JPWO2001004300A1 - Apoptosis-related factors - Google Patents

Abstract

(57) [Abstract] A novel apoptosis-related factor is provided. The apoptosis-related factor of the present invention is a novel protein containing a DED or caspase family cleavage domain. This protein induces apoptosis in cells. Therefore, this protein can be used as a target to screen for compounds useful for controlling apoptosis. Furthermore, this protein itself is useful for controlling cell proliferative disorders.

Description

[Detailed Description of the Invention] Technical Field : The present invention relates to apoptosis-related factors. Background Art : Apoptosis was once called programmed cell death. For the survival of multicellular organisms, a mechanism for eliminating unwanted or unnecessary cells from the body is necessary. Apoptosis was understood to be a pre-programmed cell death to eliminate unnecessary cells during development and cell generation. In cells that have undergone apoptosis, nuclear shrinkage and DNA fragmentation are observed, and the cell shrinks while leaving its membrane intact. In living organisms, the shrunken cells are eventually ingested and eliminated by phagocytes. In contrast to apoptosis, cell death called necrosis is accompanied by destruction of the cell structure. Therefore, apoptosis, in which cells die on their own, is often called "clean cell death." It has since become known that cell death similar to that observed during development can also be induced by stimuli from outside the cell. For example, TNF and Fas
S is a typical cytokine that induces apoptosis. Apoptosis can also be induced by stimuli such as ultraviolet light or radiation, depletion of cell growth factors, and activation of certain cancer genes. Diseases such as autoimmune diseases and cancer are now understood to be the result of the inability to kill harmful cells due to apoptosis dysfunction. Currently, molecules related to apoptosis are being identified one after another, and the overall picture of apoptosis is gradually becoming clearer. However, the mechanism of apoptosis has not yet been elucidated. At present, the process by which cells respond to external stimuli and undergo apoptosis is as follows:
It has been explained roughly as follows: It has been revealed that the caspase family, activated by various causes, leads to cell death.
It has been revealed that there are multiple caspase enzymes with different substrate specificities, and a group of caspase enzymes is collectively called the caspase family.
The Caspase family is broadly classified into three groups based on substrate specificity. Of these, Caspases in Group 2 are said to be involved in the execution of apoptosis. Caspases in Group 3 are activated in response to extracellular stimuli upstream of the apoptosis execution cascade, and have the function of activating Caspases in Group 2 further downstream. On the other hand, Caspases in Group 1
Caspase e is believed to have the effect of promoting the production and secretion of cytokines. Caspases classified into Group 2 and Group 3 and the amino acid sequences they recognize are shown below. Group 2 Caspase-2 DEHD Caspase-3 DEVD Caspase-7 DEVD Group 3 Caspase-6 VEHD Caspase-8 LETD Caspase-9 LETD Group 3 caspases are activated by ligand binding to Fas or TNF receptors, cleaving and activating Group 2 caspases.
The signal is transmitted downstream. This type of signal transmission is called the apoptosis execution cascade. It is presumed that downstream of the apoptosis execution cascade, various factors that receive the signal function as an execution force to bring about cell death. However, there are many issues to be clarified regarding what factors actually bring about cell death. For example, a certain type of caspase degrades the nuclease inhibitor ICAD (inhibitor of CAD). As a result, the nuclease CAD (Caspase Activated CAD) is degraded.
It is known that this allows the entry of nucleus-specific cleavage of chromosomal DNA, which is characteristic of apoptosis (Enari M. et al., Nature 2003, 14, 1999).
re, 391:43-50, 1998). Thus, it is believed that many of the downstream factors in the apoptosis execution cascade play important roles in the execution of apoptosis. These factors may potentially be important targets in the control of apoptosis. Disclosure of the Invention The objective of the present invention is to provide novel apoptosis-related factors. With the aim of isolating novel human genes, the present inventors isolated numerous full-length cDNA clones from a human cDNA library prepared by the oligo-capping method. These full-length cDNA clones were then analyzed for apoptosis-related genes based on alignment. As a result, one of these full-length cDNA clones, NT2RM1000558, was found to encode the Death Effector Domain (DED), which is said to be important in apoptosis signal transduction.
We found that NT2RM1000558 has a cleavage recognition sequence for the Caspase family. This full-length cDNA clone was cloned from a cDNA library of NT-2 neural progenitor cells. NT2RM1000558 encoded a protein consisting of 326 amino acid residues. Analysis of the amino acid sequence revealed that when NT2RM1000558 was digested at the cleavage recognition sequence for the Caspase family, 23 amino acid residues from the C-terminus were removed, resulting in a 303 amino acid residue.
It was predicted that the protein of the present invention would be a protein consisting of amino acid residues. In addition, the protein of the present invention contains a DED at positions 26-103, a nuclear localization signal (NLS) at positions 100-109, and a nuclear localization signal (NLS) at positions 152-174.
NLS) and other motifs were found.
h et al. 1998 The EMBO Journal 17, 20,
5974-5986. Chandra P. Leo et al., 1998 1
39,12,4838-4848) is known. DEDD is a protein consisting of 318 amino acid residues, and has a structure similar to the apoptosis-related factor of the present invention in that it contains DED, NLS, and a caspase digestion domain. However, even when comparing the amino acid sequences of both the same DED (DEDD: SEQ ID NO: 9), the homology is only 43%, and the two are clearly different proteins. Furthermore, the role of DEDD in the apoptosis execution cascade has not been fully elucidated. Next, the present inventors have investigated the relationship between the protein consisting of 326 amino acid residues (SEQ ID NO: 4) encoded by NT2RM1000558 and the 30 amino acid residues produced by digestion of this protein.
The activity of the protein consisting of three amino acid residues (SEQ ID NO: 2) was confirmed.
While a protein of 326 amino acid residues was not found to have apoptosis-inducing activity, a protein of 303 amino acid residues was confirmed to have clear apoptosis-inducing activity. The present invention was completed based on these findings. Specifically, the present invention relates to the following proteins, DNAs, and uses thereof. [1] A polynucleotide encoding a protein having apoptosis-inducing activity described in any of (a) to (d) below. (a) A polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 1; (b) A polynucleotide encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2; (c) A polynucleotide encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2 in which one or several amino acids have been substituted, deleted, inserted, and/or added; (d) A polynucleotide that hybridizes under stringent conditions with a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 1; [2] A polynucleotide having 60% or more homology with the nucleotide sequence set forth in SEQ ID NO: 1 [1]
[3] A polynucleotide encoding a precursor of a protein having apoptosis-inducing activity according to any one of (e) to (h) below: (e) a polynucleotide comprising a protein-coding region of the nucleotide sequence set forth in SEQ ID NO: 3, (f) a polynucleotide encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 4, (g) a polynucleotide encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 4 in which one or several amino acids have been substituted, deleted, inserted, and/or added, (h) a polynucleotide that hybridizes under stringent conditions with a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 3, [4] a polynucleotide having 60% or more homology with the nucleotide sequence set forth in SEQ ID NO: 3 [3]
[5] A polynucleotide encoding a partial peptide of the protein encoded by the polynucleotide of [1] or [3]. [6] A polynucleotide encoding a gene identical in terms of molecular evolution to a gene comprising the nucleotide sequence of SEQ ID NO: 3. [7] A polynucleotide encoding a protein consisting of an amino acid sequence in which one or several amino acids have been substituted, deleted, inserted, and/or added in the amino acid sequence of SEQ ID NO: 4, and which has dominant-negative properties relative to the protein consisting of the amino acid sequence of SEQ ID NO: 4. [8] The polynucleotide of [7], encoding an amino acid sequence in which the amino acid sequence of positions 300-303 in the amino acid sequence of SEQ ID NO: 4 has been modified to a caspase-resistant amino acid sequence.

[9] A protein encoded by the polynucleotide according to any one of [1], [3], [5], [6], and [7]. [10] A vector comprising the polynucleotide according to any one of [1], [3], [5], [6], and [7]. [11] A transformant harboring the polynucleotide according to any one of [1], [3], [5], [6], and [7], or the vector according to [10].
[12] A method comprising culturing the transformant according to [11] and recovering an expression product.

[13] A polynucleotide having a length of at least 15 bases, which consists of a base sequence complementary to the polynucleotide of any one of [1], [3], [5], [6], and [7], or its complementary strand.

An antibody against the protein according to [9]. [15]

[16] An immunoassay method comprising the step of observing the immunological reaction between the protein of [9] and the antibody of [14]. [16] A method for screening for a substance that controls apoptosis, comprising the steps of: (1) contacting a candidate substance with cells expressing a protein encoded by the polynucleotide of [1] or [3], and (2) culturing the cells under conditions that induce apoptosis, and selecting a candidate substance that suppresses or promotes apoptosis. [17] A method for screening for a substance that controls apoptosis, comprising the steps of: (1) contacting a candidate substance with cells into which a vector has been introduced that contains an expression control region of a gene consisting of the nucleotide sequence of SEQ ID NO: 3 and a reporter gene operably linked downstream thereof, (2) measuring the activity of the reporter gene, and (3) selecting a candidate substance that increases or decreases the reporter activity in step (2) compared to a control. [18] [16] or [17] in the control of cell proliferation or cell death.
Use of a compound obtainable by the method described in [1]. [19] A therapeutic agent for a disease characterized by cell proliferation or cell death, characterized by containing as a main component a compound obtainable by the method described in [16] or [17]. The present invention relates to a novel polynucleotide encoding a protein having apoptosis-inducing activity. The nucleotide sequence of a human-derived polynucleotide contained in the polynucleotide of the present invention is shown in SEQ ID NO: 1 or SEQ ID NO: 3. The amino acid sequence of the protein encoded by the polynucleotide of the present invention is shown in SEQ ID NO: 2 or SEQ ID NO: 4. The polynucleotide of the present invention is not particularly limited in its form as long as it can encode the protein of the present invention, and includes cDNA, genomic DNA, chemically synthesized DNA, RNA, and the like. Furthermore, polynucleotides having any nucleotide sequence based on the degeneracy of the genetic code are included, as long as they can encode the protein of the present invention. Furthermore, the polynucleotide of the present invention can be fused with a polynucleotide encoding another protein or oligopeptide. The polynucleotide encoding the protein of the present invention can be isolated by conventional methods, such as hybridization using the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3 or a portion thereof as a probe, or PCR using primers designed based on the information on these nucleotide sequences, as described above. The protein of the present invention consisting of the amino acid sequence shown in SEQ ID NO: 2 exhibits apoptosis-inducing activity in mammalian cells. On the other hand, the protein consisting of the amino acid sequence shown in SEQ ID NO: 4 does not exhibit apoptosis-inducing activity in mammalian cells. The amino acid sequence shown in SEQ ID NO: 4 contains a caspase cleavage domain at positions 300-303 from the N-terminus.
The amino acid sequence shown in SEQ ID NO: 2 corresponds to the amino acid sequence of the protein produced when digested with this caspase digestion domain. Therefore, in the present invention, proteins with apoptosis-inducing activity are called activated. On the other hand, proteins that produce activated proteins through some process, such as the protein consisting of the amino acid sequence of SEQ ID NO: 4,
The present invention also includes proteins that do not themselves exhibit apoptosis-inducing activity. Such proteins are referred to as inactive proteins in the present invention. In the present invention, an inactive protein can be defined as a protein that can generate an active protein by a protease such as caspase or by chemical treatment. The caspase digestion domain found in the amino acid sequence of SEQ ID NO: 4 consists of the amino acid sequence DEAD. Caspas that recognize this amino acid sequence
In humans, Caspase 3 and Caspase 7 are caspases belonging to group 3, and within the caspase family, they constitute the downstream of the apoptosis execution cascade. Therefore, the activated protein of the present invention can be said to be involved in an important step downstream of the apoptosis execution cascade, which is triggered by various causes. Therefore, it is believed that apoptosis can be regulated by regulating the production of the protein of the present invention or the function of this protein. Specifically, the present invention relates to substances capable of regulating apoptosis and a screening method for the same. Apoptosis can be regulated by substances capable of regulating the production of the protein of the present invention or the function of this protein. Such substances can be isolated by the screening method described below. More specifically, inhibiting the production of the activated protein in vivo can effectively suppress the progression of apoptosis. Alternatively, apoptosis can also be suppressed by inhibiting the function of the activated protein. Expression of the protein can be inhibited using antisense nucleic acid drugs or, after identifying its transcriptional regulatory region, by using decoy nucleic acids. To inhibit the function of the activated protein itself, it is effective to change the conformation of the active site by administering a compound that binds to this protein or to prevent the binding of the activated protein to its target compound. Alternatively, the function of the protein of the present invention can be reduced by a dominant-negative effect. Conversely, stimulating the production or activity of the protein in specific cells can induce apoptosis in those cells. For example, if the production of the protein can be specifically induced in cancer cells, cancer can be safely treated. Specifically, treatment based on the present invention is possible by inducing the expression of a gene encoding the apoptosis-related factor of the present invention contained in cancer cells, or by introducing a gene encoding the activated protein into cancer cells together with a promoter that is specifically expressed in cancer cells. Alternatively, a protein useful for cancer treatment can be obtained by modifying the caspase digestion domain in the amino acid sequence of the inactive protein of the present invention to an amino acid sequence that is specifically recognized by a protease highly expressed in cancer cells. Such a protein can be used as a safe pharmaceutical that produces the activated protein only in cancer cells. Furthermore, the protein of the present invention can be used to identify new factors involved in apoptosis mediated by this protein. For example, Death Effector
Proteins with the DED domain are known to be involved in the Fas-associated domain (DED).
This occurs through binding between the respective DEDs, such as the binding between earth domain protein (FADD) and caspase 8.
By using the DED of the protein of the present invention, a factor having a novel DED that binds to it can be obtained. Specifically, two yeast strains using the DED of the protein of the present invention as bait can be obtained.
- Hybrid method (A novel genetic system to d
ect protein-protein interactions. F
fields S. and Song O. (1989) Nature 340:
245-246), a novel factor having a DED that binds to the DED of the protein of the present invention can be obtained from a cDNA library derived from mammalian cells or tissues. Furthermore, for example, the inactive protein of the present invention can be used to identify the Caspas
Specifically, a labeled inactive protein is used as a substrate, and reacted with a protein solution containing activated caspase. The reaction solution is subjected to SDS-PAGE or the like to determine the transition from the inactive protein of the present invention to the activated form, thereby obtaining novel caspase that recognizes the caspase-e digestion domain.
Since the protein of the present invention is a novel protein, the caspase that digests it may be a novel caspase other than caspase 3 or 7.
Alternatively, if the activated protein of the present invention cooperates with other factors to bring about apoptosis, such factors can be isolated using the activated protein of the present invention. Such factors are thought to be proteins such as FADD and caspase 8, which play an important role in inducing the action of apoptosis signals mediated by CD95 (Fas).
Novel proteins having the above function and involved in apoptosis signaling can be isolated by measuring apoptosis after co-expression with the activated protein of the present invention in mammalian cells such as HEK293T cells. The proteins of the present invention can be prepared as recombinant proteins or natural proteins. Recombinant proteins can be prepared, for example, by introducing a vector into which DNA encoding the protein of the present invention has been inserted into an appropriate host cell, as described below, and purifying the protein expressed in the transformant. Alternatively, recombinant proteins can be prepared by in vitro translation (e.g., "On the fidelity of
f mRNA translation in the nuclease-t
Reated rabbit reticulocyte lysate sy
stem. Dasso, M. C. , Jackson, R. J. (1989) Nu
The protein of the present invention can also be prepared by, for example, an affinity column to which an antibody against the protein of the present invention is bound (see "Current Protocols in Molecular Biology" in Molecular Biology, Vol. 17, No. 17, pp. 3129-3144). On the other hand, the native protein can be prepared by, for example, an affinity column to which an antibody against the protein of the present invention is bound (see "Current Protocols in Molecular Biology, Vol. 17, No. 17, pp. 3129-3144" in Molecular Biology, Vol. 17, No. 17, pp. 3129-3144).
regular Biology edit. Ausubel et al. (
1987) Publish. Jhon Wily & Sons Section
n 16.1-16.19). The antibody used for affinity purification may be a polyclonal antibody or a monoclonal antibody. The present invention also includes polynucleotides encoding proteins functionally equivalent to the protein consisting of the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4. Here, "functionally equivalent" means that if the target protein has apoptosis-inducing activity, that protein can be said to be functionally equivalent to the activated protein of the present invention. Alternatively, a protein that can generate an activated protein with apoptosis-inducing activity through some process is functionally equivalent to the inactive protein of the present invention. The process for generating an active protein from an inactive protein is not limited. For example, in the case of a protein consisting of the amino acid sequence shown in SEQ ID NO: 4, digestion at the caspase digestion domain is necessary to generate the active protein. By replacing this region with the recognition sequence of another caspase or even a protease other than caspase, an inactive protein can be obtained that generates an active protein through the action of the corresponding enzyme. The apoptosis-inducing activity is
For example, as described in the Examples, the protein can be confirmed by expressing a gene encoding the protein in mammalian cells and observing the apoptosis of the cells. Apoptosis can be confirmed using morphological changes in the cells or cleavage of chromosomal DNA as indicators. Those skilled in the art can identify proteins functionally equivalent to the proteins identified in the Examples by, for example, methods of introducing mutations into the amino acid sequence of a protein (e.g., site-directed mutagenesis (Current Protocols in Molecules)).
ular Biology edit. Ausubel et al. (198
7) Publish. Jhon Wily & Sons Section 8
.1-8.5)). Such proteins may also be produced by amino acid mutations in nature. The present invention includes any protein having one or more amino acid substitutions in its amino acid sequence (SEQ ID NO: 2 or 4), as long as it has a function equivalent to that of the protein identified in this example.
Proteins that differ by deletion, insertion, and/or addition are also included. There is no limitation on the number or sites of amino acid mutations in a protein, as long as its function is maintained. The number of mutations is typically within 10% of the total amino acids, preferably within 5% of the total amino acids, and more preferably within 1% of the total amino acids.
From the viewpoint of maintaining the function of the protein, the substituted amino acid is preferably an amino acid having properties similar to those of the amino acid before substitution. For example, Ala, Val, Le
Since u, Ile, Pro, Met, Phe, and Trp are all classified as nonpolar amino acids, they are thought to have similar properties.
Ly, Ser, Thr, Cys, Tyr, Asn, and Gln.
Acidic amino acids include Asp and Glu. Basic amino acids include Lys, Arg, and His. Proteins functionally equivalent to the proteins identified in this example can also be isolated using hybridization techniques or gene amplification techniques well known to those skilled in the art. That is, those skilled in the art will be able to easily identify and isolate proteins functionally equivalent to the proteins identified in this example using hybridization techniques (C
Current Protocols in Molecular Biolog
y edit. Ausubel et al. (1987) Publish. J
It is common practice to isolate a polynucleotide highly homologous to the base sequence (SEQ ID NO: 1 or 3) encoding the protein identified in this example, or a portion thereof, using the DNA sequence encoding the protein identified in this example (Genome Research Letters, Vol. 1, No. 1, pp. 111-114, 1999) of the International Standard for Protein Analysis (IAS 102, pp. 111-114, 1999) (Wiley & Sons, 1999, Section 6.3-6.4), and obtain a functionally equivalent protein from this polynucleotide. The present invention also includes proteins encoded by polynucleotides that hybridize with polynucleotides encoding these proteins, so long as they have a function equivalent to that of the protein identified in this example. Organisms from which functionally equivalent proteins can be isolated include, but are not limited to, vertebrates such as humans, mice, rats, rabbits, pigs, and cows. Genes originating from the same gene in molecular evolution as the gene encoding the apoptosis-related factor of the present invention can be isolated from these animals. In the present invention, "originating from the same gene in molecular evolution" means that the protein can be identified by analysis of its DNA base sequence, physiological role, etc.
The term "apoptosis-related factor" refers to a gene that is reasonably considered to have evolved from the same gene as the apoptosis-related factor of the present invention in humans in terms of molecular evolution.
The base sequences of the two proteins maintain a high degree of homology. Stringent hybridization conditions for isolating DNA encoding functionally equivalent proteins are usually set at 1x SS as washing conditions.
The more stringent conditions are "0.5
Stringent hybridization conditions are typically about "0.1x SSC, 0.1% SDS, 42°C," and even more stringent conditions are about "0.1x SSC, 0.1% SDS, 65°C." The more stringent the hybridization conditions, the more likely it is that DNA having a higher homology to the probe sequence will be isolated. However, the above-mentioned combinations of SSC, SDS, and temperature conditions are merely examples, and those skilled in the art will be able to achieve similar stringency to the above by appropriately combining the above or other factors that determine hybridization stringency (e.g., probe concentration, probe length, hybridization reaction time, etc.). Proteins isolated using such hybridization techniques generally have a high homology in their amino acid sequence or the nucleotide sequence encoding said protein, compared to the protein of the present invention set forth in SEQ ID NO: 2 or 4. High homology refers to a sequence identity of at least 60% or more, preferably 70% or more, and more preferably 80% or more (e.g., 90% or more). Homology can be determined by B
LAST2 search algorithm (Altschul, S.F. et al., 199
7 Gapped BLAST and PSI-BLAST: a new g
energy of protein database search
programs. Nucleic Acids Res. 25:3389-
3402. ) and can be determined using gene amplification techniques (PCR) (Current protocols i
n Molecular Biology edit. Ausubel et
al. (1987) Publish. John Wiley & Sons
It is also possible to use the DNA encoding a protein of the present invention, such as a polynucleotide encoding a protein of the present invention, to design primers based on a portion of the base sequence identified in this example (SEQ ID NO: 1 or 3) using the DNA encoding the protein of the present invention. The DNA encoding a protein of the present invention has a dominant-negative trait relative to the protein of the present invention. The expression of a polynucleotide encoding a protein of the present invention has the function of eliminating or reducing the activity of an endogenous wild-type protein inherent in a cell.
For example, if the portion of the apoptosis-related factor of the present invention corresponding to the DEAD sequence is modified to an amino acid sequence that is not cleaved by caspase, it can be made inactive and will not produce an active protein. If a gene encoding such an amino acid sequence is expressed in cells, the expression of the apoptosis-related factor of the present invention will be suppressed by a dominant-negative effect (competitive inhibition of the inactive form).
Therefore, by introducing and expressing an inactive gene into a patient with the above-mentioned disease using gene transfer techniques such as viral vectors, the progression of apoptosis can be inhibited. The present invention also provides partial peptides of the protein of the present invention. The partial peptides are useful as immunogens for obtaining antibodies against the protein of the present invention. In particular, partial peptides containing an amino acid sequence unique to the protein of the present invention, which has low homology with other proteins, are expected to be immunogens that provide antibodies with high specificity to the protein of the present invention. In addition, partial peptides of the apoptosis-related factors of the present invention or their inactive proteins include partial peptides having, for example, DED. DED is a highly conserved amino acid sequence found among several apoptosis-related factors. The 78 amino acid sequence, which corresponds to positions 26-103 of the protein of the present invention (common to both the active and inactive forms),
The amino acid residues correspond to the DED. Since the DED is known to be a region responsible for signal transduction in the apoptosis execution cascade, this region in the protein of the present invention is presumed to play an important role in maintaining its activity. The nuclear localization signal (NLS) domain found in the protein of the present invention is also considered to be an important site supporting its activity. Therefore, partial peptides containing this region are useful as targets for screening compounds that modify the activity of this protein. The partial peptides of the present invention consist of an amino acid sequence of at least 7 amino acids, preferably 9 or more amino acids, more preferably 12 or more amino acids, and more preferably 15 or more amino acids. The partial peptides of the present invention are produced, for example, by genetic engineering techniques, known peptide synthesis methods, or by cleaving the protein of the present invention with an appropriate peptidase. The present invention also provides an expression vector containing any of the above DNAs. Furthermore, the present invention relates to a transformant harboring the DNA or any of the above expression vectors, as well as a method for producing an apoptosis-related factor or a partial peptide thereof, which comprises culturing the transformant and isolating the protein of the present invention from the culture. The present invention also provides an apoptosis-related factor or a partial peptide thereof produced by the above method. When producing polypeptides using recombinant DNA techniques, the type and degree of glycosylation of the target polypeptide may vary depending on the type of host cell.
It is well known to those skilled in the art that in the so-called secretory production method of polypeptides, the terminal (N-terminal and/or C-terminal) amino acid sequence of a precursor polypeptide expressed in a host cell is processed by a signal peptidase or the like to obtain polypeptides with various terminal amino acid sequences. Therefore, it is easily understood by those skilled in the art that such polypeptides are also included in the scope of the protein of the present invention. The following examples only show examples of constructing vectors that function as expression vectors in mammalian cells. However, the DNA encoding the protein of the present invention
As a result, based on these disclosures, it will be easy for a person skilled in the art to construct an expression vector that, when introduced into fungi such as yeast, or into a prokaryotic host cell, will cause the host to express and produce the protein of the present invention. Therefore, the present invention also encompasses expression vectors constructed by methods known in the art based on the DNA sequence of the present invention. Microbial cells that can be used to express a polynucleotide encoding the protein of the present invention include, for example, prokaryotic bacteria [Escherichia coli].
Examples of suitable microorganisms include bacteria belonging to the genus Escherichia and baker's yeast. Examples of suitable microorganisms include bacteria belonging to the genus Escherichia, such as E. coli HB101 (ATCC 33694), E. coli HB101-16 (FERM BP-1872), E. coli MM294 (ATCC 31446), and E. coli DH1 (ATCC 33849), as well as eukaryotic yeasts such as Saccharomyces cerevisiae. Examples of suitable microorganisms include bacteria belonging to the genus Escherichia, such as E. coli HB101 (ATCC 33694), E. coli HB101-16 (FERM BP-1872), E. coli MM294 (ATCC 31446), and E. coli DH1 (ATCC 33849), as well as baker's yeasts such as S. Examples of mammalian cells include human embryonic kidney-derived HEK293 cells and mouse L929 cells.
Examples of host cells include E. coli cells and Chinese hamster ovary (CHO) cells. When prokaryotic bacteria, particularly E. coli, are used as host cells, expression vectors typically consist of at least a promoter, an initiation codon, a polynucleotide encoding the amino acid sequence of the protein of the present invention, a stop codon, and a self-replicating unit. When eukaryotic cells, such as yeast or mammalian cells, are used as host cells, expression vectors preferably consist of at least a promoter, an initiation codon, a polynucleotide encoding the amino acid sequence of the protein of the present invention, and a stop codon. Furthermore, enhancer sequences, 5'- and 3'-noncoding regions of the protein of the present invention, a polyadenylation site, and a self-replicating unit may also be inserted. The self-replicating unit preferably contains a selectable marker for transformants (e.g., ampicillin resistance). In the case of expression vectors using bacteria as hosts, the term "promoter" refers to promoters, operators, and Shine-
This refers to a promoter-operator region consisting of a Dalgarno (SD) sequence (e.g., AAGG, etc.). Examples of such promoters include conventional promoter-operator regions (e.g., lactose operon, PL promoter, trp promoter, etc.). An example of a promoter used in an expression vector using a yeast host is the pho5 promoter. Furthermore,
For the purpose of facilitating purification, a basic amino acid having affinity for metal ion chelates can be added to either end of the protein of the present invention. When adding a basic amino acid, an oligopeptide can be added to any end of the target gene by performing PCR using a primer to which a continuous base sequence encoding the desired amino acid has been added to the 5' end. As the basic amino acid, histidine, lysine, arginine, or the like can be used. Examples of promoters used in expression vectors in mammalian cells include:
HTLV-LTR promoter, SV40 early and late promoters, CMV
Examples of suitable initiation codons include the mouse metallothionein I (MMT) promoter and the mouse metallothionein I (MMT) promoter. A preferred initiation codon is the methionine codon (ATG). A polynucleotide encoding the amino acid sequence of the protein of the present invention may be, for example,
The protein of the present invention can be obtained by partial or total synthesis using a DNA synthesizer. Alternatively, it can be obtained from a human cDNA library using probes and primers designed based on the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 3. Furthermore, genomic DNA encoding the protein of the present invention can be prepared by treating the genomic DNA using conventional methods (e.g., digestion with restriction enzymes, dephosphorylation with bacterial alkaline phosphatase, phosphorylation with T4 polynucleotide kinase, and ligation with T4 DNA ligase).
Furthermore, the genomic DNA obtained in this manner can be used to clarify the transcription start site of the gene of the present invention in the genome and identify expression control regions located further upstream. Control regions such as promoters and enhancers that control the expression of the gene encoding the protein of the present invention are useful as target regions for detecting abnormal expression of the protein of the present invention. Alternatively, expression control can be achieved by decoy nucleic acid drugs that target these regions. Of the proteins of the present invention, the active form of the protein is lethal to at least human cells. Therefore, in mammalian cells, it is desirable to express the active form of the protein in combination with an inducible promoter. Such promoters include known temperature-sensitive promoters and drug-sensitive promoters. These promoters are activated only by applying specific culture conditions. Therefore, expression can be induced when the cells have fully grown. The procedures required to carry out the present invention, such as DNA cloning, construction of each plasmid, transfection of the host, cultivation of the transformant, and recovery of the protein from the culture, can be carried out by methods known to those skilled in the art or methods described in the literature [Molecular C
loning 2nd. edition, T. Maniatis et. al,
Cold Springs Harbor Laboratory (1989)
, DNA Cloning, DM. Glover, IRL PRESS (198
5) and others]. The host cells of the present invention also include cells of interest used for functional analysis of the apoptosis-related factor of the present invention and for screening of function inhibitors or function promoters using this protein. Vector introduction into host cells can be carried out, for example, by calcium phosphate precipitation method, electroporation (Current protocols in
Molecular Biology edit. Ausubel et al.
．． (1987) Publish. John Wiley & Sons. Sec
tion 9.1-9.9), Lipofectamine method (GIBCO-BRL)
This can be done by methods such as microinjection. The protein of the present invention can be prepared from a transformant by using protein separation and purification methods known to those skilled in the art. The present invention also provides a polynucleotide comprising at least 15 nucleotides complementary to a polynucleotide consisting of the base sequence set forth in SEQ ID NO: 1 or 3, or its complementary strand. Here, the term "complementary strand" refers to the other strand of a double-stranded polynucleotide consisting of A:T (A:U) and G:C base pairs. In addition,
The term "complementary" does not necessarily mean a completely complementary sequence in a region of at least 15 consecutive nucleotides, but may mean a sequence with at least 70%, preferably at least 80%, more preferably 90%, and even more preferably 95% or more homology in base sequence. The algorithm for determining homology may be one described herein. Such polynucleotides may be DNA or RNA encoding the protein of the present invention.
The polynucleotides of the present invention can be used as probes for detecting and isolating the polynucleotides of the present invention, and as primers for amplifying the polynucleotides of the present invention. When used as primers, they typically have a chain length of 15 bp to 100 bp, preferably 15 bp to 35 bp. When used as probes, polynucleotides having at least a partial or complete sequence of the polynucleotides of the present invention and a chain length of at least 15 bp are used. When used as primers, the 3'-region must be complementary, but a restriction enzyme recognition sequence or tag can be added to the 5'-region. The polynucleotides of the present invention can be used to test and diagnose abnormalities in the proteins of the present invention. For example, expression abnormalities can be tested by Northern hybridization or RT-PCR using the polynucleotides of the present invention as probes or primers, or the polynucleotides encoding the proteins of the present invention or their expression control regions can be amplified by genomic DNA-PCR or RT-PCR using the polynucleotides of the present invention as primers, followed by RF amplification.
Sequence abnormalities can be examined and diagnosed by methods such as LP analysis, SSCP, and sequencing. In the present invention, "expression" includes transcription and/or translation. Expression analysis using the polynucleotide of the present invention allows examination and diagnosis of gene expression at the transcription level. Furthermore, using an antibody against the protein of the present invention described below, gene expression at the translation level can be examined and diagnosed. Furthermore, "a polynucleotide comprising at least 15 nucleotides complementary to a polynucleotide consisting of the base sequence set forth in SEQ ID NO: 1 or 3, or its complementary strand" includes an antisense polynucleotide for suppressing the expression of the protein of the present invention. To induce an antisense effect, the antisense polynucleotide has a chain length of at least 15 bp or more, preferably 100 bp or more, more preferably 500 bp or more, and is usually within 3000 bp, preferably 2000 bp.
The chain length is within 1 p. Such antisense polynucleotides may be applied to gene therapy for diseases caused by abnormalities (functional abnormalities or expression abnormalities) of the protein of the present invention. Specifically, apoptosis is an important step in tissue damage caused by ischemic diseases. Therefore, if the expression of the protein of the present invention can be inhibited in tissues with impaired blood flow, tissue damage associated with ischemic diseases can be alleviated. The antisense DNA can be prepared, for example, by the phosphorothioate method (Stein, 1988 Ph.D.) based on the sequence information of the polynucleotide set forth in SEQ ID NO: 1 or 3.
ysicochemical properties of phosphor
othioate oligodeoxynucleotides. Nucle
ic Acids Res 16, 3209-21 (1988)). When used in gene therapy, the polynucleotide or antisense polynucleotide of the present invention is administered to a patient by ex vivo or in vivo methods using, for example, viral vectors such as retroviral vectors, adenoviral vectors, and adeno-associated viral vectors, or non-viral vectors such as liposomes. The present invention also provides antibodies that bind to the proteins of the present invention. The form of the antibodies of the present invention is not particularly limited, and includes polyclonal antibodies, monoclonal antibodies, and portions thereof that have antigen-binding ability. Antibodies of all classes are also included. Furthermore, the antibodies of the present invention also include specialized antibodies such as humanized antibodies. In the case of polyclonal antibodies, the antibodies of the present invention can be obtained by immunizing rabbits with the proteins or partial peptides of the present invention (Current
protocols in Molecular Biology edit.
Ausubel et al. (1987) Publish. John Will
On the other hand, in the case of monoclonal antibodies, they can be obtained from hybridoma cells obtained by immunizing mice with the protein or partial peptide of the present invention and fusing spleen cells with myeloma cells (Current protocols in Molecules).
lar Biology edit. Ausubel et al. (1987
) Publish. John Wiley & Sons. Section 1
1.4 to 11.11). Antibodies that bind to the proteins of the present invention can be used not only to purify the proteins of the present invention, but also to test and diagnose abnormalities in expression or structure of these proteins. Specifically, for example, proteins can be extracted from tissues, blood, or cells, and the presence or absence of abnormalities in expression or structure can be tested and diagnosed through detection of the proteins of the present invention by methods such as Western blotting, immunoprecipitation, and ELISA. Antibodies that bind to the proteins of the present invention can also be used for purposes such as treating diseases related to the proteins of the present invention. When antibodies are used for the purpose of treating patients,
Human antibodies or humanized antibodies are preferred because they are less immunogenic. Human antibodies are antibodies derived from mice whose immune systems have been replaced with those of humans (e.g., "functional transgenic mice").
nsplant of mega base human immunoglo
bulin loci recapitulates human antib
ody response in mice, Mendez, M. J. et a
(1997) Nat. Genet. 15:146-156). Humanized antibodies can also be prepared by genetic recombination using the hypervariable regions of monoclonal antibodies (Meth.
[0003] The present invention also provides a method for screening for compounds that modulate the activity of the protein of the present invention. Since the protein of the present invention induces cell death, compounds that inhibit the activity of the product of the gene are useful as therapeutic agents or reagents for inhibiting cell death. This screening method comprises the following steps: (1) contacting a candidate compound with cells that express the protein of the present invention; and (2) culturing the cells under conditions that induce apoptosis and selecting a candidate compound that inhibits or promotes apoptosis. Cells that express the protein of the present invention include cells that express either an active or inactive form of the protein. Cells that express the active form are directly linked to apoptosis, so efficient screening cannot be expected using this method alone. Therefore, an expression plasmid in which the gene (in an active form) is linked downstream of a controllable promoter sequence can be used. An example of a controllable promoter is the metallothionein promoter. A plasmid expressing a gene encoding the active form of the protein of the present invention under the control of such a promoter is transformed into animal cells such as HEK293. The transformants thus obtained induce cell death under conditions in which the promoter is activated. For example, the transformants are seeded onto a 96-well multiplate, and a candidate compound to be screened is added to each well, after which the promoter is activated.
As a result, by selecting candidate compounds that suppress (or promote) apoptosis compared with the control, compounds that regulate the action of the protein of the present invention can be selected. This method can be used not only in animal cells but also in any host that induces cell death in a similar system, regardless of whether it is a eukaryote or a prokaryote. On the other hand, when an inactive protein is expressed, the production of the active protein is induced by placing the cells under conditions that produce the active form. Specifically, for example, when an inactive form becomes active by cleavage of the caspase recognition sequence, the necessary caspase
Alternatively, an inactive protein can be expressed in cells that express casp.
In the case of a mutant in which the amino acid sequence has been modified to be cleaved by a protease other than ATPase, cells that produce the required protease can be used. Such a protease can be artificially introduced, or an enzyme that the cell originally possesses can be used. In either case, since the protease activity required to produce the active form is directly linked to apoptosis, it is desirable that its production be controllable. The screening of the present invention is carried out using apoptosis as an indicator. Methods for detecting apoptosis are known. Specifically, methods for detecting apoptosis using changes in cell morphology or DNA fragmentation as indicators are common. Morphological changes in cells due to apoptosis are observed as changes in the stained image of Giemsa staining or fluorescent staining. Alternatively, DNA fragmentation can be detected using the TUNEL method (Gavriel Yrt)
al, 1992 J Cell Biol, 119:493-501). Furthermore, as a method for detecting the early stage of apoptosis, there is an Annexin V method in which fluorescently labeled Annexin V that binds to structurally altered cell membranes is measured using a flow cytometer (Vermes I, et al.
．． J Immunol Methods. 1995 Jul 17;184(1
): 39-51. Compounds selected by the screening method of the present invention include compounds that regulate the activity of the protein of the present invention through the following systems: (1) inhibit (or promote) the process by which the protein of the present invention is cleaved into an active form; (2) inhibit (or promote) the production of the protein of the present invention itself; (3) inhibit (or promote) the cleavage of the inactive protein of the present invention by activated caspase. Increased expression products of the gene of the present invention within cells may easily promote cell death due to some kind of stimulus. Therefore, compounds that regulate the expression of the gene or promote cell death are expected to be useful as therapeutic agents or reagents for various pathologies (e.g., cerebral infarction, cancer, etc.). Gene expression regulation can be observed using a reporter assay. The present invention relates to a method for screening for substances that regulate apoptosis by a reporter assay using the expression regulatory region of a gene consisting of the nucleotide sequence set forth in SEQ ID NO: 3, which can be obtained according to the present invention. The screening method of the present invention comprises the following steps: (1) contacting a candidate substance with a cell into which a vector containing an expression control region of a gene consisting of the base sequence of SEQ ID NO: 3 and a reporter gene functionally linked downstream of the expression control region has been introduced; (2) measuring the activity of the reporter gene; and (3) selecting a candidate substance that increases or decreases the reporter activity in step (2) compared to a control. The expression control region of the gene consisting of the base sequence of SEQ ID NO: 3 is located in the chromosomal DNA.
A can be cloned from A by a known method. For example, the S1 mapping method is known as a method for identifying the transcription start site (see "Isolation of Transcriptional Regulatory Regions" and "Identification and Purification of Transcriptional Regulatory Factors,""Cell Engineering, Special Issue 8, New Cell Engineering Experimental Protocols," edited by the Department of Cancer Research, Institute of Medical Science, University of Tokyo, Shujunsha, 1993, pp. 362-
Generally, the expression control region DNA of the gene is extracted from a human genomic library by cloning 15 to 100 of the 5' end of the gene.
By screening using a DNA probe of 100 kb or more, preferably 30-50 kb, a gene clone containing an expression control region is cloned. Clones obtained in this manner often contain 10 kb or more of the 5' untranslated region of the gene. Therefore, the 5' end of these clones is shortened or fragmented by exonuclease treatment or the like. By evaluating the expression strength and expression control of a reporter gene using a sequence containing the shortened expression control region, the minimum required unit for maintaining the activity of the expression control region can be obtained (deletion study). In addition, a program that predicts gene expression control regions using the Neural Network is known (http://www.fruitfly.org
/seq_tools/promoter.html, Reese, M. G.,
et al, “Large Scale Sequencing Specif
ic Neural Networks for Promoter and
Splice Site Recognition”Biocomputing
:Proceedings of the 1996 Pacific Sym
posium, edited by Lawrence Hunter and
Terri E. Klein, World Scientific Publ.
isching Co, Singapore, January 2-7, 1996
Alternatively, the minimum unit of activity can be predicted using a program such as Promoter Scan, which searches for transcription factor binding sequences and predicts expression control regions (http://biosci.cbs.umn.edu/softw
are/proscan/promoterscan. htm, Prestri
dge, D. S. 1995, Prediction of Pol II Pr
omoter Sequence using Transcription
Factor Binding Sites. J. Mol. Biol. 249:
923-932). In addition, deletion s
A reporter gene can also be functionally linked downstream of the thus isolated control region gene to prepare an expression plasmid, which is then introduced into suitable cells. In the present invention, functional linkage means that the two are linked so that transcription of the reporter gene is initiated by activation of the expression control region. Any gene can be used as the reporter gene, as long as it encodes a protein that allows the activation of the expression control region to be observed as gene expression. Specifically, for example, luciferase, β-galactosidase, GFP (
Genes such as GFP (Green Fluorescent Protein) are commonly used as reporter genes. Cells into which vectors are introduced can be, for example, animal cells lacking the gene. The promoter of the cell line thus obtained is activated under conditions that induce cell death (apoptosis), generating a signal from the reporter gene. The cells obtained are seeded into a 96-well multiplate, and culture is initiated under conditions that induce apoptosis. Compounds that suppress or promote the expression of the gene's expression product can be easily selected by adding the compound to be screened to each well. For example, when GFP is used as the reporter gene, selection can be performed by comparing the luminescence intensity of GFP with and without the addition of a drug. Comparison refers to a luminescence intensity ratio of 2-fold or less, preferably 5-fold or less, and more preferably 10-fold or less, or less than 1/10.
This method can be used not only in animal cells but also in any host, eukaryotic or prokaryotic, that induces cell death in a similar system. Candidate substances for use in the screening of the present invention include, but are not limited to, cell extracts, expression products of gene libraries, synthetic low-molecular-weight compounds, synthetic peptides, and natural compounds. Compounds isolated by this screening method are candidates for compounds (agonists and antagonists) that promote or inhibit the activity or expression of the protein of the present invention. They are also candidates for compounds that inhibit the interaction between the protein of the present invention and a molecule that interacts with it in vivo. These compounds may be used as pharmaceuticals for the prevention and treatment of diseases associated with the protein of the present invention. Furthermore, the present invention relates to pharmaceutical uses of substances obtainable by the screening of the present invention. Specifically, the present invention relates to the use of compounds obtainable by the screening method in controlling cell proliferation or cell death, which contain the compounds as the main component. The present invention also relates to therapeutic agents for diseases characterized by cell proliferation or cell death, which contain these compounds as the main component. In the present invention, diseases characterized by cell proliferation or cell death refer to diseases caused by abnormal cell proliferation or cell death. For example, diseases caused by abnormal cell proliferation include cancer caused by proliferation of malignant tumor cells. Diseases caused by cell death include:
This can indicate brain tissue damage caused by cerebral ischemia. Compounds of the present invention that inhibit the function or production of apoptosis-related factors can be used to control the following types of cell death: (1) cell death caused by ischemia; (2) cell death in chronic viral diseases; cell death in chronic diseases caused by other pathogenic microorganisms; (3) inflammation caused by autoimmune reactions and the resulting cell death; and (4) diseases caused by cell death and associated degeneration of unknown cause (such as Alzheimer's disease). These types of cell death can be caused by, for example, the following diseases. Therefore, compounds selected by the screening method of the present invention can be used to treat the following diseases. Cerebrovascular dementia, multiple microinfarctions, cerebral thrombosis, cerebral infarction, cerebral hemorrhage, neurodegenerative diseases (Alzheimer's disease, etc.), myocardial infarction, myocarditis, viral hepatitis, alcoholic hepatitis, liver cirrhosis, insulin-dependent diabetes mellitus (due to immunoreactive cell death of pancreatic islet cells), ischemic enteritis, cell death associated with systemic or localized autoimmune diseases; systemic generalized erythema, dermatomyositis, rheumatoid arthritis, AIDS. The proteins, nucleotides, antibodies, and substances isolated by the above screening of the present invention are useful for regulating apoptosis. When used as pharmaceuticals, they can be used as pharmaceuticals themselves, or they can be formulated using known pharmaceutical methods. For example, they can be formulated by appropriately combining them with pharmacologically acceptable carriers or vehicles, specifically, sterile water, physiological saline, vegetable oil, emulsifiers, suspending agents, etc. Administration to patients can be performed by methods known to those skilled in the art, such as intra-arterial injection, intravenous injection, or subcutaneous injection. The dosage varies depending on the patient's weight, age, administration method, etc., but a person skilled in the art can appropriately select an appropriate dosage.
If the DNA can be encoded by NA, it is possible to incorporate the DNA into a gene therapy vector and perform gene therapy. The dosage and administration method vary depending on the patient's weight, age, symptoms, etc., but those skilled in the art can select an appropriate dosage and administration method. All prior art documents cited in this specification are incorporated herein by reference. Best Mode for Carrying Out the Invention The present invention will be explained in more detail below using examples, but the present invention is not limited to these examples. [Example 1] Preparation of a cDNA Library by Oligo-Capping Method A cDNA library was prepared using NT-2 neural progenitor cells (purchased from Stratagene), which are teratocarcinoma cells derived from human fetal testis and can be differentiated into neurons by retinoic acid treatment. First, NT-2 neural progenitor cells were prepared according to the attached manual.
2 cells were cultured without induction with retinoic acid, and the cultured cells were collected and analyzed by the method described in the literature (J.S.
ambrook, E. F. Fritsch & T. Maniatis, Mol
ecular Cloning Second edition, Cold S
The mRNA was extracted by the method described in Spring Harbor Laboratory Press, 1989.
(A) ⁺ RNA was purified. Poly(A) ⁺ RNA was purified by the oligo-capping method [M. Maruyama and
d S. Sugano, Gene, 138:171-174 (1994)].
caucgagu cggccuuguu ggccuacugg/SEQ ID NO:
5) and oligo dT primer (gcggctgaag acggcctat
g tggcctttttt tttttttttttt tt/SEQ ID NO: 6) was used to elucidate the sequence of the reference [Suzuki and Sugano, Protein, Nucleic Acid, Enzyme, 41: 197-201 (1996
), Y. Suzuki et al. , Gene, 200:149-156(1
997)], BAP (Bacterial Alkaline
Phosphatase treatment, TAP (Tobacco Acid Phosphate)
sphatase treatment, RNA ligation, first-strand cDNA synthesis and RNase
A was removed. Then, 5' (agcatcgagt cggccttgt
t g/SEQ ID NO: 7) and 3' (gcggctgaag acggcctatg
PCR (polymerase chain reaction) was performed using PCR primers of
The cDNA was converted into double-stranded cDNA by chain reaction and digested with SfiI.
A was cloned by determining the direction of A to prepare a cDNA library. After excluding clones with inserted cDNA sizes of 1 kb or less from the plasmid DNA of the clones obtained, the base sequences of the 5' and 3' ends of the cDNA were determined using DNA sequencing reagents (Dye Terminator Cycle Sequencing).
ng FS Ready Reaction Kit, dRhodamine
Terminator Cycle Sequencing FS Ready
Reaction Kit or BigDye Terminator Cy
cle Sequencing FS Ready Reaction Kit
After sequencing reaction using a DNA sequencer (ABI PRISM 377, PE Biosystems) according to the manual,
DNA sequences were analyzed using a DNA sequencing system (manufactured by Sigma-Aldrich Systems). The full-length percentage of the 5'-end of the cDNA in the library prepared by the oligo-capping method was determined using the following method. For all clones whose 5'-end sequences matched those of known human mRNAs in public databases, clones whose 5'-ends were longer than those of the known mRNA sequences in the public database or clones whose 5'-ends were shorter but contained a translation initiation codon were judged to be "full-length." Those lacking a translation initiation codon were judged to be "non-full-length." The full-length percentage of the 5'-end of the cDNA clones [number of full-length clones/(number of full-length clones + number of non-full-length clones)] was determined by comparing it with known human mRNA. As a result, the full-length percentage of this library (NT2RM1) was estimated to be 69%, indicating a very high full-length percentage of the 5'-end sequences. Next, from the clones in the library created by the oligo-capping method, the predicted amino acid sequences from all ATG codons in the 5'-terminal sequence were analyzed using the protein localization prediction program "PSORT" developed by Nakai and Kanehisa to analyze the presence or absence of sequences predicted to be signal peptides characteristic of the amino termini of many secretory proteins. This allowed specific selection of clones predicted to have signal sequences (highly likely to be secretory or membrane proteins). Furthermore, for the clones selected in this way, the full-length cDNA sequences and predicted amino acid sequences were determined. The base sequences were determined by combining the following three methods, and the base sequences determined by each method were completely overlapped to determine the final confirmed base sequence. (1) Long-read sequencing from both ends of the cDNA insert fragment using a Licor DNA sequencer (the DNA base sequence was analyzed using the Licor sequencer after the sequencing reaction according to the Licor sequencer manual (sold by Aloka Co., Ltd.)), (2) Primer Island sequencing using AT2 transposon in vitro transposition.
Nested sequences by the method [S. E. Devine and J. D. Bo
eke, Nucleic Acids Res. , 22:3765-3772,
(1994)] (clones were obtained using a kit manufactured by PE Biosystems according to the manual, and then sequencing reactions were carried out using DNA sequencing reagents manufactured by PE Biosystems according to the manual, and the resulting clones were analyzed using ABI PRISM 3.
(3) Primer walking by the dideoxy terminator method using custom synthesized DNA primers (using custom synthesized DNA primers PE B
The sequencing reaction was carried out using DNA sequencing reagents manufactured by Iosystems according to the manual, and the DNA base sequence was analyzed using ABI PRISM 377. The obtained sequence was analyzed using the ATGpr [A. A. Salamov, T. Nish
ikawa, M. B. Swindells, Bioinformatics, 1
4:384-390 (1998); http://www. hri. co. jp
/atgpr/] and PSORT analysis and GenBank and SwissP
BLAST analysis was performed on rot. PSEC0004 is a clone that was determined to be a full-length cDNA clone based on ATGpr etc. from a human cDNA library created using the oligo-capping method with a high full-length ratio, and predicted by PSORT to be a secretory protein or membrane protein with a signal sequence at the N-terminus. The results are shown below. Clone name: PSEC0004 cDNA size: 1883:C-NT2RM1000558 Number of amino acids constituting the deduced amino acid sequence: 326 Number of ATG counted from the N-terminus: 1 Maximum ATGpr1 value: 0.94 Annotation: 532/852 (62%) similarity to human
man death effector domain-containing
Testicular molecule mRNA [Example 2] Function prediction by homology analysis Some genes involved in apoptosis are Death Effector Dom
It has been reported that they share the same DNA sequence (DED) (Chinnaiyan
AM, et al 1995 Cell 81:505-512. Muzio
M et al, 1996 Cell 85:817-827. Femand
es-Alnemri 1996 Proc. Natl Acad Sci U
SA 93:7464-7469). For example, DEDD has been reported to be a novel molecule containing DED, similar to FADD and caspase 8, which play important roles in the apoptosis signaling mediated by CD95 (Fas) (Al
exander H. Stegh et al. 1998 The EMBO
Journal 17, 20, 5974-5986. Peter, M. E. et
al, 1995 Cell Death Differ. , 2, 163-171
Therefore, the N-terminal 76 residues of DEDD (LYSLHRMFDIVGTHLT
HRDVRVLSFLFLVDVIDDHERGLIRNGRDFLLALER
QGRCDESNFRQVLQLLRIITRHDLL/SEQ ID NO: 9 (DED
Using the NT2RM1000558 gene as a query, a homology analysis (Blast2) was performed on the full-length cDNA clone. As a result, the amino acid sequence encoded by NT2RM1000558 showed approximately 43% homology. DEDD and some genes similarly involved in apoptosis have been reported to have a specific sequence (DEXD) that is cleaved by caspase. Caspase
pase is considered to be an effector protein of apoptosis. Therefore, using this sequence (DEXD) as a query, a homology analysis (Blast2) was performed on the full-length cDNA clone. As a result, NT2RM1000558
A DEAD sequence was found near the C-terminus of the amino acid sequence encoded by the gene. From the above results, it was predicted that the NT2RM1000558 clone is a gene involved in apoptosis. Furthermore, when the sequences of NT2RM1000558 and DEDD were compared, it was revealed that NT2RM1000558 has a sequence showing high homology to the NSL domain identified in DEDD, and it was predicted that this gene has a function involved in apoptosis. [Example 4] Distribution of expression in various human tissues The distribution of expression in various tissues of NT2RM1000558, which was predicted to have a function related to apoptosis, was investigated. Primer 1 (TCAGTGTGGATGAG
GCTGACT/SEQ ID NO: 10 (NT2RM1000558:1013-10
33)) and Primer 2 (TGCACCTGTGACAGTGCAGA/
SEQ ID NO: 11 (NT2RM1000558:1399-1380)) was used.
lontech Multiple Tissue cDNA Panels (
Using the DNA fragments (CODE K1420-1, K1426-1) as template DNA, PCR reaction (30 cycles) was carried out according to the manual. The reaction mixture was subjected to agarose gel electrophoresis, and the NT2RM100055 of each tissue was identified based on the electrophoresis results.
As a result, the expression of NT2RM1000558 was not specific to various tissues, and was expressed in many tissues (brain, heart, kidney, liver, lung, pancreas, placenta, bone marrow, lymph nodes,
Expression was confirmed in the leukocytes, spleen, thymus, and tonsils. [Example 5] Functional analysis of NT2RM1000558 In the following expression analysis, all cDNAs were cloned using pcDNA3.1vecto.
The NT2 vector inserted into the Dra III restriction enzyme site of pME18SFL3 was used.
The RM1000558 gene was excised by enzymatic digestion with the restriction enzymes EcoRI and NotI.
n) was also digested with the restriction enzymes EcoRI and NotI, and the purified fragment was ligated. The resulting pcDNA3.1 vector, into which the entire NT2RM1000558 gene was inserted, was used as an inactive expression vector. Next, a vector expressing an active protein was constructed as follows. First, the NT2
RM1000558 gene was used as a template.
PCR was performed using a PCR kit (Clontech). The sense primer used for PCR was 90-GGTTCTGAGCTTGTTCC (SEQ ID NO: 12).
consists of a base sequence corresponding to the base sequence of the 5' untranslated region of the NT2RM1000558 gene.
CTCATCCACACTGA-1013 (SEQ ID NO: 13) consists of a base sequence encoding the VSVDEAD amino acid sequence of the protein and a base sequence immediately followed by a base sequence corresponding to a stop codon. After PCR, the PCR product (active form) was cloned into the TA-cloning vector pT7Blue-2T (No.
The NT2RM10 inserted into pT7Blue-2T was ligated to a 5'-nucleotide polynucleotide (manufactured by Vagen).
The activated gene of 00558 was generated using the BigDye terminator cycle sequence with SP6 primer and M13 primer.
kit (PE Biosystems) and PE310 Genetic Ana
The base sequence was determined using a DNA lyzer (PE Biosystems) to identify NT2.
It was confirmed that there was no mutation due to PCR in the active sequence of RM1000558. Furthermore, the active gene of NT2RM1000558 inserted into pT7Blue-2T was cloned into pT7Blue-2T using the restriction enzyme sites EcoRI and X.
The expression vector pcDNA3.1 was then digested with the restriction enzymes EcoRI and XhoI using the restriction enzymes EcoRI and XhoI.
The fragment was digested with XhoI and purified, and ligated to the resulting pcDNA into which the activated gene of NT2RM1000558 had been inserted.
The A3.1 vector was used as an active expression vector. ² x 105 HEK293T cells were seeded per well on a 6-well plate and cultured overnight. 250 μl of Opti-MEM solution was added to 10 μl of Lipofectamine 2000 solution (Gibco BRL), stirred, and left at room temperature for 5 minutes. 4 μg of plasmid DNA (inactive expression vector or active expression vector) mixed with 250 μl of Opti-MEM solution was then added and left at room temperature for another 20 minutes. The resulting Lipofectamine 2000 solution was
The tamine 2000-plasmid DNA mixed solution was added to the cultured cells and cultured for 30 hours to introduce an active or inactive gene. After removing the culture medium, 2 ml of PBS-EDTA solution was added, and the cells were detached from the plate by pipetting and suspended. The cells were then centrifuged at 400 x g for 5 minutes, and the PBS supernatant was removed. 1 ml of PBS was then added to the cells to suspend them, and the cells were then centrifuged at 400 x g for 5 minutes to remove the PBS supernatant.
The cells were centrifuged at 400xg for 5 minutes, and the PBS supernatant was removed. Subsequently, 100 μl of 1% glutaraldehyde solution was added to the cells, and the cells were suspended. The cells were then left at room temperature for 30 minutes to fix the cells. The cells were then centrifuged at 400xg for 5 minutes, and the fixative supernatant was removed. Furthermore, 1 ml of PBS was added to the cells, and the cells were suspended at 400xg.
The cells were centrifuged at 1000 x g for 5 minutes, and the supernatant PBS was removed. The cells were suspended in 20 μl of PBS, and 5 μl of 1 mM Hoechst 332 was added.
A drop of the cell suspension was placed on a slide glass, a cover glass was placed on top, and the nuclear morphology (nuclear fragmentation) of the cells was observed under a fluorescence microscope.
In NT2RM1000558, the NT2RM1000558 gene lacks the DEAD portion and subsequent amino acid residues, and thus nuclear fragmentation due to apoptosis was not observed. However, cells into which NT2RM1000558 activated form (amino acid residue 303), which is thought to be an activated form in which the DEAD portion of the NT2RM1000558 gene is deleted, showed nuclear fragmentation due to apoptosis. In contrast, cells into which the Caspase 3 gene was introduced showed nuclear fragmentation due to apoptosis. The DEAD sequence of the cleavage recognition sequence by activated Caspase present in the NT2RM1000558 gene is likely to be cleaved by Poly(ADP-ribose) polymerase (PA) due to the substrate specificity of the cleavage recognition sequence of the Caspase family.
RP) and DNA fragmentation factor, as well as C
It is presumed that this molecule is cleaved and activated by aspases 3, 6, and 7, and plays a final role in apoptosis execution.
When the protein was synthesized using the ion/translation system, a 36 kd band was confirmed by SDS-PAGE.
It was confirmed that actual protein translation begins from the initiation methionine in the predicted frame. Industrial Applicability : The present invention provides a novel apoptosis-related factor. The apoptosis-related factor of the present invention contains a recognition site for group 2 caspases, which are downstream of the apoptosis execution cascade. Therefore, the apoptosis-related factor of the present invention may be an important protein involved in the final stage of apoptosis. In fact, typical symptoms of apoptosis are observed in cells in which activated molecules of the apoptosis-related factor of the present invention are forcibly expressed. Therefore, compounds that control the action of the apoptosis-related factor of the present invention can be screened using apoptosis as an indicator. Compounds obtainable by screening according to the present invention are useful as drugs for controlling cell proliferation and cell death. For example, compounds that inhibit the action of the apoptosis-related factor of the present invention can be used as drugs to prevent the progression of apoptosis. Because the apoptosis-related factor of the present invention functions in the final stage of the apoptosis execution cascade,
Compounds that inhibit its action effectively suppress apoptosis. The apoptosis-related factor of the present invention is widely distributed in various human tissues, and it is predicted that this factor plays an important role in the various pathological conditions mentioned above in which apoptosis leads to extensive damage. In particular, the apoptosis-related factor of the present invention is isolated from neurons. Drugs for preventing damage: Controlling apoptosis in brain cells associated with cerebral ischemic disease is an important issue in the prevention and treatment of brain tissue damage. The apoptosis-related factor of the present invention is important as a target molecule for the prevention and treatment of brain tissue damage. Conversely, compounds that enhance the action of the apoptosis-related factor of the present invention or stimulate its production are useful as drugs that promote cell death. The apoptosis-related factor of the present invention potently induces apoptosis. Therefore, for example, in cell proliferative diseases such as cancer, the apoptosis-related factor of the present invention itself or compounds that promote its production can be used as drugs to suppress cell proliferation. Although apoptosis is a phenomenon that occurs due to various causes, drugs that act at the final step of the execution cascade can control apoptosis regardless of the cause. Therefore, the compounds of the present invention that can control the action of apoptosis-related factors are expected to achieve control of apoptosis in a wide range of diseases.

[Sequence List]

───────────────────────────────────────────────────────── Continued from the front page (51) Int.Cl. ⁷ Identification Symbol FI C12N 1/21 C12N 1/21 5/10 C12P 21/02 C C12P 21/02 21/08 21/08 G01N 33/15 Z G01N 33/15 33/50 Z 33/50 33/53 33/53 33/577 B 33/577 C12N 5/00 B (81) Designated States EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, I T, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, K E, LS, MW, MZ, SD, SL, SZ, TZ, UG , ZW), UA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ, C A, CH, CN, CR, CU, CZ, DE, DK, DM ,DZ,EE,ES,FI,GB,GD,GE,GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor: Sosuke Miyoshi 1-4-3-602-208, Azuma, Tsukuba City, Ibaraki Prefecture (72) Inventor: Susumu Sato 6-19-11, Kinunodai, Yawara Village, Tsukuba District, Ibaraki Prefecture (Note) This publication is based on the gazette published internationally by the International Bureau of Patents (WIPO). The effect of the international publication of the Japanese patent application (Japanese utility model registration application) related to this publication arises pursuant to Article 184-10, Paragraph 1 of the Patent Act (Article 48-13, Paragraph 2 of the Utility Model Act), and is unrelated to this publication.

Claims (19)

[Claims] [Claim 1] A polynucleotide encoding a protein having apoptosis-inducing activity described in any one of (a) to (d) below: (a) a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 1, (b) a polynucleotide encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2, (c) a polynucleotide encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2 in which one or several amino acids have been substituted, deleted, inserted, and/or added, (d) a polynucleotide that hybridizes under stringent conditions with a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 1, 2. The polynucleotide according to claim 1, which has a homology of 60% or more with the base sequence set forth in SEQ ID NO: 1. [Claim 3] A polynucleotide encoding a precursor of a protein having apoptosis-inducing activity according to any one of (e) to (h) below: (e) a polynucleotide comprising a protein-coding region of the nucleotide sequence set forth in SEQ ID NO: 3, (f) a polynucleotide encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 4, (g) a polynucleotide encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 4 in which one or several amino acids have been substituted, deleted, inserted, and/or added, (h) a polynucleotide that hybridizes under stringent conditions with a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 3, 4. The polynucleotide according to claim 3, which has a homology of 60% or more with the base sequence set forth in SEQ ID NO: 3. 5. A polynucleotide encoding a partial peptide of the protein encoded by the polynucleotide according to claim 1 or 3. 6. A polynucleotide encoding a gene that is identical in terms of molecular evolution to a gene comprising the base sequence set forth in SEQ ID NO: 3. [Claim 7] A polynucleotide encoding a protein consisting of an amino acid sequence in which one or more amino acids have been substituted, deleted, inserted, and/or added in the amino acid sequence set forth in SEQ ID NO: 4, and which has dominant-negative properties relative to a protein consisting of the amino acid sequence set forth in SEQ ID NO: 4. 8. The polynucleotide according to claim 7, which encodes an amino acid sequence in which the amino acid sequence at positions 300-303 in the amino acid sequence of SEQ ID NO: 4 has been modified to a caspase-resistant amino acid sequence. 9. A protein encoded by the polynucleotide according to any one of claims 1, 3, 5, 6 and 7. 10. A vector comprising the polynucleotide according to any one of claims 1, 3, 5, 6, and 7. 11. A transformant harboring the polynucleotide according to any one of claims 1, 3, 5, 6, and 7, or the vector according to claim 10. 12. A method for producing the protein according to claim 9, comprising the steps of culturing the transformant according to claim 11 and recovering the expression product. 13. A polynucleotide having a length of at least 15 bases, which comprises a base sequence complementary to the polynucleotide according to any one of claims 1, 3, 5, 6 and 7, or its complementary strand. 14. An antibody against the protein according to claim 9. 15. An immunological assay method comprising the step of observing an immunological reaction between the protein according to claim 9 and the antibody according to claim 14. 16. A method for screening for a substance that controls apoptosis, comprising the steps of: (1) contacting a candidate substance with cells that express a protein encoded by the polynucleotide of claim 1 or 3, and (2) culturing the cells under conditions that induce apoptosis, and selecting a candidate substance that inhibits or promotes apoptosis. 17. A method for screening for a substance that controls apoptosis, comprising the steps of: (1) contacting a candidate substance with a cell into which a vector containing an expression control region of a gene consisting of the base sequence set forth in SEQ ID NO: 3 and a reporter gene operably linked downstream of the region has been introduced, (2) measuring the activity of the reporter gene, and (3) selecting a candidate substance that increases or decreases the reporter activity in step (2) compared to a control. 18. Use of a compound obtainable by the method according to claim 16 or claim 17 in controlling cell proliferation or cell death. 19. A therapeutic agent for a disease characterized by cell proliferation or cell death, comprising as a main ingredient a compound obtainable by the method according to claim 16 or 17.