WO2009136671A1 - Protéine régulatrice p53 et ses utilisations - Google Patents

Protéine régulatrice p53 et ses utilisations Download PDF

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Publication number
WO2009136671A1
WO2009136671A1 PCT/KR2008/002559 KR2008002559W WO2009136671A1 WO 2009136671 A1 WO2009136671 A1 WO 2009136671A1 KR 2008002559 W KR2008002559 W KR 2008002559W WO 2009136671 A1 WO2009136671 A1 WO 2009136671A1
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aimp2
polypeptide
seq
cell
amino acid
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PCT/KR2008/002559
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English (en)
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Sunghoon Kim
Jung Min Han
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Seoul National University Industry Foundation
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Priority to KR1020107026716A priority Critical patent/KR20110021825A/ko
Priority to PCT/KR2008/002559 priority patent/WO2009136671A1/fr
Publication of WO2009136671A1 publication Critical patent/WO2009136671A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4747—Apoptosis related proteins

Definitions

  • the present invention relates to p53 regulatory protein and uses thereof.
  • p53 which is well known as a human tumor suppressor gene, regulates expression of various downstream genes in response to DNA damage signals. Through this, p53 regulates cell cycle and induces apoptosis, thereby preventing cancer. In about 50% naturally occurring primary tumors, mutation of p53 gene is observed. In some tumors, particularly in breast cancer, lung cancer and colon cancer, their mutation rate reaches 85%. As such, the mutation of p53 gene is closely related with carcinogenesis of cells.
  • DM2 double minute 2
  • DM2 double minute 2
  • phosphorylation and acetylation modifications
  • Such stresses include those affecting DNAs such as ⁇ -ray, UV or anticancer drugs e.g. adriamycin, cisplatin, etoposide, etc., as well as nucleotide removal, heat shock, oxygen deficiency, and the like.
  • the stabilized p53 forms a tetramer in the nucleus and binds at the p53 binding site in a promoter of various downstream target genes (e.g. CD95/FAS, Bax, PUMA, caspases 1 and -6, etc.) .
  • the p53 biding site has a well-conserved base sequence of 5'- PuPuPuC (A/T) (T/A) GPyPyPy-3' .
  • the DNA-bound p53 activates the transcription of many genes mainly involved in cell cycle regulation, DNA damage repair and apoptosis regulation, and, inhibits the expression of some target genes .
  • a representative pathway of activating p53 is the interruption of the degradation of p53 through inhibition of DM2 (HDM2 or DMD2) .
  • DM2 which is a representative p53 inhibitor, binds at the transcription-activating domain of p53 and acts as ubiquitin ligase for p53, thereby inducing ubiquitination of p53. So it is mediated degradation of p53 by the 26S proteasome pathway.
  • the modulation of the bioactivity (e.g. cellular level, stability, etc.) of p53 may affect a variety of cellular activities and may provide a therapeutic means for various diseases or conditions. Accordingly, the modulation of p53 have been studied extensively over the last decade (Levine, A. J. et al., Cell, 116, 67-69, 2004; Levine, A. J. et al., Cell, 88, 323-331, 1997) .
  • AIMP2 also known as JTV-I or p38/JTVl (Nicolaides et al., Genomics, 29:329-334, 1995; Korean Patent Application No. 2003-18424), is first announced as the factor that is related with the macromolecular mass tRNA synthetase complex (Quevillon S. et al., J. MoI. Biol. 285:183-195, 1999) .
  • AIMP2 serves as a structural backbone for structural component assembly.
  • the gene encoding AIMP2 is located on human chromosome 7, and is arranged in a head-to-head fashion with a gene encoding PMS2 which is involved in DNA mismatch repair (Kolodner R. D. et al .
  • AIMP2 consists of 320 amino acid residues in mice, and 312 amino acid residues in humans (Quevillon S. et al., J. MoI. Biol., 285:183-195, 1999) .
  • AIMP2 down-regulates c-myc, which activates p53, thereby inhabiting cell proliferation during differentiation of alveolar cells in the lung.
  • AIMP2 facilitates apoptosis by binding to PDK and inhibiting phosphorylation of AKT.
  • An object of the present invention is to provide a new use of AIMP2 as a positive regulator of p53.
  • the present invention provides a method for inhibiting ubiquitination of p53 in a cell or tissue, the method comprising administering to a cell or tissue in need thereof an effective amount of one or more selected from the group consisting of: (a) an isolated (AIMP2) polypeptide having an amino acid sequence represented by SEQ ID NO: 1;
  • the present invention provides a method for modulating bioactivity of p53 in a cell or tissue, the method comprising administering to a cell or tissue in need thereof an effective amount of one or more selected from the group consisting of: (a) an isolated (AIMP2) polypeptide having an amino acid sequence represented by SEQ ID NO: 1;
  • the present invention provides a method for identifying an agent modulating p53 activity, the method comprising:
  • the present invention provides a method for identifying an agent modulating interaction between p53 and an AIMP2 polypeptide, the method comprising :
  • test agent for activity to modulate interaction between p53 and the AIMP2 polypeptide.
  • the present invention provides a method for detecting the presence of p53, the method comprising:
  • the present invention provides a method for isolating p53, the method comprising:
  • a "expression”, as used herein, refers to formation of protein or nucleic acid in cells.
  • a "host cell,” as used herein, refers to a prokaryotic or eukaryotic cell that contains heterologous DNA that has been introduced into the cell by any means, e.g., electroporation, calcium phosphate precipitation, microinjection, transformation, viral infection, and/or the like.
  • isolated means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring) .
  • a naturally-occurring nucleic acid, polypeptide, or cell present in a living animal is not isolated, but the same polynucleotide, polypeptide, or cell separated from some or all of the coexisting materials in the natural system, is isolated, even if subsequently reintroduced into the natural system.
  • nucleic acids can be part of a vector and/or such nucleic acids or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment .
  • modulate refers to a change in the cellular level or other biological activities of the p53 transcription factor. Modulation of p53 bioactivities can be up- regulation (i.e., activation or stimulation) or down- regulation (i.e. inhibition or suppression) . For example, modulation may cause a change in cellular level of p53, enzymatic modification (e.g., phosphorylation) of p53, binding characteristics (e.g., binding to a target transcription regulatory element), or any other biological, functional, or immunological properties of p53.
  • enzymatic modification e.g., phosphorylation
  • binding characteristics e.g., binding to a target transcription regulatory element
  • the change in activity can arise from, for example, an increase or decrease in expression of the p53 gene, the stability of mRNA that encodes the p53 protein, translation efficiency, or from a change in other bioactivities of the p53 transcription factor (e.g., regulating expression of a p53-responsive gene) .
  • the mode of action of a p53 modulator can be direct, e.g., through binding to the p53 protein or to genes encoding the p53 protein.
  • the change can also be indirect, e.g., through binding to and/or modifying (e.g., enzymatically) another molecule which otherwise modulates p53 (e.g., a kinase that specifically phosphorylates p53) .
  • polypeptide is used interchangeably herein with the terms “polypeptides” and “protein (s) ", and refers to a polymer of amino acid residues, e.g., as typically found in proteins in nature.
  • AIMP2 polypeptide refers to a polypeptide known as JTVl or p38/JTV.
  • the AIMP2 may be polypeptide having an amino acid sequence of SEQ ID NO: l(GenBank Accession No: AAH13630; Strausberg, R. L. et al., Proc. Natl. Acad. U.S.A. 99 (26) : 16899-16903, 2002). And the inventive AIMP2 includes functional equivalents thereof .
  • polypeptide comprising the amino acid sequence having at least 70% amino acid sequence homology (i . e. , identity), preferably at least 80%, and more preferably at least 90%, for example, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% amino acid sequence homology, that exhibit substantially identical physiological activity to the polypeptide of SEQ ID NO: 1.
  • the "substantially identical physiological activity” means p53 induction activity or binding activity to p53.
  • the functional equivalents may include, for example peptides produced by as a result of addition, substitution or deletion of some amino acid of SEQ ID NO:1.
  • Substitutions of the amino acids are preferably conservative substitutions. Examples of conservative substitutions of naturally occurring amino acids are as follows: aliphatic amino acids (GIy, Ala, Pro), hydrophobic amino acids (He, Leu, VaI), aromatic amino acids (Phe, Tyr, Trp) , acidic amino acids (Asp, GIu) , basic amino acids (His, Lys, Arg, GIn, Asn) and sulfur-containing amino acids (Cys, Met) .
  • the functional equivalents also include variants with deletion of some of the amino acid sequence of the inventive AIMP2. Deletion or substitutions of the amino acids are preferably located at regions that are not directly involved in the physiological activity of the inventive polypeptide. And deletion of the amino acids is preferably located at regions that are not directly involved in the physiological activity of the AIMP2.
  • the functional equivalents also include amino acid residues 162-225 of SEQ ID NO: 1, biding region with p53.
  • the preferable functional equivalent of the inventive polypeptide may have an amino acid sequence of SEQ ID NO: 2 (GenBank Accession No: AAC50391; Nicolaides, N. C. et al., Genomics 29 (2) : 329-334, 1995) .
  • the functional equivalents also include variants with addition of several amino acids in both terminal ends of the amino acid sequence of the AIMP2 or in the sequence.
  • inventive functional equivalents also include polypeptide derivatives which have modification of some of the chemical structure of the inventive polypeptide while maintaining the fundamental backbone and physiological activity of the inventive polypeptide. Examples of this modification include structural modifications for changing the stability, storage, volatility or solubility of the inventive polypeptide.
  • Sequence identity or homology is defined herein as the percentage of amino acid residues in the candidate sequence that are identical with amino acid sequence of AIMP2 (SEQ ID NO: 1) , after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions (as described above) as part of the sequence identity. None of N-terminal, C- terminal, or internal extensions, deletions, or insertions into the amino acid sequence of AIMP2 shall be construed as affecting sequence identity or homology. Thus, sequence identity can be determined by standard methods that are commonly used to compare the similarity in position of the amino acids of two polypeptides.
  • two polypeptides are aligned for optimal matching of their respective amino acids (either along the full length of one or both sequences or along a predetermined portion of one or both sequences) .
  • the programs provide a default opening penalty and a default gap penalty, and a scoring matrix such as PAM 250 (a standard scoring matrix; see Dayhoff et al., in Atlas of Protein Sequence and Structure, vol. 5, supp. 3 (1978)) can be used in conjunction with the computer program.
  • PAM 250 a standard scoring matrix; see Dayhoff et al., in Atlas of Protein Sequence and Structure, vol. 5, supp. 3 (1978)
  • the percent identity can be calculated as the follow. The total number of identical matches multiplied by 100 and then divided by the sum of the length of the longer sequence within the matched span and the number of gaps introduced into the longer sequences in order to align the two sequences.
  • the polypeptide according to the present invention can be prepared by separating from nature materials or genetic engineering methods.
  • a DNA molecule encoding the AIMP2 or its functional equivalents (ex: SEQ ID NO: 3 or SEQ ID NO: 4) is constructed according to any conventional method.
  • the DNA molecule may synthesize by performing PCR using suitable primers (ex: SEQ ID NO: 12, SEQ ID NO: 13) .
  • the DNA molecule may also be synthesized by a standard method known in the art, for example using an automatic DNA synthesizer (commercially available from Biosearch or Applied Biosystems) .
  • the constructed DNA molecule is inserted into a vector comprising at least one expression control sequence (ex: promoter, enhancer) that is operatively linked to the DNA sequence so as to control the expression of the DNA molecule, and host cells are transformed with the resulting recombinant expression vector.
  • the transformed cells are cultured in a medium and condition suitable to express the DNA sequence, and a substantially pure polypeptide encoded by the DNA sequence is collected from the culture medium.
  • the collection of the pure polypeptide may be performed using a method known in the art, for example, chromatography.
  • the term "substantially pure polypeptide" means the inventive polypeptide that does not substantially contain any other proteins derived from host cells.
  • inventive polypeptide can be chemically synthesized easily according to any technique known in the art (Creighton, Proteins: Structures and Molecular Principles, W. H. Freeman and Co., NY, 1983) .
  • a typical technique they are not limited to, but include liquid or solid phase synthesis, fragment condensation, F-MOC or T-BOC chemistry (Chemical Approaches to the Synthesis of Peptides and Proteins, Williams et al., Eds., CRC Press, Boca Raton Florida, 1997; A Practical Approach, Atherton & Sheppard, Eds., IRL Press, Oxford, England, 1989) .
  • nucleic acid refers to a deoxyribonucleotide or ribonucleotide polymer in either single- or double- stranded form, and unless otherwise limited, encompasses known analogues of natural nucleotides that hybridize to nucleic acids in manner similar to naturally occurring nucleotides .
  • the term “the nucleotide encoding AIMP2 or functional equivalents thereof” may have a nucleic acid encoding a polypeptide having the amino acid sequence of SEQ ID NO: 1 or an polypeptide having the amino acid sequence homology of at least 70% to the polypeptide (ex: SEQ ID N0:2) .
  • the nucleic acid includes DNA, cDNA or RNA.
  • the polynucleotide may have a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1 or an the amino acid sequence homology of at least 70% to SEQ ID NO: 1 (ex: SEQ ID N0:2) .
  • the polynucleotide comprises the nucleotide sequence of SEQ ID NO. 3 or SEQ ID NO. 4.
  • the nucleic acid can be isolated from a natural source or be prepared by a genetic engineering method known in the art.
  • analog is used herein to refer to a molecule that structurally resembles a reference molecule but which has been modified in a targeted and controlled manner, by replacing a specific substituent of the reference molecule with an alternate substituent. Compared to the reference molecule, an analog would be expected, by one skilled in the art, to exhibit the same, similar, or improved utility. Synthesis and screening of analogs, to identify variants of known compounds having improved traits (such as higher binding affinity for a target molecule) is an approach that is well known in pharmaceutical chemistry.
  • nucleic acids and/or nucleic acid sequences are homologous when they are derived, naturally or artificially, from a common ancestral nucleic acid or nucleic acid sequence.
  • the term "effective amount” refers to an amount showing an effect of the modulating p53 bioactiviy (ex: cellular levels etc.) differently to normal cells or tissues or the inhibiting the ubiquitination of p53.
  • contacting has its normal meaning and refers to combining two or more agents (e.g., two polypeptides) or combining agents and cells (e.g., a protein and a cell).
  • Contacting can occur in vitro, e.g., combining two or more agents or combining a test agent and a cell or a cell lysate in a test tube or other container.
  • Contacting can also occur in a cell or in situ, e.g., contacting two polypeptides in a cell by coexpression in the cell of recombinant polynucleotides encoding the two polypeptides, or in a cell lysate.
  • agent includes any substance, molecule, element, compound, entity, or a combination thereof. It includes, but is not limited to, e.g., protein, polypeptide, small organic molecule, polysaccharide, polynucleotide, and the like. It can be a natural product, a synthetic compound, or a chemical compound, or a combination of two or more substances. Unless otherwise specified, the terms “agent”, “substance”, and “compound” can be used interchangeably.
  • Some test agents are synthetic molecules, and others natural molecules.
  • Test agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds.
  • Combinatorial libraries can be produced for many types of compound that can be synthesized in a step- by-step fashion.
  • Large combinatorial libraries of compounds can be constructed by the encoded synthetic libraries (ESL) method described in WO 95/12608, WO 93/06121, WO 94/08051, WO 95/35503 and WO 95/30642.
  • Peptide libraries can also be generated by phage display methods (see, e.g., Devlin, WO 91/18980) .
  • Libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts can be obtained from commercial sources or collected in the field.
  • Known pharmacological agents can be subject to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification to produce structural analogs .
  • test agents can be naturally occurring proteins or their fragments. Such test agents can be obtained from a natural source, e.g., a cell or tissue lysate. Libraries of polypeptide agents can also be prepared, e.g., from a cDNA library commercially available or generated with routine methods.
  • the test agents can also be peptides, e.g., peptides of from about 5 to about 30 amino acids, with from about 5 to about 20 amino acids being preferred and from about 7 to about 15 being particularly preferred.
  • the peptides can be digests of naturally occurring proteins, random peptides, or "biased" random peptides.
  • test agents can also be "nucleic acids".
  • Nucleic acid test agents can be naturally occurring nucleic acids, random nucleic acids, or "biased” random nucleic acids. For example, digests of prokaryotic or eukaryotic genomes can be similarly used as described above for proteins.
  • the test agents are small molecules (e.g., molecules with a molecular weight of not more than about 1,000) .
  • high throughput assays are adapted and used to screen for such small molecules.
  • combinatorial libraries of small molecule test agents as described above can be readily employed to screen for small molecule modulators of p53.
  • a number of assays are available for such screening, e.g., as described in Schultz (1998) Bioorg Med Chem Lett 8:2409-2414; Weller (1997) MoI Divers. 3:61-70; Fernandes (1998) Curr Opin Chem Biol 2:597-603; and Sittampalam (1997) Curr Opin Chem Biol 1:384-91.
  • Libraries of test agents to be screened with methods of the present invention can also be generated based on structural studies of the AIMP2, its fragment or its analog. Such structural studies allow the identification of test agents that are more likely to bind to the AIMP2.
  • the three-dimensional structures of the AIMP2 can be studied in a number of ways, e.g., crystal structure and molecular modeling. Methods of studying protein structures using x-ray crystallography are well known in the literature. See Physical Biochemistry, Van Holde, K. E. (Prentice-Hall, New Jersey 1971), pp. 221-239, and Physical Chemistry with Applications to the Life Sciences, D. Eisenberg & D. C. Crothers (Benjamin Cummings, Menlo Park 1979) .
  • AIMP2 and p53 induced genotoxic stress such as adriamycin treatment, UV irradiation and apoptosis treatment (See Fig.5) . It was confirmed that the induction of p53 by genotoxic stress in AIMP2 deficient cells was inhibited remarkably (See Fig 7 and Fig 8), p53 expression increased remarkably in the cells transfected with AIMP2 (See Fig.6) . This means AIMP2 works as an upstream regulator of p53. The expression of p53 was induced by AIMP2 with a concentration-dependent manner (See Fig 9) . The induction of p53 by genotoxic stress was inhibited by transfection of AIMP2 siRNA (See Fig.10) .
  • AIMP2 siRNA See Fig.11
  • AIMP2 siRNA See Fig.11
  • the stress response of AIMP2 was regulated not at the transcriptional level but at the post- translational level (See Fig 12) .
  • AIMP2 was induced by various genotoxic stress, thereby translocation into nucleus (See Fig 13) and directly interaction with p53 in nucleus (See Fig 14 and Fig 15) .
  • the central region of AIMP2 (162- 225 of SEQ ID:1) interacts with the N-terminal region of p53 (1-132 of SEQ ID: 6) (See Fig 16 to Fig 19) .
  • the AIMP2 suppressed competitively the binding of MDM2 and p53 via the interaction with p53 and suppressed MDM2-mediated p53 ubiquitination (proteosome-mediated degradation) (See Fig 20 and Fig 21) .
  • This means AIMP2 plays an important role for stabilization of native p53.
  • AIMP2 shows a different binding affinity depending on p53 phosphorylation. That is, the binding of AIMP2 to unphosphorylated p53 has higher affinity than the binding of AIMP2 to phosphorylated p53 (See Fig 22 to Fig 24) . This result indicates that AIMP2 dissociated from p53 when it was phosphorylated by its upstream kinase. Therefore, the induced AIMP2 binds to p53 under apoptotic condition, thereby protecting p53 from the negative regulator, MDM2 until it is phosphorylated by its upstream kinase.
  • AIMP2 would be dissociated from p53 so that the phosphorylated p53 can form its active tetramer.
  • I The modulation of p53 bioactivity and inhibition of p53 ubiquitination by AIMP2
  • the present invention provides a method for modulating bioactivity of p53 and inhibiting ubiquitination of p53 in a cell or tissue.
  • P53 is a transcription factor involved various biological pathways and cell activities (Almog et al., Biochim. Biophy. Acta., 1378 : R42-R54 , 1998).
  • the broad spectrum of p53 bioactivity is disclosed in documents and specification of the present invention.
  • p53 has a pivotal role for cell cycle arrest and regulation of apoptosis.
  • the induction of p53 by DNA damage is involved in DNA repair and cell development or differentiation.
  • p53 could be involved in the cytoskeletal modification.
  • the activation of the p53 pathway can bring about trans-regulation of p53 target genes (p53 response genes) , alteration of cell adhesion, and secretion of extracellular factors (ex: growth inhibitor) .
  • p53 bioactivity refers to a biochemical and physiological role of p53 under regulation of cellular process.
  • modulation of p53 bioactivity may include other biological or cellular activities of p53 as well as regulation of intracellular level of p53.
  • p53 bioactivity may include, but is not limited, intracellular levels of p53, stabilities of p53, tetramer formation, binding activities (ex: binding to target transcription factors) , regulation of p53 responsive genes expression, interaction with other regulating proteins or molecules (ex: suppressor c-ABl or replication protein A) , regulation of cell proliferation or cell adhesion, or regulation of cell growth or apoptosis .
  • AIMP2 Modulating of p53 bioactivity or inhibiting of ubiquitination of the present invention is mediated by AIMP2. Therefore, the AIMP2 could be contacted to a cell or tissue in order to regulate p53 bioactivity and inhibit p53 ubiquitination in human. In some case, AIMP2 could be injected to a subject (ex: human or non human being) . In other case, a nucleic acid encoding AIMP2 could be introduced using retrovirus or other means (see below in detail) .
  • the methods may comprise administering to a cell or tissue in need thereof an effective amount of one or more selective from a group consisting of: (a) an isolated (AIMP2) polypeptide having an amino acid sequence represented by SEQ ID NO: 1; (b) an isolated polypeptide having an amino acid sequence homology of at least 70% with the polypeptide of (a) ; and (c) an isolated nucleotide encoding the polypeptide of (a) or (b) .
  • the inventive polypeptide can be administered by oral route or by parenteral route.
  • Oral administrations include sublingual application.
  • Parenteral administrations include injection techniques, such as subcutaneous injection, intramuscular injection and intravenous injection, as well as drip infusion.
  • the polypeptide can be formulated into various forms with a pharmaceutically acceptable carrier by a conventional method.
  • pharmaceutically acceptable carrier means a substance which is physiologically acceptable and, when administered to human beings, generally does not cause allergic reactions, such as gastrointestinal disorder and dizziness, or similar reactions thereto.
  • the pharmaceutically acceptable carriers in the case of oral administration, there may be used binders, lubricants, disintegrants, excipients, solubilizers, dispersing agents, stabilizers, suspension agents, pigments and flavors, and in case of injection agent, there can be used buffers, preservatives, analgesics, solubilizers, isotonics and stabilizers, and in case of formulations for local administration may include bases, excipients, lubricants and preservatives.
  • the inventive pharmaceutical composition containing the AIM3 protein may be formulated into various forms with the pharmaceutically acceptable carriers.
  • the inventive composition may be formulated into the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers and so on, and for injection agent, it may be formulated into unit dose ampoules or multiple dose products.
  • a total effective amount of the AIM2 protein of the present invention can be administered to patients in a single dose or can be administered by a fractionated treatment protocol, in which multiple doses are administered over a more prolonged period of time.
  • the pharmaceutical composition comprising the inventive AIMP2 polypeptide may vary an effective amount depending on the severity of diseases, the protein may be generally administered several times a day at an effective dose of 1 ⁇ g-10 mg.
  • a suitable dose of the AIM2 protein may depend on many factors, such as the age, body weight, health condition, sex, disease severity, diet and excretion of patients, as well as the route of administration and the number of treatments to be administered. In view of these factors, any person skilled in the art may determine an effective dose for increasing p53 intracellular level.
  • the inventive pharmaceutical composition containing the AIM2 protein has no special limitations on its formulation, administration route and/or administration mode insofar as it shows the effects of the present invention.
  • the nucleic acid encoding the said polypeptide (a) or (b) can be administered via an expression vector such as a plasmid or viral vector.
  • an expression vector such as a plasmid or viral vector.
  • the nucleic acid can be inserted into the expression vector, and then it can be introduced into a target cell by any method known in the art, such as infection, transfection and transduction.
  • a gene transfer method using a plasmid expression vector is a method of transferring a plasmid DNA directly to mammalian cells, which is an FDA-approved method applicable to human beings (Nabel, E. G., et al., Science, 249:1285-1288, 1990) .
  • the plasmid DNA has an advantage of being homogeneously purified.
  • Plasmid expression vectors which can be used in the present invention include mammalian expression plasmids known in the pertinent art. For example, they are not limited to, but typically include pRK5 (European Patent No. 307,247), pSVl ⁇ B (PCT Publication No. 91/08291) and pVL1392 (PharMingen) .
  • the plasmid expression vector containing the said polynucleotide may be introduced into target cells by any method known in the art, including, but not limited to, transient transfection, microinjection, transduction, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE dextran-mediated transfection, polybrene-mediated transfection, electroporation, gene gun methods, and other known methods for introducing DNA into cells (Wu et al., J. Bio. Chem. , 267:963-967, 1992; Wu and Wu, J. Bio. Chem., 263:14621-14624, 1988) .
  • virus expression vectors containing the said polynucleotide include, but are not limited to, retrovirus, adenovirus, herpes virus, avipox virus and so on.
  • the retroviral vector is so constructed that non- viral proteins can be produced within the infected cells by the viral vector in which virus genes are all removed or modified.
  • the main advantages of the retroviral vector for gene therapy are that it transfers a large amount of genes into replicative cells, precisely integrates the transferred genes into cellular DNA, and does not induce continuous infections after gene transfection (Miller, A. D., Nature, 357:455-460, 1992) .
  • the retroviral vector approved by FDA was prepared using PA317 amphotropic retrovirus packaging cells (Miller, A. D.
  • Non-retroviral vectors include adenovirus as described above (Rosenfeld et al., Cell, 68:143-155, 1992; Jaffe et al., Nature Genetics, 1:372-378, 1992; Lemarchand et al., Proc. Natl. Acad. Sci. USA, 89:6482-6486, 1992) .
  • the main advantages of adenovirus are that it transfers a large amount of DNA fragments (36kb genomes) and is capable of infecting non- replicative cells at a very high titer.
  • herpes virus may also be useful for human genetic therapy (Wolfe, J. H., et al., Nature Genetics, 1:379-384, 1992) .
  • other known suitable viral vectors can be used.
  • a vector capable of expressing the AIMP2 or their functional equivalents may be administered by a known method.
  • the vector may be administered locally, parenterally, orally, intranasally, intravenously, intramuscularly or subcutaneously, or by other suitable routes.
  • the vector may be injected directly into a target cancer or tumor cell at an effective amount for treating the tumor cell of a target tissue.
  • the inventive pharmaceutical composition can be injected directly into the hollow organ affected by the cancer or tumor using a needle, a catheter or other delivery tubes.
  • Any effective imaging device such as X-ray, sonogram, or fiberoptic visualization system, may be used to locate the target tissue and guide the needle or catheter tube.
  • the inventive pharmaceutical composition comprising the nucleic acid encoding the AIMP2 protein may be administered into the blood circulation system for treatment of a cancer or tumor which cannot be directly reached or anatomically isolated.
  • the pharmaceutical composition comprising the nucleic acid encoding the AIMP2 or their functional equivalents as an active ingredient may additionally comprise pharmaceutically acceptable carriers or excipients. These carriers or excipients include dispersing agents, wetting agents, suspending agents, diluents and fillers.
  • the ratio of the particular pharmaceutically acceptable carrier and the expression vector contained in the inventive pharmaceutical composition can be determined by the solubility and chemical properties of the composition, and the particular administration mode.
  • the therapeutic or preventive effective amount of the inventive pharmaceutical composition containing the AIMP2 protein- encoding nucleic acid may be suitably selected depending on the subject to be administered, age, individual variation and disease condition.
  • the present invention provides methods for screening an agent that modulates activity of p53.
  • the test agent is first assayed for their ability to modulate a biological activity of an AIMP2 ("the first assay step") .
  • modulating agents that modulate a biological activity of an isolated AIMP2 polypeptide may be identified by assaying a biological activity of the AIMP2 polypeptide having amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2, in the presence of a test agent.
  • the present invention may comprise:
  • Modulation of different biological activities of the AIMP2 polypeptide can be assayed in the first step.
  • a test agent can be assayed for activity to modulate expression level of the AIMP2 polypeptide, e.g., transcription or translation.
  • the test agent can also be assayed for activities in modulating cellular level or stability of the AIMP2 polypeptide, e.g., post- translational modification or proteolysis.
  • Test agents that increase a biological activity of the AIMP2 polypeptide by the first assay step are identified, the test agents are then subject to further testing for ability to modulate an activity of p53, in the presence of the AIMP2 polypeptide ("the second testing step") .
  • the test agents are then subject to further testing for ability to modulate an expression or cellular levels of p53 or its fragment.
  • the test agents may be tested for ability to modulate intracellular levels of p53.
  • the testing step may comprise:
  • test agent can be further tested for its activity on modulating transcription regulating function of p53, e.g., binding to an p53 recognition sequence or promoting expression of a gene under the control of an p53 binding sequence (i.e., p53 responsive gene) .
  • the AIMP2-modulating agents identified by the present invention can modulate cellular level of p53. If a test agent identified in the first screening step modulates cellular level (e.g., by altering transcription activity) of the AIMP2-modulating agents, it would indirectly modulate the p53. On the other hand, if a test agent modulates an activity other than cellular level of the AIMP2, then the further testing step is needed to confirm that their modulatory effect on the AIMP2 would indeed lead to modulation of p53 (e.g., cellular level of p53 or transcription regulating function of p53) .
  • p53 e.g., cellular level of p53 or transcription regulating function of p53
  • a test agent which modulates phosphorylation activity of an AIMP2, needs to be further tested in order to confirm that modulation of phosphorylation activity of the AIMP2 can result in modulation of p53 transcription regulating function or p53 cellular level.
  • AIMP2 and p53 or subunits or their fragments, analogs, or functional derivatives can be used.
  • the fragments that can be employed in these assays usually retain one or more of the biological activities of the AIMP2 and p53.
  • AIMP2 fragments may comprise 162 nd - 225 th amino acid residues of SEQ. ID:1 and p53 fragments may comprise 1 st - 32 nd amino acid residues of SEQ. ID: 6.
  • fusion proteins containing such fragments or analogs can also be used for the screening of test agents.
  • test agents usually have amino acid deletions and/or insertions and/or substitutions while maintaining one or more of the bioactivities and therefore can also be used in practicing the screening methods of the present invention.
  • a variety of well-known techniques can be used to identify test agents that modulate AIMP2 or p53.
  • the test agents are screened with a cell based assay system.
  • a construct comprising a p53 transcription regulatory element operably linked to a reporter gene is introduced into a host cell system.
  • the activity of polypeptide encoded by the reporter gene i.e., reporter polypeptide
  • an enzymatic activity in the presence of a test agent can be determined and compared to the activity of the reporter polypeptide in the absence of the test agent. An increase or decrease in the activity identifies a modulator of p53.
  • the reporter gene can encode any detectable polypeptide (response or reporter polypeptide) known in the art, e.g., detectable by fluorescence or phosphorescence or by virtue of its possessing an enzymatic activity.
  • the detectable response polypeptide can be, e.g., luciferase, alpha-glucuronidase, alpha- galactosidase, chloramphenicol acetyl transferase, green fluorescent protein, enhanced green fluorescent protein, and the human secreted alkaline phosphatase.
  • the test agent e.g., a peptide or a polypeptide
  • a library of test agents is encoded by a library of such vectors (e.g., a cDNA library; see the Example below) .
  • libraries can be generated using methods well known in the art (see, e.g., Sambrook et al. and Ausubel et al., supra) or obtained from a variety of commercial sources.
  • modulators of p53 can also be screened with non-cell based methods. These methods include, e.g., mobility shift DNA-binding assays, methylation and uracil interference assays, DNase and hydroxy radical footprinting analysis, fluorescence polarization, and UV crosslinking or chemical cross-linkers.
  • mobility shift DNA-binding assays e.g., methylation and uracil interference assays, DNase and hydroxy radical footprinting analysis, fluorescence polarization, and UV crosslinking or chemical cross-linkers.
  • One technique for isolating co-associating proteins includes use of UV crosslinking or chemical cross-linkers, including e.g., cleavable cross-linkers dithiobis (succinimidylpropionate) and 3, 3 ' -dithiobis (sulfosuccinimidyl-propionate) ; see, e.g., McLaughlin, Am. J. Hum. Genet., 59:561-569, 1996; Tang, Biochemistry, 35:8216-8225, 1996; Lingner, Proc. Natl. Acad. Sci. U.S.A., 93:10712, 1996; and Chodosh, MoI. Cell. Biol., 6:4723-4733, 1986.
  • UV crosslinking or chemical cross-linkers including e.g., cleavable cross-linkers dithiobis (succinimidylpropionate) and 3, 3 ' -dithiobis (sul
  • Fist assay step Screening test agents that modulate AIMP2
  • a number of assay systems can be employed to screen test agents for modulators of an AIMP2 polypeptide.
  • the screening can utilize an in vitro assay system or a cell-based assay system.
  • test agents can be screened for binding to the AIMP2 polypeptide, altering cellular level of the AIMP2 polypeptide, or modulating other biological activities of the AIMP2 polypeptide.
  • binding of a test agent to an AIMP2 polypeptide is determined in the first screening step. Binding of test agents to an AIMP2 polypeptide can be assayed by a number of methods including e.g., labeled in vitro protein-protein binding assays, electrophoretic mobility shift assays, immunoassays for protein binding, functional assays (phosphorylation assays, etc.), and the like. See, e.g., U.S. Pat. Nos.
  • test agent can be identified by detecting a direct binding to the AIMP2 polypeptide, e.g., co-immunoprecipitation with the AIMP2 polypeptide by an antibody directed to the AIMP2 polypeptide.
  • the test agent can also be identified by detecting a signal that indicates that the agent binds to the AIMP2 polypeptide, e.g., fluorescence quenching.
  • Competition assays provide a suitable format for identifying test agents that specifically bind to an AIMP2 polypeptide.
  • test agents are screened in competition with a compound already known to bind to the AIMP2 polypeptide.
  • the known binding compound can be a synthetic compound. It can also be an antibody, which specifically recognizes the AIMP2 polypeptide, e.g., a monoclonal antibody directed against the AIMP2 polypeptide. If the test agent inhibits binding of the compound known to bind the AIMP2 polypeptide, then the test agent also binds the AIMP2 polypeptide.
  • RIA solid phase direct or indirect radioimmunoassay
  • EIA solid phase direct or indirect enzyme immunoassay
  • sandwich competition assay see Stahli et al., Methods in Enzymology 9:242-253 (1983)
  • solid phase direct biotin-avidin EIA see Kirkland et al., J. Immunol. 137:3614-3619 (1986)
  • solid phase direct labeled assay solid phase direct labeled sandwich assay (see Harlow and Lane, "Antibodies, A Laboratory Manual,” Cold Spring Harbor Press (1988)); solid phase direct label RIA using .sup.1251 label (see Morel et al., MoI.
  • Modulating agents identified by competition assay include agents binding to the same epitope as the reference compound and agents binding to an adjacent epitope sufficiently proximal to the epitope bound by the reference compound for steric hindrance to occur. Usually, when a competing agent is present in excess, it will inhibit specific binding of a reference compound to a common target polypeptide by at least 50 or 75%.
  • the screening assays can be either in insoluble or soluble formats.
  • One example of the insoluble assays is to immobilize an AIMP2 polypeptide or its fragments onto a solid phase matrix.
  • the solid phase matrix is then put in contact with test agents, for an interval sufficient to allow the test agents to bind. Following washing away any unbound material from the solid phase matrix, the presence of the agent bound to the solid phase allows identification of the agent.
  • the methods can further include the step of eluting the bound agent from the solid phase matrix, thereby isolating the agent.
  • the test agents are bound to the solid matrix and the AIMP2 polypeptide molecule is then added.
  • Soluble assays include some of the combinatory libraries screening methods described above. Under the soluble assay formats, neither the test agents nor the AIMP2 polypeptide are bound to a solid support. Binding of an AIMP2 polypeptide or fragment thereof to a test agent can be determined by, e.g., changes in fluorescence of either the AIMP2 polypeptide or the test agents, or both. Fluorescence may be intrinsic or conferred by labeling either component with a fluorophor.
  • either the AIMP2 polypeptide, the test agent, or a third molecule can be provided as labeled entities, i.e., covalently attached or linked to a detectable label or group, or cross- linkable group, to facilitate identification, detection and quantification of the polypeptide in a given situation.
  • detectable groups can comprise a detectable polypeptide group, e.g., an assayable enzyme or antibody epitope.
  • the detectable group can be selected from a variety of other detectable groups or labels, such as radiolabels (e.g., .sup.1251, .sup.32P, .sup.35S) or a chemiluminescent or fluorescent group.
  • the detectable group can be a substrate, cofactor, inhibitor or affinity ligand.
  • Binding of a test agent to an AIMP2 polypeptide provides an indication that the agent can be a modulator of the AIMP2 polypeptide. It also suggests that the agent may modulate p53 activity (e.g., by binding to and modulate the AIMP2 polypeptide which in turn acts on p53) . Thus, a test agent that binds to an AIMP2 polypeptide can be further tested for ability to modulate p53 activities (i.e., in the second testing step outlined above) .
  • a test agent that binds to an AIMP2 polypeptide can be further examined to determine its activity on the AIMP2 polypeptide. The existence, nature, and extent of such activity can be tested by an activity assay. Such an activity assay can confirm that the test agent binding to the AIMP2 polypeptide indeed has a modulatory activity on the AIMP2 polypeptide. More often, such activity assays can be used independently to identify test agents that modulate activities of an AIMP2 polypeptide (i.e., without first assaying their ability to bind to the AIMP2 polypeptide) .
  • such methods involve adding a test agent to a sample containing an AIMP2 polypeptide in the presence or absence of other molecules or reagents which are necessary to test a biological activity of the AIMP2 polypeptide (e.g., kinase activity if the AIMP2 polypeptide is a kinase) , and determining an alteration in the biological activity of the AIMP2 polypeptide.
  • a biological activity of the AIMP2 polypeptide e.g., kinase activity if the AIMP2 polypeptide is a kinase
  • the activity assays also encompass in vitro screening and in vivo screening for alterations in expression or cellular level of the AIMP2 polypeptide.
  • Second test step Screening agents that modulate p53 activities
  • a modulating agent Once a modulating agent has been identified to bind to an AIMP2 polypeptide and/or to modulate a biological activity (including cellular level) of the AIMP2 polypeptide, it can be further tested for ability to modulate activities of p53. Modulation of activities of the p53 by the modulating agent is typically tested in the presence of the AIMP2 polypeptide.
  • the AIMP2 polypeptide can be expressed from an expression vector that has been introduced into a host cell.
  • P53 or an p53 subunit can be expressed from a second expression vector.
  • AIMP2 or p53 can be supplied endogenously by the host cell in the screening system.
  • p53 bioactivities to be monitored p53 bioactivities to be monitored in this screening step include, as noted above, cellular level of p53, stability of p53 protein, enzymatic or non-enzymatic modification (e.g., phosphorylation) of p53, tetramer formation, binding characteristics (e.g., binding to a target transcription regulatory element) , regulation of expression of p53 responsive genes, interaction with another regulatory protein or molecule that is required for a p53 bioactivity (e.g., suppressor c-Abl or replication protein A) , regulation of cellular proliferation or cell adhesion, or regulation of cell growth or apoptosis. All these bioactivities can be tested in the presence of a modulating agent that has been identified to bind to and/or modulate AIMP2.
  • enzymatic or non-enzymatic modification e.g., phosphorylation
  • binding characteristics e.g., binding to a target transcription regulatory element
  • regulation of expression of p53 responsive genes
  • the cellular level of p53 or other bioactivities of p53 can be determined in a non-cell based assay system or cell-based assays, similar to the first screening step for identifying modulators of AIMP2.
  • effects of test agents on p53 level or activities can be tested by directly measuring expression or cellular level of p53, or its transcription regulating activity in the presence of the test agents. Because the test agent is likely to exert its modulatory effect on p53 by modulating AIMP2, the AIMP2 is typically also present in the assay system.
  • modulation of p53 cellular level can be examined using methods similar to that described in Example .
  • vectors expressing a reporter gene or other linked polynucleotides under the control of a transcription regulatory element of the p53 gene (for assaying modulation of p53 expression activity) or a p53 recognition sequence (for assaying modulation of p53 transcription regulating activities) are introduced into appropriate host cells. Modulation of p53 activities are typically examined by measuring expression of the reporter genes or other linked polynucleotides. An altered activity of the reporter gene (e.g., its expression level) in the presence of a test agent would indicate that the test agent is a modulator of p53.
  • an observed modulation of the reporter gene could be due to a direct interaction between the test agents with the expression vector.
  • the modulation could also be due to an altered activity of endogenous p53 (e.g., DNA-binding activity or p53 cellular level) as a result of the presence of the test agent.
  • the test agent's activity on the endogenous p53 could be direct, e.g., by interacting directly with p53, or indirect, e.g., through interacting with another molecule (e.g., a p53 modulatory polypeptide) that in turn binds to the p53 transcription regulatory element (e.g., a p53 recognition sequence) .
  • the test agent was first identified to modulate AIMP2 in the first screening step, its modulation on p53 activities or cellular level is likely to be indirect (i.e., through its interaction with the AIMP2) .
  • Modulation of various biological activities of p53 by a test agent can also be assayed in accordance with many methods that have been disclosed in the art, e.g., Roemer et al., Proc Natl Acad Sci USA 90: 9252-6, 1993; Crook et al., Oncogene 9: 1225-30, 1994; Sabbatini et al., MoI Cell Biol 15: 1060-70, 1995; Canman et al., Genes Dev 9: 600- 11, 1995; Gobert et al . , Biochemistry 35: 5778-86, 1996; LiIl et al., Nature 387: 823-7, 1997; Sabapathy et al., EMBO J.
  • modulation of expression of a p53 responsive gene can be examined in a cell-based system by transient or stable transfection of an expression vector into cultured cell lines.
  • Assay vectors bearing a p53 recognition sequence operably linked to reporter genes can be transfected into any mammalian cell line (e.g., HCT116 cell line as described in the Examples, U2OS cell line or SAOS2 cell line) for assays of promoter activity.
  • Any readily transfectable mammalian cell line may be used to assay p53 promoter, e.g., HCT116, HEK 293, MCF-7, and HepG2 are all suitable cell lines.
  • Constructs containing a p53 recognition sequence (or a transcription regulatory element of the p53 gene) operably linked to a reporter gene can be prepared using only routinely practiced techniques and methods of molecular biology (see, e.g., Sambrook et al. and Ausubel et al., supra) .
  • expression vectors containing a reporter gene under the control of p53 response elements can also be obtained commercially (e.g., from Stratagene, San Diego, Calif.) .
  • P53 binding sites have been identified in a great number of p53 responsive genes.
  • p53 recognition sequences in p53 responsive genes have been disclosed in Yuan et al., Biochem Biophys Res Commun.
  • transcription regulatory elements of the p53 gene can be used in the screening assay. Transcription regulatory elements of the p53 gene have also been well known and characterized in the art, e.g., as disclosed in Reisman et al., Proc Natl Acad Sci USA 85: 5146-50, 1988; Tuck et al., MoI Cell Biol. 9: 2163-72, 1989; Reisman et al., Cell Growth Differ. 4: 57-65, 1993; and Roy et al., Oncogene 13: 2359-66, 1996.
  • reporter genes typically encode polypeptides with an easily assayed enzymatic activity that is naturally absent from the host cell.
  • Typical reporter polypeptides for eukaryotic promoters include, e.g., chloramphenicol acetyltransferase (CAT), luciferase, beta-galactosidase, beta-glucuronidase, alkaline phosphatase, and green fluorescent protein (GFP) .
  • Transcription driven by p53 response elements may also be detected by indirectly measuring the amount of RNA transcribed from the reporter gene.
  • the reporter gene may be any transcribable nucleic acid of known sequence that is not otherwise expressed by the host cell.
  • RNA expressed from constructs containing a p53 response element may be analyzed by techniques known in the art, e.g., reverse transcription and amplification of mRNA, isolation of total RNA or poly A+ RNA, northern blotting, dot blotting, in situ hybridization, RNase protection, primer extension, high density polynucleotide array technology and the like. These techniques are all well known and routinely practiced in the art.
  • vectors for assaying expression under the control of a p53 recognition sequence can also comprise elements necessary for propagation or maintenance in the host cell, and elements such as polyadenylation sequences and transcriptional terminators to increase expression of reporter genes or prevent cryptic transcriptional initiation elsewhere in the vector.
  • Exemplary assay vectors are the pGL3 series of vectors (Promega, Madison, Wis.; U.S. Pat. No. 5,670,356), which include a polylinker sequence 5' of a luciferase gene. P53 response elements may be inserted into the polylinker sequence and tested for luciferase activity in the appropriate host cell.
  • Assay vectors may also comprise additional enhancer or promoter sequences, depending on whether the transcription regulatory elements are sufficient to drive transcription of the reporter genes.
  • the expression vectors can contain additional promoter sequence such as a minimal promoter (e.g., a promoter derived from Herpes simplex virus thymidine kinase) .
  • a minimal promoter e.g., a promoter derived from Herpes simplex virus thymidine kinase
  • an assay vector such as pGL3-Promoter may be used. This vector has transcription initiation elements from the SV40 promoter. In such vectors, transcription initiates from a heterologous site but the rate of transcription is increased by the presence of linked p53 response elements.
  • a test agent that modulates AIMP2 can be further screened for ability to modulate cellular proliferation through modulating p53 activity.
  • the test agent can be identified based on modulation of a cellular proliferation phenotype, e.g., inhibition of cell proliferation, cell or tumor growth arrest, or cell death.
  • p53 activities in modulating cellular proliferation are well known, and methods for measuring such activities have also been described in the art (Sawhney et al., J Hum Hypertens 11: 611-4, 1997; Moretti et al., Oncogene 14: 729-40, 1997; and Onodera et al., Am J Dermatopatho 18: 580-8, 1996) .
  • a test agent modulating AIMP2 can be further examined for effects on expression of p53 responsive genes.
  • Expression of a great number of genes is known to be regulated by the p53 transcription factor. These genes are involved in many important cell processes such as DNA synthesis and repair processes, RNA transcription, and cisplatin resistance.
  • p53 responsive genes include: c-fos gene (Kley et al., Nucleic Acids Res, 20: 4083-7, 1992); herpes simplex thymidine kinase gene (Yuan et al .
  • p53 genes regulated by p53 include p21 (Wafl), Clpl, MDM2, GADD45, Cyclin G, IGF-BP3, and those described in U.S. Pat. No. 6,020,135.
  • Human p53 gene encodes a 393 amino acid residue, 53 kD phosphoprotein.
  • the protein is divided structurally and functionally into four domains.
  • the first 42 amino acids at the N-terminus constitute transcriptional activation machinery in positively regulating gene expression.
  • Amino acid residues 13-23 in the p53 protein are identical in a number of diverse species and certain amino acids in this region have been shown to be required for transcriptional activation by the protein in vivo.
  • AIMP2 can activate p53 by binding N-terminus of p53 (amino acid residues 1-132 of SEQ: 6) .
  • the present invention can provide a method for identifying an agent that modulates interaction of p53 and AIMP2.
  • the agent can activate or reinforce interaction of p53 and AIMP2; on the contrary, it can inhibit or attenuate the interaction.
  • the method for identifying can comprise the following steps:
  • the (b) step can comprise detecting a relative change of the interaction level between p53 and the AIMP2 polypeptide in the cell or the cell lysate thereof contacting the test agent compared to the interaction level between p53 and the AIMP2 polypeptide in the cell or the cell lysate thereof without contacting the test agent .
  • the method for identifying can be performed by any conventional method known in the art such as labeled in vitro protein-protein binding assays (in vitro full-down assays), EMSA (electrophoretic mobility shift assays), immunoassays for protein binding, functional assays (phosphorylation assays, etc.) , yeast two hybrid assay, assays of non-immune immunoprecipitations, Immunoprecipitation/ western blot assays, immuno-co- localization assays.
  • EMSA epidermal mobility shift assays
  • immunoassays for protein binding for protein binding
  • functional assays phosphorylation assays, etc.
  • yeast two hybrid assay assays of non-immune immunoprecipitations
  • Immunoprecipitation/ western blot assays immuno-co- localization assays.
  • yeast two hybrid analyses may be carried out using yeast expressing AIMP2 and p53, or parts or homologues of the proteins, fused with the DNA- binding domain of bacteria repressor LexA or yeast GAL4 and the transactivation domain of yeast GAL4 protein, respectively (Kim, M. J. et al . , Nat. Gent., 34:330-336, 2003) .
  • the interaction between AIMP2 and p53 reconstructs a transactivator inducing the expression of a reporter gene under the control by a promoter having a regulatory sequence binding to the DNA-binding domain of LexA or GAL4.
  • the reporter gene may be any gene known in the art encoding a detectable polypeptide.
  • chloramphenicol acetyltransferase (CAT) CAT
  • luciferase ⁇ -galactosidase
  • ⁇ -glucosidase alkaline phosphatase
  • GFP green fluorescent protein
  • a reporter gene encoding a protein which enables growth of yeast may be selected.
  • auxotropic genes encoding enzymes involved in biosynthesis for obtaining amino acids or nitrogenous bases (e.g., yeast genes such as ADE3, HIS3, etc. or similar genes from other species) may be used. If the expression of AIMP2 and p53, or parts or homologues of the proteins is inhibited or reduced by a test agent, the reporter gene is not expressed or less expressed. Accordingly, under such a condition, the growth of yeast is stopped or retarded. Such an effect on the expression of the reporter gene may be observed with eyes or using devices (e.g., a microscope) .
  • the present invention can provide a method for detecting and/or isolating p53 in a biological sample.
  • the method can comprise the following steps : (a) collecting a biological sample including p53;
  • biological sample means a sample from mammalian cells.
  • the cells may be part of an acquired tissue or organ sample (e.g., an organ sample acquired by a biopsy) . Further, the cells may be individual cells, e.g., blood cells or cells grown by tissue culture. For the purpose of detecting the presence of p53 in a sample, the sample is one conjectured to include p53.
  • a polypeptide including 162-225 amino acid sequences of SEQ ID NO: 1 may be used.
  • a part or full-length AIMP2 polypeptide including the amino acid sequences, or analogues or homologues thereof may be used.
  • the detection of p53 in a biological sample may be achieved by identifying the binding of AIMP2 and p53.
  • the binding may be detected by various known binding assays, such as non-denaturing immunoprecipitation assay, immunoprecipitation and western blot assay, yeast 2-hybrid assay, immunocolocalization assay, in vitro pull-down assay, or the like.
  • immunoprecipitation and western blot assay may be carried out as follows.
  • a biological sample is mixed with AIMP2 or a portion thereof and an anti- AIMP2 antibody under a non-denaturing condition.
  • the antibody mixture is mixed with Protein A Sepharose.
  • Protein A Sepharose binds with the anti-AIMP2 antibody, and all the proteins or peptides including AIMP2 bound to the anti-AIMP2 antibody are bound to Protein A Sepharose.
  • the proteins bound to Sepharose are isolated and subjected to electrophoresis on a denaturing gel. The gel is electroblotted onto a membrane and the membrane is mixed with an anti-p53 antibody.
  • the presence of the antibody may be detected directly by labeling the antibody, or indirectly using an enzyme bound to the antibody.
  • the anti-AIMP2 antibody may be prepared according to a common method. A peptide including amino acids of a complete AIMP2 or a portion thereof including the p53 binding site (e.g. 162-225 of SEQ ID NO: 1) may be subcutaneously injected to a goat or rabbit together with complete Freund's adjuvant (CFA) . Then, a booster may be injected subcutaneously or intra-abdominally together with CFA.
  • CFA complete Freund's adjuvant
  • the anti-AIMP2 antibody may act on one or more epitope (s) of AIMP2.
  • the antibody may be labeled with a marker, e.g. a radioactive or fluorescent marker.
  • the antibody may be indirectly labeled by binding a marker compound to a covalently bonded anti- goat or anti-rabbit antibody.
  • the anti-AIMP2 antibody may be prepared in accordance with Kim and others' method (Kim, J Y. et al., Proc. Matl. Acad.. Sci., 99, 7912-7916, 2002; Kim, M. J. et al., Nat , Genet., 34, 330-336, 2003) .
  • separation of p53 from a biological sample may be carried out in accordance with a known method utilizing the binding between AIMP2 and p53 (e.g. chromatography) .
  • affinity chromatography may be employed (Zhang Z and Zhang R, The EMBO Journal (2008) 27, 852-864) .
  • full-length AIMP2 or a portion thereof binding to p53 may be fixed on a carrier.
  • FIG.l shows the results of TUNEL staining of apoptotic cell in wild-type mouse (+/+) tissue and AIMP2- deficient mouse (-/-) tissue.
  • FIG. 2 shows the results of FACS analysis to compare the sensitivity of MEFs obtained from wild-type mouse (+/+) and AIMP2-deficient mouse (-/-) to UV irradiation.
  • the bars and numbers indicate the subGl phase cells and their percentages.
  • FIG. 3 shows the results of colony forming assay to compare the sensitivity of MEFs obtained from wild-type mouse (+/+) and AIMP2-deficient mouse (-/-) to adriamycin treatment .
  • FIG. 4 shows the results of FACS analysis to compare the sensitivity of MEFs obtained from AIMP2-deficient mouse (-/-) which is transfected using AIMP2 expression vector (+AIMP2) or empty vector (EV) to UV.
  • the bars and numbers indicate the subGl phase cells and their percentages .
  • FIG. 5 shows the results of western blotting for the induction of p53 and AIMP2 by various genotoxic stresses. Cont : No treatment
  • Adr adriamycin treatment
  • UV UV irradiation
  • Eto etoposide treatment
  • FIG. 6 shows the results of analysis of expression of p53 and AIMP2 in MEFs obtained from AIMP2-deficient mouse (-/-) which is transfected by AIMP2 expression vector (+AIMP2) or empty vector (EV) depending on UV irradiation.
  • FIG. 7 shows the results of expression pattern of p53 and AIMP2 in MEFs obtained from wild-type mouse (+/+) and AIMP2-deficient mouse (-/-) depending on adriamycin treatment.
  • FIG. 8 shows the results of analysis of expression pattern of p53 and AIMP2 in MEFs obtained from wild-type mouse (+/+) and AIMP2-deficient mouse (-/-) depending on UV irradiation.
  • FIG. 9 shows the results of western blotting analysis (A) and luciferase activity assay (B) for the p53 expression level in U2OS cell which is transfected by different amount of AIMP2 expression vector.
  • FIG. 10 shows the results of western blotting (A) and immunofluorescence staining (B) to confirm the inhibition of induction p53 by si-RNA (si-AIMP2 ) .
  • FIG. 11 shows the result (A) to examine expression pattern of p53 and AIMP2 and graph (B) of flow cytometry analysis to examine apoptosis rate in p53 positive HCT116 cell and p53 negative HCTll ⁇ cell depending on UV irradiation .
  • FIG. 12 shows the result (A) to confirm the phosphorylation of AIMP2 in the post-translation level depending on UV irradiation in HCT116 and the result (B) to confirm the phosphorylation of AIMP2 by treating alkaline phosphatase.
  • FIG. 13 shows the result of immunofluorescence staining to confirm the intracellular location of p53 and AIMP2 by UV irradiation.
  • FIG. 14 shows the result of immunoprecipitation to confirm the interaction between AIMP2 and p53 by genotoxic stress.
  • FIG. 15 shows the result of In vitro pull-down assay to confirm the direct interaction between AIMP2 and p53.
  • FIG. 16 shows the result of yeast two hybrid analysis to identify the region of p53 that interacts with AIMP2 using various p53 deletion fragments.
  • FIG. 17 shows the result of In vitro pull-down assay using p53 point mutants (22/23, 175 and 248) .
  • FIG. 18 shows the result of In vitro pull-down assay to identify the region of AIMP2 that interacts with p53 using various AIMP2 deletion fragments.
  • FIG. 19 is a schematic representation of the interaction regions of AIMP2 and p53.
  • FIG. 20 shows the result to investigate the interaction between MDM2 and p53 by the competitive inhibition of AIMP2.
  • FIG. 21 shows the result to examine the effect of AIMP2 on the p53 ubiquitination in ALLN treated HCT116 cell .
  • FIG. 22 shows the result to examine the biding affinity of AIMP2 to phosphorylated p53 and unphosphorylated p53 depeding on the time course.
  • FIG. 23 shows the result of immunoprecipitation to confirm which AIMP2 does not bind to phosphorylated p53.
  • FIG. 24 illustrates the result (INPUT) (A) to confirm phosphorylated p53 by UV before In vitro pull-down assay and the result (B) of In vitro pull-down assay with GST- AIMP2 to analyze the binding affinity of phosphorylated p53 to AIMP2.
  • FIG. 25 shows the result (A) of UV irradiation after si-AIMP2 transfection in 293 cell and the result (B) of adriamycin treatment after si-AIMP2 transfection in 293 cell to examine the effect on AIMP2 on upstream kinase activity of p53.
  • Apoptosis was compared in various tissues of wild- type mouse and AIMP2-deficient mouse.
  • AIMP2-deficient mouse (AIMP2 ⁇ / ⁇ or -/-) prepared according to the method of Kim and others (Kim et al., Proc. Natl. Acad. Sci., U. S. A., 99:7912-7916, 2002) .
  • Kidney, lung, pancreas, spleen and heart tissues were isolated from the 18.5-day embryos of wild-type mouse (C57BL/6) and AIMP2-deficient mouse, and frozen sections were prepared therefrom according to a method known in the art (Kim MJ et al., Nature Genetics, 34:330-336, 2003) .
  • the frozen section was incubated at 37 0 C in a buffer containing terminal deoxynucleotidyl transferase and biotinylated UTP for 1 hour. Then, after adding streptavidin-FITA Apoptag kit (Intergen) (65 mL/5cm 2 ) , incubation was performed at 25 0C for 1 hour. In order to detect apoptosis of cells, TUNEL staining was carried out using Apoptag kit (BMS) according to the manufacturer's instructions. The cell nucleus was stained with propidium iodide (PI) . Then, observation was made using a confocal microscope ( ⁇ -
  • the green and red colors represent TdT-positive apoptotic cells and nuclei stained with PI, respectively.
  • apoptotic cells decreased remarkably in the AIMP2-deficient tissue (-/-) as compared to the wild-type tissue (+/+) . This result means that AIMP2-deficient cells are more resistant to apoptosis .
  • AIMP2 +/+ and AIMP2 "7" MEFs were obtained from 12.5-day embryos according to the method of Kim and others (Kim et al. , Nat. Gent., 34:330-336, 2003) .
  • UV 50 J/m 2
  • UV was not irradiated to the control group. 6 hours later, cells were collected from the test group and the control group, and were fixed using 70% ethanol. After washing, the cells were stained with PI (50 ⁇ g/mL) . Thereafter, FACS analysis was performed to quantitate dying cells. 20,000 cells were analyzed per sample using FL-2A detector. After the subGl phase began, the proportion of dying cells to the total cells was calculated. The result is shown in Fig. 2.
  • AIMP2 +/+ and AIMP2 "7" MEFs were inoculated on a 35 mm plate at a concentration of 2xlO 6 cells/well. Then, after adding DMEM containing 2 ⁇ g/mL Adr (Calbiochem) , the cells were cultured for 2 days. The cells were washed and fixed with 1% PFA. After staining with Giemsa stain, the cells were washed with PBS. Colonies were counted and depicted as bar graphs.
  • AIMP2 "7" MEFs produced about 4 times more colonies than AIMP2 +/+ MEFs. This result suggest that AIMP2 ⁇ / ⁇ MEFs are less sensitive to Adr- induced apoptosis than AIMP2 +/+ MEFs.
  • AIMP2 "7" MEFs were washed with serum-free medium and incubated at 37 0 C for 3 hours under serum-free status together with DNA (pCDNA3-myc-AIMP2, Kim MJ et al.,
  • the control group was transfected under the same condition using an empty vector (EV; pCDNA3, Invitrogen) not including the AIMP2 gene.
  • EV empty vector
  • Example 2 p53 activation by AIMP2
  • the present inventors investigated response of AIMP2 to various genotoxic stresses in order to examine whether the level of AIMP2 is affected by apoptotic signals. Response of p53 was investigated together in order to determine whether AIMP2 affects p53, which is known to induce genotoxic stress-induced apoptosis.
  • AIMP2 was exogenously introduced into AIMP2- deficient cells.
  • a vector including AIMP2 gene (SEQ ID NO: 3) and an empty vector were transfected respectively into AIMP2 ⁇ / ⁇ MEFs in the same manner as Example ⁇ l-3>.
  • western blot was performed using anti-p53 antibody, anti-AIMP2 antibody and anti-actin antibody in the same manner as Example ⁇ 2-l>.
  • AIMP2 as compared to the cells transfected with an empty vector (column EV) .
  • the p53 expression was further increased by the UV irradiation.
  • Increase of p53 expression by transfection with AIMP2 was larger than that by UV irradiation.
  • This result reveals that p53 expression is increased by AIMP2 transfection, as well as by UV irradiation.
  • b) Induction of p53 in AIMP2 +/+ and AIMP2 "7" MEFs In order to confirm that AIMP2 is required to induce p53, induction of p53 in AIMP2 +/+ and AIMP2 ⁇ 7 ⁇ MEFs was compared.
  • AIMP2 +/+ and AIMP2 "7" MEFs were treated with Adr (2 ⁇ g/mL) for different time course (0, 10 and 20 minutes) . Subsequently, western blot was performed in the same manner as Example ⁇ 2-l>. The result is shown in Fig. 7.
  • AIMP2 +/+ and AIMP2 "7" MEFs were treated with UV for 30 minutes in the same manner as Example ⁇ l-2> a) . Subsequently, western blot was performed in the same manner as Example ⁇ 2-l>. The result is shown in Fig. 8.
  • U20S cells (ATCC HTB-96) were transfected with vectors including 0 ⁇ g/mL, 1 ⁇ g/mL and 2 ⁇ g/mL AIMP2, respectively, in the same manner as Example ⁇ l-3>.
  • Western blot was performed to measure expression level of p53 in the transfected cells in the same manner as Example ⁇ 2-l>. The result is shown in Fig. 9 A.
  • luciferase assay was performed using p53-dependent GADD45 promoter.
  • U2OS cells were cotransfected with vectors including 0 ⁇ g/mL, 1 ⁇ g/mL and 2 ⁇ g/mL AIMP2, respectively, and 0.5 ⁇ g/mLGADD45-luc (acquired from Dr. Roeder RG, Rockefeller Univ.) and cultured at 37 0 C for 24 hours. The cultured cells were lysed with lysis buffer (Promega) and then centrifuged.
  • the resultant cell lysate was mixed with substrate (D-luciferin, Promega) and luciferase activity was measured using a luminometer. Average was taken after 4 independent experiments. The result is shown as a bar graph in Fig . 9 B .
  • p53 expression increased depending on AIMP2 concentration. Luciferase activity was also increased by the p53-dependent GADD45 promoter. The AIMP2-dependent p53 induction was also observed in A549 cells (result not shown) . As a result, it was verified that the expression of p53 is increased by AIMP2 in a dose-dependent manner.
  • the present inventors investigated the effect of inhibition of AIMP2 expression on induction of p53 in order to reconfirm whether AIMP2 is required for induction of p53.
  • a) Western blot si-RNA for AIMP2 (SEQ ID NO: 21, ACA CCA GAU GCA GAC UUG GAU GUA A; Han JM et al., The Journal of Biological Chemistry, Dec 15; 281(50) : 38663-7, 2006) was prepared into double-stranded oligomer by Invitrogen. The resultant si-RNA was named as 'si-AIMP2'.
  • si-AIMP2 was transfected into U2OS cells (ATCC HTB-96) according to a method known in the art.
  • the control group was transfected with AIMP2-nonspecific control siRNA (Stealth RNAi Negative Control Medium GC, Cat . No.452001, Invitrogen) .
  • Genotoxic stress was induced in the transfected cells either by irradiating with UV (50 J/m 2 ) for 1 hour or by treating with Adr (2 ⁇ g/mol) for 2 hours Then, western blot was performed in the same manner as Example ⁇ 2-l>. The result is shown in Fig. 10 A.
  • b) Immunofluorescence staining In order to observe the cells transfected in a) through immunofluorescence staining, the cells were placed on a cover slip and fixed with 100% methanol at - 20 0 C for 5 minutes.
  • the cells were cultured at 25 0 C for 30 minutes. The cells were further cultured for 1 hour in blocking buffer (PBS blocking buffer containing 0.1% Triton X-IOO and 1% BSA) to which primary antibody (1:100) (rabbit polyclonal anti- p53antibody, Santa Cruz Biotechnology, Inc.) was added. Subsequently, the cells were further cultured for 30 minutes in blocking buffer to which FITC- or TRITC-bound secondary antibody (1:1000) (goat anti-rabbit IgG, Pierce) was added. After washing with PBS, the cells were immobilized. The cell nucleus was stained using PI according to a method known in the art.
  • Fig. 10 B the green fluorescence indicates p53 and the red fluorescence indicates the cell nucleus.
  • p53 was remarkably induced by UV or Adr in the control group cells transfected with nonspecific siRNA (si-Con or si-Cont), but the induction of p53 was interrupted by siRNA of AIMP2 (si-AIMP2) . This result reveals that induction of p53 in response to genotoxic stress requires AIMP2.
  • the cells irradiated with UV in ⁇ Example 3> a) were subjected to flow cytometry using a mitochondrial membrane potential detection kit (Invitrogen) .
  • a mitochondrial membrane potential detection kit Invitrogen
  • the result is shown as a bar graph in Fig. 11 B.
  • proteins were extracted from HCT116 cells exposed to UV irradiation and those that were not exposed to UV irradiation, and were analyzed by immunoprecipitation.
  • U2OS cells or mock cells were either treated with UV for 5 minutes or left untreated, and proteins were extracted from each group. After immunoprecipitation using anti-AIMP2 antibody, immunoblot was performed for phosphoserine (pSer) , phosphothreonine (pThr) and phosphotyrosine (pTyr) .
  • U2OS cells were irradiated with UV for 5 minutes, and proteins were extracted from the cells and separated by 2D gel electrophoresis (middle panel) . Proteins isolated from non-UV-treated cells were used as' control group (upper panel) . Western blot was performed using anti-AIMP2 antibody. Further, in order to verify whether AIMP2 is phosphorylated by UV, the same procedure was carried out for the cells treated with UV and then treated with alkaline ' phosphatase (lower panel, +AP) .
  • AIMP2 and p53 are located in the cytoplasm and the cell nucleus, respectively, the present inventors first identified whether AIMP2 is translocated into the nucleus by genotoxic stress.
  • A549 cells (ATCC CCL-185) were irradiated with UV (50 J/m 2 ) for 1 hour. The control group was not treated with UV. Then, immunofluorescence staining was preformed in the same manner as Example ⁇ 2- 4> b) .
  • AIMP2 was stained green and p53 was stained red. As shown in Fig. 13, whereas AIMP2 was mainly observed in the cytoplasm in the UV-non treated cells, it was observed in the nucleus in the UV-treated cells. This reveals that AIMP2 is translocated into the nucleus by genotoxic stress.
  • HCT116 cells (ATCC CCL-247) were treated with Adr (2 ⁇ g/mL) , UV (50 J/m 2 ) and etoposide (Eto, 10 ⁇ M) , respectively, for 2 hours. Then, the cells were lysed with RIPA buffer in order to extract proteins therefrom. For immunoprecipitation, after adding normal IgG (Pierce) and protein-A/G-agarose (Invitrogen) , the cell lysate was incubated at 4 0 C for 4 hours. After centrifuge, the supernatant was taken, and protein A/G-agarose and anti- p53 antibody were added. After incubation for 2 more hours, the precipitate was collected and analyzed by SDS- PAGE.
  • Adr 2 ⁇ g/mL
  • UV 50 J/m 2
  • Eto etoposide
  • the present inventors carried out in vitro pull-down assay in order to investigate whether AIMP2 and p53 interact directly.
  • Radioactively labeled p53 was synthesized using
  • GST-KRS were prepared as follows. Full-length KRS was cloned into the EcoRl/XhoI site of pGEX-4T-l vector to transfect bacteria. Then, after induction using IPTG, the vector was bound to glutathione Sepharose 4B bead (Amersham) according to the manufacturer's instructions. The radioactively labeled p53 was mixed with the GST-AIMP2 bound to the bead or with the GST-KRS bound to the bead
  • Sepharose Sepharose. After centrifuge, protein was eluted from the precipitated bead. The protein was separated by SDS-PAGE and detected by autoradiography.
  • Yeast two hybrid analysis was carried out according to a known method in order to determine the region where p53 interacts with AIMP2 (Kim, M. J. et al., Nat, Genet., 34, 330-336, 2003) .
  • Various deletion fragments of p53 consisting of 393 amino acids represented by SEQ ID NO: 5 were prepared by PCR (see Table 1) .
  • DNA was extracted from SAOS2 cells according to a method known in the art. PCR was carried out using the extracted DNA as template and using the primers listed in Table 1. PCR was carried out as follows: predenaturation of the template DNA by heating at 94 0 C for 3 minutes followed by 30 cycles of 94 0 C / 30 sec; 55 0 C / 30 sec; and 72 0 C / 1 min. Finally, reaction was performed at 72 0 C for 5 minutes. The amplified fragments were identified by agarose gel electrophoresis. They were separated, purified and fused with LexA (Lee JS et al., Biochem. Biophys. Res. Commun. 15; 291:158-64, 2002) .
  • LexA Lee JS et al., Biochem. Biophys. Res. Commun. 15; 291:158-64, 2002
  • AIMP2 was amplified by PCR using primers of SEQ ID NO: 12 and SEQ ID NO: 13.
  • the amplified AIMP2 was fused with B42 (Kim MJ et al., Nature Genetics, 34:330-336, 2003) .
  • Yeast two hybrid analysis was carried out using the prepared constructs (Lex-p53 and B42-AIMP2) .
  • the interaction between AIMP2 and p53 was measured by cell growth in the minimal medium. As shown in Fig. 16, it was identified that the 32 N-terminal amino acids (1-32) of p53 are required for interaction with AIMP2.
  • the present inventors prepared point mutants of p53.
  • Various point mutants of p53 at amino acids 22, 23, 175 and 248 positions were prepared according to a method known in the art (Lin, J. et al . , Genes Dev. , 8, 1235-1246, 1994;
  • SEQ ID NO: 1 forward CCG GAA TTC ATG CCG ATG TAC CAG GTA AAG 12
  • Example ⁇ 6-l> and ⁇ 6-2> reveal that the middle region of AIMP2 (162-225 of SEQ ID NO: 1) interacts with the N-terminal region of p53 (1-32 of SEQ ID NO: 6), as shown in Fig. 19.
  • Example ⁇ 6-l> overlaps with the binding site to mouse double minute 2 (MDM2), the negative regulator of p53, (Sherr, C. J., Nat. Rev. MoI. Cell Biol. 2,731-737, 2001), the present inventors investigated whether AIMP2 can compete with MDM2 for interaction with p53.
  • p53 and MDM2 were acquired using In vitro translation kit, mixed with purified 0 ⁇ g/mL, 1 ⁇ g/mL and 2 ⁇ g/mL GST-AIMP2, respectively, for 2 hours, and immunoprecipitated using anti-p53 antibody.
  • the coprecipitated MDM2 and AIMP2 were subjected to western blot in the same manner as Example ⁇ 2-l> using anti-AIMP2 antibody and anti-MDM2 antibody (SMP14) (Santa Cruz Biotech) .
  • the amount of MDM2 binding to p53 decreased as the amount of AIMP2 increased, whereas the amount of AIMP2 binding to p53 increased. This result reveals that the binding of MDM2 to p53 is inhibited by the increase of AIMP2.
  • HCT116 cells were treated with 20 uM ALLN (Sigma) for 2 hours in order to suppress proteasome activity.
  • the cells were transfected with expression vectors including different concentrations of AIMP2 (0 ⁇ g/mL, 1 ⁇ g/mL and 2 ⁇ g/mL) . Subsequently, the effect of AIMP2 on p53 ubiquitination was identified by western blot using anti-ubiquitin antibody (Santa Cruz Biotechnology, USA) .
  • HCTll ⁇ cells were treated with Adriamycin (Adr) , and phosphorylation of p53 and interaction with AIMP2 were investigated at different time.
  • control group was not irradiated with UV.
  • Phosphorylation at S15 which is known as a phosphorylation site for ATM, was measured by western blot using p-S15-p53 antibody (Cell Signaling) .
  • AIMP2 Since p53 is induced through phosphorylation by kinases upstream of p53 such as ATM and ATR, in order to investigate whether AIMP2 affects the activity of the kinase, AIMP was inhibited by si-AIMP and the phosphorylation of Chkl and Chk2, which are substrates of ATR and ATM, was determined.
  • Example ⁇ 2-4> 293 cells were transfected with si-AIMP2 for 24 hours in the same manner as Example ⁇ 2-4>. After treating with UV (50 J/cm 2 ) and Adr (2 ⁇ L/mL) for 30 minutes, respectively, followed by extraction of proteins, western blot was carried out in the same manner as Example ⁇ 2-l> using anti-AIMP2 antibody, phospho-Chkl antibody and phospho-Chk2 antibody (Cell Signaling) . Actin was used as loading control. As shown in Fig. 25, the inhibition of AIMP2 had little effect on the phosphorylation of Chkl (Fig. 25 A) or Chk2 (Fig. 25 B) . This indicates that AIMP2 is not related with the activity of kinases upstream of p53.
  • AIMP2 directly binds to p53 in order to protect p53 from the attack of MDM2 under various genotoxic stresses, and, after phosphorylation of p53, AIMP2 is separated from p53 and p53 is activated in tetramer form.
  • AIMP2 works as a positive regulator of p53.
  • AIMP2 can regulate activities of p53 and regulate p53- associated cellular metabolism. The interaction between
  • AIMP2 and p53 of the present invention can be effectively used for treating, preventing and/or diagnosing p53- mediated disease or disorder. It can easily identify an agent modulating p53 and an agent modulating the interaction between AIMP2 and p53 using the interaction between AIMP2 and p53.

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Abstract

La présente invention porte sur une protéine régulatrice p53 et sur ses utilisations. La présente invention porte sur un procédé pour moduler p53 dans une cellule ou un tissu, et sur un procédé pour identifier un agent modulant l'activité de p53 à l'aide d'un polypeptide AIMP2 ou d'un nucléotide codant pour le polypeptide. En outre, la présente invention porte sur un procédé pour inhiber l'ubiquitination de p53 et sur un procédé pour identifier un agent modulant une interaction entre p53 et AIMP2.
PCT/KR2008/002559 2008-05-07 2008-05-07 Protéine régulatrice p53 et ses utilisations WO2009136671A1 (fr)

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EP2566499A2 (fr) * 2010-05-04 2013-03-13 aTyr Pharma, Inc. Découverte innovante de compositions thérapeutiques, diagnostiques et à base d'anticorps liées à des fragments protéiques de complexe multi-arnt synthétase p38

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Title
HAN, J. M. ET AL.: "AIMP2/p38, the scaffold for the multi-tRNA synthetase complex, responds to genotoxic stresses via p53", PROC. NATL. ACAD. SCI. USA, vol. 105, no. 32, 12 August 2008 (2008-08-12), pages 11206 - 11211, XP055108020, DOI: doi:10.1073/pnas.0800297105 *
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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2566499A2 (fr) * 2010-05-04 2013-03-13 aTyr Pharma, Inc. Découverte innovante de compositions thérapeutiques, diagnostiques et à base d'anticorps liées à des fragments protéiques de complexe multi-arnt synthétase p38
CN102985103A (zh) * 2010-05-04 2013-03-20 Atyr医药公司 与p38多-tRNA合成酶复合物相关的治疗、诊断和抗体组合物的创新发现
EP2566499A4 (fr) * 2010-05-04 2014-08-06 Atyr Pharma Inc Découverte innovante de compositions thérapeutiques, diagnostiques et à base d'anticorps liées à des fragments protéiques de complexe multi-arnt synthétase p38
US9062302B2 (en) 2010-05-04 2015-06-23 Atyr Pharma, Inc. Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of p38 multi-tRNA synthetase complex
US9404104B2 (en) 2010-05-04 2016-08-02 Atyr Pharma, Inc. Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of P38 multi-tRNA synthetase complex
AU2011248101B2 (en) * 2010-05-04 2016-10-20 Atyr Pharma, Inc. Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of p38 multi-tRNA synthetase complex

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