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WO2007010651A1 - Nouvelles protéines et méthode pour les utiliser - Google Patents

Nouvelles protéines et méthode pour les utiliser Download PDF

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Publication number
WO2007010651A1
WO2007010651A1 PCT/JP2006/307138 JP2006307138W WO2007010651A1 WO 2007010651 A1 WO2007010651 A1 WO 2007010651A1 JP 2006307138 W JP2006307138 W JP 2006307138W WO 2007010651 A1 WO2007010651 A1 WO 2007010651A1
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Prior art keywords
protein
amino acid
serine
dna
cell
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PCT/JP2006/307138
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English (en)
Japanese (ja)
Inventor
Tatsuya Ingi
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Niigata University
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Priority to JP2007525900A priority Critical patent/JPWO2007010651A1/ja
Publication of WO2007010651A1 publication Critical patent/WO2007010651A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/82—Translation products from oncogenes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12Q1/527—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving lyase
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • 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
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer

Definitions

  • the present invention relates to a novel protein and a method for using the same, and more specifically, a novel protein having a function of increasing membrane lipid synthesis by binding to serine synthase and incorporating the amino acid serine into the cell membrane. Regarding the method.
  • Non-Patent Documents 1 and 2 describe TDE1 (member of the above-mentioned protein family with unknown functions) in human lung tumors and mouse testicular tumors. ) Is increased.
  • Non-Patent Document 3 reports that it is speculated that yeast TMS-1 (a member of the protein family with unknown function) takes a membrane topology having 11 transmembrane segments similar to amino acid transporters. Has been.
  • Non-Patent Document 4 reports that mouse TMS-1 and TMS-2 (members of the protein family with unknown function) are expressed in nerves and localized in the membrane.
  • Non-Patent Document 5 discloses that expression of TPOl (a member of the protein family with unknown function) is induced in the final differentiation stage of cultured oligodendrocytes.
  • Non-Patent Document 1 Bossolasco, M., Lebel, M., Lemieux, N., and Mes- Masson, A. M. (19 99) Mol Carcinog 26 (3), 189-200.
  • Non-Patent Document 2 Lebel, M., and Mes- Masson, A.M. (1994) DNA Seq 5 (1), 31-9.
  • Non-Patent Document 3 De Hertogh, B., Carvajal, E., Talk, E., Dujon, B., Baret, P., and Gof feau, A. (2002) Funct Integr Genomics 2 (4-5), 154-70.
  • Non-Patent Document 4 Grossman, T. R "Luque, JM, and Nelso, N. (2000) J Neurosci 13 (7), 1459-63.
  • Non-Patent Document 5 Krueger, WH, Gonye, GE, Madison, DL, Murray, KE, Kumar, M., Spoerel, N., and Pfeiffer, SE (1997) J Neurochem 69 (4), 1343-55.
  • Non-Patent Documents 1 to 5 suggest that the protein family with unknown function performs important functions in many aspects of cell biology.
  • the protein with unknown function has multiple subtypes and constitutes a protein family among organisms, but does not show homology with known proteins and its function is also clear. It was not.
  • the present invention provides a novel protein and a method for using the same, by clarifying the function of the protein family with unknown function, to obtain knowledge about cancer, and as a clue in developing an anticancer drug and a tumor marker.
  • the purpose is to provide.
  • the novel protein of the present invention has been named celink by the present inventors. Furthermore, it was found that the expression level of celink mRNA also changes during plastic regeneration of neural circuits. These facts suggest that the action of celink is necessary in the process of removing the existing cell-cell linkage and constructing a new membrane-cell junction, which led to the completion of the present invention. It was. That is, the present invention is as follows.
  • Claim 1 of the present invention is the protein according to any one of the following (a) to (b). : (a) a protein having an amino acid sequence ability according to any one of SEQ ID NOs: 1 to 9,
  • the second aspect of the present invention is the DNA according to any one of the following (a) to (b):
  • DNA that encodes the protein having the amino acid sequence ability described in any one of SEQ ID NOS: 1 to 9 is hybridized under stringent conditions and binds to serine synthase, and the amino acid serine is bound to the cell membrane.
  • Claim 3 of the present invention is a recombinant vector containing the DNA of claim 2.
  • Claim 4 of the present invention is a transformant comprising the recombinant vector according to claim 3.
  • Claim 5 of the present invention is equivalent to the protein represented by the amino acid sequence of claim 1 obtained by culturing the transformant of claim 4 and cultivating the transformant.
  • a method for producing the protein comprising collecting a protein having the following functions.
  • Claim 6 of the present invention is an antibody against the protein of claim 1.
  • Claim 7 of the present invention is an antibody against membrane lipid that increases when the protein of claim 1 is expressed on a cell membrane.
  • Claim 8 of the present invention is an antisense nucleic acid characterized in that the base sequence is a sequence complementary to all or part of the DNA of claim 2.
  • Claim 9 of the present invention is a screening method for a candidate compound for a drug, comprising the following steps (a) to (c). (a) A step of contacting the test compound with the protein according to claim 1.
  • Claim 10 of the present invention is a method for screening a candidate compound for a drug, comprising the following steps (a) to (c).
  • Claim 11 of the present invention is the claim 8, wherein the drug is any one of an anticancer agent, a cell growth regulator, and a neural circuit regenerative agent.
  • the selinic gene can be retained by obtaining a recombinant vector containing the selinic gene.
  • a selin gene can be retained by obtaining a transformant containing a recombinant vector containing a selin gene.
  • a recombinant cellulose protein can be produced.
  • an antibody against a serine protein is obtained.
  • celink This makes it possible to use it as a new tumor marker and to selectively attack cancer.
  • the activity of the serine protein can be changed by obtaining the antisense nucleic acid.
  • drugs can be efficiently screened using the activity of celink as an index.
  • the drug can be efficiently screened using the expression level of celink as an index.
  • the drug is one of an anticancer agent, a cell growth regulator, and a neural circuit regenerative agent.
  • an anticancer agent, a cell growth regulator, or a neural circuit regeneration agent can be efficiently screened.
  • FIG. 1 is a diagram showing a yeast two-hybrid experiment using rat cellulose. The results of experiments using SER3, YGP1 and PH012 as controls and the control results are shown.
  • FIG. 2 shows the alignment of amino acid sequences of the serine protein family.
  • FIG. 3 shows the separation analysis of [ 3 H] phosphatidylserine and [ 3 H] sphingolipid using thin-layer chromatography.
  • the left figure of FIG. 3A shows phosphatidylserine (PS) and phosphatidylethanolamine (PE) detected by ninhydrin staining.
  • the band in Fig. 3B is 3-ketojihide mouth-opening sphingosin (KS).
  • FIG. 4 is a diagram showing analysis of mRNA distribution and induction in rat brain of celink 1, celink 2, and celink 5 using in situ hybridization.
  • the left brain section (C-side) is derived from a normal rat, and the right brain section (S-side) is derived from a kainic acid-stimulated rat.
  • the site where the serine mRNA changes is indicated by ⁇ and *.
  • Black arrows indicate increased mRNA and open arrows indicate decreased mRNA.
  • arb cerebellar vitality
  • cc brain cerebral
  • CA hippocampus CA region
  • dg hippocampal dentate gyrus
  • ec outer envelope
  • gel cerebellar granule cell layer
  • hip hippocampus
  • neo neocortex
  • pc parietal cortex
  • pel cerebellar Purkinje cell layer
  • the present invention relates to a novel protein having a function of increasing membrane lipid synthesis by binding to serine synthase and incorporating the amino acid serine into the cell membrane, and a method for using the same.
  • the amino acid sequence of celink is represented by the sequence described in any one of SEQ ID NOs: 1 to 9. That is, the serine having the amino acid sequence ability described in any one of SEQ ID NOs: 1 to 9 is a protein having a function of increasing membrane lipid synthesis by binding to serine synthase and incorporating amino acid serine into the cell membrane. . Details of each of the amino acid sequences described in SEQ ID NOs: 1 to 9 are shown in the Examples.
  • the protein of the present invention can be produced from a eukaryotic cell or tissue (for example, cancer cell or nerve cell) by a known protein purification method, or a transformant containing DNA encoding the protein. Can also be produced by culturing. It can also be produced according to the peptide synthesis method.
  • celink provides new insights by controlling the contact between neurons and glial cells to promote the formation and regeneration of new neural circuits. These functions of celink suggest that substances that control the activity or expression of celink can be anticancer agents, cell growth control agents, and neural circuit regeneration agents.
  • the present invention includes an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence described in any one of SEQ ID NOs: 1 to 9, and binds to serine synthase, and is bound to the cell membrane. It includes proteins that have the function of increasing membrane lipid synthesis by incorporating the amino acid serine. These can be considered as serine mutants.
  • the serine mutant may have, for example, at least one amino acid deleted in the amino acid sequence described in any one of SEQ ID NOs: 1 to 9.
  • at least one amino acid may be attached, or at least one amino acid of the amino acid sequence described in any one of SEQ ID NOs: 1 to 9 is substituted with another amino acid.
  • the serine mutant is an amino acid having about 70% or more, preferably about 80% or more, particularly preferably about 90% or more homology with the amino acid sequence of any one of SEQ ID NOs: 1 to 9. Examples include sequences.
  • Such amino acid modification is caused by genetic polymorphism. In addition to being recognized in nature, such as mutations caused by, etc., artificially using methods known to those skilled in the art, such as mutagenesis using mutagens and site-specific mutagenesis using PCR, etc. It can be carried out.
  • the present invention provides a celink gene.
  • the sellin gene is a DNA encoding a protein comprising the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 9.
  • the present invention hybridizes under stringent conditions with a DNA encoding a protein having the amino acid sequence ability described in any one of SEQ ID NOS: 1 to 9, and binds to a serine synthase. It contains DNA encoding a protein that has the function of increasing membrane lipid synthesis by incorporating the amino acid serine into the cell membrane.
  • stringent conditions refer to conditions in which only specific hybridization occurs and non-specific hybridization and non-hybridization do not occur. Is 50 ° C and the salt concentration is 5 X SSC (or equivalent salt concentration).
  • the gene selected by the hybridization procedure may be a natural gene or an artificial gene by PCR. For example, it can be obtained by screening a cDNA library.
  • DNA of the present invention is prepared.
  • screening from a cDNA library is first performed.
  • MRNA forces in tissues or cells expressing celink also create a cDNA library.
  • the DNA of the present invention can be obtained.
  • the base sequence of the DNA is determined by analyzing the base sequence of the cDNA from the end using a base sequence analyzer generally used.
  • each cDNA has a new sequence can be determined by searching databases such as GenBank, EMBL, and DDBJ using a homology search program such as BLAST. Can be compared with .
  • the amino acid sequence of a protein can be specified based on the DNA base sequence.
  • the DNA of the present invention can also be obtained by chemical synthesis with a DNA synthesizer based on the determined DNA base sequence. Any of these methods are well known to those skilled in the art, and either method can be used.
  • the present invention provides a recombinant vector containing the DNA according to claim 2.
  • a recombinant vector can be obtained by inserting the DNA described in claim 2 of the present invention into an appropriate vector.
  • Such a vector is not particularly limited as long as it can replicate in the host, and examples thereof include plasmid DNA, phage DNA and the like.
  • animal viruses such as retroviruses, adenoviruses or vaccinia viruses, and insect virus vectors such as baculoviruses can also be used.
  • the purified DNA is cleaved with an appropriate restriction enzyme, and if necessary, a promoter sequence that controls transcription is added upstream, and a restriction enzyme site or multiple of the appropriate vector DNA is added. Insert into the crawling site and link to vector.
  • the expression vector preferably contains a promoter, an enhancer, a selectable marker, etc. in order to express the target DNA when introduced into a suitable host. Both of these methods are known in the art.
  • the present invention also provides a transformant comprising the above recombinant vector.
  • host cells include prokaryotic cells such as Escherichia coli and Bacillus subtilis, and cultured mammalian cells such as yeast, insect cells, monkey kidney cells COS7, and Chinese hamster ovary cells CHO.
  • a host cell can be transformed with a recombinant vector according to a conventionally known method. For example, when expressed in a microorganism such as Escherichia coli, the expression vector is introduced by introducing the above-mentioned link gene into an expression vector having an origin, promoter, ribosome binding site, DNA cloning site, terminator, etc. that can replicate in the microorganism. And a host cell can be transformed with this expression vector.
  • the present invention is the same as the protein represented by the amino acid sequence described in claim 1 from the culture obtained by culturing the transformant described in claim 4.
  • a method for producing the protein which comprises recovering a protein having a function.
  • a host transformed with an expression plasmid containing DNA encoding celink is cultured and routinely used. Therefore, it is cultured.
  • the enzyme-containing aqueous solution can be obtained by extracting the culture or cells with water or an aqueous buffer such as a phosphate buffer. In order to collect and purify the enzyme from the enzyme-containing solution, a conventional method for purifying the enzyme may be used.
  • the present invention includes not only proteins obtained by the recombinant method but also proteins isolated and purified from tissues or those produced by peptide chemical synthesis.
  • the present invention provides an antibody against the protein according to claim 1. Since antibodies specifically recognize specific molecules, they can serve as molecular markers. In addition, since a specific cell or molecule such as a cancer cell can be targeted, it can be an effective pharmaceutical that attacks only the target cell or molecule. As described above, it has been shown that celink is involved in the process of various cell types such as cancer cells and neurons that have been released from conventional cell-cell linkages and construct new cell-cell membrane junctions. Therefore, the antibody of the present invention can be used as a new tumor marker and cancer selective aggressive drug.
  • the present invention also provides an antibody against membrane lipid that increases when the protein of claim 1 is expressed on a cell membrane.
  • celink has a function of increasing membrane lipid synthesis by incorporating amino acid selenium into the cell membrane. Therefore, an antibody against membrane lipid that increases when the protein according to claim 1 is expressed on the cell membrane can be a marker for serine.
  • Specific examples of the membrane lipid that increases when the protein of claim 1 is expressed on the cell membrane include sphingolipid and phosphatidylserine. These lipids are described in detail in the following examples. These membrane lipid antibodies can also be used as new tumor markers and cancer-selective aggressive drugs, as are the antibodies against the protein described in claim 1.
  • a monoclonal antibody-producing cell is prepared.
  • animals are immunized with an antigen containing a serine protein, a fragment thereof, or a membrane lipid.
  • Immunization is carried out by administering intravenously, subcutaneously or intraperitoneally to mammals. Mammals include, for example, monkeys, rabbits, dogs, guinea pigs, mice, rats, hidges, goats, and -birds. Is preferably used.
  • Administration is usually once every 2 to 6 weeks, 2 to 10 times in total
  • Monoclonal antibody-producing hybridomas can be prepared by cell fusion with myeloma cells.
  • myeloma cells cell lines derived from animals such as mice and generally available can be used. Examples of myeloma cells include NS-1, P3U1, SP2 / 0, AP-1, and other animal myeloma cells. The cell fusion operation is performed according to a known method.
  • cell fusion can be achieved by performing pulse treatment (for example, electoral position).
  • pulse treatment for example, electoral position.
  • cell fusion operation for example, the following documents can be referred to (Kohler, G .; Milstein, C. (1975) Nature. 256: 495-97) o
  • Various methods can be used to screen for monoclonal antibody-producing hybridomas. For example, screening can be performed based on whether or not the target antibody is present in the culture supernatant of the grown hyperidoma. . At this time, a part of the culture supernatant contained in the well grown as a hyperidoma can be collected and screened by immunostaining, enzyme immunoassay (ELISA), RIA or the like. For example, add the supernatant of a hyperidoma culture to a solid phase (eg, a microplate) on which a protein antigen is adsorbed directly or with a carrier, and then add an anti-immunoglobulin antibody labeled with a radioactive substance or enzyme (for cell fusion). For example, when the cells used are mice, anti-mouse immunoglobulin antibodies are used) and monoclonal antibodies bound to the solid phase are detected.
  • a solid phase eg, a microplate
  • Cloning of the fused cells can be performed by a limiting dilution method or the like.
  • a normal cell culture method can be employed.
  • the above hyperidoma is cultured in a serum-containing medium, and the antibody is also obtained from the culture supernatant.
  • antibody purification it can be purified according to known methods. For example, ammonium sulfate fractionation, ion exchange chromatography, Purification can be performed by appropriately selecting a known method such as affinity chromatography or gel chromatography.
  • a polyclonal antibody against the protein of the present invention can be produced by collecting immune serum from an immunized animal according to a method known in the art.
  • the invention includes fragments that are monoclonal or polyclonal antibodies or portions thereof.
  • the method is not limited and a method known in the related art can be used. Examples include immunostaining, enzyme immunoassay (ELISA), RIA and the like.
  • the present invention provides an antisense nucleic acid characterized in that the base sequence is a sequence complementary to all or part of the nucleic acid of the DNA of claim 2.
  • the antisense nucleic acid means the DNA corresponding to the antisense strand of double-stranded DNA or RNA corresponding to the DNA of the antisense strand, which binds to DNA or RNA and regulates the expression of celink.
  • Antisense nucleic acid can inhibit biosynthesis of slinkin at the nucleic acid level in vivo.
  • Antisense nucleic acids are well known in the art and can be produced by known methods.
  • the antisense nucleic acid of the present invention can be obtained by PCR using a DNA that encodes serine, which is natural DNA or RNA, as a saddle.
  • the present invention provides a screening method for a candidate compound for a drug, comprising the following steps (a) to (c).
  • the protein according to claim 1 may include the protein according to claim 1 as long as the protein according to claim 1 is included. Also includes transformants that express! [0055] The activity is based on the nature of celinke when it binds to serine synthase and incorporates the amino acid serine for lipid synthesis of cell membranes.
  • a compound isolated as a compound that alters the activity of the protein of the present invention by the screening method of the present invention is a candidate compound for a therapeutic agent such as an anticancer agent, a cell growth regulator, or a neural circuit regenerative agent. It is expected.
  • the screening method of the present invention is effective as long as it is a method capable of obtaining a substance that acts on the protein of claim 1 and has an action of changing the activity of the protein. May be.
  • the protein according to claim 1 is contacted with a test compound, and the test compound is further selected using the change in the activity of the protein as an index after selecting the binding activity with the protein as an index. You can also use this method.
  • the test substance may be any substance as long as it can interact with the protein according to claim 1 and affect the activity of the protein.
  • Yo! / Specific examples include peptides, proteins, non-peptidic compounds, low molecular compounds, synthetic compounds, fermentation products, cell extracts, animal tissue extracts, and the like.
  • a method for analyzing the binding activity between the test compound and the protein a conventionally known method can be used. Specifically, for example, yeast Tsuno, hybrid method, fluorescence depolarization method, surface plasmon method, phage display method, and ribosomal display method can be mentioned.
  • the test compound can be further selected and analyzed by analyzing the change in the activity of the protein as an index. For example, by selecting candidate compounds that change the activity of taking in the amino acid serine in cytoplasmic lipid synthesis, candidate compounds such as anticancer agents, cell growth regulators, and neural circuit regenerative agents can be screened. As shown in the examples below, serine uses serine labeled with a radioactive isotope or a fluorescent dye to analyze the activity of serine uptake of amino acid serine in cytoplasmic lipid synthesis. A method for evaluating whether or not the ability to incorporate into lipid components is mentioned.
  • the expression level of celink can be used as an index. That is, the present invention provides a candidate compound for a drug comprising the following steps (a) to (c): Including a screening method.
  • the protein expression level can be measured by methods known to those skilled in the art. For example, sericin mRNA can be extracted from cells expressing sericin according to a standard method, and then the slinkin gene level can be measured by carrying out the Northern hybridization method or RT-PCR method using this mRNA as a cocoon. It can be carried out.
  • the cellin protein fraction that expresses celink can be collected, and the expression of celink can be measured by detecting the expression of cerine by electrophoresis such as SDS-PAGE.
  • it is also possible to measure the expression of celink by detecting the expression of the protein by carrying out Western blotting using an antibody against celink. As described above, by selecting a candidate compound that changes the expression level of serine, a candidate compound such as an anticancer agent, a cell growth regulator, or a neural circuit regenerative agent can be screened.
  • SELINK 1 was searched in the NCBI database.
  • Sellin 1 showed 58% and 38% homology with the reported proteins of unknown function TDE1 and TPO1, respectively, and named them Sellin 3 (SEQ ID NO: 3) and Sellin 5 (SEQ ID NO: 5), respectively.
  • a search using a database identified celink 2 (SEQ ID NO: 2), celink 3B (SEQ ID NO: 6), celink 4 (SEQ ID NO: 4), and celink 4B (SEQ ID NO: 7). These proteins showed a high homology of 31% to 58% and were found to form a serine protein family.
  • serine 4 (SEQ ID NO :) was derived from a mouse, but human 'Cerlink 4 (SEQ ID NO: 8) was later identified. Furthermore, as described above, yeast TMS1 (SEQ ID NO: 9) belongs to the serine protein family, and corresponds to serine 3. The Genbank accession number of the above link is shown below.
  • rat sericin 1 SEQ ID NO: 1
  • Yeast two-hybrid experiments were carried out using rat celite 1 as a bait against three yeast proteins (SER3, YGP1, PH012).
  • PH 012 is an acid phosphatase that directly binds to SER3.
  • the yeast two-hybrid experiment The following literature method was modified (Cagney, G., Uetz, P., and Fields, S. (2000) Methods Enzymol 328, 3-14). At this time, DNA binding domain vector pOBD 2 and Gal4 activation domain vector pOAD were used.
  • rat sericin 1 a vector in which rat sericin 1 is introduced into POBD2
  • yeast strain PJ69-4a which expresses SER3, YGP1, and PH012 as Gal4 active proteins
  • S.c EasyComp transformation kit dnvitrogen, Carlsbad, CA was used as a transformation reagent. Tryptophan 'leucine deletion synthetic culture The yeast suspension was streaked on the ground and incubated at 30 ° C for 7 days. After colony formation, the colony was streaked onto tryptophan 'leucine' histidine-deficient synthetic medium.
  • FIG. 1 shows the results of a yeast two-hybrid experiment using rat sericin.
  • SER3 and Y GP1 showed significant colony growth. No colonies were formed in the control.
  • PH012 showed a weak colony growth.
  • rat celink 1 has strong binding strength against SER3 and YGP 1, weak binding strength against PHO 12, and binding strength.
  • This result indicates that rat celinkin 1 specifically binds to serine synthase, similar to TMS1, a member of the yeast sericin family. This result revealed that the function of specific binding of serine to serine synthase is conserved among different members of the serine family.
  • Figure 2 shows the amino acid sequence alignment of the five mammalian subtypes (Cerlink 1, 2, 3, 4, 5) and yeast TMS1. Amino acid residues conserved in three or more proteins are displayed in color, and 11 regions predicted to be transmembrane sites are displayed in I-XI.
  • phosphatidylserine which is a constituent of cell membrane, is synthesized by base exchange reaction in which the head group of the existing phospholipid replaces serine.
  • Serine 'palmitoyltransferase condenses serine • palmitoyl CoA and serine to produce 3-ketodihydrate-mouthed sphingosin (KS).
  • KS 3-ketodihydrate mouth sphingosin
  • KS 3-ketodihydrate mouth sphingosin
  • SV40-transformed African green monkey kidney (COS-7) cells were cultured in DMEM medium supplemented with 10% ushi fetal serum.
  • the base exchange reaction mixture consisted of 50 mM Tris—HC1, pH 7.4, 50 mM KC1, 15 mM CaCl, 0.5 mM [ 3 H] serine (60 / z Ci) and a predetermined amount of cell disruption solution. To 0.
  • Serine 'palmitoyltransferase reaction mixture was prepared with 0.1 mM Tris-HCl, pH 8.
  • the lipid extract was analyzed by thin layer chromatography.
  • lipid extracts were dried under nitrogen, dissolved in black mouth form Z methanol (2: 1) and spotted on Silica Gel 60 HPTLC plates (Merck, Darmstadt, Germany).
  • the lipid extract was developed in thin-layer chromatography using black mouth form Z methanol Z acetic acid (65 Z25Z10).
  • 3-Ketoji Hyde Mouth Sphingosine When Atssey, Black Mouth Form Z Methanol Z2N NH
  • Lipid extracts were developed in thin layer chromatography using OH (80Z20Zl). To detect radioactive bands, plates were treated with 2-methylnaphthalene containing 0.4% 2,5-diphenyloxazole and exposed to X-OMAT film at 80 ° C for 1-4 days. . In order to quantify the radioactivity of each band, silica gel was cut out and liquid scintillation measurement was performed using Econofluor.
  • results The left figure in FIG. 3A shows phosphatidylserine (PS) and phosphatidylethanolamine (PE) detected by ninhydrin staining.
  • the right figure in FIG. 3A shows that serine labeled with [ 3 H] is incorporated into phosphatidylserine (PS).
  • Figure 4 shows that serine labeled with [ 3 H] is incorporated into 3-ketodinoid mouth sphingosine (KS).
  • PS phosphatidylserine
  • PE phosphatidylethanolamine
  • Hybridization was performed according to the method described in the following document using three rat Slink (Serlink 1, 2, and 5) riboprobes labeled with 35 S (Ingi, T , and Aoki, Y. (2002) Eur J Neurosci 15 (5), 929-36).
  • Results The brains of rats that had been treated with kainic acid and caused an epileptic seizure were removed, and changes in mRNA expression of celink 1, 2, and 5 were examined.
  • the left brain section (C-side) is derived from a normal rat
  • the right brain section (S-side) is derived from a kainic acid-stimulated rat.
  • the sites where mRNA changes are indicated by ⁇ and *.
  • Black arrows indicate an increase in mRNA
  • white arrows indicate a decrease in mRNA.
  • the results showed that the expression of cerink mRNA was greatly changed in the hippocampus and cerebral cortex where cellular plasticity changes were caused by excitatory stimuli. In neurons and glial cells, mRNA changes in the opposite direction of increasing and decreasing, respectively. This figure shows that the serine protein acts in the process of neural circuit causing plastic changes in the neuronal circuit and restructuring.
  • celink has a function of increasing membrane lipid synthesis by binding to serine synthase and incorporating amino acid serine into the cell membrane.
  • changes in the amount of serine mRNA were observed in the formation of a new circuit of nerve cells not only by abnormal growth of cancer cells.
  • the close relationship between changes in various cells such as cancer cells and nerve cells and their membrane lipid synthesis-promoting action is a process in which a wide range of cells break the conventional cell-to-cell linkage and construct new cell-cell membrane connections. This indicates that a shelling action is required.

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Abstract

L’invention a pour objet des révélations sur le cancer pouvant être obtenues en clarifiant la fonction d'une famille de protéines dont la fonction était inconnue. Donc, l’invention concerne l’obtention de nouvelles protéines qui fournissent un indice pour développer des agents anticancéreux et des marqueurs de tumeurs ainsi qu’une méthode pour les utiliser. Il s’agit notamment d’une nouvelle protéine qui a pour fonction de se lier à une sérine synthétase et d’augmenter la synthèse de la membrane lipidique par absorption de l’acide aminé (c’est-à-dire, la sérine) par la membrane cellulaire. Cette nouvelle protéine est une protéine comprenant une séquence d’acides aminés représentée par l’une quelconque des SEQ ID NO: 1 à 9. L'utilisation de cette nouvelle protéine rend possible, par exemple, l’obtention d’un anticorps dirigé contre celle-ci.
PCT/JP2006/307138 2005-07-20 2006-04-04 Nouvelles protéines et méthode pour les utiliser WO2007010651A1 (fr)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2001500007A (ja) * 1996-08-16 2001-01-09 インサイト・ファーマスーティカルズ・インコーポレイテッド ヒト誘発性腫瘍タンパク質

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2001500007A (ja) * 1996-08-16 2001-01-09 インサイト・ファーマスーティカルズ・インコーポレイテッド ヒト誘発性腫瘍タンパク質

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BOSSOLASCO M. ET AL.: "The human TDE gene homologue: localization to 20q13.1-13.3 and variable expression in human tumor cell lines and tissue", MOL. CARCINOG., vol. 26, 1999, pages 189 - 200, XP003006584 *
DATABASE GENBANK [online] STRAUSBERG R., XP003006586, accession no. EMBL Database accession no. (AAH31720) *
GROSSMAN T.R. ET AL.: "Identification of a ubiquitous family of membrane proteins and their expression in mouse brain", J. EXP. BIOL., vol. 203, 2000, pages 447 - 457, XP003006585 *
KATSURAGAWA H. ET AL.: "Monoclonal antibody against phosphatidylserine inhibits in vitro human trophoblastic hormone production and invasion", BIOL. REPROD., vol. 56, 1997, pages 50 - 58, XP002942503 *

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