2 columns, respectively, and were purified.
  • TNE buffer (10 mM Tris-HC1, pH 7.8, 1% Nonidet P-40, 150 mM NaCl, 1 mM EDTA) for immunoprecipitation.
  • the cell lysate was centrifuged and the supernatant was incubated with protein G sepharose beads (Amersham Biosciences) and pre-cleaned. The cleaned lysate was incubated with the appropriate antibody and protein G sepharose beads for 1 hour. The beads were collected by centrifugation and washed 3 times with TNE buffer. Captured proteins were boiled in SDS sample buffer and eluted from the beads and analyzed by SDS-PAGE and Western blotting.
  • Vector 1-transfected cells were grown for 2 days and treated or not with the 10 / z M proteasome inhibitor MG132! And lysed in TNE buffer after 3 hours.
  • HeLa S3 cells were cultured on coverslips and grown for 2 days. Adherent cells were washed with PBS, fixed in methanol-acetone (1: 1) for 10 minutes, dried and blocked in 2% BSA. Fixed cells were incubated with antibodies against DFRP1 or DRG1 for 1 hour. At the end of the incubation, the cells were washed in PBS containing 0.2% Tween20 and stained with Alexa 488-conjugated anti-rabbit secondary antibody (Molecular Probes) for 1 hour. The slides were examined with a laser scanning confocal microscope (Radiance 2000, Bio-Rad). The Z series consisting of three images was collected from inside the cells with an aperture setting of 2.0 and a step size of 0.5 m. Images were projected by the maximum pixel method (LaserSharp2000 software, Bio-Rad).
  • DT40 cells in RPMI medium (JRH Biosciences) supplemented with 10% fetal bovine serum (Sigma), 1% chicken serum (Sigma), penicillin, streptomycin and j8-mercaptoethanol The vesicles were allowed to grow.
  • -Avian DFRPl cDNA fragments were obtained from DT40 cDNA using the degenerate primer set (5'-GCGAATTCATGCCNCCNAARAARC-3 '(SEQ ID NO: 13) and 5'-GCCTCGAGY TTYTCYTC YTTYTTYTTRTC-3' (SEQ ID NO: 14)). Amplified.
  • the full-length DFRPl cDNA (DDBJ / EMBL / GenBank accession number AB 185935) was isolated by screening about 5 x 10 6 plaques of the DT40 cDNA library ( ⁇ Zap) using a partial cDNA fragment as a probe.
  • Genomic DNA fragments containing part of the dfrpl locus are LA Taq and forward primer 5'-AGCAAGAAGGCGGAC CAGAA-3 '(SEQ ID NO: 15) and reverse primer 5'-GAGGAAGAGCATGGCG ATAC-3' (SEQ ID NO: Isolated by long PCR using 16).
  • Xenopus laevis embryos development of about 7 X 10 5 plaques in ⁇ Zip Lox (Gibco BRL) at high stringency (0.2 X SSC, 0.1% SDS, 50 ° C) Stages 24 to 32) A cDNA library was searched.
  • the complete sequence of the isolated Xenopus dfrpl cDNA was submitted to DDBJ / EMBL / GenBank (Accession AB185934). Hole mount in situ hybridization and Northern blot analysis techniques have been previously described (Ishikawa, K., Azuma, S., Ikawa, S "et al. (2003) Cloning an d characterization of Xenopus Gene 322, 105-112.) We used cDNA covering nucleotides -68 to +1755 of the Xenopus dfr pi probe.
  • a cell lysate (homogenate) was obtained.
  • Undisrupted cells were removed by centrifugation at 700 g for 5 min, and the supernatant was separated into a supernatant (PMS) and a pellet (mitochondria) by centrifugation at 15,000 g, lOmin.
  • PMS was further ultracentrifuged at 105,000 g for 45 min to obtain P100 fraction.
  • the 1 mL PMS obtained above was layered on a discontinuous sucrose gradient of 2.0STKM and 1.5STKM, and ultracentrifuged at 10 5,000 g for 3 h. Re-dissolve the polysome pellet in 0STKM and add 15% -40% concentration Overlaid on a sugar gradient, ultracentrifugation was performed at 150,000 g for 50 min. EDTA treatment is performed at a concentration of 10 mM ave
  • GIR2 V which showed 42% homology at the acid (aa) level, was shown to be involved in the basic pathway GIR2 was significantly different from ubiquitin-conjugating enzyme (E2) and E2 variants (UEV) Three-dimensional homology (Nameki, N., Yoneyama, M., Koshiba, S., et al. (2004) Solution structu re of the RWD domain of the mouse GCN2 protein. Protein Sci. 13, 2089- 2100.) Characteristic RWD domain (Fig. 1A) (also called GI domain; Kubota, H., Sakaki, Y. and Ito, T.
  • the present inventors used PCR to amplify mouse DRG1, DRG2, and GIR2 cDNAs to construct an expression vector.
  • Expression vectors of FLAG-tagged DRG1 or DRG2 and Myc-tagged GIR2 were co-transfected into 293T cells, and the cell extract was subjected to immunoprecipitation assay using an anti-FLAG antibody. The sediment was further analyzed by Western plotting.
  • H. sapiens likely ortholog of mouse immediate early respons by BLA ST search using full-length mouse GIR2 protein sequence as a query, and an ortholog of the human immediate response erythropoietin 4 human (H. sapiens) A protein known as e erythroEoietin 4) (LEREP04) was obtained.
  • GIR2 and LEREP 04 have high lysine (K), glutamic acid (E) and aspartic acid (D) content (GIR2, K, 8.2%; E, 16.2%; D, 9.1%; LEREP04, K, 14.1%; ⁇ , 12.7%; D, 9.2%), which had a highly homologous region consisting of about 60 aa, defined by aligning multiple sequences from mice, flies and yeast ( Figure 1A, top) Section, shaded box). However, the arrangements outside this region were not structurally similar.
  • LEREP04 is the two unique CCCH-type zinc (Zn) fingers that we have named herein ZnF-1 and ZnF-2, as well as the widely recognized NES Consensus (LX-(F, I, L, V, M)-
  • BR (199b; Protein sequence requirements for function of the human T-cell leuke mia virus type 1 Rex nuclear export signal delineated by a novel in vivo randomizati on- selection assay.Mol. Cell. Biol. 16, 4207-4214.) It had a compatible leucine-rich NES sequence. LEREP04 is also highly conserved in eukaryotes (HH. Sa piens) LEREP04 and S. cerevisiae LEREP04 had 55% homology at the aa level). To investigate the interaction between LEREP04 and DRG family proteins, 293T cells were treated with FLAG-DRG1 or DRG2 expression vectors and Mvc-labeled LERE. A vector expressing PCM was co-transfected and subjected to immunoprecipitation using an anti-FLAG antibody in the cell extract, followed by Western plotting.
  • the present inventors produced a polyclonal antibody that specifically recognizes endogenous DFRP1, DRGl or DRG2.
  • anti-DRG1 and anti-DRG2 sera were adsorbed to recombinant DRG2 and DRG1, respectively, prior to final affinity purification. , Cleaned in advance. As a result, it was confirmed that the purified antibodies against DRG1 and DRG2 had high specificity and no recognizable cross-reactivity.
  • the present inventors constructed an intermediate deletion mutant (AD1) of DFRP1 that lacks a highly conserved region. This deficiency completely eliminated the ability to bind to DRG1 ( Figure 2C, lane 4). Therefore, the 25-aa peptide of the DFRP domain was found to be essential for interaction with DRG1.
  • DRG1 or DRG2 could not be overexpressed alone (FIG. 3A, lanes 5 and 6).
  • This phenomenon has been previously reported for DRG1 (Sazuka, T., Kinoshita, M., Tomooka, ⁇ ⁇ , Ikawa, ⁇ ⁇ , Noda, ⁇ . And Kumar, b. (1992b) Expression of DRu dun ng murine Biochem. Biophys. Res. Commun. 189, 371—377.; Mahajan, M. A "Park, ST and Sun, XH (1996) Association of a novel GTP binding protein, DRG, with TAL oncogenic proteins.
  • DFRP1 has two Zn finger domains that appear to interact with ubiquitin
  • DFRP2 has an RWD domain that is structurally related to ubiquitin-conjugating enzyme (E2), we Thought that the ubiquitin / proteasome system might protect DRG from degradation.
  • E2 ubiquitin-conjugating enzyme
  • the present inventors generated a DFRP1-deficient DT40- ⁇ bird B cell line.
  • PCR using degenerate primers that also designed partial forces By PCR using degenerate primers that also designed partial forces.
  • -A partial cDNA fragment of avian dfrpl was identified.
  • We designed primers to amplify partial fragments of the ⁇ chicken dfrpl locus by long PCR using the amplified cDNA sequence.
  • the isolated genomic DNA was sequenced and its locus contains seven exons, one of which is a full-length Zn-finger 1 domain (ZnF-l, illustrated in Figure 1A). It became clear that the code would be coded.
  • ZnF- ⁇ exon decay we have As illustrated in Figure 4A, the blasticidin or histidinol resistance (Bsr or HisD) gene cassette is flanked by 5'- and 3'-genomic arms located upstream or downstream of the exon ( dfrplBsr and dfrplHisD) were constructed.
  • Wild type DT40 cells were transfected with dfrplBsr and a blasticidin resistant clone was isolated. Then, one of these heterozygous clones was transfected with dfrplHisD to delete the second allele. Both targeting events were confirmed by Southern blot analysis of genomic DNA using 5'-flanking probes. dfrpl removal was additionally demonstrated by Northern blot and wet stamp lot analysis ( Figures 4C and D, respectively).
  • the vector was introduced into dfrpl ⁇ / ⁇ cells.
  • the present inventors established a stable dfrpl-deficient cell (dfrpl mfl and dfrpl + m ⁇ Dl, respectively) that stably express mDFRPlfl or mDFRPl ⁇ 1.
  • drgl was first identified as a gene that is primarily expressed in the early developmental stage of the mouse central nervous system, and we previously compared drgl and drg2 expression in Xenopus embryos and adult tissues. The analysis was reported (Ishikawa, K., Azuma, S., Ikawa, S., et al. (2003) and lonmg and characterization of Xenopus laevis drg2, a member of the developmentally regulated GTP—binding protein subfamily. Gene 322, 105-112.
  • Xenopus whole-mount in situ hybridization revealed that the expression pattern of dfrpl is very similar to the expression pattern of drgl (Fig. 6A, a-; j).
  • both genes are blood islands, somites, developing eyes, trunk nerve crest, mandibular crest segment, hyoid crest segment, and crest segment (Branchial crest segment) (FIG. 6A, a to f).
  • dfrpl and drgl were also not expressed in the prorenal primordium region, but drg2 mRNA levels were higher in the prorenal primordium (Ishikawa, K., Azuma, S., Ikawa, S., et al (2003) Cloning and characterization of Xenopus laevis drg2, a member of the developmental ly regulated GTP-binding protein subfamily. Gene 322, 105-112.), Dfrpl mRNA transcription and Z or stability similar to drgl rather than drg2 It was suggested that it was adjusted by the method described above.
  • drgl and dfrpl expression patterns were almost the same. Both genes were expressed in the otocyst, pronephros, forebrain, midbrain, hindbrain, arch, eye, lens, spinal cord and notochord (Fig. 6A, g-j). In adult tissues, dfrpl is strongly expressed in the ovary, moderately expressed in the brain, kidney, spleen, testis, intestine and colon, and rarely expressed in the heart, lung, liver, stomach and skeletal muscle. (Fig. 6B).
  • This expression pattern is more similar to the expression pattern of drgl than drg2, which is expressed at a moderate level in the heart, lung and liver (Ishikawa, K., Azuma, Ikawa, et al. (2003)).
  • the present inventors also examined temporal expression of dfrpl in the early developmental stage.
  • FIGS. 6B and 6C the present inventors have analyzed our previous paper (Ishikawa, K., Azuma, S., Ikawa, et al. (2003) Cloning and characterization or Xenopus laevis drg2, a member of the developmentally regulated GTP—binding protein subfamily. Gene 322, 105-112.). Therefore, the quality and quantity of the transcribed RNA has been confirmed.
  • HeLa S3 cell line is DMEM medium (with 10% FBS, penicillin, streptomycin), CO (
  • Kitamura of the Institute of Medical Science, the University of Tokyo followed by EcoRI and It was excised with Notl and ligated to the EcoRI / Notl site of the EF1 alpha promoter driven IRES-puro vector (provided by Dr. Kitamura, Institute of Medical Science, The University of Tokyo) (pTetR-IRES-puro).
  • Inducible RNAi vector was prepared as follows. PCR (94 ° C 15sec, 55 ° C 3) by setting primers so that TetO is added to the HI opening motor from the pSuper vector (Oligoengine). (PCR primer set, 5,-CGATAAGCTT, Osec, 72 ° C lmin, 30 cycles)
  • T7 primer (5,-TAATACGACTCACTATAGGG-3, (SEQ ID NO: 26))).
  • the amplified fragment was digested with EcoRI and Bglll and then ligated to the EcoRI / Bglll site of pSuper (pSuper- 2 ).
  • an SV40-early promoter driven EGFP vector was prepared as follows. pEGF P-CKBD Biosciences Clontech) was digested with Bglll, blunted with Klenow, and further digested with Nhel, and pRL-SV40 (Promega) was digested with Xbal, blunted with Klenow, and further digested with Nhel The fragments were ligated (pSV40-EGFP). Furthermore, a fragment obtained by digesting PSV40-EGFP with Bglll and BamHI was ligated to the BamHI site of pSuper-2 (pSuper-3).
  • a neomycin resistance gene cassette (provided by Dr. Yamamoto of the Institute of Medical Science, the University of Tokyo) that can be excised with BamHI was ligated (pHlTetO).
  • Double-stranded oligonucleotides that form shRN A capable of causing RNAi against DRG1, DRG2, DFRP1, and DFRP2 mRNA at the Bglll / Hindlll site of pHlTetO (Brummelkamp TR. ⁇ A system for stab le expression of short interfering RNAs in mammalian cells, science, 296: 550-553 (2002).) was ligated (pHlTetO-target).
  • pTetR-IRES-puro was introduced into HeLa S3 cells by the calcium phosphate method (Current Protocols in Immunology, John Wiley & Sons, Inc.). Three days later, puromycin (Sigma) was introduced at a concentration of 3 g / mL. A colony formed after 2 weeks was isolated.
  • RNAi vector (pHlTetO-target) was introduced into one clonal cell line (R6) by the calcium phosphate method (Current Protocols in Immunology, John Wiley & Sons, Inc.), and G418 (Calbiochem) was introduced 5000/2 days later. A colony that fluoresces EGFP formed after 2 weeks was isolated (R6-HlTetO-target).
  • the Northern blot method was performed in the same manner as in the following literature. (Ishikawa, K. et al. Cloning and characterization or Xenopus laevis drg2, a member of the develo pmentally regulated GTP—binding protein subfamily. ", Gene, 322: 105-112 (2003)-) .
  • the cDNA probe was amplified from a human cell-derived cDNA pool by PCR (94 ° C 15 sec, 54 ° C 30 sec, 72 ° C lmin, 30 cycles).
  • fibronectin 5'-GCGA ATTCCGCTCGATGTGGTCTG-3, (SEQ ID NO: 27), 5,-GCCTCGAGACGGGAG CCTCGAAGAG-3, (SEQ ID NO: 28); Vinculin, 5, -GCAGATCTAGGG CTGGTGGACGAAG-3 '( SEQ ID NO: 29), 5,-GCCTCGAGGCCTTGGCGATGTC-3, (SEQ ID NO: 30); Cathepsin B (CathepsinB), 5, -GCGAATTCTGCTGCCTGCTG GTG-3, (SEQ ID NO: 31), 5,-GCCTCGAGCGGCCATGATGTCCTTC-3, (sequence No .: 32); IAP, 5,-GCGGATCCACGCCGCAATACAGAG-3, (SEQ ID NO: 33), 5,-GCCTCGAGTGCTGCGGATCAGCTC-3, (SEQ ID NO: 34); Urokinas e, 5,-GCGGATCCAATTCGGAGGGCAGCAC
  • Example 9 Evaluation of knockdown cells of DRG1, DRG2, DFRP1, and DFRP2 and changes in mRNA expression of genes that control cell motility, morphology, and cytoskeleton using Doxycyclin supplements
  • HeLa S3-inducible knockdown cells (Fig. 7, lanes 3-10) and control cells (Fig. 7, lanes 1-2) suppressed by TetR fused with HI promoter to induce RNAi and shRNA expression ability to induce doxycycline Cell strength after 12 days after RNAi induction with (Dox) addition (+) (20 ng / mL) or without addition (-) every 3 days is also the protein solution (total 1 ysate) or total RNA was prepared and extracted and subjected to Western blot or Northern blot ( Figure 7).
  • factor fibronectin extracellular matrix that regulates cytoskeleton, movement, morphology, invasion, and metastasis. It binds to specific integrins and adheres to cells (Hood, JD. et al "Nature Rev., 2: 91-100 (2002)). It works negatively on the growth of cancer cells.
  • Vinculin Fecal adhesion component. For stabilizing actin filaments.
  • Contributors cathebsin B (cystine proteinase. Involved in tumor cell invasion (see, eg, Lakka, S3 ⁇ 4.
  • DRG1 and DRG2 have been shown to be present in the cytoplasm (DRG2 and DFRP2 are unpublished data) and that DRG1 and DRG2 have the ability to bind RNA in vitro (Ishikawa, K. et al. lonmg and characterization of Xenopus laevis drg2, a member of the developmentally regulated GTP-binding protein subfamily. ", Gene, 322: 105-1 12 (2003).) It was suggested that this is stable control of decomposition.
  • HeLaS3 DRG1-induced knockdown cells were cultured with doxycycline (Dox) added (+) (20 ng / mL) or non-supplemented potassium (-). DRG1 knockdown cells existed in a disparate cell population, losing intercellular adhesion and loss of colony-forming ability ( Figure 8). Shows stretched or vague cell morphology!
  • HeLaS3 DRG1-induced knockdown cells were cultured with doxycycline (Dox) added (+) (20 ng / mL) or non-added potassium (-), fixed with PBS-PFA (4%), and then Phalloidin-Rhodamin e F-actin was stained with (Molecular probes), and images were captured with a confocal laser microscope (Bio-Rad) (Fig. 9). DRG1 knockdown cells showed many stress fiber formations (white arrows) and long filopodia formations (green arrows).
  • HeLaS3 DRG1-induced knockdown cells were cultured with doxycycline (Dox) added (+) (20 ng / mL) or non-added potassium (-), and videotaped with a Timelabs microscope (Olympus) to observe cell kinetics Was measured (MetaMorph software, Molecular Devices). The average speed of 10 cells was graphed ( Figure 10). DRG1 knockdown cells by Dox (+) were about 3.3 times as fast as Dox (-). In normal cells, DRG1 was thought to negatively regulate the expression of mRNA, a factor that promotes cell motility.
  • Dox doxycycline
  • - non-added potassium
  • DFRP1 knockdown cells showed abnormal cell morphology similar to that of DRG1 knockdown ( Figure 11). This is because DRG1 protein is degraded by DFRP1 knockdown, and DRG1 expression It was thought that this phenomenon was excited by the decrease of
  • HeLaS3 DRG2 inducible knockdown cells were cultured with doxycycline (Dox) added (+) (20 ng / mL) or non-added potassium (-). DRG2 knockdown cells seemed to have lower intercellular space and lower proliferation compared to controls ( Figure 12). In addition, adhesion between cells was good.
  • Dox doxycycline
  • - non-added potassium
  • DRG2 RNAi vector and an empty vector were introduced into HeLaS3 as a control, and EGFP-expressing cells, which are introduction markers constructed on the same vector, were observed.
  • the results are shown in Fig. 13.
  • DRG2 knockdown cells have high cell-cell adhesion (blue arrows), and abnormal protrusions were extended and cells were intertwined (white arrows). This enhanced intercellular adhesion is the opposite of the DRG1 knockdown morphological abnormality described above.
  • DRG 1 and DRG2 mRNA stability It was suggested that the infiltration and transfer control are different.
  • HeLaS3 DRG1 or DRG2 inducible knockdown cells starting at 2 x 10 4 cells / mL and cultured in doxycycline (Dox) supplemented (+) (20 ng / mL) or non-supplemented (-) 3 days The cells were counted using a hemocytometer. The cumulative number of cells that were passaged is shown in the graph of FIG. DRG1 and DRG2 knockdown (Dox (+)) cells were less proliferative than R6 and Dox (-) cells. However, it continued to survive at this growth rate, where growth was not completely suppressed. If this is a high level of cancer cells and the proliferation ability is simply reduced to the normal cell level, it is expected that the side effects of the drug using this invention can be suppressed to a low level.
  • Dox doxycycline
  • Example 18 Subcellular localization of DRG1 and DRG2 The intracellular localization of DRG1 and DRG2 was examined by centrifugal fractionation. Mouse liver homogenates of DRG1 and DRG2 were roughly fractionated by centrifugation. Cell lysates were collected according to the procedure shown on the left side of Fig. 16, and solutions were prepared so that the number of cells was the same. DRG1 and DRG2 were lighter than the mitochondrial (Figure 16 circle 2) fraction and were present in the PMS (nost-mitochondria supernatant) ( Figure 16 circle 3).
  • D RG1 Due to the stronger centrifugal force, D RG1 is in the P100 fraction ( Figure 16, circle 4, smooth ER, rough ER, polynome, plasma membrane, endonom, Golgi, etc.) and DRG2 is a lighter fraction (see figure). Concentrated to 16 circles 5). From this, it became clear that DRG1 and DRG2 exist in different fractions in vivo. In addition, separate protein complexes may be formed.
  • DRG1 regulates mRNA and is concentrated in the P100 fraction in Figure 16 circle 4, so that polysomes (in the state where multiple ribosomes are simultaneously bound to a single mRNA and protein synthesis is performed) There was a possibility that it was localized.
  • the adult mouse liver homogenate was ultracentrifuged using a discontinuous sucrose gradient, the polysome pellet was redissolved, layered on a 15-40% continuous sucrose gradient, and after ultracentrifugation. Polysome profiling was performed in a rectification column equipped with a UV (254 nm) monitor. At the same time, fractions corresponding to FIG. 17 were collected, and the localization of the DRG1 protein was analyzed by Western blotting.
  • the amount of DRG1 protein was present in a pattern similar to the curve showing the ribosome (upper panel in Fig. 17, blue line) (middle panel in Fig. 17).
  • 80S ribosome has a property of dissociating into 40S and 60S subunits when the [Mg 2+ ] concentration is 5 mM or less.
  • This is achieved by adding 10 mM of EDTA (upper panel, red line)
  • the fraction in which DRG1 was present also moved toward the dissociated peak of ribosome. From these results, it was considered that DRG1 is localized on polysomes.
  • DRG1 and DRG2 does not occur in the absence of DFRP1 and DFRP2 factors under physiological conditions.
  • knocking down DRG1 in a human cancer cell line, HeLa S3 cell increases the cell's ability to infiltrate 'invasion', while knocking down DRG2 decreases it, resulting in abnormal cell adhesion. It was shown that it was strengthened. This molecular mechanism includes cytoskeletal factors, extracellular matrix factors, etc. It was found that the DRG family directly or indirectly regulates the stability and degradation of mRNA of proteins that control morphology, invasion, and metastasis.
  • DRG1 or suppressing DRG2 expression which is caused by loss of adhesion of the cell population. It is suggested.
  • the ability of cancer cells to infiltrate or metastasize may be suppressed by strongly expressing DRG1 or suppressing the expression of DRG2.
  • overexpression of DRG1 alone is impossible, co-expression with DFRP1 is necessary.
  • DRG1 expression or DFRP1 expression can be suppressed, or DRG2 can be strongly expressed.
  • DRG1 and DRG2 knockdown cells showed a decrease in proliferation to such an extent that cell death did not occur. If this is simply a reduction of the high growth potential of cancer cells to the level of normal cells, it is expected that side effects caused by cell survival and proliferation of drugs using this invention can be suppressed to a low level.
  • DRG1, DRG2, DFRP1, and DFRP2 are targeted because DRG1 and DRG2 regulate the expression of various cytoskeletal, motor, morphology, invasion, and metastasis regulators directly or indirectly at the mRNA level. It is expected that the effects and immediate effects of the selected drugs are very high.

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Abstract

Novel DRG family regulatory proteins DFRP1 and DFRP2 are identified. In a transient transduction test, DFRP1 binds specifically to DRG1 while DFRP2 binds mainly to DRG2. DFRPs block polyubiquitination seemingly occurring prior to the decomposition of the DRG proteins and thus stabilize the target DRG proteins due to physical bindings. DFRPs are highly conserved in eukaryotes. The expression patterns of dfrp1 and drg1 transcription products in the embryo and tissue of Xenopus are similar to each other, which indicates that these genes synergistically act in eukaryotic cells of various types. As the results of an immunofluorescent test, it is clarified that the interaction between DRG1 and DFRP1 would likely arise in the cytoplasm. By constructing a dfrp1-knockout cell, it is found out that the internal expression of DRG1 is regulated by DFRP1. Namely, it is confirmed that DFRP1 is a DRG1-specific up regulator in vivo. Based on these results, it is proposed that DRG1 and DRG2 are regulated in different manners, though they are close to each other in structure.

Description

明 細 書  Specification
DRG蛋白質を制御する DFRP蛋白質およびその利用  DFRP protein that regulates DRG protein and its use
技術分野  Technical field
[0001] 本発明は、 DRG蛋白質を制御する DFRP蛋白質および DRG蛋白質を制御する方法 に関する。  The present invention relates to a DFRP protein that controls DRG protein and a method for controlling DRG protein.
背景技術  Background art
[0002] 発生段階で調節される GTP-結合蛋白質 (developmentally regulated GTP-binding protein) (DRG)サブファミリ一は、 GTPaseスーパーファミリーの一部門を構成している (非特許文献 1参照)。 drgは、最初は、発生中のマウス脳において高レベルで発現さ れる遺伝子 (NEDD-3)として、サブトラクティブ cDNAクローユング方法によって同定さ れた (非特許文献 2〜4参照)。マウス drgと相同な遺伝子は、多種多様の真核生物お よび古細菌種にぉ 、て報告されて 、る。これらの DRG蛋白質は互いに顕著な類似性 を示し、 DRGの細胞における役割が非常に重要であることを示唆している。 Schenker ら（非特許文献 5参照）は DRG配列検索を実施し、 DRGサブファミリ一は密接に関連 する 2つの蛋白質、 DRG1 (オリジナルの DRG)および DRG2を含有することを報告した しかし、これら DRG1蛋白質および DRG2蛋白質の生理学的な機能およびそれらの 調節機序にっ 、ては、今まで明らかにされて 、なかった。  [0002] The developmentally regulated GTP-binding protein (DRG) subfamily constitutes a division of the GTPase superfamily (see Non-Patent Document 1). Drg was first identified by the subtractive cDNA cloning method as a gene (NEDD-3) that is expressed at high levels in the developing mouse brain (see Non-Patent Documents 2 to 4). Genes homologous to mouse drg have been reported in a wide variety of eukaryotic and archaeal species. These DRG proteins show remarkable similarities, suggesting that the role of DRG in cells is very important. Schenker et al. (See Non-Patent Document 5) conducted a DRG sequence search and reported that the DRG subfamily contains two closely related proteins, DRG1 (original DRG) and DRG2, but these DRG1 proteins And the physiological functions of DRG2 proteins and their regulatory mechanisms have not been elucidated so far.
[0003] 非特許文献 1 : Leipe, D. D.et al., (2002) Classification and evolution of P— loop GTP ases and related ATPases. J. Mol. Biol. 317, 41-72. [0003] Non-Patent Document 1: Leipe, D. D. et al., (2002) Classification and evolution of P— loop GTP ases and related ATPases. J. Mol. Biol. 317, 41-72.
非特許文献 2 : Kumar, S. et al., (1992) Identification of a set of genes with develop mentally down-regulated expression in the mouse brain. Biochem. Biophys. Res. Co mmun. 185, 1155—1161.  Non-Patent Document 2: Kumar, S. et al., (1992) Identification of a set of genes with develop mentally down-regulated expression in the mouse brain. Biochem. Biophys. Res. Commun. 185, 1155—1161.
非特許文献 3 : Sazuka, T. et al., (1992) DRG: a novel developmentally regulated G TP— binding protein. Biochem. Biophys. Res. Commun. 189, 363—370.  Non-Patent Document 3: Sazuka, T. et al., (1992) DRG: a novel developmentally regulated G TP— binding protein. Biochem. Biophys. Res. Commun. 189, 363—370.
非特許文献 4 : Sazuka, T. et al., (1992) Expression of DRG during murine embryoni c development. Biochem. Biophys. Res. Commun. 189, 371—377. 非特言午文献 5 : Schenker， T. and Trueb, B. (1997) Assignment of the gene for a dev elopmentally regulated GTP- binding protein (DRG2) to human chromosome bands 17pl3—― >pl2 by in situ hybridization. Cytogenet. Cell Genet. 79, 274-275. Non-Patent Document 4: Sazuka, T. et al., (1992) Expression of DRG during murine embryonic development. Biochem. Biophys. Res. Commun. 189, 371-377. Non-Special Publication 5: Schenker, T. and Trueb, B. (1997) Assignment of the gene for a dev elopmentally regulated GTP-binding protein (DRG2) to human chromosome bands 17pl3——>> pl2 by in situ hybridization. Cytogenet. Cell Genet. 79, 274-275.
発明の開示  Disclosure of the invention
発明が解決しょうとする課題  Problems to be solved by the invention
[0004] 本発明は、細胞増殖の重要な調節因子であると考えられる DRG蛋白質 (DRG1およ び DRG2)の生理学的な機能および調節機序を解明することを課題とする。また、 DR G1蛋白質および DRG2蛋白質に特異的に結合して、その発現を調節する新規な蛋 白質 DFRP1および DFRP2を提供することを課題とする。  [0004] An object of the present invention is to elucidate the physiological functions and regulatory mechanisms of DRG proteins (DRG1 and DRG2) which are considered to be important regulators of cell proliferation. Another object of the present invention is to provide novel proteins DFRP1 and DFRP2 that specifically bind to DRG1 protein and DRG2 protein and regulate their expression.
課題を解決するための手段  Means for solving the problem
[0005] 本発明者らは上記課題を解決するために鋭意研究を行った。  [0005] The present inventors have intensively studied to solve the above problems.
DRG1と DRG2は、全長において相同性が高い。特に強い相同性を示す領域として 、 GTPaseの活性中心となりうる特徴的な G-モチーフのドメイン、および RNA結合に関 連する C末端 TGSドメインが挙げられる（Ishikawa, K. et al.， (2003) Cloning and char acterization of Xenopus laevis drg2, a member of the developmentally regulated GT P-binding protein subfamily. Gene 322, 105-112.)。これらの類似性に基づけば、こ れらの蛋白質は同様の機能を有すると考えられる力 DRG1および DRG2の系統発生 的な分析では DRG1および DRG2は別個の進化分岐に属することが示されている（Li, B. and Trueb, B. (2000) DRG represents a family of two closely related uTP-Dindi ng proteins. Biochim. Biophys. Acta. 1491, 196- 204.および Etheridge, N., et al, (1 999) Characterization of ATDRG1, a member of a new class of GTP- binding protein s in plants. Plant Mol. Biol. 39, 1113-1126.)。従って、本発明者らは、 DRG1および DRG2は別個の機能を有すると仮定した。  DRG1 and DRG2 have high homology over the entire length. Regions with particularly strong homology include the characteristic G-motif domain that can be the active center of GTPase and the C-terminal TGS domain associated with RNA binding (Ishikawa, K. et al., (2003) Cloning and char acterization of Xenopus laevis drg2, a member of the developmentally regulated GT P-binding protein subfamily. Gene 322, 105-112.). Based on these similarities, the phylogenetic analysis of DRG1 and DRG2 indicates that these proteins are likely to have similar functions. DRG1 and DRG2 belong to separate evolutionary branches ( Li, B. and Trueb, B. (2000) DRG represents a family of two closely related uTP-Dinding proteins. Biochim. Biophys. Acta. 1491, 196-204. And Etheridge, N., et al, (1 999 ) Characterization of ATDRG1, a member of a new class of GTP-binding protein s in plants. Plant Mol. Biol. 39, 1113-1126.). Therefore, we hypothesized that DRG1 and DRG2 have distinct functions.
[0006] また本発明者らは、既存のゲノムデータベースを使用した詳細なコンピュータ分析 によって、真核生物は drglおよび drg2遺伝子の両方を有する力 古細菌は一方の dr g遺伝子だけを保有し、それは drglまたは drg2と特に対応しな 、ことを明らかにした (L 1, B. and Trueb, B. (2000) DRG represents a family of two closely related G i'P- bin ding proteins. Biocnim. Biophys. Acta. 1491, 196—204.)。 [0007] 本発明者らは以前に、 drglおよび drg2 mRNAの転写および Zまたは安定性が異な つて調節されることを示している (Ishikawa, K. et al.， (2003) Cloning and characteriz ation of Xenopus laevis drg2, a member of the developmentally regulated GTP— bindi ng protein subfamily. Gene 322, 105-112.)。し力し、 DRGlと DRG2の機能を識別する 納得の 、く証拠は得られて!/、なかった。 [0006] In addition, by detailed computer analysis using existing genomic databases, the inventors have found that eukaryotes possess both drgl and drg2 genes. Archaea possess only one drg gene, which It was clarified that it did not correspond to drgl or drg2 (L 1, B. and Trueb, B. (2000) DRG represents a family of two closely related G i'P-bind proteins. Biocnim. Biophys. Acta 1491, 196—204.). [0007] We have previously shown that transcription and Z or stability of drgl and drg2 mRNA are differentially regulated (Ishikawa, K. et al., (2003) Cloning and characterization of Xenopus laevis drg2, a member of the developmentally regulated GTP—binding protein subfamily. Gene 322, 105-112.). However, there was no convincing evidence to distinguish between DRGl and DRG2 functions!
[0008] 本発明者らは、本発明において初めて DRG蛋白質の生理学的な機能について提 唱した。さらに DRG1および DRG2に対し特異的な、別個の結合特異性を有し、 DRG 蛋白質の発現を調節する新規な 2つの DRGファミリー調節蛋白質 ( RG family regula tory nrotein) (DFRP)を同定することに成功した。そして、細胞増殖の基礎となる新た な細胞機序について提唱した。即ち、本発明者らは、真核生物で高度に保存されて おり、標的 DRG蛋白質に特異的に結合し、おそらくは分解に至らせると思われるポリ ュビキチンィ匕の遮断によって標的 DRG蛋白質の発現を調節する 2つの新規蛋白質フ アミリー、 DFRP1および DFRP2を同定した（図 1Bおよび 3)。同定された一方の蛋白質 である DFRP1と DRG1の結合は、インビボにおける DRG1の正常な発現レベルの維持 に必須であることが見いだされた力 DFRP1は DRG2に結合しな力つた。本発明者ら によるこれらの所見は、 DRG1および DRG2がインビボにおいて別個の調節によって 支配されることを示唆して 、る。  [0008] The present inventors have proposed the physiological function of DRG protein for the first time in the present invention. In addition, we have successfully identified two new DRG family regulatory proteins (DFRP) that have distinct binding specificities for DRG1 and DRG2 and regulate the expression of DRG proteins (RG family regula tory nrotein) (DFRP) did. He proposed a new cellular mechanism that is the basis of cell growth. That is, the inventors regulate the expression of the target DRG protein by blocking polyubiquitin, which is highly conserved in eukaryotes and specifically binds to the target DRG protein, possibly leading to degradation. Two new protein families, DFRP1 and DFRP2, were identified (Figures 1B and 3). The binding of one of the identified proteins, DFRP1 and DRG1, was found to be essential for maintaining normal expression levels of DRG1 in vivo. DFRP1 did not bind to DRG2. These findings by the inventors suggest that DRG1 and DRG2 are governed by distinct regulation in vivo.
[0009] DFRPの興味深!/、1つの特徴として、本発明者らが DFRPドメインと名づけた、 DFRP 間で高 、配列の同一性を示す独自のドメインの存在がある（図 1A)。この DFRPドメイ ンは DRGファミリーとの結合に必須であることが分力つた（図 2)。従って真核生物に おける進化的保存性のために、 DFRPの DRGファミリーメンバーへの結合および DRG 蛋白質の適切な発現の維持は、非常に重要と考えられる。 DRG1に結合可能で、造 血に関する発生の転写因子である SCL (TAL-l)がー過的に同時発現すると DRG1蛋 白質も安定化することは注目に値する（Mahajan, M. A., Park, S. T. and Sun, X. H. (199b; Association of a novel GTP binding protein, DRG, with TAL oncogenic prot eins. Oncogene 12, 2343-2350.)。また DRG蛋白質が安定な蛋白質複合体から放出 されると分解が生じると思われた。遊離の DRG分子のこのような分解は、ュビキチンァ クチバーゼ（activase) (El)、コンジュガーゼ（conjugase) (E2)およびリガーゼ（ligase) (E3)の協奏的作用によるポリュビキチンィ匕によって媒介される可能性がある。 DFRP の特定のドメインがこれらのュビキチン関連酵素に結合しうる。 One interesting feature of DFRP is the existence of a unique domain that is named DFRP domain by the present inventors and shows high sequence identity among DFRPs (FIG. 1A). It was found that this DFRP domain is essential for binding to the DRG family (Figure 2). Thus, for evolutionary conservation in eukaryotes, the binding of DFRP to DRG family members and the proper expression of the DRG protein is considered very important. It is noteworthy that SRG (TAL-l), a transcription factor involved in hematopoiesis that can bind to DRG1, over-expresses it also stabilizes the DRG1 protein (Mahajan, MA, Park, ST and Sun, XH (199b; Association of a novel GTP binding protein, DRG, with TAL oncogenic proteins. Oncogene 12, 2343-2350.) In addition, it is thought that degradation occurs when DRG protein is released from a stable protein complex. Such degradation of free DRG molecules can be attributed to ubiquitin activase (El), conjugase (E2) and ligase. (E3) may be mediated by polyubiquitin by concerted action. Specific domains of DFRP can bind to these ubiquitin-related enzymes.
[0010] DFRP2は、ュビキチンプロテアソーム経路による分解に関与する蛋白質に特徴的 な、 RINGフィンガー、 IBR(In Between Ring fingers)、 UBAゝ UBCおよび WD反復ドメィ ンなどの他のドメインを含有する蛋白質に見られることが多い RWDドメインを有する。 さらに、 DFRP1の CCCH-型 Znフィンガーの連続した 2つの反復は、 C3HC4-型および C3H2C3-型 RINGフィンガーに類似している。これらの 2つの Zn-フィンガー反復は RI NGフィンガー変種である可能性があると本発明者らは考えている。さらに、 1つの Zn フィンガーはュビキチンと結合することができる。例えば、新規ジンクフィンガー (novel zinc finger) (NZF)ドメイン力 ER—結合分解（EE— associated degradation) (ERAD)に 関与する NPL4において見られる（Wang, B., Alam, S. L" Meyer, H. H" et al. (2003) Structure and ubiquitin interactions of the conserved zinc finger domain of Npl4. J. Biol. Chem. 278, 20225-20234.) 0ポリュビキチン結合ジンクフィンガー (polyubiquitin -associated zinc finger) (PAZ)ドメインは微小管-結合 HDAC6にお!/、て見られる（Hoo k, b. S., Onan, A., し owley, S. M. ana disenman, R. N. (2002) Histone deacetylase 6 binds polyubiquitin through its zinc finger (PAZ domain) and copurifies with deubiq uitinating enzymes. Proc. Natl. Acad. Sci. U S A 99, 13425—13430.)。 [0010] DFRP2 is a protein that contains other domains such as RING fingers, IBR (In Between Ring fingers), UBA ゝ UBC, and WD repeat domains, which are characteristic of proteins involved in degradation by the ubiquitin proteasome pathway. Has an RWD domain often seen. In addition, two consecutive repeats of CCCH-type Zn fingers of DFRP1 are similar to C3HC4-type and C3H2C3-type RING fingers. We believe that these two Zn-finger repeats may be RI NG finger variants. In addition, one Zn finger can bind to ubiquitin. For example, it is found in NPL4 involved in novel zinc finger (NZF) domain force ER—associated degradation (ERAD) (Wang, B., Alam, S. L ”Meyer, H H "et al. (2003) Structure and ubiquitin interactions of the conserved zinc finger domain of Npl4. J. Biol. Chem. 278, 20225-20234.) 0 polyubiquitin -associated zinc finger (PAZ) The domain is found in microtubule-bound HDAC6! / (Hook, b. S., Onan, A., owley, SM ana disenman, RN (2002) Histone deacetylase 6 binds polyubiquitin through its zinc finger ( PAZ domain) and copurifies with deubiq uitinating enzymes. Proc. Natl. Acad. Sci. USA 99, 13425-13430.).
[0011] 以前の検討から、 DRG蛋白質は細胞増殖に重要な役割を果たしうることが分力つて いる。 drgl、 drg2および dfrpl転写物は、成長中のアフリカッメガエル（Xenopus)胚に おいて高いレベルで発現した（図 6 ; Ishikawa, K., Azuma, S.， Ikawa, S.， et al. (2003) Cloning and characterization of Xenopus laevis drg2, a member of the developmental ly regulated GTP— binding protein subfamily. Gene 322, 105—112.)。エンドゥマメ（Pis um sativum)およびシロイヌナズナ (Arabidopsis thaliana)では、 drg2 mRNAは主に成 長中の組織に蓄積した（Devitt, M. L., Maas, K. J. and Stafstrom, J. P. (1999) Char acterization of DRGs, developmentally regulated GTP— binding proteins, from pea an d Arabidopsis. Plant Mol. Biol. 39, 75— 82. ; Etheridge, N" Trusov, Y" Verbelen, J. P . and Botella, J. R. (1999) Characterization of ATDRGl, a member of a new class of GTP— binding proteins in plants. Plant Mol. Biol. 39, 1113-1126.)。さらに、本発明者 らカ FRP1と改名した LEREP04は、最初は、赤血白血病 SKT6細胞において C-末端 切断型 Epo受容体を介してエリスロポイエチン (Epo)シグナリングに応答して速やか に転写される遺伝子と同定されていた（Gregory, R. C, Lord, K. A., Panek, L. B.， G aines, P., Dillon, S. B. and Wojchowski, D. M. (2000) Subtraction cloning and initial characterization of novel epo- immediate response genes. Cytokine 12, 845-857.)。 Epo受容体は、成長のキーとなるシグナルを中継する（Shikama, Y., Barber, D. L.， D 'Andrea, A. D. and Sieff, C. A. (1996) A constitutively activated chimeric cytokine r eceptor confers factor-independent growth in hematopoietic cell lines. Blood 88, 45 5-464.) o DRG1の細胞機能のために安定ィ匕が必要な場合、細胞は、このような成長 シグナルによって DFRP1を誘導し、その結果 DRG1の機能を開始することが考えられ る。しかし、 DRGファミリ一は異常に発現すると、細胞の形質転換または細胞周期の 停止が生じる（Mahajan, M. A" Park, S. T. and Sun, X. H. (1996) Association of a n ovel GTP binding protein, DRG, with TAL oncogenic proteins. Oncogene 12, 2343— 2350. ; Ko, M. S.， Lee, U. H.， Kim, S. I., et al. (2004) Overexpression of DRG2 supp resses the growth of Jurkat T cells but does not induce apoptosis. Arch. Biochem. B iophys. 422, 137-144. ; Song, H., Kim, S. I., Ko, M. S., et al. (2004) Overexpression of DRG2 increases G2/M phase cells and decreases sensitivity to nocodazole- indue ed apoptosis. J. Biochem. (Tokyo) 135, 331-335.)。本発明者らのデータに基づいて 考えると、ュビキチン媒介性の分解を逃れ過剰発現した DRG蛋白質は、不適切に機 能し、正常な増殖制御の崩壊を誘発する可能性がある。 [0011] Previous studies have shown that DRG protein can play an important role in cell proliferation. drgl, drg2 and dfrpl transcripts were expressed at high levels in the growing Xenopus embryos (Figure 6; Ishikawa, K., Azuma, S., Ikawa, S., et al. (2003) Cloning and characterization of Xenopus laevis drg2, a member of the developmental ly regulated GTP—binding protein subfamily. Gene 322, 105—112.). In endum (Pis um sativum) and Arabidopsis thaliana, drg2 mRNA accumulated mainly in growing tissues (Devitt, ML, Maas, KJ and Stafstrom, JP (1999) Char acterization of DRGs, developmentally regulated GTP — Binding proteins, from pea an d Arabidopsis. Plant Mol. Biol. 39, 75— 82.; Etheridge, N "Trusov, Y" Verbelen, J. P. And Botella, JR (1999) Characterization of ATDRGl, a member of a new class of GTP—binding proteins in plants. Plant Mol. Biol. 39, 1113-1126.). In addition, the inventor LEREP04, renamed Faraca et al., Was first identified as a gene that is rapidly transcribed in response to erythropoietin (Epo) signaling via the C-terminal truncated Epo receptor in erythroleukemia SKT6 cells. (Gregory, R. C, Lord, KA, Panek, LB, Gaines, P., Dillon, SB and Wojchowski, DM (2000) Subtraction cloning and initial characterization of novel epo- immediate response genes. Cytokine 12, 845- 857.). Epo receptors relay key signals for growth (Shikama, Y., Barber, DL, D 'Andrea, AD and Sieff, CA (1996) A constitutively activated chimeric cytokine r eceptor confers factor-independent growth in hematopoietic Blood lines, Blood 88, 45 5-464.) o When stability is required for DRG1 cell function, the cell induces DFRP1 by such growth signals and consequently initiates DRG1 function. It is possible. However, abnormal expression of the DRG family results in cell transformation or cell cycle arrest (Mahajan, M. A "Park, ST and Sun, XH (1996) Association of an ovel GTP binding protein, DRG, with TAL oncogenic proteins. Oncogene 12, 2343— 2350.; Ko, MS, Lee, UH, Kim, SI, et al. (2004) Overexpression of DRG2 supp resses the growth of Jurkat T cells but does not induce apoptosis. Arch. Biochem B iophys. 422, 137-144.; Song, H., Kim, SI, Ko, MS, et al. (2004) Overexpression of DRG2 increases G2 / M phase cells and decreases sensitivity to nocodazole- indue ed apoptosis. Biochem. (Tokyo) 135, 331-335.) Based on our data, the overexpressed DRG protein that escapes ubiquitin-mediated degradation functions improperly and normal growth. There is a possibility of triggering a disruption of control.
[0012] また、 PROPHECYデータベース（http：〃 prophecy.lundberg.gu.se/Main.aspx)、 SC MD7" ~~タへ' ~~ ^ (Saccharomyces Cerevisiae Morphological Database)によると、酵 母（Yeast)の DRG1、 DRG2、 DFRP1、 DFRP2ノックアウト（K.O.)細胞の生長および形 態は野生型と劇的な差がなぐほぼ正常であることが示されている。ところが、ヒトの癌 細胞株である HeLaS3の DRG1、 DRG2、 DFRP1、 DFRP2ノックダウン（K.D.)は表 1のよ うに成長は低下し、形態も異常である。  [0012] According to the PROPHECY database (http: 〃 prophecy.lundberg.gu.se/Main.aspx), SC MD7 "~~ Tahe '~~ ^ (Saccharomyces Cerevisiae Morphological Database), The growth and morphology of DRG1, DRG2, DFRP1, and DFRP2 knockout (KO) cells have been shown to be almost normal, with no dramatic differences from wild type, but DRG1 of the human cancer cell line HeLaS3 , DRG2, DFRP1, and DFRP2 knockdown (KD), as shown in Table 1, show a decrease in growth and abnormal morphology.
[0013] [表 1] , HeLaS3 [0013] [Table 1] , HeLaS3
mU (癌細胞) 成長 正常 低下 m U (cancer cell) Growth Normal Decrease
形態 正常 異常  Form Normal Abnormal
[0014] 仮に、癌細胞でないヒトの正常細胞で DRG1、 DRG2、 DFRP1、 DFRP2がノックアウト もしくはノックダウンした時に酵母と同様に正常であるならば、 DRG1、 DRG2、 DFRP1 、 DFRP2のノックダウンによって正常細胞には何の影響も与えず、癌細胞特異的に成 長を減弱させることができる可能性がある。また、 DRG1および DFRP1の K.D.細胞で 様々な因子の mRNAの増減が見られた力 mRNA上のリボソームの安定性に関与す ることで直接あるいは間接的に mRNAの安定化'不安定ィ匕を制御していると考えられ る。  [0014] If DRG1, DRG2, DFRP1, and DFRP2 are knocked out or knocked down and normal in the same manner as yeast when normal cancer cells that are not cancer cells, normal cells can be obtained by knocking down DRG1, DRG2, DFRP1, and DFRP2. There is a possibility that growth can be attenuated specifically in cancer cells. In addition, DRG1 and DFRP1 KD cells showed changes in mRNA of various factors. Involved in the stability of ribosomes on mRNA, directly or indirectly controlled mRNA stabilization. it seems to do.
[0015] 以上、本発明者らは、 DRGファミリー（DRG1および DRG2)の特異的な制御蛋白質 である DFRP1および DFRP2をそれぞれ同定し、おそらくはュビキチンによる分解を結 合によって抑制することで、 DRG1および DRG2の蛋白質の発現を正に制御している ことを明らカにした。 DRG1と DRG2の発現はこれらの因子が存在しな!、と成立しな!ヽ ことも、生理的条件下で明らかにした。また、ヒト癌細胞株である HeLaS3を用いた DR G1のノックダウン細胞では、細胞の運動 '浸潤'転移能が上昇し、 DRG2ノックダウン 細胞では逆に低下し、細胞接着性などは異常に増強していることが示された。この分 子機構として、細胞骨格因子，細胞外マトリックス因子など、細胞運動 ·浸潤'転移な どを制御する蛋白質群の mRNAの安定化'分解の調節を DRGファミリーが行っている ためであることが見 、だされた。この本発明によって明ら力となった新たな基本機構 の原理を基にした、 DRGファミリー、 DFRPs (DFRPlおよび DFRP2)の発現調節による 美容薬の開発'癌治療法の開発が期待される。また DRG1と DRG2のノックダウン細胞 はともに細胞の死には至らしめない程度の増殖の低下が認められた。これが癌細胞 の高 、増殖能を単に正常細胞レベルまで落としたものであるならば、本発明を利用し た薬剤の副作用は低レベルに抑えられることが期待される。  [0015] As described above, the present inventors have identified DFRP1 and DFRP2, which are specific regulatory proteins of the DRG family (DRG1 and DRG2), respectively, and possibly suppressed the degradation by ubiquitin by binding, whereby DRG1 and DRG2 It was clarified that the expression of this protein is positively controlled. It was clarified that the expression of DRG1 and DRG2 did not exist! In addition, DR G1 knockdown cells using the human cancer cell line HeLaS3 have increased cell motility 'invasion' and metastasis capacity, while DRG2 knockdown cells have decreased, and cell adhesion is abnormally enhanced. It was shown that This molecular mechanism is due to the fact that the DRG family regulates the stabilization and degradation of the mRNAs of proteins that control cell motility and invasion, such as metastasis, such as cytoskeletal factors and extracellular matrix factors. I saw it. Based on the principle of a new basic mechanism that has become clear by the present invention, development of a cosmetic drug by regulating the expression of the DRG family and DFRPs (DFRPl and DFRP2) is expected to develop a cancer treatment method. Both DRG1 and DRG2 knockdown cells showed a decrease in proliferation that did not lead to cell death. If this is a high cancer cell proliferation ability simply reduced to a normal cell level, it is expected that the side effects of the drug using the present invention can be suppressed to a low level.
[0016] 即ち本発明は、細胞増殖の重要な調節因子である DRG蛋白質に特異的に結合し て、その発現を調節する新規な蛋白質 DFRP1および DFRP2に関し、詳しくは、 〔1〕 下記（1)または（2)に記載のポリペプチド、 That is, the present invention specifically binds to DRG protein which is an important regulator of cell proliferation. In particular, regarding the novel proteins DFRP1 and DFRP2 that regulate their expression, [1] the polypeptide according to (1) or (2) below,
( 1)配列番号： 2または配列番号: 4に記載のアミノ酸配列における 234位から 295位 のアミノ酸配列力もなるポリペプチド  (1) A polypeptide having an amino acid sequence from position 234 to position 295 in the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4.
(2)配列番号： 2または 4に記載のアミノ酸配列における 234位から 295位のアミノ酸 配列において 1若しくは複数のアミノ酸が置換、欠失、挿入または付加されたアミノ酸 配列からなり、 DRG1蛋白質との相互作用能力を有するポリペプチド  (2) The amino acid sequence of SEQ ID NO: 2 or 4, consisting of an amino acid sequence in which one or more amino acids are substituted, deleted, inserted or added in the amino acid sequence from position 234 to position 295, Polypeptide having action ability
〔2〕 下記（1)または（2)に記載のポリペプチド、  [2] The polypeptide according to (1) or (2) below,
(1)配列番号： 2 (ヒト DFRP1アミノ酸配列）または配列番号： 4 (マウス DFRP1アミノ酸 配列）に記載のアミノ酸配列からなるポリペプチド  (1) A polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2 (human DFRP1 amino acid sequence) or SEQ ID NO: 4 (mouse DFRP1 amino acid sequence)
(2)配列番号： 2 (ヒト DFRP1アミノ酸配列）または配列番号： 4 (マウス DFRP1アミノ酸 配列）に記載のアミノ酸配列において 1若しくは複数のアミノ酸が置換、欠失、挿入ま たは付加されたアミノ酸配列からなり、 DRG1蛋白質との相互作用能力を有するポリ ペプチド  (2) Amino acid sequence in which one or more amino acids are substituted, deleted, inserted or added in the amino acid sequence described in SEQ ID NO: 2 (human DFRP1 amino acid sequence) or SEQ ID NO: 4 (mouse DFRP1 amino acid sequence) A polypeptide comprising the ability to interact with DRG1 protein
〔3〕 〔1〕または〔2〕に記載のポリペプチドをコードした核酸、  [3] a nucleic acid encoding the polypeptide according to [1] or [2],
〔4〕 〔3〕に記載の核酸を担持したベクター、 [4] A vector carrying the nucleic acid according to [3],
〔5〕 〔1〕または〔2〕に記載のポリペプチドに対する抗体、好ましくはポリクローナル抗 体、モノクローナル抗体、キメラ抗体、ヒト型抗体またはこれらの混合物から選択され る抗体、  [5] An antibody against the polypeptide according to [1] or [2], preferably an antibody selected from a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a human antibody, or a mixture thereof,
〔6〕 下記（1)または（2)に記載の遺伝子の転写産物に対してアニーリングする活性 を有し、 15塩基長以上、好ましくは 19塩基長以上を備えた核酸、  [6] A nucleic acid having an activity of annealing to the transcription product of the gene described in (1) or (2) below, having a length of 15 bases or more, preferably 19 bases or more,
(1)配列番号： 1 (ヒト dfrplの塩基配列）または配列番号： 3 (マウス dfrplの塩基配列） に記載の塩基配列からなる遺伝子  (1) Gene consisting of the base sequence described in SEQ ID NO: 1 (base sequence of human dfrpl) or SEQ ID NO: 3 (base sequence of mouse dfrpl)
(2)配列番号： 1 (ヒト dfrplの塩基配列）または配列番号： 3 (マウス dfrplの塩基配列） に記載の塩基配列とストリンジェントな条件下でハイブリダィズする遺伝子  (2) Gene that hybridizes under stringent conditions with the nucleotide sequence of SEQ ID NO: 1 (base sequence of human dfrpl) or SEQ ID NO: 3 (base sequence of mouse dfrpl)
〔7〕 下記（1)または（2)に記載の遺伝子の転写産物に対してアニーリングする活性 を有し、 15塩基長以上、好ましくは 19塩基長以上を備えた核酸、  [7] A nucleic acid having an activity of annealing to the transcription product of the gene described in (1) or (2) below, having a length of 15 bases or more, preferably 19 bases or more,
(1)配列番号： 9 (ヒト drglの塩基配列）または配列番号： 10 (マウス drglの塩基配列） に記載の塩基配列からなる遺伝子 (1) SEQ ID NO: 9 (base sequence of human drgl) or SEQ ID NO: 10 (base sequence of mouse drgl) A gene comprising the base sequence described in
(2)配列番号： 9 (ヒト drglの塩基配列）または配列番号： 10 (マウス drglの塩基配列） に記載の塩基配列とストリンジェントな条件下でハイブリダィズする遺伝子  (2) A gene that hybridizes under stringent conditions with the nucleotide sequence of SEQ ID NO: 9 (base sequence of human drgl) or SEQ ID NO: 10 (base sequence of mouse drgl)
〔8〕 〔1〕もしくは〔2〕に記載のポリペプチドまたは〔3〕に記載の核酸を有効成分とす る、 DRG1蛋白質安定化剤、 [8] A DRG1 protein stabilizer comprising the polypeptide according to [1] or [2] or the nucleic acid according to [3] as an active ingredient,
〔9〕 〔1〕もしくは〔2〕に記載のポリペプチドまたは〔3〕に記載の核酸を有効成分とす る、細胞運動抑制剤、  [9] A cell motility inhibitor comprising the polypeptide according to [1] or [2] or the nucleic acid according to [3] as an active ingredient,
〔10〕 〔6〕または〔7〕に記載の核酸を有効成分とする、癌細胞増殖抑制剤、 〔11〕 下記（1)から（3)の工程を有する、細胞運動抑制剤のスクリーニング方法。 [10] A cancer cell growth inhibitor comprising the nucleic acid according to [6] or [7] as an active ingredient, [11] A screening method for a cell motility inhibitor comprising the following steps (1) to (3).
(1)被験物質存在下および非存在下で DRG1蛋白質および〔1〕もしくは〔2〕に記載の ポリペプチドを作用させる工程 (1) A step of allowing the DRG1 protein and the polypeptide according to [1] or [2] to act in the presence and absence of a test substance
(2) DRG1蛋白質と〔1〕もしくは〔2〕に記載のポリペプチドとの結合活性を測定するェ 程  (2) A process for measuring the binding activity between the DRG1 protein and the polypeptide according to [1] or [2]
(3)被験物質存在下における結合活性が被験物質非存在下の結合活性よりも増加 して 、る場合に、その被験物質を細胞運動抑制剤の候補として選択する工程 〔12〕 下記（1)および（2)の工程を有する細胞運動抑制剤のスクリーニング方法、 (3) When the binding activity in the presence of the test substance is greater than the binding activity in the absence of the test substance, the test substance is selected as a candidate for cell motility inhibitor [12] And a screening method for a cell motility inhibitor having the step of (2),
(1)プロモータを備えた dfrpl遺伝子を保持した細胞に被験物質を作用させる工程(1) A step of allowing a test substance to act on a cell carrying a dfrpl gene equipped with a promoter
(2) dfrpl遺伝子の発現を上昇させた被験物質を選択する工程 (2) A step of selecting a test substance with increased expression of the dfrpl gene
〔13〕 下記（1)および（2)の工程を有する細胞運動抑制剤のスクリーニング方法、 [13] A screening method for a cell motility inhibitor having the following steps (1) and (2):
(1)プロモータを備えた drgl遺伝子を保持した細胞に被験物質を作用させる工程(1) A step of allowing a test substance to act on a cell having a promoter and a drgl gene
(2) drgl遺伝子の発現を上昇させた被験物質を選択する工程 (2) A step of selecting a test substance with increased drgl gene expression
〔14〕 下記（1)から（3)の工程を有する、癌細胞増殖抑制剤のスクリーニング方法、 [14] A screening method for a cancer cell growth inhibitor, comprising the following steps (1) to (3):
(1)被験物質存在下および非存在下で DRG1蛋白質および〔1〕または〔2〕に記載の ポリペプチドを作用させる工程 (1) A step of allowing the DRG1 protein and the polypeptide according to [1] or [2] to act in the presence and absence of a test substance
(2) DRG1蛋白質と〔1〕もしくは〔2〕に記載のポリペプチドとの結合活性を測定するェ 程  (2) A process for measuring the binding activity between the DRG1 protein and the polypeptide according to [1] or [2]
(3)被験物質存在下における結合活性が被験物質非存在下の結合活性よりも低下 して 、る場合に、その被験物質を癌細胞増殖抑制剤の候補として選択する工程 〔15〕 下記（1)および（2)の工程を有する癌細胞増殖抑制剤のスクリーニング方法 (3) When the binding activity in the presence of the test substance is lower than the binding activity in the absence of the test substance, the test substance is selected as a candidate for a cancer cell growth inhibitor. [15] Screening method for cancer cell growth inhibitor having the following steps (1) and (2)
(1)プロモータを備えた dfrpl遺伝子を保持した細胞に被験物質を作用させる工程(1) A step of allowing a test substance to act on a cell carrying a dfrpl gene equipped with a promoter
(2) dfrpl遺伝子の発現を低下させた被験物質を選択する工程 (2) A step of selecting a test substance with reduced dfrpl gene expression
〔16〕 下記（1)および（2)の工程を有する癌細胞増殖抑制剤のスクリーニング方法  [16] Screening method for cancer cell growth inhibitor having the following steps (1) and (2)
(1)プロモータを備えた drgl遺伝子を保持した細胞に被験物質を作用させる工程(1) A step of allowing a test substance to act on a cell having a promoter and a drgl gene
(2) drgl遺伝子の発現を低下させた被験物質を選択する工程 (2) A step of selecting a test substance with reduced drgl gene expression
〔17〕 下記（1)または（2)に記載のポリペプチド、 [17] The polypeptide according to the following (1) or (2),
(1)配列番号: 6または配列番号: 8に記載のアミノ酸配列における 132位から 187位 のアミノ酸配列力もなるポリペプチド  (1) A polypeptide having an amino acid sequence from positions 132 to 187 in the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 8
(2)配列番号: 6または配列番号: 8に記載のアミノ酸配列における 132位から 187位 のアミノ酸配列において 1若しくは複数のアミノ酸が置換、欠失、挿入または付加され たアミノ酸配列力 なり、 DRG2蛋白質との相互作用能力を有するポリペプチド (2) A DRG2 protein comprising an amino acid sequence having one or more amino acids substituted, deleted, inserted or added in the amino acid sequence from position 132 to position 187 in the amino acid sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 8. Having the ability to interact with
〔18〕 下記（1)または（2)に記載のポリペプチド、 [18] The polypeptide according to (1) or (2) below,
(1)配列番号: 6または配列番号: 8に記載のアミノ酸配列からなるポリペプチド (1) A polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 8
(2)配列番号： 6または配列番号： 8に記載のアミノ酸配列にお!/、て 1若しくは複数の アミノ酸が置換、欠失、挿入または付加されたアミノ酸配列からなり、 DRG2蛋白質と の相互作用能力を有するポリペプチド (2) It consists of an amino acid sequence in which one or more amino acids are substituted, deleted, inserted or added to the amino acid sequence shown in SEQ ID NO: 6 or 8 and interacts with DRG2 protein Polypeptide with ability
〔19〕 〔17〕または〔18〕に記載のポリペプチドをコードした核酸、  [19] a nucleic acid encoding the polypeptide according to [17] or [18],
〔20〕 〔19〕に記載の核酸を担持したベクター、 [20] A vector carrying the nucleic acid according to [19],
〔21〕 〔17〕または〔18〕に記載のポリペプチドに対する抗体、好ましくはポリクローナ ル抗体、モノクローナル抗体、キメラ抗体、ヒト型抗体またはこれらの混合物から選択 される抗体、  [21] An antibody against the polypeptide according to [17] or [18], preferably an antibody selected from a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a humanized antibody, or a mixture thereof,
[22] 下記（1)または（2)に記載の遺伝子の転写産物に対してアニーリングする活 性を有し、 15塩基長以上、好ましくは 19塩基長以上を備えた核酸、  [22] A nucleic acid having an activity of annealing to the transcription product of the gene described in (1) or (2) below, having a length of 15 bases or more, preferably 19 bases or more,
(1)配列番号： 5 (ヒト dfrp2の塩基配列）または配列番号： 7 (マウス dfrp2の塩基配列） に記載の塩基配列からなる遺伝子 (2)配列番号： 5 (ヒト dfrp2の塩基配列）または配列番号： 7 (マウス dfrp2の塩基配列） に記載の塩基配列とストリンジェントな条件下でハイブリダィズする遺伝子 (1) A gene comprising the base sequence described in SEQ ID NO: 5 (base sequence of human dfrp2) or SEQ ID NO: 7 (base sequence of mouse dfrp2) (2) A gene that hybridizes under stringent conditions with the nucleotide sequence of SEQ ID NO: 5 (base sequence of human dfrp2) or SEQ ID NO: 7 (base sequence of mouse dfrp2)
〔23〕 下記（1)または（2)記載の遺伝子の転写産物に対してアニーリングする活性 を有し、 15塩基長以上、好ましくは 19塩基長以上を備えた核酸、 [23] a nucleic acid having an activity of annealing to the transcription product of the gene described in (1) or (2) below, having a length of 15 bases or more, preferably 19 bases or more,
(1)配列番号： 11 (ヒト drg2の塩基配列）または配列番号： 12 (マウス drg2の塩基配列 )に記載の塩基配列からなる遺伝子  (1) A gene comprising the base sequence described in SEQ ID NO: 11 (base sequence of human drg2) or SEQ ID NO: 12 (base sequence of mouse drg2)
(2)配列番号： 11 (ヒト drg2の塩基配列）または配列番号： 12 (マウス drg2の塩基配列 )に記載の塩基配列とストリンジェントな条件下でハイブリダィズする遺伝子 (2) A gene that hybridizes under stringent conditions with the nucleotide sequence of SEQ ID NO: 11 (base sequence of human drg2) or SEQ ID NO: 12 (base sequence of mouse drg2)
〔24〕 〔17〕もしくは〔18〕に記載のポリペプチドまたは〔19〕に記載の核酸を有効成 分とする、 DRG2蛋白質安定化剤、 [24] A DRG2 protein stabilizer comprising the polypeptide according to [17] or [18] or the nucleic acid according to [19] as an effective component,
〔25〕 〔22〕または〔23〕に記載の核酸を有効成分とする、細胞間接着促進剤、 〔26〕 〔22〕または〔23〕に記載の核酸を有効成分とする、細胞運動抑制剤、  [25] An intercellular adhesion promoter comprising the nucleic acid according to [22] or [23] as an active ingredient, [26] A cell movement inhibitor comprising the nucleic acid according to [22] or [23] as an active ingredient ,
[27] 〔22〕または〔23〕に記載の核酸を有効成分とする、癌細胞増殖抑制剤、 〔28〕 下記（1)力 (3)の工程を有する、細胞間接着促進剤または細胞運動抑制剤 のスクリーニング方法、 [27] A cancer cell growth inhibitor comprising the nucleic acid according to [22] or [23] as an active ingredient, [28] an intercellular adhesion promoter or cell movement having the following step (1) force (3) Screening method for inhibitors,
(1)被験物質存在下および非存在下で DRG2蛋白質および〔 17〕または〔 18〕に記載 のポリペプチドを作用させる工程  (1) A step of allowing the DRG2 protein and the polypeptide according to [17] or [18] to act in the presence and absence of a test substance
(2) DRG2蛋白質と〔 17〕もしくは〔 18〕に記載のポリペプチドとの結合活性を測定する 工程  (2) measuring the binding activity between the DRG2 protein and the polypeptide according to [17] or [18]
(3)被験物質存在下における結合活性が被験物質非存在下の結合活性よりも低下 して!ヽる場合に、その被験物質を細胞間接着促進剤または細胞運動抑制剤の候補 として選択する工程  (3) When the binding activity in the presence of the test substance is lower than the binding activity in the absence of the test substance !, the test substance is selected as a candidate for an intercellular adhesion promoter or cell motility inhibitor.
〔29〕 下記（1)および (2)の工程を有する、細胞間接着促進剤または細胞運動抑制 剤のスクリーニング方法、  [29] A screening method for an intercellular adhesion promoter or cell movement inhibitor, comprising the following steps (1) and (2):
(1)プロモータを備えた dfrp2遺伝子を保持した細胞に被験物質を作用させる工程 (1) A step of allowing a test substance to act on a cell having a dfrp2 gene having a promoter
(2) dfrp2遺伝子の発現を低下させた被験物質を選択する工程 (2) A step of selecting a test substance with reduced expression of the dfrp2 gene
〔30〕 下記（1)および (2)の工程を有する、細胞間接着促進剤または細胞運動抑制 剤のスクリーニング方法、 (1)プロモータを備えた drg2遺伝子を保持した細胞に被験物質を作用させる工程[30] A screening method for an intercellular adhesion promoter or cell motility inhibitor comprising the following steps (1) and (2): (1) A step of allowing a test substance to act on a cell having a promoter and a drg2 gene
(2) drg2遺伝子の発現を低下させた被験物質を選択する工程 (2) A step of selecting a test substance with reduced drg2 gene expression
〔31〕 下記（1)から（3)の工程を有する、癌細胞増殖抑制剤のスクリーニング方法、 [31] A screening method for a cancer cell growth inhibitor, comprising the following steps (1) to (3):
(1)被験物質存在下および非存在下で DRG2蛋白質および〔 17〕または〔 18〕に記載 のポリペプチドを作用させる工程 (1) A step of allowing the DRG2 protein and the polypeptide according to [17] or [18] to act in the presence and absence of a test substance
(2) DRG2蛋白質と〔 17〕もしくは〔 18〕に記載のポリペプチドとの結合活性を測定する 工程  (2) measuring the binding activity between the DRG2 protein and the polypeptide according to [17] or [18]
(3)被験物質存在下における結合活性が被験物質非存在下の結合活性よりも低下 して 、る場合に、その被験物質を癌細胞増殖抑制剤の候補として選択する工程 〔32〕 下記（1)および（2)の工程を有する、癌細胞増殖抑制剤のスクリーニング方 法、  (3) the step of selecting the test substance as a candidate for a cancer cell growth inhibitor when the binding activity in the presence of the test substance is lower than the binding activity in the absence of the test substance [32] ) And (2), a method for screening a cancer cell growth inhibitor,
(1)プロモータを備えた dfrp2遺伝子を保持した細胞に被験物質を作用させる工程 (1) A step of allowing a test substance to act on a cell having a dfrp2 gene having a promoter
(2) dfrp2遺伝子の発現を低下させた被験物質を選択する工程 (2) A step of selecting a test substance with reduced expression of the dfrp2 gene
〔33〕 下記（1)および（2)の工程を有する、癌細胞増殖抑制剤のスクリーニング方 法、  [33] A screening method for a cancer cell growth inhibitor comprising the following steps (1) and (2):
(1)プロモータを備えた drg2遺伝子を保持した細胞に被験物質を作用させる工程 (1) A step of allowing a test substance to act on a cell having a promoter and a drg2 gene
(2) drg2遺伝子の発現を低下させた被験物質を選択する工程 (2) A step of selecting a test substance with reduced drg2 gene expression
〔34〕 配列番号： 9または 10に記載の遺伝子にコードされたポリペプチドに対する抗 体であって、配列番号： 11または 12に記載の遺伝子にコードされたポリペプチドとク ロスリアクティビティ一を有しない、抗 DRG1蛋白質抗体、  [34] An antibody against the polypeptide encoded by the gene set forth in SEQ ID NO: 9 or 10, and having the same cross activity as the polypeptide encoded by the gene set forth in SEQ ID NO: 11 or 12. No, anti-DRG1 protein antibody,
〔35〕 配列番号： 11または 12に記載の遺伝子にコードされたポリペプチドに対する 抗体であって、配列番号： 9または 10に記載の遺伝子にコードされたポリペプチドとク ロスリアクティビティ一を有しない、抗 DRG2蛋白質抗体、  [35] An antibody against the polypeptide encoded by the gene set forth in SEQ ID NO: 11 or 12, and does not have the same cross activity as the polypeptide encoded by the gene set forth in SEQ ID NO: 9 or 10. Anti-DRG2 protein antibody,
〔36〕 〔9〕または〔26〕に記載の細胞運動抑制剤を患者に投与する工程を含む、癌 治療方法、  [36] A method for treating cancer, comprising a step of administering the cell motility inhibitor according to [9] or [26] to a patient,
〔37〕 〔10〕または〔27〕に記載の癌細胞増殖抑制剤を患者に投与する工程を含む 、癌治療方法、  [37] A method for treating cancer, comprising a step of administering the cancer cell proliferation inhibitor according to [10] or [27] to a patient,
〔38〕 [25]に記載の細胞間接着促進剤を患者に投与する工程を含む、癌治療方 法、に関する。 [38] A method for treating cancer, comprising a step of administering the intercellular adhesion promoter according to [25] to a patient. Law.
図面の簡単な説明 Brief Description of Drawings
[図 1]図 1は、 DFRPの同定に関する図および写真である。（A)マウス DFRP2 (GIR2)お よび DFRP1 (LEREP04)のドメイン構造ならびに部分配列アラインメントについて示す 図である。 DFRP1および DFRP2は、本発明者らが「DFRPドメイン」（斜線ボックス）と名 づけた相同性の高い領域を有していた。マウス（M. musculus) (Mm)、キイ口ショウジョ ゥバエ（D. melanogaster) (Dm)および S.セレビシェ（S.cerevisiae) (Sc) DFRP2および DFRP1のペプチド配列をアラインメントした。 NCBIァクセッション番号はそれぞれ、 XP —125585 (DFRP2— Mm)、 NP.651227 (DFRP2— Dm)、 NP_010436 (DFRP2— Sc)、 NP— 0812 10 (DFRPLMn)、 NP— 610401 (DFRP1— Dm)および NP— 014734 (DFRP1— Sc)である。（B) DFRP2 (GIR2)または DFRP1 (LEREP04)と DRG蛋白質の相互作用につ!/、て示す図 である。 FLAG-標識マウス DRG1もしくは DRG2、または空の対照および Myc標識マウ ス DFRP2または DFRP1の発現ベクターを同時形質移入した 293T細胞の抽出物を抗 FLAG抗体を用いて免疫沈降した。ウェスタンプロット上の免疫沈降 (IP)複合体また は細胞溶解物を抗 Myc抗体で探索して、 DFRPを検出した。このメンブレンをストリッピ ング後に再度用いて、抗 FLAG抗体で再度探索して DRG蛋白質を検出した。 （C)イン ビボにおける結合アツセィについて示す図である。 HeLa S3細胞の蛋白質抽出液を D FRP1、 DRG1および対照 IgGに対する抗体を用いて免疫沈降した。次いで、免疫沈 降複合体を抗 -DRG1抗体で探索した。このメンブレンは繰り返しストリツビングを行つ て、抗 DFRP1および抗 DRG2抗体で繰り返し再探索した。（D) DFRP1および DRG1の 細胞下局在化の免疫蛍光分析について示す写真である。 HeLa S3細胞を、 DFRP1 および DRG1に対するポリクローナル抗体で染色した。 [FIG. 1] FIG. 1 is a diagram and a photograph relating to the identification of DFRP. (A) Domain structure and partial sequence alignment of mouse DFRP2 (GIR2) and DFRP1 (LEREP04). DFRP1 and DFRP2 had a region of high homology that we named “DFRP domain” (hatched box). The peptide sequences of mouse (M. musculus) (Mm), D. melanogaster (Dm) and S. cerevisiae (Sc) DFRP2 and DFRP1 were aligned. NCBI accession numbers are XP-125585 (DFRP2—Mm), NP.651227 (DFRP2—Dm), NP_010436 (DFRP2—Sc), NP—0812 10 (DFRPLMn), NP—610401 (DFRP1—Dm) and NP—014734 (DFRP1—Sc). (B) shows the interaction between DFRP2 (GIR2) or DFRP1 (LEREP04) and DRG protein. Extracts of 293T cells co-transfected with FLAG-labeled mouse DRG1 or DRG2, or empty control and Myc-labeled mouse DFRP2 or DFRP1 expression vector were immunoprecipitated using anti-FLAG antibody. Immunoprecipitation (IP) complexes or cell lysates on the Western plot were probed with anti-Myc antibody to detect DFRP. This membrane was used again after stripping, and was searched again with an anti-FLAG antibody to detect DRG protein. (C) It is a figure shown about the joint assembly in an in vivo. The protein extract of HeLa S3 cells was immunoprecipitated using antibodies against DFRP1, DRG1 and control IgG. The immunoprecipitation complex was then probed with anti-DRG1 antibody. The membrane was repeatedly stripped and re-searched repeatedly with anti-DFRP1 and anti-DRG2 antibodies. (D) Photograph showing immunofluorescence analysis of subcellular localization of DFRP1 and DRG1. HeLa S3 cells were stained with polyclonal antibodies against DFRP1 and DRG1.
[図 2]図 2は、 DFRPと DRG蛋白質の結合のための DFRPドメインの必要性について示 す図および写真である。（A) DFRP2 (上のセクション)および DFRP1部位欠失変異体（ 下のセクション）の構造マップである。斜線ボックスは DFRPドメインを示す。グレーの 塗りつぶしのバーは、図 1 (A)に示す真核生物において DFRP1の高度に保存されて いる領域に相当する。（B) DRG2と相互作用するための DFRP2の必須領域の決定に ついて示す図である。 GST融合 DFRP2 11またはその部位欠失変異体および FLAG標 識 DRG2の発現ベクターを同時形質移入した 293T細胞の抽出物をダルタチオンセフ ァロースビーズと共にインキュベーションした。次いで、プルダウンした沈殿物または 細胞溶解液を抗 FLAG抗体で探索して、 FLAG- DRG2を検出した。同じメンブレンを ストリツビング後、抗 GST抗体で再度探索して、 GST-DFRP2 11およびその部位欠失変 異体を検出した。 （C) DRGlとの相互作用のための DFRP1における必須領域の決定 について示す図である。 FLAG標識 DFRP1 11またはその部位欠失変異体および Myc 標識 DRG1の発現ベクターを同時形質移入した 293T細胞の抽出物を抗 FLAG抗体を 用いて免疫沈降した。次いで、免疫沈降物（IP)または細胞溶解液を抗 Myc抗体で探 索して、 Myc-DRGlを検出した。同じメンブレンをストリツビング後に抗 FLAG抗体で再 度探索して FLAG-DFRP1 11およびその部位欠失変異体を検出した。 [FIG. 2] FIG. 2 is a diagram and a photograph showing the necessity of a DFRP domain for binding of DFRP and DRG protein. (A) Structural map of DFRP2 (upper section) and DFRP1 site deletion mutant (lower section). The hatched box indicates the DFRP domain. The gray filled bar corresponds to the highly conserved region of DFRP1 in eukaryotes shown in Figure 1 (A). (B) This figure shows the determination of the essential region of DFRP2 to interact with DRG2. GST fusion DFRP2 11 or its site deletion mutant and FLAG label An extract of 293T cells co-transfected with the DRG2 expression vector was incubated with dartathione sepharose beads. The pull-down precipitate or cell lysate was then probed with anti-FLAG antibody to detect FLAG-DRG2. After stripping the same membrane, GST-DFRP211 and its site deletion mutant were detected by searching again with an anti-GST antibody. (C) Determination of essential regions in DFRP1 for interaction with DRGl. Extracts of 293T cells co-transfected with FLAG-tagged DFRP11 11 or its site deletion mutant and Myc-tagged DRG1 expression vector were immunoprecipitated using anti-FLAG antibody. Subsequently, immunoprecipitate (IP) or cell lysate was searched with anti-Myc antibody to detect Myc-DRGl. The same membrane was stripped and then re-searched with anti-FLAG antibody to detect FLAG-DFRP111 and its site deletion mutants.
[図 3]図 3は、 DFRPによる DRG蛋白質の発現の調節について示す写真である。（A) D FRPとの同時発現による DRG蛋白質の発現の増加について示す写真である。 Myc標 識 DRG1もしくは DRG2と、発現ベクターまたは FLAG標識 DFRP1もしくは DFRP2の発 現ベクターを同時形質移入した 293T細胞の抽出物を、抗 Mycおよび抗 FLAG抗体を 用いるウェスタンブロット法によって分析した。（B) DFRPの結合による DRG蛋白質の ュビキチン化の阻害について示す写真である。 HA-ュビキチン（Ub) (3 /^)ぉょび1^ LAG-DRG1 (左のパネル）または DRG2 (右のパネル）（3 μ g)をコードする発現べクタ 一を Myc-DFRPlまたは DFRP2 (1または 5 μ g)と共に形質移入した 293T細胞を 10 μ Μの MG132の存在下（+)または非存在下（-)で 3時間処理してから回収した。 IPパネ ル、細胞溶解液を抗 FLAG抗体を用いて免疫沈降した。免疫沈降した複合体を抗 H A抗体で探索して、ポリュビキチン鎖を検出した。同じメンブレンを繰り返しストリツピン グ後に用いて、抗 FLAGおよび抗 Myc抗体で繰り返し再探索して、それぞれ、 DRGお よび DFRPを検出した。黒矢印および白矢印は、それぞれ、完全長の Myc-DFRPlお よび Myc- DFRP2を示す。このメンブレンでは、 FLAG- DRG1および FLAG- DRG2の量 が全てのレーンで等しくなるようにアプライするタンパク量を調整した。これを実施す るために、各試料のロード量は、同じ試料を用いて別のプロットで濃度を予備的に評 価することによって決定した。溶解液パネル、免疫沈降反応に使用した細胞溶解液 を抗 Myc抗体を用いるウェスタンブロット法で分析して、 DFRPを検出した。同じメンブ レンをストリツビング後、抗 FLAG抗体で再探索して、 DRG1および DRG2を検出した。 このメンブレンでは、本発明者らは、等しい数の細胞の抽出液を SDS-PAGEにロード した。 FIG. 3 is a photograph showing the regulation of DRG protein expression by DFRP. (A) It is a photograph showing an increase in the expression of DRG protein by co-expression with DFRP. Extracts of 293T cells co-transfected with Myc-labeled DRG1 or DRG2 and expression vector or FLAG-tagged DFRP1 or DFRP2 expression vector were analyzed by Western blotting using anti-Myc and anti-FLAG antibodies. (B) A photograph showing inhibition of ubiquitination of DRG protein by DFRP binding. The expression vector encoding HA-ubiquitin (Ub) (3 / ^) and 1 ^ LAG-DRG1 (left panel) or DRG2 (right panel) (3 μg) is Myc-DFRPl or DFRP2 ( 293T cells transfected with 1 or 5 μg) were treated for 3 hours in the presence (+) or absence (−) of 10 μM MG132 and then harvested. The IP panel and cell lysate were immunoprecipitated using an anti-FLAG antibody. The immunoprecipitated complex was searched with an anti-HA antibody to detect a polyubiquitin chain. The same membrane was used after repeated stripping and re-searched repeatedly with anti-FLAG and anti-Myc antibodies to detect DRG and DFRP, respectively. The black and white arrows indicate full-length Myc-DFRPl and Myc-DFRP2, respectively. In this membrane, the amount of protein applied was adjusted so that the amount of FLAG-DRG1 and FLAG-DRG2 was the same in all lanes. To do this, the loading of each sample was determined by preliminarily assessing the concentration on a separate plot using the same sample. The lysate panel and the cell lysate used for immunoprecipitation were analyzed by Western blot using anti-Myc antibody to detect DFRP. Same membrane After stripping the len, it was re-searched with anti-FLAG antibody to detect DRG1 and DRG2. For this membrane, we loaded an equal number of cell extracts onto SDS-PAGE.
[図 4]図 4は、 DT40細胞における dfrpl遺伝子の崩壊について示す写真である。 (A) 部分的な-ヮトリ (Gallus gallus) dfrpl遺伝子座およびノックアウト構築物の構造であ る。斜線ボックスは-ヮトリ dfrpl遺伝子のェキソンを示す。左のェキソンは ZnF-1ドメイ ンを含有する。このェキソンを標的とするために、ブラストサイジン (Bsr) -およびヒスチ ジノール (HisD) -耐性遺伝子に、上流および下流ゲノムアームを隣接させた。得られ た標的ベクターは、それぞれ、 dfrplBsrおよび dfrplHidDと名づけた。マップ以外に、 考えられる組換えクローンのサザンブロット分析に使用する Apal (A)および BamHI (B )制限部位の位置を示す。 (B) DT40野生型 (+/+)および dfrpl+/—または dfrpl 細胞か ら作製した Apal-BamHI-ダブル消化ゲノム DNAのサザンブロット分析について示す 写真である。 （C) DT40野生型（+/+)および dfrpl—細胞における dfrpl mRNA発現のノ 一ザンブロット分析にっ 、て示す写真である。臭化工チジゥム（EtBr) -染色 rRNAを口 ード対照として示す。 (D) DT40野生型（+/+)および dfrplチ細胞のウェスタンブロット 分析につ 、て示す写真である。チューブリンを内部ロード対照として染色した。 FIG. 4 is a photograph showing the disruption of the dfrpl gene in DT40 cells. (A) Structure of a partial Gallus gallus dfrpl locus and knockout construct. The hatched box indicates the exon of the duckpl dfrpl gene. The left exon contains the ZnF-1 domain. To target this exon, the blasticidin (Bsr)-and histidinol (HisD)-resistance genes were flanked by upstream and downstream genomic arms. The obtained target vectors were named dfrplBsr and dfrplHidD, respectively. In addition to the map, the location of the Apal (A) and BamHI (B) restriction sites used for Southern blot analysis of possible recombinant clones is indicated. (B) Photograph showing Southern blot analysis of Apal-BamHI-double digested genomic DNA prepared from DT40 wild type (+ / +) and dfrpl +/- or dfrpl cells. (C) Northern blot analysis of dfrpl mRNA expression in DT40 wild type (+ / +) and dfrpl-cells. Brominated chitin (EtBr) -stained rRNA is shown as a mouth control. (D) Photograph showing Western blot analysis of DT40 wild type (+ / +) and dfrpl cells. Tubulin was stained as an internal loading control.
[図 5]図 5は、インビボにおける DFRP1による DRG1発現の調節について示す写真で ある。（A) DT40野生型、 dfrpl—,—、 dfrpl"' milおよび dfrpl—,— m A Dl細胞 (mouse DFRP1 flまたは Δ Dl (図 2(A》をレスキューさせた細胞)のウェスタンブロット（上のパネル）お よびノーザンブロット（下のパネル)分析につ 、て示す写真である。全細胞抽出液を S DS- PAGEで分離し、次いで、 DRG1、 DRG2、 DFRP1およびチューブリン（内部ロード 対照用）に対する抗体で検出した。メンブレンは繰り返しストリツビングを行い、繰り返 し使用した。レーンあたりの総細胞数を等しくした (2.5 X 105細胞/レーン)。ノーザン ブロット分析のためには、ウェスタンブロット分析に使用した培養皿力 細胞を回収し た。総 RNAを単離し、 1.2%ホルムアルデヒド変性ゲルで分離し、ナイロン膜に移し、放 射性標識した-ヮトリ drgl、 drg2または gapdh (内部対照）部分 cDNAプローブで探索し た。同じ膜を繰り返し使用した。バンド強度は Fujifilm BAS2000バイオイメージングォ ートアナライザーで定量した。数値結果は、野生型細胞の発現に対する dfrPr/_、 dfrp Γ/_ milおよび dfrpr/_ m Δ Dl細胞における発現の比である。（B) DFRP1と DRG1の結合 を示す免疫沈降分析について示す写真である。
Figure imgf000016_0001
dfrpl+mflおよ び dfrpr/_ m Δ D1細胞の数は、各抽出液における DRG1蛋白質の総量を等しくするた めに、それぞれ、 1:7:1:7に調節した。細胞抽出液を抗 DFRP1抗体を用いて免疫沈降 した。次いで、ウェスタンプロット分析において免疫沈降複合体および細胞溶解液を 抗 DRG1または抗 DFRP1抗体で探索した。 FIG. 5 is a photograph showing the regulation of DRG1 expression by DFRP1 in vivo. (A) Western blot of DT40 wild type, dfrpl —, —, dfrpl "'mil and dfrpl —, — mA Dl cells (mouse DFRP1 fl or Δ Dl (cells rescued in FIG. 2 (A)) (top Panel) and Northern blot (bottom panel) analysis, whole cell extracts separated by SDS-PAGE, then DRG1, DRG2, DFRP1 and tubulin (for internal load control) were detected with antibodies against. membrane iterates Sutoritsubingu was used Shi repeatedly. was equal the total number of cells per lane (2.5 X 10 5 cells / lane) for. Northern blot analysis, Western blot analysis Culture dish used Cells were harvested Total RNA was isolated, separated on 1.2% formaldehyde denaturing gel, transferred to nylon membrane, and radiolabeled-spider drgl, drg2 or gapdh (internal control) partial cDNA probe Was searched. The same film was used repeatedly. Band intensities were quantified by Fujifilm BAS2000 bioimaging O over preparative analyzer. Numerical results, dfr P r / _ on the expression of wild-type cells, Dfrp Ratio of expression in Γ / _mil and dfrpr / _mΔDl cells. (B) A photograph showing immunoprecipitation analysis showing the binding of DFRP1 and DRG1.
Figure imgf000016_0001
The number of dfrpl + mfl and dfrpr / _mΔD1 cells was adjusted to 1: 7: 1: 7, respectively, to equalize the total amount of DRG1 protein in each extract. The cell extract was immunoprecipitated using anti-DFRP1 antibody. The immunoprecipitation complexes and cell lysates were then probed with anti-DRG1 or anti-DFRP1 antibodies in Western plot analysis.
[図 6A]図 6Aは、アフリカッメガエル（X. laevis)における dfrplおよび drglの発現分析 につ 、て示す写真である。 (A)ゼノパス（Xenopus)胚発生中の dfrplおよび drgl転写 物の空間的発現である。（a, b)腹側像、前方左；（c, d)背側像、前方左；（e〜j)側方 像、前方左；（g'および h')それぞれ、 gおよび hに図示した胚の前方部分の高倍率像 である；（i, j)ベンジルアルコール：安息香酸ベンジル（2:1)による胚の透明化である。 略語： ba,鰓弓； bcs,鰓稜セグメント（branchial crest segment)； bi,血島； de,発生中 の眼； e,眼； ft),前脳； hb,後脳； hcs,舌骨稜セグメント（hyoid crest segment) ;le,水 晶体； mb,中月 ;mcs,下顎稜セグメント (mandibular crest segment)； nc,脊索； ov,耳 胞; pr,前腎; sc,脊髄; sm,体節; tnc,体幹神経冠。  FIG. 6A is a photograph showing the expression analysis of dfrpl and drgl in Xenopus laevis (X. laevis). (A) Spatial expression of dfrpl and drgl transcripts during Xenopus embryonic development. (A, b) ventral view, front left; (c, d) dorsal view, front left; (e-j) side view, front left; (g 'and h'), shown in g and h, respectively (I, j) Benzyl alcohol: Embryo clarification with benzyl benzoate (2: 1). Abbreviations: ba, arch; bcs, branch crest segment; bi, blood island; de, developing eye; e, eye; ft), forebrain; hb, hindbrain; hcs, hyoid crest Segment (hyoid crest segment); le, crystal; mb, middle moon; mcs, mandibular crest segment; nc, notochord; ov, otocyst; pr, pronephros; sc, spinal cord; sm, segment ; tnc, trunk nerve trunk.
[図 6BC]図 6BCは、アフリカッメガエル（X. laevis)における dfrplおよび drglの発現分 祈について示す写真である。（B)成体ゼノパスにおける dfrpl mRNAの組織-特異的 発現について示す写真である。総 RNAを示した成体組織から単離し、ノーザンブロッ ト法に使用した。（C)アフリカッメガエル (X. laevis)胚発生中の dfrpl転写物の時間的 な発現である。総 RNAを示した発生段階においてゼノパス胚力 単離し、ノーザンブ ロット法に使用した。  [FIG. 6BC] FIG. 6BC is a photograph showing the expression pattern of dfrpl and drgl in Xenopus laevis (X. laevis). (B) Photograph showing the tissue-specific expression of dfrpl mRNA in an adult xenopath. Total RNA was isolated from the adult tissue shown and used in the Northern blot method. (C) Temporal expression of dfrpl transcripts during X. laevis embryo development. Xenopus embryos were isolated at the indicated developmental stage and used in the Northern blot method.
[図 7]図 7は、ドキシサイクリン（Doxycyclin)添カ卩による DRG1、 DRG2、 DFRP1、 DFRP2 のノックダウン細胞の評価、および細胞運動 ·形態 ·細胞骨格を制御する遺伝子の m RNAの発現変化を示す写真である。  [Fig. 7] Fig. 7 shows the evaluation of DRG1, DRG2, DFRP1, and DFRP2 knockdown cells by Doxycyclin supplementation and the changes in mRNA expression of genes that control cell motility, morphology and cytoskeleton It is a photograph.
[図 8]図 8は、 DRG1ノックダウンの形態変化を示す写真である。下の写真は上の写真 の拡大図である。  [Fig. 8] Fig. 8 is a photograph showing a change in morphology of DRG1 knockdown. The lower photo is an enlarged view of the upper photo.
[図 9]図 9は、 DRG1ノックダウン細胞のァクチン骨格の変化を示す写真である。  FIG. 9 is a photograph showing changes in the actin skeleton of DRG1 knockdown cells.
[図 10]図 10は、 DRG1ノックダウン細胞の細胞運動性評価を示すグラフである。 [図 11]図 11は、 DFRP1ノックダウン細胞の形態変化を示す写真である。 HeLaS3に D FRP1 RNAiベクターを導入したもの（右）、及びそのコントロールとして空ベクターを導 入したもの (左）を示す。 FIG. 10 is a graph showing cell motility evaluation of DRG1 knockdown cells. FIG. 11 is a photograph showing the morphological change of DFRP1 knockdown cells. HeLaS3 with DFRP1 RNAi vector introduced (right) and control with an empty vector introduced (left).
[図 12]図 12は、 DRG2ノックダウンの形態変化を示す写真である。  [Fig. 12] Fig. 12 is a photograph showing a change in morphology of DRG2 knockdown.
[図 13]図 13は、一過的な DRG2ノックダウン細胞の形態変化を示す写真である。 HeL aS3〖こ DRG2 RNAiベクターを導入したもの（右）及びそのコントロールとして空べクタ 一を導入したもの (左)を示す。  FIG. 13 is a photograph showing a transient morphological change of DRG2 knockdown cells. HeLas3 〖DRG2 RNAi vector introduced (right) and empty vector as a control (left) are shown.
[図 14]図 14は、 DRG1、 DRG2ノックダウン細胞の growth変化を示すグラフである。  FIG. 14 is a graph showing changes in growth of DRG1 and DRG2 knockdown cells.
[図 15]図 15は、 DRGファミリーおよび DFRPsの制御のモデル図である。  FIG. 15 is a model diagram of control of the DRG family and DFRPs.
[図 16]図 16は、 DRG1と DRG2のマウス肝臓ホモジネートを遠心により粗分画につい て示す図および写真である。左は分画手順を示す図であり、右はウェスタンプロット 分析の結果を示す写真である。  FIG. 16 is a diagram and a photograph showing a crude fraction obtained by centrifuging mouse liver homogenates of DRG1 and DRG2. The left is a diagram showing the fractionation procedure, and the right is a photograph showing the results of Western plot analysis.
[図 17]図 17は、ショ糖勾配によるポリソーム分画について示すグラフおよび写真であ る。  FIG. 17 is a graph and a photograph showing polysome fractionation by sucrose gradient.
発明を実施するための最良の形態  BEST MODE FOR CARRYING OUT THE INVENTION
[0018] 本発明者らは、細胞増殖の重要な調節因子として作用すると考えられている DRG1 蛋白質および DRG2蛋白質と特異的に結合することによって、これら蛋白質の発現を 調節することができる新規な蛋白質 DFRP1および DFRP2を同定した。  [0018] The present inventors have developed a novel protein capable of regulating the expression of these proteins by specifically binding to DRG1 protein and DRG2 protein, which are considered to act as important regulators of cell proliferation. DFRP1 and DFRP2 were identified.
[0019] 本発明では、ヒト dfrpl遺伝子の塩基配列を配列番号： 1に、ヒト DFRP1蛋白質のアミ ノ酸配列を配列番号： 2に、マウス dfrpl遺伝子の塩基配列を配列番号： 3に、マウス D FRP1蛋白質のアミノ酸配列を配列番号: 4に、ヒト dfrp2遺伝子の塩基配列を配列番 号： 5に、ヒト DFRP2蛋白質のアミノ酸配列を配列番号： 6に、マウス dfrp2遺伝子の塩 基配列を配列番号： 7に、マウス DFRP2蛋白質のアミノ酸配列を配列番号： 8に、ヒト dr glの塩基配列を配列番号： 9に、マウス drgl遺伝子の塩基配列を配列番号： 10に、ヒ ト drg2遺伝子の塩基配列を配列番号： 11に、マウス drg2遺伝子の塩基配列を配列番 号： 12にそれぞれ示す。  In the present invention, the base sequence of the human dfrpl gene is shown in SEQ ID NO: 1, the amino acid sequence of the human DFRP1 protein is shown in SEQ ID NO: 2, the base sequence of the mouse dfrpl gene is shown in SEQ ID NO: 3, and the mouse D The amino acid sequence of the FRP1 protein is SEQ ID NO: 4, the nucleotide sequence of the human dfrp2 gene is SEQ ID NO: 5, the amino acid sequence of the human DFRP2 protein is SEQ ID NO: 6, and the base sequence of the mouse dfrp2 gene is SEQ ID NO: 7 shows the amino acid sequence of the mouse DFRP2 protein in SEQ ID NO: 8, the base sequence of human drgl in SEQ ID NO: 9, the base sequence of the mouse drgl gene in SEQ ID NO: 10, and the base sequence of the human drg2 gene. SEQ ID NO: 11 shows the base sequence of mouse drg2 gene in SEQ ID NO: 12.
[0020] また上記配列番号： 1〜12に関する情報を以下に示す。  [0020] Information on SEQ ID NOs: 1 to 12 is shown below.
配列番号: 1 human DFRPl cDNA配列 SEQ ID NO: 1 human DFRPl cDNA sequence
gi| 18088891 |gb|BC021102. l| Homo sapiens likely ortholog of mouse immediate early response, erythropoietin 4, mRNA (cDNA clone MGC:31837 IMAGE:5020890), com plete cds  gi | 18088891 | gb | BC021102. l | Homo sapiens likely ortholog of mouse immediate early response, erythropoietin 4, mRNA (cDNA clone MGC: 31837 IMAGE: 5020890), com plete cds
[0021] 配列番号： 2 [0021] SEQ ID NO: 2
human DFRPlアミノ酸配列  human DFRPl amino acid sequence
> gi|l8088892|gb|AAH21102. l| LEREP04 protein [Homo sapiens]  > gi | l8088892 | gb | AAH21102. l | LEREP04 protein [Homo sapiens]
[0022] 配列番号： 3 [0022] SEQ ID NO: 3
mouse DFRPl cDNA配列  mouse DFRPl cDNA sequence
> gi|34368583|rei]NM_026934.2| Mus musculus RIKEN cDNA 2610312B22 gene (261 0312B22Rik), mRNA  > gi | 34368583 | rei] NM_026934.2 | Mus musculus RIKEN cDNA 2610312B22 gene (261 0312B22Rik), mRNA
[0023] 配列番号： 4  [0023] SEQ ID NO: 4
mouse DFRPlアミノ酸配列  mouse DFRPl amino acid sequence
> gi|34368584|rellNP_081210.2| erythropoietin 4 immediate early response [Mus mus cuius]  > gi | 34368584 | rellNP_081210.2 | erythropoietin 4 immediate early response [Mus mus cuius]
[0024] 配列番号： 5  [0024] SEQ ID NO: 5
human DFRP2 cDNA配列  human DFRP2 cDNA sequence
> gi|5138917|gb|AF092134. l|AF092134 Homo sapiens PTD013 mRNA, complete cds [0025] 配列番号： 6  > gi | 5138917 | gb | AF092134. l | AF092134 Homo sapiens PTD013 mRNA, complete cds [0025] SEQ ID NO: 6
human DFRP2アミノ酸配列  human DFRP2 amino acid sequence
> gi|5138918|gb|AAD40376. l| PTD013 [Homo sapiens]  > gi | 5138918 | gb | AAD40376. l | PTD013 [Homo sapiens]
[0026] 配列番号： 7 [0026] SEQ ID NO: 7
mouse DFRP2 cDNA配列  mouse DFRP2 cDNA sequence
> gi|21735426|reflNM_025614. l| Mus musculus RWD domain containing 1 (Rwddl), mRNA  > gi | 21735426 | reflNM_025614. l | Mus musculus RWD domain containing 1 (Rwddl), mRNA
[0027] 配列番号： 8  [0027] SEQ ID NO: 8
mouse DFRP2アミノ酸配列  mouse DFRP2 amino acid sequence
> gi|21735427|reilNP_079890. l| RWD domain containing 1 [Mus musculus] [0028] 配列番号： 9 > gi | 21735427 | reilNP_079890. l | RWD domain containing 1 [Mus musculus] [0028] SEQ ID NO: 9
human DRG1 cDNA配列  human DRG1 cDNA sequence
> gi|51093843|rellNM_004147.3| Homo sapiens developmentally regulated GTP bindi ng protein 1 (DRG1), mRNA  > gi | 51093843 | rellNM_004147.3 | Homo sapiens developmentally regulated GTP bindin ng protein 1 (DRG1), mRNA
[0029] 配列番号： 10  [0029] SEQ ID NO: 10
mouse DRG1 cDNA配列  mouse DRG1 cDNA sequence
> gi|6681224|reilNM_007879.l| Mus musculus developmentally regulated GTP bindin g protein 1 (Drgl), mRNA  > gi | 6681224 | reilNM_007879.l | Mus musculus developmentally regulated GTP bindin g protein 1 (Drgl), mRNA
[0030] 配列番号： 11  [0030] SEQ ID NO: 11
human DRG2 cDNA配列  human DRG2 cDNA sequence
> gi|34305455|rei]NM_001388.3| Homo sapiens developmentally regulated GTP bindi ng protein 2 (DRG2), mRNA  > gi | 34305455 | rei] NM_001388.3 | Homo sapiens developmentally regulated GTP bindin ng protein 2 (DRG2), mRNA
[0031] 配列番号： 12  [0031] SEQ ID NO: 12
mouse DRG2 cDNA配列  mouse DRG2 cDNA sequence
> gi|l0946677|reilNM_021354.l| Mus musculus developmentally regulated GTP bindi ng protein 2 (Drg2), mRNA  > gi | l0946677 | reilNM_021354.l | Mus musculus developmentally regulated GTP bindin ng protein 2 (Drg2), mRNA
[0032] 本発明者らは、 DFRP1および DFRP2の間で高い配列の同一性を示すドメイン（DFR Pドメイン）力 DRGファミリーとの結合に必須であることを見出だした。 DFRP1蛋白質 のアミノ酸配列（配列番号： 2または配列番号: 4)においては、 DFRPドメインは 234位 力も 295位に存在する。 DFRP2蛋白質のアミノ酸配列（配列番号： 6または配列番号: 8)においては、 DFRPドメインは 132位から 187位に存在する。  [0032] The present inventors have found that domain (DFR P domain) force showing high sequence identity between DFRP1 and DFRP2 is essential for binding to the DRG family. In the amino acid sequence of the DFRP1 protein (SEQ ID NO: 2 or SEQ ID NO: 4), the DFRP domain has a 234 position and a 295 position. In the amino acid sequence of the DFRP2 protein (SEQ ID NO: 6 or SEQ ID NO: 8), the DFRP domain is located at positions 132 to 187.
[0033] 即ち本発明はまず、下記（1)または（2)に記載のポリペプチド (DFRP1ポリペプチド )を提供する。  That is, the present invention first provides the polypeptide (DFRP1 polypeptide) described in (1) or (2) below.
( 1)配列番号： 2または配列番号: 4に記載のアミノ酸配列における 234位から 295位 のアミノ酸配列力もなるポリペプチド  (1) A polypeptide having an amino acid sequence from position 234 to position 295 in the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4.
(2)配列番号： 2または配列番号: 4に記載のアミノ酸配列における 234位から 295位 のアミノ酸配列において 1若しくは複数のアミノ酸が置換、欠失、挿入または付加され たアミノ酸配列力 なり、 DRG1蛋白質と相互作用能力を有するポリペプチド [0034] また本発明は、下記（1)または（2)に記載のポリペプチド (DFRP1ポリペプチド)を 提供する。 (2) an amino acid sequence having one or more amino acid substitutions, deletions, insertions or additions in the amino acid sequence from positions 234 to 295 in the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4, and the DRG1 protein Polypeptides that have the ability to interact with [0034] The present invention also provides the polypeptide (DFRP1 polypeptide) described in (1) or (2) below.
(1)配列番号： 2または配列番号: 4に記載のアミノ酸配列からなるポリペプチド (1) A polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4.
(2)配列番号： 2または配列番号： 4に記載のアミノ酸配列にお 、て、 1若しくは複数 のアミノ酸が欠失、挿入または付加されたアミノ酸配列力もなり、 DRG1蛋白質と相互 作用能力を有するポリペプチド (2) In the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, one or more amino acids are deleted, inserted, or added, and the amino acid sequence is also a polymorphism capable of interacting with the DRG1 protein. Peptide
[0035] また本発明は、下記（1)または（2)に記載のポリペプチド (DFRP2ポリペプチド)を 提供する。  [0035] The present invention also provides the polypeptide (DFRP2 polypeptide) described in (1) or (2) below.
(1)配列番号: 6または配列番号: 8に記載のアミノ酸配列における 132位から 187位 のアミノ酸配列力もなるポリペプチド  (1) A polypeptide having an amino acid sequence from positions 132 to 187 in the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 8
(2)配列番号: 6または配列番号: 8に記載のアミノ酸配列における 132位から 187位 のアミノ酸配列において 1若しくは複数のアミノ酸が置換、欠失、挿入または付加され たアミノ酸配列力 なり、 DRG2蛋白質と相互作用能力を有するポリペプチド  (2) A DRG2 protein comprising an amino acid sequence having one or more amino acids substituted, deleted, inserted or added in the amino acid sequence from position 132 to position 187 in the amino acid sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 8. Polypeptides that have the ability to interact with
[0036] また本発明は、下記（1)または（2)に記載のポリペプチド (DFRP2ポリペプチド)を 提供する。  [0036] The present invention also provides the polypeptide (DFRP2 polypeptide) described in (1) or (2) below.
(1)配列番号: 6または配列番号: 8に記載のアミノ酸配列からなるポリペプチド (1) A polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 8
(2)配列番号： 6または配列番号： 8に記載のアミノ酸配列にお!/、て 1若しくは複数の アミノ酸が置換、欠失、挿入または付加されたアミノ酸配列からなり、 DRG2蛋白質と 相互作用能力を有するポリペプチド (2) The amino acid sequence shown in SEQ ID NO: 6 or SEQ ID NO: 8 consists of an amino acid sequence in which one or more amino acids are substituted, deleted, inserted or added, and is capable of interacting with DRG2 protein. Polypeptide having
[0037] 本明細書において用いられる「蛋白質」は、複数のアミノ酸力 なる重合体を意味 する。従って、オリゴペプチドおよびポリペプチドもまた、蛋白質の概念に含まれる。 蛋白質は、天然に存在する状態から修飾されていないもの、および修飾されているも のの双方を含む意である。修飾としては、ァセチル化、ァシル化、 ADP-リボシル化、 アミド化、フラビンの共有結合、ヘム部分の共有結合、ヌクレオチドまたはヌクレオチ ド誘導体の共有結合、脂質または脂質誘導体の共有結合、ホスファチジルイノシトー ルの共有結合、架橋、環化、ジスルフイド結合の形成、脱メチル化、共有架橋の形成 、シスチンの开成、ピログルタメートの形成、ホルミル化、カルボキシル化、グリコシル ィ匕、 GPIアンカー形成、ヒドロキシル化、ヨウ素化、メチル化、ミリストイル化、酸化、蛋 白質分解処理、リン酸化、プレニル化、ラセミ化、セレノィル化、硫酸化、アルギニル 化のような蛋白質へのアミノ酸の転移 RNA媒介付加、ュビキチンィ匕などが含まれる。 [0037] As used herein, "protein" means a polymer having a plurality of amino acid strengths. Thus, oligopeptides and polypeptides are also included in the concept of proteins. Proteins are meant to include both those that are not modified from the naturally occurring state and those that are modified. Modifications include acetylation, acylation, ADP-ribosylation, amidation, flavin covalent bond, heme moiety covalent bond, nucleotide or nucleotide derivative covalent bond, lipid or lipid derivative covalent bond, phosphatidylinositol. Covalent bond, crosslinking, cyclization, disulfide bond formation, demethylation, covalent bridge formation, cystine formation, pyroglutamate formation, formylation, carboxylation, glycosylation, GPI anchor formation, hydroxylation, Iodination, methylation, myristoylation, oxidation, protein Examples include white matter degradation, phosphorylation, prenylation, racemization, selenoylation, sulfation, arginyl transfer of amino acids to proteins such as RNA-mediated addition, ubiquitin.
[0038] 天然の蛋白質は、例えば DRG蛋白質 (DRG1蛋白質および DRG2蛋白質を含む）あ る ヽは DFRP蛋白質 (DFRP1蛋白質および DFRP2蛋白質を含む）が発現して 、ると考 えられる細胞 (組織)の抽出液に対し、 DRG蛋白質あるいは DFRP蛋白質に対する抗 体を用いたァフィユティークロマトグラフィーを用いる方法により調製することが可能で ある。一方、組換え蛋白質は、 DRG蛋白質あるいは DFRP蛋白質をコードする DNAで 形質転換した細胞を培養することにより調製することが可能である。 [0038] Natural proteins include, for example, DRG protein (including DRG1 protein and DRG2 protein) and DFRP protein (including DFRP1 protein and DFRP2 protein) expressed in cells (tissues) that are considered to be expressed. The extract can be prepared by a method using affinity chromatography using an antibody against DRG protein or DFRP protein. On the other hand, the recombinant protein can be prepared by culturing cells transformed with DNA encoding DRG protein or DFRP protein.
[0039] 本発明にお 、て「発現」とは遺伝子からの「転写」あるいはポリペプチドへの「翻訳」 及び蛋白質の「分解抑制」によるものが含まれる。「DRG蛋白質あるいは DFRP蛋白質 の発現」とは、 DRG蛋白質あるいは DFRP蛋白質をコードする遺伝子の転写および翻 訳が生じること、またはこれらの転写 ·翻訳により DRG蛋白質あるいは DFRP蛋白質が 生成されることを意味する。 In the present invention, “expression” includes “transcription” from a gene or “translation” into a polypeptide and “degradation inhibition” of a protein. “Expression of DRG protein or DFRP protein” means that transcription and translation of a gene encoding DRG protein or DFRP protein occurs, or that transcription or translation of these genes produces DRG protein or DFRP protein. .
[0040] 当業者においては、任意のポリペプチドに対し、上記（2)の「DRG1蛋白質と相互作 用能力」または「DRG2蛋白質と相互作用能力」を有する力否かについて、適宜判定 を行うことができる。例えば、被検ポリペプチドと DRG1蛋白質あるいは DRG2蛋白質と の結合活性を指標として判定することができる。例えば、任意のポリペプチドが DRG1 蛋白質あるいは DRG2蛋白質と結合活性を有する場合、被検ポリペプチドは「DRG1 蛋白質と相互作用能力」または「DRG2蛋白質と相互作用能力」を有するものと判定さ れる。 [0040] A person skilled in the art will appropriately determine whether or not any polypeptide has the “ability to interact with DRG1 protein” or “ability to interact with DRG2 protein” in (2) above. Can do. For example, it can be determined using the binding activity between the test polypeptide and the DRG1 protein or DRG2 protein as an index. For example, when any polypeptide has binding activity with DRG1 protein or DRG2 protein, the test polypeptide is determined to have “DRG1 protein interaction ability” or “DRG2 protein interaction ability”.
[0041] また本発明は、上記ヒトまたはマウスにおける DFRP1蛋白質あるいは DFRP2蛋白質 、およびこれら蛋白質の改変体 (変異体)を提供する。  [0041] The present invention also provides the DFRP1 protein or DFRP2 protein in the human or mouse, and a variant (mutant) of these proteins.
[0042] より詳しくは、配列番号： 2または 4に記載のアミノ酸配列を有するポリペプチドまた は配列番号： 6または 8に記載のアミノ酸配列を有するポリペプチド、並びに該ァミノ 酸配列において 1若しくは複数のアミノ酸が置換、欠失、挿入、および Zまたは付カロ したアミノ酸配列力 なるポリペプチドであって、当該ポリペプチドと機能的に同等な ポリペプチドを提供する。  [0042] More specifically, the polypeptide having the amino acid sequence set forth in SEQ ID NO: 2 or 4, or the polypeptide having the amino acid sequence set forth in SEQ ID NO: 6 or 8, and one or more amino acids in the amino acid sequence Provided is a polypeptide having amino acid sequence ability in which amino acids are substituted, deleted, inserted, and Z or appended, and is functionally equivalent to the polypeptide.
[0043] ここで「機能的に同等」とは、対象となるポリペプチドが、本発明のポリペプチドと同 様の生物学的あるいは生化学的な活性あるいは能力を有することを指す。このような 能力としては、例えば DRG1蛋白質との相互作用能力あるいは DRG2蛋白質との相互 作用能力が挙げられる。 [0043] Here, "functionally equivalent" means that the target polypeptide is the same as the polypeptide of the present invention. It has the same biological or biochemical activity or ability. Examples of such ability include ability to interact with DRG1 protein or ability to interact with DRG2 protein.
[0044] このような、あるポリペプチドと機能的に同等なポリペプチドを調製するための、当 業者によく知られた方法としては、ポリペプチドに変異を導入する方法が知られて ヽ る。例えば、当業者であれば、部位特異的変異誘発法 (Hashimoto-Gotoh, T. et al. (1995) Gene 152, 271-275、 Zoller, MJ, and Smith, M.(1983) Methods Enzymol. 100, 468—500、 Kramer, W. et al. (1984) Nucleic Acids Res. 12, 9441—9456、 Kramer W, and Fritz HJ(1987) Methods. Enzymol. 154, 350—367、 Kunkel,TA(1985) Proc Natl A cad Sci USA. 82, 488-492、 Kunkel (1988) Methods Enzymol. 85, 2763- 2766)などを 用いて、本発明のポリペプチドに適宜変異を導入することにより、該ポリペプチドと機 能的に同等なポリペプチドを調製することができる。  [0044] As a method well known to those skilled in the art for preparing such a polypeptide functionally equivalent to a certain polypeptide, a method for introducing a mutation into the polypeptide is known. For example, a person skilled in the art can perform site-directed mutagenesis (Hashimoto-Gotoh, T. et al. (1995) Gene 152, 271-275, Zoller, MJ, and Smith, M. (1983) Methods Enzymol. 100 , 468-500, Kramer, W. et al. (1984) Nucleic Acids Res. 12, 9441-9456, Kramer W, and Fritz HJ (1987) Methods. Enzymol. 154, 350-367, Kunkel, TA (1985) Proc Natl Acad Sci USA. 82, 488-492, Kunkel (1988) Methods Enzymol. 85, 2762-2766), etc., by appropriately introducing mutations into the polypeptide of the present invention. Functionally equivalent polypeptides can be prepared.
[0045] また、遺伝子増幅技術（PCR) (Current protocols in Molecular Biology edit. Ausub el et al. (1987) Publish. John Wiley & Sons Section 6.1- 6.4)を用いて、本発明のポリ ペプチドをコードする DNA配列（配列番号： 1、 3、 5、または 7)の一部を基にプライマ 一を設計し、本発明のポリペプチドをコードする DNA配列と相同性の高い DNA断片 を単離し、該 DNAを基に本発明者のポリペプチドと機能的に同等なポリペプチドを得 ることち可會である。  [0045] The gene of the present invention is encoded using gene amplification technology (PCR) (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons Section 6.1-6.4). A primer is designed based on a part of the DNA sequence (SEQ ID NO: 1, 3, 5, or 7), and a DNA fragment highly homologous to the DNA sequence encoding the polypeptide of the present invention is isolated, and the DNA Based on the above, it is possible to obtain a polypeptide functionally equivalent to the polypeptide of the present inventor.
[0046] また、アミノ酸の変異は自然界においても生じうる。このように、本発明のポリべプチ ドのアミノ酸配列において 1もしくは複数のアミノ酸が変異したアミノ酸配列を有し、該 ポリペプチドと機能的に同等なポリペプチドもまた本発明のポリペプチドに含まれる。 このような変異体における、変異するアミノ酸数は、通常、 50アミノ酸以内であり、好ま しくは 30アミノ酸以内であり、さらに好ましくは 10アミノ酸以内（例えば、 5アミノ酸以内） であると考えられる。  [0046] Amino acid mutations may also occur in nature. Thus, a polypeptide having an amino acid sequence in which one or more amino acids are mutated in the amino acid sequence of the polypeptide of the present invention and functionally equivalent to the polypeptide is also included in the polypeptide of the present invention. . In such a mutant, the number of amino acids to be mutated is usually within 50 amino acids, preferably within 30 amino acids, and more preferably within 10 amino acids (for example, within 5 amino acids).
[0047] 変異するアミノ酸残基にお!、ては、アミノ酸側鎖の性質が保存されて 、る別のアミノ 酸に変異されることが望ましい。例えばアミノ酸側鎖の性質としては、疎水性アミノ酸（ A、 I、し、 M、 F、 P、 W、 Y、 V)、親水'性アミノ酸（R、 D、 N、 C、 E、 Q、 G、 H、 K、 S、 T)、月旨 肪族側鎖を有するアミノ酸 (G、 A、 V、 L、 I、 P)、水酸基含有側鎖を有するアミノ酸 (S、 T、 Y)、硫黄原子含有側鎖を有するアミノ酸 (C、 M)、カルボン酸及びアミド含有側鎖 を有するアミノ酸 (D、 N、 E、 Q)、塩基含有側鎖を有するアミノ離 (R、 K、 Η)、芳香族 含有側鎖を有するアミノ酸 (H、 F、 Y、 W)を挙げることができる (括弧内はいずれもアミ ノ酸の一文字標記を表す)。 [0047] It is desirable that the amino acid residue to be mutated should be mutated to another amino acid while preserving the properties of the amino acid side chain. For example, amino acid side chain properties include hydrophobic amino acids (A, I, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G , H, K, S, T), amino acids with lunar aliphatic side chains (G, A, V, L, I, P), amino acids with hydroxyl-containing side chains (S, T, Y), amino acids with sulfur atom-containing side chains (C, M), amino acids with carboxylic acid and amide-containing side chains (D, N, E, Q), amino acids with base-containing side chains (R, K, Η), and amino acids having aromatic side chains (H, F, Y, W) can be mentioned (the parentheses indicate single letter amino acids).
[0048] あるアミノ酸配列に対する 1又は複数個のアミノ酸残基の欠失、付加及び Z又は他 のアミノ酸による置換により修飾されたアミノ酸配列を有するポリペプチドがその生物 学的活性を維持することはすでに知られている（Mark, D. F. et al., Proc. Natl. Acad . Sci. USA (1984) 81, 5662-5666、 Zoller, M. J. & Smith, M. Nucleic Acids Research (1982) 10, 6487-6500、 Wang, A. et al" Science 224, 1431-1433、 Dalbadie- McFar land, G. et al., Proc. Natl. Acad. Sci. USA (1982) 79, 6409—6413；)。  [0048] It is already known that a polypeptide having an amino acid sequence modified by deletion or addition of one or more amino acid residues to a certain amino acid sequence and substitution by Z or another amino acid maintains its biological activity. (Mark, DF et al., Proc. Natl. Acad. Sci. USA (1984) 81, 5662-5666, Zoller, MJ & Smith, M. Nucleic Acids Research (1982) 10, 6487-6500, Wang, A. et al "Science 224, 1431-1433, Dalbadie- McFar land, G. et al., Proc. Natl. Acad. Sci. USA (1982) 79, 6409-6413;).
[0049] また本発明は、上記本発明の DFRP1ポリペプチドをコードする核酸、あるいは上記 本発明の DFRP2ポリペプチドをコードする核酸を提供する。  [0049] The present invention also provides a nucleic acid encoding the DFRP1 polypeptide of the present invention or a nucleic acid encoding the DFRP2 polypeptide of the present invention.
[0050] また本発明は、本発明の上記 DFRP1ポリペプチドをコードする核酸を担持したベタ ター、あるいは本発明の上記 DFRP2ポリペプチドをコードする核酸を担持したベクタ 一を提供する。本発明のベクターとしては、挿入した DNAを安定に保持 (担持)するも のであれば特に制限されず、例えば宿主に大腸菌を用いるのであれば、クローニン グ用ベクターとしては pBluescriptベクター (Stratagene社製）などが好まし!/、。本発明 のポリペプチドを生産する目的においてベクターを用いる場合には、特に発現べクタ 一が有用である。発現ベクターとしては、試験管内、大腸菌内、培養細胞内、生物個 体内でポリペプチドを発現するベクターであれば特に制限されないが、例えば、試験 管内発現であれば pBESTベクター（プロメガ社製）、大腸菌であれば pETベクター（In vitrogen社製）、培養細胞であれば pME18S- FL3ベクター（GenBank Accession No. A B009864)、生物個体であれば pME18Sベクター（Mol Cell Biol. 8:466-472(1988))な どが好ましい。ベクターへの本発明の DNAの挿入は、常法により、例えば、制限酵素 サイトを用いたリガーゼ反応により行うことができる（Current protocols in Molecular Bi ology edit. Ausubel et al. (1987) Publish. John Wiley & Sons. Section 11.4—11.11)。  [0050] The present invention also provides a vector carrying a nucleic acid encoding the DFRP1 polypeptide of the present invention or a vector carrying a nucleic acid encoding the DFRP2 polypeptide of the present invention. The vector of the present invention is not particularly limited as long as it can stably hold (carry) the inserted DNA. For example, when Escherichia coli is used as a host, a pBluescript vector (Stratagene) is used as a cloning vector. And so on! An expression vector is particularly useful when a vector is used for the purpose of producing the polypeptide of the present invention. The expression vector is not particularly limited as long as it is a vector that expresses a polypeptide in vitro, in E. coli, in cultured cells, or in an organism. For example, pBEST vector (manufactured by Promega), E. coli for in vitro expression. PET vector (manufactured by In vitrogen), pME18S-FL3 vector (GenBank Accession No. A B009864) for cultured cells, pME18S vector (Mol Cell Biol. 8: 466-472 (1988) for living organisms) ) Is preferred. The insertion of the DNA of the present invention into a vector can be performed by a conventional method, for example, by a ligase reaction using a restriction enzyme site (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley). & Sons. Section 11.4—11.11).
[0051] また本発明は、上記本発明の DFRP1ポリペプチドに対する (認識あるいは結合する )抗体、あるいは DFRP2ポリペプチドに対する抗体を提供する。 [0052] 本発明における「抗体」とは、好ましくは、ポリクローナル抗体、モノクローナル抗体 、キメラ抗体、ヒト型抗体またはこれらの混合物力も選択される抗体である。これら抗 体以外にも、 DFRP1蛋白質あるいは DFRP2蛋白質に対して結合する限り、例えば、ヒ ト化抗体、低分子化抗体 (抗体断片も含む)、多特異性抗体等、さらに抗体修飾物な どが含まれていてもよい。 [0051] The present invention also provides an antibody against (recognizing or binding to) the DFRP1 polypeptide of the present invention or an antibody against the DFRP2 polypeptide. [0052] The "antibody" in the present invention is preferably an antibody for which a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a human antibody or a mixture thereof is also selected. In addition to these antibodies, as long as it binds to DFRP1 protein or DFRP2 protein, for example, humanized antibodies, low molecular weight antibodies (including antibody fragments), multispecific antibodies, and antibody modified products are also available. It may be included.
[0053] 本発明の DFRP1ポリペプチドあるいは DFRP2ポリペプチド、またはその断片もしくは 類似体、またはそれらを発現する細胞は、本発明の DFRP1ポリペプチドあるいは DFR P2ポリペプチドに結合する抗体を産生するための免疫原としても使用することができ る。  [0053] The DFRP1 polypeptide or DFRP2 polypeptide of the present invention, or a fragment or analog thereof, or a cell expressing them is used for immunization to produce an antibody that binds to the DFRP1 polypeptide or DFR P2 polypeptide of the present invention. It can also be used as a source.
[0054] 本発明の DFRP1ポリペプチドあるいは DFRP2ポリペプチドに結合する抗体は、当業 者に公知の方法により調製することが可能である。ポリクローナル抗体であれば、例 えば、次のようにして得ることができる。本発明の DFRP1ポリペプチドあるいは DFRP2 ポリペプチド、またはその GSTとの融合蛋白質をゥサギ等の小動物に免疫し血清を得 る。これを、例えば、硫安沈殿、プロテイン A、プロテイン Gカラム、 DEAEイオン交換ク 口マトグラフィー、本発明の DFRP1ポリペプチドあるいは DFRP2ポリペプチドをカツプリ ングしたァフィユティーカラム等により精製することにより調製する。また、モノクローナ ル抗体であれば、例えば、本発明の DFRP1ポリペプチドあるいは DFRP2ポリペプチド をマウスなどの小動物に免疫を行い、同マウスより脾臓を摘出し、これをすりつぶして 細胞を分離し、マウスミエローマ細胞とポリエチレングリコールなどの試薬により融合 させ、これによりできた融合細胞 (ノヽイブリドーマ）の中から、本発明の DFRP1ポリぺプ チドある 、は DFRP2ポリペプチドに結合する抗体を産生するクローンを選択する。次 いで、得られたハイプリドーマをマウス腹腔内に移植し、同マウスより腹水を回収し、 得られたモノクローナル抗体を、例えば、硫安沈殿、プロテイン A、プロテイン Gカラム 、 DEAEイオン交換クロマトグラフィー、本発明の DFRP1ポリペプチドあるいは DFRP2 ポリペプチドをカップリングしたァフィユティーカラム等により精製することで、調製す ることが可能である。本発明の DFRP1ポリペプチドあるいは DFRP2ポリペプチドに対 する抗体を、本発明の DFRP蛋白質が関与する疾患の抗体治療へ応用することも考 えられる。得られた抗体を人体に投与する目的 (抗体治療)で使用する場合には、免 疫原性を低下させるため、ヒト抗体やヒト型抗体にすることが好ましい。 [0054] Antibodies that bind to the DFRP1 polypeptide or DFRP2 polypeptide of the present invention can be prepared by methods known to those skilled in the art. For example, a polyclonal antibody can be obtained as follows. Serum is obtained by immunizing small animals such as rabbits with the DFRP1 polypeptide or DFRP2 polypeptide of the present invention or a fusion protein thereof with GST. This is prepared, for example, by purifying with ammonium sulfate precipitation, protein A, protein G column, DEAE ion exchange chromatography, a DFRP1 polypeptide or DFRP2 polypeptide-coupled affinity column of the present invention, and the like. In the case of a monoclonal antibody, for example, a small animal such as a mouse is immunized with the DFRP1 polypeptide or DFRP2 polypeptide of the present invention, and the spleen is removed from the mouse, and this is ground to isolate cells. A clone that produces an antibody that binds to the DFRP2 polypeptide is selected from the fused cells (neubridoma) made by fusing myeloma cells with a reagent such as polyethylene glycol. To do. Next, the obtained hyperidoma was transplanted into the abdominal cavity of the mouse, and ascites was collected from the mouse, and the obtained monoclonal antibody was purified using, for example, ammonium sulfate precipitation, protein A, protein G column, DEAE ion exchange chromatography, The DFRP1 polypeptide or DFRP2 polypeptide of the invention can be prepared by purification using a coupled column or the like. The antibody against the DFRP1 polypeptide or DFRP2 polypeptide of the present invention may be applied to antibody therapy for diseases involving the DFRP protein of the present invention. When using the obtained antibody for the purpose of administering it to the human body (antibody therapy), In order to reduce epidemiogenicity, it is preferable to use human antibodies or human antibodies.
[0055] キメラ抗体は、異なる動物由来の配列を組み合わせて作製される抗体であり、例え ば、マウス抗体の重鎖、軽鎖の可変領域とヒト抗体の重鎖、軽鎖の定常領域からなる 抗体などである。キメラ抗体の作製は公知の方法を用いて行うことができ、例えば、抗 体 V領域をコードする DNAとヒト抗体 C領域をコードする DNAとを連結し、これを発現 ベクターに組み込んで宿主に導入し産生させることにより得られる。  [0055] A chimeric antibody is an antibody produced by combining sequences derived from different animals. For example, the antibody comprises a mouse antibody heavy chain and light chain variable region and a human antibody heavy chain and light chain constant region. Such as an antibody. A chimeric antibody can be prepared by a known method. For example, DNA encoding an antibody V region and DNA encoding a human antibody C region are ligated, incorporated into an expression vector, and introduced into a host. It is obtained by making it produce.
[0056] 低分子化抗体は、全長抗体 (whole antibody,例えば whole IgG等)の一部分が欠損 している抗体断片を含み、抗原への結合能を有していれば特に限定されない。抗体 断片は、全長抗体の一部分であれば特に限定されない。抗体断片の具体例としては 、例えば、 Fab, Fab'、 F(ab')2、 Fvなどを挙げることができる。また、低分子化抗体の 具体例としては、例えば、 Fab、 Fab'、 F(ab')2、 Fv、 scFv (single chain Fv)、ダイァボ ディ、 sc(Fv)2 (single chain (Fv)2)などを挙げることができる。  [0056] The low molecular weight antibody is not particularly limited as long as it includes an antibody fragment in which a part of a full-length antibody (such as whole IgG) is deleted and has an ability to bind to an antigen. The antibody fragment is not particularly limited as long as it is a part of a full-length antibody. Specific examples of antibody fragments include, for example, Fab, Fab ′, F (ab ′) 2, and Fv. Specific examples of the low molecular weight antibody include, for example, Fab, Fab ′, F (ab ′) 2, Fv, scFv (single chain Fv), diabodies, sc (Fv) 2 (single chain (Fv) 2 ) And the like.
[0057] ヒト化抗体は、再構成 (reshaped)ヒト抗体とも称される改変抗体である。ヒト化抗体は 、免疫動物由来の抗体の CDRを、ヒト抗体の相補性決定領域へ移植することによつ て構築される。その一般的な遺伝子組換え手法も知られて 、る。  [0057] Humanized antibodies are modified antibodies, also referred to as reshaped human antibodies. Humanized antibodies are constructed by transplanting CDRs of antibodies derived from immunized animals into complementarity determining regions of human antibodies. The general genetic recombination technique is also known.
[0058] キメラ抗体やヒト化抗体を作製した後に、可変領域 (例えば、 FR)や定常領域中の アミノ酸を他のアミノ酸で置換等してもょ 、。  [0058] After producing a chimeric antibody or a humanized antibody, amino acids in the variable region (eg, FR) or constant region may be substituted with other amino acids.
[0059] 抗体修飾物としては、例えば、ポリエチレングリコール (PEG)等の各種分子と結合し た抗体を挙げることができる。本発明の抗体修飾物においては、結合される物質は 限定されない。このような抗体修飾物を得るには、得られた抗体に化学的な修飾を施 すことによって得ることができる。これらの方法はこの分野にぉ 、て既に確立されて！ヽ る。  [0059] Examples of modified antibodies include antibodies bound to various molecules such as polyethylene glycol (PEG). In the modified antibody of the present invention, the substance to be bound is not limited. In order to obtain such a modified antibody, it can be obtained by chemically modifying the obtained antibody. These methods have already been established in this field!ヽ.
[0060] 本発明の DFRP1ポリペプチドあるいは DFRP2ポリペプチドに対する抗体は、本発明 の DFRP1ポリペプチドある!/、は DFRP2ポリペプチドやこれを発現する細胞の単離、同 定、および精製に利用することができる。  [0060] The antibody against the DFRP1 polypeptide or DFRP2 polypeptide of the present invention is the DFRP1 polypeptide of the present invention! /, Which is used for isolation, identification and purification of DFRP2 polypeptide and cells expressing the same. Can do.
[0061] また本発明者らは、 DRG1、 DFRP1、 DRG2、 DFRP2の mRNAに対し RNAiを引き起こ すことができる shRNA (ショートヘアピン RNA)を作成し、 DRG1、 DFRP1、 DRG2、 DFR[0061] In addition, the present inventors created shRNA (short hairpin RNA) capable of causing RNAi against DRG1, DFRP1, DRG2, and DFRP2 mRNA, and DRG1, DFRP1, DRG2, DFR
P2をそれぞれノックダウンすることを可能とした。 [0062] RNAiは、標的遺伝子の mRNAと相同な配列からなるセンス RNAとこれと相補的な配 列からなるアンチセンス RNAとからなる二本鎖 RNA (以下、「dsRNA」と略称する）を細 胞等に導入することにより、標的遺伝子 mRNAの破壊を誘導し、標的遺伝子の発現を 抑制し得る現象である。このように RNAiは、標的遺伝子の発現を抑制し得ることから、 従来の煩雑で効率の低 、相同組換えによる遺伝子破壊方法に代わる簡易な遺伝子 ノックアウト方法として注目^^めている。上記 RNAiは、当初、線虫において発見さ れ 7こ力 S (Fire, A. et al. Potent and specific genetic interference by double-stranded R NA in Caenorhabditis elegans. Nature 391, 806-811, (1998))、現在では、線虫のみ ならず、植物、線形動物、ショウジヨウバエ、原生動物などの種々の生物において観 察されている（Fire, A. RNA- triggered gene silencing. Trends Genet. 15, 358-363 (1 999)、 Sharp, P. A. RNA interference 2001. Genes Dev. 15, 485—490 (2001)、 Hammo nd, S. M., Caudy, A. A. & Hannon, G. J. Post— transcriptional gene silencing by dou ble— stranded RNA. Nature Rev. Genet. 2, 110—1119 (2001)、 Zamore, P. D. RNA int erference: listening to the sound of silence. Nat Struct Biol. 8, 746-750 (2001 。こ れら生物では、実際に外来より dsRNAを導入することにより標的遺伝子の発現が抑制 されることが確認され、さらにはノックアウト個体を創生する方法としても利用されつつ ある。 It was possible to knockdown each P2. [0062] RNAi is a double-stranded RNA (hereinafter abbreviated as "dsRNA") consisting of a sense RNA consisting of a sequence homologous to the mRNA of the target gene and an antisense RNA consisting of a complementary sequence. It is a phenomenon that can induce the destruction of the target gene mRNA and suppress the expression of the target gene by introducing it into the cell. RNAi is thus attracting attention as a simple gene knockout method to replace the conventional complicated, low-efficiency gene disruption method by homologous recombination because it can suppress the expression of the target gene. The above RNAi was first discovered in nematodes. 7 (S) (Fire, A. et al. Potent and specific genetic interference by double-stranded R NA in Caenorhabditis elegans.Nature 391, 806-811, (1998)) Currently, it is observed not only in nematodes but also in various organisms such as plants, linear animals, fruit flies and protozoa (Fire, A. RNA-triggered gene silencing. Trends Genet. 15, 358- 363 (1 999), Sharp, PA RNA interference 2001. Genes Dev. 15, 485-490 (2001), Hammond, SM, Caudy, AA & Hannon, GJ Post— transcriptional gene silencing by dou ble— stranded RNA. Nature Rev. Genet. 2, 110—1119 (2001), Zamore, PD RNA int erference: listening to the sound of silence. Nat Struct Biol. 8, 746-750 (2001. Has been confirmed to suppress the expression of the target gene, and is also used as a method for creating knockout individuals. A.
[0063] インビト口において、 dsRNAは、ショウジヨウバエの初期胚の溶解物または培養ショウ ジヨウバエ S2細胞の抽出物における切断に関して mRNAを標的とすることが知られて いる（Tuschl T., et al., Genes Dev. 13: 3191—3197, 1999; Hammond S. M., et al., N ature 404: 293-296, 2000; Zamore P., et al., Cell 101: 25-33, 2000)。インビトロでの RNAiの反応は ATP (Zamore P., et al., Cell 101: 25-33, 2000)を必要とする。  [0063] In vitro, dsRNA is known to target mRNA for cleavage in lysates of early Drosophila embryos or extracts of cultured Drosophila S2 cells (Tuschl T., et al. Genes Dev. 13: 3191-3197, 1999; Hammond SM, et al., Nature 404: 293-296, 2000; Zamore P., et al., Cell 101: 25-33, 2000). In vitro RNAi reactions require ATP (Zamore P., et al., Cell 101: 25-33, 2000).
[0064] 合成 RNA二本鎖による最近の研究により、それぞれの siRNA二本鎖が標的 RNAを 切断することが証明された（Elbashir S. M. et al" Genes Dev. 15: 188-200, 2001)。 si RNA二本鎖内の 2ヌクレオチドまたは 3ヌクレオチドの突出 3'末端力 効率的な標的切 断にとって必要であることが明らかになった（Elbashir S. M. et al., Genes Dev. 15: 18 8-200, 2001)。そのような 3'突出末端は、 RNァーゼ III切断反応の産物に特徴的であ り、培養ショウジヨウバエ S2細胞において、 dsRNAの siRNAへの切断は、ダイサ一とし て知られる多数のドメイン RNァーゼ III酵素を必要とする（Bernstein E. et al., Nature 409: 363-366, 2001)。その後、 siRNAは、ショウジヨウバエにおいて同定され、 RISCと 呼ばれる多成分ヌクレアーゼと会合し、 mRNAの配列特異的分解に関してこの酵素を 誘導するものと考えられる（Hammond S. M., et al., Nature 404: 293-296, 2000; Ber nstein E. et al" Nature 409: 363 - 366, 2001; Hammond S. M. et al., Science 293: 11 46-1150, 2001)。 [0064] Recent studies with synthetic RNA duplexes have demonstrated that each siRNA duplex cleaves the target RNA (Elbashir SM et al "Genes Dev. 15: 188-200, 2001). 2 or 3 nucleotide overhangs in RNA duplex 3 'end forces have been shown to be necessary for efficient target cleavage (Elbashir SM et al., Genes Dev. 15: 18 8-200, 2001), such a 3 'protruding end is characteristic of the product of the RNase III cleavage reaction, and in the cultured Drosophila S2 cells, the cleavage of dsRNA into siRNA should be a dither. Requires a number of known domain RNase III enzymes (Bernstein E. et al., Nature 409: 363-366, 2001). The siRNA was subsequently identified in Drosophila and appears to associate with a multicomponent nuclease called RISC and induce this enzyme for sequence-specific degradation of mRNA (Hammond SM, et al., Nature 404: 293 -296, 2000; Bernstein E. et al "Nature 409: 363-366, 2001; Hammond SM et al., Science 293: 11 46-1150, 2001).
[0065] RNAiは標的遺伝子を不活化する方法を提供し、このように、 C.エレガンス (C. eleg ans)、ショウジヨウバエおよび植物における遺伝子機能を研究するための強力なツー ルを提供する。遺伝子発現の特異的阻害はまた、動物および植物における dsRNAの 安定な発現および誘導型発現によって得ることができる（Kennerdell and Carthew Na ture Biotechnol. 18: 896-898, 2000; Tavernarakis N. et al., Nature Genetics 24: 18 0-183, 2000; Hammond S. M. et al" Nature Reviews Genetics 2: 110-119, 2001)。 マウス胚癌 EC細胞および胚幹 (ES)細胞では、 dsRNAによる遺伝子の不活化は成功 したが（Billy E. et al" PNAS 98: 14428-14433, 2001; Paddison P. J. et al" PNAS 99 ： 1443-1448, 2002)、培養哺乳類細胞における長い dsRNAによる RNAiの誘発は一般 的にあまり成功していない。これらの失敗は、長い dsRNA ( >30塩基対）（Stark G. R. et al., Annu. Rev. Biochem. 67: 227-264, 1998)によって活性化されるインターフエ口 ン (IFN)防御経路の一部を形成する 2つの潜在型酵素の作用によって、容易に説明 される。その一つは、 2'-5しオリゴアデ-レート（2-5A)シンターゼであり、これは dsRN Aによって活性ィ匕されて、 RNァーゼ Lと呼ばれる配列非特異的 RNァーゼの活性ィ匕に とって必要である 2-5Aの合成を増加させる（Silverman R. H. in Ribonucleases: Struct ures and Functions, eas. D'Alessio, G. and Riordan J. F. (Academic, New York) pp. 515-551)。もう一つは、蛋白質キナーゼ PKRであり、その活性型は翻訳因子真核細 胞開始因子 (eIF2)をリン酸ィ匕して、蛋白質合成の全般的な阻害と細胞死に至る（Cle mens M. J. and Elia A., J. Interferon Cytokine Res. 17: 503—524, 1997)。  [0065] RNAi provides a way to inactivate target genes and thus provides a powerful tool for studying gene function in C. eleg ans, Drosophila and plants . Specific inhibition of gene expression can also be obtained by stable and inducible expression of dsRNA in animals and plants (Kennerdell and Carthew Nature Biotechnol. 18: 896-898, 2000; Tavernarakis N. et al., Nature Genetics 24: 18 0-183, 2000; Hammond SM et al "Nature Reviews Genetics 2: 110-119, 2001). In mouse embryo cancer EC cells and embryonic stem (ES) cells, gene inactivation by dsRNA was successful. However (Billy E. et al "PNAS 98: 14428-14433, 2001; Paddison PJ et al" PNAS 99: 1443-1448, 2002), the induction of RNAi by long dsRNA in cultured mammalian cells is generally less successful. These failures are not linked to the interface (IFN) protection activated by long dsRNA (> 30 base pairs) (Stark GR et al., Annu. Rev. Biochem. 67: 227-264, 1998). It is easily explained by the action of two latent enzymes that form part of the pathway, one of which is 2'-5. Rigoadelate (2-5A) synthase, which is activated by dsRN A and synthesizes 2-5A, which is required for the activity of a sequence-specific RNase called RNase L (Silverman RH in Ribonucleases: Structures and Functions, eas. D'Alessio, G. and Riordan JF (Academic, New York) pp. 515-551) The other is protein kinase PKR, its activity The type phosphorylates the translation factor eukaryotic cell initiation factor (eIF2), leading to general inhibition of protein synthesis and cell death (Cle mens MJ and Elia A., J. Interferon Cytokine Res. 17: 503). —524, 1997).
[0066] 最近、 21ヌクレオチドの siRNA二本鎖がいくつかの哺乳類細胞において内因性遺 伝子の発現を特異的に抑制することが報告された（Elbashir S. M. et al, Nature 411: 494-498, 2001)。この場合、 21ヌクレオチド siRNA二本鎖は、 IFN防御システムから逃 れることができる。この知見は、 RNAほたは RNAi関連システムが哺乳類に存在するこ とを示唆した。実際に、 rde-l、 mut-7およびダイサ一のような RNAi関連蛋白質の哺乳 類相同体がいくつか同定されている（Tabara H. et al., Cell 99: 123-132, 1999; Ketti ng R. F. et al., Cell 99: 133—141, 1999; Bernstein E. et al., Nature 409: 363—366, 2 001)。 [0066] Recently, a 21-nucleotide siRNA duplex was reported to specifically suppress endogenous gene expression in some mammalian cells (Elbashir SM et al, Nature 411: 494-498, 2001). In this case, the 21 nucleotide siRNA duplex escapes from the IFN protection system. Can be. This finding suggested that RNA and RNAi-related systems exist in mammals. In fact, several mammalian homologues of RNAi-related proteins such as rde-l, mut-7 and Dicer have been identified (Tabara H. et al., Cell 99: 123-132, 1999; Ketting RF et al., Cell 99: 133-141, 1999; Bernstein E. et al., Nature 409: 363-366, 2 001).
[0067] また siRNAを発現する系は、タンデムタイプとステムループタイプ（またはヘアピンタ ィプ）の 2種類に大きく分類される。タンデムタイプは 2つの U6プロモータを持ち、それ ぞれ独立にセンス RNAとアンチセンス RNAを転写する。転写されたセンスおよびアン チセンス RNAは細胞内で 2本鎖 RNAを形成し、 siRNAとして働く。ステムループタイプ はひとつのプロモータを持ち、その下流にセンス鎖とアンチセンス鎖をループでつな V、だ構造を有する。このステムループ RNA構造を持った RNA (ショートヘアピン RNA、 shRNA)は切断酵素ダイサーなどによりプロセッシングされ、 siRNAが産生される。  [0067] Systems that express siRNA are roughly classified into two types, tandem type and stem loop type (or hairpin type). The tandem type has two U6 promoters, each of which independently transcribes sense RNA and antisense RNA. The transcribed sense and antisense RNA forms double-stranded RNA in the cell and acts as siRNA. The stem-loop type has a single promoter, and has a structure that is a V connecting the sense strand and the antisense strand in a loop downstream of it. RNA with this stem-loop RNA structure (short hairpin RNA, shRNA) is processed by a cleavage enzyme dicer or the like to produce siRNA.
[0068] なお上記「ステムループ」とは、一本鎖 RNA上に存在する逆方向反復配列間で水 素結合によって生じる二本鎖の部分 (ステム； stem)とそれに挟まれるループの部分 力 成る構造を言い、ヘアピンループとも呼ばれる。  [0068] The above "stem loop" is composed of a double-stranded part (stem) caused by hydrogen bonding between inverted repeats existing on single-stranded RNA and a partial force of the loop sandwiched between them. Refers to the structure, also called the hairpin loop.
[0069] 本明細書においては、このヘアピンループを形成する、 DRG1、 DFRP1、 DRG2、 DF RP2の mRNAに対し RNAiを引き起こすことができる RNA分子もまた、本発明に含まれ る。  [0069] As used herein, RNA molecules capable of inducing RNAi against DRG1, DFRP1, DRG2, and DF RP2 mRNA that form this hairpin loop are also included in the present invention.
[0070] 即ち本発明は、下記（1)または（2)に記載の遺伝子 (dfrpl遺伝子）の転写産物に 対してアニーリングする活性を有する核酸を提供する。  That is, the present invention provides a nucleic acid having an activity of annealing to the transcription product of the gene (dfrpl gene) described in (1) or (2) below.
(1)配列番号： 1または配列番号: 3に記載の塩基配列からなる遺伝子  (1) Gene consisting of the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 3
(2)配列番号：1または配列番号 : 3に記載の塩基配列とストリンジ ントな条件下で ノ、イブリダィズする遺伝子  (2) A gene that hybridizes with the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3 under stringent conditions
[0071] また本発明は、下記（1)または（2)に記載の遺伝子 (drgl遺伝子）の転写産物に対 してアニーリングする活性を有する核酸を提供する。  [0071] The present invention also provides a nucleic acid having an activity of annealing to the transcription product of the gene (drgl gene) described in (1) or (2) below.
(1)配列番号： 9または配列番号： 10に記載の塩基配列力 なる遺伝子  (1) The gene having the nucleotide sequence ability described in SEQ ID NO: 9 or SEQ ID NO: 10
(2)配列番号： 9または配列番号： 10に記載の塩基配列とストリンジ ントな条件下で ノ、イブリダィズする遺伝子 [0072] また本発明は、下記（1)または（2)に記載の遺伝子 (dfrp2遺伝子）の転写産物に対 してアニーリングする活性を有する核酸を提供する。 (2) A gene that hybridizes with the nucleotide sequence of SEQ ID NO: 9 or SEQ ID NO: 10 under stringent conditions [0072] The present invention also provides a nucleic acid having an activity of annealing to the transcription product of the gene (dfrp2 gene) described in (1) or (2) below.
(1)配列番号： 5または配列番号： 7に記載の塩基配列からなる遺伝子  (1) Gene consisting of the nucleotide sequence set forth in SEQ ID NO: 5 or SEQ ID NO: 7
(2)配列番号： 5または配列番号： 7に記載の塩基配列とストリンジ ントな条件下で ノ、イブリダィズする遺伝子  (2) A gene that hybridizes with the base sequence described in SEQ ID NO: 5 or 7 under stringent conditions
[0073] また本発明は、下記（1)または（2)に記載の遺伝子 (drg2遺伝子）の転写産物に対 してアニーリングする活性を有する核酸を提供する。  [0073] The present invention also provides a nucleic acid having an activity of annealing to the transcription product of the gene (drg2 gene) described in (1) or (2) below.
(1)配列番号： 11または配列番号： 12に記載の塩基配列力 なる遺伝子  (1) The gene having the nucleotide sequence ability described in SEQ ID NO: 11 or SEQ ID NO: 12
(2)配列番号： 11または配列番号： 12に記載の塩基配列とストリンジ ントな条件下 でノヽイブリダィズする遺伝子  (2) A gene that is hybridized under stringent conditions with the nucleotide sequence of SEQ ID NO: 11 or SEQ ID NO: 12.
[0074] 本発明における dfrpl遺伝子あるいは drgl遺伝子、または dfrp2遺伝子あるいは drg2 遺伝子の転写産物に対してアニーリング活性を有する核酸は、好ましくは 15塩基長 以上、より好ましくは 19塩基長以上を備えた核酸 (好ましくは 25塩基長以下の核酸） である。  [0074] The nucleic acid having an annealing activity for the dfrpl gene or drgl gene, or the transcription product of the dfrp2 gene or drg2 gene in the present invention is preferably a nucleic acid having a length of 15 bases or more, more preferably 19 bases or more ( A nucleic acid having a length of 25 bases or less is preferred.
[0075] ストリンジェントなハイブリダィゼーシヨン条件は、当業者であれば、適宜選択するこ とができる。一例を示せば、 25%ホルムアミド、より厳しい条件では 50%ホルムアミド、 4 X SSC、 50mM Hepes pH7.0、 10 Xデンハルト溶液、 20 g/ml変性サケ精子 DNAを 含むハイブリダィゼーシヨン溶液中、 42°Cで一晚プレハイブリダィゼーシヨンを行った 後、標識したプローブを添加し、 42°Cで一晩保温することによりハイブリダィゼーショ ンを行う。その後の洗浄における洗浄液および温度条件は、例えば「2 X SSC、 0.1% SDS、 50。C」、 「2 X SSC、 0.1%SDS、 42。C」、 「lxSSC、 0.1% SDS、 37°C」程度で、より厳 しい条件としては「2 X SSC、 0.1%SDS、 65°C」、 「0.5xSSC、 0.1% SDS、 42°C」程度で、 さらに厳しい条件としては「0.2xSSC、 0.1% SDS、 65°C」程度で実施することができる。 このようにハイブリダィゼーシヨンの洗浄の条件が厳しくなるほどプローブ配列と高!ヽ 相同性を有するポリヌクレオチド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、ノ、イブリダィゼーシヨンのス トリンジエンシー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 同様のストリンジエンシーを実現することが可能である。また、当業者においては、他 の生物における dfrpl遺伝子あるいは drgl遺伝子、または dfrp2遺伝子あるいは drg2 遺伝子に相当する内在性の遺伝子を、 dfrpl遺伝子あるいは drgl遺伝子、または dfrp 2遺伝子あるいは drg2遺伝子の塩基配列を基に適宜取得することが可能である。 [0075] Stringent hybridization conditions can be appropriately selected by those skilled in the art. For example, in a hybridization solution containing 25% formamide, 50% formamide under more severe conditions, 4 X SSC, 50 mM Hepes pH 7.0, 10 X Denhardt's solution, 20 g / ml denatured salmon sperm DNA, After prehybridization at 42 ° C, add a labeled probe and incubate overnight at 42 ° C for hybridization. For example, “2 X SSC, 0.1% SDS, 50.C”, “2 X SSC, 0.1% SDS, 42.C”, “lxSSC, 0.1% SDS, 37 ° C.” The more severe conditions are `` 2 X SSC, 0.1% SDS, 65 ° C '', `` 0.5xSSC, 0.1% SDS, 42 ° C '', and the more severe conditions are `` 0.2xSSC, 0.1% SDS '' , 65 ° C ". Thus, isolation of a polynucleotide having high homology with the probe sequence can be expected as the conditions for washing the hybridization become more severe. However, the combinations of the above SSC, SDS and temperature conditions are exemplary, and those skilled in the art will recognize the above and other factors that determine the stringency of the hybridization, such as probe concentration, By combining the probe length, hybridization reaction time, etc.) as appropriate. Similar stringency can be achieved. In addition, those skilled in the art will recognize an endogenous gene corresponding to a dfrpl gene or drgl gene, or a dfrp2 gene or drg2 gene in another organism, based on the base sequence of the dfrpl gene, drgl gene, dfrp 2 gene or drg2 gene. Can be obtained as appropriate.
[0076] また本発明者らは、 DFRP1蛋白質力 ¾RG1蛋白質を特異的に安定ィ匕し、 DFRP2蛋 白質が DRG2蛋白質を特異的に安定ィ匕することを見出した。即ち本発明は、 DFRP1 ポリペプチドあるいは DFRP1ポリペプチドをコードする核酸を有効成分とする DRG1蛋 白質安定化剤、または DFRP2ポリペプチドあるいは DFRP2ポリペプチドをコードする 核酸を有効成分とする DRG2蛋白質安定化剤を提供する。当該安定化剤は、 DRG蛋 白質に安定性を付与し、正常な細胞増殖に寄与すると考えられる。  [0076] The present inventors have also found that DFRP1 protein strength specifically stabilizes RG1 protein, and DFRP2 protein specifically stabilizes DRG2 protein. That is, the present invention relates to a DRG1 protein stabilizer comprising a DFRP1 polypeptide or a nucleic acid encoding a DFRP1 polypeptide as an active ingredient, or a DRG2 protein stabilizer comprising a nucleic acid encoding a DFRP2 polypeptide or a DFRP2 polypeptide as an active ingredient. I will provide a. The stabilizer is thought to impart stability to the DRG protein and contribute to normal cell growth.
[0077] 本発明にお ヽて「核酸を有効成分とする」とは、核酸を主要な活性成分として含む t 、う意味であり、核酸の含有率を制限するものではな 、。  [0077] In the present invention, "containing nucleic acid as an active ingredient" means t containing nucleic acid as a main active ingredient, and does not limit the content of nucleic acid.
[0078] また本発明者らによって、ドキシサイクリン（Dox)を添加することによって DRG1をノッ クダウンした細胞は、ドキシサイクリン無添加の細胞と比べ、運動'浸潤'転移能が上 昇することが見いだされた。そして、通常の細胞内では DRG1は細胞運動促進に働く 因子の mRNAの発現を統括的に負に制御していると考えられた。すなわち、 DRG1力 S 安定ィ匕して 、な 、細胞は運動速度が高くなると考えられ、 DRG1の機能を安定ィ匕する ことができる DFRP1は細胞運動速度を抑制するために利用可能と考えられる。  [0078] Further, the present inventors have found that cells in which DRG1 has been knocked down by adding doxycycline (Dox) have an increased ability to move and infiltrate compared to cells without doxycycline. . In normal cells, DRG1 is thought to negatively regulate the expression of mRNA, a factor that promotes cell motility. That is, if the DRG1 force S is stabilized, it is considered that the cell has a higher movement speed, and DFRP1, which can stabilize the function of the DRG1, can be used to suppress the cell movement speed.
[0079] 従って本発明は、上記 DFRP1ポリペプチドまたは DFRP1ポリペプチドをコードする 核酸を有効成分とする、細胞運動抑制剤 (細胞運動を抑制する作用を有する化合物 )を提供する。当該細胞運動抑制剤は、例えば細胞運動の異常 (増力 tl)に起因する 疾患、例えば癌、充実性腫瘍、腫瘍転移、良性腫瘍 (例えば血管腫、聴神経鞘腫、 神経繊維腫、トラコーマ及び化膿性肉芽腫)、血管機能不全、炎症及び免疫障害、 ベーチ ット病、痛風、関節炎、慢性関節リウマチ、乾癬、糖尿病性網膜症及び他の 眼血管由来疾患 (例えば後水晶体線維増殖症、黄斑変性、角膜移植拒絶、血管新 生緑内障)、骨粗鬆症、線溶系プロテアーゼの活性変化を伴う種々の皮膚疾患、特 に乾燥 ·洗浄剤等の刺激によって生じる肌荒れ'二キビなど表皮の増殖性異常を認 める皮膚状態、癌細胞の侵襲性および転移、炎症性腸疾患、前癌状態の結腸腺腫 、敗血性関節炎、骨関節炎、リューマチ様関節炎 (過剰 u—PA生産の直接関与が立 証されている)、ォステオポロ一シス、コレステリン腫よう並びに過剰のプラスミノーゲン 活性ィ匕が病因であることが示された若干の皮膚および角膜疾患、例えば角膜潰よう 、角膜炎、表皮水ほう症、乾せんおよび天ぼうそう、外傷、急性循環不全、炎症、血 栓症、脾炎並びに癌の転移及び浸潤等のプロテアーゼの活性亢進に起因する疾病 等の疾患の治療に利用することができると考えられる。 [0079] Therefore, the present invention provides a cell motility inhibitor (compound having an action of inhibiting cell motility) comprising the DFRP1 polypeptide or a nucleic acid encoding the DFRP1 polypeptide as an active ingredient. The cell motility inhibitor is a disease caused by, for example, abnormal cell motility (increased tl), such as cancer, solid tumor, tumor metastasis, benign tumor (eg hemangioma, acoustic schwannoma, neurofibroma, trachoma and purulent). Granulomas), vascular dysfunction, inflammation and immune disorders, Behcet's disease, gout, arthritis, rheumatoid arthritis, psoriasis, diabetic retinopathy and other ocular vascular diseases (e.g., posterior lens fibroproliferation, macular degeneration, Corneal transplant rejection, neovascular glaucoma), osteoporosis, various skin diseases accompanied by changes in the activity of fibrinolytic proteases, especially skin irritation caused by stimulation of drying / cleaning agents, etc. Skin condition, cancer cell invasiveness and metastasis, inflammatory bowel disease, precancerous colon adenoma Septic arthritis, osteoarthritis, rheumatoid arthritis (provided direct involvement of excess u-PA production), osteoporosis, cholesterin tumor and excess plasminogen activity Some skin and corneal diseases, such as corneal ulcers, keratitis, epidermolysis bullosa, psoriasis and pemphigus, trauma, acute circulatory failure, inflammation, thrombosis, splenitis and cancer metastasis and invasion It can be used for the treatment of diseases such as diseases caused by increased activity of proteases.
[0080] すなわち本発明は、当該細胞運動抑制剤を患者に投与する工程を含む、癌、充実 性腫瘍、腫瘍転移、良性腫瘍 (例えば血管腫、聴神経鞘腫、神経繊維腫、トラコーマ 及び化膿性肉芽腫)、血管機能不全、炎症及び免疫障害、ベーチ ット病、痛風、関 節炎、慢性関節リウマチ、乾癬、糖尿病性網膜症及び他の眼血管由来疾患 (例えば 後水晶体線維増殖症、黄斑変性、角膜移植拒絶、血管新生緑内障)、骨粗鬆症、線 溶系プロテアーゼの活性変化を伴う種々の皮膚疾患、特に乾燥'洗浄剤等の刺激に よって生じる肌荒れ'二キビなど表皮の増殖性異常を認める皮膚状態、癌細胞の侵 襲性および転移、炎症性腸疾患、前癌状態の結腸腺腫、敗血性関節炎、骨関節炎 、リューマチ様関節炎 (過剰 u— PA生産の直接関与が立証されている)、ォステオポロ 一シス、コレステリン腫よう並びに過剰のプラスミノーゲン活性ィ匕が病因であることが 示された若干の皮膚および角膜疾患、例えば角膜潰よう、角膜炎、表皮水ほう症、乾 せんおよび天ぼうそう、外傷、急性循環不全、炎症、血栓症、脾炎並びに癌の転移 及び浸潤等のプロテアーゼの活性亢進に起因する疾病等の疾患の治療方法に関す る。 [0080] That is, the present invention includes cancer, solid tumor, tumor metastasis, benign tumor (eg, hemangioma, acoustic schwannoma, neurofibroma, trachoma, and purulent), comprising the step of administering the cell motility inhibitor to a patient. Granulomas), vascular dysfunction, inflammation and immune disorders, Behcet's disease, gout, arthritis, rheumatoid arthritis, psoriasis, diabetic retinopathy and other ocular vascular diseases (e.g., posterior lens fibroproliferation, macular) Degeneration, corneal transplant rejection, neovascular glaucoma), osteoporosis, various skin diseases with changes in the activity of fibrinolytic proteases, particularly skin with proliferative abnormalities of the epidermis such as rough skin caused by stimulation of dry 'cleansing agents' Condition, aggressiveness and metastasis of cancer cells, inflammatory bowel disease, pre-cancerous colon adenoma, septic arthritis, osteoarthritis, rheumatoid arthritis (provided direct involvement of excess u-PA production), osteo Some skin and corneal diseases that have been shown to be pathogenic due to loci, cholesterol, and excessive plasminogen activity, such as corneal ulcers, keratitis, epidermolysis bullosa, psoriasis and celestia The present invention relates to a method for treating diseases such as bladder, trauma, acute circulatory failure, inflammation, thrombosis, splenitis and diseases caused by increased activity of proteases such as cancer metastasis and invasion.
[0081] また本発明は、 DFRP1ポリペプチドまたは DFRP1ポリペプチドをコードする核酸の、 癌、充実性腫瘍、腫瘍転移、良性腫瘍 (例えば血管腫、聴神経鞘腫、神経繊維腫、 トラコーマ及び化膿性肉芽腫)、血管機能不全、炎症及び免疫障害、ベーチ ット病 、痛風、関節炎、慢性関節リウマチ、乾癬、糖尿病性網膜症及び他の眼血管由来疾 患 (例えば後水晶体線維増殖症、黄斑変性、角膜移植拒絶、血管新生緑内障)、骨 粗鬆症、線溶系プロテアーゼの活性変化を伴う種々の皮膚疾患、特に乾燥'洗浄剤 等の刺激によって生じる肌荒れ'二キビなど表皮の増殖性異常を認める皮膚状態、 癌細胞の侵襲性および転移、炎症性腸疾患、前癌状態の結腸腺腫、敗血性関節炎 、骨関節炎、リューマチ様関節炎 (過剰 u— PA生産の直接関与が立証されている)、 ォステオポロ一シス、コレステリン腫よう並びに過剰のプラスミノーゲン活性ィ匕が病因 であることが示された若干の皮膚および角膜疾患、例えば角膜潰よう、角膜炎、表皮 水ほう症、乾せんおよび天ぼうそう、外傷、急性循環不全、炎症、血栓症、脾炎並び に癌の転移及び浸潤等のプロテアーゼの活性亢進に起因する疾病等の疾患治療剤 の製造における使用に関する。 [0081] The present invention also provides a cancer, solid tumor, tumor metastasis, benign tumor (eg, hemangioma, acoustic schwannoma, neurofibroma, trachoma, and purulent granulation) of DFRP1 polypeptide or nucleic acid encoding DFRP1 polypeptide. ), Vascular dysfunction, inflammation and immune disorders, Behcet's disease, gout, arthritis, rheumatoid arthritis, psoriasis, diabetic retinopathy and other ocular vascular diseases (e.g., posterior lens fibroproliferation, macular degeneration, Corneal transplant rejection, neovascular glaucoma), osteoporosis, various skin diseases with altered activity of fibrinolytic proteases, especially skin with proliferative abnormalities of the epidermis, such as rough skin caused by stimulation of dry 'cleansing agents' etc. Condition, cancer cell invasiveness and metastasis, inflammatory bowel disease, precancerous colon adenoma, septic arthritis Osteoarthritis, rheumatoid arthritis (provided direct involvement of excess u-PA production), osteoporosis, cholesterol tumors and excess plasminogen activity Activity of proteases such as corneal ulcers, keratitis, epidermolysis bullosa, psoriasis and pemphigus, trauma, acute circulatory failure, inflammation, thrombosis, splenitis and cancer metastasis and invasion The present invention relates to use in the manufacture of a therapeutic agent for diseases such as diseases caused by enhancement.
[0082] なお、本明細書で用いられる「治療」とは、概して、薬理学的なおよび Zまたは生理 学的な効果を得ることを意味する。効果とは、疾患や症状を完全にあるいは部分的 に妨げる点で予防的であってもよぐ疾患の症状を完全にあるいは部分的に治療す る点で治療的であっても良い。本明細書で用いられる「治療」という用語は、哺乳類、 特にヒトにおける疾患の治療すベてを含んでいる。そしてさらに、疾患の素因がある が未だ発病して 、ると診断されて 、な 、患者の発病の予防、疾患の進行を抑制する こと、または疾患を軽減させることなどもこの用語に含まれる。  [0082] As used herein, "treatment" generally means obtaining a pharmacological and Z or physiological effect. An effect may be prophylactic in that it completely or partially interferes with the disease or symptom, or may be therapeutic in that it completely or partially treats the symptom of the disease. As used herein, the term “treatment” includes all treatment of diseases in mammals, particularly humans. In addition, the term includes a predisposition to a disease but is still diagnosed and prevents the onset of the patient, suppresses the progression of the disease, or reduces the disease.
[0083] また本発明者らは、 DRG2をノックダウンした細胞は、細胞間の密着性が非常に高 いこと、それに細胞運動能が低下していることを見いだした。即ち、 DRG2の機能を安 定ィ匕する DFRP2の機能あるいは活性を抑制する dfrp2遺伝子の転写産物に対してァ ニーリングする活性を有する核酸、あるいは drg2遺伝子の転写産物に対してァニーリ ングする活性を有する核酸は、細胞間接着を促進する作用を有し、あるいは細胞の 運動能を低下させる作用を有すると考えられる。  [0083] Further, the present inventors have found that the cells in which DRG2 is knocked down have very high cell-cell adhesion and reduced cell motility. In other words, it stabilizes the function of DRG2, suppresses the function or activity of DFRP2, suppresses the function or activity of dfrp2 gene, or has the activity of annealing to the drg2 gene transcript. The nucleic acid is considered to have an action of promoting cell-cell adhesion or an action of reducing cell motility.
[0084] 従って、本発明は、上記本発明の dfrp2遺伝子あるいは drg2遺伝子の転写産物に 対してアニーリングする活性を有する核酸を有効成分とする、細胞間接着促進剤 (細 胞間接着を促進する作用を有する化合物)あるいは細胞運動抑制剤 (細胞運動を抑 制する作用を有する化合物)を提供する。当該細胞運動抑制剤は、癌、充実性腫瘍 、腫瘍転移、良性腫瘍 (例えば血管腫、聴神経鞘腫、神経繊維腫、トラコーマ及び化 膿性肉芽励、血管機能不全、炎症及び免疫障害、ベーチ ット病、痛風、関節炎、 慢性関節リウマチ、乾癬、糖尿病性網膜症及び他の眼血管由来疾患 (例えば後水 晶体線維増殖症、黄斑変性、角膜移植拒絶、血管新生緑内障)、骨粗鬆症、線溶系 プロテアーゼの活性変化を伴う種々の皮膚疾患、特に乾燥'洗浄剤等の刺激によつ て生じる肌荒れ'二キビなど表皮の増殖性異常を認める皮膚状態、癌細胞の侵襲性 および転移、炎症性腸疾患、前癌状態の結腸腺腫、敗血性関節炎、骨関節炎、リュ 一マチ様関節炎 (過剰 u—PA生産の直接関与が立証されている)、ォステオポローシ ス、コレステリン腫よう並びに過剰のプラスミノーゲン活性ィ匕が病因であることが示され た若干の皮膚および角膜疾患、例えば角膜潰よう、角膜炎、表皮水ほう症、乾せん および天ぼうそう、外傷、急性循環不全、炎症、血栓症、脾炎並びに癌の転移及び 浸潤等のプロテアーゼの活性亢進に起因する疾病等の疾患の治療に利用できると 考えられる。 [0084] Therefore, the present invention provides an intercellular adhesion promoter (an action that promotes intercellular adhesion), which comprises, as an active ingredient, a nucleic acid that has an activity of annealing to the transcription product of the dfrp2 gene or drg2 gene of the present invention. And a cell motility inhibitor (a compound having an action of inhibiting cell motility). Such cell motility inhibitors are cancers, solid tumors, tumor metastases, benign tumors (e.g. hemangiomas, acoustic schwannomas, neurofibromas, trachoma and purulent granulation, vascular dysfunction, inflammation and immune disorders, basin Disease, gout, arthritis, rheumatoid arthritis, psoriasis, diabetic retinopathy and other ocular vascular diseases (e.g., post crystalline fibrosis, macular degeneration, corneal transplant rejection, neovascular glaucoma), osteoporosis, fibrinolytic protease Various skin diseases accompanied by changes in activity, especially dryness Skin conditions that cause proliferative abnormalities of the epidermis such as rough skin, invasive and metastatic cancer cells, inflammatory bowel disease, precancerous colon adenoma, septic arthritis, osteoarthritis, rheumatoid arthritis ( Direct involvement of excess u-PA production has been demonstrated), osteoporosis, cholesterol tumors, and some skin and corneal diseases that have been shown to be caused by excessive plasminogen activity such as corneal ulcers Treatment of diseases such as keratitis, keratitis, epidermolysis bullosa, psoriasis and pemphigus, trauma, acute circulatory insufficiency, inflammation, thrombosis, splenitis and disease caused by increased protease activity such as cancer metastasis and invasion It is thought that it can be used.
[0085] 即ち本発明は、当該細胞運動抑制剤を患者に投与する工程を含む、癌、充実性 腫瘍、腫瘍転移、良性腫瘍 (例えば血管腫、聴神経鞘腫、神経繊維腫、トラコーマ及 び化膿性肉芽腫)、血管機能不全、炎症及び免疫障害、ベーチ ット病、痛風、関節 炎、慢性関節リウマチ、乾癬、糖尿病性網膜症及び他の眼血管由来疾患 (例えば後 水晶体線維増殖症、黄斑変性、角膜移植拒絶、血管新生緑内障)、骨粗鬆症、線溶 系プロテアーゼの活性変化を伴う種々の皮膚疾患、特に乾燥'洗浄剤等の刺激によ つて生じる肌荒れ'二キビなど表皮の増殖性異常を認める皮膚状態、癌細胞の侵襲 性および転移、炎症性腸疾患、前癌状態の結腸腺腫、敗血性関節炎、骨関節炎、リ ユーマチ様関節炎 (過剰 u—PA生産の直接関与が立証されている)、ォステオポロ一 シス、コレステリン腫よう並びに過剰のプラスミノーゲン活性ィ匕が病因であることが示さ れた若干の皮膚および角膜疾患、例えば角膜潰よう、角膜炎、表皮水ほう症、乾せ んおよび天ぼうそう、外傷、急性循環不全、炎症、血栓症、脾炎並びに癌の転移及 び浸潤等のプロテアーゼの活性亢進に起因する疾病等の疾患の治療方法に関する  [0085] That is, the present invention includes cancer, solid tumor, tumor metastasis, benign tumor (for example, hemangioma, acoustic schwannoma, neurofibroma, trachoma and suppuration), which comprises the step of administering the cell motility inhibitor to a patient. Granulomas), vascular dysfunction, inflammation and immune disorders, Behcet's disease, gout, arthritis, rheumatoid arthritis, psoriasis, diabetic retinopathy and other ocular vascular diseases (e.g., posterior lens fibroproliferation, macular) (Degeneration, corneal transplant rejection, neovascular glaucoma), osteoporosis, various skin diseases accompanied by changes in the activity of fibrinolytic proteases, especially rough skin caused by stimulation of dry 'cleansing agents', such as acne, proliferative abnormalities of the epidermis Perceived skin condition, cancer cell invasiveness and metastasis, inflammatory bowel disease, precancerous colon adenoma, septic arthritis, osteoarthritis, rheumatoid arthritis (provided direct involvement of excess u-PA production) , Osteopolo Some skin and corneal diseases whose etiology has been shown to be caused by cis, cholesterol tumors and excessive plasminogen activity, such as corneal ulcers, keratitis, epidermolysis bullosa, psoriasis and celestial fever Yes, it relates to methods for treating diseases such as trauma, acute circulatory failure, inflammation, thrombosis, splenitis and diseases caused by increased protease activity such as cancer metastasis and invasion.
[0086] また本発明は、 dfrp2遺伝子あるいは drg2遺伝子の転写産物に対してアニーリング する活性を有する核酸の、癌、充実性腫瘍、腫瘍転移、良性腫瘍 (例えば血管腫、 聴神経鞘腫、神経繊維腫、トラコーマ及び化膿性肉芽腫)、血管機能不全、炎症及 び免疫障害、ベーチェット病、痛風、関節炎、慢性関節リウマチ、乾癬、糖尿病性網 膜症及び他の眼血管由来疾患 (例えば後水晶体線維増殖症、黄斑変性、角膜移植 拒絶、血管新生緑内障)、骨粗鬆症、線溶系プロテアーゼの活性変化を伴う種々の 皮膚疾患、特に乾燥'洗浄剤等の刺激によって生じる肌荒れ'二キビなど表皮の増殖 性異常を認める皮膚状態、癌細胞の侵襲性および転移、炎症性腸疾患、前癌状態 の結腸腺腫、敗血性関節炎、骨関節炎、リューマチ様関節炎 (過剰 u—PA生産の直 接関与が立証されている)、ォステオポロ一シス、コレステリン腫よう並びに過剰のブラ スミノーゲン活性ィ匕が病因であることが示された若干の皮膚および角膜疾患、例えば 角膜潰よう、角膜炎、表皮水ほう症、乾せんおよび天ぼうそう、外傷、急性循環不全、 炎症、血栓症、脾炎並びに癌の転移及び浸潤等のプロテアーゼの活性亢進に起因 する疾病等の疾患治療剤の製造における使用に関する。 [0086] The present invention also relates to cancers, solid tumors, tumor metastases, benign tumors (eg, hemangiomas, acoustic schwannomas, neurofibromas) of nucleic acids having the activity of annealing to dfrp2 gene or drg2 gene transcripts. , Trachoma and purulent granuloma), vascular dysfunction, inflammation and immune disorders, Behcet's disease, gout, arthritis, rheumatoid arthritis, psoriasis, diabetic retinopathies and other ocular vascular diseases (e.g., posterior lens fiber growth) Disease, macular degeneration, corneal transplant rejection, angiogenic glaucoma), osteoporosis, various changes in fibrinolytic protease activity Skin diseases, especially dry 'rough skin caused by stimulation with detergents', acne, etc. Skin conditions with abnormal growth of the epidermis, invasiveness and metastasis of cancer cells, inflammatory bowel disease, precancerous colonic adenoma, septic Arthritis, osteoarthritis, rheumatoid arthritis (provided direct involvement in excess u-PA production), osteoporosis, cholesterol tumors, and excess blastinogen activity were shown to be pathogenic Protease activity such as some skin and corneal diseases, such as corneal ulcer, keratitis, epidermolysis bullosa, psoriasis and pemphigus, trauma, acute circulatory failure, inflammation, thrombosis, splenitis and cancer metastasis and invasion The present invention relates to use in the manufacture of a therapeutic agent for diseases such as diseases caused by enhancement.
[0087] さらに当該細胞間接着促進剤は、例えば細胞間接着の異常 (低下）に起因する疾 患、例えば癌等の治療に利用することができると考えられる。即ち本発明は、当該細 胞間接着促進剤を患者に投与する工程を含む、癌等の治療方法に関する。また本 発明は、 dfrp2遺伝子あるいは drg2遺伝子の転写産物に対してアニーリングする活性 を有する核酸の、癌等の治療剤の製造における使用に関する。  Furthermore, it is considered that the intercellular adhesion promoter can be used for the treatment of diseases caused by abnormalities (decrease) in intercellular adhesion, such as cancer. That is, the present invention relates to a method for treating cancer or the like, comprising the step of administering the intercellular adhesion promoter to a patient. The present invention also relates to the use of a nucleic acid having an activity of annealing to a transcription product of the dfrp2 gene or drg2 gene in the manufacture of a therapeutic agent for cancer or the like.
[0088] 上記以外にも、例えば本発明の細胞間接着促進剤は、細胞間の接着性減少が原 因である、皮膚の弛みあるいはしわ等にも効果を有すると考えられる。すなわち当該 細胞間接着促進剤はこれら症状を改善するための化粧品あるいは美容薬としての応 用も考えられる。  [0088] In addition to the above, for example, the intercellular adhesion promoter of the present invention is considered to have an effect on skin looseness or wrinkles caused by decreased adhesion between cells. In other words, the intercellular adhesion promoter may be applied as a cosmetic or cosmetic agent to improve these symptoms.
[0089] また、本発明者らによって、酵母細胞では DRG1、 DRG2、 DFRP1、 DFRP2ノックァゥ ト細胞の生長および形態はほぼ正常であるにも関わらず、ヒト癌細胞株 HeLaS3にお いて DRG1、 DRG2、 DFRP1、 DFRP2をノックダウンすると、細胞の成長が低下し、且つ 形態も異常になることが分かった。すなわち、 DRG1、 DRG2、 DFRP1、 DFRP2のノック アウトあるいはノックダウンは、正常細胞には何の影響も与えずに癌細胞特異的に成 長を減弱することが出来る可能性があり、癌細胞増殖抑制剤 (癌細胞の増殖を抑制 する作用を有する化合物）として機能できる可能性がある。  [0089] Furthermore, the present inventors have found that DRG1, DRG2, DFRP1, and DFRP2 knockout cell growth and morphology are almost normal in yeast cells, but in human cancer cell line HeLaS3, DRG1, DRG2, It was found that when DFRP1 and DFRP2 were knocked down, cell growth decreased and the morphology became abnormal. In other words, knockout or knockdown of DRG1, DRG2, DFRP1, and DFRP2 may be able to attenuate the growth of cancer cells specifically without affecting normal cells. There is a possibility that it can function as an agent (a compound having an action of suppressing the growth of cancer cells).
[0090] 従って、本発明は、上記 dfrpl遺伝子あるいは drgl遺伝子の転写産物に対してァ- 一リングする活性を有する核酸、または dfrp2遺伝子あるいは drg2遺伝子の転写産物 に対してアニーリングする活性を有する核酸を有効成分とする、癌細胞増殖抑制剤 を提供する。当該癌細胞増殖抑制剤は、例えば癌治療剤として、癌の治療に利用す ることができると考えられる。また本発明は、当該癌細胞増殖抑制剤を患者に投与す る工程を含む、癌治療方法に関する。また本発明は、 dfrpl遺伝子あるいは drgl遺伝 子の転写産物に対してアニーリングする活性を有する核酸、または dfrp2遺伝子ある V、は drg2遺伝子の転写産物に対してアニーリングする活性を有する核酸の、癌治療 剤の製造における使用に関する。 [0090] Therefore, the present invention provides a nucleic acid having an activity to bind to a transcription product of the dfrpl gene or drgl gene, or a nucleic acid having an activity to anneal to a transcription product of the dfrp2 gene or drg2 gene. Provided is a cancer cell growth inhibitor as an active ingredient. The cancer cell growth inhibitor is used for cancer treatment, for example, as a cancer therapeutic agent. It is thought that it can be done. The present invention also relates to a method for treating cancer comprising the step of administering the cancer cell growth inhibitor to a patient. The present invention also relates to a cancer therapeutic agent comprising a nucleic acid having an activity to anneal to a transcription product of a dfrpl gene or a drgl gene, or a nucleic acid having an activity to anneal to a transcription product of a dfrp2 gene, V or drg2 gene. Relates to the use in the manufacture of
[0091] 本発明の癌細胞増殖抑制剤の標的は癌細胞であれば特に限定されな！、。 [0091] The target of the cancer cell growth inhibitor of the present invention is not particularly limited as long as it is a cancer cell! ,.
[0092] 本明細書において「患者」とは、通常ヒトであるが、必ずしもヒトのみに限定されず、 ヒト以外の生物 (好ましくは、哺乳動物であり、より好ましくはマウス、ラット、サル、ィヌ 、ネコ等の脊椎動物）であっても構わない。 [0092] As used herein, "patient" is usually a human, but is not necessarily limited to only humans, and is a non-human organism (preferably a mammal, more preferably a mouse, rat, monkey, i.e. Vertebrates such as cats and cats).
[0093] また本発明は、細胞運動抑制剤のスクリーニング方法を提供する。本発明のスクリ 一-ング方法の好ましい態様においては、 DRG1蛋白質と本発明の DFRP1ポリぺプ チドの結合活性、あるいは DRG2蛋白質と本発明の DFRP2ポリペプチドの結合活性を 指標とする方法である。 [0093] The present invention also provides a screening method for a cell motility inhibitor. A preferred embodiment of the screening method of the present invention is a method using as an index the binding activity of the DRG1 protein and the DFRP1 polypeptide of the present invention, or the binding activity of the DRG2 protein and the DFRP2 polypeptide of the present invention.
[0094] 本発明の上記方法においては、まず、被験物質存在下あるいは非存在下で、 DRG 1蛋白質および DFRP1ポリペプチド、あるいは DRG2蛋白質および DFRP2ポリべプチ ドを作用させる。  [0094] In the above method of the present invention, first, DRG1 protein and DFRP1 polypeptide, or DRG2 protein and DFRP2 polypeptide are allowed to act in the presence or absence of a test substance.
[0095] 本発明のスクリーニング方法に供する被験物質としては、特に制限はない。例えば 天然化合物、有機化合物、無機化合物、蛋白質、ペプチドなどの単一化合物、並び に、化合物ライブラリー、遺伝子ライブラリーの発現産物、細胞抽出物、細胞培養上 清、発酵微生物産生物、海洋生物抽出物、植物抽出物等が挙げられるが、これらに 限定されない。また、これらの被験物質は必要に応じて適宜標識して用いることがで きる。標識としては、例えば、放射標識、蛍光標識等を挙げることができる。  [0095] There is no particular limitation on the test substance used in the screening method of the present invention. For example, natural compounds, organic compounds, inorganic compounds, proteins, peptides and other single compounds, as well as compound libraries, gene library expression products, cell extracts, cell culture supernatants, fermented microorganism products, marine organism extracts Products, plant extracts and the like, but are not limited thereto. In addition, these test substances can be appropriately labeled as necessary. Examples of the label include a radiolabel and a fluorescent label.
[0096] DRG1蛋白質および DFRP1ポリペプチド、あるいは DRG2蛋白質および DFRP2ポリ ペプチドは、被験物質との結合を検出するための指標に応じて、例えば DRG1蛋白 質および DFRP1ポリペプチドある!/、は DRG2蛋白質および DFRP2ポリペプチドの精製 された形態、細胞内または細胞外に発現した形態、あるいはァフィ二ティーカラムに 結合した形態であり得る。  [0096] The DRG1 protein and the DFRP1 polypeptide, or the DRG2 protein and the DFRP2 polypeptide are, for example, the DRG1 protein and the DFRP1 polypeptide depending on the indicator for detecting the binding to the test substance! /, The DRG2 protein and It can be a purified form of DFRP2 polypeptide, a form expressed intracellularly or extracellularly, or a form bound to an affinity column.
[0097] 本方法においては、次いで、 DRG1蛋白質および DFRP1ポリペプチド、あるいは DR G2蛋白質および DFRP2ポリペプチドとの結合活性を測定する。 DRG1蛋白質と DFRP 1ポリペプチドとの結合、あるいは DRG2蛋白質と DFRP2ポリペプチドとの結合は、例 えば DRG1蛋白質と DFRP1ポリペプチド、あるいは DRG2蛋白質と DFRP2ポリペプチド に結合した被験物質に付された標識によって検出することができる。 [0097] In this method, DRG1 protein and DFRP1 polypeptide, or DR Measure binding activity to G2 protein and DFRP2 polypeptide. The binding between the DRG1 protein and the DFRP 1 polypeptide, or the binding between the DRG2 protein and the DFRP2 polypeptide is performed by, for example, the label attached to the test substance bound to the DRG1 protein and the DFRP1 polypeptide or the DRG2 protein and the DFRP2 polypeptide. Can be detected.
[0098] 本方法にお!、ては次!、で、被験物質存在下における結合活性が被験物質非存在 下の結合活性よりも増加あるいは低下している場合に、その被験物質を選択する。 D RG1蛋白質および DFRP1ポリペプチドとの結合活性の場合は増加している物質、 DR G2蛋白質および DFRP2ポリペプチドとの結合活性の場合は低下して 、る物質を選択 する。本方法により選択 (単離)される物質 (化合物）は、細胞運動抑制剤の候補物質 として有用である。 [0098] In this method, if the binding activity in the presence of the test substance is greater or lower than the binding activity in the absence of the test substance, the test substance is selected. A substance that increases in the case of binding activity with DRG1 protein and DFRP1 polypeptide and a substance that decreases in the case of binding activity with DRG2 protein and DFRP2 polypeptide is selected. Substances (compounds) selected (isolated) by this method are useful as candidate substances for cell motility inhibitors.
[0099] 即ち、癌細胞転移あるいは細胞運動増加の時に、 DRG1蛋白質および DFRP1ポリ ペプチドとの結合活性が正常細胞と比べて低下して 、る場合、その癌細胞転移ある いは細胞運動増加に対する抑制剤としては、 DRG1蛋白質および DFRP1ポリべプチ ドとの結合活性を正常程度に増カロさせる被験物質を選択する。同じく癌細胞転移あ るいは細胞運動増加の時に、 DRG2蛋白質および DFRP2ポリペプチドとの結合活性 が正常細胞と比べて増加している場合、その癌細胞転移あるいは細胞運動増加に 対する抑制剤としては、 dfrp2遺伝子の発現量、 drg2遺伝子の発現量を正常程度に 低下させる被験物質を選択する。  [0099] That is, when cancer cell metastasis or cell motility is increased, the binding activity to DRG1 protein and DFRP1 polypeptide is lower than that of normal cells. In this case, inhibition of cancer cell metastasis or cell motility increase is suppressed. As the agent, select a test substance that increases the binding activity to DRG1 protein and DFRP1 polypeptide to a normal level. Similarly, when the binding activity to DRG2 protein and DFRP2 polypeptide is increased compared to normal cells when cancer cell metastasis or cell motility is increased, as an inhibitor against cancer cell metastasis or cell motility increase, Select a test substance that reduces the expression level of the dfrp2 gene and the expression level of the drg2 gene to normal levels.
[0100] また、上記にようにして選択される DRG2蛋白質および DFRP2ポリペプチドとの結合 活性を低下させる被験物質は、細胞間接着促進剤としても利用することができる。  [0100] In addition, the test substance that reduces the binding activity to the DRG2 protein and DFRP2 polypeptide selected as described above can also be used as an intercellular adhesion promoter.
[0101] また、本発明のスクリーニング方法の好ましい別の態様においては、 dfrpl遺伝子、 dfrp2遺伝子、 drgl遺伝子、あるいは drg2遺伝子の発現を指標とする方法である。即 ち、 dfrpl遺伝子の発現を上昇させる化合物、 dfrp2遺伝子の発現を低下させる化合 物、 drgl遺伝子の発現を上昇させる化合物、あるいは drg2遺伝子の発現を低下させ る化合物は、細胞運動抑制作用を有することが期待される。  [0101] In another preferred embodiment of the screening method of the present invention, the method uses expression of dfrpl gene, dfrp2 gene, drgl gene, or drg2 gene as an index. That is, a compound that increases the expression of the dfrpl gene, a compound that decreases the expression of the dfrp2 gene, a compound that increases the expression of the drgl gene, or a compound that decreases the expression of the drg2 gene must have a cell motility inhibitory effect. There is expected.
[0102] 本発明の上記方法においては、まず、プロモータを備えた dfrpl遺伝子、 dfrp2遺伝 子、 drgl遺伝子、あるいは drg2遺伝子を保持した細胞に被験物質 (化合物）を作用さ せる。 [0103] 本方法において用いる「細胞」は特に限定されないが、好ましくはヒト由来の細胞で ある。「dfrpl遺伝子を保持した細胞」、「dfrp2遺伝子を保持した細胞」、「drgl遺伝子 を保持した細胞」、あるいは「drg2遺伝子を保持した細胞」としては、内因性の dfrpl遺 伝子、 dfrp2遺伝子、 drgl遺伝子、あるいは drg2遺伝子を保持している細胞、または 外来性の dfrpl遺伝子、 dfrp2遺伝子、 drgl遺伝子、あるいは drg2遺伝子が導入され、 該遺伝子を保持している細胞を利用することができる。外来性の dfrpl遺伝子、 dfrp2 遺伝子、 drgl遺伝子、あるいは drg2遺伝子を保持している細胞は、通常、 dfrpl遺伝 子、 dfrp2遺伝子、 drgl遺伝子、あるいは drg2遺伝子が挿入された発現ベクターを宿 主細胞に導入することにより作製することができる。該発現べクタ一は、一般的な遺 伝子工学技術によって作製することができる。 [0102] In the above method of the present invention, first, a test substance (compound) is allowed to act on a dfrpl gene, dfrp2 gene, drgl gene, or drg2 gene-containing cell having a promoter. [0103] The "cell" used in the present method is not particularly limited, but is preferably a human-derived cell. `` Cells holding dfrpl gene '', `` cells holding dfrp2 gene '', `` cells holding drgl gene '', or `` cells holding drg2 gene '' include endogenous dfrpl gene, dfrp2 gene, Cells containing the drgl gene or drg2 gene, or cells into which an exogenous dfrpl gene, dfrp2 gene, drgl gene, or drg2 gene has been introduced can be used. Cells that carry exogenous dfrpl gene, dfrp2 gene, drgl gene, or drg2 gene are usually introduced into host cells with an expression vector inserted with dfrpl gene, dfrp2 gene, drgl gene, or drg2 gene It can produce by doing. The expression vector can be produced by general gene engineering techniques.
[0104] この方法に用いる被験物質は必要に応じて、適宜標識して用いることができる。標 識としては、上述したように、例えば放射標識、蛍光標識等を挙げることができる。  [0104] The test substance used in this method can be appropriately labeled as necessary. Examples of the label include radiolabels and fluorescent labels as described above.
[0105] 「被験物質を作用させる」とは、例えば dfrpl遺伝子、 dfrp2遺伝子、 drgl遺伝子、あ るいは drg2遺伝子を保持した細胞の培養液に被験物質を添加することによって行うこ とができるが、この方法に限定されない。被験物質が蛋白質等の場合には、該蛋白 質を発現する DNAベクターを、該細胞へ導入することにより、「作用させる」ことができ る。  [0105] "Making a test substance act" can be performed by adding a test substance to a culture medium of a cell retaining dfrpl gene, dfrp2 gene, drgl gene, or drg2 gene, for example. It is not limited to this method. When the test substance is a protein or the like, it can be “acted” by introducing a DNA vector expressing the protein into the cell.
[0106] 本方法においては次いで、 dfrpl遺伝子を保持した細胞における dfrpl遺伝子の発 現量、 dfrp2遺伝子を保持した細胞における dfrp2遺伝子の発現量、 drgl遺伝子を保 持した細胞における drgl遺伝子の発現量、あるいは drg2遺伝子を保持した細胞にお ける drg2遺伝子の発現量を測定する。発現量の測定は、当業者が簡便に行い得るも のであり、一般的な方法、例えばノーザンプロット法、ウェスタンプロット法等により適 宜実施することが可能である。例えば、 dfrpl遺伝子、 dfrp2遺伝子、 drgl遺伝子、ある いは drg2遺伝子を発現する細胞に作用させた被験物質に付した標識を指標にして 測定することも可能である。  [0106] Next, in this method, the expression level of the dfrpl gene in the cell holding the dfrpl gene, the expression level of the dfrp2 gene in the cell holding the dfrp2 gene, the expression level of the drgl gene in the cell holding the drgl gene, Alternatively, measure the expression level of the drg2 gene in cells that retain the drg2 gene. The expression level can be measured easily by those skilled in the art, and can be appropriately performed by a general method such as the Northern plot method or the Western plot method. For example, the measurement can be performed using as a marker the label attached to the test substance that has acted on the cells that express the dfrpl gene, dfrp2 gene, drgl gene, or drg2 gene.
[0107] 尚、本発明において「発現」とは、蛋白質をコードする遺伝子からの転写 (mRNAの 生成）、または該遺伝子の転写産物からの翻訳のいずれの場合をも意味する。  In the present invention, “expression” means either transcription from a gene encoding a protein (generation of mRNA) or translation from a transcription product of the gene.
[0108] 本方法にお!、ては次!、で、被験物質を作用させな 、場合と比較して、 dfrpl遺伝子 の発現量を上昇させた被験物質、 dfrp2遺伝子の発現量を低下させた被験物質、 drg 1遺伝子の発現量を上昇させた被験物質、ある!、は drg2遺伝子の発現量を低下させ た被験物質を選択する。 [0108] In this method, let's do the following! Test substance with increased expression level, test substance with decreased expression level of dfrp2 gene, test substance with increased expression level of drg 1 gene, or test substance with decreased expression level of drg2 gene Select.
[0109] 即ち、癌細胞転移あるいは細胞運動増加の時に、 dfrpl遺伝子の発現量、 drgl遺伝 子の発現量が正常細胞と比べて低下して 、る場合、その癌細胞転移あるいは細胞 運動増加に対する抑制剤としては、 dfrpl遺伝子の発現量、 drgl遺伝子の発現量を 正常程度に増加させる被験物質を選択する。同じく癌細胞転移あるいは細胞運動増 加の時に、 dfrp2遺伝子の発現量、 drg2遺伝子の発現量が正常細胞と比べて増加し ている場合、その癌細胞転移あるいは細胞運動増加に対する抑制剤としては、 dfrp2 遺伝子の発現量、 drg2遺伝子の発現量を正常程度に低下させる被験物質を選択す る。 [0109] That is, when cancer cell metastasis or cell motility increases, the expression level of dfrpl gene and drgl gene expression decrease compared to normal cells. As the agent, a test substance that increases the expression level of the dfrpl gene and the expression level of the drgl gene to a normal level is selected. Similarly, when the expression level of the dfrp2 gene and the expression level of the drg2 gene are increased compared to normal cells when cancer cell metastasis or cell motility is increased, dfrp2 is an inhibitor against the cancer cell metastasis or cell motility increase. Select a test substance that reduces the gene expression level and drg2 gene expression level to normal levels.
[0110] 上記スクリーニング方法によって取得される化合物は、細胞運動抑制剤として利用 できると期待される。  [0110] The compound obtained by the screening method is expected to be usable as a cell motility inhibitor.
[Oil 1] また上記 dfrp2遺伝子の発現量を低下させた被験物質、あるいは drg2遺伝子の発 現量を低下させた被験物質は、細胞間接着促進作用を有することが期待され、細胞 間接着促進剤としても利用することができる。  [Oil 1] In addition, the test substance in which the expression level of the dfrp2 gene is reduced or the test substance in which the expression level of the drg2 gene is reduced is expected to have an intercellular adhesion promoting action. Can also be used.
[0112] また本発明は、癌細胞増殖抑制剤のスクリーニング方法を提供する。本発明のスク リー-ング方法の好ましい態様においては、 DRG1蛋白質と本発明の DFRP1ポリぺプ チドの結合活性、あるいは DRG2蛋白質と本発明の DFRP2ポリペプチドの結合活性を 指標とする方法である。即ち、当該結合活性を低下させる化合物は、癌細胞増殖抑 制作用を有することが期待される。  [0112] The present invention also provides a screening method for a cancer cell growth inhibitor. A preferred embodiment of the screening method of the present invention is a method using as an index the binding activity of the DRG1 protein and the DFRP1 polypeptide of the present invention, or the binding activity of the DRG2 protein and the DFRP2 polypeptide of the present invention. That is, the compound that decreases the binding activity is expected to have a cancer cell growth inhibitory effect.
[0113] 本発明の上記方法においては、まず、被験物質存在下あるいは非存在下で、 DRG 1蛋白質および DFRP1ポリペプチドを作用させる。あるいは被験物質存在下あるいは 非存在下で、 DRG2蛋白質および DFRP2ポリペプチドを作用させる。 DRG1蛋白質お よび DFRP1ポリペプチド、あるいは DRG2蛋白質および DFRP2ポリペプチドは、被験 物質との結合を検出するための指標に応じて、例えば DRG1蛋白質および DFRP1ポ リペプチドあるいは DRG2蛋白質および DFRP2ポリペプチドの精製された形態、細胞 内または細胞外に発現した形態、あるいはァフィユティーカラムに結合した形態であ り得る。この方法に用いる被験物質は必要に応じて、適宜標識して用いることができ る。標識としては、上述したように、例えば放射標識、蛍光標識等を挙げることができ る。 [0113] In the above method of the present invention, first, DRG 1 protein and DFRP1 polypeptide are allowed to act in the presence or absence of a test substance. Alternatively, DRG2 protein and DFRP2 polypeptide are allowed to act in the presence or absence of the test substance. DRG1 protein and DFRP1 polypeptide, or DRG2 protein and DFRP2 polypeptide, for example, DRG1 protein and DFRP1 polypeptide or DRG2 protein and DFRP2 polypeptide are purified according to the indicator for detecting binding to the test substance. Morphology, expressed intracellularly or extracellularly, or bound to the affinity column. It can be. The test substance used in this method can be appropriately labeled as necessary. Examples of the label include radiolabels and fluorescent labels as described above.
[0114] 本方法においては次いで、 DRG1蛋白質と DFRP1ポリペプチドとの結合活性、ある いは DRG2蛋白質と DFRP2ポリペプチドとの結合活性を測定する。 DRG1蛋白質と DF RP1ポリペプチドとの結合、あるいは DRG2蛋白質と DFRP2ポリペプチドとの結合は、 例えば DRG1蛋白質と DFRP1ポリペプチド、あるいは DRG2蛋白質と DFRP2ポリぺプ チドに結合した被験物質に付された標識によって検出することができる。  [0114] Next, in this method, the binding activity between the DRG1 protein and the DFRP1 polypeptide or the binding activity between the DRG2 protein and the DFRP2 polypeptide is measured. The binding between the DRG1 protein and the DF RP1 polypeptide, or the binding between the DRG2 protein and the DFRP2 polypeptide, for example, the label attached to the test substance bound to the DRG1 protein and the DFRP1 polypeptide, or the DRG2 protein and the DFRP2 polypeptide. Can be detected.
[0115] 本方法においては次いで、被験物質存在下における結合活性が被験物質非存在 下の結合活性よりも低下している場合に、その被験物質を選択する。本方法により選 択 (単離)される物質 (化合物）は、癌細胞増殖抑制剤の候補物質として有用である。  [0115] Next, in this method, when the binding activity in the presence of the test substance is lower than the binding activity in the absence of the test substance, the test substance is selected. Substances (compounds) selected (isolated) by this method are useful as candidate substances for cancer cell growth inhibitors.
[0116] 即ち、癌細胞増殖の時に、 DRG1蛋白質と DFRP1ポリペプチドの結合活性が正常 細胞と比べて増加している場合、その癌細胞増殖に対する抑制剤としては、 DRG1蛋 白質と DFRP1ポリペプチドの結合活性を正常程度に低下させる被験物質を選択する 。同じく癌細胞増殖の時に、 DRG2蛋白質と DFRP2ポリペプチドの結合活性が正常細 胞と比べて増加している場合、その癌細胞増殖に対する抑制剤としては、 DRG2蛋白 質と DFRP2ポリペプチドの結合活性を正常程度に低下させる被験物質を選択する。  [0116] That is, when the binding activity of DRG1 protein and DFRP1 polypeptide is increased as compared with that of normal cells during cancer cell growth, as an inhibitor against the growth of cancer cells, DRG1 protein and DFRP1 polypeptide Select a test substance that reduces the binding activity to a normal level. Similarly, when the binding activity of DRG2 protein and DFRP2 polypeptide is increased compared to that of normal cells during cancer cell growth, the binding activity of DRG2 protein and DFRP2 polypeptide can be used as an inhibitor against that cancer cell growth. Select a test substance that reduces to a normal level.
[0117] 本発明のスクリーニング方法の他の態様は、 dfrpl遺伝子あるいは dfrp2遺伝子の発 現を指標とする方法である。 dfrpl遺伝子あるいは dfrp2遺伝子の発現量を低下させる ような物質は、癌細胞増殖を抑制するための候補ィ匕合物となることが期待される。  [0117] Another embodiment of the screening method of the present invention is a method using the expression of the dfrpl gene or the dfrp2 gene as an index. Substances that reduce the expression level of the dfrpl gene or the dfrp2 gene are expected to be candidate compounds for suppressing cancer cell growth.
[0118] 本方法においてはまず、プロモータを備えた dfrpl遺伝子あるいは dfrp2遺伝子を保 持した細胞に被験物質を作用させる。次いで、 dfrpl遺伝子あるいは dfrp2遺伝子の 発現量を低下させる被験物質を選択する。当該方法は、上述の細胞運動抑制剤の スクリーニング方法に倣って、適宜実施することができる。このようにして選択された 物質は癌細胞増殖抑制剤として有用であると考えられる。  [0118] In this method, first, a test substance is allowed to act on cells having a dfrpl gene or a dfrp2 gene having a promoter. Next, a test substance that reduces the expression level of the dfrpl gene or the dfrp2 gene is selected. This method can be appropriately performed following the above-described screening method for cell motility inhibitors. The substance thus selected is considered useful as a cancer cell growth inhibitor.
[0119] 即ち、癌細胞増殖の時に、 dfrpl遺伝子あるいは dfrp2遺伝子の発現量が正常細胞 と比べて増加している場合、その癌細胞増殖に対する抑制剤としては、 dfrpl遺伝子 あるいは dfrp2遺伝子の発現量を正常程度に低下させる被験物質を選択する。 [0120] 本発明のスクリーニング方法の他の態様は、 drgl遺伝子あるいは drg2遺伝子の発 現を指標とする方法である。 drgl遺伝子あるいは drg2遺伝子の発現量を低下させる ような物質は、癌細胞増殖を抑制するための候補ィ匕合物となることが期待される。 [0119] That is, when the expression level of the dfrpl gene or dfrp2 gene is increased compared to that of normal cells during cancer cell growth, the expression level of the dfrpl gene or dfrp2 gene is used as an inhibitor against the cancer cell growth. Select a test substance that reduces to a normal level. [0120] Another embodiment of the screening method of the present invention is a method using the expression of drgl gene or drg2 gene as an index. Substances that reduce the expression level of drgl gene or drg2 gene are expected to be candidate compounds for suppressing cancer cell growth.
[0121] 本方法においてはまず、プロモータを備えた drgl遺伝子あるいは drg2遺伝子を保 持した細胞に被験物質を作用させる。次いで、 drgl遺伝子あるいは drg2遺伝子の発 現量を低下させる被験物質を選択する。当該方法は、上述の癌細胞増殖抑制剤の スクリーニング方法に倣って、適宜実施することができる。このようにして選択された 物質は癌細胞増殖抑制剤として有用であると考えられる。  [0121] In this method, first, a test substance is allowed to act on a cell having a drgl gene or drg2 gene having a promoter. Next, a test substance that reduces the expression level of the drgl gene or drg2 gene is selected. This method can be appropriately carried out following the screening method for a cancer cell proliferation inhibitor described above. The substance thus selected is considered useful as a cancer cell growth inhibitor.
[0122] 即ち、癌細胞増殖の時に、 drgl遺伝子あるいは drg2遺伝子の発現量が正常細胞と 比べて増加している場合、その癌細胞増殖に対する抑制剤としては、 drgl遺伝子あ るいは drg2遺伝子の発現量を正常程度に低下させる被験物質を選択する。  [0122] That is, when the expression level of drgl gene or drg2 gene is increased compared to normal cells during cancer cell growth, the expression of drgl gene or drg2 gene is used as an inhibitor against the cancer cell growth. A test substance that reduces the amount to a normal level is selected.
[0123] なお、本発明の DRG1蛋白質あるいは DFRP1蛋白質の発現を増加させる場合は、 当該蛋白質のアミノ酸配列力 なるポリペプチドをコードする核酸、当該蛋白質、当 該ポリペプチドを当業者に周知の方法によって導入すること等によって実現すること ができる。また、本発明の DRG1蛋白質、 DFRP1蛋白質、 DRG2蛋白質、 DFRP2蛋白 質の発現を低下させる場合には、 RNAiを起こさせる核酸、当該蛋白質に結合する抗 体、当該蛋白質に対してドミナントネガティブの性質を有する蛋白質、当該蛋白質に 結合する低分子化合物の導入等によって実現することができる。上記「RNAiを起こさ せる核酸」とは、例えば上述したような dfrpl遺伝子、 drgl遺伝子、 dfrp2遺伝子、ある いは drg2遺伝子の転写産物に対してアニーリングする活性を有する核酸を挙げるこ とができる。また「当該蛋白質に結合する抗体」とは、例えば上述したような DFRP1ポ リペプチドあるいは DFRP2ポリペプチドに対する抗体、それに DRG1あるいは DRG2ポ リペプチドに対する抗体を挙げることができる。 DRG1あるいは DRG2ポリペプチドに対 する抗体ついては、上述したような DFRP1あるいは DFRP2ポリペプチドに対する抗体 と同様にして得ることが可能である。また「ドミナントネガティブの性質を有する蛋白質 」とは、当該タンパク質をコードする遺伝子を発現させることによって、内在性の野生 型蛋白質の活性を消失もしくは低下させる機能を有する蛋白質を指す。また「低分子 化合物 (低分子量物質)」とは、天然または人工の化合物であってもよい。通常、当業 者に公知の方法を用いることによって製造または取得可能な化合物である。 [0123] In the case of increasing the expression of the DRG1 protein or DFRP1 protein of the present invention, the nucleic acid encoding the polypeptide having the amino acid sequence ability of the protein, the protein, and the polypeptide can be obtained by methods well known to those skilled in the art. It can be realized by introducing it. In addition, when the expression of the DRG1 protein, DFRP1 protein, DRG2 protein, and DFRP2 protein of the present invention is decreased, the nucleic acid that causes RNAi, the antibody that binds to the protein, and the property of being dominant negative with respect to the protein. It can be realized by introducing a protein having a low molecular weight compound that binds to the protein. Examples of the above-mentioned “nucleic acid that causes RNAi” include nucleic acids having an activity of annealing to the dfrpl gene, drgl gene, dfrp2 gene or drg2 gene transcription product as described above. Examples of the “antibody that binds to the protein” include antibodies against the DFRP1 polypeptide or DFRP2 polypeptide as described above, and antibodies against the DRG1 or DRG2 polypeptide. An antibody against DRG1 or DRG2 polypeptide can be obtained in the same manner as the antibody against DFRP1 or DFRP2 polypeptide as described above. The “protein having a dominant negative property” refers to a protein having a function of eliminating or reducing the activity of an endogenous wild-type protein by expressing a gene encoding the protein. The “low molecular weight compound (low molecular weight substance)” may be a natural or artificial compound. Usually a person skilled in the art It is a compound that can be produced or obtained by using methods known to those skilled in the art.
[0124] 上記本発明の薬剤を医薬組成物として用いる場合には、当業者に公知の方法で 製剤化することが可能である。例えば、水もしくはそれ以外の薬学的に許容し得る液 との無菌性溶液、又は懸濁液剤の注射剤の形で非経口的に使用できる。例えば、薬 理学上許容される担体もしくは媒体、具体的には、滅菌水や生理食塩水、植物油、 乳化剤、懸濁剤、界面活性剤、安定剤、香味剤、賦形剤、べヒクル、防腐剤、結合剤 などと適宜組み合わせて、一般に認められた製薬実施に要求される単位用量形態で 混和することによって製剤化することが考えられる。これら製剤における有効成分量 は指示された範囲の適当な容量が得られるようにするものである。  [0124] When the agent of the present invention is used as a pharmaceutical composition, it can be formulated by methods known to those skilled in the art. For example, it can be used parenterally in the form of a sterile solution with water or other pharmaceutically acceptable liquid, or an injection in suspension. For example, a pharmacologically acceptable carrier or medium, such as sterilized water or physiological saline, vegetable oil, emulsifier, suspending agent, surfactant, stabilizer, flavoring agent, excipient, vehicle, preservative It is conceivable to formulate the drug by combining it in a unit dosage form that is generally required for pharmaceutical practice, in appropriate combination with drugs, binders, etc. The amount of active ingredient in these preparations is such that an appropriate volume within the indicated range is obtained.
[0125] 注射のための無菌組成物は注射用蒸留水のようなべヒクルを用いて通常の製剤実 施に従って処方することができる。  [0125] Sterile compositions for injection can be formulated according to conventional pharmaceutical practice using a vehicle such as distilled water for injection.
[0126] 注射用の水溶液としては、例えば生理食塩水、ブドウ糖やその他の補助薬を含む 等張液、例えば D-ソルビトール、 D-マンノース、 D-マン-トール、塩化ナトリウムが挙 げられ、適当な溶解補助剤、例えばアルコール、具体的にはエタノール、ポリアルコ ール、例えばプロピレングリコール、ポリエチレングリコール、非イオン性界面活性剤 、例えばポリソルベート 80 (TM)、 HCO- 50と併用してもよい。  [0126] Examples of aqueous solutions for injection include isotonic solutions containing physiological saline, glucose and other adjuvants, such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride. Other solubilizers such as alcohols, specifically ethanol, polyalcohols such as propylene glycol, polyethylene glycol, nonionic surfactants such as polysorbate 80 (TM), HCO-50 may be used in combination.
[0127] 油性液としてはゴマ油、大豆油があげられ、溶解補助剤として安息香酸ベンジル、 ベンジルアルコールと併用してもよい。また、緩衝剤、例えばリン酸塩緩衝液、酢酸 ナトリウム緩衝液、無痛化剤、例えば、塩酸プロ力イン、安定剤、例えばべンジルアル コール、フエノール、酸ィ匕防止剤と配合してもよい。調製された注射液は通常、適当 なアンプルに充填させる。  [0127] Examples of the oily liquid include sesame oil and soybean oil, which may be used in combination with benzyl benzoate or benzyl alcohol as a solubilizing agent. Further, a buffering agent such as phosphate buffer, sodium acetate buffer, a soothing agent such as hydrochloric acid pro-in, a stabilizer such as benzyl alcohol, phenol, or an acid proofing agent may be blended. The prepared injection is usually filled in a suitable ampoule.
[0128] 上記薬剤の剤型の種類としては、例えば経口剤として錠剤、粉末剤、丸剤、散剤、 顆粒剤、細粒剤、軟 '硬カプセル剤、フィルムコーティング剤、ペレット剤、舌下剤、ぺ 一スト剤等、非経口剤として注射剤、坐剤、経皮剤、経鼻剤、経肺剤、軟膏剤、硬膏 剤、外用液剤等が挙げられる。注射剤型の例としては、例えば、静脈内注射、筋肉 内注射、腹腔内注射、皮下注射などにより全身または局部的に投与することができる 。当業者においては投与経路や投与対象等に応じた最適の剤型を選ぶことができる [0129] また、 DFRP蛋白質あるいは DRG蛋白質をコードする DNAを生体内に投与する場合 には、レトロウイルス、アデノウイルス、センダイウィルスなどのウィルスベクターゃリポ ソームなどの非ウィルスベクターを利用することができる。投与方法としては、 in vivo 法および ex vivo法を例示することができる。 [0128] Examples of the dosage form of the drug include tablets, powders, pills, powders, granules, fine granules, soft and hard capsules, film coating agents, pellets, sublingual agents, Examples of parenteral preparations such as pastes include injections, suppositories, transdermal preparations, nasal preparations, pulmonary preparations, ointments, plasters, and liquids for external use. Examples of the injection form can be administered systemically or locally by, for example, intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, and the like. Persons skilled in the art can select the optimal dosage form according to the administration route and administration subject. [0129] In addition, when a DNA encoding DFRP protein or DRG protein is administered in vivo, a viral vector such as retrovirus, adenovirus, or Sendai virus, or a non-viral vector such as liposome can be used. . Examples of administration methods include in vivo methods and ex vivo methods.
[0130] また、患者の年齢、症状により適宜投与方法を選択することができる。本発明の薬 剤の投与量、投与方法は、患者の体重や年齢、症状などにより変動するが、当業者 であれば適宜選択することが可能である。  [0130] In addition, the administration method can be appropriately selected depending on the age and symptoms of the patient. The dosage and administration method of the pharmaceutical agent of the present invention vary depending on the patient's weight, age, symptoms, etc., but those skilled in the art can appropriately select them.
[0131] また本発明は、 drgl遺伝子にコードされたポリペプチドに対する抗体であって、 drg2 遺伝子にコードされたポリペプチドとクロスリアクティビティ一を有しない、 DRG1蛋白 質抗体に結合する抗体 (抗 DRG1蛋白質抗体)関する。即ち、配列番号 : 9または配 列番号： 10記載の遺伝子にコードされたポリペプチドに対する抗体であって、配列番 号： 11または配列番号： 12記載の遺伝子にコードされたポリペプチドとクロスリアクテ イビティーを有しない、抗 DRG1蛋白質抗体に関する。  [0131] The present invention also relates to an antibody against a polypeptide encoded by the drgl gene, which has no cross-reactivity with the polypeptide encoded by the drg2 gene and binds to a DRG1 protein antibody (anti-DRG1 Protein antibody). That is, an antibody against the polypeptide encoded by the gene described in SEQ ID NO: 9 or SEQ ID NO: 10, wherein the polypeptide encoded by the gene described in SEQ ID NO: 11 or SEQ ID NO: 12 is cross-reactivity. It does not have an anti-DRG1 protein antibody.
[0132] また本発明は、 drg2遺伝子にコードされたポリペプチドに対する抗体であって、 drgl 遺伝子にコードされたポリペプチドとクロスリアクティビティ一を有しない、 DRG2蛋白 質抗体に結合する抗体 (抗 DRG2蛋白質抗体)関する。即ち、配列番号 : 11または配 列番号： 12記載の遺伝子にコードされたポリペプチドに対する抗体であって、配列番 号： 9または配列番号： 10記載の遺伝子にコードされたポリペプチドとクロスリアタティ ビティーを有しない、抗 DRG2蛋白質抗体に関する。  [0132] The present invention also relates to an antibody against a polypeptide encoded by the drg2 gene, which has no cross-reactivity with the polypeptide encoded by the drgl gene and binds to a DRG2 protein antibody (anti-DRG2 Protein antibody). That is, an antibody against the polypeptide encoded by the gene described in SEQ ID NO: 11 or SEQ ID NO: 12, and the polypeptide encoded by the gene described in SEQ ID NO: 9 or SEQ ID NO: 10 and closliatati The present invention relates to an anti-DRG2 protein antibody that does not have a bite.
実施例  Example
[0133] 以下本発明を実施例により詳細に説明するが、本発明はこれら実施例により制限さ れるものではない。なお、本明細書に引用された文献は全て本明細書の一部として 組み込まれる。  [0133] Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited to these Examples. All documents cited in this specification are incorporated as part of this specification.
[0134] <試薬およびプラスミド >  <Reagents and plasmids>
ブラストサイジン、ヒスチジノールおよびピューロマイシンは Sigmaから購入した。 MG 132は Peptide Instituteから購入した。 FLAG標識、 Myc標識および GST融合発現べク ターは、 SR aプロモーター由来発現ベクター pME18Sから改変された pME- FLAG、 p ME-Mycまたは pME-GSTに cDNA断片を挿入することによって構築した（Shiio, Y., Y amamoto, T. and Yamaguchi, N. (1992) Negative regulation of Rb expression by the p53 gene product. Proc. Natl. Acad. Sci. U S A 89, 5206—5210.)。マウス DFRPlfl、 DFRP1 Δ N (aa 231〜426)、 DFRP1 Δ C (aa 1〜263)、 DFRP2、 DRG1および DRG2の c DNA断片は、 pAD- GAL4- 2.1 (Stratagene)に融合したマウス神経管 cDNAライブラリ 一から PCRによって増幅した。 DFRP2 cDNAを Ncolで消化し、 Klenow酵素で平滑末 端化し、 DFRP2の A C1および Δ Ν1部位欠失変異体を作製するために使用した。 DF RP2の A C2および Δ Ν2部位欠失変異体を作製するためには、 EcoRVを使用した。 D FRPI A DI (aa 236〜260欠損型）は、 Kunkel法によって DFRP1 cDNAの 2つの Bglll部 位の間の適切な位置に Bglll部位を挿入し、 Bglll-消ィ匕 pME- FLAG- DFRPlflベクター に Bglll-消化断片のうち長い方の断片を挿入することによって作製した。 HA標識ュビ キチンは pcDNA3.1ベクター（Invitrogen)にクローユングして作製した。薬剤耐性遺伝 子カセット、 DT40安定細胞系統を構築するための pA-puroベクターおよび DT40ゲノ ムライブラリーは Dr. Kurosaki (RIKEN、 日本）より提供を受けた。 Blasticidin, histidinol and puromycin were purchased from Sigma. MG 132 was purchased from Peptide Institute. FLAG-tagged, Myc-tagged and GST-fusion expression vectors were constructed by inserting cDNA fragments into modified pME-FLAG, pME-Myc or pME-GST from the SRa promoter-derived expression vector pME18S (Shiio, Y., Y amamoto, T. and Yamaguchi, N. (1992) Negative regulation of Rb expression by the p53 gene product. Proc. Natl. Acad. Sci. USA 89, 5206-5210.). Mouse DFRPlfl, DFRP1 Δ N (aa 231 to 426), DFRP1 Δ C (aa 1 to 263), DFRP2, DRG1 and DRG2 cDNA fragments are fused to pAD-GAL4-2.1 (Stratagene) mouse neural tube cDNA library Amplified from scratch by PCR. DFRP2 cDNA was digested with Ncol, blunt-ended with Klenow enzyme, and used to generate AC1 and Δ Δ1 site deletion mutants of DFRP2. EcoRV was used to generate the AC2 and ΔΝ2 site deletion mutants of DF RP2. D FRPI A DI (aa 236-260 deletion type) is inserted into the Bglll-extension pME-FLAG-DFRPlfl vector by inserting the Bglll site at the appropriate position between the two Bglll sites of the DFRP1 cDNA by the Kunkel method. It was created by inserting the longer of the Bglll-digested fragments. HA-labeled ubiquitin was prepared by cloning into pcDNA3.1 vector (Invitrogen). Drug resistance gene cassette, pA-puro vector and DT40 genomic library for constructing DT40 stable cell line were provided by Dr. Kurosaki (RIKEN, Japan).
<抗体 > <Antibody>
マウス DFRPl (aa 1〜270、 Δ C2と命名）、 DFRP2、 DRG1および DRG2をコードする c DNAを pGEX— 4T— 1 (Amersham Biosciences)および pMAL—c (New England BioLabs) 発現ベクターにサブクローユングした。ベクターを大腸菌（Escherichia coli) (BL21)に 形質転換した。発現した GST融合蛋白質および MBP融合蛋白質 (GST-DFRP1 Δ C2 、 GST- DFRP2、 GST- DRG1、 GST- DRG2、 MBP- DRG1および MBP- DRG2)は、製造 業者の取扱説明書により、それぞれ、グルタチオンセファロース 4Bビーズ (Amersham Biosciences)およびアミロース榭脂（New England BioLabs)を用いて細菌溶解液から 精製した。抗血清を作製するため、最初に 50%フロイント完全アジュバントに乳化し、 その後 50%フロイント不完全アジュバント（Sigma)に乳化した GST- DFRP1 A C2、 GST- DFRP2、 GST- DRG1、 MBP- DRG1または GST- DRG2で 2週間間隔で皮下投与により 免疫した。 DFRP1 A C2抗血清は、 MAbTrap (商標） GIIカラム（Amersham Biosciences )でァフィユティー精製した。 GST- DFRP2に対する抗血清は、硫安沈殿により粗精製 した。 MBP- DRG1および GST- DRG2に対する抗血清は、それぞれ、 GST- DRG1およ び MBP-DRG2カラムに最終的に吸着させてァフィユティー精製した。交差反応性 (ク ロスリアクティビティ一）を低下させるために、 DRG1および DRG2に対する抗血清は、 最終的なカラムに移す前にそれぞれ、 MBP-DRG2および GST-DRG1カラムへ事前に 吸着させて清浄化した。抗- c-Myc (A-14)および抗- HA(F- 7)抗体は Santa Cruz Bio technologyから購入し、抗 FLAG (M2)は Sigmaから購入した。抗チューブリンは Oncog eneから to入した。 CDNA encoding mouse DFRPl (aa 1-270, ΔC2), DFRP2, DRG1 and DRG2 was subcloned into pGEX—4T—1 (Amersham Biosciences) and pMAL—c (New England BioLabs) expression vectors . The vector was transformed into Escherichia coli (BL21). The expressed GST and MBP fusion proteins (GST-DFRP1 ΔC2, GST-DFRP2, GST-DRG1, GST-DRG2, MBP-DRG1, and MBP-DRG2) are each glutathione sepharose according to the manufacturer's instructions. Purified from bacterial lysates using 4B beads (Amersham Biosciences) and amylose sucrose (New England BioLabs). GST-DFRP1 A C2, GST-DFRP2, GST-DRG1, MBP-DRG1, or GST first emulsified in 50% Freund's complete adjuvant and then emulsified in 50% Freund's incomplete adjuvant (Sigma) to produce antiserum -Immunized with DRG2 subcutaneously at 2-week intervals. DFRP1 AC2 antiserum was affinity purified on a MAbTrap ™ GII column (Amersham Biosciences). Antiserum against GST-DFRP2 was roughly purified by ammonium sulfate precipitation. Antisera against MBP-DRG1 and GST-DRG2 were finally adsorbed on GST-DRG1 and MBP-DRG2 columns, respectively, and were purified. Cross-reactivity To reduce loss activity 1), anti-sera against DRG1 and DRG2 were pre-adsorbed and cleaned on MBP-DRG2 and GST-DRG1 columns, respectively, before being transferred to the final column. Anti-c-Myc (A-14) and anti-HA (F-7) antibodies were purchased from Santa Cruz Bio technology and anti-FLAG (M2) was purchased from Sigma. Anti-tubulin was entered from Oncogene.
[0136] <免疫沈降アツセィ >  [0136] <Immunoprecipitation atsey>
免疫沈降のために、培養細胞を TNE緩衝液（10 mM Tris- HC1、 pH 7.8、 1% Nonidet P-40、 150 mM NaCl、 1 mM EDTA)中で溶解した。細胞溶解液を遠心分離し、上清 を、プロテイン Gセファロースビーズ（Amersham Biosciences)と共にインキュベーショ ンし、事前に清浄化した。清浄化した溶解液を、適当な抗体およびプロテイン Gセフ ァロースビーズと共に 1時間インキュベーションした。遠心分離によってビーズを回収 し、 TNE緩衝液で 3回洗浄した。捕獲した蛋白質を SDS試料緩衝液中で煮沸してビー ズから溶出し、 SDS-PAGEおよびウェスタンブロット法によって分析した。  Cultured cells were lysed in TNE buffer (10 mM Tris-HC1, pH 7.8, 1% Nonidet P-40, 150 mM NaCl, 1 mM EDTA) for immunoprecipitation. The cell lysate was centrifuged and the supernatant was incubated with protein G sepharose beads (Amersham Biosciences) and pre-cleaned. The cleaned lysate was incubated with the appropriate antibody and protein G sepharose beads for 1 hour. The beads were collected by centrifugation and washed 3 times with TNE buffer. Captured proteins were boiled in SDS sample buffer and eluted from the beads and analyzed by SDS-PAGE and Western blotting.
ュビキチン複合体の分析は、ベクタ一-形質移入細胞を 2日間増殖させ、 10 /z Mの プロテアソーム阻害剤 MG132で処理または処理しな!、で、 3時間後に TNE緩衝液で 溶解した。  For analysis of the ubiquitin complex, Vector 1-transfected cells were grown for 2 days and treated or not with the 10 / z M proteasome inhibitor MG132! And lysed in TNE buffer after 3 hours.
[0137] <免疫蛍光分析 >  [0137] <Immunofluorescence analysis>
HeLa S3細胞をカバーガラス上で培養し、 2日間増殖させた。接着した細胞を PBSで 洗浄し、メタノール-アセトン（1:1)中で 10分間固定し、乾燥し、 2% BSA中でブロックし た。固定した細胞を DFRP1または DRG1に対する抗体と共に 1時間インキュベーション した。インキュベーション終了時に、細胞を、 0.2% Tween20を含有する PBS中で洗浄 し、 Alexa 488-結合抗ゥサギ二次抗体 (Molecular Probes)で 1時間染色した。スライド をレーザー走査共焦点顕微鏡（Radiance 2000、 Bio-Rad)で調査した。 3つの画像か らなる Zシリーズを、絞り設定 2.0およびステップサイズ 0.5 mで細胞の内部から回収 した。画像は、最大画素方法（LaserSharp2000ソフトウェア、 Bio-Rad)で投影した。  HeLa S3 cells were cultured on coverslips and grown for 2 days. Adherent cells were washed with PBS, fixed in methanol-acetone (1: 1) for 10 minutes, dried and blocked in 2% BSA. Fixed cells were incubated with antibodies against DFRP1 or DRG1 for 1 hour. At the end of the incubation, the cells were washed in PBS containing 0.2% Tween20 and stained with Alexa 488-conjugated anti-rabbit secondary antibody (Molecular Probes) for 1 hour. The slides were examined with a laser scanning confocal microscope (Radiance 2000, Bio-Rad). The Z series consisting of three images was collected from inside the cells with an aperture setting of 2.0 and a step size of 0.5 m. Images were projected by the maximum pixel method (LaserSharp2000 software, Bio-Rad).
[0138] < DT40細胞 >  [0138] <DT40 cells>
10%ゥシ胎仔血清（Sigma)、 1%ニヮトリ血清（Sigma)、ペニシリン、ストレプトマイシン および j8 -メルカプトエタノールを添カ卩した RPMI培地（JRH Biosciences)中で DT40細 胞を増殖させた。 -ヮトリ DFRPl cDNAの断片は、デジエネレートプライマーセット（5'- GCGAATTCATGCCNCCNAARAARC-3' (配列番号： 13)および 5'- GCCTCGAGY TTYTCYTC YTTYTTYTTRTC-3 ' (配列番号： 14) )を用いて DT40 cDNAから増幅し た。完全長の DFRPl cDNA(DDBJ/EMBL/GenBankァクセッション番号 AB 185935)は 、約 5 X 106プラークの DT40 cDNAライブラリー（ λ Zap)を、部分 cDNA断片をプローブ としてスクリーニングすることによって単離した。 dfrpl遺伝子座の一部を含有するゲノ ム DNA断片は、 LA Taqならびにフォーワードプライマー 5'- AGCAAGAAGGCGGAC CAGAA-3' (配列番号： 15)およびリバースプライマー 5'- GAGGAAGAGCATGGCG ATAC-3' (配列番号： 16)を用いるロング PCRで単離した。 DT40 cells in RPMI medium (JRH Biosciences) supplemented with 10% fetal bovine serum (Sigma), 1% chicken serum (Sigma), penicillin, streptomycin and j8-mercaptoethanol The vesicles were allowed to grow. -Avian DFRPl cDNA fragments were obtained from DT40 cDNA using the degenerate primer set (5'-GCGAATTCATGCCNCCNAARAARC-3 '(SEQ ID NO: 13) and 5'-GCCTCGAGY TTYTCYTC YTTYTTYTTRTC-3' (SEQ ID NO: 14)). Amplified. The full-length DFRPl cDNA (DDBJ / EMBL / GenBank accession number AB 185935) was isolated by screening about 5 x 10 6 plaques of the DT40 cDNA library (λZap) using a partial cDNA fragment as a probe. . Genomic DNA fragments containing part of the dfrpl locus are LA Taq and forward primer 5'-AGCAAGAAGGCGGAC CAGAA-3 '(SEQ ID NO: 15) and reverse primer 5'-GAGGAAGAGCATGGCG ATAC-3' (SEQ ID NO: Isolated by long PCR using 16).
[0139] -ヮトリ dfrpl遺伝子を崩壊するための標的ベクター dfrplBsrおよび dfrpHisDを構築 するため、 ZnF-1ドメインをコードするェキソンを含有する約 2.5 kbのゲノム DNAを、 dfr piの転写と逆方向のブラストサイジン-またはヒスチジノール-耐性遺伝子と交換した 。マウス DFRP1 11または A D1を挿入した pA- puroベクターのエレクト口ポレーシヨン（G ene Pulser II、 Bio- Rad、 550V, 25 μ F)による安定な形質移入体をピューロマイシン 中での増殖によって選択した。  [0139]-To construct the target vectors dfrplBsr and dfrpHisD for disrupting the dfrpl gene, approximately 2.5 kb of genomic DNA containing an exon encoding the ZnF-1 domain was blasted in the direction opposite to transcription of dfr pi. It was exchanged for a saidin- or histidinol-resistance gene. Stable transfectants of the pA-puro vector inserted with mouse DFRP111 or AD1 with the electopore position (Gene Pulser II, Bio-Rad, 550 V, 25 μF) were selected by growth in puromycin.
[0140] ノーザンブロット分析のために、総 RNAを-ヮトリ dfrpl cDNAプローブ（nt +1〜+129 0)、 -ヮトリ drgl部分 cDNAプローブ（562 bp、デジエネレートプライマー 5'- GGCAGC CTAYGAATTYAC-3' (配列番号： 17)および 5 '-AAARTTCCAGCGGTGATG- 3' (配 列番号： 18)を用いる PCRで増幅、 DDBJ/EMBL/GenBankァクセッション番号 AB186 130)または-ヮトリ drg2部分 cDNAプローブ（564 bp、プライマー 5'- GCGAATTCTGC ATCTTATGAGTTC AC-3 ' (配列番号： 19)および 5'- GCCTCGAGCCAGGTTCAAT TTCATG-3' (配列番号： 20)を用いる PCRで増殖）にハイブリダィゼーシヨンした。  [0140] For Northern blot analysis, total RNA was analyzed using-ヮ tri dfrpl cDNA probe (nt +1 to +129 0),-ヮ tri drgl partial cDNA probe (562 bp, degenerate primer 5'-GGCAGC CTAYGAATTYAC-3 ' (SEQ ID NO: 17) and 5'-AAARTTCCAGCGGTGATG-3 '(SEQ ID NO: 18) amplified by PCR, DDBJ / EMBL / GenBank accession number AB186 130) or-ヮ tri drg2 partial cDNA probe (564 bp, Hybridization was performed with primers 5′-GCGAATTCTGC ATCTTATGAGTTC AC-3 ′ (SEQ ID NO: 19) and 5′-GCCTCGAGCCAGGTTCAAT TTCATG-3 ′ (SEQ ID NO: 20).
[0141] <アフリカッメガエル（Xenopus laevis)における発現分析 >  [0141] <Expression analysis in Xenopus laevis>
ゼノパス（Xenopus) dfrpl cDNAを単離するために、本発明者らは最初に、フォーヮ 一ドプライマ一 5'- GCGGATCCATGCCGCCTAAGAAAG- 3' (配列番号： 21)および リバースプライマー 5'- GCCTCGAGGCGTCATCTGCTTCTT- 3' (配列番号： 22)を 使用してゼノパス肝臓 cDNAプールから、 PCRによってマウス DFRP1の N末端部分に 相同性の高いペプチドをコードする部分 cDNA断片を増幅した。本発明者らは、この 断片を使用して、高いストリンジエンシー（0.2 X SSC、 0.1% SDS、 50°C)において λ Zip Lox (Gibco BRL)において約 7 X 105プラークのアフリカッメガエル（Xenopus laevis)胚 (発生段階第 24〜32期） cDNAライブラリーを探索した。単離したゼノパス dfrpl cDNA の完全な配列を DDBJ/EMBL/GenBank (ァクセッション AB185934)に提出した。ホー ルマウントインサイチューハイブリダィゼーシヨンおよびノーザンブロット分析の手法は 以前に記載されている（Ishikawa, K., Azuma, S.， Ikawa, S" et al. (2003) Cloning an d characterization of Xenopus laevis drg2, a member of the developmentally regulate d GTP- binding protein subfamily. Gene 322, 105-112.)。本発明者らは、ゼノパス dfr piプローブのヌクレオチド- 68から +1755をカバーする cDNAを使用した。 In order to isolate the Xenopus dfrpl cDNA, we first developed a four-primed primer 5'- GCGGATCCATGCCGCCTAAGAAAG-3 '(SEQ ID NO: 21) and a reverse primer 5'-GCCTCGAGGCGTCATCTGCTTCTT-3' (sequence No. 22) was used to amplify a partial cDNA fragment encoding a peptide highly homologous to the N-terminal part of mouse DFRP1 from the xenopus liver cDNA pool using PCR. The inventors have Using the fragments, Xenopus laevis embryos (development) of about 7 X 10 5 plaques in λ Zip Lox (Gibco BRL) at high stringency (0.2 X SSC, 0.1% SDS, 50 ° C) Stages 24 to 32) A cDNA library was searched. The complete sequence of the isolated Xenopus dfrpl cDNA was submitted to DDBJ / EMBL / GenBank (Accession AB185934). Hole mount in situ hybridization and Northern blot analysis techniques have been previously described (Ishikawa, K., Azuma, S., Ikawa, S "et al. (2003) Cloning an d characterization of Xenopus Gene 322, 105-112.) We used cDNA covering nucleotides -68 to +1755 of the Xenopus dfr pi probe.
[0142] <コンピュータ解析 >  [0142] <Computer analysis>
多数の配列を ClustalXソフトウェア（ver. 1.81)によって整列させた。高度化のため に整列させた結果に手動による改良をカ卩えた。整列したデータは BOXSHADEソフト ウェア (ver. 3.31)で影をつけた。特徴的なドメインおよび位置（図 1A)は、 SMARTデ ータベース検索および報告されている記載（Bogerd, H. P., Fridell, R. A., Benson, R . E., Hua, J. and Cullen, B. R. (1996) Protein sequence requirements for function o f the human T— cell leukemia virus type 1 Rex nuclear export signal delineated by a n ovel in vivo randomization-selection assay. Mol. Cell. Biol. 16, 4207-4214.)によつ て決定した。  Multiple sequences were aligned by ClustalX software (ver. 1.81). A manual improvement was added to the results of alignment for sophistication. The sorted data was shaded with BOXSHADE software (ver. 3.31). Characteristic domains and locations (Fig. 1A) are derived from SMART database searches and reported descriptions (Bogerd, HP, Fridell, RA, Benson, R.E., Hua, J. and Cullen, BR (1996) Protein. sequence requirements for function of the human T—cell leukemia virus type 1 Rex nuclear export signal delineated by an in vivo randomization-selection assay. Mol. Cell. Biol. 16, 4207-4214.).
[0143] <分画分析 (fractionation analysis) >  [0143] <fractionation analysis>
マウス (メス)を 24時間以上絶食させた後、肝臓を取り出し、 0.25Mショ糖 (S)(50mM T ris-HCl, pH=7.5, 25mM KCl, 5mM MgCl , (=TKM》 buffer(0.25STKM)中で、 dounce  After fasting mice (female) for more than 24 hours, the liver is removed and 0.25 M sucrose (S) (50 mM Tris-HCl, pH = 7.5, 25 mM KCl, 5 mM MgCl, (= TKM) buffer (0.25STKM) In dounce
2  2
homogenizerを用いて 30ストロークし、細胞破砕液 (ホモジネート)を得た。未破砕の細 胞を 700g, 5minの遠心で除去し、その上清を 15,000g, lOminの遠心で、上清 (PMS)と ペレット (ミトコンドリア)に分離させた。 PMSをさらに 105,000g, 45min超遠心し、 P100画 分を得た。  Using a homogenizer for 30 strokes, a cell lysate (homogenate) was obtained. Undisrupted cells were removed by centrifugation at 700 g for 5 min, and the supernatant was separated into a supernatant (PMS) and a pellet (mitochondria) by centrifugation at 15,000 g, lOmin. PMS was further ultracentrifuged at 105,000 g for 45 min to obtain P100 fraction.
[0144] くポリソーム解析 > [0144] Polysome Analysis>
2.0STKM, 1.5STKMの不連続ショ糖勾配に、上で得られた lmLの PMSを重層し、 10 5,000g, 3h超遠心した。ポリソームペレットを 0STKMに再溶解し、 15%-40%濃度のショ 糖勾配に重層し、 150,000g , 50min超遠心した。 EDTA処理は 10mMの濃度で行つ ave The 1 mL PMS obtained above was layered on a discontinuous sucrose gradient of 2.0STKM and 1.5STKM, and ultracentrifuged at 10 5,000 g for 3 h. Re-dissolve the polysome pellet in 0STKM and add 15% -40% concentration Overlaid on a sugar gradient, ultracentrifugation was performed at 150,000 g for 50 min. EDTA treatment is performed at a concentration of 10 mM ave
た。 It was.
〔実施例 1〕 DRGファミリー結合蛋白質の同定 [Example 1] Identification of DRG family binding protein
酵母における蛋白質複合体の総合的な分析データベースを検索したところ (Uetz, P., Giot, L" Cagney, G" et al. (2000) A comprehensive analysis of protein-protein interactions in Saccharomyces cerevisiae. Nature 403, 623-627. ; Ito, T., Chiba, T., Ozawa, R., Yoshida, M., Hattori, M. and Sakaki, Y. (2001) A comprehensive two- hybrid analysis to explore the yeast protein interactome. Proc. Natl. Acad. Sci. U S A 98, 4569-4574. ; Ho, Y.， Gruhler, A., Heilbut, A., et al. (2002) Systematic identif ication of protein complexes in Saccharomyces cerevisiae by mass spectrometry. Nat ure 415, 180- 183.)、本発明者らは、サッカロミセス'セレピシェ（Saccharomyces cere visiae) DRGlおよび DRG2は (JIR2 (genetically interacting with nbosomal genes 2)と 物理的に相互作用することに気がついた。 GIR2は真核生物において高度に保存さ れており（ヒト（Homo sapiens) GIR2と S.セレビシェ（S. cerevisiae) GIR2はアミノ酸（aa) レベルにおいて 42%の相同性を有していた）、基礎的な経路に関与することを示して V、た。 GIR2は、ュビキチン結合酵素（E2)および E2変種 (UEV)と有意な三次元的相 同性を示す（Nameki, N., Yoneyama, M., Koshiba, S., et al. (2004) Solution structu re of the RWD domain of the mouse GCN2 protein. Protein Sci. 13, 2089- 2100.)特 徴的な RWDドメイン（図 1A) (GIドメインとも呼ばれる； Kubota, H.， Sakaki, Y. and Ito, T. (2000) GI domain-mediated association of the eucaryotic initiation factor 2 alpha kinase Gし N2 with its activator Gし Nl is required for general amino acid control in b udding yeast. J. Biol. Chem. 275, 20243-20246. ; Doerks, T" Copley, R. R.， Schultz , J., Ponting, C. P. and Bork, P. (2002) Systematic identification of novel protein d omain families associated with nuclear lunctions. enome Res. 12, 47- 56.)を有し、 D RGファミリーがポリュビキチンィ匕によって調節されることを示唆していた。ほ乳類 DRG ファミリー蛋白質も GIR2のほ乳類ホモログに結合することを確認するために、本発明 者らは、 PCRを用いて、マウス DRG1、 DRG2および GIR2 cDNAを増幅し、発現べクタ 一を構築した。 [0146] 293T細胞に、 FLAG標識 DRG1または DRG2および Myc標識 GIR2の発現ベクターを 同時形質移入し、細胞抽出物に、抗 FLAG抗体を用いる免疫沈降アツセィを実施し た。さらに沈降物をウェスタンプロット法で分析した。 A comprehensive analysis database of protein complexes in yeast (Uetz, P., Giot, L "Cagney, G" et al. (2000) A comprehensive analysis of protein-protein interactions in Saccharomyces cerevisiae. Nature 403, 623-627.; Ito, T., Chiba, T., Ozawa, R., Yoshida, M., Hattori, M. and Sakaki, Y. (2001) A comprehensive two- hybrid analysis to explore the yeast protein interactome. Proc. Natl. Acad. Sci. USA 98, 4569-4574.; Ho, Y., Gruhler, A., Heilbut, A., et al. (2002) Systematic identif ication of protein complexes in Saccharomyces cerevisiae by mass spectrometry. Nat ure 415, 180-183.), The present inventors found that Saccharomyces cere visiae DRGl and DRG2 physically interact with (JIR2 (genetically interacting with nbosomal genes 2)). GIR2 is highly conserved in eukaryotes (Homo sapiens GIR2 and S. cerevisiae GIR2 V, which showed 42% homology at the acid (aa) level, was shown to be involved in the basic pathway GIR2 was significantly different from ubiquitin-conjugating enzyme (E2) and E2 variants (UEV) Three-dimensional homology (Nameki, N., Yoneyama, M., Koshiba, S., et al. (2004) Solution structu re of the RWD domain of the mouse GCN2 protein. Protein Sci. 13, 2089- 2100.) Characteristic RWD domain (Fig. 1A) (also called GI domain; Kubota, H., Sakaki, Y. and Ito, T. (2000) GI domain-mediated association of the eucaryotic initiation factor 2 alpha kinase G N2 with its activator G Nl is required for general amino acid control in b udding yeast. J. Biol. Chem. 275, 20243-20246 .; Doerks, T "Copley, RR, Schultz, J., Ponting, CP and Bork, P. (2002) Systematic identification of novel protein d omain families associated with nuclear lunctions.enome Res. 12, 47-56.), D RG family is regulated by polyubiquitin It had suggested that. In order to confirm that the mammalian DRG family protein also binds to the mammalian homologue of GIR2, the present inventors used PCR to amplify mouse DRG1, DRG2, and GIR2 cDNAs to construct an expression vector. [0146] Expression vectors of FLAG-tagged DRG1 or DRG2 and Myc-tagged GIR2 were co-transfected into 293T cells, and the cell extract was subjected to immunoprecipitation assay using an anti-FLAG antibody. The sediment was further analyzed by Western plotting.
結果、 Myc-GIR2は FLAG- DRG2と同時沈降した力 FLAG- DRG1で同時沈降した Myc-GIR2は少なかった（図 1B、左のパネル）。これらの所見から、マウス GIR2が、 DR Gはりも DRG2に優先的に結合することが示唆された。  As a result, Myc-GIR2 was co-precipitated with FLAG-DRG2 and there was little Myc-GIR2 co-precipitated with FLAG-DRG1 (Figure 1B, left panel). These findings suggest that mouse GIR2 binds preferentially to DRG beam DRG2.
[0147] また、以前に報告された、 DRG1と DRG2が進化的に別個であるという系統発生デー タに基づいて、本発明者らは、 DRG1は独自の結合パートナーを有する可能性がある と仮定した。従って、本発明者らは、コンピュータで GIR2と構造的に関連のある蛋白 質をスクリーニングした。完全長のマウス GIR2蛋白質配列をクエリーとして用いた BLA ST検索により、マウス即時型応答エリスロポイエチン 4のヒト（H. sapiens)のオルソログ の 官 '性のめる 白質 H. sapiens likely ortholog of mouse immediate early respons e erythroEoietin 4) (LEREP04)として既知の蛋白質が得られた。 GIR2および LEREP 04はリジン（K)、グルタミン酸（E)およびァスパラギン酸（D)含量が高く（GIR2, K, 8.2 %;E, 16.2% ;D, 9.1%;LEREP04, K, 14.1%;Ε, 12.7% ;D, 9.2%)、マウス、ハエおよび酵 母由来の多数の配列を整列することによって規定される、約 60 aaからなる相同性の 高い領域を有していた（図 1A、上のセクション、斜線つきボックス）。し力し、この領域 以外の配列は構造的に類似していな力つた。 LEREP04は、本発明者らが本明細書 にお 、て ZnF-1および ZnF-2と名づけた 2つの独自の CCCH-型ジンク（Zn)フィンガ 一、ならびに C-末端で広く認められている NESコンセンサス [L-X - (F,I, L, V, M) - [0147] Also, based on previously reported phylogenetic data that DRG1 and DRG2 are evolutionarily distinct, we hypothesized that DRG1 may have its own binding partner did. Therefore, the present inventors screened for proteins structurally related to GIR2 by computer. H. sapiens likely ortholog of mouse immediate early respons by BLA ST search using full-length mouse GIR2 protein sequence as a query, and an ortholog of the human immediate response erythropoietin 4 human (H. sapiens) A protein known as e erythroEoietin 4) (LEREP04) was obtained. GIR2 and LEREP 04 have high lysine (K), glutamic acid (E) and aspartic acid (D) content (GIR2, K, 8.2%; E, 16.2%; D, 9.1%; LEREP04, K, 14.1%; Ε, 12.7%; D, 9.2%), which had a highly homologous region consisting of about 60 aa, defined by aligning multiple sequences from mice, flies and yeast (Figure 1A, top) Section, shaded box). However, the arrangements outside this region were not structurally similar. LEREP04 is the two unique CCCH-type zinc (Zn) fingers that we have named herein ZnF-1 and ZnF-2, as well as the widely recognized NES Consensus (LX-(F, I, L, V, M)-
2-3 2-3
X — L— X— (L, I)] ( Bogerd, H. P., Fridell, R. A" Benson, R. E" Hua, J. and Cullen, X — L— X— (L, I)] (Bogerd, H. P., Fridell, R. A "Benson, R. E" Hua, J. and Cullen,
2-3 2-3
B. R. (199b; Protein sequence requirements for function of the human T- cell leuke mia virus type 1 Rex nuclear export signal delineated by a novel in vivo randomizati on- selection assay. Mol. Cell. Biol. 16, 4207-4214.)に適合するロイシン-リッチ NES 配列を有していた。 LEREP04も真核生物において高度に保存されている（ヒ HH. sa piens) LEREP04および S.セレビシェ（S.cerevisiae) LEREP04は aaレベルで 55%の相 同性を有していた)。 LEREP04と DRGファミリー蛋白質の間の相互作用を検討するた めに、 293T細胞に、 FLAG- DRG1または DRG2の発現ベクターおよび Mvc標識 LERE PCMを発現するベクターを同時形質移入し、細胞抽出物に抗 FLAG抗体を用 ヽる免 疫沈降、次にウェスタンプロット法を実施した。 BR (199b; Protein sequence requirements for function of the human T-cell leuke mia virus type 1 Rex nuclear export signal delineated by a novel in vivo randomizati on- selection assay.Mol. Cell. Biol. 16, 4207-4214.) It had a compatible leucine-rich NES sequence. LEREP04 is also highly conserved in eukaryotes (HH. Sa piens) LEREP04 and S. cerevisiae LEREP04 had 55% homology at the aa level). To investigate the interaction between LEREP04 and DRG family proteins, 293T cells were treated with FLAG-DRG1 or DRG2 expression vectors and Mvc-labeled LERE. A vector expressing PCM was co-transfected and subjected to immunoprecipitation using an anti-FLAG antibody in the cell extract, followed by Western plotting.
[0148] 結果、 Myc- LEREP04は FLAG- DRG1と同時沈降したが、 FLAG- DRG2とは同時沈 降せず（図 1B、右のパネル）、 LEREP04は DRGlと選択的に相互作用することが示さ れた。 DRG1および DRG2に対するそれぞれ LEREP04および GIR2の独自の結合特異 性に基づいて、本発明者らは LEREP04を DRGファミリー調節蛋白質 (DFRP) 1および GIR2を DFRP2と改名した。また、 DFRP1と DFRP2の間の相同性の高い領域を DFRPド メインと名づけた（図 1A、上のセクション、斜線つきのボックス）。  [0148] As a result, Myc-LEREP04 co-precipitated with FLAG-DRG1, but not FLAG-DRG2 (Figure 1B, right panel), indicating that LEREP04 interacts selectively with DRGl. It was. Based on the unique binding specificity of LEREP04 and GIR2 for DRG1 and DRG2, respectively, we renamed LEREP04 as DRG family regulatory protein (DFRP) 1 and GIR2 as DFRP2. The region with high homology between DFRP1 and DFRP2 was named the DFRP domain (Figure 1A, upper section, box with hatching).
[0149] 〔実施例 2〕 DFRP1と DRG1のインビボにおける物理的相互作用  [Example 2] Physical interaction of DFRP1 and DRG1 in vivo
DRG1と DFRP1の特異的な相互作用をインビボにお 、て確認するために、本発明 者らは、内在性 DFRP1、 DRGlまたは DRG2を特異的に認識するポリクローナル抗体 を作製した。 DRG1および DRG2の両方を認識する交差反応性の抗体を除去するた めに、最終的なァフィユティー精製の前に、抗 DRG1血清および抗 DRG2血清を、そ れぞれ組換え DRG2および DRG1に吸着させ、事前に清浄化した。結果、 DRG1およ び DRG2に対する精製抗体は特異性が高ぐ認識可能な交差反応性がないことが確 f*i¾ れ 。  In order to confirm the specific interaction between DRG1 and DFRP1 in vivo, the present inventors produced a polyclonal antibody that specifically recognizes endogenous DFRP1, DRGl or DRG2. To remove cross-reactive antibodies that recognize both DRG1 and DRG2, anti-DRG1 and anti-DRG2 sera were adsorbed to recombinant DRG2 and DRG1, respectively, prior to final affinity purification. , Cleaned in advance. As a result, it was confirmed that the purified antibodies against DRG1 and DRG2 had high specificity and no recognizable cross-reactivity.
[0150] 次いで、本発明者らは、内在性 DFRP1が DRG1に結合するかどうかを検討するため に、 HeLa S3細胞の抽出物を使用して免疫沈降実験を実施した。結果、 DRG1は、抗 DFRP1抗体を用いた免疫沈降およびその後の抗 DRG1抗体を用いたウェスタンブロ ット分析によって検出された力 DRG2は同じ免疫沈降法において抗 DRG2抗体では 検出されなかった（図 1C、レーン 2)。逆の実験で、 DFRP1は、抗 DRG1抗体を用いた 免疫沈降により同定された（図 1C、レーン 3)。 DRG1および DFRP1は、関連のないゥ サギ対照 IgGを用いた免疫沈降法、およびその後のウェスタンプロット法では検出さ れなかった（図 1C、レーン 4)。これらの結果は、内在性 DFRP1は DRG1に特異的に結 合するが、 DRG2には結合しないことを示している。  [0150] Next, the present inventors performed an immunoprecipitation experiment using an extract of HeLa S3 cells to examine whether endogenous DFRP1 binds to DRG1. As a result, DRG1 was detected by immunoprecipitation using anti-DFRP1 antibody followed by Western blot analysis using anti-DRG1 antibody. DRG2 was not detected by anti-DRG2 antibody in the same immunoprecipitation method (Figure 1C). Lane 2). In the reverse experiment, DFRP1 was identified by immunoprecipitation using anti-DRG1 antibody (FIG. 1C, lane 3). DRG1 and DFRP1 were not detected by immunoprecipitation using an unrelated rabbit control IgG and subsequent Western plots (Figure 1C, lane 4). These results indicate that endogenous DFRP1 binds specifically to DRG1, but not DRG2.
[0151] 内在性 DFRP1と DRG1の結合をさらに確認するために、 DFRP1および DRG1の細胞 内局在につ!、て免疫蛍光顕微鏡によって検討した。 DFRP1および DRG1に対する抗 体は共にゥサギ由来なので、これらの抗体による二重染色は不可能であった。従つ て、本発明者らは、 HeLa S3細胞を、抗 DFRP1または抗 DRG1抗体を用いて、独立に 染色した。結果、どちらの抗体を用いた場合も、 HeLa S3細胞の細胞質全体において 同じパターンでシグナルが検出された（図 1D)。 [0151] In order to further confirm the binding between endogenous DFRP1 and DRG1, the intracellular localization of DFRP1 and DRG1 was examined by immunofluorescence microscopy. Since antibodies against DFRP1 and DRG1 are both from rabbits, double staining with these antibodies was not possible. Follow The present inventors independently stained HeLa S3 cells with anti-DFRP1 or anti-DRG1 antibodies. As a result, with either antibody, signals were detected in the same pattern throughout the cytoplasm of HeLa S3 cells (Fig. 1D).
[0152] 種々の器官を標的とする蛋白質をコードする細胞下局在化ベクター（BD Bioscienc es Clontech)を用いたさらに別の分析では、 DFRP1および DRG1の分布パターンが小 胞体、ゴルジ装置およびミトコンドリアの分布パターンと同じでないことが明らかになつ ており、 DFRP1および DRG1は細胞質に同時局在化していることが示唆された。これ らのデータは、 DFRP1および DRG1はインビボにおいて複合体を形成するという考え を強く裏付けるものであった。  [0152] In a further analysis using subcellular localization vectors (BD Biosciences Clontech) that encode proteins targeting various organs, the distribution pattern of DFRP1 and DRG1 was shown to be in the endoplasmic reticulum, Golgi apparatus, and mitochondrion. It became clear that the distribution pattern was not the same, suggesting that DFRP1 and DRG1 colocalize in the cytoplasm. These data strongly supported the idea that DFRP1 and DRG1 form a complex in vivo.
[0153] 〔実施例 3〕 DFRPと DRG蛋白質の相互作用のための DFRPドメインの必要性  [Example 3] Necessity of DFRP domain for interaction between DFRP and DRG protein
次に、本発明者らは、 DFRPのどの領域力 ¾RGファミリーとの相互作用に必要である かを判定した。本発明者らは、 DFRP2および DFRP1のいくつかの部位欠失変異体を 作製した（図 2A)。結果、 DFRP2の 198〜243位の C末端残基および 1〜141位の N末 端残基の欠損（それぞれ、 A C2および Δ Ν1と命名）は、 DRG2への結合に影響を与 えなかった（図 2B、レーン 1、 3、 4)。し力し、 DFRPドメインのほとんどの部分を欠損し たさらに別の部位欠失変異体（ Δ C1および Δ N2)は、 DRG2に結合しな力つた（図 2B 、レーン 2、 5)。従って、 DFRP2の DFRPドメインは DRG2との相互作用に必須であると 思われた。 DFRP1の N末端および C末端半分の欠損（それぞれ Δ Nおよび Δ C)は DR G1への結合に大きく影響しな力つた（図 2C、レーン 1〜3)。この重複領域は DFRPドメ インの一部であり、真核生物の DFRP1にお!/、て高度に保存されて!、た（図 1A)。  Next, the present inventors determined which region force of DFRP is necessary for interaction with the RG family. We have made several site deletion mutants of DFRP2 and DFRP1 (FIG. 2A). As a result, the deletion of the 198-243 C-terminal residue and the 1-141 N-terminal residue of DFRP2 (named AC2 and Δ Δ1, respectively) did not affect the binding to DRG2. (Figure 2B, lanes 1, 3, 4). However, additional site deletion mutants (ΔC1 and ΔN2) lacking most of the DFRP domain did not bind to DRG2 (FIG. 2B, lanes 2, 5). Therefore, the DFRP domain of DFRP2 appeared to be essential for interaction with DRG2. Deletions in the N-terminal and C-terminal halves of DFRP1 (ΔN and ΔC, respectively) did not significantly affect binding to DR G1 (FIG. 2C, lanes 1-3). This overlapping region is part of the DFRP domain and was highly conserved in the eukaryotic DFRP1! (Fig. 1A).
[0154] 次いで、本発明者らは、高度に保存されている領域を欠損する DFRP1の中間部欠 損変異体（A D1)を構築した。この欠損によって、 DRG1に結合する能力は完全になく なった（図 2C、レーン 4)。従って、 DFRPドメインの 25-aaペプチドは DRG1との相互作 用に必須であると分力つた。  [0154] Next, the present inventors constructed an intermediate deletion mutant (AD1) of DFRP1 that lacks a highly conserved region. This deficiency completely eliminated the ability to bind to DRG1 (Figure 2C, lane 4). Therefore, the 25-aa peptide of the DFRP domain was found to be essential for interaction with DRG1.
[0155] これらの結果は、 DFRPドメインは DRGファミリーとの相互作用に深く関与して!/、るこ とを示唆して 、る。 DFRP1および DFRP2の DFRPドメインの高度に類似して!/、る配列を 考慮すると、 DFRP1および DFRP2の DFRPドメインの構造のわずかな差が異なる結合 特異性を生じて 、ると考えられた。 [0156] 〔実施例 4〕 DFRPによる DRG蛋白質の発現の調節 [0155] These results suggest that the DFRP domain is deeply involved in the interaction with the DRG family! /. Considering the highly similar sequence of DFRP domains of DFRP1 and DFRP2! /, It was considered that slight differences in the structure of the DFRP domains of DFRP1 and DFRP2 resulted in different binding specificities. [Example 4] Regulation of DRG protein expression by DFRP
本発明者らは、 DRG1または DRG2の発現ベクターの一過的な形質移入を行ったが 、 DRG1または DRG2は単独で過剰発現させることができなかった（図 3A、レーン 5お よび 6)。この現象は以前に DRG1について報告されている（Sazuka, T., Kinoshita, M. , Tomooka, Υ·, Ikawa, Υ·, Noda, Μ. and Kumar, b. (1992b) Expression of DRu dun ng murine embryonic development. Biochem. Biophys. Res. Commun. 189, 371—377. ； Mahajan, M. A" Park, S. T. and Sun, X. H. (1996) Association of a novel GTP bin ding protein, DRG, with TAL oncogenic proteins. Oncogene 12, 2343—2350.)。し力 し、本発明者らは、 DRG1を DFRP1または DFRP2と共に同時発現させたところ、 DRG1 の発現は劇的に増加した（図 3A、レーン 1および 3)。 DFRP2と同時発現させた場合、 DRG2の発現も増加した力 DFRP1の場合はあまり増加しなかった（図 3A、レーン 2お よび 4)。  Although we performed transient transfection of DRG1 or DRG2 expression vectors, DRG1 or DRG2 could not be overexpressed alone (FIG. 3A, lanes 5 and 6). This phenomenon has been previously reported for DRG1 (Sazuka, T., Kinoshita, M., Tomooka, Υ ·, Ikawa, Υ ·, Noda, Μ. And Kumar, b. (1992b) Expression of DRu dun ng murine Biochem. Biophys. Res. Commun. 189, 371—377.; Mahajan, M. A "Park, ST and Sun, XH (1996) Association of a novel GTP binding protein, DRG, with TAL oncogenic proteins. Oncogene 12, 2343-2350.) However, when we co-expressed DRG1 with DFRP1 or DFRP2, the expression of DRG1 increased dramatically (FIG. 3A, lanes 1 and 3). When co-expressed with DFRP2, the expression of DRG2 also increased. In the case of DFRP1, it did not increase much (Figure 3A, lanes 2 and 4).
[0157] 本発明者らは、外因的または過剰発現した DRG蛋白質は未知の機序によって分解 の標的にされる可能性があること、および DFRPは DRGとの物理的な結合により、この ような分解を阻害することができると考えた。  [0157] We have shown that exogenous or overexpressed DRG proteins may be targeted for degradation by unknown mechanisms, and that DFRP is physically coupled to DRG. We thought that degradation could be inhibited.
[0158] DFRP1は、ュビキチンと相互作用すると思われる 2つの Znフィンガードメインを有し、 DFRP2はュビキチン結合酵素（E2)と構造的に関連のある RWDドメインを有するので 、本発明者らは、 DFRPは、ュビキチン/プロテアソーム系によって DRGを分解力も保 護するのではな 、かと考えた。  [0158] Since DFRP1 has two Zn finger domains that appear to interact with ubiquitin, and DFRP2 has an RWD domain that is structurally related to ubiquitin-conjugating enzyme (E2), we Thought that the ubiquitin / proteasome system might protect DRG from degradation.
[0159] この可能性を試験するために、 293T細胞に、 HA-ュビキチン、 FLAG- DRG1または DRG2の発現ベクターを同時に形質移入し、 26Sプロテアソーム阻害剤 MG132の存在 下または非存在下にお 、て 3時間インキュベーションした。細胞抽出物を抗 FLAG抗 体で免疫沈降させ、次に抗 HA抗体でウェスタンブロット分析した（図 3B、 IPブロット、 なお、 IPブロットの蛋白質試料は DRG蛋白質の量で標準化されている）。 MG132の添 加によって、 DRG1または DRG2免疫複合体中のポリュビキチン結合物の蓄積が検出 されたが（図 3B、 IPブロット、上のパネル、レーン 2、 8)、このような蓄積は MG132の非 存在下では観察されなかった（レーン 1、 7)。この結果は、 DRG蛋白質がュビキチン 化依存的機序によって構成的に分解されることを示唆するものである。 MG132添カロ 時には、未修飾サイズの DRG蛋白質の蓄積は観察されな力つたので、 DRG蛋白質の ュビキチンィ匕効率は MG132処理に影響されないことが示唆された（図 3B、溶解物ブ ロット、上のパネル、レーン 1、 2および 7、 8、なお、溶解物ブロットの蛋白質試料は細 胞数につ!/、て標準化されて!/、る)。 [0159] To test this possibility, 293T cells were co-transfected with HA-ubiquitin, FLAG-DRG1 or DRG2 expression vectors, and in the presence or absence of the 26S proteasome inhibitor MG132. Incubated for 3 hours. Cell extracts were immunoprecipitated with anti-FLAG antibody and then Western blot analyzed with anti-HA antibody (FIG. 3B, IP blot, where the protein sample of the IP blot is normalized by the amount of DRG protein). Addition of MG132 detected accumulation of polyubiquitin conjugates in DRG1 or DRG2 immune complexes (Figure 3B, IP blot, upper panel, lanes 2, 8), but such accumulation was absent in MG132 Not observed below (lanes 1, 7). This result suggests that DRG protein is constitutively degraded by a ubiquitination-dependent mechanism. MG132 Caro In some cases, accumulation of unmodified size DRG protein was not observed, suggesting that the ubiquitin efficiency of DRG protein was not affected by MG132 treatment (Figure 3B, lysate blot, upper panel, lane 1). 2 and 7, 8 and the protein sample of the lysate blot is standardized by the number of cells! /).
[0160] Myc-DFRPlと DRG1または DRG2の同時発現により、 DRG1が用量依存的に蓄積さ れた（図 3B、溶解物ブロット、上のパネル、レーン 2〜4)。 DRG2の蓄積はあまり観察さ れなかった（レーン 8〜10)。 Myc- DFRP2の同時発現により DRG1および DRG2が共に 蓄積された（レーン 2、 5、 6および 8、 11、 12)。さらに、 DFRP1発現の増加によって DR G1免疫複合体中のポリュビキチン鎖結合物のレベルが低下したが（図 3B、 IPブロット 、上のパネル、レーン 2〜4)、 DRG2免疫複合体ではあまり低下しなかった（レーン 8〜 10)。 DFRP2は、 DRG1および DRG2免疫複合体両方のポリュビキチン化を低下させ た（レーン 2、 5、 6および 8、 11、 12)。  [0160] Coexpression of Myc-DFRPl and DRG1 or DRG2 resulted in DRG1 accumulation in a dose-dependent manner (Figure 3B, lysate blot, upper panel, lanes 2-4). Little accumulation of DRG2 was observed (lanes 8-10). Coexpression of Myc-DFRP2 accumulated both DRG1 and DRG2 (lanes 2, 5, 6 and 8, 11, 12). In addition, increased DFRP1 expression reduced the level of polyubiquitin chain conjugates in the DR G1 immune complex (Figure 3B, IP blot, upper panel, lanes 2-4), but not so much in the DRG2 immune complex. (Lanes 8-10). DFRP2 reduced polyubiquitination of both DRG1 and DRG2 immune complexes (lanes 2, 5, 6 and 8, 11, 12).
[0161] 同じ免疫沈降複合体にぉ 、て、 DRGと DFRPの特異的な相互作用につ 、て、抗 -M yc抗体を用いて確認した（図 3B、 IPブロット、下のパネル）。これらの結果は、一過的 な過剰発現系の実験において、 DFRP1は DRG1を特異的に安定化し、 DFRP2は、 DR G1および DRG2をポリュビキチンィ匕および続いて起こる蛋白質分解力も保護すること によって安定ィ匕することを示唆して 、る。  [0161] Specific interactions between DRG and DFRP were confirmed using anti-Myc antibody on the same immunoprecipitation complex (Figure 3B, IP blot, lower panel). These results indicate that in transient overexpression experiments, DFRP1 specifically stabilizes DRG1, while DFRP2 stabilizes DRG1 and DRG2 by protecting both polyubiquitin and subsequent proteolytic activity. I suggest you do that.
[0162] 〔実施例 5〕 インビボにおける DFRP1による DRG1蛋白質発現の調節  [0162] [Example 5] Regulation of DRG1 protein expression by DFRP1 in vivo
インビボで DRG1蛋白質が発現する際の、 DFRP1媒介による調節について検討する ために、本発明者らは DFRP1欠損 DT40-ヮトリ B細胞系統を作製した。本発明者らは 、まず、ヒト（H. sapiens)およびキイ口ショウジヨウバエ（Drosophila melanogaster) DFR PIペプチド配列の保存されて!、る部分力もデザインされた縮重プライマーを用いた P CRによって、 -ヮトリ dfrplの部分的な cDNA断片を同定した。本発明者らは、増幅し た cDNAの配列を使用して、ロング PCRによって-ヮトリ dfrpl遺伝子座の部分的な断 片を増幅するためのプライマーをデザインした。  In order to investigate DFRP1-mediated regulation of DRG1 protein expression in vivo, the present inventors generated a DFRP1-deficient DT40- ヮ bird B cell line. We first of all conserved the human (H. sapiens) and Drosophila melanogaster DFR PI peptide sequences! By PCR using degenerate primers that also designed partial forces. -A partial cDNA fragment of avian dfrpl was identified. We designed primers to amplify partial fragments of the ヮ chicken dfrpl locus by long PCR using the amplified cDNA sequence.
[0163] 結果、単離したゲノム DNAを配列分析したところ、その遺伝子座は 7つのェキソンを 含有し、そのうちの 1つは完全長の Zn-フィンガー 1ドメイン（ZnF-l、図 1Aに例示）をコ ードすることが明らかになった。 ZnF-Ιェキソンの崩壊を目的として、本発明者らは、 図 4Aに例示するように、ブラストサイジンまたはヒスチジノール耐性 (Bsrまたは HisD) 遺伝子カセットに、ェキソンの上流または下流に位置する 5'-および 3'-ゲノムアーム が隣接して 、るターゲテイングベクター（dfrplBsrおよび dfrplHisD)を構築した。野生 型 DT40細胞に dfrplBsrを形質移入し、ブラストサイジン耐性クローンを単離した。次 いで、これらのヘテロ接合体クローンの 1つに dfrplHisDを形質移入して、第 2の対立 遺伝子を欠損させた。両方のターゲテイング事象は、 5'-隣接プローブを用いるゲノム DNAのサザンブロット分析によって確認した。 dfrpl除去は、ノーザンブロットおよびゥ エスタンプロット分析によって追加して証明された (それぞれ、図 4Cおよび D)。 [0163] As a result, the isolated genomic DNA was sequenced and its locus contains seven exons, one of which is a full-length Zn-finger 1 domain (ZnF-l, illustrated in Figure 1A). It became clear that the code would be coded. For the purpose of ZnF-Ιexon decay, we have As illustrated in Figure 4A, the blasticidin or histidinol resistance (Bsr or HisD) gene cassette is flanked by 5'- and 3'-genomic arms located upstream or downstream of the exon ( dfrplBsr and dfrplHisD) were constructed. Wild type DT40 cells were transfected with dfrplBsr and a blasticidin resistant clone was isolated. Then, one of these heterozygous clones was transfected with dfrplHisD to delete the second allele. Both targeting events were confirmed by Southern blot analysis of genomic DNA using 5'-flanking probes. dfrpl removal was additionally demonstrated by Northern blot and wet stamp lot analysis (Figures 4C and D, respectively).
[0164] dfrplの標的欠損（dfrp— /_ )により、 DRG1発現は劇的に低下した力 チューブリン対 照発現は影響なかった（図 5A、パネル a、 d)。これは、 DFRP1がインビボにおいて DR G1の正常な発現に必要であることを示している。 [0164] Target deficiency of dfrpl (dfrp — / _ ) dramatically reduced DRG1 expression. Tubulin-regulated expression was not affected (FIG. 5A, panels a, d). This indicates that DFRP1 is required for normal expression of DR G1 in vivo.
[0165] この表現型が dfrplの欠損のみによることを検証するために、本発明者らは、マウス 完全長 (A)または DRG1との相互作用に必須の aa欠損マウス（ Δ Dl) DFRP1を発現す るベクターを dfrpl -/-細胞に導入した。本発明者らは、 mDFRPlflまたは mDFRPl Δ 1を 安定して発現する-ヮトリ dfrpl-欠損細胞（それぞれ dfrpl mfl、 dfrpl+m Δ Dl)をそ れぞれ榭立した。 [0165] To verify that this phenotype is due solely to dfrpl deficiency, we expressed mouse full-length (A) or aa-deficient mice (ΔDl) DFRP1 essential for interaction with DRG1. The vector was introduced into dfrpl − / − cells. The present inventors established a stable dfrpl-deficient cell (dfrpl mfl and dfrpl + mΔDl, respectively) that stably express mDFRPlfl or mDFRPlΔ1.
[0166] 結果、 dfrpl-/-mfl細胞では、 DRG1の発現は回復しており（図 5A、パネル a、レーン 1〜3を比較のこと）、 dfrpチ細胞における DRGの低下は DFRP1の欠損によるものであ つたことが分かった。し力し、 dfrp— /_ m A Dl細胞では、 DRG1の発現は回復していなか つた（レーン 4)。 DRG1への DFRP1の結合は、 DRG1の正常なレベルの維持に必須で あることを、この結果は強く示唆していた。 [0166] As a result, DRG1 expression was restored in dfrpl-/-mfl cells (Figure 5A, panel a, compare lanes 1 to 3), and the decrease in DRG in dfrp cells was due to DFRP1 deficiency. It turned out to be a thing. However, DRG1 expression was not recovered in dfrp — / _ mA Dl cells (lane 4). The results strongly suggested that DFRP1 binding to DRG1 is essential for maintaining normal levels of DRG1.
[0167] DRG1発現の低下力 転写または翻訳後のレベルで生ずるかどうかを検討するため 、 drgl mRNAレベルをノーザンブロット分析で分析した。結果、 drgl mRNAのレベル は、 DFRP1またはその部位欠失変異体の存在下または非存在下でも、大きく異なら な力つた（図 5A、パネル e)。従って、本発明の実験において観察された DRG1発現の 変化は、 drgl発現の変化によらないものであった。これは、 DFRP1による DRG1発現 の調節が翻訳後レベルで生ずることを示唆している。一方、 DRG2レベルは、全ての 細胞種において同様であった（図 5A、パネル b)。ノーザンブロットによって、 dfrplチ および dfrplチ m A Dl細胞における drg2 mRNAレベルが、野生型および dfrpl— mil細 胞におけるレベルより高いことが明らかになつたが（図 5A、パネル f)、この現象は現時 点では明らかではない。これらの結果は、 DFRP1はインビボにおいて DRG1レベルを 特異的にアップレギュレーションして 、ることを示して 、る。 [0167] To reduce whether DRG1 expression occurs at the transcriptional or post-translational level, drgl mRNA levels were analyzed by Northern blot analysis. As a result, the level of drgl mRNA was greatly different in the presence or absence of DFRP1 or its site deletion mutant (FIG. 5A, panel e). Therefore, the change in DRG1 expression observed in the experiments of the present invention was not due to the change in drgl expression. This suggests that the regulation of DRG1 expression by DFRP1 occurs at the post-translational level. On the other hand, DRG2 levels were similar in all cell types (Figure 5A, panel b). Northern blot by dfrpl Although drg2 mRNA levels in and Dfrpl chi A Dl cells were found to be higher than those in wild-type and dfrpl-mil cells (Figure 5A, panel f), this phenomenon is not clear at this time. These results indicate that DFRP1 specifically upregulates DRG1 levels in vivo.
[0168] DRG1発現の DFRP1媒介の調節が、 DFRP1と DRG1との物理的な相互作用によって 生じるかどうかを確認するため、本発明者らはインビボにおける免疫沈降アツセィを 実施した。結果、抗 DFRP1抗体を用いた免疫沈降、および次に実施した抗 DRG1抗 体を用いるィムノブロット法によって、 DT40野生型細胞では内在性の DFRP1が DRG1 に結合することが明らかになった（図 5B、レーン 1)。安定に発現する mDFRPlflも DR G1と結合したが、 mDFRPl Δ 1は、 mDFRPl Δ 1発現レベルが十分であっても、 DRG1 に結合しな力つた（図 5B、レーン 3、 4)。逆の実験で、本発明者らは、免疫沈降のた めに抗 DRG1抗体、およびィムノブロット法のために抗 DFRP1抗体を使用したところ、 同じ結論に至った (データは示していない）。 DRG1の正常な発現には、インビボにお ける DFRP1との物理的な結合が必要なことを、これらの結果は強く示唆して 、る。  [0168] To confirm whether DFRP1-mediated regulation of DRG1 expression occurs due to the physical interaction between DFRP1 and DRG1, we performed an immunoprecipitation assay in vivo. The results revealed that endogenous DFRP1 binds to DRG1 in DT40 wild-type cells by immunoprecipitation using anti-DFRP1 antibody and immunoblotting using the next anti-DRG1 antibody (Figure 5B). Lane 1). Although stably expressed mDFRPlfl also bound to DR G1, mDFRPl Δ1 did not bind to DRG1 even when mDFRPl Δ1 expression level was sufficient (FIG. 5B, lanes 3 and 4). In the reverse experiment, we used the same anti-DRG1 antibody for immunoprecipitation and the anti-DFRP1 antibody for immunoblotting, leading to the same conclusion (data not shown). These results strongly suggest that normal expression of DRG1 requires physical binding to DFRP1 in vivo.
[0169] 〔実施例 6〕 アフリカッメガエル (X. laevis)における drglおよび dfrplの空間的および 時間的同時発現  [0169] [Example 6] Simultaneous expression of drgl and dfrpl in Xenopus laevis (X. laevis)
drglはマウス中枢神経系の早期発生段階において主に発現される遺伝子として最 初に同定されており、本発明者らは、以前に、アフリカッメガエル胚および成体組織 における drglおよび drg2発現の比較分析を報告した（Ishikawa, K., Azuma, S.， Ikawa , S., et al. (2003)し lonmg and characterization of Xenopus laevis drg2, a member of the developmentally regulated GTP— binding protein subfamily. Gene 322, 105—112. drgl was first identified as a gene that is primarily expressed in the early developmental stage of the mouse central nervous system, and we previously compared drgl and drg2 expression in Xenopus embryos and adult tissues. The analysis was reported (Ishikawa, K., Azuma, S., Ikawa, S., et al. (2003) and lonmg and characterization of Xenopus laevis drg2, a member of the developmentally regulated GTP—binding protein subfamily. Gene 322, 105-112.
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[0170] ホールマウントインサイチューハイブリダィゼーシヨンおよびノーザンブロット法によ つて、 drglおよび drg2の発現パターンのわずかな差が明らかになった。例えば、発生 段階第 22期の前腎原基では drg2発現だけが検出された。 DFRP1が DRG1の調節因 子として普遍的に作用するかどうかを判定するために、本発明者らは、アフリカッメガ エル (X. laevis)を使用して、胚発生期間中および種々の成体の組織における d¾lお よび dfrplの空間的および時間的発現について比較した。 [0171] 本発明者らは、上記するように、ゼノパス dfrpl cDNAをクローユングした。ゼノパス 胚のホールマウントインサイチューハイブリダィゼーシヨンによって、 dfrplの発現パタ ーンが drglの発現パターンに極めて類似していることが明らかになった（図 6A、 a〜； j) 。発生段階第 22期では、両方の遺伝子が血島、体節、発生中の眼、体幹神経冠、下 顎稜セグメント (mandibular crest segment)、古骨稜セグメント (hyoid crest segment) および鰓稜セグメント（branchial crest segment)において発現していた（図 6A、 a〜f) 。この発生段階では、 dfrplおよび drglも前腎原基領域では発現しな力つたが、 drg2 mRNAレベルは前腎原基では高く（Ishikawa, K., Azuma, S.， Ikawa, S.， et al. (2003) Cloning and characterization of Xenopus laevis drg2, a member of the developmental ly regulated GTP- binding protein subfamily. Gene 322, 105-112.)、 dfrpl mRNAの 転写および Zまたは安定性力 drg2よりも drglに類似した方法で調節されることが示 唆された。発生段階第 32期では、 drglおよび dfrplの発現パターンはほとんど同じで あった。両方の遺伝子は、耳胞、前腎、前脳、中脳、後脳、鰓弓、眼、水晶体、脊髄 および脊索において発現していた（図 6A、 g〜j)。成体組織では、 dfrplは卵巣で強 力に発現し、脳、腎臓、脾臓、精巣、腸および結腸で中程度に発現し、心臓、肺、肝 臓、胃および骨格筋ではほとんど発現していな力つた（図 6B)。この発現パターンは、 心臓、肺および肝臓において中程度のレベルで発現する drg2より drglの発現パター ンに類似し飞 ヽた (Ishikawa, K., Azuma, Ikawa, et al. (2003) し loning and cha racterization of Xenopus laevis drg2, a member of the developmentally regulated GT P- Dinding protein subfamily. Gene 322, 105-112.)。 [0170] Whole mount in situ hybridization and Northern blotting revealed a slight difference in the expression pattern of drgl and drg2. For example, only drg2 expression was detected in the prorenal primordium at stage 22 of development. To determine whether DFRP1 acts universally as a regulator of DRG1, we used X. laevis to develop during embryonic development and in various adult tissues. The spatial and temporal expression of d¾l and dfrpl were compared. [0171] The present inventors cloned xenopus dfrpl cDNA as described above. Xenopus whole-mount in situ hybridization revealed that the expression pattern of dfrpl is very similar to the expression pattern of drgl (Fig. 6A, a-; j). In the 22nd stage of development, both genes are blood islands, somites, developing eyes, trunk nerve crest, mandibular crest segment, hyoid crest segment, and crest segment (Branchial crest segment) (FIG. 6A, a to f). In this developmental stage, dfrpl and drgl were also not expressed in the prorenal primordium region, but drg2 mRNA levels were higher in the prorenal primordium (Ishikawa, K., Azuma, S., Ikawa, S., et al (2003) Cloning and characterization of Xenopus laevis drg2, a member of the developmental ly regulated GTP-binding protein subfamily. Gene 322, 105-112.), Dfrpl mRNA transcription and Z or stability similar to drgl rather than drg2 It was suggested that it was adjusted by the method described above. In the 32nd stage of development, drgl and dfrpl expression patterns were almost the same. Both genes were expressed in the otocyst, pronephros, forebrain, midbrain, hindbrain, arch, eye, lens, spinal cord and notochord (Fig. 6A, g-j). In adult tissues, dfrpl is strongly expressed in the ovary, moderately expressed in the brain, kidney, spleen, testis, intestine and colon, and rarely expressed in the heart, lung, liver, stomach and skeletal muscle. (Fig. 6B). This expression pattern is more similar to the expression pattern of drgl than drg2, which is expressed at a moderate level in the heart, lung and liver (Ishikawa, K., Azuma, Ikawa, et al. (2003)). cha racterization of Xenopus laevis drg2, a member of the developmentally regulated GT P-Dinding protein subfamily. Gene 322, 105-112.).
[0172] 本発明者らはまた、初期発生段階における dfrplの時間的な発現について検討した  [0172] The present inventors also examined temporal expression of dfrpl in the early developmental stage.
(図 6C)。 dfrplの発現は後期原腸胚 (発生段階第 13〜14期)では弱く誘導され、後 期神経胚 (発生段階第 20〜22期）からオタマジャクシ (発生段階第 40〜41期）までは 強力に誘導された。このパターンは drglのパターンに類似していた（Ishikawa, K., Az uma, Ikawa, S., et al. (2003) し lonmg and characterization of Xenopus laevis drg 2, a member of the developmentally regulated GTP— binding protein subfamily. Gene 322, 105-112.)。多細胞生物における dfrplと drglのこれらの空間的および時間的発 現の類似性は、 DFRP1は種々の種類の細胞において DRG1と協同するという考えを 裏付けるものであった。 (Figure 6C). Expression of dfrpl is weakly induced in late gastrulation embryos (13th to 14th stage of development), and strong from late neural embryos (20th to 22th stage of development) to tadpole (40th to 41st stage of development) Induced. This pattern was similar to that of drgl (Ishikawa, K., Azuma, Ikawa, S., et al. (2003) and lonmg and characterization of Xenopus laevis drg 2, a member of the developmentally regulated GTP—binding protein subfamily. Gene 322, 105-112.). The similarity of these spatial and temporal expression of dfrpl and drgl in multicellular organisms suggests that DFRP1 cooperates with DRG1 in various cell types. It was something to back up.
[0173] なお、図 6Bおよび図 6Cでは、本発明者らが本発明者らの以前の論文（Ishikawa, K ., Azuma, S., Ikawa, et al. (2003) Cloning and characterization or Xenopus laevis drg2, a member of the developmentally regulated GTP— binding protein subfamily. G ene 322, 105-112.)において使用したものと同じメンブレンを再探索した。従って、口 ードした RNAの品質および量は確認されて 、る。  [0173] In FIGS. 6B and 6C, the present inventors have analyzed our previous paper (Ishikawa, K., Azuma, S., Ikawa, et al. (2003) Cloning and characterization or Xenopus laevis drg2, a member of the developmentally regulated GTP—binding protein subfamily. Gene 322, 105-112.). Therefore, the quality and quantity of the transcribed RNA has been confirmed.
[0174] 〔実施例 7〕 テトラサイクリン (及びその誘導体ドキシサイクリン) -誘導性 (inducible) R NAiノックダウンシステムの構築 [Example 7] Tetracycline (and its derivative doxycycline)-construction of inducible R NAi knockdown system
HeLa S3細胞株は DMEM培地（10%FBS,ペニシリン，ストレプトマイシン入り）、 CO (  HeLa S3 cell line is DMEM medium (with 10% FBS, penicillin, streptomycin), CO (
2 2
5%)インキュベーターで培養した。 5%) Incubator.
l et— On systemにつ ヽて ίま (Yao, P . et al. Ί etracycline repressor, tetR, rather th an the tetR— mammalian cell transcription factor fusion derivatives, regulates inducibl e gene expression in mammalian cells.", Hum. Gene Ther., 9: 1939-1950 (1998) )を 参照。 Tet— On RNAi systemにつ ヽて ίま (van de Wetering M. et al. "Specific inhibiti on of gene expression using a stably integrated, inducible small— interfering— RNA vec tor.", EMBO rep., 4:609-615 (2003) )参照。  l et— On system (Yao, P. et al. Ί etracycline repressor, tetR, rather th an the tetR— mammalian cell transcription factor fusion derivatives, regulates inducibl gene expression in mammalian cells. ", Hum Gene Ther., 9: 1939-1950 (1998)) Tet—On RNAi system ヽ ί ま (van de Wetering M. et al. "Specific inhibiti on of gene expression using a stably integrated, inducible small—interfering—RNA vec tor. ”, EMBO rep., 4: 609-615 (2003)).
[0175] TetR (テトラサイクリンリプレッサー； tetracycline repressor)の cDNA fragmentを PCR  [0175] PCR of TetR (tetracycline repressor) cDNA fragment
(94°C 15sec, 55°C 30sec, 72°C lmin, 30サイクル）で pTA- Hygベクター（東京大学医 科学研究所の Dr.Yamamotoよりご提供頂!、た）から増幅（PCRプライマーセット， 5， - G CGAATTCGCCACCATGTCAAGATTAGATAAAAG-3 ' (配列番号： 23) , 5，- GCC TCGAGTTAAGACCCACTTTCACATTTAAG- 3，（配列番号： 24) )した。これを Eco RIと Xholで消化した後に、 pMXs-IRES-puroベクター（東京大学医科学研究所の Dr. Kitamuraよりご提供頂いた）の EcoRI/XhoI部位にサブクローユングし、その後に EcoR Iと Notlで切り出し、 EF1アルファプロモーター drivenの IRES- puroベクター（東京大学 医科学研究所の Dr. Kitamuraよりご提供頂いた）の EcoRI/Notl部位にライゲーシヨン した（pTetR- IRES- puro)。  (94 ° C 15sec, 55 ° C 30sec, 72 ° C lmin, 30 cycles) amplified from the pTA-Hyg vector (provided by Dr. Yamamoto, University of Tokyo) 5,-G CGAATTCGCCACCATGTCAAGATTAGATAAAAG-3 '(SEQ ID NO: 23), 5,-GCC TCGAGTTAAGACCCACTTTCACATTTAAG-3 (SEQ ID NO: 24)). This was digested with Eco RI and Xhol, then subcloned into the EcoRI / XhoI site of the pMXs-IRES-puro vector (provided by Dr. Kitamura of the Institute of Medical Science, the University of Tokyo), followed by EcoRI and It was excised with Notl and ligated to the EcoRI / Notl site of the EF1 alpha promoter driven IRES-puro vector (provided by Dr. Kitamura, Institute of Medical Science, The University of Tokyo) (pTetR-IRES-puro).
[0176] 誘導性 RNAiベクターは次の通り作製した。 pSuperベクター (Oligoengine)から HIプ 口モーターを TetOが添カ卩されるようにプライマーを設定して PCR (94°C 15sec, 55°C 3 Osec, 72°C lmin, 30サイクル）で増幅した（PCRプライマーセット， 5， - CGATAAGCTT [0176] Inducible RNAi vector was prepared as follows. PCR (94 ° C 15sec, 55 ° C 3) by setting primers so that TetO is added to the HI opening motor from the pSuper vector (Oligoengine). (PCR primer set, 5,-CGATAAGCTT, Osec, 72 ° C lmin, 30 cycles)
25) , T7プライマー（5， - TAATACGACTCACTATAGGG- 3， （配列番号： 26) ) )。増 幅された fragmentを EcoRIと Bglllで消化後、 pSuperの EcoRI/Bglll部位にライゲーショ ンした（pSuper- 2)。 25), T7 primer (5,-TAATACGACTCACTATAGGG-3, (SEQ ID NO: 26))). The amplified fragment was digested with EcoRI and Bglll and then ligated to the EcoRI / Bglll site of pSuper (pSuper- 2 ).
[0177] 別に SV40- early promoter drivenの EGFPベクターを次のようにして作製した。 pEGF P-CKBD Biosciences Clontech)を Bglllで消化後、 Klenowで平滑末端化し、さらに N helで消化した fragmentと、 pRL- SV40 (Promega)を Xbalで消化後 Klenowで平滑末端 化し、さらに Nhelで消化した fragment同士をライゲーシヨンした（pSV40-EGFP)。さら に PSV40-EGFPを Bglllと BamHIで消化した fragmentを、 pSuper-2の BamHI部位にライ ゲーシヨンした（pSuper- 3)。 pSuper- 3をさらに BamHIで消化後、 BamHIで切り出すこと ができるネオマイシン抵抗性遺伝子カセット（東京大学医科学研究所の Dr. Yamamot oよりご提供頂!、た）をライゲーシヨンした（pHlTetO)。 pHlTetOの Bglll/Hindlll部位 に DRG1、 DRG2、 DFRP1、 DFRP2の mRNAに対し RNAiを引き起こすことができる shRN Aが形成されるような二本鎖オリゴヌクレオチド（Brummelkamp TR. 〃A system for stab le expression of short interfering RNAs in mammalian cells , science, 296:550-553 ( 2002) .)をライゲーシヨンした（pHlTetO-target)。  [0177] Separately, an SV40-early promoter driven EGFP vector was prepared as follows. pEGF P-CKBD Biosciences Clontech) was digested with Bglll, blunted with Klenow, and further digested with Nhel, and pRL-SV40 (Promega) was digested with Xbal, blunted with Klenow, and further digested with Nhel The fragments were ligated (pSV40-EGFP). Furthermore, a fragment obtained by digesting PSV40-EGFP with Bglll and BamHI was ligated to the BamHI site of pSuper-2 (pSuper-3). After further digestion of pSuper-3 with BamHI, a neomycin resistance gene cassette (provided by Dr. Yamamoto of the Institute of Medical Science, the University of Tokyo) that can be excised with BamHI was ligated (pHlTetO). Double-stranded oligonucleotides that form shRN A capable of causing RNAi against DRG1, DRG2, DFRP1, and DFRP2 mRNA at the Bglll / Hindlll site of pHlTetO (Brummelkamp TR. 〃A system for stab le expression of short interfering RNAs in mammalian cells, science, 296: 550-553 (2002).) was ligated (pHlTetO-target).
[0178] HeLa S3細胞に pTetR- IRES- puroをリン酸カルシウム法（Current Protocols in Immu nology, John Wiley & Sons, Inc.)で導入し、 3日後にピューロマイシン（Sigma)を 3 g /mLの濃度でカ卩え、さらに 2週間後に形成されたコロニーを単離した。そのうち 1つの クローン細胞株（R6)に RNAiベクター（pHlTetO-target)をリン酸カルシウム法 (Curre nt Protocols in Immunology, John Wiley & Sons, Inc.)で導入し、 2日後に G418 (Calbi ochem)を 5000 /z g/mLの濃度でカ卩え、さらに 2週間後に形成された EGFPの蛍光を発 するコロニーを単離した（R6-HlTetO- target)。  [0178] pTetR-IRES-puro was introduced into HeLa S3 cells by the calcium phosphate method (Current Protocols in Immunology, John Wiley & Sons, Inc.). Three days later, puromycin (Sigma) was introduced at a concentration of 3 g / mL. A colony formed after 2 weeks was isolated. RNAi vector (pHlTetO-target) was introduced into one clonal cell line (R6) by the calcium phosphate method (Current Protocols in Immunology, John Wiley & Sons, Inc.), and G418 (Calbiochem) was introduced 5000/2 days later. A colony that fluoresces EGFP formed after 2 weeks was isolated (R6-HlTetO-target).
[0179] 〔実施例 8〕 ノーザンプロット分析  [Example 8] Northern plot analysis
ノーザンブロット法については次に示す文献と同様の操作を行った。 (Ishikawa, K. et al. Cloning and characterization or Xenopus laevis drg2, a member of the develo pmentally regulated GTP— binding protein subfamily.", Gene, 322:105-112 (2003) -) 。 cDNAプローブはヒト細胞由来 cDNAプールから PCR(94°C 15sec, 54°C 30sec, 72 °C lmin, 30サイクル）で増幅した。そのプライマーとして（フイブロネクチン， 5' - GCGA ATTCCGCTCGATGTGGTCTG- 3， （配列番号： 27) , 5， - GCCTCGAGACGGGAG CCTCGAAGAG- 3，（配列番号： 28) ;ビンキュリン（Vinculin) , 5， -GCAGATCTAGGG CTGGTGGACGAAG-3 ' (配列番号： 29) , 5，- GCCTCGAGGCCTTGGCGATGTC- 3，（配列番号： 30) ;カテブシン B (CathepsinB) , 5， -GCGAATTCTGCTGCCTGCTG GTG- 3，（配列番号： 31) , 5，- GCCTCGAGCGGCCATGATGTCCTTC- 3，（配列番 号： 32)； IAP, 5， - GCGGATCCACGCCGCAATACAGAG- 3，（配列番号： 33) , 5， - GCCTCGAGTGCTGCGGATCAGCTC- 3，（配列番号： 34) ;ゥロキナーゼ（Urokinas e) , 5，- GCGGATCCAATTCGGAGGGCAGCAC- 3，（配列番号： 35) , 5，- GCCTCG AGGGCAGGCAGATGGTC- 3，（配列番号： 36) )をそれぞれ用いた。 The Northern blot method was performed in the same manner as in the following literature. (Ishikawa, K. et al. Cloning and characterization or Xenopus laevis drg2, a member of the develo pmentally regulated GTP—binding protein subfamily. ", Gene, 322: 105-112 (2003)-) . The cDNA probe was amplified from a human cell-derived cDNA pool by PCR (94 ° C 15 sec, 54 ° C 30 sec, 72 ° C lmin, 30 cycles). As its primers (fibronectin, 5'-GCGA ATTCCGCTCGATGTGGTCTG-3, (SEQ ID NO: 27), 5,-GCCTCGAGACGGGAG CCTCGAAGAG-3, (SEQ ID NO: 28); Vinculin, 5, -GCAGATCTAGGG CTGGTGGACGAAG-3 '( SEQ ID NO: 29), 5,-GCCTCGAGGCCTTGGCGATGTC-3, (SEQ ID NO: 30); Cathepsin B (CathepsinB), 5, -GCGAATTCTGCTGCCTGCTG GTG-3, (SEQ ID NO: 31), 5,-GCCTCGAGCGGCCATGATGTCCTTC-3, (sequence No .: 32); IAP, 5,-GCGGATCCACGCCGCAATACAGAG-3, (SEQ ID NO: 33), 5,-GCCTCGAGTGCTGCGGATCAGCTC-3, (SEQ ID NO: 34); Urokinas e, 5,-GCGGATCCAATTCGGAGGGCAGCAC-3, ( SEQ ID NO: 35), 5, -GCCTCG AGGGCAGGCAGATGGTC-3, (SEQ ID NO: 36)) were used respectively.
[0180] 〔実施例 9〕 ドキシサイクリン（Doxycyclin)添カ卩による DRG1、 DRG2、 DFRP1、 DFRP2 のノックダウン細胞の評価、および細胞運動 ·形態 ·細胞骨格を制御する遺伝子の m RNAの発現変化 [Example 9] Evaluation of knockdown cells of DRG1, DRG2, DFRP1, and DFRP2 and changes in mRNA expression of genes that control cell motility, morphology, and cytoskeleton using Doxycyclin supplements
HIプロモーターに TetOを融合させ、 RNAiを引き起こす shRNAの発現力TetRで抑 制された HeLa S3誘導性ノックダウン細胞（図 7レーン 3-10)およびコントロール細胞（ 図 7,レーン 1-2)をドキシサイクリン（Dox)添加（+) (20 ng/mL)で RNAiを誘導したも の、あるいは無添加（-)で 3日置きに継代した 12日後の細胞力もタンパク溶液 (total 1 ysate)または、 total RNAを調製'抽出し、ウェスタンブロットまたはノーザンブロットを それぞれ行った（図 7)。  HeLa S3-inducible knockdown cells (Fig. 7, lanes 3-10) and control cells (Fig. 7, lanes 1-2) suppressed by TetR fused with HI promoter to induce RNAi and shRNA expression ability to induce doxycycline Cell strength after 12 days after RNAi induction with (Dox) addition (+) (20 ng / mL) or without addition (-) every 3 days is also the protein solution (total 1 ysate) or total RNA was prepared and extracted and subjected to Western blot or Northern blot (Figure 7).
[0181] ウェスタンブロットでは、抗- DRG1抗体、抗- DRG2抗体、抗- DFRP1抗体、抗- DFRP 2抗体で内在性の蛋白質をブロットした。チューブリン（Tubulin)は内部コントロールと して用いた。 DFRP1ノックダウンで DRG1の、また DFRP2ノックダウンで DRG2のタンパ クレベルが特異的に減少していた（図 7ウェスタンプロットの段)。このことは生理的条 件下において DRG1および DRG2が DFRP1および DFRP2によって蛋白質発現をそれ ぞれ特異的に正制御されていることを強く示すものである。  [0181] In Western blotting, endogenous proteins were blotted with anti-DRG1 antibody, anti-DRG2 antibody, anti-DFRP1 antibody, and anti-DFRP2 antibody. Tubulin was used as an internal control. DFRP1 knockdown resulted in a specific decrease in DRG1 and DFRP2 knockdown resulted in a specific decrease in DRG2 (FIG. 7 Western plot). This strongly indicates that DRG1 and DRG2 are specifically positively regulated by DFRP1 and DFRP2, respectively, under physiological conditions.
[0182] ノーザンプロットでは、細胞骨格 ·運動 ·形態 ·浸潤 ·転移を制御する因子フイブロネ クチン (細胞外マトリクス。特異的なインテグリンと結合し細胞を接着させる（Hood, JD. et al" Nature Rev., 2: 91-100 (2002) )。癌細胞の増殖に対しては負に働く。）、ビン キュリン (Vinculin) (Focal adhesionの構成因子。ァクチンフィラメントの安定化に寄与 。）、カテブシン B (システィンプロティナーゼ。腫瘍細胞の浸潤に関与する（例として 次の文献 照。 Lakka, S¾. et al. , Inhibition of cathepsin B and MMP-9 gene expre ssion in glioblastoma cell line via RNA interference reduces tumor cell invasion, turn or growth and angiogenesis,, Oncogene, 23: 4681-4689 (2004) , Premzl, A. et al. , "Intracellular and extracellular cathepsin B facilitate invasion of MCF-10A neoT cell s through reconstituted extracellular matrix in vitro.", Exp. Cell. Res., 283: 206—21 4 (2003) )。）、IAP (別名 CD47。膜蛋白質でインテグリンの機能を制御する （例とし て次の文献参照。 Barazi, HO. et al" "Regulation of integrin function by CD47 ligan ds. Differential effects on alpha vbeta 3 and alpha 4betal integrin— mediated adhesio n J. Biol. Chem. , 277: 42859-42866 (2002)。）、ゥロキナーゼ（例えばプラスミノー ゲンをプラスミンにタンパク分解 (proteolysis)して活性化させることで、細胞外マトリツ クス (ECM)の因子分解させる MMPや TGF- 1等を活性化させ、細胞浸潤 ·転移，運 動を Iさ起こす (Blasi, F. et al. uPAR: a versatile signalling orchestrator. ， Nat. R ev. Mol. Cell. Biol , 3: 932-943 (2002) )。この遺伝子の高発現と癌およびその浸潤 •転移の相関性が高いことを示す報告が多数存在する（例として次の文献参照。 Oht a, et al. Clinical signincance or expression of urokinase— type plasminogen activat or in patients with prostate cancer. ， Anticancer Res., 23:2945-2950 (2003)， Stabu c， B. et al. "Urokinase- type plasminogen activator and plasminogen activator inhibit or type 1 and type 2 in stage I malignant melanoma,, Oncol. Rep., 10:635-639 (20 03)， Le, DM. Et ai. Exploitation of astrocytes by glioma cells to facilitate invasiven ess: a mechanism involving matrix metalloproteinase— 2 and the urokinase— type plas minogen activator- plasmin cascade,, J. Neurosci. 23:4034-4043 (2003) , Kaneko, T. et al. "Urokinase- type plasminogen activator expression correlates with tumor an giogenesis and poor outcome in gastric cancer. ， Cancer ¾ci" 94:43-49 (2003) )の mRNAを各 cDNAプローブによって検出した。各ノックダウン依存的に各 mRNAの発現 が正及び負に制御されていた（図 7ノーザンブロットの段)。この制御は、 DRGファミリ 一および DFRPファミリーが細胞質に存在することが示されている（DRG2と DFRP2は 未公表データ）こと、また、 DRG1および DRG2がインビトロで RNA結合能を有すること（ Ishikawa, K. et al. 〃し lonmg and characterization of Xenopus laevis drg2, a member of the developmentally regulated GTP- binding protein subfamily.", Gene, 322:105-1 12 (2003) .)から、核で起こる mRNAの転写制御ではなぐ細胞質で起こる mRNAの安 定ィ匕 ·分解制御であることが示唆された。 [0182] In the Northern plot, factor fibronectin (extracellular matrix that regulates cytoskeleton, movement, morphology, invasion, and metastasis. It binds to specific integrins and adheres to cells (Hood, JD. et al "Nature Rev., 2: 91-100 (2002)). It works negatively on the growth of cancer cells.), Vinculin (Focal adhesion component. For stabilizing actin filaments. Contributors), cathebsin B (cystine proteinase. Involved in tumor cell invasion (see, eg, Lakka, S¾. Et al., Inhibition of cathepsin B and MMP-9 gene expresion in glioblastoma cell line via RNA) interference reduces tumor cell invasion, turn or growth and angiogenesis, Oncogene, 23: 4681-4689 (2004), Premzl, A. et al., "Intracellular and extracellular cathepsin B facilitate invasion of MCF-10A neoT cell s through reconstituted extracellular Matrix in vitro. ", Exp. Cell. Res., 283: 206—21 4 (2003))), IAP (aka CD47. Membrane proteins control the function of integrins (see, for example, the following literature: Barazi , HO. Et al "" Regulation of integrin function by CD47 ligan ds. Differential effects on alpha vbeta 3 and alpha 4betal integrin—mediated adhesio n J. Biol. Chem., 277: 42859-42866 (2002).), Urokinase (eg, proteolysis of plasminogen into plasmin to activate cells It activates MMP, TGF-1, etc., which cause factor degradation of external matrix (ECM), and induces cell invasion, metastasis, and movement (Blasi, F. et al. UPAR: a versatile signaling orchestrator., Nat. R ev. Mol. Cell. Biol, 3: 932-943 (2002)). There are many reports that show high correlation between this gene's high expression and cancer and its invasion • metastasis (for example, see the following literature: Oht a, et al. Clinical signincance or expression of urokinase— type plasminogen activator in patients with prostate cancer., Anticancer Res., 23: 2945-2950 (2003), Stabu c, B. et al. "Urokinase- type plasminogen activator and plasminogen activator inhibit or type 1 and type 2 in stage I malignant melanoma, , Oncol. Rep., 10: 635-639 (20 03), Le, DM. Et ai. Exploitation of astrocytes by glioma cells to facilitate invasive ess: a mechanism involving matrix metalloproteinase— 2 and the urokinase— type plas minogen activator- plasmin cascade ,, J. Neurosci. 23: 4034-4043 (2003), Kaneko, T. et al. "Urokinase- type plasminogen activator expression correlates with tumor an giogenesis and poor outcome in gastric cancer., Cancer ¾ci" 94:43 -49 (2003)) was detected by each cDNA probe. The expression of each mRNA was regulated positively and negatively in a down-dependent manner (Figure 7, Northern blot), which is controlled by the DRG family. One and the DFRP family have been shown to be present in the cytoplasm (DRG2 and DFRP2 are unpublished data) and that DRG1 and DRG2 have the ability to bind RNA in vitro (Ishikawa, K. et al. lonmg and characterization of Xenopus laevis drg2, a member of the developmentally regulated GTP-binding protein subfamily. ", Gene, 322: 105-1 12 (2003).) It was suggested that this is stable control of decomposition.
[0183] 〔実施例 10〕 DRG1ノックダウンによる細胞の形態変化  [Example 10] Morphological change of cells by DRG1 knockdown
HeLaS3 DRG1誘導性ノックダウン細胞をドキシサイクリン（Dox)添加（+) (20 ng/mL )あるいは無添カ卩 (-)で培養した。 DRG1ノックダウン細胞はバラけた細胞集団で存在 し、細胞間接着性が失われコロニー形成能が失われていた（図 8)。伸びたあるいは Vヽびつな細胞形態を示して!/ヽた。  HeLaS3 DRG1-induced knockdown cells were cultured with doxycycline (Dox) added (+) (20 ng / mL) or non-supplemented potassium (-). DRG1 knockdown cells existed in a disparate cell population, losing intercellular adhesion and loss of colony-forming ability (Figure 8). Shows stretched or vague cell morphology!
[0184] 〔実施例 11〕 DRG1ノックダウン細胞のァクチン骨格の変化  [Example 11] Changes in actin skeleton of DRG1 knockdown cells
HeLaS3 DRG1誘導性ノックダウン細胞をドキシサイクリン（Dox)添加（+) (20 ng/mL )あるいは無添カ卩 (-)で培養したのち、 PBS-PFA (4%)で固定後、 Phalloidin-Rhodamin e (Molecular probes)による F-ァクチンの染色を行い、共焦点レーザー顕微鏡（Bio- Rad)によって画像を取り込んだ（図 9)。 DRG1ノックダウン細胞は多くのストレスフアイ バーの形成（白色の矢印）また、長 ヽ Filopodiaの形成（緑色の矢印）が見られた。  HeLaS3 DRG1-induced knockdown cells were cultured with doxycycline (Dox) added (+) (20 ng / mL) or non-added potassium (-), fixed with PBS-PFA (4%), and then Phalloidin-Rhodamin e F-actin was stained with (Molecular probes), and images were captured with a confocal laser microscope (Bio-Rad) (Fig. 9). DRG1 knockdown cells showed many stress fiber formations (white arrows) and long filopodia formations (green arrows).
[0185] 〔実施例 12〕 DRG1ノックダウン細胞の細胞運動性評価  [Example 12] Cell motility evaluation of DRG1 knockdown cells
HeLaS3 DRG1誘導性ノックダウン細胞をドキシサイクリン（Dox)添加（+) (20 ng/mL )あるいは無添カ卩 (-)で培養し、タイムラブス顕微鏡 (Olympus)によるビデオ撮影を行 い、細胞の運動速度を測定した（MetaMorph software, Molecular Devices)。細胞 10 個の平均速度をグラフにした（図 10)。 Dox (+)による DRG1ノックダウン細胞は Dox (-) と比べ約 3.3倍運動速度が高力つた。通常の細胞内では DRG1は細胞運動促進に働 く因子の mRNAの発現を統括的に負に制御して 、ると考えられた。  HeLaS3 DRG1-induced knockdown cells were cultured with doxycycline (Dox) added (+) (20 ng / mL) or non-added potassium (-), and videotaped with a Timelabs microscope (Olympus) to observe cell kinetics Was measured (MetaMorph software, Molecular Devices). The average speed of 10 cells was graphed (Figure 10). DRG1 knockdown cells by Dox (+) were about 3.3 times as fast as Dox (-). In normal cells, DRG1 was thought to negatively regulate the expression of mRNA, a factor that promotes cell motility.
[0186] 〔実施例 13〕 DFRP1ノックダウン細胞のァクチン骨格の変化  [Example 13] Changes in actin skeleton of DFRP1 knockdown cells
HeLaS3 〖こ DFRP1 RNAiベクター、およびコントロールとして空ベクターを導入した。 DFRP1ノックダウン細胞は DRG1のノックダウンと同様な細胞形態の異常が見られた（ 図 11)。これは DFRP1ノックダウンによって DRG1の蛋白質が分解され、 DRG1の発現 が減少することによって励起される現象であると考えられた。 HeLaS3 Tsujiko DFRP1 RNAi vector and an empty vector were introduced as controls. DFRP1 knockdown cells showed abnormal cell morphology similar to that of DRG1 knockdown (Figure 11). This is because DRG1 protein is degraded by DFRP1 knockdown, and DRG1 expression It was thought that this phenomenon was excited by the decrease of
[0187] 〔実施例 14〕 DRG2ノックダウン細胞の形態変化  [Example 14] Morphological change of DRG2 knockdown cells
HeLaS3 DRG2誘導性ノックダウン細胞をドキシサイクリン（Dox)添加（+) (20 ng/mL )あるいは無添カ卩 (-)で培養した。 DRG2ノックダウン細胞はコントロールと比べ細胞間 スペースが広ぐ増殖が低いと思われた（図 12)。また、細胞間の密着性が良力つた。  HeLaS3 DRG2 inducible knockdown cells were cultured with doxycycline (Dox) added (+) (20 ng / mL) or non-added potassium (-). DRG2 knockdown cells seemed to have lower intercellular space and lower proliferation compared to controls (Figure 12). In addition, adhesion between cells was good.
[0188] 〔実施例 15〕 一過的な DRG2ノックダウン細胞の形態変化  [Example 15] Transient DRG2 knockdown cell morphology change
HeLaS3に DRG2 RNAiベクター及びそのコントロールとして空ベクターを導入し、同 一ベクター上に構築された導入マーカーである EGFP発現細胞を観察した。結果を 図 13に示す。 DRG2ノックダウン細胞は細胞間の密着性が高く（青色の矢印）、また、 異常な突起を伸ばして細胞同士が絡み合って 、た（白色の矢印）。このような細胞間 接着性の亢進は前述の DRG1ノックダウンの形態異常と反対の形態異常であり、 DRG 1と DRG2の mRNAの安定性 ·分解制御が弓 Iき起こす細胞形態 '運動 '接着'浸潤 ·転 移制御が異なることが示唆された。  A DRG2 RNAi vector and an empty vector were introduced into HeLaS3 as a control, and EGFP-expressing cells, which are introduction markers constructed on the same vector, were observed. The results are shown in Fig. 13. DRG2 knockdown cells have high cell-cell adhesion (blue arrows), and abnormal protrusions were extended and cells were intertwined (white arrows). This enhanced intercellular adhesion is the opposite of the DRG1 knockdown morphological abnormality described above. DRG 1 and DRG2 mRNA stability It was suggested that the infiltration and transfer control are different.
[0189] 〔実施例 16〕 DRG1、 DRG2ノックダウン細胞の成長変化  [Example 16] Change in growth of DRG1 and DRG2 knockdown cells
HeLaS3 DRG1または DRG2誘導性ノックダウン細胞を 2 x 104 cells/mLから始めて、 ドキシサイクリン (Dox)添カ卩 (+) (20 ng/mL)あるいは無添カ卩 (-)で培養し、 3日おきに 細胞を血球計算盤を用いてカウント '継代したものの細胞数の累積値を図 14のダラ フに示した。 DRG1および DRG2ノックダウン（Dox (+) )細胞は R6および Dox (-)細胞と 比べ増殖が低力つた。しかし、増殖が完全に抑制されるわけではなぐこの増殖率の まま生存し続けた。これが癌細胞の高!、増殖能を単に正常細胞レベルまで落とした ものであるならば、この発明を利用した薬の副作用は低レベルに抑えられることが期 待される。 HeLaS3 DRG1 or DRG2 inducible knockdown cells starting at 2 x 10 4 cells / mL and cultured in doxycycline (Dox) supplemented (+) (20 ng / mL) or non-supplemented (-) 3 days The cells were counted using a hemocytometer. The cumulative number of cells that were passaged is shown in the graph of FIG. DRG1 and DRG2 knockdown (Dox (+)) cells were less proliferative than R6 and Dox (-) cells. However, it continued to survive at this growth rate, where growth was not completely suppressed. If this is a high level of cancer cells and the proliferation ability is simply reduced to the normal cell level, it is expected that the side effects of the drug using this invention can be suppressed to a low level.
[0190] 〔実施例 17〕 DRGファミリーおよび DFRPsの制御のモデル  [0190] [Example 17] Control model of DRG family and DFRPs
DRGファミリー（DRG1と DRG2)の特異的な制御蛋白質（DFRPs (DFRP1と DFRP2)： DRGファミリー調節蛋白質)をそれぞれ同定し、ュビキチンによる分解を結合によって 抑制することで、 DRG1および DRG2の蛋白質の発現を正に制御して 、ることが明らか となった。図 15に制御モデル図を示す。  By identifying specific regulatory proteins (DFRPs (DFRP1 and DFRP2): DRG family regulatory proteins) of the DRG family (DRG1 and DRG2), and suppressing the degradation by ubiquitin by binding, the expression of DRG1 and DRG2 proteins is suppressed. It became clear that it was controlled positively. Figure 15 shows the control model diagram.
[0191] 〔実施例 18〕 DRG1と DRG2の細胞内局在 DRG1と DRG2の細胞内局在について、遠心分画により検討した。 DRG1と DRG2の マウス肝臓ホモジネートを遠心により粗分画した。図 16左に示した手順で細胞ライセ ートを回収し、同じ細胞数となるように溶液を調製し、ウェスタンプロットにより蛋白質 を解析した（図 16右）。 DRG1と DRG2はミトコンドリア（図 16丸 2)画分よりも軽!、PMS ( nost-mitochondria supernatant) (図 16丸 3)に存在した。さらに強い遠心力により、 D RG1は P100画分（図 16丸 4, smooth ER, rough ER,ポリノーム，プラスマメンブレン， エンドノーム，ゴルジ等が含まれる）に、 DRG2はそれよりも軽い画分（図 16丸 5)に濃 縮した。このこと力 、 DRG1と DRG2は生体内で別々の画分に存在していることが明 らかとなつた。また、別々のタンパク複合体を形成している可能性がある。 [Example 18] Subcellular localization of DRG1 and DRG2 The intracellular localization of DRG1 and DRG2 was examined by centrifugal fractionation. Mouse liver homogenates of DRG1 and DRG2 were roughly fractionated by centrifugation. Cell lysates were collected according to the procedure shown on the left side of Fig. 16, and solutions were prepared so that the number of cells was the same. DRG1 and DRG2 were lighter than the mitochondrial (Figure 16 circle 2) fraction and were present in the PMS (nost-mitochondria supernatant) (Figure 16 circle 3). Due to the stronger centrifugal force, D RG1 is in the P100 fraction (Figure 16, circle 4, smooth ER, rough ER, polynome, plasma membrane, endonom, Golgi, etc.) and DRG2 is a lighter fraction (see figure). Concentrated to 16 circles 5). From this, it became clear that DRG1 and DRG2 exist in different fractions in vivo. In addition, separate protein complexes may be formed.
[0192] 〔実施例 19〕 DRG1のポリソーム局在 [Example 19] Polysomal localization of DRG1
DRG1が mRNAを制御し、図 16丸 4において P100画分に濃縮されることから、ポリソ ーム（1本の mRNAに複数のリボソームが同時に結合してタンパク合成が行われてい る状態のもの）に局在している可能性があった。このことを確認するために、成体マウ スの肝臓ホモジネートを不連続ショ糖勾配を用いて超遠心し、そのポリソームペレット を再溶解し、 15-40%連続ショ糖勾配に重層し、超遠心後、 UV(254nm) モニター付き の精留塔でポリソームプロフアイリングを行った。同時に、図 17と対応する画分を回 収し、ウェスタンブロット法で DRG1の蛋白質の局在を解析した。リボソームを示す曲 線（図 17上のパネル,青線）とほぼ同様のパターンで DRG1のタンパク量が存在してい た（図 17真ん中のパネル)。また、 80Sリボソームは [Mg2+]濃度が 5mM以下であると 40S と 60Sのサブユニットに解離する性質がある力 これを EDTAを 10mM加えることで実現 させると（上のパネル,赤線）、リボソームの解離したピークの方へ DRG1の存在する画 分も移行した。これらのことから、 DRG1はポリソーム上に局在していると考えられた。 産業上の利用可能性 DRG1 regulates mRNA and is concentrated in the P100 fraction in Figure 16 circle 4, so that polysomes (in the state where multiple ribosomes are simultaneously bound to a single mRNA and protein synthesis is performed) There was a possibility that it was localized. To confirm this, the adult mouse liver homogenate was ultracentrifuged using a discontinuous sucrose gradient, the polysome pellet was redissolved, layered on a 15-40% continuous sucrose gradient, and after ultracentrifugation. Polysome profiling was performed in a rectification column equipped with a UV (254 nm) monitor. At the same time, fractions corresponding to FIG. 17 were collected, and the localization of the DRG1 protein was analyzed by Western blotting. The amount of DRG1 protein was present in a pattern similar to the curve showing the ribosome (upper panel in Fig. 17, blue line) (middle panel in Fig. 17). In addition, 80S ribosome has a property of dissociating into 40S and 60S subunits when the [Mg 2+ ] concentration is 5 mM or less. When this is achieved by adding 10 mM of EDTA (upper panel, red line), The fraction in which DRG1 was present also moved toward the dissociated peak of ribosome. From these results, it was considered that DRG1 is localized on polysomes. Industrial applicability
[0193] 本発明によって、 DRG1と DRG2の発現は、 DFRP1と DFRP2の因子が存在しないと成 立しないことも生理的条件下で明ら力となった。また、ヒト癌細胞株である HeLa S3細 胞内で DRG1をノックダウンさせると、細胞の運動 '浸潤'転移能が上昇し、 DRG2をノ ックダウンさせると逆に低下し、細胞接着性などは異常に増強していることが示された 。この分子機構として、細胞骨格因子，細胞外マトリックス因子など、細胞骨格 '運動' 形態 ·浸潤 ·転移などを制御する蛋白質群の mRNAの安定ィ匕 ·分解の調節を DRGファ ミリ一が直接的あるいは間接的に行っているためであることが見出された。 [0193] According to the present invention, it has become clear that the expression of DRG1 and DRG2 does not occur in the absence of DFRP1 and DFRP2 factors under physiological conditions. In addition, knocking down DRG1 in a human cancer cell line, HeLa S3 cell, increases the cell's ability to infiltrate 'invasion', while knocking down DRG2 decreases it, resulting in abnormal cell adhesion. It was shown that it was strengthened. This molecular mechanism includes cytoskeletal factors, extracellular matrix factors, etc. It was found that the DRG family directly or indirectly regulates the stability and degradation of mRNA of proteins that control morphology, invasion, and metastasis.
この新たな基本機構の原理を基にした、 DRGファミリー、 DFRPsの発現調節による 美容薬の開発'癌治療法の開発が期待される。例として、皮膚の弛み'肌荒れは細 胞集団の接着性が失われることが原因である力 DRG1を発現させる、あるいは DRG2 の発現を抑制することによって、細胞間の接着性を増強できる可能性が示唆される。 また、 DRG1を強く発現させるあるいは DRG2の発現を抑制することによって、癌細胞 の浸潤'転移能を抑制できる可能性がある。上述のように DRG1単独の過剰発現は不 可能であるので、 DFRP1との共発現させることが必要となる。また、角質化した肌を柔 らかくするには、逆に DRG1の発現または DFRP1の発現を抑制する、あるいは DRG2 を強く発現させる手法が考えられる。この際 DRG2の発現には DFRP2との共発現が必 要である。また、 DRG1と DRG2のノックダウン細胞はともに細胞の死には至らしめない 程度の増殖の低下が認められた。これが癌細胞の高い増殖能を単に正常細胞レべ ルまで落としたものであるならば、この発明を利用した薬の細胞生存 '増殖に起因す る副作用は低レベルに抑えられることが期待される。また、 DRG1と DRG2による様々 な細胞骨格 ·運動 ·形態 ·浸潤 ·転移に関する直接的あるいは間接的な制御因子の 発現調節が mRNAレベルで行われていることから、 DRG1、 DRG2、 DFRP1、 DFRP2を ターゲットとした医薬品の効果および即効性は非常に高いことが期待される。  Based on the principle of this new basic mechanism, development of beauty drugs by regulating the expression of DRG family and DFRPs is expected to develop cancer therapy. As an example, it is possible that the adhesion between cells can be enhanced by expressing DRG1 or suppressing DRG2 expression, which is caused by loss of adhesion of the cell population. It is suggested. In addition, the ability of cancer cells to infiltrate or metastasize may be suppressed by strongly expressing DRG1 or suppressing the expression of DRG2. As described above, since overexpression of DRG1 alone is impossible, co-expression with DFRP1 is necessary. To soften keratinized skin, conversely, DRG1 expression or DFRP1 expression can be suppressed, or DRG2 can be strongly expressed. In this case, co-expression with DFRP2 is required for DRG2 expression. In addition, both DRG1 and DRG2 knockdown cells showed a decrease in proliferation to such an extent that cell death did not occur. If this is simply a reduction of the high growth potential of cancer cells to the level of normal cells, it is expected that side effects caused by cell survival and proliferation of drugs using this invention can be suppressed to a low level. . In addition, DRG1, DRG2, DFRP1, and DFRP2 are targeted because DRG1 and DRG2 regulate the expression of various cytoskeletal, motor, morphology, invasion, and metastasis regulators directly or indirectly at the mRNA level. It is expected that the effects and immediate effects of the selected drugs are very high.

Claims

請求の範囲 [1] 下記（1)または（2)に記載のポリペプチド。 ( 1)配列番号： 2または配列番号: 4に記載のアミノ酸配列における 234位から 295位 のアミノ酸配列力もなるポリペプチド (2)配列番号： 2または配列番号: 4に記載のアミノ酸配列における 234位から 295位 のアミノ酸配列において 1若しくは複数のアミノ酸が置換、欠失、挿入または付加され たアミノ酸配列力 なり、 DRG1蛋白質との相互作用能力を有するポリペプチド [2] 下記（1)または（2)に記載のポリペプチド。 Claims [1] The polypeptide according to (1) or (2) below. (1) Polypeptide having an amino acid sequence from position 234 to position 295 in the amino acid sequence described in SEQ ID NO: 2 or SEQ ID NO: 4 (2) Position 234 in the amino acid sequence described in SEQ ID NO: 2 or SEQ ID NO: 4 A polypeptide having the ability to interact with DRG1 protein by substitution, deletion, insertion or addition of one or more amino acids in the amino acid sequence from position 295 to [2] (1) or (2) below The polypeptide according to 1.
(1)配列番号： 2または配列番号: 4に記載のアミノ酸配列からなるポリペプチド (1) A polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4.
(2)配列番号： 2または配列番号： 4に記載のアミノ酸配列にぉ 、て 1若しくは複数の アミノ酸が置換、欠失、挿入または付加されたアミノ酸配列からなり、 DRG1蛋白質と の相互作用能力を有するポリペプチド (2) It consists of an amino acid sequence in which one or more amino acids are substituted, deleted, inserted or added to the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4, and has the ability to interact with DRG1 protein. Polypeptide having
[3] 請求項 1または 2に記載のポリペプチドをコードした核酸。  [3] A nucleic acid encoding the polypeptide according to claim 1 or 2.
[4] 請求項 3に記載の核酸を担持したベクター。 [4] A vector carrying the nucleic acid according to claim 3.
[5] 請求項 1または 2に記載のポリペプチドに対する抗体、好ましくはポリクローナル抗 体、モノクローナル抗体、キメラ抗体、ヒト型抗体またはこれらの混合物から選択され る抗体。  [5] An antibody against the polypeptide according to claim 1 or 2, preferably an antibody selected from a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a human antibody, or a mixture thereof.
[6] 下記（1)または（2)に記載の遺伝子の転写産物に対してアニーリングする活性を有 し、 15塩基長以上、好ましくは 19塩基長以上を備えた核酸。  [6] A nucleic acid having an activity of annealing to the transcription product of the gene described in (1) or (2) below and having a length of 15 bases or more, preferably 19 bases or more.
(1)配列番号： 1または配列番号: 3に記載の塩基配列からなる遺伝子  (1) Gene consisting of the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 3
(2)配列番号：1または配列番号 : 3に記載の塩基配列とストリンジ ントな条件下で ノ、イブリダィズする遺伝子  (2) A gene that hybridizes with the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3 under stringent conditions
[7] 下記（1)または（2)に記載の遺伝子の転写産物に対してアニーリングする活性を有 し、 15塩基長以上、好ましくは 19塩基長以上を備えた核酸。  [7] A nucleic acid having an activity of annealing to the transcription product of the gene described in (1) or (2) below and having a length of 15 bases or more, preferably 19 bases or more.
(1)配列番号： 9または配列番号： 10に記載の塩基配列力 なる遺伝子  (1) The gene having the nucleotide sequence ability described in SEQ ID NO: 9 or SEQ ID NO: 10
(2)配列番号： 9または配列番号： 10に記載の塩基配列とストリンジ ントな条件下で ノ、イブリダィズする遺伝子  (2) A gene that hybridizes with the nucleotide sequence of SEQ ID NO: 9 or SEQ ID NO: 10 under stringent conditions
[8] 請求項 1もしくは 2に記載のポリペプチドまたは請求項 3に記載の核酸を有効成分と する、 DRG1蛋白質安定化剤。 [8] The polypeptide according to claim 1 or 2 or the nucleic acid according to claim 3 as an active ingredient. DRG1 protein stabilizer.
[9] 請求項 1もしくは 2に記載のポリペプチドまたは請求項 3に記載の核酸を有効成分と する、細胞運動抑制剤。 [9] A cell motility inhibitor comprising the polypeptide according to claim 1 or 2 or the nucleic acid according to claim 3 as an active ingredient.
[10] 請求項 6または請求項 7に記載の核酸を有効成分とする、癌細胞増殖抑制剤。 [10] A cancer cell growth inhibitor comprising the nucleic acid according to claim 6 or 7 as an active ingredient.
[11] 下記（1)から（3)の工程を有する、細胞運動抑制剤のスクリーニング方法。 [11] A screening method for a cell motility inhibitor comprising the following steps (1) to (3):
(1)被験物質存在下および非存在下で DRG1蛋白質および請求項 1または 2に記載 のポリペプチドを作用させる工程  (1) A step of allowing the DRG1 protein and the polypeptide of claim 1 or 2 to act in the presence and absence of a test substance
(2) DRG1蛋白質と請求項 1もしくは 2に記載のポリペプチドとの結合活性を測定する 工程  (2) measuring the binding activity between the DRG1 protein and the polypeptide according to claim 1 or 2
(3)被験物質存在下における結合活性が被験物質非存在下の結合活性よりも増加 して 、る場合に、その被験物質を細胞運動抑制剤の候補として選択する工程  (3) When the binding activity in the presence of the test substance is higher than the binding activity in the absence of the test substance, the test substance is selected as a candidate for a cell motility inhibitor.
[12] 下記（1)および（2)の工程を有する細胞運動抑制剤のスクリーニング方法。  [12] A screening method for a cell motility inhibitor comprising the following steps (1) and (2):
(1)プロモータを備えた dfrpl遺伝子を保持した細胞に被験物質を作用させる工程 (1) A step of allowing a test substance to act on a cell carrying a dfrpl gene equipped with a promoter
(2) dfrpl遺伝子の発現を上昇させた被験物質を選択する工程 (2) A step of selecting a test substance with increased expression of the dfrpl gene
[13] 下記（1)および（2)の工程を有する細胞運動抑制剤のスクリーニング方法。  [13] A screening method for a cell motility inhibitor comprising the following steps (1) and (2).
(1)プロモータを備えた drgl遺伝子を保持した細胞に被験物質を作用させる工程 (1) A step of allowing a test substance to act on a cell having a promoter and a drgl gene
(2) drgl遺伝子の発現を上昇させた被験物質を選択する工程 (2) A step of selecting a test substance with increased drgl gene expression
[14] 下記（1)から（3)の工程を有する、癌細胞増殖抑制剤のスクリーニング方法。  [14] A screening method for a cancer cell proliferation inhibitor comprising the following steps (1) to (3):
(1)被験物質存在下および非存在下で DRG1蛋白質および請求項 1または 2に記載 のポリペプチドを作用させる工程  (1) A step of allowing the DRG1 protein and the polypeptide of claim 1 or 2 to act in the presence and absence of a test substance
(2) DRG1蛋白質と請求項 1もしくは 2に記載のポリペプチドとの結合活性を測定する 工程  (2) measuring the binding activity between the DRG1 protein and the polypeptide according to claim 1 or 2
(3)被験物質存在下における結合活性が被験物質非存在下の結合活性よりも低下 して 、る場合に、その被験物質を癌細胞増殖抑制剤の候補として選択する工程 (3) When the binding activity in the presence of the test substance is lower than the binding activity in the absence of the test substance, the test substance is selected as a candidate for a cancer cell growth inhibitor.
[15] 下記（1)および（2)の工程を有する癌細胞増殖抑制剤のスクリーニング方法。 [15] A screening method for a cancer cell proliferation inhibitor comprising the following steps (1) and (2):
(1)プロモータを備えた dfrpl遺伝子を保持した細胞に被験物質を作用させる工程 (1) A step of allowing a test substance to act on a cell carrying a dfrpl gene equipped with a promoter
(2) dfrpl遺伝子の発現を低下させた被験物質を選択する工程 (2) A step of selecting a test substance with reduced dfrpl gene expression
[16] 下記（1)および（2)の工程を有する癌細胞増殖抑制剤のスクリーニング方法。 (1)プロモータを備えた drgl遺伝子を保持した細胞に被験物質を作用させる工程[16] A screening method for a cancer cell proliferation inhibitor comprising the following steps (1) and (2): (1) A step of allowing a test substance to act on a cell having a promoter and a drgl gene
(2) drgl遺伝子の発現を低下させた被験物質を選択する工程 (2) A step of selecting a test substance with reduced drgl gene expression
[17] 下記（1)または（2)に記載のポリペプチド。 [17] The polypeptide according to (1) or (2) below.
(1)配列番号: 6または配列番号: 8に記載のアミノ酸配列における 132位から 187位 のアミノ酸配列力もなるポリペプチド  (1) A polypeptide having an amino acid sequence from positions 132 to 187 in the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 8
(2)配列番号: 6または配列番号: 8に記載のアミノ酸配列における 132位から 187位 のアミノ酸配列において 1若しくは複数のアミノ酸が置換、欠失、挿入または付加され たアミノ酸配列力 なり、 DRG2蛋白質との相互作用能力を有するポリペプチド  (2) A DRG2 protein comprising an amino acid sequence having one or more amino acids substituted, deleted, inserted or added in the amino acid sequence from position 132 to position 187 in the amino acid sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 8. Having the ability to interact with
[18] 下記（1)または（2)に記載のポリペプチド。  [18] The polypeptide according to (1) or (2) below.
(1)配列番号: 6または配列番号: 8に記載のアミノ酸配列からなるポリペプチド (1) A polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 8
(2)配列番号： 6または配列番号： 8に記載のアミノ酸配列にお!/、て 1若しくは複数の アミノ酸が置換、欠失、挿入または付加されたアミノ酸配列からなり、 DRG2蛋白質と の相互作用能力を有するポリペプチド (2) It consists of an amino acid sequence in which one or more amino acids are substituted, deleted, inserted or added to the amino acid sequence shown in SEQ ID NO: 6 or 8 and interacts with DRG2 protein Polypeptide with ability
[19] 請求項 17または 18に記載のポリペプチドをコードした核酸。  [19] A nucleic acid encoding the polypeptide according to claim 17 or 18.
[20] 請求項 19に記載の核酸を担持したベクター。 [20] A vector carrying the nucleic acid according to claim 19.
[21] 請求項 17または 18に記載のポリペプチドに対する抗体、好ましくはポリクローナル 抗体、モノクローナル抗体、キメラ抗体、ヒト型抗体またはこれらの混合物から選択さ れる抗体。  [21] An antibody against the polypeptide according to claim 17 or 18, preferably an antibody selected from a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a humanized antibody, or a mixture thereof.
[22] 下記（1)または（2)に記載の遺伝子の転写産物に対してアニーリングする活性を有 し、 15塩基長以上、好ましくは 19塩基長以上を備えた核酸。  [22] A nucleic acid having an activity of annealing to the transcription product of the gene described in (1) or (2) below, having a length of 15 bases or more, preferably 19 bases or more.
(1)配列番号： 5または配列番号： 7に記載の塩基配列からなる遺伝子  (1) Gene consisting of the nucleotide sequence set forth in SEQ ID NO: 5 or SEQ ID NO: 7
(2)配列番号： 5または配列番号： 7に記載の塩基配列とストリンジ ントな条件下で ノ、イブリダィズする遺伝子  (2) A gene that hybridizes with the base sequence described in SEQ ID NO: 5 or 7 under stringent conditions
[23] 下記（1)または（2)に記載の遺伝子の転写産物に対してアニーリングする活性を有 し、 15塩基長以上、好ましくは 19塩基長以上を備えた核酸。  [23] A nucleic acid having an activity of annealing to the transcription product of the gene described in (1) or (2) below, having a length of 15 bases or more, preferably 19 bases or more.
(1)配列番号： 11または配列番号： 12に記載の塩基配列力 なる遺伝子  (1) The gene having the nucleotide sequence ability described in SEQ ID NO: 11 or SEQ ID NO: 12
(2)配列番号： 11または配列番号： 12に記載の塩基配列とストリンジ ントな条件下 でノヽイブリダィズする遺伝子 (2) A gene that is hybridized under stringent conditions with the nucleotide sequence of SEQ ID NO: 11 or SEQ ID NO: 12.
[24] 請求項 17もしくは 18に記載のポリペプチドまたは請求項 19に記載の核酸を有効 成分とする、 DRG2蛋白質安定化剤。 [24] A DRG2 protein stabilizer comprising the polypeptide according to claim 17 or 18 or the nucleic acid according to claim 19 as an active ingredient.
[25] 請求項 22または請求項 23に記載の核酸を有効成分とする、細胞間接着促進剤。 [25] An intercellular adhesion promoter comprising the nucleic acid according to claim 22 or 23 as an active ingredient.
[26] 請求項 22または請求項 23に記載の核酸を有効成分とする、細胞運動抑制剤。 [26] A cell motility inhibitor comprising the nucleic acid according to claim 22 or 23 as an active ingredient.
[27] 請求項 22または請求項 23に記載の核酸を有効成分とする、癌細胞増殖抑制剤。 [27] A cancer cell proliferation inhibitor comprising the nucleic acid according to claim 22 or 23 as an active ingredient.
[28] 下記（1)力 (3)の工程を有する、細胞間接着促進剤または細胞運動抑制剤のス クリーニング方法。 [28] A screening method for an intercellular adhesion promoter or cell motility inhibitor, comprising the following step (1) force (3):
(1)被験物質存在下および非存在下で DRG2蛋白質および請求項 17または 18に記 載のポリペプチドを作用させる工程  (1) a step of allowing the DRG2 protein and the polypeptide according to claim 17 or 18 to act in the presence or absence of a test substance
(2) DRG2蛋白質と請求項 17もしくは 18に記載のポリペプチドとの結合活性を測定 する工程  (2) a step of measuring the binding activity between the DRG2 protein and the polypeptide according to claim 17 or 18
(3)被験物質存在下における結合活性が被験物質非存在下の結合活性よりも低下 して!、る場合に、その被験物質を細胞間接着促進剤または細胞運動抑制剤の候補 として選択する工程  (3) In the case where the binding activity in the presence of the test substance is lower than the binding activity in the absence of the test substance !, the test substance is selected as a candidate for an intercellular adhesion promoter or cell motility inhibitor.
[29] 下記（1)および (2)の工程を有する、細胞間接着促進剤または細胞運動抑制剤の スクリーニング方法。  [29] A screening method for an intercellular adhesion promoter or cell movement inhibitor, comprising the following steps (1) and (2).
(1)プロモータを備えた dfrp2遺伝子を保持した細胞に被験物質を作用させる工程 (1) A step of allowing a test substance to act on a cell having a dfrp2 gene having a promoter
(2) dfrp2遺伝子の発現を低下させた被験物質を選択する工程 (2) A step of selecting a test substance with reduced expression of the dfrp2 gene
[30] 下記（1)および (2)の工程を有する、細胞間接着促進剤または細胞運動抑制剤の スクリーニング方法。  [30] A screening method for an intercellular adhesion promoter or cell movement inhibitor, comprising the following steps (1) and (2):
(1)プロモータを備えた drg2遺伝子を保持した細胞に被験物質を作用させる工程 (1) A step of allowing a test substance to act on a cell having a promoter and a drg2 gene
(2) drg2遺伝子の発現を低下させた被験物質を選択する工程 (2) A step of selecting a test substance with reduced drg2 gene expression
[31] 下記（1)から（3)の工程を有する、癌細胞増殖抑制剤のスクリーニング方法。  [31] A screening method for a cancer cell proliferation inhibitor comprising the following steps (1) to (3):
(1)被験物質存在下および非存在下で DRG2蛋白質および請求項 17または 18に記 載のポリペプチドを作用させる工程  (1) a step of allowing the DRG2 protein and the polypeptide according to claim 17 or 18 to act in the presence or absence of a test substance
(2) DRG2蛋白質と請求項 17もしくは 18に記載のポリペプチドとの結合活性を測定 する工程  (2) a step of measuring the binding activity between the DRG2 protein and the polypeptide according to claim 17 or 18
(3)被験物質存在下における結合活性が被験物質非存在下の結合活性よりも低下 して 、る場合に、その被験物質を癌細胞増殖抑制剤の候補として選択する工程(3) The binding activity in the presence of the test substance is lower than the binding activity in the absence of the test substance In this case, the step of selecting the test substance as a cancer cell growth inhibitor candidate
[32] 下記（1)および（2)の工程を有する、癌細胞増殖抑制剤のスクリーニング方法。 [32] A screening method for a cancer cell growth inhibitor comprising the following steps (1) and (2):
(1)プロモータを備えた dfrp2遺伝子を保持した細胞に被験物質を作用させる工程 (1) A step of allowing a test substance to act on a cell having a dfrp2 gene having a promoter
(2) dfrp2遺伝子の発現を低下させた被験物質を選択する工程 (2) A step of selecting a test substance with reduced expression of the dfrp2 gene
[33] 下記（1)および（2)の工程を有する、癌細胞増殖抑制剤のスクリーニング方法。  [33] A screening method for a cancer cell growth inhibitor comprising the following steps (1) and (2):
(1)プロモータを備えた drg2遺伝子を保持した細胞に被験物質を作用させる工程 (1) A step of allowing a test substance to act on a cell having a promoter and a drg2 gene
(2) drg2遺伝子の発現を低下させた被験物質を選択する工程 (2) A step of selecting a test substance with reduced drg2 gene expression
[34] 配列番号： 9または配列番号： 10に記載の遺伝子にコードされたポリペプチドに対 する抗体であって、配列番号： 11または配列番号： 12に記載の遺伝子にコードされ たポリペプチドとクロスリアクティビティ一を有しない、抗 DRG1蛋白質抗体。  [34] An antibody against the polypeptide encoded by the gene set forth in SEQ ID NO: 9 or SEQ ID NO: 10, comprising the polypeptide encoded by the gene set forth in SEQ ID NO: 11 or SEQ ID NO: 12 Anti-DRG1 protein antibody that does not have cross-reactivity.
[35] 配列番号： 11または配列番号： 12に記載の遺伝子にコードされたポリペプチドに対 する抗体であって、配列番号： 9または配列番号： 10に記載の遺伝子にコードされた ポリペプチドとクロスリアクティビティ一を有しない、抗 DRG2蛋白質抗体。  [35] An antibody against the polypeptide encoded by the gene described in SEQ ID NO: 11 or SEQ ID NO: 12, wherein the polypeptide is encoded by the gene described in SEQ ID NO: 9 or SEQ ID NO: 10. Anti-DRG2 protein antibody that does not have cross-reactivity.
PCT/JP2005/021361 2004-11-24 2005-11-21 Dfrp proteins regulating drg proteins and utilization of the same WO2006057217A1 (en)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002064786A1 (en) * 2001-02-15 2002-08-22 Mochida Pharmaceutical Co., Ltd. Novel gene tcif

Patent Citations (1)

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Publication number Priority date Publication date Assignee Title
WO2002064786A1 (en) * 2001-02-15 2002-08-22 Mochida Pharmaceutical Co., Ltd. Novel gene tcif

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