Method for identifying Xkr4 polypeptide, XRCC4 polypeptide, and genes corresponding to a desired phenotype

Abstract

To provide a method to identify molecules involved in lipid scrambling activity.SOLUTION: The present invention provides an Xkr4 polypeptide, which is (A) a polypeptide comprising a specific amino acid sequence, or (B) a polypeptide, comprising an amino acid sequence with deletion, substitution, or addition or their combination in the amino acid sequence of the (A) polypeptide, and exhibiting at least 80% sequential identity to the amino acid sequence of the (A) polypeptide, and demonstrating lipid scramble activity in a cell membrane without interaction with an XRCC4 polypeptide.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This disclosure relates to an Xkr4 polypeptide or XRCC4 polypeptide, a polynucleotide comprising an encoding nucleotide sequence, an expression vector comprising the polynucleotide, or a host cell comprising the polynucleotide or expression vector. This disclosure also relates to a method for screening substances that modulate (e.g., inhibit or induce) the lipid scrambling activity of Xkr4. This disclosure also relates to a composition for activating Xkr4. This disclosure also relates to a kit for screening substances that inhibit the lipid scrambling activity of Xkr4. This disclosure further relates to a method for identifying a gene corresponding to a desired phenotype. [Background technology]

[0002] During brain development, numerous cell deaths are observed, most of which are apoptosis. Apoptosis is a type of cell death characterized by nuclear fragmentation, cytoplasmic aggregation, and schism. Compared to other types of cell death such as necrosis, apoptosis results in the rapid removal of cells from the tissue. Deficiencies in apoptosis during brain development may be associated with neurodevelopmental or behavioral disorders, including attention deficit hyperactivity disorder (ADHD). Excessive apoptosis may be associated with neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), and amyotrophic lateral sclerosis (ALS).

[0003] The cell membrane of eukaryotes is composed of a phospholipid bilayer, with phospholipids distributed asymmetrically. This asymmetric distribution of phospholipids is thought to be maintained by flippase, an enzyme that transports aminophospholipids such as phosphatidylserine to the inside (cytoplasmic) side of the cell membrane. When a cell is stimulated by apoptosis, the asymmetric distribution of phospholipids breaks down, and phosphatidylserine, which was on the inside of the cell membrane, is exposed to the cell surface and phagocytosed by phagocytic cells. The protein Xkr8, which is involved in the exposure of phosphatidylserine due to apoptosis, has been identified (Patent Document 1). [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] WO 2014 / 077279 [Overview of the project] [Problems that the invention aims to solve]

[0005] One objective of this disclosure is to identify molecules involved in lipid scrambling activity. One objective of this disclosure is to provide a polynucleotide comprising a nucleotide sequence encoding a molecule involved in lipid scrambling activity, an expression vector comprising the polynucleotide, and a host cell comprising the polynucleotide or expression vector. One objective of this disclosure is to provide a method for screening substances that modulate (e.g., inhibit or induce) lipid scrambling activity. One objective of this disclosure is to provide a composition for activating Xkr4. One objective of this disclosure is to provide a kit for screening substances that inhibit lipid scrambling activity. One objective of this disclosure is to provide a method for identifying genes corresponding to a desired phenotype. [Means for solving the problem]

[0006] This disclosure provides the following inventions. [Section 1] Xkr4 polypeptide, (A) A polypeptide consisting of any of the amino acid sequences described in SEQ ID NOs. 3-5 and 37-39; or (B) A polypeptide having an amino acid sequence in the amino acid sequence of the polypeptide of (A) that includes deletions, substitutions, additions, or combinations thereof, exhibits at least 80% sequence identity with the amino acid sequence of (A), and can exhibit lipid scrambling activity in the cell membrane without interaction with the XRCC4 polypeptide. This is the Xkr4 polypeptide. [Section 2] XRCC4 polypeptide, (a) a polypeptide consisting of any of the amino acid sequences described in SEQ ID NOs. 7-12 and 42; or (b) A polypeptide having an amino acid sequence in the amino acid sequence of the polypeptide of (a) that includes deletions, substitutions, additions, or combinations thereof, exhibits at least 80% sequence identity with the amino acid sequence of (a), and can induce lipid scrambling activity in the cell membrane through interaction with a C-terminal cleaved Xkr4 polypeptide. This is the XRCC4 polypeptide. [Section 3] The XRCC4 polypeptide described in item 2, comprising arginine at position 270, according to the amino acid residue numbering of the amino acid sequence described in SEQ ID NO: 6. [Section 4] The XRCC4 polypeptide described in item 2 or 3, which does not contain methionine at position 265, according to the amino acid residue numbering of the amino acid sequence described in SEQ ID NO: 6. [Section 5] A polynucleotide comprising a nucleotide sequence encoding the Xkr4 polypeptide described in item 1, or the XRCC4 polypeptide described in any one of items 2 to 4. [Section 6] An expression vector containing the polynucleotides described in item 5. [Section 7] A host cell containing the polynucleotide described in item 5, or a host cell containing the expression vector described in item 6. [Section 8] A composition for activating Xkr4, comprising at least one selected from the group consisting of an XRCC4 polypeptide as described in any one of items 2 to 4; a polynucleotide comprising a nucleotide sequence encoding the XRCC4 polypeptide; and an expression vector comprising the polynucleotide. [Section 9] The composition according to item 8, for the treatment or prevention of a condition or disease in which the suppression of apoptosis is involved.

[0007] [Section 10] A method for screening a substance that regulates the lipid scrambling activity of Xkr4, comprising the following steps: (1) contacting a cell expressing the Xkr4 polypeptide according to claim 1 with a candidate substance that may regulate the lipid scrambling activity of Xkr4; (2) measuring the lipid scrambling activity in the cell contacted with the candidate substance; and (3) when the lipid scrambling activity is lower or higher compared to the lipid scrambling activity in the cell not contacted with the candidate substance, selecting the candidate substance as a substance that regulates the lipid scrambling activity of Xkr4; A screening method comprising the above steps. [Claim 11] When the substance that regulates the lipid scrambling activity of Xkr4 has a lipid scrambling activity lower than that in the cell not contacted with the candidate substance in the comparison, the substance for regulation is a drug candidate for the treatment or prevention of a condition or disease involving the promotion of apoptosis. The screening method according to claim 10. [Claim 12] A method for screening a substance that induces the lipid scrambling activity of Xkr4, comprising the following steps: (1) A C-terminal truncated Xkr4 polypeptide, (α) a C-terminal truncated Xkr4 polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2 or 36, or (β) having an amino acid sequence containing deletions, substitutions, additions, or combinations thereof in the amino acid sequence of the (α) polypeptide, and showing at least 80% sequence identity with the amino acid sequence of (α), and contacting a cell expressing a C-terminal truncated Xkr4 polypeptide that can exhibit lipid scrambling activity in the cell membrane by interaction with the XRCC4 polypeptide with a candidate substance that may induce the lipid scrambling activity of Xkr4; (2) measuring the lipid scrambling activity in the cell contacted with the candidate substance; and (3) If the lipid scrambling activity is higher than that in the cells that have not been exposed to the candidate substance, the candidate substance is selected as a substance that induces the lipid scrambling activity of Xkr4; A screening method that includes this.

[0008] [Section 13] A method for screening substances that inhibit the lipid scrambling activity of Xkr4, comprising the following steps: (1) Contacting cells expressing a C-terminally cleaved Xkr4 polypeptide, into which the XRCC4 polypeptide described in any one of items 2 to 4 has been introduced, with a candidate substance that may inhibit the lipid scrambling activity of Xkr4; (2) Measuring the lipid scrambling activity in the cells; and (3) If the lipid scrambling activity is lower than that in the cells not in contact with the candidate substance, the candidate substance is selected as a substance that inhibits the lipid scrambling activity of Xkr4; A screening method that includes this. [Section 14] A method for screening substances that inhibit the lipid scrambling activity of Xkr4, comprising the following steps: (1) Contacting the XRCC4 polypeptide described in any one of items 2 to 4 with the C-terminally cleaved Xkr4 polypeptide in the presence of a candidate substance that may inhibit the lipid scrambling activity of Xkr4; (2) Measuring the binding between the XRCC4 polypeptide and the C-terminally cleaved Xkr4 polypeptide; and (3) If the binding between the XRCC4 polypeptide and the C-terminally cleaved Xkr4 polypeptide is lower than the binding when the XRCC4 polypeptide and the C-terminally cleaved Xkr4 polypeptide are brought into contact in the absence of the candidate substance, the candidate substance is selected as a substance that inhibits the lipid scrambling activity of Xkr4; A screening method that includes this. [Section 15] The screening method according to any one of items 10 to 14, wherein the lipid scrambling activity is the uptake activity of phospholipids or glycolipids, or the exposure activity of phospholipids located on the inner side of the cell membrane.

[0009] [Section 16] A kit for screening substances that inhibit the lipid scrambling activity of Xkr4 as described in item 14 or 15, A kit comprising: an XRCC4 polypeptide as described in any one of items 2 to 4; a polynucleotide comprising a nucleotide sequence encoding the XRCC4 polypeptide; an expression vector comprising the polynucleotide; and at least one selected from the group consisting of a host cell comprising the polynucleotide or a host cell comprising the expression vector. [Section 17] A method for identifying genes corresponding to a target phenotype, (a) Prepare a guide RNA library containing guide RNAs that include at least one guide sequence corresponding to the nucleotide sequences of multiple genes in the cell's genomic DNA; (b) Obtain a population of cells into which the guide RNA library has been introduced; (c) Selecting cells from the aforementioned cell population based on the desired phenotype; (d) Recovering genomic DNA from selected cells; (e) Until steps (b) to (d) are performed a predetermined number of times, prepare a new guide RNA library from the recovered genomic DNA and perform steps (b) to (d) using the new guide RNA library; (f) Determining the guide sequence in the recovered genomic DNA when steps (b) to (d) have been performed a predetermined number of times; and (g) The step of identifying the gene corresponding to the determined guide sequence as the gene corresponding to the desired phenotype, A method for specifying the number of predetermined occurrences, wherein the number of occurrences is at least two. [Brief explanation of the drawing]

[0010] [Figure 1] Figure 1(a) is a graph showing the lipid scramble assay in parental PLB cells. Figure 1(b) is a graph showing the lipid scramble assay in PLB cells expressing Xkr4WT. Figure 1(c) is a graph showing the lipid scramble assay in PLB cells expressing Xkr4ΔC. Figure 1(d) is a graph showing the results of the fluorescent annexin V staining assay in parental PLB cells. Figure 1(e) is a graph showing the results of the fluorescent annexin V staining assay in PLB cells expressing Xkr4WT. Figure 1(f) is a graph showing the results of the fluorescent annexin V staining assay in PLB cells expressing Xkr4ΔC. Light gray indicates no STS treatment (control), and dark gray indicates STS treatment (apoptotic stimulation). Bars indicate regions positive for PC uptake and PS exposure. [Figure 2] The top row of Figure 2 shows micrographs of living PLB cells expressing Xkr4WT-GFP. The bottom row of Figure 2 shows confocal micrographs of living PLB cells expressing Xkr4ΔC-GFP. The left column of Figure 2 shows micrographs showing fluorescence derived from GFP. The middle column of Figure 2 shows micrographs showing differential interference contrast (DIC). The right side of Figure 2 shows a composite image of the fluorescence image and the DIC image. The scale bar represents 10 μm.

[0011] [Figure 3] Figure 3(a) is a graph showing the results of a lipid scramble assay in Xkr4WT-expressing cells in the absence of a caspase inhibitor. Figure 3(b) is a graph showing the results of a lipid scramble assay in Xkr4WT-expressing cells in the presence of a caspase inhibitor. Figure 3(c) is a graph showing the results of a lipid scramble assay in Xkr4Δ-expressing cells in the absence of a caspase inhibitor. Figure 3(d) is a graph showing the results of a lipid scramble assay in Xkr4ΔC-expressing cells in the presence of a caspase inhibitor. Light gray indicates no STS treatment (control), and dark gray indicates STS treatment (apoptotic stimulation). Bars indicate regions positive for PC uptake. [Figure 4] Figure 4(a) shows Western blotting after BN-PAGE of cell membrane lysates from Xkr4WT or Xkr4ΔC expressing cells. Black arrowheads indicate Xkr4 monomers, and gray arrowheads indicate Xkr4 dimers. The upper panel of Figure 4(b) shows Western blotting after SDS-PAGE of cell membrane lysates from Xkr4WT or Xkr4ΔC expressing cells. The lower panel of Figure 4(b) shows a CBB-stained PVDF membrane as a loading control.

[0012] [Figure 5] Figure 5(a) is a schematic diagram showing a strategy for establishing cells that constitutively exhibit lipid scrambling activity using Ba / F3 cells (BDKO) lacking two ubiquitous scramblases, Xkr8 and TMEM16F. Figure 5(b) is a series of graphs showing the results of a strategy for selecting cells that constitutively take up PC from a population of BDKO cells expressing Xkr4ΔC. [Figure 6] Figure 6(a) is a graph showing PC uptake in a transfectant (parental) expressing Xkr4ΔC. Figure 6(b) is a graph showing PC uptake in PC6 cells sorted six times. Figure 6(c) is a graph showing PC uptake in PC cells expressing sgRNA for Xkr4 (PC6+sgXkr4). The upper part of Figure 6(d) is a photograph showing Western blotting for detecting Xkr4. The lower part of Figure 6(d) is a photograph showing a CBB-stained PVDF membrane as a loading control. [Figure 7] Figure 7(a) is a graph showing the results of the lipid scramble assay in 1LPC0. Figure 7(b) is a graph showing the results of the lipid scramble assay in 1LPC3. Figure 7(c) is a graph showing the results of the lipid scramble assay in 2LPC0. Figure 7(d) is a graph showing the results of the lipid scramble assay in 2LPC1. The bars indicate regions where PC uptake is positive.

[0013] [Figure 8] Figure 8(a) is a graph showing the results of a PC uptake assay in BDKO cells expressing full-length Xkr4 or various Xkr4 variants. Figure 8(b) is a graph showing the results of a fluorescent annexin V staining assay. The bars indicate regions where PS exposure is positive. [Figure 9] Figure 9(a) upper panel shows Western blotting images of cell lysates of transfectants expressing an Xkr4 variant after BN-PAGE. Black arrowheads indicate Xkr4 monomers, and gray arrowheads indicate Xkr4 dimers. Figure 9(a) middle panel shows Western blotting images of cell lysates of transfectants expressing a predetermined Xkr4 variant after SDS-PAGE. Figure 9(a) lower panel shows a CBB-stained PVDF membrane as a loading control. Figure 9(b) upper panel shows Western blotting images of cell lysates of transfectants expressing a predetermined Xkr4 variant after SDS-PAGE. Figure 9(b) lower panel shows a CBB-stained PVDF membrane as a loading control.

[0014] [Figure 10] Figure 10(a) is a graph showing PC uptake under conditions where neither magnesium nor calcium ions are present extracellularly. Figure 10(b) is a graph showing PC uptake under conditions where magnesium ions are present extracellularly but calcium is not. Figure 10(c) is a graph showing PC uptake under conditions where magnesium ions are not present extracellularly but calcium ions are present. Figure 10(d) is a graph showing PC uptake under conditions where both magnesium and calcium ions are present extracellularly. In Figures 10(a) to (d), the bars indicate regions where PC uptake is positive. Figure 10(e) is a graph showing PC uptake dependent on extracellular calcium concentration. [Figure 11]Figure 11(a) is a graph showing the effect of calcium ionophores on PC uptake. Figure 11(b) is a graph showing the effect of extracellular calcium and calcium ionophores on Fluo4-AM. Figure 11(c) is a series of graphs showing PC uptake activity in transfectants expressing Xkr4WT or Xkr4ΔC. In Figure 11(c), light gray indicates no STS treatment, dark gray indicates STS treatment, and bars indicate regions where PC uptake is positive. [Figure 12] Figure 12 is a flowchart of the revival screening process.

[0015] [Figure 13] Figure 13(a) shows the sequence analysis of established CAD KO PLB cells. Underlined sequences in CAD+ / + indicate sgRNA target sequences. Dashed regions in CAD- / - indicate nucleotides (7 bps) deleted in both alleles. The upper panel of Figure 13(b) shows photographs of CAD Western blotting. Whole cell lysates from parental cells and CAD KO PLB cells were applied to SDS-PAGE and then to Western blotting using an anti-CAD antibody. The lower panel of Figure 13(b) shows photographs of CBB-stained PVDF membranes as a loading control. Figure 13(c) shows photographs of a DNA fragmentation assay. Parental cells and CAD KO PLB cells were stimulated with STS for 4 hours, solubilized with a solubilizing buffer, and applied to an agarose gel. [Figure 14]Figure 14(a) is a graph showing the flow cytometry results for a cell population (sgPC0: unsorted) containing cells that were positive for Xkr4ΔC-RFP and negative for PC uptake in the revival screening. Figure 14(b) is a graph showing the flow cytometry results for a cell population (sgPC2: sorted twice) containing cells that were positive for Xkr4ΔC-RFP and negative for PC uptake in the revival screening. Figure 14(c) is a graph showing the flow cytometry results for a cell population (sgPC3: sorted three times) containing cells that were positive for Xkr4ΔC-RFP and negative for PC uptake in the revival screening. The bars indicate regions where PC uptake is negative.

[0016] [Figure 15] Figure 15 is a graph showing the NGS analysis of all sgRNAs integrated into the genomic DNA of the sgPC4 cell population. The X-axis shows a list of genes from which more than three of the six target sgRNAs were recovered. The Y-axis shows the total number of different sgRNAs corresponding to the same gene in the reads. The terms (CYCS, APAF1) are indicated near the dots corresponding to cytochrome C (CYCS) and APAF1. [Figure 16] Figure 16(a) is a graph showing PC uptake by flow cytometry in an sgPC6 cell population. Figure 16(b) is a graph showing caspase-3 activity by flow cytometry in an sgPC6 cell population. The region enclosed by the solid line indicates the sorted region. [Figure 17] Figure 17 is a bar graph showing the ratio of the total number of target sgRNA reads in sgPC7 cells to the total number of target sgRNA reads in sgPC4 cells.

[0017] [Figure 18]Figure 18(a) upper panel shows a graph of caspase-3 activity in PLB cells. Figure 18(a) lower panel shows a graph of PC uptake in PLB cells. Figure 18(b) upper panel shows a graph of caspase-3 activity in PLB cells using sgRNA for XRCC4. Figure 18(b) lower panel shows a graph of PC uptake in PLB cells using sgRNA for XRCC4. Figure 18(c) upper panel shows a graph of caspase-3 activity in PLB cells using sgRNA for CYCS. Figure 18(c) lower panel shows a graph of PC uptake in PLB cells using sgRNA for CYCS. Figure 18(d) upper panel shows a graph of caspase-3 activity in PLB cells using sgRNA for APAF1. Figure 18(d) lower panel shows a graph of PC uptake in PLB cells using sgRNA for APAF1. The bars in the upper panels of Figures 18(a) to (d) indicate regions positive for caspase-3 activity. In Figure 18(a) to (d), the bars in the lower section indicate regions that are positive for PC uptake.

[0018] [Figure 19]Figure 19(a) shows the sequence analysis of XRCC4 knockout PLB cells. Underlined nucleotide sequences in XRCC4+ / + indicate sgRNA target sequences for XRCC4. Dashed lines in XRCC4- / - indicate deleted nucleotide (4 bp) sequences, and underlined nucleotides indicate added nucleotides (1 bp). Figure 19(b) shows photographs of Western blotting of CAD, V5, and XRCC4. Whole cell lysates were prepared from parental cells, CAD KO (CAD- / -) PLB cells, CAD / XRCC4 double KO (CAD- / - XRCC4- / -) PLB cells, and CAD- / - XRCC4- / - PLB cells expressing V5-Xkr4ΔC-FLAG. The prepared whole cell lysates were subjected to SDS-PAGE, followed by Western blotting using anti-V5 antibody, anti-CAD antibody, and anti-XRCC4 antibody. Figure 19(b) bottom panel shows a photograph of a CBB-stained PVDF membrane used as a loading control. Figure 19(c) top panel shows a photograph of Western blotting for XRCC4. Figure 19(c) middle panel shows a photograph of Western blotting for activated caspase 3. PLB cells expressing XRCC4 WT or XRCC4 2DA fused to the C-terminus with RFP were stimulated with apoptosis using STS. Whole cell lysates were prepared from these cells, subjected to SDS-PAGE, and then Western blotting was performed using a predetermined antibody. Figure 19(c) bottom panel shows a photograph of a CBB-stained PVDF membrane used as a loading control.

[0019] [Figure 20]Figure 20(a) is a graph showing PC uptake in XRCC4- / - cells. Figure 20(b) is a graph showing PC uptake in XRCC4- / - cells expressing XRCC4 WT. Figure 20(c) is a graph showing PC uptake in XRCC4- / - cells expressing XRCC4 2DA. Light gray indicates the control, and dark gray indicates STS treatment. The bars indicate regions where PC uptake is positive. Figure 20(d) is a graph showing PC uptake in Xkr4ΔC Q332E-expressing cells. Figure 20(e) is a graph showing PC uptake in Xkr4ΔC Q332E-expressing cells into which sgRNA for XRCC4 has been introduced. Figure 20(f) is a graph showing PC uptake in Xkr4ΔC Q332E-expressing cells into which sgRNA for CYCS has been introduced. The light gray area indicates PC uptake in Xkr4ΔCQ332E-expressing cells that have not been introduced with sgRNA. [Figure 21] Figure 21 (top panel) shows a photograph of Western blotting of V5 (Xkrs). Figure 21 (middle panel) shows a photograph of Western blotting of RFP (XRCC4). Total cell lysates from XRCC4 KO PLB cells expressing Xkr tagged with V5 at the N-terminus, co-expressing or not expressing XRCC4 WT-RFP, were applied to SDS-PAGE and Western blotting. Figure 21 (bottom panel) shows a photograph of a CBB-stained PVDF membrane as a loading control.

[0020] [Figure 22]Figure 22(a) is a graph showing PC uptake in XRCC4 KO PLB cells expressing Xkr4. Figure 22(b) is a graph showing PC uptake in XRCC4 KO PLB cells expressing Xkr8. Figure 22(c) is a graph showing PC uptake in XRCC4 KO PLB cells expressing Xkr9. Figure 22(d) is a graph showing PC uptake in XRCC4 KO PLB cells co-expressing Xkr4 and XCCR4 WT. Figure 22(e) is a graph showing PC uptake in XRCC4 KO PLB cells co-expressing Xkr8 and XCCR4 WT. Figure 22(f) is a graph showing PC uptake in XRCC4 KO PLB cells co-expressing Xkr9 and XCCR4 WT. In Figures 22(a) to (f), light gray indicates no STS treatment (control), dark gray indicates STS treatment (apoptotic stimulation), and bars indicate regions where PC uptake is positive. Figure 22(g) is a graph showing caspase-3 activity in XRCC4 KO PLB cells expressing Xkr4. Figure 22(h) is a graph showing caspase-3 activity in XRCC4 KO PLB cells expressing Xkr8. Figure 22(i) is a graph showing caspase-3 activity in XRCC4 KO PLB cells expressing Xkr9. Figure 22(j) is a graph showing caspase-3 activity in XRCC4 KO PLB cells co-expressing Xkr4 and XRCC4. Figure 22(k) is a graph showing caspase-3 activity in XRCC4 KO PLB cells co-expressing Xkr8 and XRCC4. Figure 22(l) is a graph showing caspase-3 activity in XRCC4 KO PLB cells co-expressing Xkr9 and XRCC4. In Figures 22(g) to (l), light gray indicates no STS treatment (control), dark gray indicates STS treatment (apoptotic stimulation), and bars indicate regions positive for caspase-3 activity.

[0021] [Figure 23]Figure 23(a) is a micrograph of a transfectant expressing XRCC4 WT (XRCC4 WT-RFP) tagged with RFP at the C-terminus. Figure 23(b) is a micrograph of a transfectant expressing XRCC4 WT (RFP-XRCC4 WT) tagged with RFP at the N-terminus. Figure 23(c) is a micrograph of a transfectant expressing XRCC4 2DA (XRCC4 2DA-RFP) tagged with RFP at the C-terminus. Figure 23(d) is a micrograph of a transfectant expressing XRCC4 2DA (RFP-XRCC4 2DA) tagged with RFP at the N-terminus. Arrowheads indicate cytoplasmic XRCC4. Scale bar indicates 10 μm.

[0022] [Figure 24] Figure 24(a) upper panel shows photographs of Western blotting of RFP-XRCC4 WT or 2DA. XRCC4 KO PLB cells expressing XRCC4 WT fused with V5-Xkr4ΔC-FLAG and RFP at the N-terminus, and XRCC4 KO PLB cells expressing caspase-uncleaved XRCC4 2DA fused with V5-Xkr4ΔC-FLAG and RFP at the N-terminus were stimulated with apoptosis using STS. Total cell lysates were prepared from these cells, subjected to SDS-PAGE, and then Western blotting with an anti-RFP antibody. Figure 24(a) lower panel shows photographs of CBB-stained PVDF membranes as a loading control. Figure 24(b) is a graph showing the PC uptake activity of XRCC4 KO PLB cells expressing V5-Xkr4ΔC-FLAG and N-terminal RFP-fused XRCC4 WT. Figure 24(c) is a graph showing the PC uptake activity of XRCC4 KO PLB cells expressing V5-Xkr4ΔC-FLAG and N-terminal RFP-fused XRCC4 2DA. In Figures 24(b) and (c), light gray indicates no STS treatment (control), dark gray indicates STS treatment (apoptotic stimulation), and bars indicate regions where PC uptake is positive. [Figure 25]Figure 25 is a schematic diagram of the XRCC4 domain. XLF indicates the XLF binding domain. Dimer indicates the dimerization domain. DNA Lig IV indicates the DNA Lig IV binding domain. The area between amino acids 265 and 266 is the caspase recognition site. Amino acids 270-275 represent the nuclear localization signal (NLS) region.

[0023] [Figure 26]Figure 26(a) is a graph showing PC uptake in XRCC4 KO PLB cells (XRCC4- / -). Figure 26(b) is a graph showing PC uptake in XRCC4 KO PLB cells expressing XRCC4 WT (1-336). Figure 26(c) is a graph showing PC uptake in XRCC4 KO PLB cells expressing XRCC4 (116-336) with a partial deletion of the N-terminus. Figure 26(d) is a graph showing PC uptake in XRCC4 KO PLB cells expressing XRCC4 (156-336) with a partial deletion of the N-terminus. Figure 26(e) is a graph showing PC uptake in XRCC4 KO PLB cells expressing XRCC4 (204-336) with a partial deletion of the N-terminus. Figure 26(f) is a graph showing PC uptake in XRCC4 KO PLB cells expressing XRCC4(248-336) with a partial deletion of the N-terminus. Figure 26(g) is a graph showing PC uptake in XRCC4 KO PLB cells expressing XRCC4(256-336) with a partial deletion of the N-terminus. Figure 26(h) is a graph showing PC uptake in XRCC4 KO PLB cells expressing XRCC4(226-336) (also called "ΔN") with a partial deletion of the N-terminus. Figure 26(i) is a graph showing PC uptake in XRCC4 KO PLB cells expressing XRCC4(1-305) with a partial deletion of the C-terminus. Figure 26(j) is a graph showing PC uptake in XRCC4 KO PLB cells expressing XRCC4(1-285) with a partial deletion of the C-terminus. Figure 26(k) is a graph showing PC uptake in XRCC4 KO PLB cells expressing XRCC4(1-265) (also known as "ΔC"), which has a partially deleted C-terminus. Figure 26(l) is a graph showing PC uptake in XRCC4 KO PLB cells expressing an XRCC4 fragment (also known as "Mini") that induces PC uptake. The PC uptake assay was performed using 10 μM STS. Light gray indicates no STS treatment (control), and dark gray indicates STS treatment (apoptotic stimulation). The bars indicate regions where PC uptake is positive.

[0024] [Figure 27]Figure 27(a) upper panel shows a photograph of Western blotting of XRCC4-RFP with a partial deletion of the N-terminus. White arrowheads indicate the expected protein size of XRCC4-RFP. Black arrowheads indicate the C-terminal fragment of XRCC4 cleaved by caspase. Asterisks indicate degradation products. Figure 27(b) shows the intrinsically disordered region (IDR) analysis of XRCC4. The amino acid sequence of XRCC4 was analyzed using the software IUPred2A, and the score is shown. Arrows indicate caspase 3 cleavage sites. Figure 27(c) shows a photograph of Western blotting of XRCC4-RFP with a partial deletion of the C-terminus. Figure 27(d) shows photographs of Western blotting of XRCC4ΔC-RFP and XRCC4ΔN-RFP. Figure 27(e) shows the alignment of the XRCC4 amino acid sequence in a given species. The underlined portion shows the isoleucine after the caspase cleavage site in human XRCC4, which is replaced by the similar hydrophobic amino acid valine in several species. The amino acid sequence enclosed by the solid line is conserved in the given species. Figure 27(f) is a photograph showing Western blotting of XRCC4(Mini)-RFP. In Figures 27(a, c, d, and f), XRCC4 KO PLB cells expressing a predetermined XRCC4 fused with Xkrc4 and RFP at the C-terminus were stimulated with apoptosis using STS. Total cell lysates were prepared from these cells, subjected to SDS-PAGE, and then Western blotting with an anti-RFP antibody. The lower panel of Figures 27(a, c, d, and f) shows a photograph of a CBB-stained PVDF membrane as a loading control. The loading amount of total cell lysate is shown below (μg).

[0025] [Figure 28]Figure 28(a) shows the amino acid sequences of synthetic peptides C20 and C21. Underlined lines indicate caspase recognition sites. Amino acid sequences enclosed by solid lines represent NLS. Figure 28(b) is a graph showing the scrambling activity of PS exposure using synthetic peptides C20 and C21. Figure 28(c) is a bar graph showing the quantification of PS exposure activity (arbitrary unit AU). 1 (AU) corresponds to the fluorescence intensity of Xkr4ΔC-expressing cells into which C20 has been introduced. [Figure 29] Figure 29(a) is a micrograph showing the intracellular distribution of XRCC4 WT with RFP fused to the C-terminus. Figure 29(b) is a micrograph showing the intracellular distribution of XRCC4 mutant (R270A) with RFP fused to the C-terminus. Figure 29(c) is a micrograph showing the intracellular distribution of XRCC4 mutant (K271A) with RFP fused to the C-terminus. Figure 29(d) is a micrograph showing the intracellular distribution of XRCC4 mutant (R272A) with RFP fused to the C-terminus. Figure 29(e) is a micrograph showing the intracellular distribution of XRCC4 mutant (R273A) with RFP fused to the C-terminus. Figure 29(f) is a micrograph showing the intracellular distribution of XRCC4 mutant (R275A) with RFP fused to the C-terminus. XRCC4 knockout PLB cells expressing V5-Xkr4ΔC-FLAG were transfected with a predetermined RFP-fused XRCC4 mutant, and the intracellular distribution of the RFP-fused XRCC4 mutant was analyzed by confocal microscopy. DRAQ5 was used for nuclear staining. DIC shows differential interference contrast. Scale bars represent 20 μm.

[0026] [Figure 30]Figure 30(a) is a graph showing PC uptake in XRCC4 KO PLB cells expressing XRCC4 WT. Figure 30(b) is a graph showing PC uptake in XRCC4 KO PLB cells expressing XRCC4 mutant (R270A). Figure 30(c) is a graph showing PC uptake in XRCC4 KO PLB cells expressing XRCC4 mutant (K271A). Figure 30(d) is a graph showing PC uptake in XRCC4 KO PLB cells expressing XRCC4 mutant (R272A). Figure 30(e) is a graph showing PC uptake in XRCC4 KO PLB cells expressing XRCC4 mutant (R273A). Figure 30(f) is a graph showing PC uptake in XRCC4 KO PLB cells expressing XRCC4 mutant (R275A). The PC uptake assay was performed using 10 μM STS. Light gray indicates no STS treatment (control), and dark gray indicates STS treatment (apoptotic stimulation). The bars indicate regions where PC uptake is positive.

[0027] [Figure 31]Figure 31(a) upper panel shows photographs of Western blotting of XRCC4 mutants. XRCC4 KO PLB cells expressing V5-Xkr4ΔC-FLAG and XRCC4-RFP were stimulated with apoptosis using STS. Whole cell lysates were prepared from these cells, subjected to SDS-PAGE, and then Western blotting with an anti-RFP antibody. Figure 31(a) lower panel shows photographs of CBB-stained PVDF membranes as a loading control. Figure 31(b) is a bar graph showing the scrambling activity for PS exposure using synthetic peptides. Underlines indicate alanine-substituted amino acid sites. XRCC4 fragment mutants C20-R270A and C20-K271A were introduced into BDKO cells or cells expressing V5-Xkr4ΔC-FLAG(ΔC) by electroporation, and PS exposure assays were performed. Quantification of PS exposure activity is shown with the fluorescence intensity of V5-Xkr4ΔC-FLAG-expressing cells with C20 introduced set to 1 (arbitrary unit AU). Error bars represent the mean ± SD of the triple sample. P-values ​​were calculated according to Student's t-test. NS indicates non-significant. [Figure 32]Figure 32(a) upper panel shows a photograph of Western blotting using an anti-FLAG antibody on XRCC4 KO PLB cells expressing SPOT-Xkr4ΔC-FLAG and XRCC4-WT-2A-RFP or XRCC4-R270A-2A-RFP. Figure 32(b) middle panel shows a photograph of Western blotting using an anti-XRCC4 antibody on XRCC4 KO PLB cells expressing SPOT-Xkr4ΔC-FLAG and XRCC4-WT-2A-RFP or XRCC4-R270A-2A-RFP. Figure 32(c) lower panel shows a photograph of a CBB-stained PVDF membrane as a loading control. Figure 32(b) is a graph showing PC uptake in XRCC4 KO PLB cells expressing SPOT-Xkr4ΔC-FLAG and XRCC4-WT-2A-RFP or XRCC4-R270A-2A-RFP. The PC uptake assay was performed using 10 μM STS. In Figure 32(b), light gray indicates no STS treatment (control), dark gray indicates STS treatment (apoptotic stimulation), and the bars indicate regions positive for PC uptake. The median fluorescence intensity was 139 in WT and 29.2 in R270A. [Figure 33] Figure 33 is a distribution map showing label-free quantification of Xkr4 interacting substances. The cell membrane fraction of PLB cells was solubilized and immunoprecipitated using anti-SPOT nanobody-binding beads to precipitate Xkr4 interacting substances. The precipitate was then subjected to mass spectrometry. The X-axis shows the number of peptides in XRCC4 WT obtained using STS divided by the number of peptides in XRCC4 WT obtained without STS. The Y-axis shows the number of peptides in XRCC4 WT obtained using STS divided by the number of peptides in XRCC4 R270A obtained using STS. [Figure 34]Figure 34(a) is a schematic diagram showing the regions of two XRCC4 peptides identified by immunoprecipitation using Xkr4. Figure 34(b) is an extracted ion chromatogram of the transition of XRCC4 peptide 1. Peptide 1 was analyzed by targeted mass spectrometry using the PRM method. Figure 34(c) is a bar graph showing the value of XRCC4 normalized by the relative amount of Xkr4. Figure 34(d) is an extracted ion chromatogram of XRCC4 peptide 2. The peptide was analyzed by targeted mass spectrometry using the PRM method. Figure 34(e) is a bar graph showing the value of XRCC4 normalized by the relative amount of Xkr4. In Figures 34(c) and (e), the relative amount of XRCC4 was normalized using the relative amount of Xkr4 so that the relative amount of XRCC4 precipitated with Xkr4 in living cells was 1. [Figure 35] Figure 35(a) is a Western blotting image showing the detection of XRCC4 in Xkr4 immunoprecipitate. XRCC4 WT immunoprecipitate recovered together with SPOT-fused Xkr4 was detected. Figure 35(b) is a micrograph showing the localization of the XRCC4 peptide. C20-TMR was introduced into HCT116 cells expressing Xkr4WT-GFP or Xkr4ΔC-GFP by electroporation. The resulting cells were observed using a confocal microscope. Figure 35(c) is a graph showing the results of line scan analysis along the white dotted arrow in the image shown in the right column (Marged) of Figure 35(b). It shows the fluorescence intensity from GFP and the fluorescence intensity from TMR. In Figure 35(c), the Y axis represents arbitrary units (AU), and the scale bar represents 20 μm. [Modes for carrying out the invention]

[0028] Xkr4 polypeptide Xkr4 is a membrane protein belonging to the Xkr family, possessing a 10-transmembrane domain. Xkr4 is expressed in specific tissues, such as the brain or skin. Xkr4 may be derived from mammals, including humans, mice, and pigs. Xkr4 may be homologous proteins of Xkr4 in birds, reptiles, amphibians, or fish. The amino acid sequence of Xkr4 is available from publicly available databases, such as GeneBnak. While not strictly theoretical, Xkr4 may be partially cleaved by caspases and form dimers in cells stimulated by apoptosis, for example. The C-terminally cleaved dimerized Xkr4 may exhibit lipid scrambling activity through interaction with the XRCC4 polypeptide, as described later.

[0029] Xkr4 is a mouse-derived Xkr4 consisting of or containing the amino acid sequence described in Sequence ID No. 1 below, for example. TIFF0007873013000001.tif81165

[0030] Xkr4 is a human-derived Xkr4 consisting of or containing the amino acid sequence described in, for example, SEQ ID NO: 35 shown below. TIFF0007873013000002.tif81165

[0031] Xkr4 consists of or includes an amino acid sequence containing 1 to 100, 1 to 50, 1 to 30, 1 to 20, 1 to 10, 1 to 5, 4, 3, 2, or 1 amino acid deletion, substitution, or addition, or a combination thereof, in the amino acid sequence described, for example, SEQ ID NO: 1 or 35. Xkr4 consists of or includes an amino acid sequence exhibiting at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with the amino acid sequence described, for example, SEQ ID NO: 1 or 35.

[0032] "Xkr4 polypeptide" is (A) a polypeptide consisting of any of the amino acid sequences described in Sequence IDs 3-5 and 37-39 (specific amino acid sequences are shown below); or (B) a polypeptide having an amino acid sequence that includes deletions, substitutions, additions, or combinations thereof in the amino acid sequence of the polypeptide in (A), exhibiting at least 80% sequence identity with the amino acid sequence of (A), and capable of exhibiting lipid scrambling activity in the cell membrane without interaction with the XRCC4 polypeptide. TIFF0007873013000003.tif187165

[0033] TIFF0007873013000004.tif197165

[0034] The amino acid sequence described in Sequence ID No. 3 is different from the amino acid sequence described in Sequence ID No. 2 in that, according to the numbering of amino acid residues in the amino acid sequence described in Sequence ID No. 1, the isoleucine (I) at position 322 is replaced with serine (S) (I322S).

[0035] The amino acid sequence described in SEQ ID NO: 4 is different from the amino acid sequence described in SEQ ID NO: 2 in that, according to the numbering of amino acid residues in the amino acid sequence described in SEQ ID NO: 1, leucine (L) at position 331 is replaced with phenylalanine (F) (L331F).

[0036] The amino acid sequence described in Sequence ID No. 5, compared to the amino acid sequence described in Sequence ID No. 2, has a substitution in which glutamine (Q) at position 332 is replaced with glutamic acid (E) (Q332E), according to the numbering of amino acid residues in the amino acid sequence described in Sequence ID No. 1.

[0037] The amino acid sequence described in SEQ ID NO: 37, compared to the amino acid sequence described in SEQ ID NO: 36, has the isoleucine (I) at position 325 replaced by serine (S) (I325S), according to the amino acid residue numbering of the amino acid sequence described in SEQ ID NO: 35.

[0038] The amino acid sequence described in SEQ ID NO: 38, compared to the amino acid sequence described in SEQ ID NO: 36, has a substitution of leucine (L) at position 334 with phenylalanine (F) according to the amino acid residue numbering of the amino acid sequence described in SEQ ID NO: 35 (L334F).

[0039] The amino acid sequence described in SEQ ID NO: 39, compared to the amino acid sequence described in SEQ ID NO: 36, has a substitution in which glutamine (Q) at position 335 is replaced with glutamic acid (E) (Q335E), according to the amino acid residue numbering of the amino acid sequence described in SEQ ID NO: 35.

[0040] The Xkr4 polypeptide can exhibit lipid scrambling activity in the cell membrane without interaction with the XRCC4 polypeptide, as described later. The lipid scrambling activity mediated by the Xkr4 polypeptide is, for example, the lipid scrambling activity (e.g., PC uptake activity) in human PLB985 cells (hereinafter also referred to as "PLB cells") (Reference 26).

[0041] "Peptides," "polypeptides," and "proteins" refer to polymers of amino acids of any length, containing amino acids linked to each other by peptide bonds or modified peptide bonds. "Peptides," "polypeptides," and "proteins" are used interchangeably. Amino acids include the 20 amino acids encoded by genes, and other modified amino acids. Peptides, polypeptides, and proteins may be derived from natural sources (cells), for example, or artificially prepared by genetic engineering techniques. Peptides, polypeptides, and proteins may be isolated or purified from natural sources or reagents after artificial preparation, for example. Methods for isolating or purifying peptides, polypeptides, and proteins are well known and may be used, for example, HPLC.

[0042] In this specification, the Xkr4 polypeptide relating to (B) above is also referred to as a "mutant of the Xkr4 polypeptide".

[0043] Xkr4 polypeptides can be prepared, for example, by genetic engineering techniques. Xkr4 polypeptides can also be prepared, for example, by transcription and translation from polynucleotides containing nucleotide sequences encoding the amino acid sequences described herein. Xkr4 polypeptides can be used, for example, in screening methods for substances that inhibit or induce the lipid scrambling activity of Xkr4, as described later.

[0044] A variant of the Xkr4 polypeptide has, for example, in the amino acid sequence described in SEQ ID NO: 1, at least one amino acid substitution selected from the group consisting of serine in place of isoleucine at position 322, phenylalanine in place of leucine at position 331, and glutamic acid in place of glutamine at position 332, according to the numbering of amino acid residues in the amino acid sequence described in SEQ ID NO: 1. A variant of the Xkr4 polypeptide has, for example, in the amino acid sequence described in SEQ ID NO: 35, at least one amino acid substitution selected from the group consisting of serine in place of isoleucine at position 325, phenylalanine in place of leucine at position 334, and glutamic acid in place of glutamine at position 335, according to the numbering of amino acid residues in the amino acid sequence described in SEQ ID NO: 35.

[0045] An amino acid "deletion" means that an amino acid residue at any position in a given amino acid sequence is lost. A variant of the Xkr4 polypeptide may, for example, have amino acids lost at its N-terminus, C-terminus, and / or the amino acid sequence between the N-terminus and C-terminus in the amino acid sequence of the Xkr4 polypeptide according to (A). In one embodiment, a variant of the Xkr4 polypeptide having the amino acid deletion may have amino acids lost at its N-terminus or the amino acid sequence between the N-terminus and C-terminus in the amino acid sequence of the Xkr4 polypeptide according to (A).

[0046] A variant of the Xkr4 polypeptide having an amino acid deletion in the amino acid sequence described in any of Sequence IDs 3 to 5 may have lengths of, for example, 500 to 563 amino acids, 520 to 563 amino acids, 540 to 563 amino acids, 550 to 563 amino acids, 560 to 563 amino acids, 561 amino acids, 562 amino acids, or 563 amino acids. A variant of the Xkr4 polypeptide having an amino acid deletion in the amino acid sequence described in any of Sequence IDs 37 to 38 may have lengths of, for example, 500 to 566 amino acids, 520 to 566 amino acids, 540 to 566 amino acids, 550 to 566 amino acids, 560 to 566 amino acids, 565, or 566 amino acids.

[0047] The "addition" of amino acids means that an amino acid residue is added or inserted at any position in a given amino acid sequence. A variant of the Xkr4 polypeptide may have amino acids added or inserted at the N-terminus, C-terminus, and / or the amino acid sequence between the N-terminus and C-terminus in the amino acid sequence of the Xkr4 polypeptide according to (A) above. In one embodiment, a variant of the Xkr4 polypeptide may have amino acids added or inserted at the N-terminus or the amino acid sequence between the N-terminus and C-terminus in the amino acid sequence of the Xkr4 polypeptide according to (A) above.

[0048] A variant of the Xkr4 polypeptide having an added amino acid in the amino acid sequence described in any of Sequence IDs 3 to 5 may have lengths of, for example, 565 to 600 amino acids, 565 to 590 amino acids, 565 to 580 amino acids, 565 to 570 amino acids, 569 amino acids, 568 amino acids, 567 amino acids, 566 amino acids, or 565 amino acids. A variant of the Xkr4 polypeptide having an added amino acid in the amino acid sequence described in any of Sequence IDs 37 to 38 may have lengths of, for example, 568 to 600 amino acids, 568 to 590 amino acids, 568 to 580 amino acids, 568 to 570 amino acids, 569 amino acids, or 568 amino acids.

[0049] Amino acid "substitution" means that an amino acid residue at any position in a given amino acid sequence is replaced by another amino acid residue. Amino acid substitutions can be, for example, conservative substitutions. Amino acid "conservative substitution" means that an amino acid residue in a polypeptide is replaced by another amino acid residue from a group of amino acids that have similar characteristics in their side chains (e.g., charge, side chain size, hydrophobic / hydrophilicity). For example, the group of amino acids with aliphatic side chains consists of glycine, alanine, valine, leucine, and isoleucine; the group of amino acids with aliphatic hydroxyl side chains consists of serine and threonine; the group of amino acids with amide-containing side chains consists of asparagine and glutamine; the group of amino acids with aromatic side chains consists of phenylalanine, tyrosine, and tryptophan; the group of amino acids with basic side chains consists of lysine, arginine, and histidine; the group of amino acids with acidic side chains consists of glutamate and aspartic acid; and the group of amino acids with sulfur-containing side chains consists of cysteine ​​and methionine. Exemplary conservative amino acid substitutions are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine.

[0050] The Xkr4 polypeptide may be fused with a functional polypeptide (e.g., a tag sequence such as SPOT or FLAG, or a labeled protein such as green fluorescent protein). A variant of the Xkr4 polypeptide in the fusion protein has an amino acid sequence obtained by removing the amino acid sequence of the functional polypeptide from the amino acid sequence of the fusion protein.

[0051] Sequence identity refers to the sequence similarity between two optimally aligned polynucleotide sequences or two amino acid sequences. If the positions of the bases or amino acids in both sequences being compared are identical, then the two molecules are identical at that position. The percentage of identity is calculated by dividing the number of identical positions in the two sequences by the total number of positions being compared and multiplying by 100. Sequence identity can be calculated using commercially or publicly available software, such as BLAST+.

[0052] In one embodiment, the sequence identity between the amino acid sequence of a variant of the Xkr4 polypeptide and the amino acid sequence of the Xkr4 polypeptide is calculated from the amino acid sequence of the variant of the Xkr4 polypeptide and the amino acid sequence described in any of SEQ ID NOs. 3-5 and 37-39 (e.g., SEQ ID NO. 37).

[0053] The numbering of amino acid residues is determined by assigning numbers in ascending order from the N-terminus (leftmost) to the C-terminus (rightmost) within a specific sequence. For example, in the ASG sequence, the leftmost amino acid, alanine (A), is assigned position 1, and the rightmost amino acid, glycine (G), is assigned position 3.

[0054] The "lipid scrambling activity" of Xkr4 includes, for example, the uptake activity of phospholipids or glycolipids, or the exposure activity of phospholipids located on the inner side of the cell membrane. "Phospholipids" are phospholipids that make up the cell membrane. Phospholipids include, for example, phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, sphingomyelin, or phosphatidylinositol. "Glycolipids" are glycolipids that make up the cell membrane. Glycolipids include, for example, glyceroglycolipids or sphingoglycolipids.

[0055] The "uptake activity" of phospholipids or glycolipids is the action of taking in phospholipids or glycolipids present outside the cell into the cell membrane of the cell. The uptake activity of phospholipids or glycolipids can be measured, for example, using fluorescently labeled phospholipids or glycolipids. This activity can be measured, for example, by adding fluorescently labeled phospholipids or glycolipids to the culture medium of cells expressing Xkr4 polypeptide (e.g., PLB cells), allowing the fluorescently labeled phospholipids or glycolipids to be taken into the cell membrane of the cell, and then detecting the fluorescence derived from the taken-in fluorescently labeled phospholipids or glycolipids. Examples of fluorescent labels that can be used include nitrobenzoxadiazole (NBD) and TopFluor. Fluorescent labeling of phospholipids or glycolipids can be carried out using known methods. Fluorescently labeled phospholipids or glycolipids are commercially available.

[0056] The uptake activity of phospholipids or glycolipids can be measured, for example, by culturing PLB cells constitutively expressing Xkr4 polypeptide in a microwell plate, adding fluorescently labeled phospholipids or glycolipids to the culture medium, and allowing them to be taken up into the cell membrane. After a predetermined time, the culture medium containing unreacted fluorescently labeled phospholipids or glycolipids is replaced with fresh culture medium, and the fluorescence derived from the fluorescently labeled phospholipids or glycolipids taken up into the cell membrane can be measured, for example, using a plate reader. The measured fluorescence intensity can be compared with the fluorescence intensity from a negative control to determine whether or not phospholipids or glycolipids have been taken up, or to what extent. If the fluorescence intensity in PLB cells constitutively expressing Xkr4 polypeptide is higher than, for example, the fluorescence intensity in the negative control, for example, at least 2 times, 3 times, 5 times, or 10 times higher, it can be determined that phospholipids or glycolipids have been taken up.

[0057] The uptake activity of phospholipids or glycolipids can be measured, for example, by collecting PLB cells constitutively expressing Xkr4 polypeptides that have incorporated fluorescently labeled phospholipids or glycolipids from a microwell plate, and measuring the collected cell suspension by flow cytometry, in the same manner as described above. The presence or degree of phospholipid or glycolipid uptake can be determined by comparing the proportion of cell populations showing a predetermined fluorescence intensity with the proportion of cell populations derived from negative controls. The proportion of cell populations showing a predetermined fluorescence intensity can be calculated, for example, by setting a predetermined gate in a distribution map of cells showing fluorescence intensity per cell and dividing the number of cells exceeding the gate by the total number of cells measured. The predetermined gate can be set, for example, at a position in a distribution map of cells showing fluorescence intensity per cell of negative controls where the proportion of cell populations showing a predetermined fluorescence intensity is 3% or less. If the proportion of the cell population in the predetermined gate in PLB cells constitutively expressing Xkr4 polypeptide is higher than, for example, the proportion of the cell population in the predetermined gate in a negative control, for example, by at least 2, 3, 5, or 10 times, then it may be determined that phospholipid or glycolipid uptake has occurred.

[0058] In one embodiment, the uptake activity of phospholipids or glycolipids may be PC or PS uptake activity using NBD-labeled phosphatidylcholine (NBD-PC) or phosphatidylserine (NBD-PS).

[0059] Phospholipid "exposure activity" is the action that exposes phosphatidylserine (PS) or phosphatidylethanolamine (PE), which are located on the inside of the cell membrane exhibiting phospholipid asymmetry, to the outside of the cell membrane. Phospholipid exposure activity can be measured, for example, using a detection reagent in which a molecule that binds to PS or PE is fluorescently labeled. Phospholipid exposure activity can be measured, for example, by adding the detection reagent to the culture medium of cells expressing Xkr4 polypeptide (e.g., PLB cells) and detecting the fluorescence derived from the detection reagent bound to the cell membrane after a predetermined time. Fluorescent labels such as Cy5 and Alexa657 can be used. Annexin V can be used as a molecule that binds to PS or PE. Fluorescent labeling of PS or PE can be carried out using known methods. Fluorescently labeled Annexin V is commercially available.

[0060] To detect phospholipid exposure, for example, PLB cells constitutively expressing Xkr4 polypeptide are cultured in a microwell plate, and a detection reagent is added to the culture medium to bind to PS or PE exposed on the outside of the cell membrane. After a predetermined time, the culture medium containing unreacted detection reagent is replaced with fresh culture medium, and the fluorescence originating from the detection reagent bound to the cell membrane can be measured, for example, using a plate reader. The measured fluorescence intensity can be compared with the fluorescence intensity from a negative control to determine the presence or degree of phospholipid exposure activity. As a negative control, for example, the results of a similar test can be performed except that wild-type PLB cells (which do not express Xkr4 polypeptide) are used.

[0061] The phospholipid exposure activity can be measured, for example, by collecting PLB cells constitutively expressing Xkr4 polypeptide conjugated with a detection reagent from a microwell plate, and measuring the collected cell suspension by flow cytometry, in the same manner as described above. The presence or degree of phospholipid exposure activity can be determined by comparing the proportion of cell populations showing a predetermined fluorescence intensity with the proportion of cell populations derived from negative controls. The proportion of cell populations showing a predetermined fluorescence intensity can be calculated, for example, by setting a predetermined gate in the distribution map of cells showing fluorescence intensity per cell and dividing the number of cells exceeding the gate by the total number of cells measured. The predetermined gate can be set, for example, at a position in the distribution map of cells showing fluorescence intensity per cell of the negative control where the proportion of cell populations showing a predetermined fluorescence intensity is 3% or less.

[0062] In one embodiment, a variant of the Xkr4 polypeptide consists of an amino acid sequence of consecutive amino acid lengths of 500-600, 520-580, 530-570, 540-570, or 550-570 amino acids in the amino acid sequence described in, for example, SEQ ID NO: 1, and the amino acid sequence of said length has at least one amino acid substitution selected from the group consisting of serine in place of isoleucine at position 322, phenylalanine in place of leucine at position 331, and glutamic acid in place of glutamine at position 332, according to the numbering of amino acid residues in the amino acid sequence described in SEQ ID NO: 1, and the amino acid sequence of the variant has at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with the amino acid sequence of the Xkr4 polypeptide; and the variant exhibits lipid scrambling activity in the cell membrane without interaction with the XRCC4 polypeptide.

[0063] In one embodiment, a variant of the Xkr4 polypeptide consists of an amino acid sequence of consecutive amino acid lengths of 500-600, 520-580, 530-570, 540-570, or 550-570 amino acids, as described in, for example, SEQ ID NO: 35, and the amino acid sequence of said length has at least one amino acid substitution selected from the group consisting of serine in place of isoleucine at position 325, phenylalanine in place of leucine at position 334, and glutamic acid in place of glutamine at position 335, according to the numbering of amino acid residues in the amino acid sequence described in SEQ ID NO: 35, and the amino acid sequence of the variant has at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with the amino acid sequence of the Xkr4 polypeptide; and the variant exhibits lipid scrambling activity in the cell membrane without interaction with the XRCC4 polypeptide.

[0064] "May exhibit lipid scrambling activity in the cell membrane without interaction with XRCC4 polypeptide" means that a variant of the Xkr4 polypeptide exhibits PC uptake activity in the cell membrane of cells expressing the Xkr4 polypeptide in the absence of the XRCC4 polypeptide described later. For example, a variant of the Xkr4 polypeptide can be identified as potentially exhibiting lipid scrambling activity in the cell membrane without interaction with XRCC4 polypeptide if, when comparing the PC uptake activity of PLB cells (subject) expressing the Xkr4 polypeptide variant (e.g., consisting of the amino acid sequence described in SEQ ID NO: 37) in the absence of the XRCC4 polypeptide with the PC uptake activity of identical PLB cells (negative control) except that they do not express the Xkr4 polypeptide variant, the subject's PC uptake activity is higher than that of the negative control, for example, if it is at least 2, 3, 4, 5, or 10 times higher. The PC uptake activity can be measured by the methods disclosed herein (e.g., methods using flow cytometry).

[0065] The term "negative control cells" in relation to Xkr4 polypeptide variants refers to the same cells that express the Xkr4 polypeptide variant used to measure PC uptake activity, except that they do not express the Xkr4 polypeptide variant. For example, the negative control cells may be wild-type PLB cells (which do not express the Xkr4 polypeptide).

[0066] The variants of the Xkr4 polypeptide have any combination of, for example, the amino acid length, amino acid substitutions, sequence identity, and lipid scrambling activity (e.g., PC uptake activity) disclosed herein.

[0067] XRCC4 polypeptide XRCC4 is a DNA repair protein encoded by the XRCC4 gene in humans. XRCC4 may be localized in the nucleus. XRCC4 may be derived from mammals, including humans, mice, and pigs. XRCC4 may be homologous proteins of XRCC4 in birds, reptiles, amphibians, or fish. The amino acid sequence of XRCC4 is available from publicly available databases, for example, from GeneBnak. While not strictly theoretical, XRCC4 may be partially cleaved by caspases in apoptotically stimulated cells, and the cleaved XRCC4 polypeptide may diffuse into the cytoplasm. The XRCC4 polypeptide may induce the lipid scrambling activity of Xkr4 through interaction with a dimerized C-terminally cleaved form of Xkr4.

[0068] XRCC4 is a human-derived XRCC4 consisting of or containing the amino acid sequence described in, for example, SEQ ID NO: 6 shown below. TIFF0007873013000005.tif49165

[0069] XRCC4 consists of or includes an amino acid sequence that includes, for example, 1 to 50, 1 to 30, 1 to 20, 1 to 10, 1 to 5, 4, 3, 2, or 1 amino acid deletion, substitution, or addition, or a combination thereof, in the amino acid sequence described in SEQ ID NO: 6. XRCC4 consists of or includes an amino acid sequence that exhibits at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with, for example, the amino acid sequence described in SEQ ID NO: 6.

[0070] "XRCC4 polypeptide" is (a) a polypeptide consisting of an amino acid sequence described in any of Sequence IDs 7-12 and 42 (specific amino acid sequences are shown below); or (b) a polypeptide having an amino acid sequence that includes deletions, substitutions, additions, or combinations thereof in the amino acid sequence of the polypeptide in (a), exhibiting at least 80% sequence identity with the amino acid sequence in (a), and capable of inducing lipid scrambling activity in the cell membrane through interaction with a C-terminal cleaved Xkr4 polypeptide. TIFF0007873013000006.tif56165

[0071] The amino acid sequence described in Sequence ID No. 7 is a 20-amino acid sequence consisting of consecutive amino acids from position 266 to 285, according to the numbering system of the amino acid sequence described in Sequence ID No. 6.

[0072] The amino acid sequence described in Sequence ID No. 8, compared to the amino acid sequence described in Sequence ID No. 7, has a substitution of lysine (K) at position 271 with alanine (A) (K271A), according to the numbering of the amino acid sequence described in Sequence ID No. 6.

[0073] The amino acid sequence described in Sequence ID No. 9, compared to the amino acid sequence described in Sequence ID No. 7, has the arginine (R) at position 272 replaced by alanine (A) (R272A), according to the numbering of the amino acid sequence described in Sequence ID No. 6.

[0074] The amino acid sequence described in SEQ ID NO: 10, compared to the amino acid sequence described in SEQ ID NO: 7, has the arginine (R) at position 273 replaced by alanine (A) (K273A), according to the numbering of the amino acid sequence described in SEQ ID NO: 6.

[0075] The amino acid sequence described in SEQ ID NO: 11 is different from the amino acid sequence described in SEQ ID NO: 7 in that, according to the numbering of the amino acid sequence described in SEQ ID NO: 6, the arginine (R) at position 275 is replaced with alanine (A) (K275A).

[0076] The amino acid sequence described in Sequence ID No. 12 is a 30-amino acid sequence consisting of consecutive amino acids from position 256 to position 285, following the numbering system described in Sequence ID No. 6.

[0077] The amino acid sequence described in Sequence ID No. 42 is an 81-amino acid sequence consisting of consecutive amino acids from position 256 to position 336, following the numbering system described in Sequence ID No. 6.

[0078] The XRCC4 polypeptide can exert Xkr4 lipid scrambling activity in the cell membrane through interaction with the C-terminally cleaved form of dimerized Xkr4. This Xkr4 lipid scrambling activity mediated by the XRCC4 polypeptide is, for example, lipid scrambling activity (e.g., PC uptake activity) in PLB cells.

[0079] In this specification, the XRCC4 polypeptide relating to (b) above is also referred to as a "mutant of the XRCC4 polypeptide".

[0080] XRCC4 polypeptides can be prepared, for example, by known methods (e.g., genetic engineering techniques or chemical synthesis techniques). XRCC4 polypeptides can also be prepared, for example, by transcription and translation from polynucleotides containing a nucleotide sequence encoding the amino acid sequence described herein. XRCC4 polypeptides can be used, for example, in a screening method for substances that inhibit the lipid scrambling activity of Xkr4, as described below, or as a component of a composition for activating Xkr4.

[0081] Variants of the XRCC4 polypeptide consist of amino acid sequences of, for example, 18-100 amino acid lengths, 18-80 amino acid lengths, 18-60 amino acid lengths, or 18-50 amino acid lengths; 19-100 amino acid lengths, 19-80 amino acid lengths, 19-60 amino acid lengths, or 19-50 amino acid lengths; or 20-100 amino acid lengths, 20-80 amino acid lengths, 20-60 amino acid lengths, or 20-50 amino acid lengths. Variants of the XRCC4 polypeptide consist of, for example, a sequence of 50-80 consecutive amino acids in the amino acid sequence described in SEQ ID NO: 42, and consist of amino acid sequences of 50-100 amino acid lengths, 60-100 amino acid lengths, or 70-100 amino acid lengths; 50-90 amino acid lengths, 60-90 amino acid lengths, or 70-90 amino acid lengths; or 70-100 amino acid lengths, 70-90 amino acid lengths, 75-90 amino acid lengths, or 75-85 amino acid lengths. The variants of the XRCC4 polypeptide include, for example, the amino acid sequence described in SEQ ID NO: 7 or 12, and consist of amino acid sequences of 18-100 amino acid lengths, 18-80 amino acid lengths, 18-60 amino acid lengths, or 18-50 amino acid lengths; 19-100 amino acid lengths, 19-80 amino acid lengths, 19-60 amino acid lengths, or 19-50 amino acid lengths; or 20-100 amino acid lengths, 20-80 amino acid lengths, 20-60 amino acid lengths, or 20-50 amino acid lengths.

[0082] For example, variants of the XRCC4 polypeptide include the amino acid sequence described in SEQ ID NO: 7 or 12, and consist of consecutive amino acid sequences of 18-100 amino acid lengths, 18-80 amino acid lengths, 18-60 amino acid lengths, or 18-50 amino acid lengths in the amino acid sequence described in SEQ ID NO: 6; or 19-100 amino acid lengths, 19-80 amino acid lengths, 19-60 amino acid lengths, or 19-50 amino acid lengths; or 20-100 amino acid lengths, 20-80 amino acid lengths, 20-60 amino acid lengths, or 20-50 amino acid lengths.

[0083] A variant of the XRCC4 polypeptide, for example, if the amino acid length of the variant is 20 to 50, includes 1 to 5, 1 to 4, 1 to 3, 2, or 1 amino acid addition, substitution, or deletion, or a combination thereof, in the amino acid sequence of the XRCC4 polypeptide according to (a) above. A variant of the XRCC4 polypeptide, for example, if the amino acid length of the variant is 51 to 100, includes 1 to 15, 1 to 10, 1 to 5, 1 to 4, 1 to 3, 2, or 1 amino acid addition, substitution, or deletion, or a combination thereof, in the amino acid sequence of the XRCC4 polypeptide according to (a) above.

[0084] In one embodiment, the variant of the XRCC4 polypeptide contains arginine at position 270, according to the amino acid residue numbering of the amino acid sequence described in SEQ ID NO: 6. In one embodiment, the variant of the XRCC4 polypeptide does not contain methionine at position 265, according to the amino acid residue numbering of the amino acid sequence described in SEQ ID NO: 6. In one embodiment, the variant of the XRCC4 polypeptide contains arginine at position 270 and does not contain methionine at position 265, according to the amino acid residue numbering of the amino acid sequence described in SEQ ID NO: 6.

[0085] In one embodiment, the variants of the XRCC4 polypeptide include at least one amino acid substitution selected from the following group: K271A, R272A, K273A, and K275A, according to the amino acid sequence numbering described in SEQ ID NO: 6. In one embodiment, the variants of the XRCC4 polypeptide include, for example, arginine at position 270 and one or both of at least one amino acid substitution selected from the following group, and do not include methionine at position 265, according to the amino acid sequence numbering described in SEQ ID NO: 6: K271A, R272A, K273A, and K275A.

[0086] The XRCC4 polypeptide may be fused with a functional polypeptide (e.g., a tag sequence such as SPOT or FLAG, or a labeled protein such as green fluorescent protein). A variant of the XRCC4 polypeptide in the fusion protein has an amino acid sequence obtained by removing the amino acid sequence of the functional polypeptide from the amino acid sequence of the fusion protein.

[0087] The variants of the XRCC4 polypeptide have amino acid sequences containing deletions, substitutions, additions, or combinations thereof in the amino acid sequences described in any of SEQ ID NOs: 7-12; the variants consist of amino acid sequences of 18-100 amino acids, 18-80 amino acids, 18-60 amino acids, or 18-50 amino acids; 19-100 amino acids, 19-80 amino acids, 19-60 amino acids, or 19-50 amino acids; or 20-100 amino acids, 20-80 amino acids, 20-60 amino acids, or 20-50 amino acids; the amino acid sequences of the variants have at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with the amino acid sequences of the XRCC4 polypeptide; and the variants can induce lipid scrambling activity in the cell membrane through interaction with the C-terminally cleaved Xkr4 polypeptide.

[0088] In one embodiment, the sequence identity between the amino acid sequence of a variant of the XRCC4 polypeptide and the amino acid sequence of the XRCC4 polypeptide is calculated from the amino acid sequence of the variant of the XRCC4 polypeptide and the amino acid sequence described in any of SEQ ID NOs. 7 to 12 (for example, SEQ ID NO. 12).

[0089] The statement "can induce lipid scrambling activity in the cell membrane through interaction with C-terminally cleaved Xkr4 polypeptide" means that in the cell membrane of cells into which a variant of the XRCC4 polypeptide has been introduced and which expresses the C-terminally cleaved Xkr4 polypeptide, the variant of the XRCC4 polypeptide induces PC uptake activity. For example, by comparing the PC uptake activity in PLB cells (subject) into which a variant of the XRCC4 polypeptide has been introduced and which expresses the C-terminally cleaved Xkr4 polypeptide consisting of the amino acid sequence described in SEQ ID NO: 2 with the PC uptake activity in identical PLB cells (negative control) except that the variant has not been introduced, if the PC uptake activity of the subject is higher than that of the negative control, for example, if it is at least 2, 3, 4, 5, or 10 times higher, then the variant of the XRCC4 polypeptide is identified as being able to induce lipid scrambling activity in the cell membrane through interaction with the C-terminally cleaved Xkr4 polypeptide. The PC uptake activity can be measured by the methods disclosed herein (e.g., methods using flow cytometry).

[0090] The variants of the XRCC4 polypeptide may have any combination of, for example, the amino acid length, amino acid substitutions, sequence identity, and lipid scrambling activity (e.g., PC uptake activity) disclosed herein.

[0091] "Introduction" or "introduction" of a peptide or protein into a cell means making the peptide or protein present within the cell. The introduction of a peptide or protein into a cell includes introducing the peptide or protein from outside the cell into the cell and expressing the peptide or protein within the cell. The introduction of a peptide or protein into a cell can be carried out according to known methods. Introducing the peptide or protein from outside the cell into the cell can be carried out, for example, by lipofection or electroporation. Expressing the peptide or protein within the cell can be carried out, for example, by transfecting the cell with a polynucleotide containing a nucleotide sequence encoding the peptide or protein, or an expression vector containing the polynucleotide, and expressing the peptide or protein within the cell. In one embodiment, an XRCC4 polypeptide or XRCC4 polypeptide can be introduced into a cell by lipofection or electroporation.

[0092] Polynucleotides A "polynucleotide" is a polymeric form of nucleotides of any length, including ribonucleotides or deoxyribonucleotides. Polynucleotides include, for example, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases or other natural, chemically or biochemically modified, unnatural, or derivatized nucleotide bases. Polynucleotides include single-stranded polynucleotides (sense or antisense, etc.) and double-stranded polynucleotides. Polynucleotides containing a given nucleotide sequence can be prepared, for example, by known methods (e.g., genetic engineering techniques or chemical synthesis techniques) or are commercially available.

[0093] In one embodiment, the polynucleotide comprises a nucleotide sequence encoding the Xkr4 polypeptide disclosed herein. In one embodiment, the polynucleotide comprises a nucleotide sequence encoding the XRCC4 polypeptide disclosed herein. In one embodiment, the polynucleotide may contain the nucleotide sequence encoding the Xkr4 polypeptide and the nucleotide sequence encoding the XRCC4 polypeptide in the same polynucleotide or in separate polynucleotides.

[0094] Expression vector An "expression vector" is a genetic element that enables the introduction, replication, or expression of an exogenous polynucleotide in a host cell. An expression vector can, for example, cause the replication, transcription, or expression of another polynucleotide (i.e., an insert) in a host cell by operably ligating the insert to a promoter. An expression vector may be stably integrated into the genome of a host cell or exist as an independent genetic element (e.g., an episome, a plasmid). Expression vectors can be, for example, plasmids, phages, viruses, or cosmids. Expression vectors for ligating another polynucleotide can be prepared, for example, by known methods (e.g., genetic engineering techniques) or are commercially available.

[0095] "Operationally linked" indicates that the linked polynucleotides are capable of functioning in the intended manner, i.e., transcribed and, if translated, in the intended manner. The intended manner refers to the conditions, or expression levels, such as high or low expression, that enable the transcription and, if translated, of the linked polynucleotides, for example, in the presence of additives such as tetracycline.

[0096] In one embodiment, the expression vector comprises a polynucleotide disclosed herein. In one embodiment, the expression vector comprises a polynucleotide comprising a nucleotide sequence encoding the Xkr4 polypeptide disclosed herein. In one embodiment, the expression vector comprises a polynucleotide comprising a nucleotide sequence encoding the XRCC4 polypeptide disclosed herein. In one embodiment, the expression vector may contain a polynucleotide comprising a nucleotide sequence encoding the Xkr4 polypeptide and a polynucleotide comprising a nucleotide sequence encoding the XRCC4 polypeptide in the same expression vector or in separate expression vectors.

[0097] host cell A "host cell" is a cell into which a vector molecule can be inserted, i.e., a eukaryotic or prokaryotic cell. Host cells are, for example, eukaryotic or prokaryotic cells. Eukaryotic cells include, for example, cells derived from mammals, birds and fish, or plant cells (including, for example, eukaryotic algal cells). Mammalian host cells include, for example, human PLB985 cells, Chinese hamster ovary (CHO) cells, COS cells, Vero cells, SP2 / 0 cells, NS / 0 myeloma cells, human embryonic kidney (HEK293) cells and juvenile hamster kidney (BHK) cells, HeLa cells, human B cells, CV-1 / EBNA cells, L cells, 3T3 cells, HEPG2 cells, PerC6 cells and MDCK cells. Prokaryotic host cells include, for example, yeast cells and Escherichia coli cells. Host cells are, for example, cells derived from the brain or skin. Host cells are, for example, cells of the nervous system. The host cells may be, for example, cells in which either or both of XRCC4 and Xkr4 are knocked out. The host cells are available, for example, from public institutions or commercially.

[0098] In one embodiment, a host cell comprises a polynucleotide or expression vector disclosed herein. The host cell comprises a genome into which, for example, some or all of the polynucleotides are incorporated. The host cell comprises a genome into which, for example, some or all of the expression vectors are incorporated. The host cell comprises the expression vector as a genetic element independent of the genome, for example.

[0099] Composition for activating Xkr4 A "composition for activating Xkr4" comprises an element capable of inducing lipid scrambling activity by Xkr4. The element is, for example, the XRCC4 polypeptide disclosed herein, a polynucleotide comprising an encoding nucleotide sequence, or an expression vector comprising the polynucleotide. The composition can be prepared, for example, by mixing the element with additives such as excipients, preservatives, buffers, and pH adjusters.

[0100] In one embodiment, a composition for activating Xkr4 comprises at least one selected from the group consisting of the XRCC4 polypeptide disclosed herein; a polynucleotide comprising a nucleotide sequence encoding the XRCC4 polypeptide; and an expression vector comprising the polynucleotide. The composition for activating Xkr4 can be used, for example, to treat or prevent conditions or diseases involving the suppression of apoptosis, as described later.

[0101] "Conditions or diseases involving the suppression of apoptosis" are conditions or diseases that can be induced by the suppression of apoptosis. Conditions or diseases involving the suppression of apoptosis are, for example, conditions or diseases that can be induced by the suppression of apoptosis in organs or cells of the nervous system. Conditions or diseases involving the suppression of apoptosis are, for example, cancer in organs or cells of the nervous system, elevated thyroid-stimulating hormone, or neurodevelopmental or behavioral disorders, including attention deficit hyperactivity disorder (ADHD).

[0102] "Treatment" means taking action with the aim of maintaining, reducing, or eliminating a given condition or symptom. Actions taken with the aim of maintaining, reducing, or eliminating a given condition or symptom include, for example, administering a given composition. A composition intended for treatment may be administered, for example, during or after the onset of a condition or disease.

[0103] "Prevention" means taking measures with the aim of suppressing the onset of a particular condition or symptom. Measures taken with the aim of suppressing the onset of a particular condition or symptom include, for example, administering a particular composition. A composition intended for prevention may be administered, for example, before the onset of a condition or disease.

[0104] A screening method for substances that modulate the lipid scrambling activity of Xkr4. A screening method for substances that modulate the lipid scrambling activity of Xkr4 comprises: (1) contacting cells expressing the Xkr4 polypeptide disclosed herein with a candidate substance that may modulate the lipid scrambling activity of Xkr4; (2) measuring the lipid scrambling activity in the cells that have been contacted with the candidate substance; and (3) selecting the candidate substance as a substance that modulates the lipid scrambling activity of Xkr4 if the lipid scrambling activity is lower or higher than that in the cells that have not been contacted with the candidate substance.

[0105] "Cells expressing the Xkr4 polypeptide" can be prepared by introducing a polynucleotide containing a nucleotide sequence encoding the Xkr4 polypeptide disclosed herein, or an expression vector containing said polynucleotide, into host cells. In one embodiment, the cells express the Xkr4 polypeptide without apoptotic stimulation (e.g., apoptotic stimulation using staurosporine (STS)).

[0106] "Candidate substances that may modulate the lipid scrambling activity of Xkr4" are substances that are expected to induce, increase, inhibit, or decrease the lipid scrambling activity of Xkr4 by directly or indirectly affecting, for example, a C-terminally cleaved Xkr4 polypeptide. The candidate substances that may modulate the activity may be, for example, small molecules, proteins (e.g., antibodies), DNA, RNA, small interfering RNA, or antisense oligonucleotides.

[0107] The aforementioned candidate substances that may modulate include candidate substances that may induce or inhibit the lipid scrambling activity of Xkr4. "Candidate substances that may induce the lipid scrambling activity of Xkr4" are substances that are expected to induce or increase the lipid scrambling activity of Xkr4 in cells or animals. "Candidate substances that may inhibit the lipid scrambling activity of Xkr4" are substances that are expected to inhibit or reduce the lipid scrambling activity of Xkr4 in cells or animals.

[0108] "Contacting" or "bringing into contact" the cells with the candidate substance means placing the cells in a situation where they can come into contact with the candidate substance which may modulate (e.g., induce or inhibit) the lipid scrambling activity of Xkr4. This contact may, for example, involve adding the candidate substance to the culture medium in which the cells are cultured.

[0109] Lipid scrambling activity is, for example, the uptake activity of phospholipids or glycolipids, or the exposure activity of phospholipids located on the inner side of the cell membrane. Lipid scrambling activity can be measured according to the methods disclosed herein (e.g., methods utilizing flow cytometry).

[0110] In one embodiment, a screening method for substances that modulate Xkr4 lipid scrambling activity is a screening method for substances that inhibit Xkr4 lipid scrambling activity. The screening method for substances that inhibit Xkr4 lipid scrambling activity includes, for example, (1) contacting cells expressing the Xkr4 polypeptide disclosed herein with a candidate substance that may inhibit Xkr4 lipid scrambling activity; (2) measuring the lipid scrambling activity in the cells that have been contacted with the candidate substance; and (3) selecting the candidate substance as a substance that inhibits Xkr4 lipid scrambling activity if the lipid scrambling activity is 50%, 40%, 30%, 20%, 10%, or 5% or less compared to the lipid scrambling activity in the cells that have not been contacted with the candidate substance. Candidate substances that may inhibit Xkr4 lipid scrambling activity can be used as drug candidates for the treatment or prevention of conditions or diseases in which the promotion of apoptosis is involved.

[0111] A "condition or disease involving the promotion of apoptosis" is a condition or disease that can be induced by the promotion of apoptosis. Examples of conditions or diseases involving the promotion of apoptosis include conditions or diseases that can be induced by the promotion of apoptosis in organs of the nervous system. Examples of conditions or diseases involving the promotion of apoptosis include neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), and amyotrophic lateral sclerosis (ALS).

[0112] In one embodiment, a screening method for substances that modulate Xkr4 lipid scrambling activity is a screening method for substances that induce Xkr4 lipid scrambling activity. The screening method for substances that induce Xkr4 lipid scrambling activity includes, for example, (1) contacting cells expressing the Xkr4 polypeptide disclosed herein with a candidate substance that may induce Xkr4 lipid scrambling activity; (2) measuring the lipid scrambling activity in the cells that have been contacted with the candidate substance; and (3) selecting the candidate substance as a substance that induces Xkr4 lipid scrambling activity if the lipid scrambling activity is at least 2, 3, 4, 5, 7, 9, or 10 times higher than the lipid scrambling activity in the cells that have not been contacted with the candidate substance. Candidate substances that may induce Xkr4 lipid scrambling activity can be used as drug candidates for the treatment or prevention of conditions or diseases in which the suppression of apoptosis is involved.

[0113] "A condition or disease involving the suppression of apoptosis" is a condition or disease that can be induced by the promotion of apoptosis. A condition or disease involving the promotion of apoptosis is, for example, a condition or disease that can be induced by the promotion of apoptosis in organs of the nervous system. A condition or disease involving the promotion of apoptosis may be, for example, a condition or disease disclosed herein, such as ADHD.

[0114] A screening method for substances that induce lipid scrambling activity of Xkr4. A screening method for substances that induce lipid scrambling activity of Xkr4 includes: (1) contacting cells expressing a C-terminally cleaved Xkr4 polypeptide, which is a C-terminally cleaved Xkr4 polypeptide comprising (α) the amino acid sequence described in SEQ ID NO: 2 or 36, or (β) an amino acid sequence having deletions, substitutions, additions or combinations thereof in the amino acid sequence of the (α) polypeptide, exhibiting at least 80% sequence identity with the (α) amino acid sequence, and capable of exhibiting lipid scrambling activity in the cell membrane through interaction with an XRCC4 polypeptide, with a candidate substance that may induce lipid scrambling activity of Xkr4; (2) measuring the lipid scrambling activity in the cells that have been contacted with the candidate substance; and (3) selecting the candidate substance as a substance that induces lipid scrambling activity of Xkr4 if the lipid scrambling activity is higher than that in the cells that have not been contacted with the candidate substance.

[0115] A "C-terminally cleaved Xkr4 polypeptide" is a fragment of Xkr4 that can form dimers in the cell membrane of a cell. While not strictly theoretical, C-terminally cleaved Xkr4 polypeptides may exhibit lipid scrambling activity through interaction with the XRCC4 polypeptide disclosed herein. A C-terminally cleaved Xkr4 polypeptide is, for example, a polypeptide obtained by caspase cleavage of Xkr4 as disclosed herein.

[0116] A C-terminally cleaved Xkr4 polypeptide is, for example, (α) a C-terminally cleaved Xkr4 polypeptide consisting of the amino acid sequence described in Sequence ID No. 2 or 36 (specific amino acid sequences are shown below), or (β) a polypeptide having an amino acid sequence that includes deletions, substitutions, additions, or combinations thereof in the amino acid sequence of the (α) polypeptide, exhibiting at least 80% sequence identity with the (α) amino acid sequence, and capable of exhibiting lipid scrambling activity in the cell membrane through interaction with the XRCC4 polypeptide. TIFF0007873013000007.tif134165

[0117] C-terminally cleaved Xkr4 polypeptides can be prepared, for example, by genetic engineering techniques. C-terminally cleaved Xkr4 polypeptides can also be prepared, for example, by transcription and translation from polynucleotides containing a nucleotide sequence encoding the amino acid sequence described herein.

[0118] In the C-terminally cleaved Xkr4 polypeptide, for example, in the amino acid sequence described in SEQ ID NO: 2, according to the amino acid residue numbering of the amino acid sequence described in SEQ ID NO: 1, position 322 is not serine, position 331 is not phenylalanine, and position 332 is not glutamine. In the C-terminally cleaved Xkr4 polypeptide, for example, in the amino acid sequence described in SEQ ID NO: 36, according to the amino acid residue numbering of the amino acid sequence described in SEQ ID NO: 35, position 325 is not serine, position 334 is not phenylalanine, and position 335 is not glutamic acid.

[0119] The C-terminally cleaved Xkr4 polypeptide consists of or includes an amino acid sequence containing 1 to 100, 1 to 50, 1 to 30, 1 to 20, 1 to 10, 1 to 5, 4, 3, 2, or 1 amino acid deletion, substitution, or addition, or a combination thereof, in the amino acid sequence described in, for example, SEQ ID NO: 2 or 36. The C-terminally cleaved Xkr4 polypeptide consists of or includes an amino acid sequence exhibiting at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with respect to the amino acid sequence described in, for example, SEQ ID NO: 2 or 36.

[0120] In this specification, the C-terminally cleaved Xkr4 polypeptide relating to (β) is also referred to as a "mutant of the C-terminally cleaved Xkr4 polypeptide".

[0121] The C-terminally cleaved Xkr4 polypeptide may have any combination of amino acid length, amino acid substitutions, and sequence identity as disclosed herein, for example.

[0122] The phrase "may exhibit lipid scrambling activity in the cell membrane through interaction with XRCC4 polypeptide" means that, in the presence of the XRCC4 polypeptide disclosed herein, the variant of the C-terminally cleaved Xkr4 polypeptide exhibits PC uptake activity in the cell membrane of cells expressing the variant. For example, a variant of the C-terminally cleaved Xkr4 polypeptide is identified as potentially exhibiting lipid scrambling activity in the cell membrane through interaction with XRCC4 polypeptide if the PC uptake activity by PLB cells expressing the variant of the C-terminally cleaved Xkr4 polypeptide in the presence of XRCC4 polypeptide is higher than the PC uptake activity under identical conditions except in the absence of XRCC4 polypeptide—for example, if it is at least 2, 3, 4, 5, or 10 times higher. The PC uptake activity can be measured by the methods disclosed herein (e.g., methods utilizing flow cytometry).

[0123] Candidate substances identified as potentially exhibiting lipid scrambling activity in cell membranes through interaction with XRCC4 polypeptides can be used as drug candidates for the treatment or prevention of conditions or diseases involving the suppression of apoptosis.

[0124] A screening method for substances that inhibit the lipid scrambling activity of Xkr4. In one embodiment, a method for screening substances that inhibit the lipid scrambling activity of Xkr4 includes (1) contacting cells expressing a C-terminally cleaved Xkr4 polypeptide, into which the XRCC4 polypeptide disclosed herein has been introduced, with a candidate substance that may inhibit the lipid scrambling activity of Xkr4; (2) measuring the lipid scrambling activity in the cells; and (3) selecting the candidate substance as a substance that inhibits the lipid scrambling activity of Xkr4 if the lipid scrambling activity is lower than the lipid scrambling activity in the cells that have not been contacted with the candidate substance.

[0125] Cells into which the XRCC4 polypeptide has been introduced and which express the C-terminally cleaved Xkr4 polypeptide can be prepared, for example, by introducing the XRCC4 polypeptide or its variants into cells expressing the C-terminally cleaved Xkr4 polypeptide via lipofection or electroporation. Cells into which the XRCC4 polypeptide or its variants have been introduced and which express the C-terminally cleaved Xkr4 polypeptide can be prepared, for example, by introducing one or more polynucleotides containing nucleotide sequences encoding the XRCC4 polypeptide or its variants and the C-terminally cleaved Xkr4 polypeptide, or one or more expression vectors containing said polynucleotides, into host cells.

[0126] Contact between the cells and a candidate substance that may inhibit the lipid scrambling activity of Xkr4 can be carried out, for example, by adding the candidate substance to the culture medium in which the cells are cultured. Contact between the cells and a candidate substance that may inhibit the lipid scrambling activity of Xkr4 may include, for example, removing the culture medium to remove unreacted candidate substances after a predetermined time has elapsed since the addition of the candidate substance.

[0127] In the above embodiment, the substance selected as a substance to inhibit the lipid scrambling activity of Xkr4 is, for example, a substance that can block or reduce the direct or indirect interaction between XRCC4 polypeptides that have formed dimers in the cell membrane and XRCC4 polypeptides.

[0128] In one embodiment, a screening method for substances that inhibit the lipid scrambling activity of Xkr4 includes: (1) contacting the XRCC4 polypeptide disclosed herein with a C-terminally cleaved Xkr4 polypeptide in the presence of a candidate substance that may inhibit the lipid scrambling activity of Xkr4; (2) measuring the binding between the XRCC4 polypeptide and the C-terminally cleaved Xkr4 polypeptide; and (3) selecting the candidate substance as a substance that inhibits the lipid scrambling activity of Xkr4 if the binding between the XRCC4 polypeptide and the C-terminally cleaved Xkr4 polypeptide is lower than the binding when the XRCC4 polypeptide and the C-terminally cleaved Xkr4 polypeptide are contacted in the absence of the candidate substance, for example, 50%, 40%, 30%, 20%, 10%, or 5% or less.

[0129] Step (1) according to the above embodiment may involve adding a candidate substance that may inhibit the lipid scrambling activity of Xkr4 and an XRCC4 polypeptide to a C-terminally cleaved Xkr4 polypeptide immobilized on a support, for example. In one example, step (1) according to the above embodiment may involve immobilizing the C-terminally cleaved Xkr4 polypeptide on the plate surface (support) of a microwell plate by adsorption or the like. A solution containing a candidate substance that may inhibit the lipid scrambling activity of Xkr4 and a fluorescently labeled XRCC4 polypeptide may be added to the microwell plate on which the C-terminally cleaved Xkr4 polypeptide is immobilized. In this example, step (2) may involve removing the unbound fluorescently labeled XRCC4 polypeptide and then measuring the fluorescence from the fluorescently labeled XRCC4 polypeptide remaining in the microwell plate.

[0130] Screening kit A kit is provided for screening substances that inhibit the lipid scrambling activity of Xkr4. The kit comprises at least one, two, or three selected from the group consisting of the XRCC4 polypeptide disclosed herein; a polynucleotide comprising a nucleotide sequence encoding the XRCC4 polypeptide; an expression vector comprising the polynucleotide; and a host cell comprising the polynucleotide, or a host cell comprising the expression vector.

[0131] The components of the kit may be, for example, located in separate containers or mixed in a single container. The kit may include instructions for use, for example, in screening for substances that inhibit the lipid scrambling activity of Xkr4.

[0132] A method for identifying genes corresponding to a desired phenotype. One embodiment of the present invention provides a method for identifying genes corresponding to a target phenotype.

[0133] "Phenotype" refers to the traits of an organism that are produced as a result of gene activity under specific environmental conditions. The target phenotype may be, for example, a reaction such as lipid scrambling activity during apoptosis, a protein induced in the presence of a specific substance, or a substance secreted after a specific stimulus.

[0134] A "gene" is the basic unit that governs traits in genetic phenomena. For example, a gene is the unit that carries the genetic information to code for proteins. Genes are broadly classified into structural genes, which code for proteins or enzymes that make up living organisms, and regulatory genes, which code for protein factors that control the expression of structural genes. A single trait is governed by multiple structural and regulatory genes.

[0135] A "corresponding gene" for a target phenotype is a gene that influences the target phenotype. The corresponding gene may act directly or indirectly on the target phenotype. If the target phenotype is a protein expressed under specific environmental conditions, the structural gene encoding that protein may be a gene that directly acts on the target phenotype. In the above example, a regulatory gene encoding a protein factor that controls the expression of that protein may be a gene that indirectly acts on the target phenotype.

[0136] <Process a> The aforementioned identification method includes (a) preparing a guide RNA library which includes a guide RNA containing at least one guide sequence corresponding to the nucleotide sequences of multiple genes in the genomic DNA of a cell.

[0137] The "genomic DNA" of a cell refers to the entire set of genes that the cell possesses. Information including sequence information for each gene related to the genomic DNA, information about the animal species, and information about the phenotype (e.g., protein) corresponding to the gene is available, for example, from publicly available databases. The genomic DNA of a cell is, for example, the genomic DNA of a cell derived from a particular species. The genomic DNA of a cell may be, for example, homogeneous genomic DNA from a particular individual of a particular species, or heterogeneous genomic DNA from multiple different individuals of a particular species. In one embodiment, the genomic DNA of a cell is homogeneous genomic DNA from a particular individual of a particular species. In one embodiment, the genomic DNA of a cell may be heterogeneous genomic DNA from multiple different individuals of a particular species.

[0138] A "guide RNA" is a single-guide RNA (sgRNA) formed by the ligation of a crRNA (CRISPR-RNA) and a tracrRNA (trans-activating crRNA) to a guide sequence. A guide RNA may contain, for example, a nucleotide sequence encoding Cas9. When referring to a guide RNA integrated into genomic DNA, the guide RNA encompasses the DNA containing the nucleotide sequence transcribed to the guide RNA.

[0139] "Cas9" is C RISPR as Cas9 is a nuclease known as sociated protein 9, with a molecular weight of approximately 160 kilodaltons. The amino acid or nucleotide sequences of Cas9 are publicly known and can be obtained from publicly available databases. Polynucleotides containing the nucleotide sequence encoding Cas9 are commercially available.

[0140] A "guide sequence" includes a sequence complementary to the target sequence that can hybridize to the target sequence within the nucleotide sequence of a given gene in the genomic DNA of a specific species. Guide sequences have lengths of, for example, 10-30 nucleotides, 15-25 nucleotides, 18-22 nucleotides, or 20-21 nucleotides. Sequence identity between the guide sequence and its corresponding target sequence is, for example, 80%-100%, over 80%, over 85%, over 90%, over 95%, over 97%, over 99%, or 100%. The optimal alignment of the guide sequence and the target sequence can be determined using any algorithm for sequence alignment (e.g., NCBIBLAST).

[0141] A "guide RNA library" contains guide RNAs for at least one target sequence in the nucleotide sequences of multiple genes in a cell's genomic DNA. Each guide RNA in the guide RNA library may be stored within, for example, a lentivirus or retrovirus. In this case, the guide RNA library is also referred to as a viral guide RNA library (e.g., a lentiviral or retroviral guide RNA library). For example, if a cell of a particular species contains 22,000 genes in its genomic DNA, and the guide RNA library contains guide RNAs for each of the genes in the genomic DNA, and each guide RNA contains guide sequences for six different target sequences, then the guide RNA library will contain 132,000 sgRNAs. Guide RNA libraries can be prepared according to known methods, for example, according to the method described in Wang, et al. (2014). Genetic screens in human cells using the CRISPR-Cas9 system. Science 343, 80. Guide RNA libraries are commercially available, for example. The lentiviral-type guide RNA library may be, for example, the Gecko sgRNA library (Addgene#1000000049).

[0142] "Preparing" or "getting ready" a guide RNA library includes, for example, placing the guide RNA library in a state where it can be introduced into cells. For example, if the guide RNA library is frozen in liquid, it includes thawing the frozen material containing the guide RNA library. Preparation of a guide RNA library may also include, for example, preparing the guide RNA library.

[0143] <Process b> The aforementioned identification method includes (b) obtaining a population of cells into which the guide RNA library has been introduced.

[0144] "Introduction" or "introduction" of a guide RNA library includes, for example, bringing the guide RNA library (e.g., a lentiviral-type guide RNA library) into contact with a cell population. In a cell population into which the guide RNA library has been introduced, the introduced guide RNA is incorporated into the cell genome. In a cell population into which the guide RNA library has been introduced, the guide RNA library is introduced at a rate such that, for example, one type of guide RNA is introduced per cell. This rate of one type of guide RNA per cell can be achieved, for example, by introducing the guide RNA library into a cell population such that the number of cells constituting the cell population is, for example, 5, 10, or 15 times the number of guide RNAs constituting the guide RNA library.

[0145] When using a viral-type guide RNA library, the guide RNA library can be introduced into a cell population by infecting the cell population with the viral-type guide RNA library. When using a lentiviral-type guide RNA library, the guide RNA can be efficiently incorporated into the cell genome. When using a lentiviral-type guide RNA library, the guide RNA can be efficiently incorporated into the cell genome even if the cell population consists of cells with low or almost no proliferation rates (e.g., nerve cells, apoptotic cells, cells in tissue). In one embodiment, the cell population into which the guide RNA library has been introduced is a population of cells with low or almost no proliferation rates.

[0146] A cell population into which a guide RNA library has been introduced expresses Cas9. Cas9 may be expressed, for example, by incorporating a guide RNA containing a nucleotide sequence encoding Cas9 into the genomic DNA of the cells in the cell population. Alternatively, Cas9 may be expressed by incorporating a nucleotide sequence encoding Cas9 into the genomic DNA of the cell population into which the guide RNA library has been introduced.

[0147] The "cell population" into which the guide RNA library is introduced may be, for example, cultured cells or a cell population in a tissue. A cell population in a tissue may be, for example, a cell population in tissue removed from the body; a cell population in tissue of non-human mammals, birds, reptiles, or fish; or a cell population in tissue constructed by tissue culture. If the cell population is cultured cells, the cultured cells may have a nucleotide sequence encoding Cas9 incorporated into them. If the cell population is a cell population in a tissue, the guide RNA in the guide RNA library introduced into the cell population may contain a nucleotide sequence encoding Cas9.

[0148] A population of cells into which the guide RNA library has been introduced may be subjected to a predetermined stimulus to induce a desired phenotype. For example, if the desired phenotype is an action observed during the apoptotic process (e.g., lipid scrambling activity), the population of cells into which the guide RNA library has been introduced may be subjected to an apoptotic stimulus (e.g., apoptotic stimulation by adding staurosporine (STS)).

[0149] <Process c> The identification method includes (c) selecting cells from the cell population based on a desired phenotype. The variety of guide RNA introduced into the cells selected in step (c) is expected to be richer in guide RNA containing sequences complementary to the gene sequences corresponding to the desired phenotype compared to the variety of guide RNA introduced into the cell population in step (b).

[0150] Step (c) includes, for example, distinguishing between cells exhibiting the desired phenotype and cells not exhibiting the desired phenotype, and recovering the cells not exhibiting the desired phenotype. If the cell population is a collection of individually isolated cells, step (c) can be carried out using, for example, fluorescence-activated cell sorting (FACS) or magnetically activated cell sorting (MACS). For example, if a fluorescent reagent that binds to cells exhibiting the desired phenotype is used, cells that show fluorescence from the fluorescent reagent above a predetermined value and cells that show a value below that value can be distinguished, and cells not exhibiting the desired phenotype can be recovered.

[0151] If the cell population is a population of cells in a tissue, step (c) can be carried out, for example, using laser microdissection. For example, if a fluorescent reagent that binds to cells that do not exhibit the desired phenotype is used, the cells or regions that emit fluorescence derived from the fluorescent reagent can be distinguished from cells or regions that do not emit fluorescence under a microscope, and the cells that do not exhibit the desired phenotype can be recovered by laser dissection.

[0152] <Process d> The identification method described above includes (d) recovering genomic DNA from the selected cells. The genomic DNA recovered in step (d) is enriched with guide RNAs containing sequences complementary to the gene sequences corresponding to the target phenotype, compared to the variety of guide RNAs introduced into the cell population in step (b).

[0153] Genomic DNA can be recovered by preparation according to known methods. Genomic DNA recovery can be carried out, for example, by ethanol precipitation or using commercially available genomic DNA extraction kits.

[0154] <Process e> The aforementioned identification method includes preparing a new guide RNA library from the recovered genomic DNA and performing steps (b) to (d) using the new guide RNA library until steps (b) to (d) are performed a predetermined number of times. By performing steps (b) to (d) a predetermined number of times, it is expected that the guide RNA containing sequences complementary to the gene sequences corresponding to the target phenotype will be further enriched.

[0155] The "predetermined number of times" is at least two, for example, two, three, four, or five or more times. A higher predetermined number of times is expected to enrich the guide RNA containing sequences complementary to the gene sequence corresponding to the target phenotype, but on the other hand, it requires more effort to implement. In one embodiment, the predetermined number of times is three.

[0156] A "new guide RNA library" refers to a guide RNA library prepared using guide RNA that has been incorporated into the genomic DNA recovered in step (d). Guide RNA incorporated into genomic DNA can be recovered, for example, by amplifying the region containing the guide RNA by PCR and recovering the amplified PCR product. In this example, the guide RNA further includes a sequence for amplifying the guide RNA by PCR. When preparing a lentiviral-type guide RNA library, the recovered guide RNA is inserted into a lentiviral vector to prepare a plasmid. A lentiviral-type guide RNA library can be prepared by transfecting host cells with the prepared plasmid and culturing the transfected host cells to generate lentiviruses.

[0157] <Process f> The aforementioned identification method includes (f) determining the guide sequence in the recovered genomic DNA when steps (b) to (d) have been performed a predetermined number of times. Step (f) can determine the guide sequence contained in the guide RNA by, for example, amplifying the region containing the guide RNA incorporated into the genomic DNA recovered in step (d) by PCR, and then sequencing the amplified PCR product.

[0158] <Process g> The identification method includes (g) identifying the gene corresponding to the determined guide sequence as the gene corresponding to the desired phenotype. Step (g) can be carried out by, for example, searching a database containing information on the genomic DNA of the cells (cells of a specific species) in step (a) for a gene that contains a sequence showing a predetermined sequence identity (e.g., 90%, 95%, 97%, 98%, 99%, or 100%) with the guide sequence of the guide RNA determined in step (f), and identifying the found gene as the gene corresponding to the desired phenotype.

[0159] By carrying out the method for identifying genes corresponding to a target phenotype disclosed herein, a cell population rich in cells in which the genes corresponding to the target phenotype have been identified can be obtained. One embodiment of the present invention provides a method for producing a cell population rich in cells in which the genes corresponding to a target phenotype have been identified. Furthermore, by carrying out the method for identifying genes corresponding to a target phenotype disclosed herein, a cell population rich in cells in which the genes corresponding to the target phenotype have been identified, and which exhibit or do not exhibit the said phenotype, can be obtained. One embodiment of the present invention provides a method for producing a cell population rich in cells in which the genes corresponding to a target phenotype have been identified, and which exhibit or do not exhibit the said phenotype.

[0160] The definitions or specific examples of terms used herein shall apply as appropriate to the corresponding terms in any aspect or embodiment disclosed herein.

[0161] The following examples illustrate the present invention, but these examples do not limit the invention in any way. The compositions and methods described in the examples constitute part of the present invention.

[0162] [Examples] material and method In the embodiments described later, the following materials and methods were used. (cell culture) Mouse interleukin-3 (IL-3)-dependent Ba / F3 cells (Reference 24) were cultured in RPMI1640 (WAKO) containing 10% FCS (Gibco), antibiotic (Nacalai), 55 μM β2-mercaptoethanol, and 45 units / ml IL-3 (Reference 25). Human PLB985 cells were cultured in RPMI1640 containing 10% FCS, antibiotic, and 55 μM β2-mercaptoethanol. HEK293T and HCT116 (RIKEN BioResource Center) cells were maintained in DMEM (WAKO) containing 10% FCS and antibiotic.

[0163] (plasmid) DNA encoding mouse Xkr4 with monomeric EGFP tagged at the N-terminus and C-terminus, and its caspase-cleaved form Xkr4ΔC, was introduced into a lentiCas9-blast vector (hereinafter also referred to as "plenti") from which Cas9 and Blast had been removed (Reference 15, Addgene#52962). DNA encoding mouse Xkr4 (V5-Xkr4-FLAG) tagged with V5 at the N-terminus and FLAG tagged at the C-terminus, Xkr4ΔC, and Xkr4 mutants having point mutations (glutamine at position 332 is substituted with glutamic acid (Q332E), leucine at position 331 is substituted with phenylalanine (L331F), and isoleucine at position 322 is substituted with serine (I322S)) was introduced into a pNEF vector (Reference 10) and a pMXs-puro vector (Reference 27), respectively. Human Xkr4, Xkr8, and Xkr9 cDNAs were introduced into plenti vectors, which have V5 tagged at their N-terminus and possess an endogenous ribosome entry site (IRES)-driven puromycin resistance gene. V5-Xkr4-FLAG and V5-Xkr4ΔC-FLAG were similarly introduced into lentiviral vectors. Mouse Xkr4ΔC (SPOTXkr4ΔC-FLAG), tagged with SPOT at the N-terminus and FLAG at the C-terminus, and mouse Xkr4ΔC (Xkr4ΔC-RFP), tagged with V5 at the N-terminus and tagRFP (Evrogen) at the C-terminus, were inserted into plenti vectors.

[0164] Human XRCC4 cDNA was amplified by PCR using the first cDNA strand generated from PLB cells as a template. The following XRCC4 mutants were fused with tagRFP at the N-terminus or C-terminus in the plenti vector: wild-type (WT), caspase-uncleaved type (2DA), caspase-cleaved N-terminal fragment (ΔC, 1-265) and C-terminal fragment (ΔN, 266-336), deletion mutants (116-336, 156-336, 204-336, 246-336, 256-336, 1-305, 1-285, 256- 285) Mutants in nuclear localization signaling (arginine at position 270 is substituted with alanine (R270A), lysine at position 271 is substituted with alanine (K271A), arginine at position 272 is substituted with alanine (R272A), arginine at position 273 is substituted with alanine (R273A), and arginine at position 275 is substituted with alanine (R275A)). The self-cleaving 2A peptide (T2A;EGRGSLLTCGDVEENPGP (SEQ ID NO: 15)) sequence was introduced between the XRCC4 cDNA and TagRFP to create XRCC4-WT-2A-RFP or XRCC4-R270A-2A-RFP. When the short guide RNA (sgRNA) for XRCC4 is stably expressed in cells, a silencing mutation is introduced into the XRCC4-sgRNA (XRCC4 sgRNA target sequence in XRCC4 cDNA 5'-tggagactgatctttataa gcgg -3' (sequence number 16) becomes 5'-ttagaaacagatctatacaaa cgt Changed to -3' (sequence number 17). (The underlined part indicates the PAM sequence).

[0165] The FLAG sequence in lentiCas9-Blast was replaced with HA, and this was used to express Cas9-HA. Lentiviral expression vectors for sgRNAs against mouse Xkr4 (5'-gcggcgctgtgcctgcgcct-3': SEQ ID NO: 18), human XRCC4 (5'-tggagactgatctttataag-3': SEQ ID NO: 19), human APAF1 (5'-agcattgtagaatgatacgt-3': SEQ ID NO: 20), and human cytochrome C (5'-acagccgccaataagaacaa-3': SEQ ID NO: 21) were constructed using the plentiGuide-Puro vector (Reference 15, Addgene#52963). To establish XRCC4 knockout cells, sgRNA for XRCC4 was inserted into a plentiGuide(puro-) vector and constructed as follows: The EF1α promoter and puromycin resistance gene in plentiGuide-puro were removed by excision at the SmaI and MluI sites, blunt ends were generated using T4 polymerase, and then ligated with T4 ligase. The pX330 vector (reference 28) was used to express sgRNA for human CAD (5'-gaacatcgcggccgagaccc-3': SEQ ID NO: 22) and mouse Xkr8 (5'-cttagacgtggtcgtaggcc-3': SEQ ID NO: 23).

[0166] (Establishment of cell lines) Ba / F3 cells lacking TMEM16F were prepared (Reference 29). A pX330 vector encoding sgRNA for Xkr8 was electroporated into TMEM16F KO Ba / F3 cells, and limiting dilution was performed 3 days later. After recovering genomic DNA from each clone, the Xkr8 locating region in the genomic DNA was amplified for sequencing to confirm Xkr8 knockout (hereinafter referred to as "BDKO"). A pX330 vector encoding sgRNA for CAD was electroporated into CRISPR / Cas9-transfected PLB cells, and limiting dilution was performed to obtain single clones. CAD deletion was confirmed by genomic sequencing, Western blotting, and DNA fragmentation assays after apoptosis stimulation. XRCC4 knockout cells were established by infection with a lentivirus encoding sgRNA for XRCC4. Single clones were obtained by limiting dilution, and XRCC4 knockout was confirmed by genome sequencing and Western blotting using an anti-XRCC4 antibody.

[0167] Retroviruses were prepared by transfecting HEK293T cells with the retroviral vectors pMXs-puro, pGag-pol IRES-bsr (donated by Dr. Toshio Kitamura), and pCMV-VSV-G (RIKEN). 48 hours after transfection, the culture supernatant containing the retroviruses was collected, filtered, concentrated by centrifugation at 6,000 × g for 16 hours, and used to infect Ba / F3 cells. Stable transfectants were obtained by selection with 2.0 μg / ml puromycin.

[0168] Lentiviruses for the expression of sgRNA or cDNA of Cas9-HA, Xkrs, XRCC4, and their variants were prepared by transfecting HEK293T cells with the lentiviral vectors pCMV-VSVG-RSV-Rev (RIKEN) and pCAG-HIVgp (RIKEN). The culture supernatant containing the lentiviruses was collected, filtered, concentrated by centrifugation at 6,000 × g for 16 hours, and used to incubate BDKO, PLB, and HCT116 cells. In several experiments, stable transfectants were selected by culturing cells with 2.0 μg / ml puromycin or 10 μg / ml blastosidine.

[0169] (Stimulation of apoptosis by staurosporine (STS)) PLB cells, 5 × 10 5 Cells were seeded at a rate of cells / ml. The next day, the cells were counted and measured at 1 × 10⁻⁶. 6 The cells were resuspended in fresh medium preheated to 37°C to achieve a cell / ml concentration, and the cell suspension was incubated in a culture incubator for 2 hours. STS (LC Laboratories) was added to the cell suspension to a final concentration of 10 μM, and incubated for the specified time. In some experiments, the cells were pretreated with 20 μM Q-VD-OPh (Cayman), a pan-caspase inhibitor, 1 hour before apoptosis stimulation.

[0170] (Lipid scrambling assay) 1 x 10 6Cells were washed with HBSS (Gibco) (HBSS / Ca / Mg) containing 1 mM CaCl2 and MgCl2, resuspended in 500 μl of HBSS / Ca / Mg, and incubated on ice for 7 minutes. An equal volume of HBSS / Ca / Mg containing phosphatidylcholine conjugated with 1 μM nitrobenzoxadiazole (also known as "NBD-PC" herein, Avanti) was added to the cell suspension and incubated on ice for 10 minutes. Subsequently, HBSS / Ca / Mg containing 5 mg / ml fatty acid-free BSA (Sigma) was mixed with 1 μM DAPI (Dojindo) for 5 minutes to remove unincorporated NBD-PC. The residual fluorescence signal with incorporated NBD-PC was measured by flow cytometry in the non-necrotic cell population (DAPI-negative). Other parameters were adjusted to maintain the same ratio depending on the cell number. NBD-PC incorporation was performed at either an incubation temperature of 4°C or 15°C.

[0171] To observe the extracellular divalent cations necessary for lipid scrambling, cells were resuspended in 500 μl of HEPES buffer (10 mM Hepes-NaOH (pH 7.4) and 140 mM NaCl) with or without predetermined concentrations of CaCl2 and / or MgCl2, incubated on ice for 10 minutes, and then subjected to the lipid scrambling assay. An equal volume of HEPES buffer containing 1 μM NBDPC was added and incubated on ice for a predetermined time. After incubation with NBD-PC, the samples were mixed with HEPES buffer containing 5 mg / ml fatty acid-free BSA and 1 μM DAPI and analyzed by flow cytometry. In some experiments, 2 μM A23187 (Sigma) was added to the cell suspension in the buffer containing NBD-PC and incubated on ice for 4 minutes.

[0172] (Measurement of intracellular calcium influx) BDKO cells expressing 2.5 million Xkr4ΔC Q332E were pretreated with 1 μM Fluo4-AM (Invitrogen) in RPMI1640 containing 10% FBS and incubated at 37°C for 30 minutes. After incubation, 5 × 10⁶ cells were obtained. 5 The cells were washed once with HEPES buffer and resuspended in HEPES buffer containing 0.125 mM or 2 mM calcium. After incubation on ice for 10 minutes, the cells were mixed for 1 minute with HEPES buffer containing 10 μg / ml PI (Dojindo), either with or without 2 μM A23187. The Fluo4 signal was observed using a flow cytometer.

[0173] (Annexin V staining) PS exposure was measured by staining with Annexin V-Cy5 (BioVision). 5 × 10 5 Cells were resuspended in 200 μl of ice-cold annexin buffer (10 mM Hepes-NaOH (pH 7.4), 140 mM NaCl, 2.5 mM CaCl2) and mixed with an equal volume of annexin buffer containing annexin V-Cy5. After incubation on ice for 15 minutes, DAPI was added to a final concentration of 1 μM. The annexin V-Cy5 signal in the non-necrotic cell population (DAPI-negative) was analyzed by flow cytometry.

[0174] (Construction of cDNA libraries and identification of Xkr4ΔC variants) To prepare the cDNA library, the total RNA was collected using the RNeasy mini-kit (Qiagen) for 10 minutes. 7PCR was prepared from individual PC6 cells, and mRNA purification was performed using the Oligotex-dT30 Super mRNA Purification Kit (Takara). A cDNA library was constructed from the purified mRNA using the In-Fusion SMARTer Directional cDNA Library Construction Kit (Clontech), with some modifications to the instructions for use. Briefly, the mRNA was applied to the first cDNA strand generation by reverse transcriptase using the In-Fusion SMARTer CDS primers and SMARTer oligos included in the kit, and then the second cDNA strand was generated using Primestar GXL DNA Polymerase (Takara) with primers 5'-AAGCAGTGGTATCAACGCAGAGT-3' (SEQ ID NO: 24) and 5'-CGGGGTACGATGAGACACCA-3' (SEQ ID NO: 25). The amplified cDNA was loaded onto a 1% agarose gel, stained with ethidium bromide, and separated into two parts under LED light: low molecular weight (LMW) (1.0–2.5 kbps) and high molecular weight (HMW) (2.5–6.0 kbps). The cDNA was purified from the excised gel and inserted into NcoI-cut pMXs-HiFi vectors using a NEBuilder HiFi DNA assembly (New England Biolabs) (Reference 30). The cells were electroporated into ElectroMAX DH10B competent cells (Thermo Fisher Scentific) and seeded onto 10 15 cm Lysogeny broth (LB) agar plates for each of the HMW and LMW cDNA libraries. The following day, colonies were collected in LB medium and applied to Maxiprep (Qiagen). The clone counts for each library were 21 million for HMW and 6.1 million for LMW. The pMXs-HiFi vector was constructed by inserting the following sequence into the BamHI and SalI sites of the pMXs vector (Reference 27) and then annealing them. 5'-ggatcccagaagcagtggtatcaaccatggtctcatcgtaccccgccagcacagtggtcgac-3'(Sequence ID 26)

[0175] (Screening of cDNA library) The HMW cDNA library was transfected into HEK293T cells (2×10 6 cells each) on 10 10-cm plates using the retroviral vectors pGag-pol IRES-bsr and pCMV-VSV-G. Two days after transfection, the supernatant was collected, filtered through a 0.22-μm filter, and centrifuged (6,000×g, 16 h, 4°C). The next day, the virus pellet was resuspended in the medium at 20-fold the concentration of the supernatant and mixed with BDKO cells at a concentration of 1×10 6 cells / ml in the presence of 8 μg / ml polybrene (Nacalai). Six hours after infection, the medium was replaced with fresh medium, and the cells were cultured for 3 days and applied to the lipid scramble assay. Then, sorting was performed using a flow cytometer FACS AriaII (Beckton Dickinson). After several rounds (3 - 4 times) of sorting, genomic DNA was prepared from 10 7 cells by phenol / chloroform extraction described below, and the integrated cDNA was amplified by PCR using Primestar GXL DNA polymerase and primers 5’-cccatatggccatatgagatctta-3’ (SEQ ID NO: 27) and 5’-caaaatggcgttacttaagctagc-3’ (SEQ ID NO: 28). Then, the amplified product was inserted into pCX4BSR (Reference 31) digested with BglII and NheI using NEBuilder. After electroporation into Escherichia coli DH10B, the second cDNA library was purified and applied to the next round of screening. After screening, genomic DNA was prepared and applied to PCR amplification of the integrated cDNA, excised from the agarose gel, and inserted into the pCX4BSR vector. The sequence inserted into the vector was sequenced, and the obtained sequence was analyzed by Blast search.

[0176] (Genomic DNA purification) After cDNA library screening, genomic DNA was prepared as follows. Briefly, 10 million cells were washed twice with PBS, resuspended in 1 ml of Genome Lysis Buffer (100 mM Tris-HCl (pH 8.5), 200 mM NaCl, 5 mM EDTA, 0.2% SDS) containing 100 μg / ml of proteinase K, and incubated at 55°C for 3 hours. Then, an equal volume of isopropanol was added and the cells were gently inverted. The resulting spool of DNA was collected, washed with 70% ethanol, resuspended in 1 ml of TE containing 50 μg / ml of RNase A, incubated at 37°C for 1 hour, and rotated overnight at room temperature. The following day, TE-saturated phenol was added to the DNA solution and mixed well. The supernatant was collected by centrifugation and then subjected to another TE-saturated phenol extraction. Next, the supernatant containing the DNA was mixed with an equal volume of phenol / chloroform / isoamyl alcohol and centrifuged. The supernatant (approximately 500 μl) was mixed with 1 / 10 the volume of 3M sodium acetate and twice the volume of 100% ethanol, and gently inverted. The sloping DNA was collected, washed with 70% ethanol, resuspended in 100 μl of TE, incubated at 65°C for 10 minutes, and stored at 4°C.

[0177] (Establishment of cells with strong lipid scrambling activity) To perform a lipid scrambling assay, BDKO cells were transfected with V5-Xkr4ΔCFLAG and Cas9-HA. 40 million cells were subjected to a lipid scrambling assay using a final concentration of 1 μM NBD-PC. After incubation with NBD-PC at 15°C for 7 minutes, lipids in the outer layer of the cell membrane were removed with fatty acid-free BSA for 5 minutes. Cells that had incorporated NBD-PC were sorted using FACS Aria II, and dead cells stained with DAPI were gated out. The collected cells were unfolded and subjected to subsequent sorting. Sorting was repeated six times to obtain cells with strong lipid scrambling activity. The obtained high lipid-scrambling cells were referred to as PC6.

[0178] (Revival screening using a CRISPR sgRNA library) CAD KO PLB cells expressing Cas9-HA and Xkr4ΔC-RFP were infected with the Gecko sgRNA library A&B (Reference 15, Addgene#1000000049) with an infection efficiency of approximately 40%. The cells were cultured with 2.0 μg / ml puromycin for 2 days and without puromycin for 3 days, after which 5 × 10⁶ cells were transferred to 150 ml of fresh medium. 5 The cells were seeded at a concentration of cells / ml. The following day, 20 million PLB cells in 20 ml of medium were cultured in a culture incubator for 3 hours, stimulated with 20 μl of 10 mM STS (final concentration 10 μM), and incubated for 3 hours. Subsequently, the cells were subjected to an NBD-PC uptake assay, and cells lacking NBD-PC uptake were collected by flow cytometry. Approximately 50,000 cells were collected in one experiment, and this was performed 5 times a day: total 1 × 10⁶ cells. 8A total of 270,000 cells were subjected to primary screening. These cells were collected by flow cytometry and applied to the Geno Plus Genomic DNA Extraction Kit (Viogene). The resulting genomic DNA (3 μg total) was subjected to PCR, and the incorporated sgRNA encoding was amplified using Primestar GXL polymerase and primers 5'-gttttaaaatggactatcatatgc-3' (SEQ ID NO: 29) and 5'-tatccatctttgcacccgggc-3' (SEQ ID NO: 30) on a 25 μl scale for 32 tubes. PCR conditions were as follows: denaturation at 95°C for 3 minutes, followed by 28 cycles of 10 seconds at 98°C, 15 seconds at 55°C, and 30 seconds at 68°C, and a final extension of 30 seconds at 68°C. The PCR reaction mixture was pooled in one 1.5 ml tube, and 160 μl was loaded onto an agarose gel to excise a 425 bps band. The PCR product was purified using a Gel / PCR extraction kit (Fastgene), mixed with plentiGuide-puro vector (250 ng) cleaved with NdeI and SmaI, incubated with NEBuilder at 50°C for 1 hour, and subjected to ethanol precipitation. The precipitate was then resuspended in 157.5 μl of 10% glycerol, and 103 μl of the solution was mixed with 63 μl of DH10B. Three 70 μl mixtures were transferred to electroporation cuvettes, pulsed at 2700 V using a NEPA Porator (Nepa Gene), resuspended in 5 ml of SOC, cultured at 37°C for 2 hours with gentle shaking, and seeded onto 15 cm LB plates (1 ml per plate). The following day, colonies were counted (2.2 million clones), collected in PBS, and subjected to Midiprep kit (Roche). The obtained plasmids were used to generate lentiviruses, which were then subjected to sorting in the next round, and this process was repeated until a clear population of NBD-PC-negative cells emerged.

[0179] Cells that were negative for NBD-PC uptake and had caspase-3 activity were collected by flow cytometry as follows: An sgRNA library was infected with CAD KO PLB cells expressing Xkr4ΔC-RFP at a low infectivity (10%), and a single sgRNA lentivirus was introduced into each cell. After 1 week of puromycin selection, RFP-positive and PC uptake-negative cells were collected by flow cytometry, fixed with 1% paraformaldehyde in PBS for 2 minutes, permeabilized with methanol, stained with anti-activated caspase-3 antibody, and selected for caspase-3 activity.

[0180] (Localization study) The localization of the EGFP fusion of Xkr4 or the tagRFP fusion of XRCC4 was observed using a Nikon A1 confocal microscope with a CFI Plan Apo 100× oil immersion objective lens, 1.40 NA, or a CFI Plan Apo 40× dry objective lens, 0.95 NA. To visualize the localization of the tagRFP fusion of XRCC4, PLB cells expressing XRCC4 and its variants were treated with or without 10 μM MSTS for 2.5 hours and resuspended in ice-cold HBSS containing 5 mg / ml fatty acid-free BSA and 20 μM DRAQ5 (BioStatus). Before observation, cells were seeded in a glass-bottom dish (IWAKI) and fluorescence images were acquired. Differential interference contrast (DIC) images were also acquired to visualize cell morphology. Images were processed with imageJ1.52p.

[0181] (DNA fragmentation assay) 5 x 10 5PLB cells and CAD KO cells were stimulated with 10 μM STS for 4 hours. Cells were collected, washed once with PBS, lysed in 500 μL of Genome Lysis buffer containing 100 μg / ml Proteinase K, and incubated overnight at 55°C. To precipitate genomic DNA, 500 μL of isopropanol was added and mixed, and the mixture was centrifuged at 20,000 × g for 5 minutes. The precipitate was rinsed with 70% ethanol and resuspended in 20 μL of TE buffer. Samples were loaded onto a 1% agarose gel, and DNA fragmentation was visualized by staining with ethidium bromide under ultraviolet light.

[0182] (Electroporation of synthetic peptides) Synthetic peptides corresponding to amino acids 266-285 of XRCC4 (C20:IAPSRKRRQRMQRNLGTEPK (SEQ ID NO: 7)), C20 with methionine at the N-terminus (C21:MIAPSRKRRQRMQRNLGTEPK (SEQ ID NO: 13)), C20 with arginine at position 270 substituted with alanine (C20-R270A:IAPSAKRRQRMQRNLGTEPK (SEQ ID NO: 14)), and C20 with lysine at position 271 substituted with alanine (C20-K271A:IAPSRARRQRMQRNLGTEPK (SEQ ID NO: 8)) were obtained from SCRUM, Inc. (Tokyo, Japan). These peptides were mixed at a final concentration of 10 μM in 100 μl of Opti-MEM (Gibco) at a concentration of 1 × 10⁶ 6 It was added to the cells. Electroporation was performed using ELEPO21 (Nepa Gene). After electroporation, the cells were immediately mixed with RPMI1640 containing 10% FBS that had been preheated to 37°C. Half of it (5 × 10) was mixed. 5 The cells were stained with Annexin V-Cy5 as described above and analyzed by flow cytometry.

[0183] The localization of C20 was observed in HCT116 expressing Xkr4WT-GFP or Xkr4ΔCGFP. In short, 2 × 10 4 The cells were treated with pepsin-solubilized rat collagen type I (Nippi) at a concentration of 5 μg / cm³. 2C20 cells were seeded in a 24-well plate with a coated glass bottom (IWAKI) and cultured for 2 days. Tetramethylrhodamine-labeled C20 (C20-TMR) was diluted in 300 μl of Opti-MEM to a final concentration of 2 μM and electroporated using an ELEPO21 equipped with a CUY900-13-3-5 adherent cell electrode. After electroporation, the cells were fixed in 4% PFA in PBS with RT for 10 minutes and observed under a confocal microscope.

[0184] (Blue Native PAGE (BN-PAGE)) BN-PAGE was performed using the NativePAGE Novex Bis-Tris Gel System (Life Technologies). To prepare the solubilized film fraction, 1 × 10⁻⁶ 8The cells were resuspended in 6 ml of chilled buffer A (10 mM Tris-HCl (pH 7.4), 1 mM CaCl2, 1 mM p-APMSF, 1 mM NaF) and disrupted using Dounce homogenization on ice (60 strokes). Then, an equal volume of chilled buffer B (10 mM Tris-HCl (pH 7.4), 0.1 M NaCl, 0.5 M sucrose, 1 mM CaCl2, 10 mM MgCl2, 1 mM p-APMSF, 1 mM NaF) was added, and the mixture was centrifuged at 800 × g at 4 °C for 10 minutes. The supernatant was collected and further centrifuged at 8,000 × g at 4 °C for 10 minutes. After removing the pellet, the supernatant was ultracentrifuged at 100,000 × g at 4 °C for 50 minutes. The pelletized membrane fraction was dissolved by incubation at 4°C for 3 hours with a solubilization buffer (20 mM Tris-HCl (pH 7.4), 100 mM 6-aminocaproic acid, 50 mM NaCl, 10% (vol / vol) glycerol, 1 mM CaCl2, 1 mM p-APMSF, protease inhibitor cocktail (Nacalai), 1 mM NaF, and 1% n-dodecyl-β-D-maltoside (DDM) (Dojindo) / 0.01% cholesteryl hemisuccinate (CHS) (Sigma)). Insoluble substances were removed by centrifugation at 4°C and 20,000 × g for 30 minutes. The amount of the solubilized membrane fraction was measured using the BCA Protein Assay Kit (Thermo Fisher Scientific) and adjusted to 0.6 μg / μl using the solubilization buffer. Next, the samples were loaded onto NativePAGE Novex 4-16% (wt / vol) Bis-Tris gels and separated by electrophoresis at 150V for 35 minutes at 4°C. The CBB G-250 concentration in the cathode buffer was changed from 0.02% to 0.002%, and the samples were further electrophoresed at 150V for 120 minutes. The gels were incubated at RT for 20 minutes using SDS running buffer (25mM Tris, 190mM glycine, 0.1% SDS, (pH 8.3)), and then transferred to a PVDF membrane at 0.1A for 1 hour for Western blotting.

[0185] (SDS-PAGE)Whole cell lysates were prepared by incubating them at 4°C for 1 hour using either a solubilizing buffer containing a protease inhibitor cocktail (Nacalai) (20 mM Tris-HCl (pH 7.4), 100 mM 6-aminocaproic acid, 50 mM NaCl, 10% (vol / vol) glycerol, 1 mM CaCl2, 1 mM p-APMSF, 1 mM NaF, and 1% DDM / 0.01% CHS) or RIPA buffer (50 mM Hepes-NaOH buffer (pH 8.0), 150 mM NaCl, 1% NP-40, 0.1% SDS, and 0.5% sodium deoxycholate). After incubation, the samples were centrifuged at 20,000 × g for 20 minutes at 4°C. The supernatant was collected, and the protein concentration was measured using the BCA Protein Assay Kit. Next, the samples were adjusted to 0.1–1.0 μg / μl using a solubilizing buffer and a 5× SDS sample buffer (200 mM Tris-HCl (pH 6.8), 10% SDS, 25% glycerol, 5% mercaptoethanol, 0.05% bromophenol blue), and loaded onto a 10% polyacrylamide gel (BIOCRAFT). The samples were electrophoresed at 35 mA for 40 minutes and transferred to a PVDF membrane at 0.1 A for 60 minutes. The membrane was then probed using a 5,000-fold dilution of anti-V5-HRP (Invitrogen), a 5,000-fold dilution of anti-FLAG-HRP (Sigma), a 4,000-fold dilution of anti-tRFP rabbit pAb (Evrogen, AB233), a 2,000-fold dilution of anti-XRCC4 mouse mAb (SantaCruz, C-4), a 500-fold dilution of anti-CAD mouse mAb (SantaCruz, F-11), a 4,000-fold dilution of anti-cleavage caspase-3 rabbit mAb (Cell Signaling Technology #9661), or a 5,000-fold dilution of antiserum against Xkr4 produced from rabbits immunized with the N-terminus (amino acids 1-110) of GST-Xkr4. Subsequently, probes were created using a 10,000-fold dilution of HRP-conjugated goat anti-mouse or rabbit IgG (Dako). The chemiluminescence signal was detected using the Immobilon Western chemiluminescent HRP substrate (Millipore) with the FUSION chemiluminescence imaging system (Vilber).The total protein on the PVDF membrane after chemiluminescence was visualized by CBB staining (WAKO). The membrane was incubated with CBB staining buffer (0.25% CBB R250, 50% methanol, 10% acetic acid) at room temperature for 2-30 minutes, and then washed with destaining buffer (10% methanol and 10% acetic acid).

[0186] (Immunoprecipitation) The SPOT tag (Reference 32) was fused to the N-terminus of Xkr4. Ten million XRCC4 KO PLB cells expressing SPOT-Xkr4ΔC-FLAG and XRCC4-RFP were harvested by centrifugation at 400g for 5 minutes, stimulated with apoptosis using 10 μM STS at 37°C for 2.5 hours, and washed with annexin buffer. The membrane fraction was then prepared and solubilized as described above. The membrane lysate was incubated with anti-SPOT nanobody-bound magnetic agarose beads (SPOT-Trap, ChromoTek) and rotated at 4°C for 2 hours. The beads were washed three times with cooled washing buffer (20 mM Tris-HCl (pH 7.5), 100 mM 6-aminocaproic acid, 50 mM NaCl, 1 mM CaCl2, and 0.03% DDM / 0.003% CHS), and then subjected to mass spectrometry or eluted by boiling at 95°C for 5 minutes in sample buffer (40 mM Tris-HCl (pH 6.8), 2% SDS, 5% glycerol, 0.01% bromophenol blue). The eluted samples were analyzed by SDS-PAGE and Western blotting.

[0187] (mass spectrometry) After immunoprecipitation, the magnetic agarose beads SPOT-Trap were washed twice more with 50 mM ammonium bicarbonate. The proteins on the beads were digested with 200 ng of trypsin / Lys-C mix (Promega) at 37°C for 16 hours. The digested product was reduced, alkylated, acidified, and desalted using GL-Tip SDB (GL Sciences). The eluate was evaporated using a SpeedVac concentrator and dissolved in 0.1% trifluoroacetic acid and 3% acetonitrile (ACN). LC-MS / MS analysis of the obtained peptides was performed using an EASY-nLC 1200 UHPLC connected to an Orbitrap Fusion mass spectrometer via a nanoelectrospray ion source (Thermo Fisher Scientific). Peptides were separated using a 75 μm × 150 mm 18 reverse-phase column (Nikkyo Technos) by applying a direct ACN gradient of 4–32% over 0–100 minutes, followed by increasing the ACN to 80% over 10 minutes. The mass spectrometer was operated in data-dependent acquisition mode with a maximum duty cycle of 3 seconds. MS1 spectra were obtained with a resolution of 120,000 and a resolution of 4 × 10⁶. 5 The automatic gain control (AGC) target was used, and measurements were taken in the mass range of 375–1,500 m / z. The HCD MS / MS spectra were obtained with the AGC target at 1 × 10⁻⁶. 4The proteins were acquired using a linear ion trap with a separation window of 1.6 m / z, a maximum injection time of 100 ms, and a normalized collision energy of 30. Dynamic exclusion was set to 20 seconds. Raw data were directly analyzed against the SwissProt database, which was limited to human (H. sapiens) and contained mouse Xkr4 protein, using Proteome Discoverer version 2.3 (Thermo Fisher Scientific) and the SequestHT search engine. The search parameters were as follows: (a) trypsin as an enzyme with a maximum of two missed cleavages; (b) precursor mass tolerance of 10 ppm; (c) fragment mass tolerance of 0.6 Da; (d) cysteine ​​carbamide methylation as a fixed modification; and (e) protein N-terminal acetylation and methionine oxidation as variable modifications. Peptides were filtered using a percolator node with a false find rate of 1%. Unlabeled precursor ion quantification was performed using a precursor ion quantification node, and the results were normalized so that the sum of abundance values ​​for each sample across all peptides was equal.

[0188] Selected peptides from human XRCC4 and mouse Xkr4 were measured by PRM, a high-resolution MS / MS-based targeted quantification method. LC-MS / MS analysis was performed on an EASY-nLC 1200 UHPLC connected to a Q Exactive Plus mass spectrometer via a nanoelectrospray ion source (Thermo Fisher Scientific). Target MS / MS scans were performed with a resolution of 70,000 and an AGC target of 2 × 10⁶. 5 The separation window was 4.0 m / z, the maximum injection time was 2 seconds, and the normalized collision energy was obtained using a time-scheduled inclusion list of 27. Transition time alignment and relative quantification were performed using PinPoint version 1.4 (Thermo Fisher Scientific).

[0189] (Mapping of NGS data) The raw data was received as a fastq file (containing sgRNA sequences and barcode sequences) and two reference CSV files (containing reference sgRNA sequences). In the reference files, each reference sgRNA sequence is listed according to the original gene from which it was obtained and assigned a unique identification tag (UID). In all cases, six unique reference sgRNA sequences (with different UIDs) originated from the same gene.

[0190] The fastq file and reference CSV file were processed using three in-house computer codes. The first code (reference 33) removes barcode sequences from the genes in the fastq file and then converts all sequences (both genes in the fastq file and sgRNA sequences in the reference file) into integer sequences (cytosine=1, adenine=2, etc.). This code outputs two lists of integer sequences, one for the genes contained in the fastq file and the other for the sgRNAs contained in the two reference files. These outputs are then read by the second code (written in C++), which counts how many times each sgRNA appears in the genes in the fastq file within the reference list. These counts are then converted to the third code (written in R), which creates a spreadsheet showing how many times each sgRNA appears in the genes in the fastq file within the reference list. To create this spreadsheet, we first group the reference sgRNAs according to the genes from which they were originally obtained. Next, the group is ranked according to the number of reference sgRNAs that appeared at least once in the genes of the fastq file.

[0191] The mapping procedure described above utilizes R's ability to read and process string data (raw gene sequences) and C++'s speed when looping through large amounts of integer data.

[0192] [Example 1] Xkr4 mutants that constitutively exhibit lipid scrambling activity During apoptosis, the C-terminus of Xkr4 is cleaved by caspases. The cleaved Xkr4 dimerizes, generating scramblase activity. This exposes phosphatidylserine (PS), located on the inner side of the cell membrane, to the outer side. However, cleavage of the C-terminus of Xkr4 alone is not sufficient to expose PS to the outer side of the cell membrane (Reference 12).

[0193] PLB cells were transfected with a plasmid containing the gene encoding wild-type Xkr4 (Xkr4WT) and a plasmid containing the gene encoding Xkr4 with its C-terminus cleaved by caspase (also referred to herein as "Xkr4ΔC") (Reference 15), respectively, to prepare transfectants that constitutively express Xkr4WT and Xkr4ΔC.

[0194] PLB cells and the transfectants were stimulated with staurosporine (STS) for apoptosis, and the uptake of phosphatidylcholine conjugated with nitrobenzoxadiazole (NBD-PC) by each cell was measured using flow cytometry (Figures 1(a)-(c)) (see (STS-mediated apoptosis stimulation) and (lipid scramble assay) in "Materials and Methods" above).

[0195] In PLB cells (parental), phosphatidylcholine (PC) uptake into cells was not observed in either the control group (without STS) or the STS-induced group (Figure 1(a)). In transfectants expressing Xkr4WT (hereinafter also referred to as "Xkr4WT-expressing cells"), PC uptake into cells was observed when stimulated with STS (Figure 1(b) STS). These results indicate that Xkr4WT generates lipid scramblase activity in response to apoptosis.

[0196] In transfectants expressing Xkr4ΔC (hereinafter also referred to as "Xkr4ΔC-expressing cells"), no uptake of PC into the cells was observed when apoptosis was not stimulated by STS (Figure 1(c) Control). This result indicates that, in cellular PC uptake, C-terminal cleavage of Xkr4 alone is not sufficient to generate scramblerase activity for taking up PC added to the culture medium. When Xkr4ΔC-expressing cells were stimulated by apoptosis with STS, PC uptake into the cells was observed (Figure 1(c) STS). This result indicates that Xkr4ΔC generates lipid scrambling activity upon apoptosis stimulation, similar to Xkr4WT, which generates scrambling activity upon apoptosis stimulation.

[0197] Annexin V staining was performed on PLB cells (parental), Xkr4WT, and Xkr4ΔC-expressing transfectants (Figures 1(d)-(f)) (see (Annexin V staining) in "Materials and Methods" above). Figures 1(e) and (f) show that PS is exposed in the transfectants Xkr4WT and Xkr4ΔC that undergo PC uptake (Figures 1(b) and (c)). These results indicate that Xkr4ΔC causes PS exposure not only through PC uptake but also through apoptosis stimulation.

[0198] PLB cells expressing Xkr4WT-GFP and Xkr4ΔC-GFP, respectively, in which EGFP was tagged to the C-terminus of Xkr4WT and Xkr4ΔC, were prepared. After apoptosis stimulation by STS, Xkr4ΔC was localized on the cell membrane, similar to Xkr4WT (Figure 2). This result indicates that Xkr4ΔC, like Xkr4WT, generates lipid scrambling activity on the cell membrane upon apoptosis stimulation.

[0199] PLB cells expressing Xkr4WT or Wkr4ΔC were cultured with the total caspase inhibitor Q-VD-OPh, and then stimulated with apoptosis using STS for 3 hours (Figure 3). Xkr4WT-expressing cells did not exhibit lipid scrambling activity upon apoptosis stimulation by STS when pretreated with a caspase inhibitor (Figures 3(a) and (b)). Similar results were obtained with Wkr4ΔC-expressing cells (Figures 3(c) and (d)). The results shown in Figures 1 to 3 indicate that Xkr4ΔC, like Xkr4WT, generates lipid scrambling activity on the cell membrane via caspases induced by apoptosis stimulation.

[0200] Cell membrane lysates of PLB cells expressing Xkr4WT or Wkr4ΔC were prepared and biochemically analyzed by blue native PAGE (BN-PAGE) or SDS-PAGE (Figure 4). BN-PAGE biochemical analysis and Western blotting with anti-Xkr4 antibody showed that before apoptosis stimulation, Xkr4WT existed as monomers and Xkr4ΔC existed as dimers (Figure 4(a)). SDS-PAGE biochemical analysis showed that Xkr4 or Xkr4ΔC had their respective molecular weights (Figure 4(b)). These results indicate that Xkr4ΔC, like Xkr4WT, forms dimers on the cell membrane, but not sufficiently to generate lipid scrambling activity.

[0201] (Establishment of cells with strong lipid scrambling activity) To understand how Xkr4 is activated, we obtained cells exhibiting constitutive lipid scrambling activity. Transfectants stably expressing Xkr4ΔC were prepared in Ba / F3 cells (BDKO) lacking two ubiquitous scramblases, Xkr8 (Reference 10) and TMEM16F (Reference 9). A lipid scrambling assay was performed on transfectants grown by culture, and transfectants with high fluorescence intensity derived from NBD-PC, i.e., cells that take up PC, were recovered by flow cytometry. This process was repeated six times (Figure 5(a)). By repeating the above process, transfectants with high fluorescence intensity derived from NBD-PC were obtained (Figure 5(b)). Each graph in Figure 5(b) shows the flow cytometry results corresponding to the number of times the above process was performed (PC0 to PC5), and the rectangular areas enclosed by solid lines in each graph indicate cells that take up PC (PC-uptake-positive cells) in the non-necrotic cell population.

[0202] As the number of repetitions of the above process increased (from PC0 to PC5), the number of transfectants included in this region increased. Starting with a transfectant expressing Xkr4ΔC (Figure 6(a)), the above process was repeated six times to obtain cells (PC6) that constitutively exhibited lipid scrambling activity without apoptosis stimulation (Figure 6(b)). PC6 cells were infected with an sgRNA lentivirus against Xkr4. No PC uptake was observed in the resulting infected PC6 cells (PC6+sgXrk4) (Figure 6(c)). The entire cell lysate of PC6 or PC6+sgXkr4 was subjected to SDS-PAGE, and Western blotting (WB) was performed using an anti-V5 antibody (Figure 6(d)). V5-Xkr4ΔC-FLAG was detected by the WB. The results of this WB indicate that PC6+sgXrk4 does not express Xkr4. These results indicate that constitutive lipid scrambling activity in PC6 cells is dependent on Xkr4.

[0203] (Construction of cDNA libraries and identification of Xkr4ΔC variants) The constitutive lipid scrambling activity dependent on Xkr4 in PC6 cells may be due to mutations introduced into the gene encoding an endogenous protein that acts on Xkr4ΔC. Total RNA was extracted from PC6 cells, and a first cDNA strand was generated using reverse transcriptase. The obtained first cDNA was used to generate a second cDNA strand by PCR using primers 5'-AAGCAGTGGTATCAACGCAGAGT-3' (SEQ ID NO: 31) and 5'-CGGGGTACGATGAGACACCA-3' (SEQ ID NO: 32). The obtained second cDNA was separated into low molecular weight (LMW) (1.0-2.5 kbps) cDNA and high molecular weight (HMW) (2.5-6.0 kbps) cDNA by agarose gel electrophoresis. ElectroMAX DH10B was transduced using plasmids containing LMW and HMW, respectively. As a result, a cDNA library of LMW containing 6.1 million transdermal clones was obtained for LMW, and a cDNA library of HMW containing 21 million transdermal clones was obtained for HMW.

[0204] (cDNA library screening) The first cDNA library of HMW was transfected into HEK293T cells using the retroviral vectors pGag-pol IRES-bsr and pCMV-VSV-G. Retroviruses were recovered from the transfectants. The recovered retroviruses infected BDKO cells to obtain a 1LPC0 cell population. The 1LPC0 cell population was subjected to a lipid scramble assay, and cells with lipid scramble activity (0.6%) were obtained by flow cytometry (Figure 7(a)). The obtained cell population was cultured and subjected to the lipid scramble assay again to obtain cells with lipid scramble activity. This process was repeated three times to obtain cells with lipid scramble activity (91.9%) in a 1LPC3 cell population (Figure 7(b)). Genomic DNA was prepared from the 1LPC3 cell population (see (Genomic DNA Generation) described in "Materials and Methods" above), and cDNA derived from the first HMW cDNA library incorporated into the genomic DNA was amplified by PCR using primers 5'-cccatatggccatatgagatctta-3' (SEQ ID NO: 33) and 5'-caaaatggcgttacttaagctagc-3' (SEQ ID NO: 34). Agarose gel electrophoresis of the resulting PCR fragments revealed several bands. Therefore, a second cDNA library was prepared using these PCR fragments.

[0205] A retrovirus was prepared from the second cDNA library in the same manner as the first cDNA library, and BDKO cells were infected with the prepared virus to obtain a 2LPC0 cell population. The 2LPC0 cell population was subjected to a lipid scramble assay, and cells with lipid scramble activity (11.7%) were obtained by flow cytometry (Figure 7(c)). The obtained cell population (2LPC1) was cultured and subjected to a lipid scramble assay, resulting in 92.5% of cells having lipid scramble activity (Figure 7(d)). The cDNA from the second cDNA library, which was incorporated into the genomic DNA of the cells obtained from the 2LPC1 cell population, was amplified by PCR. Agarose gel electrophoresis of the obtained PCR fragments revealed a nearly single band. The obtained PCR fragments were sequenced. As a result, all identified genes encoded Xkr4ΔC mutants with a single point mutation (I322S, L331F, or Q332E) in Xker4. These results indicate that the constitutive lipid scrambling activity dependent on Xkr4 in PC6 is caused by changes in Xkr4ΔC itself due to the identified point mutations.

[0206] Xkr4 has 1 to 10 transmembrane regions, I322S is a mutation in the 4th transmembrane region, and L331F and Q332E are mutations in the 5th transmembrane region.

[0207] [Example 2] Characteristics of constitutively active Xkr4 mutants Transfectants expressing full-length (FL) Xkr4, Xkr4ΔC (also referred to as "ΔC"), Xkr4ΔC with the aforementioned point mutation (referred to as "ΔC I322S", "ΔC L331F", and "ΔC Q332E", respectively), and FL Q332E, which has the point mutation Q332E in full-length Xkr4, were prepared in BDKO cells (parental).

[0208] A lipid scramble assay was performed on the prepared transfectants (Figure 8(a)). In this assay, apoptosis stimulation with STS was not applied. After starting the lipid scramble assay, an increase in PC uptake was observed over time in BDKO cells expressing ΔC I322S, ΔC L331F, and ΔC Q332E (Figure 8(a)). Annexin V staining was performed on the prepared transfectants to test whether PS, which is located on the inside of the cell membrane, is exposed on the outside of the cell membrane (Figure 8(b)) (see (Annexin V staining) in "Materials and Methods" above). Figure 8(b) shows that PS is exposed in transfectants with increased PC uptake (ΔC I322S, ΔC L331F, and ΔC Q332E). In full-length Xkr4 with Q332E (FL Q332E), neither PC uptake nor PS exposure was observed (Figures 8(a) and (b)). These results indicate that, in combination with C-terminal cleavage of Xkr4 and identified point mutations (ΔC I322S, ΔC L331F, or ΔC Q332E), Xkr4-mediated lipid scrambling activity and PS exposure occur even in living cells.

[0209] Total cell lysates were prepared from the transfected cells and applied to Western blotting using an anti-V5 antibody to detect V5-Xkr4-FLAG after SDS-PAGE to detect Xkr4. The results showed that each transfected cell expressed Xkr4 with the corresponding molecular weight (upper panel of Fig. 9(a)). Total cell lysates were prepared from transfected cells expressing Xkr4FL, Xkr4FL with Q332E (Xkr4FL-Q332E), Xkr4ΔC, and Xkr4ΔC with Q332E (Xkr4ΔC-Q332E), respectively, and applied to Western blotting using an anti-V5 antibody to detect V5-Xkr4-FLAG after BN-PAGE to detect Xkr4. As a result, Xkr4ΔC-Q322E formed a dimer, similar to Xkr4ΔC (upper panel of Fig. 9(b)). This indicates that the dimerization by Xkr4ΔC is not affected by the identified point mutation Q322E. The results shown in Figs. 8 and 9 indicate that Xkr4 that forms a dimer by C-terminal cleavage can be activated by a change in its three-dimensional structure due to the identified point mutation. These results suggest that Xkr4 that forms a dimer by C-terminal cleavage may acquire lipid scrambling activity due to a structural change regulated by some activator.

[0210] Using Xkr4 mutants with the identified point mutations, components essential for lipid scrambling activity are identified. Xkr4ΔC Q332E-expressing cells in a buffer containing neither calcium ions nor magnesium ions did not take up PC even when NBD-PC was added to the buffer (Fig. 10(a)). By including calcium ions in the buffer, Xkr4ΔC Q332E-expressing cells in the buffer took up PC (Figs. 10(c) and (d)). Increasing the concentration of calcium ions included in the buffer increased the amount of PC taken up by Xkr4ΔC Q332E-expressing cells (Fig. 10(e)).

[0211] Transfectants expressing Xkr4ΔC Q332E were mixed with NBD-PC under conditions containing extracellular calcium ion concentrations of 0 mM, 0.125 mM, or 2 mM, and PC uptake by the transfectants was examined. In the PC uptake assay, there was no change in PC uptake with or without 2 μM calcium ionophore A23187 (A23) (Figure 11(a)). Transfectants expressing Xkr4ΔC Q332E were incubated with 1 μM Fluo4-AM for 30 minutes, stimulated with 2 μM A23187 in the presence of 0.125 mM or 2 mM extracellular calcium, and analyzed by flow cytometry. There was no change in Fluo4-AM uptake with or without stimulation with A23187 (Figure 11(b)). These results indicate that the calcium ionophore A23187 does not enhance the scrambling activity by Xkr4, suggesting that calcium in the extracellular region of Xkr4 is important. Transfectants expressing Xkr4WT or Xkr4ΔC were cultured with STS for 3 hours in a medium containing 0.4 mM calcium, and then a PC uptake assay was performed with or without 2.5 mM extracellular calcium. Calcium was required for the lipid scrambling activity of both Xkr4FL and Xkr4ΔC activated by apoptosis stimulation with STS (Figure 11(c)). Thus, the importance of calcium in the extracellular region of Xkr4 for lipid scrambling activity is not specific to the Xkr4ΔC mutant in living cells.

[0212] [Example 3] Identification of Xkr4 activators by revival screening In the presence of extracellular calcium, Xkr4 is activated in two steps. One step is dimerization induced by cleavage of the C-terminus. The other step is a structural change regulated by some activator, as suggested in Example 2. To identify this activator in dying cells, we utilize a CRISPR sgRNA library. However, enriching target sgRNAs using dying cells is generally difficult because the cells do not proliferate. To overcome this difficulty, we developed a novel screening system called "revival screening" (see "Revival Screening using CRISPR sgRNA Library" in "Materials and Methods" above).

[0213] In revival screening, target sgRNAs integrated into the cell's genomic DNA after lentivirus infection are revived and enriched in the sgRNA library for the next round of screening. This occurs when lentiviral sgRNAs infect cells and are integrated into the genomic DNA of the infected cells. By selecting cells in which sgRNAs have been integrated based on specific indicators, the sgRNAs corresponding to the indicators are enriched from these cells. Genomic DNA is prepared from the selected infected cells, and the sgRNAs integrated into the genomic DNA are recovered to generate a new lentiviral sgRNA library. As a result, in revival screening, sgRNAs corresponding to specific indicators are enriched in the sgRNA library prepared for the next round of screening (Figure 12).

[0214] (Revival screening using a CRISPR sgRNA library) To facilitate revival screening, PLB cells lacking the apoptotic DNase CAD (Reference 14) were established. CAD KO PLB cells were established by electroporating PLB cells transfected with a pX330 vector encoding sgRNA for CAD using CRISPR / Cas9, and obtaining single clones through limiting dilution (Figure 13). A plasmid was prepared containing a DNA sequence encoding Xkr4ΔC (Xkr4ΔC-RFP) with tagRFP fused to the C-terminus. CAD KO PLB cells were transfected using this plasmid. The resulting transfectants were infected with a lentiviral sgRNA library. The lentiviral sgRNA library contains six different target sgRNAs for each of the entire human gene set (Reference 15).

[0215] Cells infected with a lentiviral sgRNA library were stimulated with STS for 3 hours to induce apoptosis, and then a PC uptake assay was performed. A cell population that was RFP-positive and PC uptake-negative was collected by flow cytometry (Figure 14(a)). Genomic DNA was prepared from the collected cells, and the sgRNA region incorporated into the genomic DNA was recovered to create a new lentiviral sgRNA library. The prepared lentiviral sgRNA library was used to infect the transfectant, and the infected cells were stimulated with STS to induce apoptosis, followed by a PC uptake assay. This cycle was repeated three times to obtain a cell population (sgPC3) containing 24.5% PC uptake-negative cells (Figure 13(c)). This cycle was repeated one more time to obtain the sgPC4 cell population.

[0216] Genomic DNA was recovered from an sgPC4 cell population, and the regions encoding sgRNAs embedded in the recovered genomic DNA were amplified. The amplified fragments were then subjected to next-generation sequencing (NGS) analysis for gene mapping (see "Materials and Methods" above, (Mapping of NGS Data)). After excluding genes corresponding to one or two sgRNAs, the remaining genes were ranked based on the total number of reads (Figure 13). The top 14 ranked genes are summarized in the table below. [Table 1]

[0217] As shown in Table 1, cytochrome C (CYCS) and APAF1 were found in the top 14 gene list. CYCS is released from mitochondria in response to apoptosis. Subsequently, CYCS binds to APAF1, sequentially activating caspase 9 (Casp9) and caspase 3 (Casp3). Thus, both CYCS and APAF1 are factors required for caspase 9 activation. This result indicates that revival screening functions to search for activators that modulate lipid scrambling activity by dimeric Xkr4 during the apoptotic process.

[0218] Further screening was performed to narrow down the candidate genes for the aforementioned activating factor. A cell population (sgPC5) containing cells that were RFP-positive and PC uptake-negative among the cells into which the sgRNA library had been introduced was recovered by flow cytometry. The recovered sgPC6 cells were fixed and permeabilized. Subsequently, the treated cells were stained with an anti-activated caspase-3 antibody and classified as caspase-3 activated cells. A lentiviral sgRNA library was prepared from the sgPC6 cells, and further screening was performed using the sgPC6 cell population obtained using the lentiviral sgRNA (Figure 16).

[0219] NGS analysis was performed on regions containing sgRNA integrated into the genomic DNA of sgPC7 cells obtained through the aforementioned screening, in the same manner as described above. The genes obtained from the NGS analysis were ranked based on the total number of reads, and the top 8 genes are summarized in the table below. [Table 2]

[0220] As shown in Table 2, the gene referred to as XRCC4 had higher total reads than CYCS and APAF1. The total reads of XRCC4 shown in the NGS analysis of sgPC7 (Table 2) were more than 40 times more concentrated than the total reads of XRCC4 shown in the NGS analysis of sgPC4 (Table 1) (Figure 17). This result indicates that XRCC4 is the most promising candidate as an activator of Xkr4.

[0221] [Example 4] Regulation of Xkr4 by caspase-cleaving XRCC4 In Example 3, PLB cells into which sgRNAs for the top three candidate genes identified as activators of Xkr4 were introduced were stimulated with apoptosis using STS, and the effects on caspase-3 activity and PC uptake in the cells were investigated (Figure 18). No effect on caspase-3 activity in the cells was observed when sgRNA for XRCC4 was used (Figure 18(a) upper panel and Figure 18(b) upper panel). When sgRNA for CYCS or APAF1 was used, caspase-3 activity in the cells decreased (Figure 18(c) upper panel and Figure 18(d) upper panel). These results indicate that in the apoptotic process, CYCS and APAF1 are factors that affect upstream of caspase-3, while XRCC4 is a factor that affects downstream of caspase-3. When sgRNAs for the three genes were used, PC uptake in the cells decreased (Figure 18(a)~(d) lower panel). These results indicate that XRCC4 exerts its influence downstream of caspase-3 in the apoptotic process, and consequently, is a factor that affects lipid scrambling activity.

[0222] To investigate the effect of XRCC4 on PC uptake in apoptotic-stimulated cells, XRCC4 knockout PLB cells (XRCC4 - / - Cells were prepared (Figure 19(a)). Prepared XRCC4 - / - The absence of XRCC4 expression in the cells was confirmed by Western blotting using an anti-CAD antibody (Figure 19(b)). XRCC4 has a caspase-3 cleavage site at amino acid 265 (References 16, 17) and is cleaved by apoptosis. A mutation (2DA) in which two aspartic acid molecules at this cleavage site are replaced with alanine makes the cell resistant to caspase cleavage (Figure 19(c), upper panel, XRCC4 2DA (STS +)). - / -Cells were transfected with plasmids containing genes encoding wild-type XRCC4 (WRCC4 WT) or caspase-uncleaved XRCC4 (XRCC4 2DA). The resulting transfectants were stimulated with apoptosis using STS, and the effect on PC uptake in the transfectants was examined by a lipid scramble assay using NBD-PC (Figure 20(a)-(c)). XRCC4 - / - In cells, PC uptake after apoptosis stimulation by STS was not observed (Figure 20(a)). XRCC4 expressing XRCC4 WT - / - In cells, PC uptake was observed after apoptosis stimulation by STS (Figure 20(b)). These results indicate that XRCC4 is involved in lipid scrambling activity induced by apoptosis. XRCC4 expressing XRCC4 2DA - / - In cells, PC uptake after apoptosis stimulation by STS was not observed (Figure 20(c)). This result indicates that caspase-mediated cleavage of XRCC4 regulates the lipid scrambling activity of Xkr4 in response to apoptosis.

[0223] PLB cells expressing a C-terminal cleaved Xkr4 mutant (Xkr4ΔC Q322E) with a point mutation (Q322E) that constitutively imparts lipid scrambling activity were introduced with sgRNAs for XRCC4 and CYCS. These cells were then stimulated with apoptosis using STS, and the effect on PC uptake in these cells was examined (Figure 20(e)-(f)). Using either the sgRNA for XRCC4 or the sgRNA for CYCS had no effect on PC uptake in these cells. These results support the above suggestion that caspase-mediated cleavage of XRCC4 regulates the lipid scrambling activity of Xkr4 in response to apoptosis. Furthermore, these results indicate that a point mutation in Xkr4ΔC that constitutively imparts lipid scrambling activity can avoid the involvement of XRCC4 in lipid scrambling activity in response to apoptosis.

[0224] XRCC4 KO PLB cells were transfected with Xkr4, Xkr8, and a plasmid encoding Xkr8, all tagged with V5 at the N-terminus, to create transfectants expressing each Xkr. Furthermore, these cells were transfected to express XRCC4 WT-RFP to create transfectants co-expressing each Xkr and XRCC4 WT. - / - Whole cell lysates were prepared of transfectants expressing each Xkr, and transfectants co-expressing each Xkr and XRCC4 WT. These cell lysates were then subjected to SDS-PAGE and Western blotting. Western blotting was performed using anti-V5 antibody (Figure 21, top panel) and anti-RFP antibody (Figure 21, middle panel). Figure 21 shows that the predetermined transfects were prepared.

[0225] Lipid scramble assays were performed on the prepared transfectants (Figure 22). Caspase 3 activity was observed in all tested transfects upon apoptosis stimulation by STS (Figure 22(g)-(l)). PC uptake was not observed when Xkr4 was expressed in PLB cells with XRCC4 knockout and stimulated with apoptosis by STS (Figure 22(a)). Furthermore, PC uptake was observed when XRCC4 WT was expressed (Figure 22(d)). These results support the above suggestion that caspase-mediated cleavage of XRCC4 regulates the lipid scramble activity of Xkr4 upon apoptosis stimulation.

[0226] In XRCC4 KO PLB cells expressing Xkr8 or Xkr9 instead of Xkr4, PC uptake was observed upon apoptotic stimulation by STS, even without co-expression of XRCC4 WT (Figure 22(b) and (c)). These results indicate that caspase-mediated cleavage of XRCC4 is an Xkr4-specific activator in regulating lipid scrambling activity induced by apoptosis.

[0227] XRCC4 is a component of the nuclear DNA repair complex (References 18, 19). In transfectants expressing XRCC4 WT tagged with RFP at the C-terminus (XRCC4 WT-RFP), XRCC4 was observed only in the nucleus (upper panel of Fig. 23(a)). When the above transfectants expressing XRCC4 WT-RFP were given an apoptotic stimulus with STS, XRCC4 was also observed in the cytoplasm (lower panel of Fig. 23(a)). In transfectants expressing XRCC4 WT tagged with RFP at the N-terminus (RFP-WRCC4 WT), XRCC4 was observed only in the nucleus even when given an apoptotic stimulus with STS (Fig. 23(b)). These results indicate that XRCC4 was cleaved by the apoptotic stimulus and the C-terminal fragment diffused into the cytoplasm.

[0228] In transfectants expressing caspase-non-cleavable XRCC4 tagged with RFP at the C-terminus or N-terminus (XRCC4 2DA-RFP or RGP-XRCC4 2DA), XRCC4 was observed only inside the cell whether apoptosis was given (STS+) or not (STS-) (Figs. 23(c) and (d)). Caspase-non-cleavable XRCC4 2DA was not cleaved even when given an apoptotic stimulus with STS (the fourth lane from the left in Fig. 24(a)), and no PC uptake occurred (Fig. 24(c)). XRCC4 WT was cleaved when given an apoptotic stimulus with STS (the second lane from the left in Fig. 24(a)), and PC uptake occurred (Fig. 24(b)). These results indicate that for regulating the lipid scramblase activity of Xkr4 by the apoptotic stimulus, the diffusion of the C-terminal fragment of XRCC4 generated by caspase from the nucleus into the cytoplasm is important.

[0229] [Example 5] Regulation of Xkr4 by cytoplasmic XRCC4 fragments XRCC4 / N functions as a dimerization domain and a binding domain for DNA repair proteins (References 18, 20, 21) (Figure 25). To determine the minimum region of XRCC4 that regulates Xkr4, we expressed full-length wild-type XCCR4 (1-336), XRCC4 deletion mutants with partial N-terminus deletion (116-336, 156-336, 204-336, 248-336, 256-336 and 226-336 (ΔN)), XRCC4 deletion mutants with partial C-terminus deletion (1-305, 1-285 and 1-265 (ΔC)), and XRCC4 KO expressing short-chain XRCC4 fragments (Mini). PLB cells were generated. A lipid scramble assay was performed on these cells (Figure 26). XRCC4 / 256-336, which contains the 10 amino acids N-terminal to the caspase cleavage site (between amino acids 265 and 266), showed PC uptake (Figure 26(g), Figure 27(a)). Compared to XRCC4 / N on the N-terminal side of the caspase cleavage site, XRCC4 / C on the C-terminal side of the caspase cleavage site had no known binding partners with intrinsically disordered regions (Figure 27(b)). XRCC4 / 1-285, which contains only the 20 amino acids C-terminal to the caspase cleavage site, also showed PC uptake (Figure 26(j), Figure 27(c)). Type ΔN did not show complete PC uptake (Figure 26(h)). Caspase-cleaved type ΔC did not show PC uptake (Figure 26(k)). These results indicate that isoleucine at amino acid position 266 needs to be exposed in the cleaved C-terminal fragment of XRCC4. In fact, XRCC4 / 256-285(Mini) (containing 20 amino acids C-terminal and 10 amino acids N-terminal from the caspase cleavage site) showed PC uptake (Figure 26(l), Figure 27(f)). These results indicate that the C-terminal fragment of XRCC4 containing the sequence at amino acid positions 256-285 is sufficient to induce apoptosis-stimulated lipid scrambling activity of Xkr4.

[0230] To investigate the amino acid sequence of XRCC4 / 256-285(Mini) that induces apoptosis-induced lipid scrambling activity of Xkr4, shorter peptide fragments were prepared. A peptide consisting of 20 amino acids from amino acid positions 266-285 of XRCC4 (C20) and a peptide containing methionine at the N-terminus of C20 (C21) were artificially synthesized (Figure 28(a)). The synthetic peptides C20 or C21 were introduced into BDKO cells expressing Xkr4 WT or Xkr4ΔC by electroporation, and a PS exposure assay was performed (Figure 28(b) and (c)). PS exposure was observed in combinations of synthetic peptide C20 and Xkr4ΔC. This result indicates that peptides containing the sequence from amino acid positions 266-285 of XRCC4 are sufficient to induce apoptosis-induced lipid scrambling activity of Xkr4.

[0231] Furthermore, to determine the key amino acid residues for regulating Xkr4 in synthetic peptide C20, including nuclear localization signals (NLS), peptides C20-R270A, C20-K271A, C20-R272A, C20-R273A, and C20-R275A were prepared by substituting alanine for the positively charged amino acid residues in XRCC4 (arginine at position 270, lysine at position 271, arginine at position 272, arginine at position 273, and arginine at position 275) in XRCC4. The XRCC4 fragment mutants C20-R270A, C20-K271A, C20-R272A, and C20-R273A were distributed in the cytoplasm (Figure 29(a~e)), while the XRCC4 fragment mutant C20-R275A was distributed in the nucleus (Figure 29(f)). XRCC4 KO PLB cells expressing V5-Xkr4ΔC-FLAG were transfected with plasmids encoding mutants of the aforementioned XRCC4 fragment, and apoptosis was stimulated by STS. PC uptake activity was observed in the XRCC4 fragment mutants C20-K271A, C20-R272A, C20-R273A, and C20-R275A (Figure 30(c~f), Figure 31(a)), while PC uptake activity was not observed in the XRCC4 fragment mutant C20-R270A (Figure 30(b), Figure 31(a)). These results indicate that the nuclear distribution of the XRCC4 fragment is not required for lipid scrambling activity. The XRCC4 fragment mutant C20-R270A did not completely activate Xkr4 (Figure 31(b)). This result indicates that the arginine residue at position 270 is important for regulating Xkr4.

[0232] [Example 6] Direct binding of Xkr4 to the C-terminal fragment of XRCC4 To investigate how Xkr4 is activated by cytoplasmic XRCC4 fragments, Xkr4ΔC fused with SPOT and FLAG tags (SPOT-Xkr4ΔC-FLAG) was expressed in XRCC4 WT or XRCC4 KO PLB cells expressing XRCC4 R270A (Figure 32(a)). As expected, upon apoptotic stimulation by STS, SPOT-Xkr4ΔC-FLAG, in combination with XRCC4 WT, efficiently induced lipid scrambling activity (Figure 32(b), top panel), while in combination with XRCC4 R270A, it did not sufficiently induce lipid scrambling activity (Figure 32(b), bottom panel).

[0233] Cell membrane lysates of the aforementioned cells were prepared and subjected to immunoprecipitation using anti-SPOT nanobodies, followed by mass spectrometry. This mass spectrometry was expected to identify peptides associated with SPOT-Xkr4ΔC-FLAG recovered by immunoprecipitation using anti-SPOT nanobodies. Label-free quantification revealed that XRCC4 WT is one of the most reliable apoptosis-dependent Xkr4 interacting substances (Figure 33). Two adjacent trypsin-digested peptides of XRCC4 / C, peptide 1 (amino acid positions 286-296: MAPQENQLQEK (SEQ ID NO: 40)) and peptide 2 (amino acid positions 297-310: ENSRPDSSLPETSK (SEQ ID NO: 41)), were identified by mass spectrometry (Figure 34(a)). Targeted quantification using parallel reaction monitoring (PRM) showed that the two peptides increased after apoptosis stimulation by STS in cells expressing XRCC4 WT (Figure 34(b~e)).

[0234] The interaction between peptide fragments derived from XRCC4 WT and Xkr4 was confirmed by Western blotting to detect XRCC4 in the precipitate obtained by Xkr4 immunoprecipitation of a whole cell library from cells stimulated with STS apoptosis (Figure 35(a)). To confirm the above interaction in intact cells, C20 peptide conjugated with fluorescent TMR (C20-TMR) was introduced into HCT116 cells expressing Xkr4 WT-GFP or Xkr4ΔC-GFP by electroporation, and the localization of the introduced C20-TMR with Xkr4 WT-GFP or Xkr4ΔC-GFP was observed (Figures 35(b) and (c)). The fluorescence intensity derived from GFP reflects the intracellular distribution of Xkr4. The peaks in the fluorescence intensity derived from GFP in the middle and lower panels of Figure 35(c) reflect the cell membrane. The lower panel of Figure 35(d) shows that the fluorescence intensity peaks derived from C20-TEM and Xkr4ΔC-GFP overlap. This result indicates that peptide fragments of each protein XRCC4 induce lipid scrambling activity by directly associating with the plasma membrane protein Xkr4 in cells undergoing cell death.

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Claims

1. A method for screening substances that modulate the lipid scrambling activity of Xkr4, comprising the following steps: (1) Contacting cells expressing the Xkr4 polypeptide with candidate substances that may modulate the lipid scrambling activity of Xkr4; (2) Measuring the lipid scrambling activity in the cells that have been brought into contact with the candidate substance; and (3) If the lipid scrambling activity is lower or higher than the lipid scrambling activity in the cells that have not been contacted with the candidate substance, select the candidate substance as a substance to regulate the lipid scrambling activity of Xkr4; including, and The aforementioned Xkr4 polypeptide (A) Consists of the amino acid sequence described in Sequence ID No. 1 or 35; or (B) A method comprising having an amino acid sequence that includes deletions, substitutions, additions, or combinations thereof in the amino acid sequence of (A), exhibiting at least 90% sequence identity with the amino acid sequence of (A), and exhibiting lipid scrambling activity in the cell membrane of the cell.

2. A method for screening substances that modulate the lipid scrambling activity of Xkr4, comprising the following steps: (1) Contacting cells expressing the Xkr4 polypeptide with candidate substances that may modulate the lipid scrambling activity of Xkr4; (2) Measuring the lipid scrambling activity in the cells that have been brought into contact with the candidate substance; and (3) If the lipid scrambling activity is lower or higher than the lipid scrambling activity in the cells that have not been contacted with the candidate substance, select the candidate substance as a substance to regulate the lipid scrambling activity of Xkr4; including, and The aforementioned Xkr4 polypeptide (A) Consists of any of the amino acid sequences described in SEQ ID NOs: 3-5 and 37-39; or (B) The amino acid sequence of (A) above, having an amino acid sequence that includes deletions, substitutions, additions, or combinations thereof, The amino acid sequence of (A) above exhibits at least 90% sequence identity, The amino acid sequence described in any of Sequence IDs 3 to 5 includes the substitution of I322S, L331F, or Q332E, or a conservative amino acid substitution thereof. The amino acid sequence described in any of Sequence IDs 37 to 39 includes the substitution of I325S, L334F, or Q335E, or their conserved amino acid substitutions, and A method for exhibiting lipid scrambling activity in the cell membrane of the aforementioned cells.

3. The method according to claim 1 or 2, wherein, in the comparison, the substance that modulates the lipid scrambling activity of Xkr4 is a candidate drug for the treatment or prevention of a condition or disease in which the promotion of apoptosis is involved.

4. A method for screening substances that modulate the lipid scrambling activity of Xkr4, comprising the following steps: (1) Contacting cells expressing the Xkr4 polypeptide with candidate substances that may modulate the lipid scrambling activity of Xkr4; (2) Measuring the lipid scrambling activity in the cells that have been brought into contact with the candidate substance; and (3) If the lipid scrambling activity is lower or higher than the lipid scrambling activity in the cells that have not been contacted with the candidate substance, select the candidate substance as a substance to regulate the lipid scrambling activity of Xkr4; Includes, The aforementioned Xkr4 polypeptide (α) Consists of the amino acid sequence described in SEQ ID NO: 2 or 36; or (β) A method comprising having an amino acid sequence in which deletions, substitutions, additions, or combinations thereof are present in the amino acid sequence of (α), exhibiting at least 90% sequence identity with the amino acid sequence of (α), and exhibiting lipid scrambling activity in the cell membrane of the cell.

5. The method according to claim 1, 2, or 4, wherein the cells expressing the Xkr4 polypeptide are cells transfected with an expression vector comprising a polynucleotide containing a nucleotide sequence encoding the Xkr4 polypeptide described in claim 1, 2, or 4.

6. The method according to any one of claims 1 to 5, wherein the lipid scrambling activity is the uptake activity of phospholipids or glycolipids, or the exposure activity of phospholipids located on the inside of the cell membrane.

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