JP2012046470A - Enzyme activity inhibitor for gapdh - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an enzyme activity inhibitor for GAPDH.SOLUTION: The enzyme activity inhibitor, oxidative modification inhibitor, nuclear translocation inhibitor for GAPDH, and a pharmaceutical composition for cell protection or cytotoxicity treating comprises carbon monoxide and/or heme.

Description

  The present invention relates to an enzyme activity inhibitor of GAPDH containing heme or carbon monoxide and the like.

  Although oxidative stress response is thought to be related to aging and neurological diseases, control methods using antioxidants such as vitamin C and vitamin E are generally used to suppress such oxidative stress response. (Non-Patent Document 1).

  In recent years, it has been found that carbon monoxide or protoheme increases cytoprotective amino acids in vivo, and it has been shown that carbon monoxide and protoheme can be used as cytoprotective agents (Patent Document 1: WO2007-73006). . According to Patent Document 1, methionine, which is a cytoprotective amino acid, is one of the essential amino acids, metabolized to S-adenosylmethionine (SAM), and after transferring the methyl group to DNA, protein, etc. It becomes homocysteine and is further converted to homocysteine. This homocysteine is metabolized as a raw material for cysteine and used as a raw material for glutathione, which is an antioxidant. Therefore, methionine and homocysteine can be used as an ameliorating agent for various oxidative stress conditions, and carbon monoxide (CO) induces increased methionine and the like. On the other hand, since a gene-deficient model of heme-degrading enzyme (heme oxygenase) that produces CO gas shows toxicity, it has been considered that heme is cytotoxic (Non-patent Document 2).

International Publication No. 2007/73006 Pamphlet

Pratico D. Evidence of oxidative stress in Alzheimer's disease brain and antioxidant therapy: lights and shadows. Ann N Y Acad Sci. 1147: 70-8, 2008. Farombi EO, Surh YJ. Heme oxygenase-1 as a potential therapeutic target for hepatoprotection. J Biochem Mol Biol. 39 (5): 479-91, 2006.

  An object of the present invention is to provide an enzyme activity inhibitor of GAPDH and the like.

  GAPDH, one of the glycolytic metabolic enzymes, has received attention because it is modified by oxidative stress such as NO and active oxygen to control its activity and induce cell death such as nerve cells. As a result of intensive studies to solve the above problems, the present inventors have found that GAPDH binds to heme, and that heme and carbon monoxide (CO) gas specifically suppress the oxidative modification of GAPDH. It has been newly found that it exhibits a protective effect on nerve cells. Furthermore, the present inventors have found that CO gas binds on GAPDH via heme and enhances this cytoprotective action.

The present invention is based on the elucidation of a new molecular mechanism in which heme, which has been considered to be toxic in the past, exhibits a cytoprotective action in cooperation with CO.
The present invention has been completed based on such findings.
That is, the present invention is as follows.
(1) An enzyme activity inhibitor of GAPDH containing heme and / or carbon monoxide.
(2) An oxidation modification inhibitor of GAPDH containing heme and / or carbon monoxide.
(3) A nuclear translocation inhibitor of GAPDH containing heme and / or carbon monoxide.
(4) A protein s-nitrosylation modification inhibitor containing heme and / or carbon monoxide.
(5) The s-nitrosylation modification inhibitor according to (4), wherein the protein is GAPDH.
(6) A pharmaceutical composition for cell protection or cell injury treatment, comprising heme.
(7) A method for inhibiting GAPDH enzyme activity, comprising incubating GAPDH and its substrate in the presence of heme and / or carbon monoxide to inhibit the catalytic action of the substrate by GAPDH.
(8) A method for inhibiting GAPDH enzyme activity, which comprises incubating GAPDH in the presence of heme and / or carbon monoxide to inhibit GAPDH enzyme activity.
(9) A method for inhibiting GAPDH oxidative modification, which comprises incubating GAPDH in the presence of heme and / or carbon monoxide to inhibit oxidative modification of GAPDH.
(10) A method for inhibiting GAPDH enzyme activity, comprising treating cells with heme and / or carbon monoxide to inhibit GAPDH enzyme activity in the cells.
(11) A method for inhibiting GAPDH oxidative modification, comprising treating a cell with heme and / or carbon monoxide to inhibit oxidative modification of GAPDH in the cell.
(12) A method for inhibiting GAPDH translocation into the nucleus, comprising treating cells with heme and / or carbon monoxide to inhibit GAPDH in the cells from translocating from the cytoplasm into the nucleus.
(13) The enzyme activity inhibition, oxidation modification inhibition or nuclear translocation inhibition of GAPDH is caused by binding of heme to the NAD recognition site of GAPDH, according to any one of (8) to (12) the method of.
(14) The method according to any one of (8) to (12), wherein carbon monoxide enhances GAPDH enzyme activity inhibition, oxidative modification inhibition, or nuclear translocation inhibition by heme.

  According to the present invention, an enzyme activity inhibitor, an oxidation modification inhibitor, and a nuclear translocation inhibitor of GAPDH containing heme and / or carbon monoxide are provided. By using heme and carbon monoxide, inhibition of oxidative modification of GAPDH can be caused, and thereby, a neuron protective effect and a cytotoxic effect can be obtained.

  In the present invention, heme and carbon monoxide each alone or in combination with each other exhibit a cytoprotective effect by selectively regulating the function of GAPDH directly controlled by oxidative stress. The action of the factor concerned can be specifically blocked.

  Therefore, heme and / or carbon monoxide can be used for the purpose of cell protection or as a pharmaceutical composition for cell disorders including refractory neurodegenerative diseases such as Alzheimer and Parkinson's disease.

It is a figure which shows the model of the cell defense mechanism through GAPDH by heme / CO. It is a figure which shows an affinity nano bead and hem b. It is a figure which shows the refinement | purification and identification result of GAPDH. It is a figure which shows the CO gas response between heme and GAPDH. It is a figure which shows the effect of heme and CO with respect to GAPDH activity. It is a figure which shows the spectrum between heme and GAPDH. It is a figure which shows the amino acid sequence of GAPDH, and a mutation introduction site. It is a figure which shows the binding activity with the heme of the point mutant of GAPDH. It is a figure which shows the inhibitory effect with respect to the oxidative modification of GAPDH by heme / CO. It is a figure which shows the cytoprotective effect by heme / CO. It is a figure which shows inhibition of the nuclear translocation of GAPDH by heme / CO.

Hereinafter, the present invention will be described in detail.
1. Outline The present inventor has so far developed a unique affinity purification technique using a nanoscale carrier and established a protein purification system that selectively binds to a low molecular weight compound. In order to search for novel heme-binding proteins, we used exhaustive extracts from mouse livers and comprehensively investigated gas-responsive factors that specifically bind to heme, a gas-responsive prosthetic group, as a ligand. Was isolated and identified.
As a result, Glyceraldehyde 3-phophate dehydrogenase (GAPDH), an enzyme known to act on the glycolytic system of metabolic systems, was newly found to selectively bind to heme, and GAPDH was selected as a new target molecule for heme. It was identified as a heme protein of approximately 35.7 kDa.

GPADH is an enzyme that converts Glyceraldehyde 3-phosphate to Glycerate 1,3bisphosphate depending on the coenzyme NAD (Nicotinamide adenine dinucleotide) in the glycolytic system.
GAPDH is a sugar-metabolizing enzyme that is well preserved from bacteria, protozoa, and humans, and is modified by oxidative stress such as nitric oxide (NO) and active oxygen to control its activity, thereby killing cells such as neurons. Since it induces, it attracts attention as a determinant of neuronal cell death (Hara MR et al., Nat Cell Biol. 7 (7): 665-74, 2005.).
On the other hand, GAPDH has recently been clarified to be functionally modified with gas mediators such as NO and H ₂ S (Mustafa AK et al., Sci Signal. 2 (96): ra72, 2009.).

  As a result of analyzing the function of GAPDH by focusing on this GAPDH, the present inventor has revealed that heme binds to GAPDH. Biochemical kinetics analysis showed that CO gas enhances the binding activity of heme to GAPDH. Further, in the present invention, it is found that heme inhibits GAPDH enzyme activity by binding to GAPDH, and CO gas enhances inhibition of GAPDH enzyme activity by promoting the binding between heme and GAPDH. It was.

  Further, in the present invention, CO gas suppresses cell death due to oxidative stress by significantly inhibiting the oxidative modification of GAPDH by NO gas via heme, which is a molecular mechanism of the cell defense action by CO gas. Revealed one end. Furthermore, CO gas alone was found to inhibit oxidative modification of GAPDH. This may be due to the mechanism that CO gas promotes the binding of endogenous heme and GAPDH in the cell.

  In one embodiment of the present invention, S-nitrosylation is performed as an example for oxidative modification of a protein, and the oxidative modification of the protein is inhibited by CO, heme, or the like. However, protein oxidation modification is not limited to the above method.

  Here, S-nitrosylation of a protein is a special form of oxidative modification targeting a cysteine residue (SH group) of the protein, and CO and heme recognize the SH group and inhibit the oxidative modification. It is unexpected that CO or heme inhibits such oxidative modification, and is unexpectedly found for the first time in the present invention. Therefore, the present invention also provides an inhibitor and a method for inhibiting S-nitrosylation using CO and / or heme.

  In recent years, GAPDH has been attracting attention as it induces apoptotic cell death in nerve cells in response to oxidative stress such as NO gas. The point which shows is interesting. Therefore, the present invention provides a GAPDH enzyme activity inhibitor and a method for inhibiting GAPDH enzyme activity using heme and / or CO.

Furthermore, GAPDH has been reported to be involved in cell death in response to oxidative stress such as NO gas as well as enzymatic activity in glycolysis. As for this molecular mechanism, NO gas specifically oxidizes and modifies Cys152 residue of GAPDH (S-nitrosylation), so that the nuclear translocation protein SIAH1 and GAPDH form a complex, and GAPDH exists in the cytoplasm. Is believed to translocate into the nucleus and activate transcription factor p53 to induce apoptotic cell death. This means that by suppressing the oxidative modification of GAPDH using heme or CO, the transfer of GAPDH to the nucleus can be suppressed, and as a result, cells can be protected.
Accordingly, the present invention relates to an oxidation modification inhibitor of GAPDH and a GAPDH nuclear translocation inhibitor containing heme and / or CO, a method of inhibiting oxidative modification of GAPDH using heme and / or CO, and a nucleus of GAPDH. A method of inhibiting internalization is provided.

  Furthermore, conventionally, heme has been known to have cytotoxicity when it comes into contact with cells alone, but the present invention overturns such common general knowledge and shows that heme alone has a cytoprotective effect. It was. Therefore, the present invention provides a pharmaceutical composition for cell protection containing heme.

2. The inhibitor and pharmaceutical composition of the present invention The present invention relates to an enzyme activity inhibitor of GAPDH, an oxidation modification inhibitor, and a nuclear translocation inhibitor (hereinafter referred to as “inhibitor of the present invention”). The present invention relates to a method for inhibiting enzyme activity, oxidative modification and nuclear translocation (referred to as the method of the present invention). Furthermore, the present invention relates to a pharmaceutical composition for cell protection or cell damage treatment.

  Heme is a complex composed of a divalent iron atom and porphyrin, and generally means ferroheme, which is a protoheme composed of divalent iron and type IX protoporphyrin. Examples of the heme used in the present invention include ferroheme and other porphyrin iron complexes such as ferriheme, hemochrome, hemin, and hematin. It is also possible to increase the amount of heme in the body by administration of the substrate 5 aminolevulinic acid which is the rate-limiting of heme biosynthesis with the heme precursor in vivo. These hems can be obtained from Sigma Aldrich and Porphyrin Science.

CO gas is produced in vivo by stress-induced heme oxygenase 1 (HO-1) or resident HO-2. It is also possible to induce CO gas in the living body by controlling the expression and activity of these enzymes.
In the present invention, CO and heme can be used alone or in combination.

  In addition, CO and heme may be used as they are, or a liposome containing CO, a heme and heme protein containing CO, a red blood cell containing CO, a CO-containing complex capable of releasing CO in an aqueous solution, etc. Modified carbon monoxide or a compound that is metabolized in the body to release CO (eg, allyl hydrocarbon) may be used. In the present invention, it is preferable to use gaseous CO. From the viewpoint of operational safety, it is preferable to use modified carbon monoxide.

Here, since CO binds to hemoglobin and is transported to peripheral tissues, regarding the method of administering CO to a living body,
(a) Inhalation at a low concentration (0.1 ppm to 300 ppm, preferably about 100 ppm),
(b) binding to red blood cells, modified hemoglobin, hemoglobin encapsulated in liposomes, etc.
(c) Administration of protoheme IX and protoporphyrin IX or 5-aminolevulinic acid into the body (oral administration or intraperitoneal or intramuscular injection)
Is preferred.

As used herein, the term “carbon monoxide” or “CO” refers to CO molecules in a gaseous state, CO molecules compressed to a liquid state, or dissolved in an aqueous solution.
CO can be a gaseous CO composition. The CO gas used in the present invention can be obtained from any commercially available source (for example, a container storing compressed CO gas).
CO can also be a liquid CO composition. The liquid CO composition can be obtained by any method in the art, such as dissolving a gas in a liquid, for example by bubbling CO gas until the liquid reaches a desired CO concentration.

In the present invention, when CO and / or heme is used as the inhibitor of the present invention, for example, in addition to use as a reagent for experiments, etc., in addition to research on cytoprotective effects, clinical studies such as Alzheimer's disease and Parkinson's disease. Can be used. Here, examples of the cell protection target include nerve cells, hepatocytes and the like, but are not limited to these cells.
As used herein, the term “pharmaceutical composition” refers to a gas or liquid composition containing heme that can be administered to patients with cell death, such as Alzheimer's disease patients, Parkinson's disease patients, etc. Means.
In the present invention, when heme is used as a pharmaceutical composition, its dosage varies depending on age, sex, symptom, route of administration, frequency of administration, and dosage form.

Heme includes ferrohem, iron complexes of other porphyrins such as ferriheme, hemochrome, hemin, and hematin, and can be administered with reference to the amount of endogenous heme contained in the living body. For example, the daily dose for an adult is 50 μM / kg or less, preferably 40 μM / kg or less.
In the present invention, the active ingredient of the pharmaceutical composition for cell protection or cell damage treatment is heme alone, but CO can also be used in combination.

When using gaseous CO, adults (60 kg) at a concentration of 0.1 to 300 ppm per day, preferably 10 to 200 ppm, more preferably 100 ppm for 1 minute to 24 hours, preferably 5 minutes to 12 hours, more preferably 10 Administer by inhalation intermittently or continuously for min to 6 hours. Inhalation can be performed using a mask, respiratory tube, nasal catheter, nasal cannula connected to a carbon monoxide source (eg, a carbon monoxide cylinder or concentrator). It is also possible to administer a saturated solution of CO from the blood vessel. The liquid is generally an aqueous solution suitable for administration to a patient. Examples of the aqueous solution include physiological saline, phosphate buffer (PBS), Collins solution, citric acid solution, Ringer's solution, University of Wisconsin (UW) solution, and the like.
The administration period of CO can be appropriately set such as 1 day, 2 days, 7 days, 10 days, 14 days, 20 days, or 20 days or more.
Moreover, when using protoheme IX or protoporphyrin IX, you may use as it is and may use what was formulated. Moreover, administration of 5-aminolevulinic acid, which is a heme precursor, is also possible.

  In the present invention, when heme is used as a pharmaceutical composition, these can be included in a pharmaceutically acceptable carrier. Examples of pharmaceutically acceptable carriers include excipients, binders, disintegrants, lubricants, lubricants, emulsifiers, colorants, flavoring agents, surfactants, solubilizers, which are commonly used in medicine. Agents, suspending agents, isotonic agents, stabilizers, buffers, antioxidants and the like.

Examples of the preparation include oral preparations such as inhalants, tablets, powders, granules, capsules and syrups, external preparations such as suppositories, ointments, eye drops, and poultices, and injections.
The above injection can be used by methods such as infusion, intramuscular injection, subcutaneous injection, and intravenous injection. The injection may be formulated as a liposome preparation by appropriately combining the above pharmacologically acceptable carriers.

  In the present invention, since heme has a cytoprotective action, applicable diseases include cytotoxic diseases including neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, or hepatitis.

  In addition, the inhibitor of the present invention is a reagent for inhibiting GAPDH enzyme activity, a reagent for inhibiting GAPDH oxidation modification, or for inhibiting GAPDH nuclear translocation in order to conduct in vitro experiments or animal experiments for non-human subjects. Although it can be used as a reagent, in that case, in addition to CO and heme, it can be in the form of a kit containing at least one selected from a buffer solution, a cell culture solution, an antibody against GAPDH, a fluorescent dye, and the like. . The kit can also include instructions for use that describe GAPDH enzyme activity, oxidative modification, methods for testing nuclear translocation, and the like.

3. Method of the present invention The present invention provides a method for inhibiting GAPDH enzyme activity, which comprises incubating GAPDH and its substrate in the presence of heme and / or CO to inhibit the catalytic action of the substrate by GAPDH. provide.
The present invention also relates to a method for inhibiting GAPDH enzymatic activity by incubating GAPDH in the presence of heme and / or carbon monoxide, or treating cells with heme and / or CO, and oxidation of GAPDH. Methods of inhibiting modification and methods of inhibiting GAPDH nuclear translocation are provided.

Here, in order to understand the action of heme or CO of the present invention on GAPDH, the mechanism of action of heme or CO will be described (FIG. 1).
FIG. 1 is a diagram showing the oxidative modification of GAPDH in cells and the behavior of cells at that time. As shown in FIG. 1, GAPDH is subjected to oxidative stress due to NO, and the 152st cysteine (Cys152) in the amino acid sequence of GAPDH is oxidatively modified. When GAPDH is oxidized and modified, a nuclear translocation protein called SIAH1 present in the cytoplasm binds to GAPDH, and GAPDH translocates into the nucleus. When GAPDH moves into the nucleus, GAPDH activates p53, a transcription factor. Since p53 has functions such as maintaining cell homeostasis and inducing apoptosis, activation of p53 induces apoptotic cell death.

On the other hand, oxidatively modified GAPDH forms an abnormal SS bond with each other to form an abnormal complex of GAPDH. This complex promotes the formation of aggregates such as β-amyloid and α-synuclein, and induces neuronal cell death.
As described above, in the molecular mechanism of GAPDH, oxidative modification of GAPDH is one of the causes for apoptosis and neurological diseases. Therefore, in the present invention, oxidative modification of GAPDH in the cytoplasm can be inhibited, and inhibition of oxidative modification can inhibit GAPDH translocation into the nucleus, thereby protecting cells from apoptosis and the like.

  In order to inhibit the enzyme activity of GAPDH in the method of the present invention, GAPDH and its substrate are incubated in the presence of heme, CO, or both to inhibit the catalytic action of the substrate by GAPDH, or Samples may be treated with heme, CO, or both. When both heme and CO are used, one can be processed first and the other can be processed later, or both can be processed simultaneously. This prevents the reaction of GAPDH with its substrates glyceraldehyde 3-phosphate and coenzyme NAD.

  In the method of the present invention, in order to inhibit oxidative modification of GAPDH, GAPDH is incubated in the presence of heme, CO, or both, or a test cell is treated with heme, CO, or both. Good. Furthermore, in the method of the present invention, inhibition of GAPDH translocation into the nucleus can be carried out by treating a test cell with heme, CO, or both.

“Incubation” refers to reacting GAPDH with a substrate in the presence or absence of heme and / or CO in a reaction system such as in a test tube so that GAPDH is exposed to heme, CO, or both. means.
“Treatment” means contacting GAPDH with CO and / or heme such that GAPDH is exposed to heme, CO, or both. Contact means, for example, culturing cells in the presence of heme and / or CO, adding heme and / or CO to a solution containing isolated GAPDH, and making GAPDH and heme and / or CO identical. It means to be mixed in the reaction system.

  In the above method, each of the above inhibitory effects of GAPDH is caused by binding of heme to the NAD recognition site of GAPDH. Moreover, CO can synergistically enhance the inhibitory activity of GAPDH by heme.

Whether heme and / or CO has inhibited the enzyme activity of GAPDH is determined by a commonly used biochemical method such as reacting GAPDH with a substrate in the presence of a coenzyme, and the product (NADH) produced by the reaction. Can be confirmed by measuring at a predetermined absorbance (340 nm).
Whether heme and / or CO inhibited the oxidative modification of GAPDH or whether GAPDH was translocated into the nucleus was determined by commonly used biochemical techniques such as SDS-polyacrylamide gel electrophoresis (SDS- PAGE), Western blotting, immunoprecipitation, etc.

4). Summary As mentioned above, a large amount of endogenous gas molecules such as CO are produced in vivo, but the molecular mechanisms such as metabolism, stress response, and diseases via CO gas in the intestine are still unclear. Many. In the present invention, a novel heme protein GAPDH responsive to CO gas was successfully identified using a unique screening technique.

CO gas has been shown to exhibit an interesting molecular response that promotes the formation of the heme / GAPDH complex. Furthermore, heme / CO not only inhibits GAPDH enzyme activity, but also blocks cellular damage due to stress by selectively protecting the oxidative modification of the oxidative stress responsive residue (Cys152) contained in GAPDH. It was possible to clarify the dynamic biological defense reaction. Elucidation of the molecular mechanism of biological function through gas molecules can be applied as a diagnostic marker for stress states and diseases, and can lead to the establishment of treatments for diseases using this as an index. Therefore, the knowledge obtained in the present invention is considered to be a very interesting result, and further development is expected.
Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

Identification of heme-binding protein GAPDH In general, affinity chromatography is known as a technique for analyzing a protein that selectively binds to a ligand. Affinity chromatography uses a carrier on which a ligand is immobilized (porous agarose beads or the like), and the beads are packed in a column and the target protein is affinity purified.
However, in this method, it is difficult to purify the target ligand-binding protein from the crude cell extract with a single operation.
Therefore, in the present invention, affinity nanobeads capable of achieving high recovery and high purity were used instead of affinity chromatography (Nishio K, et al., Colloids Surf B Biointerfaces. 64 (2): 162-9, 2008. ).

This affinity nanobead is a bead having a particle size of about 200 nm with styrene as a core and the surface thereof covered with glycidyl methacrylate (GMA), and is called “SG bead” (FIG. 2).
Heme binds to the bead by binding of the epoxy group of GMA that forms the surface of the SG bead and the hydroxyl group of heme (the “OH” portion of COOH in the structural formula of heme in FIG. 2). Next, when the beads and the cell extract are mixed, a protein having heme as a ligand specifically binds. Thereafter, the beads are washed, and the protein bound to the beads is eluted (elution can be performed by changing the salt concentration or pH) to obtain a protein group bound to heme. The obtained protein group is separated by SDS-polyacrylamide gel electrophoresis to obtain the target protein.

Specifically, it was performed as follows.
Nanoaffinity beads (FG-NEGDEN beads) for immobilizing heme were those described in the following literature.
Nishio K, et al., Colloids Surf B Biointerfaces. 64 (2): 162-9, 2008.
For immobilization of heme on beads, hemin (Porphyrin Scinece) 1 mM (in DMF) was reacted with equimolar N-hydroxysucciimide (Wako Pure Chemical) and WSC (Wako Pure Chemical) for 2 hours at room temperature. This reaction solution was stirred with 2 mg of equilibrated FG-NEGDEN beads and reacted at room temperature for 12 hours, then washed three times with DMF, reacted with 1 mM acetic anhydride (Nacalai Tesque) for 2 hours at room temperature, and washed again. And stored as hem-immobilized beads at 4 ° C.

For purification of heme-binding factor, 1 mg / ml of protein extract prepared from mouse liver in buffer (20 mM Hepes (pH 7.9), 100 mM NaCl, 1 mM MgCl ₂ , 0.2 mM EDTA, 10% glycerol, 0.1% NP40) The resulting mixture was diluted with 0.2 mg of beads and allowed to react at 4 ° C. for 1 hour.
After washing the beads with a buffer, the protein bound to the beads was recovered, and the protein solution suspended with SDS-Loading dye was developed on a 12.5% SDS-polyacrylamide gel and detected by silver staining (FIG. 3). .
The obtained protein was decomposed into peptide fragments by Trypsin in-gel digestion method and analyzed by liquid chromatography / mass spectrometry (LC-MS) apparatus (Hitachi Nano Frontier).
As a result, the band appearing around 35.7 kDa was found to be a heme protein, and the obtained protein was identified as GAPDH.

GAPDH Gas Response A model showing inhibition of GAPDH enzyme activity by heme and / or CO is shown in FIG.
GAPDH uses NAD (nicotinamide adenine dinucleotide) as a coenzyme to convert glyceraldehyde 3-phosphate to glycerate 1,3 diphosphate and has a catalytic action to convert NAD to NADH (reduced form of NAD) . If heme is added to this, the catalytic action of GAPDH is inhibited, and conversion from NAD to NADH is not performed. Therefore, the enzymatic activity of GAPDH can be measured using the amount of NADH produced as an index.

Therefore, the enzyme activity was measured as follows.
GAPDH enzyme activity was measured using 400 U of erythrocyte-derived GAPDH (Sigma) in 400 ml of a reaction solution (100 mM Tris (pH 8.3), 3.75 mM GA3P, 1 mM NAD, 5 mM DTT, 5 mM sodium arsenate, 0.5 mM MgCl ₂ ). The production of NADH was detected by measuring the absorbance at 340 nm.

Hemin was added to this reaction system at a predetermined concentration (FIG. 5) and incubated for 5 minutes to detect the inhibitory effect. In addition, Ruthenium Chloride or Tricarbonyl dichloro ruthenium (CO donor) (both Sigma) was added together with hemin to verify this inhibitory effect.
As a result, it was found that the enzyme activity of GAPDH was inhibited by the addition of heme, and CO further enhanced this effect (FIG. 5).

  In FIG. 5, the activity of GAPDH (vertical axis of the graph) shows the activity of GAPDH as a control when heme (hemin in this example) is not added, that is, the amount of GAPDH when NADH production is measured without adding heme. The values are shown as relative values when the enzyme activity (measured value at an absorbance of 340 nm) is 100%. It can be seen that increasing the amount of heme added inhibited the enzyme activity in a dose-dependent manner. Also, when CO was added, the activity was the same as with heme, and the activity decreased depending on the CO concentration.

  This indicates that the enzyme activity of GAPDH was inhibited by heme and / or CO.

As for the absorption spectrum of GAPDH and heme, the absorbance from 350 nm to 700 nm was measured by adding 10 mM hemin to a final concentration of 10 mM of erythrocyte-derived GAPDH in 100 mM Tris (pH 8.3). Under reducing conditions (Fe _(II) ), dithionite (Sigma) was added at a final concentration of 5 mM, and the absorbance was measured in the same manner. In addition, CO gas in a gas cylinder was sprayed on the reaction solution to analyze the CO-bonded spectrum. As a result, maximum wavelengths were observed at 390, 410 nm for oxidation conditions, 426, 540, 565, 630 nm for reduction conditions, 420, 540, 565, and 630 nm for CO types, and significant wavelength changes were observed for reduction and CO types. As can be seen, the heme on GAPDH responds to CO gas (Fig. 6).

  As a result of absorption spectrum analysis of GAPDH bound to heme, the absorption maximum wavelength in the Soret region is shifted from 426 nm to 420 nm by CO addition in the reduced state, so that CO gas responds through heme bound to GAPDH. Things became clear.

GAPDH heme binding site
In order to verify the binding site of heme to GAPDH, a point mutant recombinant protein in which histidine residue (His) or cysteine residue (Cys) in GAPDH was substituted with alanine was prepared. The binding activity was verified by reacting 1 mg of these proteins with heme-immobilized beads.

  It is known that heme proteins usually recognize Fe atoms in heme molecules and bind to heme via cysteine residues (Cys) or histidine residues (His). Therefore, in order to examine the heme binding site of GAPDH, a point mutant protein at the Cys or His residue in the GAPDH molecule was prepared and the heme binding activity was examined.

The binding activity was measured as follows.
GAPDH having the amino acid sequence of Accession No. P04406 (FIG. 7, SEQ ID No. 1) was used. In the amino acid sequence of GAPDH, the portion of the amino acid residue into which a mutation has been introduced is shown by the underline in FIG. Various GAPDH point mutants were prepared by PCR.
As a result, it was found that the binding activity was completely lost in the mutant in which the 152nd Cys residue (Cys152) was substituted with alanine (FIG. 8).

  In FIG. 8, the “WT” band represents wild type GAPDH. The notation of alphabets, numbers, and combinations of alphabets represents the mode of amino acid sequence variation. The alphabet before the number is the amino acid sequence before the mutation at the position of the number in the amino acid sequence of GAPDH, and the alphabet after the number is the amino acid after the mutation at the position of the number in the amino acid sequence of GAPDH Is an array. For example, “H53A” is a mutant in which the 53rd histidine (His) is replaced with alanine (Ala) in the amino acid sequence of GAPDH (FIG. 7). Other mutants are also represented in the same manner, and “C152A” is a mutant in which the 152nd cysteine (Cys) is substituted with alanine (Ala) in the amino acid sequence of GAPDH. “Input” is a band of GAPDH before the reaction between GAPDH and heme, and is a control diagram showing that GAPDH was indeed present before the reaction.

As can be seen from the electrophoresis image shown in FIG. 8, the band disappears in the C152A mutant (lane “C152A”). This indicates that GAPDH of the C152A mutant did not bind to heme, that is, heme did not bind when the amino acid residue at position 152 of GAPDH was mutated.
Therefore, it was shown that the amino acid residue at position 152 of GAPDH is an essential residue for binding to heme.

By the way, in the said experiment, although the binding test of LDH (lactate dehydrogenase) and GAPDH was conducted, the binding activity with GAPDH was not recognized even if heme was present ("LDH-A" in Fig. 3). Lane).
This indicates that the enzyme to which heme binds has specificity, and the same NAD-dependent oxidoreductase binds to GAPDH but not to LDH.
Cys152 is a residue that serves as an active center for transferring protons to coenzyme NAD in the enzyme activity of GAPDH.
As a result of biochemical analysis of the inhibitory effect of heme on the enzyme activity of GAPDH, it was found that heme was competitively competitive with NAD and inhibited. In addition, heme / GAPDH docking simulation was performed using the crystal structure of GAPDH as an index, and it was shown that heme also binds to the NAD recognition site of GAPDH.

Inhibition of oxidative modification and cytoprotective action of GAPDH by heme / CO (1) Inhibition of oxidative modification
Since GAPDH reacted with NO gas and Cys152 residue was oxidatively modified, the effect of heme / CO on this action was verified.
The recombinant vector expressing the GAPDH recombinant fused with the FLAG tag is cultivated by transfection (Lipofectamin2000, INVITROGEN) into the neural cell line SH-SY5Y cells and culturing with the resistance marker G418. A cell line expressed in To this was added 1 mM of NOR3 (DOJINDO), which is a NO donor, and incubated for 12 hours.
Thereafter, the cells were disrupted and extracted, and FLAG-GAPDH was purified by a resin (Sigma) to which an anti-FLAG antibody was conjugated.

Since GAPDH forms a homotetramer in vivo, endogenous GAPDH (endogenous GAPDH) is simultaneously purified together with FLAG-GAPDH. This was developed on SDS-PAGE and detected using anti-GAPDH antibody or anti-S-nitrosylated GAPDH antibody (provided by Johns Hopkins University, Dr. Akira Sawa).
The anti-GAPDH antibody is an antibody that binds to GAPDH, and the anti-S-nitrosylated GAPDH antibody is an antibody that binds to S-nitrosylated GAPDH, that is, oxidatively modified GAPDH.
The results are shown in FIG. In FIG. 9, it can be seen that when heme (2 μM and 5 μM) is reacted with GAPDH, the band becomes thinner in a dose-dependent manner. This indicates that the amount of GAPDH oxidatively modified by heme is reduced, that is, the oxidative modification of GAPDH is inhibited. As with heme, the band was thin when CO was added, indicating that CO inhibited oxidative modification of GAPDH.

  As a result, it was found that the oxidative modification of GAPDH observed with the addition of NO donor (NOR3) was significantly suppressed by the addition of heme or CO donor (FIG. 9). In addition, GADPH binds to nuclear migratory SIAH1 by oxidative modification and localizes in the nucleus, but it has also been found that addition of heme or CO inhibits these effects (Example 5).

(2) Cell protective effect
Since GAPDH is known to induce cell death by oxidative modification with NO, the cytoprotective effect of heme / CO was examined.
0.5 mM NOR3 was added to SH-SY5Y cells and incubated for 12 hours. The survival rate when heme or CO was added to this was detected. The survival rate was detected using a cell death detection kit (Wako Pure Chemical Industries) based on LDH activity.
The results are shown in FIG. In FIG. 10, the vertical axis indicates the rate of cell death, a high bar indicates that cell death has occurred, and a low bar indicates that cell death has not occurred and the cells are protected. . It can be seen that GAPDH is oxidized by NO treatment, which causes cell death, whereas treatment of heme or CO suppresses cell death in a dose-dependent manner.
As a result, cell death was observed with the addition of NO, but it became clear that cell death was remarkably suppressed by the simultaneous addition of hemin or CO donor (FIG. 10), and heme / CO suppressed the oxidative modification of GAPDH. It was shown to show a cytoprotective action.

Inhibition of GAPDH nuclear translocation In this example, it was examined whether heme / CO inhibits GAPDH nuclear translocation.
This test was performed as follows.
SH-SY5Y cells expressed by transfection with the SIAH1 expression vector were disrupted using a buffer containing 0.1% NP40, 1 mM phenyomethylsulfonyl fluoride, 40 mM Tris-HCl (pH 8.0), 150 mM NaCl. A cytoplasmic fraction was prepared. The insoluble fraction was centrifuged to prepare the nuclear component, and the nuclear extract was prepared using a buffer containing 0.1% NP40, 1 mM phenyomethylsulfonyl fluoride, 40 mM Tris-HCl (pH 8.0), 400 mM NaCl. did. These fractions were fractionated by SDS-PAGE and then detected using an antibody of the indicated target protein.

As a result, in FIG. 11, when GAPDH is oxidized and modified by NO treatment, the GAPDH band in the nucleus becomes deeper, indicating that GAPDH has migrated into the nucleus due to the oxidation modification. In contrast, when hemin was added, the GAPDH band became thinner in a dose-dependent manner, indicating that GAPDH was inhibited from nuclear translocation by heme.
As described above, since heme binds to GAPDH via Cys152 residue subjected to oxidative modification, the present inventor thinks that heme and CO gas may antagonize such oxidative stress response of GAPDH. It was.

  Therefore, the effect of heme or CO gas on the oxidation modification of GAPDH by adding NO was investigated. As a result, it was found that heme / CO remarkably suppressed oxidative modification (S-nitrosylation) of GAPDH at the cellular level. In vitro experiments using recombinant proteins have also revealed that heme / CO specifically inhibits oxidative modification of Cys152 of GAPDH. Furthermore, heme / CO was found to significantly inhibit cell death induction by NO gas.

  According to the present invention, an enzyme activity inhibitor, an oxidation modification inhibitor, and a nuclear translocation inhibitor of GAPDH containing heme or carbon monoxide are provided. These inhibitors can be used for neurodegenerative diseases and the like.

Claims (14)

An enzyme activity inhibitor of GAPDH containing heme and / or carbon monoxide. An oxidation modification inhibitor of GAPDH containing heme and / or carbon monoxide. A nuclear translocation inhibitor of GAPDH containing heme and / or carbon monoxide. An inhibitor of s-nitrosylation modification of protein comprising heme and / or carbon monoxide. The s-nitrosylation modification inhibitor according to claim 4, wherein the protein is GAPDH. A pharmaceutical composition for cell protection or cell injury treatment, comprising heme. A method for inhibiting GAPDH enzyme activity, which comprises incubating GAPDH and its substrate in the presence of heme and / or carbon monoxide to inhibit the catalytic action of the substrate by GAPDH. A method for inhibiting GAPDH enzyme activity, comprising incubating GAPDH in the presence of heme and / or carbon monoxide to inhibit GAPDH enzyme activity. A method for inhibiting oxidative modification of GAPDH, wherein GAPDH is incubated in the presence of heme and / or carbon monoxide to inhibit oxidative modification of GAPDH. A method for inhibiting GAPDH enzyme activity, comprising treating a cell with heme and / or carbon monoxide to inhibit GAPDH enzyme activity in the cell. A method for inhibiting oxidative modification of GAPDH, comprising treating a cell with heme and / or carbon monoxide to inhibit oxidative modification of GAPDH in the cell. A method for inhibiting GAPDH translocation into the nucleus, comprising treating cells with heme and / or carbon monoxide to inhibit GAPDH in the cells from translocating from the cytoplasm into the nucleus. The method according to any one of claims 8 to 12, wherein the enzyme activity inhibition, oxidation modification inhibition or nuclear translocation inhibition of GAPDH is caused by binding of heme to the NAD recognition site of GAPDH. The method according to any one of claims 8 to 12, wherein carbon monoxide enhances enzyme activity inhibition, oxidation modification inhibition, or nuclear translocation inhibition of GAPDH by heme.