JP2005082557A - Medicinal composition - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a medicinal composition that has an endoplasmic reticulum stress-inducing apoptosis-suppressive action and contains, as active ingredients, a protein (an enzyme) useful as an ameliorator for neurodegenerative diseases, for example, Alzheimer's disease and diabetes, and to provide a screening system for screening candidates that inhibit or promote the apoptosis-suppressing activity, or the like. <P>SOLUTION: The medicinal composition contains, as active ingredients, a polypeptide (a) (not shown) that comprises an amino acid sequence represented by an amino acid sequence shown in a sequence number: 1, and/or an amino acid sequence (b) (not shown) that is given by deleting one or more amino acid from, inserting one or more amino acids into, substituting one or more amino acids for or adding one or more amino acids to the amino acid sequence of the polypeptide (a). <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a pharmaceutical composition having an action of suppressing endoplasmic reticulum stress-induced apoptosis. The present invention also relates to a method for screening a compound having an activity of enhancing or suppressing endoplasmic reticulum stress-induced apoptosis-suppressing action.

  Mammalian cells, like other eukaryotic and prokaryotic cells, have developed a number of homeostatic mechanisms to successfully address the diverse physiological and environmental conditions that threaten their survival. Among these, endoplasmic reticulum-associated degradation (hereinafter referred to as “ERAD”) is well known as one of the mechanisms ubiquitously found in all living organisms. The ERAD is one of mechanisms for reducing stress (endoplasmic reticulum stress) caused by accumulation of proteins that are not folded or misfolded in the endoplasmic reticulum. That is, ERAD is a mechanism for transporting and degrading proteins that are not folded or misfolded to the outside of the endoplasmic reticulum.

  However, it is well known that mammalian cells subjected to endoplasmic reticulum stress cause cell death regardless of ERAD (endoplasmic reticulum stress-responsive cell death). Further, it has been suggested that this endoplasmic reticulum stress-responsive cell death is involved in diseases such as neurodegenerative diseases (Alzheimer's disease, Parkinson's disease, etc.) and diabetes (for example, Non-Patent Document 1, Non-Patent Document 2, (See Non-Patent Document 3, etc.).

  Suppressing endoplasmic reticulum stress-responsive cell death caused by endoplasmic reticulum stress is thought to contribute to the treatment of these diseases. Therefore, if a new protein responsible for ERAD, that is, a new protein involved in the mechanism of reducing endoplasmic reticulum stress, can be found, this protein can be used for new treatments such as neurodegenerative diseases related to endoplasmic reticulum stress-responsive cell death. It is considered effective. Such a protein provides useful information on elucidation of endoplasmic reticulum stress-responsive cell death itself, elucidation of the regulation mechanism of cell death by ERAD in the pathway, and the like.

  The inventors of the present invention previously expressed the ubiquitin E3-linked enzyme HRD1, which is a membrane protein localized in the endoplasmic reticulum whose expression is induced by endoplasmic reticulum stress and is an ERAD-related protein, in a cultured cell by high expression. It was revealed that endoplasmic reticulum stress-responsive cell death can be suppressed (see Non-Patent Document 4).

  In addition, ASK1 (patent document 1) composed of 1375 amino acids belonging to the MAPKK kinase family is composed of 66 amino acids induced by endoplasmic reticulum stress as proteins that induce endoplasmic reticulum stress-induced apoptosis. ER membrane protein SERP1 (Patent Document 2) and ER stress sensor protein hERRj-1 consisting of 793 amino acids whose expression decreases by AIP treatment (Patent Document 3) have been reported.

Thus, a novel protein capable of suppressing endoplasmic reticulum stress-responsive cell death for the treatment of neurodegenerative diseases and cytopathic diseases including diabetes is desired.
International Publication No. WO WO02 / 38179 Pamphlet JP 2000-245474 A JP 2002-335980 JP-A-9-215495 Kudo, T., et.al., Ann. NY Acad. Sci., 977: 349-355 (2002) Ryu, EJ, et.al., J. Neurosci., 22 (24): 10690-10698 (2002) Harding, HP, et al., Diabetes, 51 (3) S455-S461 (2002) Kaneko, M., et al., FEBS Lett., 532: 147-152 (2002) Imai, Y., et al., J. Biol. Chem., 275 (46): 35661-35664 (2000) Yasojima, K., et.al., BBRC, 231 (2): 481-487 (1997) Lorick, KL, et.al., PNAS USA., 96 (20): 11364-11369 (1999) Yamada, M., et.al., BBRC, 278: 150-157 (2000) Kakiuchi, C., et al., Nature Genetics, 1-5 (2003/08/03, online version)

  As described above, if a novel protein having an inhibitory action on endoplasmic reticulum stress-responsive cell death or a gene encoding the same can be provided, these can be used to elucidate the regulatory mechanism by ERAD in the pathway of endoplasmic reticulum stress-responsive cell death, It is useful for elucidating the mechanism of stress-responsive cell death and suppressing the endoplasmic reticulum stress-induced apoptotic action. Further, these genes and proteins can be expected as therapeutic and preventive agents for neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, and cytodegenerative diseases such as diabetes, based on their apoptosis-suppressing action. Furthermore, the use of these genes and proteins makes it possible to screen for candidate compounds that promote or suppress endoplasmic reticulum stress-induced apoptosis.

  From the above viewpoint, the present inventors first searched for a gene having a ubiquitin E3-binding enzyme-like domain using a public database for the purpose of searching for a novel gene having an endoplasmic reticulum stress-induced apoptosis inhibitory action. It was. As a result, we found a gene (KF-1 gene) encoding a new ubiquitin E3-linked enzyme (named KF-1), which is different from proteins that have been reported to suppress endoplasmic reticulum stress-responsive cell death. It was. KF-1 has been reported to be expressed in the cerebral cortex of Alzheimer patients (see Patent Document 4, Non-Patent Document 6 and Non-Patent Document 7).

  Next, the inventors expressed KF-1 gene-expressing cells as `` thapsigargin '' which is a calcium ATPase inhibitor, `` tunicamycin '' which is a reagent for an N-glucoside sugar chain synthesis inhibitor, etc. It was found that the expression level of KF-1 increases when treated with an endoplasmic reticulum stress-inducing agent.

  Furthermore, the present inventors induced endoplasmic reticulum stress in human KF-1 stably expressing cells (neuroblasts), and hKF-1 expression in the cells has an inhibitory activity against apoptosis (anti-apoptotic activity). It was confirmed.

  From the above, the present inventors have obtained a new finding that KF-1 suppresses endoplasmic reticulum stress-induced apoptosis. The present invention has been completed as a result of further research based on this knowledge.

  The present invention provides the inventions described in the following items 1-16.

Item 1. A pharmaceutical composition comprising as an active ingredient at least one polypeptide selected from the group consisting of the following (a) and (b):
(a) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1,
(b) a polymorphic amino acid sequence in which one or more amino acids are deleted, inserted, substituted or added in the amino acid sequence of (a) above, and having an action of suppressing endoplasmic reticulum stress-induced apoptosis in target cells peptide.

Item 2. A pharmaceutical composition comprising, as an active ingredient, an expression product of at least one polynucleotide selected from the group consisting of the following (c), (d) and (e):
(c) a polynucleotide comprising the DNA sequence represented by SEQ ID NO: 2,
(d) a polynucleotide capable of hybridizing with the polynucleotide of (c) above under stringent conditions and capable of expressing an effect of suppressing endoplasmic reticulum stress-induced apoptosis in a target cell,
(e) a polynucleotide comprising a DNA sequence which is 80% or more homologous to the polynucleotide of (c) above and capable of expressing an action of suppressing endoplasmic reticulum stress-induced apoptosis in a target cell.

  Item 3. Item 3. The pharmaceutical composition according to Item 1 or 2, which is an endoplasmic reticulum stress-induced apoptosis inhibitor.

  Item 4. Item 3. The pharmaceutical composition according to Item 1 or 2, wherein the target cell is selected from the group consisting of nerve cells, neuroblasts, pancreatic β cells, hepatocytes, epithelial cells, skeletal muscle cells, smooth muscle cells, and lymphocytes. .

  Item 5. Alzheimer's disease, Parkinson's disease, Huntington's disease, polyglutamine disease, ischemic encephalopathy, amyotrophic lateral sclerosis, multiple sclerosis, HIV-related neuropathy, diffuse Lewy body disease, type I diabetes, type II Item 3. The pharmaceutical composition according to Item 1 or 2, which is a therapeutic agent for a disease selected from the group consisting of diabetes, manic depression and depression.

  Item 6. An endoplasmic reticulum stress-induced apoptosis inhibitor containing, as an active ingredient, at least one selected from the group consisting of the polypeptide of Item 1 and the expressed product of Item 2.

  Item 7. The pharmaceutical composition according to Item 1 or 2 is administered to a patient who requires treatment, Alzheimer's disease, Parkinson's disease, Huntington's disease, polyglutamine disease, ischemic brain injury, amyotrophic lateral sclerosis For treating a disease selected from the group consisting of infectious disease, multiple sclerosis, HIV-related neuropathy, diffuse Lewy body disease, type I diabetes, type II diabetes, manic depression and depression.

Item 8. In the presence and absence of the test substance, measure the degree of inhibition of endoplasmic reticulum stress-induced apoptosis in the target cells of the polypeptide (a) or (b) below, and test the measured value in the presence of the test substance. In contrast to the measured value in the absence of a substance, an agonist that enhances the inhibitory action or an antagonist that reduces the inhibitory action is selected. Agonist or antagonist screening methods:
(a) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1,
(b) a polypeptide comprising an amino acid sequence in which one or more amino acids are deleted, inserted, substituted or added in the amino acid sequence of (a) above and having an action of suppressing endoplasmic reticulum stress-induced apoptosis in target cells peptide.

  Item 9. Item 9. The method according to Item 8, wherein the degree of inhibition of endoplasmic reticulum stress-induced apoptosis in the target cells of the polypeptide is obtained by counting the number of target cells in which nuclei have aggregated, and obtaining the number of reductions.

Item 10. In the presence and absence of the test substance, the degree of inhibition of endoplasmic reticulum stress-induced apoptosis in the target cell of the expression product of the following polynucleotide (c), (d) or (e) is measured, and the test substance ER in a target cell, wherein an agonist that enhances the inhibitory action or an antagonist that reduces the inhibitory action is selected by comparing a measured value in the presence of a test substance with a measured value in the absence of a test substance Methods for screening for agonists or antagonists for stress-induced apoptosis inhibition:
(c) a polynucleotide comprising the DNA sequence represented by SEQ ID NO: 2,
(d) a polynucleotide capable of hybridizing with the polynucleotide of (c) above under stringent conditions and capable of expressing an action of suppressing endoplasmic reticulum stress-induced apoptosis in a target cell,
(e) a polynucleotide comprising a DNA sequence which is 80% or more homologous to the polynucleotide of (c) and capable of expressing an action of suppressing endoplasmic reticulum stress-induced apoptosis in a target cell.

  Item 11. Item 11. The method according to Item 10, wherein the degree of suppression of endoplasmic reticulum stress-induced apoptosis in the target cells of the expressed product is obtained as a decrease in the number of target cells in which nuclei have been aggregated.

  Item 12. Item 11. The method according to Item 8 or 10, wherein the degree of inhibition of apoptosis is determined by (1) agarose gel electrophoresis, (2) pulse field electrophoresis, (3) TUNEL method, or (4) flow cytometry.

Item 13. A screening method for a candidate substance that enhances endoplasmic reticulum stress-induced apoptosis-suppressing action in a target cell of the polypeptide according to Item 1, comprising the following steps (1) to (5):
(1) a step of preparing a target cell transformed with the polypeptide expression vector according to Item 1,
(2) Inducing apoptosis by applying endoplasmic reticulum stress to the target cell in the presence or absence of a candidate substance,
(3) a step of determining the survival rate of the target cell in the presence and absence of the candidate substance or the proportion of cells in the apoptotic form,
(4) Each value obtained in (3) above, using as a reference value the survival rate of cells or the proportion of cells in an apoptotic form obtained by applying endoplasmic reticulum stress load to non-transformed cells in the absence of a candidate substance Obtaining a difference between the reference value and the reference value;
(5) When the difference obtained in the presence of the candidate substance is larger in the survival rate or smaller in the apoptotic form cell ratio than the difference obtained in the absence, the candidate substance is Process to select.

Item 14. A method for screening a candidate compound that enhances the expression in a target cell of a polynucleotide capable of expressing an action of suppressing endoplasmic reticulum stress-induced apoptosis according to Item 2, comprising the following steps (1) to (5):
(1) preparing a target cell transformed with the polynucleotide expression vector according to claim 2,
(2) subjecting the target cell to endoplasmic reticulum stress load in the presence or absence of a candidate substance,
(3) a step of determining the expression level of the polynucleotide of the target cell in the presence and absence of the candidate substance,
(4) a step of comparing the expression level in the absence of the candidate substance with the same expression level in the presence of the candidate substance,
(5) A step of selecting the candidate substance when the expression level obtained in the presence of the candidate substance is increased as compared with that obtained in the absence.

Item 15. A method for screening a candidate substance that enhances the action of the polypeptide of Item 1 in a target cell comprising the following steps (1) to (5):
(1) A step of preparing a target cell transformed with an expression vector of a gene encoding the polypeptide according to item 1,
(2) subjecting the target cell to endoplasmic reticulum stress load in the presence or absence of a candidate substance,
(3) a step of determining the amount of polypeptide produced by the target cell in the presence and absence of a candidate substance using an antibody against the polypeptide,
(4) a step of comparing the production amount obtained in the absence of the candidate substance with the production amount obtained in the presence of the candidate substance,
(5) A step of selecting the candidate substance when the production amount in the presence of the candidate compound is increased as compared with that in the absence.

  Item 16. The polypeptide comprising at least one selected from the group consisting of the polypeptide according to Item 1 and the expression product according to Item 2 in a target cell and an endoplasmic reticulum stress load as constituents Alternatively, a screening kit for screening an agonist or antagonist for the action of suppressing the endoplasmic reticulum stress-induced apoptosis in target cells of the expressed product.

  The abbreviations of amino acids, peptides, base sequences, nucleic acids, etc. in this specification are defined by IUPAC-IUB (IUPAC-IUB Communication on Biological Nomenclature, Eur. J. Biochem., 138: 9 (1984)), “Base "Guidelines for the preparation of specifications including sequences or amino acid sequences" (edited by the Patent Office) and common symbols in the field.

  In the present specification, the term "gene" is intended to include not only double-stranded DNA but also each single-stranded DNA comprising sense strand and antisense strand constituting the same, and is not limited to the length thereof. . Therefore, unless otherwise specified, the gene (DNA molecule) of the present invention has double-stranded DNA containing human genomic DNA and single-stranded DNA containing cDNA (sense strand) and a sequence complementary to the sense strand. Both double-stranded DNA (antisense strand) and fragments thereof are included.

  Further, in the present specification, the gene (DNA molecule) does not ask whether the functional region is different, and can include at least one of an expression suppression region, a coding region, a leader sequence, an exon, and an intron. Polynucleotides include RNA and DNA. DNA molecules include cDNA, genomic DNA and synthetic DNA. Polypeptides having a specific amino acid sequence and polynucleotides encoding the same include fragments, homologues, derivatives and variants thereof.

  Polynucleotide variants (variant DNA) include naturally occurring allelic variants; non-naturally occurring variants; variants with deletions, substitutions, additions and insertions. However, these variants shall encode a polypeptide having substantially the same function as that of the polypeptide encoded by the polynucleotide before mutation.

  Polypeptide mutations (amino acid sequence alterations) do not have to occur in nature, such as mutations, post-translational modifications, etc., but naturally occurring genes (e.g., the KF-1 gene of the present invention) It may be generated artificially by using. Variants of the above polypeptides are alleles, homologs, natural variants, etc., and at least 80%, preferably 95% or more, more preferably 98% or more are homologous to the polypeptide before mutation. included.

Variant DNA is silent (no change in the amino acid residue encoded by the mutated nucleic acid sequence) or conserved with respect to the amino acid encoded thereby. Examples of conservative amino acid substitutions are shown below.
Original amino acid residue Conservatively substituted amino acid residue
Ala Ser
Arg Lys
Asn Gln or His
Asp Glu
Cys Ser
Gln Asn
Glu Asp
Gly Pro
His Asn or Gln
Ile Leu or Val
Leu Ile or Val
Lys Arg, Asn or Glu
Met Leu or Ile
Phe Met, Leu or Tyr
Ser Thr
Thr Ser
Trp Tyr
Tyr Trp or Phe
Val Ile or Leu.

  In general, one or more codons encoding a Cys residue affect the disulfide bond of a particular polypeptide.

Substitutions of amino acid residues that are generally considered to change the properties of proteins include the following.
a) replacement of hydrophobic residues with hydrophilic residues, e.g. replacement of Leu, Ile, Phe, Val or Ala with Ser or Thr;
b) substitution of amino acid residues other than Cys and Pro to Cys or Pro,
c) substitution of residues having electropositive side chains, for example Lys, Arg, His, to electronegative residues, for example Glu or Asp,
d) Substitution to amino acid residues with very large side chains, eg amino acid residues without side chains such as Ply's Gly.

(1) Polypeptide as an active ingredient of the pharmaceutical composition of the present invention Polypeptide (and polypeptide used in the screening method of the present invention) as an active ingredient in the pharmaceutical composition of the present invention (hereinafter simply referred to as “the polypeptide of the present invention”) ) Consists of (a) the amino acid sequence shown in SEQ ID NO: 1, or (b) consists of an amino acid sequence in which one or more amino acids are deleted, inserted, substituted or added in SEQ ID NO: 1 and It has an endoplasmic reticulum stress-induced apoptosis inhibitory action.

  The polypeptide of the present invention is, for example, human cells, particularly human neurons, neuroblasts, pancreatic cells, pancreatic β cells, or human tissues, particularly cerebrum (cerebral cortex), cerebellum, pancreas, kidney, liver, spleen, lung, It may be a polypeptide derived from human cells or tissues originating from the stomach, small intestine, large intestine, etc., or may be a polypeptide obtained by chemical synthesis according to the amino acid sequence information.

  As a specific example of the amino acid sequence of the polypeptide of the present invention, the amino acid sequence represented by SEQ ID NO: 1 can be exemplified. The amino acid sequence of the polypeptide of the present invention is not limited to that shown in SEQ ID NO: 1, and can have a certain degree of homology (homologous substance). The homologue includes a polymorphic amino acid sequence having an amino acid sequence in which one or more amino acids are deleted, inserted, substituted or added in the amino acid sequence represented by SEQ ID NO: 1 and having an endoplasmic reticulum stress-induced apoptosis inhibitory action. Peptides can be mentioned.

  More specifically, the apoptosis-inhibiting action is an action of inhibiting cell apoptosis induced by endoplasmic reticulum stress. As a result, this action is an action of reducing apoptosis (cell death) of nerve cells, pancreatic β cells and the like. The action includes an action of suppressing or improving cell degeneration caused by apoptosis, that is, an action of suppressing cell degeneration. The action of the homologue may be substantially the same as that of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1. Here, “substantially the same quality” indicates that the action is the same in physiochemical properties or pharmacological properties, and the amount of the apoptosis-inhibiting action, the molecular weight of the protein, etc. Target elements may be different.

  In the polypeptide of the present invention shown as (b) above, the degree of amino acid modification, that is, “deletion, insertion, substitution or addition” and the position thereof are as follows. There is no particular limitation as long as it has substantially the same quality of activity as the polypeptide having the amino acid sequence shown (same effect). The degree of modification is usually preferably about 1 to several. In particular, the degree of modification is such that the homology in the amino acid sequence is, for example, about 80% or more, preferably about 95% or more, more preferably about 98% or more.

  The polypeptide of the present invention consisting of the modified amino acid sequence shown in (b) above has, for example, the intrinsic activity of the protein (for example, the enzyme activity when the protein is an enzyme), antigenicity, Also in the immunogenic activity and the like, it is preferably substantially the same as the polypeptide consisting of the amino acid sequence before modification, that is, the amino acid sequence represented by SEQ ID NO: 1.

  In the present specification, a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1, and an amino acid sequence in which one or more amino acids are deleted, inserted, substituted or added in SEQ ID NO: 1, and endoplasmic reticulum stress The above-mentioned common activity possessed by polypeptides having an inducing apoptosis-inhibiting action is hereinafter simply referred to as “KF-1 activity”.

  Homologues of the polypeptide of the present invention consisting of the amino acid sequence represented by SEQ ID NO: 1 include proteins originating from yeast, nematodes, plant tissues and mammals other than human having KF-1 activity. Examples of the mammal include horses, sheep, cows, dogs, monkeys, cats, bears, mice, rats, rabbits and the like.

  The apoptosis-inhibiting action of the polypeptide of the present invention is the original (not imparting expression ability) when the apoptosis-inducing substance is administered to the cell or tissue imparted with the expression ability of the polypeptide of the present invention. In contrast to cells and tissues, it means that apoptosis is suppressed. More specifically, the apoptosis-inhibiting action of the polypeptide of the present invention is a substance that induces endoplasmic reticulum stress such as calcium ATPase inhibitor (thapsigargin), N-glucoside sugar chain synthesis inhibitor (tunicamycin), etc. It can be said to be an inhibitory action against endoplasmic reticulum stress-induced apoptosis in cells or tissues induced by.

  Apoptosis and its inhibitory action can be measured and detected by a known method, for example, the TUNEL method, using as an index the number (ratio) of cells that have undergone nuclear aggregation in Hoechst-stained cells. For example, it can be measured and detected by a method (MTT method) using a change in absorbance due to reduction of methylthiazole / tetrazolium as an index.

  More specifically, for example, as described in Examples below, neuroblasts expressing recombinant hKF-1 labeled with a Myc epitope (hKF-1-expressing cells) are treated with drugs that induce endoplasmic reticulum stress such as tunicamycin. After induction of endoplasmic reticulum stress, Hoechst staining was performed, and the number (ratio) of stained cells (cells that caused nuclear aggregation, apoptotic cells) was counted. The presence or absence of inhibition of apoptosis can be detected by comparing with the same measurement value of the expression cell). Apoptotic cells can be identified by morphological observation with an electron microscope or an optical microscope. That is, apoptotic cells can be detected by the disappearance of cell surface microvilli, aggregation of chromatin in the nucleus, changes in intracellular microstructure, and the like.

The homology of a polypeptide or polynucleotide can be analyzed by measurement using sequence analysis software, for example, FASTA program (Clustal, V., Methods Mol. Biol., 25, 307-318 (1994)). . The most preferred and simple method of homology analysis is to store the sequence on a computer readable medium (eg flexible disk, CD-ROM, hard disk drive, external disk drive, DVD, etc.) and then store the sequence. Examples of how to use a sequence to search a known sequence database according to well-known search means can be exemplified. Specific examples of known sequence databases include:
・ DNA Database of Japan (DDBJ) (http://www.ddbj.nig.ac.jp/);
Genebank (http://www.ncbi.nlm.nih.gov/web/Genebank/Index.htlm); and the European Molecular Biology Laboratory Nucleic Acid Sequence Database (EMBL) (http://www.ebi.ac .uk / ebi docs / embl db.html).

  Many search algorithms are available to those skilled in the art for homology analysis. One example is a program called a BLAST program. This program has 5 BLAST tools. Three of them (BLASTN, BLASTX and TBLASTX) are designed for querying nucleotide sequences. The remaining two (BLASTP and TBLASTN) are designed for protein sequence queries (Coulson, Trends in Biotechnology, 12: 76-80 (1994); Birren, et al., Genome Analysis, 1: 543-559 (1997)).

  Further additional programs are available in the art for analysis of identified sequences, such as sequence alignment programs, programs for identifying more distant sequences.

  As a specific example of the polypeptide of the present invention, a polypeptide encoded by a PCR product named “hKF-1” shown in Examples described later can be mentioned. The DNA sequence of hKF-1 is as shown in SEQ ID NO: 3 (full length 3423 bp), and the amino acid sequence encoded by the ORF (2058 bp) is as shown in SEQ ID NO: 1. That is, hKF-1 has a 686 amino acid sequence (DDBJ accession number D76444).

  Another specific example of the polypeptide of the present invention is mouse KF-1 (mKF-1). The DNA sequence is as shown in SEQ ID NO: 6 (full length 3263 bp), and the amino acid sequence encoded by the ORF (2052 bp) is as shown in SEQ ID NO: 4. That is, mKF-1 has a 684 amino acid sequence (DDBJ accession number D76445). This mKF-1 has 93% identity and 95% similarity to hKF-1 at the amino acid level. It also has 92% identity at the nucleic acid level.

  hKF-1 is expressed in normal cerebellum and cerebral cortex from Alzheimer patients. In addition, mKF-1 is expressed in the cerebellum, cerebral hippocampus, midbrain and subbrain cone. As for mKF-1, the gene contains a zinc-binding ring finger motif at the carboxy terminus, as found in acetylcholine receptor-related proteins (Non-patent Document 6), and the C-terminal side of the gene ( The detection of E2-dependent ubiquitin ligase activity was confirmed using a GST fusion protein at the ring fingers (540-686) (Non-patent document 7), and this gene was detected in the rat brain by long-term administration of an antidepressant. It has been reported that the expression of arginine is abnormally increased (Non-patent Document 8).

  Patent Document 4 describes that a human KF-1 gene and a mouse KF-1 gene having homology to the gene were isolated as genes that are specifically expressed in the cerebrum of Alzheimer-type dementia patients. Column 5 suggests that a substance that suppresses the function of the KF-1 protein or an expression product of a gene whose transcription is regulated by the KF-1 protein may be a therapeutic drug.

  Contrary to the description in Patent Document 4, the present invention is based on the fact that KF-1 itself exerts an action of suppressing neuronal cell death due to endoplasmic reticulum stress observed in Alzheimer's disease, Parkinson's disease and the like. Yes. That is, the present invention provides that KF-1 (polypeptide) has an endoplasmic reticulum stress-induced apoptosis inhibitory action, and therefore, for example, as a pharmaceutical composition such as an apoptosis inhibitor disclosed herein, a neurological disease or Findings that are effective in the treatment and prevention of cytopathic diseases such as pancreatic β cells, especially various disease symptoms caused by endoplasmic reticulum stress-induced apoptosis, such as Alzheimer's disease (Alzheimer-type senile dementia), Parkinson's disease, Huntington's disease , Machado-Joseph disease, polyglutamine disease, ischemic brain injury (brain ischemia), amyotrophic lateral sclerosis, multiple sclerosis, HIV-related neuropathy, diffuse Lewy body disease, type I diabetes, It was completed based on the knowledge that it can be used for the treatment and prevention of type II diabetes, depression, manic depression and the like.

  Regarding the relationship between endoplasmic reticulum stress induction and diseases, several reports have recently been made along with reports of endoplasmic reticulum stress sensor molecules. For example, Oyadomari et al. Show that Akita mice, which are diabetes model mice, induce the endoplasmic reticulum (ER, Endoplasmic Reticulum) stress-related apoptotic factor Chop in the pancreas, and the induction of Chop expression induces apoptosis. We report that targeted inhibition of the Chop gene delays onset of diabetes until 8-10 weeks in heterozygous Akita mice. It has also been reported that excessive abnormal protein accumulation in β cells causes ER stress and induces apoptosis through induction of Chop. Furthermore, a new therapeutic approach has been proposed to prevent diabetes onset by suppressing Chop induction in the ER or by increasing chaperone capacity (Oyadomari, S., et al. , J. Clin. Invest., 109, 525-532 (2002)).

  Pancreatic beta cells are insulin-producing cells with highly developed endoplasmic reticulum, and abnormal functioning of the endoplasmic reticulum due to abnormalities of PERK, called endoplasmic reticulum stress sensor molecule, can cause pancreatic abnormalities and diabetes with beta cell death. Has been reported (see Delepine, M., et al., Nature Genet., 25, 406-409 (2000)).

  As described in the above literature, in mice that produce abnormal insulin and develop diabetes, endoplasmic reticulum stress caused by accumulation of abnormal insulin causes diabetes and suppresses apoptosis caused by the endoplasmic reticulum stress. By doing so, it is considered that diabetes caused by functional abnormality due to cytopathicity of pancreatic β cells can be suppressed. From this, as shown in Examples below, by introducing and administering KF-1 or a KF-1 gene expression product having an inhibitory effect on apoptosis induced by endoplasmic reticulum stress, treatment of diabetes and The development of anti-diabetic drugs with substances that promote or increase KF-1 gene expression in target cells such as pancreatic β cells is also expected.

  Patent Document 1 discloses that a host cell transformed with a chemical substance having an inhibitory or inhibitory action on ASK1 associated with the induction of apoptosis by endoplasmic reticulum stress, a sense oligonucleotide of ASK1, or a vector containing such a nucleotide is used. It describes that it can be a preventive and therapeutic agent for neurodegenerative diseases caused by endoplasmic reticulum stress, such as Alzheimer's disease, Parkinson's disease, polyglutamine disease, and amyotrophic lateral sclerosis.

  Imaizumi et al. Have a stress response system that eliminates abnormal proteins starting from stress sensors in the endoplasmic reticulum, but in Alzheimer's disease, this system fails, and abnormal protein accumulation and resistance to various cellular stresses occurs. Report that neuronal cell death is triggered from the decrease in phenotype and that they are caused by denatured presenin 1 or exon 5 deficient presenin 2 (Imaizumi, K., et al., Nature Cell Biol ., 3, E104 (1999); Katayama, T., et al., J. Biol. Chem., 276, 43446-43454 (2001); Cell engineering, 21 (4), 382-388 (2002), etc. ).

  Regarding the relationship between polyglutamine disease represented by Huntington's dream and Machado-Joseph disease and induction of endoplasmic reticulum stress, Nishigami et al. Conducted an experiment to express polyglutamine protein using adenovirus in primary mouse neurons. The system has confirmed that IRE1 and PERK activity, which is said to be an endoplasmic reticulum stress sensor molecule, and Bip and CHOP that are induced to occur during endoplasmic reticulum stress are induced, and that endoplasmic reticulum stress is induced. In addition, the ASK1-JNK pathway is strongly activated, and the accumulation of polyglutamine aggregates suppresses intracellular proteasome activity in a relatively gentle time. It induces stress and shows that the ASK1-JNK pathway is strongly activated as described above. In polyglutamine disease, it is speculated that polyglutamine protein causes neuronal cell death through endoplasmic reticulum stress (see Hideaki Nishigami, Cell Engineering, 21 (4), 377-381 (2002)).

  The causative gene parkin of familial Parkinson's disease is responsible for degrading abnormal proteins in the endoplasmic reticulum and is thought to be involved in cell death from endoplasmic reticulum stress. It has also been reported that the genes responsible for familial Alzheimer presenilin-1 (presenilin-1) and presenilin-2 (presenilin-2) are involved in cell death from endoplasmic reticulum stress. (See Imaizumi et al., Supra), whereby neuronal cell death causes these diseases. That is, Alzheimer's disease and Parkinson's disease are considered to be caused by functional abnormalities caused by neuronal cell degeneration.

  Imai et al. Reported that parkin, a causative gene for autosomal recessive juvenile Parkinson's disease, suppresses cell death of cultured cells (SH-SY5Y cells, which have the ability to produce dopamine when differentiated) due to abnormal proteins. (See Imai, Y., et al., Cell, 105, 891-902 (2001)). Also, pael receptor, one of parkin's substrates, has the property of causing abnormal protein folding in the endoplasmic reticulum, which is the cause of endoplasmic reticulum stress. Due to the fact that cell death caused by aggregated pael receptors is suppressed in the body, loss of parkin activity leads to accumulation of pael receptor, which is a substrate protein, in the endoplasmic reticulum, and subsequent apoptosis induced by endoplasmic reticulum stress in dopamine neurons. And the hypothesis that Parkinson's disease develops has been proposed (see Joe Imai, Ryosuke Takahashi, Cell Engineering, 21 (4), 389-394 (2002)).

  XBP1 is a protein downstream of the endoplasmic reticulum transmembrane protein ATF6, and it is said that it plays a role in promoting normalization of proteins with abnormal structures in the endoplasmic reticulum. Kakiuchi et al. Have revealed that mutations in the promoter portion of the XBP1 gene are frequently seen in patients with manic depression. As a result of analyzing the expression of the gene in twin patients with bipolar disorder (manic depression), the XBP1 gene is highly expressed in the human frontal cortex, but in the affected twin, the expression of the XBP1 gene is The decreased expression of this XBP1 gene prevents the crisis management mechanism from working, prevents normalization of the intracellular environment, causes this mutation to damage the body's signal transduction mechanism, and suffers from bipolar disorder Estimated to increase the probability. Furthermore, among the three existing drugs for the treatment of manic-depressive diseases, we investigated the effects of reducing the expression of the gene due to the polymorphism in the XBP1 promoter region. Of the lithium preparations, carbabazepine and valproic acid, only valproic acid was found. It has been reported that abnormalities in information transmission associated with decreased expression of XBP1 are improved (see Non-Patent Document 9).

  As described above, the relationship between the onset of Alzheimer's disease, Parkinson's disease, polyglutamine disease, amyotrophic lateral sclerosis, depression, manic depression, etc. and neuronal cell death due to endoplasmic reticulum stress is involved. The action of KF-1 in the present invention is to suppress the neuronal cell death and to treat and prevent the above various diseases. Suggests usefulness.

(2) Gene encoding the polypeptide of the present invention As a specific example of the gene encoding the polypeptide of the present invention (hereinafter referred to as “the gene of the present invention”), the DNA sequence represented by SEQ ID NO: 2 or its complementary strand The gene which consists of a polynucleotide to include can be mentioned.

  Other genes (polynucleotides) of the present invention include polynucleotides that can hybridize with the above-mentioned polynucleotides under stringent conditions and can express an endoplasmic reticulum stress-induced apoptosis inhibitory action. Here, “stringent conditions” means conditions that hybridize at 50 ° C. in 0.2 × SSC containing 0.1% SDS and do not desorb by washing at 60 ° C. in 1 × SSC containing 0.1% SDS. Can be mentioned.

  Still another gene of the present invention (polynucleotide) comprises 80% or more, preferably 95% or more, more preferably about 98% or more of a homologous DNA sequence to the above-mentioned polynucleotide and has an apoptosis inhibitory action (particularly endoplasmic reticulum stress). A polynucleotide capable of expressing an inducible apoptosis inhibitory action) is included.

  These polynucleotides (hereinafter sometimes referred to as “variants”) are required to be capable of expressing an apoptosis-inhibiting action (particularly, an endoplasmic reticulum stress-induced apoptosis-inhibiting action). In other words, the requirement is that a transformant transformed with a recombinant expression vector into which these polynucleotides (variants) have been inserted is expressed as KF-1 or an apoptosis inhibitory effect (especially endoplasmic reticulum stress). It is possible to express a protein having an inducing apoptosis-inhibiting action).

  The variant includes a gene comprising a DNA sequence encoding an amino acid sequence (modified amino acid sequence) in which one or several amino acids are deleted, inserted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1. Is included. The variant may be one that can detect the gene of the present invention before modification by its use.

  The gene of the present invention further includes a homologue of a polynucleotide (KF-1 gene) comprising the DNA sequence represented by SEQ ID NO: 2. Here, “KF-1 gene homologue” refers to a gene family that has sequence homology with the KF-1 gene and is based on structural features, commonality in gene expression patterns, and similarity in biological function. A series of related genes recognized as This includes alleles (alleles) of the KF-1 gene.

  The alteration (mutation) of the amino acid sequence may occur in nature, for example, by mutation or post-translational modification. It can also be artificially modified based on a naturally derived gene (for example, the KF-1 gene of the present invention). The gene of the present invention encompasses all modified genes having the above characteristics regardless of the cause and means of such modification and mutation.

  Examples of the artificial means include cytospecific mutagenesis [Methods in Enzymology, 154, 350, 367-382 (1987); 100, 468 (1983); Nucleic Acids Res., 12, 9441 (1984); Genetic engineering methods such as Secondary Biochemistry Experiment Course 1 “Gene Research Method II”, edited by Japanese Biochemical Society, p105 (1986)]; Chemical synthesis methods such as phosphate triester method and phosphate amidite method [J. Am. Chem. Soc., 89, 4801 (1967); 91, 3350 (1969); Science, 150, 178 (1968); Tetrahedron Lett., 22, 1859 (1981); 24, 245 (1983)] and those The combination method can be illustrated. More specifically, DNA synthesis can be performed by chemical synthesis by the phosphoramidite method or triester method, and can also be performed on a commercially available automatic oligonucleotide synthesizer. Double-stranded fragments can be synthesized by synthesizing the complementary strand and annealing the complementary strand with the chemically synthesized single strand under appropriate conditions, or by chemically synthesizing the complementary strand using DNA polymerase with an appropriate primer sequence. It can be obtained by adding to a single strand.

  The DNA sequence of the gene having the DNA sequence represented by SEQ ID NO: 2 which is a specific embodiment of the gene of the present invention (for example, human KF-1 gene) is a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1. It is an example of one combination of each codon encoding an amino acid residue. The gene of the present invention is not limited to a DNA molecule having such a specific DNA sequence, but can also have a selected DNA sequence by combining arbitrary codons for each amino acid residue. Codon selection can follow conventional methods. In that case, for example, the codon usage of the host to be used can be considered [Ncleic Acids Res., 9, 43 (1981)].

(3) Production of the gene of the present invention The gene of the present invention can be easily produced and obtained by a general genetic engineering technique based on the sequence information of the specific examples of the gene of the present invention disclosed in the present specification. [See Molecular Cloning 2d Ed, Cold Spring Harbor Lab. Press (1989); Secondary Biochemistry Experiment Course “Gene Research Methods I, II, III”, Japanese Biochemical Society (1986), etc.)].

  Specifically, in the production, a cDNA library is prepared according to a conventional method from an appropriate source from which the gene of the present invention is expressed, and desired using an appropriate probe or antibody peculiar to the gene of the present invention from the library. Clones can be selected and performed (Proc. Natl. Acad. Sci., USA., 78, 6613 (1981); Science, 222, 778 (1983), etc.).

  In the above, examples of the origin of cDNA include various cells and tissues that express the gene of the present invention, cultured cells derived therefrom, and the like. Isolation of total RNA from these sources, isolation and purification of mRNA, acquisition of cDNA, and cloning thereof can all be performed according to conventional methods. MRNA or cDNA libraries are commercially available. The gene of the present invention can be produced by using such commercially available mRNA or cDNA libraries, for example, various mRNA or cDNA libraries commercially available from Clontech Lab. Inc., particularly cerebral cortex derived from human cerebellum or Alzheimer patients. It can also be performed using mRNA or cDNA libraries.

  The method for screening the gene of the present invention from a cDNA library is not particularly limited, and can be performed according to a usual method. Specific methods include, for example, a method of selecting a corresponding cDNA clone by immunoscreening using a specific antibody of a protein produced by cDNA, and a probe that selectively binds to a target DNA sequence. The plaque hybridization method, the colony hybridization method, etc. which were used, and these combinations can be illustrated.

  The probe used in the above is generally a DNA chemically synthesized based on information on the DNA sequence of the gene of the present invention. Moreover, the gene of the present invention already obtained and fragments thereof can be advantageously used as the probe. Furthermore, sense primers and antisense primers set based on the DNA sequence information of the gene of the present invention can be used as screening probes.

  The nucleotide used as a probe is a partial nucleotide corresponding to SEQ ID NO: 2, and is at least 15 contiguous DNAs, preferably at least 100 contiguous DNAs, more preferably at least 150 contiguous DNAs, most Those having at least 200 continuous DNAs are preferred. The positive clone itself for producing the above-described gene of the present invention can also be used as a probe.

  In obtaining the gene of the present invention, a DNA / RNA amplification method by PCR [Science, 230, 1350 (1985)] can be preferably used. In particular, when it is difficult to obtain a full-length cDNA from a library, the RACE method [Rapid amplification of cDNA ends; experimental medicine, 12 (6), 35 (1994)], particularly the 5′-RACE method [MA Frohman, et al ., Proc. Natl. Acad. Sci., USA., 8, 8998 (1988)].

  Primers used in carrying out the PCR method can be appropriately set based on the sequence information of the gene of the present invention clarified by the present invention, and can be synthesized according to a conventional method. The DNA / RNA fragment amplified by the PCR method can be isolated and purified according to a conventional method, for example, by gel electrophoresis.

  The gene of the present invention or various DNA fragments obtained above can be obtained by conventional methods such as the dideoxy method [Proc. Natl. Acad. Sci., USA., 74, 5463 (1977)], the Maxam-Gilbert method [Methods in Enzymology, 65 , 499 (1980)], etc., or simply using a commercially available sequence kit or the like, the DNA sequence can be determined.

(4) Production of the polypeptide of the present invention by genetic engineering The polypeptide of the present invention (expressed product) can be produced by utilizing the gene of the present invention in the usual genetic engineering methods (for example, Science, 224, 1431 (1984); Biochem. Biophys Res. Comm., 130, 692 (1985); Proc. Natl. Acad. Sci., USA., 80, 5990 (1983), etc.) and easily as an expression of the gene or as a protein containing the same. It can be manufactured in large quantities and stably. More specifically, a recombinant DNA (expression vector) capable of expressing a gene encoding a desired protein in a host cell is prepared, introduced into the host cell, transformed, the transformant is cultured, Subsequently, the polypeptide of the present invention (expressed product) can be obtained by recovering the target protein from the obtained culture.

  The present invention relates to a polypeptide encoded by a gene of the present invention, a vector containing the gene of the present invention for the production of the polypeptide, a host cell transformed with the vector, Also provided are methods for producing the inventive polypeptides.

  In the above, any of prokaryotes and eukaryotes can be used as the host cell. For example, the prokaryotic host may be any commonly used host such as Escherichia coli or Bacillus subtilis. Preferably, Escherichia coli, especially Escherichia coli K12 strain can be used. Eukaryotic host cells include cells such as vertebrates and yeast. Examples of the former include COS cells that are monkey cells (Cell, 23: 175 (1981)), Chinese hamster ovary cells and their dihydrofolate reductase-deficient strains [Proc. Natl. Acad. Sci., USA., 77: 4216 (1980)], etc., and the latter is preferably Saccharomyces yeast cells. Of course, it is not necessarily limited to these.

  When a prokaryotic cell is used as a host, a promoter and SD (Shine and Dalgarno) are used upstream of the gene so that the gene of the present invention can be expressed in the vector. An expression plasmid provided with a sequence and an initiation codon necessary for initiation of protein synthesis (for example, ATG) can be preferably used. As the vector, E. coli-derived plasmids such as pBR322, pBR325, pUC12, pUC13 and the like are generally used, but not limited to these, various known vectors can be used. Commercially available products of the above vectors used in expression systems using E. coli include, for example, pGEX-4T (Amersham Pharmacia Biotech), pMAL-C2, pMAL-P2 (New England Biolabs), pET21, pET21 / lacq (Invitrogen) And pBAD / His (Invitrogen).

  Examples of the expression vector in the case of using a vertebrate cell as a host typically include a promoter located upstream of the gene of the present invention to be expressed, an RNA splice site, a polyadenylation site and a transcription termination sequence. If necessary, this may have a replication origin. Specific examples of the expression vector include pSV2dhfr [Mol. Cell. Biol., 1: 854 (1981)] carrying the SV40 early promoter. In addition to the above, various known commercial vectors can be used. Examples of commercially available vectors used in expression systems using animal cells include animal cells such as pEGFP-N, pEGFP-C (Clontrech), pIND (Invitrogen), pcDNA3.1 / His (Invitrogen) Vectors for insect cells such as pFastBac HT (GibciBRL), pAcGHLT (PharMingen), pAc5 / V5-His, pMT / V5-His, pMT / Bip / V5-his (Invitrogen) .

  Specific examples of expression vectors in the case of using yeast cells as a host include, for example, pAM82 (Proc. Natl. Acad. Sci., USA., 80: 1 (1983)) having a promoter for the acid phosphatase gene. it can. Examples of commercially available expression vectors for yeast cells include pPICZ (Invitrogen) and pPICZα (Invitrogen).

  The promoter is not particularly limited, and when a bacterium belonging to the genus Escherichia is used as a host, for example, tryptophan (trp) promoter, lpp promoter, lac promoter, recA promoter, PL / PR promoter and the like can be preferably used. When the host is Bacillus, SP01 promoter, SP02 promoter, penP promoter and the like are preferable. As a promoter in the case of using yeast as a host, for example, pH05 promoter, PGK promoter, GAP promoter, ADH promoter and the like can be suitably used. Preferred promoters when animal cells are used as hosts include SV40-derived promoters, retrovirus promoters, metallothionein promoters, heat shock promoters, cytomegalovirus promoters, SRα promoters and the like.

  As the expression vector of the gene of the present invention, a normal fusion protein expression vector can also be preferably used. Specific examples of the vector include pGEX (Promega) for expression as a fusion protein with glutathione-S-transferase (GST).

  Moreover, examples of the polynucleotide sequence that helps the expression and secretion of the polypeptide from the host cell by the coding sequence of the mature polypeptide include a secretory sequence and a leader sequence. These sequences include a marker sequence (hexahistidine tag, histidine tag) used for purification of the fusion mature polypeptide against a bacterial host, and a hemagglutinin (HA) tag in the case of mammalian cells.

  A method for introducing a desired recombinant DNA (expression vector) into a host cell and a transformation method using the method are not particularly limited, and various general methods can be employed.

  The obtained transformant can be cultured according to a conventional method, and the target protein encoded by the gene designed as desired by the culture is expressed, produced (accumulated, accumulated) in the cell, extracellular or on the cell membrane of the transformant. Secreted).

  As the medium used for the culture, various media commonly used according to the employed host cells can be appropriately selected and used, and the culture can also be performed under conditions suitable for the growth of the host cells.

  The recombinant protein thus obtained (the polypeptide of the present invention) can be subjected to various separation operations utilizing its physical properties, chemical properties, etc. ["Biochemical Data Book II", pages 1175-1259, first edition, as desired. 1st printing, June 23, 1980, published by Tokyo Chemical Co., Ltd .; see Biochemistry, 25 (25), 8274 (1986); Eur. J. Biochem., 163, 313 (1987), etc.) .

  Specific examples of the method include ordinary reconstitution treatment, treatment with a protein precipitating agent (salting out method), centrifugation, osmotic shock method, ultrasonic crushing, ultrafiltration, molecular sieve chromatography (gel filtration). , Adsorption chromatography, ion exchange chromatography, affinity chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography, dialysis methods, and combinations thereof can be exemplified, and particularly preferred methods are specific for the polypeptide of the present invention. An affinity chromatography using a column to which a typical antibody is bound can be exemplified.

  In designing a gene encoding the polypeptide of the present invention, the DNA sequence of the KF-1 gene shown in SEQ ID NO: 2 can be used. The gene can be used by appropriately selecting and changing a codon indicating each amino acid residue as desired.

  Further, in the amino acid sequence encoded by the KF-1 gene, when a part of the amino acid residue or amino acid sequence is modified by substitution, insertion, deletion, addition, etc., for example, the above-mentioned cytospecific mutagenesis and the like Various methods can be employed.

(5) DNA molecule expression product as an active ingredient of the pharmaceutical composition of the present invention An expression product of a specific DNA molecule as an active ingredient in the pharmaceutical composition of the present invention (and the same expression product used in the screening method of the present invention) (Simply referred to as “the product of the present invention”) can be obtained by the genetic engineering technique described in (4) above.

  The apoptosis inhibitory action of the expression product of the present invention is synonymous with the apoptosis inhibitory action of the polypeptide of the present invention described in the above section (1). The detection of this inhibitory action can be performed by a known method as shown in the above item (1).

  More specifically, apoptosis and its inhibitory action can be detected by, for example, (1) agarose gel electrophoresis for detecting a fragment of DNA cleaved at a nucleosome unit as a DNA ladder, and (2) tens of bp to hundreds of kilobp. (3) TUNEL method (Gavrieli, Y., et al., J. Cell Biol.), Which detects DNA breaks to detect apoptosis in tissues. , 119, 493-501 (1992)), (4) After staining cells with fluorescent dyes, a method for detecting cells with changes in cell size or DNA content decreased, (5) After staining cells with fluorescent dyes, The detection of viable and dead cells can be performed by a method of performing flow cytometry. In addition, the activity of activated caspase characteristic of apoptosis is measured by the cleavage amount of the labeled substrate, or the amount of the activated caspase itself is measured using an antibody that specifically recognizes activated caspase. The apoptotic effect and its inhibitory effect can also be detected by the method. For details of the method, for example, CaspSELECT Caspase immunoassay Kits (manufactured by Bio Vison) can be referred to.

In addition, cell viability is determined by using a cell staining dye to separate viable and dead cells; enzyme activity measurement method; MTT method; radioisotope method for measuring free ⁵¹ Cr from cells; colony formation method, etc. It can be carried out according to various known methods. Apoptosis inhibitory action can also be detected by these methods. For details of the above-described detection methods for apoptosis and its inhibitory action, see Enomoto Hide et al., “Latest Apoptosis Experiment Method” (Yodosha, Tokyo, 1995); Yamada, H., Surgery Frontier, 3, 119 (1996). Etc. can be referred to.

  As the fluorescent dye for cell staining, various known dyes such as “Hoechst33342”, “DAPI”, “Calcein-AM”, FDA (Fluorescein diacetate), CFSE, propidium iodide (PI) Ethidium bromide (EB) can be used.

(6) Chemical synthesis of the polypeptide of the present invention The polypeptide of the present invention can also be produced by a general chemical synthesis method according to the amino acid sequence information shown in SEQ ID NO: 1. The method includes peptide synthesis methods by a normal liquid phase method and a solid phase method.

  More specifically, the peptide synthesis method is based on the amino acid sequence information, and a so-called stepwise elongation method in which each amino acid is sequentially linked one by one to extend the chain, and a fragment consisting of several amino acids is synthesized in advance. And then a fragment condensation method in which each fragment is subjected to a coupling reaction. The polypeptide of the present invention may be synthesized by any of them.

  Condensation methods employed for peptide 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 + additive (1-hydroxybenzotriazole, N-hydroxysuccinamide, N-hydroxy-5 -Norbornene-2,3-dicarboximide etc.) method, Woodward method etc. can be followed.

  The solvent used in each of these methods can also be appropriately selected from general solvents that are known to be used in this kind of peptide condensation reaction. Examples thereof include dimethylformamide (DMF), dimethyl sulfoxide (DMSO), hexaphosphoroamide, dioxane, tetrahydrofuran (THF), ethyl acetate, and a mixed solvent thereof.

  In the peptide synthesis reaction, an amino acid or a carboxyl group in a peptide that is not involved in the reaction is generally esterified, for example, a lower alkyl ester such as methyl ester, ethyl ester or tertiary butyl ester, such as benzyl ester, p- It can be protected as aralkyl esters such as methoxybenzyl ester and p-nitrobenzyl ester.

  An amino acid having a functional group in the side chain, for example, a hydroxyl group of a tyrosine residue may be protected with an acetyl group, a benzyl group, a benzyloxycarbonyl group, a tertiary butyl group, etc., but such protection is necessarily required. There is no. Furthermore, for example, the guanidino group of the arginine residue may be a suitable nitro group, tosyl group, p-methoxybenzenesulfonyl group, methylene-2-sulfonyl group, benzyloxycarbonyl group, isobornyloxycarbonyl group, adamantyloxycarbonyl group, etc. Can be protected by various protecting groups.

  The deprotection reaction of these protecting groups in amino acids having a protecting group, peptides and finally obtained polypeptides of the present invention is also carried out by conventional methods such as catalytic reduction, liquid ammonia / sodium, hydrogen fluoride, bromide. It can be carried out according to a method using hydrogen, hydrogen chloride, trifluoroacetic acid, acetic acid, formic acid, methanesulfonic acid or the like.

  The polypeptide of the present invention thus obtained can be appropriately purified according to the various methods described above, for example, methods widely used in the field of peptide chemistry such as ion exchange resin, distribution chromatography, gel chromatography, and countercurrent distribution. it can.

(7) Pharmaceutical composition of the present invention The pharmaceutical composition of the present invention comprises the polypeptide of the present invention and the expressed product of the present invention as active ingredients. The pharmaceutical composition is useful, for example, as an inhibitor of apoptosis induced by endoplasmic reticulum stress, and as an agent for improving cell degeneration such as nerve cells and pancreatic β cells caused by the apoptosis. In addition, using the above-mentioned action as an apoptosis inhibitor and cytopathic agent, (familial or sporadic) Alzheimer's disease (Alzheimer-type senile dementia), Parkinson's disease, Huntington's disease, polyglutamine disease, ischemic brain Treatment for disorders (cerebral ischemia), amyotrophic lateral sclerosis, multiple sclerosis, HIV-related neuropathy, diffuse Lewy body disease, type I diabetes, type II diabetes, depression, manic depression, etc. It is also useful as an agent and prophylactic agent.

  More specifically, the polypeptide of the present invention and the expression product of the present invention (for example, KF-1) as an active ingredient in the pharmaceutical composition of the present invention have an action of suppressing apoptosis induced by ER stress peculiar to them. This action or activity can be used to treat diseases related to apoptosis and cell degeneration of various cells. Examples of cells affected by apoptosis include nerve cells, neuroblasts, pancreatic β cells, hepatocytes, epithelial cells, skeletal muscle cells, smooth muscle cells, fibroblasts, lymphocytes and the like. Important tissues containing these cells include cerebellum, cerebral cortex, midbrain, pituitary gland, pancreas, stomach, small intestine, lung, liver, kidney, large intestine, especially cerebellum, cerebral cortex, pituitary gland, pancreas, etc. Can be mentioned. Specific examples of the cells include neuroblastoma, glioma cells, and other neurons such as N2aSK-N-SH cells (ATCC No. HTB-11), N18TG-2, IMR-32 (ATCC No. CCL-127 ), GOTO, NBI, C6BU-1, U251, KNS42, KNS81, NG108-15 cells, PC12 cells (ATCC No. CRL-1721) and the like. Human fetal kidney-derived cells HEK293T cells are also included in the specific examples of the above cells.

  The polypeptide (expressed product) as an active ingredient in the pharmaceutical composition of the present invention also includes pharmaceutically acceptable salts thereof. Such salts include non-toxic alkali metal salts such as sodium, potassium, lithium, calcium, magnesium, barium, ammonium, alkaline earth metal salts, ammonium salts and the like prepared by methods well known in the art. The Further, the above salts include nontoxic acid addition salts obtained by reacting the polypeptide of the present invention (expressed product) with an appropriate organic acid or inorganic acid. Representative non-toxic acid addition salts include, for example, hydrochloride, hydrochloride, hydrobromide, sulfate, bisulfate, acetate, oxalate, valerate, oleate, laurate, Borate, benzoate, lactate, phosphate, p-toluenesulfonate (tosylate), citrate, maleate, fumarate, succinate, tartrate, sulfonate, glycolate , Maleate, ascorbate, benzenesulfonate, napsilate and the like.

  The pharmaceutical composition of the present invention is prepared into a pharmaceutical preparation form containing the polypeptide (expressed product) of the present invention and a salt thereof as an active ingredient and a pharmaceutically effective amount thereof together with an appropriate pharmaceutical carrier or diluent.

  As a pharmaceutical carrier used for the preparation of the pharmaceutical preparation, dilutions such as a filler, a bulking agent, a binder, a moistening agent, a disintegrant, a surfactant, a lubricant, etc., which are usually used depending on the form of the preparation Agents or excipients can be exemplified. These are appropriately selected and used depending on the dosage unit form of the preparation to be obtained. Particularly preferred pharmaceutical preparations are various ingredients used in ordinary protein preparations, such as stabilizers, bactericides, buffers, isotonic agents, chelating agents, pH adjusters, surfactants and the like as appropriate. Prepared.

  In the above, examples of the stabilizer include human serum albumin, ordinary L-amino acids, saccharides, and cellulose derivatives. 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.

  The saccharide is 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, chondroitin Polysaccharides such as sulfuric acid and hyaluronic acid, and derivatives thereof can be used.

  The surfactant is not particularly limited, and both ionic and nonionic surfactants can be used. Specific examples thereof include polyoxyethylene glycol sorbitan alkyl ester, polyoxyethylene alkyl ether, sorbitan monoacyl ester, and fatty acid glyceride.

  The cellulose derivative is not particularly limited, and methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and the like can be used.

  The amount of the saccharide added is appropriately about 0.0001 mg or more, preferably about 0.01 to 10 mg per 1 μg of the active ingredient. The amount of the surfactant added is suitably about 0.00001 mg or more, preferably about 0.0001-0.01 mg per μg of active ingredient. The amount of human serum albumin added is suitably about 0.0001 mg or more, preferably about 0.001-0.1 mg per 1 μg of active ingredient. The amount of L-amino acid added is suitably about 0.001-10 mg per 1 μg of active ingredient. The addition amount of the cellulose derivative is appropriately about 0.00001 mg or more, preferably about 0.001-0.1 mg per 1 μg of the active ingredient.

  The amount of the active ingredient contained in the pharmaceutical preparation of the present invention is appropriately selected from a wide range. Usually, it is appropriate that the preparation contains about 0.00001-70% by weight, preferably about 0.0001-5% by weight.

  Various additives such as buffers, isotonic agents, chelating agents and the like can also be added to the pharmaceutical preparation of the present invention. Buffering agents include boric acid, phosphoric acid, acetic acid, citric acid, ε-aminocaproic acid, glutamic acid and / or their corresponding salts (e.g., alkali metal salts such as sodium, potassium, calcium and magnesium salts thereof) And alkaline earth metal salts). Examples of the tonicity agent include sodium chloride, potassium chloride, saccharides, glycerin and the like. Examples of chelating agents include sodium edetate and citric acid.

  The pharmaceutical preparation of the present invention can be prepared as a solution preparation, lyophilized and stored, and then dissolved in a buffer solution containing water for use, saline solution, etc. to an appropriate concentration. It can also be in the form of a lyophilized agent to be prepared.

  The dosage unit form of the pharmaceutical preparation of the present invention can be appropriately selected depending on the therapeutic purpose. Representative examples include solid dosage forms such as tablets, pills, powders, powders, granules, capsules, and liquid dosage forms such as solutions, suspensions, emulsions, syrups, elixirs and the like. These are further classified into oral preparations, parenteral preparations, nasal preparations, vaginal preparations, suppositories, sublingual preparations, ointments and the like according to the administration route, and can be prepared, molded or prepared according to usual methods. it can. In addition, in the chemical | medical agent of this invention, a coloring agent, a preservative, a fragrance | flavor, a flavoring agent, a sweetening agent, other pharmaceuticals, etc. can also be contained as needed.

  The administration method of the pharmaceutical preparation is not particularly limited, and is determined according to various preparation forms, patient age, sex and other conditions, the degree of disease, and the like. For example, tablets, pills, solutions, suspensions, emulsions, granules, and capsules are administered orally, and injections are administered intravenously alone or mixed with normal fluids such as glucose and amino acids. Independently administered intramuscularly, intradermally, subcutaneously or intraperitoneally, suppositories are administered rectally, vaginal agents are administered intravaginally, nasal agents are administered intranasally, sublingual agents are administered orally, Ointments are topically administered transdermally.

  The amount of the active ingredient to be contained in the pharmaceutical preparation and the dose thereof are not particularly limited, and are appropriately selected from a wide range depending on the desired therapeutic effect, administration method, treatment period, patient age, sex, and other conditions. Selected. In general, the dose is usually about 0.01 μg to 10 mg, preferably about 0.1 μg to 1 mg per kg body weight per day, and the preparation is once to several times a day. Can be administered separately.

(8) Gene therapy As shown in the Examples below, the expression of the gene of the present invention (KF-1 gene) is observed in tissues such as the cerebellum, midbrain, pituitary gland, pancreatic β cell, Alzheimer patient cerebral cortex, etc. Therefore, all or part of the sense DNA of the KF-1 gene according to the present invention is directly or in the form of any gene expression vector containing the same, and a virus or liposome is placed in a tissue cell such as the cerebellum or pancreas. By introducing it, it is possible to produce RNA having a complementary sequence in the cell and forcibly increase the expression of the KF-1 gene by promoting translation. Thus, it is possible to suppress apoptosis in tissues such as cerebellum and pancreas, and as a result suppress cytopathy or cell death caused by apoptosis, thereby suppressing or improving pathological progress of diseases related to cell degeneration or cell death caused by apoptosis. it can. These effects can cause (familial or sporadic) Alzheimer's disease (Alzheimer-type senile dementia), Parkinson's disease, Huntington's disease, polyglutamine disease, ischemic brain injury (brain ischemia), amyotrophic lateral sclerosis It can be used for the suppression or improvement of the development of infectious disease, multiple sclerosis, HIV-related neuropathy, diffuse Lewy body disease, type I diabetes, type II diabetes, depression, manic depression and the like. The present invention also provides a gene therapy composition or gene therapy agent having such an apoptosis-inhibiting action.

  As described above, the present invention provides a gene therapy vector containing all or part of the KF-1 gene and a pharmaceutical composition comprising as an active ingredient a cell into which the KF-1 gene has been introduced. More specifically, the present invention relates to a gene therapy vector containing a sense gene containing the entire or part of the sense DNA of the DNA sequence represented by SEQ ID NO: 2, a cell into which the sense gene has been introduced, and the cell Provided are a gene therapy vector and a gene therapy agent comprising as an active ingredient a cell into which a sense gene has been introduced. In the above and the following, “partial sequence” means a partial sequence including a sequence capable of expressing apoptosis-inhibiting activity.

  The present invention also relates to a gene therapy vector containing a sense gene containing the entire or part of the sense DNA of the DNA sequence represented by SEQ ID NO: 2, and a cell into which the sense gene has been introduced (family). Or sporadic) Alzheimer's disease (Alzheimer-type senile dementia), Parkinson's disease, Huntington's disease, polyglutamine disease, ischemic brain injury (brain ischemia), amyotrophic lateral sclerosis, multiple sclerosis, HIV Cells produced by apoptosis in these tissues when administered to skeletal muscle cells or muscle tissue sites of patients with associated neuropathy, diffuse Lewy body disease, type I diabetes, type II diabetes, depression, manic depression, etc. Provided is a method for suppressing or improving degeneration and suppressing or improving the progression of pathological conditions in each of the above diseases, that is, a method for improving cytopathy, particularly a method for suppressing apoptosis. The

  Hereinafter, such gene therapy will be described in detail. In the following gene therapy, conventional methods of chemistry, molecular biology, microbiology, recombinant DNA, genetics and immunology can be used. These include, for example, Maniatis (Tania, T., et al., Molecular Cloning: A laboratory manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1982)), Sambrook (J., et al. , Molecular Cloning: A laboratory manual, 2nd Ed. (Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1981)), Ausbel (FM, et al., Current Protocols in Molecular Biology, John Wiley and Sons, New York, (1992)), Glover (Glover, D., DNA Cloning, I and II (Oxford Press) (1985)), Anand (Anand, Techniques for the Analysis of Complex Genomes, Academic Press (1992)), Goosely ( Guthrie, G., et al., Guide to Yeast Genetics and Molecular Biology, Academic Press (1991)) and Fink (Fink, et al., Hum. Gene Ther., 3, 11-19 (1992)) Has been.

  The gene therapy of the present invention is a sense pharmaceutical for producing RNA having a sequence complementary to intracellular mRNA, inhibiting translation and suppressing expression of KF-1 gene in cells expressing KF-1 gene Inhibition method of apoptosis, particularly, a suppression method of apoptosis caused by induction of endoplasmic reticulum stress, or a method of suppressing cell degeneration in cells or tissues. The therapeutic method provides, for example, a KF-1 expressing cell original amount or an excessive amount of mRNA to a KF-1 gene expressing cell in which expression of the KF-1 gene is reduced or significantly reduced. Thus, the transcription or translation process is enhanced to increase the expression of the target gene. In this method, a sense oligonucleotide complementary to the mRNA of the target gene is produced, and the sense oligonucleotide is supplied to the target cell. As described above, if the action of promoting the expression function of the KF-1 gene is supplied to the target cell, apoptosis in the recipient cell / target cell can be suppressed, and cytopathy can be suppressed.

  Specific examples of supplying the sense oligonucleotide to the target cell include a method of introducing a vector or plasmid containing the sense oligonucleotide into the target cell and maintaining the sense oligonucleotide outside the chromosome. it can.

  According to the gene therapy for cytopathic inhibition based on the inhibitory action of apoptosis using the sense oligonucleotide according to the present invention, the sense oligonucleotide is incorporated into a retrovirus, adenovirus, AAV-derived vector, etc. By infecting 1-expressing cells and overexpressing the sense oligonucleotide, the desired apoptosis-inhibiting action can be increased or enhanced, and thus the apoptosis-inhibiting effect or cytopathic-inhibiting effect can be obtained.

  When a sense oligonucleotide is introduced into a target cell having a KF-1 gene to suppress the expression of KF-1, the sense oligonucleotide need not correspond to the full length of the KF-1 gene. As long as it retains substantially the same function as the function of suppressing the expression function of the KF-1 gene, for example, the above-described variant may be a gene consisting of a partial sequence retaining a specific function. May be.

  Any of various vectors already known in the art can be used as a vector for introducing a desired gene for both incorporation of a sense oligonucleotide into a vector and maintenance of the target cell extrachromosomally. The vector is, for example, a viral vector or plasmid vector that contains a copy of the sense oligonucleotide of KF-1 linked to an expression control element and can express the sense oligonucleotide product in the target cell. Specific examples thereof include, for example, expression vectors (pWP-7A, pwP-19, pWU-1, pWP-8A, pWP-21, and the like disclosed in US Pat. No. 5,252,479 and PCT International Publication WO 93/07282. And / or pRSVL), pRC / CMV (Invitrogen) and the like. Suitable vectors are various virus vectors described below.

  In addition, as a promoter used for a vector used in gene transfer therapy, a promoter specific to a diseased tissue to be treated for various diseases can be suitably used.

  As specific examples, for the brain, for example, glial fibrillary acidic protein, mature astrocyte-specific protein, myelin, tyrosine hydroxylase pancreatic villin, glucagon, Langerhans islet amyloid polypeptide and the like can be exemplified.

  Examples of the thyroid gland include thyroglobulin and calcitonin.

  For bone, α1 collagen, osteocalcin, bone sialoglycoprotein and the like can be exemplified.

  For the kidney, renin, liver / bone / kidney alkaline phosphatase, erythropoietin and the like can be exemplified.

  Examples of the pancreas include amylase and PAP1.

  For the small intestine, villin, lactase, adenosine deamidase, calbindinin, small intestine fatty acid binding protein, sodium glucose cotransporter and the like can be exemplified.

  For the large intestine, colon carboxylic acid dehydrogenase can be exemplified.

  For the liver, albumin, α-fetoprotein, α1-antitrypsin, transferrin, transstyrene and the like can be exemplified.

  For the colon, carboxylate anhydrase I, an antigen of carcinoenbrogen, and the like can be exemplified.

  For the uterus and placenta, estrogen, aromatase cytochrome P450, cholesterol side chain cleavage P450, 17 alpha hydroxylase P450 and the like can be exemplified.

  For the prostate, prostate antigen, gp91-fox gene, prostate-specific kallikrein and the like can be exemplified.

  Examples of the breast include erb-B2, erb-B3, β-casein, β-lactoglobin, and whey protein.

  For the lung, active agent protein C uroglobulin and the like can be exemplified.

  For the skin, K-14-keratin, human keratin 1 or 6, leucline and the like can be exemplified.

  In addition, in the production of the sense oligonucleotide introduction vector, the sense oligonucleotide (all or part of the complementary sequence corresponding to the KF-1 gene) to be introduced, based on the DNA sequence information of the KF-1 gene, As described above, it can be easily produced and obtained by a general genetic engineering technique.

  The introduction of the sense oligonucleotide vector into the target cell should be carried out according to various methods already known in the art for introducing DNA into the cell, such as electroporation, calcium phosphate coprecipitation, viral transduction, etc. Can do. Cells transformed with the KF-1 gene sense oligonucleotide can be used as pharmaceuticals to suppress apoptosis or cytopathic effects in isolation, and as a model system for therapeutic research. it can.

  The sense oligonucleotide introduction vector can be introduced into a target cell of a patient by local or systemic injection administration to a tissue site of the patient. Systemic administration allows the desired vector to reach any cell capable of expressing KF-1 mRNA. If the transduced gene is not permanently incorporated into the chromosome of each target cell, the administration can be repeated periodically.

  The gene therapy method of the present invention includes an in vivo method in which a sense oligonucleotide introduction vector is directly administered into the body, a target cell is once removed from the patient's body, the gene is introduced outside the body, and then And both ex vivo methods of returning the cells to the body.

  The gene therapy of the present invention is preferably used as suppression of apoptosis particularly for patients with cytopathic diseases caused by apoptosis. The gene therapy (treatment) of the present invention includes (familial or sporadic) Alzheimer's disease (Alzheimer-type senile dementia), Parkinson's disease, Huntington's disease, polyglutamine disease, ischemic brain injury (brain ischemia), muscle For the treatment of amyotrophic lateral sclerosis, multiple sclerosis, HIV-related neuropathy, diffuse Lewy body disease, type I diabetes, type II diabetes, depression, manic depression and their complications You can also Furthermore, the present invention is not limited to gene therapy, and for example, gene markers can also be used for that purpose.

  The target cell into which the sense oligonucleotide is introduced can be appropriately selected depending on the target of gene therapy (treatment). Examples of the target cells include various cells in which KF-1 expression is observed, and tissues that are aggregates thereof, particularly the cerebellum, cerebral cortex, pituitary gland, liver, pancreas, lung, small intestine tissue, lymphocytes, nerve cells, Neuroblasts, pancreatic β cells, adipocytes, hepatocytes, fibroblasts, mucosal epithelial cells, hematopoietic stem cells, epidermal cells and the like can be mentioned.

  Methods for introducing sense oligonucleotides in gene therapy include viral introduction methods and non-viral introduction methods.

  As a viral introduction method, for example, in view of the fact that the sense oligonucleotide of the KF-1 gene is a foreign substance expressed in normal cells, a method using a retroviral vector as a vector can be mentioned. Other viral vectors include adenovirus vectors, HIV (human immunodeficiency virus) vectors, adeno-associated virus vectors (AAV, adeno-associated virus), herpes virus vectors, herpes simplex virus (HSV) vectors, Epstein-Barr virus (EBV , Epstein-Barr virus) vector.

  As non-viral introduction method, calcium phosphate coprecipitation method: fusion of liposome encapsulated with DNA and inactivated Sendai virus that had previously destroyed the gene with ultraviolet rays, and created a membrane fusion liposome, which was directly fused to the cell membrane Membrane fusion liposome method for introducing DNA into cells (Kato, K., et al., J. Biol. Chem., 266, 22071-22074 (1991)); A method of physically introducing DNA into cells [Yang, NS et al., Proc. Natl. Acad. Sci., USA., 87, 9568-9572 (1990)]; Naked DNA method for injection into tissue [Wolff, JA, et al., Science, 247,1465-1467 (1990)]; Cationic liposome method for introducing genes embedded in multilamellar positively charged liposomes into cells [Kunio Yagi, History of Medicine, Vol.175, No.9, 635-637 (1995)]; inherited only in specific cells Ligand-DNA complex method in which a ligand that binds to a receptor expressed in the target cell is bound to DNA and administered (Frindeis, et al. , Trends Biotechnol., 11, 202 (1993); Miller, et al., FASEB J., 9,190 (1995)].

  In the ligand-DNA complex method, for example, an asialoglycoprotein receptor expressed in hepatocytes is used as a target, and asialoglycoprotein is used as a ligand [Wu, et al., J. Biol. Chem., 266, 14338 (1991 Ferkol, et al., FASEB. J., 7, 1081-1091 (1993)], a method using transferrin as a ligand targeting a transferrin receptor strongly expressed in the target cell [Wagner et al., Proc Natl. Acad. Sci., USA., 87, 3410 (1990)].

  Further, the gene transfer method may be a combination of various biological and physical gene transfer methods as described above as appropriate. Examples of the combination method include a method of combining a plasmid DNA of a certain size with a polylysine-conjugated antibody specific for an adenovirus hexon protein. According to this method, the resulting complex binds to an adenovirus vector, and the cells are infected with the thus obtained trimolecular complex, whereby the sense oligonucleotide of the present invention can be introduced into the cell. This method allows for efficient binding, internalization and endosomal degradation before the DNA coupled to the adenovirus vector is damaged. Liposomes / DNA complexes can also mediate gene transfer directly in vivo.

  Each gene transfer vector can be prepared by a known method. For example, EBV vectors can be produced according to the method of Shimizu et al. [Norio Shimizu et al., Cell Engineering, 14 (3), 280-287 (1995)].

  The non-viral introduction method is a method for introducing a desired sense oligonucleotide into a target cell without using a recombinant viral vector. One of the methods is a gene transfer method using membrane-fused liposomes, for example, by the method of Nakanishi et al. [Nakanishi, M., et al., Exp. Cell Res., 159, 399-499 (1985) ; Nakanishi, M., et al., Gene introduction into animal tissues.In Trends and Future Perspectives in Peptide and Protein Drug Delivery (ed. By Lee, VH et al.), Harwood Academic Publishers Gmbh.Amsterdam, 1995, pp. 337-349].

  In the gene therapy of the present invention, methods for introducing a desired gene into a target cell or target tissue typically include two methods.

  The first method is to collect target cells from a patient to be treated, and then culture the cells outside the body under the addition of, for example, interleukin-2 (IL-2). This is a method (ex vivo method) in which the obtained cells are re-transplanted after introducing the sense oligonucleotide of the KF-1 gene.

  The second method is a direct gene introduction method (direct method) in which a target sense oligonucleotide (sense oligonucleotide of KF-1 gene) is directly injected into a patient's body or a target site such as skeletal muscle.

  More specifically, the first method of gene therapy is performed as follows, for example. That is, mononuclear cells collected from a patient are separated from monocytes using a blood separator, and the sorted cells are cultured in an appropriate medium such as AIM-V medium for about 72 hours in the presence of IL-2 and introduced. A vector containing the sense oligonucleotide (sense oligonucleotide of the KF-1 gene) is added. In order to increase the efficiency of introduction of the sense oligonucleotide, it may be centrifuged at 2500 rpm for 1 hour at 32 ° C. in the presence of protamine, and then cultured at 37 ° C. under 10% carbon dioxide for 24 hours. After repeating this operation several times, the cells were further cultured for 48 hours in AIM-V medium in the presence of IL-2, the cells were washed with physiological saline, the number of viable cells was calculated, and the efficiency of introduction of sense oligonucleotide was improved. The target sense oligonucleotide can be obtained by the in situ PCR, for example, by measuring the degree of apoptosis inhibition by the desired target as in the present invention, or by measuring the inhibition of apoptosis caused by endoplasmic reticulum stress induction. Confirm the introduction effect of.

In addition, after confirming safety by searching for bacterial / fungal culture in the cultured cells, presence of mycoplasma infection, presence of endotoxin, etc. Cultured cells into which nucleotides (sense oligonucleotides of KF-1 gene) have been introduced are returned to the patient by intravenous infusion. Gene therapy is performed by repeating such a method, for example, at intervals of several weeks to several months. The dosage of the viral vector is appropriately selected depending on the target cell to be introduced. Usually, as the virus titer, for example, a dose in the range of 1 × 10 ³ cfu to 1 × 10 ⁸ cfu is preferably employed for 1 × 10 ⁸ target cells.

  As an alternative to the first method, a virus-producing cell containing a retroviral vector containing a target sense oligonucleotide (sense oligonucleotide of the KF-1 gene) and a patient cell, for example, are co-cultured and the target It is also possible to adopt a method of introducing a sense oligonucleotide (sense oligonucleotide of the KF-1 gene) into the cells.

  In conducting the second method (direct method) of gene therapy, whether or not the target sense oligonucleotide (sense oligonucleotide of the KF-1 gene) is actually introduced by the gene transfer method is determined in advance. Inhibition of apoptosis, which is a desired therapeutic effect based on experiments, for example, confirmation in advance by PCR of vector gene cDNA or in situ PCR, or introduction of the desired sense oligonucleotide (sense oligonucleotide of KF-1 gene) It is desirable to confirm in advance the action, decrease in apoptosis of target cells, decrease in the number of degenerated cells, and the like. When viral vectors are used, the sense of gene therapy can be determined by searching for proliferative retroviruses by PCR, measuring reverse transcriptase activity, or monitoring the membrane protein (env) gene by PCR. -It is important to confirm the safety of oligonucleotide introduction.

  In the gene therapy of the present invention, when targeting brain neurons of patients with Alzheimer's disease, Parkinson's disease, depression, etc., bone marrow stem cells (KF-1 expressing cells) are collected from the patient's bone marrow and then subjected to enzyme treatment and the like. After establishing cultured cells, for example, introduced into cultured bone marrow stem cells (KF-1 expressing cells) targeting the desired sense oligonucleotide with retrovirus, screened with G418 cells, IL-12 etc. A preferred method is to measure the expression level of (in vivo) and then inject the cells into the patient's brain after radiation treatment.

 When targeting patients with type I and type II diabetes, after collecting pancreatic β cells (KF-1 expressing cells) from the patient's pancreas, applying cultured enzymes, etc. Introduced into cultured pancreatic β-cells (KF-1 expressing cells) targeting the sense oligonucleotide, and screened with G418 cells, measured the expression level of IL-12 etc. (in vivo), then A preferred method is to inoculate the patient's pancreas after the radiation treatment.

(9) Preparation of gene therapy agent The present invention comprises a cell into which a sense oligonucleotide introduction vector or a sense oligonucleotide (sense oligonucleotide of the KF-1 gene) has been introduced as an active ingredient, and their pharmaceutically effective Provided is a pharmaceutical composition or pharmaceutical formulation (gene therapy agent) containing the amount together with a suitable non-toxic pharmaceutical carrier or diluent.

  As a pharmaceutical carrier that can be used in the pharmaceutical composition (pharmaceutical preparation) of the present invention, a filler, a bulking agent, a binder, a moistening agent, a disintegrant, a surfactant, which are usually used according to the use form of the preparation, Diluents or excipients such as lubricants can be exemplified, and these can be appropriately selected and used depending on the dosage unit form of the resulting preparation.

  Examples of the dosage unit form of the pharmaceutical preparation of the present invention include those similar to those detailed in the above item (7). This can be appropriately selected from various forms depending on the purpose of treatment.

  For example, a pharmaceutical preparation containing a vector for introducing a sense oligonucleotide is in a form in which the vector is embedded in a liposome or a cultured cell infected with a virus containing a retroviral vector containing the desired sense oligonucleotide. To be prepared.

  These can also be prepared in the form of a phosphate buffered saline solution (pH 7.4), Ringer's solution, an injection for intracellular composition solution, etc., and administered together with a substance that enhances gene transfer efficiency such as protamine. It can also be prepared in such a form.

  The administration method of the pharmaceutical preparation is not particularly limited, and is determined according to various preparation forms, patient age, sex and other conditions, the degree of disease, and the like.

  The amount of the active ingredient to be contained in the pharmaceutical preparation and the dose thereof are not particularly limited, and are appropriately selected from a wide range depending on the desired therapeutic effect, administration method, treatment period, patient age, sex, and other conditions. Selected.

In general, the dose of the desired sense oligonucleotide-containing retroviral vector as a pharmaceutical preparation is about 1 × 10 ³ pfu to 1 × 10 ¹⁵ pfu per kg body weight per day, for example, as the titer of retrovirus. Is good.

In the case of a cell into which a desired sense oligonucleotide for introduction is introduced, it is appropriate to select from a range of about 1 × 10 ⁴ cells / body to 1 × 10 ¹⁵ cells / body.

  The preparation can be administered once or divided into several times a day, or can be intermittently administered at intervals of 1 to several weeks. In addition, Preferably, it can administer together with the substance which improves gene transfer efficiency, such as a protamine, or a formulation containing this.

(10) Detection of KF-1 gene The present invention relates to KF-1 having a cell apoptosis inhibitory effect (particularly, an apoptosis inhibitory effect induced by induction of endoplasmic reticulum stress) and a gene encoding the same (in particular, this gene is an endoplasmic reticulum). A method for detecting the presence of high expression by stress induction is provided.

  The method prepares a biological sample such as blood or serum, extracts nucleic acid from the sample, if desired, and analyzes whether or not a susceptibility gene for KF-1 is present in the sample and nucleic acid. .

  In addition, the present invention detects the level of KF-1 activity in cells or tissues by detecting the KF-1 gene (apoptosis inhibitory action, in particular, apoptosis inhibitory action induced by endoplasmic reticulum stress induction, expression level of KF-1 mRNA, etc.) Is provided to determine the degree of apoptosis, the degree of progression of cell degeneration (apoptotic cells) caused by apoptosis, the degree of increase in cell death, or the prognosis. The method comprises preparing a biological sample in which apoptosis is induced in a cell (by endoplasmic reticulum stress) and causing cell degeneration, and analyzing whether the KF-1 gene is present in the sample, or KF The basic operation is to analyze the amount of mRNA of -1 gene.

  By carrying out the method of detecting the KF-1 gene of the present invention, apoptosis suppression effect induced by endoplasmic reticulum stress induction in cells or tissues, expression level of KF-1 mRNA, enhancement of apoptosis or cell degeneration caused by apoptosis (apoptotic cells) It is possible to obtain a degree of progression, an increase in cell death, or a prognostic indicator. In addition, for example, the diagnosis of cell degeneration (apoptotic cells), cell death, etc. caused by increased apoptosis or apoptosis, (familial or sporadic) Alzheimer's disease (Alzheimer type senile dementia), Parkinson's disease, Huntington's disease, polyglutamine Disease, ischemic brain injury (brain ischemia), amyotrophic lateral sclerosis, multiple sclerosis, HIV-related neuropathy, diffuse Lewy body disease, type I diabetes, type II diabetes, depression, epilepsy It is possible to determine a therapeutic effect such as depression or predict a prognosis.

  In this detection method, first, screening and / or amplification of the KF-1 gene is performed based on information on the KF-1 gene obtained from a sample of apoptotic cells or degenerated cells caused by apoptosis induced by endoplasmic reticulum stress. Create a DNA fragment designed for use. More specifically, a DNA fragment having a property as a probe that can be used in, for example, plaque hybridization, colony hybridization, Southern blotting, Northern blotting or the like is prepared. In addition, a DNA fragment having a property as a probe for obtaining all or part of the DNA fragment of the KF-1 gene amplified by polymerase chain reaction (PCR) in which the DNA sequence is amplified by polymerase is prepared.

  Next, for example, a primer having the same sequence as that of the KF-1 gene is used as a screening probe and reacted with a biological sample (nucleic acid sample) to confirm the presence of the KF-1 gene in the sample. The sample may be prepared by various methods that facilitate detection of the target sequence, such as denaturation, restriction digestion, electrophoresis or dot blotting.

  As a screening method for confirming the presence of the KF-1 gene, PCR is particularly preferred from the viewpoint of sensitivity. The method uses a KF-1 gene fragment as a primer, a conventionally known method (Science, 230, 1350-1354 (1985)), or a newly developed or used PCR method (for example, Yoshiyuki Tsuji) , Et al., Yodosha, Experimental Medicine, Special Issue, 8 (9) (1990); Protein / Nucleic Acid / Enzyme, Extra Special Issue, Kyoritsu Publishing Co., Ltd., 35 (17) (1990)) .

  The DNA fragment used as a primer is not particularly limited as long as it is unique to the gene capable of specifically amplifying the gene of the present invention (KF-1 gene). This may be a chemically synthesized oligo DNA. The oligo DNA can be synthesized using an automatic DNA synthesizer, for example, a DNA synthesizer (Pharmacia LKB Gene Assembler Plus: Pharmacia). The length of the synthesized primer (sense or antisense primer) is preferably about 10-50 nucleotides, more preferably about 15-30 nucleotides. In addition, the primer to be synthesized is preferably a sequence that can distinguish other known similar DNA sequences.

  The probe used in the screening method is usually labeled, but may be unlabeled, or may be detected by specific binding with a directly or indirectly labeled ligand. Methods for labeling probes and ligands are known in the art and can follow methods such as nick translation, random priming, kinase treatment, for example. As the labeling agent, for example, any of a radiolabeling agent, biotin, a fluorescent group, a chemiluminescent group, an enzyme, an antibody and the like can be used.

  PCR used for detection is, for example, RT-PCR (Reverse transcribed-Polymerase chain reaction; ES Kawasaki, et al., Amplification of RNA.In PCR Protocol, A Guide to methods and applications, Academic Press, Inc., San Diego, 21-27 (1991)] is preferable, but it is of course possible to appropriately apply various methods used in this field. Examples of the method include Northern blotting analysis [Molecular Cloning, Cold Spring Harbor Lab. (1989)], in situ RT-PCR [Nucl. Acids Res., 21, 3159-3166 (1993)], in situ hybridization. Examples include measurement at the cell level using NASBA, NASBA method [Nucleic acid sequence-based amplification, Nature, 350, 91-92 (1991)], and other various methods.

(11) KF-1 gene detection and diagnostic kit The detection method of the present invention can be more easily carried out by using a kit. The present invention also provides a kit for detecting a KF-1 gene containing a DNA fragment of the KF-1 gene.

  The kit must include at least a DNA fragment that hybridizes to a part or all of the DNA sequence represented by SEQ ID NO: 2 or its complementary DNA sequence as an essential component. Others can contain, for example, a labeling agent, a reagent essential for PCR (for example, Taq DNA polymerase, deoxynucleotide triphosphate, primer) and the like, as in the usual reagent kit of this kind.

  Examples of the labeling agent include chemically modified substances such as radioisotopes and fluorescent substances, but the DNA fragment itself may be conjugated with the labeling agent in advance. Further, the reagent kit may contain an appropriate reaction diluent, standard antibody, buffer solution, detergent, reaction stop solution and the like for the convenience in carrying out the measurement.

  The kit for detecting a KF-1 gene of the present invention can be used not only for measurement and detection of the KF-1 gene but also for detecting the degree of cell damage due to apoptosis based on the detection result. In particular, it is possible to diagnose the state of apoptosis induced by endoplasmic reticulum stress or the progress of cell degeneration. Therefore, the present invention also provides a kit for diagnosing cell damage due to apoptosis.

  Further, in the regulation and detection method according to the present invention, the DNA sequence of the KF-1 gene obtained from the test sample can be directly or indirectly sequenced, and thereby homologous to the wild type KF-1 gene. It is also possible to find related genes related to the new KF-1 gene, which is a high homologue. The present invention also provides a method for screening related genes related to the human KF-1 gene in the test sample by such measurement, detection and determination of the DNA sequence of the KF-1 gene in the test sample.

(12) Antibody against the polypeptide of the present invention The present invention also provides an antibody (polyclonal antibody and monoclonal antibody) that binds to a polypeptide (expressed product) having the amino acid sequence represented by SEQ ID NO: 1. The antibody is a polypeptide (expressed product) encoded by the human KF-1 gene represented by SEQ ID NO: 1, or an amino acid sequence in which one or several amino acids are deleted, inserted, substituted or added in SEQ ID NO: 1. Or a fragment of the polypeptide can be used as an antigen.

  Antibody production methods and purification methods are well understood by those skilled in the art, and these conventional methods can be appropriately employed in the present invention. "See Japan Biochemical Society (1986) etc.].

  The obtained antibody can be used, for example, for purification of the polypeptide of the present invention such as KF-1 and measurement and identification by immunological techniques thereof. More specifically, since the expression of the gene of the present invention has been confirmed in tissues such as nerve cells, particularly human cerebellum, cerebral cortex derived from Alzheimer patients and rat hippocampus administered with a long-term antidepressant, It can be used for measurement of KF-1 concentration in these tissues.

  In addition, the antibody or a fragment of the antibody can exert an action of inhibiting or reducing the KF-1 activity, that is, the apoptosis-suppressing action of KF-1 through binding with KF-1, for example.

  The antibody of the present invention is useful as a substance that inhibits or reduces KF-1 activity as described above, and can also be used for measurement of, for example, wild type KF-1 and / or mutant KF-1. Therefore, the present invention also provides a method for measuring wild-type KF-1 and / or mutant KF-1 using an antibody that binds to the polypeptide of the present invention.

  By this measurement method, it is also possible to detect the degree of KF-1 activity, the degree of cell damage due to apoptosis, the degree of cell degeneration due to apoptosis, the degree of cell death, and the like based on changes in wild-type KF-1. Specific examples of the measurement method using the antibody of the present invention include, for example, a method of immunoprecipitation of KF-1 protein from a solution containing a biomaterial sample collected from a human using KF-1 antibody, or a biomaterial sample And a method of directly reacting KF-1 protein in a solution containing a KF-1 antibody on a Western blot or immunoblot of a polyacrylamide gel. Alternatively, KF-1 antibody can be used to detect KF-1 in paraffin or frozen tissue sections according to immunohistochemical techniques.

  More preferred methods for measuring wild type KF-1 or mutant KF-1 include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), including sandwich methods using monoclonal and / or polyclonal antibodies. ), Immunoradioassay (IRMA) and immunoenzymatic method (IEMA).

(13) Screening for KF-1 Activity (Apoptosis Inhibitory Action) Promoter (Agonist) and Inhibitor (Antagonist) The present invention promotes or inhibits KF-1 activity (inhibition of apoptosis induced by endoplasmic reticulum stress). A method for screening candidate compounds is also provided.

  In the method, a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1 or one or more amino acids in the amino acid sequence is deleted, inserted, substituted or added in the presence and absence of a test substance The degree of inhibition of endoplasmic reticulum stress-induced apoptosis in a target cell of a polypeptide having an amino acid sequence and having KF-1 activity is measured, and the measured value in the presence of the test substance is defined as the measured value in the absence of the test substance. In contrast, an agonist that enhances the inhibitory action or an antagonist that reduces the action is selected. Here, the degree of inhibition of endoplasmic reticulum stress-induced apoptosis in the target cells of the polypeptide is obtained, for example, by counting the number of target cells in which nuclei are aggregated and reducing the number. Polypeptides also include gene expression products having KF-1 activity.

The method also comprises a polynucleotide comprising the DNA sequence represented by SEQ ID NO: 2 below, in the presence and absence of a test substance, which can hybridize with the polynucleotide under stringent conditions and in a target cell. A polynucleotide that can express an action that suppresses endoplasmic reticulum stress-induced apoptosis, or a DNA sequence that is 80% or more homologous to the polynucleotide, and can express an action that suppresses endoplasmic reticulum stress-induced apoptosis in target cells. Measures the degree of inhibition of endoplasmic reticulum stress-induced apoptosis in target cells by the expression of various polynucleotides, and compares the measured value in the presence of the test substance with the measured value in the absence of the test substance. The selection of an agonist that enhances or an antagonist that reduces the inhibitory action. To. Here, the degree of suppression of endoplasmic reticulum stress-induced apoptosis in the target cells of the expressed product is obtained, for example, by counting the number of target cells in which nuclei have aggregated and reducing the number. Apoptosis in cells can be detected by, for example, (1) agarose gel electrophoresis (detected as a DNA ladder), (2) pulse field electrophoresis, (3) TUNEL (Gavrieli, Y., et al. al., J. Cell Biol., 119, 493-501 (1992)), (4) Method for detecting cell size changes and cells with reduced DNA content after staining with fluorescent dyes, (5) Fluorescence After cell staining using a dye, it can be carried out by a method of detecting viable and dead cells by flow cytometry. In addition, cell viability is determined by (6) a staining method that separates viable and dead cells using a cell staining dye, (7) enzyme activity measurement method, (8) MTT method, and (9) free ⁵¹ Cr from cells. It can be carried out according to various known methods such as a radioisotope method for measuring selenium and (10) a colony formation method. Apoptosis inhibitory action can also be detected by these methods.

The present invention also provides a method for screening a candidate substance that enhances the endoplasmic reticulum stress-induced apoptosis-suppressing action of a polypeptide of the present invention in a target cell, comprising the following steps (1) to (5).
(1) a step of preparing a target cell transformed with an expression vector of a gene encoding the polypeptide of the present invention,
(2) Inducing apoptosis by applying endoplasmic reticulum stress to the target cell in the presence or absence of a candidate substance,
(3) a step of determining the survival rate of the target cell in the presence and absence of the candidate substance or the proportion of cells in the apoptotic form,
(4) With each cell value obtained in (3) above as a reference value, the survival rate of cells or the proportion of cells in an apoptotic form obtained by applying endoplasmic reticulum stress load to non-transformed cells in the absence of a candidate substance, Obtaining a difference from the reference value;
(5) Select the candidate substance when the difference obtained in the presence of the candidate compound is larger in the survival rate or smaller in the apoptotic form cell ratio than the difference obtained in the absence Process.

Furthermore, the present invention provides a method for screening a candidate compound that enhances the expression in a target cell of a polynucleotide (the gene of the present invention) encoding the expression product of the present invention comprising the following steps (1) to (5): .
(1) a step of preparing a target cell transformed with an expression vector of the gene of the present invention,
(2) subjecting the target cell to endoplasmic reticulum stress load in the presence or absence of a candidate substance,
(3) a step of determining the expression level of the gene of the present invention in the target cell in the presence and absence of the candidate substance,
(4) a step of comparing the expression level in the absence of the candidate substance with the same expression level in the presence of the candidate substance,
(5) A step of selecting the candidate substance when the expression level obtained in the presence of the candidate compound is increased as compared with that obtained in the absence of the candidate substance.

Furthermore, the present invention provides a method for screening a candidate substance that enhances the action of the polypeptide of the present invention in a target cell, comprising the following steps (1) to (5).
(1) a step of preparing a target cell transformed with an expression vector of the gene of the present invention,
(2) Inducing apoptosis by applying endoplasmic reticulum stress to the target cell in the presence or absence of a candidate substance,
(3) a step of determining the amount of polypeptide produced by the target cell in the presence and absence of a candidate substance using an antibody against the polypeptide,
(4) a step of comparing the production amount obtained in the absence of the candidate substance with the production amount obtained in the presence of the candidate substance,
(5) A step of selecting the candidate substance when the production amount in the presence of the candidate compound is increased as compared with that in the absence.

  In these screening methods, the expression level or production level of the polypeptide of the present invention (e.g., KF-1 or KF-1 gene expression product, hereinafter simply referred to as `` KF-1 '') is determined by an antibody against the polypeptide. It can be measured as the ratio of labeled KF-1 bound to the antibody by competitively reacting with labeled KF-1 in a test solution containing a test substance. In addition, by reacting the test solution with the antibody insolubilized on the carrier and another labeled anti-KF-1 antibody simultaneously or successively, the activity of the labeling agent on the insolubilized carrier is measured. In addition, it is possible to quantify KF-1 in the test solution.

  According to the screening method of the present invention, the binding of KF-1 to a ligand can be evaluated, for example, in cells, cell-free preparations, chemical libraries, and natural product mixtures.

  An agent for promoting or enhancing or inhibiting (inhibiting) or reducing apoptosis of KF-1 may be a natural substrate or ligand, or a structural or functional mimic thereof.

  The screening method of the present invention can apply a high-throughput screening technique. According to the application of this technology, for example, in screening for apoptosis inhibitors or pro-apoptotic agents, synthetic reaction mixtures, cell fractions such as endoplasmic reticulum, membranes, cell envelopes, cell walls or any of these preparations (KF -1 and a labeled ligand of the polypeptide) are incubated in the presence or absence of the candidate compound to be screened. Whether the candidate compound agonizes KF-1 or antagonizes KF-1 action is detected by a decrease in the binding of the labeled ligand. A candidate compound that suppresses apoptosis without inducing the action of KF-1 is likely to be a good apoptosis inhibitor. On the other hand, compounds that bind to KF-1 and increase apoptosis are considered KF-1 inhibitors. Detection of KF-1 activity levels includes reporter systems such as (but not limited to) colorimetric labels, incorporation of reporter genes in response to changes in polynucleotide or polypeptide activity and known in the art. It can be performed by using a binding assay.

  Competition assays combining KF-1 and other apoptosis inhibitors with compounds that bind to them can also be used to screen candidate compounds as apoptosis inhibitors or promoters.

  The test substance (drug candidate compound) to be screened by the screening method of the present invention includes both a KF-1 activity promoting substance (particularly an apoptosis inhibiting substance: agonist) and a KF-1 activity inhibiting substance (particularly an apoptosis promoting substance: antagonist). It is out. That is, an agonist can be a substance that increases KF-1 activity measured by its presence in the screening system of the present invention, and an antagonist can be a substance that decreases KF-1 activity measured by its presence. .

  Specific examples of these test substances include, for example, oligopeptides, proteins, antibodies, RNA molecules, siRNA, non-peptide compounds (synthetic compounds), fermentation products, cell extracts (plant extracts, animal tissue extracts, etc.), Plasma is included. These substances may be newly developed or known substances.

  Apoptosis inhibitors include inorganic or organic low molecular weight compounds that bind to KF-1 and increase its activity, peptides, polypeptides, etc., natural or synthetic or prepared by genetic recombination techniques. Also included are sense DNA molecules for KF-1 DNA that are administered directly in vivo or in a form inserted into a recombinant vector.

  Apoptosis-promoting substances bind to the same site of the molecule to which KF-1 binds without inducing the activity induced by KF-1 (apoptosis-inhibiting activity), thereby inhibiting the action of KF-1 Organic or inorganic low molecular weight compounds, peptides, polypeptides, antibodies, antisense DNA molecules that can interfere with the action (e.g. Okano, J. Neurochem, 56: 560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press , Boca Raton, FL (1988)), RNA molecules having an RNA interference effect (see, for example, McCaffrey, AP, et al., Nature, 418, 38-39 (2002)) and the like.

  Evaluation of candidate substances having an action of activating or inhibiting KF-1 activity screened according to the screening method of the present invention can be performed as follows. That is, when the KF-1 activity is promoted by about 20% or more, preferably about 30% or more, more preferably about 50% or more compared to the control (no test substance present), the candidate substance is KF. -1 can be selected as a compound that promotes activity. On the other hand, for example, when the KF-1 activity is inhibited by about 20% or more, preferably about 30% or more, more preferably about 50% or more, compared to the case of control (no test substance present), the test substance Can be selected as a compound that inhibits KF-1 activity.

  KF-1 is expressed in tissues such as the cerebellum, cerebral cortex, midbrain, pituitary gland, and pancreas, so cell degeneration caused by apoptosis induced in these sites, especially nerve cells, neuroblasts Promoters and inhibitors of KF-1 activity are useful for diseases related to cell degeneration such as cells and pancreatic β cells. For example, compounds that promote KF-1 activity in the cerebellum, cerebral cortex, mesencephalon, pituitary and pancreatic tissues can suppress apoptosis induced by endoplasmic reticulum stress (familial or sporadic) Alzheimer's disease ( Alzheimer type senile dementia), Parkinson's disease, Huntington's disease, ischemic brain injury (brain ischemia), amyotrophic lateral sclerosis, multiple sclerosis, HIV-related neuropathy, diffuse Lewy body disease, I It is thought to inhibit the improvement or progress of patients with type 2 diabetes, type 2 diabetes, depression and manic depression. In addition, a compound that inhibits KF-1 activity promotes apoptosis in nerve cells and neuroblasts, and thus is considered useful for cancer treatment of brain tumors, pancreatic cancer, autoimmune diseases, and rheumatism.

  The substance that promotes KF-1 activity obtained by the screening method of the present invention is considered to increase the KF-1 concentration in cells and suppress apoptosis in the cells or decrease the amount of apoptotic cells. As a KF-1 extender, it is useful in various fields including the pharmaceutical field.

  The KF-1 bulking agent comprising the KF-1 activity promoting substance as an active ingredient is particularly used as an endoplasmic reticulum stress-induced apoptosis inhibitor such as (familial or sporadic) Alzheimer's disease (Alzheimer type senile dementia), Parkinson Disease, Huntington's disease, polyglutamine disease, ischemic brain injury (brain ischemia), amyotrophic lateral sclerosis, multiple sclerosis, HIV-related neuropathy, diffuse Lewy body disease, type I diabetes, II It can be effectively used as a medicament for treatment / prevention that inhibits the improvement or progress of patients with type 2 diabetes, depression, and manic depression.

  The substance that inhibits KF-1 activity obtained by the screening method of the present invention is considered to reduce the KF-1 concentration of cells and promote apoptosis in the cells or increase the number of apoptotic cells. As a KF-1 lowering agent, it is useful in various fields including the pharmaceutical field.

  The KF-1 lowering agent containing the KF-1 activity inhibitor as an active ingredient is particularly used as an endoplasmic reticulum stress-induced apoptosis promoter, for example, for cancer treatment of brain tumors, pancreatic cancer, and prevention / treatment of autoimmune diseases and rheumatic diseases. Therefore, it can be effectively used as a medicine for

  The above-mentioned KF-1 bulking agent, endoplasmic reticulum stress-induced apoptosis inhibitor, KF-1 lowering agent, endoplasmic reticulum stress-induced apoptosis promoter is a pharmaceutical comprising an effective amount exhibiting each action together with an appropriate pharmaceutical carrier. The composition can be applied to each of the above diseases.

  The KF-1 promoter or inhibitor used as an active ingredient in the pharmaceutical composition (pharmaceutical preparation) can be a pharmaceutically acceptable salt. The type of salt, its preparation method, preparation method of pharmaceutical composition, diluent used in the pharmaceutical composition, pharmaceutical carrier such as excipient, dosage unit form of pharmaceutical composition, administration route, administration method, etc. The above-mentioned pharmaceutical composition containing the polypeptide of the present invention as an active ingredient can be substantially the same as those detailed above.

  The amount of the active ingredient to be contained in the pharmaceutical preparation and the dose thereof are not particularly limited, and are appropriately selected from a wide range according to the desired therapeutic effect, administration method, treatment period, patient age, sex, and other conditions. The In general, the dosage should be about 0.01 μg-10 mg, preferably about 0.1 μg-1 mg per kg body weight per day, and the preparation should be divided into 1 to several times a day. Can do.

(14) Screening Kit The present invention provides a screening kit for screening for a substance that promotes or inhibits (decreases) KF-1 apoptosis-suppressing action (KF-1 activity).

  One specific example of the screening kit of the present invention is a polypeptide of the present invention (for example, a polypeptide having the amino acid sequence represented by SEQ ID NO: 1) or an expression product of the present invention (for example, the DNA sequence represented by SEQ ID NO: 2). Polynucleotide expression product) and endoplasmic reticulum stress load, ie, a substance that induces apoptosis in cells by promoting stress induction in the endoplasmic reticulum, such as calcium ATPase inhibitor (thapsigargin), N-glucoside sugar chain synthesis inhibition It is important to include an agent (tunicamycin) as an essential component, and the others can be the same as this kind of screening kit. For example, a kit containing various reagents such as a cell culture solution, a reaction diluent, a staining agent, a buffer solution, a fixing solution, and a washing agent as optional components together with KF-1 and tunicamycin having KF-1 activity. it can.

Specific examples of the kit of the present invention include those comprising the following constitutions 1-4 or 1-5.
Configuration 1. Target cells (target KF-1 expressing cells), cells transformed with the KF-1 gene introduced into the cells to be measured), Dulbecco's modified Eagles medium at 0.5-1 × 10 ⁵ cells / well Cultivated at 37 ° C under 5% carbon dioxide in a 60mm dish using
Composition 2. Endoplasmic reticulum stress load (apoptosis inducer such as tunicamycin, tabsigargin, etc.)
Composition 3. Nuclear stain (eg “Hoechst 33258”)
Configuration 4. Buffer for measurement (0.05% EDTA-PBS and 1% glutaraldehyde-PBS, etc.)
Configuration 5. Detection Microscope for Observation In the screening method using the screening kit of the present invention, typical observation of apoptosis of cells per well unit visual field to which a test compound (test substance, drug candidate compound) is added. The number of cells in which the nucleus as an image is aggregated is counted as the number of stained cells, and a significant difference test with the same number of cells in a control well to which no test compound is added may be performed.

  Apoptosis (dead cells due to apoptosis) can be evaluated and detected in addition to the above, for example, as a degree of cell damage or a decrease in the number of living cells. Furthermore, apoptosis can be evaluated by observing cell changes (such as atrophy and degeneration) using a microscope or the like. The degree of cell damage can be evaluated, for example, by measuring the activity of LDH (lactate dehydrogenase) leaked into the medium after culturing the cells for a certain period of time. The number of viable cells can be evaluated by a dye exclusion test using, for example, trypan blue. These measurements and evaluations can be carried out according to conventional methods.

  As described in detail above, the present invention provides a pharmaceutical composition comprising a polypeptide (human KF-1) having an endoplasmic reticulum stress-induced apoptosis-inhibiting action. The composition of the present invention can be used for various diseases involving KF-1 gene and its products (diseases related to cell degeneration or cell death caused by apoptosis) such as (familial or sporadic) Alzheimer's disease (Alzheimer type aging). Dementia), Parkinson's disease, Huntington's disease, polyglutamine disease, ischemic brain injury (brain ischemia), amyotrophic lateral sclerosis, multiple sclerosis, HIV-related neuropathy, diffuse Lewy body disease, For the treatment of type I diabetes, type II diabetes, depression, manic depression and the like, it is useful as an apoptosis inhibitor, an endoplasmic reticulum stress-induced apoptosis inhibitor, a cytopathic agent, and the like.

  The elucidation of the relationship between KF-1 (KF-1 gene expression product) found by the present invention and apoptosis inhibitory action (particularly, apoptosis inhibitory action induced by endoplasmic reticulum stress) Various pathological conditions caused by cytopathy induced by endoplasmic reticulum stress such as β cells, especially apoptosis in nerve cells, pancreatic β cells, such as (familial or sporadic) Alzheimer's disease (Alzheimer type senile dementia) ), Parkinson's disease, Huntington's disease, polyglutamine disease, ischemic brain injury (brain ischemia), amyotrophic lateral sclerosis, multiple sclerosis, HIV-related neuropathy, diffuse Lewy body disease, type I Useful information or means for elucidating, understanding, diagnosing, preventing, treating, etc. for diabetes, type II diabetes, depression, manic depression, etc.

  The present invention is also directed to the development of a new drug that promotes or suppresses the expression of the gene encoding the polypeptide of the present invention (KF-1 gene) or the apoptosis inhibitory action of the polypeptide of the present invention (the gene expression product of the present invention). A screening technique for candidate compounds is also provided.

  Furthermore, the present invention also provides a technique for detecting the expression of the polypeptide of the present invention in an individual or tissue, and a technique for detecting gene mutation (deletion or point mutation) or expression abnormality for the expression. Such detection technology can be suitably used for elucidation and diagnosis of pathological conditions such as Alzheimer's disease, Parkinson's disease, polyglutamine disease, diabetes, depression, manic depression.

  The present invention further provides a vector for gene transfer containing a KF-1 sense oligonucleotide, a cell into which the vector has been introduced, and the like. These are useful for gene therapy.

  Examples are given below to illustrate the present invention in more detail. These examples are illustrative only and do not limit the invention.

1. Identification of human KF-1 Identification of human KF-1 was carried out by homology search using a database for human genes having a ring finger motif. That is, a protein containing a ring-finger motif using the amino acid sequence of HRD1 (GenBank Accession No. NP # 115807.1) and SMART program (http://smart.embl-heidelberg.de/) Using the PSORT program (http://psort.ims.u-tokyo.ac.jp/) that predicts subcellular localization of the human full-length sequence obtained, We searched for endoplasmic reticulum localization proteins.

  As a result, an endoplasmic reticulum protein hKF-1 (DDBJ Accession No: D76444) having a ring-figer motif was found. In addition, a mouse homologue (mKF-1; DDBJ Accession No: D76445) was found.

2. Induction of gene expression by endoplasmic reticulum stress induction
Whether or not the hKF-1 gene shows expression level fluctuation by treatment with an endoplasmic reticulum stress-inducing agent was examined as follows. That is, human hepatocytes HepG2 (ATCC Accession No: HB-8065) 2 × 10 ⁶ cells were seeded in a 60 mm diameter culture dish, 0.1 μM tapsigargin (made by Wako Pure Chemical Industries) and 2 μg / mL tunicamycin (tunicamycin) : Wako Pure Chemical Industries, Ltd.) is added to the culture dish and cultured for 1, 3, 6, 12, 24, or 48 hours, then total RNA is prepared, cDNA synthesis is performed, and the expression level is changed by RT-PCR. Was observed.

  RT-PCR was performed using cDNA synthesized using Total RNA extracted from HepG2 cells as a template. The expression level of the hKF-1 gene was determined by RT-PCR using hKF-1up having the sequence shown in SEQ ID NO: 7 and hKF-1low having the sequence shown in SEQ ID NO: 8 as primers.

  C / EBP homologous protein (hereinafter abbreviated as “CHOP”) is a gene that induces endoplasmic reticulum stress-induced expression (Price and Calderwood, Cancer Res., 52: 3814-3817 (1992); Halleck et al ., Cell Stress Chaper. 2: 31-40 (1997)), and used as a positive control for expression induction.

  As for the expression level of the CHOP gene, RT-PCR was performed using the CHOP-upper-primer of SEQ ID NO: 9 and the CHOP-lower-primer of SEQ ID NO: 10 as primers, and the expression level of the gene was observed. Glyceraldehyde 3-phosphate dehydrogenase (hereinafter abbreviated as “GAPDH”) is a gene generally used for quantitative correction of samples, and was used to confirm the amount of cDNA. The amount of cDNA derived from GAPDH mRNA was subjected to RT-PCR using GAPDH-upper-primer of SEQ ID NO: 11 and GAPDH-lower-primer of SEQ ID NO: 12 as primers, and the expression level of the gene was observed. PCR conditions for each gene are as follows.

For any gene, 2 μL of 10 × PCR buffer (10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl ₂ manufactured by SIGMA), 0.4 μL of 10 μM each dNTP mixture, 0.1 μL of 20 μM An upper primer (0.1 mM), 2 μL of 20 μM lower primer (0.1 mM) and 0.1 μL of 5U / μL Taq polymerase (manufactured by SIGMA) were added, the total volume was adjusted to 18 μL with sterile purified water, and 2 μL of cDNA sample was added. PCR reaction was performed using Gene Amp PCR system 2400-R (Perkin Elmer) under the following conditions for each gene.
KF-1:
94 ° C / 3 min, [(94 ° C / 30 sec, 55.1 ° C / 30 sec, 72 ° C / 30 sec) x 25 cycles], 72 ° C / 5 min
CHOP:
94 ° C / 3 minutes, [(94 ° C / 30 seconds, 55.2 ° C / 30 seconds, 72 ° C / 30 seconds) x 24 cycles], 72 ° C / 5 minutes
GAPDH:
94 ° C / 3 minutes, [(94 ° C / 30 seconds, 59.0 ° C / 30 seconds, 72 ° C / 30 seconds) x 18 cycles], 72 ° C / 5 minutes
The PCR product was subjected to agarose gel electrophoresis, and the expression level was determined from the intensity of the band.

  The obtained results are shown in FIG.

  In FIG. 1, the top row (indicated as KF-1) is the control (control; no endoplasmic reticulum stress-inducing agent added), thapsigargin 0.1 μM added, and the time shown in the top row, and tunicamycin (Tunicamycin) Induction of endoplasmic reticulum stress-responsive expression of the hKF-1 gene in human hepatocytes when treated for 2 hours with the addition of 2 μg / mL for the time indicated in the upper panel is shown. The second row from the top (labeled as CHOP) shows the induction of expression in each case of CHOP known as a gene showing endoplasmic reticulum stress-responsive expression induction (see the above-mentioned document) (positive control). The bottom row (indicated as GAPDH) is the result of induction of expression in each case in GAPDH, and was used as quantitative correction of cDNA derived from HepG2 cells used for PCR.

  From the results shown in FIG. 1, KF-1 (see the top row) shows an increase in the expression level such that the expression level increases most after 12 hours in the tabcigargin treatment compared to the untreated control group. confirmed. Moreover, in tunicamycin treatment, it was confirmed that the expression level increased so that the expression level increased most after 3 hours.

3. Construction of mammalian cell expression vector ATF6 N-terminal transcriptional activation region, a sensor protein of endoplasmic reticulum stress, known to activate transcription of genes with various endoplasmic reticulum stress response elements ( Haze, K., et al., Mol. Biol. Cell., 10: 3787-3799 (1999)) mRNA expressed from human embryonic kidney cells HEK293T cells (ATCC Accession No: B-8065) All the coding except for the stop codon of hKF-1 by PCR using the cDNA of the sequence shown in SEQ ID NO: 13 and the primer of the sequence shown in SEQ ID NO: 14 using the cDNA as a template. The sequence containing the region was amplified.

  After the amplified fragment was digested with restriction enzymes Kpn I and Xba I, pcDNA6-Myc / His vector expressing Myc (c-Myc) and polyhistidine (6 x His) epitopes at the C-terminus of the inserted sequence ( The digest obtained above was inserted into the Kpn I / Xba I site of Invitrogen (Invitrogen) to obtain an expression vector pcDNA6-Myc / His-hKF-1 expressing hKF-1.

4. Endoplasmic reticulum stress-induced cell death inhibitory activity
8 μg of pcDNA6-Myc / His-hKF-1 expressing hKF-1 labeled with Myc epitope and 15 μL of Lipofectamine ^TM 2000 (Invitrogen) were mixed and allowed to stand at room temperature for 20 minutes. The gene was introduced into the cells by mixing with blast cells Neuro-2a (ATCC Accession No: CCL-131, hereinafter abbreviated as “N2a”). After culturing the cells for 1 day, Dulbecco's modified Eagle's medium (10% (v / v) heat inactivated) supplemented with Blasticidin (Invitrogen: Invitrogen) in a final concentration of 10 μg / mL Using the fetal bovine serum, 100 U / mL penicillin and 100 μg / mL streptomycin), transgenic cells were selected to establish two hKF-1 stable expression strains. These were named KF-1 stable N2a (# 2) and KF-1 stable N2a (# 3), respectively.

  Using these two strains, the expression and localization of KF-1 protein in cells and the resistance to endoplasmic reticulum stress-responsive cell death (apoptosis) were confirmed as follows. That is, in N2a cells stably expressing hKF-1 labeled with Myc epitope, immunostaining was performed using an anti-Myc primary antibody and an anti-mouse-Alexia488 secondary antibody. Its intracellular localization was confirmed.

  The obtained results are shown in FIG.

In FIG. 2, KF-1 stable N2a (# 2) and (# 3) are N2a cells stably expressing hKF-1 labeled with the Myc epitope prepared above, and Normal N2a is used as a comparative control. It is an untreated N2a cell that has not introduced the hKF-1 gene (KF-1 non-expressing N2a cell) .From the results shown in FIG. 2, in the established KF-1 stable N2a (# 2) and (# 3), Intracellular fluorescence could be observed. The fluorescence was observed in the endoplasmic reticulum. This revealed that KF-1 was overexpressed in the two stably expressing cell lines, and that the KF-1 protein was localized in the endoplasmic reticulum.

Apoptosis was detected as follows. Specifically, 0.5-1 × 10 ⁵ cells were seeded on a 60 mm diameter dish and treated with 5 μg / mL tunicamycin for 42 hours to induce endoplasmic reticulum stress. Next, the cells were collected using 0.05% EDTA-PBS, fixed with 1% glutaraldehyde-PBS for 10 minutes, and then, nuclear aggregation, which is a typical observation image of apoptosis, was subjected to Hoechst staining [Hoechst 33258 (Bisbenzimide H 33258: Wako Pure Chemical Industries, Ltd. use] and observed.

  Approximately 30-70 stained cells observed in the visual field were counted in three different visual fields for one sample, and the average value was calculated. The experiment was repeated 5 times for each sample.

  The stained images of the cells observed above are shown in FIG. 3 and FIG. FIG. 5 shows the result of comparison of the calculated cell number (ratio).

  In FIG. 3 and FIG. 4, normal-, normal Tm, No.2-, No.2Tm, No.3-, No.3Tm are KF-1 non-expressing N2a cells (without endoplasmic reticulum stress drug treatment) and KF, respectively. -1 non-expressing N2a cells (with endoplasmic reticulum stress drug (tunicamycin; Tm) treatment), KF-1 stable N2a (# 2) (no endoplasmic reticulum stress drug treatment), KF-1 stable N2a (# 2) (endoplasmic reticulum) Stress drug (with Tm treatment), KF-1 stable N2a (# 3) (without endoplasmic reticulum stress drug treatment), KF-1 stable N2a (# 3) (with endoplasmic reticulum stress drug (Tm) treatment). In each figure, the cells indicated by arrows are the cells that have caused the aggregation of the nucleus (stained apoptotic cells).

  In FIG. 5, the vertical axis represents the ratio of cells with cell death (Cell death) to the total cells (Cell death (%)), and the horizontal axis represents “KF-1 non-expressing N2a cells” (normal and Display), “KF-1 stable N2a (# 2)” (displayed as KF-1 stable No. 2) and “KF-1 stable N2a (# 3)” (displayed as KF-1 stable No. 3). Each bar graph shows no tunicamycin added (black bar) and tunicamycin 5 μg / mL added (white bar), respectively.

  As shown in Fig. 3 to Fig. 5, KF-1 stable N2a (# 2) and (# 3), which are hKF-1 expressing cell lines, are more prone to nuclear aggregation than non-expressing cells. The ratio was small and it was confirmed that clear cell death resistance was acquired. From this, it was confirmed that KF-1 of the present invention suppresses apoptosis caused by endoplasmic reticulum stress induction.

  From the above test results, it has been clarified that hKF-1 is an endoplasmic reticulum protein whose expression is enhanced by inducing endoplasmic reticulum stress. In addition, it was revealed that N2a cells stably expressing KF-1 showed remarkable resistance to apoptosis when ER stress was induced by tunicamycin.

  The above results indicate that the enhanced function of KF-1 protein remarkably suppresses endoplasmic reticulum stress-induced apoptosis in nerve cells, and promotes functions such as enhanced expression and stabilization of hKF-1 The substance is for central diseases associated with neuronal degeneration such as Alzheimer's disease, Parkinson's disease, Huntington's disease, polyglutamine disease, and apoptosis-related diseases such as type I and type II diabetes including pancreatic β-cell degeneration. This proves that an effective treatment effect can be obtained.

  Recently, it has been clarified that the expression of KF-1 gene is enhanced by continuous administration of antidepressants, electric shock therapy, etc. It can be expected to become a new target for treatment.

  Furthermore, suppression of KF-1 gene function suggests that cell death is enhanced, so hKF-1 inhibitors are also effective in the treatment of cancer, autoimmune diseases including cell hyperproliferation, and rheumatism. it is conceivable that.

It is a figure which shows the result (result of having investigated the expression level of hKF-1) which prepared total RNA after processing human hepatocyte HepG2 with a tab cigargin and tunicamycin, and analyzed by RT-PCR. It is a figure which shows the fluorescence-microscope observation result which investigated the intracellular localization of hKF-1 in the mouse neuroblast Neuro-2a which introduce | transduced hKF-1 expression vector pcDNA6-KF-1 / Myc by immuno-staining. It is a figure which shows the result (stained image of the cell which has caused the nuclear aggregation) which investigated the tolerance degree with respect to the endoplasmic reticulum stress-responsive cell death of the neuroblastoma cell Neuro-2a which introduce | transduced the hKF-1 expression vector. It is a figure which shows the result (stained image of the cell which has caused the nuclear aggregation) which investigated the tolerance degree with respect to the endoplasmic reticulum stress-responsive cell death of the neuroblastoma cell Neuro-2a which introduce | transduced the hKF-1 expression vector. A graph showing the results of examining the degree of resistance of the neuroblastoma neuro-2a introduced with the hKF-1 expression vector to endoplasmic reticulum stress-responsive cell death (the ratio of the number of cells causing nuclear aggregation to the total cell quality). is there.

SEQ ID NO: 7-8: Primer sequence for hKF-1 SEQ ID NO: 9-10: Primer sequence for CHOP SEQ ID NO: 11-12: Primer sequence for GAPDH SEQ ID NO: 13-14: Primer sequence for hKF-1

Claims (16)

A pharmaceutical composition comprising as an active ingredient at least one polypeptide selected from the group consisting of the following (a) and (b):
(a) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1,
(b) a polymorphic amino acid sequence in which one or more amino acids are deleted, inserted, substituted or added in the amino acid sequence of (a) above, and having an action of suppressing endoplasmic reticulum stress-induced apoptosis in target cells peptide. A pharmaceutical composition comprising, as an active ingredient, an expression product of at least one polynucleotide selected from the group consisting of the following (c), (d) and (e):
(c) a polynucleotide comprising the DNA sequence represented by SEQ ID NO: 2,
(d) a polynucleotide capable of hybridizing with the polynucleotide of (c) above under stringent conditions and capable of expressing an effect of suppressing endoplasmic reticulum stress-induced apoptosis in a target cell,
(e) a polynucleotide comprising a DNA sequence which is 80% or more homologous to the polynucleotide of (c) above and capable of expressing an action of suppressing endoplasmic reticulum stress-induced apoptosis in a target cell. 3. The pharmaceutical composition according to claim 1 or 2, which is an endoplasmic reticulum stress-induced apoptosis inhibitor. 3. The pharmaceutical composition according to claim 1 or 2, wherein the target cell is selected from the group consisting of nerve cells, neuroblasts, pancreatic β cells, hepatocytes, epithelial cells, skeletal muscle cells, smooth muscle cells and lymphocytes. Stuff. Alzheimer's disease, Parkinson's disease, Huntington's disease, polyglutamine disease, ischemic encephalopathy, amyotrophic lateral sclerosis, multiple sclerosis, HIV-related neuropathy, diffuse Lewy body disease, type I diabetes, type II 3. The pharmaceutical composition according to claim 1 or 2, which is a therapeutic agent for a disease selected from the group consisting of diabetes, manic depression and depression. An endoplasmic reticulum stress-induced apoptosis inhibitor containing as an active ingredient at least one selected from the group consisting of the polypeptide of claim 1 and the expressed product of claim 2. Alzheimer's disease, Parkinson's disease, Huntington's disease, polyglutamine disease, ischemic brain injury, amyotrophic lateral cord, characterized in that the pharmaceutical composition according to claim 1 or 2 is administered to a patient in need of treatment A method for treating a disease selected from the group consisting of sclerosis, multiple sclerosis, HIV-related neuropathy, diffuse Lewy body disease, type I diabetes, type II diabetes, manic depression and depression. In the presence and absence of the test substance, measure the degree of inhibition of endoplasmic reticulum stress-induced apoptosis in the target cells of the polypeptide (a) or (b) below, and test the measured value in the presence of the test substance. In contrast to the measured value in the absence of a substance, an agonist that enhances the inhibitory action or an antagonist that reduces the inhibitory action is selected. Agonist or antagonist screening methods:
(a) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1,
(b) a polypeptide comprising an amino acid sequence in which one or more amino acids are deleted, inserted, substituted or added in the amino acid sequence of (a) above and having an action of suppressing endoplasmic reticulum stress-induced apoptosis in target cells peptide. 9. The method according to claim 8, wherein the degree of inhibition of endoplasmic reticulum stress-induced apoptosis in the target cells of the polypeptide is obtained as a decrease in the number by counting the number of target cells in which nuclei have aggregated. In the presence and absence of the test substance, the degree of inhibition of endoplasmic reticulum stress-induced apoptosis in the target cell of the expression product of the following polynucleotide (c), (d) or (e) is measured, and the test substance ER in a target cell, wherein an agonist that enhances the inhibitory action or an antagonist that reduces the inhibitory action is selected by comparing a measured value in the presence of a test substance with a measured value in the absence of a test substance Methods for screening for agonists or antagonists for stress-induced apoptosis inhibition:
(c) a polynucleotide comprising the DNA sequence represented by SEQ ID NO: 2,
(d) a polynucleotide capable of hybridizing with the polynucleotide of (c) above under stringent conditions and capable of expressing an action of suppressing endoplasmic reticulum stress-induced apoptosis in a target cell,
(e) a polynucleotide comprising a DNA sequence which is 80% or more homologous to the polynucleotide of (c) and capable of expressing an action of suppressing endoplasmic reticulum stress-induced apoptosis in a target cell. 11. The method according to claim 10, wherein the degree of suppression of endoplasmic reticulum stress-induced apoptosis in the target cells of the expressed product is obtained as a decrease in the number by counting the number of target cells in which nuclei have aggregated. The method according to claim 8 or 10, wherein the degree of inhibition of apoptosis is determined by (1) agarose gel electrophoresis, (2) pulse field electrophoresis, (3) TUNEL method, or (4) flow cytometry. A method for screening a candidate substance that enhances endoplasmic reticulum stress-induced apoptosis-suppressing action in a target cell of the polypeptide according to claim 1, comprising the following steps (1) to (5):
(1) preparing a target cell transformed with an expression vector of a gene encoding the polypeptide according to claim 1,
(2) Inducing apoptosis by applying endoplasmic reticulum stress to the target cell in the presence or absence of a candidate substance,
(3) a step of determining the survival rate of the target cell in the presence and absence of the candidate substance or the proportion of cells in the apoptotic form,
(4) Each value obtained in (3) above, using as a reference value the survival rate of cells or the proportion of cells in an apoptotic form obtained by applying endoplasmic reticulum stress load to non-transformed cells in the absence of a candidate substance Obtaining a difference between the reference value and the reference value;
(5) When the difference obtained in the presence of the candidate substance is larger in the survival rate or smaller in the apoptotic form cell ratio than the difference obtained in the absence, the candidate substance is Process to select. A method for screening a candidate compound that enhances the expression in a target cell of a polynucleotide capable of expressing an effect of suppressing endoplasmic reticulum stress-induced apoptosis according to claim 2, comprising the following steps (1) to (5):
(1) preparing a target cell transformed with the polynucleotide expression vector according to claim 2,
(2) subjecting the target cell to endoplasmic reticulum stress load in the presence or absence of a candidate substance,
(3) a step of determining the expression level of the polynucleotide of the target cell in the presence and absence of the candidate substance,
(4) a step of comparing the expression level in the absence of the candidate substance with the same expression level in the presence of the candidate substance,
(5) A step of selecting the candidate substance when the expression level obtained in the presence of the candidate substance is increased as compared with that obtained in the absence. A method for screening a candidate substance that enhances the action of the polypeptide of claim 1 in a target cell comprising the following steps (1) to (5):
(1) preparing a target cell transformed with an expression vector of a gene encoding the polypeptide according to claim 1,
(2) subjecting the target cell to endoplasmic reticulum stress load in the presence or absence of a candidate substance,
(3) a step of determining the amount of polypeptide produced by the target cell in the presence and absence of a candidate substance using an antibody against the polypeptide,
(4) a step of comparing the production amount obtained in the absence of the candidate substance with the production amount obtained in the presence of the candidate substance,
(5) A step of selecting the candidate substance when the production amount in the presence of the candidate compound is increased as compared with that in the absence. Comprising at least one selected from the group consisting of the polypeptide according to claim 1 and the expression product according to claim 2 in a target cell, and an endoplasmic reticulum stress load as a constituent, A screening kit for screening for an agonist or an antagonist for an action of suppressing endoplasmic reticulum stress-induced apoptosis in a target cell of a polypeptide or an expression product.