KR20050107007A - Human daxx protein and a fragment thereof as binding agents of human nhe1 ion channel protein - Google Patents

Abstract

The present invention relates to the use of human Daxx protein (death-associated protein 6) as a human sodium hydrogen exchanger 1 (NHE1) ion channel protein binder, and to fragments of amino acids 571 to 625 of human Daxx protein and human NHE1 ion channel protein binder thereof. It is about a use.

Description

Human Daxx protein and a fragment according as binding agents of human NHE1 ion channel protein

The present invention relates to novel uses of human Daxx protein (death-asscoaited protein 6), and novel fragments of human Daxx protein and its use. More specifically, the present invention relates to the use of human Daxx protein as a human sodium hydrogen exchanger 1 (NHE1) ion channel protein binder, and to fragments of amino acids 571 to 625 region of human Daxx protein and its human NHE1 ion channel protein binder. It is about.

Life control phenomena caused by cell death appear in the development, differentiation, or cancer, immunodeficiency, degenerative disease or ischemic disease of the organism, which is called apoptosis. In ischemic diseases, necrosis and apoptosis of cells are caused by ischemia. In particular, apoptosis after reperfusion is a major cause of myocardial injury. In addition, the release of CD95L, TRAIL, and TNFα, known as death inducing ligand (DIL) during ischemia, increases, and thus, regulation of apoptosis in ischemic heart disease is very important. The apoptosis during ischemia is caused by the acidification of the pH of the cells by the change of various ion channels present in the cell membrane. Representative ion channels related to ischemia are K ⁺ -ATPase (sodium-potassium channel) and NHE (sodium- hydrogen exchanger) and sodium-calcium exchanger (NCX).

As a protein related to ischemic apoptosis, there are several isoforms in the NHE ion channel protein, among which the human NHE1 ion channel is composed of 815 amino acids. The N-terminus of the NHE ion channel protein is hydrophobic and has 12 transmembrane regions, the C-terminus is hydrophilic and regulated by hormones, and several isoforms have sequence similarity with each other at the C-terminus. It is known to have. NHE ion channel proteins do not work in normal cellular conditions, but are activated by excessive acidification in cells during ischemia, increasing intracellular Na ⁺ ions. At this time, the sodium calcium exchanger (NCX) operates in reverse to discharge excessively increased Na ⁺ ions in the cell, thereby increasing the intracellular calcium concentration, and the excessive increase of intracellular calcium causes cell death. It is reported.

As described above, ischemic apoptosis due to NHE ion channel activation is known to perform its function by correlation with signal transmitters by NHE ion channel composed of about 815 amino acids. In addition, the presence of an H ⁺ ion sensor at the membrane protein site has been reported to be involved in the regulation of NHE ion channel activity. In addition, it is reported that calcium plays an important role in the ischemic cell death signaling system by the NHE ion channel, and calcium binding proteins such as calmodulin are involved. In particular, it has recently been found that tescalcin, a calcium binding protein, binds to the cytoplasmic region of NHE ion channel proteins and is involved in the regulation of NHE channel activity. In other words, it is reported that the structural change of the NHE ion channel and the sensitivity to H ⁺ are controlled by calcium and pH change, and tescalin is closely related.

In addition, NHE is known to be associated with arrhythmia, diabetic heart disease, heart hypertrophy and heart failure.

Oxygen supply from the periphery of tissues is the most important factor in the growth, development and homeostasis of tissues. This oxygen is regulated by the cardiovascular system to supply vastly listed blood vessels in the body. However, cells affected by the lack of oxygen and energy due to arterial stenosis have a fatal effect on their viability, and ischemic disease is a representative disease of such cell damage. Ischemic diseases have already been known as the most common cause of death in the West for a long time, while in the East including Korea, it is increasing rapidly with recent changes in eating habits. Ischemic diseases are involved in cell death in important areas that are regulated by the nerves, such as the brain, colon, and lower extremities, as well as the heart, and thus are primarily linked to life or directly affect physical activity. Nevertheless, the development of effective therapeutic or preventive agents for ischemic diseases has not been achieved until now. In recent years, attempts have been made to control ischemic apoptosis by regulating the activity of ion channels, but have not revealed a clear regulatory mechanism. In addition, an NHE ion channel inhibitor was developed to suppress ischemic apoptosis, but it does not have a clear effect due to the incomplete establishment of an accurate cell signaling system.

On the other hand, Fas, like other members of the tumor necrosis factor (TNF) receptor gene family, binds to Fas to form three-membrane receptors, which form a trimer, and the Fas-associated death domain with a death domain (DD). The adapter protein called) binds to the death domain of the cytoplasmic site of Fas through its death domain. FADD also has a death effector domain that can bind to similar domains present in caspase-8. Collected by FADD, caspase-8 is activated by its cleavage and activates caspase that executes apoptosis. Human Daxx (death-associated protein 6) protein (SEQ ID NOs: 1 and 2, Figure 6) recognizes and binds to the Fas death domain in receptor-mediated apoptosis by this Fas, and is activated by stress, a death pathway independent of FADD. It is known to act to activate c-jun amino terminal kinase (JNK). However, the fact that human Daxx binds to human NHE1 and is involved in ischemic cell death has not been revealed at all.

The inventors have focused on the development of new anti-ischemic agents by detecting proteins that specifically bind to the cytoplasmic domain of NHE ion channels known to be involved in ischemic apoptosis as described above. Was performed. As a result, we confirmed that human Daxx protein, which is known to be involved in receptor mediated apoptosis, binds to human NHE1 ion channel protein, and further finds a fragment of human Daxx protein having the minimum site of activity that binds to human NHE1 ion channel protein. Came out.

It is therefore an object of the present invention to provide human NHE1 ion channel protein binders containing human Daxx proteins.

Another object of the present invention is to provide a fragment of a specific site of human Daxx protein having human NHE1 ion channel binding activity and a human NHE1 ion channel protein binding agent containing the same.

The human NHE1 ion channel protein binding agent may be usefully used as an anti-ischemic agent.

First, the present invention relates to a human NHE1 ion channel protein binder containing human Daxx protein as an active ingredient.

Secondly, the present invention relates to fragments of amino acids 571 to 625 of human Daxx protein having an activity of binding to NHE1 ion channel protein.

Third, the present invention relates to an NHE1 ion channel protein binder containing the human Daxx fragment.

Human NHE1 ion channel protein binding agents of the present invention can be used as a prophylactic or therapeutic agent for ischemic diseases such as cardiac ischemia, retinal ischemia, diabetic vascular heart disease, heart failure or myocardial hypertrophy.

Hereinafter, the present invention will be described in detail.

In the present invention, yeast two-hybrid screening was performed to search for factors that bind to the intracellular region (amino acids 508-815) of the human NHE1 ion channel protein. Yeast 2-Hybrid Screening Labels All cDNA Libraries of Target Genes and Cells and Puts it in Yeast for Yeast Colors to Be Changed When the Binding to the Target Protein Occurs and Easily Recognizes the Binding As a method of obtaining information on the binding protein by revealing, in the present invention, NHE1 was used as a target protein. As a result, it was confirmed that the human Daxx protein, which is known to be involved in receptor mediated apoptosis, directly binds to the cytoplasmic region of the NHE1 ion channel protein, which was reconfirmed using in vitro and in vivo binding assays. That is, in the present invention, in order to detect a protein that binds to an NHE ion channel, a GST (glutathion S-transferase) fusion protein in a region of the NHE cytoplasm is prepared, and specifically bound to NHE using coprecipitation. Proteins were identified by two-dimensional electrophoresis. Immunoprecipitation using GST has the advantage that it is possible to settle a large amount of protein to visualize the NHE binding protein present in the cell by using a large amount of GST fusion protein. In addition, NAL binding proteins can be identified using MALDI-TOF MS.

Furthermore, the inventors have found that amino acid sites 571-625 (SEQ ID NOs: 3 and 4) of the human Daxx protein are the minimum sites having the activity of binding to the intracellular region of the NHE1 ion channel protein. Based on these results, it was confirmed that when intracellular injection of human Daxx protein increases intracellular pH, it affects ischemic apoptosis.

Therefore, it can be seen that the human Daxx protein and amino acids 571 to 625 thereof can be usefully used as a prophylactic or therapeutic agent for ischemic disease. In addition, it is thought that the human Daxx protein, which is known to be involved in receptor mediated apoptosis, has a specific binding activity to NHE ion channel protein in terms of NHE ion channel activity regulation.

The total daily dose to be administered to a patient when administered for clinical purposes in the Daxx protein or fragment of the invention ranges from 0.05 to 200 mg / kg body weight, preferably 0.05 mg / kg body weight. However, the dosage for a particular patient may be increased or decreased depending on the weight, sex, health condition, diet, time of administration, method of administration, and severity of the disease.

Daxx proteins or fragments of the invention can be used directly infused or formulated in solution or micelle form. The anti-ischemic agent according to the present invention may be applied to the human body by parenteral or topical administration, and is preferably administered by injection such as intravenous injection, subcutaneous injection, endothelial injection, intramuscular injection, and the like. For this purpose, the active ingredient of the present invention may be suspended or dissolved in a pharmaceutically acceptable water soluble carrier.

Hereinafter, the present invention will be described in more detail with reference to Examples, which are intended to help the understanding of the present invention, but do not limit the scope of the present invention in any way.

Example 1 Screening of NHE1 Ion Channel Binding Proteins Through Yeast 2-Hybrid Screening

Proteins that bind to the intracellular region of the NHE1 ion channel were searched using the yeast two-hybrid screening method. That is, the region (amino acid No. 508-815) of the NHE1 ion channel cytoplasm was cloned into a pLexA vector (BD bioscience Clontech, California, USA) and searched from the HeLa cDNA library.

The results are shown in FIG. As shown in FIG. 1, human Daxx protein was detected with NHE1 binding protein. As a positive control, FADD and TRADD, which are already known to bind to NHE1, were used. As a negative control, FADD and Daxx were used.

Example 2: GST-Daxx (1 to 740), GST-Daxx (1 to 130), GST-Daxx (1 to 400), GST-Daxx (1 to 571), GST-Daxx (1 to 625), GST Preparation of -Daxx (575-740 ) and GST-Daxx ( 625-740 )

Human Daxx, which was detected as binding to the NHE1 ion channel in Example 1, was cloned into a pGEX-4T-1 vector (Amersham Pharmacia Biotech, Piscataway, USA), which is a GST-fusion vector, to obtain seven types of cleaved proteins. To clone Daxx deletion mutants into a GST fusion vector, PCR (polymerase chain reaction) was used as primers for Eco R I and Xho I. Primers containing the enzyme linker were performed using Daxx full length cDNA as a template. The recombinant vector was transformed into E. coli BL21 (DE3) strain (Novagen) to obtain transformed colonies. To isolate the GST fusion protein, cells were cultured at 37 ° C. such that the OD ₆₀₀ was 0.5 to 0.6, followed by the expression of GST-Daxx deletion mutants by adding isopropyl-β-D-galactopyranoside (IPTG) at a concentration of 0.5 mM. Induced. The cells were sonicated in buffer (200 mM Tris-HCl, pH 8.0, 0.5 M NaCl, 100 μM EDTA, 0.1% Triton X-100, 0.4 mM PMSF), centrifuged at 13,000 rpm, and the supernatants were harvested. Glutathione agarose beads were added to an amount of supernatant, left at 4 ° C. for 4 hours, and GST fused Daxx deletion proteins were separated and quantified on an SDS-PAGE gel.

Example 3 In Vitro Binding of Human NHE1 Ion Channels and Daxx

In order to confirm whether the NHE1 ion channel and Daxx mutual interaction is direct, NHE1.cd (cytoplasmic domain) was labeled with ³⁵ S-methionine, and the mutual binding between the GST fused Daxx proteins prepared in Example 2 was investigated. Both proteins were added to binding buffer (50 mM HEPES, pH 7.6, 50 mM NaCl, 5 mM EDTA, 0.1% Nonidet P-40 and 10% glycerol) and left for 3 hours at 4 ° C. Washed twice. The washed protein-binding complexes were separated on an SDS-PAGE gel and radioactive images were obtained after exposure to the X-ray film. The results are shown in FIG. As shown in Figure 2, Daxx protein was confirmed to specifically bind to the region of the NHE ion channel cytoplasm.

Example 4 In Vivo Binding of Daxx with Human NHE1 Ion Channels

As observed in Example 3, in view of the fact that Daxx specifically binds to NHE1.cd in vitro, we attempted to reconfirm intracellular interaction. In order to clone the Daxx full-length cDNA sequence into a FLAG-fused pcDNA3 (myc / FLAG) vector (Invitrogen, California, USA), a primer comprising Eco R I and Xho I enzyme linkers was prepared, and Daxx full-length cDNA was used as a template. Was carried out. The cloned FLAG-Daxx clone was introduced into the BOSC23 cell line (ATCC CRL-11554), a human kidney derived cell line. After incubation for 48 hours, cells were harvested, sonicated in lysis buffer (50 mM Tris-Cl (pH8.0), 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.4 mM PMSF) and centrifuged. The supernatant was taken to isolate and remove cell debris. From this supernatant, FLAG antibody (Sigma, Missouri, USA) was added, and immunoprecipitation was performed for 4 hours at 4 ° C. In order to identify protein complexes immunoprecipitated with Daxx protein, binding to NHE1 was confirmed by separation on an SDS-PAGE gel. The results are shown in Figure 3, from which it can be confirmed that Daxx protein binds to NHE1.cd.

Example 5 Determination of Binding Sites of Daxx Binding to Human NHE1 Ion Channels

As confirmed in the above examples, Daxx specifically binds to NHE1.cd, so in this example, various GSTs prepared in Example 2 were determined in order to determine binding sites with NHE1.cd by in vitro binding assays. A fused Daxx deletion mutant protein was obtained. Regions of NHE1 cytoplasm were cloned into pcDNA3 vector (Invitrogen, California, USA) and labeled with ³⁵ S-methionine in vitro. NHE1.cd labeled with ³⁵ S-methionine and respective GST-Daxx-deficient mutant proteins (GST-Daxx (1-740), GST-Daxx (1-130), GST-Daxx (1-400), GST- Daxx (1 ~ 571), GST-Daxx (1 ~ 625), GST-Daxx (575 ~ 740), GST-Daxx (625 ~ 740)) were added to the binding buffer and reacted at 4 ° C for 4 hours. , Separated on SDS-PAGE gel, and then exposed to X-ray film.

The results are shown in FIG. 4, from which it can be seen that the NHE1.cd protein labeled with ³⁵ S-methionine was detected. Therefore, it was confirmed that amino acids 571 to 625 of Daxx play an important role in the binding of NHE1.cd.

Example 6: Measurement of pH change in cells after introduction of human Daxx into cells

In order to evaluate the ability to inhibit ischemic apoptosis by Daxx protein, the pcDNA3 (myc / FLAG) / Daxx clone prepared in Example 4 was analyzed by introducing into the myocardial cell line H9c2 cell line (ATCC CRL-1446). For the analysis, Daxx-introduced cells were incubated for 12 hours by adding 5 × 10 ⁴ cells to a 60 mm cell culture vessel, followed by ischemia / reperfusion (I / R) (2 hours ischemia and 12 hours reperfusion). Treatment resulted in ischemic apoptosis. Then, PBS buffer and the non--Na ⁺ washing with buffer, the intracellular pH measured fluorescence instructions solution of BCECF-AM (2 ', 7' -bis- (2-carboxyethyl) -5 (6 for) - carboxyfluorescein, Acetoxymethyl ester) was treated with 10 μM to treat stimulation (excitation) at 440 nm and 490 nm in the fluorometer, and the emission (emission) was measured at 530 nm.

The results are shown in FIG. As shown in Figure 5, the cells introduced Daxx in the steady state was observed to increase the intracellular pH. On the other hand, in ischemia / reperfusion, it was observed that the intracellular pH was maintained in the normal state of the cells into which Daxx was introduced, whereas the intracellular pH was increased by the NHE1 channel activation. This suggests that NHE1 channel activation by intracellular acidification during ischemia / reperfusion was inhibited by direct binding of Daxx.

As described above, it was confirmed that the human Daxx protein, particularly its 571-625 amino acid site, specifically binds to NHE1, and the intracellular pH is increased in the Daxx-introduced cell line. Therefore, human Daxx protein is expected to be useful for the development of anti-ischemic drugs. In addition, it will provide a basis for research on ischemia at the protein level, thereby providing an opportunity for more active molecular and cellular biological studies on ischemia. In addition, it is considered that human Daxx, which is found to be involved in receptor mediated apoptosis, has specific binding activity for NHE ion channel in terms of regulation of NHE ion channel activity.

1 is a diagram showing that the human Daxx protein binds to human NHE1 ion channel protein by the yeast two-hybrid screening method;

FIG. 2 shows Daxx protein binds to NHE1 ion channel protein in vitro; FIG.

FIG. 3 shows that Daxx protein binds to the cytoplasmic region of the NHE1 ion channel protein in vivo; FIG.

FIG. 4 shows that amino acids 571-625 of Daxx protein bind to NHE1 ion channel protein in vivo; FIG.

5 is a graph showing intracellular pH change by ischemia / reperfusion after introduction of human Daxx protein into cardiomyocytes:

6 shows the base and amino acid sequences of human Daxx proteins.

<110> Dongbu Hannong Chemical Co., Ltd. <120> Human Daxx protein and a fragment according as binding agents of human NHE1 ion channel protein <130> PC04-0078-BJHD <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 2223 <212> DNA <213> Homo sapiens <220> <221> CDS (222) (1) .. (2220) <400> 1 atg gcc acc gct aac agc atc atc gtg ctg gat gat gat gac gaa gat 48 Met Ala Thr Ala Asn Ser Ile Ile Val Leu Asp Asp Asp Asp Glu Asp 1 5 10 15 gaa gca gct gct cag cca ggg ccc tcc cac cca ctc ccc aat gcg gcc 96 Glu Ala Ala Ala Gln Pro Gly Pro Ser His Pro Leu Pro Asn Ala Ala 20 25 30 tca cct ggg gca gaa gcc cct agc tcc tct gag cct cat ggg gcc aga 144 Ser Pro Gly Ala Glu Ala Pro Ser Ser Ser Glu Pro His Gly Ala Arg 35 40 45 aga agc agt agt tcg ggc ggc aag aaa tgc tac aag ctg gag aat gag 192 Arg Ser Ser Ser Ser Gly Gly Lys Lys Cys Tyr Lys Leu Glu Asn Glu 50 55 60 aag ctg ttc gaa aag ttc ctt gaa ctt tgt aag atg cag aca gca gac 240 Lys Leu Phe Glu Lys Phe Leu Glu Leu Cys Lys Met Gln Thr Ala Asp 65 70 75 80 cat cct gag gtg gtc cca ttc ctc tat aac cgg cag caa cgt gcc cac 288 His Pro Glu Val Val Pro Phe Leu Tyr Asn Arg Gln Gln Arg Ala His 85 90 95 tct ctg ttt ttg gcc tcg gcg gag ttc tgc aac atc ctc tct agg gtc 336 Ser Leu Phe Leu Ala Ser Ala Glu Phe Cys Asn Ile Leu Ser Arg Val 100 105 110 ctg tct cgg gcc cgg agc cgg cca gcc aag ctc tat gtc tac atc aat 384 Leu Ser Arg Ala Arg Ser Arg Pro Ala Lys Leu Tyr Val Tyr Ile Asn 115 120 125 gag ctc tgc act gtt ctc aag gcc cac tca gcc aaa aag aag ctg aac 432 Glu Leu Cys Thr Val Leu Lys Ala His Ser Ala Lys Lys Lys Leu Asn 130 135 140 ttg gcc cct gcc gcc acc acc tcc aat gag ccc tct ggg aat aac cct 480 Leu Ala Pro Ala Ala Thr Thr Ser Asn Glu Pro Ser Gly Asn Asn Pro 145 150 155 160 ccc aca cac ctc tcc ttg gac ccc aca aat gct gaa aac act gcc tct 528 Pro Thr His Leu Ser Leu Asp Pro Thr Asn Ala Glu Asn Thr Ala Ser 165 170 175 cag tct cca agg acc cgt ggt tcc cgg cgg cag atc cag cgt ttg gag 576 Gln Ser Pro Arg Thr Arg Gly Ser Arg Arg Gln Ile Gln Arg Leu Glu 180 185 190 cag ctg ctg gcg ctc tat gtg gca gag atc cgg cgg ctg cag gaa aag 624 Gln Leu Leu Ala Leu Tyr Val Ala Glu Ile Arg Arg Leu Gln Glu Lys 195 200 205 gag ttg gat ctc tca gaa ttg gat gac cca gac tcc gca tac ctg cag 672 Glu Leu Asp Leu Ser Glu Leu Asp Asp Pro Asp Ser Ala Tyr Leu Gln 210 215 220 gag gca cgg ttg aag cgt aag ctg atc cgc ctc ttt ggg cga cta tgt 720 Glu Ala Arg Leu Lys Arg Lys Leu Ile Arg Leu Phe Gly Arg Leu Cys 225 230 235 240 gag ctg aaa gac tgc tct tca ctg acc ggc cgt gtc ata gag cag cgc 768 Glu Leu Lys Asp Cys Ser Ser Leu Thr Gly Arg Val Ile Glu Gln Arg 245 250 255 atc ccc tac cgt ggc acc cgc tac cca gag gtt aac agg cgc att gag 816 Ile Pro Tyr Arg Gly Thr Arg Tyr Pro Glu Val Asn Arg Arg Ile Glu 260 265 270 cgg ctc atc aac aag cca ggg cct gat acc ttc cct gac tat ggg gat 864 Arg Leu Ile Asn Lys Pro Gly Pro Asp Thr Phe Pro Asp Tyr Gly Asp 275 280 285 gtg ctt cgg gct gta gag aag gca gct gcc cga cac agc ctt ggc ctc 912 Val Leu Arg Ala Val Glu Lys Ala Ala Ala Arg His Ser Leu Gly Leu 290 295 300 ccc cga cag cag ctc cag ctc atg gct cag gat gcc ttc cga gat gtg 960 Pro Arg Gln Gln Leu Gln Leu Met Ala Gln Asp Ala Phe Arg Asp Val 305 310 315 320 ggc atc agg tta cag gag cga cgt cac ctc gat ctc att tac aac ttt 1008 Gly Ile Arg Leu Gln Glu Arg Arg His Leu Asp Leu Ile Tyr Asn Phe 325 330 335 ggc tgc cac ctc aca gat gac tat agg cca ggc gtt gac cct gca cta 1056 Gly Cys His Leu Thr Asp Asp Tyr Arg Pro Gly Val Asp Pro Ala Leu 340 345 350 tct tat cct gtg tcg gcc cgg cgc ctt cgg gaa aac cgg att ttg gcc 1104 Ser Tyr Pro Val Ser Ala Arg Arg Leu Arg Glu Asn Arg Ile Leu Ala 355 360 365 ttg agt cgg ctg gat cag gtc atc tcc ttt tat gca atg ttg caa gac 1152 Leu Ser Arg Leu Asp Gln Val Ile Ser Phe Tyr Ala Met Leu Gln Asp 370 375 380 ggg ggt gag gag ggc aaa aaa aaa aag aga aga gct cgg ctc cac ggc 1200 Gly Gly Glu Glu Gly Lys Lys Lys Lys Arg Arg Ala Arg Leu His Gly 385 390 395 400 ccc tct tcc cac tct gca aac ccc ccc gaa ccc tcc ttg gat tct ggt 1248 Pro Ser Ser His Ser Ala Asn Pro Pro Glu Pro Ser Leu Asp Ser Gly 405 410 415 gag ggc cct att gga atg gca tcc cag ggg tgc cct tct gcc tcc aga 1296 Glu Gly Pro Ile Gly Met Ala Ser Gln Gly Cys Pro Ser Ala Ser Arg 420 425 430 gct gag aca gat gac gaa gac gat gag gag agt gat gag gaa gag gag 1344 Ala Glu Thr Asp Asp Glu Asp Asp Glu Glu Ser Asp Glu Glu Glu Glu 435 440 445 gag gag gag gaa gaa gaa gag gag gag gcc aca gat tct gaa gag gag 1392 Glu Glu Glu Glu Glu Glu Glu Glu Glu Ala Thr Asp Ser Glu Glu Glu 450 455 460 gag gat ttg gaa cag atg cag gag ggt cag gag gat gat gaa gag gag 1440 Glu Asp Leu Glu Gln Met Gln Glu Gly Gln Glu Asp Asp Glu Glu Glu 465 470 475 480 gac gaa gag gaa gaa gca gca gca ggt aaa gat gga gac aag agc ccc 1488 Asp Glu Glu Glu Glu Ala Ala Ala Gly Lys Asp Gly Asp Lys Ser Pro 485 490 495 atg tcc tca cta cag att tcc aat gaa aag aaa ctg gaa cct ggc aaa 1536 Met Ser Ser Leu Gln Ile Ser Asn Glu Lys Lys Leu Glu Pro Gly Lys 500 505 510 cag atc agc aga ttt tca ggg gag cag caa aac aaa gga cgc ata gtg 1584 Gln Ile Ser Arg Phe Ser Gly Glu Gln Gln Asn Lys Gly Arg Ile Val 515 520 525 tca cca tcg tta ctg tca gaa gaa ccc ctg gcc ccc tcc agc ata gat 1632 Ser Pro Ser Leu Leu Ser Glu Glu Pro Leu Ala Pro Ser Ser Ile Asp 530 535 540 gct gaa agc aat gga gaa cag cct gag gag ctg acc ctg gag gaa gaa 1680 Ala Glu Ser Asn Gly Glu Gln Pro Glu Glu Leu Thr Leu Glu Glu Glu 545 550 555 560 agc cct gtg tct cag ctc ttt gag cta gag att gaa gct ttg ccc ctg 1728 Ser Pro Val Ser Gln Leu Phe Glu Leu Glu Ile Glu Ala Leu Pro Leu 565 570 575 gat acc cct tcc tct gtg gag acg gac att tcc tct tcc agg aag caa 1776 Asp Thr Pro Ser Ser Val Glu Thr Asp Ile Ser Ser Ser Arg Lys Gln 580 585 590 tca gag gag ccc ttc acc act gtt tta gag aat gga gca ggc atg gtc 1824 Ser Glu Glu Pro Phe Thr Thr Val Leu Glu Asn Gly Ala Gly Met Val 595 600 605 tct tct act tcc ttc aat gga ggc gtc tct cct cac aac tgg gga gat 1872 Ser Ser Thr Ser Phe Asn Gly Gly Val Ser Pro His Asn Trp Gly Asp 610 615 620 tct ggt ccc ccc tgc aaa aaa tct cgg aag gag aag aag caa aca gga 1920 Ser Gly Pro Pro Cys Lys Lys Ser Arg Lys Glu Lys Lys Gln Thr Gly 625 630 635 640 tca ggg cca tta gga aac agc tat gtg gaa agg caa agg tca gtg cat 1968 Ser Gly Pro Leu Gly Asn Ser Tyr Val Glu Arg Gln Arg Ser Val His 645 650 655 gag aag aat ggg aaa aag ata tgt acc ctg ccc agc cca cct tcc ccc 2016 Glu Lys Asn Gly Lys Lys Ile Cys Thr Leu Pro Ser Pro Pro Ser Pro 660 665 670 ttg gct tcc ttg gcc cca gtt gct gat tcc tcc acg agg gtg gac tct 2064 Leu Ala Ser Leu Ala Pro Val Ala Asp Ser Ser Thr Arg Val Asp Ser 675 680 685 ccc agc cat ggc ctg gtg acc agc tcc ctc tgc atc cct tct cca gcc 2112 Pro Ser His Gly Leu Val Thr Ser Ser Leu Cys Ile Pro Ser Pro Ala 690 695 700 cgg ctg tcc caa acc ccc cat tca cag cct cct cgg cct ggt act tgc 2160 Arg Leu Ser Gln Thr Pro His Ser Gln Pro Pro Arg Pro Gly Thr Cys 705 710 715 720 aag aca agt gtg gcc aca caa tgc gat cca gaa gag atc atc gtg ctc 2208 Lys Thr Ser Val Ala Thr Gln Cys Asp Pro Glu Glu Ile Ile Val Leu 725 730 735 tca gac tct gat tag 2223 Ser Asp Ser Asp 740 <210> 2 <211> 740 <212> PRT <213> Homo sapiens <400> 2 Met Ala Thr Ala Asn Ser Ile Ile Val Leu Asp Asp Asp Asp Glu Asp 1 5 10 15 Glu Ala Ala Ala Gln Pro Gly Pro Ser His Pro Leu Pro Asn Ala Ala 20 25 30 Ser Pro Gly Ala Glu Ala Pro Ser Ser Ser Glu Pro His Gly Ala Arg 35 40 45 Arg Ser Ser Ser Ser Gly Gly Lys Lys Cys Tyr Lys Leu Glu Asn Glu 50 55 60 Lys Leu Phe Glu Lys Phe Leu Glu Leu Cys Lys Met Gln Thr Ala Asp 65 70 75 80 His Pro Glu Val Val Pro Phe Leu Tyr Asn Arg Gln Gln Arg Ala His 85 90 95 Ser Leu Phe Leu Ala Ser Ala Glu Phe Cys Asn Ile Leu Ser Arg Val 100 105 110 Leu Ser Arg Ala Arg Ser Arg Pro Ala Lys Leu Tyr Val Tyr Ile Asn 115 120 125 Glu Leu Cys Thr Val Leu Lys Ala His Ser Ala Lys Lys Lys Leu Asn 130 135 140 Leu Ala Pro Ala Ala Thr Thr Ser Asn Glu Pro Ser Gly Asn Asn Pro 145 150 155 160 Pro Thr His Leu Ser Leu Asp Pro Thr Asn Ala Glu Asn Thr Ala Ser 165 170 175 Gln Ser Pro Arg Thr Arg Gly Ser Arg Arg Gln Ile Gln Arg Leu Glu 180 185 190 Gln Leu Leu Ala Leu Tyr Val Ala Glu Ile Arg Arg Leu Gln Glu Lys 195 200 205 Glu Leu Asp Leu Ser Glu Leu Asp Asp Pro Asp Ser Ala Tyr Leu Gln 210 215 220 Glu Ala Arg Leu Lys Arg Lys Leu Ile Arg Leu Phe Gly Arg Leu Cys 225 230 235 240 Glu Leu Lys Asp Cys Ser Ser Leu Thr Gly Arg Val Ile Glu Gln Arg 245 250 255 Ile Pro Tyr Arg Gly Thr Arg Tyr Pro Glu Val Asn Arg Arg Ile Glu 260 265 270 Arg Leu Ile Asn Lys Pro Gly Pro Asp Thr Phe Pro Asp Tyr Gly Asp 275 280 285 Val Leu Arg Ala Val Glu Lys Ala Ala Ala Arg His Ser Leu Gly Leu 290 295 300 Pro Arg Gln Gln Leu Gln Leu Met Ala Gln Asp Ala Phe Arg Asp Val 305 310 315 320 Gly Ile Arg Leu Gln Glu Arg Arg His Leu Asp Leu Ile Tyr Asn Phe 325 330 335 Gly Cys His Leu Thr Asp Asp Tyr Arg Pro Gly Val Asp Pro Ala Leu 340 345 350 Ser Tyr Pro Val Ser Ala Arg Arg Leu Arg Glu Asn Arg Ile Leu Ala 355 360 365 Leu Ser Arg Leu Asp Gln Val Ile Ser Phe Tyr Ala Met Leu Gln Asp 370 375 380 Gly Gly Glu Glu Gly Lys Lys Lys Lys Arg Arg Ala Arg Leu His Gly 385 390 395 400 Pro Ser Ser His Ser Ala Asn Pro Pro Glu Pro Ser Leu Asp Ser Gly 405 410 415 Glu Gly Pro Ile Gly Met Ala Ser Gln Gly Cys Pro Ser Ala Ser Arg 420 425 430 Ala Glu Thr Asp Asp Glu Asp Asp Glu Glu Ser Asp Glu Glu Glu Glu 435 440 445 Glu Glu Glu Glu Glu Glu Glu Glu Glu Ala Thr Asp Ser Glu Glu Glu 450 455 460 Glu Asp Leu Glu Gln Met Gln Glu Gly Gln Glu Asp Asp Glu Glu Glu 465 470 475 480 Asp Glu Glu Glu Glu Ala Ala Ala Gly Lys Asp Gly Asp Lys Ser Pro 485 490 495 Met Ser Ser Leu Gln Ile Ser Asn Glu Lys Lys Leu Glu Pro Gly Lys 500 505 510 Gln Ile Ser Arg Phe Ser Gly Glu Gln Gln Asn Lys Gly Arg Ile Val 515 520 525 Ser Pro Ser Leu Leu Ser Glu Glu Pro Leu Ala Pro Ser Ser Ile Asp 530 535 540 Ala Glu Ser Asn Gly Glu Gln Pro Glu Glu Leu Thr Leu Glu Glu Glu 545 550 555 560 Ser Pro Val Ser Gln Leu Phe Glu Leu Glu Ile Glu Ala Leu Pro Leu 565 570 575 Asp Thr Pro Ser Ser Val Glu Thr Asp Ile Ser Ser Ser Arg Lys Gln 580 585 590 Ser Glu Glu Pro Phe Thr Thr Val Leu Glu Asn Gly Ala Gly Met Val 595 600 605 Ser Ser Thr Ser Phe Asn Gly Gly Val Ser Pro His Asn Trp Gly Asp 610 615 620 Ser Gly Pro Pro Cys Lys Lys Ser Arg Lys Glu Lys Lys Gln Thr Gly 625 630 635 640 Ser Gly Pro Leu Gly Asn Ser Tyr Val Glu Arg Gln Arg Ser Val His 645 650 655 Glu Lys Asn Gly Lys Lys Ile Cys Thr Leu Pro Ser Pro Pro Ser Pro 660 665 670 Leu Ala Ser Leu Ala Pro Val Ala Asp Ser Ser Thr Arg Val Asp Ser 675 680 685 Pro Ser His Gly Leu Val Thr Ser Ser Leu Cys Ile Pro Ser Pro Ala 690 695 700 Arg Leu Ser Gln Thr Pro His Ser Gln Pro Pro Arg Pro Gly Thr Cys 705 710 715 720 Lys Thr Ser Val Ala Thr Gln Cys Asp Pro Glu Glu Ile Ile Val Leu 725 730 735 Ser Asp Ser Asp 740 <210> 3 <211> 165 <212> DNA <213> Homo sapiens <220> <221> CDS (222) (1) .. (165) <400> 3 att gaa gct ttg ccc ctg gat acc cct tcc tct gtg gag acg gac att 48 Ile Glu Ala Leu Pro Leu Asp Thr Pro Ser Ser Val Glu Thr Asp Ile 1 5 10 15 tcc tct tcc agg aag caa tca gag gag ccc ttc acc act gtt tta gag 96 Ser Ser Ser Arg Lys Gln Ser Glu Glu Pro Phe Thr Thr Val Leu Glu 20 25 30 aat gga gca ggc atg gtc tct tct act tcc ttc aat gga ggc gtc tct 144 Asn Gly Ala Gly Met Val Ser Ser Thr Ser Phe Asn Gly Gly Val Ser 35 40 45 cct cac aac tgg gga gat tct 165 Pro His Asn Trp Gly Asp Ser 50 55 <210> 4 <211> 55 <212> PRT <213> Homo sapiens <400> 4 Ile Glu Ala Leu Pro Leu Asp Thr Pro Ser Ser Val Glu Thr Asp Ile 1 5 10 15 Ser Ser Ser Arg Lys Gln Ser Glu Glu Pro Phe Thr Thr Val Leu Glu 20 25 30 Asn Gly Ala Gly Met Val Ser Ser Thr Ser Phe Asn Gly Gly Val Ser 35 40 45 Pro His Asn Trp Gly Asp Ser 50 55

Claims (5)

A human sodium hydrogen exchanger 1 (NHE1) ion channel protein binder containing human Daxx protein (death-associated protein 6) as an active ingredient. A fragment of amino acids 571 to 625 of a human Daxx protein having activity of binding to NHE1 ion channel protein. An NHE1 ion channel protein binder containing the human Daxx fragment according to claim 2 as an active ingredient. The binder according to claim 1 or 3, which is used as a prophylactic or therapeutic agent for ischemic disease. The binder according to claim 4, wherein the ischemic disease is cardiac ischemia, retinal ischemia, diabetic vascular heart disease, heart failure or myocardial hypertrophy.