KR20160057941A - Screening method for therapeutic agent of mitochondria dysfunction using TRAP1 gene - Google Patents

Abstract

The present invention relates to a method for screening an agent for preventing or treating diseases caused by mitochondrial dysfunction, which comprises a step of determining whether a tested formulation inhibits expression of TRAP1 or not. The present invention also relates to use of antisense RNA, siRNA and miRNA to TRAP1 genes, a TRAP1-specific antibody and a TRAP1 inhibitor for preventing or treating diseases caused by mitochondrial dysfunction. The method according to the present invention allows effective screening of materials having an effect of preventing and treating diseases caused by mitochondrial dysfunction. In addition, the composition capable of inhibiting TRAP1 and genes thereof has the effects of increasing resistance against oxidative stress and restoring mitochondrial dysfunction into a normal condition.

Description

{Screening method for therapeutic agent for mitochondrial dysfunction using TRAP1 gene}

The present invention relates to a method for screening for a therapeutic agent for mitochondrial dysfunction using the TRAP1 gene, and more particularly, to a method for screening a therapeutic agent for mitochondrial dysfunction using the TRAP1 gene, comprising: (a) culturing TRAP1- And (b) measuring the level of expression of TRAP1 in cells incubated with the test agent and comparing the level of expression to the cultured cells without the test agent to determine whether the test agent inhibits the expression of TRAP1 A method for screening a drug for the prevention or treatment of diseases caused by mitochondrial dysfunction, antisense RNA for TRAP1 gene, siRNA and miRNA, antibodies specific for TRAP1 and mitochondrial dysfunction of TRAP1 inhibitor (TRAP1 inhibitor) For the prevention or treatment of diseases caused by cancer.

Although mitochondria have been regarded as cell organelles that simply supply energy to cells, they have been actively studied for various biological functions of mitochondria in the 1990s as a key cell signal regulator for cell death. Recently, it has been reported that the dysfunction of mitochondria is responsible for various diseases related to aging process and aging - important chronic diseases such as cancer and diabetes, functional abnormalities of important organs such as circulatory system, respiratory system, internal organs and endocrine system and Alzheimer's disease or Parkinson's disease Have been reported to be closely related to the onset and worsening of neurodegenerative diseases. It has been reported that the function of the mitochondrial electron transport system is reduced in muscle and liver tissues of type 2 diabetic patients with insulin resistance [1], and it has been reported that the mitochondrial electron transport system in the lesion tissue of a representative degenerative neurological disease, It has been observed that the activity of complex I is decreased [2]. Furthermore, when Parkinson and Parkinson's genes, Parkin and PINK1, were deleted in the Drosophila model, mitochondria in muscles and dopaminergic neurons were severely damaged, resulting in loss of motility and dopaminergic neuron loss similar to human patients. Mitochondrial dysfunction And the mechanism of disease development and deterioration [3, 4]. Therefore, studies have been actively conducted to develop the preventive and therapeutic methods for the various mitochondria-related diseases mentioned above. Currently, attempts to normalize the function of mitochondria degraded in the patient's body by controlling the activities of important components constituting mitochondria are underway at home and abroad .

PINK1 and Parkin have been cloned into a Parkinson-related gene that induces autosomal recessive juvenile parkinsonism (AR-JP) at the time of deficiency [5, 6], and vigorous studies have been conducted on their biological functions and pathophysiological roles. In the early days, Parkin focused on the ubiquitin ligase, which binds ubiquitin to a specific protein. Much research has been carried out to explore the link between Parkin and Parkinson's disease, And the pathophysiology of Parkinson 's disease was not clear. However, when the gene of PINK1, a phosphorylating enzyme located in mitochondria, was deleted in Drosophila, Parkinsonism-related symptoms (dopaminergic neurons decrease, motility decrease) accompanied with mitochondrial dysfunction occurred, and when Parkin was expressed in PINK1 gene deletion Drosophila, In the present study, the effects of PINK1 and Parkin in the mitochondria were investigated. In the present study, the membrane potential of the inner membrane was decreased. PINK1, which is located in the mitochondria, specifically activates Parkin to the damaged mitochondria, triggering remodeling of the damaged mitochondria and repairing or eliminating them to maintain intracellular mitochondrial function [7-9]. In addition, in a variety of genetic and cellular biology experiments using the PINK1 gene, it has been shown that PINK1 maintains mitochondrial function through other signaling pathways other than Parkin [10,11], suggesting that PINK1 inhibits neuronal cell protection and aging It has been shown that genes Sir2 and FOXO play important roles in maintaining mitochondrial function and protecting dopamine neurons. [12].

TRAP1 (TNF receptor-associated protein 1) is very similar to the 90-kDa heat shock protein (HSP90) present in the cytoplasm as a heat shock protein (HSP) specifically present in mitochondria [13]. Previous studies have shown that when TRAP1 is present, it protects cells from reactive oxygen species in various animal cell lines [14], but the specific molecular mechanism of cell protection by TRAP1 has not been elucidated.

[1] Lowell BB, Shulman GI. (2005) Mitochondrial dysfunction and type 2 diabetes. Science 307: 384-387. [2] Hoepken HH, Gispert S, Morales B, Wingerter O, Del Turco D, et al. (2007) Mitochondrial dysfunction, peroxidation damage and changes in glutathione metabolism in PARK6. Neurobiol Dis 25: 401-411. [3] Cha GH, Kim S, Park J, Lee E, Kim M, et al. (2005) Parkin negatively regulates JNK pathway in the dopaminergic neurons of Drosophila. Proc Natl Acad Sci U SE 102: 10345-10350. [4] Park J, Lee SB, Lee S, Kim Y, Song S, et al. (2006) Mitochondrial dysfunction in Drosophila PINK1 mutants is complemented by parkin. Nature 441: 1157-1161. [5] Kitada T, Asakawa S, Hattori N, Matsumine H, Yamamura Y, et al. (1998) Mutations in the parkin gene cause autosomal recessive juvenile parkinsonism. Nature 392: 605-608. [6] Valente EM, Abou-Sleiman PM, Caputo V, Muqit MMK, Harvey K, et al. (2004) Hereditary early-onset Parkinson's disease caused by mutations in PINK1. Science 304: 1158-1160. [7] Park J, Lee G, Chung J (2009) The PINK1-Parkin pathway is involved in the regulation of mitochondrial remodeling process. Biochem Biophys Res Commun 378: 518-523. [8] Kim Y, Park J, Kim S, Song S, Won SK, et al. (2008) PINK1 controls mitochondrial localization of Parkin through direct phosphorylation. Biochem Biophys Res Commun 377: 975-980. [9] Geisler S, Holmstrom KM, Skujat D, Fiesel FC, Rothfuss OC, et al. (2010) PINK1 / Parkin-mediated mitophagy is dependent on VDAC1 and p62 / SQSTM1. Nature Cell Biol 12: 119-131. [10] Pridgeon JW, Olzmann JA, Chin LS, Li L (2007) PINK1 protects against oxidative stress by phosphorylating mitochondrial chaperone TRAP1. Plos Biol 5: E172. doi: 10.1371 / journal.pbio.0050172. [11] Imai Y, Kanao T, Sawada T, Kobayashi Y, Moriwaki Y, et al. (2010) The Loss of PGAM5 Suppresses the Mitochondrial Degeneration Caused by Inactivation of PINK1 in Drosophila. Plos Genet 6: e1001229. doi: 10.1371 / journal.pgen.1001229. [12] Koh H, Kim H, Kim JJ, Park J, Chung J (2012) Sir2 and FOXO Complement Mitochondrial Dysfunction and Dopaminergic Neuron Loss in Drosophila PINK1 Mutants. J Biol Chem 287: 12750-12758. [13] Kang BH (2012) TRAP1 regulation of mitochondrial life or death decisions in cancer cells and ccsmitochondria-targeted TRAP1 inhibitors. BMB Rep 45: 1-6. [14] Montesano Gesualdi N, Chirico G, Pirozzi G, Costantino E, Landriscina M, Esposito F. Tumor necrosis factor-associated protein 1 (TRAP-1) protects cells from oxidative stress and apoptosis. Stress 2007; 10: 342-350.

Accordingly, the inventors of the present invention found that inhibiting TRAP1 activity or expression while studying the intracellular role and molecular mechanism of TRAP1 not only improves the resistance of cells to reactive oxygen, but also the function of mitochondrial dysfunction induced by PINK1 deficiency And the present invention was completed.

Accordingly, it is an object of the present invention to provide a method for producing a tumor necrosis factor receptor-associated protein 1 (TRAP1) comprising: (a) culturing cells expressing tumor necrosis factor receptor-associated protein 1 (TRAP1) And (b) measuring the activity of TRAP1 or the expression level of TRAP1 in the cells incubated with the test agent, and comparing the activity of TRAP1 or the level of expression of TRAP1 in cells cultured without the test agent, Or inhibiting the expression of TRAP1 in a mammal, which comprises administering a therapeutically effective amount of TRAP1 to a subject in need thereof.

It is another object of the present invention to provide a composition for preventing and treating diseases caused by mitochondrial dysfunction, which comprises as an active ingredient any one selected from the group consisting of antisense RNA, siRNA and miRNA for the TRAP1 gene.

It is still another object of the present invention to provide a composition for preventing and treating diseases caused by mitochondrial dysfunction which comprises an antibody specific for TRAP1 as an active ingredient.

It is still another object of the present invention to provide a composition for preventing and treating a disease caused by a mitochondrial dysfunction which comprises a TRAP1 inhibitor (TRAP1 inhibitor) as an active ingredient.

(A) culturing cells expressing tumor necrosis factor receptor-associated protein 1 (TRAP1) with or without a test agent; And (b) measuring the activity of TRAP1 or the expression level of TRAP1 in the cells incubated with the test agent, and comparing the activity of TRAP1 or the level of expression of TRAP1 in cells cultured without the test agent, Or inhibiting the expression of TRAP1 in a mammal, which comprises administering an effective amount of TRAP1 to a subject in need thereof.

In order to accomplish another object of the present invention, the present invention provides a method for preventing and treating diseases caused by mitochondrial dysfunction, which comprises, as an active ingredient, any one selected from the group consisting of antisense RNA, siRNA and miRNA for the TRAP1 gene ≪ / RTI >

In order to accomplish still another object of the present invention, the present invention provides a composition for preventing and treating diseases caused by mitochondrial dysfunction comprising an antibody specific for TRAP1 as an active ingredient.

In order to accomplish still another object of the present invention, the present invention provides a composition for preventing and treating diseases caused by mitochondrial dysfunction comprising TRAP1 inhibitor (TRAP1 inhibitor) as an active ingredient.

Hereinafter, the present invention will be described in detail.

The present inventors first discovered that when the activity or expression of TRAP1 is inhibited in a cell, not only the ability of the cell to react with reactive oxygen is enhanced, but also the functional disorder of the mitochondria induced by PINK1 deficiency is recovered. Is disclosed first in the specification.

Justice

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The following references provide one of the skills with a general definition of the various terms used in the specification of the present invention. Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY (2d ed. 1994); THE CAMBRIDGE DICTIONARY OF SCIENCE AND TECHNOLOGY (Walker ed., 1988); And Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY. The following definitions are also provided to assist the reader in the practice of the present invention.

As used herein, the term "protein" is used interchangeably with "polypeptide" or "peptide" and refers to a polymer of amino acid residues as commonly found in natural state proteins.

In the present invention, "nucleic acid", "DNA sequence" or "polynucleotide" refers to deoxyribonucleotides or ribonucleotides in the form of single- or double-stranded. Unless otherwise constrained, also includes known analogs of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides.

The term "homologues " as used herein refers to those derived from a common ancestral protein or protein sequence naturally or artificially when referring to protein and / or protein sequences. Similarly, nucleic acid 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 "analog" as used herein refers to a substance that is structurally similar to a reference molecule, but whose target or control method has been modified by substitution of a specific substituent of the reference material. Compared to standard materials, analogs have the same, similar, or improved utility as can be expected by those skilled in the art. Synthesis and screening of analogs for identifying known compound variants with improved properties (e.g., higher binding affinity for the target substance) are methods known in the pharmacochemical field.

The present invention

(a) culturing cells expressing tumor necrosis factor receptor-associated protein 1 (TRAP1) with or without a test agent; And

(b) measuring the level of expression of TRAP1 in the cells incubated with the test agent, comparing the expression level of TRAP1 with the cells cultured without the test agent, and confirming whether the test agent inhibits the expression of TRAP1 A method for screening a medicament for preventing or treating a disease caused by mitochondrial dysfunction.

(a) is a step of culturing cells expressing tumor necrosis factor receptor-associated protein 1 (TRAP1) with or without a test agent.

The 'TRAP1' of the present invention has high homology with hsp90 and binds to a type 1 tumor necrosis factor receptor (see Song et al., J. Biol. Chem., 270: 3574-3581 (1995 )). The yeast 2-hybrid screen and 5-prime RACE of the Gal4 / HeLa cDNA library were performed using the intracellular domain of TNFR1 as bait, Song et al. (1995) isolated cDNA fragments encoding TRAP1. The deduced 661-amino acid protein is 60% similar to the HSP90 family member, but lacks the highly charged domain found in the HSP90 protein. See, for example, Felts et al., J Biol Chem. 2000; 275 (5): 3305-12. Also, TRAP1 is a heat-shock protein 75-KD (HSP75); Tumor necrosis factor receptor-associated protein 1; TRAP1; And TNFR-associated protein 1.

The 'TRAP1' of the present invention is not particularly limited as long as it has an amino acid sequence of a polypeptide known as TRAP1, but preferably a polypeptide having the amino acid sequence of SEQ ID NO: 1 (Genbank Accession No. NP_057376.2) or SEQ ID NO: 3 (Genbank Accession No. NP_477439. 2). ≪ / RTI > TRAP1 of the present invention also includes functional equivalents thereof.

The functional equivalent means a polypeptide having at least 70%, preferably at least 80%, more preferably at least 90% sequence homology (i.e., identity) with the amino acid sequence of TRAP1. For example, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84% Sequence homology to the sequences of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, Quot; refers to a polypeptide having substantially the same physiological activity as the polypeptide represented by SEQ ID NO: 1 or SEQ ID NO: 3. The functional equivalents may result in some of the amino acid sequence of TRAP1 being added, substituted or deleted. The substitution of the amino acid is preferably a conservative substitution. Examples of conservative substitutions of amino acids present in nature are as follows: (Gly, Ala, Pro), hydrophobic amino acids (Ile, Leu, Val), aromatic amino acids (Phe, Tyr, Trp), acidic amino acids (Asp, Glu), basic amino acids (His, Lys, Arg, Gln, Asn ) And sulfur-containing amino acids (Cys, Met). Also included in such functional equivalents are variants in which a portion of the amino acid is deleted in the amino acid sequence of the TRAP1 polypeptide of the invention. The deletion or substitution of the amino acid is preferably located in a region that is not directly related to the physiological activity of the polypeptide of the present invention. In addition, deletion of the amino acid is preferably located at a site that is not directly involved in the physiological activity of the TRAP1 polypeptide. Also included are variants in which several amino acids have been added at both ends or sequences of the amino acid sequence of the TRAP1 polypeptide. Also included within the scope of the functional equivalents of the present invention are polypeptide derivatives in which some of the chemical structures of the polypeptides are modified while maintaining the basic skeleton of the polypeptide according to the present invention and its physiological activity. This includes, for example, structural modifications to alter the stability, shelf stability, volatility, or solubility of the polypeptides of the present invention.

As used herein, sequence homology and homology refers to the percentage of amino acid residues of the candidate sequence to the amino acid sequence of TRAP1 after aligning the candidate sequence with the amino acid sequence of TRAP1 (SEQ ID NO: 1 or SEQ ID NO: 3) and introducing gaps Is defined. If necessary, conservative substitutions as part of sequence homology are not considered to obtain maximum percent sequence homology. Also, the N-terminus, C-terminus, or internal stretch, deletion, or insertion of the amino acid sequence of TRAP1 is not interpreted as a sequence that affects sequence homology or homology. In addition, such homology can be determined by standard standard methods used to compare similar portions of amino acid sequences of two polypeptides. A computer program, such as BLAST, aligns two polypeptides so that each amino acid is optimally matched (either along the full length sequence of one or two sequences or along the predicted portion of one or two sequences). The program provides a PAM 250 (standard scoring matrix; Dayhoff et al., In Atlas of Protein (R)), which provides a default opening penalty and a default gap penalty and can be used in conjunction with a computer program Sequence and Structure, vol 5, supp 3, 1978). For example, percentage homogeneity can be calculated as: By multiplying the total number of identical matches by 100 and then multiplying by the sum of the length of the longer sequence in the matched span and the number of gaps introduced into the longer sequence to align the two sequences Share it.

The proteins (polypeptides) according to the present invention can be extracted naturally or constructed by genetic engineering methods. For example, a nucleic acid encoding the polypeptide or a functional equivalent thereof (e.g., SEQ ID NO: 2 or SEQ ID NO: 4) is constructed according to a conventional method. The nucleic acid can be constructed by PCR amplification using an appropriate primer. Alternatively, DNA sequences may be synthesized by standard methods known in the art, for example, using an automated DNA synthesizer (commercially available from Biosearch or Applied Biosystems). The constructed nucleic acid is inserted into a vector comprising one or more expression control sequences (e.g., promoters, enhancers, etc.) operatively linked to the expression of the nucleic acid, and the recombinant The host cell is transformed with an expression vector. The resulting transformant is cultured under culture medium and conditions suitable for expression of the nucleic acid to recover a substantially pure polypeptide expressed by the nucleic acid from the culture. The recovery can be carried out using methods known in the art (for example, chromatography). By "substantially pure polypeptide" as used herein is meant that the polypeptide according to the invention is substantially free of any other proteins derived from the host cell. Genetic engineering methods for the synthesis of polypeptides of the present invention can be found in Maniatis et al., Molecular Cloning; A laboratory Manual, Cold Spring Harbor Laboratory, 1982; Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, NY, Second (1998) and Third (2000) Editions; Gene Expression Technology, Method in Enzymology, Genetics and Molecular Biology, Method in Enzymology, Guthrie & Fink (eds.), Academic Press, San Diego, Calif., 1991; And Hitzeman et al., J. Biol. Chem., 255: 12073-12080,1990.

Also, the polypeptide of the present invention can be easily prepared by chemical synthesis (Creighton, Proteins, Structures and Molecular Principles, WH Freeman and Co., NY, 1983) known in the art. Representative methods include, but are not limited to, liquid or solid phase synthesis, fractional condensation, F-MOC or T-BOC chemistry (see for example Chemical Approaches to the Synthesis of Peptides and Proteins, Williams et al., Eds., CRC Press , Boca Raton Florida, 1997; A Practical Approach, Atherton and Sheppard, Eds., IRL Press, Oxford, England, 1989).

As used herein, the term " promoter " refers to a DNA sequence that regulates the expression of a nucleic acid sequence operatively linked to a particular host cell. The term 'operably linked' means that a nucleic acid fragment is different from other nucleic acid fragments Quot; is meant that the function or expression thereof is influenced by other nucleic acid fragments. In addition, it may further comprise any operator sequence for regulating transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence controlling the termination of transcription and translation. As the promoter, a constitutive promoter that induces expression of a target gene at all times or an inducible promoter that induces expression of a target gene at a specific position and time can be used.

In the present invention, the term "host cell" includes heterologous DNA introduced into cells by any means (for example, electric shock method, calcium phosphatase precipitation method, microinjection method, transformation method, virus infection, etc.) Refers to prokaryotic or eukaryotic cells.

In the present invention, the 'polynucleotide encoding TRAP1' may have an amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 3, or a nucleotide sequence encoding an amino acid sequence having at least 70% or more sequence homology. The nucleic acid includes both DNA, cDNA, and RNA sequences. That is, the polynucleotide may have a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3, or an amino acid sequence having at least 70% or more homology thereto, or may have a nucleotide sequence complementary to the nucleotide sequence. Preferably, it has a nucleotide sequence represented by SEQ ID NO: 2 (Genbank Accession No. NM_016292.2) or SEQ ID NO: 4 (Genbank Accession No. NM_058091.3). The nucleic acid may be isolated from nature or may be prepared by genetic engineering methods as described above.

As used herein, the term "expression " refers to the production of a protein or nucleic acid in a cell, and includes both transcription and translation of a specific nucleotide sequence caused by a promoter. Thus, the expression " TRAP1 expression " of the present invention is meant to include both transcription and translation of a nucleotide sequence (for example, SEQ ID NO: 2 or SEQ ID NO: 4) encoding a TRAP1 protein.

The 'TRAP1-expressing cell' is not limited to the cell in which the TRAP1 gene is expressed in the cell, but it may preferably be a eukaryotic cell, more preferably a mitochondria (mitochondria) .

In the present invention, the term "agent" or "test agent" refers to any substance, molecule, element, compound, Combinations. But are not limited to, proteins, polypeptides, small organic molecules, polysaccharides, polynucleotides, and the like. It may also be a natural product, a synthetic compound or chemical compound, or a combination of two or more substances. Unless otherwise specified, the agents, substances and compounds may be used interchangeably.

Test agents that can be screened or identified by the methods of the invention include but are not limited to polypeptides, beta-turn mimetics, polysaccharides, phospholipids, hormones, prostaglandins, steroids, aromatics, heterocyclic compounds, benzodiazepines Oligomeric N-substituted glycines, oligocarbamates, saccharides, fatty acids, purines, pyrimidines or derivatives, structural analogs or combinations thereof. Some test agents may be synthetic and other test agents may be natural. The test agent can be obtained from a wide variety of sources including libraries of synthetic or natural compounds. A combinatorial library can be produced with a variety of compounds that can be synthesized step-by-step. Compounds of multiple combinatorial libraries can be produced by the encoded synthetic libraries (ESL) method (WO 95/12608, WO 93/06121, WO 94/08051, WO 95/395503 and WO 95/30642). 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 applied to direct or random chemical modifications such as acylation, alkylation, esterification, amidification to produce structural analogs. The test agent may be a naturally occurring protein or a fragment thereof. Such test agents can be obtained from natural sources, such as cell or tissue lysates. Libraries of polypeptide preparations can be obtained, for example, by conventional methods or from commercially available cDNA libraries. The test agent may be a peptide, for example, a peptide having about 5-30 amino acids, preferably about 5-20 amino acids, and more preferably about 7-15 amino acids. The peptide may be a naturally occurring protein, a random peptide or a cleavage of a "biased" random peptide.

The test agent may also be a "nucleic acid. &Quot; The nucleic acid test agent may be a naturally occurring nucleic acid, a random nucleic acid, or a" biased " random nucleic acid. For example, cuts of prokaryotic or eukaryotic genomes can be used similar to those described above.

The test agent may also be a small molecule (e.g., a molecule having a molecular weight of about 1,000 or less). Preferably, a high throughput assay can be applied to the method for screening a control preparation of a small molecule. A combination library of small molecule test preparations as described above can be easily applied to the screening method of the present invention. Many assays are useful in the screening (Shultz, Bioorg. Med. Chem. Lett., 8: 2409-2414, 1998; Weller, Mol. Drivers., 3: 61-70, 1997; Fernandes, Curr. Opin. Chem. Biol., 2: 597-603, 1998; and Sittampalam, Curr. Opin. Chem. Biol., 1: 384-91, 1997).

Libraries of test agents screened in the methods of the present invention can be prepared based on structural studies of TRAP1 proteins or analogs. This structural study enables the identification of test agents potentially binding to TRAP1. The three-dimensional structure of TRAP1 can be studied in a number of ways, such as crystal structure and molecular modeling. Methods of studying protein structures using X-ray crystallography are well known in the literature: Physical Bio-Chemistry, Van Holde, KE (Prentice-Hall, New Jersey 1971), pp. 221-239, and Physical Chemistry with Applications to the Life Sciences, D. Eisengerg & D.Crothers (Benjamin Cummings, Menlo Park 1979). Computer modeling of the structure of TRAP1 provides other means for the design of test agents for screening. Molecular modeling methods are described in the literature: US Pat. No. 612,894 and US Pat. No. 5,583,973. Protein structures can also be determined by neutron diffraction and nuclear magnetic resonance (NMR): Physical Chemistry, 4th Ed. Moore, WJ (Prentice-Hall, New Jersey 1972) and NMR of Proteins and Nucleic Acids, K. Wuthrich (Wiley-Interscience, New York 1986).

In the present invention, 'culturing cells expressing TRAP1 together with a test agent' means a process of inducing contact between TRAP1 itself and factors affecting its expression and the test agent. The method of contacting the test agent may be, for example, by in vitro administration of the test agent to the outside of the cell membrane to be introduced into the cell, or by a known standard recombinant DNA and molecular cloning technique But the present invention is not limited thereto.

(b) comprises measuring the expression level of TRAP1 in the cells incubated with the test agent of step (a), comparing the expression level of TRAP1 with the level of expression of TRAP1 in the cells cultured without the test agent, TRAP1 < / RTI >

The drug screening method for prevention or treatment of diseases caused by the mitochondrial dysfunction of the present invention can be performed by measuring the expression level of TRAP1, wherein the 'measurement of the expression level of TRAP1' means the measurement of the expression level of the TRAP1 gene Quot; is meant to include both mRNA expression level measurement and protein expression level measurement (protein amount change measurement). The 'measurement of mRNA expression level' refers to measuring the amount of mRNA by measuring the presence and expression level of TRAP1 gene in the target cell. The 'measurement of protein expression level' is a process of confirming the presence and the expression level of a protein expressed from a TRAP1 gene in a target cell, preferably using an antibody that specifically binds to the TRAP1 gene, But is not limited to.

As described above, when the screening method of the present invention is carried out by analyzing the expression of TRAP1, the change in the expression level of the TRAP1 gene can be measured by various methods known in the art. For example, mRNA expression levels can be measured by RT-PCR (Sambrook et al., Molecular Cloning, A Laboratory Manual, 3rd ed. Cold Spring Harbor Press (2001)), competitive RT- Real-time RT-PCR, RNase protection assay (RPA), northern blotting (Peter B. Kaufma et al., Molecular and Cellular Methods in Biology and Medicine, 102-108, CRC press) (Sambrook et al., Molecular Cloning, A Laboratory Manual, 3rd Ed. Cold Spring Harbor Press (2001)) or in situ hybridization reaction (Sambrook et al., Molecular Cloning. ed. Cold Spring Harbor Press (2001)), DNA chips, and the like, but the present invention is not limited thereto.

When the RT-PCR is performed according to the RT-PCR protocol, total RNA is isolated from cells treated with the test substance, and first strand cDNA is prepared using oligo dT primer and reverse transcriptase. Next, the first-strand cDNA is used as a template and a PCR reaction is carried out using a TRAP1 gene-specific primer set. Then, the PCR amplification product is electrophoresed, and the band formed is analyzed to measure the change amount of TRAP1 gene expression.

In addition, measurement of protein expression levels (i. E., Changes in the amount of TRAP1 protein) may be performed through a variety of immunoassay methods known in the art. For example, changes in the amount of TRAP1 protein can be measured by radioimmunoassay, radioimmunoprecipitation, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), capture-ELISA, inhibition or competitive assay, and sandwich assay Ouchterlony immunodiffusion, rocket immunoelectrophoresis, tissue immuno staining, Complement Fixation Assay, Fluorescence Activated Cell Sorter (FACS), Western blot, protein chip ) May be used, but the present invention is not limited thereto.

The 'mitochondrial dysfunction' of the present invention is a state in which the biological activity of mitochondria appearing in normal cells is reduced, but not limited thereto. For example, mitochondrial enzyme activity, electron transport chain activity, Increased production of reactive oxygen species, mitochondrial fragmentation, calcium regulation disorders, and mutation of mitochondrial DNA (mtDNA).

The 'disease caused by mitochondrial dysfunction' of the present invention is preferably a disease due to a mitochondrial dysfunction caused by a PINK1 gene deletion.

The 'deletion' means that the function of the gene is deleted. The 'PINK1 gene deletion' of the present invention includes cases in which a functional deletion type mutation occurs in the PINK1 gene and a case in which the PINK1 gene is deleted. The functional deletion mutation of the PINK1 gene may be caused by a disruption of the normal expression of the PINK1 gene through an arbitrary manipulation (mutation such as substitution, inversion, addition or redundancy) added to the gene, or a protein expressed from the gene is inherently biologically Is meant to include those that do not have activity.

The PTEN-induced putative kinase 1 (PINK1) gene may preferably have a polynucleotide sequence represented by SEQ ID NO: 6 (Genbank Accession No. NM_032409.2) or SEQ ID NO: 8 (Genbank Accession No. NM_132112.4) , But is not limited thereto. Also, the PINK1 protein expressed by the polynucleotide may have an amino acid sequence represented by SEQ ID NO: 5 (Genbank Accession No. NP_115785.1) or SEQ ID NO: 7 (Genbank Accession No. NP_572340.2).

Specifically, the 'diseases caused by mitochondrial dysfunction' include degenerative brain diseases, NARP syndrome, Multiple Sclerosis-like Syndrome, Maternally Inherited Cardio Myopathy, Progressive External Ophthalmoplegia, Myoclonic Epilepsy with Ragged-Red Fibers syndrome, Myoneurogastrointestinal disorder and encephalopathy, Pearson Marrow syndrome, Kearns-Sayre syndrome ), Leber hereditary optic neuropathy, aminoglycoside-associated deafness, diabetes with deafness, Luft disease, Leigh syndrome, Alpers Disease, medium chain acyl-CoA dehydrogenase (MCAD) defici ency), Segmental colitis associated with diverticular (SCAD) disease, Short chain 3-hydroxyacyl CoA dehydrogenase [SCHAD] deficiency, High-chain acyl coacidase (Long chain acyl-CoA dehydrogenase [LCHAD] deficiency), glutaric aciduria II (long chain acyl-CoA dehydrogenase [LCHAD] And lethal infantile cardiomyopathy. ≪ Desc / Clms Page number 2 >

The degenerative brain disease is characterized by being selected from the group consisting of Parkinson's disease, Alzheimer's disease, Huntington's disease, and dementia.

 Further, the screening method of the present invention can be carried out after the steps (a) and (b)

(c) a step of inspecting whether or not a test agent confirmed to inhibit the expression of TRAP1 in step (b) is administered to an animal having a disease caused by mitochondrial dysfunction to show a therapeutic effect ' .

In step (c), the animal is preferably a non-human animal. The term " therapeutic effect " includes a part or all of the effect of alleviating or ameliorating the disease due to the mitochondrial dysfunction and its symptoms, or suppressing the progress of the mitochondrial dysfunction.

(A) culturing TRAP1 or a cell expressing said TRAP1 and a test agent; And

(b) measuring the activity of TRAP1, and confirming whether the test agent inhibits the activity of TRAP1, and screening the agent for the prevention or treatment of diseases caused by mitochondrial dysfunction.

Whether the test agent inhibits the activity of TRAP1 in step (b) can be determined by measuring the activity of TRAP1 in the cells cultured with the test agent and comparing the level of TRAP1 activity with that of cultured cells without the test agent It can be judged according to the decrease.

The screening method may be carried out after the steps (a) and (b)

(c) a step of inspecting whether the test agent confirmed to inhibit the activity of TRAP1 in step (b) is administered to an animal having a disease caused by mitochondrial dysfunction to show a therapeutic effect ' .

In the method for screening drugs for the prevention or treatment of diseases caused by the mitochondrial dysfunction of the present invention, screening methods for agents that inhibit the activity of TRAP1 protein are not limited thereto. For example, Or by screening for a substance that binds to the TRAP1 protein. In the latter case, any type of TRAP1 protein can be used, such as isolated TRAP1 or TRAP1 protein contained in the cells.

The screening method of the present invention can be carried out in various ways, and can be carried out in a high throughput manner according to various binding assays known in the art. In the screening method of the present invention, the test substance or TRAP1 protein may be labeled with a detectable label. For example, the detectable label may be a chemical label (e.g., biotin), an enzyme label (such as horseradish peroxidase, alkaline phosphatase, peroxidase, luciferase,? -Galactosidase, (E.g., ¹⁴ C, ¹²⁵ I, ³² P and ³⁵ S), fluorescent labels (e.g., coumarin, fluorescein, FITC (fluoresein isothiocyanate), rhodamine 6G, rhodamine B , TAMRA (6-carboxy-tetramethyl-rhodamine), Cy-3, Cy-5, Texas Red, Alexa Fluor, DAPI, HEX, TET, Dabsyl and FAM, A chemiluminescent label, a fluorescence resonance energy transfer (FRET) label or a metal label (e.g., gold and silver).

When a detectable marker labeled TRAP1 protein or test substance is used, whether or not the binding between the TRAP1 protein and the test substance is detected can be detected by detecting a signal from the label. For example, when alkaline phosphatase is used as a label, it is possible to use bromochloroindoleyl phosphate (BCIP), nitroblue tetrazolium (NBT), naphthol-AS-B1-phosphate and Signals are detected using chromogenic substrates such as enhanced chemifluorescence (ECF). When horseradish peroxidase is used as a label, it is preferable to use chlorinaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, lucigenin (bis-N-methylacridinium nitrate), resorpine benzyl ether, luminol, HYR (p-phenylenediamine-HCl and pyrocatechol), TMB (tetramethylbenzidine), ABTS (2,2'-Azine-di [3-ethylbenzthiazoline sulfonate]), o-phenylenediamine (OPD), and naphthol / pyronin.

Alternatively, the binding of the test substance to the TRAP1 protein may be assayed without the labeling of the interactants. For example, a microphysiometer can be used to analyze whether a test substance binds to TRAP1 protein. The microphysiometer is an analytical tool that uses a light-addressable potentiometric sensor (LAPS) to measure the rate at which a cell acidifies its environment. A change in the rate of acidification can be used as an indicator of the binding between the test substance and the TRAP1 protein (McConnell et al., Science 257: 19061912 (1992)).

The ability of the test substances to bind to the TRAP1 protein can be analyzed using real-time bimolecular interaction analysis (Sjolander & Urbaniczky, Anal. Chem., 63: 2338-2345 (1991), and Szabo et al. , Curr. Opin. Struct Biol., 5: 699-705 (1995)). BIA is a technique for analyzing specific interactions in real time and can be performed without the labeling of interactants (e.g., BIAcore ™). Changes in surface plasmon resonance (SPR) can be used as indicators for real-time reactions between molecules.

The screening method of the present invention can be carried out according to a two-hybrid analysis or a three-hybrid analysis method (US Pat. No. 5,283,317; Zervos et al., Cell 72, 223232, 1993; Madura et al. Bartel et al., BioTechniques 14, 920924, 1993; Iwabuchi et al., Oncogene 8, 16931696, 1993; and WO 94/10300). In this case, the TRAP1 protein can be used as a "bait" protein. According to this method, a substance binding to TRAP1 protein, particularly a protein, can be screened. To-hybrid system is based on the modular nature of the transcription factor consisting of segmentable DNA-binding and activation domains. Briefly, this analysis method uses two DNA constructs. For example, in one construct, a TRAP1-coding polynucleotide is fused to a DNA binding domain-coding polynucleotide of a known transcription factor (e.g., GAL4). In another construct, a DNA sequence encoding a protein of interest ("prey" or "sample (test preparation)") is fused to a polynucleotide encoding the activation domain of the known transcription factor. If the bait and the fray interact in vivo to form a complex, the DNA-binding and activation domains of the transcription factor become adjacent, which triggers the transcription of the reporter gene (e.g., LacZ). The expression of the reporter gene can be detected. This indicates that the protein to be analyzed can bind to the TRAP1 protein. As a result, it can be used as an agent for preventing and treating diseases caused by increased antioxidant ability of cells and mitochondrial dysfunction Lt; / RTI >

Next, the activity of the TRAP1 protein treated with the test substance is measured. As a result of the measurement, when it is measured that the activity of TRAP1 protein is down-regulated, the test substance can be determined as a preventive and therapeutic agent for a disease caused by mitochondrial dysfunction.

The screening method of the present invention is characterized in that the test agent is selected to inhibit (or inhibit) the expression of TRAP1 or TRAP1, thereby selecting a substance having a preventive and therapeutic effect against a disease caused by mitochondrial dysfunction do. The above method utilizes the principle that TRAP1 activity or TRAP1 gene expression is inhibited, resistance to oxidative stress is increased, and mitochondrial dysfunction caused by PINK1 gene deletion is restored. This fact is disclosed in the present invention will be.

This is well illustrated in the specification of the present invention.

In one embodiment of the present invention, a Drosophila model lacking TRAP1 gene was prepared, and paraquat and rotenone, known as oxidative stress agents, were treated with TRAP1 deficient Drosophila and the survival rate of Drosophila was measured. As a result, the survival rate of TRAP1 deficient Drosophila was higher than that of normal Drosophila. The same results were obtained in the Drosophila that inhibited TRAP1 mRNA expression. Thus, it was confirmed that the resistance to oxidative stress is increased by inhibiting the TRAP1 gene.

In another embodiment of the present invention, resistance to oxidative stress and recovery of mitochondrial dysfunction were evaluated in the absence of TRAP1 gene in Drosophila expressing mitochondrial dysfunction due to deletion of PINK1 gene. As a result, we confirmed that the TRAP1 gene deletion normalizes the loss of rotenone resistance due to the PINK1 gene deletion, and the PINK1 gene deletion such as chest muscle apoptosis and mitochondrial dysplasia, mitochondrial amount and ATP level in the chest muscle, decreased motility and dopaminergic neuronal cell death mutant phenotypes were normalized when the TRAP1 gene was deleted.

In another embodiment of the present invention, gammitrinib-TPP (G-TPP), which is known as a TRAP1 specific inhibitor, is administered to Drosophila to determine whether mitochondrial dysfunction caused by PINK1 deficiency can be normalized. As a result, the mobility of the PINK1 gene-deficient Drosophila melanogaster treated with the G-TPP solution was statistically significantly restored, and that the administration of the G-TPP solution for 30 days inhibited the death of dopaminergic neurons by the PINK1 deficiency. In contrast, in the PINK1-deficient cell line, the mitochondrial membrane potential decreased when the carbon source was changed from gulcose to galactose. G-TPP treatment decreased the membrane potential.

As described above, the inhibitor of TRAP1 gene expression or the activity of TRAP1 protein prevents and treats dysfunction of mitochondria, particularly mitochondrial dysfunction caused by PINK1 gene deletion and diseases thereof, and elevates antioxidant power of cells.

Accordingly, the present invention provides a method for preventing and / or preventing diseases caused by mitochondrial dysfunction, which comprises, as an active ingredient, any one selected from the group consisting of antisense RNAs, siRNAs and miRNAs specific for the TRAP1 gene (polynucleotide encoding TRAP1) Thereby providing a therapeutic composition. The " specific " means the ability to suppress only the target gene without affecting other genes in the cell.

The "antisense RNA" refers to an RNA transcript that is complementary to all or a portion of a first transcript of a target gene and that blocks the expression of the target gene by interfering with the processing, transport and / or translation of the first transcript or mRNA . The antisense RNA may be complementary to a portion of a particular gene transcript in a 5 'noncoding sequence, a 3' noncoding sequence, an intron, or a coding sequence. The term " ribozyme " as used herein refers to a catalytic RNA, and refers to a sequence specific endoribonucleic acid Lt; / RTI > The 'target gene' is TRAP1 for the purpose of the present invention.

The 'RNA transcript' refers to a product resulting from transcription catalyzed by an RNA polymerase of the DNA sequence. When the RNA transcript is a complete complementary copy of the DNA sequence, it may be an RNA sequence derived from the first transcript or from post-transcription processing of the first transcript. "Messenger RNA (mRNA)" refers to RNA that can be translated into a protein by a cell.

The term " siRNA " means a double-stranded RNA capable of inducing RNA interference (RNA interference) through cleavage of mRNA of a target gene, and includes a sense RNA strand having a sequence homologous to the mRNA of the target gene And antisense RNA strands having complementary sequences. siRNA is provided as an efficient gene knockdown method or gene therapy method because it can inhibit the expression of a target gene. The siRNA is not limited to a complete pair of double-stranded RNA portions that are paired with each other, but is paired by a mismatch (the corresponding base is not complementary), a bulge (no base corresponding to one chain) May be included. The siRNA termini are both blunt or cohesive termini. The adhesive end structure can be a structure having a 3-terminal protruding structure and a 5-terminal protruding structure, and the number of protruding bases is not limited. In addition, the siRNA can be added to a protruding portion of one end in a range where the effect of suppressing the expression of the target gene can be maintained, for example, a small RNA (for example, a natural RNA molecule such as tRNA, rRNA, ). The siRNA terminal structure does not need to have a cleavage structure on both sides, and a stem loop structure in which one terminal region of double-stranded RNA is connected by linker RNA may be used. The length of the linker is not particularly limited as long as it does not interfere with the pairing of the stem portions.

The siRNA used in the present invention may be a complete form having polynucleotide pairing itself, that is, a form in which siRNA is directly synthesized in a test tube and introduced into a cell through two transformation processes, Single chain oligonucleotide fragments and their reverse-phase trefoil can be derived from single-stranded polynucleotides separated by a spacer, for example, a form of siRNA expression vector or PCR-derived siRNA prepared so that the siRNA is expressed in a cell The expression cassette may be introduced into the cell through a transformation or infection process. The determination of how to prepare siRNAs and introduce them into cells or animals may depend on the objective and the cellular biological function of the target gene product.

In the present invention, "miRNA (microRNA)" means a short non-coding RNA derived from an endogenous gene, which functions as a post-transcriptional regulator of gene expression. miRNAs serve as post-transcriptional regulators of gene expression by forming base pairs with the mRNA of the target gene. As long as the miRNA of the present invention has an activity of inhibiting the expression of the TRAP1 gene, its nucleotide sequence and its length are not particularly limited.

The antisense RNA, siRNA and miRNA for the TRAP1 gene of the present invention can be introduced into cells through a known method. For example, the antisense RNA, siRNA and miRNA for the TRAP1 gene of the present invention may be contained in a vector and provided to an individual. A promoter; And a polynucleotide encoding any one selected from the group consisting of antisense RNAs, siRNAs and miRNAs against the TRAP1 gene operably linked thereto, in the form of a recombinant expression vector .

The expression vector includes, but is not limited to, a plasmid vector, a cosmid vector, a bacteriophage vector, and a viral vector. Suitable expression vectors may include promoter, operator, initiation codon, termination codon, expression modulating elements such as polyadenylation signal and enhancer, and the like, and may be prepared variously according to the purpose.

The first combination vector may be introduced into a host cell using methods known in the art. Methods for introduction into host cells are preferably selected from the group consisting of calcium chloride method, microprojectile bombardment, electroporation, PEG-mediated fusion, microinjection, liposome mediated method liposome-mediated method) can be used.

Examples of host cells include prokaryotic cells such as Escherichia coli, Bacillus subtilis, Streptomyces, Pseudomonas, Proteus mirabilis or Staphylococcus. A host cell, a fungus (e.g., Aspergillus), a yeast (e.g., Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces ), Neurospora crassa and the like, eukaryotic cells such as insect cells, plant cells, mammals and the like can be used as a host cell, but not limited thereto, and preferably May be a human cell.

Meanwhile, the standard recombinant DNA and molecular cloning techniques used in the present invention are well known in the art and are described in Sambrook, J., Fritsch, EF and Maniatis, T., Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Laboratory, NY (1984), 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989); by Silhavy, TJ, Bennan, ML and Enquist, LW, Experiments with Gene Fusions, ; and by Ausubel, FM et al., Current Protocols in Molecular Biology, published by Greene Publishing Assoc. and Wiley-lnterscience (1987)).

The 'mitochondrial dysfunction' and 'diseases caused by mitochondrial dysfunction' are as described above in the screening method.

The present invention provides a composition for preventing and treating diseases caused by mitochondrial dysfunction, which comprises an antibody specific for TRAP1 as an active ingredient.

The term " specific " or " specific " as used herein refers to the specificity of an antibody capable of binding to only one antigen within a cell, and also refers to a specific epitope among a plurality of epitopes, It is also used in specific cases. In the present invention, the antigen is TRAP1.

The term " antibody " refers to a specific protein molecule directed against an antigenic site. For purposes of the present invention, an antibody refers to an antibody that specifically recognizes TRAP1, including both polyclonal and monoclonal antibodies.

Since the sequence and structure of the TRAP1 protein have already been identified in the field, the production of the antibody using the TRAP1 protein can be easily made using techniques well known in the art.

Polyclonal antibodies can be produced by methods well known in the art for obtaining sera containing antibodies by injection of the above-mentioned TRAP1 protein antigen into an animal and blood sampling from the animal. Such polyclonal antibodies can be prepared from any animal species host, such as goats, rabbits, sheep, monkeys, horses, pigs, small dogs, and the like.

Monoclonal antibodies can be prepared by methods known in the art such as the fusion method (see Kohler and Milstein (1976) European Jounal of Immunology 6: 511-519), the recombinant DNA method (US Patent No. 4,816,567) or the phage antibody library et al., Nature, 352: 624-628, 1991; Marks et al., J. Mol. Biol., 222: 58, 1-597, 1991).

Antibodies used in the prevention and treatment of diseases due to mitochondrial dysfunction through TRAP1 inhibition of the present invention include functional fragments of antibody molecules as well as complete forms having two full-length light chains and two full-length heavy chains . A functional fragment of an antibody molecule refers to a fragment having at least an antigen-binding function, and includes, for example, Fab, F (ab ') 2, F (ab') 2 and Fv.

The antibody may be indirectly coupled (e.g., covalently linked) to a therapeutic agent for a disease caused by existing mitochondrial dysfunction, either directly or through a linker. The type of the therapeutic agent that can be coupled is not limited as long as it has an activity of restoring mitochondrial dysfunction, for example, Geldanamycin and the like.

The antibody may be administered by itself or in a composition comprising the antibody. In the case of a therapeutic composition, an appropriate preparation is prepared, including an acceptable carrier depending on the mode of administration. Formulations suitable for the mode of administration are well known and typically comprise a surfactant which facilitates migration through the membrane. Such surfactants are those derived from steroids or cationic lipids such as N- [1- (2,3-dioloyl) propyl-N, N, N-trimethylammonium chloride (DOTMA), or cholesterol hemi- , Phosphatidylglycerol, and the like.

The composition comprising the antibody of the present invention may be administered in a pharmaceutically effective amount to treat diseases caused by mitochondrial dysfunction. The pharmaceutical composition may be administered singly or multiply. Compositions comprising the antibody are administered by any suitable method including, but not limited to, parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal, and, if necessary, for localized immunosuppressive therapy. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. The preferred modes of administration and formulations are intravenous, subcutaneous, intradermal, intramuscular, and drip injections. The pH of the formulation is generally in the range of pH 4 and pH 8, in order to balance antibody stability (chemical and physical stability) and make it suitable for the patient being administered. In addition, other techniques for administration such as transdermal administration and oral administration may be suitably modified and used to devise a suitable preparation. Typical dosage levels can be optimized using standard clinical techniques.

In addition, the antibody of the present invention may be administered in the form of a nucleic acid encoding an antibody to produce an antibody in the cell (WO 96/07321).

The 'mitochondrial dysfunction' and 'diseases caused by mitochondrial dysfunction' are as described above in the screening method.

The present invention provides a composition for preventing and treating a disease caused by a mitochondrial dysfunction which comprises a TRAP1 inhibitor (TRAP1 inhibitor) as an active ingredient.

In the present invention, the TRAP1 inhibitory substance means a substance that partially or completely (completely) inhibits the activity of TRAP1 in mitochondria. Therefore, the TRAP1 inhibitor is meant to include both a substance that inhibits the expression of TRAP1 or an inhibitor of the activity of TRAP1 expressed. Since the TRAP1 is known to have high homology with Hsp90, a substance that binds to Hsp90 and inhibits its activity is highly likely to bind to TRAP1 and inhibit its activity. Therefore, the TRAP1 inhibitory substance in the present invention is mitochondria Penetrable Hsp90 activity inhibiting material.

The inhibition of the expression can be inhibited through regulation in the transcription stage or in the post transcription stage. Modulation in the transcription step may be a method for inhibiting the expression of a gene known to a person skilled in the art, for example, a method of inducing a mutation of a promoter or a gene site to inhibit the promoter activity or the function of the protein, a method of expressing an antisense gene, an iRNA (iRNA means an interfering RNA having various forms or titles known in the art such as an siRNA, a shRNA, etc.) or a microRNA (microRNA) method.

Modulation in the post-transcriptional step may be carried out by a method known to those skilled in the art for inhibiting protein expression, for example, a gene encoding the TRAP1 protein represented by SEQ ID NO: 1 or 3, or a functional equivalent thereto (for example, 2 or SEQ ID NO: 4) of the present invention may be carried out by a method of inhibiting the stability of the mRNA transcribed with the template, or a method of inhibiting the stability of the protein or polypeptide.

Substances which specifically inhibit Hsp90 or TRAP1 are known in the art, and WO 2009/036092 can be referred to regarding the types of Hsp90 or TRAP1 inhibitory substances.

Preferably, the TRAP1 inhibitory substance of the present invention is any one selected from the group consisting of gamitrinib, geldanamycin and Shepherdin conjugated with an antigen-pediatric cell-penetrating sequence (ANT).

The gamitriinib is composed of a site (carrier) for transporting the drug to the mitochondria, a site (geldanamycin) for inhibiting the target protein, and a linker for linking the sites. The gamitriinib G1 , G2, G3, G4 or TPP. All gamitri nips are transported to the mitochondria and have a common mechanism of action that inhibits Hsp90 / TRAP1, but there is a slight difference in transport efficiency and drug reaction rate depending on the carrier.

In the present invention, 'gamitrinib' binds to mitochondrial through-going moieties (for example, tetraguanidinium (G4), trianguidinium (G3), diguanidinium (G2) Refers to a geldanamycin analog (e. G., 17-AAG) conjugated to a monoguanidinium (Mono guanidinium (G1), or triphenylphosphonium (TPP) moiety). Through this application, the mitochondrial through-going moiety, which is part of a particular gmitrinib, is sometimes indicated. For example, gamitriinib-G4 refers to gamitriinib in which the tetraguanidinium moiety is present. For example, Kamitri nip-TPP refers to kamitriinib in which a triphenylphosphonium moiety is present.

Preferably, the paramitriin of the present invention may be at least one selected from the group consisting of kamitriinib-G1, kamitriinib-G2, kamitriinib-G3, kamitriinib-G4 and kamitriinib-TPP, Preferably gammitriin-G4 or gametrienib-TPP.

Gamitirinib-G1 preferably has a structure of the following formula (1), and may be isolated or purified from nature, commercially purchased, or produced by a chemical synthesis method known in the art.

≪ Formula 1 >

Gamitirinib-G2 preferably has a structure of the following formula (2), and may be isolated from natural sources, commercially purchased, or produced by a chemical synthesis method known in the art.

(2)

Gamitirinib-G3 preferably has a structure of the following formula (3), and may be isolated from natural sources, commercially purchased, or produced by a chemical synthesis method known in the art.

(3)

Gamitirinib-G4 preferably has a structure of the following formula (4), and may be isolated and purified from nature, commercially obtained, or produced by a chemical synthesis method known in the art.

≪ Formula 4 >

The germitriin-TPP preferably has a structure of the following formula (5), and may be isolated from natural sources, commercially obtained, or produced by a chemical synthesis method known in the art.

≪ Formula 5 >

The gamitirinib-TPP used in one embodiment of the present invention is a compound capable of an excellent drug reaction rate and economical synthesis as compared to other gamitirinib. The organic synthesis of the gamitirinib can be carried out as described in J. Med. Clin. Invest., 2009, Mar. 119 (3): 454-64; US 2009/0099080. Although the present invention has been carried out using gamitirinib-TPP, one skilled in the art will have the same effect even if another gamitirinib is used instead of gamitirib TPP because the other gamitirinib also has the same mechanism of action As will be appreciated by those skilled in the art. Therefore, pharmaceutical compositions using gamitriibs G1, G2, G3 and G4 are also included in the scope of the present invention.

In the present invention, the 'ANT' is not limited to the ANT, but may preferably be the peptide sequence shown in SEQ ID NO: 11.

The geldanamycin (denoted by ANT-GA) conjugated with the ANT and the ANP preferably has the following structure (see FIG. 12).

(6)

The Shepherdin refers to a peptide comprising an Hsp90 (or TRAP1) inhibitory peptide sequence derived from an Antennapedia cell penetration sequence and survivin. For the HSP90 (or TRAP1) inhibition peptide sequence derived from the male vivin, WO 2009/036092 can be referred to, but not limited thereto, preferably the Hsp90 (or TRAP1) Amino acid sequence.

The shaperdine sequence is not limited thereto, but may preferably be the amino acid sequence represented by SEQ ID NO: 13.

The 'mitochondrial dysfunction' and 'diseases caused by mitochondrial dysfunction' are as described above in the screening method.

The term 'composition' of the present invention may include both a pharmaceutical composition and a food composition and a cosmetic composition, but preferably means a pharmaceutical composition. The pharmaceutical composition according to the present invention may contain the TRAP1 inhibitory active ingredients of the present invention alone or in combination with one or more pharmaceutically acceptable carriers, excipients or diluents. The term "pharmaceutically acceptable" as used herein means a non-toxic composition which is physiologically acceptable and which, when administered to humans, does not inhibit the action of the active ingredient and does not normally cause an allergic reaction such as gastrointestinal disorder, dizziness, .

The 'TRAP1 inhibitory active ingredient' includes any one selected from the group consisting of antisense RNA, antisense RNA, siRNA and miRNA for the TRAP1 gene, an antibody specific for TRAP1, gamitriinib, ANT-GA, Lt; RTI ID = 0.0 > TRAP1 < / RTI > inhibitors.

The pharmaceutically acceptable carrier may further include, for example, a carrier for oral administration or a carrier for parenteral administration. Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. In addition, it may contain various drug delivery materials used for oral administration to peptide preparations. In addition, the carrier for parenteral administration may contain water, a suitable oil, a saline solution, an aqueous glucose and a glycol, and may further contain a stabilizer and a preservative. Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, etc. in addition to the above components. Other pharmaceutically acceptable carriers and preparations can be found in Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, Pa., 1995).

The composition of the present invention can be administered to mammals including humans by any method. For example, it can be administered orally or parenterally. Parenteral administration methods include, but are not limited to, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual or rectal administration Lt; / RTI >

The pharmaceutical composition of the present invention can be formulated into oral preparations or parenteral administration preparations according to the administration route as described above.

In the case of a preparation for oral administration, the composition of the present invention may be formulated into a powder, a granule, a tablet, a pill, a sugar, a tablet, a liquid, a gel, a syrup, a slurry, . For example, an oral preparation can be obtained by combining the active ingredient with a solid excipient, then milling it, adding suitable auxiliaries, and then processing the mixture into a granular mixture. Examples of suitable excipients include sugars including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol and maltitol, and starches including corn starch, wheat starch, rice starch and potato starch, Cellulose such as methylcellulose, sodium carboxymethylcellulose and hydroxypropylmethyl-cellulose and the like, fillers such as gelatin, polyvinylpyrrolidone and the like. In addition, crosslinked polyvinylpyrrolidone, agar, alginic acid, or sodium alginate may optionally be added as a disintegrant. Further, the pharmaceutical composition of the present invention may further comprise an anti-coagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent and an antiseptic agent.

In the case of a preparation for parenteral administration, it can be formulated by a method known in the art in the form of injection, cream, lotion, external ointment, oil, moisturizer, gel, aerosol and nasal aspirate. These formulations are described in Remington's Pharmaceutical Science, 19th ed., Mack Publishing Company, Easton, Pa., 1995, which is a commonly known formulary for all pharmaceutical chemistries.

The total effective amount of the composition of the present invention may be administered to a patient in a single dose and may be administered by a fractionated treatment protocol administered over a prolonged period of time in multiple doses. In the pharmaceutical composition of the present invention, the content of the active ingredient may be varied depending on the degree of the disease. Preferably, the total preferred dose of the pharmaceutical composition of the present invention may be from about 0.01 μg to about 10,000 mg, and most preferably from about 0.1 μg to 500 mg, per kilogram of patient body weight per day. However, the dosage of the pharmaceutical composition may be determined depending on various factors such as the formulation method, administration route and frequency of treatment, as well as the patient's age, body weight, health condition, sex, severity of disease, diet and excretion rate, It will be possible to determine the appropriate effective dose of the composition of the present invention by those of ordinary skill in the art in view of this point. The pharmaceutical composition according to the present invention is not particularly limited to its formulation, administration route and administration method as long as the effect of the present invention is exhibited.

The screening method of the present invention can effectively screen for substances having the function of preventing and treating diseases caused by mitochondrial dysfunction. In the present invention, compositions having an inhibitory function against TRAP1 and its gene have an effect of increasing resistance to oxidative stress and restoring mitochondrial dysfunction to a normal state.

FIG. 1 is a schematic diagram showing the relative positions and manipulation states of TRAP1 gene and peripheral genes in TRAP1 ^P Drosophila (a) and TRAP1 ^D6 Drosophila (b), respectively. Specifically, (a) represents the positions of p-element insertion sites and peripheral genes centered on the TRAP1 gene in TRAP1 ^P Drosophila, and (b) indicates the position where the C-terminal coding sequence of TRAP1 is lost in TRAP1 ^D6 Drosophila .
Be B of Figure 2 using the fruit fly TRAP1 antibody produced by using a TRAP1 recombinant protein, The results confirm whether TRAP1 protein expression by Western blot to TRAP1 ^P and TRAP1 ^D6 Drosophila body (ACT: β-actin (loading control) , TRAP1: TRAP1 protein, RV: normal Drosophila, TRAP1 ^P : Drosophila with p-element inserted into exon of TRAP1 , TRAP1 ^D6 : Drosophila with loss of C-terminal coding sequence of TRAP1, hs> TRAP1: TRAP1 overexpressing Drosophila hs-GAL4 and UAS-TRAP1 express TRAP1 throughout the fruit fly).
FIG. 2C shows the results of RT-PCR analysis of TRAP1 mRNA expression in TRAP1 ^P and TRAP1 ^D6 Drosophila (RV: normal Drosophila, TRAP1 ^P : Drosophila with p-element inserted into exon of TRAP1 , TRAP1 ^D6 : Drosophila in which the C-terminal coding sequence of TRAP1 is lost).
FIG. 3D shows the results of RT-PCR analysis of the mRNA expression level of Vha 16-1, a gene adjacent to the TRAP1 expression gene, in TRAP1 ^P and TRAP1 ^D6 Drosophila (RV: normal Drosophila, TRAP1 ^P : TRAP1 TRAP1 ^D6 : Drosophila in which the C-terminal coding sequence of TRAP1 is lost).
The upper line is a Drosophila S2 cell line in Fig. 3 E TRAP1 ^WT when sikyeoteul expressing the cDNA of the (wild-type Drosophila TRAP1 protein) shows a state that both the TRAP1 protein specific to go to the mitochondria, the bottom line is a Drosophila S2 cell line TRAP1 ^{△ N} (TRAP1 protein, TRAP1 protein with the N-terminal mitochondrial transport signal peptide removed in TRAP ^WT ) is present in the cell matrix (HA: hamagglutinin, mito: mitochondria, merge: image integration).
FIG. 4A shows the results of examining the lifespan of the normal (RV) fruit fly and the TRAP1 mutant (TRAP1 ^D6 ).
FIG. 5B shows the survival rate (RV: normal fruit flora) of each fruit floss group after treatment with paraquat which is an oxidative stress agonist.
FIG. 5C shows survival rates (RV: normal fruit flies) of each Drosophila group after treatment with the oxidative stress agonist rotenone.
FIG. 6D shows the degree of resistance to oxidative stress in Drosophila in which TRAP1-specific RNAi expressed in SEQ ID NO: 10 was expressed in the whole body and inhibited TRAP1 mRNA expression (da / +: male: da- GAL4 alone, da / + ♀: da-GAL4 only as a female control, da> TRAP1i♂: Male TRAIL1 RNAi expressing strain expressing TRAP1 RNAi in whole body using da-GAL4 and UAS-TRAP1 RNAi , da> TRAP1i♀: a female experimental group expressing TRAP1 RNAi, using da-GAL4 and UAS-TRAP1 RNAi to express TRAP1 RNAi throughout the body).
FIG. 7A shows the effect of the oxidative stress agonist rrotenone, which is an oxidative stress agonist, in the wild type Drosophila (WT), PINK1 gene deficient Drosophila (B9), PINK1 and TRAP1 gene simultaneous deficient Drosophila ('B9, TRAP1 ^D6 ' and B9, TRAP1 ^P ' (rotenone), respectively.
FIG. 8B shows the results of examining the proportion of individuals having a defective phenotype for the wing and thorax muscles in each of the fruit fly groups.
The upper line in FIG. 8C shows the morphology of the mitochondria in the Drosophila chest muscle tissues by the toluidine blue staining in each of the Drosophila group and the lower line shows the degree of death of the Drosophila chest muscle cells in the Drosophila group by the TUNEL assay .
FIG. 9D shows the result of performing a motility test on each of the Drosophila groups.
FIG. 10E shows the result of measurement of mitochondrial amount (mitochondrial DNA amount) in the chest muscle in each of the Drosophila group (cox I: cytochrome c oxidase subunit I, coxIII: cytochrome oxidase III gene, cyt B: cytochrome B) .
Fig. 10F shows the result of measuring the ATP level in the chest muscle in each of the Drosophila groups.
FIG. 11G shows the result of confirming the dopaminergic neurons of the brain tissue by anti-tyrosine hydroxylase (TH) antibody staining in each of the Drosophila group.
H in FIG. 11 shows the number of dopaminergic neurons located in the dorsal-lateral (DL) region of the brain in each of the Drosophila group.
Figure 12 schematically illustrates the structure of TRAP1 inhibitor materials (Kang BH, BMB Rep. 45, 1-6).
FIG. 13A shows the result of a test for the movement of fruit flies according to the concentration of G-TPP when G-TPP was administered for 3 days to freshly grown adult flies by each flock group (WT: wilde type Drosophila, B6: PINK1 gene Deficient fruit flies).
FIG. 14B shows the results of confirming the state of dopaminergic neurons according to the concentration of G-TPP administration by anti-tyrosine hydroxylase (TH) antibody staining when G-TPP was administered for 30 days for each of the fruit flies.
FIG. 14C shows the number of dopaminergic neurons according to the concentration of G-TPP when G-TPP was administered for 30 days in each Drosophila group.
In Figure 15, each cell line D is a carbon source (glucose or galactose), G-TPP, 17- AAG is a result showing the processing condition by the mitochondrial membrane potential (PINK1 ^{+ / +:} the normal cell line, PINK1 ^{- / -:} PINK1 gene-deficient Cell line).
FIG. 16A shows the result of performing a mobility test on each of the Drosophila groups.
FIG. 16B shows the result of measuring the ATP level in the chest muscle for each of the fruit flies.
FIG. 16C shows the result of measurement of mitochondrial amount (mitochondrial DNA amount) in the chest muscle for each of the fruit flies group (cox I: cytochrome c oxidase subunit I, cox III: cytochrome oxidase III gene, cyt B: cytochrome B ).

Hereinafter, the present invention will be described in detail.

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

≪ Example 1 >

Production of TRAP1 gene manipulation model

<1-1> TRAP1 deficient Drosophila model and Drosophila breeding

To identify the biological function of TRAP1, Drosophila ( TRAP1 ^P ), in which a p-element, a transposon of Drosophila, was inserted into the exon of TRAP1 was obtained from Bloomington Drosophila Stock Center (BDSC) Indiana, USA (a)). This cross-breeding of the Drosophila with the transgenic Drosophila expressing p-element excision induces impaired excision of the TRAP1 gene through the imprecise excision of the p-element, resulting in a 1 kb deletion of the p-element insertion site, ( TRAP1 ^D6 ) in which the terminal coding sequence was lost (Fig. 1 (b)).

Unless otherwise noted, in all experiments, Drosophila was grown on standard Conmeal media (7% sucrose, 5% yeast, 2.5% agar )).

<1-2> Identification of TRAP1 protein expression in TRAP1-deficient Drosophila model

To confirm the expression of the TRAP1 gene in the TRAP1 ^P and the Drosophila mutant TRAP1 ^{D6 prepared} in the above Example <1-1>, the Drosophila TRAP1 recombinant protein (SEQ ID NO: 9) was administered to New Zealand white rabbit Western blotting was performed with the Drosophila TRAP1 antibody obtained by the above-described method, and qRT-PCR was also performed.

As a control, normal flora (RV) and hs > TRAP1 were used, and hs > TRAP1 was produced by TRAP1 overexpressing Drosophila. Briefly, a UAS-TRAP1 expression vector was prepared by inserting the TRAP1-expressing nucleic acid (SEQ ID NO: 4, NM_058091.3) into a pUAST vector (Drosophila genome resource center, DGRC, Indiana, USA). The UAS-TRAP1 expression vector was microinjected into a w-Drosophila (obtained from BDSC) as a known P-element-mediated germ line transformation method and transformed into a UAS-TRAP1 expression vector Were prepared. Using the UAS / Gal4 system (AH Brand and N. Perrimon., Development 1993 118, 401-15), the Drosophila transformed with the UAS-TRAP1 expression vector was mated with hs-GAL4 Drosophila (obtained from BDSC) In the next generation, hs > TRAP1 Drosophila in which TRAP1 was expressed throughout the fruit fly body was obtained.

The Western blot was performed in the following manner. Briefly, the protein samples obtained from each of the experimental flies were SDS-PAGE and electro-blotted on a nitrocellulose membrane. Membrane was blocked with 2% BSA TBS-T (Tris-buffered saline with 0.1% TWEEN20) for 30 min. Then, membrane was added to the solution of the Drosophila TRAP1 antibody diluted 1: 1000 in blocking solution. Lt; / RTI &gt; After incubation for 1 hour at room temperature with TBS-T diluted 1: 5000 with anti-rabbit IgG HRP 2 ^nd antibody (Rockland), the cells were incubated with ECL kit (Amersham) ).

In addition, qRT-PCR (Quantitative RT-PCR) was performed briefly as follows. Total RNA was isolated from five adult 3 day old Drosophila melanogaster using the Easy-Blue kit (Intron) and cDNA was synthesized with random hexamer (Promega) and MMLV reverse transcriptase (Promega). Quantitative real-time PCR was performed using SYBR Premix Ex Taq (Takara) kit and Prism 7000 real-time PCR system (ABI) at 94 ° C for 30 seconds, 55 ° C for 30 seconds, and 72 ° C for 30 seconds . The TRAP1-specific primer sequences used in this experiment are shown in Table 1 below.

SEQ ID NO: target Primer sequence (5 'to 3') 14 TRAP1-Forward GCA GCG TTC AAT ATC ACC ATT G 15 TRAP1-Reverse GAC CTC GTG GTC GGA GTC GTA TAA GG

As a result of Western blot and qRT-PCR, it was confirmed that TRAP1 protein and mRNA expression were successfully inhibited in both TRAP1 ^P and TRAP1 ^D6 (Fig. 2B and Fig. 2C).

<1-3> Evaluation of effect on TRAP1 peripheral gene in TRAP1 deficient Drosophila model

In the TRAP1 ^P and the Drosophila mutant TRAP1 ^{D6 prepared} in Example <1-1>, the degree of mRNA expression of Vha-16 gene next to TRAP1 was examined by qRT-PCR technique. The qRT-PCR was carried out in the same manner as described in the above Example < 1-2 &gt;. The Vha-16 specific primers used herein have the sequence shown in Table 2 below.

SEQ ID NO: target Primer sequence (5 'to 3') 16 Vha16-1-Forward GTC TTC TGA AGT GAG CAG CGA C 17 Vha16-1-Reverse CAT CAC TGA CAT GGC AGC AAT ACC

As a result, it was confirmed that the expression of Vha-16 was not significantly affected in the body of the produced Drosophila mutant TRAP1 ^D6 (FIG. 3 D). In other words, TRAP1 ^{D6, a} fruit fly mutant, was found to be deficient in TRAP1 gene, and the functions of other genes around TRAP1 were normal.

<1-4> Identification of the presence of TRAP1 protein in cells

Drosophila TRAP1 protein is a Drosophila cell line to confirm that the mitochondrial protein Drosophila Schneider 2 (S2), respectively in the cell TRAP1 ^WT (wild-type Drosophila TRAP1 protein) of the cDNA and TRAP1 ^{△ N} (TRAP1 removing the mitochondrial movement signal peptide of the N-terminal in the TRAP ^WT Protein) was expressed to confirm the presence in the cell. Specific experimental methods are as follows. the pUAST vector C-terminal hamagglutinin (HA) tagging the TRAP1 full length cDNA (TRAP1 ^WT) and ^{N-terminal deletion mutant (TRAP1 △} N) cloning the cDNA, and Han (DDAB method with these plasmid and pMT-GAL4 plasmid K , Nucleic Acids Res., 1996). After 48 hours from transfection, 0.5 mM CuSO _{4 was added} to the cells to activate the metallothionein promoter of pMT-GAL4 to express GAL4, thereby expressing TRAP1 protein. After 24 hours, 5 mg / mL MitoTracker Red CMXRos (Molecular Probes) And treated with time to stain mitochondria. Then, the cells were fixed with PBS containing 4% paraformaldehyde and then blocked with 3% BSA PBS-T (0.3% Triton X-100 in PBS) for 1 hour at room temperature. Then, the cells were blocked with 1: 200 anti- HA monoclonal antibody solution was incubated overnight at 4 ° C. The cells were then incubated in PBS-T containing 1: 200 anti-mouse FITC antibody for 2 hours, and then the intracellular TRAP1 protein was localized using an LSM 700 confocal fluorescence microscope (Carl Zeiss).

As a result, when wild-type Drosophila TRAP1 cDNA was obtained and expressed in Drosophila Schneider 2 (S2) cell of Drosophila cell line, all of TRAP1 protein migrated specifically to mitochondria. When N-terminal mitochondrial transfer signal peptide was removed, Drosophila TRAP1 was also confirmed to be a mitochondrial protein (Fig. 3E).

&Lt; Example 2 >

Oxidative stress resistance due to TRAP1 gene deletion

<2-1> Resistance to Oxidative Stress in TRAP1 Deficient Mutants

To investigate the biological functions of the TRAP1 gene, we investigated the lifespan and oxidative stress resistance of normal flora (RV) and TRAP1 mutant TRAP1 ^D6 . The normal Drosophila (RV) has a p-element in the TRAP1 ^P Drosophila It is a fruit fly that normalizes the function of the TRAP1 gene by accurately extracting the surrounding DNA without damaging it.

Specifically, 120 rabbits per each experimental group genotype of RV and TRAP1 ^D6 were divided into 30 rabbits. The rabbits were transferred into vials containing 10 ml of Drosophila culture medium. The rabbits were transferred to new vials every 3-4 days, And the number of dead flies. When the test was completed, the Kaplan-Meier estimator and the log-rank test were used to verify the statistically significant difference in survival rates between genotypes.

To measure the resistance to oxidative stress, 120 rabbits (3 day old) per genotype of RV, TRAP1 ^p and TRAP1 ^D6 were divided into 4 groups of 30 rats each, starvation was carried out for 6 hours, and 5% Survival rate is measured every 30 hours by placing 30 fruit flies in oxidative agent vial containing 3 ml of PBS gel containing sucrose, 1% agar and 20 mM paraquat (or 5 mM rotenone). The oxidative agent vials are replaced with new ones once a day. When the test was completed, the Kaplan-Meier estimator and the log-rank test were used to verify the statistically significant difference in survival rates between genotypes.

As a result, there was no significant difference in the lifespan between the normal flora (RV) and the TRAP1 mutant TRAP1 ^D6 (Fig. 4A). However, surprisingly, oxidative stress agents such as paraquat (B in Fig. 5) and rotenone (C in Fig. 5) were treated with Drosophila to show that TRAP1 gene deletion enhances resistance to oxidative stress .

<2-2> Resistance to oxidative stress in TRAP1 knock-down fruit flies

TRAP1-specific RNAi (SEQ ID NO: 10) was expressed in the whole body in a cancer-male da-GAL4 Drosophila strain (denoted by da / +) obtained from BDSC to inhibit the expression of TRAP1 mRNA Respectively. Briefly, a transgenic Drosophila in which the TRAP1 specific RNAi (SEQ ID NO: 10) with the UAS promoter attached was inserted into the chromosome was obtained from the Vienna Drosophila Resource Center (VDRC) (Vienna, Austria) , We constructed da> TRAP1 Drosophila in which the expression of TRAP1 mRNA was suppressed in whole body in the next generation. Using the same method as described in Example 2-1 above using the male / female da-GAL4 Drosophila (da / +) as the control group and rotenone as the oxidizing substance in the female and female da> TRAP1 Drosophila, oxidative stress resistance Were measured.

As shown in FIG. 6, TRAP1 gene deletion was found to enhance resistance to oxidative stress even in experiments using Drosophila in which TRAP1-specific RNAi (SEQ ID NO: 10) was expressed throughout the body and TRAP1 mRNA expression was inhibited 6, D).

&Lt; Example 3 >

In the PINK1 gene-deficient Drosophila model, resistance to oxidative stress by TRAP1 gene deletion and hyperactivity of mitochondrial function

It has been reported that PINK1 gene deletion decreases resistance to oxidaive stress (Clark et al., (2006) Drosophila pink1 is required for mitochondrial function and interacts genetically with parkin. Nature 441: 1162-6), PINK1 and TRAP1 gene was simultaneously produced and the following experiment was carried out.

<3-1> Drosophila line and PINK1 deficient cell line (cell line)

The PINK1 gene-deficient Drosophila (B9) was obtained from Professor Jong-Kyung Chung of Seoul National University. The PINK1 and TRAP1 gene simultaneous deletion fruit fly 'B9, TRAP1 ^D6 ', and B9 and TRAP1 ^p 'were prepared by crossing the provided PINK1 gene deletion fruit fly with each of TRAP1 ^D6 and TRAP1 ^p of the above Example <1-1>. TRAP1-overexpressed Drosophila was produced using the cDNA prepared in Example <1-2>, and was submitted to the KAIST Drosophila Research Support Office (see Example <3-8 below).

Obtained from the PINK1 gene-deficient mice produced in Xiaoxi Zhuang Professor Laboratory of Chicago Medical School mouse embryonic fibroblast (MEF) [Xiong et al., J Clin Invest., 2009] to get to 37 ° C, 10% at 5% CO ₂ incubator The cells were cultured in DMEM containing FBS and used for experiments.

<3-2> Evaluation of Resistance to Oxidative Stress Agents (rotenone) in Drosophila with Simultaneous Loss of PINK1 and TRAP1 Gene

B9, TRAP1 ^D6 ', and B9 and TRAP1 ^p ', which are the normal fruit flies 'WT', the PINK1 gene deletion fruit fly 'B9', the PINK1 and TRAP1 genes of Example <3-1> Oxidative stress resistance to rotenone was evaluated. The oxidative stress resistance was performed in the same manner as described in the above Example <2-1>.

As a result, it was confirmed that the loss of rotenone resistance due to the PINK1 gene deletion normalizes the TRAP1 gene deletion (Fig. 7A).

<3-3> Recovery of defective phenotype

B9, TRAP1 ^D6 ', and B9 and TRAP1 ^{p in which the} normal Drosophila' WT ', the PINK1 gene deletion Drosophila' B9 ', the PINK1 and TRAP1 genes of Example <3-1> The ratio of the phenotype of Drosophila and Drosophila melanogaster was calculated by calculating the percentage of deficient phenotype.

Drosophila deficient in PINK1 gene showed that the wings were sagging and the chest area worsened as the chest muscles were lost. The TRAP1 gene deletion confirmed the wings of the PINK1 gene-deficient Drosophila and the wormy chest recovered to normal (See Fig. 8B).

<3-4> Measurement of the degree of apoptosis in muscle tissue and observation of mitochondrial appearance

It has been reported that mitochondrial dysfunction in muscle tissue causes muscle cell death, and the degree of apoptosis of muscle cells was examined by the following method. B9, TRAP1 ^D6 ', and B9 and TRAP1 ^{p in which the} normal Drosophila' WT ', the PINK1 gene deletion Drosophila' B9 ', the PINK1 and TRAP1 genes of Example <3-1> The muscle tissue was dissected from the chest area of Drosophila, fixed with 4% formaldehyde, and then subjected to TUNEL assay to investigate the degree of apoptosis in the chest muscle. In order to identify the intramuscular mitochondrial outline, the Drosophila chest was separated, placed in Spurr's resin, and coagulated. A 10-micron thick slice was prepared using a rotary microtome, stained with toluidine blue stain, The outline was observed.

As a result, PINK1 mutant phenotypes of PINK1 gene deletion (bottom line in C in FIG. 8) and mitochondrial dysplasia (in C line in FIG. 8) were all normalized at the time of TRAP1 gene deletion.

<3-5> Model animal motility test

B9, TRAP1 ^D6 ', and B9 and TRAP1 ^{p in which the} normal Drosophila' WT ', the PINK1 gene deletion Drosophila' B9 ', the PINK1 and TRAP1 genes of Example <3-1> A climbing assay was performed to measure the motility of Drosophila. The Drosophila has the property of moving in the opposite direction of gravity by sensing gravity. An experimental technique for measuring the motility of Drosophila is called climbing assay. Once the flies were kept in a dark room for one hour to stabilize them, the flies were placed on the vial floor by tapping the vials containing the flies, counting the number of flies rising above 15 cm for 10 seconds, The percentage of fruit flies exhibiting motility satisfying these criteria was obtained, and the average value was obtained by repeating this five times.

As a result, the PINK1 mutant phenotype of the reduced motility due to the PINK1 gene deletion (FIG. 9D) was normalized when the TRAP1 gene was deleted.

<3-6> Measurement of mitochondria amount and ATP concentration in muscle tissue

B9, TRAP1 ^D6 ', and B9 and TRAP1 ^{p in which the} normal Drosophila' WT ', the PINK1 gene deletion Drosophila' B9 ', the PINK1 and TRAP1 genes of Example <3-1> The amount and function of mitochondria in tissues were measured. Specifically, quantitative real-time PCR was used to measure the amount of mitochondrial DNA (mtDNA) in the tissues to determine the amount of mitochondria. In addition, the amount of ATP, an energy transfer material produced by mitochondria, was measured to examine the function of mitochondria. The amount of mtDNA was determined by collecting 5 chick flies from each experimental group and then isolating total DNA in muscle tissue using qiagen DNeasy Blood & Tissue Kit (# 69506). The primers for the three mitochondrial protein genes cox I, cox III and cyt B in mtDNA were prepared (see Table 3), then the SYBR Premix Ex Taq (Takara) kit and the Prism 7000 Real-Time PCR system Quantitative real-time PCR was performed at 94 ° C for 30 seconds, 55 ° C for 30 seconds, and 72 ° C for 30 seconds to determine the amount of mtDNA. In addition, the amount of nDNA was determined using primers (see Table 3) for rp49 gene located in the nuclear DNA (nDNA), and the amount of mtDNA was accurately confirmed by normalization.

To determine the amount of ATP in the muscle tissue, 5 mouse flies of the chest muscles were homogenized in 0.1 ml extraction buffer (6M guanidine-HCl, 10 mM Tris, 4 mM EDTA, pH 7.8) and rapidly frozen with liquid nitrogen After boiling for 3 minutes, the supernatant was collected by centrifugation to prepare an extract. The concentration of ATP in the extract was determined by using Enlighten Kit (Promega) in the extract, and normalized to the protein concentration in the extract obtained through BCA assay to confirm intramuscular ATP levels.

SEQ ID NO: target Primer sequence (5'to 3 ') 18 COX I-Forward GTG CCT TTA ATA TTA GGT GCT C 19 COX I-Reverse GTA ATA GCT CCT GCT AGT ACT G 20 COX III-Forward CGA GAT GTA TCA CGA GAA GG 21 COX III-Reverse GAA TTC CGT GGA ATC CTG TTG 22 CytB-Forward CGA ACT TTA CAT GCT AAC GGT G 23 CytB-Reverse CCG ATA GGA TTA TTA GAT CCT G 24 rp49-Forward GCT TCA AGA TGA CCA TCC GCC C 25 rp49-Reverse GGT GCG CTT GTT CGA TCC GTA AC

As a result, PINK1 mutant phenotypes in the chest muscle due to PINK1 gene deletion (E in FIG. 10) and ATP level decrease (FIG. 10 F) were normalized at all TRAP1 gene deletion.

<3-7> Measurement of Drosophila Dopamine Nerve Cell Death Level

B9, TRAP1 ^D6 ', and B9 and TRAP1 ^{p in which the} normal Drosophila' WT ', the PINK1 gene deletion Drosophila' B9 ', the PINK1 and TRAP1 genes of Example <3-1> After 30 days of fermentation, the brain of the Drosophila melanogaster was isolated, fixed in 4% paraformaldehyde solution, stained with anti-tyrosine hydroxylase (TH) antibody from Pel-freez, (N = 40) (Koh H, Kim H, Kim JJ, Park J, Chung J (2012) Sir2 and FOXO Complement Mitochondrial Dysfunction and Dopaminergic Neuron Loss in Drosophila PINK1 Mutants J Biol Chem 287: 12750-12758 ).

As a result, PINK1 mutant phenotypes of dopaminergic neuronal apoptosis (G and H in FIG. 11) due to PINK1 gene deletion were normalized when TRAP1 gene was deleted.

<3-8> Tissue-specific overexpression of TRAP1 gene and its effect

B9, da 'fruit flies obtained through the mating of da-GAL4 fruit flies (denoted by da) obtained from BDSC, the PINK1 gene-deficient Drosophila (B9) of Example <3-1> above and da-GAL4 Drosophila, 'B9, da TRAP1', which is overexpressed in TRAP1 gene in the next generation through mating of 'B9, da' Drosophila and the UAS-TRAP1 expression vector prepared in Example <1-2> Were evaluated for mobility, mitochondria in muscle tissue and ATP concentration.

In addition, the mobility was evaluated by the method described in Example <3-5>, and the amount of mitochondria in muscle tissue and the ATP concentration were measured by the method described in Example <3-6> above.

16A), decrease in intracellular ATP concentration (Fig. 16B) and decrease in intramuscular mitochondria content (Fig. 16C) observed in the PINK1 mutant (B9, da) It was confirmed that it was not normalized at all in the PINK1 deficient mutant (B9, da TRAP1). In other words, overexpression of TRAP1 gene did not affect the PINK1 deletion mutant phenotype.

The results of Examples 3-1 to 3-8 found that TRAP1 gene deletion increases resistance to oxidative stress and normalizes mitochondrial dysfunction due to PINK1 gene deletion.

<Example 4>

Normalization of mitochondrial dysfunction by TRAP1 specific inhibitor

In order to inhibit TRAP1 specifically located in mitochondria, geldanamycin (17-AAG), an HSP90 inhibitor, and gammitrinib (G-TPP) (see FIG. 12), a TRAP1 inhibitor combined with mitochondria targeting groups, To normalize mitochondrial dysfunction.

<4-1> TRAP1 specific inhibitor treatment and mobility test

Using the normal Drosophila (WT) and the PINK1-deficient Drosophila model (B9), G-TPP was administered to freshly grown adult flies for 3 days before mobility.

G-TPP administration was carried out in the following manner. A vial containing 5 ml of Drosophila culture medium was prepared, and 50 or more holes were drilled in the medium with 18 G needle. Then, 0.2 ml of distilled water containing 1 mM or 5 mM concentration of G-TPP was added and the mixture was allowed to stand at room temperature overnight. . 30 vials of Drosophila melanogaster were added to the completed vial containing G-TPP and cultured for 3 days. The mobility test method was performed in the same manner as described in Example <3-5>.

As a result, the mobility of the PINK1 gene-deficient Drosophila melanogaster treated with the 5 mM G-TPP solution was statistically recovered (Fig. 13A).

<4-2> Treatment of TRAP1 specific inhibitor and inhibition of dopaminergic neuronal cell death

The G-TPP-containing vials prepared by the method described in Example <4-1> were replaced with fresh vials every 3-4 days, and the G-TPP solution was added to the normal Drosophila WT and PINK1-deficient Drosophila B9 for 30 days , And its effect on dopaminergic neuronal cell death was evaluated by the method described in the above Example <3-7>.

It was confirmed that administration of G-TPP solution for 30 days inhibited the death of dopaminergic neurons due to PINK1 deficiency (Fig. 14B and Fig. 14C).

<4-3> Treatment of TRAP1 specific inhibitor and measurement of mitochondrial membrane potential

Normal (PINK1 ^{+ / +)} and PINK1 gene-deficient MEF cell (PINK1 ^{- / -)} in the conditions described in Figure 15 on glucose (4.5g / L) containing DMEM, Galactose (4.5g / L) containing DMEM, G-TPP, 17-AAG and DMSO were treated for each test group and cultured for 16 hours. Cells were then treated with trypsin and incubated in DMEM containing 1 mg / mL JC-1 for 20 min at 37 ° C. The red and green fluorescence were measured with an EPICS XL cytometer (Beckman Coulter), and the red fluorescence intensity was divided by the green fluorescence intensity to determine the mitochondrial membrane potential.

As shown in FIG. 15, the PINK1-deficient MEF cell line showed a decrease in mitochondrial membrane potential when the carbon source was changed from gulcose to galactose, unlike the normal cell line. G-TPP treatment increased the decreased membrane potential 15 D). On the other hand, when treated with 17-AAG, there was no significant change in reduced membrane potential (Fig. 15D).

Experimental results of the examples <4-1> to <4-3> above confirmed the results of the TRAP1 gene deletion by a pharmacological experimental technique, and TRAP1 as a treatment for mitochondrial dysfunction related diseases such as Parkinson's disease inhibitor was used in the study.

The screening method of the present invention can effectively screen for substances having the function of preventing and treating diseases caused by mitochondrial dysfunction. In the present invention, compositions having an inhibitory function against TRAP1 and its gene are highly industrially applicable because they increase resistance to oxidative stress and restore mitochondrial dysfunction to a normal state.

<110> Dong-A University Research Foundation For Industry-Academy Cooperation <120> Screening method for therapeutic agent of mitochondria          dysfunction using TRAP1 gene <130> NP13-0046 <160> 25 <170> Kopatentin 2.0 <210> 1 <211> 704 <212> PRT <213> Artificial Sequence <220> <223> human TRAP1 amino acid sequence (NP_057376.2) <400> 1 Met Ala Arg Glu Leu Arg Ala Leu Leu Leu Trp Gly Arg Arg Leu Arg   1 5 10 15 Pro Leu Leu Arg Ala Leu Ala Ala Val Pro Gly Gly Lys Pro              20 25 30 Ile Leu Cys Pro Arg Arg Thr Thr Ala Gln Leu Gly Pro Arg Arg Asn          35 40 45 Pro Ala Trp Ser Leu Gln Ala Gly Arg Leu Phe Ser Thr Gln Thr Ala      50 55 60 Glu Asp Lys Glu Glu Pro Leu His Ser Ile Ile Ser Ser Thr Glu Ser  65 70 75 80 Val Gln Gly Ser Thr Ser Lys His Glu Phe Gln Ala Glu Thr Lys Lys                  85 90 95 Leu Leu Asp Ile Val Ala Arg Ser Leu Tyr Ser Glu Lys Glu Val Phe             100 105 110 Ile Arg Glu Leu Ile Ser Asn Ala Ser Asp Ala Leu Glu Lys Leu Arg         115 120 125 His Lys Leu Val Ser Asp Gly Gln Ala Leu Pro Glu Met Glu Ile His     130 135 140 Leu Gln Thr Asn Ala Glu Lys Gly Thr Ile Thr Ile Gln Asp Thr Gly 145 150 155 160 Ile Gly Met Thr Gln Glu Glu Leu Val Ser Asn Leu Gly Thr Ile Ala                 165 170 175 Arg Ser Gly Ser Lys Ala Phe Leu Asp Ala Leu Gln Asn Gln Ala Glu             180 185 190 Ala Ser Ser Lys Ile Ile Gly Gln Phe Gly Val Gly Phe Tyr Ser Ala         195 200 205 Phe Met Val Ala Asp Arg Val Glu Val Tyr Ser Arg Ser Ala Ala Pro     210 215 220 Gly Ser Leu Gly Tyr Gln Trp Leu Ser Asp Gly Ser Gly Val Phe Glu 225 230 235 240 Ile Ala Glu Ala Ser Gly Val Arg Thr Gly Thr Lys Ile Ile Ile His                 245 250 255 Leu Lys Ser Asp Cys Lys Glu Phe Ser Ser Glu Ala Arg Val Arg Asp             260 265 270 Val Val Thr Lys Tyr Ser Asn Phe Val Ser Phe Pro Leu Tyr Leu Asn         275 280 285 Gly Arg Arg Meth Met Asn Thr Leu Gln Ala Ile Trp Met Met Asp Pro Lys     290 295 300 Asp Val Arg Glu Trp Gln His Glu Glu Phe Tyr Arg Tyr Val Ala Gln 305 310 315 320 Ala His Asp Lys Pro Arg Tyr Thr Leu His Tyr Lys Thr Asp Ala Pro                 325 330 335 Leu Asn Ile Arg Ser Ile Phe Tyr Val Pro Asp Met Lys Pro Ser Met             340 345 350 Phe Asp Val Ser Arg Glu Leu Gly Ser Ser Val Ala Leu Tyr Ser Arg         355 360 365 Lys Val Leu Ile Gln Thr Lys Ala Thr Asp Ile Leu Pro Lys Trp Leu     370 375 380 Arg Phe Ile Arg Gly Val Val Asp Ser Glu Asp Ile Pro Leu Asn Leu 385 390 395 400 Ser Arg Glu Leu Leu Gln Glu Ser Ala Leu Ile Arg Lys Leu Arg Asp                 405 410 415 Val Leu Gln Gln Arg Leu Ile Lys Phe Phe Ile Asp Gln Ser Lys Lys             420 425 430 Asp Ala Glu Lys Tyr Ala Lys Phe Phe Glu Asp Tyr Gly Leu Phe Met         435 440 445 Arg Glu Gly Ile Val Thr Ala Thr Glu Gln Glu Val Lys Glu Asp Ile     450 455 460 Ala Lys Leu Leu Arg Tyr Glu Ser Ser Ala Leu Pro Ser Gly Gln Leu 465 470 475 480 Thr Ser Leu Ser Glu Tyr Ala Ser Arg Met Arg Ala Gly Thr Arg Asn                 485 490 495 Ile Tyr Tyr Leu Cys Ala Pro Asn Arg His Leu Ala Glu His Ser Pro             500 505 510 Tyr Tyr Glu Ala Met Lys Lys Lys Asp Thr Glu Val Leu Phe Cys Phe         515 520 525 Glu Gln Phe Asp Glu Leu Thr Leu Leu His Leu Arg Glu Phe Asp Lys     530 535 540 Lys Lys Leu Ile Ser Val Glu Thr Asp Ile Val Val Asp His Tyr Lys 545 550 555 560 Glu Glu Lys Phe Glu Asp Arg Ser Pro Ala Ala Glu Cys Leu Ser Glu                 565 570 575 Lys Glu Thr Glu Glu Leu Met Ala Trp Met Arg Asn Val Leu Gly Ser             580 585 590 Arg Val Thr Asn Val Lys Val Thr Leu Arg Leu Asp Thr His Pro Ala         595 600 605 Met Val Thr Val Leu Glu Met Gly Ala Ala Arg His Phe Leu Arg Met     610 615 620 Gln Gln Leu Ala Lys Thr Gln Glu Glu Arg Ala Gln Leu Leu Gln Pro 625 630 635 640 Thr Leu Glu Ile Asn Pro Arg His Ala Leu Ile Lys Lys Leu Asn Gln                 645 650 655 Leu Arg Ala Ser Glu Pro Gly Leu Ala Gln Leu Leu Val Asp Gln Ile             660 665 670 Tyr Glu Asn Ala Met Ile Ala Ala Gly Leu Val Asp Asp Pro Arg Ala         675 680 685 Met Val Gly Arg Leu Asn Glu Leu Leu Val Lys Ala Leu Glu Arg His     690 695 700 <210> 2 <211> 2310 <212> DNA <213> Artificial Sequence <220> Human TRAP1 cDNA (NM_016292.2) <400> 2 gaggaagccc cgccccgcgc agccccgtcc cgccccttcc catcgtgtac ggtcccgcgt 60 ggctgcgcgc ggcgctctgg gagtacgaca tggcgcgcga gctgcgggcg ctgctgctgt 120 ggggccgccg cctgcggcct ttgctgcggg cgccggcgct ggcggccgtg ccgggaggaa 180 aaccaattct gtgtcctcgg aggaccacag cccagttggg ccccaggcga aacccagcct 240 ggagcttgca ggcaggacga ctgttcagca cgcagaccgc cgaggacaag gaggaacccc 300 tgcactcgat tatcagcagc acagagagcg tgcagggttc cacttccaaa catgagttcc 360 aggccgagac aaagaagctt ttggacattg ttgcccggtc cctgtactca gaaaaagagg 420 tgtttatacg ggagctgatc tccaatgcca gcgatgcctt ggaaaaactg cgtcacaaac 480 tggtgtctga cggccaagca ctgccagaaa tggagattca cttgcagacc aatgccgaga 540 aaggcaccat caccatccag gatactggta tcgggatgac acaggaagag ctggtgtcca 600 acctggggac gattgccaga tcggggtcaa aggccttcct ggatgctctg cagaaccagg 660 ctgaggccag cagcaagatc atcggccagt ttggagtggg tttctactca gctttcatgg 720 tggctgacag agtggaggtc tattcccgct cggcagcccc ggggagcctg ggttaccagt 780 ggctttcaga tggttctgga gtgtttgaaa tcgccgaagc ttcgggagtt agaaccggga 840 caaaaatcat catccacctg aaatccgact gcaaggagtt ttccagcgag gcccgggtgc 900 gagatgtggt aacgaagtac agcaacttcg tcagcttccc cttgtacttg aatggaaggc 960 ggatgaacac cttgcaggcc atctggatga tggaccccaa ggatgtccgt gagtggcaac 1020 gccccgctac ataagacgga cgcaccgctc aacatccgca gcatcttcta cgtgcccgac atgaaaccgt 1140 ccatgtttga tgtgagccgg gagctgggct ccagcgttgc actgtacagc cgcaaagtcc 1200 tcatccagac caaggccacg gacatcctgc ccaagtggct gcgcttcatc cgaggtgtgg 1260 tggacagtga ggacattccc ctgaacctca gccgggagct gctgcaggag agcgcactca 1320 tcaggaaact ccgggacgtt ttacagcaga ggctgatcaa attcttcatt gaccagagta 1380 aaaaagatgc tgagaagtat gcaaagtttt ttgaagatta cggcctgttc atgcgggagg 1440 gcattgtgac cgccaccgag caggaggtca aggaggacat agcaaagctg ctgcgctacg 1500 agtcctcggc gctgccctcc gggcagctaa ccagcctctc agaatacgcc agccgcatgc 1560 gggccggcac ccgcaacatc tactacctgt gcgcccccaa ccgtcacctg gcagagcact 1620 caccctacta tgaggccatg aagaagaaag acacagaggt tctcttctgc tttgagcagt 1680 ttgatgagct caccctgctg caccttcgtg agtttgacaa gaagaagctg atctctgtgg 1740 agacggacat agtcgtggat cactacaagg aggagaagtt tgaggacagg tccccagccg 1800 ccgagtgcct atcagagaag gagacggagg agctcatggc ctggatgaga aatgtgctgg 1860 ggtcgcgtgt caccaacgtg aaggtgaccc tccgactgga cacccaccct gccatggtca 1920 ccgtgctgga gatgggggct gcccgccact tcctgcgcat gcagcagctg gccaagaccc 1980 aggaggagcg cgcacagctc ctgcagccca cgctggagat caaccccagg cacgcgctca 2040 tcaagaagct gaatcagctg cgcgcaagcg agcctggcct ggctcagctg ctggtggatc 2100 agatatacga gaacgccatg attgctgctg gacttgttga cgaccctagg gccatggtgg 2160 gccgcttgaa tgagctgctt gtcaaggccc tggagcgaca ctgacagcca gggggccaga 2220 aggactgaca ccacagatga cagccccacc tccttgagct ttatttacct aaatttaaag 2280 gtatttctta acccgaaaaa aaaaaaaaaa 2310 <210> 3 <211> 691 <212> PRT <213> Artificial Sequence <220> <223> Drosophila TRAP1 amino acid sequence (NP_477439.2) <400> 3 Met Ser Val Arg Ala Met Gly Val Leu Gly Gln Ala Cys Arg Leu Gly   1 5 10 15 Arg Cys Ala Gln Val Leu Arg Gln Ser His Arg Ser Ala Pro Ala Ala              20 25 30 Phe Asn Ile Thr Ile Gly Ser Gln His Ser Pro Gly Ala Leu Ser Leu          35 40 45 Arg Arg Tyr Ser Thr Glu Thr Lys Gln Ala Ser Gly Ser Val Val Asp      50 55 60 Lys His Glu Phe Gln Ala Glu Thr Arg Gln Leu Leu Asp Ile Val Ala  65 70 75 80 Arg Ser Leu Tyr Ser Asp His Glu Val Phe Val Arg Glu Leu Ile Ser                  85 90 95 Asn Ala Ser Asp Ala Leu Glu Lys Phe Arg Tyr Thr Ser Leu Ser Ala             100 105 110 Gly Gly Glu Asn Leu Ala Gly Lys Asp Arg Pro Leu Glu Ile Arg Ile         115 120 125 Thr Thr Asp Lys Pro Leu Met Gln Leu Ile Ile Gln Asp Thr Gly Ile     130 135 140 Gly Met Thr Lys Glu Glu Leu Val Ser Asn Leu Gly Thr Ile Ala Arg 145 150 155 160 Ser Gly Ser Lys Lys Phe Leu Glu Gln Met Lys Gly Thr Gln Gln Gly                 165 170 175 Ala Ser Ser Glu Ala Ser Ser Asn Ile Ile Gly Gln Phe Gly Val Gly             180 185 190 Phe Tyr Ser Ser Phe Ile Val Ala Asn Lys Val Glu Val Phe Thr Arg         195 200 205 Ala Ala Val Pro Asn Ala Pro Gly Leu Arg Trp Ser Thr Asp Gly Ser     210 215 220 Gly Thr Tyr Glu Ile Glu Glu Val Pro Asp Val Glu Leu Gly Thr Arg 225 230 235 240 Ile Val Leu His Leu Lys Thr Asp Cys Arg Glu Tyr Ala Asp Glu Glu                 245 250 255 Arg Ile Lys Ala Val Ile Lys Lys Tyr Ser Asn Phe Val Gly Ser Pro             260 265 270 Ile Leu Leu Asn Gly Lys Gln Ala Asn Glu Ile Lys Pro Leu Trp Leu         275 280 285 Leu Glu Pro Gln Ser Ile Ser Lys Glu Gln His His Asp Phe Tyr Arg     290 295 300 Phe Ile Ser Asn Ser Phe Asp Val Pro Arg Phe Thr Leu His Tyr Asn 305 310 315 320 Ala Asp Val Pro Leu Ser Ile His Ala Leu Leu Tyr Phe Pro Glu Gly                 325 330 335 Lys Pro Gly Leu Phe Glu Met Ser Arg Asp Gly Asn Thr Gly Val Ala             340 345 350 Leu Tyr Thr Arg Lys Val Leu Ile Gln Ser Lys Thr Glu His Leu Leu         355 360 365 Pro Lys Trp Leu Arg Phe Val Lys Gly Val Val Asp Ser Glu Asp Ile     370 375 380 Pro Leu Asn Leu Ser Arg Glu Leu Leu Gln Asn Ser Ser Leu Ile Arg 385 390 395 400 Lys Leu Ser Ser Val Ile Ser Thr Arg Val Ile Arg Phe Leu Gln Glu                 405 410 415 Arg Ser Lys Gln Pro Glu Glu Tyr Glu Ala Phe Tyr Arg Asp Tyr             420 425 430 Gly Leu Phe Leu Lys Glu Gly Ile Val Thr Ser Ser Asp Ala Ser Glu         435 440 445 Lys Glu Glu Ile Ala Lys Leu Leu Arg Phe Glu Ser Ser Lys Ser Glu     450 455 460 Thr Ala Ser Gly Arg Met Ser Leu Glu Glu Tyr Tyr Asn Ala Val Pro 465 470 475 480 Ala Glu Gln Gln Asn Ile Tyr Tyr Leu Ala Ala Pro Asn Arg Val Leu                 485 490 495 Ala Glu Ser Ser Pro Tyr Tyr Glu Ser Leu Lys Lys Arg Asn Glu Leu             500 505 510 Val Leu Phe Cys Tyr Glu Pro Tyr Asp Glu Leu Val Leu Met Gln Leu         515 520 525 Gly Lys Phe Lys Ser Lys Lys Leu Val Ser Val Glu Lys Glu Met Arg     530 535 540 Glu Glu Ser Lys Glu Thr Thr Ala Thr Thr Asp Phe Gly Glu Gly Ser 545 550 555 560 Leu Leu Arg Ser Glu Leu Asp Thr Leu Ile Pro Trp Leu Glu Glu Glu                 565 570 575 Leu Arg Gly Gln Val Ile Lys Val Lys Ala Thr Thr Arg Leu Asp Thr             580 585 590 His Pro Cys Val Ile Thr Val Glu Glu Met Ala Ala Ala Arg His Phe         595 600 605 Ile Arg Thr Gln Ser His Gln Val Pro Glu Gln Asn Arg Phe Ala Leu     610 615 620 Leu Gln Pro Glu Leu Glu Ile Asn Pro Lys His Pro Ile Ile Lys Lys 625 630 635 640 Leu Asn Lys Leu Arg Glu Ser Asp Lys Asp Leu Ala Gln Leu Ile Ala                 645 650 655 Lys Gln Leu Phe Ala Asn Ala Met Val Gly Ala Gly Leu Ala Glu Asp             660 665 670 Pro Arg Met Leu Leu Thr Asn Met Asn Thr Leu Leu Ser Arg Ala Leu         675 680 685 Glu Lys Tyr     690 <210> 4 <211> 2478 <212> DNA <213> Artificial Sequence <220> <223> Drosophila TRAP1 cDNA (NM_058091.3) <400> 4 ggcagtttgc ttcagttttg agctgaataa tgtgatgaaa ctgaactctc cattacatgt 60 agtaactctt attcaaacca actaagcatt tgagtccaaa attcattaca gcccagaaat 120 ttggaagaaa acaccgttat gagtagggtt ttaccagttt actattatcg aatacttaca 180 atcgattgat ttggctacac tgtgcacaca tgtgcgctgg aacaaacttc gtagttgggt 240 aacattaaag ttgggcaaaa cattaaaaca acaagatgtc tgtacgagcg atgggagtgc 300 tgggccaggc gtgccgcctc ggtcgctgtg cccaagtgtt gaggcagagt caccgatccg 360 ccccagcagc gttcaatatc accattggat cgcagcattc gccgggtgct ctctcgctcc 420 gtcgctactc cacggagacc aagcaggcat caggatcagt ggtggacaag catgagttcc 480 aggcagagac gcgccagcta cttgacattg tggctcgttc cttatactcc gaccacgagg 540 tctttgtgcg cgagcttatt tcgaatgcaa gcgatgcgct ggagaaattt cggtacacct 600 cgttgagcgc tggaggcgag aatctggctg gaaaggaccg gcctctggag attcgtatca 660 ccaccgacaa gccactgatg cagctaatca tccaggacac gggcatcggc atgaccaagg 720 aggagctggt cagcaacctg ggcaccattg cacgcagcgg ctcgaagaag ttcctggagc 780 agatgaaggg gacccagcag ggagccagct cggaggcatc ctcgaacatt attggtcagt 840 tcggcgtggg cttttactct tccttcattg tcgccaataa ggtggaggtg ttcacgaggg 900 cggctgtgcc taatgcaccc ggactgcgtt ggtccacgga tggcagtgga acctacgaga 960 tcgaagaggt gcctgacgtt gagttgggaa cgcgaatcgt gcttcattta aagaccgatt 1020 gccgtgaata tgcggacgag gagaggatca aggcggtaat caagaaatac agcaactttg 1080 tgggctcacc catcttgctt aacggaaagc aggccaatga gatcaagcct ctgtggcttc 1140 ttgagccaca aagcatcagc aaggagcagc accatgactt ctatcggttc attagcaata 1200 gcttcgatgt cccgcgcttc accttacact ataatgctga cgtacccttg agcatccatg 1260 cactgctcta cttccccgaa ggaaagcctg gtttattcga gatgtctcgg gacggaaaca 1320 ccggtgtggc tctttacacc cgcaaggtgc ttattcagtc caagactgag catctgctgc 1380 ccaagtggct gcgttttgta aagggtgttg tcgactctga ggacattcca ctcaacctga 1440 gtcgcgagct gctccagaac agcagcctga ttcgcaaact gtccagtgtg atttccaccc 1500 gcgttatccg tttcctgcaa gagcggtcca agaagcagcc agaagagtac gaggccttct 1560 accgggacta tgggcttttc ctgaaggaag gaattgtaac ctccagcgac gcctccgaga 1620 aagaagaaat agctaaactt ctgcgttttg aatcctccaa gtcggagacc gccagtgggc 1680 gcatgtcgct ggaggagtac tacaacgccg ttcccgcaga acagcagaat atttactact 1740 tggcagcacc caaccgcgtt cttgccgagt cctcgcccta ctacgagagt ctgaagaagc 1800 gcaacgagct ggtgctcttc tgctacgaac cctatgatga gctggtgctc atgcagctgg 1860 gcaagttcaa aagcaaaaaa ctcgtttccg tggagaagga gatgcgcgaa gagtccaagg 1920 agacaacagc gaccaccgac tttggcgagg gcagtctgct gcgctcagag ctcgacactt 1980 tgattccctg gctggaggag gagctcaggg ggcaggttat taaagtaaaa gccaccactc 2040 gtctagacac ccatccctgt gtaatcaccg tagaggaaat ggctgccgct cgccacttca 2100 ttcgcaccca gagtcaccag gtgccagaac agaatcgatt tgccctgctt caaccagaac 2160 tggagatcaa cccaaagcac cccatcatca agaagcttaa caagctccgt gagagcgaca 2220 aggatctggc ccaacttatc gccaagcagc tctttgcaaa cgcaatggtg ggcgccggtt 2280 tggctgagga tccccgcatg ctgctaacca acatgaacac tctgctatcg cgggccctgg 2340 agaaatacta gagtgcgccg tagaaggaac agtgtagata taccgactat gtaatattaa 2400 tttctataat taatttcata aaacgtgttg aaatagtttt aaatatgtat aattaaacgt 2460 tttaaatatt ttaattcc 2478 <210> 5 <211> 581 <212> PRT <213> Artificial Sequence <220> Human PINK1 amino acid sequence (NP_115785.1) <400> 5 Met Ala Val Arg Gln Ala Leu Gly Arg Gly Leu Gln Leu Gly Arg Ala   1 5 10 15 Leu Leu Leu Arg Phe Thr Gly Lys Pro Gly Arg Ala Tyr Gly Leu Gly              20 25 30 Arg Pro Gly Pro Ala Ala Gly Cys Val Arg Gly Glu Arg Pro Gly Trp          35 40 45 Ala Ala Gly Pro Gly Ala Gly Pro      50 55 60 Asn Arg Leu Arg Phe Phe Arg Gln Ser Val Ala Gly Leu Ala Ala Arg  65 70 75 80 Leu Gln Arg Gln Phe Val Val Arg Ala Trp Gly Cys Ala Gly Pro Cys                  85 90 95 Gly Arg Ala Val Phe Leu Ala Phe Gly Leu Gly Leu Gly Leu Ile Glu             100 105 110 Glu Lys Gln Ala Glu Ser Arg Arg Ala Val Ser Ala Cys Gln Glu Ile         115 120 125 Gln Ala Ile Phe Thr Gln Lys Ser Lys Pro Gly Pro Asp Pro Leu Asp     130 135 140 Thr Arg Leu Gln Gly Phe Arg Leu Glu Glu Tyr Leu Ile Gly Gln 145 150 155 160 Ser Ile Gly Lys Gly Cys Ser Ala Ala Val Tyr Glu Ala Thr Met Pro                 165 170 175 Thr Leu Pro Gln Asn Leu Glu Val Thr Lys Ser Thr Gly Leu Leu Pro             180 185 190 Gly Arg Gly Pro Gly Thr Ser Ala Pro Gly Gly Gly Gln Glu Arg Ala         195 200 205 Pro Gly Ala Pro Ala Phe Pro Leu Ala Ile Lys Met Met Trp Asn Ile     210 215 220 Ser Ala Gly Ser Ser Ser Glu Ala Ile Leu Asn Thr Ser Ser Gln Glu 225 230 235 240 Leu Val Pro Ala Ser Arg Val Ala Leu Ala Gly Glu Tyr Gly Ala Val                 245 250 255 Thr Tyr Arg Lys Ser Lys Arg Gly Pro Lys Gln Leu Ala Pro His Pro             260 265 270 Asn Ile Ile Arg Val Leu Arg Ala Phe Thr Ser Ser Val Leu Leu         275 280 285 Pro Gly Ala Leu Val Asp Tyr Pro Asp Val Leu Pro Ser Arg Leu His     290 295 300 Pro Glu Gly Leu Gly His Gly Arg Thr Leu Phe Leu Val Met Lys Asn 305 310 315 320 Tyr Pro Cys Thr Leu Arg Gln Tyr Leu Cys Val Asn Thr Pro Ser Pro                 325 330 335 Arg Leu Ala Met Met Leu Leu Gln Leu Leu Glu Gly Val Asp His             340 345 350 Leu Val Gln Gln Gly Ile Ala His Arg Asp Leu Lys Ser Asp Asn Ile         355 360 365 Leu Val Glu Leu Asp Pro Asp Gly Cys Pro Trp Leu Val Ile Ala Asp     370 375 380 Phe Gly Cys Cys Leu Ala Asp Glu Ser Ile Gly Leu Gln Leu Pro Phe 385 390 395 400 Ser Ser Trp Tyr Val Asp Arg Gly Gly Asn Gly Cys Leu Met Ala Pro                 405 410 415 Glu Val Ser Thr Ala Arg Pro Gly Pro Arg Ala Val Ile Asp Tyr Ser             420 425 430 Lys Ala Asp Ala Trp Ala Val Gly Ala Ile Ala Tyr Glu Ile Phe Gly         435 440 445 Leu Val Asn Pro Phe Tyr Gly Gln Gly Lys Ala His Leu Glu Ser Arg     450 455 460 Ser Tyr Gln Glu Ala Gln Leu Pro Ala Leu Pro Glu Ser Val Pro Pro 465 470 475 480 Asp Val Arg Gln Leu Val Arg Ala Leu Leu Gln Arg Glu Ala Ser Lys                 485 490 495 Arg Pro Ser Ala Arg Val Ala Asn Val Leu His Leu Ser Leu Trp             500 505 510 Gly Glu His Ile Leu Ala Leu Lys Asn Leu Lys Leu Asp Lys Met Val         515 520 525 Gly Trp Leu Leu Gln Gln Ser Ala Ala Thr Leu Leu Ala Asn Arg Leu     530 535 540 Thr Glu Lys Cys Cys Val Glu Thr Lys Met Lys Met Leu Phe Leu Ala 545 550 555 560 Asn Leu Glu Cys Glu Thr Leu Cys Gln Ala Leu Leu Leu Cys Ser                 565 570 575 Trp Arg Ala Ala Leu             580 <210> 6 <211> 2680 <212> DNA <213> Artificial Sequence <220> Human PINK1 cDNA (NM_032409.2) <400> 6 cgcagaggca ccgccccaag tttgttgtga ccggcggggg acgccggtgg tggcggcagc 60 ggcggctgcg ggggcaccgg gccgcggcgc caccatggcg gtgcgacagg cgctgggccg 120 cggcctgcag ctgggtcgag cgctgctgct gcgcttcacg ggcaagcccg gccgggccta 180 cggcttgggg cggccgggcc cggcggcggg ctgtgtccgc ggggagcgtc caggctgggc 240 cgcaggaccg ggcgcggagc ctcgcagggt cgggctcggg ctccctaacc gtctccgctt 300 cttccgccag tcggtggccg ggctggcggc gcggttgcag cggcagttcg tggtgcgggc 360 ctggggctgc gcgggccctt gcggccgggc agtctttctg gccttcgggc tagggctggg 420 cctcatcgag gaaaaacagg cggagagccg gcgggcggtc tcggcctgtc aggagatcca 480 gccaattttt acccagaaaa gcaagccggg gcctgacccg ttggacacga gacgcttgca 540 gggctttcgg ctggaggagt atctgatagg gcagtccatt ggtaagggct gcagtgctgc 600 tgtgtatgaa gccaccatgc ctacattgcc ccagaacctg gaggtgacaa agagcaccgg 660 gttgcttcca gggagaggcc caggtaccag tgcaccagga gaagggcagg agcgagctcc 720 gggggcccct gccttcccct tggccatcaa gatgatgtgg aacatctcgg caggttcctc 780 cagcgaagcc atcttgaaca caatgagcca ggagctggtc ccagcgagcc gagtggcctt 840 ggctggggag tatggagcag tcacttacag aaaatccaag agaggtccca agcaactagc 900 ccctcacccc aacatcatcc gggttctccg cgccttcacc tcttccgtgc cgctgctgcc 960 aggggccctg gtcgactacc ctgatgtgct gccctcacgc ctccaccctg aaggcctggg 1020 ccatggccgg acgctgttcc tcgttatgaa gaactatccc tgtaccctgc gccagtacct 1080 ttgtgtgaac acacccagcc cccgcctcgc cgccatgatg ctgctgcagc tgctggaagg 1140 cgtggaccat ctggttcaac agggcatcgc gcacagagac ctgaaatccg acaacatcct 1200 tgtggagctg gacccagacg gctgcccctg gctggtgatc gcagattttg gctgctgcct 1260 ggctgatgag agcatcggcc tgcagttgcc cttcagcagc tggtacgtgg atcggggcgg 1320 aaacggctgt ctgatggccc cagaggtgtc cacggcccgt cctggcccca gggcagtgat 1380 tgactacagc aaggctgatg cctgggcagt gggagccatc gcctatgaaa tcttcgggct 1440 tgtcaatccc ttctacggcc agggcaaggc ccaccttgaa agccgcagct accaagaggc 1500 tcagctacct gcactgcccg agtcagtgcc tccagacgtg agacagttgg tgagggcact 1560 gctccagcga gaggccagca agagaccatc tgcccgagta gccgcaaatg tgcttcatct 1620 aagcctctgg ggtgaacata ttctagccct gaagaatctg aagttagaca agatggttgg 1680 ctggctcctc caacaatcgg ccgccacttt gttggccaac aggctcacag agaagtgttg 1740 tgtggaaaca aaaatgaaga tgctctttct ggctaacctg gagtgtgaaa cgctctgcca 1800 ggcagccctc ctcctctgct catggagggc agccctgtga tgtccctgca tggagctggt 1860 gaattactaa aagaacatgg catcctctgt gtcgtgatgg tctgtgaatg gtgagggtgg 1920 gagtcaggag acaagacagc gcagagaggg ctggttagcc ggaaaaggcc tcgggcttgg 1980 caaatggaag aacttgagtg agagttcagt ctgcagtcct ctgctcacag acatctgaaa 2040 agtgaatggc caagctggtc tagtagatga ggctggactg aggaggggta ggcctgcatc 2100 cacagagagg atccaggcca aggcactggc tgtcagtggc agagtttggc tgtgaccttt 2160 gcccctaaca cgaggaactc gtttgaaggg ggcagcgtag catgtctgat ttgccacctg 2220 gatgaaggca gacatcaaca tgggtcagca cgttcagtta cgggagtggg aaattacatg 2280 aggcctgggc ctctgcgttc ccaagctgtg cgttctggac cagctactga attattaatc 2340 tcacttagcg aaagtgacgg atgagcagta agtaagtaag tgtggggatt taaacttgag 2400 ggtttccctc ctgactagcc tctcttacag gaattgtgaa atattaaatg caaatttaca 2460 actgcagatg acgtatgtgc cttgaactga atatttggct ttaagaatga ttcttatact 2520 ctgaaggtga gaatattttg tgggcaggta tcaacattgg ggaagagatt tcatgtctaa 2580 ctaactaact ttatacatga tttttaggaa gctattgcct aaatcagcgt caacatgcag 2640 taaaggttgt cttcaactga aaaaaaaaaa aaaaaaaaaa 2680 <210> 7 <211> 721 <212> PRT <213> Artificial Sequence <220> <223> Drosophila PINK1 amino acid sequence (NP_572340.2) <400> 7 Met Ser Val Arg Leu Leu Thr Val Arg Leu Ile Lys His Gly Arg Tyr   1 5 10 15 Ile Leu Arg Ser Tyr Cys Lys Arg Asp Ile His Ala Asn Ile Leu Asp              20 25 30 Gln Asn Gln Leu Lys Thr Arg Ser Lys Arg Gly Phe Pro Leu Pro Ser          35 40 45 Thr Ala Asn Val Leu Arg Thr Thr Pro Gln Gln Ala Ala Lys Ser      50 55 60 Val Val Asn Val Val Pro Arg Thr Ile Asn Ser Pro Ser Gly Ser Pro  65 70 75 80 Phe Asn Gly Ser Gly Ser Ser Pro Thr Ser Ser Gly Ile Phe Arg                  85 90 95 Val Gly Gln His Ala Arg Lys Leu Phe Ile Asp Asn Ile Leu Ser Arg             100 105 110 Val Thr Thr Thyr Ser Glu Asp Leu Arg Gln Arg Ala Thr Arg Lys         115 120 125 Leu Phe Phe Gly Asp Ser Ala Pro Phe Phe Ala Leu Ile Gly Val Ser     130 135 140 Leu Ala Ser Gly Ser Gly Val Leu Ser Lys Glu Asp Glu Leu Glu Gly 145 150 155 160 Val Cys Trp Glu Ile Arg Glu Ala Ala Ser Arg Leu Gln Asn Ala Trp                 165 170 175 Asn His Asp Glu Ile Ser Asp Thr Leu Asp Ser Lys Phe Thr Ile Asp             180 185 190 Asp Leu Glu Ile Gly Pro Pro Ile Ala Lys Gly Cys Ala Ala Val Val         195 200 205 Tyr Ala Ala Asp Phe Lys Lys Asp Val Ala Ser Asp Gly Ala Ser Leu     210 215 220 His Thr Asp Ala Gln Pro Gln Ala Thr Pro Ala Phe Ala Pro Asn Ser 225 230 235 240 Trp Ser Thr His Glu Met Met Ser Pro Leu Gln Asn Met Ser Arg Phe                 245 250 255 Val His Asn Phe Gly Gly Ser Val Asp Asn Val Phe His Tyr Ser Gln             260 265 270 Pro Ser Ala Ala Ser Asp Phe Val Gly Ala Gln Ser Arg Glu Gln Asp         275 280 285 Gln Arg His His Glu Gln Gln Gln His Gln Asn Gln Glu Gln Glu Gln     290 295 300 His Gln Asn Gln Glu Pro Ser Ser Ser Ala Phe Asn Val Thr Ser Pro 305 310 315 320 Ala Asn Ser Asn Ile Asn Ser Ser Val Asp Ser Tyr Pro Leu Ala Leu                 325 330 335 Lys Met Met Phe Asn Tyr Asp Ile Gln Ser Asn Ala Leu Ser Ile Leu             340 345 350 Arg Ala Met Tyr Lys Glu Thr Val Pro Ala Arg Gln Arg Gly Met Asn         355 360 365 Glu Ala Ala Asp Glu Trp Glu Arg Leu Leu Gln Asn Gln Thr Val His     370 375 380 Leu Pro Arg His Pro Asn Ile Val Cys Met Phe Gly Phe Phe Cys Asp 385 390 395 400 Glu Val Arg Asn Phe Pro Asp Gly His Leu Leu Tyr Pro Val Ala Gln                 405 410 415 Pro Gln Arg Ile Asn Pro Gln Gly Tyr Gly Arg Asn Met Ser Leu Tyr             420 425 430 Leu Leu Met Lys Arg Tyr Asp His Ser Leu Arg Gly Leu Leu Asp Ser         435 440 445 Gln Asp Leu Ser Thr Arg Asn Arg Ile Leu Leu Leu Ala Gln Met Leu     450 455 460 Glu Ala Val Asn His Leu Ser Arg His Gly Val Ala His Arg Asp Leu 465 470 475 480 Lys Ser Asp Asn Val Leu Ile Glu Leu Gln Asp Asp Ala Ala Pro Val                 485 490 495 Leu Val Leu Ser Asp Phe Gly Cys Cys Leu Ala Asp Lys Val His Gly             500 505 510 Leu Arg Leu Pro Tyr Val Ser His Asp Val Asp Lys Gly Gly Asn Ala         515 520 525 Ala Leu Met Ala Pro Glu Ile Phe Asn Thr Met Pro Gly Pro Phe Ala     530 535 540 Val Leu Asn Tyr Gly Lys Ala Asp Leu Trp Ala Cys Gly Ala Leu Ala 545 550 555 560 Tyr Glu Ile Phe Gly Asn Arg Asn Pro Phe Tyr Ser Ser Ser Gly Gly                 565 570 575 Met Ala Arg Glu Arg Gly Glu Met Thr Leu Ser Leu Arg Asn Ser Asp             580 585 590 Tyr Arg Gln Asp Gln Leu Pro Pro Met Ser Asp Ala Cys Pro Pro Leu         595 600 605 Leu Gln Gln Leu Val Tyr Asn Ile Leu Asn Pro Asn Pro Ser Lys Arg     610 615 620 Val Ser Pro Asp Ile Ala Ala Asn Val Val Gln Leu Phe Leu Trp Ala 625 630 635 640 Pro Ser Asn Trp Leu Lys Ala Gly Gly Met Pro Asn Ser Pro Glu Ile                 645 650 655 Leu Gln Trp Leu Leu Ser Leu Thr Thr Lys Ile Met Cys Glu Gly Arg             660 665 670 Pro Gln Met Gly Ala Gly Leu Met Pro Val Ala Ser Cys Gly Asn Arg         675 680 685 Arg Ala Tyr Val Glu Tyr Leu Leu Ile Cys Ser Phe Leu Ala Arg Ala     690 695 700 Arg Leu Arg Arg Ile Arg Gly Ala Leu Asn Trp Ile Gln Asn Val Val 705 710 715 720 Ala     <210> 8 <211> 2884 <212> DNA <213> Artificial Sequence <220> <223> Drosophila PINK1 cDNA (NM_132112.4) <400> 8 tataccgtaa gtgtggtcac actaacgcct tgctgtttct aaattattcc gttgttttta 60 tcgtaaattc ttgcacactt agctattatt ttatagcgaa atataagcaa agcaagcttt 120 taaacaagtg tttattgcgg cgcacatttt ccgtgacaaa tgaaaagatc cagctgacca 180 gcgattatct aacgaccaaa tcgaatcgaa acgaaacgtt gaagtaggcg cattacatta 240 aaaattggct gccagcccac acagatatcg aaaaaggcga tatatctttg gataatcatt 300 gcaaaaataa accctttcga ttcgacgatt tcgccttgta gttctgccac caccgccccc 360 acacttcgcc ccgcagctgc accacaacca ccaccaccca caaacagaaa atttatttgc 420 gacgcaaaaa caaaacaatc gaatcgaaac gaaccgaacc gaaacgaaac gacagcagct 480 gtttggttat aaatcggcgc tatcgatcgc acacatgagc aaaattggcg gagaagaaga 540 ggaagcagca gcaagagtta agcaccacaa atcttaaaga ataggcacta ggagccgagg 600 tctttgcggg ccgaaagatg tctgtgagac tgctgaccgt gcgactgatc aaacatggtc 660 gctacatttt gcgcagctat tgtaaacgtg atatacacgc caacattttg gaccagaatc 720 aattagagac aagaagcaag cgaggctttc ccctaccctc caccgctgcc aatgtgctgc 780 ggacaacgcc ccagcaggcg gccaaatcgg tggtcaatgt agtgccccgc accatcaact 840 ccccgtcggg atcaccgttt aatggcagcg gcagtagccc caccagcagc agtggaatct 900 tccgagtggg ccagcatgcg cgcaaattgt tcatcgacaa catcctcagc cgggtgacca 960 ccacctactc ggaggatctg cgccagcgcg ctacccgcaa gctattcttt ggcgattcag 1020 cgcccttctt cgccctgatt ggcgttagtc tggcctccgg atcgggtgtg ctcagcaagg 1080 aggatgaact ggagggcgtg tgctgggaga ttcgggaggc agctagccgg ctgcagaacg 1140 cctggaatca cgacgagatc tccgatacgc tagacagcaa gttcaccatc gatgacttgg 1200 aaatcggtcc gcccatagcc aaaggttgtg ccgctgtcgt ctatgcagcg gatttcaaga 1260 aagatgttgc ctcggatggt gcatccctgc ataccgatgc gcagccgcag gcaacgccag 1320 cctttgcgcc gaatagctgg agtacccacg agatgatgtc gccgctgcag aacatgtcgc 1380 gctttgttca caactttggc ggctctgtgg acaacgtctt ccactacagc cagccatctg 1440 cggccagtga tttcgtgggc gcccagtcga gggaacagga ccagcggcac cacgagcaac 1500 agcagcatca gaatcaggaa caagagcagc atcagaacca ggagcccagc agcagtgcct 1560 tcaatgtgac ttctccagcg aattcaaaca tcaacagctc tgtggacagt tatccactgg 1620 cactcaaaat gatgttcaac tacgacatcc agagcaacgc tctgtccata ctgcgtgcca 1680 tgtacaagga gacggtaccg gcacgccagc gcggcatgaa tgaggccgcc gatgagtggg 1740 aacgtttgct gcagaatcaa actgttcacc tgccgcggca tccgaacatt gtctgtatgt 1800 ttggcttctt ttgcgacgag gtgcgcaact ttcccgatgg ccatctcctg tatccggtgg 1860 cgcagccgca acgcatcaat ccacagggct atggccgcaa tatgtcgctg tatctgctga 1920 tgaaacgcta cgatcacagt cttcgcggcc tgctcgatag ccaggatctg agcacgcgca 1980 atcgcatcct gctgctggct caaatgctcg aggctgtcaa ccatttgagc cgtcacggcg 2040 ttgcccatcg tgacctaaag tctgataatg tgctaatcga gctgcaggac gatgcggcgc 2100 cggtgctagt gctctccgac tttggctgtt gtctggcgga caaggtgcat ggcctgcgcc 2160 tgccgtacgt ttcgcacgac gtcgacaagg gcggcaatgc ggcgttgatg gcgccagaga 2220 tcttcaatac gatgcccggt ccgtttgccg tactcaatta tggcaaggcc gatctgtggg 2280 cttgcggtgc cctggcttac gaaatctttg gcaaccgcaa tccgttctat tcgagcagcg 2340 gtgggatggc gcgcgaacgc ggtgagatga cgctttcgct gaggaacagc gattaccgac 2400 aggaccaatt gccgccaatg agcgatgctt gcccgccgct gctgcaacag ctggtctaca 2460 acatcctcaa tcccaacccg tccaagcggg tcagtccgga tattgcggca aatgtggtgc 2520 agctgttcct ctgggcccca tccaattggc ttaaggcggg cggcatgccc aacagtccgg 2580 agatcctaca gtggctcctt tcgctgacca ctaagattat gtgcgagggt cgtccacaga 2640 tgggcgccgg attgatgcca gtggccagct gtggcaatag gcgcgcctat gtggagtacc 2700 tgctcatatg cagctttctg gcacgcgccc ggctgcgtcg cattcgcggc gccctcaact 2760 ggatccagaa tgtggtggcg taggagaaaa gccttctact tgttactctg agaaaatcaa 2820 atggaaaagc gtgtgatacc tatattttac atatgtattt ataaatgaaa tcaaaattaa 2880 cgcc 2884 <210> 9 <211> 699 <212> PRT <213> Artificial Sequence <220> <223> TRAP1 recombinant protein amino acid sequence <400> 9 Met Ser Val Arg Ala Met Gly Val Leu Gly Gln Ala Cys Arg Leu Gly   1 5 10 15 Arg Cys Ala Gln Val Leu Arg Gln Ser His Arg Ser Ala Pro Ala Ala              20 25 30 Phe Asn Ile Thr Ile Gly Ser Gln His Ser Pro Gly Ala Leu Ser Leu          35 40 45 Arg Arg Tyr Ser Thr Glu Thr Lys Gln Ala Ser Gly Ser Val Val Asp      50 55 60 Lys His Glu Phe Gln Ala Glu Thr Arg Gln Leu Leu Asp Ile Val Ala  65 70 75 80 Arg Ser Leu Tyr Ser Asp His Glu Val Phe Val Arg Glu Leu Ile Ser                  85 90 95 Asn Ala Ser Asp Ala Leu Glu Lys Phe Arg Tyr Thr Ser Leu Ser Ala             100 105 110 Gly Gly Glu Asn Leu Ala Gly Lys Asp Arg Pro Leu Glu Ile Arg Ile         115 120 125 Thr Thr Asp Lys Pro Leu Met Gln Leu Ile Ile Gln Asp Thr Gly Ile     130 135 140 Gly Met Thr Lys Glu Glu Leu Val Ser Asn Leu Gly Thr Ile Ala Arg 145 150 155 160 Ser Gly Ser Lys Lys Phe Leu Glu Gln Met Lys Gly Thr Gln Gln Gly                 165 170 175 Ala Ser Ser Glu Ala Ser Ser Asn Ile Ile Gly Gln Phe Gly Val Gly             180 185 190 Phe Tyr Ser Ser Phe Ile Val Ala Asn Lys Val Glu Val Phe Thr Arg         195 200 205 Ala Ala Val Pro Asn Ala Pro Gly Leu Arg Trp Ser Thr Asp Gly Ser     210 215 220 Gly Thr Tyr Glu Ile Glu Glu Val Pro Asp Val Glu Leu Gly Thr Arg 225 230 235 240 Ile Val Leu His Leu Lys Thr Asp Cys Arg Glu Tyr Ala Asp Glu Glu                 245 250 255 Arg Ile Lys Ala Val Ile Lys Lys Tyr Ser Asn Phe Val Gly Ser Pro             260 265 270 Ile Leu Leu Asn Gly Lys Gln Ala Asn Glu Ile Lys Pro Leu Trp Leu         275 280 285 Leu Glu Pro Gln Ser Ile Ser Lys Glu Gln His His Asp Phe Tyr Arg     290 295 300 Phe Ile Ser Asn Ser Phe Asp Val Pro Arg Phe Thr Leu His Tyr Asn 305 310 315 320 Ala Asp Val Pro Leu Ser Ile His Ala Leu Leu Tyr Phe Pro Glu Gly                 325 330 335 Lys Pro Gly Leu Phe Glu Met Ser Arg Asp Gly Asn Thr Gly Val Ala             340 345 350 Leu Tyr Thr Arg Lys Val Leu Ile Gln Ser Lys Thr Glu His Leu Leu         355 360 365 Pro Lys Trp Leu Arg Phe Val Lys Gly Val Val Asp Ser Glu Asp Ile     370 375 380 Pro Leu Asn Leu Ser Arg Glu Leu Leu Gln Asn Ser Ser Leu Ile Arg 385 390 395 400 Lys Leu Ser Ser Val Ile Ser Thr Arg Val Ile Arg Phe Leu Gln Glu                 405 410 415 Arg Ser Lys Gln Pro Glu Glu Tyr Glu Ala Phe Tyr Arg Asp Tyr             420 425 430 Gly Leu Phe Leu Lys Glu Gly Ile Val Thr Ser Ser Asp Ala Ser Glu         435 440 445 Lys Glu Glu Ile Ala Lys Leu Leu Arg Phe Glu Ser Ser Lys Ser Glu     450 455 460 Thr Ala Ser Gly Arg Met Ser Leu Glu Glu Tyr Tyr Asn Ala Val Pro 465 470 475 480 Ala Glu Gln Gln Asn Ile Tyr Tyr Leu Ala Ala Pro Asn Arg Val Leu                 485 490 495 Ala Glu Ser Ser Pro Tyr Tyr Glu Ser Leu Lys Lys Arg Asn Glu Leu             500 505 510 Val Leu Phe Cys Tyr Glu Pro Tyr Asp Glu Leu Val Leu Met Gln Leu         515 520 525 Gly Lys Phe Lys Ser Lys Lys Leu Val Ser Val Glu Lys Glu Met Arg     530 535 540 Glu Glu Ser Lys Glu Thr Thr Ala Thr Thr Asp Phe Gly Glu Gly Ser 545 550 555 560 Leu Leu Arg Ser Glu Leu Asp Thr Leu Ile Pro Trp Leu Glu Glu Glu                 565 570 575 Leu Arg Gly Gln Val Ile Lys Val Lys Ala Thr Thr Arg Leu Asp Thr             580 585 590 His Pro Cys Val Ile Thr Val Glu Glu Met Ala Ala Ala Arg His Phe         595 600 605 Ile Arg Thr Gln Ser His Gln Val Pro Glu Gln Asn Arg Phe Ala Leu     610 615 620 Leu Gln Pro Glu Leu Glu Ile Asn Pro Lys His Pro Ile Ile Lys Lys 625 630 635 640 Leu Asn Lys Leu Arg Glu Ser Asp Lys Asp Leu Ala Gln Leu Ile Ala                 645 650 655 Lys Gln Leu Phe Ala Asn Ala Met Val Gly Ala Gly Leu Ala Glu Asp             660 665 670 Pro Arg Met Leu Leu Thr Asn Met Asn Thr Leu Leu Ser Arg Ala Leu         675 680 685 Glu Lys Tyr Leu Gly His His His His His     690 695 <210> 10 <211> 346 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence for expressing TRAP1 specific RNAi <400> 10 ccgacttgga ggattcaaaa cgcagaagtt tagctatttc ttctttctcg gaggcgtcgc 60 tggaggttac aattccttcc ttcaggaaaa gcccatagtc ccggtagaag gcctcgtact 120 cttctggctg cttcttggac cgctcttgca ggaaacggat aacgcgggtg gaaatcacac 180 tggacagttt gcgaatcagg ctgctgttct ggagcagctc gcgactcagg ttgagtggaa 240 tgtcctcaga gtcgacaaca ccctttacaa aacgcagcca cttgggcagc agatgctcag 300 tcttggactg aataagcacc ttgcgggtgt aaagagccac accggt 346 <210> 11 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Antennapedia (ANT0) <400> 11 Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys   1 5 10 15 <210> 12 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> survivin-derived peptide inhibiting activity of Hsp90 (or          TRAP1) <400> 12 Lys His Ser Ser Gly Cys Ala Phe Leu   1 5 <210> 13 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> Shepherdin amino acid sequence <400> 13 Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys   1 5 10 15 Lys His Ser Ser Gly Cys Ala Phe Leu              20 25 <210> 14 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> TRAP1-Forward primer <400> 14 gcagcgttca atatcaccat tg 22 <210> 15 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> TRAP1-Reverse primer <400> 15 gacctcgtgg tcggagtcta agg 23 <210> 16 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Vha16-1-Forward primer <400> 16 gtcttctgaa gtgagcagcg ac 22 <210> 17 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Vha16-1-Reverse primer <400> 17 catcactgac atggcagcaa tacc 24 <210> 18 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> COX I-Forward primer <400> 18 gtgcctttaa tattaggtgc tc 22 <210> 19 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> COX I-Reverse primer <400> 19 gtaatagctc ctgctagtac tg 22 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> COX III-Forward primer <400> 20 cgagatgtat cacgagaagg 20 <210> 21 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> COX III-Reverse primer <400> 21 gaattccgtg gaatcctgtt g 21 <210> 22 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> CytB-Forward primer <400> 22 cgaactttac atgctaacgg tg 22 <210> 23 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> CytB-Reverse primer <400> 23 ccgataggat tattagatcc tg 22 <210> 24 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> rp49-Forward primer <400> 24 gcttcaagat gaccatccgc cc 22 <210> 25 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> rp49-Reverse primer <400> 25 ggtgcgcttg ttcgatccgt aac 23

Claims (12)

(a) culturing cells expressing tumor necrosis factor receptor-associated protein 1 (TRAP1) with or without a test agent; And
(b) measuring the level of expression of TRAP1 in the cells incubated with the test agent, comparing the expression level of TRAP1 with the cells cultured without the test agent, and confirming whether the test agent inhibits the expression of TRAP1 A method for screening a medicament for preventing or treating a disease caused by mitochondrial dysfunction.
The method according to claim 1, wherein after step (b)
(c) inspecting whether the test agent confirmed to inhibit the expression of TRAP1 in step (b) is administered to an animal having a disease caused by mitochondrial dysfunction to show a therapeutic effect.
3. The screening method according to claim 1 or 2, wherein the disease caused by the mitochondrial dysfunction is a disease due to a mitochondrial dysfunction caused by a PINK1 gene deletion.
3. The method of claim 1 or 2, wherein the mitochondrial dysfunction is selected from the group consisting of mitochondrial enzyme activity reduction, electron transport chain activity reduction, membrane potential reduction, increase of reactive oxygen species production, mitochondrial fragmentation fragmentation, calcium regulation disorder, and mutation of mitochondrial DNA &lt; RTI ID = 0.0 &gt; (mtDNA). &lt; / RTI &gt;
The method according to claim 1 or 2, wherein the disease caused by the mitochondrial dysfunction is selected from the group consisting of degenerative brain diseases, NARP syndrome, Multiple Sclerosis-like Syndrome, Maternally Inherited CardioMyopathy, Progressive External Ophthalmoplegia, Myoclonic Epilepsy with Ragged-Red Fibers Syndrome, Myoneurogastrointestinal disorder and encephalopathy, Pearson Marrow syndrome, Kearns-Sayre syndrome, Leber hereditary optic neuropathy, aminoglycoside-associated deafness, diabetes with deafness, Luft disease, Syndrome (Leigh syndrome), Alpers Disease, medium chain acyl-CoA dehydrogenase (SCAD) deficiency, short chain 3-hydroxyacyl CoA dehydrogenase (SCHAD) deficiency, and short-chain acylase deficiency (VLCAD) deficiency, long chain 3-hydroxy acyl-CoA dehydrogenase (LCHAD) deficiency, glutaric aciduria type II Glutaric aciduria II), and lethal infantile cardiomyopathy.
6. The screening method according to claim 5, wherein the degenerative brain disease is one selected from the group consisting of Parkinson's disease, Alzheimer's disease, Huntington's disease, and dementia. .
A composition for preventing and treating diseases caused by mitochondrial dysfunction, which comprises as an active ingredient any one selected from the group consisting of antisense RNA, siRNA and miRNA against the TRAP1 gene.
A composition for preventing and treating diseases caused by mitochondrial dysfunction comprising an antibody specific for TRAP1 as an active ingredient.
A composition for preventing and treating diseases caused by mitochondrial dysfunction comprising TRAP1 inhibitor (TRAP1 inhibitor) as an active ingredient.
10. The method according to claim 9, wherein the TRAP1 inhibitory substance is geldanamycin conjugated with gamitrinib or an antigen-pediatric cell-penetrating sequence (ANT), for the prevention and treatment of diseases caused by mitochondrial dysfunction Composition.
The composition for preventing and treating diseases caused by mitochondrial dysfunction according to claim 9, wherein the TRAP1 inhibitor is Shepherdin (SEQ ID NO: 13).
(a) culturing TRAP1 or a cell expressing said TRAP1 and a test agent; And
(b) measuring the activity of said TRAP1, and confirming whether said test agent inhibits the activity of TRAP1; and screening for a drug for preventing or treating diseases caused by mitochondrial dysfunction.

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