WO2005116082A1 - Complexe de 2-cysteine peroxyredoxine a fonction agissant comme une chaperone moleculaire et ses utilisations - Google Patents

Complexe de 2-cysteine peroxyredoxine a fonction agissant comme une chaperone moleculaire et ses utilisations Download PDF

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WO2005116082A1
WO2005116082A1 PCT/KR2005/001568 KR2005001568W WO2005116082A1 WO 2005116082 A1 WO2005116082 A1 WO 2005116082A1 KR 2005001568 W KR2005001568 W KR 2005001568W WO 2005116082 A1 WO2005116082 A1 WO 2005116082A1
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proteins
peroxiredoxin
cprxi
clam
cysteine
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PCT/KR2005/001568
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English (en)
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Sang Yeol Lee
Jeong Chan Moon
Jin Ho Park
Sun Young Kim
Young Mi Lee
Ho Hee Jang
Jung Ro Lee
Soo Kwon Park
Seoung Sik Lee
Kyun Oh LEE
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Industry-Academic Cooperation Foundation Gyeong Sang National University
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Priority claimed from KR1020050037546A external-priority patent/KR100675342B1/ko
Application filed by Industry-Academic Cooperation Foundation Gyeong Sang National University filed Critical Industry-Academic Cooperation Foundation Gyeong Sang National University
Publication of WO2005116082A1 publication Critical patent/WO2005116082A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0065—Oxidoreductases (1.) acting on hydrogen peroxide as acceptor (1.11)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes

Definitions

  • 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.
  • ROS Reactive oxygen species
  • 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).
  • SOD superoxide dismutase
  • HSP heat shock protein
  • HSP HSP70
  • HSP60 HSP60
  • HSP40 small HSPs
  • Prx-dependent peroxidase 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.
  • 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:
  • 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 H2 ⁇ 2-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.
  • 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).
  • 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.
  • PDI protein disulfide isomerase
  • 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.
  • cPrxI 2-cysteine peroxiredoxin
  • 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.
  • a high molecular weight complex comprised of microorganism, plant or animal 2-cysteine peroxiredoxin proteins linked together by intermolecular interaction, having chaperone activity.
  • 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.
  • 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.
  • a monoclonal antibody specifically binding to the complex.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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. c ⁇ ////pGEX-hPrxII) was deposited at the Korean Collection for Type Cultures on April 25,
  • the transformants are preferably stored in, for example, liquid nitrogen with a storage buffer until reuse.
  • 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.
  • 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.
  • 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.
  • 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.
  • sample proteins obtained by rapturing or lysing cells from tissues or humors of animals, plants and microorganisms may be used as test samples.
  • 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.
  • subject matter as used herein means whole blood, sera, plasmas, lymph fluids, intercellular fluids, or tissues such as organs.
  • test sample 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
  • 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.
  • 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).
  • an ELISA method using 96- or 386-well plates may be effective (D' Ercole et al, J.
  • 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.
  • an antibody binding specifically to a high molecular weight peroxiredoxin complex is added, and an immunoblotting method is conducted for quick diagnosis.
  • 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.
  • 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.
  • the diagnostic kit in accordance with the
  • 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.
  • an antibody prepared according to the present invention is immobilized.
  • 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.
  • the absorbance is detected and analyzed by an ELISA method so as to determine whether the subject is afflicted with the diseases.
  • 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.
  • 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.
  • Hie pharmaceutical composition of the present invention may be prepared in various dosage forms.
  • 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.
  • 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.
  • 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.
  • 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.
  • a transformant transformed cell strain
  • the dosage of the pharmaceutical composition of the present invention can vary within the range in which no desensitization happens.
  • peroxiredoxin is the second or the third in blood level among the proteins found in blood (Moore et al, J. Biol.
  • 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.
  • 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. 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 (c
  • 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 ( ⁇ 2-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 ⁇ ;
  • 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).
  • 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.
  • 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.
  • 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.
  • 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 co ⁇ esponding 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.
  • 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.
  • subject matter as used herein means whole blood, sera, plasmas, lymph fluids, intercellular fluids, or tissues such as organs.
  • test sample 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
  • 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.
  • 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).
  • an ELISA method using 96- or 386-well plates may be effective (D' Ercole et al, J.
  • 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.
  • an antibody binding specifically to a high molecular weight peroxiredoxin complex is added, and an immunoblotting method is conducted for quick diagnosis.
  • 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.
  • the diagnostic kit in accordance with the present invention is illustrated.
  • 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.
  • a control unit (12) for monitoring the normal operation of the kit are positioned at predetermined positions.
  • an antibody prepared according to the present invention is immobilized.
  • 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.
  • the absorbance is detected and analyzed by an ELISA method so as to determine whether the subject is afflicted with the diseases.
  • 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.
  • 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.
  • 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.
  • the pharmaceutical composition of the present invention may be prepared in various dosage forms.
  • 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.
  • 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.
  • 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.
  • 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.
  • a transformant transformed cell strain
  • the dosage of the pharmaceutical composition of the present invention can vary within the range in which no desensitization happens.
  • peroxiredoxin is the second or Hie third in blood level among the proteins found in blood (Moore et al, J. Biol.
  • 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.
  • EXAMPLE 1 Expression of the Native and Peroxidase-Inactive Forms of cPrxI Ameliorates the Hypersensitivityoffhe ⁇ cPrxI/II Yeast to Heat Shock.
  • 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.
  • almost no peroxidase acricity was found in tlie Cys-less cPrxI.
  • 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 ⁇ -sheet, a cylinder for an ⁇ -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).
  • 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 ⁇ -crystallin, showed about 15- and 3-folds weaker activity than that of cPrxI, respectively.
  • 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).
  • 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).
  • Prxs including the human NKEF, AhpC, calpromotin, HBP23, and Prx-B form HMW complexes with masses of 230-500 kDa (Schroder et al.
  • 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).
  • F-II electron microscopy and image processing
  • 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).
  • 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 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
  • 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).
  • 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% CO 2 ) instead of H 2 O 2 (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).
  • 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, ⁇ trxl/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
  • 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).
  • H2O2 the HMW complexes of cPrxI reverted to their original stmctures after the oxygen tension was reduced.
  • the H2 ⁇ 2-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).
  • 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.
  • 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 ⁇ cPrxIJJ 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.
  • RNA was first separated from Arabidopsis and used as a template to synthesize cDNA with the aid of a reverse transcriptase.
  • 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.
  • 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.
  • "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
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • the 2-Cys Prx I (basl) transformed plant was thermaUy treated at 45 ° C for 6 hours or treated with a pathogen (Pseudomonas syringe).
  • a pathogen Pseudomonas syringe
  • EXAMPLE 8 Diagnosis for Human Cancer CeUs Using Monoclonal Antibody Specific for High Molecular Weight Complex of 2-Cys Prx I
  • 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.
  • 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 ⁇ g of the protein with an equal volume of an adjuvant.
  • 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.
  • FIG.8A showing that the monoclonal antibody specificaUy recognized the high molecular weight complex of 2-Cys Prxs (lane 2).
  • 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.
  • cPrxI 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 H 2 O 2 .
  • 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.
  • E. coti was spread on an LB plate containing 50 ⁇ g/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.
  • 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.
  • E. coti was spread on an LB plate containing 50 ⁇ g/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.
  • EXAMPLE 12 Chaperone function of a human Prx isotype II, hPrxI
  • 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 ⁇ , which were previously shown to have dual functions as both peroxidases and molecular chaperones (Jang et al, CeU, 117: 625-635, 2004).
  • yeast cPrxI and ⁇ which were previously shown to have dual functions as both peroxidases and molecular chaperones (Jang et al, CeU, 117: 625-635, 2004).
  • 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 ⁇ -synuclein as substrates as shown in FIG. 10.
  • 10 ⁇ M hPrxII was incubated with 5 ⁇ M ⁇ -synuclein at 37 °C for 2h and the aggregation of ⁇ - synuclein was analyzed by immunoblotting using an anti- ⁇ -synuclein antibody.
  • Lane 1 ⁇ - synuclein; lane 2, ⁇ -synuclein incubated with 10 ⁇ M Cu, Zn-SOD and 300 ⁇ M H 2 O 2 ; lane3, ⁇ -synuclein was added to the mixture containing 10 ⁇ M hPrxII, 10 ⁇ M Cu, Zn-SOD and 300 uMH 2 O 2 .
  • hPrxII protects the insulin ⁇ - chain from DTT-induced precipitation (FIG. 10C).
  • the chaperone activity of hPrxII was tested against ⁇ -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).
  • 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.
  • FIG. 11 shows tlie relationship between the structure and the dual functions, peroxidase and chaperone functions, of hPrxU with the aid of SEC.
  • 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.
  • F-H high molecular weight
  • F-IVfl medium molecular weight
  • F-L low molecular weight
  • EXAMPLE 14 Effect of the N-te ⁇ ninal peroxidatic Cys and C-terminal domain of hPrxII on its protein structure and functions in response to H 2 O 2 During the H 2 O 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).
  • Cys-SOH sulfenic acid
  • 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 ⁇ .
  • native hPrxU Native
  • three mutant hPrxU C51S, ⁇ C-ter, DM
  • C stands for cysteine
  • S serine
  • YF the motif consisting of 6 carboxy terminal amino acid residues.
  • mutant proteins of a C51S-hPrx ⁇ , a C-terminal truncated ( ⁇ C-ter) protein and a double mutant (DM) form of hPrxU were prepared.
  • H 2 O 2 - catalyzing peroxidase activity of the C-terminal truncated hPrxU was not inactivated by high concentrations of H 2 O 2 , whUe the peroxidase activity of native hPrxU was significantly decreased by H 2 O 2 (FIG. 12C).
  • the C-terminal truncation converted the oxidation-sensitive form of hPrxU to an overoxidation-resistant protein.
  • the C51S mutant-hPrx ⁇ proteins were totaUy unable to catalyze H 2 O 2 .
  • ⁇ 2O2 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).
  • the bacterial 2-Cys Prxs that lack C-terminal domains are robust to oxidative-inactivation (Wood et al. Science, 300: 650-653, 2003).
  • H 2 O 2 -mediated HMW complexes of hPrxU can be formed, but not dissociated into its original structure in yeast ceUs
  • 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.
  • HMW complexes of hPrxU did not return to their original stmctures within 40min after the removal of H 2 O 2 from the yeasts under the same experimental conditions (FIG. 14A, lane 3).
  • the yeast Trx can be used for the H 2 O 2 -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 H 2 ⁇ 2 -induced ceU death
  • 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).
  • the C51S mutant, C-terminal truncated ( ⁇ C-ter) protein, and double mutant form (DM) of hPrx ⁇ 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 H 2 Q 2 .
  • 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 H 2 O 2 treatment (FIG.
  • 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).
  • H2O2 treatment FIG. 15E
  • the level of 85- kDa fragment was significantly increased.
  • the expression of native-hPrx ⁇ remarkably inhibited the cleavage of PARP, even in the presence of H2O2.
  • 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 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.
  • the protein of interest shows super chaperone activity when most low molecular weight species are multimerization to the protein complexes.
  • hPrxU is also demonstrated to be capable of preventing ⁇ - 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.

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Abstract

L'invention concerne un complexe de poids moléculaire élevé, comprenant des protéines de 2-cystéine peroxyrédoxine interconnectées par interaction intramoléculaire, à activité de chaperone, ainsi que ses utilisations. Sur la base de la découverte et du fait que les protéines de 2-cystéine peroxyrédoxine sous diverses formes sont différentes en termes de structure et de poids moléculaire, fonctionnant comme une peroxydase dans une forme structurale de faible poids moléculaire et comme chaperone moléculaire, dans une forme structurale de poids moléculaire élevé, peuvent être appliquées à une médecine diagnostique pour des troubles neurodégénératifs, la maladie d'Alzheimer, le syndrome de Down, le cancer du sein, le cancer du poumon, etc. L'invention concerne également une composition pharmaceutique pour assurer la prophylaxie et le traitement de pathologies, ainsi qu'un animal ou une plante transgénique, résistant au stress environnemental ou à de telles pathologies.
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WO2008142163A2 (fr) 2007-05-23 2008-11-27 Cropdesign N.V. Plantes possédant des traits de rendement améliorés et procédé de fabrication
CN103733069A (zh) * 2011-08-24 2014-04-16 浦项工科大学校产学协力团 筛选伴侣蛋白调节物的方法
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CN103733069B (zh) * 2011-08-24 2016-06-08 浦项工科大学校产学协力团 筛选伴侣蛋白调节物的方法
EP2659897A1 (fr) * 2012-05-02 2013-11-06 Philipps-Universität Marburg Composition dérivée de cellules souches pour le traitement d'une lésion aiguë et des maladies dégénératives
WO2014208157A1 (fr) * 2013-06-28 2014-12-31 栄研化学株式会社 Nouveau marqueur du cancer du poumon (prdx4)
EP3358355A1 (fr) 2017-02-04 2018-08-08 Warszawski Uniwersytet Medyczny Utilisation des péroxyrédoxines 2-cystéine (2-cys-prdx) sériques comme biomarqueurs de maladies rénales chroniques (ckd) telles que le lupus nephritis (ln), la néphropathie à iga (igan) et la polykystose rénale type dominant (adpkd) pour le diagnostic, le suivi et le pronostic de ces maladies et pour leur différenciation
WO2018141975A1 (fr) 2017-02-04 2018-08-09 Warszawski Uniwersytet Medyczny Utilisation de peroxyrédoxines 2-cystéine (2-cys-prdx) sériques en tant que biomarqueurs de maladies rénales chroniques

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