KR100779007B1 - Method for inducing the expression of structurally mismatched protein in cell membrane by intracellular alkalization - Google Patents

Abstract

The present invention relates to a method for increasing cell membrane expression of structurally misfolded proteins through intracellular alkalizing.

Misfolded protein, alkalized, endoplasmic reticulum, Golgi apparatus, Nadrium bicarbonate (NaHCO3), Vapilomycin A

Description

Method for inducing the expression of structurally mismatched protein in cell membrane by intracellular alkalization}

Figure 1 is a schematic diagram showing the degradation of the ΔF508-CFTR protein structurally misfolded protein and cell membrane expression process by intracellular alkalinization.

2 is Cl in CFPAC-1 ^cells were measured using a quenched (puenching) dyes in ^{MQAE-exchange} active Cl ^- / NO ₃ ^- Cl by the cAMP stimulus to be measured by exchange activity ^- / NO _3. FIG. 2a shows the change of Cl ⁻ / NO ₃ ⁻ exchange activity by cAMP stimulation after incubating CFPAC-1 cells expressing ΔF508-CFTR protein in a 2% CO ₂ incubator for 12 hours, and FIG. After treatment with A (bafilomycin A) for 12 hours, cAMP stimulation was performed to measure changes in Cl ⁻ / NO ₃ ⁻ exchange activity. Figure 2c shows the average value of the MQAE fluorescence of Figures 2a and 2b increased after 10 seconds after cAMP stimulation, * means p <0.05.

FIG. 3 shows intracellular pHi changed by Cl ⁻ / HCO ₃ ⁻ exchange using BCECF fluorescent material after differentiating CFPAC-1 cells. FIG. 3a shows CFPAC − cultured for 12 hours in a 5% CO ₂ incubator. For 1 cell, FIG. 3B measures changes in Cl ⁻ / HCO ₃ ⁻ exchange activity before and after cAMP stimulation on CFPAC-1 cells treated for 12 hours in a 2% CO ₂ incubator. Figure 3c shows the mean value of the pH change of Figures 3a and 3b increased before and after cAMP stimulation, ** means p <0.01.

Figure 4 shows the wild type and ΔF508-CFTR protein expression changes by intracellular alkalinization, Figure 4a shows the cells after expressing the wild type-CFTR protein in Chinese Hamster Ovary (CHO) cells 5 % CO to the culture at ₂ incubator, would one divided by one cultured in 2% CO ₂ incubator and bar Philo azithromycin group treated with a (BA) observing the CFTR protein expression patterns, Figure 4b is CHO the ΔF508-CFTR protein After expression in cells and cultured in the same conditions as above to observe the expression of CFTR protein. PNG treated with Peptide: N-glycosidase and CHX treated with cycloheximide.

5 is Cl by CFTR according to intracellular alkalizing ^- as a measure of the change in current, Figure 5a is a WT-CFTR, Figure 5c ΔF508-CFTR of at _2% CO 2 incubator the cells was expressed in CHO cells After 12 hours of incubation, the Cl ⁻ current of CFTR by forskolin stimulation was measured using whole cell patch clamp technique. Figure 5b is a WT-CFTR, Figure 5d is a plotted by dividing the current by ΔF508-CFTR by the cell surface area ratio. 5E records the I / V curve of the CFTR Cl ⁻ channel in CHO cells expressing ΔF508-CFTR.

Figure 6a is the alkaline culture medium after expressing the pEYFP-Golgi plasmid in CHO cells, Figure 6b is a measure of the pH change of Golgi by Baphyllomycin A (BA). Figure 6c is an alkaline culture medium using BCECF fluorescent material in CHO cells, Figure 6d was measured the pH change of the cytoplasm by batillomycin A

Figure 7 shows the change in pH of the cytoplasm and Golgi body and the expression of ΔF508-CFTR protein by intracellular alkalinization over time, Figure 7a shows the time using BCECF while culturing CHO cells continuously in a 2% CO ₂ incubator The change in pH of the cytoplasm was measured, and FIG. 7B shows the expression change of ΔF508-CFTR over time while continuously culturing CHO cells expressing ΔF508-CFTR in a 2% CO ₂ incubator. Figure 7c and 7d is the pH of the Golgi body with time after the saturation of the culture medium in the 2% CO ₂ incubator and the treatment with Baphylomycin A was measured using the pEYFP-Golgi plasmid.

Figure 8 shows the change in the amount of molecular chaperone expression and the change of ubiquitinylated protein according to the intracellular alkalinization, Figure 8a is a group treated with the Bamplomycin A and the group of CHO cells cultured in a 2% CO ₂ incubator In order to determine the intracellular expression changes of Hsp70 and Hsc70 in the group, Western blot was performed using Hsp70 and Hsc70-specific primary antibodies. 8b is a western blot using ubiquitinated protein-specific primary antibodies to determine the degree of ubiquitination of the protein.

Figure 9 is a measure of the change in ^proteosome activity according to intracellular alkalinization, after the expression of Ub ^{G76V -GFP in} CHO cells to determine the activity of the ^proteosome a. Treated with a group cultured in a 5% CO ₂ incubator (FIG. 9A), a group treated with the proteasome inhibitor lactacystin (FIG. 9B), a group cultured in a 2% CO ₂ incubator (FIG. 9C) and a bacillomycin A In one group (FIG. 9D), fluorescence of Ub ^G76V -GFP was measured under the same conditions using a confocal microscope.

FIG. 10 shows changes in the expression of CFTR gene according to intracellular alkalinization. After expression of the WT-CFTR and ΔF508-CFTR genes in CHO cells, the cells were incubated for 12 hours in 5% CO ₂ and 2% CO ₂ incubators, respectively. Next, the expression level of CFTR mRNA was measured.

Figure 11 is a measure of the change in pH and HCO ₃ ^- concentration of the mouse urine according to NaHCO ₃ intake, Figure 11a is measured by using a Glass Micro-pH Combination Electrode immediately after receiving the urine of each group of mice, FIG. 11B shows the amount of HCO ₃ ⁻ contained in urine using CO ₂ , HCO ₃ ⁻ SIGMA Diagnostics Kit immediately after urine of each group of mice.

FIG. 12 shows the survival rate of ΔF508 homozygous mice according to induction of metabolic alkalosis and CFTR-dependent Cl ⁻ current in the small intestine. FIG. 12A shows water containing 2% sucrose in a group fed with ΔF508 homozygous mice. Survival rate of 4 months after birth was divided into groups fed, group fed with water containing 2% sucrose and 0.9% NaCl, and group fed with water containing 2% sucrose and 280 mM NaHCO ₃ , and FIG. The small intestine of the ΔF508 homozygous mice of the group was extracted and measured for CFTR-dependent short circuit current (SSC) that is activated by forskolin and IBMX and inhibited by DPC.

FIG. 13 shows changes in the expression of CFTR protein in the large intestine of the mouse. The colon of the ΔF508 homozygous mouse group and the large intestine of the WT mouse were respectively extracted and then immunized with CFTR-specific primary antibody (24-1). Fluorescent staining was performed.

The present invention relates to a method for increasing cell membrane expression of structurally misfolded proteins through intracellular alkalizing.

Cystic fibrosis, a representative genetic disease in Caucasians, is manifested by a structurally misfolded protein called ΔF508-CFTR. Although ΔF508-CFTR retains the Cl ^- channel function of normal CFTR (Pasyk and Foskett, J Biol Chem. 270 (21): 12347-50, 1995), 100% of the synthesized ΔF508-CFTR is abnormal because of its abnormal structure. The disease is caused by degradation by the Endoplasmic reticulum (ER) quality control system (ERQCS) and unable to migrate to the cell membrane (Ward et al. Cell 83: 121-127, 1995).

Currently, three methods are known to increase cell membrane expression of misfolded ΔF508-CFTR protein at the cellular level:

First, ΔF508-CFTR, a misfolded protein, binds a large amount of molecular chaperones and regulates its expression and degradation.It treats cells with chemical chaperones such as glycerol to inhibit the binding of ΔF508-CFTR to molecular chaperones. There is a method of increasing cell membrane expression of ΔF508-CFTR (Sachiko et. Al. J. Biol. Chem. 271: 635-638, 1996).

Second, there is a method of increasing cell membrane expression of ΔF508-CFTR by culturing cells expressing ΔF508-CFTR at low temperature below 30 ° C. to inhibit ERQCS (Randell et. Al. J. Clin. Invest. 99: 1432- 1444, 1997).

Third, since the endoplasmic reticulum contains high concentrations of calcium, molecular chaperones, which are dependent on high concentrations of calcium such as Calnexin, bind to ΔF508-CFTR to inhibit cell membrane expression. At this time, the antifoller calcium pump inhibitor thapsigargin may be used to lower the vesicle calcium concentration, thereby weakening the binding of these chaperones to ΔF508-CFTR, thereby increasing cell membrane expression of ΔF508-CFTR (Egan et. Al. Nat Med. 8: 485-492, 2002).

However, these methods are effective at the cellular level but are limited because they are highly toxic for use in the treatment of actual patients.

In the present invention, to find a method of increasing the cell membrane expression of misfolded protein while giving less toxicity to the cell, structurally misfolded protein by inhibiting the process of degradation through intracellular alkalizing to escape from the endoplasmic reticulum The present invention was completed by confirming that the protein can be expressed on the cell membrane.

Accordingly, it is an object of the present invention to provide a method for increasing cell membrane expression of misfolded proteins via intracellular alkalizing.

The present invention relates to a method of inducing cell membrane expression of a protein comprising treating an alkalizing agent to a cell having a structurally misfolded protein.

As used herein, the term “structurally misfolded protein” refers to folding that differs from the folding form in its natural form by substitution, insertion, deletion, or combination of one or more amino acids in the primary structure of a wild-type (natural) protein. Means a protein whose steric structure has changed. Such proteins include mutant proteins of CFTR, such as ΔF508-CFTR, mutant proteins of α1-AT, mutant proteins of aqaporin, mutant proteins of apoliproteins, and mutant proteins of polycystins. , Mutant protein of side chain α-ketosan dehydrogenase, mutant protein of phenylalanine hydroxylase, mutant protein of V2-vasopressin receptor, mutant protein of transthyretin, mutant protein of β-glucosidase, mutant protein of α-galactosidase, mutant protein of opoid receptor, mutant protein of konadotropin-releasing hormone receptor, mutant protein of α-synuclein, mutant protein of prion, etc. have.

CFTR (cystic fibrosis transmembrane conductance regulator) protein cAMP-dependent Cl ^- is a protein that plays an important role in the secretion and secretion regulation of epithelial secretions ^- HCO ₃ in the passage of respiratory and epithelial cells of the stomach through interaction with various proteins in . Loss of CFTR function due to genetic variation causes cystic fibrosis.

The most common genetic variation of the CFTR protein is CFTR in the form of deletion of the 508th phenylalanine of CFTR, ΔF508-CFTR (William J. Welch / Seminars in Cell & Developmental Biology 15 (2004) 31-38). The ΔF508-CFTR has the Cl ⁻ channel function of normal CFTR, but since the newly synthesized ΔF508-CFTR has an abnormal structure, 100% of the ΔF508-CFTR is decomposed by ERQCS and cannot be moved to the cell membrane, causing disease.

Diseases caused by the inability to express structurally misfolded proteins on cell membranes include cystic fibrosis, Class2 familial hypercholesterolemia, and diabetes insipidus.

Treatment of cells with structurally misfolded proteins with alkalizing agents in accordance with the method of the present invention results in attenuation of ERQCS (weakening the binding of KDEL receptor and chaperone) and inhibition of Golgi from vesicles to inhibition of COPI vesicle formation. Structurally misfolded proteins can be normally expressed.

Alkaliating agents that can be used in the process of the present invention can exemplify carbonates, bicarbonates, phosphates, hydroxylates and the like. Of the above alkalizing agents, bicarbonate can be preferably used.

In addition, a cystic (large) H ⁺ -ATPase inhibitor may be used as an alkalizing agent in the method of the present invention. Inhibitors of cystic H ⁺ -ATPase include bafilomycin A, salicylihalamide A, concanamycin A, derivatives thereof, and the like.

Bapilomycin is known as Streptomyces griseus sp. It is a macrolide antifungal compound isolated from sulphurus, and there are known batillomycin A1, A2, B1, B2, C1, C2 and the like.

As used herein, the term "expression" as used in connection with the ΔF508-CFTR protein, unless otherwise stated, means that the protein is transcribed and translated and then secreted through the Golgi apparatus and inserted into the cell membrane.

We selected ΔF508-CFTR protein, one of the structurally misfolded proteins, to confirm the expression of ΔF508-CFTR protein at the cellular level by intracellular alkalizing and the expression of ΔF508-CFTR protein in animal models.

First, alkaline cells were cultured by making an alkaline culture medium by adjusting CO ₂ gas concentration in CFPAC-1 cells expressing endogenous ΔF508-CFTR protein, and Bafilomycin A (Bafilomycin A), one of Vacuolar H ⁺ -ATPase inhibitors. ) To alkalinize the pH of the Golgi lumen. As a result, as shown in Figures 2 and 3, it was confirmed that the activity of the CFTR functioning in the cell membrane is significantly increased in the group that the cells were alkalized, which means that ΔF508-CFTR protein misfolded by intracellular alkalinization It is expressed in.

In addition, after expression of the ΔF508-CFTR protein in CHO cells through transduction, the cell was alkalized using CO ₂ gas concentration control and batillomycin A in the same manner as the above method. As shown in Fig. 4, it was confirmed that band C (form of CFTR protein expressed in cell membrane), which was not seen in the control group, appeared only in the group in which the cells were alkalized. As a result of confirming the Cl ⁻ channel function of CFTR protein, as shown in FIG. 5, CFTR-dependent current that was not seen in the control group could be recorded only when the cells were alkalized. The above results indicate that the misfolded ΔF508-CFTR protein in CHO cells expresses and functions in the cell membrane.

At the cellular level, CO ₂ gas concentration control and bacillopein to confirm that increased cell membrane expression of misfolded ΔF508-CFTR protein by intracellular alkalinization is not due to quantitative increase or change in activity of molecular chaperones or proteasomes As a result of observing these changes after alkalizing the cells with mycin A, it was confirmed that they did not affect their activity as shown in FIGS. 8 and 9.

Real time PCR was performed to confirm whether intracellular alkalinity affected the expression of CFTR gene. As a result, intracellular alkalinization did not affect the expression of CFTR gene. It was.

In order to confirm that the above results are applicable not only to the cellular level but also to animal models, induction of mild metabolic alkalosis by ingesting NaHCO ₃ in ΔF508 homozygous mice confirmed the survival rate and the expression pattern of CFTR cell membrane during 4 months of life. It was. As a result, as shown in FIG. 12, the survival rate was significantly increased in the group inducing mild metabolic alkalosis by ingesting NaHCO ₃ and confirmed the CFTR-dependent current in the small intestine which was not seen in other groups. This indicates that ΔF508-CFTR is expressed on the cell membrane and functions in the small intestine of the mouse. As shown in FIG. 13, it was confirmed that ΔF508-CFTR was expressed on the colon cell membrane only in the group inducing metabolic alkalosis.

The results indicate that the method of increasing expression of misfolded proteins by intracellular alkalizing provides a very useful method that can be used at the cellular level as well as at the individual level.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are merely to illustrate the invention, the present invention is not limited to the following examples.

< Example  1> cell culture and Misfolded  ΔF508- CFTR  Protein gene introduction

CFPAC-1 and CHO-K1 cell lines were purchased from the American Type Culture Collection, Rockville, MD, USA, and were used in 10% FBS and phenylsilin (50 units / ml) / streptomycin in IMDM and RPMI 1640 cultures, respectively. 50 ug / ml) was added and then incubated in a 5% CO ₂ incubator. The CFPAC-1 cell line was cultured using trypsin / EDTA after incubation and about 4 to 5 on a permeable support made using a Transwell-Clear Pol membrane (0.4 m pore diameter, Costar) at 3 × 10 ⁵ cells / cm ² . The cells were grown for one day and cultured with functionally differentiated pancreatic duct cells forming a tight junction in a single layer. Expression of WT-CFTR and ΔF508-CFTR were transfected with CPO cells using LipofectAMINE Reagent (Life Technologies) and the pCMV plasmid containing the WT-CFTR and ΔF508-CFTR genes, and for 24 to 48 hours after transfection. After incubation, the experiment was performed. The pCMV plasmid containing the ΔF508-CFTR gene was made by PCR mutagenesis. The primers used were as follows.

Sense: 5´-CCA TTA AAG AAA ATA TCA TTG GTG TTT CCT ATG ATG AAT ATA G-3´ (SEQ ID NO: 1)

Antisense: 5´-CTA TAT TCA TCA TAG GAA ACA CCA ATG ATA TTT TCT TTA ATG G-3´ (SEQ ID NO: 2)

< Example  2> CFTR Membrane permeability of Cl ions

The cell membrane permeation of cAMP dependent Cl ions was mainly by CFTR and was measured by MQAE with Cl ^- quenching fluorescent salts. First, CFPAC-1 cells expressing ΔF508-CFTR were incubated for 48 hours on coverslips, and then incubated for 30 minutes in PBS containing MQAE, which is 40 μM Cl ⁻ quenching dye, at room temperature for 30 minutes and then again on ice for 40 minutes. Cells loaded with MQAE dye were mounted in the perfusion chapter and then measured for the change in MQAE fluorescence from excitation wavelength 350 nm and emission (wavelength 455 nm. Cl ⁻ secretion measurement principle is as follows: about 145 mM Cl judaga flowing the perfusate containing the 0mM Cl ^{- -} by entering into the ^cell-and 145mM of NO ₃ ^- the perfusion fluid flowing main surface, Cl in the cells contain ^- instead of NO ₃ ^- the cells to be secreted out and secreted Cl Faster Cl ⁻ movements can be measured.

As a result, as shown in FIG. 2, CFPAC-1 cells were cultured in a 2% CO ₂ incubator, and the Cl ⁻ / NO ₃ ⁻ exchange activity was significantly increased than cells cultured in a 5% CO ₂ incubator. In the cells treated with batillomycin A, Cl ⁻ / NO ₃ ⁻ exchange activity was increased in proportion to the dose of batillomycin A.

< Example  3> Intracellular  Cl by Alkalization ^- Of HCO ₃ ^-  Exchange change measurement

Cells were incubated on a cover slip and a permeable support, loaded for 10 minutes in a PBS containing 4 μM of BCECF / AM, a pH measuring fluorescent substance, and mounted in a perfusion chamber. After the cells were mounted in the perfusion chamber, intracellular pH change was measured by observing the ratio of BCECF fluorescence at the excitation wavelength 477 nm and 442 nm and the emission wavelength 510 nm in the perfusion solution containing HCO ₃ ⁻ .

Cl ^- / HCO ₃ ^- exchange activity measurement was performed in the following way. The perfusion solution of the lumen and the basal membrane was flowed into the HCO ₃ ⁻ perfusate containing 0 mM Cl ⁻ to the side membrane side of the chamber. Luminal membrane side is 130mM Cl ^- is contained HCO ₃ ^- perfusion fluid flow judaga 0mM Cl a ^- major surface flush with perfusate, Cl ^{- -} the HCO ₃ containing the / HCO ₃ ^- Cl in through the heat exchanger ^cells are lumens makjjok perfusion The HCO ₃ ⁻ in the luminal perfusate enters the cell and increases the intracellular pH. Thus, Cl ⁻ / HCO ₃ ⁻ exchange activity can be measured by intracellular pH changes that increase in unit time.

As a result, as in the 3 CFPAC-1 cells luminal membrane Cl ^- / HCO ₃ ^- when cultured in a measure of the exchange result _2% CO 2 incubator Cl than in a cell culture in a 5% CO ₂ incubator ^- / HCO ₃ ^- exchange activity was increased.

< Example  4> Weston Blot  Protein expression level measurement

The cells were washed three times with phosphate-buffered saline (PBS) solution followed by lysis buffer (50 mM Tris-HCl, pH 7.4), 1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, complete Protease inhibitor mixture (Roche Applied Scinence, Mannheim, Germany) was added and allowed to react on ice for 10 minutes. After 10 minutes, a sample was obtained by sonication, and the supernatant was separated by centrifugation at 13000 rpm for 15 minutes at 4 ° C., and the protein was quantified using a Bio-Rad assay kit. 50-120 ug of protein was separated by electrophoresis on 6% acrylamide gel and then transferred to nitrocellulose membrane. The protein-transferred nitrocellulose membrane was blocked with PBS containing 3% non-fat milk, 0.1% Tween 20 for 1 hour at room temperature, and then the primary antibody specific to each protein (CFTR-specific M3A7 and 24-1 primary antibodies are purchased from Upstate (Lake Placid, NY) and R & D system (Minneapolis, Minn.) And primary antibodies specific for Hsp70 and Hsc70 are purchased from Santa Cruz Biotechnology (Santa Cruz, CA). And reacted at 4 ° C for 12 hours. After the reaction was washed twice with distilled water and reacted with a secondary antibody (purchased from Zymed Laboratories Inc (San Francisco, CA)) at room temperature for one hour. After one hour, the solution was washed with PBS containing 0.05% Tween 20 for 10 minutes and developed using an ECL solution kit.

< Example  5> N- Glucosidase  Used CFTR  Sugar removal from protein

Since CFTR is an N-linked glycosylated protein, N-glycosidase separates N-linked carbohydrates. N-glycosidase F (New England Bioblabs) was used to degrade the N-linked carbohydrates of CFTR. First, proteins were isolated from lysis buffer in CFTR-expressing CHO cells, and then made into a buffer containing 5% SDS and 1% β-mercaptoethanol and incubated at 37 ° C. for 10 minutes to release the tertiary structure of the protein. Then, N-glycosidase was added to decompose the N-linked carbohydrates through incubation at 37 ° C. for 2 hours.

As a result, N-linked carbohydrates were separated from the CFTR protein as shown in FIG. 4A.

< Example  6> Using patch-clamp technology CFTR Cl ^-  Current measurement

After transfecting CHO cells with wild type and ΔF508-CFTR, the cells were incubated for 24 hours in a 5% CO ₂ incubator and then cultured for 12-18 hours in a 2% CO ₂ incubator using trypsin / EDTA. After separation into unit cell states, the cells were fixed in a patch-clamp perfusion chapter and the Cl ⁻ current of the CFTR was measured using whole cell patch-clamp technology. The pipette solution included 145 mM KCl, 5 mM NaCl, 1 mM EGTA, 1 mM MgCl ₂ , 0.4 mM CaCl ₂ , 2 mM Mg-ATP, 10 mM HEPES and the pH was adjusted to 7.4. Bath solution contains 150 mM NaCl, 5 mM KCl, 1 mM CaCl ₂ , 2 mM MgCl ₂ , 5 mM glucose, 10 mM HEPES and pH was adjusted to 7.4. All experiments were conducted at 22 to 25 ° C. and the pipettes had a resistance of 3 to 5 MΩ. CFTR-specific Cl ⁻ currents were measured via currents generated by 5 μM forskolin and 100 μM IBMX stimulation. The holding potential (HP) was -30 mV, and the Cl ^- current was recorded by the AxoScope 8.1 system and the Digidata 1322A AC / DC converter.

As a result, as shown in FIGS. 5A and 5B, when the WT-CFTR was cultured in the 2% CO ₂ incubator, Cl ⁻ current was slightly increased than in the cells cultured in the 5% CO ₂ incubator. In the case of ΔF508-CFTR, Cl ⁻ current, which was not observed when incubated in a 5% CO ₂ incubator as measured in FIGS. 5C and 5D, was measured. As shown in FIG. 5E, a CFTR-specific linear I / V curve was shown. .

< Example  7> Intracellular  By alkalizing Golgi apparatus  pH change measurement

The pH of Golgi body was measured using pEYFP-Golgi vector (BD Biosciences, CA). The pEYFP-Golgi vector expresses a protein in which YFP and a portion of the protein residing in Golgi are combined with fluorescence as the pH changes, thereby measuring Golgi-specific pH. After expressing the pEYFP-Golgi vector in CHO cells, the fluorescence was measured at 480 nm of excitation wavelength and 530 nm of YFP expressed in Golgi apparatus using Zeiss LSM510 confocal fluorescence microscope. Fluorescence was measured by flowing a neutral culture and a basic culture to cells mounted on a confocal fluorescence microscope, and consisted of a pH of 145 mM KCl, 10 mM HEPES, 5 μM nigericin and 5 μM monensin. The pH of the Golgi apparatus was measured by converting the degree of fluorescence to pH based on the measured fluorescence by flowing the pH titration solutions of 5.1, 5.8, 6.5 and 7.2.

As a result, as shown in FIGS. 6A and 6B, the pH of the Golgi apparatus was increased from pH 6.2 to pH 7.0 by both the alkaline culture medium and the bacillomycin A.

< Example  8> Intracellular  By alkalizing Proteasome  Active change measurement

Ub ^G76V- GFP (Dantuma et. Al. Nat Biotechnol. 18: 538-543, 2000) was used to measure proteasome activity. Ub ^G76V- GFP acts as a substrate of the proteasome. When the function of the proteasome is inhibited, the fluorescence of the GFP increases, so the degree of activity of the proteasome can be measured. First, when ^Gb expresses Ub ^G76V- GFP in CHO cells and then cultures the cells in a 2% CO ₂ incubator or treated with 5% CO _{2 in} the case of ^Bafilomycin A, the fluorescence of each GFP is confocal Zeiss LSM510 confocal. It was measured using a fluorescence microscope. GFP fluorescence was measured at excitation wavelength 480 nm and emission wavelength 510 nm.

As a result, as shown in FIG. 9, the fluorescence degree of the GFP did not appear to be different from that of the culture in the 2% CO ₂ incubator or the treatment with the bacillomycin A in the 5% CO ₂ incubator.

< Example  9> Real time PCR Using CFTR Gene expression

After expressing wild-type and ΔF508-CFTR genes in CHO cells, the cells were incubated for 12 hours in a 2% CO ₂ incubator and a 5% CO ₂ incubator after 24 hours. The cells were then washed twice with PBS, followed by extraction of total RNA using Trizol (Invitrogen, CA) and RNase H-reverse transcriptase (Invitrogen, CA) using random hexa-primer and oligo-dT primers. CA) Reverse transcription was performed with reverse transcriptase to synthesize cDNA. The synthesized cDNA was used as a template, and real-time PCR was performed using a QuantiTect SYBR Green PCR Kit (QIAGEN, CA, USA) and a Rotor Gene 2072D (Corbett Research, Australia) real-time PCR machine. The degree of β-actin mRNA expression was used as an internal control. The base sequence of the primer used and the size of the PCR product is as follows.

CFTR, sense: 5'-GCT TCT TTG GTT GTG CTG TGG C-3 '(SEQ ID NO: 3),

CFTR antisense: 5'-AGT GTC GGC TAC TCC CAC GTA AAT G-3 '(SEQ ID NO: 4)

β-actin, sense: 5'-GAC CCA GAT CAT GTT TGA GAC C-3 '(SEQ ID NO: 5),

β-actin, antisense: 5'-CTC GTA GAT GGG CAC AGT GTG-3 '(SEQ ID NO: 6)

As a result, when the cells were cultured in a 2% CO ₂ incubator as shown in FIG. 10, the mRNA expression levels of wild type and ΔF508-CFTR tended to decrease slightly, but there was no statistical significance.

< Example  10> ΔF508 homozygotes In mice Alkalosis  Induction and Survival rate  Measure

ΔF508 homozygous mice were given no water other than water containing 2% sucrose and 280 mM NaHCO ₃ to induce mild metabolic alkalosis. The solid food except for water containing NaHCO ₃ was to eat standard food. In the present invention, a group fed with tap water, a group fed with water containing 2% sucrose, a group fed with water containing 2% sucrose and 0.9% NaCl, and fed with a water containing 2% sucrose and 280 mM NaHCO ₃ The survival rate for 4 months after birth was observed.

As a result, the survival rate of ΔF508 homozygous mice fed with water containing 2% sucrose and 280 mM NaHCO ₃ was significantly increased compared to other groups as shown in FIG. 12A.

< Example  11> mouse urine HCO ₃ ^- Volume measurement and pH measurement

CO ₂ , HCO ₃ ^- SIGMA Diagnostics Kit (132-A) was used to determine the amount of HCO ₃ ^- contained in urine. The CO ₂ sample starting reagent was brought to 37 ° C. and the spectrophotometer was adjusted to 340 nm and kept at 37 ° C. 1 ml of CO ₂ sample initiating reagent was placed in a tube, 5 μl of urine was obtained from the mouse, immediately mixed, and incubated at 37 ° C. for 5 minutes. Measuring the concentration ^- the HCO ₃ containing the rat urine out the standard for ^- HCO ₃ using a spectrophotometer to obtained the OD value using a 20, 40, 42, 44, 46, 48 mM NaHCO 3 in After cultivation 340 nm It was. The urine pH of the mice was measured using Glass Micro-pH Combination Electrode (9802BN, Thermo electron co., MA) immediately after receiving urine from mice.

As a result, as shown in FIGS. 11A and 11B, the pH and HCO ₃ ⁻ concentrations in the NaHCO ₃ ingested group were significantly increased compared to the other groups.

< Example  12> CFTR  Dependent SCC (short-circuit current) measurement

Mice were not fed at night except water, and then the small intestine (duodenal region) of the mice was extracted. The isolated small intestine was mounted in a Classic Tissue Ussing Chamber (Warner instruments, Handen, CT) and then stabilized for 10-20 minutes in a perfusion solution containing 25 mM NaHCO ₃ maintained at 37 ° C, followed by 10 μM forskolin and 100 μM IBMX. Induce cAMP stimulation to measure CFTR dependent SCC (I _sc ). The small intestine tissue was fixed at 0 mV and the changing current was measured using a PowerLab data acquisition system (AD Instruments).

As a result, it was a CFTR-dependent is about 21% of I _sc time measurement in 2% sucrose and 280mM NaHCO ₃ in rat small intestine fed water expressing WT-CFTR only in the group including, as shown in Figure 12b .

< Example  13> Immunofluorescence staining  Through the colon tissue CFTR Expression measurement

Mouse colon tissue was frozen in liquid nitrogen using OCT compounds and stored at −70 ° C., and then tissue was cut into 4 μm thickness using a microtome and attached to slide glass. Tissues were fixed with -20 ° C. for 10 minutes in methanol, permeabilized, washed twice with PBS and then at room temperature with blocking solution (5% goat serum, 1% bovine serum albumin, 0.1% gelatin in PBS). Incubate for 1 hour. After washing the tissue twice with PBS, CFTR specific primary antibody (24-1, R & D system (Minneapolis, MN)) was diluted 1: 100 in the blocking solution and incubated at room temperature for 90 minutes. After incubation, the tissues were washed three times with PBS and the secondary antibody (Zymed Laboratories Inc (San Francisco, Calif.)) Diluted 1: 100 in blocking solution and incubated for 1 hour at room temperature. After washing, the water was well removed, and then the cover slip was fixed to the slide glass attached with the tissue using a mounting medium and dried overnight, and then observed with a Zeiss LSM510 confocal fluorescence microscope.

As a result, as shown in FIG. 13, it can be seen that a large amount of CFTR was expressed in the mice expressing WT-CFTR as shown by the arrowed regions, but not in ΔF508 homozygous mice fed with tap water. In the large intestine of mice given CFTR to NaHCO ₃ , a large amount was expressed in the cell membrane.

Alkaline treatment of structurally misfolded proteins according to the present invention can be usefully applied to various diseases according to the impaired cell membrane expression of these proteins.

Attach sequence list electronic file

Claims (6)

In cells with structurally misfolded proteins that are destroyed by endoplasmic reticulum-associated degradation (ERAD), carbonates, bicarbonates, phosphates, hydroxylates and vacuolar H ⁺ -ATPase inhibitors A method of inducing cell membrane expression of a protein comprising the step of treating an alkalizing agent selected from the group consisting of: The method of claim 1, wherein the structurally misfolded protein is ΔF508-CFTR (cystic fibrosis transmembrane conductance regulator in the form of 508th phenylalanine deletion). delete The method of claim 1 wherein the alkalizing agent is bicarbonate. delete The method of claim 1, wherein the alkalizing agent is batillomycin A.

Images