WO2005116082A1 - 2-cysteine peroxiredoxin complex exhibiting function acting as molecular chaperone and uses thereof - Google Patents

Abstract

Disclosed herein are a high molecular weight complex, comprised of 2-cysteine peroxiredoxin proteins linked together by intermolecular interaction, having chaperone activity, and its uses. On the basis of the finding of the fact that 2-cysteine peroxiredoxin proteins in various forms are different in structure and molecular weight, functioning as a peroxidase in a low molecular weight structural form and as a molecular chaperone in a high molecular weight structural form, the 2-cysteine peroxiredoxin proteins can be applied to a diagnostic medicine for neurodegenerative disorders, Alzheimer's disease, Down's disease, breast cancer, lung cancer, etc., a pharmaceutical composition for the prophylaxis and treatment of the diseases, and a transgenic animal or plant that is resistant to environment stress or such diseases.

Description

[Invention Title] 2-CYSTEH E PEROXIREDOXIN COMPLEX EXHJBΠTNG FUNCΗON ACTING AS A MOLECULAR CHAPERONE AND USES THEREOF

[Technical Field] The present invention relates to a high molecular weight complex of 2-cysteine peroxiredoxins, which has chaperone activity. More particularly, the present invention relates to a high molecular weight complex of 2-cysteine peroxiredoxins and its application to a diagnostic medicine for neurodegenerative disorders, Alzheimer's disease, Down's syndrome, breast cancer, lung cancer, etc., a pharmaceutical composition for the prophylaxis and treatment of the diseases, and a transgenic animal or plant that is resistant to environmental stress or such diseases, on the basis of the finding the fact that 2-cysteine peroxiredoxin proteins in various forms are different in structure and molecular weight, functioning as a peroxidase in a low molecular weight structural form and as a molecular chaperone in a high molecular weight structural form. [Background Art] Reactive oxygen species (ROS) can be produced during the course of normal aerobic metabolism or when an organism is exposed to a variety of stress conditions (Finkel T, Curr. Opin. Cell Biol. 15: 247-254, 2003). ROS can cause widespread damage to biological macromolecules and are believed to play a causal role in many degenerative diseases (Neumann et al., Nature, 424: 561-565, 2003). To protect themselves from oxidative stress and ROS-mediated protein unfolding and aggregation, all aerobic organisms are equipped with a wide range of antioxidant proteins, including superoxide dismutase (SOD), catalase, many kinds of peroxidases (Bryk et al., Science, 295: 1073-1077, 2002), and diverse forms of molecular chaperones, such as heat shock protein (HSP) 90, HSP70, HSP60, HSP40, and small HSPs (sHSPs). Recently, a new type of antioxidant protein that reduces H2 2, peroxinitrite, and organic hydroperoxide with thioredoxin Trx) as an electron donor was discovered in most prokaryotic and eukaryotic cells. It was initially called Trx-dependent peroxidase and later renamed peroxiredoxin (Prx) (Chae et al., J. Biol. Chem., 269: 27670-27678, 1994). Although the overall sequence homology of Prx proteins (Prxs) with other proteins that contain the Trx-folding motif is low, Prxs have nevertheless been classified as novel members of the Trx-fold superfamily (Schroder et al., Protein Sci., 7: 2465-2468, 1998). The Prxs were initially divided into two groups, namely 1-Cys Prxs and 2-Cys Prxs, based on the number of conserved Cys residues in their sequences. In addition to the report that Prxs regulate peroxide-mediated signaling cascades, a large number of Prxs are associated with diverse cellular functions, such as cell proliferation, differentiation, immune response, growth control, tumor promotion, apoptotic process, and numerous unidentified functions (Neumann et al., Nature, 424: 561-565, 2003; Hirotsu et al., Proc. Natl. Acad. Sci. USA, 96:

12333-12338, 1999). Specifically, it was reported that 2-Cys Prxs were overexpressed in several cancers (Noh et al., Anticancer Res., 21: 2085-2090, 2001; Yanagawa et al., Cancer Letter, 145: 127- 135, 1999; Kinnulaetal., J. Pathol., 196: 316-323, 2002; Chang etal, Biochem. Biophys. Res. Comm., 289: 507-512, 2001) and in neurodegenerative disorders such as Alzheimer's disease,

Pick's disease and Down syndrome (Multhaup et al., Biochem. Phamacol., 54: 533-539, 1997; Krapfenbauer et al., Brain Res., 967: 152-160, 2003). In cancer cells, their expression was also found to correlate positively with the degree of resistance to apoptosis induced by radiation therapy (Chen et al., J. Neurosci. Res., 70: 794-798, 2002) or the anticancer dmg imexon (Nonn et al., Mol. Cancer Res., 1: 682-689, 2003). With regard to neurodegenerative disorders, it is well known that the generation of toxic reactive oxygen species (ROS) is involved in the development of age-dependent neurodegeneration (Mu et al., J. Biol. Chem., 277: 43175-43184, 2002; Kim et al., J. Biol. Chem., 275: 18266-18270, 2000). It was therefore speculated that antioxidant proteins, including 2-Cys Prxs, are upregulated in these disorders in an attempt by the body to protect the neural cells from oxidative stress. Despite the multitude of these studies, the true cellular function of Prx proteins remains unclear. Particularly perplexing is that Prxs have low catalytic efficiencies compared to those of catalases or glutathione peroxidases (GPxs). In addition, they show a marked susceptibility to being inactivated during the H2θ2-catalytic process (Yang et al., J. Biol. Chem., 277: 38029- 38036, 2002). These observations cast seriously doubt upon the notion that the sole purpose of Prxs in cells is to act as peroxidases. In addition to functioning in hydrogen peroxide catalysis, 2-Cys Prxs have been shown to regulate mitogen-activated protein kinase activity (Veal et al., Mol. Cell., 15: 129-139,2004) and to modulate the activity of NF-kB, a master transcription factor (Kang et al., J. Biol. Chem., 279: 2535-2543, 2004; Barford et al., Curr. Opin. Struct. Biol., 14: 679-686, 2004). X-ray crystal stmctures of the 2-Cys Prxs found in human erythrocytes and Crithidia fasciculate show that they form high molecular weight (HMW) toroid stmctures comprised of five dimers linked together by hydrophobic interactions (Schrder et al., Structure, 8: 605-615, 2000). External conditions can induce these structures to undergo large conformational changes. With regard to these properties, the 2-Cys Prxs resemble sHSPs, most of which are well-ordered oligomers with a defined number of subunits and molecular chaperones (Kim et al., Nature, 394: 595-599,1998; Haley et al, J. Mol. Biol., 277: 27-35, 1998). Moreover, certain non-Prx proteins harboring the Trx-folding motif have also been shown to be potent molecular chaperones. These include protein disulfide isomerase (PDI), E. coli Trx, Trx reductase (TR), and bacterial HSP33 protein (Wang, Methods Enzymol, 348: 66-75, 2002; Kem et al, Biochem. J, 371 : 965-972, 2003; Jakob et al. Cell, 96: 341-352, 1999). [Disclosure] [Technical Problem] Based on the description given above, the present inventors expected 2-cysteine peroxiredoxins to have chaperone activity and intended to use them for industrial applications. For this purpose, 2-cysteine peroxiredoxin genes were cloned from animals (human cells), plants (Arabidopsis thaliana) and microorganisms (yeast) and expressed in E. coli. The proteins were isolated and assayed for chaperone activity. Also, the proteins of interest were analyzed for their ability to diagnose diseases from which the subject suffers, treat diseases, and be used to prepare a transgenic plant which can be made resistant to diseases or environmental stress through gene transduction and expression control. Herein, only 2-cysteine peroxiredoxin (cPrxI), obtained from yeast, will be described because the proteins obtained from the animal, the plant and the microorganism are similar in structure and function (Prak et al, J. Biol. Chem, 275: 5723-5732, 2000). Therefore, it should be noted that properties of the 2-cysteine peroxiredoxin protein and a gene coding for the protein, obtained from yeast, are similar to those of the 2-cysteine peroxiredoxin proteins and genes obtained from animals or plants. Accordingly, the object of the present invention is to provide a kit and a method for diagnosing a disease using a high molecular weight complex, comprised of 2-cysteine peroxiredoxins ∞ntaining the thioredoxin motif, having chaperone activity. It is another object of the present invention to provide a pharmaceutical composition for the prophylaxis and treatment of neurodegeneration, Alzheimer's disease, Down's syndrome, Parkinson's disease, heart cancer, breast cancer, lung cancer, tumors, etc. It is a further object of the present invention to provide a transgenic cell or organism having resistance to diseases and environmental stresses. [Technical Solution] In accordance with an aspect of the present invention, the above objects could be accomplished by a provision of a high molecular weight complex, comprised of microorganism, plant or animal 2-cysteine peroxiredoxin proteins linked together by intermolecular interaction, having chaperone activity. In accordance with another aspect of the present invention, there is provided a transformed cell capable of overexpressing 2-cysteine peroxiredoxin, which is prepared by introducing a recombinant vector overexpressing at least one 2-cysteine peroxiredoxin protein represented by SEQ. ID. NOS.2, 4, 6, 8, 10, 11 and 12 into a microorganism cell. In accordance with a further aspect of the present invention, there is provided a transgenic plant cell strain capable of overexpressing 2-cysteine peroxiredoxin, which harbor the mutant Agrobacterium tumefaciencs transformed by introducing a recombinant vector overexpressing at least one 2-cysteine peroxiredoxin protein selected from a group consisting of polypeptides represented by SEQ. ID. NOS.2, 4, 6 and 8 into Agrobacterium tumefaciencs. In accordance with still a further aspect of the present invention, there is provided a monoclonal antibody, specifically binding to the complex. In accordance with still another aspect of the present invention, there is provided a method for diagnosing a disease from which a subject suffers, in which a monoclonal antibody is subjected to immunoreaction with a sample from the subject and the sample is quantitatively analyzed for peroxiredoxin complex in comparison to a control. In accordance with yet another aspect of the present invention, there is provided a kit for diagnosing a disease from which a subject suffers, comprising: a test sample; a gel on which positive and negative control samples are separated by native-PAGE; a comb card to which nitrocellulose membranes as many as lanes of the gel are attached; an electric device for supplying electricity to transfer the proteins on the gel onto the nitrocellulose membranes; the monoclonal antibody on the nitrocellulose membranes; and an incubation tray for allowing the monoclonal antibody to recognize the proteins transferred onto the nitrocellulose membranes. In accordance with yet a further aspect of the present invention, there is provided a kit for diagnosing a disease from which a subject suffers, comprising: a strip (1) comprising: a developing membrane 13 on which a reaction unit (11) for immobilizing an antibody thereon, and a control unit (12) for monitoring the normal operation of the kit are positioned at predetermined positions; and a housing (2) for accommodating Hie strip (1), comprised of: a sample feeder (21): and indication windows (22) through which reaction results in the reaction unit (11) and the control unit (12) are visualized with the naked eye. In accordance with yet still another aspect of the present invention, there is provided a medicine for use in diagnosing a disease from which a subject suffers, comprising: (a) an immobilized sample obtained by processing a subject matter; (b) the monoclonal antibody of claim 11 or 12, recognizing a high molecular weight complex of 2-Cys Prxs as an antigen; (c) a coloring enzyme-labeled complement binding specifically to the antibody; (d) a substrate solution undergoing a color change upon reaction with the coloring enzyme; and (e) a buffer for the reaction for the color change and an incubation tray. In accordance with yet still a further aspect of the present invention, there is provided a pharmaceutical composition for the prophylaxis and treatment of neurodegeneration, Alzheimer's disease, Down's syndrome, Parkinson's disease, thyroid cancer, heart cancer, breast cancer, lung cancer, and tumors, comprising a peroxiredoxin complex or its salt as an effective ingredient, in combination with a pharmaceutically acceptable carrier, the peroxiredoxin complex consisting of 2-cysteine peroxiredoxin proteins composed of at least one selected from a group consisting of polypeptides represented by SEQ. ID. NOS.2, 4, 6, 8, 10, 11 and 12. In accordance with yet another aspect of the present invention, there is provided a pharmaceutical composition for the prophylaxis and treatment of neurodegeneration, Alzheimer's disease, Down's syndrome, Parkinson's disease, thyroid cancer, heart cancer, breast cancer, lung cancer, and tumors, comprising a transformed cell of claim and a pharmaceutically acceptable carrier, the transformed cell being prepared by introducing a recombinant vector overexpressing at least one 2-cysteine peroxiredoxin protein selected from a group consisting of polypeptides represented by SEQ. ID. NOS. 2, 4, 6 and 8 into a human cell. In the present invention, examples of the disease suitable to be diagnosed include neurodegeneration, Alzheimer's disease, Down's syndrome, thyroid cancer, lung cancer, tumors, heat shock-induced diseases, reactive oxygen species-induced diseases, liver cancer, breast cancer, womb cancer, etc. The microorganism transformed by introducing a cPrx I gene into E. coli (E. co/i/pGEX-cPrxi) was deposited at the Korean Collection for Type Cultures on May 24, 2004, with the accessionNo. KCTC 10645BP. The microorganism transformed by introducing a hPrxIJ gene into E. coli (E. cø///pGEX-hPrxII) was deposited at the Korean Collection for Type Cultures on April 25,

2005 with the accessionNo. KCTC 10793BP. As storages for the peroxiredoxin genes anchored at vectors, the transformants are preferably stored in, for example, liquid nitrogen with a storage buffer until reuse.

[Best Mode] Below, a detailed description will be given of the present invention. Technical and scientific terminology, as used herein, lias the same meanings as understood by those skilled in the art, if not specifically defined otherwise. The 2-cysteine peroxiredoxin complexes useful in the present invention include complexes resulting from the association of heterogeneous peroxiredoxins derived from microorganisms, higher animals and higher plants, as well as homogeneous peroxiredoxins. Because 2-cysteine peroxiredoxins derived from microorganisms, higher animals and higher plants are similar in structure and function, most 2-cysteine peroxiredoxins may be used in the present invention. Referring to the accompanying sequence list, 2-cysteine peroxiredoxins used in the present invention are represented by SEQ. ID. NOS. 2, 4, 6 and 8, isomers derived from humans, by SEQ. ID. NO. 10, derived from Arabidos thaliana, and by SEQ. ID. NOS. 11 and 12, derived from Saccharomyces cerevisiae. As is generally recognized in the art, a chaperone is defined as any of a class of proteins that helps proteins fold or escorts proteins or other molecules throughout the cell. For example, proteins, when under stress, such as heat shock, have unfolded tertiary stmctures so that they cannot perform their functions. Chaperones recognize and bind to such unfolded proteins and provide an environment that allows them to refold. Based on the finding that, of peroxiredoxin proteins present in the cytoplasm of animals, plants and microorganisms, 2-cysteine peroxiredoxin (2-Cys Prx), having two cysteine groups, functions as a chaperone component as well as a peroxidase, the present invention provides applications for the chaperone functions possessed by 2-cysteine peroxiredoxin. In addition^ observing that chaperone functions predominate in high molecular complexes of peroxiredoxin proteins, the present inventors speculate that senile diseases, various cancers, and exposure to oxidative stress and heat induce 2-cysteine peroxiredoxin (2- Cys Prx) proteins to undergo a conformational change from low molecular weight stmctures to high molecular weight complexes and that the structural change leads to a functional switching from a peroxidase function, which is dominant in isolated peroxiredoxin, to a chaperone function. It is also believed that the chaperone function of 2-Cys Prxs improves the resistance to various external environmental stresses, heat shocks, oxidative stresses, diseases, etc. because the functional change is mostly attributed to changes in the cysteine located at the active site of peroxidase, for example, residue 47 (Cys47) in yeast and residue 51 (Cys51) in humans, which acts as an effective recognizer of hydrogen peroxide in cells. Properties of this mechanism were identified in experiments, which examined whether various oxidative stresses induce low molecular weight 2-Cys Prxs to change into high molecular weight protein stmctures, and whether this conformational change results in a functional change from peroxidase to a novel chaperone function. With these structural and functional changes, plants, animals and microorganisms were found to show high resistance to external stress. Thus, it is expected that plants or microorganisms, if transformed with a gene coding for the protein of interest and controlled in the expression of the gene, will have resistance to various environmental stresses, and that the pursuit of the chaperone function will allow the application of the protein in the diagnosis and treatment of various human diseases, such as cancers, Alzheimer's disease, Parkinson's disease, etc. This is highly plausible, in light of H e fact that the dysfunction or functional disability of 2-Cys Prx due to mutations or deletions is reported to cause degeneration in cranial nerves (Krapfenbauer et al, Electrophoresis.23: 2541-2547, 2002), Alzheimer's disease (Kim et al, J

Neural Transm Suppl. 61 : 223-235, 2001), Down's syndrome (Krapfenbauer et al. Brain Res.

967: 152-160, 2003), thyroid cancer (Yanagawa et al,Cancer Lett, 145;127-132, 1999), heart cancer, lung cancer (Kim et al. Cell Biol Toxicol. 19: 285-298, 2003.), tumors (Neumann et al.

Nature, 424: 561-565, 2003), etc. In order to apply 2-Cys Prxs to diagnostic fields, including diagnostic medicines, diagnostic technology and diagnostic kits, and to pharmaceutical compositions, advantage is taken of the chaperone functions thereof. Preferably, the diagnostic method of the present invention uses monoclonal antibodies that are specific for peroxiredoxin complexes having chaperone activity which result from the association of proteins selected from a family group of 2-Cys Prxs consisting of peptide sequences of SEQ. ID. NOS. 2, 4, 6, 8, 10, 11 and 12. More preferable are monoclonal antibodies corresponding to the human 2-cysteine peroxiredoxin proteins of SEQ. ID. NOS.2, 4, 6 and 8. Proteins obtained by rapturing or lysing cells from tissues or humors of animals, plants and microorganisms may be used as test samples. For the immunoassay with the monoclonal antibodies, sample proteins, which may be prepared by separation from cell lysates on native- PAGE, are transferred onto predetermined positions of nitrocellulose membranes and reacted with the monoclonal bodies, which are then visualized for position and amount by dyeing with enzymes or dyes, so as to determine the amount of peroxiredoxin complexes in test samples. In accordance with one embodiment of the present invention, a diagnostic composition that is capable of quantitatively analyzing high molecular weight complexes of 2-Cys Prx isomers in the subject to determine disease occurrence comprises the following components: (a) an immobilized sample obtained by processing a subject matter; (b) an antibody recognizing a high molecular weight complex of 2-Cys Prxs as an antigen; (c) a coloring enzyme-labeled complement binding specifically to the antibody; (d) a substrate solution undergoing a color change upon reaction with the coloring enzyme; and (e) a buffer for the reaction for the color change. The term "subject matter" as used herein means whole blood, sera, plasmas, lymph fluids, intercellular fluids, or tissues such as organs. The term "test sample", as used herein, means a protein separated/extracted from the subject matter. The complement used in the present invention is an enzyme-labeled secondary antibody against the antibody specific for the high molecular weight complex of 2-Cyst Prx isomers. The coloring enzyme linked to the secondary antibody may be peroxidase or alkaline phosphatase, with the requirement for corresponding substrates, e.g., TMB or

NBT/BCIP, respectively. However, the present invention is not limited to this method, but includes any method within the scope thereof if it can use an antibody against the high molecular weight complex of 2-Cys Prx isomers to detect the diseases. Any may be used for the antibody, the complement and the coloring enzyme, instead of those mentioned above, if they show high binding specificity and reaction affinity and are so susceptible as to react with each other even at very low concentrations. In accordance with another embodiment of the present invention, a diagnostic composition comprises a Western blotting set adapted to separate test samples as proteins using native-PAGE, blot the test samples onto predetermined positions of a nitrocellulose membrane, treat the membrane with an antibody and visualize the position and concentration of the antibody bound onto the membrane with an enzyme or dye; an antibody binding specifically to a high molecular weight complex of 2-Cys Prxs and a coloring enzyme linked to the antibody; and a substrate solution for the enzyme (Jang et al. Cell. 117; 625-635, 2004). For quick and mass examination, an ELISA method using 96- or 386-well plates may be effective (D' Ercole et al, J. Immunoassay Immunochem, 26: 43-56, 2005). In this case, 96 or 386 test samples are added into respective wells of ELISA plates made from polystyrene so as to readily bind proteins thereto, and sufficiently coated on the wells, followed by washing the wells to remove non-bound materials. Into each of the wells that contain a test sample, an antibody binding specifically to a high molecular weight peroxiredoxin complex is added, and an immunoblotting method is conducted for quick diagnosis. In this regard, the antibody binding specifically to the high molecular weight peroxiredoxin complex is reacted with a secondary antibody against this antibody and the color development of the second antibody is analyzed. Preferably, peroxidase or alkaline phosphatase is conjugated with the secondary antibody as a coloring enzyme while TMB or NBT BCIP is used as a corresponding substrate, respectively. In accordance with a further embodiment of the present invention, a diagnostic kit, for use in the diagnosis of diseases comprises a test sample; a gel, on which positive and negative control samples are separated by native-PAGE; a comb card, to which are attached a number of nitrocellulose membranes corresponding to the number of lanes of gel; an electric device, for supplying electricity to transfer the proteins on the gel onto the nitrocellulose membranes; a monoclonal antibody on the nitrocellulose membranes, defined in claim 11 or 12; and an incubation tray, for allowing the monoclonal antibody to recognize the proteins transferred onto the nitrocellulose membranes. For the diagnosis, a Western blotting method is applied. Using a typical ELISA, a coloring enzyme may be used to detect the antibody bound to the transferred proteins. In FIG. 16, the diagnostic kit in accordance with the present invention is illustrated.

As seen in FIG. 16, the diagnostic kit is comprised of a strip (1) and a housing (2). The strip (1) comprises a developing membrane 13 on which a reaction unit (11) and a control unit (12) for monitoring the normal operation of the kit are positioned at predetermined positions. On the reaction unit (11), an antibody prepared according to the present invention is immobilized. In addition to accommodating the strip (1), the housing (2) comprises a sample feeder (21) and indication windows (22) through which reaction results in the reaction unit (11) and the control unit (12) are visualized with the naked eye. In the kit, the absorbance is detected and analyzed by an ELISA method so as to determine whether the subject is afflicted with the diseases. In accordance with another aspect of the present invention, a pharmaceutical composition for the prophylaxis and treatment of neurodegeneration, Alzheimer's disease, Down's syndrome, Parkinson's disease, thyroid cancer, heart cancer, breast cancer, lung cancer and tumors is provided. The pharmaceutical composition of the present invention comprises a high molecular weight complex of the above-mentioned human 2-cysteine peroxiredoxin isomers or its salt as an effective ingredient, in combination with a pharmaceutically acceptable carrier. As described, high molecular complexes of human 2-cysteine peroxiredoxin isomers according to the present invention themselves can be used as they are or in a pharmaceutically acceptable acid addition salt or metal complex form. Examples of the pharmaceutically acceptable acid addition salt include hydrochloride salts, hydrobromide salts, sulfuric acid salts, phosphoric acid salts, maleic acid salts, acetic acid salts, citric acid salts, benzoic acid salts, succinic acid salts, maleinic acid salts, ascorbic acid salts, and tartaric acid salts. The metal complex useful in tl e present invention may contain zinc or iron. Depending on administration routes, administration forms and treatment purposes, Hie pharmaceutical composition of the present invention may be prepared in various dosage forms. For example, a diluent for use as the carrier may be selected from among, but is not limited to, saline, buffers, dextrose, water, glycerol, ringer's solution, lactose, sucrose, calcium silicate, methyl cellulose, ethanol, and combinations thereof. Using these diluents, H e pliarmaceutical composition of the present invention can be prepared in dosage forms for oral or parenteral administration, such as powder, granules, injection solutions, syrups, tablets, suppositories, pessaries, ointments, creams, aerosols, and etc. Parenteral administration means the administration of the dosage forms according to the present invention through rectal, intravenous, intraperitoneal, intramuscular, intra-arterial, subcutaneous, or intranasal routes, or the like. In addition, the pharmaceutical composition may further comprise an additive, such as a filler, an anticoagulant, a lubricant, a wetting agent, a flavor, an emulsifier, a preservative, etc, and may be formulated to show quick, sustained or delayed release. In another embodiment of the present invention, a pharmaceutical composition for injection comprises a transformant (transformed cell strain), obtained by introducing a 2- cysteine peroxiredoxin isomer-expressing recombinant vector into a human cell strain, capable of overexpressing 2-cysteine peroxiredoxin; and a pharmaceutically acceptable carrier. Depending on degrees of disease severity, administration routes and dosage forms, the dosage of the pharmaceutical composition of the present invention can vary within the range in which no desensitization happens. In consideration of the fact that peroxiredoxin is the second or the third in blood level among the proteins found in blood (Moore et al, J. Biol. Chem.266: 18964-18968, 1991) and is known to amount to as much as 0.1-0.8% of the water- soluble proteins in animal cells (Chae et al. Diabetes Res. Clin. Pract. 45: 101-112, 1999), the effective ingredient, that is, 2-cysteine peroxiredoxin or its salts, are preferably administered in a dose of 10 ~ 2,000mg per kg of body weight a day, in a sporadic or continuous manner.

[Description of Drawings] FIG. 1 shows the heat shock resistance of 2-cysteine peroxiredoxin I and U-deficient yeast strains, into which have been inserted genes coding for a native structural 2-cysteine peroxiredoxin I or Cysteine mutant C47/170S-2-cysteine peroxiredoxin I; FIG. 2 shows an amino acid sequence of 2-cysteine peroxiredoxin I (cPrxI) having dual functions of peroxidase and chaperone, along with a secondary structure thereof (A), and the chaperone activity of the 2-cysteine peroxiredoxin I from yeast; FIG. 3 shows properties of 2-cysteine peroxiredoxin I after separation using size chromatography in an absorbance spectrum, in an optical photograph after separation using native-PAGE, and in an electronmicrophotograph; FIG. 4 shows the structural and functional switching of 2-cysteine peroxiredoxin I, induced by oxidative stress and heat shock; FIG. 5 shows the reversible structural switching of 2-cysteine peroxiredoxin I according to oxidative stress and heat shock; FIG. 6 shows the influence of the expression of various types of 2-cysteine peroxidoxin in 2-cysteine peroxiredoxin I and II-free yeast (Δ2-cysteine peroxiredoxin I/H) on heat shock and protein solubility upon treatment with hydrogen peroxide; FIG.7 shows a recombinant DNA vector carrying a 2-cysteine peroxiredoxin I (basl) gene, suitable for transfection into Arabidopsis, and the stress resistance of transgenic Arabidopsis; FIG. 8 shows the immumo-specificity of a monoclonal antibody for a high molecular weight complex of human 2-cysteine peroxiredoxins, and the expression of the high molecular weight complex in various cancer cells; FIG. 9 shows the physiological mechanism of oxidative stress-dependent structural and functional change in the peroxidase and chaperone activity of 2-cysteine peroxiredoxin I; FIG. 10 shows the chaperone activity of a native structure of human 2-cysteine peroxiredoxin II against heat shock; FIG. 11 shows the relationship between the structure and the dual functions of peroxidase and chaperone of human peroxiredoxin II using size chromatography; FIG. 12 shows the influence of the amino terminal Cys51, responsible for the peroxidase function, and the carboxy tail on the structure and function of human peroxiredoxin π; FIG. 13 shows the reversible formation of the oxidative stress-dependent high molecular weight complex of human peroxiredoxin II in HeLa cells; FIG. 14 shows the hydrogen peroxide-dependent structural change of human peroxiredoxin II and 2-cysteine peroxiredoxin (cPrxi) in yeast; FIG. 15 shows the chaperone function of human peroxiredoxin II to protect HeLa cells upon hydrogen peroxide-induced apoptosis; and FIG. 16 shows a diagnostic kit according to an embodiment of the present invention in an exploded view (A) and a strip (B).

[Best Mode] Below, a detailed description will be given of the present invention. Technical and scientific terminology, as used herein, has the same meanings as understood by those skilled in the art, if not specifically defined otherwise. The 2-cysteine peroxiredoxin complexes useful in the present invention include complexes resulting from the association of heterogeneous peroxiredoxins derived from microorganisms, higher animals and higher plants, as well as homogeneous peroxiredoxins. Because 2-cysteine peroxiredoxins derived from microorganisms, higher animals and higher plants are similar in structure and function, most 2-cysteine peroxiredoxins may be used in the present invention. Referring to the accompanying sequence list, 2-cysteine peroxiredoxins used in the present invention are represented by SEQ. ID. NOS. 2, 4, 6 and 8, isomers derived from humans, by SEQ. ID. NO. 10, derived from Arabidos thaliana, and by SEQ. ID. NOS. 11 and 12, derived from Saccharomyces cerevisiae. As is generally recognized in the art, a chaperone is defined as any of a class of proteins that helps proteins fold or escorts proteins or other molecules throughout the cell. For example, proteins, when under stress, such as heat shock, have unfolded tertiary stmctures so that they cannot perform their functions. Chaperones recognize and bind to such unfolded proteins and provide an environment that allows them to refold. Based on the finding that, of peroxiredoxin proteins present in the cytoplasm of animals, plants and microorganisms, 2-cysteine peroxiredoxin (2-Cys Prx), having two cysteine groups, functions as a chaperone component as well as a peroxidase, the present invention provides applications for the chaperone functions possessed by 2-cysteine peroxiredoxin. In addition, observing that chaperone functions predominate in high molecular complexes of peroxiredoxin proteins, the present inventors speculate that senile diseases, various cancers, and exposure to oxidative stress and heat induce 2-cysteine peroxiredoxin (2- Cys Prx) proteins to undergo a conformational change from low molecular weight stmctures to high molecular weight complexes and that the structural change leads to a functional switching from a peroxidase function, which is dominant in isolated peroxiredoxin, to a chaperone function. It is also believed that the chaperone function of 2-Cys Prxs improves the resistance to various external environmental stresses, heat shocks, oxidative stresses, diseases, etc. because the functional change is mostly attributed to changes in the cysteine located at the active site of peroxidase, for example, residue 47 (Cys47) in yeast and residue 51 (Cys51) in humans, which acts as an effective recognizer of hydrogen peroxide in cells. Properties of this mechanism were identified in experiments, which examined whether various oxidative stresses induce low molecular weight 2-Cys Prxs to change into high molecular weight protein structures, and whether this conformational change results in a functional change from peroxidase to a novel chaperone function. With these structural and functional changes, plants, animals and microorganisms were found to show high resistance to external stress. Thus, it is expected that plants or miαoorganisms, if transfonned with a gene coding for the protein of interest and controlled in the expression of the gene, will have resistance to various environmental stresses, and that the pursuit of the chaperone function will allow the application of the protein in the diagnosis and treatment of various human diseases, such as cancers, Alzheimer's disease, Parkinson's disease, etc. This is highly plausible, in light of the fact that the dysfunction or functional disability of 2-Cys Prx due to mutations or deletions is reported to cause degeneration in cranial nerves (Krapfenbauer et al, Electrophoresis.23: 2541-2547, 2002), Alzheimer's disease (Kim et al, J Neural Transm Suppl. 61: 223-235, 2001), Down's syndrome (Krapfenbauer et al. Brain Res. 967: 152-160, 2003), thyroid cancer (Yanagawa et al,Cancer Lett, 145;127-132, 1999), heart cancer, lung cancer (Kim et al. Cell Biol Toxicol. 19: 285-298, 2003.), tumors (Neumann et al, Nature, 424: 561-565, 2003), etc. In order to apply 2-Cys Prxs to diagnostic fields, including diagnostic medicines, diagnostic technology and diagnostic kits, and to pharmaceutical compositions, advantage is taken of the chaperone functions thereof. Preferably, the diagnostic method of the present invention uses monoclonal antibodies that are specific for peroxiredoxin complexes having chaperone activity which result from the association of proteins selected from a family group of 2-Cys Prxs consisting of peptide sequences of SEQ. ID. NOS. 2, 4, 6, 8, 10, 11 and 12. More preferable are monoclonal antibodies coπesponding to the human 2-cysteine peroxiredoxin proteins of SEQ. ID. NOS.2, 4, 6 and 8. Proteins obtained by mpturing or lysing cells from tissues or humors of animals, plants and microorganisms may be used as test samples. For tl e immunoassay with the monoclonal antibodies, sample proteins, which may be prepared by separation from cell lysates on native- PAGE, are transferred onto predetermined positions of nitrocellulose membranes and reacted with the monoclonal bodies, which are then visualized for position and amount by dyeing with enzymes or dyes, so as to determine the amount of peroxiredoxin complexes in test samples. In accordance with one embodiment of the present invention, a diagnostic composition that is capable of quantitatively analyzing high molecular weight complexes of 2-Cys Prx isomers in the subject to determine disease occurrence comprises the following components: (a) an immobilized sample obtained by processing a subject matter; (b) an antibody recognizing a high molecular weight complex of 2-Cys Prxs as an antigen; (c) a coloring enzyme-labeled complement binding specifically to the antibody; (d) a substrate solution undergoing a color change upon reaction with the coloring enzyme; and (e) a buffer for the reaction for the color change. The term "subject matter" as used herein means whole blood, sera, plasmas, lymph fluids, intercellular fluids, or tissues such as organs. The term "test sample", as used herein, means aprotein separated/extracted from the subject matter. The complement used in the present invention is an enzyme-labeled secondary antibody against Hie antibody specific for the high molecular weight complex of 2-Cyst Prx isomers. The coloring enzyme linked to the secondary antibody may be peroxidase or aUcaline phosphatase, with the requirement for corresponding substrates, e.g, TMB or

NBT/BCIP, respectively. However, the present invention is not limited to this method, but includes any method within the scope thereof if it can use an antibody against the high molecular weight complex of 2-Cys Prx isomers to detect the diseases. Any may be used for the antibody, the complement and the coloring enzyme, instead of those mentioned above, if they show high binding specificity and reaction affinity and are so susceptible as to react with each other even at very low concentrations. In accordance with another embodiment of the present invention, a diagnostic composition comprises a Western blotting set adapted to separate test samples as proteins using native-PAGE, blot the test samples onto predetermined positions of a nitrocellulose membrane, treat the membrane with an antibody and visualize the position and concentration of the antibody bound onto the membrane with an enzyme or dye; an antibody binding specifically to a high molecular weight complex of 2-Cys Prxs and a coloring enzyme linked to the antibody; and a substrate solution for the enzyme (Jang et al. Cell, 117; 625-635, 2004). For quick and mass examination, an ELISA method using 96- or 386-well plates may be effective (D' Ercole et al, J. Immunoassay Immunochem, 26: 43-56, 2005). fa this case, 96 or 386 test samples are added into respective wells of ELISA plates made from polystyrene so as to readily bind proteins thereto, and sufficiently coated on the wells, followed by washing the wells to remove non-bound materials. Into each of the wells that contain a test sample, an antibody binding specifically to a high molecular weight peroxiredoxin complex is added, and an immunoblotting method is conducted for quick diagnosis. In this regard, the antibody binding specifically to the high molecular weight peroxiredoxin complex is reacted with a secondary antibody against this antibody and the color development of the second antibody is analyzed. Preferably, peroxidase or alkaline phosphatase is conjugated with the secondary antibody as a coloring enzyme while TMB or NBT/BCIP is used as a corresponding substrate, respectively. In accordance with a further embodiment of the present invention, a diagnostic kit, for use in the diagnosis of diseases comprises a test sample; a gel, on which positive and negative control samples are separated by native-PAGE; a comb card, to which are attached a number of nitrocellulose membranes corresponding to tl e number of lanes of gel; an electric device, for supplying electricity to transfer Hie proteins on the gel onto the nitrocellulose membranes; a monoclonal antibody on the nitrocellulose membranes, defined in claim 11 or 12; and an incubation tray, for allowing the monoclonal antibody to recognize the proteins transferred onto the nitrocellulose membranes. For the diagnosis, a Western blotting method is applied. Using a typical ELISA, a coloring enzyme may be used to detect the antibody bound to the transferred proteins. In FIG. 16, the diagnostic kit in accordance with the present invention is illustrated. As seen in FIG. 16, the diagnostic kit is comprised of a strip (1) and a housing (2). The strip (1) comprises a developing membrane 13 on which a reaction unit (11) and a control unit (12) for monitoring the normal operation of the kit are positioned at predetermined positions. On the reaction unit (11), an antibody prepared according to the present invention is immobilized. In addition to accommodating the strip (1), the housing (2) comprises a sample feeder (21) and indication windows (22) through which reaction results in the reaction unit (11) and the control unit (12) are visualized with the naked eye. In the kit, the absorbance is detected and analyzed by an ELISA method so as to determine whether the subject is afflicted with the diseases. In accordance with another aspect of the present invention, a pharmaceutical composition for the prophylaxis and treatment of neurodegeneration, Alzheimer's disease, Down's syndrome, Parkinson's disease, thyroid cancer, heart cancer, breast cancer, lung cancer and tumors is provided. The pharmaceutical composition of the present invention comprises a high molecular weight complex of the above-mentioned human 2-cysteine peroxiredoxin isomers or its salt as an effective ingredient, in combination with a pharmaceutically acceptable carrier. As described, high molecular complexes of human 2-cysteine peroxiredoxin isomers according to the present invention themselves can be used as they are or in a pharmaceutically acceptable acid addition salt or metal complex form. Examples of the pharmaceutically acceptable acid addition salt include hydrochloride salts, hydrobromide salts, sulfuric acid salts, phosphoric acid salts, maleic acid salts, acetic acid salts, citric acid salts, benzoic acid salts, succinic acid salts, maleinic acid salts, ascorbic acid salts, and tartaric acid salts. The metal complex useful in the present invention may contain zinc or iron. Depending on administration routes, administration forms and treatment purposes, the pharmaceutical composition of the present invention may be prepared in various dosage forms. For example, a diluent for use as the carrier may be selected from among, but is not limited to, saline, buffers, dextrose, water, glycerol, ringer's solution, lactose, sucrose, calcium silicate, methyl cellulose, ethanol, and combinations thereof. Using these diluents, the pharmaceutical composition of the present invention can be prepared in dosage forms for oral or parenteral administration, such as powder, granules, injection solutions, syrups, tablets, suppositories, pessaries, ointments, creams, aerosols, and etc. Parenteral administration means the administration of the dosage forms according to the present invention through rectal, intravenous, intraperitoneal, intramuscular, intra-arterial, subcutaneous, or intranasal routes, or tlie like. In addition, the pharmaceutical composition may further comprise an additive, such as a filler, an anticoagulant, a lubricant, a wetting agent, a flavor, an emulsifier, a preservative, etc, and may be formulated to show quick, sustained or delayed release. In another embodiment of the present invention, a pharmaceutical composition for injection comprises a transformant (transformed cell strain), obtained by introducing a 2- cysteine peroxiredoxin isomer-expressing recombinant vector into a human cell strain, capable of overexpressing 2-cysteine peroxiredoxin; and a pharmaceutically acceptable carrier. Depending on degrees of disease severity, administration routes and dosage forms, the dosage of the pharmaceutical composition of the present invention can vary within the range in which no desensitization happens. In consideration of the fact that peroxiredoxin is the second or Hie third in blood level among the proteins found in blood (Moore et al, J. Biol. Chem.266: 18964-18968, 1991)andisl nownto amountto asmuchas 0.1-0.8%ofthewater- soluble proteins in animal cells (Chae et al. Diabetes Res. Clin. Pract.45: 101-112, 1999), the effective ingredient, that is, 2-cysteine peroxiredoxin or its salts, are preferably administered in a dose of 10 ~ 2,000 mg per kg of body weight a day, in a sporadic or continuous manner.

[Mode for Invention] A better understanding of the present invention may be obtained through the following examples which are set forth to illustrate, but are not to be construed as the limit of the present invention.

EXAMPLE 1 : Expression of the Native and Peroxidase-Inactive Forms of cPrxI Ameliorates the HypersensitivityoffheΔcPrxI/II Yeast to Heat Shock.

A ΔcPrxI H yeast strain lacking both of the most abundant cytosolic yeast Prxs, namely, cPrxI and A, was prepared (Kang et al, J. Biol. Chem, In press 2003). The introduction of the native cPrxI or Cys-less cPrxI (C47/170S-cPrxι) into the prepared mutant yeast strain to construct isogenic mutant strains that express the native or Cys- less cPrxI gene under the control of its own promoter on the pRS416 plasmid. Referring to FIG. 1A, the activity of thioredoxin-dependent peroxidase is compared between the native cPrxI and the Cys-less cPrxI which were extracted from the mutant strains harboring the native cPrxI gene and the Cys-less cPrx gene, respectively and then reacted with thioredoxin. As seen in the figure, almost no peroxidase acricity was found in tlie Cys-less cPrxI.

Equal densities of yeasts (10 μJl) grown to the exponential phase were serially diluted by lO^lO⁵ fold, spotted onto YPD plates, followed by heat shock at 43°C for 30 min, and their colony-forming ability was analyzed after 4 days incubation. Significantly, the expression of C47/170S-cPrxI in the ΔcPrxI/II yeast, which lacks peroxidase activity, consistently conferred ΔcPrxI/II cells with heat shock resistance, although the level of resistance was still lower than that observed for the wt cell (FIG. IB). In FIG. IB, a wild type yeast (1), a ΔcPrxI/II yeast strain (2), a ΔcPrxI/U yeast strain that expresses the native cPrxI under the control of its own promoter (3), and a ΔcPrxI/II yeast strain that expresses C47/170S-cPrxI under the control of its own promoter (4) were usedlhis suggests that the ability of cPrxI to protect the ΔcPrxI/11 cell from heat shock is not exclusively due to its peroxidase activity. Instead, part of the resistance appears to be due to another as yet unidentified function of tlie protein. These data support the notion that cPrxI has another function that is unrelated to its antioxidant activity and that is involved in other cellular processes.

EXAMPLE 2: cPrxI has dual functions as it acts both as a peroxidase and as a molecular chaperone

FIG. 2 shows the amino acid sequence and secondary structure of cPrxI having the dual function of a peroxidase and a molecular chaperone. The amino acid sequence of cPrxI was compared to that of its homologues using the Clustal method. The secondary structure was predicted using the GOR IV prediction tool on the NPS@ server. These stmctures are denoted by an arrow for a β-sheet, a cylinder for an α-helix, and a line for a random coil. Two conserved cysteines that are essential for peroxidase function are boxed in black. Stars(*) and dots(») indicate the positions of perfectly- and well-conserved residues, respectively (FIG.2A). Amino acid numbers are shown to tl e right. Genbank numbers of the compared clones are cPrxI(NP_013684), cPrxH (NP_010741), HsPrxI(NP_002565), HsPrxIJ(NP_00580), and tbasl(NP_l 87769). Sequence- and structure-based multiple alignments of the 2-Cys Prxs indicate that they contain highly conserved Cys residues, similar to the Ecoli chaperone ΗSP33, as well as a Trx motif, which has been shown to have chaperone activity (Jakob et al. Cell, 96: 341-352, 1999; Kem et al, Biochem. J.371 :965-972, 2003) (FIG.2A). To identify the novel function of 2-Cys Prxs that confers the ΔcPrxI/π yeast with resistance to heat shock (FIG. 1), the chaperone activity of the five yeast Prxs was investigated by assessing their ability to inhibit the thermal aggregation of the model substrate CS. lμM CS(*-») in 50mM Hepes, pH 7.0, was incubated in a spectre-photometer cell thermostatted at 43 ^°C with 2μM BSA( A- A), cPrxI(α-π), CPTXIIOM), or cPrxDI, mPrx, and nPrx(Δ-Δ). Then, cPrxI and II efficiently suppressed the thermal aggregation ofCS at 43 ^°C, whereas no inhibition of CS aggregation was observed when the other yeast Prx isotypes or BS A were added (FIG.2B). Incubation of CS with increasing amounts of cPrxI resulted in a concomitant decrease in CS aggregation, and at a subunit molar ratio of cPrxI to CS of 2:1, CS aggregation could be completely suppressed (FIG. 2C), indicating that cPrxI functions as a protein molecular chaperone. Effects of cPrxI concentrations on the thermal aggregation of

CS at 43 ^"C (FIG. 2C) or on the lOmM DTT-induced chemical aggregation of insulin at 25 ^°C (FIG. 2D) in the absence(*-«) of or in the presence of cPrxI at molar ratios of cPrxI to substrates of 0.25:1 (Δ-Δ), 0.5:1 (A- A), 1:1 (D-D), and 2:1(M) were examined. As in FIG. 2C, the aggregation of the substrates was monitored by measuring the apparent light scattering at A₃₆₀. Besides CS, cPrxI can efficiently protect the insulin β-chain from DTT-induced precipitation (FIG.2D). Under the assay conditions of the present invention, the chaperone activities of the well-known chaperone proteins, such as HSP16.5 of Methanococcus jannaschi(Kim et al. Nature, 394: 595-599, 1998) and α-crystallin, showed about 15- and 3-folds weaker activity than that of cPrxI, respectively. Moreover, it was reported that an 8-fold molar excess of goat tubulin and 14-15 moles of HSP16 in C. elegans are required to protect one mole of CS (Manna et al, J. Biol. Chem, 276: 39742-39747, 2001). The interaction of non-native substrates to cPrxI was confirmed using the fluorescent compound bis-ANS that binds to hydrophobic regions of amino acyl residues (Sharma et al, J. Biol. Chem, 273 : 8965-8970, 1998). Its emission maximum was shifted from about 520mm to a shorter wavelength, ~470nm(FIG.2E). The fluorescence intensity was significantly increased by heat treatment, which indicates that the more hydrophobic patches of cPrxI are exposed by the treatment. From these results, it can be concluded that cPrxI and II function both as a peroxidase and as an efficient molecular chaperone. Indeed, these proteins can be classified into a novel group of the chaperone family because their amino acid sequences do not share much similarity with other chaperone sequences.

EXAMPLE 3 : cPrxI forms a variety of differently-sized HMW protein structures

A well-conserved feature of molecular chaperones is their tendency to associate into dimers, trimers, and high oligomers in a reversible fashion (Hendrick et al, Annu. Rev. Biochem, 62: 349-384, 1993). In particular, many sHSPs are known to form HMW complexes in vivo, which is a prerequisite for their chaperone activity (Haley et al, J. Mol. Biol, 277: 27-35, 1998). Since several Prxs, including the human NKEF, AhpC, calpromotin, HBP23, and Prx-B form HMW complexes with masses of 230-500 kDa (Schroder et al. Structure, 8: 605-615, 2000; Hirotsu et al, Proc. Natl. Acad. Sci, 96: 12333-12338, 1999), it was determined that the molecular masses of purified yeast Prxs by SEC. SEC was performed using a TSK G4000SWX_L column (Tosoh, Japan). The separated proteins were divided and pooled into three fractions (F-I, F-IJ, and F-IU) for further analysis through native-PAGE and electron microscopy. Each fraction in SEC was subjected to Western blotting by using a polyclonal anti- cPrxI antibody after separating the protein on 10% native-PAGE (upper panel) and 12% SDS- PAGE(lover panel). Western blotting was performed using a polyclonal anti-cPrxI antibody. It was found that cPrxI and II produced multiple forms of differently-sized HMW protein complexes (FIG. 3 A), whereas cPrxiπ, mPrx, and nPrx appeared at their monomer or dimer positions in SEC (data not shown). The molecular masses of the cPrxI complexes separated by SEC ranged from about 40 kDa to more than 1000 kDa The molecular size- range of the proteins in each fraction from SEC was analyzed again by Western blotting on a native-PAGE. Multiple forms of differently sized protein complexes were detected by the cPrxI-specific antibody in each protein fraction (FIG. 3B, upper image). The molecular sizes of the proteins in the first SEC fraction, which contained the largest cPrxI complexes, were too high to penetrate the pores of a 10% native-polyacrylamide gel, and thus were retained at the top of the separating gel. In contrast, the last fraction contained proteins with molecular masses ranging from about 40 to 120 kDa Unlike the result of native-PAGE analysis, SDS-PAGE of all the fractions revealed a single band with a MW of 23 kDa, which is the monomeric size of the cPrxI protein (FIG.3B, lower image). These observations suggest that in its native condition, cPrxI exists as a homopolymeric complex made up of various numbers of cPrxI monomer. To analyze the oligomeric structures of cPrxI, it was examined that the proteins that had been separated by SEC by electron microscopy (EM). EM of the negatively stained protein fractions revealed three different configurations, namely, spherically shaped and ring-shaped stmctures and irregularly shaped small particles (FIG. 3C). The spherically shaped particles observed in the F-I fraction were subjected to rotational and translational alignment and could be classified into three groups based on eigenvector-eigenvalue data analysis (FIG.3D, F-I). Remarkably, the projected images of the class averages revealed spherical particles with diameters ranging from 22 to 28 nm, possibly reflecting the number of cPrxI molecules in each particle. The oligomeric stmctures of the F- II proteins were also determined by electron microscopy and image processing (FIG. 3D, F-II). Depending on the orientation of the grid, EM of the F-II fraction showed two basic views, namely, ring-shaped stmctures with an end-on orientation (F-IJ, 1-4) and double-dot stmctures with a side-on orientation (F-II, 5-8). A total of 409 end-on views of well-stained particles were translationally and rotationally aligned and subjected to multivariate statistical analysis (van Heel el at, Ultramicroscopy, 6: 187-194, 1981). For purposes of classification, the four most significant eigenvectors were selected (I)uιr,Ultramicroscopy, 38: 135-141, 1991). By using this approach, three classes according to feature similarity were defined. Unsymmetrized class averages of end-on views revealed an unequivocal 5-fold symmetry. The averaged image of end-on views of F-IJ complexes showed five centers of mass arranged as a ring with heavy stain accumulation in the center of the ring (F-IJ, 4). The diameters of the ring and the central hole were approximately 14 and 5 nm, respectively. The averaged image of 170 side-on views of F-IJ complexes is shown in FIG. 3D (F-II, 8). This average revealed a double-dot structure with an equal distribution of mass across the horizontal plane of the complex. Unlike the protein stmctures of F-I and F-II, the proteins in the last fraction (FIG. 3C, F-LT) did not form a regular structure. The SEC and native-PAGE analyses of the molecular sizes of F-JJJ suggest that they consist of dimers or tetramers of cPrxI.

EXAMPLE 4: Heat Shock and Oxidative Stress Cause cPrxI to Undergo Stmctural Changes Accompanied by a Functional Switch from a Peroxidase to a Molecular Chaperone In Vitro

As cPrxI forms HMW complexes ranging in size from 40 to 1000 kDa and functions both as a peroxidase and as a molecular chaperone, the specific activities of the two functions in the three protein fractions separated by SEC (FIG. 3 A) was explored. To test whether the protein stmctures of each fraction were stable, the protein fractions were subjected to rechromatography under the same conditions described in FIG. 3A. It was found that the proteins were eluted with nearly the same retention times as in the first SEC, which suggests that the protein stmctures are sufficiently stable so as not to be changed during experimentation in vitro (FIG. 4A, a). This was also confirmed by Western blotting after native- and SDS- PAGE (data not shown). When the chaperone and peroxidase activities of the F-I to F-IU fractions were investigated, remarkable results were obtained. The highest MW protein complexes (F-I) exhibited only chaperone activity and the lowest MW fraction (F-Ifl) displayed only peroxidase activity, whereas the middle-sized proteins (F-II) displayed both peroxidase and chaperone activities, as did total protein (Tot) (FIG. 4A, b). These results suggest that multimerization of the protein complexes promotes chaperone activity and that dissociation of the complexes into low MW (LMW) species enhances peroxidase activity. This notion was confirmed by analysis of the bis-ANS binding to each protein fraction which showed that more bis-ANS bound as the molecular weight of the cPrxI complex increased (FIG.4A,b, inset). Thus, ti e dual functions of cPrxI are associated with its ability to form distinct protein stmctures. sHSPs exhibit chaperone activity by forming HMW complexes during heat shock (Haley et al, J. Mol. Biol, 277: 27-35, 1998; Hendrick et al, Annu. Rev. Biochem, 62: 349- 384, 1993). It was found that, like sHSPs, the protein structure and functions of native-cPrxI were significantly changed when they were incubated for 30 min at various temperatures (FIG. 4B, a). As the incubation temperature increased, the HMW protein peak also increased significantly. This was matched by a concomitant decrease in the levels of the LMW protein fraction. This suggests that the lower MW proteins are assembled into higher MW complexes during heat stress. C47/170S-cPrxI also formed HMW complexes during heat shock (data not shown). The chaperone and peroxidase activity of the protein that underwent heat shock was comparable to that of 2-Cys Prxl measured at 25°C. In addition to this structural change, the chaperone activity of cPrxI was also greatly increased following heat treatment (FIG. 4B, b). A temperature shift from 55°C to 65°C caused about a 3-fold increase in cPrxI chaperone activity, while at 75°C the activity was remarkably increased about 30-fold. In contrast, the peroxidase activity of heat-treated cPrxI gradually decreased with the increase in temperature and almost no peroxidase activity was detected in the sample incubated at 75°C for 30 min. Significant stiuctural and functional changes of cPrxI were also obtained when cPrxI was treated with H2O2 in the presence of a Trx system containing Trx, TR, and NADPH in vitro (FIG. 4C). When more than 2 mM H2O2 was present, the chaperone activity of cPrxI was increased about 10-fold, whereas its peroxidase activity was greatly decreased (FIG.4C, a). However, in contrast to the temperature-dependent control, the H2θ2-mediated switching process occurred only when native cPrxI protein was used with the Trx system, whereas C47S- or C47/170S-cPrxI did not show this H2O2 effect when investigated under the same assay conditions (data not shown). This suggests that the Cys47 residue of cPrxI and the Trx system are indispensable for the FfcC -dependent regulation of its chaperone activity. The result was confirmed again by analyzing FLCfc-dependent structural changes by

Western blotting on a native PAGE gel (FIG.4C, b). From these results, it can be concluded that the heat shock-dependent in vitro regulation of cPrxI activity that occurs under nonphysiological conditions must involve a mechanism that differs at least partly from the mechanism that regulates cPrxI activity during oxidative stress. Since molecular chaperones have been reported to associate physically with denatured substrates to inhibit nonproductive aggregation (Hendrick et al, Anna Rev. Biochem, 62: 349-384, 1993), it was investigated whether cPrxI binds to partially denatured proteins. Although cPrxI displayed partial hydrophobic domain exposition at 25°C (FIG. 2E), CS did not interact with cPrxI at all at 25°C, probably because CS was still in its native conformation at this temperature (FIG.4D, a and b). However, when cPrxI was incubated with CS at 43 °C for 30 min and then subjected to SEC, there was a significant increase in the levels of CS that coeluted with the highest MW form of cPrxI. This was associated with a concomitant decrease in the amount of free CS. The physical association of cPrxI with normative CS was verified by SDS-PAGE, which showed that cPrxI and CS subunits were present in the highest

MW fraction resulting from SEC (FIG. 4D, c and d). In contrast, CS that had been preincubated at 43°C for 30 min in the absence of cPrxI was nearly completely precipitated, thereby showing no protein peak during SEC (data not shown). The dual functions of cPrxI led to the enforcement of investigating whether the conserved Cys residues, which are essential for its peroxidase activity, are also required for its chaperone function. The in vitro chaperone activity of the C47/170S-cPrxI mutant that lacked peroxidase activity (FIG. 1 A) was about 90% that of native cPrxI (FIG. 4E), which suggests that the Cys residues of cPrxI are not critical for in vitro chaperone activity at 43°C.

EXAMPLE 5: Exposure of Yeasts to Oxidative Stress or Heat Shock Induces Reversible Changes in cPrxI Structure In Vivo Caused by Oxidation of the Catalytic Cys-thiol into Cys- SulfinicAcid

Based on a report that showed the peroxidase activity of Prxs was rapidly inactivated by bursts of intraceUular peroxide production (Yang et al, J. Biol. Chem, 277: 38029-38036, 2002), the sttuctural changes of cPrxI that occurred in vivo in wt and mutant yeasts exposed to oxidative stress and heat shock were examined (FIG.5). Yeast cells were grown to the exponential phase and divided into three equal portions. At time zero, each aliquot was challenged with different patterns of H2Q2 as depicted. The white and grey boxes indicate the duration of cell culture in YPD medium in the presence (■) or in the absence (D) of 0.5mM H2O2 (FIG. 5A). At the end of culture (30min) in Scheme FIG. 5A, crude proteins extracted from WT, Δtrxl/2, Δsrxl cell, or ΔcPrxI/U cell expressing _cPrχ]c⁴⁷s _{or cPrxI}c^i7θs _{weiβ separated on m}tive-PAGE (FIG. 5B and D), 2D-gels(FIG. 5C) and subjected to immunoblotting with an anti-cPrxI antibody. The isoelectric points of the oxidized(Ox) and reduced(Re) cPrxI in panel C are 4.7 and 4.9, respectively. The immunoblot of cPrxI extracted from WT yeast was treated with hyperaerobic stress (95% 0^5% CO₂) instead of H₂O₂ (FIG. 5E). The challenge and recovery times of the stress were changed to lh each EM stmctures of cPrxI proteins purified from exponentially grown WT yeast were pretreated in the absence (a) or presence (b) of ImM H^for 10 min (FIG. 5F). The in vitro effect of heat shock and/or oxidative stress applied for 10 min on the HMW complex formation of cPrxI in WT yeast was analyzed by immunoblotting on native-PAGE (FIG. 5G). After exposing various conditions of heat shock and/or oxidative stresses, the ROS generated from the cells was analyzed by reacting with 1 mM 2,7-DCFH-DA under a confocal fluorescence microscope(FIG. 5H). The relative intensity (Rel. int.) was calculated for 200 cells from each group. The light and DCF fluorescence fields are in the upper and lower panel, respectively. After culturing the yeasts with or without 0.5 mM H2O2 as shown by the scheme depicted in FIG. 5A, crude extracts prepared from the cells were subjected to Western blot analysis on a native-PAGE gel (FIG. 5B). The cPrxI proteins obtained from H_θ2-untreated wt or native cPrxI-expressing ΔcPrxI/JJ yeast cells consisted of multiple forms of low and oligomeric stmctures along with a small amount of HMW complexes (wt-a). When these cells were challenged with 0.5 mM H2O2 for 10 min, most of the LMW proteins were converted into HMW complexes, although some of the middle-sized oligomeric proteins did not respond to the treatment (wt-b). However, the HMW complexes returned to their original stmctures within 20 min after removal of H e H2O2 (wt-c). Although stmctural change of the middle-sized proteins was not detected even at higher concentrations of H2O2, it was found that the H_O2-mediated HMW complex formation of cPrxI is dependent on the concentration of H2O2 (data not shown). In particular, no ILCfe-induced stmctural change of cPrxI was found in a Trx-deficient yeast (FIG. 5B, Δtrxl/2), which suggests that Trx is essential for the HMW complex formation of cPrxI in vivo. A previous report indicated that the active Cys-thiol of cPrxI is hyperoxidized to Cys- sulfinic acid during H2O2 exposure and that the hyperoxidized protein is reduced by srxl

(Biteau et al. Nature, 425: 980-984, 2003). On the basis of these observations, the oxidation status of the cPrxI after H2O2 treatment was investigated and the role of srxl in its H2O2- dependent stmctural changes was assessed. It was found that although cPrxI in Δsrxl cells was oligomerized into HMW complexes by H2O2, the protein did not revert to its original stmctures even after the removal of H2O2 (FIG. 5B, Δsrxl). This suggests that srxl plays a critical role in the recovery of cPrxI stmctures. Upon the analysis of the oxidation status of cPrxI by immunoblotting of 2D-gels, it was found that most of the cPrxI existed in a reduced form in wt yeast but that H2O2 treatment significantly increased the amounts of oxidized cPrxI (FIG. 5C, wt). When H2O2 was removed, the proportion of the reduced enzyme increased rapidly along with a concomitant decrease in the oxidized enzyme levels. However, the removal of H2O2 was not sufficient to reduce the oxidized form of cPrxI in Δsrxl cells (FIG. 5C;mutant lacking a sulfyredoxin gene, Δsrxl). These results strongly suggest that the redox- dependent association of cPrxI with Trx and srxl plays an important role in the switching of cPrxI protein structures and functions. fn contrast to native cPrxI, Cys47-laclάng cPrxI isolated from H Oa-untreated ΔcPrxI U cells expressing C47S- or C47/170S-cPrxI consisted of low and small oligomeric proteins that did not respond to H2O2 stress at all (FIG. 5D, cPrxIC47S). This suggests that the peroxidase active site, Cys47, is essential for H-Ck-mediated HMW complex formation of cPrxI in vivo. However, the cPrxI molecules extracted from isogenic cells expressing C170S-cPrxI consisted of LMW and oligomeric proteins along with large amounts of HMW complexes that formed even before H2O2 treatment (FIG.5D, cPrxIC170S). Given the data showing the catalytic Cys oxidation of cPrxI in FIG. 5C, it is likely that in the absence of Cysl70, the Cys47 residue of C170S-cPrxI becomes so sensitive to oxidative stress that it may be hyperoxidized, resulting in the formation of HMW complexes even in normally growing yeast cells in vivo. These results together with the observed normal in vitro chaperone activity of C47/170S-cPrxI (FIG. 4E) suggest that LMW forms of cPrxI are multimerized by two different pathways. One is H2θ2-sensitive and the other is H2O2- insensitive. In the former reaction, the active peroxidase site Cys47 is critically involved in HMW complex formation, whereas the ϋCwndependent process does not require Cys47 to form oligomeric proteins from LMW species. HMW complexes of cPrxI were also formed by different oxygen tensions. Yeast cells grown aerobically contained low and oligomeric forms of cPrxI as major stmctures but a significant portion of the protein was oligomerized into HMW complexes when the cells were cultured in hyperaerobic atmospheres (95% 025% CO2) (FIG. 5E). As with H2O2, the HMW complexes of cPrxI reverted to their original stmctures after the oxygen tension was reduced. The H2θ2-dependent formation of HMW cPrxI complexes in vivo was confirmed by EM analysis of the proteins purified from exponentially grown wt yeast that had been exposed to 1 mM H2O2 (FIG.5F). Since a large body of evidence supports the notion that heat shock is a form of oxidative stress (Davidson et al, Proc. Natl. Acad. Sci. USA, 93: 5116-5121, 1996), the effect of temperature on the stmctural changes of cPrxI in vivo was analyzed. Whereas yeast cells that were incubated at 43°C for 10 min caused no stmctural changes in cPrxI, a significant stmctural transition from LMW to HMW complexes was induced by heat shock exposure of the cells at 46°C for 10 min (FIG. 5G). These results suggest that, like the effect of oxidative stress, the stmctural and functional regulation of cPrxI is sensitively tuned in vivo to the degree of heat shock imposed on the cells. In particular, it was also found that the addition of low amounts of H2O2 (50 M) to the cells exposed at 43°C caused cPrxI to shift remarkably from an LMW complex to an HMW complex within 10 min. To investigate the relationship between heat shock and oxidative stress in vivo, the intraceUular levels of ROS generated by heat shock were measured using DCF staining and confocal fluorescence microscopy (FIG. 5H). It was found that the stmctural shift in cPrxI is closely related to the amount of ROS generated by the heat shock treatment. This strongly indicates that tl e protein stmctures and functions of cPrxI are mainly regulated by the levels of ceUular ROS.

EXAMPLE 6: Expression of cPrxI Endows Yeast CeUs with Heat Shock Resistance

It is important to investigate the physiological significance of the heat shock- or oxidative stress-mediated stmctural and functional switcliing of cPrxI from a peroxidase to a molecular chaperone. Moreover, the fact that the H2θ2-dependent regulation of cPrxI is already activated by the presence of less than 0.5 mM H2O2, which does not affect ceU viability or damage ceUular components, is intriguing. In this example, attention is paid to the chaperone activity of 2-Cys Prxl because its peroxidase function is already identified against defense systems of various cells (Wong et al, J. Biol. Chem, 277:5385-5394, 2002). Heat shock resistance of wt yeast (1), ΔcPrxJΛI ceUs transformed with the vector pRS416 (2), or isogenic mutant strains expressing native (3), C47S- (4), C170S- (5), or C47/170S-cPrxI (6) under its own promoter was analyzed. The yeasts grown in YPD medium to 5 x IO⁷ ceUs/ml were preincubated at 30°C for 10 min in the absence (FIG.6A) or presence (FIG. 6B) of 0.2 mM H₂O₂. After the treatments, an aHquot from each strain was placed in a test tube and incubated at 43 °C. The ceU viabiUty of the samples taken at the times indicated was then measured. CeU survival is expressed as the percentage of the number of viable ceUs incubated at 43°C to the number of ceUs before heat shock exposure. The inset in (FIG. 6A) shows Western blotting with a cPrxI-specific antibody of the total extracts obtained from the yeast ceUs (FIG. A and B 1-6). The prevention of heat shock-induced protein aggregation in the yeast cultures which had pretreated with 0.2 mM hydrogen peroxide for 10 min was examined (FIG. 6C). A modification of the Tomoyasu et al. (2001) method was appUed to this examination. At time zero and 20 min at 43°C, culture aUquots from each yeast strain were withdrawn and analyzed for the amount of insoluble protein by ceU lysis and centrifugation. The insoluble fractions were subjected to SDS-PAGE foUowed by sUver staining. The ΔcPrxIJJ yeasts transformed with the Cys-mutant cPrxI constructs expressed corresponding proteins at levels that were simUar to that of cPrxI expressed by wt yeast (FIG. 6A, inset). The capacity of cPrxI to prevent heat shock-induced loss of ceU viability and protein aggregation was investigated using yeasts grown to the middle of the exponential phase.

To measure heat tolerance, equal numbers of yeast ceUs were preincubated in a medium with or without 0.2 mM H2O2 for 10 min and the ceUs were then transferred to 43°C incubation. After harvesting at different time points, the ceUs were plated on YPD plates to determine colony-forming abitity. Upon exposure of the ceUs to 43 °C for 30 min in the absence of H2O2 (FIG. 6A), survival rates of the ΔcPrxI/II ceUs and the isogenic lines expressing C47S- or

C47/170S-cPrxI were greatly reduced, to 12% and 48%, respectively, of ceU numbers counted before the heat shock treatment at 43°C. In contrast, wt yeast and the ΔcPrxI/TJ strain that expressed native or C170S-cPrxI showed about 62%-73% survival. These observations suggest that the chaperone function of cPrxI remarkably enhances yeast resistance to heat shock. In addition, it was found that the preincubation of the ceUs with 0.2 mM H2O2 for 10 min significantly improves the heat shock resistance of the wt or ΔcPrxl/II ceUs that express native-cPrxI (FIG. 673). However, H2O2 preincubation did not improve the resistance conferred by the cPrxI mutants that lacked Cys47, namely C47S- and C47/170S-cPrxI, as expected from the previous observation that this amino acid is essential for the stmctural and functional switching of cPrxI in vivo. The difference in the ceU survival rates of the ΔcPrxI/U yeasts expressing native cPrxI and Cys47-deficient-cPrxI can be attributed to their different chaperone activities. Thus, the data presented in FIG. 6A and 6B rønfirm that the Cys47-mediated H2O2- sensitive chaperone activity of cPrxI in vivo is much stronger compared to the chaperone activity of Cys47-deficient-cPrxI, which is independent of H2O2 stress. Also, it was observed that the chaperone function of cPrxI induced by 0.2 mM H2O2 pretreatment strongly protects yeast ceUs from oxidative stress against nonphysiologicaUy high concentrations of H2O2 (more than 10 mM H2O2) (data not shown). These results suggest that a low, nonstressful level of H2O2 induces a functional transition of cPrxI from a peroxidase to a molecular chaperone, thereby eUciting an adaptive response that counteracts the toxicity at much higher levels of other stresses, as described previously (Demple et al, CeU, 67: 837-839, 1991). Based on the report that the loss of ceU viabitity may be related to the aggregation of thermolabUe proteins witliin the cells (Tomoyasu et al, Mol. Microbiol, 40: 397-413, 2001), the abitity of cPrxI to prevent protein aggregation in heat shocked yeast strains after pretreatment with 0.2 mM H2O2 for 10 min was analyzed. To detect the proteins that were aggregated after stress, a protocol developed by Tomoyasu et al. (2001) was adopted, which reduces background levels and greatly increases the sensitivity of aggregate detection. Only a few aggregated protein bands were induced by a 10 min preincubation with 0.2 mM H2O2 in any of the yeast strains at 30°C (FIG. 6C). In contrast, significant amounts of aggregated proteins were observed in ΔcPrxIII ceUs and the isogenic mutant yeast expressing C47S- or C47/170S-cPrxI after heat shock for 20 min. The expression of native or C170S-cPrxI protein in ΔcPrxI/II yeast remarkably prevented the stress-induced aggregation of soluble proteins. These results show that the protection of the yeasts from heat shock does not depend on the peroxidase activity of cPrxI but on its chaperone function, which is greatly increased by mUd oxidative stress.

EXAMPLE 7: Assay for Disease and Stress Resistance of Transgenic Plant Carrying 2- Cysteine Peroxiredoxin I (basl) Gene of Arabidopsis

In order to construct a plant vector for use in introducing the 2-cysteine peroxiredoxin I gene (basl) of Arabidopsis in vivo, total RNA was first separated from Arabidopsis and used as a template to synthesize cDNA with the aid of a reverse transcriptase. Using the cDNA thus obtained, PCR was performed in the presence of a set of primers specific for plant 2-Cys Prx I (NM_111995) to clone a plant 2-Cys Prx gene. At this time, the primers contained the sites of the restriction enzymes Xbal and Sad therein to be inserted into a pBI121 vector. The cloned 2-Cys Prx gene was inserted into a pBI121 vector to afford a recombinant plant transformation vector pBI121 ::Prx (FIG. 7A) whose expression is regulated under the control of cauliflower mosaic virus 35S promoter and 2'/7' transcription terminator. Base sequencing identified the cloned 2-Cys Prx gene as having the same nucleotide sequence as the NM_111995 sequence registered in the NCBI. In FIG. TA, "kan" stands for kanamycin used as an antibiotic marker for selecting transformed plant ceUs, "RB" for right board, "LB" for left board, "Prx" for a gene coding for

2-Cys Prx I (basl) of Arabidopsis, and "2'/7"' for the untranslated region of the 277' gene of Agrobacterium tumefaciens. Then, the recombinant vector was introduced into Agrobacterium tumefaciens GV3101 bacterial ceUs using electroporation, foUowed by the selection of the transformants. For the selection of the transformants, the ceUs that had undergone electric shock were spread on an agar plate containing kanamycin, rifampicin and gentamicin and incubated at 30 ^°C (rifampicin and gentamicin were used as antibiotic markers for Agrobacterium and lcanamycin was used as an antibiotic marker for the pBI121 vector). DNA of Agrobacterium was separated from a colony grown on a YEP medium and was amplified by PCR to identify the transformation of the Agrobacterium strain. After Arabidopsis was cultured for three weeks, first bolts were cUpped to encourage the proliferation of many secondary bolts. Then, the Arabidopsis was subjected to floral dip transformation in which the plant was dipped in a solution containing transformed ceUs. Seeds were harvested from the transgenic Arabidopsis. In order to select transgenic Arabidopsis from the seeds obtained, a two-step selection procedure was carried out. In the first selection step, seeds of the Arabidopsis were allowed to mature in a medium containing kanamycin, an antibiotic selectable marker, and cefotaxime used for excluding Agrobacterium, and three kinds of Arabidopsis growing therein were selected and cultured to harvest seeds therefrom (Generation 1). In the second selection step, the seeds of the three kinds were aUowed to mature so as to select four Arabidopsis plants per kind (Generation 2). Proteins were isolated from leaves of the Arabidopsis plants, foUowed by sequential SDD-PAGE and Western blotting to identify the overexpression of the protein of interest. As seen in FIG. 7B, the Arabidopsis plant in lane 3 was found to express 2-Cys Prx I (basl) in the largest amount. Accordingly, the transgenic Arabidopsis in lane 3 was utilized for assays for stress resistance. To monitor stress resistance, the 2-Cys Prx I (basl) transformed plant was thermaUy treated at 45 ^°C for 6 hours or treated with a pathogen (Pseudomonas syringe). As a result, the transgenic plant in lane 3 was found to have more potential resistance to heat shock and pathogens than did the wfld type (refer to FIG.7C).

EXAMPLE 8: Diagnosis for Human Cancer CeUs Using Monoclonal Antibody Specific for High Molecular Weight Complex of 2-Cys Prx I

Using a set of primers containing BamHI and Xhol sites, a PCR for cloning a fuU length peroxiredoxin I DNA from a human placenta cDNA tibrary started with 94 °C pre- denaturation for 7 min and was carried out with 30 cycles of deraturing temperature at 94 °C for 30 sec, annealing temperature at 63 °C for 30 sec and extending temperature at 72 °C for 1 min, finaUy foUowed by 72 °C extension for an additional 1 min. The PCR product thus amplified (about 600 bp) was recovered from the agarose gel and inserted into a GST-fused pGEX vector, which was then introduced into E. coti and subjected to overexpression. From the GST-fused protein thus obtained, the fused GST was removed using a protease and the remainder was identified to be a pure human peroxiredoxin I (HPrxl) as analyzed by SDS- PAGE. Using size exclusion chromatography, the HPrxl was separated according to molecular weight to obtain fractions of a low molecular weight protein and a high molecular weight complex, which were used to prepare respective monoclonal antibodies (Harlow, E. and Lane D. Antibodies-A laboratory Manual, Cold Spring Harbour Laboratory, 1998; MisheU B.B and Shugi S.M. Selected Methods in CeUular Immunology W.H. Freeman and Company, 1980). In detaU, the high molecular weight complex of HPrxl was injected five times at intervals of two weeks into mice. Each injection was prepared by mixing 500 βg of the protein with an equal volume of an adjuvant One week after the final injection, B ceUs were obtained from the spleen of the mice and fused with SP2 myeloma ceUs. (Mturing in a HAT medium was foUowed by ELISA (Enzyme-linked immunosorbent assay) for the selection of a suitable monoclone. The high molecular weight complex, used as an antigen, was dUuted in a coating buffer (carbonate-bicarbonate) and placed coated into each of the weUs of 96-weU plates. The antigen-coated plates were incubated at 4^°C for 24 hours or longer and washed with a low concentration salt buffer. A blocking solution containing 3% skim mUk was appUed to the plates and incubated at room temperature for one hour or more, after which the plates were again washed in a low concentration salt buffer. A ceU-free hybridoma supernatant was added to the weUs of the plate, foUowed by incubation for one hour or longer. The plates were then washed with a high concentration salt buffer and a secondary antibody (donkey anti-mouse IgG-HRP) was added to the weUs of the plates which were then incubated at room temperature for one hour and again washed with a high concentration salt buffer. A coloring reagent was added to the plates and incubated at room temperature for about 10 min. After treatment with a stop solution, the plates were subjected to ELISA. By this analysis, selection could be made for clones which could produce a monoclonal antibody, which was identified to bind specificaUy to the high molecular complex as measured by Western blotting. The specificity of the monoclonal antibody to the high molecular weight complex of human 2-Cys Prxs was examined. A protein solution prepared from a human tissue was appUed to 10% native-PAGE to separate proteins which were then reacted with the monoclonal antibody. Reaction results are given in FIG. 8 A, showing that the monoclonal antibody specificaUy recognized the high molecular weight complex of 2-Cys Prxs (lane 2). In FIG.8A, lane 1 was visualized with a sUver dye solution after native-PAGE. Accordingly, the monoclonal antibody prepared using the high molecular weight complex of 2-Cys Prxs was found to have binding specificity only to the high molecular weight complex of 2-Cys Prxs. Based on this result, the monoclonal antibody binding specificaUy to the high molecular weight complex was examined for its abiUty to be used in the diagnosis of various cancers through Western blot analysis using proteins separated from samples of the subjects. In this regard, protein solutions prepared from ceUs exercised from tissues of patients with tiver cancer, womb cancer, breast cancer, or lung cancer were subjected to native- electrophoresis, foUowed by Western blotting using the monoclonal antibody against the high molecular weight complex of 2-Cys Prxs. As seen in FIG. 8B, 2-Cys Prxl was expressed excessively enough to be visualized with the naked eye in the patients unlike normal persons. Therefore, the monoclonal antibody that recognizes high molecular weight complex of 2-Cys Prxl is beUeved to aUow the diagnosis of various human cancers. In addition, a diagnostic kit and a simple diagnostic method, together with a diagnostic medicine, can be developed on the basis of the monoclonal antibody prepared according to the present invention.

EXAMPLE 9: Physiological Mechanism on Stress-Dependent Stmctural and Functional Change of 2-Cysteine Peroxiredoxin I (cPrxI) to Both Peroxidase and Chaperone Activities

It was possible to draw a comprehensive model that shows how cPrxI can function both as a peroxidase and as a molecular chaperone during oxidative stress (FIG. 9). This model shows that cPrxI can reversibly change its protein stmctures in vivo from LMW species to oUgomeric and HMW complexes by two different pathways. At low concentrations of ROS generated under normal conditions, cPrxI forms mainly low and oUgomeric protein stmctures with a smaU amount of HMW complex. cPrxI in these forms possesses the dual activities that remove low levels of ROS and protect proteins from denaturation. The Cys47 residue of cPrxI is not involved in the formation of these low and oUgomeric protein stmctures. However, under oxidative stress conditions, cPrxI rapidly undergoes stmctural changes and the LMW form is converted into HMW complexes with the help of the Trx system including Trx, TR and NADPH. In the presence of srxl, which can reduce Cys- sulfinic acid to Cys-thiol protein , the dissociation of the HMW complexes into LMW species occurs upon the removal of H₂O₂. In conclusion, it has been found that Cys47-dependent and -independent reactions of cPrxI participate in the association or dissociation of the protein and that this regulates cPrxI function during heat shock or oxidative stress. These changes are induced by Cys47, which acts as a highly efficient "HbOa-sensor" in ceUs experiencing oxidative stress.

EXAMPLE 10: Mass Production of Yeast 2-Cysteine Peroxiredoxin 1

Using a set of primers containing BamHI and Xhol sites, a PCR was performed to amplify a 2-Cys Prx gene from yeast genomic DNA. The PCR product was inserted into a GST-fused pGEX vector and identified to be a peroxiredoxin gene as analyzed by base sequencing. After being transformed with the recombinant vector pGEX: :cPrxI, E. coti was spread on an LB plate containing 50 μg/ml ampiciUn (antibiotic selectable marker for pGEX vector), and then incubated at 37 ^°C for 12 hours to form colonies. GST-fused peroxiredoxin was overexpressed in the transformant and isolated through affinity chromatography. The removal of the GST from the fusion protein using thrombin resulted in a peroxiredoxin protein which was then identified by SDS-PAGE. The microorganism capable of overexpressing peroxiredoxin (E. co/z/pGEX-cPrxi) was deposited at the Korean CoUection for Type Cultures onMay 24, 2004, with the accessionNo. KCTC 10645BP.

EXAMPLE 11 : Mass Production of Human 2-Cysteine Peroxiredoxin 2

Using a set of primers containing BamHI and Xhol sites, a PCR was performed to amplify a 2-Cys Prx gene 2 from human genomic DNA. The PCR product was inserted into a GST-fused pGEX vector, and identified to be a peroxiredoxin gene as analyzed by base sequencing. After being transformed with the recombinant vector pGEX: :hPrxII, E. coti was spread on an LB plate containing 50 μg/ml ampicilin (antibiotic selectable marker for pGEX vector), and then incubated at 37 ^°C for 12 hours to form colonies. GST-fused peroxiredoxin was overexpressed in the transformant and isolated through affinity chromatography. The removal of the GST from the fusion protein using thrombin resulted in a peroxiredoxin protein which was then identified by SDS-PAGE. The microorganism capable of overexpressing peroxiredoxin (E co //pGEX-hPrxII) was deposited at the Korean CoUection for Type Cultures on April 25, 2005, with the accession No. KCTC 10793BP.

EXAMPLE 12: Chaperone function of a human Prx isotype II, hPrxI

Importantly, hPrxII has a high degree of sequence homology and shares simUar biochemical properties (Wood et al. Science, 300: 650-653, 2003) with yeast cPrxI and π, which were previously shown to have dual functions as both peroxidases and molecular chaperones (Jang et al, CeU, 117: 625-635, 2004). Thus, the potential chaperone activity of hPrxII was investigated by assessing its abitity to inhibit the thermal aggregation of CS. The chaperone function of hPrxII was measured using CS, insulin, and α-synuclein as substrates as shown in FIG. 10. 1 μM CS (o-o) in 50 mM Hepes, pH 7.0, was incubated in a spectrophotometer ceU at 43 °C with 2 μM BSA (•-•), hPrxIJ (M-B) and yeast cPrxI (A- A) used as a control (FIG. 10A). Effects of hPrxII concentrations on the thermal aggregation of CS at 43 °C (FIG. 10B) or on the 10 mM DTT-induced chemical aggregation of insulin at 25 °C (FIG. 10C), in the absence (o-o), or presence of hPrxU at molar ratios of hPrxII to substrate of 1:1 (■-■), 2:1 (•-•), and 3:1 (A-A) were examined. Substrate aggregation was monitored by measuring the tight scattering at A ₆₀. hPrxII-mediated protection of human α- synuclein aggregation from oxidative damage in vitro was also examined (FIG. 10D). 10 μM hPrxII was incubated with 5 μM α-synuclein at 37 °C for 2h and the aggregation of α- synuclein was analyzed by immunoblotting using an anti-α-synuclein antibody. Lane 1, α- synuclein; lane 2, α-synuclein incubated with 10 μM Cu, Zn-SOD and 300 μM H₂O₂; lane3, α-synuclein was added to the mixture containing 10 μM hPrxII, 10 μM Cu, Zn-SOD and 300 uMH₂O₂. This revealed that hPrxII efficiently suppressed the thermal aggregation of CS at 43 °C, simUar to yeast cPrxI which was used as a control (FIG. 10 A). No inhibition of CS aggregation was observed when BSA was used instead of hPrxII. Incubation of CS with increasing concentrations of hPrxI produced a concomitant concentration-dependent decrease in CS aggregation and at a subunit molar ratio of hPrxJJ to CS of 3 : 1, CS aggregation was completely suppressed (FIG. 10B), suggesting that hPrxII can indeed act as an efficient molecular chaperone. Besides CS, it was also demonstrated that hPrxII protects the insulin β- chain from DTT-induced precipitation (FIG. 10C). In addition to these model substrates, the chaperone activity of hPrxII was tested against α-synuclein, which is a key component of Lewy bodies in Parkinson's and Alzheimer's diseased brains, and is abnormaUy aggregated by oxidative stress (Kim et al. Free Radic. Biol. Med, 32: 544-550, 2002, Matsuzaki et al. Brain Research, 1004: 83-89, 2004). Immunoblot analysis indicated that ROS generated by superoxide dismutase (SOD) and H₂O₂ induce extensive aggregation of α-synuclein, in agreement with previous reports (Kim et al. Free Radic. Biol. Med, 32: 544-550, 2002). However, the denaturation and aggregation of α- synuclein induced by ROS was prevented by hPrxII (FIG. 10D). This suggests that the chaperone function of hPrxU is effective in preventing ROS-induced aggregation of a cytosoUc protein in mammaUan ceUs. From these results, it can be concluded that, in addition to its weU-known peroxidase function, hPrxU exhibits highly efficient molecular chaperone activity, as is observed for the yeast cPrxI and II proteins (Jang et al, CeU, 117: 625-635, 2004). EXAMPLE 13: The dual functions of hPrxII are associated with its protein stmctures

Yeast cPrxI and II produce multiple homo-polymeric complex forms and previously it was found that their dual peroxidase and chaperone functions were closely associated with the degree of polymerization in their protein stmctures (Janget al, CeU, 117: 625-635, 2004). Since hPrxII exhibits strong chaperone activity (FIG. 10), and high molecular weight (HMW) complex formation of a protein is a typical feature of molecular chaperones (Haley et al, J. Mol. Biol, 277: 27-35, 1998), the molecular stmctures of purified hPrxTJ were analyzed using SEC. FIG. 11 shows tlie relationship between the structure and the dual functions, peroxidase and chaperone functions, of hPrxU with the aid of SEC. In FIG. 11 A, the numerals represent expected molecular weights of proteins and the separated total proteins (Tot) were divided and pooled into three fractions (F-H: high molecular weight, F-IVfl: medium molecular weight, and F-L: low molecular weight ) for further analysis.This analysis confirmed that hPrxU produces multiple forms of HMW protein complex (FIG. 11 A) that have molecular masses ranging from 100 kDa to in excess of 1 ,000 kDa When the molecular sizes of each protein fraction in SEC (FIG. 11 A) were further examined by Western blotting with an anti- hPrxπ antibody on a native-PAGE, multiple forms of polydisperse protein complex were detected in each fraction (FIG. 11B, upper panel). The protein sizes of hPrxU in native-

PAGE were sirnUar to those estimated by SEC. However, in contrast to the data obtained from native-PAGE analysis, a single protein band with a MW of 22 kDa was observed upon SDS-PAGE of aU the SEC-separated protein fractions. This strongly suggests that under native conditions, hPrxII exists as a homo-polymeric complex consisting of variable copies of its subunit. Since it was previously estabUshed that the dual functions of yeast cPrxI and fl^" are regulated by their protein stmctures (Jang et al, CeU, 117: 625-635, 2004), it was sought to determine whether this is the case for the human PrxII protein using the stmctures separated from SEC (FIG. 11 C). The molecular stmctures in each of the SEC-separated fractions were stable enough to faciUtate analysis of their biochemical properties in vitro (data, not shown). The highest MW hPrxII protein complex (F-H) predominantly exhibited chaperone activity with a negUgible amount of peroxidase activity, whereas the last fraction, containing the lowest MW proteins (F-L) displayed significant peroxidase activity and virtuaUy no chaperone activity. In contrast, the middle-sized protein complex (F-M) demonstrated both peroxidase and chaperone activities, as did the total protein (Tot). These combined observations strongly suggest that the peroxidase and chaperone activities of hPrxII are regulated by the degree of homo-polymerization in its protein stmctures.

EXAMPLE 14: Effect of the N-teπninal peroxidatic Cys and C-terminal domain of hPrxII on its protein structure and functions in response to H₂O₂ During the H₂O catalytic process, the peroxidatic Cys of 2-Cys Prxs is oxidized to sulfenic acid (Cys-SOH), which typicaUy reacts with a proximal thiol to form an intemiolecular disulfide bond (Chae et al, J. Biol. Chem, 269: 27670-27678, 1994). However, upon exposure of yeast ceUs to elevated levels of H₂O₂, the peroxidatic Cys is hyperoxidized to sulfinic or sulfonic acid (Woo et al. Science, 300: 653-656, 2003, Yang et al, J. Biol. Chem, 277: 38029-38036, 2002). This reaction induces significant stmctural changes of yeast 2-Cys Prxs and mediates their functional switching from peroxidases to molecular chaperones(Jang et al, CeU, 117: 625-635, 2004). The functional regulation of 2-Cys Prxs is also controUed by phosphorylation and limited proteolysis (Chang et al, J. Biol. Chem, 277: 25370-25376, 2002, Koo et al. Arch. Biochem. Biophys, 397: 312-318, 2002). Recently, two 2-Cys Prxs have been identified from Schistosomα mαnsoni, one of which is sensitive to hyperoxidation by H₂O₂, with the other being remarkably resistant to hyperoxidation (Sayedet al, J. Biol. Chem, 279: 26159-26166, 2004). In that study, the authors concluded that the susceptibiUty of the sensitive Prx to oxidative inactivation was due to the presence of a C- terminal taU containing the ' YF-motif . FIG. 12 shows the influence of the amino terminal Cys51, responsible for the peroxidase function, and the carboxy taU on the structure and function of human peroxiredoxin π. In FIG. 12A, native hPrxU (Native) and three mutant hPrxU (C51S, ΔC-ter, DM) are shown while C stands for cysteine, S for serine and YF for the motif consisting of 6 carboxy terminal amino acid residues. On the basis of these observations, mutant proteins of a C51S-hPrxπ, a C-terminal truncated (ΔC-ter) protein and a double mutant (DM) form of hPrxU (FIG. 12A) were prepared. Using the recombinant proteins expressed in E. coli, stmctural and functional switching of the proteins in the presence of various concentrations of H O₂ was investigated. In contrast to the native form of hPrxU, the C51 S mutant, C-terminal truncated (ΔC-ter) protein, and double mutant (DM) form of hPrxU did not respond to H₂O₂, even in the presence of a Trx system containing Trx, Trx reductase and NADPH (FIG. 12B). Furthermore, the H₂O₂- catalyzing peroxidase activity of the C-terminal truncated hPrxU was not inactivated by high concentrations of H₂O₂, whUe the peroxidase activity of native hPrxU was significantly decreased by H₂O₂ (FIG. 12C). This suggests that the C-terminal truncation converted the oxidation-sensitive form of hPrxU to an overoxidation-resistant protein. As expected, the C51S mutant-hPrxπ proteins were totaUy unable to catalyze H₂O₂. On the contrary, in the presence of a Trx system, the chaperone activity of native hPrxI increased significantly upon exposure to increasing concentrations of H₂O₂, whereas the chaperone functions of the C51S mutant, C-terminal truncated (ΔC-ter) protein, and double mutant hPrxII (DM) were not influenced by H₂O₂ (FIG. 12D). These results strongly suggest that the catalytic Cys of hPrxII can act as an efficient

Η2O2 sensor' to induce the structural and functional switching of the protein and that the stmctural changes mediated by the 'YF-mottf -containing C-terminal taU are also critical for responding to oxidative stress in vitro. The result is consistent with the data of yeast cPrxI, whose chaperone activity is regulated by two different processes, such as Cys-dependent and - independent processes (Jang et al, CeU, 117: 625-635, 2004). Notably, the bacterial 2-Cys Prxs that lack C-terminal domains are robust to oxidative-inactivation (Wood et al. Science, 300: 650-653, 2003). Thus, the existence of mammaUan oxidative stress-sensitive 2-Cys Prxs, that harbor an extra C-terminal YF-mσtif, indicates that this mechanism of stmctural and functional regulation emerged selectively during the evolution of eukaryotic ceUs.

EXAMPLE 15 : Oxidative stress-dependent HMW complex formation of hPrxU in vivo

Given that the protein stmctures and functions of yeast cPrxI and II are exquisitely sensitive to oxidative stress in vivo (Jang et al, CeU, 117: 625-635, 2004), it was examined whether hPrxII is regulated simUarly. To address this question, HeLa ceUs were treated with various concentrations of H₂O₂, extracted ceU lysates and subjected them to native-PAGE and immunoblotting using an anti-hPrxII antibody. HPrxII protein extracted from H₂θ₂-untreated HeLa ceUs consisted of multiple forms of low and oUgomeric protein stmctures (FIG. 13 A, lane 1 in left panel), whereas exposure to H₂O₂ resulted in the majority of proteins being converted into HMW complexes within 20 min in a concentration dependent manner (FIG. 13A, leftpanel). Using a sulfinylation-sulfonylation-specific Prx antibody (Prx-SO₃-Ab), it was estabUshed that the H₂O₂-induced HMW complex resulted from the hyperoxidation of the Cys thiol of hPrxU (FIG. 13A, lane 2 and 3 in right panel) (Woo et al, J. Biol. Chem, 278: 47361- 47364, 2003). Thus, to analyze the oxidation status of hPrxU, protein extracts of HeLa ceU on 2D-gels were separated and subjected to immunoblotting with hPrxU- and Prx-SO₃-antibodies. Most of the hPrxU existed in a reduced form in the absence of H₂O , whereas significant amounts of hPrxU were oxidized by exposure to 0.2 mM H₂O₂, with a corresponding decrease in the reduced form of the protein (FIG. 13B, left panel). The H₂θ₂-induced hyperoxidation of hPrxU was reconfirmed using the Prx-SO₃ antibody (FIG. 13B, right panel). When H₂θ₂ was removed from the media using the scheme depicted in FIG. 13C, most of the H₂θ₂-mediated HMW complexes of hPrxU restored to their original stmctures in vivo within 40 min (FIG. 13D). From the results obtained here it is clear that the protein stmctures of hPrxU are precisely regulated by oxidative stress in a reversible manner. Moreover, since stmctural changes of hPrxU are foUowed by functional switching, it is possible that the ROS-dependent inactivation of its peroxidase function (FIG. 12C) does not constitute the termination of its catalytic cycle, but rather serves as a regulatory mechanism for exhibiting its alternative molecular chaperone function.

EXAMPLE 16: H₂O₂-mediated HMW complexes of hPrxU can be formed, but not dissociated into its original structure in yeast ceUs

To examine whether H₂θ₂-mediated stmctural changes of hPrxU can also occur in yeasts, hPrxU was expressed in yeasts under the control of Gall promoter. The protein stmctures of hPrxU expressed in yeast ceUs were sUghtly different from those expressed in HeLa ceUs analyzed by Western blotting on a native-PAGE with an anti-hPrxU antibody (FIG. 14A). A lower amount of middle-sized hPrxU protein structure was detected in yeast ceUs.

When the yeast ceUs expressing hPrxU were exposed to 0.2 mM H₂O₂ according to the scheme shown in panel C of FIG. 13, significant amounts of LMW proteins were converted into HMW complexes by H₂O₂ within 20 min. Comparing the yeast cPrxI used as a positive control (Jang et al, CeU, 117: 625-635, 2004) (FIG. 14B), less hPrxU was migrated into HMW complexes by the H₂O₂ treatment. Particularly, in contrast to the reversible regulation of cPrxI protein stmctures (FIG. 14B, lane 3), HMW complexes of hPrxU did not return to their original stmctures within 40min after the removal of H₂O₂ from the yeasts under the same experimental conditions (FIG. 14A, lane 3). These results propose that the yeast Trx can be used for the H₂O₂-dependent HMW complex formation of both cPrxI and hPrxU, but the Srxl in yeast can not replace the function of its mammaUan counterpart, such as mammaUan Srx (Chang et al, J. Biol. Chem, 279: 50994-51001, 2004) or sestrin (Budanov et al. Science, 304: 596-600, 2004), in the dissociation of the HMW complexes in vivo.

EXAMPLE 17: Chaperone function of hPrxU protects HeLa ceUs from H₂θ₂-induced ceU death

Even low concentrations of H₂O₂, that are non-toxic to ceUs, induce the switching of hPrxU structure and function in wvo(FIG. 13). To investigate the physiological significance of the functional switching of hPrxU from a peroxidase to a molecular chaperone, four hPrxII DNA constructs containing 6 x His-tag at their N-termini were prepared (FIG. 12A) and Ugated into the pcDNA3.1 vector. The DNA constructs were individuaUy transfected into

HeLa ceUs and the ceU lines showing stable overexpression of the four hPrxU proteins were selected. Immunoblot analysis of total ceU lysates revealed that the expression level of hPrxU in transfected HeLa ceUs was about 2- to 3- fold higher than the endogeneous level observed in control ceUs transfected with the empty vector (data not shown). In addition, the C51S mutant, C-terminal truncated (ΔC-ter) protein, and double mutant form (DM) of hPrxπ proteins were expressed at levels simUar to the native hPrxU protein, as determined by Western blotting on SDS-PAGE using an anti-His-tag antibody (FIG. 15 A, lower panel). Equal loading of the proteins onto PAGE-gel was confirmed by immunoblotting the membrane with an anti-b-actin antibody. The protein stmctures of hPrxU and ceU viabiUty measured using a TUNEL assay were examined, foUowing exposure of HeLa ceUs to 1 mM H₂Q₂. Immunoblot analysis with an anti-His-tag antibody foUowing native-PAGE confirmed that in HeLa ceU lysates, native hPrxU shifts from low and oUgomeric stmctures to HMW complexes within 20 min of H₂O₂ treatment (FIG. 15A, upper panel), with these HMW stmctures being maintained for at least 12 h in vivo (data not shown). However, the low MW protein structures of the C51S, ΔC-ter, and double mutant (DM) hPrxU proteins were not influenced by H₂O₂. This result strongly suggests that both Hie catalytic Cys and the C-terminal domain of hPrxU containing 'YF motif are indispensable for H₂O₂-mediated HMW complex formation both in vitro (FIG. 12B) and in vivo (FIG. 15 A). With regard to ceU survival/death, HeLa ceUs transfected with or without the empty vector were quite sensitive to H₂O₂ chaUenge, with approximately 70% of ceUs becoming TUNEL-positive within 12 h (FIG. 15B & D). In contrast, ceUs over-expressing native hPrxU showed remarkable resistance to H₂O₂-induced ceU death under the same conditions (FIG. 15C). This may be because the chaperone function of hPrxU prevents denaturation of substrates due to oxidative stress, or alternatively it may reflect the activation of downstream defense signaling cascades by the HMW form of hPrxU, which prevents H₂O - mediated ceU death. These results are consistent with previous studies that described a cytoprotective function of mammaUan Prxl and U in FRTL-5 rat thyroid (Kim et al, J. Biol. Chem, 275: 18266-18270, 2000) and ratla fϊbroblast ceU lines (Mu et al, J. Biol. Chem, 277: 43175-43184, 2002), although in those reports, cytoprotection was not linked to the chaperone function of these proteins. The survival rates of HeLa ceUs expressing C51S, ΔC-ter, and double mutant (DM) form of hPrxU were higher than vector-transfected control cells, but were significantly lower than those of ceUs expressing native hPrxU (FIG. 15C & D). The result suggests that the basal level of chaperone activity (FIG. 12D) in mutant hPrxU, which is neither dependent on active Cys residue nor sensitive to external stress, plays a role to a certain degree in protecting ceUs against oxidative stress. In a paraUel study, the cleavage pattern of PARP in HeLa ceUs expressing various forms of hPrxU was examined using Western blotting with an anti-PARP antibody after treatment with 1 mM H2O2. The PARP catalyzing the poly(ADP-ribosyl)ation of nuclear histone and non-histone proteins has been impUcated in the induction of p53 and apoptosis (Tewari et al, CeU, 81: 801809, 1995). Thus, the specific proteolysis of PARP into 85- and 24-kDa fragments has been closely associated with programmed ceU death (Nicholson et al, Nature, 376: 3743, 1995). In an experiment according to the present invention, an intact PARP, of which MW was 116-kDa, was present in HeLa ceUs without the treatment of H2O2, but significant amounts of the protein were cleaved into 85kDa by H2O2 treatment (FIG. 15E). When HeLa ceUs transfected with control vector were exposed to ImM H2O2, the level of 85- kDa fragment was significantly increased. However, the expression of native-hPrxπ remarkably inhibited the cleavage of PARP, even in the presence of H2O2. In contrast, the C51S, ΔC-ter, and double mutant (DM) hPrxU proteins cleaved the PARP in between the range of ceUs expressing empty vector and the native-hPrxU. In conclusion, taken together, the observation data suggests that the chaperone function of hPrxU significantly protects human ceUs against ROS-mediated ceU death. [Industrial Applicability] As described hereinbefore, 2-cysteine peroxiredoxin functions both as a peroxidase of scavenging reactive oxygen species and as a chaperone of preventing protein unfolding, acting as a chaperone, which is proven to be associated with the dynamic conversion in the tertiary and quaternary stmctures in accordance with the present invention. The stmctural and functional switching of 2-cysteine peroxiredoxin I (cPrxI) and human peroxiredoxin I-IV

(hPrxI-IV) is also found to be highly susceptible for ROS that is typicaUy produced by heat shock and oxidative stress in accordance with the present invention. It is revealed that the chaperone function of 2-cysteine peroxiredoxin is activated when low molecular weight 2-cysteine peroxiredoxin undergoes a stmctural change to high molecular weight complex. Evidences for the fact that the expression of 2-Cys Prx and its formation to a high molecular weight complex is highly involved in the occurrence of various diseases, including neurodegeneration, Alzheimer's disease, Down's syndrome, thyroid cancer, heart cancer, lung cancer, tumor, etc, as weU as in the resistance against heat shock and oxidative stresses, as reveled in the present invention, afford a highly plausible deduction that the protein protect ceUs from various diseases. In addition, when switched to high molecular weight multiple fomis under the condition of oxidative stresses, 2-cysteine peroxiredoxin acts as a probe for reactive oxygen species protein. The C-terminal YF-motif, found only in the 2-Cys Prx of eukaryotic ceUs, is highly responsible for the mediation of stmctural changes to multimerized forms. In addition, the protein of interest shows super chaperone activity when most low molecular weight species are multimerization to the protein complexes. In the present invention, hPrxU is also demonstrated to be capable of preventing α- synuclein, a key component of Lewy bodies found in Parkinson's and Alzheimer's diseased brains, from being aggregated upon oxidative stress as weU as preventing oxidative stress- induced protein unfolding. It is also found that oxidative stress-induced apoptosis is significantly decreased in HeLa ceUs where hPrxl is overexpressed. Consequently, the high molecular complex of 2-Cys Prx proteins and the antibody specificaUy binding to the complex can be appUed for the development of a diagnostic method and a diagnostic medicine for various diseases including neurodegneration, Alzheimer's disease, Down's syndrome, thyroid cancer, heart cancer, lung cancer, tumors, etc. Transgenic animal ceUs harboring the gene coding for the protein are expected to aUow the creation of transgenic animals which are resistant to various diseases and environmental stresses. MeanwhUe, this is true of plants as demonstrated by the transgenic Arabidopsis carrying 2-cysteine peroxiredoxin gene, which is resistant to various diseases and environment stress.

Claims

[CLAIMS] [CLA 1] A high molecular weight complex, comprised of microorganism, plant or animal 2- cysteine peroxiredoxin proteins linked together by intermolecular interaction, having chaperone activity. [CLA 2] The high molecular weight complex as defined in claim 1, wherein the 2-cycteine peroxiredoxin proteins are of a holotype or an isotype. [CLA 3] The high molecular weight complex as defined in claim 2, wherein the 2-cysteine peroxiredoxin proteins are composed of at least one selected from a group consisting of polypeptides represented by SEQ. ID. NOS.2, 4, 6, 8, 10, 11 and 12. [CLAM 4] A transformed ceU capable of overexpressing 2-cysteine peroxfredoxin, which is prepared by introducing a recombinant vector overexpressing at least one 2-cysteine peroxiredoxin protein represented by SEQ. UD. NOS. 2, 4, 6, 8, 10, 11 and 12 into a microorganism ceU. [CLAM 5] The transformed ceU as defined in claim 4, wherein the ceU is E.cø/z/pGEX-cPrxI (AccessionNo. KCTC 10645BP). [CLAM 6] The transformed ceU as defined in claim 4, wherein the ceU is Eco/z/pGEX-hPrxU (AccessionNo. KCTC 10793BP). [CLAM 7] The transformed ceU as defined in claim 4, wherein the ceU is prepared by introducing a recombinant vector overexpressing at least one 2-cysteine peroxiredoxin protein selected from a group consisting of polypeptides represented by SEQ. ID. NOS.2, 4, 6 and 8 into a human ceU. [CLAM 8] The transformed ceU as defined in claim 4, wherein the ceU is Agrobacterium tumefaciencs EHAl 01. [CLAM 9] A transgenic plant ceU strain capable of overexpressing 2-cysteine peroxiredoxin, which harbors the Agrobacterium tumefaciencs of claim 9. [CLAM 10] A monoclonal antibody, specificaUy binding to the complex of claim 1. [CLAM 11] A monoclonal antibody, specificaUy binding to a complex of 2-cysteine peroxiredoxin proteins Unked together by intermolecular interaction, wherein the 2-cycteine peroxiredoxin proteins are comprised of at least one selected from a group of 2-cysteine peroxiredoxin isotypes represented by SEQ. ID. NOS.2, 4, 6, and 8. [CLAM 12] A method for diagnosing a disease from which a subject suffers, in which the monoclonal antibody of claim 10 is subjected to immunoreaction with a sample from the subject and the sample is quantitatively analyzed for peroxiredoxin complex in comparison to a control. [CLAM 13] The method as defined in claim 11, wherein the monoclonal antibody is able to specificaUy recognize, as an antigen, a peroxiredoxin complex having chaperone activity, said peroxiredoxin complex being comprised of 2-cysteine peroxiredoxin proteins Unked together by intermolecular interaction, said 2-cysteine peroxiredoxin proteins being comprised of at least one selected from a group consisting of 2-cysteine peroxiredoxin isotypes represented by SEQ. ID. NOS.2, 4, 6 and 8. [CLAM 14] The method as defined in claim 10 or 11, wherein the sample is proteins obtained by mpturing or lysing ceUs from tissues or humors of animals, plants and microorganisms. [CLAM 15] The method as defined in claim 10 or 11, wherein the immunoreaction is conducted by dividing proteins of the sample through native-PAGE, blotting the proteins onto predetermined positions of a nifroceUulose membrane, and visualizing positions and amounts of the proteins with an enzyme or dye to determine the level of the proteins in the sample. [CLAM 16] The method as defined in claim 10 or 11, wherein the disease is selected from a group consisting of neurodegeneration, Alzheimer's disease, Down's syndrome, thyroid cancer, heart cancer, lung cancer, tumors, heat shock-induced diseases, reactive oxygen species-induced diseases, Uver cancer, breast cancer, and womb cancer. [CLAM 17] A kit for diagnosing a disease from which a subject suffers, comprising: a test sample; a gel on which positive and negative control samples are separated by native-PAGE; a comb card to which the same number of nitroceUulose membranes as a number of lanes of the gel are attached; an electric device for supplying electricity to transfer the proteins on the gel onto the nitroceUulose membranes; a monoclonal antibody on the nitroceUulose membranes, defined in claim 10 or 11; and an incubation tray for aUowing the monoclonal antibody to recognize the proteins transferred onto the nitroceUulose membranes. [CLAM 18] A kit for diagnosing a disease from which a subject suffers, comprising: a strip (1) comprising: a developing membrane (13) on which a reaction unit (11) for immobilizing an antibody thereon, and a confrol unit (12) for monitoring the normal operation of the kit are positioned at predetermined positions; and a housing (2) for accommodating the strip (1), comprised of: a sample feeder (21): and indicator windows (22) through which reaction results in the reaction unit (11) and the control unit (12) may be visualized with the naked eye. [CLAM 19] A medicine for use in diagnosing a disease from which a subject suffers, comprising: (a) an immobilized sample obtained by processing a subject matter; (b) an antibody of claim 10 or 11, recognizing a high molecular weight complex of 2-Cys Prxs as an antigen; (c) a coloring enzyme-labeled complement binding specificaUy to the antibody; (d) a substrate solution undergoing a color change upon reaction with tl e coloring enzyme; and (e) a buffer for the reaction for the color change and an incubation tray. [CLAM 20] The medicine as defined in claim 19, wherein the immobilized sample is obtained by applying the subject matter to native-PAGE to divide proteins and transferring the proteins from the gel onto a nitroceUulose membrane in the presence of an electric field. [CLAM 21] A pharmaceutical composition for the prophylaxis and treatment of neurodegeneration, Alzheimer's disease, Down's syndrome, Parkinson's disease, thyroid cancer, heart cancer, breast cancer, lung cancer, and tumors, comprising the peroxiredoxin complex of claim 3 or its salt as an effective ingredient, in combination with a pharmaceuticaUy acceptable carrier. [CLAM 22] A phamiaceutical composition for the prophylaxis and treatment of neurodegeneration, Alzheimer's disease, Down's syndrome, Parkinson's disease, thyroid cancer, heart cancer, breast cancer, lung cancer, and tumors, comprising the transformed ceU of claim 7 and a pharmaceuticaUy acceptable carrier.