KR20040097851A - Novel protein inhibiting Apaf-1 medicated activation - Google Patents

Abstract

PURPOSE: A protein inhibiting apoptotic protease activating factor-1(Apaf-1) medicated activation is provided, thereby treating or preventing diseases caused by over-activation of Apaf-1 or under-activation of Apaf-1. CONSTITUTION: The isolated protein APIP inhibiting Apaf-1 medicated activation comprises the amino acid sequence with a more than 80% or 90% homology to the amino acid sequence of SEQ ID NO:2. The isolated protein APIP2 inhibiting Apaf-1 medicated activation comprises the amino acid sequence with a more than 80% or 90% homology to the amino acid sequence of SEQ ID NO:4. A gene encoding the protein APIP has the nucleotide sequence of SEQ ID NO:1. A gene encoding the protein APIP2 has the nucleotide sequence of SEQ ID NO:3. A vector containing the APIP gene or APIP2 gene is provided. A host cell transformed with the vector is provided. The method for producing the protein APIP or APIP2 comprises culturing the transformed host cell and purifying the protein APIP or APIP2.

Description

Novel protein inhibiting Apaf-1 medicated activation

Apaf-1 plays an important role in the general apoptosis program of normal development and oncology (Hengartner, MO et al., Nature . 356, 494-499,1992; Yuan, JY et al., Dev. Biol . 138, 33-41, 1990; Vaux, DL, Cell . 90, 389-390, 1997; Zou, H. et al., Cell . 90, 405-413, 1997). Apaf-1 comprises a CED-3 homology region (CARD) at its N-terminus, followed by a CED-4 homology region, and 12 WD-40 repeats at the C-terminus. The crystal structure of CARD in Apaf-1 alone and in a complex indicates that each molecule of Apaf-1 interacts with a molecule of procaspase-9 (Qin, H. et al ., Nature . 399, 549-557, 1999). In stress-induced cell death where the mitochondrial pathway is required for cell death, mitochondria release cell death factors such as cytochrome c, AIF, Smac / Diablo and serine protease HtrA2 (Yuan, JY et al., Dev. Biol) 138, 33-41, 1990; Vaux, DL et al., Cell . 90, 389-390, 1997; Zou, H. et al., Cell . 90, 405-413, 1997; Qin, H. et al , Nature . 399, 549-557, 1999; Susin, SA et al ., Nature . 397, 441-446, 1999). Pro-cell death members of the Bcl-2 family, such as Bax and Bid, induce release of cell death factors, and anti-cell death members such as Bcl-2 or Bcl-X _L inhibit their release (Desagher, S. et al., Trends Cell Biol . 10, 369-377, 2000). Bid is degraded by caspase-8 and cleaved Bid (tBid) migrates to the mitochondria to activate Bax (Li, H. et al., Cell . 94, 491-501, 1998; Eskes, R. et al., Mol. Cell Biol . 20, 929-935, 2000). Cytochrome c binds to Apaf-1 but does not induce an oligomeric complex of Apaf-1 in the absence of dATP. However, when dATP is added to the cytochrome c-linked Apaf-1 complex, complex oligomerization occurs (Saleh, A. et al., J. Biol. Chem . 274, 17941-17945, 1999). The oligomerization of Apaf-1 induces caspase-9 to form apoptosomes and activate caspase-9 (Zou, H. et al., J. Biol. Chem . 274, 11549-11556, 1999 ).

Previous reports on Apaf-1 and caspase-9 knock-out mice suggest that Apaf-1 is involved in the regulation of cell numbers in the developing brain, retina, face and limbs. Apaf-1 and caspase-9 are important for the activation of caspase-3 and play an important role in normal development (Cecconi, F. et al., Cell . 94, 727-737, 1998; Yoshida, H. et al. , Cell . 94, 739-750, 1998; Kuida, K. et al ., Cell . 94, 325-337, 1998). Apaf-1 deficient mice show reduced cell death in brains and facial malformations with neuroproliferation. Thymus bulbs from Apaf-1 knock-out mice do not show cell death after treatment with various cell death stimuli, including etoposide and γ-irradiation, but are sensitive to Fas-induced cell death. Apaf-1 and caspase-9 have been reported to modulate tumor development by functionally interacting with p53 (Soengas, MS et al . Science . 284, 156-159, 1999). These observations indicate that Apaf-1 plays an important role in the general event of the mitochondrial dependent cell death pathway. Therefore, the identification of molecules such as Boo and Hsp70 as regulators of Apaf-1 mediated cell death is of great importance (Song, Q. et al., EMBO J. 18, 167-178, 1999; Beere, HM. Et . al . Nat. Cell Biol . 2, 469-475, 2000; Saleh, A. et al., Nat. Cell Biol . 8, 476-483 (2000).

The present inventors isolated and identified the new gene of APIP (Apaf-1-Interactiong Protein), a novel protein that inhibits Apaf-1 mediated activation. We present several evidences that APIP interacts with Apaf-1 and consequently inhibits Apaf-1 mediated activation of caspase-9, namely (i) binding to Apaf-1 in vitro and in vivo. To; (ii) inhibit cytochrome c-induced activation of caspase-9; (iii) inhibits cell death induced by mitochondrial injury including expression of cleaved Bid, expression of etoposide, cisplastin, hypoxia, etc., resulting from expression of APIP; (iv) We have found that mitochondrial mediated cell death increases due to down-regulation of APIP expression, thereby suggesting a method for treating or preventing diseases associated with Apaf-1 activation using newly identified APIP. I can do it.

The gist of the invention

The present invention is based on the isolation and characterization of genes of API-1 (Apaf-1-Interactiong Protein), a novel protein that inhibits Apaf-1 mediated activation. More specifically, the present invention utilizes a yeast 2-hybrid system to isolate cDNAs encoding APIP proteins whose sequences are known but whose function is unknown, and express and characterize the encoded APIP proteins. Is based on.

The present invention provides novel APIP proteins and functional derivatives thereof that inhibit Apaf-1 mediated activation with the sequence set forth in SEQ ID NO: 2. The present invention also provides novel APIP2 proteins and functional derivatives thereof that inhibit Apaf-1 mediated activation with the sequence set forth in SEQ ID NO: 4.

The present invention also provides novel nucleic acid sequence molecules encoding novel APIP proteins and functional derivatives thereof that inhibit Apaf-1 mediated activation with the sequence set forth in SEQ ID NO: 2. Specifically, the present invention provides novel nucleic acid sequence molecules having the nucleic acid sequence set forth in SEQ ID NO: 1. The present invention also provides novel nucleic acid sequence molecules encoding novel APIP2 proteins and functional derivatives thereof that inhibit Apaf-1 mediated activation with the sequence set forth in SEQ ID NO: 4. Specifically, the present invention provides novel nucleic acid sequence molecules having the nucleic acid sequence set forth in SEQ ID NO: 3. In addition, the nucleic acid sequence molecules of the present invention include all gDNA, cDNA and RNA.

The present invention also provides a plasmid containing the nucleic acid sequence molecule, for example a cloning vector or a recombinant plasmid expressing the APIP or APIP2 inhibitory protein.

The present invention also provides cells and cell lines containing the vector.

The present invention also provides a method for preparing the APIP or APIP2 protein, particularly preferably a method for producing the APIP or APIP2 protein by genetic engineering method.

The present invention also provides for the separation and / or purification of substances that selectively bind to APIP or APIP proteins, for example Apaf-1 or fragments thereof, using APIP or APIP2 proteins, in particular largely expressed APIP or APIP proteins. Provide a method.

The present invention also provides a method for screening a substance that modulates the activity of an APIP or APIP2 protein or Apaf-1, in particular a method for screening a substance that enhances or inhibits binding of Apaf-1 / APIP or APIP2 protein.

The present invention also provides a therapeutically or prophylactically effective amount of an active ingredient selected from the group consisting of APIP or APIP2 protein expression vectors, isolated APIP or APIP2 protein mRNA or isolated APIP or APIP2 protein and mixtures of two or more thereof. Provided is a pharmaceutical composition useful for treating or preventing a disease caused by excessive activity of Apaf-1, comprising the active ingredient alone or in a pharmaceutically acceptable carrier.

The present invention also provides a therapeutically or prophylactically effective amount of an active ingredient selected from the group consisting of APIP or APIP2 protein expression vectors, isolated APIP or APIP2 protein mRNA or isolated APIP or APIP2 protein and mixtures of two or more thereof. A method of treating or preventing a disease caused by excessive activity of Apaf-1 is provided by administering to the animal a pharmaceutical composition comprising the active ingredient alone or a pharmaceutically acceptable carrier.

The present invention also provides an active ingredient selected from the group consisting of antisense RNA or DNA of APIP or APIP2 protein or antibodies to APIP or APIP2 protein and mixtures of two or more thereof, and a therapeutic or prophylactically effective amount of the active ingredient alone Or a pharmaceutically acceptable carrier, a pharmaceutical composition useful for treating or preventing a disease caused by under-activity of Apaf-1.

The present invention also provides an active ingredient selected from the group consisting of antisense RNA or DNA of APIP or APIP2 protein or antibodies to APIP or APIP2 protein and mixtures of two or more thereof, and a therapeutic or prophylactically effective amount of the active ingredient alone Or by administering a pharmaceutical composition comprising an pharmaceutically acceptable carrier to an animal, thereby treating or preventing a disease caused by the under activity of Apaf-1.

The present invention also provides antibodies that selectively recognize APIP or APIP2 proteins.

In addition, the present invention is characterized in that it comprises an active component selected from the group consisting of APIP or APIP2 protein expression plasmid, isolated APIP or APIP2 protein mRNA or isolated APIP or APIP2 protein and mixtures of two or more thereof. Provided is a pharmaceutical composition for preventing or treating hypoxia.

The present invention also provides an animal with a pharmaceutical composition comprising as an active ingredient a pharmaceutical composition selected from the group consisting of an APIP or APIP2 protein expression plasmid, an isolated APIP or APIP2 protein mRNA or an isolated APIP or APIP2 protein and a mixture of two or more thereof. Characterized in that the administration, provides a method for preventing or treating ischemia or hypoxia.

1 shows the amino acid sequences of APIP and APIP2.

2 shows the expression of APIP in various tissues. Northern blot analysis using mRNA prepared from human adult tissue. β-actin was used as a control.

3 shows the interaction of APIP with CARD region of Apaf-1. As a result of the in vitro binding assay, the GST and GST-CARD proteins were expressed and purified, and the proteins bound to their affinity (equivalent to 20 μg of protein) were incubated with the in vitro translated APIP protein in the presence of ³⁵ S-methionine. It was. Caspase-9S protein was used as a positive control. After separation by 12% SDS-PAGE, the bound protein was detected by autoradiography (top) and the resin-coupled protein was visualized by Coomassie blue staining (bottom).

4 shows inhibition of etoposide-caused cell death by APIP without affecting the release of cytochrome c from mitochondria. During the C2C12 Cells C2C12 or untreated stably expressing APIP or 24 h and then exposed to etoposide (25μg ml ^-1) or soluble Fas ligand (0.2μg ml ^-1). 4A shows the percentage of apoptosis of C2C12 cell line and muscle cell line C2C12 / APIP transformed to overexpress APIP. Trypan blue assay. 4B shows the results of staining C2C12 cell line and C2C12 / APIP cell line with anti-cytochrome c antibody and Hoechst 33258.

FIG. 5 shows the inhibition of cytochrome c-induced activation of caspase-9 by APIP in vitro with competitive binding of APIP and caspade-9 to CARD of Apaf-1. FIG. 5a shows the reaction of purified GST-CARD with radio-labeled caspase-9 while increasing the amount of radio-labeled caspase-9, and precipitation of GST protein to separate precipitated proteins by SDS-PAGE. The result exposed to the film is shown. Figure 5b shows the result of measuring the amount of GST-CARD-bound APIP with a densitometer.

6 shows protection of muscle cells from ischemic injury by APIP induced expression. Figure 6a shows the result of Western blot analysis of the amount of APIP expression after injecting control vector and antisense APIP into the hind limb muscle of the mouse and inducing ischemia. 6B shows the results of the TUNEL assay.

7 shows inhibition of ischemia / hypoxia injury by increased expression of APIP in C2C12 skeletal muscle cells. Figure 6a shows the result of measuring the degree of cell death through trypan blue staining after exposing APIP overexpressing C2C12 cell line and control C2C12 cell line to hypoxic conditions. 7B shows the cell state observed with an optical microscope.

In one aspect, the present invention provides novel APIPs or APIP2s and functional derivatives thereof that inhibit Apaf-1 mediated activation.

As used herein, the term “APIP or APIP2 protein” refers to an isolated native polypeptide that interacts with Apaf-1 to inhibit Apaf-1 mediated physiological activity. The APIP or APIP2 proteins of the present invention include polypeptides having 204 and 242 amino acid sequences specifically shown in SEQ ID NOs: 2 and 4, and their splicing and allelic variants thereof.

The term “natural polypeptide” is a polypeptide present in all cell types of human or non-human mammal species, including everything with or without starting methionine, as well as purified, chemically synthesized, or DNA from natural sources. It includes everything produced by recombinant technology or made by a combination of these and / or other methods. In this respect, natural polypeptides obtained from humans as well as other mammalian species such as pigs, dogs, horses and the like are also included in the present invention.

APIP or APIP2 proteins of the present invention include functional derivatives thereof. The term "functional derivative" typically refers to a compound that exhibits biological activity substantially comparable to a natural polypeptide. For the purposes of the present invention, the functional derivative of a native APIP or APIP2 protein having biological activity equivalent to that of native APIP or APIP2 protein means that the derivative binds to Apaf-1 and inhibits Apaf-1 mediated physiological activity. it means. Preferably, the functional derivatives of the native APIP and APIP2 proteins of the present invention are functional derivatives of polypeptides having the amino acid sequences set forth in SEQ ID NOs: 2 and 4, respectively. The functional derivative is preferably at least about 60%, more preferably at least about 70%, even more preferably at least about 80%, and most preferably about natural APIP or APIP2 protein, preferably human APIP or APIP2 protein. At least 90% amino acid sequence identity.

In addition, functional derivatives of APIP or APIP2 proteins according to the present invention include amino acid sequence variants in which some of the natural protein amino acids have been substituted, deleted or added unless the native biological activity is lost. Substitutions of amino acids are preferably conservative substitutions. Examples of conservative substitutions of naturally occurring amino acids are as follows: aliphatic amino acids (Gly, Ala, Pro); Hydrophobic or aromatic amino acids (Ile, Leu, Val, Phe, Tyr, Trp); Acidic amino acids (Asp, Glu); Basic amino acids (His, Lys, Arg, Gln, Asn); And sulfur-containing amino acids (Cys, Met). The deletion of the amino acid is preferably located at the site where the APIP or APIP2 protein does not interact with Apaf-1.

Polypeptides having a biological activity substantially equivalent to native APIP or APIP2 protein as fragments of the native APIP or APIP2 protein from several mammalian species form another preferred group of functional derivatives of the native APIP or APIP2 protein. do. The term “fragment” refers to an amino acid sequence corresponding to a portion of a protein, which has a common element of origin, structure and mechanism of action within the scope of the present invention and which is either naturally present or is cleaved using a protease. Or chemically cleaved. In addition, modifications are made to alter the stability, shelf life, solubility, etc. of the native APIP or APIP2 protein, or functional derivatives which have been modified to alter the relationship with substances that interact with the APIP or APIP2 protein, such as, for example, Apaf-1. Another preferred group of functional derivatives of the native APIP or APIP2 protein according to the invention is formed.

Another preferred group of functional derivatives of native APIP or APIP2 proteins are polypeptides encoded by nucleic acids that hybridize under stringent conditions with complementary sequences of nucleic acids encoding native APIP or APIP2 proteins.

The term "identity" or "homology" as used in connection with a natural protein or functional derivative thereof refers to a gap to incubate corresponding candidate sequences identical to amino acid residues of the natural protein or functional derivative thereof and to obtain the maximum homology ratio if necessary. Refers to the ratio of amino acid residues in the candidate sequence after the introduction of (considering no conservative substitution as part of the sequence identity). Methods and computer programs used for the arrangement of sequences are well known in the art.

The term "strain conditions" can be provided by several methods, one example of which is 20% formamide, 5 x SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6). ), 5 x Denhardt solution, 10% dextran sulfate and 20 μg / ml denatured and sheared salmon sperm DNA, incubated overnight at 42 ° C. Another method for constriction conditions is hybridization in a buffer comprising 30% formamide in 5 × SSPE (0.18 M NaCl, 0.01 M NaPO 4 (pH 7.7), 0.0001 M EDTA) buffer at a temperature of 42 ° C. followed by 42 ° C. Wash with 0.2 x SSPE. Preferably, the constriction conditions are to use a hybridization buffer containing 50% formamide in 5 x SSPE at 42 ° C. and wash with 0.2 x SSPE at the same temperature.

The term "isolated" as used in connection with a protein or polypeptide or nucleic acid refers to a state in which the protein or polypeptide or nucleic acid is released from at least some of the substances associated with the protein or polypeptide or nucleic acid in its natural environment.

We screened a human cDNA library (Jurkat cDNA library) by the yeast 2-hybrid screening method using the CARD region of Apaf-1 as a bait to clones that bind particularly strongly to the region. The sequence of SEQ ID NO: 1 was obtained by collecting cDNA of the positive clone and analyzing the sequence. Sequence analysis using Genebank BLAST homology studies revealed that the sequence was a new protein that has not been reported yet. In addition, a novel protein APIP2 having an amino-terminal amino acid sequence different from APIP was identified through the expressed-sequenced tag (EST) database (SEQ ID NO: 4). The novel APIP or APIP2 protein of the present invention thus obtained was shown in the yeast two-hybrid assay to show that APIP binds to Apaf-1 but not to FADD protein. In vitro binding experiments showed that APIP and APIP2 proteins did not bind to GST protein, but similar to Caspase-9S protein with GST-CARD protein (FIG. 3). The binding of APIP and Apaf-1 protein was confirmed to bind to Apaf-1 in HeLa cell line through immunoprecipitation assay using anti-APIP antibody. The intracellular location of APIP was confirmed in the cytoplasm, as in Apaf-1, by immunochemical analysis and subcellular fractional analysis. These experimental results demonstrate that APIP selectively binds with Apaf-1.

Native APIP or APIP2 proteins are coprecipitated with immunoprecipitated Apaf-1. In a preferred embodiment, radiolabeled Apaf-1 is immunoprecipitated with Protein A-Agarose (Oncogene Science) or Protein A-Sepharose (Pharmacia). The immunoprecipitates are then analyzed by autoradiography, etc., depending on the radiolabel used. The APIP or APIP2 proteins of the invention can be purified by methods known in the art, such as by co-precipitation with Apaf-1 or derivatives thereof and purification on an affinity column. For large scale purification, methods similar to those disclosed in Smith and Johnson, Gene 67, 31-40, 1998 can be used. For example, cell lysates containing the APIP or APIP2 protein to be purified can be purified by application to a glutathione-S-transferase (GST) -CARD fusion protein affinity column.

The APIP or APIP2 proteins, functional derivatives, fragments thereof and all of the variants described above can be prepared using one of the known organic chemical methods for peptide synthesis. Organic chemical methods of peptide synthesis include coupling the necessary amino acids by a condensation reaction either in homogeneous phase or with the aid of the so-called solid phase. The most common methods for condensation reactions include the carbodiimide method, the azide method, mixed anhydride methods and methods using active esters, which are described in The Peptides Analysis, Synthesis, Biology Vol. 1-3 (Gross, E. and Meienhofer, J. Edit), 1979-1981 (Academic Press Inc.).

Particularly suitable solid phases include p-alkoxybenzyl alcohol resins (4-hydroxy-methyl-phenoxy-methyl-copolystyrene-1% di) described in Wang, J. Am. Chem. Soc., 95, 132, 1974. Vinylbenzene resins). After synthesis, peptides can be separated from the solid phase under moderate conditions. After synthesis of the desired amino acid sequence, the detachment of the peptide from the resin is carried out using trifluoroacetic acid containing a scavenger such as triisopropyl silane, anisole or ethanedithiol, thioanisole. Reactive groups that cannot be involved in the condensation reaction are effectively protected by groups that can be removed very easily again by hydrolysis or reduction with acids, bases. For protecting groups that may possibly be used, see Peptides, Analysis, Synthesis, Biology, Vol. 1-9 (Gross, Udenfriend and Meienhofer edited) 1979-1987 (Academic Press Inc.). In addition, the synthesized peptides may be combined with each other by the sequences synthesized in fragments to obtain the desired full length sequence.

Particularly preferred APIP or APIP2 proteins, functional derivatives, fragments thereof and methods of making all of the variants described above are by means of genetic recombination techniques. The gene of a native APIP or APIP2 protein can be expressed in a suitable host cell to make the cell lysate or the APIP or APIP2 protein mRNA can be read in vitro and purified by protein isolation methods known in the art. have. Typically, the cell lysate or in vitro detox is centrifuged to remove cell debris and the like, followed by precipitation, dialysis, various column chromatography, and the like. Examples are ion exchange chromatography, gel-permeation chromatography, HPLC, reverse phase-HPLC, SDS-PAGE for preparation, affinity columns and the like. Affinity columns can be made using, for example, anti-APIP or APIP2 protein antibodies or Apaf-1. APIP or APIP2 proteins may also be expressed in the form of secreted proteins and harvested from cell culture. General recombinant DNA techniques are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition (Cold Spring Harbor, N.Y .: Cold Spring Harbor Laboratory Press) 1989.

Amino acid sequence variants, including functional derivatives of native APIP or APIP2 proteins, are prepared by introducing appropriate nucleotide changes into native APIP or APIP2 protein DNA or by in vitro synthesis of the polypeptide of interest according to methods known in the art. Amino acid sequence variants have two main variables: the location of the mutation site and the nature of the mutation. Except for natural alleles that do not require manipulation of the DNA sequence encoding the APIP or APIP2 protein, amino acid sequence variants of the APIP or APIP2 protein are preferably directed to form DNA such that allelic or amino acid sequence variants that do not naturally exist are formed. I make it by mutation. Methods of identifying target residues in natural proteins and preparing amino acid sequence variants are well known in the art and described, for example, in US Pat. No. 5,108,901. Preferred techniques include alanine-scanning mutagenesis, PCR mutagenesis, cassette mutagenesis and the like, all of which are described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition (Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press) 1989 and Current Protocols in Molecular Biology, in Molecular Biology, Ausubel et al eds., Greene Publishing Associates and Wiley-Interscience 1991.

The present invention provides nucleic acid sequences encoding APIP or APIP2 proteins capable of inhibiting Apaf-1 mediated activity and binding to the CARD region of Apaf-1. Nucleic acid sequences include all gDNA, cDNA and RNA. Nucleic acid sequences of the invention include both nucleic acid sequences encoding native proteins and their functional equivalents and functional derivatives, and sequences that hybridize with these sequences under high stringent conditions. High-tension conditions are as described in known literature (Molecular Cloning, Cold Spring Harbor, New York, Cold Spring Harbor Laboratory Press, 1989).

The nucleic acid sequence encoding the APIP or APIP2 protein is of mammalian origin, preferably human. The nucleic acid sequence encoding the APIP or APIP2 protein is more preferably the nucleic acid sequence encoding the protein of the amino acid sequence set forth in SEQ ID NOS: 2 and 4 by codon degeneracy, particularly preferably shown in SEQ ID NOs: 1 and 3 Nucleic acid sequence. For the purposes of the present invention, a nucleic acid sequence encoding a protein APIP or APIP2 protein, for example, is a cDNA prepared from tissues that possess and express detectable levels of mRNA or APIP or APIP2 protein mRNA of the APIP or APIP2 protein. You can get it from the library. For example, cDNA libraries can be constructed by obtaining polyadenylation of mRNA from cell lines known to express APIP or APIP2 proteins and using this mRNA as a template to synthesize double-stranded cDNA. In addition, DNA encoding APIP or APIP2 proteins can be obtained from genomic libraries such as human genomic cosmid libraries.

Suitable probes for the cDNA library include, for example, oligonucleotide probes encoding portions of APIP or APIP2 polypeptides from the same or different species, base sequences usually of about 20 to 80 bases in length. Probes suitable for screening genomic DNA libraries include, but are not limited to, oligonucleotides, cDNAs or fragments thereof and / or homologous genomic DNA or fragments thereof that encode the same or similar polypeptides. Screening cDNA or genomic libraries with selected probes can be performed according to standard methods described in Sambrook et al., Molecular Cloning: A Laboratory Manual, New York, Cold Spring Harbor Laboratory Press, (1989).

In addition, cDNAs encoding APIP or APIP2 proteins may be polymerized as described in other known recombinant DNA techniques, such as Current Protocols in Molecular Biology, Ausubel et aleds., Greene Publishing Associates and Wiley-Interscience 1991. Identification and separation can be accomplished using chain reaction (PCR) or by direct expression cloning. This method requires the use of oligonucleotide probes that hybridize with DNA encoding APIP or APIP2 proteins.

As the present invention illustrates, nucleic acid sequences encoding APIP or APIP2 proteins can be obtained using the yeast two-hybrid system method [Fields and Song, Nature (London) 340, 245-246 (1989); Chien et al., Proc. Natl. Acad. Sci. USA 88, 9578-9582 (1991); Chevray and Nathans, Proc. Natl. Acad. Sci. USA 89, 5789-5793 (1992). The yeast two-hybrid method uses two kinds of fusion proteins. Many transcriptional activators, such as GAL4 in yeast, are composed of two physically separate module domains, a DNA-binding domain and a transcriptional activation domain. Apaf-1, preferably Apaf-1, CARD is fused to the DNA-binding domain of GAL4, and the protein to be screened is fused to the GAL4 transcriptional active domain. When GAL4 activity is reconstitution by binding between proteins, the GAL1-lacZ reporter gene under the GAL4-promoter is expressed. Thus, colonies with binding polypeptides can be detected by measuring β-galactosidase activity. Yeast two-hybrid systems are commercially available [Matchmaker (Clontech)]. The present inventors screened a human cDNA library (Jurket cDNA library) by the yeast two-hybrid screening method by means of CARD of Apaf-1 to obtain a clone that binds particularly strongly with Apaf-1, and cDNA of the positive clone. The nucleotide sequence of FIG. 1 was obtained by collecting and analyzing the sequence.

As the sequence of the APIP or APIP2 protein gene is revealed by the present invention, this gene is also described in Engels and Uhlmann, Angew. Chem. Int. Ed. Engl. 28, 716 (1989)] can be prepared by chemical synthesis according to any of the methods described. These methods include tryster, phosphite, phosphoramidite and H-phosphate methods, PCR and other autoprimer methods and oligonucleotide synthesis on solid supports.

The present invention provides a vector containing a nucleic acid sequence encoding an APIP or APIP2 protein, for example a cloning vector or an expression vector expressing an APIP or APIP2 protein. By definition, a vector contains DNA sequences linked to operably regulate regulatory sequences capable of controlling the expression of a protein in a suitable host cell and other sequences introduced to facilitate expression or to optimize the expression of the protein. DNA construct. Such regulatory sequences include promoters for regulating transcription, operators optionally added to modulate transcription, appropriate mRNA ribosomal binding sites, and sequences regulating termination of transcription / detoxification. The vector may be inserted into a plasmid, virus, phage or genome. Particularly illustrated in the present invention are vectors such as pcDNA3, pEGFP, pGEX5X.

However, to express DNA sequences encoding the APIP or APIP2 proteins of the invention, any of a wide variety of expression control sequences may be used in the vector. Examples of useful expression control sequences include, for example, SV40 or adenoviruses. Early and late promoters, lac system, trp system, TAC or TRC system, T3 and T7 promoters, major operator and promoter region of phage lambda, regulatory region of fd code protein, 3-phosphoglycerate kinase or other glycol Promoters for degradation enzymes, promoters of the phosphatase, such as Pho5, promoters of yeast alpha-crossing systems and other sequences of construction and induction known to modulate the expression of genes of prokaryotic or eukaryotic cells or viruses thereof And various combinations thereof.

Nucleic acids are “operably linked” when placed in a functional relationship with other nucleic acid sequences. This may be genes and regulatory sequence (s) linked in such a way as to enable gene expression when appropriate molecules (eg, transcriptional activating proteins) bind to regulatory sequence (s). For example, the DNA for a pre-sequence or secretion leader is operably linked to the DNA for the polypeptide when expressed as a shear protein that participates in the secretion of the polypeptide and the promoter or enhancer is sequenced. Operably linked to a coding sequence when affecting the transcription of a ribosome binding site is operably linked to a coding sequence when affecting the transcription of a sequence or a ribosome binding site is arranged to facilitate translation Operably linked to the sequence. In general, “operably linked” means that the linked DNA sequences are in contact, and in the case of a secretory leader, are in contact and present within the reading frame. However, enhancers do not need to touch. Linking of these sequences is performed by ligation (linking) at convenient restriction enzyme sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers according to conventional methods are used.

The present invention provides host cells to cell lines (hereinafter, host cells) containing the vector. Host cells transformed or transfected with the expression vectors described above constitute another aspect of the present invention. As used herein, the term “transformation” means introducing DNA into a host so that the DNA is replicable as an extrachromosomal factor or by chromosomal integration. As used herein, the term "transfection" means that the expression vector is accepted by the host cell whether or not any coding sequence is actually expressed. Host cells of the invention are prokaryotic or eukaryotic cells. A host having a high DNA introduction efficiency and a high expression efficiency of the introduced DNA is usually used. this. Well-known eukaryotic and prokaryotic hosts such as E. coli, Pseudomonas, Bacillus, Streptomyces, fungi, yeast, insect cells such as Spodoptera pruperifera (SF9), animals such as CHO, HeLa, HEK293, C2C12, mouse cells Cells, African green monkey cells such as COS 1, COS 7, BSC 1, BSC 40 and BMT 10, and tissue cultured human cells are examples of host cells that can be used. Host cells exemplified in the present invention are E. coli. E. coli, yeast, HeLa, HEK293, C2C12 and the like.

The APIP or APIP2 proteins of the present invention can be used to isolate and purify native Apaf-1 proteins or their functional derivatives or various variants and fragments based on their ability to bind to Apaf-1. Thus, as a further aspect, the present invention relates to molecules which selectively bind to APIP or APIP2 proteins, for example Apaf-1 proteins or derivatives, variants or the like, using APIP or APIP2 proteins, in particular largely expressed APIP or APIP2 proteins. Provided are methods for isolating and / or purifying fragments. The method includes incubating a mixture of APIP or APIP2 protein with a candidate molecule, separating the complex, and freely removing the APIP or APIP2 protein from the separated complex. Separation of the complex can be carried out by various conventional separation methods. Examples are affinity columns immobilized with APIP or APIP2 proteins, affinity columns with anti-idiote type antibodies, yeast two-hybrid systems, and the like.

The present invention may be usefully used for assays to identify API compounds of therapeutically active substances that modulate the binding of Apaf-1 / APIP or APIP2 proteins. Specifically, lead compounds that inhibit the formation of Apaf-1 / APIP or APIP2 protein complexes or prevent or inhibit the dissociation of already formed Apaf-1 / APIP or APIP2 protein complexes can be identified by conventional methods. In this regard, the present invention provides a method for screening a molecule that modulates the activity of an APIP or APIP2 protein or Apaf-1, in particular a method for screening a molecule that modulates the binding of Apaf-1 / APIP or APIP2 protein. Preferably, the screened molecules are those that prevent or inhibit dissociation of Apaf-1 / APIP or APIP2 protein complexes or inhibit the interaction of Apaf-1 / APIP or APIP2 protein complexes. This method incubates a mixture comprising Apaf-1 and APIP or APIP2 proteins with a candidate molecule and the ability of the candidate molecule to regulate Apaf-1 / APIP or APIP2 protein binding, e.g., of Apaf-1 and APIP or APIP2 proteins. Detecting the ability to inhibit interaction or to prevent or inhibit dissociation of Apaf-1 / APIP or APIP2 protein complexes.

Molecules that prevent the interaction of Apaf-1 with APIP or APIP2 proteins may be useful in situations where the immune system needs to be strengthened. Dissociation inhibitors of APIP or APIP2 protein / Apaf-1 complexes may be useful as immunosuppressants or anti-inflammatory agents.

Assays can be performed in a variety of formats and include, for example, protein-protein binding assays, biochemical screening assays, immunoassays, cell-based assays, and the like. Such assay formats are well known in the art.

When screening inhibitors of Apaf-1 / APIP or APIP2 protein binding, the assay mixture is subjected to conditions such that the APIP or APIP2 protein specifically binds Apaf-1 with standard binding affinity except when a candidate molecule is present. Incubate. The components of the mixture may be added in any order as long as they provide the necessary binding. Incubation may be carried out at any temperature that promotes optimal binding and is typically about 4 ° C. to 40 ° C., more typically about 15 ° C. to 40 ° C. Incubation time should be chosen for optimal binding but minimized to facilitate high speed screening. Typical incubation times are about 0.1 to 10 hours, preferably less than 5 hours, and more preferably less than 2 hours. The effect of candidate molecules on Apaf-1 / APIP or APIP2 protein binding after incubation is determined by conventional methods. For cell-free binding-type assays, a separation step is often used to separate the binding and non-binding components. Separation can be carried out, for example, by precipitation (eg TCA precipitation, immunoprecipitation), immobilization (eg immobilization on a solid substrate) and then washing. The bound protein is conveniently detected by using a detection label attached thereto, for example, by measuring radioactivity release, absorbance, or the like, or indirectly using an antibody conjugate.

Compounds that inhibit or prevent dissociation of Apaf-1 / APIP or APIP2 protein complexes conveniently form Apaf-1 / APIP or APIP2 protein complexes in the absence of candidate compounds, add candidate compounds to the mixture formed, and candidate compounds Except where present, it can be identified by changing the conditions such that Apaf-1 is released from the complex. This can be achieved, for example, by changing the incubation temperature or by adding a candidate compound to the mixture which releases Apaf-1 from the complex in the absence of the candidate compound.

The APIP or APIP2 proteins of the invention can be used to produce antagonistic or potent anti-APIP or APIP2 protein antibodies that block or mimic the ability of the protein to inhibit Apaf-1-mediated signal transduction. Polyclonal or monoclonal antibodies against APIP or APIP2 proteins can be readily prepared using known techniques (Monoclonal Antibodies: Principles and Practice, Second Ed., James W. Goding, Academic Press (1986)). . Antibodies to APIP or APIP2 proteins, fragments of antibodies (eg, Fab fragments), humanized antibodies, antibodies conjugated to cytotoxic substances, and the like are included in the antibody scope of the present invention.

Antibodies to the APIP or APIP2 proteins of the invention can be obtained by the following methods in certain embodiments. That is, as the antigen for immunity required for the production of monoclonal antibodies, native APIP or APIP2 protein or recombinant APIP or APIP2 protein can be used. Lymphocytes immunized with each antigen by each antigen or immunized with the myeloma cell line are fused to produce hybridomas by conventional methods. Each antigen highly purified against this hybridoma culture is used to select antibody producing hybridomas that recognize each antigen by solid phase ELISA. The obtained hybridomas are cloned and each of the obtained stable antibody-producing hybridomas is cultured to obtain the desired antibody. Mouse and rat can be used to make hybridomas.

Immunization is achieved by diluting the antigen to an appropriate concentration in an appropriate solvent such as physiological saline, administering this solution intravenously or intraperitoneally, co-administering Freund's complete adjuvant as needed, and in animals at intervals of 1-2 weeks. It is common to administer about 3 to 4 times. The immunized animal is dissected 3-4 days after the final immunization, and splenocytes obtained from the extracted spleen are used as immune cells. As myeloma cells derived from mice that are cell-fused with immune cells, for example, p3 / x63-Ag8, p3-U1, NS-1, MPC-11, SP-2 / 0, F0, P3x63 Ag8, 653, S194 and the like can be used. Can be. As a cell derived from a rat, a cell line such as R-210 can be used. Human antibodies can be prepared by immunizing human B lymphocytes in vitro and fusion with human myeloma cells and cell lines transformed with EB virus.

Fusion of immunized cells with myeloma cell lines is commonly used by known methods, such as the method of Koehler and Milstein (Koehler et al., Nature 256, 495-497, 1975). An electric pulse method using a pulse can also be used. Immune lymphocytes and myomeric cell lines are mixed at a ratio of the number of cells normally performed, and fusion treatment is performed by adding polyethyleneglycol to a commonly used fetal bovine serum (FCS) -free cell culture medium. The cells are cultured with fusion cells (hybridomas) to select them. Subsequently, the hybridoma producing the antibody is selected by a commonly used antibody detection method such as ELISA, plague method, ouchterlony method, aggregation method, and stable hybridomas are established. The hybridoma thus established can be subcultured by a conventional culture method and can be cryopreserved as necessary. The hybridomas can be cultured by a conventional method to recover the culture solution, or the culture solution can be transplanted intraperitoneally of a mammal to recover the antibody in ascites. Antibodies in the culture solution or ascites can be purified by commonly used methods such as salting out, ion exchange and gel filtration chromatography, Protein A or Protein G affinity chromatography. Almost all of the monoclonal antibodies obtained by this method can specifically recognize native APIP or APIP2 proteins as well as functional analogs, variants and fragments thereof. These antibodies can be measured by radiometric or enzyme labeling by measurement methods known as radioimmunoassay (RIA) and enzyme immunoassay (EIA).

In certain embodiments of the invention, polyclonal antibodies that recognize APIP or APIP2 proteins can be generated as follows. By pulverizing and purifying host cells expressing APIP or APIP2 proteins, native APIP or APIP2 proteins to be used as antigens for immunization can be obtained. In addition, a recombinant recombinant APIP or APIP2 protein can be obtained by inserting the APIP or APIP2 protein cDNA into an expression vector by a conventional method and expressing the formed recombinant plasmid in a host cell and purifying it in the same manner as above. It can also be used as an antigen. As another method, a base sequence encoding a signal sequence derived from another secreted protein is added upstream of the 5 'end of the APIP or APIP2 protein cDNA, and inserted into an expression vector by a genetic engineering method in the same manner as above. By expressing and purifying these proteins, an antigen for immunization can be obtained. The immunizing antigen thus obtained is dissolved in phosphate-buffered saline (PBS), emulsified in the same dose of Freund's complete adjuvant, and then subcutaneously administered to the animal at about 1 week intervals. Immunity several times. Antibody titers are measured and boosted at the point when the highest antibody titers are reached, and all blood is collected 10 days after administration. The obtained antiserum was precipitated with ammonium sulfate fraction and the globulin fraction was purified by anion exchange chromatography, or the antiserum was diluted twice with binding buffer (Biorad), and the diluted antiserum was converted into Protein A or Protein G Sepharose column. Purification by chromatography can yield anti-APIP or APIP2 protein polyclonal antibodies.

The present invention also provides antisense oligonucleotides that inhibit the expression of APIP or APIP2 proteins. Antisense technology can be used to regulate gene expression via antisense DNA or RNA or through triple-helix formation. Antisense techniques are described, for example, in Okano, J. Neurochem. 56: 560 (1991); "Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression." CRCPress, Boca Raton, FL (1988). Triple helix formation is described, for example, in Lee et al., Nucleic Acids Research 6: 3073 (1979); Cooney et al., Science 241: 456 (1988); And Dervan et al., Science 251: 1360 (1991). This method is based on the binding of polynucleotides to complementary DNA or RNA. DNA oligonucleotides are designed to complement regions of genes involved in transcription and thereby inhibit the transcription and production of APIP or APIP2 proteins. Antisense RNA oligonucleotides hybridize with mRNA in vivo and block the translation of mRNA molecules into APIP or APIP2 polypeptides. The oligonucleotides described above can also be delivered to cells so that antisense RNA or DNA can be expressed in vivo to inhibit the production of APIP or APIP2 proteins.

Antisense nucleic acids of the invention comprise sequences complementary to at least a portion of an RNA transcript of an APIP or APIP2 protein gene. However, absolute complementarity is desirable but not necessarily required. As referred to herein, a “complementary to at least a portion of an RNA” refers to a sequence that has sufficient complementarity to hybridize with the RNA to form a stable double strand. Thus, in one embodiment, in the case of a double stranded APIP or APIP2 protein antisense nucleic acid, the single strand of the double stranded DNA can be tested or assayed for triple strand formation. The ability to hybridize depends on both the degree of complementarity and the length of the antisense nucleic acid. In general, the larger the hybridizing nucleic acid, the more base mismatch with the APIP or APIP2 protein RNA, which can form a stable double strand (or triple strand). One skilled in the art can determine the acceptable level of mismatch by using standard procedures to determine the melting point of the hybridized complex.

Oligonucleotides complementary to the 5 'end of the message (e.g., the previous 5' nontranslated sequence including the AUG start codon) should work most efficiently when inhibiting translation. However, sequences complementary to the 3 ′ non-toxic sequences of mRNA have also been shown to be effective in inhibiting the translation of mRNA (Wagner, R., 1994, Nature 372: 333-335). Thus, oligonucleotides complementary to the 5'- or 3'-non-toxin noncoding region of the NSP can be used in antisense methods that inhibit the translation of endogenous APIP or APIP2 protein mRNAs. Oligonucleotides complementary to the 5 ′ non-toxic region of the mRNA should include the complement of the AUG start codon. Antisense oligonucleotides complementary to the mRNA coding region are less efficient translation inhibitors but can be used in accordance with the present invention. The antisense nucleic acid, whether designed to hybridize to the 5'-, 3'- or code region of the APIP or APIP2 protein mRNA, should be at least 6 nucleotides in length and preferably oligonucleotides consisting of 6 to about 50 nucleotides in length. to be. In certain aspects oligonucleotides consist of at least 10 nucleotides, at least 17 nucleotides, at least 25 nucleotides or at least 50 nucleotides.

Antisense oligonucleotides include, but are not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- (carboxyhydrate Oxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylquinosine, inosine, N6-isopentenyladenin, 1- Methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil , 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylquiosine, 5-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenin, uracil- 5-oxyacetic acid (v), Waibutoxin, pseudouracil, quiocin, 2-thiocytosine, 5-methyl-2-thiouracil, 2- Uracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3- (3-amino-3- One or more modified base residues selected from the group consisting of N-2-carboxypropyl) uracil, (acp3) w and 2,6-diaminopurine.

In addition, the antisense oligonucleotides may include, but are not limited to, one or more modified sugar moieties selected from the group consisting of arabinose, 2-fluoroarabinose, xylulose and hexose.

In another embodiment, antisense oligonucleotides include, but are not limited to, phosphorothioate, phosphorodithioate, phosphoramidothioate, phosphoramidate, phosphoramidate, methylphosphonate, alkyl It may include one or more modified phosphate backbones selected from the group consisting of phosphoester and formacetal or analogs thereof.

The present invention is an active ingredient selected from the group consisting of APIP or APIP2 protein expression vector, APIP or APIP2 protein mRNA or APIP or APIP2 protein and mixtures of two or more thereof, and the therapeutic or prophylactically effective amount of the active ingredient alone or in pharmaceutical Provided is a pharmaceutical composition useful for treating or preventing a disease caused by excessive activity of Apaf-1, characterized in that it comprises a pharmaceutically acceptable carrier.

In a further aspect, the invention provides an active ingredient selected from the group consisting of an APIP or APIP2 protein expression vector, an APIP or APIP2 protein mRNA or an APIP or APIP2 protein, and mixtures of two or more thereof, and an effective amount of an active ingredient alone or in a pharmaceutical A pharmaceutical composition comprising a pharmaceutically acceptable carrier is administered to an animal, thereby providing a method for treating or preventing a disease caused by excessive activity of Apaf-1.

In the present invention, diseases caused by caspase activation due to excessive activity of Apaf-1 are not limited thereto, but are not limited to these diseases. Dopaminergic cell death (Mochizuki H. et al., Methods, 28 (2), 248- 252, Oct. 2002; Dodel RC et al., Mol Brain Res 64 (1): 141-8, 1999; Takai N. et al., J Neurosci Res 54 (2): 214-22, 1998),, Alzheimer's Disease Gervais FG et al., Cell, 97 (3): 395-406, 1999; Walter J. et al., Proc Natl Acad Sci USA 96 (4): 1391-6, 1999; Barnes NY et al., J Neurosci 18 (15): 5869-80,1998; Kim TW et al., Science 277 (5324): 373-6, 1997), Huntington's disease (Goldberg YP et al., Nat Genet. 13 (4): 442-9 , 1996; Wellington CL et al., J Biol Chem. 273 (15): 9158-67, 1998; Sanchez I. et al., Neuron. 22 (3): 623-33, 1999), amyltrophic lateral sclerosis (Pasinelli P. et al., Proc Natl Acad Sci US A. 95 (26): 15763-8, 1998), AIDS (Kruman II et al., Exp Neurol. 154 (2): 276-88, 1998) Stroke / ischemia (Hara H. et al., Proc Na tl Acad Sci US A. 94 (5): 2007-12, 1997; Namura S. et al., J Neurosci. 18 (10): 3659-68, 1998; Schulz JB et al., Ann Neurol. 45 (4 : 421-9, 1999), traumatic brain injury (Yakovlev AG et al., J Neurosci. 17 (19): 7415-24, 1997; Kermer P. et al., J Neurosci. 18 (12): 4656-62, 1998; Chaudhary P. et al, Mol Brain Res. 67 (1): 36-45, 1999), spinal cord injury (Crowe MJ et al., Nat Med. 3 (1): 73-6, 1997; Shuman SL et al., J Neurosci Res. 50 (5): 798-808, 1997) and diseases such as osteoarthritis.

In another aspect, the present invention provides an active ingredient selected from the group consisting of an antibody against APIP or APIP2 protein antisense RNA or APIP or APIP2 protein and a mixture of two or more thereof, and the therapeutic or prophylactically effective amount of the active ingredient alone Or a pharmaceutically acceptable carrier, which provides a pharmaceutical composition useful for treating or preventing a disease caused by under-activity of Apaf-1.

As a still further aspect, the present invention provides a therapeutic or prophylactically effective amount of an active ingredient, wherein the active ingredient is selected from the group consisting of APIP or APIP2 protein antisense RNA or antibodies to APIP or APIP2 protein and mixtures of two or more thereof It provides a method of treating or preventing a disease caused by the under-activity of Apaf-1, characterized in that to administer to the animal a pharmaceutical composition comprising alone or a pharmaceutically acceptable carrier.

In the present invention, diseases caused by the under-activation of Apaf-1 are not limited to these, but are not limited to cancer (Hahn C. et al., Biochemical and Biophysical Research Communications 261, 746-749, 1999).

In particular, the expression of the APIP or APIP2 protein of the present invention is significantly increased in ischemia-induced muscle cells. Accordingly, in another aspect, the present invention provides an active ingredient selected from the group consisting of APIP or APIP2 protein, APIP or APIP2 protein mRNA or APIP or APIP2 protein expression plasmid, and mixtures of two or more thereof, with a therapeutically or prophylactically effective amount. It relates to a pharmaceutical composition for the prevention or treatment of ischemia or hypoxia, characterized in that it comprises the active ingredient alone or a pharmaceutically acceptable carrier.

As a still further aspect, the present invention provides a therapeutic or prophylactically effective amount of an active ingredient selected from the group consisting of APIP or APIP2 protein, APIP or APIP2 protein mRNA or APIP or APIP2 protein expression plasmid and mixtures of two or more thereof. A method of treating or preventing ischemia or hypoxia is provided by administering the active ingredient alone or to an animal comprising a pharmaceutically acceptable carrier.

Carriers used in the pharmaceutical compositions of the present invention include carriers, adjuvants and vehicles commonly used in the pharmaceutical art and are collectively referred to as "pharmaceutically acceptable carriers." Pharmaceutically acceptable carriers that can be used in the pharmaceutical compositions of the invention include, but are not limited to, ion exchange, alumina, aluminum stearate, lecithin, serum proteins (eg, human serum albumin), buffer materials (eg, Various phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids), water, salts or electrolytes (e.g. protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride and zinc salts), colloidal Silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substrates, polyethylene glycols, sodium carboxymethylcellulose, polyarylates, waxes, polyethylene-polyoxypropylene-blocking polymers, polyethylene glycols and wool, and the like.

The pharmaceutical composition of the present invention may be administered via any general route as long as it can reach the desired tissue. Accordingly, the pharmaceutical compositions of the present invention may be administered topically, orally, parenterally, intraocularly, transdermally, rectally, intestine, and the like, and may be formulated into solutions, suspensions, tablets, pills, capsules, sustained release preparations and the like. As used herein, the term “parenteral” includes subcutaneous, intranasal, intravenous, intraperitoneal, intramuscular, intraarticular, synovial, intrasternal, intracardiac, intradural, intralesional, and intracranial injection or infusion techniques. .

In a preferred embodiment, for parenteral administration the pharmaceutical compositions of the present invention may be prepared in an aqueous solution. Preferably, a physically suitable buffer such as Hanks' solution, Ringer's solution, or physically buffered saline may be used. Aqueous injection suspensions may add a substrate that can increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. In addition, suspensions of the active ingredient may be prepared as suitable oily injection suspensions. Suitable lipophilic solvents or carriers include fatty acids such as sesame oil or synthetic fatty acid esters such as ethyl oleate, triglycerides or liposomes. Polycationic amino polymers can also be used as carriers. Optionally, the suspension may use a suitable stabilizer or agent to increase the solubility of the compound and to prepare a high concentration of solution.

Preferred pharmaceutical compositions of the invention may be in the form of sterile injectable preparations as sterile injectable aqueous or oily suspensions. This suspension may be formulated according to techniques known in the art using suitable dispersing or wetting agents (eg, Tween 80) and suspending agents. Sterile injectable preparations may also be sterile injectable solutions or suspensions (eg, solutions in 1,3-butanediol) in nontoxic parenterally acceptable diluents or solvents. Vehicles and solvents that may be used that are acceptable include mannitol, water, ring gel solution, and isotonic sodium chloride solution. In addition, sterile nonvolatile oils are conventionally employed as a solvent or suspending medium. For this purpose, any non-irritating oil may be used including synthetic mono or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives, are useful in injection formulations as well as pharmaceutically acceptable natural oils (eg olive oil or castor oil), especially their polyoxyethylated ones.

The therapeutically effective and prophylactically effective amounts of the term active ingredients used in connection with the compositions of the present invention can be readily determined by one skilled in the art.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited by the examples.

Example

reagent

Etopside and Cisplatin (cis-PLATINUM (II) -Diammine dichloride) were purchased from Sigma (St. Louis, Mo.). Soluble Fas ligand was obtained from the culture supernatant of CHO-K1-Fas cells grown in medium without serum (CHO-S-SFM II Life Technology). Anti-α-tubulin, anti-Apaf-1, anti-rabbit IgG-HRP (horseradish peroxidase), anti-mouse IgG-HRP, anti-rat IgG-HRP, and anti-goat IgG-HRP antibodies are described in Santa Cruz ( Santa Cruz, CA). Anti-Apaf-1 monoclonal antibodies were purchased from Alexis (Lausen, Switzerland), anti-HA antibodies from Boehringer Mannheim (BM, Germany), and cytochrome c antibodies from BD (Franklin Lakes, NJ).

Example 1 Construction of Plasmids

Polymerase chain reaction (PCR) using the Apaf-1 gene as a medium using the front primer: 5'-cgggatcccgatggatgcaaaagctcgaaa-3 '(SEQ ID NO: 6) and the back primer: 5'-cgggatcccgagggcagcaagatctttata-3' (SEQ ID NO: 7) The CARD site (SEQ ID NO: 12) of Apaf-1 amplified through was subcloned into the BamH1 site of the pLexA vector (CLONTECH) (pLexA-CARD).

CDNA encoding the full open reading gene of APIP using the front primer of APIP: 5'-cgcggatccgcgcatgtctggctgtgatg-3 '(SEQ ID NO: 8) and the back primer: 5'-cgcggatccgcgttagacaattccattttc-3' (SEQ ID NO: 9). Clontech: www.BD.com) to amplify by polymerase reaction and each to HAD sequence (atg cgg tcc tac cct tat gat gtg cca gat tat gcc) to pcDNA3 vector (Invitrogen) Kpan1 and EcoR1 sites of pcDNA3 vector Subcloned into the BamH1 site of the pcDNA3-HA vector constructed by insertion into (pcDAN3-APIP). In addition, cDNA encoding the full open reading gene of APIP2 using the front primer: 5'-cgcggatccgcgcatgctcgggagggagactgt-3 '(SEQ ID NO: 10) and the back primer: 5'-cgcggatccgcgttagacaattccattttc-3' (SEQ ID NO: 9) of APIP2. Amplify by polymerase reaction from the library (Clontech: www.BD.com) and each HA tag sequence (atg cgg tcc tac cct tat gat gtg cca gat tat gcc) to pcDNA3 vector (Invitrogen) Kpan1 and Subcloned into the BamH1 site of the pcDNA3-HA vector constructed by inserting into the EcoR1 site (pcDAN3-APIP2).

CDNA encoding the full open reading gene of APIP using the front primer of APIP: 5'-cgcggatccgcgcatgtctggctgtgatg-3 '(SEQ ID NO: 8) and the back primer: 5'-cgcggatccgcgttagacaattccattttc-3' (SEQ ID NO: 9). Clontech: www.BD.com), respectively, and amplified through the polymerase chain reaction from the pEGFP-C1 vector (Clontech:. was sub-cloned into the BamH1 site of the www BD.com) (pGFP-APIP) . In addition, cDNA library encoding the APIP2 full open reading gene using the front primer: 5'-cgcggatccgcgcatgctcgggagggagactgt-3 '(SEQ ID NO: 10) and the back primer: 5'-cgcggatccgcgttagacaattccattttc-3' (SEQ ID NO: 9) of APIP2. amplification through the polymerase chain reaction from: (Clontech BD.com www.) and each of pEGFP-C1 vector: was subcloned into BamH1 site of the (Clontech. www BD.com) (pGFP -APIP2).

Front primer of APIP: 5'-cgcggatccgcgcatgtctggctgtgatg-3 '(SEQ ID NO: 8) and rear primer of APIP: 5'-cgcggatccgcgttagacaattccattttc-3' (SEQ ID NO: 9) using 3'- at the site of XhoI of the pcDNA3-HA vector Plasmids cloned in the 5 'direction were used as antisense plasmids (pcDHA3-APIP / AS).

CARD frontal primer of Apaf-1: 5'-cgggatcccgatggatgcaaaagctcgaaa-3 '(SEQ ID NO: 6) and CARD rear primer of Apaf-1: 5'-cgggatcccgagggcagcaagatctttata-3' (SEQ ID NO: 7) using the pGEX5X-1 vector (Amersham) Pharmacia Biotech: www.apbiotech.com) subcloned to the BamH I site (pGEX5X-CARD). Subcloned to the BamH I site of the pGEX5X-1 vector using APIP Common Anterior Primer: 5'-cggatcccggatgaaatctacattgctc-3 '(SEQ ID NO: 11) and APIP Common Anterior Primer: 5'-cgcggatccgcgttagacaattccattttc-3' (SEQ ID NO: 9). (pGEX5X-APIP)

Example 2: Antibody Production

To produce APIP antibodies, pGEX5X-APIP plasmids were purified from E. coli. Coli BL21 (DE3), And expressed in large quantities in bacteria by addition of 0.3 mM IPTG (isopropyl-1-thio-a-D-galactopyranoside), and purified using Glutathion-Sepharose ™ 4B beads (Amersham Pharmacia Biotech, NJ). Purified protein was injected four times into rabbits. APIP antibodies made in rabbits were purified using only APIP antibodies using antigen-affinity chromatography using SulfoLink kit (PIERCE, IL).

Example 3: Identification of APIP as Apaf-1 Binding Protein

To identify proteins that bind to the CARD domain of Apaf-1, a yeast two-hybrid screening system using a pLexA-CARD vector constructed by binding the ApARD-1 CARD domain to a pLexA vector Human thymic cDNA libraries were examined. Specifically, the CARD region (amino acids 1-89) of Apaf-1 was used in conventional yeast two-hybrid screening using pLexA vector (CLONTECH, Palo Alto, Calif.) And Jurkat cDNA library (Stratagene, La Jolla, Calif.). bait).

As a result, a new Apaf-1 binding protein named APIP was identified. APIP, located on chromosome 11q13.11-11q13.12, consists of 204 amino acids (FIG. 1). In addition, an APIP2 consisting of 242 amino acids having a different amino terminal sequence from APIP was identified through an expression-sequence tag (EST) human gene database (FIG. 1). As a result of sequence alignment, the amino acid sequence of APIP was well preserved in other species.

Tissue distribution of APIP was examined with Northern blot membrane (Clontech) and Northern blot assay using ³² P-radiolabeled APIP and actin as probes. APIP is expressed in all tissues, especially skeletal muscle, heart and kidney. It was confirmed that much expression in the back (Fig. 2).

Yeast two-hybrid assays showed that APIP binds to Apaf-1 but not to FADD protein.

In vitro binding test results showed that in vitro transcription and protein synthesis using the pcDAN3-APIP, pcDNA3-APIP2 plasmids and radiolabeled APIP and APIP2 proteins were performed by E. coli. Caspase-9S protein synthesized in vitro from the pcDNA3-Caspase9S plasmid was not able to bind to GST protein, which was purified by expressing pGEX5X-1 plasmid in E. coli, but was purified by expressing pGEX5X-CARD plasmid. Combined to a similar degree as The binding of the APIP and Apaf-1 proteins was also confirmed that the binding to Apaf-1 through the immunoprecipitation assay using anti-APIP antibody in HeLa cell line. Specifically, HeLa cell lines were prepared using RIPA buffer [50 mM Tris-HCl (pH 7.4), 1% Nonidet P-40, 0.25% Na-deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 μg ml each. ^-1 of aprotinin, leupeptin and pepstatin, 1 mM Na3VO4, and 1 mM NaF], and then the anti-APIP antibody and protein-G-Sepharose (Amersham Pharmacia Biotech) were added to the lysed cell masses. And reacted for 3 hours at 4 ° C. to immunoprecipitate APIP from the cell lysate, centrifuge the immuno-complex, wash three or more times with cold lysis buffer to remove nonspecific proteins, and obtain the SDS-PAGE protein. Separation was carried out and confirmed by Western blot experiment.

The intracellular location of APIP was found to be present in the cytoplasm, as in Apaf-1, by immunochemical analysis and subcellular fractional analysis. Cell fractionation experiments were performed as follows: HEK293 cell line was stored in storage buffer [5 mM Tris-HCl (pH 7.4), 5 mM KCl, 1.5 mM MgCl 2, 0.1 mM EDTA, 1 mM DTT, 0.2 mM PMSF, 5 μg ml ^{− 1} leupetin, 5 μg ml ⁻¹ aprotinin], centrifuged at 500 g for 5 minutes to remove nuclei, and the supernatant was centrifuged at 10,000 g for 30 minutes to collect heavy membrane pellets. , The supernatant was centrifuged at 150,000 g for 90 minutes to obtain a cytoplasmic fraction and Western blotting using each antibody.

These experimental results suggest that APIP selectively binds with Apaf-1.

Example 4 Inhibition of Apoptosis by APIP in Apoptosis Via Mitochondria

Apaf-1 is a protein with the function of activating caspase-9 dependent on cytochrome c. To determine the function of APIP in cell death, pcDAN3-APIP was introduced into C2C12 cell line using LipofectAMINE (Invtrogen Carsbad, CA) reagent, incubated for two weeks in selection medium containing 1 mg / ml G418, and After cell cloning, colonies were screened by Western blot analysis to produce C2C12 cell lines that overexpress APIP (C2C12 / APIP). C2C12 cell line and C2C12 / APIP cell line were treated with etoposide and Fas ligand to measure cell death. Overexpression of APIP inhibited cell death by etoposide, which is known to activate the mitochondrial death pathway. , Did not affect apoptosis induced by Fas (FIG. 4). This inhibitory effect was also confirmed in HeLa and HEK293 cell lines transformed with pGFP-APIP.

Treatment of etoposide with a C2C12 cell line releases cytochrome c in the mitochondria into the cytoplasm during cell death. However, overexpression of APIP in C2C12 cells protected C2C12 cell lines from cell death by etoposide despite the release of cytochrome c from mitochondria. This suppression of cell death was also observed in HeLa and HEK293 cell lines. These results indicate that APIP protects cells from apoptosis by inhibiting the next step in cytochrome c activation from mitochondria.

To confirm the function of APIP as a cell death inhibitor, Bid (tBid) in a form cleaved by caspase-8 was expressed in HeLa cell line. Cell death by tBid was effectively inhibited in cells expressed with APIP. In addition, the function of APIP as a cell death inhibitory protein was confirmed by antisense test using pcDNA3-APIP / AS plasmid. Expression of antisense (SEQ ID NO: 5) significantly increased cell death induced by cisplatin, but did not affect cell death by Fas.

In addition, Western blot experiments confirmed that expression of APIP antisense in HEK293 cell line significantly inhibited APIP expression.

These results suggest that APIP is a protein that inhibits cell death in the mitochondrial mediated cell death pathway.

Example 5 Inhibition of Activation of Cytochrome c Mediated Caspase-9 and Caspase-3 by APIP

Caspase-9 is a protein having its activity through the interaction with Apard-1 CARD, and examined the competitive competition between caspase-9 and APIP in binding to Apaf-1.

As a result of in vitro binding experiments, increasing the amount of in vitro synthesized caspase-9 in the reaction mixture increased the amount of caspase-9 binding to CARD of Apaf-1 and vice versa: Purified GST and GST-CARD bound to Glutathione-Sepharose 4B beads were treated with ³⁵ S-radiolabeled APIP protein, its deletion mutant and caspase-9S for 3 hours at 4 ° C. ((San Luis Obispo, CA) Binding reaction with TNT-coupled transcription / detoxification system from GST precipitate reaction using beads in binding buffer [50 mM Tris-HCl (pH 6.8), 100 mM NaCl, 1 mM DTT, 2 mM EDTA, 0.05% Triton X-100, 1 mM PMSF, and 5% (v / v) glycerol], washed three times or more to remove nonspecific binding protein, separated by 12% SDS PAGE and detected by radioactivity.

These results suggest that APIP and caspase-9 can be mutually competitive in binding to Apaf-1 (FIG. 5).

In addition, to study the ability of APIP to inhibit Apaf-1 mediated caspase activation, a cell free system comprising an S-100 fraction of cells was used.

The cell free system was performed as follows: In vitro, a 3 μl aliquot of Caspase-3 decoded was prepared from 1 mM dATP, 2 μg ml ⁻¹ cytochrome c, and 2 μg or 10 μg purified GST or GST-APIP protein. Incubate with 80 μg of HeLa S-100 fraction (Zou, H., et al., Cell, 90, 405-413, 1997) for 1 hour at 30 ° C., with or without the reaction product and 15% SDS Caspase activation was examined by exposure to X-ray film after PAGE.

When cytochrome c and dATP were added to the S-100 fraction, detoxified caspase-9 and caspase-3 were activated. However, the addition of purified GST-APIP protein to the S-100 fraction inhibited the activation of caspase-9 and caspase-3, but did not activate caspase-9 and caspase-3 when only GST was added.

These results indicate that APIP inhibits the activation of cytochrome c mediated caspases and acts in the next step of cytochrome c released from mitochondria.

Example 6: Apoptosis Inhibitory Activity of the APIP Core Region

In order to investigate functionally important sites in the apoptosis inhibitory activity of APIP, various forms of deletion mutant APIP genes were constructed. The ability of the radiolabeled deletion mutants to bind Apaf-1 to the CARD protein was investigated.

As a result, although the mutants having only the carbosyl end portion showed weak binding ability, all the deletion mutants bound with GST-CARD. To investigate the effect of these mutants on cell death, each was introduced and expressed in the HeLa cell line. Only mutants comprising the common core regions of APIP and APIP2 (amino acids 16-204 of APIP) had cell death inhibitory activity, while other forms did not inhibit cell death.

These results indicate that although both the amino and carboxyl termini of APIP can be associated with Apaf-1, the core part plays an important role in cell death suppression.

Example 7: Inhibition of Ischemic Injury by Accumulation of APIP Expression

APIP is most expressed in skeletal muscle. To investigate whether APIP is involved in ischemic injury, the expression regulation of APIP in ischemia-induced mouse muscle tissue was investigated.

Western blot and immunostaining experiments showed a dramatic increase in the expression of APIP in ischemic conditions induced by the hindlimb's femoral artery in 20-25 g male C57BL / 6 mice (FIG. 6). The expression level of APIP protein increased up to 8-fold in one day of ischemic condition and then returned to normal level on day 7. In ischemic conditions, few cells were killed by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining and Hoechst staining. However, in the muscles of mice expressed after injecting APIP antisense (pcDAN3-APIP / AS 25ug), APIP accumulation was significantly reduced in ischemic conditions, and many cells were killed during TUNEL staining.

These results suggest that accumulation of APIP in muscle cells inhibits cell death induced by ischemia.

To confirm the inhibitory effect of APIP on ischemic injury, we investigated the ischemic injury in mouse muscle cell line C2C12 cells. Unlike differentiated muscle tissue, the undifferentiated C2C12 cell line did not change the expression level of APIP even in hypoxic conditions, and about 50% cell death was observed in 24 hours under hypoxic conditions. However, expression of APIP completely inhibited the death of C2C12 cells that occur under hypoxic conditions (FIG. 7).

These experimental results support that increased APIP expression protects cells from apoptosis induced by ischemia / hypoxia.

APIP or APIP2 which inhibits Apaf-1 mediated activity of the present invention is a novel protein, which can be used to treat or prevent diseases caused by excessive activity of Apaf-1, in particular ischemia / hypoxia, and antisense of APIP protein Antibodies to DNA or RNA or APIP proteins can be used to treat or prevent diseases caused by the underactivity of Apaf-1.

<110> Kwangju Institute of Science and Technology <120> Novel protein inhibiting Apaf-1 mediated activation <130> PR / KJIS / 030428/134 <160> 12 <170> KopatentIn 1.71 <210> 1 <211> 615 <212> DNA <213> homo sapiens <220> <221> CDS (222) (1) .. (612) <400> 1 atg ctc ggg agg gag act gtt gtt ccc gga gat gcg gcg cgc agc gat 48 Met Leu Gly Arg Glu Thr Val Val Pro Gly Asp Ala Ala Arg Ser Asp   1 5 10 15 gaa atc tac att gct cct tca gga gtg caa aag gaa cga att cag cct 96 Glu Ile Tyr Ile Ala Pro Ser Gly Val Gln Lys Glu Arg Ile Gln Pro              20 25 30 gaa gac atg ttt gtt tgt gat ata aat gaa aag gac ata agt gga cct 144 Glu Asp Met Phe Val Cys Asp Ile Asn Glu Lys Asp Ile Ser Gly Pro          35 40 45 tcg cca tcg aag aag cta aaa aaa agc cag tgt act cct ctt ttc atg 192 Ser Pro Ser Lys Lys Leu Lys Lys Ser Gln Cys Thr Pro Leu Phe Met      50 55 60 aat gct tac aca atg aga gga gca ggt gca gtg att cat acc cac tct 240 Asn Ala Tyr Thr Met Arg Gly Ala Gly Ala Val Ile His Thr His Ser  65 70 75 80 aaa gct gct gtg atg gcc acc ctt ctc ttt cca gga cgg gag ttt aaa 288 Lys Ala Ala Val Met Ala Thr Leu Leu Phe Pro Gly Arg Glu Phe Lys                  85 90 95 att aca cat caa gag atg ata aaa gga ata aag aaa tgt act tcc gga 336 Ile Thr His Gln Glu Met Ile Lys Gly Ile Lys Lys Cys Thr Ser Gly             100 105 110 ggg tat tat aga tat gat gat atg tta gtg gta ccc att att gag aat 384 Gly Tyr Tyr Arg Tyr Asp Asp Met Leu Val Val Pro Ile Ilu Glu Asn         115 120 125 aca cct gag gag aaa gac ctc aaa gat aga atg gct cat gca atg aat 432 Thr Pro Glu Glu Lys Asp Leu Lys Asp Arg Met Ala His Ala Met Asn     130 135 140 gaa tac cca gac tcc tgt gca gta ctg gtc aga cgt cat gga gta tat 480 Glu Tyr Pro Asp Ser Cys Ala Val Leu Val Arg Arg His Gly Val Tyr 145 150 155 160 gtg tgg ggg gaa aca tgg gag aag gcc aaa acc atg tgt gag tgt tat 528 Val Trp Gly Glu Thr Trp Glu Lys Ala Lys Thr Met Cys Glu Cys Tyr                 165 170 175 gac tat tta ttt gat att gcc gta tca atg aag aaa gta gga ctt gat 576 Asp Tyr Leu Phe Asp Ile Ala Val Ser Met Lys Lys Val Gly Leu Asp             180 185 190 cct tca cag ctc cca gtt gga gaa aat gga att gtc taa 615 Pro Ser Gln Leu Pro Val Gly Glu Asn Gly Ile Val         195 200 <210> 2 <211> 204 <212> PRT <213> homo sapiens <400> 2 Met Leu Gly Arg Glu Thr Val Val Pro Gly Asp Ala Ala Arg Ser Asp   1 5 10 15 Glu Ile Tyr Ile Ala Pro Ser Gly Val Gln Lys Glu Arg Ile Gln Pro              20 25 30 Glu Asp Met Phe Val Cys Asp Ile Asn Glu Lys Asp Ile Ser Gly Pro          35 40 45 Ser Pro Ser Lys Lys Leu Lys Lys Ser Gln Cys Thr Pro Leu Phe Met      50 55 60 Asn Ala Tyr Thr Met Arg Gly Ala Gly Ala Val Ile His Thr His Ser  65 70 75 80 Lys Ala Ala Val Met Ala Thr Leu Leu Phe Pro Gly Arg Glu Phe Lys                  85 90 95 Ile Thr His Gln Glu Met Ile Lys Gly Ile Lys Lys Cys Thr Ser Gly             100 105 110 Gly Tyr Tyr Arg Tyr Asp Asp Met Leu Val Val Pro Ile Ilu Glu Asn         115 120 125 Thr Pro Glu Glu Lys Asp Leu Lys Asp Arg Met Ala His Ala Met Asn     130 135 140 Glu Tyr Pro Asp Ser Cys Ala Val Leu Val Arg Arg His Gly Val Tyr 145 150 155 160 Val Trp Gly Glu Thr Trp Glu Lys Ala Lys Thr Met Cys Glu Cys Tyr                 165 170 175 Asp Tyr Leu Phe Asp Ile Ala Val Ser Met Lys Lys Val Gly Leu Asp             180 185 190 Pro Ser Gln Leu Pro Val Gly Glu Asn Gly Ile Val         195 200 <210> 3 <211> 729 <212> DNA <213> homo sapiens <220> <221> CDS (222) (1) .. (726) <400> 3 atg tct ggc tgt gat gct ggg gag gga gac tgt tgt tcc cgg aga tgc 48 Met Ser Gly Cys Asp Ala Gly Glu Gly Asp Cys Cys Ser Arg Arg Cys   1 5 10 15 ggc gcg cag gac aag gag cat cca aga tac ctg atc cca gaa ctt tgc 96 Gly Ala Gln Asp Lys Glu His Pro Arg Tyr Leu Ile Pro Glu Leu Cys              20 25 30 aaa cag ttt tac cat tta ggc tgg gtc act ggg act gga gga gga att 144 Lys Gln Phe Tyr His Leu Gly Trp Val Thr Gly Thr Gly Gly Gle Ile          35 40 45 agc ttg aag cat ggc gat gaa atc tac att gct cct tca gga gtg caa 192 Ser Leu Lys His Gly Asp Glu Ile Tyr Ile Ala Pro Ser Gly Val Gln      50 55 60 aag gaa cga att cag cct gaa gac atg ttt gtt tgt gat ata aat gaa 240 Lys Glu Arg Ile Gln Pro Glu Asp Met Phe Val Cys Asp Ile Asn Glu  65 70 75 80 aag gac ata agt gga cct tcg cca tcg aag aag cta aaa aaa agc cag 288 Lys Asp Ile Ser Gly Pro Ser Pro Ser Lys Lys Leu Lys Lys Ser Gln                  85 90 95 tgt act cct ctt ttc atg aat gct tac aca atg aga gga gca ggt gca 336 Cys Thr Pro Leu Phe Met Asn Ala Tyr Thr Met Arg Gly Ala Gly Ala             100 105 110 gtg att cat acc cac tct aaa gct gct gtg atg gcc aca ctt ctc ttt 384 Val Ile His Thr His Ser Lys Ala Ala Val Met Ala Thr Leu Leu Phe         115 120 125 cca gga cgg gag ttt aaa att aca cat caa gag atg ata aaa gga ata 432 Pro Gly Arg Glu Phe Lys Ile Thr His Gln Glu Met Ile Lys Gly Ile     130 135 140 aag aaa tgt act tcc gga ggg tat tat aga tat gat gat atg tta gtg 480 Lys Lys Cys Thr Ser Gly Gly Tyr Tyr Arg Tyr Asp Asp Met Leu Val 145 150 155 160 gta ccc att att gag aat aca cct gag gag aag acc ctc aaa gat aga 528 Val Pro Ile Ile Glu Asn Thr Pro Glu Glu Lys Thr Leu Lys Asp Arg                 165 170 175 atg gct cat gca atg aat gaa tac cca gac tcc tgt gca gta ctg gtc 576 Met Ala His Ala Met Asn Glu Tyr Pro Asp Ser Cys Ala Val Leu Val             180 185 190 aga cgt cat gga gta tat gtg tgg ggg gaa aca tgg gag aag gcc aaa 624 Arg Arg His Gly Val Tyr Val Trp Gly Glu Thr Trp Glu Lys Ala Lys         195 200 205 acc atg tgt gag tgt tat gac tat tta ttt gat att gcc gta tca atg 672 Thr Met Cys Glu Cys Tyr Asp Tyr Leu Phe Asp Ile Ala Val Ser Met     210 215 220 aag aaa gta gga ctt gat cct tca cag ctc cca gtt gga gaa aat gga 720 Lys Lys Val Gly Leu Asp Pro Ser Gln Leu Pro Val Gly Glu Asn Gly 225 230 235 240 att gtc taa 729 Ile val <210> 4 <211> 242 <212> PRT <213> homo sapiens <400> 4 Met Ser Gly Cys Asp Ala Gly Glu Gly Asp Cys Cys Ser Arg Arg Cys   1 5 10 15 Gly Ala Gln Asp Lys Glu His Pro Arg Tyr Leu Ile Pro Glu Leu Cys              20 25 30 Lys Gln Phe Tyr His Leu Gly Trp Val Thr Gly Thr Gly Gly Gle Ile          35 40 45 Ser Leu Lys His Gly Asp Glu Ile Tyr Ile Ala Pro Ser Gly Val Gln      50 55 60 Lys Glu Arg Ile Gln Pro Glu Asp Met Phe Val Cys Asp Ile Asn Glu  65 70 75 80 Lys Asp Ile Ser Gly Pro Ser Pro Ser Lys Lys Leu Lys Lys Ser Gln                  85 90 95 Cys Thr Pro Leu Phe Met Asn Ala Tyr Thr Met Arg Gly Ala Gly Ala             100 105 110 Val Ile His Thr His Ser Lys Ala Ala Val Met Ala Thr Leu Leu Phe         115 120 125 Pro Gly Arg Glu Phe Lys Ile Thr His Gln Glu Met Ile Lys Gly Ile     130 135 140 Lys Lys Cys Thr Ser Gly Gly Tyr Tyr Arg Tyr Asp Asp Met Leu Val 145 150 155 160 Val Pro Ile Ile Glu Asn Thr Pro Glu Glu Lys Thr Leu Lys Asp Arg                 165 170 175 Met Ala His Ala Met Asn Glu Tyr Pro Asp Ser Cys Ala Val Leu Val             180 185 190 Arg Arg His Gly Val Tyr Val Trp Gly Glu Thr Trp Glu Lys Ala Lys         195 200 205 Thr Met Cys Glu Cys Tyr Asp Tyr Leu Phe Asp Ile Ala Val Ser Met     210 215 220 Lys Lys Val Gly Leu Asp Pro Ser Gln Leu Pro Val Gly Glu Asn Gly 225 230 235 240 Ile val <210> 5 <211> 615 <212> DNA <213> Artificial Sequence <220> <223> antisense of APIP gene <400> 5 ttagacaatt ccattttctc caactgggag ctgtgaagga tcaagtccta ctttcttcat 60 tgatacggca atatcaaata aatagtcata acactcacac atggttttgg ccttctccca 120 tgtttccccc cacacatata ctccatgacg tctgaccagt actgcacagg agtctgggta 180 ttcattcatt gcatgagcca ttctatcttt gaggtctttc tcctcaggtg tattctcaat 240 aatgggtacc actaacatat catcatatct ataataccct ccggaagtac atttctttat 300 tccttttatc atctcttgat gtgtaatttt aaactcccgt cctggaaaga gaagggtggc 360 catcacagca gctttagagt gggtatgaat cactgcacct gctcctctca ttgtgtaagc 420 attcatgaaa agaggagtac actggctttt ttttagcttc ttcgatggcg aaggtccact 480 tatgtccttt tcatttatat cacaaacaaa catgtcttca ggctgaattc gttccttttg 540 cactcctgaa ggagcaatgt agatttcatc gctgcgcgcc gcatctccgg gaacaacagt 600 ctccctcccg agcat 615 <210> 6 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 cgggatcccg atggatgcaa aagctcgaaa 30 <210> 7 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 cgggatcccg agggcagcaa gatctttata 30 <210> 8 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 cgcggatccg cgcatgtctg gctgtgatg 29 <210> 9 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 cgcggatccg cgttagacaa ttccattttc 30 <210> 10 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 cgcggatccg cgcatgctcg ggagggagac tgt 33 <210> 11 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 cggatcccgg atgaaatcta cattgctc 28 <210> 12 <211> 267 <212> PRT <213> homo sapiens <220> <221> PEPTIDE (222) (1) .. (267) <223> Apaf-1CARD <400> 12 Ala Thr Gly Gly Ala Thr Gly Cys Ala Ala Ala Ala Ala Gly Cys Thr Cys   1 5 10 15 Gly Ala Ala Ala Thr Thr Gly Thr Thr Thr Gly Cys Thr Thr Cys Ala              20 25 30 Ala Cys Ala Thr Ala Gly Ala Gly Ala Ala Gly Cys Thr Cys Thr Gly          35 40 45 Gly Ala Ala Ala Ala Gly Gly Ala Cys Ala Thr Cys Ala Ala Gly Ala      50 55 60 Cys Ala Thr Cys Cys Thr Ala Cys Ala Thr Cys Ala Thr Gly Gly Ala  65 70 75 80 Thr Cys Ala Cys Ala Thr Gly Ala Thr Thr Ala Gly Thr Gly Ala Thr                  85 90 95 Gly Gly Ala Thr Thr Thr Thr Thr Ala Ala Cys Ala Ala Thr Ala Thr             100 105 110 Cys Ala Gly Ala Ala Gly Ala Gly Gly Ala Ala Ala Ala Ala Gly Thr         115 120 125 Ala Ala Gly Ala Ala Ala Thr Gly Ala Gly Cys Cys Cys Ala Cys Thr     130 135 140 Cys Ala Ala Cys Ala Gly Cys Ala Ala Ala Gly Ala Gly Cys Ala Gly 145 150 155 160 Cys Thr Ala Thr Gly Cys Thr Gly Ala Thr Thr Ala Ala Ala Ala Thr                 165 170 175 Gly Ala Thr Ala Cys Thr Thr Ala Ala Ala Ala Ala Ala Gly Ala Thr             180 185 190 Ala Ala Thr Gly Ala Thr Thr Cys Cys Thr Ala Cys Gly Thr Ala Thr         195 200 205 Cys Ala Thr Thr Cys Thr Ala Cys Ala Ala Thr Gly Cys Thr Cys Thr     210 215 220 Ala Cys Thr Ala Cys Ala Thr Gly Ala Ala Gly Gly Ala Thr Ala Thr 225 230 235 240 Ala Ala Ala Gly Ala Thr Cys Thr Thr Gly Cys Thr Gly Cys Cys Cys                 245 250 255 Thr Thr Cys Thr Cys Cys Ala Thr Gly Ala Thr             260 265

Claims (25)

An isolated APIP protein comprising a sequence having at least 80% homology with the amino acid sequence set forth in SEQ ID NO: 2 and which inhibits Apaf-1 mediated activation. The isolated APIP protein of claim 1, comprising a sequence having at least 90% homology with the sequence set forth in SEQ ID NO: 2. An isolated APIP2 protein comprising a sequence having at least 80% homology with the amino acid sequence set forth in SEQ ID NO: 4 and which inhibits Apaf-1 mediated activation. The isolated APIP2 protein of claim 3, comprising a sequence having at least 90% homology with the sequence set forth in SEQ ID NO: 4. A nucleic acid sequence encoding the APIP protein according to claim 1. The nucleic acid sequence of claim 5 wherein said nucleic acid sequence is gDNA, cDNA or RNA. The nucleic acid sequence of claim 5 comprising the sequence as set forth in SEQ ID NO: 1. A nucleic acid sequence encoding the APIP2 protein according to claim 3 or 4. The nucleic acid sequence of claim 8 wherein the nucleic acid sequence is gDNA, cDNA or RNA. The nucleic acid sequence of claim 8 comprising the sequence as set forth in SEQ ID NO: 3. A vector comprising a nucleic acid sequence encoding the APIP protein according to claim 1. The vector of claim 11, wherein the nucleic acid sequence is the sequence set forth in SEQ ID NO: 1. A vector comprising a nucleic acid sequence encoding the APIP2 protein according to claim 3. The vector of claim 12, wherein the nucleic acid sequence is the sequence set forth in SEQ ID NO: 3. A host cell transformed or transfected with the vector according to claim 11. A host cell transformed or transfected with the vector according to claim 13 or 14. A method for producing an isolated APIP protein, comprising culturing the transformed or transfected host cell according to claim 15 and purifying the APIP protein from the culture. A method for producing an isolated APIP2 protein, comprising culturing the transformed or transfected host cell according to claim 16 and purifying the APIP2 protein from the culture. An antibody against the APIP protein of claim 1. An antibody against the APIP2 protein of claim 3. The active ingredient is selected from the group consisting of the APIP protein expression vector of claim 11, the isolated APIP protein of claim 1, or an mRNA thereof and a mixture of two or more thereof, and a therapeutically or prophylactically effective amount of the active ingredient alone or pharmaceutically A pharmaceutical composition useful for treating or preventing a disease caused by excessive activity of Apaf-1, comprising an acceptable carrier. The active ingredient is selected from the group consisting of the APIP2 protein expression vector of claim 13, the isolated APIP2 protein of claim 3, or an mRNA thereof and a mixture of two or more thereof, and a therapeutically or prophylactically effective amount of the active ingredient alone or pharmaceutically A pharmaceutical composition useful for treating or preventing a disease caused by excessive activity of Apaf-1, comprising an acceptable carrier. An antisense DNA or RNA of the APIP protein of claim 1 or an antibody against the APIP protein of claim 19 and a mixture of two or more thereof as the active ingredient and a therapeutically or prophylactically effective amount of the active ingredient alone or in a medicament A pharmaceutical composition useful for treating or preventing a disease caused by under-activity of Apaf-1, characterized in that it comprises a pharmaceutically acceptable carrier. A pharmaceutical composition for preventing or treating ischemia or hypoxia, comprising the APIP protein expression plasmid of claim 11, the isolated APIP protein of claim 1, or an mRNA or a mixture of two or more thereof as an active ingredient. A pharmaceutical composition for preventing or treating ischemia or hypoxia, comprising the APIP2 protein expression plasmid of claim 13, the isolated APIP2 protein of claim 3, or an mRNA thereof or a mixture of two or more thereof as an active ingredient.