WO2007010651A1 - Novel proteins and method of using the same - Google Patents

Abstract

By clarifying the function of a protein family the function of which has been unknown, findings relating to cancer can be obtained. Thus, it is intended to provide novel proteins providing a clue to develop anticancer agents and tumor markers and a method of using the same. Namely, a novel protein which has a function of binding to serine synthase and enhancing membrane lipid synthesis via the absorption of the amino acid (i.e., serine) into the cell membrane. This novel protein is a protein comprising an amino acid sequence represented by any one of SEQ ID NOS:1 to 9. Use of this novel protein makes it possible to, for example, obtain an antibody against it.

Description

 Specification

 Novel protein and method of using the same

 Technical field

 TECHNICAL FIELD [0001] 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.

 Background art

 [0002] Now that the human genome has been deciphered and many unknown genes have been discovered, the emphasis of research that has so far been on genome analysis is shifting to functional analysis of proteins expressed from genes. Therefore, the three-dimensional structure of proteins has been elucidated, and the functions of each protein are estimated and elucidated based on the three-dimensional structure. However, there are proteins whose functions are still unknown.

 [0003] In recent years, proteins with unknown functions whose expression increases in cancer cells have been reported. For example, 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. Furthermore, 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.

 Disclosure of the invention

 Problems to be solved by the invention

[0004] Reports such as Non-Patent Documents 1 to 5 mentioned above suggest that the protein family with unknown function performs important functions in many aspects of cell biology. However, it is known that 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.

 [0005] In addition, the action of many anticancer agents known so far has been one that acts at each step of cancer cell DNA synthesis, transcription to RNA, and translation into protein. It has been suggested that the protein of unknown function whose expression is increased may be a material for developing a new anticancer agent.

 [0006] Therefore, 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.

 Means for solving the problem

 As a result of intensive studies in view of the above problems, a novel protein that has the function of increasing membrane lipid synthesis by incorporating the amino acid serine into the cell membrane and that can promote membrane lipid synthesis has been found. 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.

[0008] 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,

(b) the amino acid sequence of SEQ ID NOS: 1 to 9, consisting of an amino acid sequence in which one or more amino acids are deleted, substituted or added, and binds to serine synthase, Proteins that have the function of increasing membrane lipid synthesis by incorporating the amino acid serine into the cell membrane

[0009] The second aspect of the present invention is the DNA according to any one of the following (a) to (b):

 (a) DNA encoding a protein having the amino acid sequence ability according to any one of SEQ ID NOS: 1 to 9,

 (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. DNA that encodes a protein that has the function of increasing membrane lipid synthesis by incorporating.

 [0010] Claim 3 of the present invention is a recombinant vector containing the DNA of claim 2.

 [0011] Claim 4 of the present invention is a transformant comprising the recombinant vector according to claim 3.

 [0012] 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.

 [0013] Claim 6 of the present invention is an antibody against the protein of claim 1.

 [0014] 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.

[0015] 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.

[0016] 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.

 (b) A step of measuring the activity of the protein according to claim 1.

 (c) A step of selecting a compound that changes the activity as compared with the case where the test compound is not contacted.

 [0017] Claim 10 of the present invention is a method for screening a candidate compound for a drug, comprising the following steps (a) to (c).

 (a) contacting the test compound with a cell expressing the protein according to claim 1

 (b) a step of measuring the expression level of the protein according to claim 1

 (c) A step of selecting a compound that changes the expression level as compared to the case without contacting the test compound.

 [0018] 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 screening method according to the item.

 The invention's effect

 [0019] According to claim 1 of the present invention, by clarifying the nature and function of the serine protein family whose function is unknown, a substance that is a clue in developing anticancer agents, tumor markers, etc. Can be provided.

According to claim 2 of the present invention, by clarifying the nature and function of the serine protein family whose function is unknown, a substance that is a clue in developing anticancer agents, tumor markers, etc. Can be provided.

[0021] According to claim 3 of the present invention, the selinic gene can be retained by obtaining a recombinant vector containing the selinic gene.

[0022] According to claim 4 of the present invention, a selin gene can be retained by obtaining a transformant containing a recombinant vector containing a selin gene.

[0023] According to claim 5 of the present invention, a recombinant cellulose protein can be produced.

[0024] According to claim 6 of the present invention, an antibody against a serine protein is obtained. Thus, it is possible to specifically recognize celink. This makes it possible to use it as a new tumor marker and to selectively attack cancer.

 [0025] According to claim 7 of the present invention, utilization as a new tumor marker and selective attack against cancer are possible.

[0026] According to claim 8 of the present invention, the activity of the serine protein can be changed by obtaining the antisense nucleic acid.

[0027] According to Claim 9 of the present invention, drugs can be efficiently screened using the activity of celink as an index.

 [0028] According to claim 10 of the present invention, the drug can be efficiently screened using the expression level of celink as an index.

[0029] According to claim 11 of the present invention, in claim 9 or 10, the drug is one of an anticancer agent, a cell growth regulator, and a neural circuit regenerative agent. In addition, an anticancer agent, a cell growth regulator, or a neural circuit regeneration agent can be efficiently screened.

 Brief Description of Drawings

 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] FIG. 2 shows the alignment of amino acid sequences of the serine protein family.

[FIG. 3] 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] 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. Abbreviations: 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

 BEST MODE FOR CARRYING OUT THE INVENTION

[0031] The present invention will be described below.

 [0032] 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.

 [0033] (Novel protein (Cellin protein)) The present inventor believes that the function of a mammalian protein defined by one of the unknown genes whose expression is increased in cancer cells is related to the lipid synthesis of cell membranes. It was discovered that the amino acid serine was taken up, and the protein was named “selink” (hereinafter referred to as a protein having a function of increasing membrane lipid synthesis by binding to the serine synthase and taking up the amino acid serine into the cell membrane. Is referred to as Sellin). Sellin has a unique structure that has multiple subtypes and has the ability to form a protein family. Its amino acid sequence does not show homology with known proteins. NCBI's database search identified sericin 1, 2, 3, 3B, 4, 4B, and 5 as subtypes of mammalian sericin (the above-mentioned TMS-1, TMS-2, TDE1, and TPOl are respectively Corresponds to Selllink 3, Selllink 1, Selllink 3, Selllink 5). 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.

[0034] (Functions of celink) Regarding the interaction between rat cerine 1 and serine synthase (SER3, YGP1, PH012), we conducted a yeast 2-hybrid experiment and found that celink 1 binds to each serine synthase. Indicated. Moreover, when CELIN-1 was expressed in COS7 cells, the activity of synthesizing phosphatidylserine and sphingolipid, which are constituents of cell membrane, was promoted. From these facts, cerink binds to serine synthase and cell membrane It has been clarified that it has a function of increasing membrane lipid synthesis by incorporating the amino acid serine into.

 [0035] Furthermore, when the changes in the expression levels of mRNA for celink 1, 2, and 5 were examined in the brains of rats that were artificially caused by seizures after administration of kainate, excitatory stimuli were examined. In the hippocampus and cerebral cortex where cell reversibility occurs, the expression of celink mRNA was significantly changed. From this, it was clarified that the serine protein acts in the process of reconstructing the neuronal circuit by neural activity. The changes in various cells such as cancer cells and nerve cells and their membrane lipid synthesis-promoting action are due to the process in which various cells detach from conventional cell-cell linkages and construct new cell-cell membrane connections. It suggests that it is needed. For this reason, celink provides new insights into the mechanisms of cancer invasion and metastasis. In the nervous system, 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.

[0036] (Celinc mutant) In order for an enzyme to exert its enzymatic activity, not all of the native amino acid sequence is necessary as it is. Even if the amino acid sequence is modified by addition, substitution or the like, it is known to exhibit the original enzyme activity. Therefore, 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. In addition, 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. Also good. Specifically, 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.

 [0037] (DNA encoding celink) The present invention provides a celink gene. Specifically, 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.

 [0038] Furthermore, 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.

 [0039] Here, "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.

(Preparation of DNA encoding celink) In order to prepare the DNA of the present invention, screening from a cDNA library is first performed. MRNA forces in tissues or cells expressing celink also create a cDNA library. Isolate the clone of interest using a probe labeled with a radioactive isotope. Using a portion of the DNA of the present invention as a probe, colony hybridization or plaque hybridization for cDNA or cDNA library synthesized with mRNA force contained in tissues or cells of human or non-human animals. Etc., the DNA of the present invention can be obtained. For each clone, 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. Whether 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.

 [0041] (Recombinant vector) 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. In addition, animal viruses such as retroviruses, adenoviruses or vaccinia viruses, and insect virus vectors such as baculoviruses can also be used. To insert the DNA into the vector, 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.

 (Transformant) The present invention also provides a transformant comprising the above recombinant vector. Examples of 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.

[0043] (Production method) 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. Provided is 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. For example, salting-out, gel filtration chromatography, ion exchange chromatography, adsorption chromatography, high performance liquid chromatography (HPLC), etc. can be used in combination. 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.

 [0044] (Antibodies to Sellin) 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.

 [0045] The present invention also provides an antibody against membrane lipid that increases when the protein of claim 1 is expressed on a cell membrane. As described above, 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.

In order to obtain the monoclonal antibody of the present invention described in claim 6 or 7, a monoclonal antibody-producing cell is prepared. First, 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

[0047] Individuals with an antibody titer selected from animals immunized with the antigen, spleen or lymph nodes are collected 2 to 5 days after the final immunization, and antibody-producing cells contained therein are collected from allogeneic or allogeneic animals. Monoclonal antibody-producing hybridomas can be prepared by cell fusion with myeloma cells. As 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. For example, in an animal cell culture medium, antibody-producing cells and myeloma cells are mixed at a predetermined ratio (for example, 3: 1), and in the presence of a cell fusion promoter such as polyethylene glycol, certain silkworms are electrically Cell fusion can be achieved by performing pulse treatment (for example, electoral position). Regarding cell fusion operation, for example, the following documents can be referred to (Kohler, G .; Milstein, C. (1975) Nature. 256: 495-97) o

 [0048] 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.

[0049] Cloning of the fused cells can be performed by a limiting dilution method or the like. In order to collect a monoclonal antibody that produces an isolated monoclonal antibody, a normal cell culture method can be employed. For example, the above hyperidoma is cultured in a serum-containing medium, and the antibody is also obtained from the culture supernatant. When antibody purification is required, 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.

 [0050] In the same manner as the monoclonal antibody described above, 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.

 [0051] As a method of using the antibody for detecting a membrane lipid that increases when the protein according to claim 1 or the protein according to claim 1 is expressed on a cell membrane, 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.

 [0052] (Antisense nucleic acid) 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. . Here, 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.

 (Screening method) The present invention provides 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.

 (b) A step of measuring the activity of the protein according to claim 1.

 (c) A step of selecting a compound that changes the activity as compared with the case where the test compound is not contacted.

[0054] In the screening method, 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.

 [0056] 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.

 [0057] 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. For example, first, 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.

 [0058] 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. As 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. After selecting the binding activity with the protein according to claim 1 as an index, 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.

[0059] In the screening method of the present invention, 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.

 (a) contacting the test compound with a cell expressing the protein according to claim 1

 (b) a step of measuring the expression level of the protein according to claim 1

 (c) A step of selecting a compound that changes the expression level as compared to the case without contacting the test compound.

 [0060] 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. In addition, 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. Furthermore, 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.

 [0061] Specific examples will be described below, but the present invention is not limited to the following examples.

 Example

 [0062] (Experiment 1: Identification of a serine protein)

ZAP—Create a cDNA library using 5 μg poly (A) TRNA isolated from rat hippocampal force stimulated with kainic acid using cDNA synthesis kit (Strategene, La Jolla, CA) did. Screening of the above cDNA library using [ ³² P] cDNA prepared by reverse transcription of poly (A) + RNA from rats stimulated with kainic acid and kainic acid, and from rat hippocampus. did. Analysis of the nucleotide sequence of cDNAs that are expressed differently in rats stimulated with kainic acid and kainic acid, and that are expressed differently in rats. Genbank accession number DQ103 708) was identified and named Sellinc 1 (SEQ ID NO: 1).

 [0063] Next, 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. Furthermore, 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. In addition, the above 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. Sellin 1 (SEQ ID NO: 1): DQ103708, Sellin 2 (SEQ ID NO: 2): DQ103709, Sellin 3 (SEQ ID NO: 3): NM—006811, Sellin 4 (SEQ ID NO: 4): BC026459, Sellin 5 (SEQ ID NO: 5) : DQ103710, Serin 3B (SEQ ID NO: 6): U49188, Serin 4B (SEQ ID NO: 7): AK044479, Haze-Serink 4 (SEQ ID NO: 8): DQ103711, Yeast 'Serin 3 (TMS1) (SEQ ID NO: 9): NP_010390.

 [0064] (Experiment 2: Yeast two-hybrid experiment using rat celink)

 Experiment: Sasaki et al. Developed and analyzed a system that investigates two-hybrid interactions between about 6000 proteins of Saccharomyces cervisiae (Ito, T., Chiba, T "Ozawa, R., Yoshida, M., Hattori, M., and Sasaki, Y. (2001) Proc Natl Acad Sci USA 98 (8), 4569-74) Family members: SEQ ID NO: 9) were found to interact with two yeast proteins, SER3 and YGP1, which are involved in serine biosynthesis and function as serine synthases. Therefore, it was suggested that TMS1 is involved in intracellular serine synthesis.

[0065] In order to clarify the function of mammalian sericin, the interaction between rat sericin 1 (SEQ ID NO: 1) and the above protein was examined. Yeast two-hybrid experiments were carried out using rat celite 1 as a bait against three yeast proteins (SER3, YGP1, PH012). Here, 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. By transforming pOBD2—rat sericin 1 (a vector in which rat sericin 1 is introduced into POBD2) into yeast strain PJ69-4a, which expresses SER3, YGP1, and PH012 as Gal4 active proteins, went. At this time, 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. Since only cells expressing his3'2 and hybrid reporter genes can grow on the medium, the activity of the two hybrids was detected. As a control, the yeast strain PJ69-4a that expresses only Gal4 active protein was used. In order to show the growth rate, an equal amount of colonies grown on the synthetic medium lacking tributophane 'leucine' histidine was streaked on the medium.

 [0066] Results: 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. From this, it was shown that 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.

[0067] (Experiment ³ : Separation analysis of [¾] phosphatidylserine and [¾] sphingolipid using thin layer chromatography)

In mammalian cells, 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. [0068] In mammals, sphingolipids different from phosphatidylserine are also known as components of cell membranes. Serine 'palmitoyltransferase condenses serine • palmitoyl CoA and serine to produce 3-ketodihydrate-mouthed sphingosin (KS). The production of 3-ketodihydrate mouth sphingosin (KS) is a rate-limiting step that determines the rate of the entire sphingolipid synthesis pathway.

[0069] The purpose of Experiment 3 is to confirm that celink has the function of increasing membrane lipid synthesis by incorporating the amino acid serine into the cell membrane. Therefore, we investigated the incorporation of [ ³ H] serine into phosphatidylserine and 3-ketodihydrate mouth sphingosine by cerink.

 [0070] The method of Experiment 3 is described below.

[0071] SV40-transformed African green monkey kidney (COS-7) cells were cultured in DMEM medium supplemented with 10% ushi fetal serum. 8 g of pcDNA3.2 / V5DEST- Serine 1 (a vector in which rat sericin 1 gene was introduced into pcD NA3.2ZV5DEST) and an empty vector (Invitrogen) in 1 x 10 ⁶ COS7 cells in a 10 cm culture dish The gene was introduced into. After gene transfer, the cells were incubated for 60 hours and suspended in Atsy buffer 2401. 0.25 ml of cells suspended on ice using a microprobe was sonicated for 1 minute.

[0072] 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.

 2

 35ml was included. After incubating at 37 ° C for 20 minutes, 1.5 ml of chloroform / methanol (1Z2, vZv) was added to stop the reaction. Phospholipids were extracted by the method of Bligh and Dyer in the following literature (Bligh, EG, and Dyer, W. J. (1959) Can J Biochem Phisol 37 (8), 911-7).

 [0073] Serine 'palmitoyltransferase reaction mixture was prepared with 0.1 mM Tris-HCl, pH 8.

3, 5 mM dithiothreitol, 2.5 mM EDTA, 50 M pyridoxal phosphate, 0.2 mM palmitoyl CoA, ImM [ ³ H] serine (60 Ci), A total of 0.3 ml of the cell disruption solution was contained. After incubation at 37 ° C for 10 minutes, 1.5 ml of Kuroguchi Form Z methanol (1Z2, v / v) was added to stop the reaction. 25 μg Finganin was added as a carrier, and phospholipid products were extracted by the method described in the following literature (Williams, RD, Wang, E. and Merrill, AH, Jr. (1984) Arch Biochem Biophys 228 (1), 282 -91) o

[0074] The lipid extract was analyzed by thin layer chromatography.

 [0075] First, 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). In the case of phosphatidylserine, 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

 Four

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).

[0076] From FIG. 3, it was revealed that the expression of celink 1 increases the synthesis of [ ³ H] phosphatidylserine 2.8-fold. This indicates that the expression of celink 1 promotes the synthesis of phosphatidylserine. In addition, Fig. 3 revealed that the expression of celink 1 increased the incorporation of [ ³ H] serine into the 3-ketodihydrate mouthpiece sphingosin by 2.1 times or more. From this, it was found that increasing the expression of celink 1 promotes the production of 3-ketodihydrate mouth sphingosine. Since 3-ketodihydride mouth sphingosine production is the rate-limiting step in the overall synthesis of sphingolipid,

Increased expression of 1 has been shown to promote biosynthesis of sphingolipids [0077] (Experiment 4: Rat brain link 1, serine 2, and link 5 mRNA distribution and induction analysis using in situ hybridization)

Experiment: Adult rats (Sprague Dawley) were used in studies on the control of cerink. Stimulation with strength ninnic acid was carried out as follows: kainic acid (8 kg body weight per kg) was injected subcutaneously into rats. 3.5 hours later, they were killed under a large amount of anesthesia. The cut brain sections were placed on dry ice and immediately frozen in a plastic mold. The control and test tissues were frozen in the same tissue block, and care was taken to ensure that the conditions were completely the same during storage and in-situ hybridization. Frozen sections of 10 μm thickness were mounted on gelatin-coated slide glass, dried at 20 ° C. and stored until use. 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).

 [0078] 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, and 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, and 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.

 [0079] From the results of Experiment 2 to Experiment 4, it has been clarified that celink has a function of increasing membrane lipid synthesis by binding to serine synthase and incorporating amino acid serine into the cell membrane. In addition, it became clear that 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.

Claims

The scope of the claims  [1] 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,  (b) the amino acid sequence of SEQ ID NOS: 1 to 9, consisting of an amino acid sequence in which one or more amino acids are deleted, substituted or added, and binds to serine synthase, Proteins that have the function of increasing membrane lipid synthesis by incorporating the amino acid serine into the cell membrane [2] The DNA according to any one of the following (a) to (b):  (a) DNA encoding a protein having the amino acid sequence ability according to any one of SEQ ID NOS: 1 to 9,  (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. DNA that encodes a protein that has the function of increasing membrane lipid synthesis by incorporating.  [3] A recombinant vector containing the DNA according to claim 2.  [4] A transformant comprising the recombinant vector according to claim 3.  [5] The transformant according to claim 4 is cultured, and from the culture obtained, A method for producing the protein, comprising recovering a protein having a function equivalent to that of the protein represented by the amino acid sequence according to item 1. [6] An antibody against the protein according to claim 1.  [7] An antibody against membrane lipid that increases when the protein according to claim 1 is expressed on a cell membrane.  [8] An antisense nucleic acid, wherein the base sequence is a sequence that is complementary to all or part of the DNA according to claim 2. [9] 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;  (b) measuring the activity of the protein according to claim 1, (c) Do not contact the test compound! Process to choose.  [10] A method for screening a candidate compound for a drug, comprising the following steps (a) to (c):  (a) contacting the test compound with a cell expressing the protein according to claim 1  (b) a step of measuring the expression level of the protein according to claim 1  (c) A step of selecting a compound that changes the expression level as compared to the case without contacting the test compound.  [11] The screening method according to claim 9 or 10, wherein the drug is any one of an anticancer drug, a cell growth regulator, and a neural circuit regenerative agent.