WO2005075513A1 - Dj-1 derivative having acidified cysteine residue - Google Patents

Abstract

A DJ-1 derivative wherein the cysteine residue at the 106-position in DJ-1 has been acidified into cysteinesulfonate. This DJ-1 derivative is useful as a diagnostic marker for neurodegenerative diseases.

Description

 Specification

 DJ-1 derivative with oxidized cysteine residue

 Technical field

 The present invention relates to an oxidized derivative of DJ-1, an antibody against the derivative, and use of the oxidized DJ-1 derivative as an indicator of oxidative stress.

 Background art

 [0002] DJ-1 was discovered as an oncogene that transforms NIH3T3 cells (Non-Patent Document 1).

[0003] Further, PARK7 was identified as a causative gene of Parkinson's disease, and it was found that this gene was the same as a DJ-1 point mutation (leucine at position 166 was mutated to proline) (Non-patent Document 2 ).

 [0004] Furthermore, it has been suggested that spots on two-dimensional electrophoresis of DJ-1 are shifted by oxidizing stress due to hydrogen peroxide and paraquat, and that the spot position changing force is also due to oxidization of cysteine. Patent Document 3).

 [0005] Further, Non-Patent Document 4 is a paper on X-ray crystal structure analysis of DJ-1, which shows that the introduction of the L166P mutation related to Parkinson's disease causes a change in the dimer structure, Suggests that the 106th cysteine is oxidized to sulfinic acid (SOH)

 2

[0006] By the way, oxidative stress is considered to be a cause of important diseases such as aging and arteriosclerosis, and identification of oxidative stress marker molecules that are early markers of these diseases is extremely important.

 [0007] However, at present, only low-molecular-weight markers such as 8-oxoguadin are known as oxidative stress markers, and useful oxidative stress markers are not substantially known.

Non-noon literature 1: Nagakubo D, Taira T, Kitaura H, Ikeda M, Tamai K, Igucni- Anga SM, Ariga HJ-l, a novel oncogene which transforms mouse NIH3T3 cells in cooperation with ras.Biochem Biophys Res Commun. (1997 ) 231, 509-13 Non-Patent Document 2: Bonifati V, Rizzu P, van Baren MJ, Schaap O, Breedveld GJ, Krieger E, Dekker MC, Squitieri F, Ibanez P, Joosse M, van Dongen JW, Vanacore N, van Swieten JC, Brice A, Meco G, van Duijn CM, Oostra BA, Heutink P. Mutations in the DJ-1 gene associated with autosomal recessive early-onset parkinsonism. Science. (2003) 299, 256-9.

 Non-Patent Document 3: Mitsumoto A, Nakagawa Y, Takeuchi A, Okawa K, Iwamatsu A, Takanezawa Y. Oxidized forms of peroxiredoxins and DJ-1 on two-dimensional gels increased in response to sublethal levels of paraquat.Free Radic Res. 2001) 35, 301-10.

 Non-Patent Document 4: Wilson MA, Collins JL, Hod Y, Ringe D, Petsko GA.The 1.1-A resolution crystal structure of DJ-1, the protein mutated in autosomal recessive early onset Parkinson's disease.Proc Natl Acad Sci US A. (2003) 100, 9256-61. Brief description of the drawings.

FIG. 1 shows the results of two-dimensional electrophoresis.

 Disclosure of the invention

 Problems to be solved by the invention

[0009] It is an object of the present invention to provide a technique for evaluating oxidative stress in a living body, and eventually finding a useful diagnostic marker for a neurodegenerative disease such as Parkinson's disease and Alzheimer's disease.

 Means for solving the problem

[0010] As a result of repeated studies in view of the above problems, the present inventors have determined the structure of a DJ-1 derivative, and thereby established a useful technique for diagnosing biological oxidative stress or a disease based thereon.

 [0011] That is, the present invention relates to the following inventions.

 1. A DJ-1 derivative in which the cysteine residue at position 106 in DJ-1 is oxidized to cysteinesulfonic acid and Z or cysteinesulfonic acid.

 2. An antibody against the DJ-1 derivative according to item 1.

3. The stress marker according to item 1, which is a DJ-1 derivative. 4. In samples from humans or mammals,

 (3) a step of measuring the amount of the DJ-1 derivative according to item 1; or

 (4) Step of measuring the ratio of DJ-1 derivative to DJ-1 according to item 1

 A method for evaluating an oxidative stress state in a human or mammal, comprising:

 5. A disease marker for a neurodegenerative disease comprising the DJ-1 derivative according to item 1 or 2.

6. The mask according to item 5, wherein the neurodegenerative disease is Parkinson's disease or Alzheimer's disease.

'~~ power'

 7. In human-derived samples,

 (1) the step of measuring the amount of the DJ-1 derivative according to item 1; or

 (2) Step of measuring the ratio of DJ-1 derivative to DJ-1 according to item 1

 A method for diagnosing a neurodegenerative disease in a human, comprising:

 8. The method according to Item 7, wherein the neurodegenerative disease is Parkinson's disease or Alzheimer's disease.

 The invention's effect

 [0012] According to the present invention, the structure of DJ-1, which is an index of neurodegenerative diseases such as oxidative stress and Parkinson's disease, is determined, so that the degree of these pathological conditions can be easily evaluated. I came to be able to.

 BEST MODE FOR CARRYING OUT THE INVENTION

[0013] In the present specification, human DJ-1 is a protein having the amino acid sequence shown in SEQ ID NO: 1. In the case of human DJ-1, the DJ-1 derivative has the cysteine residue at position 106 (Cys) oxidized to cysteinesulfonic acid (Cys (SOH) and Z or cysteinesulfonic acid (Cys (SOH)). Oxidized derivatives (Cys (SOH) ^1Q6 DJ_1) and (Cys (SOH) ^iee DJ_l) In the case of mammals other than humans, the cysteine residue corresponding to position 106 of DJ-1 is an acid. It means an oxidized DJ-1 derivative that has been subjected to an oxidation.

[0014] In the following, a description will be given of human DJ-1 as an example of DJ-1. Then, those skilled in the art can easily implement the present invention. [0015] Oxidative stress causes the cysteine residue at position 106 of DJ-1 to be oxidized to cysteine sulfonic acid or cysteine sulfinic acid, an intermediate thereof, to form an oxidized DJ-1 derivative. This oxidation is due to other peroxides, such as tert-butyl hydroperoxide, which are not limited to the hydrogen peroxide present in the body, and biologically-derived peroxides, such as PLPC-OOH (a peroxide of phosphatidylcholine). It has been confirmed by the present inventor that the treatment proceeds even when the dang lipid is used. Therefore, this oxidation of DJ-1 occurs under physiological conditions. Although not shown as data, the present inventor has found that the oxidized form of DJ-1 Cys (SOH) ^{1 ()} 6DJ-1 is present in the blood of patients with neurodegenerative diseases such as Alzheimer's disease and healthy subjects. The present inventors have confirmed that the abundance of this oxidized DJ-1 tends to increase with age.

 [0016] The antibody (monoclonal antibody or polyclonal antibody) recognizing the DJ-1 derivative of the present invention can be used to immunize a suitable milk animal such as rat, mouse, rabbit, and goat using the DJ-1 derivative as an immunogen. Conventional methods, such as collecting antibody-producing cells of the immunized mammal, screening cells producing monoclonal or polyclonal antibodies as necessary, and fusing the obtained immunized cells with mammalian myeloma cells. Obtained according to

 [0017] The antibody can specifically recognize an oxidized DJ-1 derivative as long as it recognizes cysteinesulfonic acid at position 106.

 [0018] Human-derived samples for measuring the amount of the DJ-1 derivative or the ratio of the DJ-1 derivative to DJ-1 include blood, urine, saliva, lymph, and the like, preferably blood. .

 [0019] The amount of the DJ-1 derivative or the ratio of the DJ-1 derivative to DJ-1 is measured using an antibody to the DJ-1 derivative and, if necessary, an antibody to DJ-1 by an assay such as ELISA. Preferably, V can be performed by two-dimensional electrophoresis.

 [0020] According to the present invention, the degree of oxidative stress and the neurodegenerative diseases that can be diagnosed by the DJ-1 oxidized derivative include Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), spinal cord Examples include muscular atrophy syndrome (SMA), Huntington's disease, and dementia following cerebrovascular disease.

[0021] Regarding the relationship between the strength of oxidative stress and neurodegenerative diseases (especially Parkinson's disease and Alzheimer's disease), the amount (concentration) of a DJ-1 derivative or the induction of DJ-1 in the form of an iridescent type against DJ-1 The higher the proportion of conductors, the more oxidative stress is the force and It is considered that the efficiency is high.

[0022] When measuring the degree of oxidative stress, it is only necessary to measure the amount of DJ-1 in which position 106 is oxidized to cysteine sulfonic acid and Z or cysteine sulfinic acid. This can also be used to diagnose Parkinson's disease. Can be.

 Example

 Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples.

 Example 1

 (1) Sample preparation

 Cultured Human Umbilical Vein Endothelial Cell:

HUVEC, a primary culture from Sanko Junyaku) was cultured in a dish with a diameter of 6 cm. Using EGM-2 (manufactured by Sanko Junyaku) as a culture medium, culture in a CO incubator at 37 ° C and 5% CO. Cells when they become 4 passages to 80- 90% Konfuruento was continued and 3.4 _mu 1 added 88.2 mM of hydrogen peroxide in the medium (final concentration 100 mu Micromax) cultured in 1 hour CO incubator . The cells subjected to the hydrogen peroxide treatment were collected, washed with a phosphate buffer, and subjected to two-dimensional electrophoresis.

 (2) Two-dimensional electrophoresis

 The collected cells are dissolved in 280 μl of isoelectric focusing solution (9 M urea, 2% CHAPS, 65 mM DTE, 0.5% IPG Buffer (Amersham Bioscience)), and centrifuged at 15000 rpm for 20 min to remove insoluble components. Except 250 μl of Immobiline Dry Strip (13 cm long, pi range 4-7,

Amersham Bioscience) and swelled for 12 hours. After swelling, isoelectric focusing (first dimension) was performed with a time program of 500 V for 1 hour, 1000 V for 1 hour, and 8000 V for 6 hours using an IPGPhor electrophoresis apparatus (Amersham Bioscience). After the electrophoresis, the gel subjected to isoelectric focusing was equilibrated with 50 mM Tris-HCl (pH 6.8), 6 M urea, 30% glycerol, 2% SDS, 20 mM DTE, and electrophoresed on 12.5% SDS polyacrylamide gel. For the second dimension electrophoresis. The two-dimensional electrophoresis gel was fluorescently stained with SyproRuby (Molecular Probes) to visualize the spots, and the spots that moved depending on the presence or absence of hydrogen peroxide load on HUVEC were cut out. [0024] The sample before hydrogen peroxide addition (control) was subjected to two-dimensional electrophoresis in the same manner. The results are shown in Figure 1.

 (3) Protein identification and structural analysis by mass spectrometry

 The spots that responded to hydrogen peroxide loading (the positions of which moved on two-dimensional electrophoresis) were converted into peptide fragments by in-gel digestion using trypsin. This peptide mixture was introduced into a nanospray ionization LC-MS / MS system. (Solvent A: 0.1% formic acid, 5% aceto-tolyl aqueous solution; solvent B: 0.1% formic acid, 98% acetonitrile aqueous solution; 5% power of solvent A up to 65% with a linear gradient of 40 μm for 40 min. The column was subjected to measurement in a data dependent scan mode by an ion trap mass spectrometer (LCQ-DECA, ThermoElectron) through a reversed-phase C18 column (MAGIC C18, MichromBioresources). ) The measurement data was used for protein identification using the MASCOT system (Matrix Science) in combination with peptide-mass fingerprinting and MS / MS data search. These database searches identified 68% and 93% of the native and DJ-1 derivative tryptic digestion fragments.

 [0025] As a result, the structure of a spot (DJ-1 derivative) that appeared on the acidic side on two-dimensional electrophoresis was determined by LC-MS / MS. The residue was found to be oxidized to cysteine sulfonic acid (Cys-SO H).

Claims

The scope of the claims  [1] A DJ-1 derivative in which the cysteine residue at position 106 in DJ-1 is oxidized to cysteine sulfonic acid and / or cystine sulphinic acid. [2] An antibody against the DJ-1 derivative according to claim 1. [3] An oxidative stress marker comprising the DJ-1 derivative according to claim 1. [4] In a sample derived from a human or mammal,  (1) a step of measuring the amount of the DJ-1 derivative according to claim 1; or  (2) a step of measuring the ratio of the DJ-1 derivative and DJ-1 according to claim 1  A method for evaluating an oxidative stress state in a human or mammal, comprising:  [5] The disease marker for a neurodegenerative disease according to claim 1 or 2, wherein the DJ-1 derivative is a neurodegenerative disease.  [6] The method according to claim 5, wherein the neurodegenerative disease is Parkinson's disease or Alzheimer's disease. '~~ power'.  [7] In human-derived samples,  (1) a step of measuring the amount of the DJ-1 derivative according to claim 1; or  (2) a step of measuring the ratio of the DJ-1 derivative and DJ-1 according to claim 1  A method for diagnosing a neurodegenerative disease in a human, comprising: [8] The method according to claim 7, wherein the neurodegenerative disease is Parkinson's disease or Alzheimer's disease.