JP2005330231A - Pharmaceutical composition - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a pharmaceutical composition containing as active ingredient a protein (enzyme) that has APP(amyloid precursor protein) production inhibitory activity or Aβ(amyloidβ) production inhibitory activity and is useful as an agent for ameliorating neurodegenerative diseases including Alzheimer's disease, and to provide a screening system for candidate substances that promote APP production inhibitory activity or Aβ production inhibitory activity. <P>SOLUTION: The pharmaceutical composition contains as the active ingredient (a) a polypeptide consisting of an amino acid sequence represented by the sequence No.1 and/or (b) a polypeptide consisting of an amino acid sequence where one or several amino acids are deleted, inserted, substituted or added in the amino acid sequence mentioned in (a) and having APP production inhibitory activity or Aβ production inhibitory activity. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a pharmaceutical composition having an action of suppressing production of amyloid β peptide and its precursor. The present invention also relates to a screening method for a substance that enhances (enhances) the action of suppressing the production of amyloid β peptide and its precursor.

  Alzheimer's Disease is one of the dementias in the elderly population. As a classic pathological feature of Alzheimer's disease, accumulation of a large number of senile plaques containing an amyloid β peptide (hereinafter referred to as “Aβ”) consisting of 40 to 42 amino acids is recognized. . Genetic and cell biology findings both suggest that Aβ accumulation in the brain is responsible for Alzheimer's disease (Yankner, (1996), Neuron, 16: 921-932; Selkoe, (1997 ), Science, 275: 630-631).

  Strong genetic evidence supporting the pathological role of Aβ is that individuals who inherit mutations in amyloid precursor protein (hereinafter abbreviated as “APP”) always develop Alzheimer's disease almost at an early age. Arises from the observation that This mutation increases the production of long variants of Aβ that form senile plaques in the brain (Goate et al. (1991), Nature, 349: 704-706).

  Mutations and allelic variants (including presenilin and apolipoprotein E) in other genes than the APP that cause Alzheimer's disease also result in increased production and deposition of Aβ. Furthermore, individuals belonging to Down's syndrome overexpress APP, cause Aβ deposition in the brain at a young age, and develop Alzheimer's disease at a young age.

  Aβ formation is a normal physiological process. That is, this peptide has been found to be naturally produced in the case of cells cultured in vitro (Haass et al., (1992), Nature, 359: 322-325; Seubert et al., (1992), Nature, 359: 325-327; Shoji et al. (1992), Science, 258: 126-129). Various similar reports are also observed in vivo (Seubert et al., (1992), Nature, 359: 325-327; Vigo-Pelfrey et al., (1993), J. Neurochem., 61: 1965-1968; Tabaton et al., (1994), Biochem. Biophys. Res. Commun., 200: 1598-1603; Teller et al. (1996), Nature Med., 2: 93-95).

  Aβ is considered to be a denatured byproduct of intracellular catabolism of β-APP, which is a non-secreted form (see Non-Patent Document 1). In vitro, it has been shown that it does not show cytotoxicity by inhibiting the formation of APP. Furthermore, Aβ has been demonstrated to be highly toxic to neurons. Therefore, for example, a drug that reduces the amount of Aβ produced in the brain is considered to be able to prevent Alzheimer's disease.

  The present inventors previously found an E3 ubiquitin-binding enzyme localized in the endoplasmic reticulum called HRD1, which consists of a 616 amino acid sequence consisting of a nucleotide sequence whose ORF region is 1851 (including a stop codon) ( Non-patent document 2). The present inventors have found that this HRD1 is a membrane protein localized in the endoplasmic reticulum, the expression of which is induced by endoplasmic reticulum stress, and when this HRD1 is highly expressed in cultured cells, It has been clarified that death can be suppressed (see Non-Patent Document 3).

As described above, Alzheimer's disease is known to be caused by increased production and deposition of Aβ. In addition to such Alzheimer's disease, other neurodegenerative diseases such as Parkinson's disease and cell degeneration such as diabetes. For the treatment of diseases and the like, substances that have an action to suppress APP production or substances that have an action to reduce Aβ production are considered to be effective. A substance having an action to reduce the production amount of Aβ is not found. If a new substance (protein) having such an APP production inhibitory action or Aβ production inhibitory action is found, a new therapeutic method for the above-mentioned diseases can be established.
Higaki., Et al., Neuron, 14: 651-659, (1995) Kaneko, M., et al., FEBS Lett., 532: 147-152, (2002) Nadav, E., et al., BBRC, 303, (1): 91-97 (2003)

  As described above, if a new protein having an APP production inhibitory action or Aβ production inhibitory action or a gene encoding the same can be provided, the mechanism of action such as neurodegenerative diseases and cell degenerative diseases accompanying APP production or Aβ production can be clarified. It is possible, and based on the action of suppressing APP production or Aβ production, it can be used effectively for the treatment of these diseases. In particular, such a protein having an inhibitory effect on APP production or Aβ production and a gene encoding the same are used as therapeutic agents and preventive agents for neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, and gene mutation diseases such as Down's syndrome. New applications can be expected. Furthermore, by using these genes and proteins, it is possible to screen for candidate compounds as new therapeutic agents and preventive agents that enhance (enhance) the action of suppressing APP production action or Aβ production action.

  From the above viewpoint, the inventors focused on the HRD1 that the inventors found earlier in the course of earnest research for the purpose of searching for a novel gene having an APP production inhibitory action or an Aβ production inhibitory action. . That is, the present inventors show that the expression of the HRD1 is induced by endoplasmic reticulum stress, that the HRD1 is a membrane protein localized in the endoplasmic reticulum, and that the HRD1 is small when highly expressed in cultured cells. Focusing on the ability to suppress ER stress-responsive cell death, assuming that the expression of the HRD1 is the result of an APP production inhibitory effect or an Aβ production inhibitory action, further expression of APP in the previously established human HRD1 stable expression cells The APP expression level and Aβ production level of the obtained APP expression-induced cells were examined. As a result, it was confirmed that the APP expression level and Aβ production level were suppressed (decreased) in cells co-expressing HRD1 and APP as compared to normal APP-expressing cells. That is, new findings were obtained that HRD1 has an activity to suppress APP production and an activity to inhibit Aβ production.

  The present invention has been completed as a result of further research based on this knowledge.

  The present invention provides the invention of the gist shown in the following items 1-12.

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) It consists of an amino acid sequence in which one or several amino acids are deleted, inserted, substituted or added in the amino acid sequence of (a) above, and has an action to suppress APP production action or Aβ production action Polypeptide.

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 action of suppressing an APP production action or an action of inhibiting an Aβ production action,
(e) A polynucleotide comprising a DNA sequence that is 80% or more homologous to the polynucleotide of (c) above and capable of expressing an action of suppressing APP production or an action of inhibiting Aβ production.

  Item 3. An APP production inhibitor or an Aβ production 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 4. In the presence and absence of the test substance, the degree of inhibition of APP production or Aβ production of the polypeptide (a) or (b) below is measured, and the presence of the test substance is measured. The action of suppressing APP production action or Aβ production action characterized by selecting a substance that increases the degree of inhibition by comparing the measurement value in the above with the measurement value in the absence of the test substance For screening for substances that enhance
(a) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1,
(b) It consists of an amino acid sequence in which one or several amino acids are deleted, inserted, substituted or added in the amino acid sequence of (a) above, and has an action to suppress APP production action or Aβ production action Polypeptide.

  Item 5. The method according to Item 4, wherein the degree of suppression of the APP production action or the degree of inhibition of the Aβ production action is determined as the degree of decrease in APP expression level or the degree of decrease in Aβ production amount in the target cell.

Item 6. In the presence and absence of a test substance, the degree of inhibition of APP production or Aβ production of the following expression (c), (d) or (e) Measuring APP in the presence of the test substance, and comparing the measured value in the absence of the test substance, and selecting a substance that increases the degree of suppression of the production action. A method for screening a substance that enhances an inhibitory action or an Aβ producing action:
(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 an APP production action or an action of inhibiting an Aβ production action,
(e) A polynucleotide comprising a DNA sequence that is 80% or more homologous to the polynucleotide of (c) above and capable of expressing an action of suppressing APP production or an action of inhibiting Aβ production.

  Item 7. The method according to Item 6, wherein the degree of suppression of the APP production action or the degree of inhibition of the Aβ production action is determined as the degree of decrease in APP expression level or the degree of decrease in Aβ production amount in the target cell.

  Item 8. The method according to Item 4 or 6, wherein the action of suppressing the APP production action is determined by Western blotting analysis, and the action of suppressing the Aβ production action is determined by ELISA.

Item 9. A screening method for a substance that enhances the action of suppressing the APP production action or the action of inhibiting Aβ production of the polypeptide according to Item 1, comprising the following steps (1) to (5):
(1) preparing a target cell transformed with an expression vector containing each of the polynucleotide encoding the polypeptide of Item 1 and the labeled APP encoding polynucleotide;
(2) culturing each target cell of (1) in the presence or absence of a candidate substance,
(3) a step of determining the amount of APP expression or Aβ production in the target cells in the presence and absence of the candidate substance,
(4) a step of comparing the APP expression amount or Aβ production amount in the absence of the candidate substance with the APP expression amount or Aβ production amount in the presence of the candidate substance, respectively.
(5) Select at least one of the APP expression level and Aβ production level obtained in the presence of the candidate substance as compared to those obtained in the absence. Process.

Item 10. A method for screening for a compound that enhances an action of suppressing an APP production action or an action of suppressing an Aβ production action of an expression product of a polynucleotide according to Item 2 comprising the following steps (1) to (5): :
(1) providing a target cell transformed with an expression vector comprising each of the polynucleotide according to item 2 and a polynucleotide encoding labeled APP,
(2) culturing each target cell of (1) in the presence or absence of a candidate substance,
(3) a step of determining the APP expression level or Aβ production level of the target cell in the presence and absence of the candidate substance,
(4) a step of comparing the APP expression amount or Aβ production amount in the absence of the candidate substance with the APP expression amount or Aβ production amount in the presence of the candidate substance, respectively.
(5) a step of selecting the candidate substance when at least one of the APP expression level and the Aβ production level obtained in the presence of the candidate substance is lower than those obtained in the absence .

Item 11. A method for screening for a substance that enhances the action of suppressing the APP production action or the action of inhibiting Aβ production of the polypeptide according to Item 1, comprising the following steps (1) to (5):
(1) preparing a target cell transformed with an expression vector comprising each of the polynucleotide encoding the polypeptide of item 1 and the polynucleotide encoding APP;
(2) culturing each target cell of (1) in the presence or absence of a candidate substance,
(3) The step of determining the polypeptide production amount according to claim 1 of the target cell in the presence and absence of a candidate substance using an antibody against the polypeptide,
(4) a step of comparing the polypeptide production amount determined in the absence of the candidate substance and the polypeptide production amount determined in the presence of the candidate substance,
(5) A step of selecting the candidate substance when the amount of polypeptide production obtained in the presence of the candidate substance is increased as compared with that obtained in the absence.

  Item 12. A composition comprising at least one selected from the group consisting of the polypeptide according to Item 1 and the expression product according to Item 2, and an expression product of a polynucleotide encoding a labeled APP as a constituent component A screening kit for screening for a substance that enhances the action of suppressing the APP production action of the polypeptide or expressed product or a substance that enhances the action of suppressing the Aβ production action.

  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 polynucleotide includes RNA and DNA. DNA includes 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 naturally, for example, due to mutations, post-translational modifications, etc., but artificially using naturally occurring genes (for example, the HRD1 gene) It may be generated in the above. Variants of the above polypeptides are alleles, homologs, natural variants, etc., and at least 80%, preferably 95% or more, more preferably 99% or more are homologous to the polypeptide before mutation. included.

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.

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 represented by SEQ ID NO: 1, or (b) one or several amino acids are deleted, inserted, substituted or added in the amino acid sequence represented by SEQ ID NO: 1. And has an APP production inhibitory action or Aβ production 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, Polypeptides derived from human cells or tissues originating from the stomach, small intestine, large intestine, etc. are preferred. The polypeptide of the present invention may be a polypeptide obtained by chemical synthesis according to the amino acid sequence information of the preferred polypeptide.

  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 polyamino acid sequence having an amino acid sequence in which one or several amino acids are deleted, inserted, substituted or added in the amino acid sequence represented by SEQ ID NO: 1 and has an APP production inhibitory action or an Aβ production inhibitory action. Peptides can be mentioned.

  The APP production inhibitory action or Aβ production inhibitory action refers to an action that suppresses APP production action or Aβ production action of APP-expressing cells. More specifically, this action can be confirmed, for example, by examining the APP production action or Aβ production action in APP-expressing cells transformed so as to express the polypeptide of the present invention as shown in Examples below. For example, the APP production action (its inhibitory action) can be measured and detected by Western blotting analysis of labeled APP-expressing cells. In addition, the Aβ production action (its inhibitory action) can be measured and detected by the ELISA method using an antibody against Aβ (anti-Aβ antibody) with respect to the amount of Aβ produced in the culture solution.

  Inhibition of the APP production action or Aβ production action results in prevention or improvement of progression of neuronal degeneration. From this, it can also be said that the inhibitory action is an action of suppressing or improving cytopathy caused by APP or Aβ accumulation, that is, cytopathic inhibitory action.

  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” means that the action is the same in physiochemical or pharmacological properties (referred to as “same effect”), and this APP production inhibitory action Alternatively, quantitative factors such as the degree of Aβ production inhibitory action and the molecular weight of the protein having these actions 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 as (b) above has, in addition to the APP production inhibitory action or Aβ production inhibitory action, for example, the original protein activity of the polypeptide (for example, its enzyme activity), antigen Also in terms of sex and immunogenic activity, it is preferable that the amino acid sequence is substantially the same as the amino acid sequence before modification, that is, the polypeptide consisting of 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 several amino acids are deleted, inserted, substituted or added in SEQ ID NO: 1, and APP production suppression The above-mentioned common activity possessed by the polypeptide having the action or Aβ production inhibitory action (the polypeptide of the present invention) may be simply referred to as “APP production inhibitory activity” hereinafter.

  The homologues of the polypeptide of the present invention consisting of the amino acid sequence represented by SEQ ID NO: 1 include proteins originating from mammals other than yeast, nematodes, plant tissues and humans, which have APP production inhibitory activity. Examples of the mammal include horses, sheep, cows, dogs, monkeys, cats, bears, mice, rats, rabbits and the like.

  The APP production inhibitory activity possessed by the polypeptide of the present invention is a system (co-culture system) in which both genes are introduced into cells or tissues capable of simultaneously expressing the polypeptide of the present invention and APP, ie, cells of appropriate origin, and the cells are cultured. ) And a system (APP expression system) for culturing APP-expressing cells not imparting the expression ability of the polypeptide of the present invention, the co-culture system shows lower APP production or Aβ production. That is, that APP production and / or Aβ production is suppressed.

  The APP production-suppressing action can be measured and detected by a known method using labeled blot expression analysis by Western blotting analysis, with the decrease / decrease in the expression level as an index. The Aβ production inhibitory action can be measured and detected by a conventional method as a decrease / decrease in the amount of Aβ production by ELISA using an Aβ antibody against Aβ produced in the culture medium.

  More specifically, for example, as described in Examples below, recombinant HRD1 and recombinant APP labeled with specific epitopes such as Myc and flag are expressed by gene transfer into HEK293 cells, and after a predetermined time after gene transfer. About the collected cell lysate, the amount of APP was measured by Western blotting using an epitope recognition antibody, and this measurement value was compared with the same measurement value of non-recombinant cells (HRD1 non-expressing cells). The presence or absence of APP production inhibitory action or the degree of the action can be detected.

  In addition, after culturing HEK293 cells that express HRD1 and HEK293 cells that do not express HRD1 for a predetermined time, the medium is replaced with Opti-MEM, and after incubation for each predetermined time, the culture supernatant is recovered. Inhibition of Aβ production by measuring the amount of Aβ (production amount) in the supernatant using an anti-Aβ antibody and comparing this measurement value with that of non-recombinant cells (HRD1 non-expressing cells) It is possible to detect the presence or absence or the extent.

  As a specific example of the polypeptide of the present invention, a polypeptide encoded by a PCR product named “HRD1” shown in Examples described later can be mentioned. The DNA sequence of HRD1 is as shown in SEQ ID NO: 2 (1851 bp), and the amino acid sequence encoded by the ORF is as shown in SEQ ID NO: 1. That is, HRD1 has a 616 amino acid sequence (GenBank accession number AB085847).

  The above HRD1 was discovered by the present inventors as an E3 ubiquitin-binding enzyme localized in the endoplasmic reticulum. This HRD1 is a membrane protein localized in the endoplasmic reticulum whose expression is induced by endoplasmic reticulum stress.When this HRD1 is highly expressed in cultured cells, it can suppress ER stress-responsive cell death (see above). Non-patent document 2).

  Another specific example of the polypeptide of the present invention includes HRD1 isoform consisting of 616 amino acid sequence of GenBank Accession No. NP 115807. The DNA sequence is as shown in SEQ ID NO: 4 (1851 bp), and the amino acid sequence encoded by the ORF is as shown in SEQ ID NO: 3. This isoform of HRD1 has 99% identity with HRD1 at the amino acid level.

  Examples of the substance having high homology with HRD1 include a Synoviolin-related protein specifically expressed in the synovial tissue of a rheumatic patient consisting of 616 amino acid sequences. The synoviolin-related protein and HRD1 are both 99% homologous in amino acid sequence and nucleic acid sequence (WO200252007).

  Next, human gene 4 encoding human secretory protein HDLWF88 consisting of 617 amino acid sequences useful for diagnosis and treatment of diseases such as cancer, arteriosclerosis, anemia, hypertension, infection, multiple myeloma, and wounds Can be mentioned. This human secreted protein and HRD1 are 99% homologous in amino acid sequence and nucleic acid sequence (WO2003038038).

  Furthermore, human albumin fusion protein consisting of 617 amino acid sequences useful for diagnosis and treatment of diseases such as diabetes, cancer, ulcerative colitis, immunodeficiency syndrome, AIDS, Alzheimer's disease, Parkinson's disease, Creutzfeldt-Jakob disease, encephalitis Can be mentioned. This human albumin fusion protein and HRD1 are 99% homologous in amino acid sequence and nucleic acid sequence (WO200177137).

  Diagnosis and treatment of diseases such as actinic keratosis, arteriosclerosis, cirrhosis, cancer, autoimmune disease, inflammatory disease, asthma, allergy, Alzheimer's disease, myocardial infarction, anemia, chronic heart failure And human secreted protein SECP17 consisting of 617 amino acid sequences useful for the above. This human secreted protein SECP17 and HRD1 are both 99% homologous in amino acid sequence and nucleic acid sequence (WO200262841).

  Furthermore, it consists of 617 amino acid sequences as a nucleic acid sequence encoding one of 21 human secreted proteins, for improving, preventing and treating medical conditions such as autoimmune diseases and cancer, or as a food additive Human gene 4 encoding human secreted protein HRABV43 has also been reported. The human secreted protein HRABV43 and HRD1 are both 99% homologous in amino acid sequence and nucleic acid sequence (WO200134629).

  As mentioned above, HRD1 and its isoforms have been reported several times, but these reports indicate that HRD1 and its isoforms have an APP production inhibitory effect or an Aβ production inhibitory action. There is no teaching or suggestion about.

  The present invention has been completed based on the discovery of the fact that HRD1 has an APP production inhibitory action or an Aβ production inhibitory action. The present invention provides a pharmaceutical composition comprising the HRD1 as an active ingredient, particularly an APP production inhibitor and an Aβ production inhibitor.

  According to the APP production inhibitory action or Aβ production inhibitory action of the APP production inhibitor and Aβ production inhibitor of the present invention, for example, when the inhibitory action can be shown in the cerebrum of a familial Alzheimer's disease patient, Can be expected to prevent or improve

  The cell that can express the APP production inhibitory effect or the Aβ production inhibitory effect may be any cell that can express APP and / or Aβ. Examples of the cells include nerve cells, mononuclear blood cells, oocytes, embryo cells, pancreatic β cells, hepatocytes, epithelial cells, skeletal muscle cells, smooth muscle cells, fibroblasts, lymphocytes and the like. [Autillio-Gambetti, L., et al., FEBS Lett., 241 (1-2): 94-98 (1988); Erratum, FEBS Lett., 244 (2): 509 (1989); Allen , JS, et al., Neurosci. Lett., 132 (1): 109-112 (1991); Fisher, S., Proc. Natl. Acad. Sci. USA, 88 (5): 1779-1782 (1991) ].

  In particular, Alzheimer's disease, Down's syndrome and the like can be exemplified as diseases that have been reported to be associated with high APP expression.

  Mutations in the causative gene PS1 of familial Alzheimer have been reported to enhance Aβ production (Citron M, Westaway D, Xia W., et al., Nat. Med., (1): 67-72 ( 1997)).

  In relation to Down's syndrome and APP, APP expression, Aβ deposition, Aβ deposition, Aβ deposition, and Alzheimer's disease-like symptoms are observed in Down's syndrome caused by trisomy on chromosome 21 where APP is present. It is thought that the increase in the expression level of is involved in the onset of Down's syndrome (Neve. RL, et al., Brain Res., 15: 886 (1-2): 54-66 (2000)).

  Therefore, increased APP expression and Aβ deposition in patients with chromosome 21 trisomy can be improved by administration of the APP production inhibitor or Aβ production inhibitor according to the present invention, and thus in the patient. It is possible to prevent or delay the onset of Down's syndrome or reduce the symptoms. Therefore, the pharmaceutical composition of the present invention may be expected as an agent for preventing or improving Down's syndrome.

  As described above, according to the present invention, an APP production inhibitor or an Aβ production inhibitor can be provided, and these prevent or improve the progress of symptoms in various neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease and genetic diseases such as Down's syndrome. Can be expected. That is, the present invention provides a pharmaceutical product having a specific pharmacological action directed to these diseases, and techniques for preventing and treating these diseases.

  Examples of the disease that can be prevented and treated by the APP production inhibitory action or Aβ production inhibitory action, which is a pharmacological action specific to the present invention, include, in addition to the various neurodegenerative diseases, for example, Huntington's disease, Machado-Joseph Disease, polyglutamine disease, ischemic brain injury (brain ischemia), amyotrophic lateral sclerosis, multiple sclerosis, HIV-related neuropathy, diabetes, diffuse Lewy body disease, depression, manic depression And various cell degenerative diseases. The present invention provides pharmaceutical compositions and prevention and treatment techniques useful for preventing or ameliorating the progression of symptoms for these diseases.

(2) Polynucleotide encoding the polypeptide of the present invention As a specific example of the polynucleotide encoding the polypeptide of the present invention (hereinafter sometimes referred to as “the gene of the present invention”), a DNA sequence represented by SEQ ID NO: 2 Or the polynucleotide containing the complementary strand can be mentioned.

  Other polynucleotides of the present invention include polynucleotides that can hybridize with the above-mentioned polynucleotides under stringent conditions and can express an APP production inhibitory action or an Aβ production 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 polynucleotide of the present invention comprises a DNA sequence that is 80% or more, preferably 95% or more, more preferably about 98% or more, homologous to the above-mentioned polynucleotide, and exhibits APP production inhibitory action or Aβ production inhibitory action. Possible polynucleotides are included.

  These other polynucleotides (hereinafter sometimes referred to as “variants”) are required to be capable of expressing an APP production inhibitory action or an Aβ production inhibitory 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 used as an expression product of the polypeptide of the present invention, ie, HRD1 protein or APP production suppression. It is possible to express a protein having an action or an Aβ production inhibitory action.

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

  The polynucleotide of the present invention further includes a homologue of a polynucleotide (HRD1 gene) comprising the DNA sequence represented by SEQ ID NO: 2. Here, "HRD1 gene homologue" has sequence homology with HRD1 gene and is recognized as one gene family due to structural features, commonality in gene expression pattern and similarity of biological function Means a series of related genes. This includes alleles (alleles) of the HRD1 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 occurring gene (for example, the HRD1 gene). 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, for example, 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 using a DNA polymerase with an appropriate primer sequence to chemically synthesize the complementary strand. Can be obtained by adding to the single strand.

  The DNA sequence 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 HRD1 gene), is the amino acid residue of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1. It is one combination example of each codon to encode. 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) Genetic engineering production of the polypeptide of the present invention The polypeptide of the present invention (expressed product) can be obtained by using the gene of the present invention to perform a conventional genetic engineering technique (for example, Science, 224, 1431 (1984); Biochem. 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 polynucleotide encoding the polypeptide of the present invention, the DNA sequence of the HRD1 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.

  In addition, in the amino acid sequence encoded by the HRD1 gene, in the case where a part of the amino acid residue or amino acid sequence is modified by substitution, insertion, deletion, addition, etc., for example, the above-mentioned various types such as cytospecific mutagenesis The method can be adopted.

(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 APP production inhibitory action or Aβ production inhibitory action of the expression product of the present invention is synonymous with the APP production inhibitory action or Aβ production 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).

  The APP production-suppressing action can be measured and detected by a known method using labeled blotting cells expressing APP by Western blotting analysis, with the decrease / decrease in the expression level as an index. The Aβ production inhibitory action can be measured and detected by a conventional method as a decrease / decrease in the amount of Aβ production by ELISA using an Aβ antibody against Aβ produced in the culture medium.

  More specifically, recombinant HRD1 labeled with Myc epitope and recombinant APP labeled with flag epitope were introduced into HEK293 cells, and cell lysates collected at 13 hours and 37 hours after gene transfer were used for anti-flag antibody western・ Measure the amount of APP in cells by blotting and compare the measured value with the same measured value of non-recombinant cells (HRD1 non-expressing cells) to detect the presence or level of APP production inhibitory effect. Can do.

  In addition, HEK293 cells expressing HRD1 in the above and HEK293 transformed cells not expressing HRD1 were cultured for 10 hours, and then the medium was replaced with Opti-MEM, followed by incubation for 3 hours and 27 hours, respectively. Collect the supernatant, and measure the amount of Aβ produced from the cells using Aβ1-40 ELISA kit or Aβ1-42 ELISA kit. By comparing with the measured value, it is possible to detect the presence or absence or the extent of the Aβ production amount suppressing action.

(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 APP production inhibitor or Aβ production inhibitor, and as a prophylactic / ameliorating agent for cell degeneration against cell degeneration such as nerve cells caused by APP production or Aβ accumulation in the brain neurons.

  In addition, by using the above-mentioned action as an APP production inhibitor or Aβ production inhibitor and cytopathic prevention / amelioration agent, (familial or sporadic) Alzheimer's disease (Alzheimer-type senile dementia), Parkinson's disease, Down's syndrome, 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 It is also useful as a therapeutic and prophylactic agent for depression, manic depression and the like.

  More specifically, the polypeptide of the present invention and the expression product of the present invention (for example, recombinant HRD1) as active ingredients in the pharmaceutical composition of the present invention are produced by cell degeneration such as nerve cells caused by APP production or accumulation of Aβ unique to them. It has the effect of suppressing or improving various induced symptoms by APP production inhibitory action or Aβ production inhibitory action, and using this action or activity, APP production of various cells or accumulation of Aβ and cells It can be used to treat diseases associated with degeneration. The cells affected by this APP production inhibitory effect or Aβ production inhibitory effect are nerve cells, neuroblasts, mononuclear blood cells, oocytes, embryonic cells, pancreatic β cells, hepatocytes, epithelial cells, skeletons. Examples include 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 addition amount of the surfactant is suitably about 0.00001 mg or more, preferably about 0.0001-0.01 mg per 1 μg of the 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 (HRD1 gene) is observed in tissues such as neurons, neuroblasts, Alzheimer's patient cerebral cortex, etc. All or part of the sense DNA of a gene is directly or directly in the form of any gene expression vector containing it, and uses viruses or liposomes in tissue cells such as the cerebrum, midbrain, cerebellum, and cerebral cortex. Thus, the expression of the HRD1 gene can be forcibly increased by generating RNA having a complementary sequence in the cell and promoting translation. Thus, it suppresses APP production and Aβ accumulation in tissues such as cerebrum, cerebellum, etc., and consequently suppresses neuronal degeneration caused by APP production or Aβ accumulation and is related to cell degeneration caused by APP production and Aβ accumulation It is possible to suppress or improve the progression of the disease state. Such effects can cause (familial or sporadic) Alzheimer's disease (Alzheimer-type senile dementia), Parkinson's disease, Down's syndrome, Huntington's disease, polyglutamine disease, ischemic brain injury (brain ischemia), muscle atrophic side It can be used to suppress or improve the progress of cord sclerosis, 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 APP production inhibitory action or Aβ production inhibitory action.

  As described above, the present invention provides a gene therapy vector containing all or part of the HRD1 gene and a pharmaceutical composition comprising as an active ingredient a cell into which the HRD1 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 APP production inhibitory activity or Aβ production inhibitory activity.

  The present invention also relates to a gene therapy vector containing a sense gene containing the whole 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 by the vector (family Or sporadic) Alzheimer's disease (Alzheimer-type senile dementia), Parkinson's disease, Down's syndrome, Huntington's disease, polyglutamine disease, ischemic brain injury (brain ischemia), amyotrophic lateral sclerosis, multiple sclerosis Desired from these tissues by administration to skeletal muscle cells or muscle tissue sites in patients with HIV-related neuropathy, diffuse Lewy body disease, type I diabetes, type II diabetes, depression, manic depression, etc. A method for suppressing or improving the progression of disability caused by APP production or Aβ accumulation in the target tissue and suppressing or improving the progression of the pathological condition in each of the above diseases, that is, suppression of APP production. A control method or a method for suppressing Aβ production is provided.

  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 (Maniatis, T., et al., Molecular Cloning: A laboratory manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1982))), Sambrook (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)) 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)) It is described in.

  In the gene therapy of the present invention, in a cell that expresses the HRD1 gene, RNA having a complementary sequence to the mRNA in the cell is produced, translation is inhibited, and APP production by a sense drug for suppressing the expression of the HRD1 gene Or a method for suppressing Aβ accumulation, particularly a method for inhibiting or improving the progression of disability caused by APP production or Aβ accumulation in brain neurons, or a method for inhibiting neuronal degeneration caused by APP production or Aβ accumulation in cells or tissues To do. For example, the therapeutic method provides transcription or translation by providing the HRD1 gene-expressing cells with the original or excessive amount of mRNA to HRD1 gene-expressing cells in which expression of the HRD1 gene is reduced or significantly reduced. It is a method of increasing the expression of a target gene by enhancing the process.

  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. Thus, if the target cell is supplied with an action that promotes the expression function of the HRD1 gene, APP production or Aβ accumulation in the recipient / target cell can be suppressed, and it is based on APP production or Aβ accumulation in the nerve cell. Neuronal degeneration 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 APP production inhibitory action or Aβ production inhibitory action using the sense oligonucleotide according to the present invention, retrovirus, adenovirus, adeno-associated virus vector (AAV) Incorporate a sense oligonucleotide into a vector derived from this, infect it with HRD1-expressing cells, and overexpress the sense oligonucleotide to increase or enhance the desired APP production inhibitory effect or Aβ production inhibitory effect Thus, an APP production inhibitory effect or an Aβ production inhibitory effect can be obtained.

  When a sense oligonucleotide is introduced into a target cell having an HRD1 gene to increase HRD1 expression, the sense oligonucleotide does not need to correspond to the full length of the HRD1 gene, and the expression function of the HRD1 gene As long as it retains a function that is substantially the same as the function that suppresses the above, it may be, for example, the aforementioned variant or a gene consisting of a partial sequence that retains such a specific function.

  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 HRD1 sense oligonucleotide linked to an expression control element and is capable of expressing the sense oligonucleotide product in the target cell. Specific examples thereof include, for example, the 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.

  Specific examples thereof include, for example, glial fibrillary acidic protein, mature astrocyte specific protein, myelin, tyrosine hydroxylase pancreatic villin, glucagon, and Langerhans islet amyloid polypeptide.

  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 the production of the sense oligonucleotide introduction vector, the sense oligonucleotide (all or part of the complementary sequence corresponding to the HRD1 gene) to be introduced is based on the DNA sequence information of the HRD1 gene as described above. It can be easily produced and obtained by general genetic engineering techniques.

  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 a sense oligonucleotide of the HRD1 gene. It can be used as a pharmaceutical for suppressing APP production or Aβ production in its isolated state, and as a model system for therapeutic research.

  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 HRD1 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 APP production or Aβ production particularly for a disease patient having a physical disorder caused by APP production or Aβ accumulation in brain neurons. Further, the gene therapy (treatment) of the present invention includes (familial or sporadic) Alzheimer's disease (Alzheimer-type senile dementia), Parkinson's disease, Down's syndrome, Huntington's disease, polyglutamine disease, ischemic brain injury (brain ischemia) 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 It can also be targeted. 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 expression of HRD1 is observed, and tissues that are aggregates thereof, particularly cerebellum, cerebral cortex, pituitary gland, neurons, neuroblasts, mononuclear blood cells, oocytes And embryonic cells, liver, pancreas, lung, small intestine tissue, lymphocytes, pancreatic β cells, adipocytes, hepatocytes, fibroblasts, mucosal epithelial cells, hematopoietic stem cells, epidermal cells, and the like.

  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 HRD1 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) of reimplanting the resulting cells after introducing the sense oligonucleotide of the HRD1 gene.

  The second method is a direct gene introduction method (direct method) in which a target sense oligonucleotide (sense oligonucleotide of the HRD1 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 (HRD1 gene sense oligonucleotide) 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 in situ PCR, for example, by measuring the degree of APP production inhibitory action or Aβ production inhibitory action of the desired subject as in the present invention, or by measuring the inhibitory action of apoptosis caused by endoplasmic reticulum stress induction. Confirm the introduction effect of the target sense oligonucleotide.

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 introduced with nucleotides (HRD1 gene sense oligonucleotide) 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 above first method, a virus-producing cell containing a retroviral vector containing the target sense oligonucleotide (sense oligonucleotide of the HRD1 gene) and a patient cell, for example, are co-cultured and the target A method of introducing a sense oligonucleotide (sense oligonucleotide of the HRD1 gene) into cells can also be employed.

  In conducting the second method (direct method) of gene therapy, preliminary experiments in vitro whether or not the target sense oligonucleotide (sense oligonucleotide of the HRD1 gene) is actually introduced by the gene transfer method, For example, it can be confirmed in advance by PCR or in situ PCR of vector gene cDNA, or it can suppress APP production or Aβ, which is the desired therapeutic effect based on the introduction of the target sense oligonucleotide (sense oligonucleotide of HRD1 gene) It is desirable to confirm beforehand the production inhibitory effect, the decrease in APP production in the target cells, the decrease in Aβ accumulation, 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, Down's syndrome, etc., bone marrow stem cells (HRD1-expressing cells) are collected from the patient's bone marrow and then subjected to enzyme treatment etc. to culture cells. After establishment, introduce into cultured bone marrow stem cells (HRD1-expressing cells) that target the desired sense oligonucleotide with retrovirus, for example, and screen for G418 cells, then measure the expression level of IL-12, etc. A preferred method is to inoculate the patient's brain after in vivo treatment and then radiation treatment.

 When targeting Alzheimer's disease patients, bone marrow stem cells (HRD1-expressing cells) are collected from the patient's bone marrow and then treated with enzymes to establish cultured cells, and then target the desired sense oligonucleotides using, for example, liposomes After being introduced into cultured bone marrow stem cells (HRD1-expressing cells) and screened with G418 cells, the expression level of IL-12 and the like is measured (in vivo), and then subjected to radiation treatment, A preferred method is to inoculate the bone marrow of the patient.

(9) Preparation of gene therapeutic agent The present invention comprises a cell into which a sense oligonucleotide introduction vector or a sense oligonucleotide (HRD1 gene sense oligonucleotide) has been introduced as an active ingredient, and a pharmaceutically effective amount thereof. The present invention provides a pharmaceutical composition or pharmaceutical preparation (gene therapy agent) containing 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 HRD1 gene The present invention provides a method for detecting the presence of an HRD1 protein having an APP production inhibitory action or an Aβ production inhibitory action in a target cell and a gene encoding the same.

  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 HRD1 protein is present in the sample or nucleic acid.

  Further, the present invention detects the level of HRD1 in cells or tissues by detecting the HRD1 gene (APP production inhibitory action or Aβ production inhibitory action, particularly APP production or Aβ production inhibitory action in brain nerve tissue, expression level of HRD1 mRNA, etc.) The degree of APP production or Aβ production (accumulation) or the degree of progression of neuronal degeneration (increase in Aβ accumulation cells) caused by APP production or Aβ production, APP production cells or Aβ production cells (Aβ accumulation cells) A method of determining the degree of increase or prognosis is provided. The method involves preparing a biological sample that causes neuronal degeneration in which APP production or Aβ production (accumulation) occurs in the cell and analyzing whether the HRD1 gene is present in the sample, or HRD1 The basic operation is to analyze the amount of mRNA of a gene.

  By carrying out the method of detecting the HRD1 gene of the present invention, it is possible to know the APP production inhibitory effect or Aβ production inhibitory effect in cells or tissues, and thus the expression level of HRD1 mRNA, neuronal degeneration caused by APP production or Aβ production. It is possible to obtain an indication of the degree of progression or prognosis (increase in Aβ accumulating cells). Also, for example, diagnosis of neuronal degeneration (Aβ accumulating cells), cell death, etc. caused by increased APP production or Aβ production or increase of Aβ accumulating cells, (familial or sporadic) Alzheimer's disease (Alzheimer type senile dementia) ), Parkinson's disease, Down syndrome, Huntington's disease, polyglutamine disease, ischemic brain injury (brain ischemia), amyotrophic lateral sclerosis, multiple sclerosis, HIV-related neuropathy, diffuse Lewy body disease, It is possible to determine the therapeutic effects and predict the prognosis of type I diabetes, type II diabetes, depression, manic depression and the like.

  In this detection method, first, information on the HRD1 gene obtained from a sample of neurodegenerative (Aβ accumulating cells) cells or degenerated cells caused by increased APP production or Aβ production or an increase in Aβ accumulating cells in the target cells is used. In addition, a DNA fragment designed to be used for screening of the HRD1 gene and / or its amplification is prepared. 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. This DNA fragment may have a property as a probe used in, for example, polymerase chain reaction (PCR).

  Next, for example, a primer having a partial sequence of the HRD1 gene is used as a screening probe and reacted with a biological sample (nucleic acid sample) to confirm the presence of the HRD1 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 HRD1 gene, it is particularly preferable to use PCR from the viewpoint of sensitivity. The method uses a HRD1 gene fragment as a primer, and a conventionally known method (Science, 230, 1350-1354 (1985)) or a newly developed or used PCR method (for example, Yoshiyuki Tsuji, et al.) Hen, 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 (HRD1 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) Kit for HRD1 gene detection and diagnosis The detection method according to the present invention can be carried out more easily by using a kit. The present invention also provides a kit for detecting an HRD1 gene containing a DNA fragment of the HRD1 gene.

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

  Examples of the labeling agent include chemical modifiers such as radioisotopes and fluorescent substances. Instead of including these labeling agents, a conjugate obtained by conjugating the DNA fragment itself with these labeling agents in advance is used. It can also be used as a component. Further, the reagent kit may contain an appropriate reaction diluent, a standard antibody, a buffer solution, a cleaning agent, a reaction stop solution, etc. for the convenience in carrying out the measurement.

  According to the use of the kit for detecting the HRD1 gene of the present invention, not only the HRD1 gene can be measured and detected, but also based on the detection result, cell damage caused by increased APP production or Aβ production or increased Aβ accumulating cells. Can be detected. Therefore, the present invention also provides a diagnostic kit for diagnosing cell damage caused by increased APP production or Aβ production or an increase in Aβ accumulating cells.

  Further, in the detection method according to the present invention, the DNA sequence of the HRD1 gene obtained from the test sample can be directly or indirectly sequenced, whereby a new homologue having a high homology with the wild type HRD1 gene can be obtained. Related genes related to the HRD1 gene can also be found. The present invention also provides a method for screening related genes related to the human HRD1 gene in a test sample by such measurement, detection and determination of the DNA sequence of the HRD1 gene in the test sample.

(12) Antibody against the polypeptide of the present invention The present invention 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 consists of a polypeptide (expressed product) encoded by the human HRD1 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. It can be produced using a polypeptide or a fragment of the polypeptide 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, for example, purification of the polypeptide of the present invention such as HRD1 and measurement and identification by immunological techniques. More specifically, the expression of the gene of the present invention is confirmed in tissues such as cerebral neurons, particularly cerebellum, cerebral cortex, pituitary gland, neurons, neuroblasts, mononuclear blood cells, oocytes, embryo cells, etc. Therefore, the antibody can be used for measurement of HRD1 concentration in these tissues.

  The present invention also provides a method for measuring wild-type HRD1 and / or mutant HRD1 using an antibody that binds to the polypeptide of the present invention.

  By this measurement method, the degree of HRD1 activity, the degree of APP production inhibition or Aβ production inhibition, the degree of improvement of cell damage by these inhibitions, the degree of cytopathic death based on APP production or Aβ production (accumulation) Can also be detected based on changes in wild-type HRD1. Specific examples of the measurement method using the antibody of the present invention include, for example, a method of immunoprecipitation of HRD1 protein from a solution containing a biomaterial sample collected from a human using a HRD1 antibody, or a solution containing a biomaterial sample The HRD1 protein can be directly reacted with the HRD1 antibody on a Western blot or immunoblot of a polyacrylamide gel. The HRD1 antibody can also be used to detect HRD1 in paraffin or frozen tissue sections according to immunohistochemical techniques.

  More preferred methods for measuring wild type HRD1 or mutant HRD1 include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoradiation, including sandwich methods using monoclonal and / or polyclonal antibodies. Assays (IRMA) and immunoenzymatic methods (IEMA) are included.

(13) Screening for Substance that Promotes (Enhances) APP Production Suppressing Action or Aβ Production Suppressing Action The present invention also provides a method for screening a substance that promotes (enhances) APP production inhibiting action or Aβ production inhibiting action.

  In the method, in the presence and absence of a test substance, (a) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1 or (b) one or several amino acids in the amino acid sequence of the polypeptide is missing. Measures the degree of APP production inhibitory effect or Aβ production inhibitory effect, and compares the measured value in the presence of the test substance with the measured value in the absence of the test substance. And selecting a substance that enhances the inhibitory action.

  Here, the degree of the APP production inhibitory action of the polypeptide of the present invention is determined as a decrease in the APP expression level in the target cell, and the degree of the Aβ production inhibitory action is determined as a decrease in the Aβ production amount in the target cell.

  The screening method can also be carried out by using an expression product of a polynucleotide encoding the polypeptide instead of the polypeptide of the present invention. That is, the present invention allows (c) a polynucleotide comprising the DNA sequence represented by SEQ ID NO: 2 in the presence and absence of a test substance, and (d) to hybridize with the polynucleotide under stringent conditions. And a polynucleotide capable of expressing an APP production inhibitory action or Aβ production inhibitory action, or (e) a polynucleotide comprising a DNA sequence that is 80% or more homologous to the polynucleotide and capable of expressing an APP production inhibitory action or Aβ production inhibitory action A substance that increases the degree of inhibition by measuring the degree of APP production inhibitory action or Aβ production inhibitory action of the expressed product, and comparing the measured value in the presence of the test substance with the measured value in the absence of the test substance A screening method for a substance that enhances the action of suppressing the APP production action or the Aβ production action is provided.

  Here, the degree of the APP production inhibitory action of the expressed product is determined as a decrease in the APP expression level in the target cell, and the degree of the Aβ production inhibitory action is obtained as a decrease in the Aβ production amount in the target cell.

  In the screening method of the present invention, the APP production inhibitory action and its degree can be detected by Western blotting analysis as described above, for example. In addition, the Aβ production inhibitory action and its degree can be detected by ELISA as described above.

A preferred embodiment of the screening method of the present invention includes the following steps (1) to (5).
(1) a step of preparing target cells transformed with respective expression vectors of a gene encoding the polypeptide of the present invention and a gene encoding labeled APP,
(2) culturing each target cell of (1) in the presence or absence of a candidate substance,
(3) a step of determining a decrease in the APP expression level of the target cell or a decrease in the Aβ production amount of the target cell in the presence and absence of the candidate substance,
(4) a step of comparing the APP expression amount or Aβ production amount in the absence of the candidate substance with the APP expression amount or Aβ production amount in the presence of the candidate substance, respectively.
(5) a step of selecting the candidate substance when at least one of the APP expression level and the Aβ production level obtained in the presence of the candidate substance is lower than those obtained in the absence .

Further, a preferred embodiment of the screening method of the present invention using a polynucleotide includes the following steps (1) to (5).
(1) preparing a target cell transformed with an expression vector comprising each of a polynucleotide encoding the polypeptide of the present invention and a polynucleotide encoding labeled APP,
(2) culturing each target cell of (1) in the presence or absence of a candidate substance,
(3) determining the APP expression level of the target cell or the Aβ production level of the target cell in the presence and absence of the candidate substance,
(4) a step of comparing the APP expression amount or Aβ production amount in the absence of the candidate substance with the APP expression amount or Aβ production amount in the presence of the candidate substance, respectively.
(5) a step of selecting the candidate substance when at least one of the APP expression level and the Aβ production level obtained in the presence of the candidate substance is lower than those obtained in the absence .

Furthermore, a preferred embodiment of the screening method of the present invention includes the following steps (1) to (5).
(1) preparing a target cell transformed with an expression vector comprising each of a polynucleotide encoding the polypeptide of the present invention and a polynucleotide encoding APP;
(2) culturing each target cell of (1) in the presence or absence of a candidate substance,
(3) a step of determining the amount of the polypeptide of the present invention 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 polypeptide production amount determined in the absence of the candidate substance with the polypeptide production amount determined in the presence of the candidate substance,
(5) A step of selecting the candidate substance when the amount of APP production obtained in the presence of the candidate substance is increased as compared with that obtained in the absence.

  In the screening method described above, the expression level or production level of the polypeptide of the present invention (for example, HRD1 or HRD1 gene expression product, hereinafter simply referred to as “HRD1”) is the test solution containing an antibody against the polypeptide and the test substance. It can be reacted competitively with HRD1 labeled therein and measured as the proportion of labeled HRD1 bound to the antibody. In addition, by reacting the test solution with the antibody insolubilized on the carrier and another labeled different anti-HRD1 antibody simultaneously or successively, by measuring the activity of the labeling agent on the insolubilized carrier, It is possible to quantify HRD1 in the test solution.

  According to the screening method of the present invention, the binding of HRD1 to a ligand can be evaluated in, for example, cells, cell-free preparations, chemical libraries, and natural product mixtures.

  Candidate substances selected by the method of the present invention are useful as promoters or enhancers of APP production inhibitory action or Aβ production inhibitory action. These promoters or enhancers may be natural substrates or ligands, or structural or functional mimics thereof.

  In the screening method of the present invention, a high-throughput screening technique can be applied. According to the application of this technology, for example, in screening for APP production inhibitors or Aβ production inhibitors, synthesis reaction mixtures, cell fractions such as cytoplasm, endoplasmic reticulum, membrane, cell envelope, cell wall or any of these The preparation (containing HRD1 and a labeled ligand of the polypeptide) is incubated in the presence or absence of the candidate compound to be screened. Whether the candidate compound activates HRD1 or promotes HRD1 action can be detected by a decrease in binding of the labeled ligand. Detection of the activity level of HRD1 utilizes reporter systems such as (but not limited to) colorimetric labels and integration of reporter genes in response to changes in polynucleotide or polypeptide activity and the art Can be carried out by binding assays known in the art.

  Competitive assays combining HRD1 and other APP production inhibitors or Aβ production inhibitors with compounds that bind to them can also be used to screen candidate compounds as APP production inhibition promoters or Aβ production inhibition promoters.

  The test substance (candidate compound) to be screened by the screening method of the present invention is a substance that enhances the activity of HRD1, particularly an APP production inhibitor or Aβ production inhibitor. That is, such an enhancing substance is a substance that increases the activity of HRD1 measured by its presence in the screening system of the present invention.

  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.

  APP production inhibitors or Aβ production inhibitors include inorganic or organic low-molecular compounds that bind to HRD1 and increase its activity, peptides, polypeptides, etc., natural or synthetic or prepared by genetic engineering techniques It is. Also included are sense DNA molecules for HRD1 DNA that are administered directly in vivo or in a form inserted into a recombinant vector.

  Evaluation of candidate substances having an action of activating or inhibiting HRD1 activity screened according to the screening method of the present invention can be performed as follows. That is, when the HRD1 activity promotes 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 promotes HRD1 activity. It can be selected as a promoting compound.

  HRD1 is expressed especially in cerebral nerve cells, especially cerebellum, cerebral cortex, pituitary gland, neurons, neuroblasts, mononuclear blood cells, oocytes, embryonic cells and other tissue cells, Promoter of HRD1 activity against diseases related to neurodegeneration caused by APP production or Aβ production (Aβ accumulation in tissues) induced by these cells, especially neurodegeneration such as neurons and neuroblasts Is useful. For example, compounds that promote HRD1 activity in the cerebrum, cerebellum, cerebral cortex, mesencephalon and pituitary tissues (familial or sporadic) by inhibiting APP production or Aβ production (Aβ accumulation in tissues) Alzheimer's disease (Alzheimer-type senile dementia), Parkinson's disease, Down's syndrome, Huntington's disease, ischemic brain injury (brain ischemia), amyotrophic lateral sclerosis, multiple sclerosis, HIV-related neuropathy, diffuse Lewy It is thought to inhibit the improvement or progress of patients with body disease, type I diabetes, type II diabetes, depression and manic depression.

  Substances that promote HRD1 activity obtained by the screening method of the present invention are thought to increase HRD1 concentration in cells and suppress APP production or decrease Aβ accumulation in cells. It is useful in various fields.

The HRD1 bulking agent is an APP production inhibitor or Aβ production inhibitor, for example, (familial or sporadic) Alzheimer's disease (Alzheimer type senile dementia), Parkinson's disease, Down's syndrome Huntington's disease, polyglutamine disease, ischemic Brain disorders (brain ischemia), amyotrophic lateral sclerosis, multiple sclerosis, HIV-related neuropathy, diffuse Lewy body disease, type I diabetes, type II diabetes, depression, manic depression It can be effectively used as a medicament for treatment / prevention that inhibits improvement or progression of disease state.

  The above HRD1 bulking agent, APP production inhibitor or Aβ production inhibitor can be applied to each of the above diseases as a pharmaceutical composition containing an effective amount of each action together with an appropriate pharmaceutical carrier.

  The HRD1 promoter 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 a substance that promotes or inhibits (reduces) the HRD1 activity of HRD1.

  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). It is important to include a polynucleotide expression product) and an expression product of a gene encoding labeled APP as essential components, and the others can be the same as this kind of screening kit. For example, a kit containing various reagents such as cell culture solution, reaction diluent, staining agent, buffer solution, fixing solution, and washing agent as optional components together with HRD1 having HRD1 activity and labeled APP can be mentioned. .

Specific examples of the kit of the present invention include those having the following constitutions 1-4 or 1-5.
Configuration 1.Target cells (target HRD1-expressing cells), that is, cells to be measured, which are cells transformed by introduction of APP gene labeled with HRD1 gene and flag epitope, 0.5-1 × 10 ⁵ cells / well, cultured in a 60mm dish at 37 ° C in 5% carbon dioxide using Dulbecco's modified Eagles medium,
Configuration 2. Cell lysate,
Configuration 3. Anti-flag epitope antibody, anti-myc antibody (for Western blot analysis),
Composition 4.Immunoprecipitation buffer (2 × SDS, 6% 2-Mercaptoethanol, 10% Glycerol, 0.005% Bromophenol blue (BPB, etc.),
Configuration 5. Detection electrophoresis device for observation.

  In the screening method using the screening kit of the present invention, the amount of APP per lane unit visual field to which a test compound (test substance, drug candidate compound) is added is counted, and the control lane unit visual field to which no test compound is added. What is necessary is just to perform a significant difference test with the amount of APP per hit. In addition, in the case of inhibiting Aβ production, in addition to the above, for example, after culturing cells transformed with the HRD1 gene and the APP gene introduced, the culture supernatant is recovered, and the Aβ1-40 or Aβ1-42 ELISA kit is used. When measuring the amount of Aβ produced from the cells, correcting the measurement result of this Aβ production amount with the amount of APP mRNA determined by, for example, real-time PCR, and adding a test compound (test substance, drug candidate compound) A significant difference test may be performed between the Aβ production amount of the control and the Aβ production amount of the control to which the test compound is not added. These measurements and evaluations themselves can be carried out according to conventional methods.

  As described in detail above, the present invention provides a pharmaceutical composition comprising a polypeptide (human HRD1) having an APP production inhibitory action or an Aβ production inhibitory action. The composition of the present invention is used for various diseases involving HRD1 gene and its products (diseases related to neuronal degeneration caused by APP production or Aβ accumulation (production)), for example, (familial or sporadic) Alzheimer Disease (Alzheimer type senile dementia), Parkinson's disease, Down's syndrome, Huntington's disease, polyglutamine disease, ischemic brain injury (brain ischemia), amyotrophic lateral sclerosis, multiple sclerosis, HIV-related neuropathy, For the treatment of diffuse Lewy body disease, type I diabetes, type II diabetes, depression, manic depression, etc., it is useful as an APP production inhibitor, an Aβ production inhibitor, a neuronal degeneration improving agent, or the like.

  The elucidation of the relationship between HRD1 (HRD1 gene expression product) found by the present invention and APP production inhibitory action or Aβ production inhibitory action is a nerve that appears to be caused by APP production or Aβ accumulation in neurons, neuroblasts, etc. Various pathological conditions caused by APP production or Aβ accumulation caused by cell degeneration, such as (familial or sporadic) Alzheimer's disease (Alzheimer-type senile dementia), Parkinson's disease, Down's syndrome, Huntington's disease, polyglutamine disease, false Hematological encephalopathy (brain ischemia), amyotrophic lateral sclerosis, multiple sclerosis, HIV-related neuropathy, diffuse Lewy body disease, type I diabetes, type II diabetes, depression, manic depression, etc. Useful information or means for understanding, understanding, diagnosing, preventing, treating, etc.

  The present invention also relates to the development of a new drug that promotes the APP production inhibitory action or Aβ production inhibitory action of the expression product of the gene encoding the polypeptide (HRD1 gene) or the polypeptide of the invention (the gene expression product of the invention). A screening technology for candidate substances 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 elucidating and diagnosing pathological conditions such as Alzheimer's disease, Parkinson's disease, Down's syndrome, polyglutamine disease, diabetes, depression and manic depression.

  Furthermore, the present invention provides a vector for gene introduction containing HRD1 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. Construction of wild-type and mutant HRD1 protein mammalian cell expression vectors
MRNA extracted from HEK293 cells (ATCC Accession No: CRL-1573) is reverse transcribed to obtain cDNA, and using the cDNA as a template, a primer having the sequence shown in SEQ ID NO: 4 and a primer having the sequence shown in SEQ ID NO: 5 are used. The sequence including the entire coding region excluding only the stop codon of HRD1 was amplified by PCR.

  After the amplified fragment was digested with restriction enzymes Kpn I and Xho I, pcDNA6-Myc / His vector expressing Myc (c-Myc) and polyhistidine (6 × His) epitope at the C-terminus of the inserted sequence ( The digest obtained above was inserted into the Kpn I / Xho I site of Invitrogen (Invitrogen) to obtain an expression vector pcDNA6-Myc / His-HRD1wt expressing HRD1.

  A region lacking the RING-Finger motif present at the C-terminus of the HRD1 protein by PCR using the same cDNA as a template and a primer having the sequence shown in SEQ ID NO: 6 and a primer having the sequence shown in SEQ ID NO: 7. A sequence containing was amplified.

  After the amplified fragment was digested with restriction enzymes Kpn I and Xho I, pcDNA6-Myc / His vector expressing Myc (c-Myc) and polyhistidine (6 × His) epitope at the C-terminus of the inserted sequence ( The digest obtained above was inserted into the Kpn I / Xho I site of Invitrogen (Invitrogen) to obtain a RING finger motif-deficient HRD1 expression vector, pcDNA6-Myc / His-ΔRING-HRD1.

HEK293 cells are Dulbecco's modified Eagles supplemented with 10% (v / v) heat-inactivated fetal calf serum, 100 U / mL penicillin and 100 μg / mL streptomycin in 37 ° C, 5% carbon dioxide. It was maintained in a medium (Dulbecco's modified Eagle's medium; hereinafter abbreviated as DMEM).
2. Construction of amyloid precursor protein mammalian cell expression vector
A mammalian cell expression vector (named pcDNA3.1-flag vector) that inserts a flag sequence into the Xba I / Apa I site of pcDNA3.1 (Invitrogen: Invitrogen) and expresses a flag epitope at the C-terminus of the inserted sequence. Produced.

  The mRNA extracted from SH-SY5Y cells (ATCC Accession No: CRL-2266) is reverse transcribed to obtain cDNA, and using the cDNA as a template, the primer of the sequence shown in SEQ ID NO: 8 and the primer of the sequence shown in SEQ ID NO: 9 The sequence including the entire coding region excluding only the APP stop codon was amplified by PCR.

  After digesting the amplified fragment with restriction enzymes Nhe I and Not I, the digest obtained above is inserted into the Nhe I / Not I site of the pcDNA3.1-flag vector, and the flag epitope-labeled amyloid precursor A body protein expression vector, pcDNA3.1-APP, was obtained.

HEK293 cells are maintained in DMEM supplemented with 10% (v / v) heat-inactivated fetal bovine serum, 100 U / mL penicillin and 100 μg / mL streptomycin in 37% at 5% CO2. did.
3. APP-HRD1 interaction 10 μg and flag of pcDNA6-Myc / His-HRD1 expressing wild-type HRD1 labeled with Myc epitope in HEK293 cells that became 80% confluent after 1 day in culture on a 6 cm diameter culture dish Ten μg of pcDNA3.1-APP expressing APP labeled with an epitope was transfected by the calcium phosphate method.

Three hours before the transfection operation, the medium was changed with DMEM not containing antibiotics, and then the following two solutions were prepared.
(1) A solution in which 50 μL of 2.5 M CaCl ₂ is added to ₂₀ μg of plasmid DNA, and the total volume is 490 mL with sterile purified water,
(2) A solution in which 10 μL of 70 mM Na ₂ HPO ₄ is added to 500 mL of 2 × Hebs buffer (42 mM HEPES, 290 mM NaCl, 1.4 mM NaHPO ₄ , pH 7.05),
While gently stirring the solution of (2), the solution of (1) was dropped into the solution, and the mixed solution was allowed to stand at room temperature for 30 minutes. Thereafter, 0.5 mL of the mixture was mixed with HEK293 cells to introduce the gene into the cells.

  The medium was exchanged with DMEM 16 hours after the transfection operation. After culturing the cells for 1 day, 400 μL of lysis buffer (20 mM Hepes-NaOH (pH 7.4), 150 mM NaCl, 5 mM EDTA, 1% glycerol, 1% Triton X-100, 10 μg / mL aprotinin, 10 μg / mL leupeptin, 1 μM The cell lysate was obtained by lysis with PMSF).

  The collected cell lysate was subjected to immunoprecipitation (IP) with APP using an anti-flag antibody (SIGMA; M2), and then the precipitate was washed with 2 × SDS sample buffer (50 mM Tris-HCl (pH 6.8)). , 2% SDS, 6% 2-mercaptoethanol, 10% glycerol, 0.005% BPB).

  Perform 8% SDS-PAGE using 13 μL of the lysate, transfer it to a nitrocellulose membrane, and then use the anti-myc antibody (Oncogene; 9E10) to detect the presence or absence of HRD1 protein by Western blotting (Western blotting: WB) Was examined.

  The obtained results are shown in FIG.

  In the figure,-in the HRD1-myc and APP-flag indicates the pcDNA6-Myc / His-HRD1 (indicated as HRD-1-myc) and pcDNA3.1-APP (indicated as APP-flag) expression vectors shown on the left. It shows that it was not transfected, and + mark shows that it was transfected. 150, 100 and 75 kDa on the vertical axis indicate the molecular weight (kDa) using a molecular weight marker, and the left figure (IP: flag, WB: myc) performs IP with an anti-flag antibody and uses the precipitate to generate WB. Is a diagram showing the results of detection with an anti-myc antibody, the central diagram (Input, WB: myc) is a lysate (Input) similar to the left diagram, WB was performed, anti-myc antibody The right figure (IP: flag, WB: flag) shows the result of the detection by the anti-flag antibody, performs the WB using the precipitate, and performs the detection by the anti-flag antibody. It is a figure which shows the result. The positions indicated by arrows at the left end of each figure are the expected molecular weights of flag-labeled APP protein (indicated as APP-flag) and myc-labeled HRD1 protein (indicated as HRD1-myc), respectively.

  The results shown in FIG. 1 indicate the following. That is, as shown in the third lane from the left in the left diagram of FIG. 1, a protein that reacts with the myc antibody that matches the molecular weight of HRD1-myc was detected, and it was clear that HRD1 was co-immunoprecipitated with APP. became. In the center of Fig. 1, in the lysate of cells transfected with pcDNA6-Myc / His-HRD1 (indicated as +), HRD1-myc protein that reacts with anti-myc antibody can be confirmed, and cells that have not been transfected Since no expression of the protein was observed in the lysate (denoted as-), the expression of the HRD1-myc protein from the pcDNA6-Myc / His-HRD1 expression vector was confirmed. In addition, in the figure on the right, in the lysate of cells transfected with pcDNA3.1-APP (indicated as +), APP-flag protein that reacts with anti-flag antibody can be confirmed. Since no expression of the protein was observed in the lysate (labeled +), it was confirmed that APP-flag was expressed from the pcDNA3.1-APP expression vector.

  Using the same cell lysate, immunoprecipitation (IP) of HRD1 using anti-Myc antibody (manufactured by Oncogene; 9E10), the precipitate was added to 2 × SDS sample buffer (50 mM Tris-HCl (pH 6.8), (2% SDS, 6% 2-mercaptoethanol, 10% glycerol and 0.005% BPB).

  7% SDS-PAGE was performed using 13 μL of the lysate, transferred to a nitrocellulose membrane, and the presence or absence of APP protein was examined by Western blotting (WB) using an anti-flag antibody (SIGMA; M2). .

  The obtained results are shown in FIG. 2 in the same manner as FIG.

  In the figure, “-” in HRD1-myc and APP-flag is the transfection of pcDNA6-Myc / His-HRD1 (indicated as HRD1-myc) and pcDNA3.1-APP (indicated as APP-frag) expression vector shown on the left. The + symbol indicates that the cells were transfected. 150, 100 and 75 kDa on the vertical axis indicate the molecular weight (kDa) using the molecular weight marker, and the left figure (IP: myc, WB: flag) performs IP with an anti-myc antibody and uses the precipitate to generate WB. And the center figure (Input, WB: flag) is WB using the same cell lysate (Input) as in the left figure, and detected with the anti-flag antibody. The figure on the right (IP: myc, WB: myc) is a figure in which IP was performed using an anti-myc antibody, WB was performed using the precipitate, and detection was performed using an anti-myc antibody. The positions indicated by arrows at the left end of each figure are the expected molecular weights of flag-labeled APP protein (indicated as APP-flag) and myc-labeled HRD1 protein (indicated as HRD1-myc), respectively.

  The results shown in FIG. 2 indicate the following. That is, as shown in the third lane from the left in FIG. 2, a protein that reacts with a flag antibody that matches the molecular weight of APP-flag was detected, and it was reconfirmed that APP was co-immunoprecipitated with HRD1. It was. In the lysate of cells transfected with pcDNA3.1-APP in the center of Fig. 2 (labeled +), APP-flag protein reacting with anti-flag antibody can be confirmed, and lysate of cells not transfected In (-), since the expression of the protein was not observed, the expression of APP-flag protein from the pcDNA3.1-APP expression vector was confirmed. Also, in the right figure, HRD1-myc protein that reacts with anti-myc antibody can be confirmed in the lysate of cells transfected with pcDNA6-Myc / His-HRD1 (indicated as +), and no transfection was performed. In the cell lysate (denoted as-), the expression of the protein was not observed, so it was also confirmed that HRD1-myc was expressed from the pcDNA6-Myc / His-HRD1 expression vector.

  From these results, it was clarified that APP-flag and HRD1-myc co-precipitated by immunoprecipitation, and it was confirmed that APP and HRD1 interacted.

Inhibition of APP production by HRD1 10 μg of pcDNA6-Myc / His-HRD1 expressing wild-type HRD1 labeled with Myc epitope and flag epitope on HEK293 cells that became 80% confluent after 1 day in a 6 cm diameter culture dish 10 μg of pcDNA3.1-APP expressing labeled APP was transfected by the calcium phosphate method.

Three hours before the transfection operation, the medium was changed with DMEM not containing antibiotics, and then the following two solutions were prepared.
(1) A solution in which 50 μL of 2.5 M CaCl ₂ is added to 20 μg of plasmid DNA, and the total amount is 490 mL with sterile purified water,
(2) A solution in which 10 μL of 70 mM Na ₂ HPO ₄ is added to 500 mL of 2 × Hebs buffer (42 mM HEPES, 290 mM NaCl, 1.4 mM NaHPO ₄ , pH 7.05),
While gently stirring the solution of (2), the solution of (1) was added dropwise thereto, and the mixed solution was allowed to stand at room temperature for 30 minutes. Thereafter, 0.5 mL of the mixture was mixed with HEK293 cells to introduce the gene into the cells.

  The medium was exchanged with DMEM 16 hours after the transfection operation.

  After culturing the cells for 1 day, DMEM (10% (v / v) heat-inactivated fetal bovine serum to which Blasticidin (Blastcidin: Invitrogen: Invitrogen) in an amount of final concentration of 8 μg / mL was added. , 100 U / mL penicillin and 100 μg / mL streptomycin) were used to select transgenic cells, and two HRD1 stable expression strains were established. The established expression strains were named wt-HRD1 # 3 and wt-HRD1 # 4, respectively.

  PcDNA6-Myc / His-ΔRING-HRD1 expressing mutant HRD1 labeled with Myc epitope was introduced into cells, the same selection was performed to establish a mutant HRD1 stable expression strain, and this strain was named ΔRING-HRD1 did.

  8 μg of pcDNA-APP-flag expressing APP labeled with the flag epitope and 20 μL of Lipofectamine ™ 2000 (Invitrogen) were mixed and allowed to stand at room temperature for 20 minutes, and then the mixture was mixed with 2 wt-HRD1 HEK293 cells that stably express -myc (indicated as wt-HRD1 # 3 and wt-HRD1 # 4), ΔRING-HRD1-myc that lacks the RING-finger motif at the CRD end of HRD1 protein The HEK293 cells (expressed as ΔRING-HRD1) and HEK293 cells (expressed as APP) in which nothing was expressed were mixed with each other to introduce the gene into the cells.

  Using the cell lysates collected at 13 hours and 37 hours after gene transfer, the amount of APP in the cells was observed by anti-flag antibody western blotting. As a negative target, a lysate of cells in which neither wt-HRD1-myc, ΔRING-HRD1-myc69v nor APP-flag was expressed (indicated as Mock) was used. In addition, after gene transfer, the proteasome inhibitor MG132 (Peptide Institute) was added to the medium at a final concentration of 10 μM to simultaneously produce a protein with suppressed ubiquitin-dependent degradation of the protein at 37 hours of APP protein. The total amount was observed.

  The obtained results are shown in FIG.

  In the figure, wt-HRD1 # 3 / APP represents a sample from a cell transfected with pcDNA3.1-APP to a HRD1-myc stable expression strain (# 3 strain), and wt-HRD1 # 4 / APP represents HRD1 Samples from cells transfected with pcDNA3.1-APP in the -myc stable expression strain (# 4 strain) are shown, and ΔRING-HRD1 / APP is a RING-finger-deficient ΔRING-HRD1-myc stable expression strain. Samples from cells transfected with pcDNA3.1-APP are shown. The + and-signs in MG132 (10 μM) indicate the presence (+) and absence (-) of addition of the proteasome inhibitor MG132 to the medium, respectively.APP-FLAG is the APP-flag detected by anti-flag antibody. HRD1-Myc indicates the HRD1-myc protein detected by the anti-myc antibody, and ΔRING-HRD1-Myc indicates the ΔRING-HRD1-myc protein detected by the anti-myc antibody. The amount of intracellular APP transiently expressed can be confirmed as the amount of protein (band density) detected by the anti-flag antibody (indicated as APP-FLAG in the figure).

  As shown in Fig. 3, in the HRD1 stable expression cell lines (wt-HRD1 # 3 / APP, indicated as wt-HRD1 # 4 / APP), the amount of intracellular APP transiently expressed was stable for mutant HRD1 Compared with the expression cell line (ΔRING-HRD1 / APP), HRD1 non-expressing cells expressing APP (denoted APP), after 13 hours and 37 hours (denoted 13, 37), wt-HRD1 # In 3 / APP and wt-HRD1 # 4 / APP, it was confirmed that the amount of APP-flag was clearly decreased.

In addition, 17 hours after gene transfer, 10 μM MG132 inhibitor of proteasome proteolysis was added to these cells, and analysis was performed using cell lysates collected after incubation for 20 hours (in a CO ₂ incubator at 37 ° C.). As a result, in the mutant HRD1 stable expression cell line (indicated as ΔRING-HRD1 / APP), in the HRD1 non-expressing cells in which APP was expressed (indicated as APP), the amount of intracellular APP transiently expressed (37, MG132 + Compared with no addition of MG132 at the same incubation time (labeled 37, MG132-), there is no significant difference in the amount of protein with a molecular weight greater than 100 kDa that reacts with anti-flag antibody, whereas HRD1 In the stable expression cell lines (wt-HRD1 # 3 / APP, wt-HRD1 # 4 / APP), the amount of intracellular APP transiently expressed (37, MG132 +) is the same as the MG132 of the same incubation time. Compared with no addition (indicated as 37, MG132-), it has a molecular weight greater than 100 kDa that reacts with anti-flag antibodies. It was found that the increase in the protein amount was enhanced, and it was confirmed that the degradation of APP protein by the ubiquitin-proteasome degradation system was enhanced by the high expression of HRD1 protein. It was confirmed that HRD1 ubiquitinates APP, and that ubiquitinated APP is degraded by the ubiquitin-proteasome degradation system, thereby reducing the amount of APP in the cell. The wide distribution of the molecular weight of APP-FLAG recognized by the anti-flag antibody is due to the APP protein undergoing sugar chain modification in multiple stages.

  The bottom row of FIG. 3 confirms the expression of HRD1 and ΔRING-HRD1 protein with anti-Myc antibody using the lysate.

  In HRD1 stable expression cell lines (indicated as wt-HRD1 # 3 / APP and wt-HRD1 # 4 / APP), the expression of HRD1-Myc could be confirmed at a position of about 75 kDa. In the mutant HRD1 stably expressing cell line (referred to as ΔRING-HRD1 / APP), the expression of RING-finger-deficient RING-HRD1-myc was confirmed at about 30 kDa.

ARD production inhibitory effect by HRD1 Expression vector pcDNA3.1-APP, HEK293 cells not expressing HRD1 (labeled 293-normal), wt-HRD1- # 3 cell line, wt-HRD1- # 4 cell line and ΔRING -After 10 hours of transfection into each of the HRD1 cell lines, the culture medium of each cell was replaced with Opti-MEM (Invitrogen). After further incubation for 3 hours and 27 hours, respectively, the culture supernatant was recovered and The amount of Aβ produced in the clean was measured using Aβ1-40 ELISA kit and Aβ1-42 ELISA kit (both manufactured by Immunobiological Laboratories). These kits measure Aβ1-40 and Aβ1-42, respectively, which are the main constituent Aβ peptides of the elderly population in Alzheimer's disease.

  Further, in order to measure the expression level of APP in the cells, TRI-reagent (SIGMA) was added to the cells after collecting the supernatant, and the total RNA was collected after the cells were lysed. Collect 2 μg of total RNA from the reaction solution (100 U SuperScript II RNase H-reverse transcriptase (Invitrogen); 0.25 μg oligo (dT) 12-18 primer (Invitrogen); 10 mM DTT; 0.5 μM 4dNTP and 1 × first strand buffer ( Invitrogen)], reverse transcription was performed to synthesize cDNA.

The synthesized cDNA was diluted 64 times and used as a template. APP (F) (SEQ ID NO: 10) and APP (R) (SEQ ID NO: 11) was used to measure the APP mRNA amount in each cell by real-time PCR using ABI7700 ^TM (Applied Biosystems), the value Used to correct the gene transfer efficiency.

  For real-time PCR, use the reaction solution [5μL cDNA, 200nM APP (F), 200nM APP (R), 1 × TaqMan Universal Master Mix (Applied Biosystems), 1 × SYBER Green (TAKARA)] and heat at 95 ° C for 10 minutes. Thereafter, the reaction was carried out at 95 ° C., 10 seconds to 60 ° C. for 40 minutes, and the amplified product was analyzed.

In addition, correction of APP mRNA in each cell was performed by real-time PCR using ABI7700 ^™ (Applied Biosystems) using GAPDH (F) (SEQ ID NO: 12) and GAPDH (R) (SEQ ID NO: 13) ( (Same conditions).

  The concentration of Aβ1-40 and Aβ1-42 peptides in the culture supernatant obtained by ELISA was corrected using the amount of APP mRNA determined by real-time PCR, and the Aβ1-40 or Aβ1-42 peptide of each cell was corrected. The production amount was determined. In each experiment, the same operation was repeated three times.

  The results obtained are shown in FIG. 4 (Aβ1-40 peptide amount, after 13 hours culture), FIG. 5 (Aβ1-40 peptide amount, after twelve hour culture) and FIG. 6 (Aβ1-42 peptide amount, after 37 hour culture). Show.

  In each figure, the vertical axis represents an arbitrary unit and the horizontal axis represents each cell.

  As shown in FIG. 4, the amount of Aβ1-40 peptide was determined after the introduction of pcDNA3.1-APP into cells at 13 hours after two wild-type HRD1 stably expressing HEK293 strains (wt-HRD1- # 3 and wt-HRD1 -# 4) was found to be significantly lower than that of the mutant HRD1 stably expressing cell line (designated ΔRING-HRD1).

  As shown in FIG. 5, the two wild-type HRD1 stably expressing HEK293 strains (indicated as wt-HRD1- # 3 and wt-HRD1- # 4) have mutant HRD1 stably expressing cell lines (indicated as ΔRING-HRD1). ), A significant inhibitory effect on the amount of Aβ1-40 peptide was also confirmed at 37 hours after introducing pcDNA3.1-APP into the cells.

  Further, as shown in FIG. 6, regarding the production of Aβ1-42 peptide, two hours of wild type HRD1 stably expressing HEK293 strains (wt-HRD1- # 3, wt- In HRD1- # 4), it was revealed that the production amount was significantly reduced as compared with the mutant HRD1 stably expressing cell line (indicated as ΔRING-HRD1).

  These results indicate that the hyperfunction of HRD1 protein remarkably suppresses the production of Aβ, which is highly toxic in nerve cells. From this, it is proved that a substance responsible for promoting functions such as expression enhancement and stabilization of HRD1 can exert an effective treatment effect on Alzheimer's disease.

  In addition, referring to the above-described Examples of the present invention, methods for screening for substances that promote HRD1 expression enhancement, HRD1 APP production inhibitory action or HRD1 Aβ production inhibitory action are obvious to those skilled in the art. is there.

In the test according to Example 1, it is a figure which shows the result of the immunoprecipitation of each expression by the western blotting in the cell which co-expressed HRD1 and APP transiently. In the test according to Example 1, it is a figure which shows the result of the immunoprecipitation of each expression by the western blotting in the cell which co-expressed HRD1 and APP transiently. FIG. 4 is a diagram showing the results of Western blotting showing the effect of HRD1 on inhibiting APP production in the test shown in Example 2. 4 is a graph showing the effect of inhibiting HRD1 production of Aβ1-40 peptide in the test shown in Example 3. 4 is a graph showing the effect of inhibiting HRD1 production of Aβ1-40 peptide in the test shown in Example 3. 2 is a graph showing the effect of HRD1 on Aβ1-42 peptide production suppression in the test shown in Example 3.

  SEQ ID NOs: 5 to 14 show primer sequences for PCR, respectively.

Claims (12)

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) It consists of an amino acid sequence in which one or several amino acids are deleted, inserted, substituted or added in the amino acid sequence of (a) above, and has an action to suppress APP production action or Aβ production action Polypeptide. 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 action of suppressing an APP production action or an action of inhibiting an Aβ production action,
(e) A polynucleotide comprising a DNA sequence that is 80% or more homologous to the polynucleotide of (c) above and capable of expressing an action of suppressing APP production or an action of inhibiting Aβ production. An APP production inhibitor or an Aβ production inhibitor comprising, 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. In the presence and absence of the test substance, measure the degree of inhibition of APP production or Aβ production of the polypeptide (a) or (b) below, and the measured value in the presence of the test substance In contrast to the measured value in the absence of the test substance, a substance that increases the degree of inhibition is selected, and this enhances the action to suppress the APP production action or the Aβ production action Substance screening method:
(a) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1,
(b) It consists of an amino acid sequence in which one or several amino acids are deleted, inserted, substituted or added in the amino acid sequence of (a) above, and has an action to suppress APP production action or Aβ production action Polypeptide. 5. The method according to claim 4, wherein the degree of inhibition of the APP production action or the degree of inhibition of the Aβ production action is determined as the degree of decrease in the APP expression level or the degree of reduction in the Aβ production amount in the target cell. In the presence and absence of the test substance, the degree of suppression of APP production action or the degree of inhibition of Aβ production action of the expression product of the following polynucleotide (c), (d) or (e) is measured, The action of suppressing the APP production action, characterized by selecting a substance that increases the degree of inhibition of the production action by comparing the measurement value in the presence of the test substance with the measurement value in the absence of the test substance Alternatively, a method of screening for a substance that enhances the action of suppressing Aβ production action:
(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 an APP production action or an action of inhibiting an Aβ production action,
(e) A polynucleotide comprising a DNA sequence that is 80% or more homologous to the polynucleotide of (c) above and capable of expressing an action of suppressing APP production or an action of inhibiting Aβ production. 7. The method according to claim 6, wherein the degree of suppression of the APP production action or the degree of inhibition of the Aβ production action is determined as the degree of decrease in the APP expression level or the degree of reduction in the Aβ production amount in the target cell. 7. The method according to claim 4 or 6, wherein the action of suppressing the APP production action is determined by Western blotting analysis, and the action of suppressing the Aβ production action is determined by ELISA. The screening method of the substance which enhances the effect | action which suppresses the APP production effect | action or the Abeta production effect | action of the polypeptide of Claim 1 including the process of the following (1)-(5):
(1) preparing a target cell transformed with an expression vector containing each of the polynucleotide encoding the polypeptide of claim 1 and the labeled APP encoding polynucleotide;
(2) culturing each target cell of (1) in the presence or absence of a candidate substance,
(3) a step of determining the amount of APP expression or Aβ production in the target cells in the presence and absence of the candidate substance,
(4) a step of comparing the APP expression amount or Aβ production amount in the absence of the candidate substance with the APP expression amount or Aβ production amount in the presence of the candidate substance, respectively.
(5) Select at least one of the APP expression level and Aβ production level obtained in the presence of the candidate substance as compared to those obtained in the absence. Process. A method for screening for a compound that enhances an action of suppressing an APP production action or an action of suppressing an Aβ production action of an expression product of a polynucleotide according to claim 2, comprising the following steps (1) to (5):
(1) providing a target cell transformed with an expression vector comprising each of the polynucleotide according to claim 2 and a polynucleotide encoding labeled APP,
(2) culturing each target cell of (1) in the presence or absence of a candidate substance,
(3) a step of determining the APP expression level or Aβ production level of the target cell in the presence and absence of the candidate substance,
(4) a step of comparing the APP expression amount or Aβ production amount in the absence of the candidate substance with the APP expression amount or Aβ production amount in the presence of the candidate substance, respectively.
(5) a step of selecting the candidate substance when at least one of the APP expression level and the Aβ production level obtained in the presence of the candidate substance is lower than those obtained in the absence . A method for screening for a substance that enhances the action of suppressing the APP production action or the action of inhibiting Aβ production of the polypeptide according to claim 1, comprising the following steps (1) to (5):
(1) preparing a target cell transformed with an expression vector comprising each of the polynucleotide encoding the polypeptide of claim 1 and the polynucleotide encoding APP,
(2) culturing each target cell of (1) in the presence or absence of a candidate substance,
(3) The step of determining the polypeptide production amount according to claim 1 of the target cell in the presence and absence of a candidate substance using an antibody against the polypeptide,
(4) a step of comparing the polypeptide production amount determined in the absence of the candidate substance with the polypeptide production amount determined in the presence of the candidate substance,
(5) A step of selecting the candidate substance when the amount of polypeptide production obtained in the presence of the candidate substance is increased as compared with that obtained 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, and an expression product of a polynucleotide encoding a labeled APP as a constituent, A screening kit for screening a substance that enhances the action of suppressing the APP production action of the polypeptide or expression product or a substance that enhances the action of suppressing the Aβ production action.

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