Method and kit for screening translation modifiers

Abstract

This invention provides a screening method for translation regulators that can be obtained to control the accumulation of structurally abnormal proteins. [Solution] A screening method for translation regulators of substrates that are degraded by a translation-coupled proteolytic degradation mechanism under endoplasmic reticulum stress conditions includes a binding evaluation step of selecting a test substance that promotes or inhibits the binding of Sec61β to ARIH1.

Description

[Technical Field]

[0001] The present invention relates to a screening method for translation modifiers and a screening kit for translation modifiers. [Background technology]

[0002] In living organisms, the correct three-dimensional structure and quality control of proteins are essential for various life activities. While much research has been conducted on the mechanisms of protein homeostasis (proteostasis) in the cytoplasm and individual organelles, and the molecular mechanisms related to quality control have been elucidated, many aspects of the proteostasis maintenance mechanisms beyond organelle membranes remain unclear.

[0003] Of all proteins in eukaryotic cells, secretory proteins and membrane proteins containing signal sequences are inserted into the endoplasmic reticulum (ER), folded correctly, and transported to their appropriate locations for function. However, under ER stress conditions, where structurally abnormal proteins accumulate in the ER, to reduce the transport burden on the ER, the destination of certain secretory proteins is changed from the ER to the cytoplasm by a translation-coupled proteolytic degradation mechanism (ER stress-induced preemptive quality control: ERpQC), and the proteins are immediately degraded by the ubiquitin-proteasome (UPS) after translation is complete.

[0004] As reported in Non-Patent Documents 1 and 2, substrate recognition involves the endoplasmic reticulum membrane protein Derlin, which binds to the translocon in an ER stress-dependent manner. The binding of signal recognition particles (SRPs) to the cytoplasmic domain of Derlin is necessary for a shift in translational transport in the ER to translational degradation in the cytoplasm. Next, Sec61β, one of the translocon components, suppresses translation to minimize the synthesis of substrates degraded by the no longer functional ERpQC (ERpQC substrates). On the other hand, any ERpQC substrates that have been generated are degraded by the proteasome via the endoplasmic reticulum membrane E3 ligase HRD1, the triple ATPase p97, and the chaperone molecule Bag6. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Hisae Kadowaki, 12 others, Pre-emptive Quality Control Protects the ER from Protein Overload via the Proximity of ERAD Components and SRP, Cell Reports, 2015, 13, 944-956 [Non-Patent Document 2] Hisae Kadowaki, and 3 others, Molecular mechanism of ER stress-induced pre-emptive quality control involving association of the translocon, Derlin-1, and HRD1, SCIENTIFIC REPORTS, 2018, 8, 7317 [Overview of the project] [Problems that the invention aims to solve]

[0006] The accumulation of structurally abnormal proteins resulting from decreased proteasome activity is known to cause conformational diseases, such as neurodegenerative diseases. Furthermore, a link between structurally abnormal proteins and ER stress has also been reported. For example, it has been shown that an ER stress response is necessary for viral replication in virus-infected cells. Additionally, cancerous tissue is subject to ischemia and hypoxia, which induces ER stress, and the ER stress response has been reported to be involved in cancer survival or its spread to surrounding tissues.

[0007] Until now, it has been technically difficult to capture proteins degraded by ERpQC, so the molecular mechanism of ERpQC has remained largely unknown. If we can elucidate the molecular mechanism of ERpQC in detail and control the accumulation of structurally abnormal proteins, it will lead to overcoming various diseases.

[0008] The present invention has been made in view of the above circumstances, and aims to provide a screening method for translation regulators and a screening kit for translation regulators that can obtain substances that control the accumulation of structurally abnormal proteins. [Means for solving the problem]

[0009] The inventors have diligently researched the molecular mechanism of ERpQC and have revealed that disruption of ERpQC regulation not only impairs ER but also cytoplasmic proteostasis. Based on this newly obtained knowledge, the inventors have completed an invention relating to the evaluation of substances that control the accumulation of structurally abnormal proteins by suppressing or promoting the translation of ERpQC substrates.

[0010] A screening method for translation regulators of substrates that are degraded by a translation-coupled proteolytic degradation mechanism under endoplasmic reticulum stress conditions, according to the first aspect of the present invention, The process includes a binding evaluation step of selecting a test substance that promotes or inhibits the binding of Sec61β to ARIH1.

[0011] The aforementioned coupling evaluation step is, An exposure step of exposing cells expressing Sec61β with a first tag added to the N-terminus and ARIH1 with a second tag different from the first tag added to the N-terminus to the test substance, A treatment step of bringing the cells into an endoplasmic reticulum stress state, For a control lysate obtained by solubilizing the cells that were not exposed to the test substance and were brought into an endoplasmic reticulum stress state, and a lysate obtained by solubilizing the cells that were exposed to the test substance and were brought into an endoplasmic reticulum stress state, a detection step of detecting luminescence based on chemic energy transfer that occurs when the distance between a first bead having an antibody that binds to the first tag and a second bead having an antibody that binds to the second tag is close, A selection step of selecting the test substance based on a comparison between the intensity of the luminescence in the control lysate and the intensity of the luminescence in the lysate, may also be included.

[0012] The binding evaluation step includes A treatment step of bringing cells expressing Sec61β with a first tag added to the N-terminus and ARIH1 with a second tag different from the first tag added to the N-terminus into an endoplasmic reticulum stress state, A mixing step of obtaining a reaction solution containing a lysate obtained by solubilizing the cells in an endoplasmic reticulum stress state and the test substance, For a control lysate obtained by solubilizing the cells that do not contain the test substance and were brought into an endoplasmic reticulum stress state, and the reaction solution, a detection step of detecting luminescence based on chemic energy transfer that occurs when the distance between a first bead having an antibody that binds to the first tag and a second bead having an antibody that binds to the second tag is close, A selection step of selecting the test substance based on a comparison between the intensity of the luminescence in the control lysate and the intensity of the luminescence in the lysate, may also be included.

[0013] The screening method according to the present invention described above Prior to the binding evaluation step, the method further includes an accumulation evaluation step of selecting a test substance that suppresses or promotes the accumulation of the substrate in a cell under endoplasmic reticulum stress, In the aforementioned integration evaluation step, From among the test substances that suppress the accumulation of the substrate selected in the accumulation evaluation step, a test substance that promotes the binding of Sec61β and ARIH1 is selected. From among the test substances that promote the accumulation of the substrate selected in the accumulation evaluation step, a test substance that inhibits the binding of Sec61β and ARIH1 is selected. It would be acceptable to do so.

[0014] In the accumulation evaluation step, null Hong Kong or transthyretin of α1-antitrypsin is expressed in the cells as the substrate. Select a test substance that suppresses or promotes the accumulation of the substrate whose signal sequence is not cleaved. It would be acceptable to do so.

[0015] The screening method according to the present invention described above is The method further includes, after the binding evaluation step, an accumulation evaluation step of selecting a test substance that suppresses or promotes the accumulation of the substrate in a cell under endoplasmic reticulum stress, In the aforementioned accumulation evaluation step, From among the test substances that promote the binding of Sec61β and ARIH1 selected in the binding evaluation step, a test substance that suppresses the accumulation of the substrate is selected. From among the test substances that inhibit the binding of Sec61β and ARIH1 selected in the binding evaluation step, a test substance that promotes the accumulation of the substrate is selected. It would be acceptable to do so.

[0016] In the accumulation evaluation step, null Hong Kong or transthyretin of α1-antitrypsin is expressed in the cells as the substrate. Select a test substance that suppresses or promotes the accumulation of the substrate whose signal sequence is not cleaved. It would be acceptable to do so.

[0017] A screening kit for translation regulators of substrates that are degraded by a translation-coupled proteolytic degradation mechanism under endoplasmic reticulum stress conditions, according to a second aspect of the present invention, The present invention comprises cells expressing Sec61β with a first tag attached to its N-terminus and ARIH1 with a second tag, different from the first tag, attached to its N-terminus. [Effects of the Invention]

[0018] According to the present invention, a substance that controls the accumulation of structurally abnormal proteins can be obtained. [Brief explanation of the drawing]

[0019] [Figure 1] Figure A shows the results of Western blotting (WB) detecting null Hong Kong (NHKQQQ), a genetic variant lacking the N-glycosylation site of α1-antitrypsin (α1AT), in Test Example 1. Figure B shows the results of WB detecting the endogenous secretory protein transthyretin (TTR) in Test Example 1. [Figure 2] Figure A shows the results of the autoradiography analysis to detect NHKQQQ in Test Example 2. Figure B shows the relative radiation dose of non-cut NHKQQQ in Test Example 2. Figure C shows the relative radiation dose of cut NHKQQQ in Test Example 2. [Figure 3] Figure A shows the results of the autoradiography analysis to detect TTR in Test Example 2. Figure B shows the relative radiation dose of the non-cutting type TTR in Test Example 2. Figure C shows the relative radiation dose of the cutting type TTR in Test Example 2. [Figure 4] Figure A shows the results of the autoradiography analysis to detect BiP in Test Example 2. Figure B shows the relative radiation dose of the cleavage-type BiP in Test Example 2. [Figure 5]Figure A shows the results of the autoradiography analysis to detect CL1 in Test Example 2. Figure B shows the relative radiation dose of the segmented CL1 in Test Example 2. [Figure 6] Figure A shows the domain structures of Sec61β and ARIH1, respectively. Figure B shows the WB results for cells expressing ariadne RING-in-between-RING (RBR) E3 ubiquitin protein ligase 1 (ARIH1) in Test Example 3. Figure C shows the WB results for cells expressing eukaryotic translation initiation factor (eIF) 4E-homologous protein (4EHP) in Test Example 3. [Figure 7] This figure shows the results of Western blotting (WB) for Sec61β-expressing cells and Sec61β-expressing cells lacking the IDR region in Test Example 3. [Figure 8] This figure shows the results of Western blotting (WB) detection of NHKQQQ in NHKQQQ-expressing cells in which ARIH1 or Sec61β was knocked down in Test Example 4. [Figure 9] This figure shows the results of Western blotting (WB) detection for NHKQQQ-expressing cells in which 4EHP or Sec61β was knocked down, as in Test Example 4. [Figure 10] This figure shows the results of Western blotting (WB) for cells in which ARIH1 was knocked down in Test Example 4, expressing NHKQQQ and either Ariadne domain deletion active ARIH1 or Ariadne domain deletion inactive ARIH1. [Figure 11] This figure shows the results of Western blotting (WB) for NHKQQQ-expressing cells in which 4EHP was knocked down and expressed in Test Example 4. [Figure 12] This figure shows the results of Western blotting (WB) for cells in which Sec61β was knocked down in Test Example 4, and which expressed NHKQQQ and either Ariadne domain deletion active ARIH1 or Ariadne domain deletion inactive ARIH1. [Figure 13]This figure shows the results of Western blotting (WB) studies involving cells in which Sec61β was knocked down in Test Example 4, and which expressed NHKQQQ, Ariadne domain-deficient active ARIH1, and 4EHP. [Figure 14] This figure shows the results of Western blotting (WB) studies on cells expressing Sec61β in which Sec61β was knocked down, resulting in a Sec61β lacking NHKQQQ and the IDR region, as in Test Example 4. [Figure 15] Figure A shows the proteasome activity of cells treated with thapsigargin (Tg), an ER stressor, in Test Example 5. Figure B shows the proteasome activity of cells treated with tunicamycin (Tun), an ER stressor, in Test Example 5. [Figure 16] This figure shows the results of a pulse chase experiment on cells expressing CL1 after Sec61β was knocked down in Test Example 5. [Figure 17] This figure shows the results of a pulse chase experiment on cells expressing NHKQQQ after Sec61β was knocked down in Test Example 5. [Figure 18] This figure shows the detection results of aggregated proteins in cells in which Sec61β was knocked down in Test Example 6. [Figure 19] This figure shows the detection results for aggregated proteins in cells where Sec61β was knocked down in Test Example 6, and which expressed wild-type Sec61β or Sec61β lacking the IDR region. [Figure 20] This figure shows the fluorescence intensity of cells expressing NHKQQQ in Example 1. [Figure 21] This figure shows the fluorescence intensity of cells expressing Sec61β and ARIH1 in Example 1. [Modes for carrying out the invention]

[0020] Embodiments of the present invention will be described below with reference to the drawings. In each drawing, the same or equivalent parts are denoted by the same reference numerals. However, the present invention is not limited to the embodiments and drawings described below. In the embodiments described below, expressions such as “having,” “including,” or “containing” also include the meaning of “consisting of” or “composed of.”

[0021] The screening method according to this embodiment is suitable for searching for, selecting, or identifying translation regulators of substrates that are degraded by a translation-coupled proteolytic mechanism under ER stress conditions, i.e., ERpQC substrates. The screening method includes a binding evaluation step of selecting a test substance that promotes or inhibits the binding of Sec61β to ARIH1.

[0022] Sec61β is located opposite the lateral gate of the translocon, a region necessary for the lateral movement of the polypeptide transmembrane domain. Sec61β binds to Derlin under ER stress. ARIH1 is known to contribute to translation inhibition during DNA damage by binding to and modifying 4EHP, which interacts with the 5' cap structure of mRNA and inhibits translation, instead of eIF4E.

[0023] In the following examples, it was revealed that Sec61β, ARIH1, and 4EHP bind to Derlin in an ER stress-dependent manner, that the translation of ERpQC substrates is suppressed by the binding of Sec61β and ARIH1, and that an increase in ERpQC substrates leads to the formation of abnormal aggregated proteins in the cytoplasm. Since the quality of cytoplasmic proteins is maintained through the binding of Sec61β and ARIH1, promoting or suppressing the binding of Sec61β and ARIH1 can regulate the translation of ERpQC substrates and control the accumulation of structurally abnormal proteins. More specifically, promoting the binding of Sec61β and ARIH1 can suppress the translation of ERpQC substrates and the accumulation of structurally abnormal proteins. On the other hand, suppressing the binding of Sec61β and ARIH1 can promote the translation of ERpQC substrates and the accumulation of structurally abnormal proteins.

[0024] "The binding of Sec61β to ARIH1" refers to a specific protein-protein interaction (PPI) between Sec61β and ARIH1. PPIs include electrostatic interactions, van der Waals forces, dipole interactions, dispersion forces, hydrogen bonds, hydrophobic interactions, etc., that act between Sec61β and ARIH1. In the binding evaluation step, it is sufficient to determine whether the test substance promotes or inhibits the binding of Sec61β to ARIH1 by measuring or evaluating the PPI between Sec61β and ARIH1 using a known method for measuring or evaluating the PPI between Sec61β and ARIH1 in the presence of at least Sec61β, ARIH1, and the test substance. The method for measuring or evaluating PPIs is not particularly limited and includes, for example, co-immunoprecipitation, pull-down assay, far-western blotting, cross-linking, label transfer, interaction mapping, surface plasmon resonance, FRET (Fluorescence resonance energy transfer), and Alpha (Amplified Luminescence Proximity Homogeneous Assay) assays. A method for evaluating PPIs can also be in silico screening using computers.

[0025] In the binding evaluation step, for example, the effect of the test substance on the binding of purified Sec61β expressed by genetic engineering technology to ARIH1 may be measured, or cells that stably express Sec61β and ARIH1 by plasmid introduction may be used. The cells are not particularly limited, but known cells such as HEK293 cells and HepG2 cells can be used. When using cells, the binding of Sec61β to ARIH1 may be measured in a lysate obtained by solubilizing cells that have been subjected to ER stress in the presence of the test substance (in cell screening), or the binding of Sec61β to ARIH1 may be measured by adding the test substance to a lysate obtained by solubilizing cells that have been subjected to ER stress (in vitro screening).

[0026] Preferably, in the binding evaluation step, the results of measuring the binding of Sec61β to ARIH1 in the presence of the test substance are compared with the results of measuring the binding of Sec61β to ARIH1 in a control that underwent the same treatment except in the absence of the test substance. This makes it possible to determine with greater accuracy whether the test substance promotes or inhibits the binding of Sec61β to ARIH1.

[0027] The test substance may be an organic or inorganic compound, and may be derived from natural products or be a synthetic compound. The test substance may also be a plant extract, cell extract, culture supernatant, etc. Examples of test substances include compounds, antibodies, proteins, peptides, DNA aptamers, RNA aptamers, etc. The test substance may also be an existing drug.

[0028] Cells can be put into an ER stress state by treating them with, for example, an ER stressor such as Tg or a proteasome inhibitor. This treatment can be carried out by, for example, adding at least one of the ER stressor and the proteasome inhibitor to the culture medium and incubating it for 10 to 24 hours, 12 to 22 hours, or 14 to 20 hours, preferably 16 hours.

[0029] Here, we will explain in-cell screening in more detail using the alpha assay method as an example. The binding evaluation step includes an exposure step, a treatment step, a detection step, and a selection step. In the exposure step, cells expressing Sec61β with tag 1 (first tag) attached to the N-terminus and ARIH1 with tag 2 (second tag) different from tag 1 attached to the N-terminus are exposed to the test substance. Tags 1 and 2 are polypeptide chains of several to several tens of amino acids, and are preferably epitope tags recognized by antibodies. Any known tags of different types can be used as tag 1 and tag 2. Examples of tags include DDDDK (Flag(trademark)) tag, HA tag, 6×His (6His) tag, Myc tag, V5 tag, S tag, E tag, T7 tag, etc. For example, tag 1 is a Flag tag and tag 2 is an HA tag.

[0030] In the treatment step, cells are treated with at least one of an ER stressor and a proteasome inhibitor to induce an ER stress state. The treatment step may be performed before or after the exposure step.

[0031] In the detection step, luminescence based on chemical energy transfer occurs when beads 3 (first beads) containing an antibody bound to tag 1 and beads 4 (second beads) containing an antibody bound to tag 2 are in close proximity in a lysate obtained by solubilizing cells exposed to the test substance and subjected to ER stress. Commercially available kits and measuring devices such as AlphaLISA® can be used to detect cell solubilization and luminescence. In the detection step, as a control, luminescence based on chemical energy transfer is also detected in a control lysate obtained by solubilizing cells that were not exposed to the test substance and subjected to ER stress, as described above.

[0032] In the selection step, the test substance is selected based on a comparison of the luminescence intensity in the control solution and the luminescence intensity in the test solution. For example, if the luminescence intensity in the test solution is greater than that in the control solution, the test substance promotes (enhances) the binding of Sec61β to ARIH1. On the other hand, if the luminescence intensity in the test solution is less than that in the control solution, the test substance inhibits (suppresses) the binding of Sec61β to ARIH1.

[0033] Next, we will mainly explain the differences between in vitro screening using the alpha assay method and in cell screening. The binding evaluation step includes a processing step, a mixing step, a detection step, and a selection step. In the processing step, cells expressing Sec61β with tag 1 attached to the N-terminus and ARIH1 with tag 2, which is different from tag 1, attached to the N-terminus are subjected to ER stress.

[0034] In the mixing step, a reaction solution is obtained containing a lysate obtained by solubilizing cells subjected to ER stress and the test substance. For example, the reaction solution can be obtained by adding the test substance to the lysate and allowing it to react for a certain period of time.

[0035] In the detection step, luminescence based on the chemical energy transfer described above is detected in the reaction solution and the control solution. The selection step is the same as in cell screening.

[0036] To more efficiently obtain substances that control the accumulation of structurally abnormal proteins, the screening method according to this embodiment may include an accumulation evaluation step of selecting a test substance that suppresses or promotes the accumulation of ERpQC in cells under ER stress.

[0037] In the accumulation evaluation step, an ERpQC substrate is expressed in cells, and a test substance is selected that suppresses or promotes the accumulation of the ERpQC substrate, which has not had its signal sequence cleaved. The ERpQC substrate can be any known secreted protein whose destination is changed from the ER to the cytoplasm by ERpQC and which is degraded immediately after translation is complete. Examples of ERpQC substrates include NHK QQQ Examples include TTR, etc.

[0038] The accumulation of ERpQC substrates with uncleaved signal sequences may be identified by molecular weight using a Western blotting (WB) method with an antibody against ERpQC substrates, for example, by using a lysate obtained by solubilizing cells as a sample, or by measuring it using an alpha assay method.

[0039] For example, the accumulation evaluation step using the alpha assay method includes an exposure step, a treatment step, a detection step, and a selection step. In the exposure step, cells expressing ERpQC with tags 1 and 2 attached to the N-terminus and C-terminus, respectively, are exposed to the test substance. The treatment step, detection step, and selection step are the same as those in the in-cell screening using the alpha assay method described above.

[0040] The accumulation evaluation step may be performed before or after the binding evaluation step. If the accumulation evaluation step is performed before the binding evaluation step, the binding evaluation step selects a test substance from the test substances selected in the accumulation evaluation step that controls the binding of Sec61β to ARIH1. If the accumulation evaluation step is performed after the binding evaluation step, the accumulation evaluation step selects a test substance from the test substances selected in the binding evaluation step that controls the accumulation of ERpQC substrates.

[0041] More specifically, if the accumulation evaluation step is performed before the binding evaluation step, in the binding evaluation step, a test substance that promotes the binding of Sec61β to ARIH1 is selected from among the test substances that suppress the accumulation of ERpQC substrates selected in the accumulation evaluation step, and a test substance that suppresses the binding of Sec61β to ARIH1 is selected from among the test substances that promote the accumulation of ERpQC substrates selected in the accumulation evaluation step.

[0042] If the accumulation evaluation step is performed after the binding evaluation step, in the accumulation evaluation step, a test substance that suppresses the accumulation of ERpQC substrates is selected from among the test substances that promote the binding of Sec61β and ARIH1 selected in the binding evaluation step, and a test substance that promotes the accumulation of ERpQC substrates is selected from among the test substances that suppress the binding of Sec61β and ARIH1 selected in the binding evaluation step.

[0043] The translation regulator selected by the screening method according to this embodiment can suppress the translation of ERpQC substrates and the accumulation of structurally abnormal proteins by promoting the binding of Sec61β to ARIH1. Furthermore, the test substance can promote the translation of ERpQC substrates and the accumulation of structurally abnormal proteins by inhibiting the binding of Sec61β to ARIH1.

[0044] In conformational diseases, including neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, spinocerebellar degeneration, and amyotrophic lateral sclerosis, as well as diabetes, ischemic diseases, and inflammatory diseases, it has been suggested that ER stress-induced apoptosis contributes to the onset and exacerbation of these diseases. Translational modulators that promote the binding of Sec61β to ARIH1, thereby suppressing the translation and accumulation of structurally abnormal proteins of ERpQC substrates, are useful as therapeutic or prophylactic agents for these diseases. On the other hand, translational modulators that inhibit the binding of Sec61β to ARIH1, thereby promoting the translation and accumulation of structurally abnormal proteins of ERpQC substrates, are useful as therapeutic or prophylactic agents for viral replication that utilizes the ER stress response, viral diseases, and cancers in which ER stress is induced.

[0045] The in silico screening described above uses the three-dimensional structural data of Sec61β, ARIH1, and the test substance. Three-dimensional structural data refers to information indicating the position of each atom constituting each molecule in three-dimensional space. Specifically, the three-dimensional structural data of Sec61β and ARIH1 are coordinate data of each atom constituting each molecule, determined by X-ray crystallography, NMR, etc. Three-dimensional structural data can be obtained from sources such as the Protein Data Bank (PDB). The three-dimensional structural data of the test substance is coordinate data of each atom set based on theoretical interatomic distances according to the structural formula of the test substance. Coordinate data of a three-dimensional structure optimized using a known force field may also be used as the three-dimensional structural data.

[0046] In in silico screening, the test substance is placed at the site where Sec61β and ARIH1 interact, and the intermolecular interaction between Sec61β and ARIH1 can be evaluated using computational chemistry. By comparing the evaluation of the intermolecular interaction between Sec61β and ARIH1 using computational chemistry with the evaluation of the intermolecular interaction between Sec61β and ARIH1 without the test substance, it is possible to select test substances that contribute to stabilizing or destabilizing the binding between Sec61β and ARIH1. In silico screening is preferable because it allows for virtual screening that comprehensively evaluates many test substances.

[0047] In another aspect of this embodiment, a screening kit for translation regulators of ERpQC substrates is provided. The screening kit comprises cells expressing Sec61β with tag 1 attached to its N-terminus and ARIH1 with a different tag 2 attached to its N-terminus. The screening kit is suitable for use, for example, in the screening method described above. In addition to the cells, the screening kit may also comprise an ER stressor, a proteasome inhibitor, a culture medium, a buffer, a cell lysis reagent, beads 3 having an antibody that binds to tag 1, beads 4 having an antibody that binds to tag 2, and the like.

[0048] The present invention will be described in more detail by the following examples, but the present invention is not limited to these examples. [Examples]

[0049] [Cell culture] HEK293 cells (Invitrogen) were cultured in Dulbecco's modified Eagle medium (DMEM, Nacalai Tesque; 08459-64) containing 10% fetal bovine serum (FBS) and penicillin-streptomycin solution (Nacalai Tesque; 09367-34). HepG2 cells (ATCC; HB-8065) ​​were cultured in Eagle's minimal essential medium (MEM, Nacalai Tesque; 21443-15) containing 10% FBS and penicillin-streptomycin solution (Nacalai Tesque; 09367-34). Cell culture was performed at 37°C under 5% CO2.

[0050] [Genetic transfer] For plasmid transfection, polyethyleneimine (PEI)-Max (Polysciences; 24765) was used. In knockdown experiments, Lipofectamine RNAiMAX (Invitrogen; 13778150) was used according to the manufacturer's instructions to reverse transfect HEK293 cells or HepG2 cells with the siRNAs shown in Table 1 (Invitrogen, Stealth® siRNA) at a final concentration of 10–40 nM for 48–72 hours.

[0051] [Table 1]

[0052] [ER stress processing] In ER stress treatment, HEK293 cells were treated for 16 hours with the ER stressor 50 nM Tg (Nacalai Tesque; 33637-31) and the proteasome inhibitor 200 nM MG132 (Calbiochem; 474790).

[0053] [Immunoprecipitation (IP)] Cells were lysed on ice in a lysis buffer containing 5 μg / mL leupeptin (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EGTA, 1% Triton X-100, 12 mM β-glycerophosphate). For secreted TTR, culture samples were prepared by centrifuging the culture medium. Cell lysates or culture samples were immunoprecipitated with anti-Flag M2 antibody affinity gel or HA-tagged protein purification gel. Beads were washed with a high-salt buffer (20 mM Tris-HCl (pH 7.5), 500 mM NaCl, 5 mM EGTA, 1% Triton X-100) or a low-salt buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EGTA), and samples were separated by SDS-PAGE and subjected to antibody-based Western blotting. Proteins were detected using an ECL system.

[0054] [Western blotting] Cell lysates were separated by SDS-PAGE and transferred to a PVDF membrane. After blocking with TBS-T containing 5% skim milk (50mM Tris-HCl (pH 8.0), 150mM NaCl, 0.05% Tween-20), the cells were reacted with antibodies. Proteins were detected using an ECL system. Band intensity was measured using ImageQuant TL (GE Healthcare).

[0055] [Pulse labeling assay] Cells transfected with plasmids and siRNAs were immersed in DMEM (Gibco; 21013024) lacking methionine and cysteine, and containing 1 mM sodium pyruvate and 2 mM L-glutamine for 3 minutes or 10 minutes. 35 S]-methionine / cysteine ​​labeled (EXPRE 35 S 35 S Protein Labeling Mix 35S], manufactured by Revvity Health Sciences; NEG772). In autoradiography analysis, band radioactivity was measured using a Typhoon FLA7000 image analyzer (manufactured by GE Healthcare) and quantified using ImageQuant TL.

[0056] In the pulse-chase experiment, cells were stimulated with 50 nM Tg for 16 hours, 35 labeled with [S]-methionine / cysteine for 15 minutes, washed with PBS, and chased for a predetermined time in a medium containing 50 nM Tg, methionine, and cysteine. Cells were immediately washed with PBS and lysed on ice in a lysis buffer containing 5 μg / mL leupeptin. Immunoprecipitation was performed, and the beads were washed with a high-salt buffer and then a low-salt buffer, and the samples were separated by SDS-PAGE.

[0057] Test Example 1: Functional analysis of Sec61β Sec61β was knocked down with siRNA, and the behavior of the signal sequence (SS) non-cleaved type of the ERpQC substrate was examined by WB. The ERpQC substrates are the secreted protein NHK QQQ and the endogenous secreted protein TTR. To have little effect on the behavior and be easily identified by molecular weight and enable isolation of the SS non-cleaved type, Flag-6His-NHK QQQ expressing NHK with a Flag tag and a His tag added to the N-terminus and a HA tag added to the C-terminus QQQ -HA (pcDNA3 / GW Flag-6His-NHK QQQ -HA, see the above Non-Patent Document 1) and Flag-TTR-HA (pcDNA3 / GW Flag-TTR-HA, see the above Non-Patent Document 1) expressing TTR with a Flag tag added to the N-terminus and a HA tag added to the C-terminus were used as plasmids.

[0058] Plasmids and siRNAs (at least one of siCtrl, siDerl1, siDerl2, siDerl3, siSec61β #1, and siSec61β #2) were introduced, and cell lysates prepared from HEK293 cells with or without ER stress treatment were subjected to Western blotting (WB).

[0059] Rabbit polyclonal anti-α1AT antibody (1:2000, DAKO; A0012) or rabbit polyclonal anti-TTR antibody (1:2000, DAKO; A0002) was used as the primary antibody in Western blotting, and HRP-labeled anti-rabbit IgG antibody (1:4000, Cell Signaling Technology; 7074) was used as the secondary antibody.

[0060] (result) As shown in Figure 1A, in cells in which Derlin-1, Derlin-2, and Derlin-3, which are involved in ERpQC induction, were knocked down, SS non-cleavage type NHK was observed under ER stress. QQQ While the amount decreased, both types of siRNA against Sec61β produced SS-uncleaved NHK QQQ The accumulation of was enhanced. As shown in Figure 1B, SS non-cleavage type NHK due to Sec61β knockdown. QQQ Enhanced accumulation was also observed in the SS-noncleaved form of TTR. These results indicate that Sec61β suppresses the accumulation of ERpQC substrates under ER stress.

[0061] Test Example 2: Investigation of the specificity of Sec61β-mediated suppression of ERpQC substrate accumulation. Next, we investigated whether the suppression of ERpQC substrate accumulation by Sec61β affected substrate translation. HEK293 cells transduced with siRNA (siSec61β #1 or siSec61β #2) and plasmid were subjected to stress treatment and pulse labeling assays. After immunoprecipitation, the beads were washed with low-salt buffer and separated by SDS-PAGE.

[0062] As a plasmid, NHK has a Flag tag attached to its C-terminus. QQQ NHKQQQ -Flag(pcDNA3 / GW NHK QQQ The following were used: Flag (see Non-Patent Document 1 above), Flag-TTR-HA, BiP-2×Flag-KDEL (pcDNA3 / GW BiP-2×Flag-KDEL, see Non-Patent Document 1 above) expressing ER chaperone BiP with a Flag-KDEL tag attached to the C-terminus, and Venus-CL1-Flag (pcDNA3 / GW Venus-CL1-Flag, see Genes Dev. 2008, 22(11), 1451-1464) expressing CL1 with Venus and Flag tags attached to the N-terminus and C-terminus, respectively. CL1 is a degradation model substrate containing a degron sequence localized in the cytoplasm.

[0063] (result) Figure 2A is NHK QQQ The results of autoradiography analysis for the following are shown. Figures 2B and 2C show the results of detection by pulse labeling for 3 minutes in cells introduced with siCtrl. 35 [S] Radioactivity of labeled protein detected by 10-minute pulse labeling [ 35 This shows the radioactivity of the [S]-labeled protein. As shown in Figure 2B, SS-uncleaved NHK is observed between 3 and 10 minutes. QQQ The amount synthesized increased due to the knockdown of Sec61β, whereas, as shown in Figure 2C, SS-cleaved NHK QQQ The amount of protein did not change.

[0064] Figure 3A shows the results of autoradiographic analysis for TTR. Figures 3B and 3C show the detection of siCtrl in cells with 3 minutes of pulsed labeling. 35 [S] Radioactivity of labeled protein detected by 10-minute pulse labeling [ 35 The radioactivity of the [S]-labeled protein is shown. As shown in Figure 3B, the amount of SS-uncleaved TTR synthesized increased between 3 and 10 minutes after Sec61β knockdown, whereas as shown in Figure 3C, the amount of SS-cleaved TTR protein remained unchanged. This indicates that Sec61β specifically suppresses the translation of ERpQC substrates.

[0065] Figures 4A and 5A show the results of autoradiographic analysis for BiP and CL1, respectively. As shown in Figures 4B and 5B, neither BiP nor CL1, which are substrates of ERpQC, were affected by Sec61β knockdown. Therefore, it was confirmed that Sec61β-mediated translational repression is specific to the ERpQC substrate.

[0066] Test Example 3: Analysis of Sec61β-binding molecules in a stress-dependent manner within the ER The interactions between endogenous Sec61β and Derlin-1 and ARIH1, as well as the interactions between endogenous Sec61β, Derlin-1, and ARIH1 and 4EHP, were investigated. First, EcoRI restriction enzyme sites and XhoI restriction enzyme sites were introduced into the forward and reverse primers shown in Table 2, respectively. PCR was performed on the template, and the resulting PCR products were incorporated into pcDNA3.0 (Invitrogen) treated with EcoRI and XhoI to construct each plasmid. HA-ARIH1ΔAri(1-407) C357S was constructed using site-directed mutagenesis.

[0067] [Table 2]

[0068] Flag-ARIH1 is a plasmid (pcDNA3 / GW Flag-ARIH1) that expresses ARIH1 (CDS:NM_005744.3) with a Flag tag attached to the N-terminus. Flag-4EHP is a plasmid (pcDNA3 / GW Flag-4EHP) that expresses 4EHP (CDS:NM_004837.1) with a Flag tag attached to the N-terminus. Flag-Sec61β WT is a plasmid (pcDNA3 / GW Flag-Sec61β) that expresses Sec61β (CDS:NM_006808.2) with a Flag tag attached to the N-terminus. Flag-Sec61β ΔIDR is a plasmid (pcDNA3 / GW Flag-Sec61βΔIDR(55-96)) that expresses Sec61β with a Flag tag attached to the N-terminus and a deletion of the IDR region from position 55 to 96 from the N-terminus. The active form of HA-ARIH1 ΔAri is a plasmid (pcDNA3 / GW HA-ARIH1ΔAri(1-407)) in which an HA tag is added to the N-terminus and ARIH1 is expressed from the N-terminus to position 407 (C357S). The inactive form of HA-ARIH1 ΔAri is a plasmid (pcDNA3 / GW HA-ARIH1ΔAri(1-407) C357S) in which an HA tag is added to the N-terminus and ARIH1 is expressed from the N-terminus to position 407 (C357S). In addition, the plasmid HA-4EHP (pcDNA3 / GW HA-4EHP), which expresses 4EHP with an HA tag added to the N-terminus, was constructed by replacing Flag-4EHP with EcoRI / XhoI.

[0069] After immunoprecipitation of cell lysates prepared from HEK293 cells transfected with or without ER stress treatment, the beads were washed with a low-salt buffer and subjected to Western blotting. Rabbit polyclonal anti-Sec61β antibody (1:500, Proteintech; SC-514551), rabbit polyclonal Derlin-1 antibody (1:4000, see Genes Dev. 2008, 22(11), 1451-1464), mouse monoclonal anti-ARIH1 antibody (Clone C-7, 1:100, Santa Cruz Biotechnology; PM019), rabbit polyclonal anti-4EHP antibody (1:1000, Gene Tex; GTX103977), and mouse monoclonal anti-DYKDDDDK tag antibody (clone FLA-1, 1:5000, MBL; 185-3L) were used as primary antibodies, and HRP-labeled anti-rabbit IgG antibody or HRP-labeled anti-mouse IgG antibody (1:5000, Cytiva; NA931) were used as secondary antibodies. The amount of protein co-immunoprecipitated with ARIH1 or 4EHP was normalized by the input amount of each protein and shown as a multiple compared to the control.

[0070] (result) Figure 6A shows the domain structures of Sec61β and ARIH1, respectively. ARIH1 is known to bind to 4EHP, which suppresses the recruitment of the translation initiation complex by binding to the cap structure of mRNA. ARIH1 modifies 4EHP in an E3 ligase activity-dependent manner, thereby promoting 4EHP's cap-binding ability and repressing translation. As shown in Figure 6B, increased binding of endocellular Sec61β, which binds to Flag-ARIH1 in an ER stress-dependent manner, and Derlin1 was confirmed. As shown in Figure 6C, increased binding of Sec61β, which binds to Flag-4EHP, and Derlin1 was confirmed in an ER stress-dependent manner.

[0071] Regarding the binding region, as shown in Figure 7, Sec61β was shown to bind to ARIH1 at the intrinsically disordered region (IDR) localized in the cytoplasm at its N-terminus.

[0072] Test Example 4: Investigation of suppression of ERpQC substrate accumulation in ARIH1 and 4EHP We investigated whether ARIH1 and 4EHP are involved in the translational repression of ERpQC substrates. Flag-6His-NHK QQQ Cell lysates prepared from HEK293 cells, either with or without ER stress treatment, after introducing HA and siRNA (siCtrl, siSec61β #1, siARIH1 #1, siARIH1 #2, si4EHP #1, or si4EHP #2), were placed in Western water (WB).

[0073] (result) As shown in Figure 8, similar to the knockdown of Sec61β, knockdown of ARIH1 enhanced the accumulation of SS-uncleaved NHK, an ERpQC substrate, in both of the two siRNAs. As shown in Figure 9, knockdown of 4EHP also enhanced the accumulation of SS-uncleaved NHK.

[0074] In the steady state, ARIH1 activity is negatively regulated because the C-terminal Ariadne domain masks the Ring domain, which has E3 ligase activity. Therefore, we evaluated the accumulation of SS-uncleaved NHK by returning either the ΔAri active form lacking the Ariadne domain or the ΔAri inactive form lacking the Ariadne domain and having a mutation (C357S) in the Ring2 domain to ARIH1 knockdown cells. The following were used: HA-ARIH1 ΔAri active form (pcDNA3 / GW HA-ARIH1ΔAri(1-407)) which has an HA tag attached to the N-terminus and expresses ARIH1 from the N-terminus to position 407 (CDS NM_005744.3); HA-ARIH1 ΔAri inactive form (pcDNA3 / GW HA-ARIH1ΔAri(1-407) C357S) which has an HA tag attached to the N-terminus and expresses ARIH1 from the N-terminus to position 407 (with the cysteine ​​residue at position 357 from the N-terminus replaced by a serine residue); and HA-4EHP (pcDNA3 / GW HA-4EHP) which expresses 4EHP (CDS:NM_004837.1) with an HA tag attached to the N-terminus.

[0075] Flag-6His-NHK QQQCell lysates prepared from HEK293 cells, with or without ER stress treatment, were subjected to Western blotting after introducing HA, siRNA (siCtrl, siARIH1 UTR, si4EHP #1, or siSec61β #1), and at least one of HA-ARIH1 ΔAri active form, HA-ARIH1 ΔAri inactive form, and HA-4EHP.

[0076] (result) As shown in Figure 10, in cells with ARIH1 knocked down, ERpQC substrate accumulation was suppressed in the ΔAri active form, but the suppression was weaker in the ΔAri inactive form. As shown in Figure 11, ERpQC substrate accumulation was also suppressed in cells with 4EHP knocked down when 4EHP was returned to the cells. This indicates that ARIH1 and 4EHP suppress the translation of ERpQC substrates.

[0077] Next, we investigated whether translation repression by Sec61β was mediated by ARIH1 and 4EHP. As shown in Figure 12, the enhancement of ERpQC substrates by knockdown of Sec61β was suppressed by the expression of ARIH1 ΔAri activated form, not ARIH1 ΔAri inactive form. As shown in Figure 13, the enhancement of ERpQC substrates by knockdown of Sec61β was also suppressed by overexpression of 4EHP, and a further suppressive effect was confirmed with the simultaneous expression of ARIH1 ΔAri activated form and 4EHP.

[0078] Furthermore, we investigated the importance of the IDR of Sec61β, the binding region to ARIH1. Flag-6His-NHK QQQ Cell lysates prepared from HEK293 cells, either with or without ER stress treatment, after introducing -HA, siRNA (siSec61β UTR #2), and Flag-Sec61β WT or Flag-Sec61β ΔIDR, were subjected to Western blotting (WB).

[0079] As shown in Figure 14, the enhancement of ERpQC substrates in cells with Sec61β knockdown was suppressed in Flag-Sec61β WT, but the suppressive effect was weakened in Flag-Sec61β ΔIDR. These findings indicate that Sec61β suppresses ERpQC substrate translation via ARIH1-4EHP.

[0080] Study Example 5: Examination of the physiological significance of translational repression of ERpQC substrates. It was hypothesized that ERpQC translation repression would be disrupted due to Sec61β deletion, leading to an increase in ERpQC substrates and thus placing a burden on the degradation of proteins in the cytoplasm that are normally quality-controlled. Therefore, proteasome activity was investigated. HepG2 cells were introduced with siRNA (siCtrl, siSec61β #1, siSec61β #2, or siSec61β #3) and treated with 200 nM Tg or 2 μg / mL Tun (Nacalai Tesque; 35638-74) for 16 hours. These cells were washed with PBS, lysed in CHAPS buffer (50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 0.2% CHAPS, 5 mM EDTA, 1 mM EGTA) containing a 5 μg / mL leupeptin and protease inhibitor cocktail (Nacalai Tesque; 25955-11), and the cell extracts were centrifuged. To measure the chymotrypsin-like peptidase activity of the proteasome, cell extracts were incubated at 37°C for 20 minutes with CHAPS buffer containing 100 μM Succinil-Leu-Leu-Val-Tyr-7 amino-4-methylcoumarin (Suc-LLVY-AMC, Calbiochem; 539142) and 1 mM DTT. The fluorescence of the released AMC was measured using a multimode detector DTX800 (Beckman Coulter) at an excitation wavelength of 365 nm and an emission wavelength of 465 nm. Fluorescence measurements were normalized by cell viability under each condition. Cell viability was quantified using a Cell Counting Kit-8 (Dojindo Laboratories) and a multimode detector DTX800 according to the manufacturer's protocol. Proteasome activity is expressed as a multiple compared to cells introduced with siCtrl.

[0081] (result) Figures 15A and 15B show proteasome activity in cells treated with Tg or Tun, respectively. Both Tg and Tun ER stressors reduced proteasome activity upon knockdown of Sec61β.

[0082] Next, the degradation rate of CL1, a cytoplasmic protein degradation model substrate, was investigated using pulse-chase experiments. This involved siRNA (siCtrl or siSec61β #1) and Venus-CL1-Flag or Flag-6His-NHK. QQQ A pulse chase experiment was performed on HEK293 cells introduced with -HA. The relative radiation dose is expressed as a multiple of the relative radiation dose at time 0.

[0083] (result) As shown in Figure 16, knockdown of Sec61β suppressed the degradation of CL1. On the other hand, as shown in Figure 17, although the amount of SS-uncleaved NHK, an ERpQC substrate, synthesized increased with Sec61β knockdown, the degradation rate was not affected. These results suggest that when ERpQC substrates are excessively generated in the cytoplasm by knockdown of Sec61β, the degradation of ERpQC substrates occurs preferentially, loading the proteasome, reducing proteasome activity, and suppressing the degradation of proteins localized in the cytoplasm.

[0084] Test Example 6: Investigation of aggregated protein accumulation by knockdown of Sec61β To investigate the quality of intracellular proteins, proteostat analysis was performed to detect aggregated proteins (aggresome) in HepG2 cells. HepG2 cells were analyzed by flow cytometry using the ProteoStat Aggresome Detection Kit (Enzo Life Sciences; ENZ-51035-K100). HepG2 cells were transfected with siRNA (siCtrl or siSec61β #1) using Lipofectamine RNAiMAX, or with siRNA and Flag-Sec61β WT or Flag-Sec61β ΔIDR using Lipofectamine 3000, and cultured on a 10cm plate for 3 days. After stimulating with 50nM Tg or DMSO for 4 hours, the cells were washed with PBS, triedpsinized, collected in 15mL tubes, washed with PBS, and pelleted by centrifugation. Cells suspended in 150μL of PBS were gradually added to 1mL of 4% paraformaldehyde while vortexing, and fixed at room temperature for 20 minutes. After washing with PBS by centrifugation, the cell pellet was suspended in 150 μL of PBS and gradually added to 1 mL of permeabilization buffer (0.5% Triton X-100 and 3 mM EDTA) while vortexing, and incubated on ice for 20 minutes. After washing three times with PBS, the cells were resuspended in 500 μL of PBS and counted under a microscope. For quantification by flow cytometry, 1 × 10⁶ cells per mL of PBS were used. 6Cells prepared to the specified state were incubated for 30 minutes in the dark at room temperature with ProteoStat dye (1:5,000-1:10,000 dilution) and APC anti-DYKDDDDK Tag antibody (1:450, Biolegend; 637308), or without the APC anti-DYKDDDDK Tag antibody. After washing with PBS, the cells were filtered through a cell strainer cap (BD Biosciences; 352235) and analyzed using a FACSVerse flow cytometer (BD Biosciences) or a CytoFLEX flow cytometer (Beckman Coulter). Flow cytometry data were analyzed using FlowJo software (Treestar) or CytExpert software (Beckman Coulter).

[0085] (result) As shown in Figure 18, knockdown of Sec61 significantly increased aggregated proteins in Tg-stimulated cells (see figure below). When we investigated whether the accumulation of aggregated proteins due to Sec61β knockdown was suppressed by Sec61β expression, as shown in Figure 19, expression of Sec61β ΔIDR, which lacks the IDR (a binding site to ARIH1), showed a lower inhibitory effect on accumulation compared to the wild type (WT). Therefore, it became clear that increasing ERpQC substrates due to Sec61β knockdown leads to the generation of abnormal aggregated proteins in the cytoplasm. Sec61β is thought to maintain the quality of cytoplasmic proteins through its binding to ARIH1.

[0086] Example 1: Screening system for translation regulators of ERpQC substrates As an example of a screening system for translation modulators of ERpQC substrates, we investigated the Alpha assay (PerkinElmer).

[0087] Wild-type HEK293 cells (WT) or Flag-6His-NHK QQQHEK293 cells (clone N1) stably expressing -HA were seeded in 100 μL of medium in a 96-well plate. After 20 hours, 100 nM Tg and 200 nM MG132 were added and incubated for 14 hours (ER stress treatment). 25 μL of AlphaLISA® Lysis Buffer (5×) (Levity Japan; AL003C) was added and the cells were solubilized at 4°C for 20 minutes. 20 μL of the resulting sample was transferred to a 96-well plate for measurement. 10 μL of anti-Flag antibody donor beads (Levity Japan; AS103D) and 10 μL of anti-HA antibody acceptor beads (Levity Japan; AL170C) were added in the dark and reacted at 4°C for 2 hours. Fluorescence intensity was measured using a Nivo multimode microplate reader (PerkinElmer Japan).

[0088] (result) In the Alpha assay, when the photosensitizer in the donor beads is energetically excited by a 680nm laser, the surrounding oxygen is converted to singlet oxygen. This singlet oxygen reacts with the thioxine derivative in the adjacent acceptor beads, initiating a chemiluminescent reaction, causing the fluorescent substance in the acceptor beads to emit fluorescence (615nm). (Non-cleavage type NHK) QQQ Fluorescence is emitted when anti-Flag antibody donor beads and anti-HA antibody acceptor beads bind to Flag and HA, respectively, thus the non-cleaved NHK is an ERpQC substrate. QQQ As the value increases, the fluorescence intensity increases. As shown in Figure 20, the fluorescence intensity of N1 treated with ER stress increased compared to N1 treated with WT and N1 treated without ER stress. This screening system allows us to evaluate whether the test substance suppresses or promotes the accumulation of ERpQC substrates based on the fluorescence intensity of N1 treated with ER stress in the presence of the test substance.

[0089] HEK293 cells (WT) or HEK293 cells (clone #6) stably expressing both ARIH1 with an N-terminal HA tag (HA-ARIH1) and Sec61β with an N-terminal Flag tag (Flag-Sec61β WT) were seeded in 100 μL of medium in a 96-well plate. After 20 hours, 100 nM Tg and 200 nM MG132 were added and incubated for 14 hours. 25 μL of AlphaLISA Lysis Buffer (5×) was added and solubilized at 4°C for 20 minutes. 20 μL of the sample was transferred to a 96-well plate for measurement. 10 μL of anti-Flag antibody donor beads and 10 μL of anti-HA antibody acceptor beads were added in the dark and reacted at 4°C for 2 hours. Fluorescence intensity was measured using a Nivo multimode microplate reader.

[0090] (result) As shown in Figure 21, the fluorescence intensity at #6 increased compared to WT. This screening system allows us to evaluate whether the test substance inhibits or promotes the binding of Sec61β to ARIH1, which contributes to the suppression of ERpQC substrate accumulation, based on the fluorescence intensity at N1 after ER stress treatment in the presence of the test substance.

[0091] The embodiments described above are for illustrative purposes only and do not limit the scope of the present invention. That is, the scope of the present invention is defined not by the embodiments, but by the claims. Various modifications made within the scope of the claims and equivalent inventive meaning are considered to be within the scope of the present invention. [Industrial applicability]

[0092] This invention is useful in the development of pharmaceuticals. [Explanation of symbols]

[0093] 1,2 Tags 3,4 beads

Claims

1. The process includes a binding evaluation step of selecting a test substance that promotes or inhibits the binding of Sec61β and ARIH1. A screening method for translation regulators of substrates that are degraded by translation-coupled proteolytic degradation mechanisms under endoplasmic reticulum stress.

2. The aforementioned coupling evaluation step is, An exposure step of exposing cells expressing Sec61β with a first tag attached to its N-terminus and ARIH1 with a second tag different from the first tag attached to its N-terminus to the test substance, A processing step that puts the aforementioned cells into an endoplasmic reticulum stress state, A detection step involves detecting luminescence based on chemical energy transfer that occurs when the distance between a first bead having an antibody bound to the first tag and a second bead having an antibody bound to the second tag is close to that of a control lysate obtained by solubilizing cells that have not been exposed to the test substance and have been subjected to endoplasmic reticulum stress, and a lysate obtained by solubilizing cells that have been exposed to the test substance and have been subjected to endoplasmic reticulum stress, A selection step of selecting the test substance based on a comparison of the intensity of the luminescence in the control solution and the intensity of the luminescence in the solution, The screening method according to claim 1, including the following:

3. The aforementioned coupling evaluation step is, A processing step of putting cells expressing Sec61β with a first tag attached to its N-terminus and ARIH1 with a second tag different from the first tag attached to its N-terminus into an endoplasmic reticulum stress state, A mixing step to obtain a reaction solution containing a lysate obtained by solubilizing the cells that have been subjected to endoplasmic reticulum stress and the test substance, A detection step involves detecting luminescence based on chemical energy transfer that occurs when the distance between a first bead having an antibody bound to the first tag and a second bead having an antibody bound to the second tag is close to that of a control lysis solution and the reaction solution, which do not contain the test substance and in which the cells have been subjected to endoplasmic reticulum stress have been solubilized, and A selection step of selecting the test substance based on a comparison of the intensity of the luminescence in the control solution and the intensity of the luminescence in the solution, The screening method according to claim 1, including the following:

4. Prior to the binding evaluation step, the method further includes an accumulation evaluation step of selecting a test substance that suppresses or promotes the accumulation of the substrate in a cell under endoplasmic reticulum stress, In the aforementioned integration evaluation step, From among the test substances that suppress the accumulation of the substrate selected in the accumulation evaluation step, a test substance that promotes the binding of Sec61β and ARIH1 is selected. From among the test substances that promote the accumulation of the substrate selected in the accumulation evaluation step, a test substance that inhibits the binding of Sec61β and ARIH1 is selected. The screening method according to any one of claims 1 to 3.

5. In the accumulation evaluation step, null Hong Kong or transthyretin of α1-antitrypsin is expressed in the cells as the substrate. Select a test substance that suppresses or promotes the accumulation of the substrate whose signal sequence is not cleaved. The screening method according to claim 4.

6. The method further includes, after the binding evaluation step, an accumulation evaluation step of selecting a test substance that suppresses or promotes the accumulation of the substrate in a cell under endoplasmic reticulum stress, In the aforementioned accumulation evaluation step, From among the test substances that promote the binding of Sec61β and ARIH1 selected in the binding evaluation step, a test substance that suppresses the accumulation of the substrate is selected. From among the test substances that inhibit the binding of Sec61β and ARIH1 selected in the binding evaluation step, a test substance that promotes the accumulation of the substrate is selected. The screening method according to any one of claims 1 to 3.

7. In the accumulation evaluation step, null Hong Kong or transthyretin of α1-antitrypsin is expressed in the cells as the substrate. Select a test substance that suppresses or promotes the accumulation of the substrate whose signal sequence is not cleaved. The screening method according to claim 6.

8. The present invention comprises cells expressing Sec61β with a first tag attached to its N-terminus and ARIH1 with a second tag different from the first tag attached to its N-terminus. A screening kit for translational regulators of substrates that are degraded by translation-coupled proteolytic degradation mechanisms under endoplasmic reticulum stress.

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