JP6475550B2 - Method and kit for detecting phospholipid hydroperoxide-dependent cell death, inhibitor of phospholipid hydroperoxide-dependent cell death, and preventive or therapeutic agent for diseases associated with phospholipid hydroperoxide-dependent cell death - Google Patents

Description

  The present invention relates to a method and kit for detecting phospholipid hydroperoxide-dependent cell death, an inhibitor of phospholipid hydroperoxide-dependent cell death, and a preventive or therapeutic agent for diseases associated with phospholipid hydroperoxide-dependent cell death.

  The phospholipid constituting the biological membrane has an unsaturated fatty acid at the 2-position. As shown in FIG. 1, it is known that a phospholipid hydroperoxide is produced when a hydroxy radical generated from superoxide or hydrogen peroxide reacts with the unsaturated fatty acid by a Fenton reaction via iron.

  Phospholipid hydroperoxide glutathione peroxidase (hereinafter sometimes referred to as “PHGPx”) is an enzyme that directly reduces the produced phospholipid hydroperoxide and converts it into a phospholipid hydroxy form using glutathione as a coenzyme. is there. This PHGPx gene knockout mouse was known to be embryonic lethal (see, for example, Non-Patent Document 1). However, the mechanism of cell death due to PHGPx deficiency is unknown.

Imai H, et al., Early embryonic lethality caused by targeted disruption of the mouse PHGPx gene, Biochem Biophys Res Commun., 305, 278-86, 2003.

  The present invention clarifies factors involved in the pathway of cell death caused by PHGPx deficiency, and a method and kit for detecting the cell death (hereinafter referred to as “phospholipid hydroperoxide-dependent cell death”), phospholipid hydroperoxide dependency An object of the present invention is to provide a cell death inhibitor and a preventive or therapeutic agent for a disease associated with phospholipid hydroperoxide-dependent cell death.

The present invention is as follows.
(1) A step of detecting an activation state of at least one protein selected from the group consisting of a proteasome-related protein, a heat shock protein, an iron / sulfur cluster-related protein, a nucleolar protein, and a WD repeat family protein in a cell. A method for detecting phospholipid hydroperoxide-dependent cell death, comprising:
(2) The detection includes measuring the intracellular localization of the protein, measuring the expression level of the protein at the mRNA level, measuring the expression level of the protein at the protein level, The detection method according to (1), wherein the detection method is performed by at least one selected from the group consisting of measuring a phosphorylation rate of a protein and measuring suppression of cell death due to suppression of protein expression.
(3) The detection method according to (1) or (2), wherein the proteasome-related protein is at least one protein selected from the group consisting of Rbx1, Ube2d1, and Ube2a.
(4) The detection method according to any one of (1) to (3), wherein the heat shock protein is Dnajc9.
(5) The detection method according to any one of (1) to (4), wherein the iron / sulfur cluster-related protein is Nubp2.
(6) The detection method according to any one of (1) to (5), wherein the nucleolar protein is Fbl.
(7) The detection method according to any one of (1) to (6), wherein the WD repeat family protein is Wdfy1.
(8) An antibody against at least one protein selected from the group consisting of phosphorylated or non-phosphorylated proteasome-related protein, heat shock protein, iron / sulfur cluster-related protein, nucleolar protein, and WD repeat family protein , A primer for amplifying mRNA of at least one protein selected from the group consisting of proteasome-related protein, heat shock protein, iron / sulfur cluster-related protein, nucleolar protein and WD repeat family protein, or proteasome-related protein, heat shock protein , MRNA of at least one protein selected from the group consisting of iron-sulfur cluster-related proteins, nucleolar proteins and WD repeat family proteins Against, siRNA, shRNA, ribozymes or antisense nucleic acids comprise a kit for detecting phospholipid hydroperoxide-dependent cell death.
(9) The kit according to (8), wherein the proteasome-related protein is at least one selected from the group consisting of Rbx1, Ube2d1, and Ube2a.
(10) The kit according to (8) or (9), wherein the heat shock protein is Dnajc9.
(11) The kit according to any one of (8) to (10), wherein the iron / sulfur cluster-related protein is Nubp2.
(12) The kit according to any one of (8) to (11), wherein the nucleolar protein is Fbl.
(13) The kit according to any one of (8) to (12), wherein the WD repeat family protein is Wdfy1.
(14) siRNA, shRNA, ribozyme or antisense against mRNA of at least one protein selected from the group consisting of proteasome-related protein, heat shock protein, iron / sulfur cluster-related protein, nucleolar protein and WD repeat family protein A phospholipid hydroperoxide-dependent cell death inhibitor comprising a nucleic acid as an active ingredient.
(15) The inhibitor of phospholipid hydroperoxide-dependent cell death according to (14), wherein the proteasome-related protein is at least one protein selected from the group consisting of Rbx1, Ube2d1 and Ube2a.
(16) The inhibitor of phospholipid hydroperoxide-dependent cell death according to (14) or (15), wherein the heat shock protein is Dnajc9.
(17) The inhibitor of phospholipid hydroperoxide-dependent cell death according to any one of (14) to (16), wherein the iron / sulfur cluster-related protein is Nubp2.
(18) The inhibitor of phospholipid hydroperoxide-dependent cell death according to any one of (14) to (17), wherein the nucleolar protein is Fbl.
(19) The inhibitor of phospholipid hydroperoxide-dependent cell death according to any one of (14) to (18), wherein the WD repeat family protein is Wdfy1.
(20) siRNA, shRNA, ribozyme or antisense against mRNA of at least one protein selected from the group consisting of proteasome-related protein, heat shock protein, iron / sulfur cluster-related protein, nucleolar protein and WD repeat family protein A preventive or therapeutic agent for a disease associated with phospholipid hydroperoxide-dependent cell death, comprising a nucleic acid as an active ingredient.
(21) The prophylactic or therapeutic agent according to (20), wherein the proteasome-related protein is at least one protein selected from the group consisting of Rbx1, Ube2d1, and Ube2a.
(22) The preventive or therapeutic agent according to (20) or (21), wherein the heat shock protein is Dnajc9.
(23) The preventive or therapeutic agent according to any one of (20) to (22), wherein the iron / sulfur cluster-related protein is Nubp2.
(24) The prophylactic or therapeutic agent according to any one of (20) to (23), wherein the nucleolar protein is Fbl.
(25) The preventive or therapeutic agent according to any one of (20) to (24), wherein the WD repeat family protein is Wdfy1.
(26) The preventive or therapeutic agent according to any one of (20) to (25), wherein the disease associated with phospholipid hydroperoxide-dependent cell death is heart failure.

  According to the present invention, a method and a kit for detecting phospholipid hydroperoxide-dependent cell death can be provided. In addition, an inhibitor of phospholipid hydroperoxide-dependent cell death can be provided. In addition, a prophylactic or therapeutic agent for a disease associated with phospholipid hydroperoxide-dependent cell death can be provided.

It is a figure explaining a biological membrane new lipid oxidation reaction and its metabolic pathway. It is a photograph which shows the result of the western blotting which detects PHGPx protein. It is a graph which shows a time-dependent change of the quantity of the phospholipid hydroperoxide (The hydroperoxide derived from 18: 0, 20: 4 phosphatidylcholine) produced | generated in PHGPx deficient MEF cell. It is a graph which shows the result of the MTT assay in which PHGPx-deficient MEF cells are lethal when tamoxifen is added. (A) is a graph showing the results of cell viability (%) showing the inhibitory effect of phospholipid hydroperoxide-dependent cell death when an apoptosis inhibitor is used. (B) is a photograph showing the result of fluorescent immunostaining of an index of apoptosis during staurosporine treatment and tamoxifen treatment. (A) is a photograph showing the result of fluorescent immunostaining of HMGB1 30 minutes after addition of tert-butyl hydroperoxide, and FIG. 6 (b) is a photograph showing the result of fluorescent immunostaining of HMGB1 48 hours after addition of tamoxifen. It is. (A) is a graph showing the results of cell viability (%) for phospholipid hydroperoxide-dependent cell death by knockdown of autophagy-related factor ATG5. (B) is a photograph showing the results of suppression of ATG5 expression by RT-PCR. (A) is a graph showing the results of cell viability (%) for phospholipid hydroperoxide-dependent cell death by knockdown cells of necrotosis-related factor Rip1. (B) is a photograph showing the result of Rip1 expression reduction by Western blotting. It is a model figure of the phospholipid hydroperoxide dependent cell death pathway which became clear by analysis of inventors. It is a figure which shows the outline | summary of the screening procedure of shRNA library. It is a graph which shows the result arranged about 151 candidate genes in order with the high inhibitory effect of phospholipid hydroperoxide dependence cell death by knockdown. It is a graph which shows the result of the cell viability (%) with respect to the phospholipid hydroperoxide dependent cell death by knockdown of Rbx1. (A) is a figure which shows the structure of a piMG-drU6 vector. (B) is a model diagram showing a process in which shRNA is expressed, siRNA is formed, and target RNA is destroyed. It is a figure which shows the structure of a pMXs-IR vector. It is a graph which shows the result of the cell viability (%) with respect to the phospholipid hydroperoxide dependent cell death by knockdown of Ube2d1. It is a graph which shows the result of the cell viability (%) with respect to the phospholipid hydroperoxide dependent cell death by knockdown of Ube2a. It is a graph which shows the result of the cell viability (%) with respect to the phospholipid hydroperoxide dependent cell death by knockdown of Dnajc9. It is a graph which shows the result of the cell viability (%) with respect to the phospholipid hydroperoxide dependent cell death by Knup2 knockdown. It is a graph which shows the result of the cell viability (%) with respect to the phospholipid hydroperoxide dependent cell death by knockdown of Fbl. It is a graph which shows the result of the cell viability (%) with respect to the phospholipid hydroperoxide dependent cell death by knockdown of Wdfy1. (A) is a photograph of wild-type mice (Control) and cardiac-specific PHGPx-deficient mice (KO) on development day 17.5 and 18.5. (B) is a photograph showing the result of DAPI and TUNEL staining (TUNEL) of a tissue section of the heart of a mouse on the 17.5th day of development. (C) is a photograph showing the result of detecting the expression of PHGPx protein in the heart of a mouse on the 17.5th day of development by Western blotting. (A)-(c) is a photograph of a mouse fetus on the developmental process 18.5 days when the effect of 3-ATA was examined. (A) is an explanatory diagram of a model mouse of sudden arrhythmic death (heart failure) caused by a decrease in the amount of vitamin E in the diet, and shows that sudden death occurs in about 10 days when a vitamin E-added diet is changed to a normal diet It is a graph which shows the survival rate of a mouse | mouth. (B) is a graph showing the results of measuring the amount of vitamin E in the heart. (C) is a graph showing an electrocardiogram of a mouse. It is the graph which showed the inhibitory effect of 3-ATA with respect to the sudden death mouse model by the heart failure caused by the vitamin E fall. It is the graph which showed the inhibitory effect of U-0126 with respect to the sudden death mouse model by the heart failure caused by the vitamin E fall.

[Method for detecting phospholipid hydroperoxide-dependent cell death]
In one embodiment, the present invention relates to the activity of at least one protein selected from the group consisting of a proteasome-related protein, a heat shock protein, an iron / sulfur cluster-related protein, a nucleolar protein, and a WD repeat family protein in a cell. A method for detecting phospholipid hydroperoxide-dependent cell death comprising the step of detecting a phosphorylation state is provided.

  As described later, the inventors have found cell death (phospholipid hydroperoxide-dependent cell death) by a novel pathway distinct from apoptosis, necrosis, autophagic cell death, and necrosis. Furthermore, it was clarified that proteasome-related proteins, heat shock proteins, iron / sulfur cluster-related proteins, nucleolar proteins and WD repeat family proteins are involved in phospholipid hydroperoxide-dependent cell death.

  In the detection method of the present embodiment, as proteasome-related proteins, Rbx1 (ring-box 1, GenBank accession number: BC027396), Ube2d1 (ubiquitin-conjugating enzyme E2D1, GenBank accession number: BC019464u, Ub194e) conjugating enzyme E2A, GenBank accession number: NM019686), and the like.

  Rbx1 protein is known to interact with cullin. Rbx1 protein has been shown to play a unique role in the ubiquitination reaction and catalyze the polymerization of ubiquitin by forming a heterodimer with curin-1. It has also been pointed out that it may be involved in the regulation of protein turnover.

  Ube2d1 protein is a ubiquitin E2 ligase involved in ubiquitination during the degradation of proteins with fast turnover. It has been reported to be involved in the metabolism of P53 and HIF-1α. Ube2a protein is ubiquitin E2 ligase.

  In the detection method of this embodiment, examples of the heat shock protein include Dnajc9 (DnaJ (Hsp40) homolog, subfamily C, member 9, GenBank accession number: NM_134081). The Dnajc9 protein has been reported to interact with HSP70 and act as a chaperone.

  In the detection method of this embodiment, examples of the iron / sulfur cluster-related protein include Nubp2 (nucleoside binding protein 2, GenBank accession number: NM_011956). Nubp2 protein is a member of the NUBP / MRP superfamily and is an ATP-binding protein. Known as Cytosolic Fe-S cluster assembly factor.

  In the detection method of the present embodiment, examples of the nucleolar protein include Fbl (rRNA 2'-O-methyltransferase fibrialin, GenBank accession number: NM_007991). The Fbl protein is involved in the excision of the first step of preribosomal RNA (pre-rRNA) and is present in the nucleolus. It has a glycine- and arginine-rich domain at the N-terminal part.

  In the detection method of the present embodiment, examples of the WD repeat family protein include Wdfy1 (WD repeat and FYVE domain containing 1, GenBank accession number: NM_001111279). The FYVE domain of Wdfy1 protein is known to mediate transport to a membrane containing PI3P (phosphatidylinositol triphosphate). Wdfy1 protein is said to localize in endosomes.

  By detecting the activation state of these proteins in the cell, phospholipid hydroperoxide-dependent cell death can be detected. According to the method of this embodiment, it is possible to detect whether or not phospholipid hydroperoxide-dependent cell death is in progress, and after cell death has occurred, the cell death is a phospholipid hydroperoxide-dependent cell death pathway. It is also possible to determine whether or not it was through.

  Examples of the cells include human, mouse, rat, guinea pig, hamster, rabbit, goat, pig, dog, cat, monkey and the like. In these cells, as a protein for detecting the activation state, a homologue in each species of the protein described above may be detected.

  The detection of the activation state of the protein includes, for example, measuring the intracellular localization of the protein, measuring the expression level of the protein at the mRNA level, and determining the expression level of the protein at the protein level. It can be performed by at least one selected from the group consisting of measuring, measuring the phosphorylation rate of the protein, and measuring suppression of cell death due to suppression of expression of the protein.

  The intracellular localization state can be measured, for example, by fixing a target cell, staining with a fluorescently labeled antibody against the above factor, and observing with a fluorescent microscope. Alternatively, the localization state of the factor in the cell can be measured by expressing a fusion protein of the factor and the fluorescent protein in the target cell and observing the fluorescence of the fusion protein with a fluorescence microscope. Here, examples of the fluorescent protein include green fluorescent protein (GFP), blue fluorescent protein (BFP), yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), and red fluorescent protein (RFP).

  As a result of measuring the localization state of the protein in the cell, for example, when a change is observed in the localization state of the protein compared to normal cells in which phospholipid hydroperoxide-dependent cell death is not in progress, Phospholipid hydroperoxide-dependent cell death can be detected.

  The expression level of the protein at the mRNA level can be measured by, for example, real-time PCR, Northern blotting, or the like. For example, phospholipid hydroperoxide-dependent cell death is detected when there is a change in the expression level of the protein at the mRNA level compared to normal cells in which phospholipid hydroperoxide-dependent cell death is not ongoing be able to.

  The expression level of the protein at the protein level can be measured, for example, by Western blotting using an antibody against the protein. For example, phospholipid hydroperoxide-dependent cell death is detected when there is a change in the expression level of the protein at the protein level compared to normal cells in which phospholipid hydroperoxide-dependent cell death is not ongoing be able to.

  The phosphorylation rate of the protein can be measured, for example, by Western blotting using an antibody specific for the phosphorylated factor. For example, phospholipid hydroperoxide-dependent cell death can be detected when there is a change in the phosphorylation rate of the protein compared to normal cells in which phospholipid hydroperoxide-dependent cell death is not ongoing .

  Moreover, when cell death can be suppressed by suppressing the expression of the protein, it can be determined that the protein is involved in the cell death. Therefore, in such a case, phospholipid hydroperoxide-dependent cell death can be detected.

  Suppression of the expression of the protein can be performed, for example, by introducing siRNA, shRNA, ribozyme, antisense nucleic acid or the like against the factor into the cell. Moreover, the suppression of cell death can be measured, for example, by measuring the cell viability when the expression of the protein is suppressed or not. The cell viability can be measured by, for example, calculating the change in the number of living cells by MTT assay or microscopic photography.

  siRNA (small interfering RNA) is a small double-stranded RNA of 21 to 23 base pairs used for gene silencing by RNA interference. The siRNA introduced into the cell binds to the RNA-induced silencing complex (RISC). This complex binds to and cleaves mRNA having a sequence complementary to siRNA. This suppresses gene expression in a sequence-specific manner.

  For siRNA, sense strand and antisense strand oligonucleotides are respectively synthesized by a DNA / RNA automatic synthesizer and denatured at about 90 to about 95 ° C. for about 1 minute in an appropriate annealing buffer, and then about 30 minutes. It can be prepared by annealing at ˜70 ° C. for about 1-8 hours.

  shRNA (short hairpin RNA) is a hairpin RNA sequence used for gene silencing by RNA interference. shRNA may be introduced into cells by a vector and expressed by U6 promoter or H1 promoter, or an oligonucleotide having shRNA sequence may be synthesized by a DNA / RNA automatic synthesizer and self-annealed in the same manner as siRNA. May also be prepared. The shRNA hairpin structure introduced into the cell is cleaved into siRNA and binds to the RNA-induced silencing complex (RISC). This complex binds to and cleaves mRNA having a sequence complementary to siRNA. This suppresses gene expression in a sequence-specific manner.

  Ribozymes are RNAs that have catalytic activity. Although some ribozymes have various activities, research on ribozymes as enzymes that cleave RNA has made it possible to design ribozymes for the purpose of site-specific cleavage of RNA. The ribozyme may be a group I intron type, a size of 400 nucleotides or more such as M1 RNA contained in RNaseP, or may be about 40 nucleotides called a hammerhead type, a hairpin type, or the like.

  An antisense nucleic acid is a nucleic acid that is complementary to a target sequence. Antisense nucleic acid inhibits transcription initiation by triplex formation, suppresses transcription by hybridization with a site where an open loop structure is locally formed by RNA polymerase, inhibits transcription by hybridization with RNA that is being synthesized, Inhibition of splicing by hybridization at the junction of intron and exon, suppression of splicing by hybridization with spliceosome formation site, suppression of transition from nucleus to cytoplasm by hybridization with mRNA, capping site and poly (A) addition site Suppression of splicing by hybridization with a protein, suppression of translation initiation by hybridization with a translation initiation factor binding site, suppression of translation by hybridization with a ribosome binding site near the initiation codon, translation region of mRNA and polysome binding site Hybridization outgrowth inhibitory peptide chain by the, by gene silencing due hybridization interaction site between a nucleic acid and a protein, it is possible to suppress the expression of a target gene.

  In this embodiment, siRNA, shRNA, ribozyme, and antisense nucleic acid may contain various chemical modifications in order to improve stability and activity. For example, in order to prevent degradation by a hydrolase such as nuclease, the phosphate residue may be substituted with a chemically modified phosphate residue such as phosphorothioate (PS), methylphosphonate, phosphorodithionate, and the like. Moreover, at least one part may be comprised with nucleic acid analogs, such as a peptide nucleic acid (PNA).

[Kit for detection of phospholipid hydroperoxide-dependent cell death]
In one embodiment, the present invention is selected from the group consisting of phosphorylated or non-phosphorylated proteasome related proteins, heat shock proteins, iron / sulfur cluster related proteins, nucleolar proteins and WD repeat family proteins. An antibody against at least one protein; a primer for amplifying mRNA of at least one protein selected from the group consisting of a proteasome-related protein, a heat shock protein, an iron-sulfur cluster-related protein, a nucleolar protein, and a WD repeat family protein; or a proteasome At least one selected from the group consisting of related proteins, heat shock proteins, iron / sulfur cluster related proteins, nucleolar proteins, and WD repeat family proteins For the mRNA of the protein, siRNA, shRNA, ribozymes or antisense nucleic acids; comprises, provides a kit for the detection of phospholipid hydroperoxide-dependent cell death.

  In the kit of this embodiment, examples of the proteasome-related protein include Rbx1, Ube2d1, Ube2a, and the like. Examples of heat shock proteins include Dnajc9. Examples of the iron / sulfur cluster-related protein include Nubp2. Examples of the nucleolar protein include Fbl. Examples of the WD repeat family protein include Wdfy1.

  An antibody against the above-mentioned protein that is phosphorylated or non-phosphorylated is suitable for measuring the intracellular localization of the protein, measuring the expression level of the protein at the protein level, measuring the phosphorylation rate of the protein, etc. It is.

  The primer for amplifying mRNA of the protein is suitable for measuring the expression level of the protein at the mRNA level.

  SiRNA, shRNA, ribozyme or antisense nucleic acid against the protein is suitable for suppressing the expression of the protein.

  By using the kit of the present embodiment, phospholipid hydroperoxide-dependent cell death in a target cell can be easily detected.

[Inhibitor of phospholipid hydroperoxide-dependent cell death]
In one embodiment, the present invention provides siRNA against mRNA of at least one protein selected from the group consisting of proteasome-related protein, heat shock protein, iron-sulfur cluster-related protein, nucleolar protein, and WD repeat family protein, Disclosed is a phospholipid hydroperoxide-dependent cell death inhibitor comprising shRNA, ribozyme or antisense nucleic acid as an active ingredient.

  As will be described later, the inventors have revealed that proteasome-related proteins, heat shock proteins, iron-sulfur cluster-related proteins, nucleolar proteins, and WD repeat family proteins are involved in phospholipid hydroperoxide-dependent cell death. did.

  Therefore, expression inhibitors of proteasome-related proteins, heat shock proteins, iron / sulfur cluster-related proteins, nucleolar proteins and WD repeat family proteins can be used as inhibitors of phospholipid hydroperoxide-dependent cell death.

  In the inhibitor of this embodiment, examples of proteasome-related proteins include Rbx1, Ube2d1, Ube2a, and the like. Examples of heat shock proteins include Dnajc9. Examples of the iron / sulfur cluster-related protein include Nubp2. Examples of the nucleolar protein include Fbl. Examples of the WD repeat family protein include Wdfy1.

  In the inhibitor of this embodiment, siRNA, shRNA, ribozyme or antisense nucleic acid is selected from the group consisting of proteasome-related proteins, heat shock proteins, iron / sulfur cluster-related proteins, nucleolar proteins, and WD repeat family proteins. There is no particular limitation as long as the expression of at least one protein can be suppressed.

  In the inhibitor of the present embodiment, the dosage of siRNA, shRNA, ribozyme or antisense nucleic acid is preferably 50 nM to 10 μM, for example 50 to 200 nM, when added to the culture medium of cultured cells. Moreover, when administering to an animal, it is preferable to administer 50-500 mg / kg body weight once a day, for example.

  In the inhibitor of the present embodiment, siRNA, shRNA, ribozyme or antisense nucleic acid may be administered as it is or formulated as a pharmaceutical composition mixed with appropriate pharmacologically acceptable additives. Things may be administered.

(Pharmaceutical composition)
Examples of the dosage form of the pharmaceutical composition include injections. The pharmaceutical composition may contain vehicles such as cationic lipids, additives such as stabilizers, antioxidants, preservatives, and the like. In addition, the pharmaceutical composition may be in a powder state from which the solvent is removed by freeze drying or the like, or in a liquid state. When the pharmaceutical composition is in powder form, it can be suspended or dissolved in a pharmaceutically acceptable medium before use and used as an injection. When the pharmaceutical composition is in a liquid state, it can be used as an injection as it is or suspended or dissolved in a pharmaceutically acceptable medium.

  Examples of pharmaceutically acceptable media include isotonic solutions containing, for example, physiological saline, glucose, and other adjuvants such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride. An agent such as alcohol, specifically ethanol, polyalcohol such as propylene glycol, polyethylene glycol, a nonionic surfactant such as polysorbate 80 (trademark), HCO-50 may be used in combination.

  Administration of the composition to a patient can be performed, for example, by intraarterial injection, intravenous injection, subcutaneous injection, or the like. The dose of the composition varies depending on the administration subject, target organ, symptom, administration method and the like, but those skilled in the art can appropriately select an appropriate dose.

  Suitable examples of the therapeutic effect expected from the inhibitor of this embodiment include heart failure and sudden arrhythmic death.

[Prophylactic or therapeutic agent for diseases associated with phospholipid hydroperoxide-dependent cell death]
In one embodiment, the present invention provides siRNA against mRNA of at least one protein selected from the group consisting of proteasome-related protein, heat shock protein, iron-sulfur cluster-related protein, nucleolar protein, and WD repeat family protein, Provided is a prophylactic or therapeutic agent for a disease associated with phospholipid hydroperoxide-dependent cell death, comprising shRNA, ribozyme or antisense nucleic acid as an active ingredient.

  In another embodiment, the present invention provides a proteasome-related protein, a heat shock protein, an iron-sulfur cluster-related protein, a nucleolus protein, and a proteasome-related protein for the prevention or treatment of a disease associated with phospholipid hydroperoxide-dependent cell death. Provided is a siRNA, shRNA, ribozyme or antisense nucleic acid against mRNA of at least one protein selected from the group consisting of WD repeat family proteins.

  In another embodiment, the present invention provides a proteasome-related protein, a heat shock protein, an iron / sulfur cluster-related protein, a nucleolus for the manufacture of a prophylactic or therapeutic agent for a disease associated with phospholipid hydroperoxide-dependent cell death. Provided is the use of siRNA, shRNA, ribozyme or antisense nucleic acid for mRNA of at least one protein selected from the group consisting of proteins and WD repeat family proteins.

  In another embodiment, the present invention provides a proteasome-related protein, a heat shock protein, an iron / sulfur cluster-related protein, a nucleolus for the manufacture of a prophylactic or therapeutic agent for a disease associated with phospholipid hydroperoxide-dependent cell death. A phospholipid comprising administering to a patient in need of treatment an effective amount of siRNA, shRNA, ribozyme or antisense nucleic acid against mRNA of at least one protein selected from the group consisting of protein and WD repeat family protein Provided are methods for preventing or treating diseases associated with hydroperoxide-dependent cell death.

  As will be described later, the inventors have revealed that proteasome-related proteins, heat shock proteins, iron-sulfur cluster-related proteins, nucleolar proteins, and WD repeat family proteins are involved in phospholipid hydroperoxide-dependent cell death. did. Therefore, it consists of proteasome-related protein, heat shock protein, iron / sulfur cluster-related protein, nucleolar protein, and WD repeat family protein for the manufacture of preventive or therapeutic agents for diseases related to phospholipid hydroperoxide-dependent cell death SiRNA, shRNA, ribozyme or antisense nucleic acid against mRNA of at least one protein selected from the group can be used as a preventive or therapeutic agent for diseases associated with phospholipid hydroperoxide-dependent cell death.

  In the prophylactic or therapeutic agent of this embodiment, examples of the proteasome-related protein include Rbx1, Ube2d1, Ube2a, and the like. Examples of heat shock proteins include Dnajc9. Examples of the iron / sulfur cluster-related protein include Nubp2. Examples of the nucleolar protein include Fbl. Examples of the WD repeat family protein include Wdfy1.

  For siRNA, shRNA, ribozyme or antisense nucleic acid against proteasome-related protein, heat shock protein, iron / sulfur cluster-related protein, nucleolar protein or WD repeat family protein, it may be administered itself or an appropriate drug Formulated as a pharmaceutical composition mixed with a physically acceptable additive may be administered. Preferred forms of the pharmaceutical composition are the same as those described above.

  Examples of the above-mentioned diseases associated with phospholipid hydroperoxide-dependent cell death include heart failure and sudden arrhythmic death.

  EXAMPLES Next, although an Example is shown and this invention is demonstrated further in detail, this invention is not limited to a following example.

[Experimental Example 1]
(Establishment of Tamoxifen-induced PHGPx-deficient MEF cell line)
In order to elucidate the mechanism of PHGPx-deficient cell death, a tamoxifen-induced PHGPx-deficient MEF cell line was established. In this cell, the PHGPx genomic gene is deleted by the Cre-loxP system within 24 hours when tamoxifen is added to the medium.

Specifically, a PHGPx gene (loxP-PHGPx) sandwiched between loxP sequences was introduced (Tg) into a PHGPx gene knockout mouse (PHGPx ^{− / −} ), and Tg (loxP-PHGPx): PHGPx ^{− / −} mouse And PHGPx ^+/− mice were mated. Subsequently, mouse embryo fibroblasts (MEF) were prepared from 13.5 day embryos whose genotype was Tg (loxP-PHGPx): PHGPx ^{− / −} . Subsequently, the obtained cells were immortalized by introducing a T antigen gene of SV40 virus. Subsequently, the tamoxifen-inducible CreERT2 gene was introduced into the obtained cells. Thereby, a tamoxifen-induced PHGPx-deficient MEF cell line was obtained.

  CreERT2 is obtained by mutating Cre (CreER), which is a fusion protein with an estrogen receptor, so that it does not react with estrogen but reacts with tamoxifen. When tamoxifen is added to the tamoxifen-induced PHGPx-deficient MEF cell line medium, tamoxifen binds to the estrogen receptor portion of CreER2, and the nuclear translocation signal of the estrogen receptor is exposed. As a result, CreER2 moves into the nucleus and the PHGPx genomic gene sandwiched between loxPs is destroyed. The concentration of tamoxifen to be added may be about 1 μM final concentration.

[Experiment 2]
(Administration of Tamoxifen to Tamoxifen-Induced PHGPx-Deficient MEF Cell Line)
Tamoxifen with a final concentration of 1 μM was added to the medium of the Tamoxifen-induced PHGPx-deficient MEF cell line. Subsequently, cells at 0, 24 and 48 hours after tamoxifen addition were collected, and the amount of PHGPx protein in each cell sample was measured by Western blotting using an anti-PHGPx antibody.

  FIG. 2 is a photograph showing the results of Western blotting for detecting PHGPx protein. As a result, it was confirmed that the expression level of PHGPx protein decreased within 24 hours after tamoxifen administration.

[Experiment 3]
(Detection of phospholipid hydroperoxide in PHGPx-deficient cells)
Tamoxifen at a final concentration of 1 μM was added to the tamoxifen-induced PHGPx-deficient MEF cell line medium, and the phospholipid hydroperoxide produced was detected. For detection, liquid chromatography (LC) -electrospray ionization mass spectrometry (ESI-MS) / mass spectrometry (MS) was used.

  An ACQUITY UPLC apparatus (Waters) was used for liquid chromatography, and a quadrupole linear ion trap mass spectrometry system (trade name 4000 Q-TRAP, AB Sciex) was used for mass analysis. An ACQUITY UPLC ™ BEH C18 column (0.17 μm, 150 mm × 1.0 mm) was used as the liquid chromatography column.

  Samples of the total phospholipid fraction extracted from each cell at 0, 12, 24, 36 and 48 hours after addition of tamoxifen were subjected to an autosampler, and mobile phase A (acetonitrile / methanol / water = 2: 2: 1 ( v / v), 0.1% boric acid, 0.028% ammonia): mobile phase B (isopropanol, 0.1% boric acid, 0.028% ammonia) 100: 0 (0-5 min), 50:50 (5-25 min), 50:50 (25-49 min), 100: 0 (59-60 min) and 100: 0 (60-75 min) step gradient, flow rate 70 μL / min, column temperature 30 ° C. Separated by condition.

  The MS / MS analysis was performed in a negative ion mode using a MRM (Multi Reaction Monitoring) technique. The ion spray voltage was set to -4500V. Nitrogen gas was used as a barrier gas and a collision gas. In the detection of oxidized phospholipids, the collision energy was set to 20-65 eV. The scan range of the apparatus was set to m / z 50-950 and a scan speed of 1000 Th / sec. Q0 trapping was turned on and the linear ion trap filter time was set to 10 milliseconds. The declustering potential was set to -105V. The resolution of Q1 was set to “unit”. The characteristic fragmentation pattern of phosphatidylcholine hydroperoxide (from 18: 0: 20: 4) was set at a dwell time of 50 milliseconds and a declustering potential of −80V. The resolution of Q1 and O3 was set to unit, Q1 was set to 886 m / z, and O3 was set to 283 m / z (collision energy −60 eV) and 335 m / z (collision energy −45 eV).

  FIG. 3 shows that after adding tamoxifen having a final concentration of 1 μM to the tamoxifen-induced PHGPx-deficient MEF cell line, cells at 0, 12, 24, 36, and 48 hours, 18: 0 (stearic acid), 20: It is the graph which showed the production amount of phospholipid hydroperoxide (PCOOH) which is an oxidation primary product produced | generated by oxidation from phosphatidylcholine (PC) which has 4 (arachidonic acid) with time. Phospholipid hydroperoxide, a primary oxidation product of phospholipid containing arachidonic acid, was produced at a peak 24 hours after the addition of tamoxifen. Similar results were obtained with phospholipid hydroperoxide, which is a primary oxidation product of phospholipids containing DHA (not shown).

[Experimental Example 4]
(MTT assay of PHGPx-deficient cells)
Tamoxifen having a final concentration of 1 μM was added to the tamoxifen-induced PHGPx-deficient MEF cell line, and cell viability was measured by MTT assay. The same study was performed for the vitamin E addition group having a final concentration of 200 μM simultaneously with tamoxifen.

  More specifically, MTT (3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazole bromide) is added to the cell culture medium 24, 48 and 72 hours after the addition of tamoxifen, After 4 hours, each cell was dissolved in dimethyl sulfoxide (DMSO), and the absorbance at 540 nm was measured.

  FIG. 4 is a graph showing the results of the MTT assay. It was revealed that cell death occurred between 72 hours and 48 hours after the addition of tamoxifen. Moreover, it became clear that cell death was completely suppressed by addition of vitamin E.

[Experimental Example 5]
(Comparison between phospholipid hydroperoxide-dependent cell death and apoptosis)
It was examined in detail whether phospholipid hydroperoxide-dependent cell death (cell death due to PHGPx deficiency) is via a cell death pathway due to apoptosis.

  Tamoxifen-induced PHGPx-deficient MEF cell line has a final concentration of 50 μM Z-VAD-FMK, a caspase inhibitor, and a final concentration of 300 μM, DHIQ, a release inhibitor of apoptosis-inducing factor (AIF), a mitochondrial protein. An E derivative, Trolox, having a final concentration of 400 μM and a cell to which nothing was added were prepared as controls. Tamoxifen with a final concentration of 1 μM was simultaneously added to each cell. Three days after the addition of tamoxifen and inhibitors, 20 photographs were taken at random with an all-in-one microscope from Keyence, and the number of viable cells attached was counted. Observation was performed at 10 times using Plan Floor ELWD 20 × 0.45 (Nicon) as an objective lens (one visual field: 877.5 μm × 661.2 μm). The cell viability (%) was expressed as 100% when the number of cells cultured for 3 days without adding tamoxifen.

  The results are shown in FIG. Caspase inhibitors and AIF inhibitors could not suppress phospholipid hydroperoxide-dependent cell death. On the other hand, Trolox, a vitamin E derivative, was able to suppress cell death.

  Next, a tamoxifen-inducible PHGPx-deficient MEF cell line was prepared by adding an apoptosis-inducing reagent, staurosporine, at a final concentration of 0.5 μM, and tamoxifen at a final concentration of 1 μM. After 5 hours, 24 hours, and 48 hours, each cell was fluorescently immunostained to stain cytochrome C and activated caspase-3. Further, DNA fragmentation, which is a feature of apoptosis, was detected by the TUNEL (TdT-mediated dUTP nick end labeling) method.

  The results are shown in FIG. In staurosporine treatment, cytochrome C release was observed after 5 hours, caspase 3 was activated, and TUNEL-positive cell death (apoptosis) was observed after 24 hours. On the other hand, no release of cytochrome C or activation of caspase 3 was observed in phospholipid hydroperoxide-dependent cell death (the figure shows 48 hours later, but was not observed even after 60 hours). Only in TUNEL staining, dot-like staining was observed in the nucleus, but no DNA ladder was detected.

  From the above, it became clear that phospholipid hydroperoxide-dependent cell death is different from typical apoptosis.

[Experimental Example 6]
(Comparison between phospholipid hydroperoxide-dependent cell death and necrosis)
We examined in detail whether phospholipid hydroperoxide-dependent cell death was due to necrosis.

  Tamoxifen was added to the tamoxifen-induced PHGPx-deficient MEF cell line at a final concentration of 1 μM, and the moment of cell death was examined by time-lapse analysis. However, the swelling of cells characteristic of necrosis at the moment of cell death was not observed. The cell deformed just before the cell death, and finally it was peeled off from the petri dish and suddenly lethal (not shown).

  Subsequently, a tamoxifen-induced PHGPx-deficient MEF cell line was prepared by adding a necrosis-inducing agent, tert-butyl hydroperoxide, at a final concentration of 300 mM and a tamoxifen-added tamoxifen at a final concentration of 1 μM. After 30 minutes (tert-butyl hydroperoxide addition group) and 48 hours (tamoxifen addition group), each cell was fluorescent immunostained, and the release of HMGB1 (High Mobility Group Box 1) protein, which is characteristic of necrosis, into the cytoplasm Was observed. Nuclei were stained with Hoechst.

  FIG. 6 (a) is a photograph showing the result 30 minutes after the addition of tert-butyl hydroperoxide, and FIG. 6 (b) is a photograph showing the result 48 hours after the addition of tamoxifen. As a result, in the cell death caused by the addition of tert-butyl hydroperoxide, release of HMGB1, which is an indicator of necrosis, from the nucleus to the cytoplasm was observed, whereas in phospholipid hydroperoxide-dependent cell death, HMGB1 remained in the nucleus. And release into the cytoplasm was not observed.

  From the above, it became clear that phospholipid hydroperoxide-dependent cell death is different from necrosis.

[Experimental Example 7]
(Comparison between phospholipid hydroperoxide-dependent cell death and autophagic cell death)
Whether phospholipid hydroperoxide-dependent cell death was due to autophagic cell death was examined in detail. It is known that the ATG5 gene is essential for causing autophagic cell death.

  ATG5-specific shRNA (short hairpin RNA) was introduced into a tamoxifen-induced PHGPx-deficient MEF cell line to prepare cells in which the expression of ATG5 was knocked down. As the ATG5-specific shRNA, two types of shRNAs, ATG5 shRNA-3 (SEQ ID NO: 1) and ATG5 shRNA-4 (SEQ ID NO: 2), were used. As a control, cells into which only the shRNA expression vector was introduced were prepared. Tamoxifen was added to the culture medium of these cells at a final concentration of 1 μM, and the cells after 24, 36, 48, 60 and 72 hours were randomly taken with a Keyence all-in-one microscope and 20 photographs were attached. The number of live cells that have been counted. The cell viability (%) was expressed as the cell viability (%), with the number of cells 24 hours after adding tamoxifen being 100 (%) and the change in the ratio of the number of viable cells at each time.

  The results are shown in FIGS. 7 (a) and 7 (b). Even when ATG5 expression was knocked down, phospholipid hydroperoxide-dependent cell death could not be suppressed. Moreover, as shown in FIG.7 (b), it was confirmed from the result of RT-PCR that the expression of ATG5 gene was knocked down by introduction of shRNA.

  From the above, it became clear that phospholipid hydroperoxide-dependent cell death is different from autophagic cell death.

[Experimental Example 8]
(Comparison between phospholipid hydroperoxide-dependent cell death and necrosis)
We examined in detail whether phospholipid hydroperoxide-dependent cell death was due to necrosis. Necrosis is a form of cell death known as programmed necrosis. In addition, it is known that knockdown of receptor-interacting protein kinase 1 (Rip1) significantly suppresses necrosis.

  Rip1-specific shRNA (small hairpin RNA) was introduced into a tamoxifen-induced PHGPx-deficient MEF cell line to prepare cells in which Rip1 expression was knocked down. Rip1 shRNA-4 (SEQ ID NO: 3) was used as the Rip1 specific shRNA. Further, as a control, cells into which only the shRNA expression vector was introduced and cells in which Trolox was added at a final concentration of 400 μM in the medium were prepared. Tamoxifen was added to the culture medium of these cells at a final concentration of 1 μM, and after 24 hours, 72 hours later, the living cells were randomly taken with an all-in-one microscope from Keyence, and 20 attached living cells. Counted the number of. The cell viability (%) was expressed as the cell viability (%), where the cell number 24 hours after addition of tamoxifen was 100 (%) and the viable cell number after 72 hours was attached.

  The results are shown in FIGS. 8 (a) and 8 (b). Even when Rip1 expression was knocked down, cell death due to PHGPx deficiency could not be suppressed. Moreover, as shown in FIG.8 (b), it was confirmed from the result of Western blotting that the expression of Rip1 protein was knocked down by introduction of shRNA.

  From the above, it became clear that phospholipid hydroperoxide-dependent cell death is different from necrosis. That is, it has been clarified that phospholipid hydroperoxide-dependent cell death is a novel cell death different from apoptosis, necrosis, autophagic cell death, and necrosis.

  FIG. 9 shows a model diagram of the phospholipid hydroperoxide-dependent cell death pathway, which was clarified by the inventors' analysis. When PHGPx is deficient, phospholipid hydroperoxide, which is a primary oxidation product of phospholipid, increases after 24 hours. Downstream of that, CDK4 is activated, and after 36 hours, MEK and ERK of the MAP kinase pathway are phosphorylated, and cell death occurs between 48 hours and 72 hours.

  Further, as a result of studies using a tamoxifen-induced PHGPx-deficient MEF cell line, the inventors have found that vitamin E, a CDK4 inhibitor 3-ATA (3-amino-9-thio [10H] -acridone) or a MEK inhibitor. Phospholipids induced by PHGPx deficiency by adding U-0126 (1,4-diamino-2,3-dicyano-1,4-bis [2-amino-phenylthio] butadiene) to the medium It was found that hydroperoxide-dependent cell death can be suppressed.

[Experimental Example 9]
(Identification of execution factors for phospholipid hydroperoxide-dependent cell death using lentiviral shRNA library)
In order to identify an execution factor of phospholipid hydroperoxide-dependent cell death, screening using a lentivirus comprehensive shRNA library (SBI) was performed. In this system, since the sequence of shRNA is created to bind to Genechip (trade name, Affymetrix), Genechip can be used to identify the sequence of shRNA.

  FIG. 10 is a diagram showing an outline of the screening procedure for the shRNA library. First, tamoxifen-induced PHGPx-deficient MEF cells were infected with a pool-type lentivirus capable of expressing exhaustive shRNA to produce shRNA-expressing cells. Tamoxifen having a final concentration of 1 μM was added to the medium of the shRNA-expressing cells, and normally, cells that survived 96 hours after complete cell death were collected. RNA was extracted from the collected cells, cDNA containing the shRNA sequence that had been introduced into the cells was amplified several times, a Genechip probe was prepared, and microarray analysis was performed.

  According to two experiments, the shRNA sequences contained in the two lentivirus-infected cells that survived 96 hours after addition of tamoxifen and all the cells that survived 96 hours in the absence of tamoxifen. Genechip analysis was carried out using the probe amplified, and a probe enriched in the tamoxifen-added cells than in the non-added cells was detected. As a result of trying to identify common cell death performing factors concentrated in two experiments, 151 genes were obtained as candidates for cell death performing factors.

[Experimental Example 10]
(Determination of candidate genes for execution factors of phospholipid hydroperoxide-dependent cell death by expression of single shRNA using a retrovirus infection system)
Each of the identified candidate genes 151 of exemplifying factors for phospholipid hydroperoxide-dependent cell death was knocked down, and the inhibitory effect on phospholipid hydroperoxide-dependent cell death was examined.

  One type of shRNA expression vector of a candidate gene was introduced into tamoxifen-inducible PHGPx-deficient MEF cells one by one using a retrovirus infection system to prepare cells in which each candidate gene was knocked down. Tamoxifen with a final concentration of 1 μM was added to the medium of each cell, and the effect of suppressing cell death after 96 hours was measured. Specifically, 24 hours after addition of tamoxifen, 96 hours later, 20 cells were randomly taken with an all-in-one microscope from Keyence, and the number of viable cells attached was counted. The cell viability (%) was calculated as the cell viability (%) with the number of cells 24 hours after adding tamoxifen as 100 (%) and the ratio of the viable cell number after 96 hours.

  FIG. 11 shows the results obtained by arranging the ratio of the number of cells (cell survival rate (%)) after 96 hours from the addition of tamoxifen with respect to the number of cells of 151 candidate genes in the order of the number of cells 24 hours after the addition of tamoxifen. It is a graph to show.

  Inhibition of expression of the target gene was confirmed by real-time PCR, and 37 genes that could suppress phospholipid hydroperoxide-dependent cell death by knockdown were found. These 37 genes did not contain any genes involved in known cell death such as apoptosis. This result further supports that phospholipid hydroperoxide-dependent cell death is a novel cell death. Hereinafter, among these candidate genes, Rbx1, Ube2d1 and Ube2a which are proteasome-related proteins; Dnajc9 which is a heat shock protein; Nubp2 which is an iron / sulfur cluster-related protein; Fbl which is a nucleolar protein; WD repeat family The protein Wdfy1 was examined in detail.

[Experimental Example 11]
(Rbxl protein is a related factor in phospholipid hydroperoxide-dependent cell death)
By expressing shRNA with a retrovirus, knockdown cells of Rbx1 were prepared, and it was examined whether Rbx1 protein is a factor related to phospholipid hydroperoxide-dependent cell death.

  FIG. 12 is a graph showing the examination results. First, Rbx1-specific shRNA (SEQ ID NO: 4) was introduced into a tamoxifen-induced PHGPx-deficient MEF cell line to prepare cells in which Rbx1 was knocked down (Rbx1 KD). As a control, cells into which only the vector was introduced were prepared (Control). Furthermore, Rbx1 expression vector (Rbx1) or vector only (mock) was introduced into these cells as a control. The cDNA of Rbx1 incorporated into the Rbx1 expression vector was one in which a silent mutation was introduced so as not to be knocked down by Rbx1-specific shRNA.

  Subsequently, tamoxifen at a final concentration of 1 μM was added to each cell. Twenty (24) hours after the addition of tamoxifen, 96 photos of living cells were randomly taken with Keyence's all-in-one microscope, and the number of attached living cells was counted. The cell viability (%) was calculated as the cell viability (%) with the number of cells 24 hours after adding tamoxifen as 100 (%) and the ratio of the viable cell number after 96 hours.

  As a result, phospholipid hydroperoxide-dependent cell death was suppressed in Rbx1 knockdown cells (Rbx1 KD / mock). In addition, phospholipid hydroperoxide-dependent cell death was recovered in cells (Rbx1 KD / Rbx1) in which Rbx1 gene was reintroduced after knocking down Rbx1.

  From the above results, it was revealed that Rbx1 protein is an execution factor of phospholipid hydroperoxide-dependent cell death.

  Here, the knockdown and reintroduction (re-expression) of Rbx1 will be described in more detail. The knockdown of Rbx1 was performed as follows. First, a DNA fragment encoding an Rbx1-specific shRNA (SEQ ID NO: 4) was incorporated downstream of the U6 promoter of the shRNA vector (piMG-drU6) shown in FIG. 13 (a). Subsequently, the retrovirus infection system was used to integrate the above shRNA expression cassette into the genome of the target cell, to express Rbx1-specific shRNA, and to obtain a cell in which Rbx1 was knocked down.

  FIG. 13 (b) is a model diagram showing a process in which siRNA is formed from expressed shRNA and target RNA is destroyed. shRNA vectors were obtained from Prof. Kenzo Hirose, School of Medicine, The University of Tokyo, and Prof. Atsutaro Sugao, School of Medicine, Nagoya University. The ATG5 and Rip1 knockdown cells used in Experimental Examples 7 and 8 described above, and cells in which genes other than Rbx1 described below were knocked down were also prepared in the same manner.

(Re-expression of Rbx1)
The pMXs-IR vector shown in FIG. 14 was used as a high expression vector of the retrovirus infection system used in the reexpression experiment. The pMXs-IR vector was obtained from Dr. Toshio Kitamura, Institute of Medical Science, University of Tokyo. The expression of genes other than Rbx1 described later was also performed in the same manner.

(Gene transfer to PlatE cells)
PlatE cells were used for the preparation of retroviruses from shRNA and pMXs-IR vectors. The Plat E cell is a cell in which the gag-pol gene and the env gene are incorporated into 293T cells, which are human embryonic kidney epithelial cells.

PlatE cells were cultured at 37 ° C. using Dulbecco's modified Eagle medium (DMEM, Nissui Pharmaceutical) containing 10% FCS, 2 mM glutamine (GIBCO), 100 units / ml penicillin (GIBCO), 100 μg / ml streptomycin (GIBCO). And cultured in a CO ₂ incubator.

PlatE cells were seeded in a 6 cm petri dish (CORNING) with 5 × 10 ⁵ cells / dish and cultured for 1 day. Add 200 μL of DMEM FCS (−) to the 1.5 mL tube, 2 μg / 2 μL of the generated plasmid DNA (shRNA vector or pMXs-IR vector incorporating the target gene), and 8 μL of Plus Reagent (invitrogen, product number 11514-015). And left at room temperature for 15 minutes. Next, 50 μL of DMEM FCS (−) and 12 μL of Lipofectamine Reagent (Invitrogen, product number 18324-020) were mixed in advance and the mixture was allowed to stand at room temperature for 15 minutes. The cells were washed with a phosphate buffer (PBS), and 1.728 mL of DMEM FCS (−) was added. Subsequently, the above reaction solution was added to the cells and allowed to stand in a CO ₂ incubator at 37 ° C. for 3 hours for gene transfer. Thereafter, the reaction solution was replaced with 6 mL of DMEM 10% FCS, and cultured at 37 ° C. for about 48 hours.

(Preparation of virus solution)
Subsequently, the culture supernatant was transferred to a 15 mL tube (CORNING) and centrifuged at 3,000 rpm for 5 minutes at 4 ° C. The supernatant was transferred to a new tube and used as a virus solution.

(Virus infection)
The day before virus infection, tamoxifen-inducible PHGPx-deficient cells were seeded in a 6 cm petri dish (CORNING) at 1 × 10 ⁵ cells / dish and cultured for 1 day. 5 mL of virus solution to which Polybrene ™ (Hexadimethine bromide, SIGMA, catalog number H9268) was added to a final concentration of 8 μg / mL was infected by replacing the cell culture medium, and the culture medium was changed after 24 hours of culture. . In addition, Polybrene (trademark) was dissolved in sterile water so as to be 10 mg / mL in a clean bench, sterilized by filter, and diluted at the time of use.

[Experimental example 12]
(Ube2d1 protein is a related factor in phospholipid hydroperoxide-dependent cell death)
Whether the Ube2d1 protein is a related factor of phospholipid hydroperoxide-dependent cell death was examined by the same method as in Experimental Example 11.

  FIG. 15 is a graph showing the examination results. First, Ube2d1-specific shRNA (SEQ ID NO: 5) was introduced into a tamoxifen-inducible PHGPx-deficient MEF cell line to prepare cells in which Ube2d1 was knocked down (Ube2d1 KD). As a control, cells into which only the vector was introduced were prepared (Control). Furthermore, a Ube2d1 expression vector (Ube2d1) or a vector alone (mock) was introduced into these cells as a control. The Ube2d1 cDNA incorporated into the Ube2d1 expression vector was used with a silent mutation introduced so as not to be knocked down by Ube2d1-specific shRNA.

  Subsequently, tamoxifen at a final concentration of 1 μM was added to each cell. Twenty (24) hours after the addition of tamoxifen, 96 photos of living cells were randomly taken with Keyence's all-in-one microscope, and the number of attached living cells was counted. The cell viability (%) was calculated as the cell viability (%) with the number of cells 24 hours after adding tamoxifen as 100 (%) and the ratio of the viable cell number after 96 hours.

  As a result, phospholipid hydroperoxide-dependent cell death was suppressed in Ube2d1 knockdown cells (Ube2d1 KD / mock). In addition, phospholipid hydroperoxide-dependent cell death was recovered in the cells (Ube2d1 KD / Ube2d1) into which Ube2d1 gene was reintroduced after knocking down Ube2d1.

  From the above results, it was revealed that the Ube2d1 protein is an execution factor of phospholipid hydroperoxide-dependent cell death.

[Experimental Example 13]
(Ube2a protein is a related factor in phospholipid hydroperoxide-dependent cell death)
Whether the Ube2a protein is a factor related to phospholipid hydroperoxide-dependent cell death was examined by the same method as in Experimental Example 11.

  FIG. 16 is a graph showing the examination results. First, Ube2a-specific shRNA (SEQ ID NO: 6) was introduced into a tamoxifen-inducible PHGPx-deficient MEF cell line to prepare cells in which Ube2a was knocked down (Ube2a KD). As a control, cells into which only the vector was introduced were prepared (Control).

  Subsequently, tamoxifen at a final concentration of 1 μM was added to each cell. Twenty (24) hours after the addition of tamoxifen, 96 photos of living cells were randomly taken with Keyence's all-in-one microscope, and the number of attached living cells was counted. The cell viability (%) was calculated as the cell viability (%) with the number of cells 24 hours after adding tamoxifen as 100 (%) and the ratio of the viable cell number after 96 hours.

  As a result, phospholipid hydroperoxide-dependent cell death was suppressed in Ube2a knockdown cells (Ube2a KD).

  Subsequently, after knocking down Ube2a, the Ube2a gene was reintroduced into the cells. The Ube2a cDNA incorporated into the Ube2a expression vector was used with a silent mutation introduced so as not to be knocked down by Ube2a-specific shRNA. As a result, cell death was induced only by reintroducing the Ube2a gene into the cell, even though tamoxifen was not added to the cell culture medium.

  The reason why cell death is induced by reintroduction of the Ube2a gene is not clear, but the above results revealed that the Ube2a protein is a factor associated with phospholipid hydroperoxide-dependent cell death.

[Experimental Example 14]
(Dnajc9 protein is a related factor in phospholipid hydroperoxide-dependent cell death)
Whether the Dnajc9 protein is a factor related to phospholipid hydroperoxide-dependent cell death was examined by the same method as in Experimental Example 11.

  FIG. 17 is a graph showing the examination results. First, a cell in which Dnajc9-specific shRNA (SEQ ID NO: 7) was introduced into a tamoxifen-induced PHGPx-deficient MEF cell line to knockdown Dnajc9 was prepared (Dnajc9 KD). As a control, cells into which only the vector was introduced were prepared (Control). Furthermore, a Dnajc9 expression vector (Dnajc9) or a vector only (mock) was introduced into these cells as a control. As the cDNA of Dnajc9 incorporated into the Dnajc9 expression vector, a cDNA in which a silent mutation was introduced so as not to be knocked down by Dnajc9-specific shRNA was used.

  Subsequently, tamoxifen at a final concentration of 1 μM was added to each cell. Twenty (24) hours after the addition of tamoxifen, 96 photos of living cells were randomly taken with Keyence's all-in-one microscope, and the number of attached living cells was counted. The cell viability (%) was calculated as the cell viability (%) with the number of cells 24 hours after adding tamoxifen as 100 (%) and the ratio of the viable cell number after 96 hours.

  As a result, phospholipid hydroperoxide-dependent cell death was suppressed in Dnajc9 knockdown cells (Dnajc9 KD / mock). Moreover, in the cells (Dnajc9 KD / Dnajc9) into which Dnajc9 gene was reintroduced after knocking down Dnajc9, phospholipid hydroperoxide-dependent cell death was recovered.

  From the above results, it was revealed that Dnajc9 protein is an execution factor of phospholipid hydroperoxide-dependent cell death.

[Experimental Example 15]
(Nubp2 protein is a related factor in phospholipid hydroperoxide-dependent cell death)
Whether the Nubp2 protein is a related factor of phospholipid hydroperoxide-dependent cell death was examined by the same method as in Experimental Example 11.

  FIG. 18 is a graph showing the examination results. First, Nubp2-specific shRNA (SEQ ID NO: 8) was introduced into a tamoxifen-inducible PHGPx-deficient MEF cell line to prepare a cell in which Nubp2 was knocked down (Nubp2 KD). As a control, cells into which only the vector was introduced were prepared (Control).

  Subsequently, tamoxifen at a final concentration of 1 μM was added to each cell. Twenty (24) hours after the addition of tamoxifen, 96 photos of living cells were randomly taken with Keyence's all-in-one microscope, and the number of attached living cells was counted. The cell viability (%) was calculated as the cell viability (%) with the number of cells 24 hours after adding tamoxifen as 100 (%) and the ratio of the viable cell number after 96 hours.

  As a result, phospholipid hydroperoxide-dependent cell death was suppressed in Nubp2 knockdown cells (Nubp2 KD).

  Subsequently, Nubp2 was knocked down and the Nubp2 gene was reintroduced into the cells. The Nubp2 cDNA incorporated into the Nubp2 expression vector used was introduced with a silent mutation so as not to be knocked down by Nubp2-specific shRNA. As a result, cell death was induced only by reintroducing the Nubp2 gene into the cells, even though tamoxifen was not added to the cell culture medium.

  The reason why cell death is induced by reintroduction of Nubp2 gene is not clear, but the above results revealed that Nubp2 protein is a factor related to phospholipid hydroperoxide-dependent cell death.

[Experimental Example 16]
(Fbl protein is a related factor in phospholipid hydroperoxide-dependent cell death)
Whether the Fbl protein is a factor related to phospholipid hydroperoxide-dependent cell death was examined by the same method as in Experimental Example 11.

  FIG. 19 is a graph showing the examination results. First, Fbl-specific shRNA (SEQ ID NO: 9) was introduced into a tamoxifen-induced PHGPx-deficient MEF cell line, and Fbl knocked-down cells were prepared (Fbl KD). As a control, cells into which only the vector was introduced were prepared (Control).

  Subsequently, tamoxifen at a final concentration of 1 μM was added to each cell. Twenty (24) hours after the addition of tamoxifen, 96 photos of living cells were randomly taken with Keyence's all-in-one microscope, and the number of attached living cells was counted. The cell viability (%) was calculated as the cell viability (%) with the number of cells 24 hours after adding tamoxifen as 100 (%) and the ratio of the viable cell number after 96 hours.

  As a result, phospholipid hydroperoxide-dependent cell death was suppressed in Fbl knockdown cells (Fbl KD).

  Subsequently, after knocking down Fbl, the Fbl gene was reintroduced into the cells. As the Fbl cDNA incorporated into the Fbl expression vector, a cDNA having a silent mutation introduced so as not to be knocked down by Fbl-specific shRNA was used. As a result, cell death was induced only by reintroducing the Fbl gene into the cells, even though tamoxifen was not added to the cell culture medium.

  The reason why cell death is induced by reintroduction of the Fbl gene is not clear, but the above results revealed that the Fbl protein is a factor associated with phospholipid hydroperoxide-dependent cell death.

[Experimental Example 17]
(Wdfy1 protein is a related factor in phospholipid hydroperoxide-dependent cell death)
Whether the Wdfy1 protein is a related factor of phospholipid hydroperoxide-dependent cell death was examined by the same method as in Experimental Example 11.

  FIG. 20 is a graph showing the examination results. First, Wdfy1-specific shRNA (SEQ ID NO: 10) was introduced into a tamoxifen-inducible PHGPx-deficient MEF cell line to prepare cells in which Wdfy1 was knocked down (Wdfy1 KD). As a control, cells into which only the vector was introduced were prepared (Control).

  Subsequently, tamoxifen at a final concentration of 1 μM was added to each cell. Twenty (24) hours after the addition of tamoxifen, 96 photos of living cells were randomly taken with Keyence's all-in-one microscope, and the number of attached living cells was counted. The cell viability (%) was calculated as the cell viability (%) with the number of cells 24 hours after adding tamoxifen as 100 (%) and the ratio of the viable cell number after 96 hours.

  As a result, phospholipid hydroperoxide-dependent cell death was suppressed in Wdfy1 knockdown cells (Wdfy1 KD).

  Subsequently, after knocking down Wdfy1, the Wdfy1 gene was reintroduced into the cells. The cDNA of Wdfy1 incorporated into the Wdfy1 expression vector was one in which a silent mutation was introduced so as not to be knocked down by Wdfy1-specific shRNA. As a result, cell death was induced only by reintroducing the Wdfy1 gene into the cells, even though tamoxifen was not added to the cell culture medium.

  The reason why cell death is induced by reintroduction of the Wdfy1 gene is not clear, but the above results revealed that the Wdfy1 protein is a factor associated with phospholipid hydroperoxide-dependent cell death.

[Experiment 18]
(Examination of the formation of phospholipid hydroperoxide)
Production of phospholipid hydroperoxide by PHGPx deficiency when tamoxifen is knocked down in tamoxifen-induced PHGPx-deficient MEF cell line and Rbx1, Ube2d1, Ube2a, Dnajc9, Nubp2, Fbl, and Wdfy1 are respectively added to the medium and tamoxifen at a final concentration of 1 μM is added. Was examined.

  First, tamoxifen-induced PHGPx-deficient MEF cells in which Rbx1, Ube2d1, Ube2a, Dnajc9, Nubp2, Fbl, and Wdfy1 were respectively knocked down and a tamoxifen-induced PHGPx-deficient MEF cell as a control were prepared.

  Subsequently, tamoxifen at a final concentration of 1 μM was added to the culture medium of these cells. After incubation for 26 hours, the cells were stained with H2DCFDA, which is a fluorescent dye capable of detecting the production of phospholipid hydroperoxide, and phospholipid hydrogel was analyzed using flow cytometry. The formation of peroxide was studied.

  The results are shown in Table 1. In Table 1, “◯” indicates that the generation of phospholipid hydroperoxide due to PHGPx deficiency is suppressed by knockdown, and “x” indicates the case where phospholipid hydroperoxide is generated due to PHGPx deficiency.

  As a result, it was revealed that the production of phospholipid hydroperoxide due to PHGPx deficiency was suppressed in the knockdown cells of Ube2a and Nubp2. In knockdown cells other than these, production of phospholipid hydroperoxide due to PHGPx deficiency was observed.

[Experimental Example 19]
(Investigation of phosphorylation of ERK)
When Tamoxifen is added to tamoxifen-induced PHGPx-deficient MEF cell line and tamoxifen is stained with anti-phosphorylated ERK antibody, cells with enhanced ERK phosphorylation are detected 36 hours after addition of tamoxifen. be able to.

  Therefore, in Tamoxifen-induced PHGPx-deficient MEF cell lines, Rbx1, Ube2d1, Ube2a, Dnajc9, Nubp2, Fbl and Wdfy1 were knocked down, respectively, and enhanced phosphorylation of ERK when tamoxifen at a final concentration of 1 μM was added to the medium. investigated.

  First, tamoxifen-induced PHGPx-deficient MEF cells in which Rbx1, Ube2d1, Ube2a, Dnajc9, Nubp2, Fbl, and Wdfy1 were respectively knocked down and a tamoxifen-induced PHGPx-deficient MEF cell as a control were prepared.

  Subsequently, tamoxifen having a final concentration of 1 μM was added to the medium of each cell, and unadded cells were prepared. After 36 hours, the cells were fluorescently stained with an anti-phosphorylated ERK antibody. Subsequently, phosphorylation of ERK was examined by observation with a fluorescence microscope.

  The results are shown in Table 1. In Table 1, the case where ERK phosphorylation due to PHGPx deficiency is suppressed by knockdown is indicated as “◯”, and the case where ERK is phosphorylated due to PHGPx deficiency is indicated as “x”.

  As a result, enhanced ERK phosphorylation was confirmed in the knockdown cells of Dnajc9 and the knockdown cells of Wdfy1. In knockdown cells other than these, suppression of ERK phosphorylation was observed.

  From the results in Table 1, it was considered that the Rbx1 protein, Ube2d1 protein, and Fbl protein function downstream of the production of phospholipid hydroperoxide by PHGPx deficiency and upstream of the phosphorylation of ERK.

  In addition, Ube2a protein and Nubp2 protein were considered to function upstream of the production of phospholipid hydroperoxide by PHGPx deficiency.

  It is also possible that the Dnajc9 protein and Wdfy1 protein function in a pathway that is not related to the ERK phosphorylation pathway or downstream of ERK phosphorylation or phospholipid hydroperoxide generation due to PHGPx deficiency. It was.

[Experiment 20]
(Myocardium-specific PHGPx-deficient mice are killed by cardiomyocyte death at 17.5 days of development)
PHGPx-deficient mice are known to be lethal at 7.5 days of development. Here, a mouse deficient in PHGPx in a heart-specific manner was prepared, and the effect was analyzed. First, mating of a PHGPx ^+/− mouse with a mouse (Cre ^{+ / +} ) having a Cre gene downstream of a muscle creatine kinase promoter which is a heart-specific promoter is repeated, and Cre ^{+ / +} PHGPx ^{+ //-} mice were obtained.

Subsequently, Cre ^{+ / +} PHGPx ^+/− mice and the above-described Tg (loxP-PHGPx) ^{+ / +} : PHGPx ^{− / −} mice are crossed to create Cre ^+/− Tg (loxP-PHGPx) ^{+ / −} : PHGPx ^{− / −} mice were obtained. This mouse is heart-specific and lacks PHGPx.

  When the heart-specific PHGPx-deficient mouse thus obtained was observed, it grew normally until 16.5 days of development, but the cardiomyocytes suddenly died on 17.5 days, and 18.5 days. Was fatal, causing edema. FIG. 21 (a) is a photograph of wild-type mice (Control) and heart-specific PHGPx-deficient mice (KO) at 17.5 days (17.5 dpc) and 18.5 days (18.5 dpc) during development. is there.

  FIG. 21 (b) is a photograph showing the results of DAPI staining and TUNEL staining (TUNEL) of a heart tissue section of a mouse on the 17.5th day of development. In the heart tissue of heart-specific PHGPx-deficient mice (KO), many cells (cell death) positive for TUNEL staining were observed. When the mother was given a dietary supplement with vitamin E (50mg vitamin E / 100g diet) every day, cardiac death of heart-specific PHGPx-deficient mice (KO) on development day 17.5 was completely suppressed and normal In addition, heartbeat-specific PHGPx-deficient mice were born. No caspase 3 activation or DNA ladder was observed in cardiomyocyte death that occurred in the fetus of a heart-specific PHGPx-deficient mouse on day 17.5 when a mother mouse was raised on a normal diet. Oxidant accumulation was observed (not shown). This suggests that this cardiomyocyte death is also induced by a novel cell death via lipid oxidation.

  FIG. 21 (c) is a photograph showing the result of detecting the expression of PHGPx protein in the heart of a mouse on the 17.5th day of development by Western blotting. It was confirmed that PHGPx protein is deficient in heart-specific PHGPx-deficient mice (KO).

[Experimental example 21]
(Administration of CDK4 inhibitor 3-ATA to the abdominal cavity of mother mice suppresses death of heartbeat-specific PHGPx-deficient mice at 18.5 days)
It was examined whether or not lethality of heartbeat-specific PHGPx-deficient mice can be suppressed by administering CDK4 inhibitor 3-ATA into the abdominal cavity of mother mice.

  Developmental process of heartbeat-specific PHGPx-deficient mouse embryos 14.5 days, 15.5 days, 16.5 days, 17.5 days, 1.5 mg / kg body weight of 3-ATA was administered into the abdominal cavity of the mother mice, The fetus on the 18.5th day of the development process was observed.

  As a result, edema was suppressed in 8 out of 10 heartbeat-specific PHGPx-deficient mice. Edema was observed in the remaining two animals. Of the 8 animals in which edema was suppressed, 2 animals were alive. The remaining six were dead.

  22 (a) to 22 (c) are photographs of a mouse fetus on the development stage 18.5. FIG. 22 (a) is a photograph of a wild-type mouse, and FIG. 22 (b) is a photograph of a heartbeat-specific PHGPx-deficient mouse to which 3-ATA was not administered (heart PHGPx KO 3-ATA (−). FIG. 29 (c) is a photograph of a heartbeat-specific PHGPx-deficient mouse administered with 3-ATA (heart PHGPx KO 3-ATA (+)).

[Experimental example 22]
(Heart-specific PHGPx-deficient mice grow normally on a diet supplemented with vitamin E, but suddenly die about 10 days after changing to a normal diet.)
In the fetal stage of heartbeat-specific PHGPx-deficient mice, when the mother was fed a dietary supplement with vitamin E, lethality was completely suppressed. In addition, it was revealed that when a heartbeat-specific PHGPx-deficient mouse was continuously fed with a diet containing vitamin E (50 mg vitamin E / 100 g diet) every day, the mice grew normally.

  Here, the vitamin E supplemented diet contained 130 mg / kg body weight of vitamin E per day. The normal diet also contained 13 mg / kg body weight of vitamin E per day.

  FIG. 23 (a) is a graph showing the survival rate after changing the diet of heartbeat-specific PHGPx-deficient mice that grew normally by feeding a vitamin E-added diet to a normal diet. It was found that sudden death occurred around 10 days when the diet of normally grown heartbeat-specific PHGPx-deficient mice was changed to a normal diet.

  FIG. 23 (b) shows a heartbeat-specific PHGPx-deficient mouse fed with a vitamin E-added diet (vitamin E-added diet) and a heartbeat-specific PHGPx-deficient mouse that died by changing the diet to a normal diet (normal diet KO (dead)). ) And the results of measuring the amount of vitamin E in the heart of a wild-type mouse (normal diet wild) fed a normal diet. The heart of a heartbeat-specific PHGPx-deficient mouse fed a dietary supplement with vitamin E contained about 24 nmol of vitamin E per gram of heart. The normal diet contained about 4 nmol of vitamin E per gram of heart. In this mouse lethal model, cardiomyocyte death due to lipid oxidation is induced by a decrease in the amount of vitamin E in the heartbeat (not shown).

  FIG. 23 (c) shows a heartbeat-specific PHGPx-deficient mouse (vitamin E-added diet heart KO) fed with a vitamin E-added diet, and a heartbeat-specific PHGPx-deficient mouse (daily diet) on the 10th day after changing the diet to a normal diet. It is a graph which shows the electrocardiogram just before death of heart KO). An arrhythmia was observed in the electrocardiogram of heartbeat-specific PHGPx-deficient mice in which the diet was changed to a normal diet, and sudden arrhythmic death (heart failure) was observed.

[Experimental example 23]
(CDK4 inhibitor 3-ATA can prolong the sudden death due to heart failure in heartbeat-specific PHGPx-deficient mice, which is caused by changing from a vitamin E-added diet to a normal diet)
After changing the diet of heartbeat-specific PHGPx-deficient mice fed with a vitamin E-added diet to a normal diet, the effect of administering the CDK4 inhibitor 3-ATA was examined.

  FIG. 23 shows a case where the heartbeat-specific PHGPx-deficient mouse diet was changed to a normal diet (Vit.E-), the heartbeat-specific PHGPx-deficient mouse diet was changed to a normal diet, and the diet was changed to a normal diet. Vitamin E supplemented diet was given to mice (Vit.E−, 3-ATA +) and heartbeat-specific PHGPx-deficient mice administered intraperitoneally with 2 mg / kg body weight of 3-ATA once a day from the day. It is a graph which shows the survival rate of the mouse | mouth (Vit.E +, 3-ATA +) intraperitoneally administered 2 mg / kg body weight 3-ATA once a day from the 4th day after the start.

  The median survival time from the start of the experiment for mice (Vit. E−) (n = 23) in which the heartbeat-specific PHGPx-deficient mice were changed to a normal diet was 10 days. In addition, the heartbeat-specific PHGPx-deficient mouse diet was changed to a normal diet, and the mice were administered intraperitoneally with 2 mg / kg body weight of 3-ATA once a day from the fourth day after changing the diet to a normal diet (Vit The median survival time from the start of the experiment for E-, 3-ATA +) (n = 6) was 18 days. These results were significantly different with a risk rate of less than 1%.

  From the above results, it was shown that CDK4 inhibitor 3-ATA can prolong the sudden death due to heart failure that occurs when heartbeat-specific PHGPx-deficient mice are changed from a vitamin E-added diet to a normal diet.

  In another study by the inventors, when a heartbeat-specific PHGPx-deficient mouse was changed from a vitamin E-added diet to a normal diet even when the MEK inhibitor U-0126 was administered instead of 3-ATA. It has been shown that it can prolong the sudden death caused by heart failure.

  The above results occur when a heartbeat-specific PHGPx-deficient mouse is changed from a vitamin E-added diet to a normal diet by administering Rbx1, Ube2d1, Ube2a, Dnajc9, Nubp2, Fbl, and Wdfy1 expression inhibitors. Indicates that sudden death from heart failure can be prolonged.

  According to the present invention, a method and a kit for detecting phospholipid hydroperoxide-dependent cell death can be provided. In addition, an inhibitor of phospholipid hydroperoxide-dependent cell death can be provided. In addition, a prophylactic or therapeutic agent for a disease associated with phospholipid hydroperoxide-dependent cell death can be provided.

Claims (5)

Detecting the activation state of at least one protein selected from the group consisting of proteasome-related protein, heat shock protein, iron / sulfur cluster-related protein, nucleolar protein and WD repeat family protein in the cell ,
The proteasome-related protein is at least one protein selected from the group consisting of Rbx1, Ube2d1 and Ube2a;
The heat shock protein is Dnajc9;
The iron / sulfur cluster-related protein is Nubp2.
The nucleolar protein is Fbl;
The WD repeat family protein is Wdfy1,
The detection is performed by measuring the expression level of the protein at the mRNA level, measuring the expression level of the protein at the protein level, or measuring suppression of cell death due to suppression of the expression of the protein. Called
Detection method for re phospholipid hydroperoxide-dependent cell death. An antibody to at least one protein selected from the group consisting of phosphorylated or non-phosphorylated proteasome-related protein, heat shock protein, iron-sulfur cluster-related protein, nucleolar protein and WD repeat family protein,
A primer for amplifying mRNA of at least one protein selected from the group consisting of a proteasome-related protein, a heat shock protein, an iron / sulfur cluster-related protein, a nucleolar protein and a WD repeat family protein, or a proteasome-related protein, a heat shock protein, SiRNA, shRNA, ribozyme or antisense nucleic acid against mRNA of at least one protein selected from the group consisting of iron / sulfur cluster-related proteins, nucleolar proteins and WD repeat family proteins,
Equipped with a,
The proteasome-related protein is at least one protein selected from the group consisting of Rbx1, Ube2d1 and Ube2a;
The heat shock protein is Dnajc9;
The iron / sulfur cluster-related protein is Nubp2.
The nucleolar protein is Fbl;
The WD repeat family protein is Wdfy1;
Li phospholipid hydroperoxides dependent cell death detection kit. Effective siRNA, shRNA, ribozyme or antisense nucleic acid against mRNA of at least one protein selected from the group consisting of proteasome-related protein, heat shock protein, iron / sulfur cluster-related protein, nucleolar protein and WD repeat family protein As an ingredient ,
The proteasome-related protein is at least one protein selected from the group consisting of Rbx1, Ube2d1 and Ube2a;
The heat shock protein is Dnajc9;
The iron / sulfur cluster-related protein is Nubp2.
The nucleolar protein is Fbl;
The WD repeat family protein is Wdfy1;
Inhibitors of the re-phospholipid hydroperoxide-dependent cell death. Effective siRNA, shRNA, ribozyme or antisense nucleic acid against mRNA of at least one protein selected from the group consisting of proteasome-related protein, heat shock protein, iron / sulfur cluster-related protein, nucleolar protein and WD repeat family protein As an ingredient ,
The proteasome-related protein is at least one protein selected from the group consisting of Rbx1, Ube2d1 and Ube2a;
The heat shock protein is Dnajc9;
The iron / sulfur cluster-related protein is Nubp2.
The nucleolar protein is Fbl;
The WD repeat family protein is Wdfy1;
Prophylactic or therapeutic agent for Li phospholipid hydroperoxides dependent cell death related diseases. The preventive or therapeutic agent according to claim 4 , wherein the disease associated with phospholipid hydroperoxide-dependent cell death is heart failure.