Methods for analyzing the intracellular dynamics of fatty acid transporters, and utilization of modified fatty acid transporter proteins.

Abstract

To provide methods for analyzing intracellular kinetics of fatty acid transporters.SOLUTION: Disclosed is a method for analyzing intracellular kinetics of a fatty acid transporter comprising the steps of: stimulating a cell expressing a fatty acid transporter modified protein so that the modified protein transfers to the cell membrane; and measuring the modified protein moved to the cell membrane, where the modified fatty acid transporter protein comprises a labelling protein moiety in the intracellular region and at least one tag peptide in the extracellular region.SELECTED DRAWING: Figure 4A

Description

Technical Field

[0001] The present invention relates to a method for analyzing intracellular dynamics of a fatty acid transporter. The present invention relates to a vector comprising a polynucleotide encoding a modified fatty acid transporter protein and a recombinant cell comprising the same. The present invention relates to a reagent for analyzing intracellular dynamics of a fatty acid transporter. The present invention relates to a method for screening a test substance. The present invention relates to a method for analyzing a receptor.

Background Art

[0002] A fatty acid transporter is a protein molecule involved in the transport of fatty acids from outside to inside of a cell. A fatty acid transporter is a transmembrane protein, but it does not always exist on the cell surface, and translocates from inside the cell to the cell membrane in a stimulation-dependent manner. For example, Fatty acid transport protein 1 (FATP1) protein, which is a fatty acid transporter, translocates to the cell membrane when cells are stimulated with insulin (Non-Patent Document 1).

Prior Art Literature

Non-Patent Literature

[0003]

Non-Patent Document 1

Summary of the Invention

Problem to be Solved by the Invention

[0004] As described in Non-Patent Document 1, many parts of the intracellular dynamics and intracellular signal transduction mechanism of fatty acid transporters remain unelucidated. An object of the present invention is to provide a means that enables analysis of intracellular dynamics of a fatty acid transporter.

Means for Solving the Problem

[0005] As a result of diligent research, the inventors of the present invention have found that the intracellular dynamics of fatty acid transporters can be analyzed by expressing a modified fatty acid transporter protein in cells and stimulating the expressing cells, thereby completing the present invention. Specifically, the present invention provides a method for analyzing the intracellular dynamics of fatty acid transporters, comprising the steps of stimulating cells expressing a modified fatty acid transporter protein to cause the modified protein to migrate to the cell membrane, and measuring the modified protein that has migrated to the cell membrane, wherein the modified protein contains a labeled protein portion in the intracellular region of the fatty acid transporter and contains at least one tag peptide in the extracellular region.

[0006] The present invention provides a vector comprising a fatty acid transporter modified protein and a polynucleotide encoding the fatty acid transporter modified protein, the vector comprising a protein containing a labeled protein moiety in the intracellular region of the modified protein and at least one tag peptide in the extracellular region.

[0007] The present invention provides recombinant cells into which a vector containing a polynucleotide encoding a fatty acid transporter modified protein has been introduced, the vector comprising a fatty acid transporter modified protein and a protein having a labeled protein moiety in the intracellular region of the modified protein and at least one tag peptide in the extracellular region.

[0008] The present invention provides recombinant cells having a fatty acid transporter modified protein comprising a fatty acid transporter, a labeled protein portion located in the intracellular domain of the fatty acid transporter, and at least one tag peptide located in the extracellular domain of the fatty acid transporter.

[0009] The present invention provides a reagent for analyzing the intracellular dynamics of fatty acid transporters, comprising a modified fatty acid transporter protein, a vector containing a polynucleotide encoding the modified fatty acid transporter protein (the vector comprising a protein containing a labeled protein portion in the intracellular region of the modified protein and at least one tag peptide in the extracellular region), and a ligand.

[0010] The present invention provides a screening method for a test substance, comprising the steps of: adding a test substance to cells expressing a modified fatty acid transporter protein; measuring the tag peptide of the modified protein in the cells; and, if the tag peptide is detected in the measurement step, determining that the test substance is a ligand that causes the fatty acid transporter to move to the cell membrane, wherein the modified protein contains a labeled protein portion in the intracellular region of the fatty acid transporter and contains at least one tag peptide in the extracellular region.

[0011] The present invention provides a method for screening a test substance, comprising the steps of: adding a test substance to cells expressing a modified fatty acid transporter protein; stimulating the cells; measuring the tag peptide of the modified protein in the cells; and, if the tag peptide is detected in the measurement step, determining that the state of the cell membrane changes due to stimulation, causing the test substance to bind to a receptor and the fatty acid transporter to migrate to the cell membrane, wherein the modified protein contains a labeled protein portion in the intracellular region of the fatty acid transporter and contains at least one tag peptide in the extracellular region.

[0012] The present invention provides a method for analyzing receptors, comprising the steps of adding a ligand to cells expressing a modified fatty acid transporter protein and measuring the tag peptide of the modified protein in the cells, wherein the modified protein includes a labeled protein moiety in the intracellular region of the fatty acid transporter and at least one tag peptide in the extracellular region, and the method for evaluating the receptor in the cell based on the measurement value obtained in the measurement step. [Effects of the Invention]

[0013] The present invention provides a simple method for analyzing the intracellular dynamics of fatty acid transporters. [Brief explanation of the drawing]

[0014] [Figure 1]Fig. 1 is a schematic diagram of a fatty acid transporter-modified protein using CD36 as the fatty acid transporter. [Figure 2A] Fig. 2 is a schematic diagram of the seamless cloning method for constructing pCAGGS-CD36-9myc-EGFP. [Figure 2B] Fig. 3 is a schematic diagram of the seamless cloning method for constructing pCAGGS-7myc-FATP1-EGFP. [Figure 3A] Fig. 4 shows the results of observing EGFP in L6 cells into which pCAGGS-CD36-9myc-EGFP has been introduced. [Figure 3B] Fig. 5 shows the results of observing EGFP in L6 cells into which pCAGGS-7myc-FATP1-EGFP has been introduced. [Figure 4A] Fig. 6 shows the results of immunostaining performed on L6 cells into which pCAGGS-CD36-9myc-EGFP has been introduced after insulin stimulation. [Figure 4B] Fig. 7 shows the results of measuring Myc tag by immunostaining performed on L6 cells into which pCAGGS-CD36-9myc-EGFP has been introduced after insulin stimulation. [Figure 5A] Fig. 8 shows the results of immunostaining performed on L6 cells into which pCAGGS-7myc-FATP1-EGFP has been introduced after insulin stimulation. [Figure 5B] Fig. 9 shows the results of measuring Myc tag by immunostaining performed on L6 cells into which pCAGGS-7myc-FATP1-EGFP has been introduced after insulin stimulation. [Figure 6A] Fig. 10 shows the results of immunostaining performed on 3T3-L1 cells into which pCAGGS-CD36-9myc-EGFP has been introduced after insulin stimulation. [Figure 6B] Fig. 11 shows the results of measuring Myc tag by immunostaining performed on 3T3-L1 cells into which pCAGGS-CD36-9myc-EGFP has been introduced after insulin stimulation. [Figure 7] Fig. 12 is an image showing an example of injection of a solution for in vivo introduction into a mouse. [Figure 8] Fig. 13 is an image showing an example of electroporation into a mouse. [Figure 9] These are the results of insulin stimulation and immunostaining performed on mouse-derived white adipocytes into which pCAGGS-CD36-9myc-EGFP has been introduced. [Figure 10] These are the results of insulin stimulation and immunostaining performed on mouse-derived white adipocytes into which pCAGGS-7myc-FATP1-EGFP has been introduced. MODE FOR CARRYING OUT THE INVENTION

[0015] (Method for analyzing intracellular dynamics of fatty acid transporter) In the method for analyzing intracellular dynamics of a fatty acid transporter according to the present invention (hereinafter, also simply referred to as "the method"), cells expressing a modified fatty acid transporter protein are stimulated to translocate the modified fatty acid transporter protein to the cell membrane, and the translocated modified protein is measured. In the present specification, "measurement" may include both qualitative (detection) and quantitative measurements. For example, by measuring the translocation of the modified fatty acid transporter protein to the cell membrane induced by stimulation, qualitative information about the translocation of the fatty acid transporter to the cell membrane can be obtained. Qualitative information is information indicating the presence or absence of translocation of the modified fatty acid transporter protein to the cell membrane. The information indicating the presence or absence of translocation may be optical information, or may be numerical information (measured values) obtained by a measuring instrument. Quantitative information is numerical information such as numerical information (measured values) obtained by a measuring instrument, and values calculated from the measured values. Examples of optical information include luminescence, fluorescence, absorbance, turbidity, and the like. Examples of numerical information include information digitized by a measuring instrument based on optical information. Examples of such numerical values include signal intensity from luminescence or fluorescence, absorbance, turbidity, and the like. Based on this information, a person skilled in the art can analyze the function of intracellular fatty acid transporters and responses to stimuli (intracellular dynamics).

[0016] (Modified fatty acid transporter protein) A modified fatty acid transporter protein (hereinafter also referred to as "modified protein") is a recombinant protein in which a labeled protein portion and a tag peptide are fused to a fatty acid transporter. The fatty acid transporter that forms the basis of the modified protein has a region that is exposed outside the cell when it translocates to the cell membrane (extracellular region) and a region that remains inside the cell (intracellular region). The fatty acid transporter may be a two-pass transmembrane protein or a one-pass transmembrane protein. CD36 is an example of a two-pass transmembrane protein. FATP1 is an example of a one-pass transmembrane protein. Figure 1 shows a schematic diagram of the modified protein. Referring to Figure 1, the labeled protein portion is located in the intracellular region of the modified protein. Also referring to Figure 1, the tag peptide is located in the extracellular region of the modified protein.

[0017] The fatty acid transporter used in the modified protein is preferably the CD36 protein, the FATP1 protein, or a protein in which 1 to 20 amino acid residues are deleted, substituted, or added to the amino acid sequence of the CD36 or FATP1 protein. Here, the deletion, substitution, and addition of amino acid residues refer to deletions, substitutions, and additions to the amino acid sequence of the fatty acid transporter itself, and are separate from the labeled protein portion and tag peptide fused to the modified protein. The number of amino acid residues deleted, substituted, or added may be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. The number of amino acid residues deleted, substituted, or added is 1 to 20, preferably 1 to 15, more preferably 1 to 10, more preferably 1 to 7, more preferably 1 to 5, and more preferably 1 to 3. Conservative substitutions are preferred. Conservative substitutions refer to substitutions in which the acidic or basic properties of an amino acid residue do not substantially change between the original amino acid residue and the substituted amino acid residue. Specifically, these include substitutions between F, W, and Y; L, I, and V; K, R, and H; D and E; and S and T.

[0018] (cell) The cells expressing the modified protein may be cells isolated from a living organism, or cells that make up a living organism other than a human. The species from which the cells originate is not particularly limited and may be any animal, plant, insect, or microorganism, but it is preferable that they be animals, and more preferably mammals. Examples of mammals include humans, monkeys, cattle, sheep, goats, horses, pigs, rabbits, dogs, cats, rats, mice, and guinea pigs. The type of cell is not particularly limited and may include somatic cells, germ cells, stem cells, etc. Somatic cells are not particularly limited, but muscle cells or adipocytes are preferred. The cells may be cells collected from a living organism, or cultured cells. These cells may have the same genetic sequence as the species from which they originate, or they may be mutant cells such as cancer cells or recombinant cells with modified genetic information. The cells may or may not express an endogenous fatty acid transporter corresponding to the modified protein. It is also preferable that the cells have receptors for the ligand described later.

[0019] The method of this embodiment may express the modified protein in cells by introducing an expression vector containing a polynucleotide encoding the modified protein into the cells. The method of introducing the expression vector is not particularly limited and may be either infection or transfection, but transfection is preferred. Infection is not particularly limited and can be carried out by methods known to those skilled in the art. Examples include gene transfer methods using adenoviruses or lentiviruses, or the Agrobacterium method. Transfection is not particularly limited and can be carried out by methods known to those skilled in the art. Examples include lipofection, electroporation, calcium phosphate coprecipitation, DEAE-dextran method, or methods using a particle gun, but lipofection or electroporation is preferred. The modified protein may be introduced into cells, into biological tissues, or directly into living organisms other than humans. The expression vector may be transient or stable. Both transient and stable expression cells of the modified protein can be used in the method of the present invention.

[0020] The modified protein of the present invention translocates from inside the cell to the cell membrane in response to a stimulus. The stimulus is not particularly limited as long as it can translocate the fatty acid transporter used in the modified protein to the cell membrane. Examples of stimuli include ligand stimulation, acid or base stimulation, temperature stimulation, nutrient excess or deficiency stimulation, antibiotic stimulation, or electrical stimulation. Ligands are substances that bind to receptors on the cell surface, and examples include low molecular weight compounds with a molecular weight of 1000 or less, nucleic acids, oligosaccharides, lipopolysaccharides, peptides, glycopeptides, proteins, glycoproteins, antibodies, etc. Extracellular signaling molecules are preferred as ligands for proteins, and examples include hormones such as insulin, glucagon, and adrenaline, cytokines such as interferon, cell growth factors such as EGF, and G proteins.

[0021] The tag peptide is not particularly limited as long as it is a polypeptide that can be specifically detected. Examples of tag peptides include Myc tag (EQKLISEEDL: SEQ ID NO: 1), HA tag (YPYDVPDYA: SEQ ID NO: 2), PA tag (GVAMPGAEDDVV: SEQ ID NO: 3), V5 tag (GKPIPNPLLGLDST: SEQ ID NO: 4), S tag (KETAAAKFERQHMDS: SEQ ID NO: 5), E tag (GAPVPYPDPLEPR: SEQ ID NO: 6), T7 tag (MASMTGGQQMG: SEQ ID NO: 7), VSV-G tag (YTDIEMNRLGK: SEQ ID NO: 8), Glu-Glu tag (EEEEYMPME: SEQ ID NO: 9), Strep-tag II tag (WSHPQFEK: SEQ ID NO: 10), HSV tag (QPELAPEDPED: SEQ ID NO: 11), 6×His tag (HHHHHH: SEQ ID NO: 12), or DYKDDDDK tag (DYKDDDDK: Examples include SEQ ID NO: 13). The tag peptide in the modified protein may be one type or multiple types.

[0022] A modified protein may contain two or more repeated fusions of the same tag peptide. The number of tag peptides fused to the modified protein may be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and it is preferable that there are between 6 and 9 of them. Including multiple copies of the same tag peptide can improve the signal intensity when immunostaining targeting the tag peptide is performed.

[0023] If the fatty acid transporter is CD36, the tag peptide is preferably substituted or inserted between amino acid positions 358 and 364 of the CD36 amino acid sequence, and more preferably inserted between positions 360 and 361. If the fatty acid transporter is a protein in which 1 to 20 amino acid residues are deleted, substituted, or added to the amino acid sequence of the CD36 protein, the tag peptide is preferably substituted or inserted in the region corresponding to positions 358 to 364, referring to the amino acid sequence of the CD36 protein before the deletion, substitution, or addition, and more preferably inserted between positions 360 and 361. Furthermore, if the fatty acid transporter is FATP1, the tag peptide is preferably attached to the N-terminus of the amino acid sequence of the FATP1 protein. If the fatty acid transporter is a protein in which 1 to 20 amino acid residues are deleted, substituted, or added to the amino acid sequence of the FATP1 protein, the tag peptide is preferably attached to the N-terminus of the amino acid sequence of the protein in which 1 to 20 amino acid residues are deleted, substituted, or added.

[0024] The labeled protein portion is not particularly limited, but is preferably a fluorescent protein, such as GFP protein, BFP protein, CFP protein, YFP protein, EYFP protein, PA-GFP protein, mCherry protein, etc.

[0025] In the method of this embodiment, the modified protein that has migrated to the cell membrane is measured. The modified protein may be measured using an antibody that specifically binds to the tag peptide. The antibody that specifically binds to the tag peptide may be a commercially available antibody or an antibody prepared by a method known in this art. The antibody may be a monoclonal antibody, a polyclonal antibody, or a fragment thereof (e.g., Fab, F(ab')2, Fab', etc.), but a monoclonal antibody is preferred.

[0026] The step of measuring the modified protein that has migrated to the cell membrane is preferably immunostaining. By measuring the labeled protein portion fused with the antibody, information about the modified protein that has migrated to the cell membrane can be obtained. Immunostaining may be a direct method in which the antibody that binds to the tag peptide (primary antibody) is labeled, or an indirect method in which a labeled antibody that binds to the primary antibody (secondary antibody) is further bound to the primary antibody for measurement. The measurement of modified proteins by immunostaining is described below.

[0027] The measurement process preferably includes a step of fixing the cells before immunostaining. The method of cell fixation is not particularly limited and can be carried out by methods known to those skilled in the art. Examples of fixatives used for fixation include formaldehyde, paraformaldehyde (PFA), 4% (v / v) PFA / PBS, glutaraldehyde, acetone, methanol, and ethanol, and these can be used in appropriate combinations.

[0028] A primary antibody that specifically binds to the tag peptide is added to the fixed cells. In the direct method, a labeled primary antibody is used. By adding a primary antibody that specifically binds to the modified protein, a first complex between the cells and the primary antibody can be formed. The temperature and time conditions for mixing the cells and primary antibody are not particularly limited. For example, the cells to which the antibody has been added may be incubated at 4-40°C for 5 minutes to 24 hours. During incubation, the cells to which the antibody has been added may be left undisturbed, stirred, or shaken.

[0029] Examples of antibody labels include fluorescent substances, enzymes, and radioisotopes. Of these, fluorescent substances are particularly preferred. Examples of fluorescent substances include CF(registered trademark) 543 Dye, CF(registered trademark) 647 Dye, fluorescein isothiocyanate (FITC), rhodamine, Texas Red (registered trademark), phycoerythrin (PE), Cy(registered trademark), DyLight(registered trademark), ATTO(trademark), TRITC or Alexa Fluor(registered trademark), and quantum dots such as Qdot(registered trademark). In addition, as the label attached to the antibody, a label protein that can be used in the label protein portion as described above may be used, but in that case, it is desirable that it be different from the label protein portion attached to the modified protein. Examples of radioisotopes include: 125 I, 14 C, 32 Examples include P. Enzymes such as alkaline phosphatase, peroxidase, β-galactosidase, glucose oxidase, tyrosinase, acid phosphatase, and luciferase.

[0030] The substrate of the enzyme can be appropriately selected from known substrates depending on the type of enzyme. For example, when using peroxidase as the enzyme, examples of substrates include chemiluminescent substrates such as luminol and its derivatives, and chromogenic substrates such as 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonate ammonium) (ABTS), 1,2-phenylenediamine (OPD), and 3,3',5,5'-tetramethylbenzidine (TMB). Furthermore, when alkaline phosphatase is used as the enzyme, suitable substrates include chemiluminescent substrates such as CDP-Star(registered trademark) (2-chloro-5-(4-methoxyspiro[1,2-dioxetane-3,2'-(5-chlorotricyclo[3.3.1.13.7]decane])-4-yl]-1-phenyl phosphate disodium) and CSPD(registered trademark) (3-(4-methoxyspiro[1,2-dioxetane-3,2-(5'-chloro)tricyclo[3.3.1.13,7]decane]-4-yl)phenyl phosphate disodium), and chromogenic substrates such as 5-bromo-4-chloro-3-indolyl phosphate (BCIP), 5-bromo-6-chloro-indolyl phosphate disodium, and p-nitrophenyl phosphate.

[0031] Biotin may be used as an antibody label. The modified protein can be measured by adding avidin (or streptavidin) conjugated with a fluorescent substance or enzyme to the modified protein to which the biotin-labeled antibody is bound. The fluorescent substance or enzyme to be conjugated to avidin (or streptavidin) is as described above.

[0032] When a fluorescent substance is used as a label, the signal intensity due to fluorescence can be measured using known measuring devices such as a confocal laser microscope, fluorescence microscope, spectrofluorometer, or fluorescence plate reader. The signal intensity can be obtained, for example, by acquiring an image of the cell using a confocal laser microscope and processing the fluorescence in the image using an image processing program or image analysis software downloaded to the confocal laser microscope or computer. An example of image analysis software is Image J. Alternatively, the fluorescence may be processed within the instrument, such as a spectrofluorometer, and calculated as a measured value. Based on the obtained measured value, the amount of modified protein on the cell membrane can be calculated. The excitation wavelength and fluorescence wavelength can be appropriately determined depending on the fluorescent substance used. The light source of the measuring device is not particularly limited, and a light source with a wavelength suitable for exciting the fluorescent substance can be appropriately selected. Examples of light sources include mercury arc lamps, xenon lamps, LED lamps, or combinations of these with optical filters. When the label is a radioactive isotope, the radiation as a signal can be measured using known devices such as a scintillation counter. If the label is an enzyme, the reaction can be measured, for example, by adding the substrate described above to the enzyme and measuring the reaction of the substrate. The reaction of the substrate can be measured using a commercially available measurement kit, or by measuring the wavelength specific to the substrate or enzyme reactant using an absorbance meter, or by any other method known in this art, depending on the enzyme and substrate used.

[0033] When immunohistochemistry is performed indirectly, the first complex is mixed with a labeled secondary antibody that binds to the primary antibody. This yields a second complex in which the first complex and the secondary antibody are bound. The temperature and time conditions for mixing the first complex and the secondary antibody are not particularly limited. For example, cells to which the antibody has been added may be incubated at 4-40°C for 5 minutes to 24 hours. During incubation, the cells to which the antibody has been added may be left undisturbed, agitated, or shaken.

[0034] (Washing and blocking) Cell washing may be performed after cell fixation or after the formation of the first or second complex. Washing can remove fixative and unreacted antibodies. A suitable aqueous medium should be used for washing. Examples of such aqueous mediums include physiological saline, PBS, Tris-HCl, and Good's buffer. Cell blocking may also be performed before antibody addition. A known blocking agent should be used for blocking. Examples of such blocking agents include solutions of normal goat serum (NGS), BSA, and casein.

[0035] (Measurement of labeled protein portion) The method of this embodiment may include the measurement of the labeled protein portion. By measuring the labeled protein portion in the expressed modified protein, the expression of the modified protein in cells can be confirmed. It is preferable that the fluorescence wavelength of the labeled protein portion of the modified protein and the fluorescence wavelength of the fluorescent substance attached to the antibody are different.

[0036] If the labeled protein is a fluorescent protein, the fluorescence can be measured using known measuring devices such as a fluorescence microscope, spectrofluorometer, or fluorescence plate reader. Fluorescence may also be confirmed visually through a fluorescence microscope. Furthermore, signal intensity can be obtained based on the optical information from the fluorescence. The method for obtaining signal intensity is as described above. Based on the obtained measurements, the amount of modified protein contained in the cells can be calculated.

[0037] Both measurement of the labeled protein portion in the modified protein and measurement of the tag peptide in the modified protein by immunostaining may be performed. By measuring the labeled protein portion in addition to immunostaining, it is possible to obtain values ​​for both the amount of modified protein contained in the cell and the amount of modified protein that has migrated to the cell membrane. This makes it possible to evaluate the migration of modified protein to the cell membrane. For example, by dividing the amount of modified protein that has migrated to the cell membrane by the amount of modified protein contained in the cell, it is possible to calculate a ratio that shows what amount of modified protein in the cell has migrated to the cell membrane.

[0038] Cells may be subjected to a flow cytometer. By using a flow cytometer, optical information can be obtained from each individual cell in a cell population. Examples of flow cytometers include flow cytometers and imaging flow cytometers. An imaging flow cytometer is a flow cytometer equipped with an imaging unit such as a CCD camera, and is a device capable of acquiring images of cells flowing through a liquid. More specifically, an imaging flow cytometer can acquire fluorescence-based optical information and fluorescence images from thousands to millions of cells in a short time of a few seconds to a few minutes. By processing the acquired images, information from each individual cell can be extracted, or quantitative information can be obtained. The light source for a flow cytometer is not particularly limited, and a light source with a wavelength suitable for exciting fluorescent substances can be appropriately selected. For example, blue semiconductor lasers, red semiconductor lasers, argon lasers, He-Ne lasers, and mercury arc lamps can be used.

[0039] When using a flow cytometer, immunostaining may be performed without fixing the cells. By subjecting unfixed cells to the flow cytometer, information about living cells can be obtained without damaging them.

[0040] The method of this embodiment allows for obtaining information about molecules involved in the intracellular translocation of fatty acid transporters. For example, when transfecting a modified protein in which the fatty acid transporter is CD36, the effect of molecules involved in the intracellular translocation of CD36 can be measured by adding these molecules. If the molecule is a protein, the effect on the intracellular translocation of the modified protein can be measured by simultaneously expressing the two proteins in the cell by transfecting a vector capable of expressing that protein along with the modified protein.

[0041] (A vector containing polynucleotides encoding a modified protein) Another embodiment is a vector encoding a modified protein. The vector is not particularly limited as long as it is a known nucleic acid vector capable of incorporating a polynucleotide encoding a modified protein. Depending on the cell type into which it is introduced, the vector may be a protein expression vector with a promoter and other components appropriately designed to express the protein in the cell, or it may simply be a carrier vector. By introducing a protein expression vector containing a polynucleotide encoding a modified protein into cells, the modified protein can be expressed in the cells and used in the above method. Examples of vectors include plasmids, cosmids, phagemids, or artificial chromosome vectors. In addition to the polynucleotide encoding the modified protein, the vector may also encode proteins other than the modified protein, such as origins of replication or polynucleotides encoding drug resistance genes.

[0042] Polynucleotides encoding modified proteins can be obtained by techniques known to those skilled in the art. For example, they can be obtained by directly synthesizing the polynucleotide encoding the modified protein, or by incorporating a polynucleotide encoding a tag peptide and a polynucleotide encoding the labeled protein portion into a polynucleotide encoding a fatty acid transporter. The method of incorporating a polynucleotide encoding a tag peptide and a polynucleotide encoding the labeled protein portion into a polynucleotide encoding a fatty acid transporter can be carried out using DNA recombination techniques known to those skilled in the art. Known DNA recombination techniques include, for example, subcloning and seamless cloning. Seamless cloning is a technique for joining DNA fragments by giving each end of the DNA fragments to be joined a DNA homology region of about 15 bases.

[0043] Besides cells, vectors containing polynucleotides encoding modified proteins may also be introduced into microorganisms such as E. coli, yeast, actinomycetes, algae, lactic acid bacteria, and Bacillus subtilis. For example, if the vector is a plasmid that can replicate within the microorganism into which it is introduced, the above-mentioned vector can be easily produced by introducing it into E. coli or yeast.

[0044] (Cells containing a vector with polynucleotides encoding a modified protein) Another embodiment involves a cell containing a vector comprising a polynucleotide encoding the modified protein. The host cell is not particularly limited, and the above-mentioned cells can be used. Preferably, the host cell is a cell that lacks or expresses very little of the fatty acid transporter corresponding to the modified protein.

[0045] (Reagent for analyzing the intracellular dynamics of fatty acid transporters) Another embodiment is a reagent for analyzing the intracellular dynamics of fatty acid transporters. The reagent comprises at least a vector encoding a modified protein and a ligand. The vector encoding the modified protein and ligand are as described above. The vector encoding the modified protein and ligand may be in solution or in a dry state. The reagent may separately contain a solvent for suspending and diluting the vector encoding the modified protein and ligand. Examples of such solvents include aqueous media such as water, physiological saline, PBS, Tris-HCl, and Good's buffer. The reagent of this embodiment is usually provided to the user in a container. The container containing the reagent may be provided to the user in a box or bag. The box or bag may include an accompanying document describing how to use the reagent.

[0046] The analytical reagent of this embodiment may further contain a transfection reagent. The transfection method is as described above, and the analytical reagent may contain appropriate reagents depending on the transfection method used. For example, if the transfection method is lipofection, the analytical reagent may contain a cationic lipid such as DOTAP (N-[1-(2,3-dioreyloxy)propyl]-N,N,N,-trimethylammonium methyl sulfate); if it is calcium phosphate coprecipitation, the analytical reagent may contain calcium chloride; and if it is DEAE-dextran method, the analytical reagent may contain diethylaminoethyl-dextran.

[0047] The analytical reagent of this embodiment may include an antibody that captures the tag peptide of the modified protein. The antibody may also be labeled. The antibody is as described above. The form of the antibody is not particularly limited and may be solid (e.g., powder, crystal, lyophilized product, etc.) or liquid (e.g., solution, suspension, emulsion, etc.). The antibody and label may be integrated or separate. If they are separate, the reagent may further include a reagent for binding the antibody and label. The antibody may be a primary antibody that captures the tag peptide of the modified protein, or it may include a secondary antibody that captures the primary antibody. The reagent may also include a washing solution for removing unbound antibody after binding. Examples of washing solutions include physiological saline, PBS, Tris-HCl, and Good's buffer.

[0048] The analytical reagent in this embodiment may include a fixative for fixing cells. Fixing the cells allows the state of the cells after stimulation to be preserved. The fixative is as described above.

[0049] (Screening method for test substances) Another embodiment is a screening method for test substances using a modified protein. The modified protein in this embodiment migrates to the cell membrane upon stimulation. This property can be used to add a test substance to cells expressing the modified protein, and to determine whether the modified protein migrates to the cell membrane by measuring the tag peptide of the modified protein. For example, if the tag peptide is detected by measurement, it can be determined that the test substance is a ligand that causes fatty acid transporters to migrate to the cell membrane. In another example, by applying stimulation along with the test substance, if the tag peptide is detected by measurement, it can be determined that the stimulation changes the state of the cell membrane, causing the test substance to bind to a receptor and the fatty acid transporter to migrate to the cell membrane. Furthermore, if the tag peptide is not detected by measurement, it can be determined that the test substance is not a ligand that causes fatty acid transporters to migrate to the cell membrane, or that the cell does not have a receptor for the ligand. The modified protein, its measurement, and stimulation are as described above.

[0050] Examples of test substances include, but are not limited to, physiologically active substances such as amino acids, sugars, nucleic acids, metal ions, peptides, neurotransmitters, or hormones.

[0051] (Methods for analyzing receptors) Another embodiment is a method for analyzing receptors using a modified protein. The modified protein in this embodiment translocates to the cell membrane upon cell stimulation by a ligand. This property can be used to evaluate the receptors in cells. For example, a ligand known to specifically bind to a certain receptor (let's call it receptor A) and cause a fatty acid transporter to translocate to the cell membrane is prepared. The modified protein is expressed in cells in which receptor A is to be investigated, and the ligand is added. The tag peptide of the cells after ligand addition is measured, and if the tag peptide can be detected, it can be determined that the cells possess receptor A. Conversely, if the tag peptide cannot be detected, it can be determined that the cells do not possess receptor A.

[0052] In another example, an experiment was conducted to eliminate receptor A from cells that possessed receptor A. Afterward, a modified protein was expressed in these cells, and a ligand was added. By measuring the tag peptide of the cells after ligand addition, it was possible to confirm whether the cells had lost receptor A.

[0053] In yet another example, in cells possessing receptor A and expressing a modified protein, the test substance is added along with the ligand. If the amount of modified protein translocated to the cell membrane decreases after the addition of the test substance compared to when only the ligand is added, it indicates that the test substance caused competitive inhibition of ligand-receptor binding. Thus, by using the modified protein of the present invention, it is possible to evaluate the corresponding receptor. [Examples]

[0054] The present invention will be described in more detail below with reference to examples, but the present invention is not limited in any way.

[0055] Example 1: Confirmation of modified protein expression In the following experiment, a vector for expressing the modified protein was created and introduced into cells to confirm whether the modified protein was expressed within the cells.

[0056] (1) Production of fatty acid transporter modified protein expression vectors 1 Using a seamless cloning method, multiple DNA fragments were combined and cloned to create vectors for expressing modified proteins in cells. The DNA fragments used were the pCAGGS vector fragment (sequence number 14) treated with restriction enzyme XhoI, and the following insert fragments: CD36 fragment (1-360) (nucleotide sequence portion 1-360 of CD36: sequence number 15), 9-Myc fragment (sequence number 16) consisting of nine consecutive Myc sequences, CD36 fragment (361-472) (nucleotide sequence portion 361-472 of CD36: sequence number 17), and an EGFP fragment (sequence number 18) which was a modified GFP protein. For each insert fragment, the DNA homologous region was adjusted so that each insert fragment was incorporated into the pCAGGS vector fragment as shown in Figure 2A. Using the quantities of each DNA fragment shown in Table 1 below, and the In-Fusion HD Cloning Kit (Takara Bio Inc.), seamless cloning was performed to create a modified protein expression vector, pCAGGS-CD36-9myc-EGFP, which utilizes the fatty acid transporter CD36. The prepared pCAGGS-CD36-9myc-EGFP was introduced into competent cells (E. coli DH5α) and stored.

[0057] [Table 1]

[0058] (2) Preparation of modified protein expression vectors Instead of CD36 in (1), a modified protein expression vector using the fatty acid transporter FATP1 was prepared. The DNA fragments used were the pCAGGS vector fragment and, as insert fragments, a 7-Myc fragment (sequence number 19) consisting of seven consecutive Myc sequences, a FATP1 fragment (sequence number 20), and an EGFP fragment. Each insert fragment was prepared to be incorporated into the pCAGGS vector fragment as shown in Figure 2B. Using the quantities of each DNA fragment shown in Table 2 below and the In-Fusion HD Cloning Kit (Takara Bio Inc.), seamless cloning was performed to produce the modified protein expression vector pCAGGS-7myc-FATP1-EGFP using the fatty acid transporter CD36. The prepared pCAGGS-7myc-FATP1-EGFP was introduced into competent cells (E. coli DH5α) and stored.

[0059] [Table 2]

[0060] (3) Confirmation of modified protein expression in rat-derived myoblast culture cells (L6 cells) The modified protein expression vector prepared above was introduced into cells, and its expression within the cells was confirmed. Rat-derived myoblast cultured cells, specifically L6 cells (hereinafter also referred to as L6 cells), were used. L6 cells were cultured in DMEM(+ / +) medium (Fujifilm Wako Pure Chemical Industries) in the presence of 5% CO2 at 37°C. L6 cells were seeded in 24-well plates and cultured overnight.

[0061] 0.5 μg of pCAGGS-CD36-9myc-EGFP or pCAGGS-7myc-FATP1-EGFP was added to serum- and antibiotic-free OPTI-MEM medium (Thermo Fisher Scientific), and then Lipofectamine 3000 (Thermo Fisher Scientific) was added according to the manufacturer's instructions to prepare the transfection reagent.

[0062] After culturing L6 cells in the 24-well plate described above, the culture medium was removed, the plate was washed with 1×PBS, and one of the two transfection reagents described above was added. The plate was then incubated at 37°C for 48 hours in the presence of 5% CO2.

[0063] After transfection, L6 cells were washed twice with 1×PBS and then fixed in 4% (v / v) PFA / PBS for 5 minutes. After fixation, the L6 cells were washed three times with 1×PBS and mounted using 50% (v / v) glycerol / 1×PBS. Cells were observed using a fluorescence microscope BZ-8100 (KEYENCE), and EGFP fluorescence was measured.

[0064] Figure 3A shows the results of observing L6 cells transfected with pCAGGS-CD36-9myc-EGFP, and Figure 3B shows the results of observing L6 cells transfected with pCAGGS-7myc-FATP1-EGFP. In Figures 3A and 3B, "Untreated" refers to L6 cells that have not been transfected. As shown in Figures 3A and 3B, it was demonstrated that the modified protein can be expressed in cells using the constructed vector.

[0065] (4) Introduction of a modified protein expression vector into 3T3-L1 mature adipocytes In addition to the L6 cells mentioned above, the cells to be introduced were replaced with 3T3-L1 mature adipocytes (hereinafter also referred to as 3T3-L1 cells), and the modified protein expression vector pCAGGS-CD36-9myc-EGFP, prepared in the same manner as in (3) above, was introduced into the cells. Unless otherwise specified, the cells were cultured in DMEM(+ / +) medium, in the presence of 5% CO2, at 37°C. 2 × 10 53T3-L1 cells were suspended in OPTI-MEM, and 300 μg of pCAGGS-CD36-9myc-EGFP was added to the cell suspension and mixed. This mixture was transferred to a cuvette electrode, and electroporation was performed under the conditions shown in Table 3 below. After electroporation, 10% fetal bovine serum-containing DMEM medium was added to the mixture, and the mixture was incubated for 72 hours to produce 3T3-L1 cells transfected with pCAGGS-CD36-9myc-EGFP.

[0066] [Table 3]

[0067] Example 2: Investigation of cell membrane translocation of modified proteins We investigated the translocation of modified proteins to the cell membrane in response to insulin stimulation using four types of transfected cells. Cell membrane translocation was confirmed using immunohistochemistry.

[0068] (Insulin stimulation) After washing the cells twice with 1×PBS, they were cultured in serum and antibiotic-free DMEM for 5 hours to starve the cells. The culture medium was removed, and serum supplemented with 100 nM insulin and antibiotic-free DMEM medium were added, and the cells were incubated for 45 minutes.

[0069] (immunostaining) After incubation, the cells were washed twice with 1×PBS. After washing, 4% (v / v) PFA / PBS was added to the cells, and they were fixed at room temperature for 15 minutes. After fixation, the cells were washed three times with 1×PBS. 2% (v / v) normal goat serum (NGS) / 1×PBS was added to the washed cells, and they were blocked at room temperature for 30 minutes. After blocking, the 2% (v / v) NGS / 1×PBS was removed, and a 500-fold dilution of anti-Myc antibody (mouse-derived monoclonal antibody, clone 4A6: Merck Millipore) was added to the cells, and they were reacted overnight at 4°C. After the reaction, the cells were washed three times with 1×PBS, and a 1,000-fold dilution of secondary antibody (CF(registered trademark) 543 Dye-labeled anti-mouse IgG antibody: Biotium) was added, and they were reacted at room temperature for 1 hour under light protection. Subsequently, the cells were washed three times with 1×PBST and mounted in 50% (v / v) glycerol / 1×PBS. Cells were observed using a fluorescence microscope, model BZ-8100.

[0070] (Image analysis) The ratio of the signal intensity of modified protein migrated to the cell membrane to the total amount of modified protein in the cell (Myc / GFP) was evaluated using the image analysis software ImageJ. The signal intensity value of the Myc tag indicates the amount of modified protein in the cell membrane, and the signal intensity value of GFP indicates the total amount of modified protein in the cell. For statistical analysis, a t-test was performed using the values ​​from six cells.

[0071] Figures 4A and 5A show the results of insulin stimulation of transfected L6 cells. Furthermore, Figures 4B and 5B show the results of measuring the ratio of the signal intensity of the modified protein that migrated to the cell membrane to the total signal intensity of the modified protein in the cell, as measured by image analysis. Figures 4A and 4B show L6 cells introduced with pCAGGS-CD36-9myc-EGFP, and Figures 5A and 5B show L6 cells introduced with pCAGGS-7myc-FATP1-EGFP, respectively. From Figures 4A and 5A, it was confirmed that the modified protein migrated to the cell surface upon insulin stimulation. From Figures 4B and 5B, the migration of the modified protein to the cell membrane could be quantitatively evaluated from the ratio of the signal intensity of GFP and Myc tags in the acquired images, and it was shown that there was a significant difference in the amount of modified protein migrated to the cell membrane between insulin-stimulated cells and insulin-unstimulated cells.

[0072] Figures 6A and 6B show the results of the insulin stimulation test performed on transfected 3T3-L1 cells, similar to that performed on L6 cells (10 cells were used for image analysis). Figures 6A and 6B show 3T3-L1 cells into which pCAGGS-CD36-9myc-EGFP was introduced. These results confirm that, similar to L6 cells, the modified protein migrated to the cell surface in 3T3-L1 cells upon insulin stimulation. The migration of the modified protein to the cell membrane could be quantitatively evaluated from the ratio of the signal intensities of GFP and Myc tags in the acquired images, and a significant difference was observed between insulin-stimulated cells and insulin-unstimulated cells in terms of the amount of modified protein migrated to the cell membrane.

[0073] Example 3: Introduction of modified protein into biological tissue Two modified protein expression vectors were introduced into living tissue. A mouse and a biointroduction solution (physiological saline containing 80 μg of pCAGGS-CD36-9myc-EGFP or pCAGGS-7myc-FATP1-EGFP) were prepared, and the biointroduction solution was injected into the subcutaneous adipose tissue of the mouse (see Figure 7). After injection, terminals were attached to the subcutaneous adipose tissue area where the biointroduction solution had been injected so that an electric current could flow (see Figure 8), and electroporation was performed under the conditions in Table 4 below to introduce the modified protein expression vector into white adipocytes in the subcutaneous adipose tissue of the mouse.

[0074] [Table 4]

[0075] After electroporation, mice were kept under normal conditions for 5 days, followed by a 12-hour fast. Following fasting, the mice were euthanized, and a portion of the white adipocytes in the subcutaneous adipose tissue into which the modified protein expression vector had been introduced were excised to obtain insulin-unstimulated cells. Alternatively, after fasting, the mice were administered an insulin solution (saline solution containing 35.1 μg / kg (1 U / kg) of insulin) via the tail vein, and insulin stimulation was performed for 45 minutes. A portion of the white adipocytes in the subcutaneous adipose tissue into which the modified protein expression vector had been introduced was then excised from these mice to obtain insulin-stimulated cells.

[0076] After cutting these insulin-unstimulated and insulin-stimulated cells into small pieces, immunostaining was performed in the same manner as in Example 2, and the cells were observed using a confocal laser microscope (Olympus Corporation) to observe the migration of the modified protein to the cell membrane surface.

[0077] The results for mice introduced with two different modified protein expression vectors are shown in Figures 9 and 10, respectively. Figure 9 shows the results for mice introduced with pCAGGS-CD36-9myc-EGFP, and Figure 10 shows the results for mice introduced with pCAGGS-7myc-FATP1-EGFP. Figures 9 and 10 show that fluorescence was observed on the cell surface upon insulin stimulation, indicating that the modified protein migrated to the cell membrane upon insulin stimulation.

Claims

1. A step of stimulating recombinant myoblasts that express a modified fatty acid transporter protein and have an insulin receptor with insulin to translocate the modified protein to the cell membrane, The process of measuring the modified protein that has migrated to the cell membrane, The modified protein includes a labeled protein moiety in the intracellular region of the CD36 protein or FATP1 protein, and includes at least one tag peptide in the extracellular region. The myoblasts are myoblasts that express the modified protein by introducing an expression vector containing a polynucleotide encoding the modified protein. Methods for analyzing the intracellular dynamics of fatty acid transporters.

2. The method according to claim 1, wherein the tag peptide is at least one selected from Myc peptide, HA peptide, PA peptide, V5 peptide, S peptide, E peptide, T7 peptide, VSV-G peptide, Glu-Glu peptide, Strep-tag II peptide, HSV peptide, 6×His peptide, and DYKDDDDK peptide.

3. The method according to claim 1 or 2, wherein the tag peptide is contained in six to nine quantities in the modified protein.

4. The method according to any one of claims 1 to 3, wherein the fatty acid transporter of the modified protein is a CD36 protein, and the tag peptide is a protein inserted between amino acid positions 358 and 364 of the amino acid sequence of the CD36 protein.

5. The method according to any one of claims 1 to 4, wherein the fatty acid transporter of the modified protein is FATP1 and the tag peptide is attached to the N-terminus.

6. The aforementioned measurement step is The process of fixing the cells, The fixed cells are then subjected to immunostaining using an antibody that binds to a tag peptide. The method according to any one of claims 1 to 5, including the method described in any one of claims 1 to 5.

7. The method according to any one of claims 1 to 6, further comprising the step of measuring the labeled protein portion of the modified protein expressed from a vector containing a polynucleotide encoding the modified protein.

8. The method according to claim 7, referencing claim 6, wherein the migration of the modified protein to the cell membrane is evaluated based on the measured value of the labeled protein portion and the value based on the immunostaining.

9. The method according to any one of claims 1 to 8, wherein the labeled protein portion is at least one selected from GFP protein, BFP protein, CFP protein, YFP protein, EYFP protein, PA-GFP protein, and mCherry protein.

10. A vector for use in the method of claim 1, comprising a polynucleotide encoding a fatty acid transporter modified protein, the polynucleotide comprising a CD36 protein or FATP1 protein, a labeled protein portion located in the intracellular region of the protein, and at least one tag peptide located in the extracellular region of the protein.

11. Recombinant myoblasts into which the vector according to claim 10 has been introduced.

12. fatty acid transporters, The labeled protein portion located in the intracellular region of the fatty acid transporter, and At least one tag peptide present in the extracellular domain of the fatty acid transporter. Recombinant myoblasts having a fatty acid transporter-modified protein and an insulin receptor, wherein the recombinant myoblasts are for use in the method described in claim 1.

13. An analytical reagent for use in the method according to claim 1, comprising the vector according to claim 10 and insulin.

14. The reagent according to claim 13, further comprising a transfection reagent.

15. The reagent according to claim 13 or 14, further comprising an antibody that captures a tag peptide.

16. A step of adding a test substance to recombinant myoblasts that express a modified fatty acid transporter protein and have an insulin receptor, A step of measuring the tag peptide of the modified protein in the aforementioned cells, If a tag peptide is detected in the measurement step, the step of determining that the test substance is a ligand that causes fatty acid transporters to move to the cell membrane is as follows: Includes, The myoblasts are myoblasts that express the modified protein by introducing an expression vector containing a polynucleotide encoding the modified protein. The modified protein comprises a labeled protein moiety in the intracellular region of the CD36 protein or FATP1 protein, and at least one tag peptide in the extracellular region. A screening method for test substances.

17. A step of adding insulin to recombinant myoblasts that express a modified fatty acid transporter protein and have an insulin receptor, A step of measuring the tag peptide of the modified protein in the aforementioned cells, Includes, The myoblasts are myoblasts that express the modified protein by introducing an expression vector containing a polynucleotide encoding the modified protein. The modified protein comprises a labeled protein moiety in the intracellular region of the CD36 protein or FATP1 protein, and at least one tag peptide in the extracellular region. A method for analyzing receptors, comprising evaluating the insulin receptors of the cells based on the measurements taken in the measurement step.

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