JP7353631B2 - Probe, kit for determining mitochondrial status, method for determining mitochondrial status, and screening method for mitochondrial function improving agents - Google Patents

Description

The present invention relates to a probe, a kit for determining mitochondrial status, a method for determining mitochondrial status, and a method for screening mitochondrial function improving agents.

Human obesity is an urgent issue that must be solved. In order to suppress obesity, it is possible to enhance the function of brown fat cells and beige cells that dissipate accumulated energy, that is, fat, into heat. Mitochondria are important organelles for heat production by brown fat cells. If there is a drug that activates mitochondria, it would be possible to suppress obesity and treat mitochondrial diseases.

Patent Document 1 discloses a mitochondrial activator containing carnosine or anserine as an active ingredient. The mitochondrial activator promotes mitochondrial biogenesis.

Japanese Patent Application Publication No. 2015-097507

In order to search for a drug that activates individual mitochondria rather than increasing the amount of mitochondria as the mitochondrial activator disclosed in Patent Document 1 mentioned above, it is necessary to activate mitochondrial activation, especially heat production in mitochondria. An index that reflects the activation of energy metabolism is needed. However, the regulatory mechanism of mitochondria is not very well understood, and no promising markers related to mitochondrial activation have currently been identified.

The present invention has been made in view of the above circumstances, and provides a probe capable of monitoring mitochondrial activation and determining the mitochondrial state, a kit for determining the mitochondrial state, a method for determining the mitochondrial state, and a method for determining the mitochondrial state. The purpose of the present invention is to provide a screening method for mitochondrial function improving agents that can evaluate test substances based on activation.

The present inventor has conducted extensive research and found that specific amino acid residues of PKR-like endoplasmic reticulum kinase (hereinafter also referred to as "PERK"), a sensor molecule present in the endoplasmic reticulum (ER), are phosphorylated in a mitochondrial stress-dependent manner. We have completed the present invention by discovering that brown fat cells are oxidized and that this phosphorylation is essential for activation of heat production and energy metabolism in brown fat cells.

The probe according to the first aspect of the present invention is
Recognizing at least one phosphorylated serine residue in the amino acid sequence shown in RSRSFSV from the N-terminal side to the C-terminal side of the kinase insert of the PKR-like endoplasmic reticulum kinase, the PKR-like endoplasmic reticulum kinase join to.

The probe according to the first aspect of the present invention includes:
is an antibody,
It may also be a thing.

The mitochondrial status determination kit according to the second aspect of the present invention includes:
The probe according to the first aspect of the present invention is provided.

The method for determining the state of mitochondria according to the third aspect of the present invention includes:
The method includes a detection step of detecting at least one phosphorylated serine residue in the amino acid sequence represented by RSRSFSV from the N-terminal side to the C-terminal side of the kinase insert of PKR-like endoplasmic reticulum kinase in the cell.

In this case, the cell is
brown fat cells,
It may also be a thing.

The method for screening mitochondrial function improving agents according to the fourth aspect of the present invention includes:
an exposure step of exposing the cells to the test substance;
a detection step of detecting at least one phosphorylated serine residue in the amino acid sequence represented by RSRSFSV from the N-terminal side to the C-terminal side of the kinase insert of PKR-like endoplasmic reticulum kinase in the cell;
a selection step of selecting the test substance based on the result of the detection step;
including.

In this case, the cell is
brown fat cells,
It may also be a thing.

According to the probe, mitochondrial state determination kit, and mitochondrial state determination method of the present invention, mitochondrial activation can be monitored and mitochondrial state can be determined. Furthermore, according to the screening method for mitochondrial function improving agents according to the present invention, test substances can be evaluated based on mitochondrial activation.

FIG. 2 is a diagram showing the structure of mouse PERK. FIG. 2 is a diagram showing the results of electron microscopic analysis of undifferentiated cells and mature brown adipocytes according to Test Example 1. (A) and (B) are diagrams showing electron microscopic images of undifferentiated cells and mature brown adipocytes, respectively. (C) is a diagram showing the average length of the mitochondria-ER proximal region. FIG. 2 is a diagram showing the structure of a mouse PERK and a PERK mutant in which the ER luminal region is deleted according to Test Example 2. FIG. 3 is a diagram showing the expression of PERK and the like during the differentiation process of brown adipocytes according to Test Example 2. FIG. 3 is a diagram showing the expression of PERK during the differentiation process of brown adipocytes expressing the PERK mutant according to Test Example 2. FIG. 7 is a diagram showing abnormal mitochondrial morphology due to suppression of PERK expression according to Test Example 3. (A) and (B) show electron microscopy images of cells transfected with siRNA without a target (sictrl) and siRNA targeting PERK (siPERK) as controls, respectively. (C) and (D) show enlarged images of the parts surrounded by white frames in (A) and (B), respectively. (E) shows the percentage of developed mitochondria per cell. FIG. 4 is a diagram showing the expression of mitochondrial-related proteins via PERK according to Test Example 4. FIG. 6 is a diagram showing changes over time in the rate of ATP production due to oxidative phosphorylation according to Test Example 5. FIG. 2 is a diagram showing phosphorylated PERK detected with the antibody according to Example 1. FIG. 3 is a diagram showing the expression of PERK and the like during the differentiation process of brown adipocytes according to Example 2. FIG. 3 is a diagram showing the analysis results of PERK phosphorylation induced by β3 adrenergic receptor stimulation in Example 3. FIG. 4 is a diagram showing mitochondrial stress-dependent phosphorylation of PERK by cell staining in Example 4. (A) and (B) are diagrams showing microscopic images of cells to which mitochondrial stress has not been applied and cells to which mitochondrial stress has been applied, respectively. FIG. 7 is a diagram showing the results of the dot blotting method in Example 5. FIG. 7 is a diagram showing the analysis results of heat production due to β3 adrenergic receptor stimulation in Test Example 6.

Embodiments according to the present invention will be described. Note that the present invention is not limited to the embodiments described below.

(Embodiment 1)
The probe according to this embodiment recognizes the phosphorylated serine residue contained in the kinase insert of PERK and binds to PERK. Figure 1 shows the structure of mouse PERK, which consists of a total length of 1114 residues. PERK includes an ER lumen region located in the ER lumen, a transmembrane region, and a kinase region. The kinase region includes a kinase insert. The serine residues mentioned above are the three serine residues in the kinase insert. The amino acid sequence of mouse PERK is shown in SEQ ID NO: 1. In the case of mouse PERK, the three serine residues mentioned above are the 715th, 717th, and 719th serine residues from the N-terminus of PERK.

In mammalian PERK, the amino acid sequence of the portion containing the serine residue is highly conserved. For example, the amino acid sequence from positions 714 to 720 from the N-terminus of mouse PERK, the amino acid sequence from positions 718 to 724 from the N-terminus of human PERK, and the amino acid sequence from positions 714 to 720 from the N-terminus of rat PERK. The amino acid sequence of both is "RSRSFSV" (SEQ ID NO: 2).

The probe is not limited as long as it recognizes at least one phosphorylated serine residue in the amino acid sequence shown by "RSRSFSV" and binds to PERK. Examples of the probe include antibodies, aptamers, and compounds that recognize the above-mentioned phosphorylated serine residues and bind to PERK. Aptamers can be obtained by known methods such as in vitro selection methods and SELEX methods that target a partial sequence (peptide) of PERK containing at least the phosphorylated serine residue. Compounds can be selected by binding assays, such as affinity chromatography, spectroscopic methods, gel filtration methods and surface plasmon resonance measurements.

Preferably the probe is an antibody. Antibodies can be produced using the above-mentioned peptides as antigen peptides by known methods. Animals targeted for immunization include mice, rats, rabbits, goats, and chickens. Preferably, said peptide comprises three phosphorylated serine residues as described above.

The antibody may be a polyclonal antibody or a monoclonal antibody. In producing polyclonal antibodies, an antigen peptide may be repeatedly injected into an animal, and the antibody may be purified from blood collected after a predetermined period of time. Preferably, the polyclonal antibody is obtained by collecting blood from an animal such as a rabbit that has been sensitized with an antigenic peptide together with an adjuvant, and after affinity purification with the antigenic peptide, the polyclonal antibody has the same amino acid sequence as the antigenic peptide and has no serine residue phosphorylated. obtained by absorption work using peptides that do not contain peptides.

In producing monoclonal antibodies, B cells are collected from the spleen or lymph nodes of animals immunized with antigenic peptides, and hybridomas are obtained by cell fusion. Hybridomas that react with the antigen peptide may be selected and cloned. Monoclonal antibodies can be produced by the hybridoma.

The number of amino acid residues in the antigenic peptide is not particularly limited, but is, for example, 10 to 20, 11 to 15, or 12 to 14, including the three phosphorylated serines. The amino acid sequence of the antigenic peptide is, for example, PSPERSRSFSVGI (SEQ ID NO: 3), which includes the amino acid sequence shown in SEQ ID NO: 2.

As shown in Test Example 2 and Example 1 below, the probe recognizes the serine residue that is phosphorylated in an ER stress-independent manner during the differentiation process of brown adipocytes and binds to PERK. According to Test Example 2 and Test Example 4 below, phosphorylation of the serine residue of PERK is essential for the expression of thermogenic factor UCP1 in the mitochondria of brown adipocytes. In addition, phosphorylation of the serine residue of PERK is important for mitochondrial development in differentiated brown adipocytes (see Test Example 3 below), and is also important for ATP production through oxidative phosphorylation in mitochondria during the differentiation process. (See Test Example 5 below). Furthermore, according to Test Example 6 below, mitochondrial development controlled by phosphorylation of the serine residues is essential for thermogenesis. Therefore, phosphorylation of the serine residue of PERK is involved in activation of heat production and energy metabolism in mitochondria.

The probe according to this embodiment recognizes the phosphorylated serine residue of PERK, which is involved in the activation of thermogenesis and energy metabolism in cells, and binds to PERK. Therefore, by detecting the probe, Mitochondrial activation can be monitored.

The probe can be detected by a known method. For example, to detect a probe, the probe may be directly labeled. When directly labeling an antibody as a probe, the antibody is labeled with a radioactive isotope, a fluorescent dye, an enzyme used in a coloring reaction, or the like. The labeled antibody binds to the phosphorylated serine residue of PERK by immunoreaction. When using an antibody labeled with a radioisotope, the radiation of the radioisotope may be detected after the immune reaction. When using an antibody labeled with a fluorescent dye, after the immunoreaction, light at an excitation wavelength for the fluorescent dye may be applied and the fluorescence detected. When using an antibody labeled with an enzyme used in a chromogenic reaction, a chromogenic substrate for the enzyme may be reacted after the immunoreaction, and PERK may be detected based on the resulting color.

Examples of radioactive isotopes include deuterium ( ² H), tritium ( ³ H), ¹⁰ B, ¹¹ B, ¹³ C, ¹⁵ N, and ¹⁸ O. Enzymes used in the color reaction are known enzymes, such as alkaline phosphatase and peroxidase. Any known fluorescent dye can be used, such as FITC, Cy2, Cy3, fluorescein, rhodamine, dansyl, and carbocyanine derivatives. The fluorescent dye may be a fluorescent protein, such as green fluorescent protein and variants thereof.

When an antibody is employed as a probe, the probe may be detected by an indirect fluorescent antibody method using a primary antibody as the probe and a secondary antibody that specifically binds to the primary antibody. The secondary antibody is labeled with, for example, a fluorescent dye. As the primary antibody, a goat antibody against PERK prepared in a non-human animal, for example, a goat, in which the serine residue is phosphorylated, is used, and as the secondary antibody, a donkey anti-goat antibody labeled with a fluorescent dye is used. good. Note that an avidin-biotin system may be used to label the antibody.

The probe according to this embodiment binds to PERK via the phosphorylated serine residue, which is necessary for activation of heat production and energy metabolism in mitochondria. Therefore, the state of mitochondria can be determined using the probe. Therefore, in another embodiment, a kit for determining the state of mitochondria is provided, which includes the above probe.

As a method for determining the state of mitochondria, a method of using a kit for determining the state of mitochondria will be described. The method includes a detection step. In the detection step, at least one phosphorylated serine residue of PERK is detected in the cell. The cells are not particularly limited, and include various cells of mammals such as humans, monkeys, cows, horses, rabbits, mice, rats, guinea pigs, and hamsters. Cells include cardiomyocytes, smooth muscle cells, adipocytes, fibroblasts, osteocytes, chondrocytes, osteoclasts, parenchymal cells, epidermal keratinocytes, epithelial cells, endothelial cells, nerve cells, glial cells, and splenocytes. , pancreatic β cells, mesangial cells, Langerhans cells, hepatocytes, and their precursor cells. The cells may be mesenchymal stem cells, embryonic stem cells, embryonic germ cells, adult stem cells, induced pluripotent stem cells (iPS cells), and the like. In addition to normal cells, cells include cells such as cancer cells, established cells such as HeLa cells, CHO cells, Vero cells, HEK293 cells, HepG2 cells, Cos-7 cells, NIH3T3 cells, and Sf9 cells. It may be.

Preferably, the cell is a cell in which the at least one serine residue of PERK is phosphorylated by differentiation induction or stimulation. For example, the cells are brown adipocytes that are induced to differentiate from undifferentiated cells isolated from brown adipose tissue. Before the detection step, the differentiated brown adipocytes may be stimulated by exposing them to CCCP (carbonylcyanide m-chlorophenylhydrazone), which imparts mitochondrial stress. Additionally, prior to the detection step, the differentiated brown adipocytes may be stimulated with a β3 adrenergic receptor agonist, such as CL316,243. This enhances phosphorylation of the serine residues.

The phosphorylated serine residue can be detected by a known method using the probe described above. For example, the primary antibody as the probe may be detected with a secondary antibody using Western blotting or the like. In this case, the mitochondrial status determination kit may further include a secondary antibody. Cells may be observed under a microscope using fluorescent labels to detect signals. Proximity Ligation Assay (PLA) may be used to detect the signal.

Subsequently, for example, the signal or band detected using the above probe is quantified by a known method. Since phosphorylation of the serine residue of PERK correlates with mitochondrial activation, the degree of mitochondrial activation can be determined as the mitochondrial state based on the magnitude of the signal. For example, it can be determined that a cell with a large signal has a large degree of mitochondrial activation compared to a cell with a small signal. Further, according to the mitochondrial state determination kit according to the present embodiment, by evaluating the degree of mitochondrial activation after stimulation, the mitochondrial activation potential of the cell before stimulation is determined as the mitochondrial state. Can be judged.

Note that the kit for determining the state of mitochondria may include a buffer, a diluent, and other reagents in addition to the reagent for inducing differentiation of undifferentiated cells. The cells to be stimulated with CCCP are not limited to differentiation-induced brown adipocytes, but any cell can be used as long as at least one serine residue of PERK is phosphorylated by stimulation with CCCP. can.

Further, since determining the state of mitochondria means determining the state of cells, the kit for determining the state of mitochondria can also be used as a kit for determining the state of cells. Furthermore, by applying the above-described mitochondrial state determination method to, for example, iPS cells or cardiomyocytes constituting a cardiomyocyte sheet, it is also possible to evaluate the quality of the cells in the iPS cells or cardiomyocyte sheet. This application is useful for quality control of iPS cells or cardiomyocyte sheets.

Note that by applying the above mitochondrial state determination method to brown cells collected from humans, it is also possible to evaluate the degree of heat production by brown fat cells. Further, by applying the above method for determining the state of mitochondria to any cells collected from humans, it is also possible to predict or diagnose susceptibility to mitochondrial diseases.

(Embodiment 2)
Next, Embodiment 2 will be described. The method for screening mitochondrial function improving agents according to the present embodiment includes an exposure step, a detection step, and a selection step. The exposure step exposes the cells to the test substance. For example, in the exposure step, the test substance may be added to the cell culture medium. The cells used in the exposure step are the same as those used in the method for using the mitochondrial status determination kit described above. Prior to exposure to the test substance, the cells may be stimulated with CCCP or a β3 adrenergic receptor agonist. The test substance is not particularly limited, and can be any substance such as a compound, peptide, or nucleic acid. The detection step is the same as the detection step in the method for using the mitochondrial state determination kit described above.

In the selection step, a test substance is selected based on the results of the detection step. Based on the magnitude of the signal detected using the probe, a test substance that improves mitochondrial function, particularly activates thermogenesis and energy metabolism, can be selected. For example, the magnitude of the signal may be compared between cells that were not exposed to the test substance and cells that were exposed to the test substance. In this case, a test substance for which a signal exceeding that of cells not exposed to the test substance is detected may be selected as a mitochondrial function improving agent.

In addition, in the selection step, a β3 adrenergic receptor agonist, etc., which is known to enhance the phosphorylation of the serine residue of PERK, is used as a positive control, and the magnitude of the signal is compared between the positive control and the test substance. You may. In this case, a test substance for which a signal equivalent to or higher than that of the positive control is detected may be selected as a mitochondrial function improving agent.

According to the method for screening mitochondrial function improving agents according to the present embodiment, a test substance is selected based on the degree of phosphorylation of the serine residue of PERK. The degree of phosphorylation of the serine residues is an indicator of the activation of heat production and energy metabolism, so the test substance can be evaluated based on mitochondrial activation.

Note that in the selection step, the magnitude of the signal may be compared with a reference value set in advance as a reference. Further, the signal magnitude may be compared after being corrected using a signal such as β-actin.

Note that the mitochondrial function improving agent according to the present embodiment can also be used as a mitochondrial activator, an anti-obesity drug, and an obesity drug for mitochondrial disease.

The present invention will be explained in more detail with reference to the following test examples and examples, but the present invention is not limited by the test examples and examples.

(Test Example 1: Proximity of mitochondria and ER associated with induction of differentiation of brown fat cells)
Undifferentiated cells were isolated from the brown adipose tissue of a newborn ICR mouse, and after culturing for 8 days (day 0), the medium was replaced with a differentiation-inducing medium to induce differentiation. The composition of the differentiation induction medium was DMEM (Dulbecco's modified Eagle medium) containing 20% FBS, 20 nM insulin, 1 nM triiodothyronine (T3), 5 μM dexamethasone, 0.125 mM indomethacin, 0.5 mM IBMX, and 1 μM rosiglitazone. . Two and four days after differentiation induction, the medium was replaced with differentiation-enhancing medium to maintain the cells. The composition of the differentiation-enhancing medium was DMEM containing 20% FBS (fetal bovine serum), 20 nM insulin, and 1 nM T3. Cells on day 6 of differentiation induction were designated as mature brown adipocytes (day 6).

Cells on day 0 and cells on day 6 were washed with PBS and fixed with Karnovsky's fixative (a mixture of paraformaldehyde and glutaraldehyde). Cells were dehydrated with ethanol, stained with uranyl acetate, and observed with an electron microscope. Using image analysis software (Image J), the length of a region where mitochondria and ER are close to each other within 30 nm (mitochondria-ER proximal region) in the electron microscopy image was calculated.

(result)
FIGS. 2(A) and 2(B) show electron microscope images of cells on day 0 and cells on day 6, respectively. The dotted line in the observed image is the mitochondria-ER proximal region. FIG. 2(C) shows the average length of the mitochondria-ER proximal region in 26 observed images. It was shown that the length of the mitochondria-ER proximal region increased significantly with induction of differentiation, that is, mitochondria and ER were brought into close proximity (t-test, n=3).

(Test Example 2: ER stress-independent PERK phosphorylation during differentiation process)
Undifferentiated cells (day -2) were seeded in a 24-well plate at 0.5 x ¹⁰ cells/well, 2 days later (day 0), differentiation was induced by changing the medium to differentiation-inducing medium, and 4 days later (day 0), differentiation was induced by changing the medium to differentiation-inducing medium. 2) and 6 days later (day 4), the medium was replaced with a differentiation-enhancing medium. Cells were collected before changing the medium to the differentiation-enhancing medium on day 2 of differentiation induction, and 6 hours and 12 hours after changing the medium.

On the other hand, siRNA targeting PERK (siPERK) was introduced into the cells on day -2. Specifically, MSS203819 (SEQ ID NO: 4) manufactured by Invitrogen as siPERK was introduced into cells using Lipofectamine (trademark) RNAiMAX Transfection Reagent (manufactured by Thermo Fisher Scientific).

The following genes to be introduced into cells were incorporated into the pMXs-IP plasmid. The plasmid was introduced into a retrovirus (Platinum-GP) packaged in an ecotropic envelope using PEI-Max. 24 hours after introducing siPERK into cells (day -1), culture medium containing retrovirus and 7.5 μg/mL (final concentration) of polybrene were added to the cell culture medium, and after 24 hours, the culture medium was replaced. did.

The gene introduced into cells by retrovirus infection is a gene encoding a kinase-inactive form of PERK (PERK-ΔLD-KA) with a FLAG tag added to the C-terminus. Figure 3 shows the structures of mammalian PERK and PERK-ΔLD-KA. PERK is autophosphorylated when ATP binds to the ATP-binding region of the kinase domain. Therefore, in PERK-ΔLD-KA, lysine (K618) in the ATP binding region, which is the 618th position from the N-terminus, was replaced with alanine to prevent autophosphorylation. PERK-ΔLD-KA lacks a portion of the ER luminal region consisting of the 29th to 502nd amino acid sequence counting from the N-terminus of PERK. By lacking this portion, PERK's response to ER stress is suppressed.

Cells expressing PERK-ΔLD-KA were collected on day 0 and on the second day of differentiation induction (day 2). After adding 200 μL of lysis buffer per well, the cells were lysed at 4° C. for 30 minutes, and centrifuged at 4° C., 15,000 rpm for 10 minutes to collect 150 μL of supernatant. The composition of the lysis buffer was 20mM Tris-HCl pH 7.5, 150mM NaCl, 10mM EDTA pH 8.0, 1% sodium deoxycholate and 1% TritonX-100.

150 μL of 2×SDS sample buffer (containing 20 mM dithiothreitol) was added to the cell lysate, boiled at 98° C. for 5 minutes, and subjected to Western blotting analysis. After SDS-PAGE electrophoresis, proteins were transferred from the gel to polyvinylidene difluoride membrane (0.45 μm, manufactured by Pall), and TBS-T containing 1% skim milk (150 mM NaCl, 50 mM Tris-HCl pH 8.0 and After blocking with 0.05% Tween 20) at room temperature for 1 hour, the cells were reacted overnight at 4°C with each primary antibody diluted in TBS-T containing 5% bovine serum albumin and 0.1% _NaN3 . . After washing three times with TBS-T, the plate was reacted with a secondary antibody diluted in TBS-T containing 1% skim milk for 1 hour at room temperature, and washed with TBS-T for 30 minutes. The band was detected using a high-sensitivity chemiluminescence method.

(result)
FIG. 4 shows the bands of PERK, IRE1α, and GRP78 in the cells before (0 hr) and 6 hours (6 hr) and 12 hours (12 hr) after the medium exchange to the differentiation-enhancing medium on day 2. In PERK, a band shifted toward higher molecular weight was observed. This showed that PERK was phosphorylated during the differentiation process. There are three sensor proteins (PERK, IRE1α, and ATF6) in the ER, all of which span the ER membrane. No change was observed in the band for GRP78, whose expression is induced downstream of IRE1α and ATF6. This suggested that phosphorylation during the differentiation process is specific to PERK.

FIG. 5 shows PERK bands on day 0 and day 2 of cells in which PERK-ΔLD-KA was expressed in cells in which PERK was knocked down with siPERK. A shift in the band indicating phosphorylation of PERK was observed during the differentiation process. In the known PERK pathway, PERK senses ER stress in the ER lumen region, is phosphorylated, and suppresses translation through phosphorylation of eIF2α. PERK-ΔLD-KA lacks the ER lumen region of PERK and is a kinase-inactive type. Therefore, the results in FIG. 5 indicate that phosphorylation of PERK during the differentiation process shown in FIG. 4 is independent of ER stress. It was suggested that PERK plays an unknown function in the differentiation process.

(Test Example 3: Abnormal mitochondrial morphology due to suppression of PERK expression)
Similarly to Test Example 2, on day -2, undifferentiated cells were treated with siRNA (Stealth RNAi (trademark) Negative Control Medium GC Duplex, manufactured by Invitrogen, hereinafter also referred to as "sictrl") or siPERK (MSS203821) without a target as a control. , Invitrogen (SEQ ID NO: 5)) was introduced, differentiation was induced, and cultured until day 6. As in Test Example 1, cells were fixed, stained, and observed with an electron microscope. Mitochondria with a parallel and dense cristae structure were counted as developed mitochondria.

(result)
FIGS. 6(A) and (B) show electron microscopy images of cells into which sictrl and siPERK were introduced, respectively. The scale bar indicates 2 μm. FIGS. 6(C) and (D) show enlarged images of the portions surrounded by white frames in FIGS. 5(A) and (B), respectively. Figure 6(E) shows the percentage of developed mitochondria per cell. The percentage is an average value of 50 cells. Suppression of PERK expression decreased the proportion of parallel, dense cristae found in normal mitochondria and increased the proportion of abnormally shaped cristae. It was shown that PERK is important for the development of mitochondria in differentiated cells.

(Test Example 4: Expression of mitochondrial-related proteins via PERK)
As in Test Example 2, sictrl or siPERK (MSS203821) was introduced into undifferentiated cells on day -2, differentiation was induced, and cells were collected on days 0, 2, 4, and 6. Western blotting analysis was performed on mitochondria, thermogenesis-related proteins, and ER stress-related proteins in the same manner as in Test Example 2.

(result)
As shown in FIG. 7, by suppressing the expression of PERK during the differentiation process, the expression of UCP1, a protein that mainly constitutes the mitochondrial inner membrane and that is associated with thermogenesis, was reduced.

(Test Example 5: ATP production by oxidative phosphorylation via PERK)
As in Test Example 2, sictrl or siPERK (MSS203821) was introduced into undifferentiated cells on day -2, differentiation was induced, and cells were collected on days 0, 2, 4, and 6. The collected cells were treated with or without treatment with 1 μg/mL oligomycin A for 45 minutes, washed with PBS, added with 500 μL of ATP assay buffer (100 mM Tris-HCl pH 7.5, 4 mM EDTA pH 7.5), and collected. . The cell suspension was boiled at 98°C for 3 minutes, and the supernatant was collected after centrifugation at 4°C and 15,000 rpm for 1 minute. ATP in the supernatant was measured using ATP Bioluminescence Assay Kit HS II (manufactured by Roche). The amount of intracellular ATP in the untreated group that was not treated with oligomycin A was defined as the total amount of intracellular ATP, and the amount of ATP produced in the treated group was defined as the glycolysis-dependent ATP production amount. The amount of ATP production dependent on oxidative phosphorylation in mitochondria was calculated from the total amount of intracellular ATP and the measured value of the amount of ATP production dependent on glycolysis.

(result)
As shown in FIG. 8, in cells introduced with sictrl, the rate of oxidative phosphorylation-dependent ATP production increased as differentiation was induced. On the other hand, by suppressing the expression of PERK, the proportion of ATP production dependent on oxidative phosphorylation did not increase. This indicates that PERK is involved in ATP production through oxidative phosphorylation in mitochondria during the differentiation process.

(Example 1: Production of PERK phosphorylated antibody that specifically reacts with mitochondrial stress)
The predicted sites of the serine residues that are phosphorylated in an ER stress-independent manner shown in Test Example 2 are the three serine residues in the kinase insert shown in Figure 1 (715, 717, and 717 from the N-terminal side). 719th). Therefore, an antibody (pPERK (3S) antibody) was produced using a peptide (SEQ ID NO: 3) consisting of 13 amino acids containing phosphorylated serine residues at positions 715, 717, and 719 as an antigen. For the production of pPERK (3S) antibody, rabbits were sensitized by intradermally injecting the above peptide together with an adjuvant four times, whole blood was collected after 9 weeks, and after affinity purification with the above peptide, a rabbit with the same amino acid sequence as the above peptide was sensitized. , and the absorption work was carried out using a peptide in which the three serine residues were not phosphorylated.

Sictrl or siPERK (MSS203819) was introduced into undifferentiated cells on day -2, and Venus-FLAG or PERK-ΔLD-KA was expressed using a retrovirus on day -1 to induce differentiation. Cells on day 3 of differentiation induction were collected before and after stimulation with 10 μM CCCP, and subjected to Western blotting analysis in the same manner as Test Example 2.

(result)
Figure 9 shows the results of Western blotting analysis. PERK was phosphorylated by mitochondrial stress caused by CCCP, and a band indicating the phosphorylation could be detected using the pPERK (3S) antibody. A band could be detected with the pPERK(3S) antibody even in cells in which PERK was knocked down with siPERK and PERK-ΔLD-KA was expressed. This demonstrated that ER stress-independent phosphorylation of PERK caused by mitochondrial stress can be detected using the pPERK(3S) antibody.

(Example 2: Analysis of PERK phosphorylation induced during the differentiation process)
sictrl or siPERK (MSS203819) is introduced into undifferentiated cells on day -2, and 24 hours later (day -1), Venus-FLAG, PERK-ΔLD or PERK-ΔLD-3SA is expressed using a retrovirus, Differentiation was induced. On the second day of differentiation induction, the medium was replaced with a differentiation-enhancing medium, and the cells were collected 12 hours later. The collected cells were subjected to Western blotting analysis in the same manner as in Test Example 2.

PERK-ΔLD lacks a portion of the ER luminal region consisting of the 29th to 502nd amino acid sequence counting from the N-terminus of PERK. Note that a FLAG tag is also added to the C-terminus of PERK-ΔLD.

PERK-ΔLD-3SA, like PERK-ΔLD, lacks a portion in the ER lumen region consisting of the amino acid sequence from the 29th to the 502nd amino acid sequence counting from the N-terminus of PERK. Furthermore, in PERK-ΔLD-3SA, the above three serine residues are replaced with alanine residues. Note that a FLAG tag is also added to the C-terminus of PERK-ΔLD-3SA.

(result)
Figure 10 shows the results of Western blotting analysis. Deletion of the ER luminal region did not affect phosphorylation of PERK (see "PERK"). Phosphorylation of at least one of the three serine residues during brown adipocyte differentiation could be detected in PERK and PERK-ΔLD using the pPERK(3S) antibody, but not in PERK-ΔLD-3SA. Using the pPERK (3S) antibody, it was shown that the kinase insert of PERK is physiologically phosphorylated during brown adipocyte differentiation.

(Example 3: Analysis of PERK phosphorylation induced by β3 adrenergic receptor stimulation)
siPERK (MSS203819) was introduced into undifferentiated cells on day -2, and PERK-ΔLD-KA or PERK-ΔLD-3SA was expressed on day -1 to induce differentiation.

Cells on day 6 of differentiation induction were collected before or 60 minutes after stimulation with 50 nM CL316,243. After adding 550 μL of lysis buffer per well, cells were lysed at 4° C. for 30 minutes, centrifuged at 4° C. and 15,000 rpm for 10 minutes, and 500 μL of supernatant was collected. 20 μL of anti-FLAG M2 affinity gel was added to the cell lysate and reacted at 4° C. for 30 minutes. After removing the supernatant, wash twice with washing buffer (20mM Tris-HCl pH 7.5, 5mM EGTA pH 8.0, 5M NaCl, 1% Triton (containing toll) was added thereto, boiled at 98°C for 5 minutes, and subjected to Western blotting analysis.

(result)
Figure 11 shows the results of Western blotting analysis. In cells expressing PERK-ΔLD-KA, stimulation with CL316,243 induced phosphorylation of three serine residues. On the other hand, it was confirmed that no band was detected with the pPERK(3S) antibody in cells expressing PERK-ΔLD-3SA. This demonstrated that PERK was phosphorylated in an ER stress-independent manner by stimulation of β3 adrenergic receptors.

(Example 4: Detection of mitochondrial stress-dependent PERK phosphorylation by PLA)
HeLa cells maintained in DMEM containing 10% FBS were seeded in a 6-well plate at 2.0 x 10 ⁵ cells/well, and 12 to 16 hours later, 0.5 μg/well of the gene encoding PERK-ΔLD-KA was added. It was introduced in 24 hours after gene introduction, cells were seeded at 0.5 x ¹⁰ cells/well in a 24-well plate containing one collagen-coated coverslip, and 24 hours later, cells were seeded in a glucose-free medium (10% FBS, 1 mM pyruvate). The medium was replaced with glucose-free DMEM containing sodium and 10 mM galactose. 24 hours after medium exchange, cells were stimulated with 10 μM CCCP for 1 hour, washed twice with PBS, and fixed with 4% paraformaldehyde.

After fixation, cells were washed three times with PBS and cell membrane permeabilized with 100 μg/mL digitonin for 5 minutes. After washing five times with PBS, blocking was performed with a 1% BSA solution, and a primary antibody reaction was performed with pPERK (3S) antibody and anti-FLAG antibody. For the secondary antibody reaction, Duolink (trademark) In situ PLA (trademark) probe Anti-Mouse PLUS and Duolink (trademark) In situ PLA (trademark) probe Anti-Rabbit Minus were used (both manufactured by Sigma-Aldrich). After the secondary reaction, the cells were treated with Duolink (trademark) in Situ Detection Green (manufactured by Sigma-Aldrich) and analyzed using a confocal laser microscope. Nuclei were visualized by DAPI staining.

(result)
FIG. 12(A) shows a microscopic image of cells that were not stimulated with CCCP. In the absence of stimulation by CCCP, no PLA signal was detected. FIG. 12(B) shows a microscopic image of cells stimulated with CCCP. Using the pPERK(3S) antibody, it was possible to detect ER stress-independent phosphorylation of PERK induced by CCCP stimulation.

(Example 5: Construction of mitochondrial function improving agent assay system)
An assay system was constructed to identify a PERK3S phosphorylation inducer, that is, a mitochondrial function improving agent, using the dot blotting method. 1×10 ⁶ PERK-FLAG-knockin HEK293 cells maintained in DMEM containing 10% FBS were seeded in a 35 mm dish. A 3×FLAG tag is added to the C-terminus of cell-intrinsic PERK in PERK-FLAG-knockin HEK293 cells. The amino acid sequence of the 3×FLAG tag and the base sequence encoding the 3×FLAG tag are shown in SEQ ID NO: 6 and SEQ ID NO: 7, respectively.

12 to 16 hours after seeding, the medium was replaced with glucose-free medium (DMEM containing 10% FBS, 1mM sodium pyruvate, and 10mM galactose). Sixteen hours after medium change, cells were stimulated with 10 μM CCCP for 30 minutes. Cells were lysed with lysis buffer in the same manner as in Test Example 2 above, immunoprecipitated with anti-Flag antibody (DDDDK-tag antibody, manufactured by MBL Life Sciences), and then in 2x SDS sample buffer in the same manner as in Test Example 2 above. It eluted. 1/10 of the eluate was spotted on a nitrocellulose membrane, and the spot was detected by immunoreaction using pPERK (3S) antibody and DDDDK-tag antibody in the same manner as in Test Example 2 above.

(result)
As shown in FIG. 13, no signal from the pPERK(3S) antibody was detected in unstimulated cells (lane 1) or cells adapted to glucose-free medium (lane 2). On the other hand, in cells stimulated with CCCP (lane 3), a clear signal due to the pPERK(3S) antibody was detected. By allowing a test substance to act instead of CCCP and detecting the pPERK(3S) antibody signal, the test substance can be identified as a PERK3S phosphorylation inducer or mitochondrial function improving agent.

(Test Example 6: Control of thermogenesis via PERK)
The function of PERK in thermogenesis upon stimulation of β3 adrenergic receptors was analyzed. On day -2, sictrl or siPERK (MSS203819) was introduced into undifferentiated cells. After 24 hours, siPERK cells were induced to differentiate by expressing PERK-ΔLD or PERK-ΔLD-3SA using a retrovirus. The cells were cultured until day 5, and the cells were seeded in a glass bottom dish. After 24 hours (day 6), the cells were washed once with PBS and once with 0.5% glucose solution, and then treated with a temperature sensor probe solution (0.04% Cellular Thermoprobe for Fluorescence (manufactured by Funakoshi), 5% glucose). The probe was incorporated into the cells by allowing it to stand at room temperature while shielding from light for 10 minutes.

After stimulating the cells with 0.5 μM CL316,243, images were taken every 10 minutes using a confocal laser microscope (TSC-SP8, manufactured by Leica) to obtain fluorescent images. Fluorescence intensity was measured from the fluorescent image using image analysis software (Image J), and intracellular temperature changes were calculated (20 cells for sictrl, 19 cells for siPERK, 21 cells for siPERK+PERK-ΔLD, siPERK+PERK-ΔLD-3SA). (18 cells). A repeated measures analysis was performed in the testing process.

(result)
Figure 14 shows the fluorescence intensity of each cell. The increase in intracellular temperature caused by CL316,243 stimulation, which was confirmed with sictrl, was suppressed by suppressing the expression of PERK (siPERK). The intracellular temperature, whose rise was suppressed by suppressing PERK expression, increased by expression of exogenous PERK-ΔLD (siPERK+PERK-ΔLD), but hardly increased by PERK-ΔLD-3SA (siPERK+PERK-ΔLD-3SA). ). The results of this test example and the results of test example 4 showed that mitochondrial development, which is controlled by phosphorylation of three serine residues within the kinase insert of PERK, is essential for thermogenesis.

The present invention is capable of various embodiments and modifications without departing from the broad spirit and scope of the invention. Further, the embodiments described above are for explaining the present invention, and do not limit the scope of the present invention. That is, the scope of the present invention is indicated by the claims rather than the embodiments. Various modifications made within the scope of the claims and within the meaning of the invention equivalent thereto are considered to be within the scope of the present invention.

The present invention is suitable for cell state determination, quality control, and drug screening methods.

Claims (7)

Recognizing at least one phosphorylated serine residue in the amino acid sequence shown in RSRSFSV from the N-terminal side to the C-terminal side of the kinase insert of the PKR-like endoplasmic reticulum kinase, the PKR-like endoplasmic reticulum kinase join to,
probe. is an antibody,
The probe according to claim 1. comprising the probe according to claim 1 or 2;
Kit for determining mitochondrial status. a detection step of detecting at least one phosphorylated serine residue in the amino acid sequence represented by RSRSFSV from the N-terminal side to the C-terminal side of the kinase insert of PKR-like endoplasmic reticulum kinase in the cell;
Method for determining mitochondrial status. The cell is
brown fat cells,
The method for determining the state of mitochondria according to claim 4. an exposure step of exposing the cells to the test substance;
a detection step of detecting at least one phosphorylated serine residue in the amino acid sequence represented by RSRSFSV from the N-terminal side to the C-terminal side of the kinase insert of PKR-like endoplasmic reticulum kinase in the cell;
a selection step of selecting the test substance based on the result of the detection step;
A screening method for mitochondrial function improving agents, including: The cell is
brown fat cells,
The method of screening for a mitochondrial function improving agent according to claim 6.