WO2006132248A1 - Procédé d’ubiquitination du runx - Google Patents

Procédé d’ubiquitination du runx Download PDF

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Publication number
WO2006132248A1
WO2006132248A1 PCT/JP2006/311333 JP2006311333W WO2006132248A1 WO 2006132248 A1 WO2006132248 A1 WO 2006132248A1 JP 2006311333 W JP2006311333 W JP 2006311333W WO 2006132248 A1 WO2006132248 A1 WO 2006132248A1
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nedd4
runx
seq
polynucleotide
represented
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PCT/JP2006/311333
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English (en)
Japanese (ja)
Inventor
Gen Kudo
Takehiko Takata
Haruna Hayasaka
Hirofumi Doi
Yasuhiro Kikuchi
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Daiichi Pharmaceutical Co., Ltd.
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Priority claimed from JP2006024521A external-priority patent/JP2008206398A/ja
Application filed by Daiichi Pharmaceutical Co., Ltd. filed Critical Daiichi Pharmaceutical Co., Ltd.
Publication of WO2006132248A1 publication Critical patent/WO2006132248A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/25—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving enzymes not classifiable in groups C12Q1/26 - C12Q1/66
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/9015—Ligases (6)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • the present invention relates to a RUNX (Runt—related transcription factor) ubiquitination method and degradation method. More specifically, the present invention relates to a RUNX ubiquitin method and decomposition method characterized by coexistence of RUNX and NEDD4 (Neural precursor cell Expressed, developmentally down-regulated 4).
  • the present invention also relates to a RUNX ubiquitinating agent and degrading agent.
  • the present invention further relates to a method for inhibiting RUNX ubiquitin and degradation. More specifically, the present invention relates to a method for inhibiting RUNX ubiquitin and degradation by NEDD4, characterized by inhibiting at least one of the binding of NEDD4 and RUNX, the enzyme activity of NEDD4, and the expression of NEDD4. .
  • the present invention also relates to a RUNX ubiquitin inhibitor and degradation inhibitor of NEDD4.
  • the present invention further relates to a polynucleotide having a partial base sequence ability of NEDD4.
  • the present invention also relates to a polynucleotide comprising a partial base sequence of NEDD4 and a double-stranded polynucleotide comprising a complementary base sequence of the base sequence.
  • the present invention also relates to a method for promoting osteogenesis and an agent for promoting osteogenesis. More specifically, the present invention relates to an osteogenesis promoting method and an osteogenesis promoter characterized by inhibiting the expression and Z or function of NEDD4.
  • the present invention further relates to a method for inhibiting tumor growth and a tumor growth inhibitor. More details
  • the present invention relates to a method for inhibiting tumor growth and a tumor growth inhibitor, characterized by inhibiting the expression and Z or function of NEDD4.
  • the present invention also relates to a method for identifying a compound that inhibits or promotes RUNX ubiquitin by NEDD4, and a method for identifying a compound that inhibits or promotes the binding of NEDD4 to RUNX.
  • the present invention further relates to a method for identifying a compound capable of promoting bone formation.
  • the present invention also relates to a method for identifying a compound capable of suppressing tumor growth.
  • the present invention further includes NEDD4, a polynucleotide encoding NEDD4, a vector containing the polynucleotide, and a transformant containing the vector, and a polynucleotide encoding RUNX and RUNX.
  • a reagent kit comprising at least one of a vector containing the polynucleotide and a transformant containing the vector.
  • the present invention also relates to a disease caused by an abnormality in RUNX, for example, a disease caused by an increase or decrease in the function and Z or expression of RUNX, specifically a preventive and Z or therapeutic agent for cancer diseases, etc. , And prevention and Z or treatment methods.
  • a disease caused by an abnormality in RUNX for example, a disease caused by an increase or decrease in the function and Z or expression of RUNX, specifically a preventive and Z or therapeutic agent for cancer diseases, etc. , And prevention and Z or treatment methods.
  • the present invention further relates to a preventive and Z or therapeutic agent for diseases associated with bone loss, and a preventive and Z or therapeutic method.
  • RUNX is a protein characterized by having a Runt domain, and many family proteins are known.
  • the Runt domain has been identified as (1) AML1 associated with acute myeloid leukemia in humans, acute myelocytic leukemia 1), (2) Retronoles hennoenser ⁇ [a transcription factor that binds to a common core. CBF (core binding factor), and (3) the alpha subunit of PEBP2 (polyomavirus enhancer binding protein 2), a transcription factor identified as an enzyme that regulates gene expression and DNA replication in poliovirus. It is a highly homologous sequence with about 130 amino acid residues.
  • RUNX is known to form a heterodimer with ⁇ 2 CBF j8 subunit (PEBP2 j8 ZCBF j8) and act as a transcription factor.
  • the Runt domain is responsible for both DNA binding and heterodimerization with the j8 subunit, and is an important domain for the action of RUNX as a transcription factor.
  • RUNX is known to have three types of family proteins in mammals, namely RUNX1, RUNX2 and RUNX3 (Non-patent Documents 1 and 2). Homology between human RUNX3 and human RUNX1 and human RUNX2 is 57% and 54%, respectively, at the amino acid level
  • the RUNX family acts as a transcription factor and plays an important role in normal differentiation and tumorigenesis Fulfill.
  • RUNX1 is present on human chromosome 21 (21q22) and has been identified as the gene with the most frequent chromosomal translocation in acute myeloid leukemia (AML) (Non-patent Document 3). On the other hand, it has been reported that RUNX1 also plays a crucial role in regulating the differentiation of hematopoietic cells (Non-patent Documents 4 and 5). In AML, a reciprocal translocation (t (8; 21)) in which the long arms of chromosomes 8 and 21 are interchanged is frequently observed, and this chromosomal translocation acts on dominant negative for RUNX1. It is known that chimeric proteins are produced.
  • Non-patent Document 6 It has been reported that when RUNX1 is expressed in large quantities in proliferating cells expressing this chimeric protein, cell proliferation is suppressed and differentiation is promoted. On the other hand, even in leukemia cells without chromosomal translocation, loss of RUNX1 transcriptional activity due to point mutations has been reported, and this loss of function seems to play an important role in the development of leukemia. (Non-patent document 7).
  • RUNX2 is an important transcription factor involved in bone formation, and is essential for chondrocyte differentiation 'maturation, osteoblast differentiation and bone marrow formation (Non-patent Documents 26 and 27).
  • both membranous ossification and endochondral ossification are deficient, so the skeleton is formed only from cartilage (Non-patent Document 8).
  • the skeleton is constructed by membranous ossification directly formed by osteoblasts and endochondral ossification where the cartilage is replaced by bone after the cartilage is formed. Both osteoblasts and chondrocytes differentiate from mesenchymal stem cells.
  • RUNX2 site force-in such as BMP (bone morphogenetic protein) and various transcription factors. Since RUNX2 binds to the transcription factor Smadl, which is involved in the induction of bone morphogenetic gene expression by BMP stimulation, it is thought to be involved in bone formation signals via BMP stimulation (Non-patent Document 28). ). RUNX2 affects the differentiation of bone cells and also expresses bone matrix genes (Collal, Colla2, osteopontin, osteocalcin, bone sialoprotein). It has been shown to be active (Non-patent Document 29).
  • BMP bone morphogenetic protein
  • Non-patent Document 9 Since RUNX3 binds to Smad, an intracellular mediator of TGF-j8 (tumor growth factor-j8), it is considered to be involved in TGF- ⁇ signaling (Non-patent Document 10). Mutations of TGF- ⁇ receptor and Smad are known in many cancers (Non-patent Document 11), and RUNX3 is also considered to be involved in cancer.
  • RUNX3 is highly expressed in normal gastric mucosal epithelium (Non-patent Document 12), but when RUNX3 is knocked out, mucosal thickening due to increased proliferation of gastric mucosal epithelial cells is observed. This increase in cell proliferation has been shown to be due to suppression of apoptosis.
  • gastric mucosal epithelial cells of RUNX3 knockout mice are resistant to the growth-inhibiting action of TGF- ⁇ , suggesting that mucosal thickening is caused by abnormal TGF- ⁇ signaling due to RUNX3 deficiency (non- Patent Document 13).
  • Non-patent Documents 14 mucosal thickening due to proliferation of gastric mucosal epithelial cells is also observed in TGF- ⁇ knockout mice.
  • RUNX3 is deficient in human esophageal cancer SEG-1 cells and is resistant to the production of TGF- ⁇ .
  • the reactivity is restored (Non-Patent Document 15). .
  • RUNX3 is located on chromosome 1 (1 ⁇ 36), where many tumor suppressor genes are present. This region is frequently defective in gastric cancer, colon cancer, bile duct cancer, and spleen cancer.
  • the expression of RUNX3 in gastric cancer cells decreased with the progression of cancer, and the expression of RUNX3 gene decreased in about 90% of stage 4 gastric cancer (Non-patent Document 13).
  • RUNX3 expression is thought to be due to hemizygous deletion and methylation of the promoter region of RUNX3.
  • the tumor formation inhibitory effect of RUN X3 has been confirmed in animal experiments using a transplant model.When RUNX3 gene was expressed in RU NX3-deficient human gastric cancer cells, tumor growth after transplantation of nude mice was significantly suppressed (non- Patent Document 13).
  • NEDD4 is an H £ LC ⁇ (homologous to the papillomavirus K6-associated protein car boxyl terminus) type ubiquitin ligase.
  • NEDD4 is a C2 domain involved in binding to membrane lipids in the N-terminal region, four WW domains involved in binding to the substrate in the middle, and a ubiquitin ⁇ catalytic domain in the C-terminal region and ubiquitin ligase activity It has a HECT domain that indicates (Non-patent Document 18).
  • the ubiquitin proteasome system is a selective and active proteolytic mechanism that regulates various physiological phenomena such as the cell cycle and signal transduction, and is thus involved in maintaining homeostasis of proteins and cells.
  • Ubiquitin is an evolutionarily conserved protein with 76 amino acid residues existing in eukaryotes, which is covalently linked in a chain to the target protein and acts as a degradation signal.
  • the binding of ubiquitin to the target protein occurs by the continuous catalysis of ubiquitin activity enzyme (E1), ubiquitin-conjugating enzyme (E2) and ubiquitin ligase (E3).
  • ubiquitin ligase (E3) is important as an enzyme responsible for substrate selectivity.
  • the ubiquitinated target protein is degraded by the proteasome that recognizes ubiquitin bound in a chain to the protein.
  • Abnormalities in the ubiquitin proteasome system lead to protein deficiency due to excessive degradation of the target protein, or protein accumulation due to inhibition of target protein degradation, thereby causing various diseases.
  • the involvement of the ubiquitin proteasome system in cancer diseases has been reported (Non-patent Document 20).
  • NEDD4 The tissue distribution of NEDD4 is highly expressed in muscle in normal tissues. Also, in some cancer cell lines, such as the human eclampsia cancer cell line HeLa, the human lung cancer cell line A549, and the human leukemia cell line K562, the expression of NEDD4 is increased at the mRNA level! It has been reported (Non-patent Document 21). In addition, NEDD4 is known to decrease in expression with neuronal differentiation (Non-patent Document 22).
  • Non-patent Document 23 As a substrate for NEDD4, Bel 10 which is a regulator of NF- ⁇ (nuclear factor- ⁇ ) has been reported (Non-patent Document 23). Another substrate for NEDD4 is amyloid-sensitive epithelial sodium channel (ENaC) (Non-patent Document 24).
  • ENaC amyloid-sensitive epithelial sodium channel
  • Non-patent Document 30 HECT type E already as E3 ligase of RUNX2 Smurfl (Smad ubiquitination regulatory factor 1) known as 3 ligase has been reported (Non-patent Document 31). It has been reported that treatment with proteasome inhibitors induces osteoblast differentiation in vitro and promotes bone formation in individuals (Non-patent Documents 31 and 32).
  • Non-Patent Document 1 Lund A. H. et al., “Cancer Cell”, 2002, No. 1, No. 3, p. 213-215.
  • Non-Patent Document 2 Otto F. et al., “Journal of Cellular Biochemistry”, 2003, No. 89, No. 1, p. 9-1
  • Non-Patent Document 3 Miyoshi H. et al., "Proceedings of the National Academy of Sciences of fhe United 3 ⁇ 4 tates of America” 1991, 88th, 23rd, p. 10431-10434.
  • Non-Patent Document 4 Okuda T. et al., “Cell”, 1996, 84, No. 2, p. 321—330.
  • Non-Patent Document 5 Wang Q. et al., “Cell”, 1996, 87th, No. 4, p. 6 97—708.
  • Non-Patent Document 6 Kitabayashi I. et al., “EMBO Journal”, 1998, Vol. 17, No. 11, p. 2994—3004.
  • Non-Patent Document 7 Osato M. et al., “Blood”, 1999, No. 93, No. 6, p. 1817-1824.
  • Non-Patent Document 8 Otto F. et al., “Cell”, 1997, 89th, No. 5, p. 765—771.
  • Non-Patent Document 9 Valliant F. et al., “Oncogene”, 1999, No. 18, No. 50, p. 7124-7134.
  • Non-Patent Document 10 Ito Y. et al., “Current Opinion in Genetics and Development”, 2000, 13th, No. 1, p. 43— 47.
  • Non-Patent Document 11 Kawabata M. et al., “Journal of Biochemistry J, 1999, 125th, No. 1, p. 9-16.
  • Non-Patent Document 12 Osaki M. et al., “European Journal of Clinical Investigation”, 2004, No. 34, No. 9, p. 605 — 612.
  • Non-Patent Document 13 Li Q. L. et al., “Cell”, 2002, No. 109, No. 1, p. 11 3-124.
  • Non-Patent Document 14 Crawford S. E. et al., “Cell”, 1998, 93rd, No. 7, p. 1159-1170.
  • Non-Patent Document 15 Torquati A. et al., “Surgery”, 2004, Vol. 136, No. 2, p. 310-316.
  • Non-Patent Document 16 Wada M. et al., “Oncogene”, 2004, 23rd, No. 13, p. 2401—2407.
  • Non-Patent Document 17 Goel A. et al., “International Journal of Cancer”, 2004, 112, 5, p. 7 54-759.
  • Non-Patent Document 18 Harvey K. F. et al., “Trends in-cell biology (Trends in-cell biology (Trends in-cell biology (Trends in-cell biology (Trends in-cell biology (Trends in-cell biology (Trends in-cell biology (Trends in-cell biology (Trends in-cell biology (Trends in-cell biology (Trends in-cell biology (
  • Non-Patent Document 19 Hershko A. et al., “Annual Review of Biochemistry”, 1998, 67th, p. 425-4
  • Non-Patent Document 20 Burger AM et al., “Eurpoean Journal of Cancer”, 2004, No. 40, No. 15, p. 22 17-2229.
  • Non-Patent Document 21 Anan T. et al., “Genes to Cells”, 1998, No. 3, No. 11, p. 751-763.
  • Non-Patent Document 22 Kumar S. et al., “Biochemical and Biophysical Research Communications”, 1992, No. 185, No. 3, p. 1155-1161.
  • Non-Patent Document 23 Scharschmidt E et al., “Molecular and Cellular Biology”, 2004, Vol. 24, No. 9, p. 3860-3873.
  • Non-Patent Document 24 Staub O. et al., “EMBO Journal J, 1996, Vol. 15, No. 10, p. 2371-2380.
  • Non-Patent Document 25 Jin Y. H. et al., “The Journal of Biological Chemistry”, 2004, No. 279, No. 28, p. 29409-29417.
  • Non-Patent Document 26 Komori, “Journal of Cellular Biochemistry”, 2005, Vol. 95, p. 445-453.
  • Non-Patent Document 27 Himeno, M. et al., “Journal of Bone and Mineral Research", 2002, 17th, p. 1297-1305.
  • Non-Patent Document 28 Hanai, J. I. et al., “The Journal of Biological Chemistry”, 1999, No. 274, p. 3 1577-31582.
  • Non-Patent Document 29 Komori, T., “Journal of Cellular Biochemistry”, 2002, 87th, p. 1-8.
  • Non-Patent Document 30 HTintut, Y.) et al., “The Journal of Biological Chemistry”, 1999, 274th p. 28875—28879.
  • Non-Patent Document 31 Zhao M. et al., “The Journal of Biological Chemistry”, 2003, No. 278, p. 279 39-27944.
  • Non-Patent Document 32 Garrett, L R. et al., “The Journal of Clinical Investigation”, 2003, 111, p. 1771—1782. .
  • Non-Patent Document 33 Yamashita, M. et al., “Cell”, 2005, 121, p. 101-113.
  • RUNX is a protein that acts as a transcription factor and plays an important role in normal differentiation and tumorigenesis. Abnormalities in RUNX cause, for example, abnormal differentiation and tumor formation. Therefore, by regulating the action of RUNX, it is possible to prevent and Z or treat diseases caused by abnormal RUNX.
  • An object of the present invention is to find and provide a protein that interacts with RUNX to regulate its action.
  • the subject of the present invention includes providing means for adjusting the action of RUNX.
  • the subject of the present invention includes providing a useful means for the prevention and Z or treatment of diseases caused by abnormalities in RUNX, such as cancer diseases. Means for solving the problem
  • NE DD4 which is a HECT-type E3 ligase, interacts with RUNX3.
  • NEDD4 ubiquitinated RUNX, thereby reducing the stability of RUNX.
  • D NEDD4 binds to RUNX3 intracellularly
  • NEDD4 binds to RUNX1 in cells
  • NEDD4 reduces RUNX1 stability.
  • NEDD4 binds to RUNX to catalyze the ubiquitination of RUNX, and as a result, the ubiquitinated RUNX is degraded by the ubiquitin proteasome system and its stability decreases Can do.
  • RUNX degradation is regulated by the ubiquitin proteasome system involving NEDD4, thereby regulating the function of RUNX, for example, as a transcription factor, resulting in various physiological phenomena involving RUNX, such as the cell cycle. And signal transduction are regulated.
  • inhibition of RUNX ubiquitination by NEDD4 can inhibit degradation of RUNX by the ubiquitin proteasome system, thereby inhibiting reduction of RUNX.
  • the physiological phenomena involved in RUNX can be recovered, so that diseases caused by the reduction of RUNX and its function can be prevented and / or treated.
  • by promoting RUNX ubiquitin by NEDD4 degradation of RUNX by the ubiquitin proteasome system can be promoted, so RUNX can be reduced.
  • physiological phenomena involving RUNX can be inhibited, so that diseases caused by increased RUNX or enhanced function can be prevented and / or treated.
  • NEDD4 or short-chain NEDD4 also binds to RUNX to catalyze the ubiquitination of RU NX, whereas it binds to RUNX but does not have E3 ligase activity.
  • Type NEDD4 mutants and inactive short chain NEDD4 mutants have demonstrated that RUNX is not ubiquitinous.
  • the inactive NEDD4 mutant and the inactive short NEDD4 mutant all bind to RUNX but have no E3 ligase activity.
  • the inventors believe that antagonistic inhibition of binding between NEDD4 or short-chain NEDD4 and RUNX results in inhibition of RUNX ubiquitination by NEDD4 or short-chain NEDD4.
  • NEDD4 E3 ligase activity is important for RUNX ubiquitination by NEDD4, and it binds to RUNX but does not have E3 ligase activity! /, NEDD4 inactive mutant Revealed that RUNX ubiquitin can be inhibited.
  • a double-stranded polynucleotide capable of inhibiting the expression of NEDD4 was identified.
  • the double-stranded polynucleotide is specifically a double-stranded RNA having a polynucleotide strength consisting of a polynucleotide having a partial base sequence ability of a polynucleotide encoding NEDD4 and a complementary base sequence of the partial base sequence, Reduced the expression of NEDD4.
  • NEDD4 siRNA such double-stranded RNA is sometimes referred to as NEDD4 siRNA or Nedd4 siRNA.
  • NEDD4 inactive mutant that binds to a double-stranded polynucleotide and RUNX that can inhibit NEDD4 expression but does not have E3 ligase activity. It was found that the model cell promotes bone breakdown by BMP stimulation. Based on this, the present inventors believe that inhibiting the expression and Z or function of NEDD4 promotes bone differentiation and, as a result, promotes bone formation.
  • the present invention relates to a RUNX ubiquity method characterized in that RUNX and NEDD4 coexist.
  • the present invention also relates to the above-mentioned RUNX ubiquitination method, wherein RUNX is any one selected from human RUNX1, human RUNX2 and human RUNX3.
  • the present invention relates to a RUNX ubiquitinating agent comprising NEDD4.
  • RUNX is derived from human RUNX1, human RUNX2, and human RUNX3.
  • the RUNX ubiquitinating agent is any one selected.
  • the present invention uses the RUNX ubiquitin method described above.
  • the present invention relates to a method for decomposing RUNX, characterized in that RUNX is processed using the RUNX ubiquitinating agent.
  • the present invention relates to a decomposing agent for RUNX comprising NEDD4.
  • the present invention also relates to a method for inhibiting RUNX ubiquitin, which comprises inhibiting the binding of RUNX and NEDD4.
  • the present invention uses the protein represented by the amino acid sequence set forth in SEQ ID NO: 11 of the sequence listing and the protein represented by Z or the amino acid sequence set forth in SEQ ID NO: 12 above.
  • the present invention relates to a method for inhibiting RUNX ubiquitination.
  • the present invention relates to a method for inhibiting RUNX ubiquitin ⁇ , which comprises inhibiting the enzyme activity of NEDD4.
  • the present invention also relates to a RUNX ubiquitin-inhibiting method characterized by inhibiting the expression of NEDD4.
  • the present invention relates to a RUNX ubiquitin-inhibiting method characterized by using a double-stranded polynucleotide capable of inhibiting NEDD4 expression.
  • the present invention relates to a method of inhibiting RUNX ubiquitin ⁇ characterized by using any one double-stranded polynucleotide selected from the following group:
  • a double-stranded polynucleotide comprising a polynucleotide represented by the base sequence set forth in SEQ ID NO: 21 and a polynucleotide represented by the base sequence set forth in SEQ ID NO: 22,
  • a double-stranded polynucleotide comprising a polynucleotide represented by the base sequence set forth in SEQ ID NO: 23 and a polynucleotide represented by the base sequence set forth in SEQ ID NO: 24, and
  • a double-stranded polynucleotide comprising a polynucleotide represented by the base sequence set forth in SEQ ID NO: 25 and a polynucleotide represented by the base sequence set forth in SEQ ID NO: 26.
  • the present invention also relates to any one of the aforementioned RUNX ubiquitination inhibiting methods, wherein RUNX is any force 1 selected from human RUNX1, human RUNX2 and human RUNX3.
  • the present invention provides a partial base sequence of a polynucleotide encoding NEDD4, wherein The present invention relates to a polynucleotide having any one base sequence ability selected from the base sequences described in column numbers 21 to 26.
  • the present invention provides a double-stranded polynucleotide comprising a polynucleotide having a partial base sequence ability of a polynucleotide encoding NEDD4, and a polynucleotide having a complementary base sequence ability of the partial base sequence, With respect to any one double-stranded polynucleotide selected from the group of:
  • a double-stranded polynucleotide comprising a polynucleotide represented by the base sequence set forth in SEQ ID NO: 21 and a polynucleotide represented by the base sequence set forth in SEQ ID NO: 22,
  • a double-stranded polynucleotide comprising a polynucleotide represented by the base sequence set forth in SEQ ID NO: 23 and a polynucleotide represented by the base sequence set forth in SEQ ID NO: 24, and
  • a double-stranded polynucleotide comprising a polynucleotide represented by the base sequence set forth in SEQ ID NO: 25 and a polynucleotide represented by the base sequence set forth in SEQ ID NO: 26.
  • the present invention provides a protein represented by the amino acid sequence set forth in SEQ ID NO: 11 in the sequence listing, and a protein represented by Z or a protein represented by the amino acid sequence set forth in SEQ ID NO: 12.
  • the present invention further comprises a double-stranded polynucleotide capable of inhibiting the expression of NEDD4.
  • the present invention relates to a RUNX ubiquitination inhibitor comprising the double-stranded polynucleotide.
  • the present invention also relates to the RUNX ubiquitination inhibitor, wherein RUNX is any one selected from human RUNX1, human RUNX2 and human RUNX3.
  • the present invention relates to a method for inhibiting RUNX degradation, characterized by using any one of the aforementioned methods for inhibiting RUNX ubiquitination.
  • the present invention relates to a method for inhibiting RUNX degradation, characterized by using the RUNX ubiquitination inhibitor.
  • the present invention also includes a protein represented by the amino acid sequence set forth in SEQ ID NO: 11 in the sequence listing, and a protein represented by Z or a protein represented by the amino acid sequence set forth in SEQ ID NO: 12.
  • the present invention further comprises a double-stranded polynucleotide capable of inhibiting the expression of NEDD4.
  • the present invention relates to a RUNX degradation inhibitor comprising the double-stranded polynucleotide.
  • the present invention also relates to a method for promoting osteogenesis, which comprises inhibiting the expression and Z or function of NEDD4.
  • the present invention uses a protein represented by the amino acid sequence set forth in SEQ ID NO: 11 of the sequence listing and a protein represented by Z or the amino acid sequence set forth in SEQ ID NO: 12
  • the present invention relates to a formation promotion method.
  • the present invention relates to a method for promoting osteogenesis, comprising using a double-stranded polynucleotide capable of inhibiting the expression of NEDD4.
  • the present invention also relates to a method for promoting osteogenesis, comprising using the double-stranded polynucleotide.
  • the present invention provides a bone formation promoter comprising a protein represented by the amino acid sequence set forth in SEQ ID NO: 11 of the sequence listing and a protein represented by Z or the amino acid sequence set forth in SEQ ID NO: 12. About.
  • the present invention relates to an osteogenesis promoter comprising a double-stranded polynucleotide capable of inhibiting NEDD4 expression.
  • the present invention also relates to an osteogenesis promoter comprising the double-stranded polynucleotide.
  • the present invention relates to a method for suppressing tumor growth, which comprises inhibiting the expression and Z or function of NEDD4.
  • the present invention is characterized by using a protein represented by the amino acid sequence set forth in SEQ ID NO: 11 of the sequence listing and a protein represented by Z or the amino acid sequence set forth in SEQ ID NO: 12.
  • the present invention relates to a method for inhibiting tumor growth.
  • the present invention also relates to a method for inhibiting tumor growth, comprising using a double-stranded polynucleotide capable of inhibiting NEDD4 expression.
  • the present invention relates to a method for inhibiting tumor growth, characterized by using the double-stranded polynucleotide.
  • the present invention provides a tumor growth inhibitor comprising a protein represented by the amino acid sequence represented by SEQ ID NO: 11 in the sequence listing and a protein represented by Z or the amino acid sequence represented by SEQ ID NO: 12. About.
  • the present invention also relates to a tumor growth inhibitor comprising a double-stranded polynucleotide capable of inhibiting NEDD4 expression.
  • the present invention relates to a tumor growth inhibitor comprising the double-stranded polynucleotide.
  • the present invention provides a method for identifying a compound that inhibits or promotes RUNX ubiquitin by NEDD4, comprising contacting NEDD4 and Z or RUNX with a compound (test compound), Using a system that uses a signal that detects RUNX ubiquitin by NEDD4 and a system that uses Z or a marker, and detects the presence or absence or change of this signal and Z or marker, the test compound is The present invention relates to an identification method including a step of determining whether or not to inhibit or promote RUNX ubiquitin.
  • the present invention also relates to a method for identifying a compound that inhibits or promotes the binding of NEDD4 and RUNX, wherein NEDD4 and Z or RUNX are contacted with a compound, and then! The ability to inhibit the binding of NEDD4 and RUNX by detecting the presence or absence or change of the signal and Z or marker using a system that uses the signal and Z or marker resulting from binding.
  • the present invention relates to an identification method including a step of determining whether or not to promote power.
  • the present invention relates to a method for identifying a compound capable of promoting bone formation, which comprises the step of measuring whether or not a test compound has a force that inhibits the expression and Z or function of SNEDD4.
  • the present invention is a process power for measuring whether a test compound is capable of inhibiting the expression and Z or function of NEDD4.
  • the bone formation is any one process selected from the following group: Relates to methods for identifying compounds that can promote:
  • NEDD4 and Z or RUNX is brought into contact with the test compound, and then the presence or absence of the signal and Z or the marker using a system using the signal and Z or marker generated by the binding of NEDD4 and RUNX Alternatively, a step of determining whether or not the test compound is capable of inhibiting the binding of NEDD4 and RUNX by detecting a change.
  • the present invention further includes the step of measuring whether or not a test compound that has been shown to inhibit the expression and Z or function of NEDD4 can promote bone formation.
  • the present invention relates to a method for identifying a compound that can be promoted.
  • the present invention relates to a method for identifying a compound capable of suppressing tumor growth, which comprises a step of measuring whether or not a test compound is capable of inhibiting the expression and Z or function of NEDD4.
  • the present invention is a process power for measuring whether or not a test compound is capable of inhibiting the expression and Z or function of NEDD4.
  • the tumor growth is any one process selected from the following group: Relates to a method for identifying compounds capable of inhibiting
  • test compound (D. The step of determining whether or not the test compound is capable of inhibiting the expression of NEDD4 by contacting the test compound with a polynucleotide encoding D NEDD4 and then measuring NED D4,
  • NEDD4 and Z or RUNX are brought into contact with the test compound, and then the presence or absence of the signal and Z or marker using a system using the signal and Z or marker generated by binding of NEDD4 and RUNX Alternatively, a step of determining whether or not the test compound is capable of inhibiting the binding of NEDD4 and RUNX by detecting a change.
  • the present invention further comprises the step of measuring whether or not a test compound that has been shown to inhibit the expression and Z or function of NEDD4 can suppress tumor growth.
  • the present invention relates to a method for identifying a compound that can be produced.
  • the present invention relates to NEDD4, a polynucleotide encoding NEDD4, a recombinant vector containing the polynucleotide and a transformant containing the recombinant vector, and at least one of RUNX, RUNX And a reagent kit containing at least one of a recombinant vector containing the polynucleotide and a transformant containing the recombinant vector.
  • the present invention provides a preventive and Z or therapeutic agent for a disease caused by increased RUNX function and Z or expression, comprising an effective amount of the RUNX ubiquitinating agent and Z or the RUNX degrading agent. About.
  • the present invention also relates to prevention and Z or treatment of a disease caused by a decrease in RUNX function and Z or expression, comprising an effective amount of the RUNX ubiquitination inhibitor and Z or the RUNX degradation inhibitor. It relates to the agent.
  • the present invention uses the method or agent of at least one of the RUNX ubiquitination method, the RUNX ubiquitinating agent, the RUNX decomposition method, and the RUNX decomposition agent.
  • the present invention relates to a method for the prevention and Z or treatment of diseases caused by RUNX function and increased Z or expression.
  • the present invention provides at least any one of the RUNX ubiquitination inhibiting method, the RU NX ubiquitination inhibitor, the RUNX degradation inhibiting method, and the RUNX degradation inhibitor.
  • RU characterized by using any method or agent
  • the present invention relates to a method for preventing and / or treating a disease caused by decreased function or Z or expression of NX.
  • the present invention also relates to a preventive and Z or therapeutic agent for diseases associated with bone loss, comprising an effective amount of the osteogenesis promoter.
  • the present invention relates to prevention and Z or treatment of a disease accompanied by bone loss, characterized by using at least one agent or method of the osteogenesis promoting agent and the osteogenesis promoting method. Regarding the method.
  • the present invention relates to a preventive and Z or therapeutic agent for cancer diseases comprising an effective amount of the tumor growth inhibitor.
  • the present invention also relates to a method for the prevention and Z or treatment of cancer diseases, characterized by using at least one agent or method of the tumor growth inhibitor and the tumor growth inhibition method. .
  • a RUNX ubiquitin method and a decomposition method can be provided.
  • a RUNX ubiquitination method and decomposition method characterized by coexistence of RUNX and NEDD4 can be provided. It can also provide RUNX ubiquitinating and degrading agents.
  • the present invention can provide a method for inhibiting RUNX ubiquitination and degradation by NEDD4.
  • a method for inhibiting RUNX ubiquitination and degradation which comprises inhibiting at least one of binding of NEDD4 and RUNX, enzyme activity of NEDD4, and expression of NE DD4.
  • an inhibitor of RUNX ubiquitin by NEDD4 and an inhibitor of RUNX degradation by NEDD4 can be provided. Further, it is possible to provide a method for identifying a compound that inhibits or promotes RUNX ubiquitin by NED D4, and a method for identifying a compound that inhibits or promotes the binding of NEDD4 and RUNX.
  • NEDD4 a polynucleotide encoding NEDD4, a vector containing the polynucleotide, and at least one of the transformants containing the vector, a polynucleotide encoding RUNX, RUNX, and the polynucleotide
  • a reagent kit comprising at least one of a vector containing the vector and a transformant containing the vector.
  • a preventive and Z or therapeutic agent for cancer diseases and the like, and a preventive and Z or therapeutic method can be provided.
  • RUNX ubiquitination can be performed using, for example, NEDD4.
  • RUNX ubiquitin by NEDD4 can be promoted using compounds obtained by the method of identifying compounds that promote RUNX ubiquitin by NEDD4.
  • degradation of RUNX can be promoted. Therefore, prevention and Z or treatment of diseases caused by increased RUNX and enhanced function can be expected.
  • RUNX ubiquitination by NEDD4 for example, by inhibiting the binding of NEDD4 to RUNX using an inactive NEDD4 mutant that binds to RUNX but has no E3 ligase activity, Can inhibit.
  • RUNX ubiquitin caused by NEDD4 can be inhibited using a compound obtained by the method for identifying a compound that inhibits RUNX ubiquitin caused by NEDD4.
  • By inhibiting RUNX ubiquitination by NEDD4 degradation of RUNX can be inhibited. Therefore, prevention and Z or treatment of diseases caused by reduction of RUNX and its function can be expected.
  • the ability to bind bone formation for example, bone formation caused by BMP-2 stimulation, for example, the ability to bind to RUNX 3 ⁇ 43 Inactive NEDD4 mutant that does not have ligase activity, or a duplex that can inhibit the expression of NEDD4 It can be facilitated using polynucleotides.
  • bone formation for example, bone formation by BMP-2 stimulation, can be promoted using the compound obtained by the method for identifying a compound capable of promoting bone formation according to the present invention.
  • tumor growth for example, inactive NEDD4 mutants that bind to RUNX but have E3 ligase activity, or double-stranded polynucleotides that can inhibit the expression of NEDD4 can be suppressed using tides.
  • tumor growth can be suppressed using a compound obtained by the method for identifying a compound capable of suppressing tumor growth according to the present invention. By using these compounds capable of suppressing tumor growth, prevention and Z or treatment of diseases associated with tumor growth, such as cancer diseases, can be expected.
  • RUNX ubiquitination by NEDD4 or short-chain NEDD4 degradation of RUNX can be regulated, and as a result, RUNX functions, for example, functions as transcription factors can be regulated.
  • RUNX functions for example, functions as transcription factors
  • prevention and Z or treatment of diseases caused by abnormal RUNX can be expected.
  • bone formation can be promoted, and prevention and Z or treatment of diseases associated with bone loss can be expected.
  • tumor growth can be suppressed, and prevention and Z or treatment of diseases associated with tumor growth such as cancer diseases can be expected.
  • FIG. 1 is a diagram showing the results of in silico prediction of the interaction between RUNX3 and NEDD4. A local alignment was done between RUNX3 and NEDD4, and the area showing the high! And score was shown.
  • the amino acid sequence is represented by one letter. The numbers in the figure mean the position of the N-terminal amino acid in each region shown in the amino acid sequence of RUNX3 or NED D4. (Example 1)
  • FIG. Panels A and B show the results of immunoblotting using anti-Myc antibody and anti-FLAG antibody, respectively, for immunoprecipitates with anti-FLAG antibody.
  • + and 1 indicate the presence or absence of each expression plasmid
  • IP indicates a sample immunoprecipitated using anti-FLAG M2 antibody
  • cell lysate indicates a V-cell lysate sample that has not been immunoprecipitated.
  • the numbers listed in the left column of the figure are the molecular weights of the molecular weight markers. Only in samples prepared from cells co-expressing Myc-NEDD4 and FLAG-RUNX3, coprecipitation of Myc-NEDD4 and FLAG-RUNX3 was observed by immunoprecipitation using anti-FLAG M2 antibody (Panel A). On the other hand, from FLAG—RUNX3 non-expressing cells No coprecipitation of Myc-NEDD4 was observed in the prepared sample. My C—NEDD4 expression was similar in both samples (Panel A). It was also confirmed that FLAG-RUNX3 expressed in the cells was recovered by anti-FLAG M2 antibody (panel B). (Example 2)
  • FIG. 3 In vivo ubiquitination of human RUNX3 by human NEDD4, immunoprecipitation using human cultured cells transiently co-expressing FLAG—RUN X3, Myc—NEDD4 and HA—ubiquitin It is a figure which shows the result examined by the method. Panels A and B show the results of immunoblotting using an anti-FLAG antibody and an anti-HA antibody, respectively, for the immunoprecipitates obtained from the anti-FLAG antibody. Panel C shows the results of immunoblotting using anti-Myc antibody for cell lysate. In the figure, + and indicate the presence or absence of each expression plasmid. The numbers listed in the left column are the molecular weight markers.
  • FIG. 4 Results of Western blotting on the effects of human NEDD4 on the stability of human RUNX3 using human cultured cells in which FLAG-RUNX3 and Myc-NEDD4 were transiently co-expressed.
  • FIG. Panels A, B and C show the results of immunoblotting using anti-FLAG antibody, anti-Myc antibody and anti-actin antibody, respectively.
  • + and 1 indicate the presence or absence of each expression plasmid, and the number indicates the amount of DNA introduced.
  • the numbers listed in the right column of the figure are the molecular weights of the molecular weight markers. is there.
  • FIG. 5 Human NEDD4 binding to human RUNX1 and human NEDD4 in vitro ubiquitin 4 using human cultured cells transiently co-expressing FLAG—RUNX1, Myc—NEDD4 and HA—ubiquitin. It is a figure which shows the result examined by the immunoprecipitation method. Panels A, B and C show the results of immunoblotting with anti-Myc antibody, anti-FLAG antibody and anti-HA antibody, respectively, for immunoprecipitates with anti-FLAG antibody. Panel D shows the results of immuno plotting of cell lysate with anti-Myc antibody. In the figure, + and-indicate the presence or absence of each expression plasmid.
  • the numerical value described in the left column of the figure is the molecular weight of the molecular weight marker.
  • Myc—NEDD4 and FLAG—RUNX1 co-precipitated in samples prepared from cells co-expressed with Myc-NED D4, FLAG—RUNX1 and HA—Ub (panel A), higher molecular weight than FLAG—RUNX1 (Panel B) and an increase in FLAG-RUNX1 with HA-Ub attached (Panel C).
  • Myc—NEDD4 (C967A), FLAG—RUNX1 and HA which have inactivated E3 ligase activity.
  • FIG. Panels A, B and C show the results of immunoblotting using anti-FLAG antibody, anti-Myc antibody and anti-actin antibody, respectively.
  • + and 1 indicate the presence or absence of each expression plasmid, and the number indicates the amount of DNA introduced.
  • FIG. 7 The size of endogenous NEDD4 detected in human cancer cell lines was compared with human NEDD4 transiently expressed in human cell lines and short-chain human NEDD4 by Western blotting. It is a figure which shows a result.
  • Panel A shows HEK293T cells expressing short-chain NEDD4 (lane 1), short-chain NEDD4 (C867A) with inactivated E3 ligase activity (lane 2), or Myc—NEDD4 (lane 3).
  • the results of immunoblotting using an anti-NEDD4 antibody are shown for cell lysate and human breast cancer cell line T-4 7D cell lysate (lane 4).
  • Panel B shows the results of immunoblotting using anti-NEDD4 antibody for cell lysates from various human cancer cell lines.
  • the numbers in the left column of the figure are the molecular weights of the molecular weight markers. (Example 7)
  • FIG. 4 is a diagram showing the results of examination by immunoprecipitation using cultured human cells in which ubiquitin is transiently co-expressed.
  • Panels A, B and C show anti-Myc antibody, anti-FLAG antibody and anti-HA anti-HA antibody anti-FLAG antibody immunoprecipitates, respectively. The result of having performed immunoblotting by a body is shown.
  • Panel D shows the results of immunoblotting with anti-Myc antibody for cell lysate.
  • + and 1 indicate the presence or absence of each expression plasmid.
  • the numbers listed in the left column of the figure are the molecular weights of the molecular weight markers.
  • Myc—short chain type NEDD4, FLAG—RUNX1 and HA—Ub were co-expressed in samples prepared from cells co-precipitated with Myc—short chain type NEDD4 and FLAG—RUNX1 (Panel A).
  • Multiple proteins with a molecular weight higher than RUNX1 were detected (Panel B), and an increase in FLAG—RUNX1 with HA—Ub added was observed (Panel C).
  • FIG. 5 is a diagram showing the results of examination by immunoprecipitation using human cultured cells in which HA-ubiquitin is transiently co-expressed.
  • Panels A, B and C show the results of immunoblotting with anti-Myc antibody, anti-FLAG antibody and anti-HA antibody, respectively, for the immunoprecipitates with anti-FLAG antibody.
  • Panel D shows the results of immunoblotting with anti-Myc antibody for cell lysate.
  • + and 1 indicate the presence or absence of each expression plasmid.
  • the numbers listed in the left column of the figure are the molecular weights of the molecular weight markers.
  • FIG. 6 is a view showing the results of examination by immunoprecipitation using cultured human cells.
  • Panel A, Panel B and Panel C show the results of immunoblotting with anti-Myc antibody, anti-FLAG antibody and anti-HA antibody, respectively, for the immunoprecipitates with anti-FLAG antibody.
  • Panel D shows the results of immunoblotting with anti-Myc antibody for cell lysate.
  • + and 1 indicate the presence or absence of each expression plasmid, respectively.
  • the numbers in the left column of the figure are the molecular weight markers.
  • Samples prepared from cells co-expressed with Myc short-chain NEDD4, FLAG-RUNX2 and HA-Ub showed coprecipitation of Myc short-chain NEDD4 and FLAG-RUNX2 (Panel A).
  • Several proteins with higher molecular weight than FLAG—RUNX2 were detected (Panel B), and increased FLAG—RUNX2 with HA—Ub added (Panel C).
  • samples prepared from cells co-expressed with Myc-short-chain NE DD4 (C867A), FLAG-RUNX2 and HA-Ub co-expressed with E3 ligase activity were inactive.
  • FIG. 11 In mouse C2C12 cells, short-chain human NE DD4 (C867A) with inactivated E3 ligase activity enhances alkaline phosphatase (ALP) activity under BMP-2 stimulation.
  • FIG. An empty vector (Empty vector), short-chain human NEDD4 expression plasmid or short-chain human NEDD4 (C867A) expression plasmid was introduced into cells, cultured for 3 days under 300 ⁇ gZml BMP-2 stimulation, and ALP activity in the cells was measured. Each data shows the relative value of ALP activity in BMP-2 untreated empty vector-introduced cells (mean value SD, n 6).
  • short human NEDD4 and short human NEDD4 are simply indicated as NEDD4 and NEDD4 (C867A), respectively.
  • ALP activity in BMP 2 treated short-chain human NEDD4 (C867A) expression plasmid-introduced cells and ALP activity in BMP 2-treated empty vector-introduced cells (*: P ⁇ 0. 05).
  • Nedd4 knockdown by mouse Nedd4 siRNA inhibited the expression of endogenous Nedd4 (Panel B), and as a result, ALP activity of the cells was enhanced under BMP-2 stimulation.
  • Nedd4 knockdown effect by mouse Nedd4 siRNA was evaluated by Western blotting (Panel B). Intensity indicates the relative value of the concentration of Nedd4 band detected in each cell with respect to the concentration of Nedd4 band detected in BMP-2 untreated negative control siRNA-introduced cells. When calculating the relative value, it is detected in each cell. The concentration of Nedd4 band was corrected by the concentration of Actin band. (Example 11)
  • F-test statistical analysis using Student's t-test or Welch's t-test showed that the negative control siRNA treatment group and the human NEDD4 siRNA treatment group Significant differences were observed (*: p ⁇ 0.05).
  • the endogenous NEDD4 knockdown effect of human NEDD4 siRNA was evaluated by Western blotting (panel)
  • Intensity in the figure represents the relative value of the concentration of the human NEDD4 band detected in the human NEDD4 siRNA treatment group relative to the concentration of the human NEDD4 band detected in the negative control siRNA treatment group.
  • protein As used herein generically, it means an isolated or synthetic full-length protein; an isolated or synthetic full-length polypeptide; or an isolated or synthetic full-length oligopeptide.
  • protein may be used.
  • the protein, polypeptide or oligopeptide includes two or more amino acids linked to each other by peptide bonds or modified peptide bonds. In the following, amino acids may be represented by one or three letters.
  • isolated full length DNA and Z or RNA synthetic full length DNA and Z or RNA; isolated DNA and Z or RNA oligonucleotides; or synthetic DNA and Z or RNA
  • polynucleotide is sometimes used as a generic term to refer to RNA oligonucleotides. Where such DNA and Z or RNA have a minimum size of 2 nucleotides
  • RUNX By regulating the degradation of RUNX by the ubiquitin proteasome system involving NEDD4, the functions of RUNX, for example, functions as a transcription factor, are regulated. As a result, various physiological phenomena involving RUNX, For example, the cell cycle and signal transduction are regulated.
  • RUNX ubiquitination by NEDD4 by regulating RUNX ubiquitination by NEDD4, degradation of RUNX can be regulated, thereby regulating the function of RUNX, for example, as a transcription factor.
  • RUNX By regulating the function of RUNX, diseases caused by abnormal RUNX can be prevented and Z or treated.
  • Ubiquitination means modification of a protein by ubiquitin by covalently binding one or more ubiquitins to one molecule of protein. Ubiquitin is covalently bound to a lysine residue in the target protein. Similar to protein ubiquitination, multiple ubiquitins are bound to a protein in a chain by further covalently binding another ubiquitin to a lysine residue in ubiquitin bound to a lysine residue in the target protein.
  • Ubiquitin is a protein consisting of 76 amino acids that is universally present in eukaryotes.
  • Ubiquitin is linked to the target protein by covalent bonds in a chain in the ubiquitin proteasome system known as the proteolytic mechanism.
  • ubiquitin-active enzyme E1
  • ubiquitin-conjugating enzyme E2
  • ubiquitin ligase E3
  • ubiquitin ligase E3 is important as an enzyme responsible for substrate selectivity.
  • Ubiquitin ligase (E3) is also referred to as E3 ligase.
  • target protein means a protein to be ubiquitinated.
  • substrate means a molecule that is catalyzed by an enzyme.
  • the "ubiquitin proteasome system” is a selective and active proteolytic mechanism. Yes, it regulates various physiological phenomena such as cell cycle and signal transduction, and is involved in maintaining homeostasis of proteins and cells (Non-patent Document 19).
  • the ubiquitinated target protein is degraded by the proteasome that recognizes ubiquitin bound in a chain to the protein. Abnormalities in the ubiquitin proteasome system lead to protein deficiency due to excessive degradation of the target protein, or protein accumulation due to inhibition of target protein degradation, thereby causing various diseases. Specifically, for example, the involvement of the ubiquitin proteasome system in cancer diseases has been reported (Non-patent Document 20).
  • NEDD4 is a HECT-type E3 ligase involved in the ubiquitin proteasome system, a proteolytic mechanism. NEDD4 has a C2 domain that is involved in binding to membrane lipids, four WW domains that are involved in binding to substrates, and a HECT domain that is the catalytic domain of ubiquitin and exhibits E3 ligase activity (Non-patent Document 18). .
  • NEDD4 substrates include RUNX.
  • E3 ligases since many of E3 ligases have self-ubiquitin activity, self-protein can be mentioned as a substrate for NEDD4.
  • NEDD4 wild-type NEDD4 having E3 ligase activity.
  • E3 ligase activity refers to ubiquitin that recognizes a target protein as a substrate in ubiquitin protein, and is activated by ubiquitin activity enzyme (E1) and bound to ubiquitin-binding enzyme (E2). It means the action of binding to the target protein.
  • the E3 ligase activity of NEDD4 recognizes RUNX as a substrate and is activated by ubiquitin activating enzyme (E1). This is the action of NEDD4 that binds ubiquitin bound to) to RUNX.
  • the E3 ligase activity of NEDD4 specifically refers to, for example, self-protein ubiquitin, which recognizes self-protein as a substrate and is activated by ubiquitin-activating enzyme (E1). This is the action of NEDD4 that binds ubiquitin bound to) to self-proteins.
  • the measurement of E3 ligase activity can be performed using the binding of ubiquitin to the target protein by E3 ligase as an index.
  • the binding of ubiquitin to the target protein was ubiquitinated. It can be measured by detecting the target protein. Detection of the ubiquitinated target protein can be performed by a known method such as Western blotting (see Examples 3, 5, 8 and 9).
  • the measurement of the EDD ligase activity of NEDD4 can be specifically performed by, for example, using RUNX as a substrate and detecting RUNX that has been ubiquitinated.
  • RUNX as a substrate
  • self-protein can be used as a substrate and ubiquitinated self-protein can be detected.
  • NEDD4 is preferably a protein encoded by a human-derived polynucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 1, for example.
  • the protein encoded by the human-derived polynucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 1 is preferably a human-derived protein represented by the amino acid sequence set forth in SEQ ID NO: 2.
  • the amino acid sequence described in SEQ ID NO: 2 is registered as P46934 in the Swiss plot database.
  • NEDD4 is not limited to the above protein, has sequence homology with the protein encoded by the polynucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 1, and has the same structural features and the same as the protein. Any protein is included as long as it is a protein having a biological function.
  • the polynucleotide encoding NEDD4 is not limited to the above-mentioned polynucleotide, and has a sequence homology with the polynucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 1 and a protein encoded by the polynucleotide. As long as it is a polynucleotide encoding a protein having similar structural characteristics or biological functions, any polynucleotide is also included.
  • sequence homology is usually 50% or more of the entire amino acid sequence or base sequence, preferably at least 70%. More preferably, it is 70% or more, more preferably 80% or more, even more preferably 90% or more, and even more preferably 95% or more.
  • the protein having sequence homology with the protein encoded by the polynucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 1 contains one or more amino acid sequences in the amino acid sequence of the protein encoded by the polynucleotide For example, 1 to: L00, preferably 1 to 30, more preferably 1 to 20, more preferably 1 to 10, particularly preferably 1 to several amino acid residues. It includes proteins represented by amino acid sequences with mutations such as deletion, substitution, addition or insertion.
  • the polynucleotide having sequence homology with the polynucleotide represented by the nucleotide sequence shown in SEQ ID NO: 1 in the nucleotide sequence, one or more, for example, 1 to 300, preferably 1 to 90, Preferably, it includes a polynucleotide represented by a base sequence having a mutation such as deletion, substitution, addition or insertion of 1 to 60 nucleotides, more preferably 1 to 30, particularly preferably 1 to several nucleotides. It is.
  • the degree of mutation and the position thereof are the same as the protein encoded by the polynucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 1 in the protein having the mutation or the protein encoded by the polynucleotide having the mutation. As long as it has the following structural features and biological functions, it is not particularly limited. Proteins and polynucleotides having mutations may be naturally occurring or artificially introduced with mutations.
  • the structural features of the protein encoded by the polynucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 1 include, for example, the C2 domain involved in binding to membrane lipids, and the WW domain involved in binding to substrates It is a catalytic domain of ubiquitin and a HECT domain that exhibits E3 ligase activity.
  • the structural features similar to those of the protein encoded by the polynucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 1 are domains having sequence homology and similar functions to the above-described domain existing in the protein. Means.
  • the sequence homology of the domain is preferably at least 70%, more preferably 70% or more, even more preferably 80% or more, even more preferably 90% or more, and even more preferably 95% or more. It is.
  • Examples of the biological function of the protein encoded by the polynucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 1 include E3 ligase activity.
  • the biological function of this protein includes binding to RUNX.
  • Preferred examples of RUNX that binds to this protein include human-derived RUNX1, RUNX2, and RUNX3.
  • the RUNX that binds to the present protein is not limited to those exemplified, and can be any RUNX family protein as long as it binds to the present protein.
  • the E3 ligase activity of the protein to be examined can be measured by measuring the ubiquitination of the target protein of the E3 ligase, for example, RUNX. RUNX conversion to ubiquitin This measurement can be performed using the RUNX ubiquitin method described later.
  • the measurement of the binding between the protein to be examined and RUNX can be performed using a protein binding assay known per se. Specifically, the protein and RUNX are allowed to coexist in vivo or in vitro, and then complex formation of the protein and RUNX is performed by Western blotting, immunoprecipitation, pull-down, two-hybrid, and fluorescence resonance energy. The binding between the protein and RUNX can be measured by measuring using a known method such as the Luge-Issage transfer method.
  • a protein having sequence homology with the protein encoded by the polynucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 1 and having the same structural characteristics and biological function as the protein is:
  • a protein encoded by the polynucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 3 is preferable.
  • the protein encoded by the polynucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 3 is preferably a protein represented by the amino acid sequence set forth in SEQ ID NO: 4.
  • the nucleotide sequence set forth in SEQ ID NO: 3 and the amino acid sequence set forth in SEQ ID NO: 4 are registered in the NCBI database as accession numbers NM-0 06154 and NP-006145, respectively.
  • the protein encoded by the polynucleotide represented by the nucleotide sequence represented by SEQ ID NO: 3 is the N-terminal first protein of the protein encoded by the polynucleotide represented by the nucleotide sequence represented by SEQ ID NO: 1.
  • the second power is a protein from which the 100th amino acid residue at the 100th is deleted.
  • the protein encoded by the polynucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 3 is the N-terminal side first force of the protein represented by the amino acid sequence set forth in SEQ ID NO: 2 and the 100th 100 It is a protein in which one amino acid residue is deleted.
  • the protein encoded by the polynucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 3 may be referred to as "short-chain NEDD4".
  • a protein encoded by a polynucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 1 may be referred to as “full-length NEDD4J”.
  • Short-chain NEDD4J has a full-length NEDD4 with a deleted amino acid residue at the N-terminal side, but, like full-length NEDD4, has a C2 domain, a WW domain, and a HECT domain.
  • a protein that functions as a HECT-type E3 ligase in the present specification, when simply referred to as short-chain NEDD4, it means short-chain NEDD4 having E3 ligase activity.
  • RUNX is a protein characterized by having a Runt domain, forms a heterodimer with PEBP2 ⁇ / C BF
  • RUNX has a number of family proteins. In mammals, three types of family proteins are known, namely RUNX 1, RUNX2 and RUNX3 (Non-patent Documents 1 and 2). Homology between human RUNX3 and human RUNX1 and human RUNX2 is 57% and 54%, respectively, at the amino acid level.
  • RUNX is preferably RUNX1, RUNX2, and RUNX3.
  • RUNX is not limited to those exemplified, and may be a RUNX family protein as long as it is bound to NEDD4 and ubiquitinated.
  • RUNX1 is preferably a protein encoded by a human-derived polynucleotide represented by the base sequence described in SEQ ID NO: 5, for example.
  • the protein encoded by the human-derived polynucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 5 is preferably a human-derived protein represented by the amino acid sequence set forth in SEQ ID NO: 6.
  • the base sequence described in SEQ ID NO: 5 and the amino acid sequence described in SEQ ID NO: 6 are registered in the NCBI database as accession numbers NM-001754 and NP-001745, respectively.
  • RUNX2 is preferably a protein encoded by a human-derived polynucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 7, for example.
  • the protein encoded by the human-derived polynucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 7 is preferably a human-derived protein represented by the amino acid sequence set forth in SEQ ID NO: 8.
  • the base sequence described in SEQ ID NO: 7 and the amino acid sequence described in SEQ ID NO: 8 are registered in the NCBI database as accession numbers NM-004348 and NP-004339, respectively.
  • RUNX3 is preferably a protein encoded by a human-derived polynucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 9, for example.
  • the protein encoded by the human-derived polynucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 9 is preferably a human-derived protein represented by the amino acid sequence set forth in SEQ ID NO: 10.
  • the base sequence described in SEQ ID NO: 9 and the amino acid sequence described in SEQ ID NO: 10 are respectively stored in the NCBI database.
  • the session numbers are registered as NM-004350 and NP-004341.
  • RUNX is not limited to the above protein, and has sequence homology with the protein encoded by the polynucleotide represented by the nucleotide sequence set forth in any one of SEQ ID NOS: 5, 7, and 9. Any protein is included as long as the protein has the same structural characteristics and biological function as the protein.
  • the polynucleotide encoding RUNX is not limited to the above-mentioned polynucleotide, and has sequence homology with the polynucleotide represented by the nucleotide sequence of SEQ ID NO: 5, 7, and 9, and As long as the polynucleotide encodes a protein having the same structural characteristics and biological function as the protein encoded by the polynucleotide, any of the polynucleotides is also included.
  • sequence homology is usually 50% or more of the entire amino acid sequence or base sequence, preferably at least 70%. More preferably, it is 70% or more, more preferably 80% or more, even more preferably 90% or more, and even more preferably 95% or more.
  • a protein having sequence homology with the protein encoded by the polynucleotide represented by the nucleotide sequence set forth in any one of SEQ ID NOs: 5, 7, and 9 includes a protein encoded by the polynucleotide. 1 or more, such as 1 to: LOO, preferably 1 to 30, more preferably 1 to 20, more preferably 1 to 10, particularly preferably 1 to several amino acid residues. Proteins represented by amino acid sequences in which mutations such as deletions, substitutions, additions or insertions exist are included.
  • polynucleotide having sequence homology with the polynucleotide represented by the nucleotide sequence described in any one of SEQ ID NOs: 5, 7, and 9, one or more, for example, 1 to 300, in the nucleotide sequence Preferably, it is represented by a nucleotide sequence having a mutation such as deletion, substitution, addition or insertion of 1 to 90, more preferably 1 to 60, still more preferably 1 to 30, and particularly preferably 1 to several nucleotides. Polynucleotides are included.
  • the degree of mutation and the position thereof are represented by the nucleotide sequence of SEQ ID NO: 5, 7, and 9, wherein the protein having the mutation or the protein encoded by the polynucleotide having the mutation is selected from
  • the protein is not particularly limited as long as it has the same structural characteristics and biological function as the protein encoded by the polynucleotide. Proteins and polynucleotides with mutations can be naturally occurring In addition, an artificially introduced mutation may be used.
  • the structural feature of the protein encoded by the polynucleotide represented by the nucleotide sequence set forth in any one of SEQ ID NOs: 5, 7, and 9 is, for example, the Runt domain.
  • the structural characteristics of the protein encoded by the polynucleotide represented by the nucleotide sequence set forth in any one of SEQ ID NOs: 5, 7, and 9 are similar to the Runt domain present in the protein and have the same sequence homology. This means a Runt domain with the following functions.
  • the sequence homology of the Runt domain is preferably at least 70%, more preferably 70% or more, further preferably 80% or more, more preferably 90% or more, and even more preferably 95% or more. It is.
  • Examples of the biological function of the protein encoded by the polynucleotide represented by the nucleotide sequence set forth in any one of SEQ ID NOs: 5, 7, and 9 include transcription factor activity.
  • Transcription factor activity refers to a transcription device that synthesizes RNA from DNA, recognizes and binds to a characteristic nucleotide sequence in the DNA, and directly or indirectly (in eukaryotes). This means the action of regulating transcription positively or negatively (including basic transcription factors).
  • the transcription factor activity can be measured using a known transcription activity measurement method.
  • RUN X forms a dimer with PEBP2 ⁇ / CBF ⁇ and is involved in the expression of various genes as a transcription factor (Non-patent Document 2).
  • RUNX transcription factor activity can be measured, for example, by creating a vector in which a reporter gene is linked in place of the gene downstream of the promoter or enhancer site of the gene in which the dimer acts as a transcription factor. It can be carried out by contacting RUNX with an excised cell such as a eukaryotic cell and measuring the presence or absence and change of the reporter gene.
  • a reporter gene a gene generally used in reporter assembly can be used.
  • a gene having an enzyme activity such as luciferase, 13 galactosidase, or chloramphee-cholacetyl transferase can be used.
  • the expression of the reporter gene can be measured by detecting the gene product itself or the activity of the gene product.
  • the expression of a reporter gene as described above is up to the gene product itself. Alternatively, it can be measured by detecting the enzyme activity of the gene product.
  • One embodiment of the present invention relates to a RUNX ubiquitin method using NEDD4, and a RUNX decomposition method using the method.
  • one embodiment of the present invention is a RUNX ubiquitinating agent comprising NEDD4 and
  • one embodiment of the present invention relates to a method for decomposing RUNX, characterized by treating RUNX using a RUNX ubiquitinating agent.
  • the RUNX ubiquitin method characterized by using NEDD4 and the RUNX decomposition method characterized by using the method can be carried out by coexisting NEDD4 and RUNX.
  • Coexistence of NEDD4 and RUNX can preferably be performed in cells. Specifically, eukaryotic cells or cultured cell lines that have been found to express RUNX are used, and a vector containing a polynucleotide encoding NEDD4 is transfected into the cells or cell lines. By allowing NEDD4 and RUNX to coexist in a cell, a RUNX ubiquitination method and a RUNX degradation method characterized by using this method can be carried out (see Examples 3, 5, 8 and 9).
  • both the vector containing the polynucleotide encoding NEDD4 and the vector containing the polynucleotide encoding RUNX are transfected into the cells or cell lines.
  • a RUNX ubiquitination method and a RUNX degradation method characterized by using the method can be carried out.
  • NEDD4 and RUNX are combined as described above.
  • a vector containing a polynucleotide encoding ubiquitin can be further transfected into the expressed or expressed cells.
  • ubiquitin-active enzyme (E1), ubiquitin-binding enzyme (E2) and ubiquitin are required in addition to NEDD4 for the ubiquitination reaction of the target protein. Therefore, it is appropriate to use these enzymes and ubiquitin together with NEDD4.
  • proteasomes can also be prepared with cellular power with proteasomes.
  • preferred cells for example, human erythrocytes can be used.
  • the proteanome can be prepared by the method of Emma Rich et al. (“The Journal of Biological Chemistry”, 2000, Vol. 275, p. 21140-21148). Yes! / ⁇ can also use a commercially available proteasome.
  • proteasomes can be purchased from AFFINITI Research Products Ltd.
  • Detection of ubiquitin can be measured by detection of a ubiquitinated target protein.
  • Detection of the ubiquitinated target protein can be performed by a known method such as Western blotting (see Examples 3, 5, 8 and 9).
  • a target protein having an increased molecular weight is detected after the ubiquitin reaction, compared to before the ubiquitin reaction of the target protein, it can be determined that the target protein has been ubiquitinated.
  • Detection of protein degradation can be performed by a known method such as Western blotting (see Examples 4, 6, 8 and 9). It can be determined that the target protein has been degraded when the amount of the target protein is reduced after the ubiquitin reaction, compared to before the ubiquitin reaction of the target protein.
  • RUNX ubiquitinating agent and RUNX degrading agent are characterized by comprising NEDD4.
  • the RUNX ubiquitinating agent according to the present invention the RUNX ubiquitination method and the RUNX decomposition method can be carried out.
  • One embodiment of the present invention provides a method for inhibiting RUNX ubiquitin and uses the inhibition method
  • the present invention relates to a method for inhibiting RUNX degradation.
  • the method for inhibiting RUNX ubiquitin and the method for inhibiting degradation of RUNX, which is characterized by using the inhibition method, can be carried out under in vitro and in vitro conditions.
  • one embodiment of the present invention relates to a RUNX ubiquitin-inhibitor and a degradation inhibitor.
  • one embodiment of the present invention relates to a method for inhibiting RUNX degradation, which comprises using a RUNX ubiquitination inhibitor.
  • NEDD4 binds to RUNX to catalyze the ubiquitination of RUNX. It was found that the stability of the product is degraded by the degradation.
  • Inhibiting NEDD4's action on RUNX can be achieved by inhibiting NEDD4 expression and Z or function.
  • NEDD4 functions means the functions that NEDD4 has! As described above, NEDD4 functions as follows: NEDD4 enzyme activity, and NEDD4 and other proteins such as
  • Inhibiting NEDD4 function means reducing or eliminating the function of NEDD4. Inhibiting the function of NEDD4 can be exemplified by inhibiting the enzyme activity of NEDD4 or binding of NEDD4 to other proteins such as RUNX.
  • the method for inhibiting RUNX ubiquitination can be carried out by inhibiting at least one of the binding of RUNX and NEDD4, the enzyme activity of NEDD4, and the expression of NEDD4.
  • the method for inhibiting RUNX ubiquitination includes, for example, a compound that inhibits the binding of RUNX to NEDD4, a compound that inhibits the enzyme activity of NEDD4, and a compound that inhibits the expression of NEDD4. This can be done by using at least 1.
  • compounds having such an inhibitory effect for example, polypeptides having competitive inhibitory effects, antibodies, low-molecular compounds, etc. are referred to as inhibitors.
  • RUNX ubiquitination inhibitor and RUNX degradation inhibitor are compounds that inhibit the binding of RUNX and NEDD4, compounds that inhibit the enzyme activity of NEDD4, and compounds that inhibit the expression of NEDD 4 1 At least.
  • RUNX and NEDD4 Binding of RUNX and NEDD4 means that RUNX and NEDD4 interact with each other by a non-covalent bond such as a hydrogen bond, a hydrophobic bond, or an electrostatic interaction so as to form a complex. Means. In this case, it is sufficient that RUNX and NEDD4 are partly connected.
  • the amino acids that make up RUNX or NEDD4 may contain amino acids that are not involved in the binding of RUNX and NEDD4! /.
  • “Inhibiting the binding of RUNX and NEDD4” means reducing or eliminating the amount of the complex formed from RUNX and NEDD4.
  • the measurement of the binding between RUNX and NEDD4 can be carried out by a method known per se, such as Western blotting, immunoprecipitation, pull-down, two-hybrid, and fluorescence resonance energy transfer. These methods can be used in combination.
  • the compound that inhibits the binding of RUNX and NEDD4 is preferably a compound that specifically inhibits the binding, and more preferably a low molecular weight compound that specifically inhibits the binding.
  • a compound that specifically inhibits the binding is preferably a compound that specifically inhibits the binding, and more preferably a low molecular weight compound that specifically inhibits the binding.
  • To specifically inhibit the binding of RUNX and NEDD4 means that the binding is strongly inhibited, but the binding between other proteins is not inhibited or is weakly inhibited.
  • the compound that inhibits the binding of RUNX and NEDD4 can be, for example, an inactive mutant of NEDD4 (hereinafter sometimes referred to as inactive NEDD4).
  • inactive NEDD4 an inactive mutant of NEDD4 having the ability to bind to RUNX and having no E3 ligase activity can be used.
  • Such inactive NEDD4 can inhibit the action of NEDD4 on RUNX by binding to RUNX in competition with wild-type NEDD4. Therefore, such inactive NEDD4 can inhibit RUNX ubiquitination.
  • Inactive NEDD4 is designed by designing a desired protein based on the amino acid sequence of NEDD4 and producing it using a known method. It can obtain by sorting using the method of.
  • an "inactive mutant of NEDD4" is a NEDD4 mutant in which mutations such as amino acid deletion, substitution, addition or insertion have been introduced into NEDD4, compared to wild-type NEDD4. This means a NEDD4 mutant whose E3 ligase activity is attenuated or lost. Inactive NEDD4 may be naturally occurring or artificially mutated.
  • the mutation site in inactive NEDD4 is, for example, a site necessary for NEDD4 E3 ligase activity in the amino acid sequence of NEDD4.
  • this site is, for example, the 967th cysteine residue in the amino acid sequence set forth in SEQ ID NO: 2.
  • the cysteine residue corresponds to the 867th cysteine residue in the amino acid sequence shown in SEQ ID NO: 4.
  • the cysteine residue is an active site of E3 ligase activity to which ubiquitin binds, and is an amino acid residue essential for NEDD4 E3 ligase activity.
  • Inactive NEDD4 is preferably a protein represented by the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 12, for example.
  • the protein represented by the amino acid sequence shown in SEQ ID NO: 11 is a protein represented by the amino acid sequence in which the 967th cysteine residue is substituted with an alanine residue in the amino acid sequence shown in SEQ ID NO: 2.
  • the protein represented by the amino acid sequence shown in SEQ ID NO: 12 is a protein represented by the amino acid sequence in which the 867th cysteine residue is substituted with an alanine residue in the amino acid sequence shown in SEQ ID NO: 4 (hereinafter referred to as the amino acid sequence shown below).
  • inactive short chain NEDD4 is sometimes referred to as inactive short chain NEDD4.
  • the compound that inhibits the binding of RUNX and NEDD4 may be a polypeptide that also has an amino acid sequence at the site where RUNX and NEDD4 bind.
  • Such polypeptides are Binding between proteins can be competitively inhibited.
  • Such a polypeptide can be obtained by designing the amino acid sequence of RUNX or NEDD4 and selecting one that inhibits the binding of RUNX and NEDD4 from those synthesized by a peptide synthesis method known per se.
  • a polypeptide in which mutations such as deletion, substitution, addition or insertion of one to several amino acid residues are introduced into the identified polypeptide is also encompassed in the scope of the present invention.
  • the polypeptide into which such a mutation is introduced preferably inhibits the binding of RUNX and NEDD4.
  • Polypeptides having mutations may be naturally occurring or artificially introduced with mutations. These polypeptides can be obtained by a general production method described later.
  • the compound that inhibits the binding of RUNX and NEDD4 may be an antibody that recognizes RUNX or NEDD4, and an antibody that inhibits the binding of RUNX and NEDD4, or a fragment thereof.
  • an antibody can be obtained by a known antibody production method using RUNX or NEDD4 itself, or a fragment thereof, preferably a polypeptide consisting of the amino acid sequence of the site where RUNX and NEDD4 bind to each other as an antigen.
  • the compound that inhibits the binding of RUNX and NEDD4 may also be an aptamer that specifically recognizes RUNX or NEDD4 and inhibits the binding of RUNX and NEDD4.
  • the aptamer can be a nucleic acid aptamer or a peptide aptamer, and a powerful aptamer can be a known method (eg, Hermann T. et al., “Science”, 2000, 287th, No. 5454, p. 820-825; Burgstaller P. et al., “Current Opinion in Drug Discovery and Development”, 2002, V. 5, 690-700; and Hop pe-Seyler F. et al., "Current Molecular Medicine", 2004, IV, 5, p. 529-538. ) Can be obtained using the method described in).
  • Enzyme activity of NEDD4 means the E3 ligase activity of NEDD4.
  • “Inhibiting NEDD4 enzyme activity” means reducing or eliminating NEDD4 E3 ligase activity.
  • a compound that inhibits the enzyme activity of NEDD4 can be obtained using the compound identification method described below.
  • the compound that inhibits the enzyme activity of NEDD4 may be, for example, an antibody or a fragment thereof that inhibits the enzyme activity of NEDD4, and is an abutama that specifically binds to NEDD4 and has an action of inhibiting the activity.
  • You can also An antibody or aptamer can be obtained by the above-mentioned method. By measuring the inhibitory action of these enzyme activities of NEDD4 by the method described in the compound identification method described later, the action of inhibiting the enzyme activity of NEDD4 can be obtained. Antibody or abutama can be obtained.
  • NEDD4 "Expression of NEDD4" means that gene information of DNA encoding NEDD4 is transferred to mRNA, or is transcribed into mRNA and translated as the amino acid sequence of protein (NEDD4) Say.
  • NEDD4 expression means that the gene information of DNA encoding NEDD4 is transcribed into mRNA, or is transcribed into mRNA and translated as the amino acid sequence of protein (NEDD4) By interfering with at least one of the various reactions that occur during the process, the transcription of the NEDD4 gene means that the production of NEDD4 by translation is prevented.
  • a compound that inhibits the expression of NEDD4 can be obtained using the compound identification method described below.
  • the compound that inhibits the expression of NEDD4 can be, for example, an antisense oligonucleotide of a polynucleotide encoding NEDD4 or a double-stranded polynucleotide that can inhibit the expression of NEDD4.
  • a double-stranded polynucleotide capable of inhibiting the expression of NEDD4 has been identified.
  • this double-stranded polynucleotide is a double-stranded RNA that also has the power of a polynucleotide having a partial base sequence ability of a polynucleotide encoding NEDD4 and a polynucleotide comprising a complementary base sequence of the partial base sequence.
  • double-stranded polynucleotide capable of inhibiting the expression of NEDD4 include the following double-stranded polynucleotide: (i) a polynucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 21 A double-stranded polynucleotide comprising a nucleotide and a polynucleotide represented by the base sequence set forth in SEQ ID NO: 22, (ii) a polynucleotide represented by the base sequence set forth in SEQ ID NO: 23 and the base sequence set forth in SEQ ID NO: 24 (Iii) a polynucleotide represented by the base sequence set forth in SEQ ID NO: 25 And a double-stranded polynucleotide comprising the polynucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 26.
  • Each of these double-stranded polynucleotides is a double-stranded RNA comprising a polynucleotide consisting of a partial sequence of a polynucleotide encoding NEDD4 derived from human and a polynucleotide consisting of a complementary base sequence of the partial base sequence.
  • the double-stranded polynucleotide that can be used in the present invention is not limited to those exemplified above, and any double-stranded polynucleotide can be used as long as it is a double-stranded polynucleotide that can inhibit the expression of NEDD4. But it can be.
  • double-stranded polynucleotides that can inhibit NEDD4 expression include short double-stranded polynucleotides that can inhibit NED D4 expression by RNA interference, ie, siRNA (small interfering RNA) against NEDD4 ( Elbashir SM et al., “Nature”, 2001, 411, p. 494-498; and Pad dison PJ et al., “Genes and Development”, 2002 Year 16th, p. 948-958).
  • siRNA small interfering RNA
  • the siRNA for NEDD4 is composed of RNA consisting of a partial sequence of NEDD4 mRNA (sense RNA) and RNA consisting of a base sequence complementary to the base sequence of the RNA (antisense RNA) based on the sequence of NEDD4 mRNA.
  • a double-stranded RNA is produced by designing and synthesizing by a chemical synthesis method known per se, and making the obtained RNA noislative, and its intermediate force can also inhibit the expression of NEDD4. It can be acquired by selecting. Selection of siRNA that can inhibit NEDD4 expression involves transfecting the double-stranded RNA to be examined in cells expressing NEDD4, measuring the expression level of endogenous NEDD4, and inhibiting the expression level.
  • the sense RNA and the antisense RNA constituting the siRNA each consist of several or about ten or more nucleotides.
  • one or several nucleotide sequences called an overhang sequence are bound to the 3 ′ end of each nucleotide.
  • the overhang sequence has the effect of protecting RNA from nuclease power.
  • the overhang sequence is not particularly limited as long as it does not inhibit the RNA interference effect of the RNA, preferably 1 to 10, more preferably 1 to 4, more preferably 2 Any of those having nucleotide strength can be used.
  • a sequence that also includes deoxythymidylate eg, TT
  • a sequence that also includes uridylate eg, UU
  • a sequence that is linked to deoxythymidylate, followed by any nucleotide eg, TN
  • an array can be illustrated.
  • two deoxythymidylate sequences are used as overhang sequences because they can be synthesized inexpensively and are more resistant to nucleases.
  • the overhang sequence is bound to the ribose hydroxyl group at the 3 'end of each of sense RNA and antisense RNA by a diester bond.
  • shRNA is a short double-stranded RNA with a hairpin structure and, like siRNA, suppresses gene expression by RNA interference (Paddison PJ et al., “Genes and Development (Genes and Development). ”, 2002, 16th pp. 948-958).
  • shRNA has a hairpin-like structure because sense RNA and antisense RNA are linked by, for example, an oligonucleotide, and a sense RNA-derived portion and an antisense RNA-derived portion form a double strand.
  • shRNA is designed based on the nucleotide sequence of NEDD4 mRNA by designing RNA containing oligonucleotides that link these two RNAs and form a loop structure. Can be obtained by producing a short double-stranded RNA having a hairpin structure and selecting one that can inhibit the expression of NEDD4. The selection of shRNAs that can inhibit NEDD 4 expression is achieved by transfecting short double-stranded RNA with a hairpin structure that expresses NEDD4! It can be carried out by measuring the amount and selecting one that can inhibit the expression level.
  • oligonucleotide that forms a loop structure means an oligonucleotide that exists between a sense RNA and an antisense RNA and that can link both RNAs and itself forms a loop structure.
  • the design of such oligonucleotides is described in the literature (Paddison PJ et al., “Genes and Development”, 2002, Vol. 16, p.
  • RNA-derived portion 948-95. It can be implemented with reference to the description in 8). Preferably 4 to 23, more preferably 4 or 8 nucleotides are desirable. For example, sequences such as TTCAAGAGA (Ambion or Oligoengine), AACGTT, TTAA, CAAGCTTC and the like can be mentioned. Formation of a duplex having a hairpin structure can be carried out by annealing a sense RNA-derived portion and an antisense RNA-derived portion by a conventional method.
  • An embodiment of the present invention includes the double-stranded polynucleotide.
  • the following double-stranded polynucleotide can be exemplified: (i) consisting of a polynucleotide represented by the base sequence set forth in SEQ ID NO: 21 and a polynucleotide represented by the base sequence set forth in SEQ ID NO: 22 A heavy chain polynucleotide, (ii) a double-stranded polynucleotide comprising a polynucleotide represented by the base sequence set forth in SEQ ID NO: 23 and a polynucleotide represented by the base sequence set forth in SEQ ID NO: 24, and (iii) a sequence A polynucleotide represented by the nucleotide sequence represented by No. 25 and a double-stranded polynucleotide comprising the polynucleotide represented by the nucleotide sequence represented by SEQ ID No. 26
  • polypeptide constituting the double-stranded polynucleotide for example, a polynucleotide represented by the nucleotide sequence set forth in any one of SEQ ID NOs: 21 to 26. included.
  • the RUNX ubiquitin inhibition method and the RUNX degradation inhibition method according to the present invention are preferably performed using the NEDD4 inactive mutant and the double-stranded polynucleotide capable of inhibiting the expression of NEDD4. it can.
  • the method for inhibiting RUNX ubiquitination and the method for inhibiting RUNX degradation according to the present invention can be performed using at least one selected from the following proteins and double-stranded polynucleotides: SEQ ID NO: 1 A protein represented by the amino acid sequence described; a protein represented by the amino acid sequence represented by SEQ ID NO: 12; a polynucleotide represented by the base sequence represented by SEQ ID NO: 21 and the base sequence represented by SEQ ID NO: 22.
  • a double-stranded polynucleotide comprising the polynucleotide represented by the base sequence set forth in SEQ ID NO: 23 and a polynucleotide represented by the base sequence set forth in SEQ ID NO: 24; and A duplex comprising the polynucleotide represented by the base sequence set forth in SEQ ID NO: 25 and the polynucleotide represented by the base sequence set forth in SEQ ID NO: 26 Strand polynucleotide.
  • the RUNX ubiquitin inhibitor and the RUNX degradation inhibitor according to the present invention are preferably selected for the NEDD4 inactive mutant and the double-stranded polynucleotide force capable of inhibiting the expression of NEDD4.
  • the RUNX ubiquitin inhibitor and the RUNX degradation inhibitor according to the present invention comprise an effective amount of at least one selected from the following proteins and double-stranded polynucleotides: SEQ ID NO: 11 A protein represented by the amino acid sequence described; a protein represented by the amino acid sequence represented by SEQ ID NO: 12; a polynucleotide represented by the nucleotide sequence represented by SEQ ID NO: 21; and a base sequence represented by SEQ ID NO: 22.
  • a double-stranded polynucleotide comprising the polynucleotide represented; a polynucleotide represented by the base sequence set forth in SEQ ID NO: 23 and a polynucleotide represented by the polynucleotide represented by the base sequence set forth in SEQ ID NO: 24 And a duplex comprising the polynucleotide represented by the base sequence set forth in SEQ ID NO: 25 and the polynucleotide represented by the base sequence set forth in SEQ ID NO: 26 Li nucleotide.
  • the RUNX ubiquitination inhibition method and the RUNX degradation inhibition method can be carried out.
  • One aspect of the present invention relates to a pharmaceutical composition comprising an effective amount of the RUNX ubiquitinating agent according to the present invention and Z or the RUNX decomposing agent according to the present invention as active ingredients.
  • Another aspect of the present invention relates to a pharmaceutical composition comprising the RUNX ubiquitin inhibitor according to the present invention and Z or the RUNX degradation inhibitor according to the present invention as an effective ingredient.
  • a RUNX ubiquitinating agent according to the present invention a RUNX decomposing agent according to the present invention, and a pharmaceutical composition comprising the ubiquitinating agent and an effective amount of Z or the RUNX decomposing agent, In addition, it can be used as a preventive and Z or therapeutic agent for diseases caused by reduction of RUNX degradation by the ubiquitination.
  • These drugs and pharmaceutical compositions can be used to implement prevention and Z or treatment methods for such diseases.
  • the RUNX ubiquitin inhibitor according to the present invention, the RUNX degradation inhibitor according to the present invention, and the pharmaceutical composition comprising the ubiquitin inhibitor and Z or the RUNX degradation inhibitor in an effective amount, RUNX ubiquitination and RUNX by ubiquitination It can be used as a preventive and z or therapeutic agent for diseases caused by increased degradation of These drugs and pharmaceutical compositions can be used to implement prevention and Z or treatment methods for such diseases.
  • RUNX1 is involved in hematopoietic cell sorting and is a causative gene of leukemia (Non-patent Documents 4 and 5). Specifically, there are reports showing that loss of RUNX1 function is involved in the development of leukemia (Non-patent Documents 6 and 7). Based on this, the inventors believe that reduction of RUNX1 is involved in the onset and exacerbation of cancer diseases such as leukemia.
  • RUNX2 is an important transcription factor involved in bone formation, and is essential for chondrocyte differentiation 'maturation, osteoblast differentiation and bone marrow formation (Non-patent Documents 26 and 27). For example, since bone formation is not observed in RUNX2 knockout mice, it is considered that the defect causes bone formation failure (Non-patent Document 8). Since RUNX2 binds to the transcription factor Smadl, which is involved in the induction of bone morphogenetic gene expression by BMP stimulation, it is thought that RUNX2 is involved in bone formation signals via BMP stimulation (Non-Patent Documents). 28).
  • RU NX2 acts on the differentiation of bone cells, and also expresses bone matrix genes (Collal, Colla2, osteopontin, osteocalcin), bone sialoprotein, etc. Has been shown to be active (Non-patent Document 29). From these, the inventors believe that reduction of RUNX2 is associated with abnormal bone formation, such as bone loss.
  • osteogenesis model cells stimulated with BMP-2 are inactivated NEDD4 that binds to RUNX but does not have E3 ligase activity, or siRNA that can inhibit the expression of NEDD4.
  • NEDD4 expression and Z or function increases alkaline phosphatase activity, which is an indicator of bone formation (see Examples 10 and 11).
  • Bone-type alkaline phosphatase present in the osteoblastic membrane reflects early osteoblast activity, for example, when pre-osteoblasts differentiate into osteoblasts and then transition from the proliferative phase to the substrate synthesis phase. ing. That is, NEDD4 Inhibition of expression and Z or function can be thought to promote osteoblast ossification by BMP-2 stimulation.
  • BMP is a site force-in that induces bone tissue by differentiating and proliferating undifferentiated mesenchymal stem cells into chondrocytes and osteoblasts in vivo.
  • BMP-2 has been shown to be expressed early in the fracture healing process and is involved in the progression of a series of cascades in bone repair.
  • BMP-2 is applied to muscles, bone is formed ectopically, and when it is applied to the bone surface, callus-like osteogenesis occurs. Based on these facts, it is considered that BMP-2 is used to repair bone tissues such as fractures, bone defects, and root surgery (Chen, D. et al., “Growth Factor”). 2004, 22nd, 4th, p. 233-241).
  • osteoporosis is a disease in which the bone balance is unbalanced due to decreased osteoblast function and increased bone resorption, resulting in bone loss and increased fractures. Therefore, the enhancement of BMP-2 signal is It is thought to reinforce both the restoration of osteoblast function and the promotion of fracture healing for the disease.
  • inhibition of NEDD4 expression and Z or function increases the alkaline phosphatase activity of bone formation model cells stimulated with BMP-2, and, as described above, the short chain type.
  • NEDD4 and RUNX2 combined. Based on these findings, inhibition of NEDD4 expression and Z or function resulted in bowel I inhibition of RUNX2 ubiquitination by NEDD4 in RUNX2, which is involved in osteogenesis signals by BMP-2 stimulation. It can be considered that this promoted the osteogenic signal.
  • the bone differentiation of the bone formation model cell is promoted and its alkaline phosphatase activity is increased.
  • RUNX 2 ubiquitination by NEDD4 and degradation of RUNX2 by the ubiquitination can be inhibited, and RUNX2 can be stabilized.
  • osteogenesis signals involving RUNX2 can be promoted, and osteoblast differentiation and osteogenesis can be further promoted.
  • the inventors believe that by promoting osteoblast differentiation and bone formation in this way, it is possible to prevent and Z or treat abnormal bone formation, such as bone loss disease, more specifically osteoporosis, for example. ing.
  • One embodiment of the present invention based on this finding relates to an osteogenesis promoter characterized by inhibiting the expression and Z or function of NEDD4.
  • the osteogenesis promoter according to the present invention is preferably an osteogenesis promoter by RUNX, more preferably RUNX2.
  • the bone formation promoter according to the present invention comprises at least one of a compound that inhibits the function of NEDD4 and a compound that inhibits the expression of NEDD4.
  • the osteogenesis promoter according to the present invention is represented by an inactive NEDD4 that binds to RUNX but does not have E3 ligase activity, for example, the amino acid sequence set forth in SEQ ID NO: 11 in the Sequence Listing.
  • An osteogenesis promoter comprising an effective amount of a protein and a protein represented by Z or the amino acid sequence set forth in SEQ ID NO: 12.
  • the osteogenesis promoter according to the present invention may be an osteogenesis promoter comprising a double-stranded polynucleotide capable of inhibiting the expression of NEDD4.
  • Preferred examples of the double-stranded polynucleotide contained in the osteogenesis promoter according to the present invention include the following double-stranded polynucleotides: (i) a polynucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 21 A double-stranded polynucleotide comprising a nucleotide and a polynucleotide represented by the base sequence set forth in SEQ ID NO: 22; (ii) a polynucleotide represented by the base sequence set forth in SEQ ID NO: 23 and the base set forth in SEQ ID NO: 24 A double-stranded polynucleotide comprising a polynucleotide represented by the sequence; and (iii) a polynucleotide
  • the promotion of bone formation by these three types of double-stranded polynucleotides has not been studied.
  • these three types of double-stranded polynucleotides inhibited NEDD4 expression (see Example 12), and also derived from mouse-derived double-stranded polynucleotides (represented by the nucleotide sequence set forth in SEQ ID NO: 19).
  • Inhibition of NEDD4 expression using a polynucleotide and a polynucleotide represented by the nucleotide sequence of SEQ ID NO: 20 promoted alkaline phosphatase activity, an indicator of bone formation, in mouse cell lines (Example 10). Therefore, the inventors consider that the three types of double-stranded polynucleotides have a bone formation promoting action.
  • the osteogenesis promoting method of the present invention can be used to implement the osteogenesis promoting method.
  • One aspect of the present invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising an effective amount of the osteogenesis promoter according to the present invention as an active ingredient.
  • Such a pharmaceutical composition can be used as a preventive and Z or therapeutic agent for abnormal bone formation, such as bone loss diseases, more specifically osteoporosis.
  • These agents and pharmaceutical compositions can be used to implement prevention and Z or treatment methods for such diseases.
  • RUNX2 has also been reported that its overexpression causes abnormalities in T cell differentiation and is involved in lymphoma formation through synergistic action with c-myc (Non-patent Document 9). Based on this, the inventors think that increased RUNX2 calorie is involved in the development and exacerbation of lymphoma.
  • RUNX2 can be degraded by ubiquitinating RUNX2 with NEDD4, resulting in prevention and Z or treatment of lymphoma.
  • Non-patent Document 10 Since RUNX3 binds to Smad, an intracellular mediator of TGF- ⁇ , it is considered to be involved in TGF- ⁇ signaling (Non-patent Document 10). Mutations in the TGF- ⁇ receptor and Smad have been observed in many cancers (Non-patent document 11), and RUNX3 is thought to be involved in cancer via the TGF-
  • Non-patent Document 13 the tumor formation inhibitory effect of RUN X3 has been confirmed in animal experiments using a transplant model. From these, the inventors think that the reduction of RUNX3 is involved in the onset and exacerbation of cancer diseases such as gastric cancer, colon cancer, bile duct cancer, and spleen cancer.
  • RUNX2 has been reported to be involved in lymphoma formation through synergism with c-myc (Non-patent Document 9), while RUNX3 has been reported to be involved in tumor growth inhibition (Non-patent Document 9).
  • References 10-17 the extent of RUNX's involvement in tumorigenesis and tumor growth inhibition and its mechanism are not clear.
  • NEDD4 ubiquitinates RUNX but the difference in the degree of ubiquitin by NEDD4 for each RUNX family protein such as RUNX1, RUNX2, and RUNX3 is not clear.
  • RUNX is ubiquitinated by NEDD4. (See Examples 3, 5, 8 and 9), and in human uterine cervical cancer cell line HeLa and human gastric cancer cell line NCI-N87, using siRNA that can inhibit the expression of NEDD4, It was found that tumor growth was suppressed by inhibiting expression (see Example 12).
  • NEDD4 expression is increased at the mRNA level in several cancer cell lines, such as the human eclampsia cancer cell line HeLa, the human lung cancer cell line A549, and the human leukemia cell line K562! Has been reported (Non-patent Document 21). However, there are no reports suggesting an increase in the expression of NEDD4 in human clinical specimens or a link between NEDD4 and cancer diseases.
  • inhibition of NEDD4 expression suppresses tumor growth, and as described above, short-chain NEDD4 binds to RUNX, and RUNX force SNEDD4 causes ubiquitin secretion.
  • RUNX3 is thought to be involved in tumor growth inhibition.
  • the inventors believe that suppression of tumor growth by inhibiting the expression of NEDD4 may be related to the inhibition of RUNX3 ubiquitination by NEDD4 and stabilization of RUNX3. Therefore, the inventors think that tumor growth can be suppressed not only by the expression of NEDD4 but also by inhibiting the function of NEDD4.
  • One embodiment of the present invention based on this finding relates to a tumor growth inhibitor characterized by inhibiting the expression and Z or function of NEDD4.
  • the tumor growth inhibitor according to the present invention comprises at least any one of a compound that inhibits the function of NEDD4 and a compound that inhibits the expression of NEDD4.
  • the tumor growth inhibitor according to the present invention binds to RUNX but does not have E3 ligase activity, and is represented by inactive NEDD4, for example, the amino acid sequence set forth in SEQ ID NO: 11 of the Sequence Listing.
  • a tumor growth inhibitor comprising an effective amount of the protein represented by Z or the amino acid sequence represented by SEQ ID NO: 12.
  • the tumor growth inhibitor according to the present invention may be a tumor growth inhibitor comprising a double-stranded polynucleotide capable of inhibiting the expression of NEDD4.
  • Preferred examples of the double-stranded polynucleotide contained in the tumor growth inhibitor according to the present invention include the following double-stranded polynucleotide: (i) a polynucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 21 Nucleotides and sequences A double-stranded polynucleotide comprising the polynucleotide represented by the nucleotide sequence represented by No.
  • double-stranded polynucleotides are double-stranded polynucleotides having a human-derived polynucleotide ability. All of these three types of double-stranded polynucleotides inhibited cell growth as a result of transfection into human cancer cell lines (see Example 12).
  • the method for inhibiting tumor growth can be carried out using the tumor growth inhibitor according to the present invention.
  • One aspect of the present invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising an effective amount of the tumor growth inhibitor according to the present invention as an active ingredient.
  • Such a pharmaceutical composition can be used as a preventive and Z or therapeutic agent for diseases associated with tumor growth, such as cancer diseases, more specifically, cancer diseases such as stomach cancer, colon cancer, bile duct cancer, liver cancer and the like. .
  • cancer diseases such as stomach cancer, colon cancer, bile duct cancer, liver cancer and the like.
  • These agents and pharmaceutical compositions can be used to implement prevention and Z or treatment methods for such diseases.
  • inhibition of NEDD4 expression and Z or function can inhibit RUNX3 degradation by RUNX3 ubiquitination by NED D4 and stabilize RUNX3.
  • tumor growth is suppressed by RUNX3.
  • cancer diseases more specifically cancer diseases such as gastric cancer, colon cancer, bile duct cancer, and liver cancer.
  • the pharmaceutical composition according to the present invention is usually preferably prepared as a pharmaceutical composition containing one or more pharmaceutical carriers in addition to the active ingredient.
  • the amount of the active ingredient contained in the pharmaceutical preparation according to the present invention is appropriately selected from a wide range. Usually, it is appropriate that the amount is in the range of about 0.0001 to 70% by weight, preferably about 0.0001 to 5% by weight.
  • the pharmaceutical carrier includes a filler, a bulking agent, a binder, a moistening agent, a disintegrant, a lubricant, a diluent, an excipient, and the like that are generally used according to the form of use of the preparation. used. They are It is appropriately selected and used depending on the administration form of the resulting preparation.
  • water pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polybutylpyrrolidone, carboxyvinyl polymer, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin , Xanthan gum, gum arabic gum, casein, gelatin, agar, glycerin, propylene glycol, polyethylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin, mannitol, sorbitol, ratatose, etc. .
  • These may be used singly or in combination of two or more according to the dosage form of the pharmaceutical composition.
  • the stabilizer for example, human serum albumin, ordinary L-amino acid, saccharide, cellulose derivative and the like can be used. These can be used alone or in combination with a surfactant or the like. In particular, according to this combination, the stability of the active ingredient may be further improved.
  • the L amino acid is not particularly limited, and may be any of glycine, cysteine, glutamic acid and the like.
  • Sugars are not particularly limited, for example, monosaccharides such as glucose, mannose, galactose and fructose, sugar alcohols such as mannitol, inositol and xylitol, disaccharides such as sucrose, maltose and lactose, dextran, hydroxypropyl starch, Any of polysaccharides such as chondroitin sulfate and hyaluronic acid, and derivatives thereof may be used.
  • monosaccharides such as glucose, mannose, galactose and fructose
  • sugar alcohols such as mannitol, inositol and xylitol
  • disaccharides such as sucrose, maltose and lactose
  • dextran hydroxypropyl starch
  • Any of polysaccharides such as chondroitin sulfate and hyaluronic acid, and derivatives thereof may be used.
  • the cellulose derivative is not particularly limited, and may be any of methyl cellulose, ethyl cenololose, hydroxy ethino reseno relose, hydroxypropino reseno relose, hydroxy open propyl methyl cellulose, carboxymethyl cellulose sodium and the like.
  • surfactant any of an ionic surfactant and a nonionic surfactant can be used.
  • Surfactants include, for example, polyoxyethylene glycol sorbitan alkyl ester, polyoxyethylene alkyl ether, sorbitan monoacyl ester, and fatty acid glyceride.
  • Buffering agents include, for example, boric acid, phosphoric acid, acetic acid, citrate, ⁇ -aminocaproic acid, dartamic acid and ⁇ or a salt thereof (for example, sodium salt, potassium salt, calcium salt, magnesium salt thereof) Alkali metal salts such as alkaline earth metal salts).
  • tonicity agent for example, sodium chloride sodium, potassium salt sodium, saccharide, and glycerin can be used.
  • chelating agent for example, sodium edetate or quenate can be used.
  • the medicine and pharmaceutical composition according to the present invention can be used as a solution preparation, and after freezing, drying and storing it, it contains water, physiological saline, etc. It can also be used after it is dissolved in a buffer solution or the like to prepare an appropriate concentration.
  • the dose range of the medicine and the pharmaceutical composition is not particularly limited, and the effectiveness of the contained ingredient, the administration form, the administration route, the type of the disease, the nature of the subject (weight, age, medical condition and use of other medicines) Or the like) and the judgment of the doctor in charge.
  • a suitable dose is, for example, in the range of about 0.01 ⁇ g to 100 mg, preferably about 0.1: g to lmg, per kg of body weight of the subject.
  • these dosage changes can be made using general routine experimentation for optimization well known in the art.
  • the above dosage can be divided into once to several times a day, and may be administered intermittently at a rate of once every several days or weeks! /.
  • the pharmaceutical composition When administering the pharmaceutical composition according to the present invention, the pharmaceutical composition may be used alone or in combination with other compounds or medicines necessary for the prevention and Z or treatment of the target disease. May be. For example, you may mix
  • the route of administration can be selected from systemic administration or local administration!
  • an appropriate administration route is selected according to the disease, symptom and the like.
  • parenteral routes include normal intravenous administration and intraarterial administration, as well as subcutaneous, intradermal, intramuscular administration and the like. It can also be administered by the oral route.
  • transmucosal administration or transdermal administration is possible. When used for cancer diseases, it can be administered directly to the tumor by injection or the like.
  • various forms can be selected according to the purpose. Typical examples include solid dosage forms such as tablets, pills, powders, powders, fine granules, granules, capsules, aqueous preparations, ethanol solution preparations, suspensions, fat emulsions, and ribosome preparations. , Cyclodextrin, etc. Liquid dosage forms such as clathrate, syrup, elixir and the like.
  • these can be further administered orally, parenterally (instillations, injections), nasal preparations, inhalants, vaginal preparations, suppositories, sublingual, eye drops, ear drops, ointments, creams And can be prepared, molded and prepared according to conventional methods.
  • One embodiment of the present invention relates to a method for identifying a compound that inhibits or promotes RUNX ubiquitin.
  • RUNX ubiquitination can be thought to be performed by NEDD4 participating as an E3 ligase and binding to RUNX to catalyze RUNX ubiquitination.
  • the method for identifying a compound that inhibits or promotes RUNX ubiquitination is preferably a method for identifying a compound that inhibits or promotes RUNX ubiquitination by NEDD4.
  • the method for identifying a compound according to the present invention can be carried out using a pharmaceutical screening system known per se.
  • a method for identifying a compound that inhibits or promotes RUNX ubiquitin by NEDD4 can be carried out using the method generally used in screening for inhibitors of E3 ligase.
  • the substrate in vivo or E3 ligase itself is used as the substrate for E3 ligase.
  • RUNX or NEDD4 itself can be used as a substrate for NEDD4.
  • a compound that inhibits the binding of NEDD4 and RUNX can be obtained, so it is more preferable to use RUNX as the substrate. ,.
  • a method for identifying a compound that inhibits or promotes RUNX ubiquitin can be performed using, for example, an experimental system using the RUNX ubiquitin method according to NEDD4 of the present invention.
  • the experimental system using the RUNX ubiquitination method by NEDD4 according to the present invention refers to an experimental system in which NEDD4 and RUNX coexist and RUNX is ubiquitinated by NEDD4.
  • the compound identified by the identification method using such an experimental system includes a compound that inhibits or promotes the enzyme activity of NEDD4, and a compound that inhibits or promotes the binding of NEDD4 to RUNX.
  • a signal (hereinafter referred to as a test compound) is brought into contact with NEDD4 and / or RUNX, and a signal capable of detecting RUNX ubiquitination by NEDD4 and Use a system that uses Z or a marker to detect the presence or absence or change of this signal and Z or marker and whether or not the test compound inhibits RUNX ubiquitination by NEDD4 By determining whether to do so, compounds that inhibit or promote RUNX ubiquitination by NEDD4 can be identified.
  • the test compound can coexist in the RUNX ubiquitin reaction by NEDD4, or the test compound can be previously contacted with NEDD4 and Z or RUNX, followed by the RUNX ubiquitination reaction by NEDD 4. it can. Signal produced by RUNX ubiquitin by NEDD4 or marker power of ubiquitin ⁇ ⁇ ⁇ When test compound is contacted with NE DD4 and Z or RUNX, compared to when test compound is not contacted If it shows a change such as decrease or disappearance, it can be determined that the test compound inhibits RUNX ubiquitination by NED D4.
  • the test compound when the test compound is brought into contact with NEDD4 and Z or RUNX, the signal or marker force is increased or generated compared to when the test compound is not brought into contact with the test compound. If indicated, it can be judged that the test compound promotes RUNX ubiquitination by NEDD4.
  • Such an experimental system can be performed under both in vivo and in vitro conditions.
  • ubiquitin activity enzyme (E1) ubiquitin activity enzyme
  • E2 ubiquitin-conjugating enzyme
  • ubiquitin are required in addition to NEDD4 in the ubiquitination reaction of the target protein.
  • the in vivo experimental system may preferably be an experimental system using eukaryotic cells or cultured cell lines expressing both NEDD4 and RUNX, for example.
  • eukaryotic cells or cultured cell lines expressing RUNX or NEDD4 can be used.
  • a eukaryotic cell or cultured cell line that expresses both NEDD4 and RUNX, or a cell that further expresses ubiquitin in a eukaryotic cell or cultured cell line that expresses NEDD4 or RUNX can be used.
  • the expression of these proteins in cells uses appropriate vectors containing a polynucleotide encoding NEDD4, appropriate vectors containing a polynucleotide encoding RUNX, and appropriate vectors containing a polynucleotide encoding Z or ubiquitin. This can be achieved by transfecting these vectors into cells using conventional genetic engineering techniques.
  • test compound can be determined to inhibit RUNX ubiquitination by NEDD4.
  • signal or marker shows a change such as an increase or occurrence when the cell is treated with the test compound compared to when the cell is not treated with the test compound.
  • Test compounds can be judged to promote RUNX ubiquitination by NEDD4.
  • compounds that inhibit the enzyme activity of NEDD4 can be identified by measuring self-ubiquitin activity by NEDD4 without using RUNX.
  • Compounds that inhibit the enzymatic activity of NEDD4 can inhibit RUNX ubiquitination by NEDD4. Therefore, compounds that can inhibit RUNX ubiquitination by NEDD4 can also be identified by measuring self-ubiquitination by NEDD4 without using RUNX in the above experimental system.
  • Detection of ubiquitin can be measured by detection of a ubiquitinated target protein.
  • Detection of the ubiquitinated target protein can be performed by a known method such as Western blotting (see Examples 3, 5, 8 and 9).
  • the ubiquitin reaction of the target protein If a target protein having an increased molecular weight is detected after the ubiquitin reaction compared to before, it can be determined that the target protein has been ubiquitinated.
  • ubiquitinated labeled protein can be easily detected using the labeling substance as an index, and thus the use of such a labeled ubiquitin is useful. .
  • tag peptides such as HA-tag and FLAG-tag as labeling substances is not limited to these, and as long as it is a substance that does not inhibit NEDD4 ubiquitination of RUNX Can also be used.
  • the labeling substance can be detected using a detection method known per se.
  • tag peptides can be detected by anti-tag peptide antibodies. In this case, detection can be more easily carried out by using an antibody labeled with HRP (Hosradish peroxidase), alkaline phosphatase, a radioisotope, a fluorescent substance or piotin as an anti-tag peptide antibody.
  • HRP Hosradish peroxidase
  • alkaline phosphatase a radioisotope
  • a fluorescent substance or piotin as an anti-tag peptide antibody.
  • a secondary antibody labeled with the above enzyme, radioisotope, fluorescent substance, piotin or the like may be used.
  • methods for identifying a compound that inhibits or promotes RUNX ubiquitination by NEDD4 include, for example, an appropriate vector containing a polynucleotide encoding RUNX, an appropriate vector containing NEDD4, and an HA-tag.
  • the test compound When ubiquitinated RUNX is reduced or eliminated compared to untreated cells, the test compound can be determined as a compound that inhibits RUNX ubiquitination by NEDD4. In contrast, when ubiquitination RUNX increases, the test compound can be determined as a compound that promotes RU NX ubiquitination by NEDD4.
  • the ubiquitination RUNX contained in the cell lysate can be measured, for example, by detecting ubiquitin bound to RUNX with an HRP-labeled anti-HA antibody.
  • any method can be used without limitation as long as it is generally used in screening for inhibitors of ubiquitin E3 ligase.
  • a system using a fluorescence resonance energy transfer assay (FRET Assay), a dissociation-enhanced lanthanum-defluorinated assay (DELFIA Assay), or electrochemiluminescence (ECL) should be used.
  • FRET Assay fluorescence resonance energy transfer assay
  • DELFIA Assay dissociation-enhanced lanthanum-defluorinated assay
  • ECL electrochemiluminescence
  • Applied systems, systems that apply Scintillation Kimi City 1 Atsei (SPA), etc. can be used suitably (Yi Sun, “Methods in Enzymology”, 2005, No. 3 99) , P. 654—663).
  • RUNX can be used as a substrate for ubiquitination, and self-ubiquitin activity using NEDD4 itself as a substrate can be
  • One embodiment of the present invention also relates to a method for identifying a compound that inhibits or promotes the binding of NEDD4 to RUNX.
  • a method for identifying a compound that inhibits or promotes the binding between NEDD4 and RUNX can be carried out using a protein binding assay generally used in screening for binding inhibitors for IJ.
  • a method for identifying a compound that inhibits or promotes the binding of NEDD4 and RUNX can be carried out, for example, using an experimental system in which NEDD4 and RUNX coexist and NEDD4 and RUNX are combined.
  • a test compound is brought into contact with NEDD4 and Z or RUNX, and a system using a signal and Z or marker that can detect the binding of NEDD4 and RUNX is used. By detecting the presence or absence or change of this signal and Z or marker and determining whether the test compound inhibits or promotes the binding of NEDD4 to RUNX. And compounds that inhibit or promote the binding of RUNX.
  • the test compound can coexist in the binding reaction of NEDD4 and RUNX, or the test compound can be previously contacted with NEDD4 and Z or RUNX, and then the binding reaction of NEDD4 and RUNX can be performed.
  • Signal or binding marker force generated by binding of NEDD4 and RUNX When test compound is brought into contact with NEDD4 and Z or RUNX, it decreases or disappears compared to when test compound is not in contact with force If there is a change, it can be determined that the test compound inhibits the binding of NEDD4 and RUNX.
  • the signal or marker increases or occurs when the test compound is brought into contact with NEDD4 and Z or RU NX compared to when the test compound is not brought into contact with the test compound. If it shows a change, it can be determined that the test compound promotes the binding of NEDD4 and RUNX.
  • An in vitro experimental system can be carried out with reference to identification methods generally used in screening for protein binding inhibitors.
  • the in vitro experimental system can be, for example, an experimental system in which NE DD4 and RUNX are reacted in vitro and the binding of both proteins is detected by a pull-down method.
  • the in vivo experimental system may preferably be an experimental system using eukaryotic cells or cultured cell lines expressing both NEDD4 and RUNX, for example.
  • Eukaryotic cells or cultured cell lines expressing RUNX or NEDD4 can also be used in such experimental systems. Expression of these proteins in the cells is accomplished by conventional genetic engineering techniques using appropriate vectors containing a polynucleotide encoding NEDD4 and appropriate vectors containing a polynucleotide encoding Z or RUNX. This can be achieved by transformation.
  • the binding between NEDD4 and RUNX can be performed by a known protein detection method such as immunoprecipitation method, pull-down method, two-hybrid method, western blotting and fluorescence resonance energy transfer method, or a combination of these methods. In addition, it can be carried out by detecting the complex formed by NEDD4 and RUNX.
  • NEDD4 and Z or RUNX are preferably labeled with an appropriate labeling substance.
  • tag peptides such as FLAG-tag, Myc tag and HA-tag can be preferably used. Detection of the labeling substance can be performed using a detection method known per se.
  • tag peptides can be detected by anti-tag peptide antibodies.
  • detection can be performed more easily by using an antibody labeled with HRP (horseradish peroxidase), alkaline phosphatase, radioisotope, fluorescent substance or piotin as an anti-tag peptide antibody.
  • HRP horseradish peroxidase
  • alkaline phosphatase alkaline phosphatase
  • radioisotope fluorescent substance or piotin
  • a secondary antibody labeled with the above enzyme, radioisotope, fluorescent substance, piotin or the like may be used.
  • a method for identifying a compound that inhibits or promotes the binding of NEDD4 and RUNX includes, for example, an appropriate vector containing a polynucleotide encoding RUNX with a FLAG-tag added to the N-terminus, and This can be carried out using cells transfected with an appropriate vector containing a polynucleotide encoding NEDD4 with a Myc-tag attached to the N-terminus (see Example 2). After the cells are treated with the test compound, the cells are collected, lysed by an appropriate method to prepare cell lysate, and the complex of NEDD4 and RUNX contained in the cell lysate is detected.
  • Measurement of the complex contained in the cell lysate can be carried out by immunoprecipitation using an anti-FLAG antibody followed by Western blotting using an anti-Myc antibody. Quantities of NEDD4 and RUNX complex detected when cells are treated with test compound If cells are not treated with test compound, sometimes reduced or disappeared compared to the amount of complex detected On the other hand, it can be determined that the test compound inhibits the binding of NE DD4 and RUNX. In contrast, the amount of complex of NEDD4 and RUNX detected when cells are treated with the test compound Increased compared to the amount of complex detected when cells are not treated with the test compound It can be determined that the test compound promotes the binding of NEDD4 and RUNX.
  • a method for identifying a compound that inhibits or promotes the binding of NEDD4 and RUNX can also be performed using a known two-hybrid method.
  • a plasmid that expresses NE DD4 and a DNA binding protein as a fusion protein a plasmid that expresses RUNX and a transcriptional activation protein as a fusion protein
  • a plasmid containing a reporter gene such as lacZ connected to an appropriate promoter gene can be used in yeast or true.
  • the expression level of the reporter gene when the cells are treated with a test compound after transfection into a cell such as a nuclear cell, and the expression level of the reporter gene when the cell is not treated with the test compound Compare.
  • the test compound may be NEDD4 and RUNX. It can be determined that binding is inhibited. In contrast, the expression level of the reporter gene in the cells treated with the test compound is Not treated with the compound! When the expression level of the reporter gene in the cell increases, it can be determined that the test compound promotes the binding between NEDD4 and RUNX.
  • the compound identified by the identification method is a compound that inhibits or promotes the binding of NEDD4 and RUNX.
  • a method for identifying a compound that inhibits or promotes the expression of NEDD4 can also be carried out.
  • a method for identifying a compound that inhibits the expression of NEDD4 can be carried out using an experimental system capable of measuring the expression of NEDD4.
  • an experimental system capable of measuring the expression of NEDD4.
  • a polynucleotide encoding NEDD4 and a test compound were allowed to coexist and their expression was measured, and the change in expression compared to the measurement result in the absence of the test compound. By detecting (decrease, disappearance or increase), compounds that inhibit or promote NEDD4 expression can be identified.
  • the experimental system that can measure the expression of NEDD4 can be specifically an experimental system that expresses NEDD4 using cells transfected with an expression vector containing a polynucleotide encoding NEDD4.
  • the cells are collected, lysed by an appropriate method to prepare cell lysate, and NEDD4 contained in the cell lysate is detected. .
  • the amount of NEDD4 detected when cells are treated with the test compound If the amount of NEDD4 is reduced or disappears compared to the amount of NEDD4 detected when cells are not treated with the test compound, the test compound is It can be determined that expression is inhibited. In contrast, the amount of NEDD4 detected when cells are treated with the test compound. When the amount of NEDD4 detected when cells are not treated with the test compound increases, Can be determined to promote the expression of NEDD4.
  • the expression of NEDD4 can be measured by directly detecting NEDD4 by a known protein detection method such as Western blotting.
  • NEDD4 can be easily measured by introducing a signal serving as an expression index into an experimental system and detecting the signal.
  • a signal serving as an expression index for example, a labeling substance can be used.
  • NEDD4 can be easily measured by labeling NEDD4 with a labeling substance and measuring the labeling substance.
  • FLAG -tag, Myc— tag And tag peptides such as HA-tag can be preferably used. Detection of the labeling substance can be carried out using a known detection method. For example, tag peptides can be detected with anti-tag peptide antibodies.
  • detection can be more easily carried out by using an antibody labeled with HRP (horseradish bar oxidase), alkaline phosphatase, radioisotope, fluorescent substance or piotin as an anti-tag peptide antibody.
  • HRP horseradish bar oxidase
  • alkaline phosphatase alkaline phosphatase
  • radioisotope fluorescent substance or piotin
  • secondary antibody labeled with the above enzyme radioisotope, fluorescent substance, or piotin.
  • an experimental system that can measure the expression of NEDD4 creates a vector in which a reporter gene is linked instead of the polynucleotide downstream of the promoter region of the polynucleotide encoding NEDD4, It may be an experimental system using a cell that has been cleaved, such as a eukaryotic cell. In such an experimental system, the expression level of the reporter gene when the cells are treated with the test compound is compared with the expression level of the reporter gene when the cells are not treated with the test compound.
  • the test compound is NEDD4 Can be determined to inhibit the expression of.
  • the test sample The compound can be determined to promote the expression of NEDD4.
  • a reporter gene a gene generally used in reporter assembly can be used, and for example, a gene having an enzyme activity such as luciferase, j8-galactosidase, or chloramphee-cholase transferase can be used.
  • the expression of the reporter gene can be detected by detecting the activity of the gene product, for example, the enzyme activity in the case of the reporter gene.
  • test compound for example, a chemical library, a compound derived from a natural product, or a compound obtained by drug design based on the primary structure or three-dimensional structure of NEDD 4 and RUNX can be used. .
  • a compound obtained by drug design based on the structure of a polypeptide consisting of the amino acid sequence of the binding site of NEDD4 and RUNX is also suitable as the test compound.
  • Another embodiment of the present invention relates to a method for identifying a compound capable of promoting bone formation. The method for identifying a compound capable of promoting bone formation according to the present invention is characterized by measuring whether a test compound is capable of inhibiting the expression and Z or function of NEDD4.
  • BMP is inhibited by inhibiting the expression and Z or function of NEDD4.
  • a compound that inhibits the expression and Z or function of NEDD4 may cause inhibition of RUNX2's ubiquitination by NEDD4, thereby promoting RUNX2's stabilization and the resulting osteogenic signal. it can.
  • Such compounds can be thought to promote bone differentiation and bone formation by promoting bone formation signals.
  • a method for identifying a compound capable of promoting bone formation characterized by measuring whether a test compound inhibits NEDD4 expression, is a method for identifying a compound that inhibits the above-mentioned NEDD4 expression. Can be implemented. When the test compound inhibits the expression of NEDD4, it can be determined that the test compound can promote bone formation.
  • the function of NEDD4 can be evaluated, for example, by measuring its E3 ligase activity and its ability to bind to RUNX.
  • the E3 ligase activity of NEDD4 can be measured, for example, by detecting RUNX or self-ubiquitin using RUNX or NED D4 as a substrate.
  • the ubiquitin activity is detected using RUNX as a substrate.
  • a method for identifying a compound capable of promoting bone formation characterized by measuring the power of the test compound to inhibit the function of NEDD4, is described, for example, by NEDD4. It can be carried out using a method for identifying a compound that inhibits RUNX ubiquitin.
  • NEDD4 itself can be used as a substrate instead of RUNX, and a compound that inhibits self-ubiquitination by NEDD4 can be identified.
  • the test compound inhibits RUNX ubiquitin or auto-ubiquitin caused by NEDD4
  • the method for identifying a compound capable of promoting bone formation is characterized in that the test compound measures the force or inability to inhibit the function of NEDD4.
  • the above-mentioned binding method of NEDD4 and RUNX is inhibited. It can carry out using the identification method of the compound to do.
  • the test compound inhibits the binding of NEDD 4 and RUNX it can be determined that the test compound can promote bone formation.
  • RUNX2 is known to play an important role in bone formation, and therefore it is characterized by measuring whether or not a test compound inhibits the function of NEDD4. In the method for identifying a compound that can promote bone formation, RUNX2 is preferably used.
  • the test compound that has been shown to inhibit NEDD4 expression and Z or function by the above-described identification method is further used. It may be an identification method that further includes a step of measuring force dynamism capable of promoting the above.
  • mouse C2C12 cells can be used as osteogenesis model cells.
  • Mouse C2C12 cells are myoblasts and are derived from the same mesenchymal stem cells as osteoblasts and chondrocytes.
  • C2C12 cells show typical osteoblastic phenotypes upon stimulation with BMP-2, such as increased alkaline phosphatase activity and osteocalcin production. It has been used as a model cell (Katagiri T. et al., “The Journal of Cell Biology”, 1994, 127, p. 1755—1766).
  • BMP-2 can be preferably used as a site force-in that promotes bone formation.
  • a bone formation marker for example, alkaline phosphatase or osteocalcin can be used.
  • the site force-in and the bone formation marker that promote bone formation are not limited to these, and any site force-in or marker that is generally used for evaluation of bone formation can be used.
  • the bone formation marker can be measured by a generally used method for measuring a bone formation marker.
  • a test compound that can promote bone formation can be identified by treating the cells with the test compound.
  • the bone formation marker in the cell treated with the test compound increases with respect to the cell compared with the bone formation marker when treated with the test compound, it is judged that the bone differentiation and bone formation of the cell are promoted. it can.
  • One embodiment of the present invention also relates to a method for identifying a compound capable of suppressing tumor growth.
  • the method for identifying a compound capable of suppressing tumor growth according to the present invention is characterized in that the test compound measures the power and inability to inhibit the expression and Z or function of NEDD4.
  • NEDD4 binds to RUNX3 and ubiquitinates RUNX3 (Examples 2 and 3). It was also found that inhibition of NEDD4 expression suppresses the growth of human cancer cell tumor lines (see Example 12).
  • a method for identifying a compound capable of suppressing tumor growth characterized by measuring whether a test compound inhibits the expression of NEDD4, is a compound that inhibits the expression of NEDD4 as described above.
  • the identification method can be used. When NEDD4 expression is inhibited by the test compound, it can be determined that the test compound can suppress tumor growth.
  • NEDD4 functions by, for example, measuring its E3 ligase activity and its ability to bind to RUNX. Can be evaluated.
  • the ligase activity of NEDD4 can be measured, for example, by detecting RUNX ubiquitination or self-ubiquitination using RUNX or NEDD4 as a substrate.
  • the ubiquitin activity is detected using RUNX as a substrate.
  • a method for identifying a compound capable of suppressing tumor growth characterized by measuring the power of the test compound to inhibit NEDD4 function, is, for example, RUNX ubiquitination by NEDD4. It can be carried out by using a method for identifying a compound that inhibits the above.
  • NEDD4 itself can be used as a substrate instead of RUNX, and it can be carried out by identifying compounds that inhibit self-ubiquitination by NEDD4.
  • the test compound inhibits RUNX ubiquitin or self-ubiquitin caused by NEDD 4, it can be determined that the test compound can suppress tumor growth.
  • the binding ability of NEDD4 and RUNX is generally used in screening for binding inhibitors, and can be performed using the protein binding assay.
  • a method for identifying a compound capable of suppressing tumor growth characterized by measuring the power of the test compound to inhibit the function of NEDD4, includes, for example, the above-mentioned binding of NEDD4 and RUNX. This can be carried out using a method for identifying a compound to be inhibited.
  • the test compound inhibits the binding of NEDD4 and RUNX, it can be determined that the test compound can suppress tumor growth.
  • RUNX3 is known to play an important role in tumor growth suppression. Therefore, it is characterized by measuring the power of the test compound to inhibit the function of NEDD4. It is preferable to use RUNX3 as RUNX in the method for identifying compounds that can suppress tumor growth! /.
  • the test compound that has been shown to inhibit the expression and Z or function of NEDD4 by the above-described identification method is further used.
  • the identification method may further include a step of measuring a force force that can be suppressed.
  • an experimental system capable of measuring suppression of tumor growth for example, an experimental system using a human cancer cell line can be used.
  • an experimental system using a human cancer cell line can be used.
  • by treating cancer cells with a test compound when the growth of cancer cells is suppressed as compared with cancer cells not treated with the test compound, it can be determined that the test compound can suppress tumor growth.
  • One embodiment of the present invention relates to a reagent kit.
  • This reagent kit encodes at least one of NEDD4, a polynucleotide encoding NEDD4, a recombinant vector containing the polynucleotide, and a transformant containing the recombinant vector, and RUNX and RUN X.
  • a reagent kit comprising at least one of a polynucleotide containing the polynucleotide, a recombinant vector containing the polynucleotide, and a transformant containing the recombinant vector.
  • the reagent kit further comprises at least one of ubiquitin, a polynucleotide encoding ubiquitin, a recombinant vector containing the polynucleotide, and a transformant containing the recombinant vector. It can be.
  • This reagent kit can be used, for example, in the method for identifying a compound according to the present invention.
  • the reagent kit according to the present invention may contain a required substance such as a signal and Z or a marker, a buffer, and a salt used in the identification method.
  • a required substance such as a signal and Z or a marker
  • a buffer such as a buffer
  • a salt used in the identification method.
  • stabilizers and substances such as Z or preservatives may be included.
  • NEDD4, RUNX and ubiquitin are preferably human-derived proteins.
  • mammalian-derived proteins having the same functions and structural homology as the human-derived proteins such as mice, It can be a protein such as mah, hidge, ushi, nu, monkey, cat, bear, rat or rabbit.
  • the polynucleotides encoding NEDD4, RUNX and ubiquitin are preferably human-derived polynucleotides, but mammals having the same functions and structural homology as the human-derived proteins.
  • NEDD4 As long as it is a polynucleotide encoding a protein derived from, it can be a polynucleotide derived from, for example, mouse, horse, hidge, ushi, dog, monkey, cat, bear, rat, or rabbit.
  • the properties and functions of NEDD4, RUNX and ubiquitin include, for example, the binding of NEDD4 and RU NX, the binding of ubiquitin to RUNX by NEDD4 (ubiquitination), and the ligase activity of NEDD 4. [0317] NEDD4, RUNX and ubiquitin were either expressed in genetically engineered cells or biological samples prepared, cell-free or chemically synthesized products, or further purified from them It may be a thing.
  • NEDD4, RU NX and ubiquitin are expressed by a genetic engineering technique.
  • NEDD4, RUNX, and ubiquitin are genetically linked to other proteins or polypeptides on the N-terminal side or C-terminal side, either directly or indirectly through linker peptides, as long as their properties and functions are affected. It may be attached using engineering techniques.
  • proteins and polypeptides include, for example, enzymes such as dartathione S-transferase (GST), ⁇ -galatatosidase, horseradish peroxidase (HR ⁇ ) or alkaline phosphatase (ALP), His-tag, Myc-tag Tag peptides such as HA-tag, FLAG-tag or Xpress-tag can be used.
  • GST dartathione S-transferase
  • HR ⁇ horseradish peroxidase
  • ALP alkaline phosphatase
  • His-tag His-tag
  • Myc-tag Tag peptides such as HA-tag, FLAG-tag or Xpress-tag can be used.
  • Any known method can be used as a genetic engineering technique.
  • the method described in the book (edited by Sambrook et al., “Molecular Cloning, Laboratory Manual, Second Edition”, 1989, Cold Spring Harbor Laboratory; edited by Masami Muramatsu, “Laboratory-Genetic Engineering” 1988, Maruzen Co., Ltd .; Ulmer, KM, Science, 1983, 219th, p. 666-671; edited by Ehrlich, HA, PCR Technology, DNA Amplification Principles and Applications ”(see 1989, Stotton Press etc.) can be used.
  • Polynucleotides encoding NEDD4, RUNX, and ubiquitin can be easily obtained from, for example, appropriate sources in which the expression of each polynucleotide is observed using a cloning method known per se.
  • As the origin of these polynucleotides various cells and tissues in which the expression of the polynucleotide has been confirmed, or cultured cells derived therefrom can be used.
  • As a source of a polynucleotide encoding NEDD4 for example, cells derived from human muscle or muscle tissue can be used.
  • RUNX for example, various cancer cell lines can be used.
  • As the origin of the polynucleotide encoding ubiquitin for example, cells derived from human brain and brain tissue can be used.
  • Isolation of total RNA from origin, isolation and purification of mRNA, acquisition of cDNA and its cloning, etc. can all be performed according to conventional methods.
  • commercially available cDNA libraries Can also be used.
  • the method for selecting a desired clone as well as the cDNA library is not particularly limited, and a conventional method can be used. For example, a plaque hybridization method using a probe that selectively binds to a target DNA sequence, a colony hybridization method, or a combination of these methods can be used.
  • DNA chemically synthesized based on information on the nucleotide sequences of polynucleotides encoding NEDD4, RUNX and ubiquitin, respectively, can be generally used.
  • sense primers and antisense primers designed based on the nucleotide sequence information of the polynucleotide can be used as such probes.
  • Selection of the target clone from the cDNA library can be performed, for example, by confirming the expressed protein for each clone using a known protein expression system and using the biological function as an index.
  • Primers used for PCR can be appropriately designed based on the DNA sequence information of DNA, and can be obtained by synthesis according to conventional methods. Isolation and purification of the amplified DNAZRNA fragment can be performed by a conventional method. For example, DNAZRNA fragments can be isolated and purified by gel electrophoresis or the like.
  • the polynucleotide may be, for example, GST, ⁇ -galactosidase, HRP or ALP. Enzymes such as His-tag, Myc-tag, or HA-tag, FLAG-tag or Xpress-tag etc.
  • the nucleotide can be one or more added polynucleotides. The attachment of these polynucleotides can be performed by conventional genetic engineering techniques.
  • NEDD4, RUNX and ubiquitin By constructing a recombinant vector containing a polynucleotide encoding NEDD4, RUNX and ubiquitin, respectively, and expressing the polynucleotide in an appropriate host cell using the recombinant vector, NEDD4, RUNX and ubiquitin are used. Among them, cells expressing at least 1 can be obtained. Moreover, NEDD4, RUNX and ubiquitin can be prepared from the cells by a known method.
  • the vector DNA is not particularly limited as long as it can replicate in the host, and is appropriately selected depending on the type of host and intended use.
  • the vector DNA may be one obtained by extracting a naturally occurring one, or a part of the DNA other than that required for replication.
  • Representative vector DNAs are, for example, vector DNAs derived from plasmids, butteriophages and viruses.
  • plasmid DNA for example, a plasmid derived from E. coli, a plasmid derived from Bacillus subtilis, or a plasmid derived from yeast can be used.
  • ⁇ phage can be used as the pacteriophage DNA.
  • virus-derived vector DNA examples include vectors derived from animal viruses such as retrovirus, vaccinia virus, adenovirus, papovavirus, SV40, fowlpox virus, and pseudorabies virus, or insect viruses such as baculovirus. You can use vectors.
  • vector DNA derived from a transposon, an insertion element, or a yeast chromosomal element can be used.
  • a vector DNA prepared by combining them for example, a vector DNA (such as cosmid phage phagemid) prepared by combining genetic elements of plasmid and butteriophage can be used.
  • an expression vector or a cloning vector can be used as desired.
  • a gene sequence carrying information regarding replication and control for example, a ribosome binding sequence, a terminator, a signal sequence, a cis-element such as an enhancer, a splicing signal, and a selectable marker may be selected as desired.
  • a selection marker for example, a dihydrofolate reductase gene, an ampicillin resistance gene, or a neomycin resistance gene can be used.
  • the target gene sequence is treated with an appropriate restriction enzyme, cleaved at a specific site, mixed with vector DNA treated in the same manner in V, and religated by ligase.
  • the desired recombinant vector can also be obtained by ligating an appropriate linker to the target gene sequence and inserting it into the multicloning site of the appropriate vector.
  • a transformant can be obtained by transfecting a recombinant vector containing polynucleotides encoding NEDD4, RUNX and ubiquitin, respectively, into a host. If an expression vector is used as the vector DNA, cells expressing at least one of these polynucleotides can be obtained, and NEDD4, RUNX or ubiquitin can be produced by using the cells by a known method.
  • the transformant can be further transfected with one or more vector DNAs containing a desired polynucleotide other than polynucleotides encoding NEDD4, RUNX and ubiquitin, respectively.
  • Prokaryotic and eukaryotic! / Slack can also be used as hosts.
  • Prokaryotes include, for example, the genus Escherichia such as Escherichia coli, the Bacillus genus such as Bacillus subtilis, the Syudomonas genus such as Pseudomonas putida, and the Rhizobium meriroti (Rhizobium). Bacteria belonging to the genus Rhizobium such as meliloti) can be used.
  • yeasts such as Saccharomyces cerevisiae and Schizosaccharomyces pombe
  • insect cells such as Sf9 and Sf21
  • COS cells monkey kidney-derived cells
  • Vero cells monkey kidney-derived cells
  • Chinese hamsters Animal cells such as ovary cells (CHO cells), mouse L cells, rat GH3 cells, and human HEK293T cells can be used.
  • animal cells are used.
  • Transfection of vector DNA into a host cell can be carried out by a method known per se.
  • a standard method described in a book (Sambrook et al. Ng, Laboratories-Second Edition ", 1989, Cold Sp It can be implemented by the Ringnober Laboratory.
  • an integration method into a chromosome can be used in consideration of gene stability.
  • an autonomous replication system using extranuclear genes can be used. Specific methods include, for example, calcium phosphate transfer, DEAE-dextran mediated transfer, microinjection, cationic lipid mediated transfer, electoral position, transduction, scrape loading. ), Ballistic introduction and infection.
  • NEDD4, RUNX, and ubiquitin are cells, biological samples, or cell-free or chemically synthesized products that express genetically engineered polynucleotides encoding NEDD4, RUNX, and ubiquitin, respectively. However, it may be purified or further purified from these. In addition, these proteins can be expressed in cells containing a polynucleotide encoding the protein.
  • the cell may be a transformant obtained by transfecting a vector containing a polynucleotide encoding RUNX and ubiquitin, respectively.
  • NEDD4, RUNX, and ubiquitin can be further modified without significant changes in function, such as modification of its constituent amino groups or carboxyl groups, for example, by amidation.
  • it is labeled by adding another protein or the like to the N-terminal side or C-terminal side directly or indirectly using a genetic engineering method through a linker peptide or the like. May be.
  • a label that does not inhibit the basic properties of the protein is desirable.
  • proteins to be added include enzymes such as GST, ⁇ -galatatosidase, HRP or ALP, tag peptides such as His-tag, Myc-tag, HA-tag, FLAG-tag or Xpress-tag, fluoro Fluorescent dyes such as fluorescein isothiocyanate or phycoerythrin, maltose binding protein, Fc fragment of immunoglobulin or piotin can be used, but not limited thereto. It can also be labeled with a radioisotope. One or a combination of two or more substances can be added for labeling.
  • these proteins are obtained by culturing a transformant obtained by transfecting a vector DNA containing NEDD4, RUNX or ubiquitin, and then recovering the target protein from the obtained culture. Can be manufactured.
  • the transformant can be cultured using culture conditions and culture methods known per se optimal for each host. Culturing can be performed using the present protein expressed by the transformant itself or its function as an index. Some may be cultured using the present protein itself or the amount of the protein produced in or outside the host as an index, and subculture or batch culture may be performed using the amount of transformant in the medium as an index. You may go.
  • the target protein When the target protein is expressed in the cell or on the cell membrane of the transformant, the target protein is extracted by crushing the transformant. When the target protein is secreted outside the transformant, use the culture solution as it is, or use a culture solution from which the transformant has been removed by centrifugation or the like.
  • NEDD4, RUNX or ubiquitin can also be produced by common chemical synthesis methods.
  • the methods described in the book (“Peptide Synthesis”, Maruzen Co., Ltd., 1975 and “Peptide Synthesis”, Interscience, New York, 1996) are used. Not limited to these, known methods can be widely used.
  • Known methods for chemically synthesizing proteins include solid-phase synthesis methods and liquid-phase synthesis methods. Any of these methods can be used.
  • such a protein synthesis method is based on amino acid sequence information, in which each amino acid is sequentially linked one by one to extend the chain, and the so-called stepwise longong method,
  • the condensation methods used in the above protein synthesis can also follow conventional methods, for example, azide method, mixed acid anhydride method, DCC method, active ester method, redox method, DPPA (diphenylphosphoryl azide) method, DCC +
  • the methods such as potassium (1-hydroxybenzotriazole, ⁇ -hydroxysuccinamide, ⁇ -hydroxy-5-norbornene-1,2,3-dicarboximide), and the Wordword method can be used.
  • these proteins can be produced using a commercially available amino acid synthesizer.
  • NEDD4, RUNX or ubiquitin having a mutation introduced therein can also be used in the present invention.
  • Proteins, polypeptides and means for introducing mutations into polypeptides are known per se, such as Ulmer's technology (KM Ulmer, Science), 1983, 219, p. 666-671. ). From the viewpoint of not changing the basic properties (physical properties, functions, immunological activity, etc.) in introducing such mutations, for example, homologous amino acids (polar amino acids, nonpolar amino acids, hydrophobic amino acids). Mutual substitution between acids, hydrophilic amino acids, positively charged amino acids, negatively charged amino acids, aromatic amino acids, etc.) is readily envisioned. Furthermore, these usable polypeptides can be altered to a degree without significant or altered functions, such as modification of their constituent amino groups or carboxyl groups, for example by amido.
  • NEDD4, RUNX or ubiquitin can be purified and / or separated by various separation methods utilizing its physical properties, chemical properties, etc., if desired. Separation and Z or purification can be performed using the function of the protein as an indicator.
  • separation operation method for example, ammonium sulfate precipitation, ultrafiltration, gel chromatography, ion exchange chromatography, affinity chromatography, high performance liquid chromatography, dialysis method and the like can be used alone or in appropriate combination.
  • a specific antibody against them is prepared and specifically adsorbed using the antibody, for example, affinity chromatography using a column to which the antibody is bound. It is recommended to use graphy.
  • Prediction of a protein having a function of interacting with RUNX3 was carried out as follows according to the in silico prediction method described in International Publication No. WO01Z67299: (i) Oligopeptide having a certain length of amino acid sequence of RUNX3 (Ii) search the database for proteins having the amino acid sequence of each oligopeptide or an amino acid sequence homologous to the amino acid sequence, and (iii) local alignment between the obtained protein and RUNX3. (Iv) Predict that a protein with a high local alignment score interacts with RUNX3.
  • NEDD4 was found as a protein predicted to have a function of interacting with RUNX3.
  • Oligopeptides consisting of partial amino acid sequences of RUNX3 FPYSA T (SEQ ID NO: 13), AELRNA (SEQ ID NO: 15) and LSVAGM (SEQ ID NO: 17), homologous oligopeptides FEYSAT (SEQ ID NO: 14), AEELNA (SEQ ID NO: 16) ) And GRVAGM (SEQ ID NO: 18) were found in the amino acid sequence of NEDD4.
  • a human RUNX3 expression plasmid was constructed as described below.
  • Human RUNX3 cDNA was purchased from Open Biosystems (clone number MHS1011-74936).
  • the ORF was amplified by PCR using the purchased human RUNX3 cDNA in a cage, primers with EcoRI and Xhol sites attached, and the nucleotide sequence was confirmed by sequencing. Subsequently, the obtained ORF region was incorporated into an animal cell expression plasmid pCMV-Tag2 (STRATAGENE) for expressing an N-terminal FLAG-tag binding protein at the EcoRlZXhoI site to construct a human RUNX3 expression plasmid.
  • STRATAGENE animal cell expression plasmid pCMV-Tag2
  • FLAG-RUNX3 N-terminal FLAG-tag binding human RUNX3 (hereinafter referred to as FLAG-RUNX3) is expressed.
  • the deduced amino acid sequence encoded by the cloned human RUNX3 cDNA was the same as that published in the NCBI protein database accession number NP-004341 (registered gene name is RUNX3).
  • a human NEDD4 expression plasmid was constructed as described below.
  • Human NEDD4 cDNA was prepared based on the nucleotide sequence of the KIAA0093 clone (NCBI accession number D42055). The base sequence of the KIAA0093 clone was considered to have a deletion of 218 bp on the end side compared to the genomic sequence. In view of this, cDNA was prepared by further extending 218 bp to the ⁇ -terminal side of the base sequence of the KIAA0093 clone with reference to the genome sequence.
  • Extension 218 bp per minute was obtained from human skeletal muscle-derived cDNA (QUICK-clone cDNA ⁇ Clontech) by PCR, and the nucleotide sequence was confirmed by sequencing.
  • Acquired human NEDD4 cDNA after adding the Myc-tag coding sequence at the 5 'end was incorporated into the expression for animal cell bra plasmid P CI (Promega Corp.) to construct a human NEDD4 expression plasmid.
  • This human NEDD4 expression plasmid expresses N-terminal Myc-tag-linked human NEDD4 (hereinafter referred to as Myc-NEDD4).
  • Myc-NEDD4 N-terminal Myc-tag-linked human NEDD4
  • the cells were suspended in the suspension and allowed to stand for 20 minutes on ice to lyse the cells. Thereafter, the supernatant was collected by centrifugation at 15, 0 OOrpm for 15 minutes at 4 ° C and used as a cell lysate. Subsequently, 50% of ⁇ 50% of protein G sepharose 4 FastFlow (manufactured by Amersham Biosciences) was added to the cell lysate and mixed by inversion at 4 ° C for 1 hour. After centrifugation at 10, OOOrpm for 15 seconds at 4 ° C, add 0.5 L of anti-FLAG M2 antibody (Sigma) to the collected supernatant, and mix by inverting at 4 ° C for 2 hours.
  • anti-FLAG M2 antibody Sigma
  • Protein G sepharos e 4 FastFlow 50% slurry was newly added, and the mixture was mixed again by inversion at 4 ° C for 2 hours.
  • Protein G sepharos e 4 FastFlow was collected by centrifugation, washed 3 times with lysis buffer, and then 20 / zL of 2X SDS sample buffer was prepared and heat-treated at 100 ° C for 5 minutes as an SDS-PAGE sample. used.
  • E3 ligase inactive human NEDD4 In vivo ubiquitination of RUNX3 by NEDD4 was examined by immunoprecipitation using human cultured cells in which human RUNX3, human NEDD4 and human ubiquitin were transiently co-expressed. In addition, using a human NEDD4 mutant whose E3 ligase activity was inactivated by the introduction of the mutation (hereinafter sometimes referred to as E3 ligase inactive human NEDD4) was examined.
  • the expression plasmid prepared in Example 2 was used as the human RUNX3 expression plasmid.
  • This human RUNX3 expression plasmid expresses FLAG-RUNX3.
  • the expression plasmid prepared in Example 2 was used as the human NEDD4 expression plasmid. Myc-NEDD4 is expressed by this human NEDD4 expression plasmid.
  • Human NEDD4 (C967A), which is a human NEDD4 mutant in which a mutation was introduced into the HECT domain, was used as a human NEDD4 mutant.
  • Human NEDD4 (C967A) is a human NEDD4 mutant in which the 967th cysteine residue is substituted with an alanine residue in the amino acid sequence of human NEDD4, thereby inactivating the E3 ligase activity.
  • the human NEDD4 (C967A) expression plasmid is a human NEDD4 expression plasmid (see Example 2), and is a primer that can introduce a substitution of the 967th cysteine residue of human NEDD4 to an alanine residue.
  • This human NEDD4 (C967 A) expression plasmid expresses N-terminal Myc-tag binding human NEDD4 (C967A) (hereinafter referred to as Myc-NEDD4 (C967A)).
  • a human ubiquitin (Ub) expression plasmid was constructed as described below.
  • Human Ub cDNA was obtained by PCR from human brain-derived cDNA (QUICK-clone cDNA ⁇ Clontech), and the nucleotide sequence was confirmed by sequencing.
  • the Ub expression plasmid was constructed by incorporating human Ub cDNA into an animal cell expression plasmid pCMV-HA (Clontech) for expressing the N-terminal HA tag binding protein.
  • This Ub expression plasmid expresses N-terminal HA-tag binding Ub (hereinafter referred to as HA-Ub).
  • the deduced amino acid sequence encoded by the cloned Ub cDNA was identical to that published in NCBI protein database accession number NP-0666289 (registered gene name is UBC).
  • human NEDD4 expression plasmid 2.0 g, and human Ub expression plasmid 0.25 ⁇ g were transfected into cells using FuGENE6 (Roche diagnostics).
  • human NEDD4 expression plasmid instead of the human NEDD4 expression plasmid, cells transfected with human NEDD4 (C967A) expression plasmid were prepared in the same manner.
  • cells transfected with only the human RUNX3 expression plasmid and cells transfected with the human RUNX3 expression plasmid and the human Ub expression plasmid were prepared in the same manner and used as controls. The total amount of DNA introduced was corrected with an empty vector. Cells cultured for 2 days after transfection were treated in the same manner as in Example 2 to prepare cell lysate.
  • Protein G sepharose 4 FastFlow is collected by centrifugation, washed 3 times with a lysis buffer (same composition as in Example 2), 20 ⁇ L of 2 X SDS sample buffer is added, and heated at 100 ° C for 5 minutes The treated sample was used as an SDS-PAGE sample. Samples were separated by 5-20% SDS-PAGE, and then Western blotting was performed using peroxidase-labeled anti-c-Myc antibody (manufactured by Nacalai Testa) for Myc-NEDD 4 and Myc-NEDD4 (C967A).
  • FLAG-RUNX3 was detected with a FLAG M2 antibody (manufactured by Sigma), and HA-Ub was detected with a peroxidase-labeled anti-HA antibody (manufactured by Roche di agnostics). Detection was performed using 7 ECL western blotting detection kits (Amersham Biosciences).
  • the expression plasmids prepared in Examples 2 and 3 were used as the human NEDD4 expression plasmid and the human NEDD4 (C967A) expression plasmid.
  • Myc-NEDD4 is expressed by this human NEDD4 expression plasmid.
  • Myc—NEDD4 (C967A) is expressed by this human NEDD4 (C967A) expression plasmid.
  • l; z g was transfected into cells using FuGENE6 (Roche di agnostics) with human NEDD4 expression plasmid 1.0-2.
  • cells were prepared by transfecting 2.0 g of a human NEDD4 (C967A) expression plasmid in the same manner instead of the human NEDD4 expression plasmid.
  • cells transfected with only the human RUNX3 expression plasmid were prepared in the same manner and used as a control. The total amount of DNA introduced was corrected with an empty vector. The day after transfection, cells were washed with PBS and harvested using trypsin Z ethylenediaminetetraacetic acid (EDTA) solution.
  • EDTA trypsin Z ethylenediaminetetraacetic acid
  • the collected cells were lysed by adding 60 ⁇ L of lysis buffer (Cell Signaling), suspending with pipetting, and then allowing to stand on ice for 5 minutes. Thereafter, the supernatant was collected by centrifugation at 15, OOOrpm for 10 minutes at 4 ° C and used as cell lysate. Next, an equal volume of 2 X SDS sample buffer was added to the cell lysate and calcined at 100 ° C for 5 minutes and used as an SDS-PAGE sample.
  • lysis buffer Cell Signaling
  • 2 X SDS sample buffer was added to the cell lysate and calcined at 100 ° C for 5 minutes and used as an SDS-PAGE sample.
  • the human RUNX1 expression plasmid was constructed as described below.
  • Human RUNX1 cDNA was obtained from human kidney-derived cDNA (QUCIK-clone cDNA Clontech) by PCR using primers with EcoRI and Xhol sites added, and the nucleotide sequence was confirmed by sequencing. The obtained cDNA was incorporated at the EcoRl / XhoI site into an animal cell expression plasmid pCMV-Tag2 (STRATAGENE) for expressing an N-terminal FLAG-tag binding protein to construct a human RUNX1 expression plasmid.
  • STRATAGENE animal cell expression plasmid pCMV-Tag2
  • This human RU NX1 expression plasmid expresses N-terminal FLAG-tag binding human RUNXl (hereinafter referred to as FLAG-RUNX1).
  • the deduced amino acid sequence encoded by the cloned human RUNX1 cDNA was the same as that published in the NCBI protein database accession number NP-001745 (registered gene RUNX1).
  • the expression plasmids prepared in Examples 2 and 3 were used as the human NEDD4 expression plasmid and the human NEDD4 (C967A) expression plasmid, respectively.
  • This human NEDD4 expression plasmid expresses Myc—NEDD4.
  • Myc—NEDD4 (C967A) is expressed by this human NEDD4 (C967A) expression plasmid.
  • the expression plasmid prepared in Example 3 was used as the human ubiquitin (Ub) expression plasmid. This human Ub expression plasmid expresses HA-Ub.
  • human NEDD4 expression plasmid 2.0 g, and human Ub expression plasmid 0.25 ⁇ g were transfected into cells using FuGENE6 (Roche diagnostics).
  • human NEDD4 expression plasmid instead of the human NEDD4 expression plasmid, cells transfected with human NEDD4 (C967A) expression plasmid were prepared in the same manner.
  • cells transfected with human RUNX1 expression plasmid and human Ub expression plasmid were prepared in the same manner and used as a control. The total amount of DNA introduced was corrected with an empty vector. Cells cultured for 2 days after transfection were treated in the same manner as in Example 2 to prepare cell lysates.
  • Protein G sepharose 4 FastFlow is collected by centrifugation, washed 3 times with lysis buffer (same composition as in Example 2), then 20 / z L 2 X SDS sample buffer is prepared, and 5 ° C at 100 ° C. What was heat-processed for minutes was used as a SDS-PAGE sample. Samples were separated by 5-20% SDS-PAGE, and Western blotting was performed using peroxidase-labeled anti-c-Myc antibody (manufactured by Nacalai Testa) for Myc-NEDD4 and Myc-NEDD4 (C967A), and peroxidase-labeled anti-FLAG.
  • M2 antibody manufactured by Sigma
  • FLAG—RUNX1 and peroxidase-labeled anti-HA antibody were detected.
  • Detection was carried out using an ECL western blotting detection kit (Amersham Biosciences).
  • samples prepared from cells co-expressing FLAG-RUNX1, Myc-NEDD4 and HA-Ub were subjected to immunoprecipitation using anti-FLAG M2 antibody to Myc-NEDD4. And FLAG—RUNX1 coprecipitation was observed.
  • coprecipitation of Myc-NEDD4 (C967A) and FLAG-RUNX1 was observed in samples prepared from cells co-expressed with Myc-NEDD4 (C967A), FLAG-RUNX1 and HA-Ub.
  • samples prepared from cells co-expressing FLAG—RUNX1, Myc—NEDD4 and HA—Ub showed immunoprecipitation using anti-FLAG M2 antibody and The protein strength detected by immunoblotting using anti-HA antibody Myc-NE DD4 was also significantly increased compared to the prepared sample.
  • the detected protein is FLAG-RUNX1 with HA-Ub added. That is, in samples prepared from cells co-expressing FLAG-RUNX1, Myc-NEDD4 and HA-Ub, an increase in FLAG-RUNX1 with HA-Ub attached was observed.
  • the expression plasmid prepared in Example 5 was used as the human RUNX1 expression plasmid.
  • This human RUNX1 expression plasmid expresses FLAG-RUNX1.
  • the expression plasmids prepared in Examples 2 and 3 were used as the human NEDD4 expression plasmid and the human NEDD4 (C967A) expression plasmid, respectively. With this human NEDD4 expression plasmid, Myc—NEDD4 is expressed. In addition, Myc—NEDD4 (C967A) is expressed by this human NEDD4 (C967A) expression plasmid.
  • HEK293T cells were transfected with 0.1 g of human RUN XI expression plasmid and 1.0 to 2.0 g of human NEDD4 expression plasmid in the same manner as described in Example 4.
  • human NEDD4 expression plasmid cells were prepared by transfecting 2.0 g of human NEDD4 (C967A) expression plasmid in the same manner.
  • cells transfected with only the human RUNX1 expression plasmid were prepared in the same manner and used as a control. The total amount of DNA introduced was corrected with an empty vector. On the day after the transfer, cells were collected by the same method as described in Example 4 to prepare cell lysate.
  • the size of endogenous NEDD4 detected in human cancer cell lines was compared with the size of human NEDD4 transiently expressed in cultured human cell lines and the size of short human NEDD4 by Western blotting.
  • the expression plasmid prepared in Example 2 was used as the human NEDD4 expression plasmid. This human
  • Myc—NEDD4 is expressed by the NEDD4 expression plasmid.
  • the first force on the N-terminal side of human NEDD4 is also 10th.
  • Short-chain human NEDD4 whose E3 ligase activity was inactivated by mutation Mutants were used.
  • a short-chain human NEDD4 mutant a short-chain human NEDD4 (C867A), which is a short-chain human NEDD4 mutant with a mutation introduced in the HECT domain, was prepared.
  • Short-chain human NEDD4 (C867A) is a short-chain human in which the 867th cysteine residue was replaced with an alanine residue in the amino acid sequence of short-chain human NEDD4, thereby inactivating E3 ligase activity.
  • NEDD4 mutant The 867th cysteine residue in short-chain human NEDD4 corresponds to the 967th cysteine residue in human NEDD4.
  • a short human NEDD4 expression plasmid was constructed as described below. Of the ORF region of the human NEDD4 gene, a region encoding the 101st to 1000th amino acid sequences of human NEDD4 (hereinafter referred to as short human NEDD4 cDNA) is amplified by PCR and sequenced. The base sequence was confirmed. PCR was performed using the human NEDD4 expression plasmid prepared in Example 2 as a saddle and primers added with EcoRI site and Xhol site. The acquired short human NEDD4 cDNA was incorporated into an animal cell expression plasmid pCI (Promega) to construct a short human NEDD4 expression plasmid.
  • pCI Promega
  • the deduced amino acid sequence encoded by the cloned short-chain human NEDD4 cDNA is identical to the amino acid sequence published in the N CBI database under the accession number NP-006145 (KIAA0093), and Swiss-Prot It matched the amino acid sequence from the 101st to the 1000th amino acid sequence published in the database accession number P46934 (registered gene name is NEDD4).
  • the short-chain human NEDD4 (C867A) expression plasmid is a short-chain human NEDD4 expression plasmid, and the substitution of the 867th cysteine residue of a short-chain human NEDD4 with an alanine residue is introduced.
  • the primers obtained were designed and synthesized and used to construct the QuikChange Site—Directed Mutagenesis kit (manufactured by STRATAGENE). The sequence of the constructed expression plasmid was performed, and it was confirmed that a mutation was introduced into the expression plasmid.
  • HEK293T cells with 1.0 x 10 6 cells are seeded in 6cm dishes and cultured in spear culture at 37 ° C under 5% CO / 95% air in DMEM medium containing 10% FBS.
  • Short human NEDD4 expression plasmid and short human NEDD4 (C867A) expression plasmid One of the plasmids, 1.0 g, was transfected into cells using FuGENE6 (Roche diagnostics). Using cells cultured for 2 days after transfection, cells were collected by the same method as described in Example 4 to prepare cell lysate.
  • Endogenous NEDD4 expressed in human cancer cell lines was detected in breast cancer-derived cell lines (T-4 7D cells, MDA-MB-468 cells and BT-20 cells), colon cancer cell lines (SW480 cells).
  • Cell lysates lung cancer cell lines (A549 cells), myeloid leukemia cell lines (K562 cells and HL-60 cells), lymphoid leukemia cells (MOLT-4 cells), gastric cancer cell lines (MKN28 cells and MKN74 cells) was carried out.
  • Cell lysates of cancer cell lines other than MKN28 and MKN74 cells were purchased from SantaCruz.
  • MKN28 cells and MKN74 cells are treated with RIPA buffer (50 mM Tris-HCl (pH 8.0) / 150 mM NaCl / 1% Triton X-100 / 0.1% SDS) containing the protease inhibitor cocktail Complete Mini (Roche di agnostics). / 0. 1% DOC) to prepare a cell lysate.
  • RIPA buffer 50 mM Tris-HCl (pH 8.0) / 150 mM NaCl / 1% Triton X-100 / 0.1% SDS
  • protease inhibitor cocktail Complete Mini (Roche di agnostics).
  • 0. 1% DOC / 0. 1% DOC
  • Short-chain human NEDD4 was detected in HEK293 cells transfected with a short-chain human NEDD4 expression plasmid (Panel A in FIG. 7, lane 1).
  • Short-chain human NEDD4 (C867A) was detected in HEK293 cells transfected with a short-chain human NE DD4 (C867A) expression plasmid (Panel A in FIG. 7, lane 2).
  • NEDD4 expressed mainly in cancer cell lines is considered to be short-chain NEDD4.
  • the expression plasmid prepared in Example 5 was used as the human RUNX1 expression plasmid.
  • This human RUNX1 expression plasmid expresses FLAG-RUNX1.
  • the expression plasmid prepared in Example 2 was used as the human RUNX3 expression plasmid.
  • This human RUNX3 expression plasmid expresses FLAG-RUNX3.
  • a Myc-tag binding short human NEDD4 expression plasmid was constructed as described below.
  • Short-chain human NEDD4 cDNA (see Example 7) was added to the ⁇ -end with a Myc-tag coding sequence, and then incorporated into an animal cell expression plasmid pC Promega).
  • a tag-binding short human NEDD4 expression plasmid (hereinafter referred to as Myc short human NEDD4 expression plasmid) was constructed.
  • This Myc short-chain human NEDD4 expression plasmid expresses Myc short-chain human NEDD4 (hereinafter referred to as Myc short-chain NEDD4).
  • a Myc-tag binding short human NEDD4 (C867A) expression plasmid was constructed as described below.
  • Short-chain human NEDD4 (C867A) cDNA (see Example 7) was added with a Myc-tag coding sequence at its 5 'end, and then incorporated into an animal cell expression plasmid pCI (manufactured by Promega).
  • a tag-binding short human NEDD4 (C867A) expression plasmid hereinafter referred to as Myc short human NEDD4 (C867A) expression plasmid was constructed.
  • the Myc-short human NEDD4 (C867A) expression plasmid expresses Myc-short human NEDD4 (C867A) (hereinafter referred to as Myc short-chain NEDD4 (C867A)).
  • the expression plasmid prepared in Example 3 was used as the human ubiquitin (Ub) expression plasmid.
  • This human Ub expression plasmid expresses N-terminal HA-tag-linked human Ub (hereinafter referred to as HA-Ub).
  • Myc short chain was obtained by immunoprecipitation using anti-FLAG M2 antibody in a sample prepared by co-expression of FLAG-RUNX1, Myc short chain type NEDD4 and HA-Ub. Co-precipitation of type NEDD4 and FLAG—RUNX1 was observed. In addition, coprecipitation of Myc short chain type NEDD4 (C867A) and FL AG— RUNX1 was observed in samples prepared with cell force co-expressing Myc short chain type NEDD4 (C867A), FLAG—RUNX1 and HA—Ub. .
  • FLAG—RUNX1, Myc short chain NEDD4 and H as shown in Panel C of Figure 8
  • the protein detected by immunoprecipitation using anti-FLAG M2 antibody and immunoblotting using anti-HA antibody expressed My c—short-chain NEDD4 Compared with the prepared sample, the increased cell force was significantly increased.
  • the detected protein is FLAG-RUNX1 with HA-Ub added.
  • samples prepared from cells coexpressed with FLAG-RUNX1, Myc short-chain NEDD4 and HA-Ub showed an increase in FLAG-RUNX1 with HA-Ub added.
  • FLAG-RUNX1 with HA-Ub added was also detected in a sample prepared by co-expression of Myc short-chain NEDD4 (C86 7A) instead of Myc short-chain NEDD4.
  • the amount and the degree of ubiquitination were significantly lower than those of the cell-force-prepared samples expressing Myc short-chain NEDD4.
  • anti-FLAG M2 antibody was used in samples prepared by co-expression of FLAG-RUNX3, Myc short chain type NEDD4 and HA-Ub. Proteins detected by immunoprecipitation and immunoblotting using anti-HA antibody increased significantly compared to samples prepared with strong cell strength that did not express Myc-short chain type NEDD4. The detected protein is FLAG-RUNX3 with HA-Ub added. In other words, samples prepared from cells co-expressed with FLAG—RUNX3, Myc—short-chain NEDD4, and HA—Ub showed an increase in FLAG—RUNX3 with HA-Ub added.
  • short-chain human NEDD4 and short-chain human NEDD4 (C867A) bind to human RU NX3 intracellularly (Panel A in Fig. 9). It was also revealed that human RUNX3 is ubiquitinated in cells by short-chain human NEDD4 (Panel C in Figure 9), and that human RUNX3 is polymerized (Panel B in Figure 9). Furthermore, it was found that E3 ligase activity of short-chain human NEDD4 is important for ubiquitination and high molecularization of human RUNX3 by short-chain human NEDD4.
  • the human RUNX2 expression plasmid was constructed as described below.
  • Human RUNX2 cDNA was obtained from human bone marrow-derived cDNA (QUCIK—clone cDNA ⁇ Clontech) by PCR using primers with EcoRV site and Xhol site added, and the nucleotide sequence was confirmed by sequencing.
  • the obtained cDNA was incorporated at the EcoRVZXhoI site into an animal cell expression plasmid pCMV-Tag2 (STRATAGENE) for expressing an N-terminal FLAG-tag binding protein to construct a human RUNX2 expression plasmid.
  • the human RUNX2 expression plasmid expresses N-terminal FLAG-tag-linked human RUNX2 (hereinafter referred to as FLAG-RUNX2).
  • FLAG-RUNX2 N-terminal FLAG-tag-linked human RUNX2
  • the deduced amino acid sequence encoded by the cloned human RUNX2 cDNA was identical to that disclosed in the NCBI protein database accession number NP-004339 (registered gene RUNX2).
  • the expression plasmid prepared in Example 8 was used as the Myc short-chain human NEDD4 expression plasmid and the Myc short-chain human NEDD4 (C867 A) expression plasmid.
  • This Myc short human NEDD4 expression plasmid expresses Myc short human NEDD4.
  • This Myc short human NEDD4 (C867A) expression plasmid expresses Myc short human NE DD4 (C867A).
  • the expression plasmid prepared in Example 3 was used as the human ubiquitin (Ub) expression plasmid. This human Ub expression plasmid expresses HA-Ub.
  • Cell lysate was prepared by treating cells cultured for 2 days after transfection in the same manner as in Example 2. Next, Senolehuise ⁇ Tokoko protein G sepharose 4 FastFlow (Amersham Bioscienc es) 50% slurry was mixed at 40 / z L and mixed by inversion at 4 ° C for 1 hour. Then, centrifuge at 1,00 Orpm for 15 seconds at 4 ° C, add anti-FLAG M2 antibody (Sigma) 0. to the collected supernatant, and mix by inverting for 2.5 hours at 4 ° C.
  • Protein G sepharose 4 FastFlow 50% slurry was newly added, and the mixture was mixed again by inversion at 4 ° C for 2 hours.
  • Protein G sepharose 4 FastFlow is collected by centrifugation, washed 3 times with lysis buffer (same composition as described in Example 2), and then 20 ⁇ L of 2 X SDS sample buffer is added, and 100 ° A sample heat-treated for 5 minutes at C was used as an SDS-PAGE sample.
  • samples prepared from cells co-expressing FLAG-RUNX2, Myc short-chain NEDD4 and HA-Ub were subjected to Myc-by immunoprecipitation using anti-FLAG M2 antibody.
  • Short-chain NEDD4 and FLAG-RUNX2 were co-precipitated.
  • coprecipitation of Myc short-chain NEDD4 (C867A) and FLAG-RUNX2 was observed in cells prepared using cell force co-expressed Myc short-chain NEDD4 (C867A), FLAG-RUNX2 and HA-Ub. It was.
  • FLAG-RUNX2, Myc-short-chain NEDD4 and HA-Ub co-expressed samples were immunized with anti-FLAG M2 antibody. Proteins detected by immunoblotting using sedimentation and anti-HA antibody increased significantly compared to samples prepared with strong cell strength that did not express Myc-short chain type NEDD4. The detected protein is FLAG-RUNX2 with HA-Ub added. In other words, samples prepared from cells co-expressing FLAG—RUNX2, Myc—short-chain NEDD4, and HA—Ub showed an increase in FLAG—RUNX2 with HA-Ub added. On the other hand, in samples prepared from cells co-expressed with Myc—short chain type NEDD4 (C86 7A) instead of Myc—short chain type NEDD4, 1 ⁇ -1 ⁇ UNX2 No band was detected.
  • E3 ligase-inactive human NEDD4 was transiently expressed in cultured mouse cells, and alkaline phosphatase (hereinafter referred to as bone formation marker) (Abbreviated as ALP) activity was measured
  • C2C12 cells are mouse myoblasts and are derived from the same mesenchymal stem cells as osteoblasts and chondrocytes.
  • C2 C12 cells are stimulated by BMP-2 stimulation and are typically osteoblastic phenotypes such as ALP activity. It has been used as a model cell for osteogenesis depending on BMP-2 signal (Katagiri T. et al., “The Journal of Cell Biology (The Journal of Cell Biology), 1994, 127, p. 1755—1766).
  • BMP is a site force-in that induces bone tissue by differentiating and proliferating undifferentiated mesenchymal stem cells into chondrocytes and osteoblasts in vivo.
  • BMP-2 has been shown to be expressed early in the fracture healing process and is involved in the progression of a series of cascades in bone repair.
  • Short-chain human NEDD4 (C867A) was used as E3 ligase inactive human NEDD4.
  • the Myc-tag-linked short human NEDD4 (C867A) expression plasmid is designed and synthesized using primers that can introduce substitution of the 867th cysteine residue of a short human NEDD 4 to an alanine residue. It was constructed using a Quick Change Site Directed Mutagenesis Kit (manufactured by STRATAGENE). The constructed expression plasmid was sequenced to confirm that the mutation was introduced into the expression plasmid.
  • the Myc short-chain human NEDD4 (C867A) expression plasmid expresses N-terminal Myc-tag-linked short-chain human NEDD4 (C867A) (hereinafter referred to as Myc-short chain NEDD4 (C867A)).
  • Myc short-chain NEDD4 N-terminal Myc-tag binding short-chain human NEDD4 (hereinafter referred to as Myc short-chain NEDD4).
  • Myc-short human NEDD4 (C867A) expression plasmid was transfected into cells by electroporation using Nucleofector (Amaxa).
  • Myc short-chain human NEDD 4 (C867A) expression plasmid cells transfected with the Myc short-chain human NEDD4 expression plasmid were prepared in the same manner.
  • these expression plasmids instead, cells transfected with an empty vector (animal cell expression plasmid pCI) were prepared in the same manner and used as a control.
  • 1.6 ⁇ 10 5 cells were seeded on each well of the 6-well plate, and after culturing, the culture medium was treated with phenol red-free DMEMZ 5% Thiacol-dextran-treated FCS (Hyclone The medium was replaced with a medium supplemented with BMP-2 (R & D Systems) to a final concentration of 300 ngZml. After culturing for 3 days in the presence of BMP-2, the cells were collected, and a cell lysate was prepared with a lysis buffer (20 mM Tris (pH 8.0) /0.1% Triton X-100). After measuring the protein concentration of the cell lysate, ALP activity (Ab595nmZminZg) in each 20 g protein was measured using BluPhos Microwell Phosphatase Substrate System (KPL).
  • KPL BluPhos Microwell Phosphatase Substrate System
  • F-test was performed for the variance of ALP activity data of each treatment group, and Student's t test (Welch's t-test) or Welch's t test (Welch's t-test) was performed for the difference between the mean values.
  • Short human NEDD4 (C867A) is E3 ligase inactive short human NEDD4.
  • NM-010890-stealth-360 purchased from Invitrogen was used as the siRNA for mouse NEDD4 (published in NCBI database with accession number NM-010890!).
  • a control siRNA a negative control siRNA manufactured by QIAGEN was used.
  • Nedd4 The nucleotide sequences of sense RNA and antisense RNA constituting siRNA of mouse NEDD4 (hereinafter referred to as Nedd4) are shown below.
  • Sense RNA 5'— GGAGUUGAAUCCGAAUUCCCUGGAA— 3 ′ (SEQ ID NO: 19)
  • Antisense RNA 5 '-UUCCAGGGAAUUCGGAUUCAACUCC-3' (SEQ ID NO: 20)
  • siRNA was introduced into cells as described below.
  • Mouse myoblast C2C12 cells with 4 ⁇ 10 5 cells were seeded in a 6 cm dish and cultured in a DMEM medium containing 10% FBS at 37 ° C. under conditions of 5% CO 2/95% air.
  • Lipofectamione 2000 (Lipofectamione 2000, manufactured by Invitrogen) was added to the M medium and incubated at room temperature for 5 minutes. Then mix with 500 1 OPTI-MEM medium supplemented with 250 pmol Nedd4 siRNA or negative control siRNA (total lml) and incubate for additional 20 min at room temperature to prepare siRNA / 1 ipof ectamine2000 mixture did. The culture medium for the above cells was replaced with 3 ml of OPTI-MEM, and siRNAZlipofectamine 2000 mixture was added.
  • Nedd4 siRNA was examined as described below. After siRNA is introduced into the cells and cultured for 6 hours, the culture medium is treated with phenol red-free Caro DMEMZ 5% Thiacol-dextran treated FCS (Hyclone) and BMP-2 (R & D Systems) to a final concentration of 600 ngZml. The medium was replaced with After culturing for 3 days in the presence of BMP-2, the cells were collected and lysis buffer (20 mM Tris (p A cell lysate was prepared with H8.0) /0.1% Triton X-100). After measuring the protein concentration in the cell lysate, the ALP activity (Ab595nmZminZg) in each 20 ⁇ g protein was measured using Blu Phos Microwell Phosphatase Substrate System (KPL).
  • KPL Blu Phos Microwell Phosphatase Substrate System
  • ALP activity of cell lysate prepared from cells transfected with Nedd4 siRNA compared to ALP activity of cell lysate prepared from cells transfected with negative control siRNA under no addition of BMP-2
  • the relative value was calculated, and the effect of Nedd4 siRNA on the bone differentiation effect of BMP-2 was evaluated based on the obtained relative value.
  • Statistical processing was performed on the obtained data. For the variance of ALP activity data of each treatment group, F test was performed, and for difference in mean value, Student's t test or Welch's t test was performed.
  • the relative value of the concentration of Nedd4 band detected in each cell relative to the concentration of Nedd4 band detected in BMP-2 untreated negative control siRNA-introduced cells was calculated.
  • the concentration of the Nedd 4 band detected in each cell was corrected by the concentration of the actin band.
  • Nedd4 siRNA-treated cells ALP activity by BMP-2 stimulation was increased approximately 2-fold compared to negative control siRNA-treated cells (Panel A in Fig. 12).
  • Nedd4 expression was markedly inhibited in Nedd4 siRNA-treated cells (panel B in Figure 12).
  • Nedd4 knockdown rate by Nedd4 siRNA was more than 50%.
  • endogenous NEDD4 was knocked down by transfection of NEDD4 siRNA in human cancer cell lines, and the proliferation of the cancer cell lines was measured.
  • Human eclampsia cancer cell line HeLa and human gastric cancer cell line NCI-N87 were used as human cancer cell lines.
  • siRNA of human NEDD4 (published in NCBI database with accession number NM—006154) was purchased from Invitrogen using D42055—stealth—757, D42 055—stealth—1053 and D42055—stealth—1376 did. In addition, QIAGEN's negative control siRNA was used as the control siRNA.
  • Sense RNA 5, — GGACAACCUAACAGAUGCUGAGAAU— 3, (SEQ ID NO: 2 1)
  • Antisense RNA 5'— AUUCUCAGCAUCUGUUAGGUUGUCC— 3 '(SEQ ID NO: 22)
  • Sense RNA 5'— GCAGAAGAGGCAGCUUACAAGCCUA— 3 ′ (SEQ ID NO: 2 3)
  • Antisense RNA 5 '-UAGGCUUGUAAGCUGCCUCUUCUGC-3' (SEQ ID NO: 24)
  • Sense RNA 5'—GCACCAAAUGGGAGGCCUUUCUUUA—3 '(SEQ ID NO: 25)
  • Antisense RNA 5 '-U AAAG AAAGGCCUCCC AUUUGGUGC-3' (Column number 26)
  • NCI-N87 cells were seeded on a 24 well plate at 5 X 10 4 Zwell, and in 10% FBS-containing RPMI164 0 medium at 37 ° C, 5% CO / 95% air for 2 days. After incubation, transfer
  • siRNA introduction amount is equivalent to 2 OpmolZwell
  • This sample was separated on a 5-20% acrylamide gel, and the primary antibody was labeled with anti-NEDD4 antibody H-135 and anti-beta-tubulin antibody H-235 (Santa Cruz Biotechnology).
  • Western blotting using a goat anti-rabbit IgG antibody Alexa Fluor 680, goat anti-rab bit IgG, manufactured by Molecular Probe was performed.
  • the detection and quantification of the protein band was performed by an Odyssey imaging system (Aloka).
  • the knockdown effect of NEDD4 by siRNA was evaluated based on the ratio of the concentration of protein band detected in each NEDD 4 siRNA treatment group to the concentration of NEDD4 protein band detected in the negative control siRNA treatment group.
  • a method for ubiquitinating RUNX using NEDD4 or short-chain NEDD4 can be provided.
  • the identification method can be provided.
  • NEDD4, or a compound that promotes RUNX ubiquitination by NEDD4 or short-chain NEDD4 can be used to promote RUNX ubiquitination by NEDD4 or short-chain NED D4, thereby promoting RUNX degradation it can.
  • RUNX ubiquitination with NEDD4 or short-chain NEDD4 using inactive NEDD4, a double-stranded polynucleotide that inhibits the expression of NEDD4, or a compound that inhibits RUNX ubiquitination with NEDD4 or short-chain NEDD4 Can be inhibited, thereby inhibiting the degradation of RUNX.
  • RUNX ubiquitin and its degradation it is possible to prevent and Z or treat diseases caused by RU NX abnormalities. Since RUNX is considered to be involved in tumor formation and exacerbation of cancer diseases, various cancer diseases can be prevented and / or treated according to the present invention. Further, since RUNX2 is considered to be involved in bone formation, the present invention can prevent and Z or treat bone loss diseases.
  • the present invention it is possible to provide a method for identifying a compound capable of promoting bone formation, characterized by identifying a compound that inhibits the function of NEDD4 or short-chain NEDD4. Since the compound obtained by this identification method is a compound capable of promoting bone formation, it can be used as an effective component for prevention and Z or treatment of osteoporosis such as bone loss disease.
  • the present invention is useful for basic research and drug development regarding RUNX degradation mechanism, transcription mechanism involving RUNX, and diseases caused by RUNX abnormality. Furthermore, the present invention can be used for the prevention and Z or treatment of diseases caused by abnormalities in RUNX, such as cancer diseases. In addition, the present invention is useful for the prevention of bone loss diseases such as osteoporosis and the development of Z or therapeutic agents.
  • SEQ ID NO: 1 Polynucleotide encoding human NEDD4 (SEQ ID NO: 2).
  • SEQ ID NO: 2 human NEDD4.
  • SEQ ID NO: 3 A polynucleotide encoding a protein (SEQ ID NO: 4) in which the 100th amino acid residue at the N-terminal side of human NEDD4 (SEQ ID NO: 2) has also been deleted.
  • SEQ ID NO: 4 A protein from which the 100th amino acid residue at the N-terminal side of human NEDD4 (SEQ ID NO: 2) has also been deleted.
  • SEQ ID NO: 5 Polynucleotide encoding human RUNX1 (SEQ ID NO: 6).
  • SEQ ID NO: 6 human RUNX1.
  • SEQ ID NO: 7 Polynucleotide encoding human RUNX2 (SEQ ID NO: 8).
  • SEQ ID NO: 8 human RUNX2.
  • SEQ ID NO: 9 Polynucleotide encoding human RUNX3 (SEQ ID NO: 10).
  • SEQ ID NO: 10 human RUNX3.
  • SEQ ID NO: 11 Inactive mutant of human NEDD4 (SEQ ID NO: 2).
  • SEQ ID NO: 12 Inactive mutant of a protein (SEQ ID NO: 4) in which the 100th amino acid residue in the N-terminal side of human NEDD4 (SEQ ID NO: 2) is deleted in the 100th amino acid.
  • SEQ ID NO: 13 Human RU having high homology with a partial sequence of human NEDD4 (SEQ ID NO: 2)
  • SEQ ID NO: 14 Partial sequence of human NE DD4 (SEQ ID NO: 2) having high homology with the partial sequence of human RUNX3 (SEQ ID NO: 10).
  • SEQ ID NO: 15 Partial sequence of human RU NX3 (SEQ ID NO: 10) having high homology with the partial sequence of human NEDD4 (SEQ ID NO: 2).
  • SEQ ID NO: 16 Partial sequence of human NE DD4 (SEQ ID NO: 2) having high homology with the partial sequence of human RUNX3 (SEQ ID NO: 10).
  • SEQ ID NO: 17 Partial sequence of human RU NX3 (SEQ ID NO: 10) having high homology with the partial sequence of human NEDD4 (SEQ ID NO: 2).
  • SEQ ID NO: 18 Partial sequence of human NE DD4 (SEQ ID NO: 2) having high homology with the partial sequence of human RUNX3 (SEQ ID NO: 10).
  • SEQ ID NO: 19 sense RNA constituting siRNA for mouse Nedd4
  • SEQ ID NO: 20 Antisense RNA constituting siRNA against mouse Nedd4
  • SEQ ID NO: 21 sense RNA constituting siRNA against human NEDD4
  • Sequence number 22 Antisense RNA which comprises siRNA with respect to human NEDD4.
  • Sequence number 23 The sense RNA which comprises siRNA with respect to human NEDD4.
  • SEQ ID NO: 24 antisense RNA constituting siRNA against human NEDD4
  • SEQ ID NO: 25 sense RNA constituting siRNA against human NEDD4
  • SEQ ID NO: 26 antisense RNA constituting siRNA against human NEDD4

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Abstract

Le problème à résoudre dans le cadre de la présente invention consiste à trouver une protéine capable d’interagir avec du RUNX afin de réguler l’action du RUNX, de procurer un moyen de régulation de l’action du RUNX et de procurer un moyen utile pour la prévention et/ou le traitement d’une maladie causée par une anormalité du RUNX. La solution proposée consiste en le NEDD5 qui est une ligase HECT E3 qui peut ubiquitiner le RUNX de manière à causer une dégradation du RUNX. Sur la base de cette découverte, l’invention concerne un procédé d’ubiquitination du RUNX ou un procédé de dégradation du RUNX caractérisé en ce qu’il utilise du NEDD4 ; un agent d’ubiquitination ou un agent de dégradation de RUNX ; un procédé d’inhibition de l’ubiquitination ou de la dégradation de RUNX par NEDD4 ; un inhibiteur d’ubiquitination ou un inhibiteur de dégradation pour le RUNX, un procédé d’identification d’un composé capable d’inhiber ou d’améliorer l’ubiquitination de RUNX ; un procédé d’identification d’un composé capable d’inhiber ou d’améliorer la liaison entre RUNX et NEDD4 ; un kit de réactifs ; et un procédé de prévention et/ou de traitement d’une maladie causée par une anormalité du RUNX.
PCT/JP2006/311333 2005-06-07 2006-06-06 Procédé d’ubiquitination du runx WO2006132248A1 (fr)

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JP2004529619A (ja) * 2001-01-29 2004-09-30 スク−チュル バエ 抗癌活性を示すｒｕｎｘ３遺伝子及びその使用方法
WO2002090549A2 (fr) * 2001-03-12 2002-11-14 Proteologics Inc. Compositions et methodes destinees a la modulation de la maturation virale
JP2005204557A (ja) * 2004-01-22 2005-08-04 Teijin Pharma Ltd 変形性関節症関連遺伝子

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008153814A2 (fr) * 2007-05-29 2008-12-18 President And Fellows Of Harvard College Molécules impliquées dans la régulation de l'activité des ostéoblastes et des ostéoclastes, et leurs méthodes d'utilisation
WO2008153814A3 (fr) * 2007-05-29 2009-02-05 Harvard College Molécules impliquées dans la régulation de l'activité des ostéoblastes et des ostéoclastes, et leurs méthodes d'utilisation
US8357637B2 (en) 2007-05-29 2013-01-22 Cornell University Molecules involved in regulation of osteoblast activity and osteoclast activity, and methods of use thereof
US9745589B2 (en) 2010-01-14 2017-08-29 Cornell University Methods for modulating skeletal remodeling and patterning by modulating SHN2 activity, SHN3 activity, or SHN2 and SHN3 activity in combination

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