JP2012127694A - Detection method of intracellular ubiquitination - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for quantitatively detecting polyubiquitination and/or deubiquitination of a target protein in a cell.SOLUTION: The method for detecting polyubiquitination and/or deubiquitination of a target protein in a cell includes the following steps of: (1) preparing a transformed cell, which expresses both of a fusion protein (X) of a target protein (A) and a tag (T) and a fusion protein (Y) of ubiquitin (B) and a tag (T), in which the fusion protein (X) is labeled with a fluorescent pigment (D) and the fusion protein (Y) is labeled with a fluorescent pigment (D); (2) incubating the transformed cell under the conditions to promote polyubiquitination and/or deubiquitination of the target proteins; (3) irradiating the transformed cell with laser light; and (4) measuring a fluorescent life of fluorescence emitted by the transformed cell.

Description

  The present invention relates to a method for detecting intracellular ubiquitination. More specifically, the present invention relates to a detection method using the fluorescence lifetime of intracellular ubiquitination.

  Ubiquitin is a low molecular protein consisting of 76 amino acids and having a molecular weight of about 8.6 kDa, and is present in all eukaryotic cells. Moreover, the primary structure of ubiquitin is evolutionarily well preserved, for example, there is about 96% homology between human and baker's yeast.

  Ubiquitin is linked to a cysteine residue of E1 (ubiquitin activating enzyme) at the C-terminal glycine residue in an APT-dependent manner, and then transferred from E1 to E2 (ubiquitin conjugating enzyme) and recognized by E3 (ubiquitin ligase). Is linked to the ε-amino group of the lysine side chain of the target protein to control the function of the polyubiquitinated protein. In many cases, the function of a target protein is controlled by a polyubiquitin chain to which ubiquitin is continuously bound.

  In the case of proteolysis, a polyubiquitin chain is formed by repeating the isopeptide bond of the ubiquitin C-terminal to the 48th lysine residue of ubiquitin bound to the lysine residue of the target protein. The polyubiquitin chain formed via lysine 48 (K48) functions as a recognition signal by the regulatory subunit of the 26S proteasome, and the polyubiquitinated protein is degraded by the proteasome.

  Numerous deubiquitinases that remove ubiquitin from substrate proteins have been identified, and ubiquitin modification functions as a ligand-dependent internalization signal for growth factor receptors, protein localization signals, etc. New physiological functions other than proteolysis of the system are becoming clearer one after another. Therefore, the ubiquitin modification system is now positioned as a reversible protein modification system that widely controls protein functions as well as proteolysis.

  The ubiquitin-proteasome system, which consists of the ubiquitin system that catalyzes polyubiquitination and the proteasome, a degradation machine, is involved in various life phenomena such as cell cycle progression, signal transduction, gene expression control, and protein quality control. Yes. Therefore, the breakdown of this degradation system leads to illness, while this degradation system is a target for drug discovery. Therefore, research on the mechanism of the ubiquitin-proteasome system is actively conducted in the field of life science.

  As a method for detecting polyubiquitination of a target protein, conventionally, a tissue or a cell is ground to form a tissue homogenate or a cell lysate, dissolved in a buffer solution, developed by SDS-PAGE, and then applied to a membrane such as nitrocellulose. It was common to detect the polyubiquitinated target protein by transferring and immunostaining the membrane (Western blotting). However, this method has a complicated procedure and low throughput, and it has not been possible to detect polyubiquitination for each cell.

  In recent years, throughput has been improved by flow cytometry (FCM, Fluorescence-Activated Cell Sorting), and polyubiquitination can be detected for each cell. In the FCM method, as a method for detecting polyubiquitination, that is, the interaction between a target protein and ubiquitin, a method of detecting FRET (Fluorescence Resonance Energy Transfer), BiFC (Bimolecular Fluorescence Complementation: Fluorescent protein reactivation) Configuration) method etc. are used.

  FRET is a phenomenon that occurs when two types of fluorescent molecule pairs (donor and acceptor) approach a distance of 10 nm or less, and a part of the excitation energy received by the donor from the laser is transferred to the acceptor without interposing fluorescence. It refers to the phenomenon of passing. The occurrence of FRET causes a decrease in donor fluorescence intensity and an increase in acceptor fluorescence intensity.

  In a method of detecting FRET by measuring fluorescence intensity, a fluorescence intensity ratio between donor fluorescence intensity and acceptor fluorescence intensity is usually used as an index of FRET. At this time, in the fluorescence channel for detecting the acceptor, in addition to acceptor fluorescence derived from FRET, leakage of donor fluorescence and acceptor fluorescence directly excited by the laser are measured. Further, since the fluorescence intensity measured at this time depends on the number of molecules of the expressed fluorescent protein, complicated correction is required. Therefore, it is difficult to quantitatively measure the polyubiquitination of proteins in cells, and the difference in polyubiquitination for each cell cannot be compared.

  In addition, a TR-FRET method that combines FRET and TRF (Time-Resolved Fluorescence) is also used for FRET detection. The point that FRET is detected based on the fluorescence intensity ratio between donor fluorescence and acceptor fluorescence is the same as the FRET method described above. The TR-FRET method is characterized in that a lanthanoid complex that is a fluorescent substance having a very long fluorescence lifetime is used as a donor and a fluorophore such as fluorescein or GFP is used as an acceptor. When both are close to each other, the energy excited by the light absorption of the lanthanoid complex is transferred to the acceptor fluorophore by resonance energy transfer. Here, the lanthanoid complex is a fluorescent material having a very long fluorescence lifetime and has a time resolution, so that the background of short-lived fluorescence from other compounds and acceptors in the sample by the excitation light before detecting the signal by FRET. Can be suppressed. The TR-FRET method can detect intracellular polyubiquitination with high sensitivity (low background).

  For example, in Patent Document 1, polyubiquitination of a fusion protein of GFP expressed in cells and IκBα is performed by terbium (Tb) -labeled anti- (poly) ubiquitin antibody bound to ubiquitin and FRET between Tb and GFP. And polyubiquitination is detected on the microplate by the TR-FRET method (Example 23, FIG. 29, etc.).

  However, the TR-FRET method must measure fluorescence intensity. If the donor and acceptor labeling amounts are different or the molar extinction coefficient changes, it is directly affected and quantitatively measured. It becomes difficult. Further, since donor fluorescence is limited to lanthanoid complexes such as terbium and europium which are so-called rare metals, there is a problem that handling is required and it cannot be carried out easily. Furthermore, since the TR-FRET method performs detection in the time domain where the fluorescence intensity is attenuated, it is difficult to obtain a sufficient amount of fluorescence for measurement when the amount of fluorescent dye labeling or the amount of detection target is small, There is also a problem that the detectable range is limited.

Special table 2009-513681

  Therefore, an object of the present invention is to provide a method for quantitatively detecting polyubiquitination and / or deubiquitination of a target protein in a cell.

As a result of intensive studies to solve the above problems, the present inventors expressed both a fusion protein of a target protein and a protein tag and a fusion protein of a ubiquitin and a protein tag in a transformed cell, It is labeled with a fluorescent dye. By irradiating a cell with laser light and measuring the fluorescence lifetime, it is possible to quantitatively detect polyubiquitination and / or deubiquitination of the target protein in the cell. I knew it.
That is, the present invention is as follows.

(1) A method for detecting intracellular polyubiquitination and / or deubiquitination of a target protein, comprising the following steps:
1) A transformed cell that expresses both a fusion protein (X) of a target protein (A) and a tag (T ₁ ) and a fusion protein (Y) of a ubiquitin (B) and a tag (T ₂ ), Preparing a transformed cell in which the fusion protein (X) is labeled with a fluorescent dye (D ₁ ) and the fusion protein (Y) is labeled with a fluorescent dye (D ₂ );
2) Incubating the transformed cell under conditions where polyubiquitination and / or deubiquitination of the target protein is performed,
3) The transformed cells are irradiated with laser light, and 4) the fluorescence lifetime of the fluorescence emitted by the transformed cells is measured.

(2) The method according to (1), wherein the fluorescence intensity is simultaneously measured in the step of measuring the fluorescence lifetime.

(3) The method according to (1) or (2) above, wherein the step of irradiating the laser beam and the step of measuring the fluorescence lifetime are performed using a flow cytometer.

(4) The method according to any one of (1) to (3), wherein fluorescence energy transfer (FRET) occurs between the fluorescent dye (D ₁ ) and the fluorescent dye (D ₂ ).

(5) The method according to (4) above, wherein the fluorescent dye (D ₁ ) is a donor and the fluorescent dye (D ₂ ) is an acceptor.

(6) The above (1) to (5), wherein the tag (T ₁ ) is an affinity tag (AT ₁ ) and the fluorescent dye (D ₁ ) is bound to the affinity tag (AT ₁ ). The method in any one of.

(7) The tag (T ₂ ) is an affinity tag (AT ₂ ), and the fluorescent dye (D ₂ ) is bound to the affinity tag (AT ₂ ), (1) to (5) The method in any one of.

(8) The tag (T ₁ ) and the tag (T ₂ ) are an affinity tag (AT ₁ ) and an affinity tag (AT ₂ ), respectively, and the fluorescent dye (D ₁ ) and the fluorescent dye ( The method according to any one of (1) to (5) above, wherein D ₂ ) is bound to the affinity tag quality (AT ₁ ) and the affinity tag quality (AT ₂ ), respectively.

(9) The fluorescent dye (D ₁ ) is an organic synthetic fluorescent dye (C ₁ ) or fluorescent protein (P ₁ ), and / or the fluorescent dye (D ₂ ) is an organic synthetic dye (C ₂ ) or fluorescent protein The method according to any one of (1) to (8) above, which is (P ₂ ).

(10) The fluorescent dye (D ₁ ) is a fluorescent protein (P ₁ ), and the tag (T ₂ ) is a fluorescent protein tag (PT ₁ ) containing the fluorescent protein (P ₁ ) The method according to any one of 1) to (5).

(11) The above (Fluorescent dye (D ₂ ) is a fluorescent protein (P ₂ ), and the tag (T ₂ ) is a fluorescent protein tag (PT ₂ ) containing the fluorescent protein (P ₂ ) The method according to any one of 1) to (5).

(12) The fluorescent dye (D ₁ ) and the fluorescent dye (D ₂ ) are a fluorescent protein (P ₁ ) and a fluorescent protein (P ₂ ), respectively, and the tag (T ₁ ) and the tag ( ( ₂ ) to (5), wherein T ₂ ) are a fluorescent protein tag (PT ₁ ) containing the fluorescent protein (P ₁ ) and a fluorescent protein tag (PT ₂ ) containing the fluorescent protein (P ₂ ), respectively. ) Any one of the methods.

(13) The target protein is carbamoyl phosphate synthase I (CPSI), glutathione S-transferase (GST), fructose-1,6-bisphosphate aldolase, electron transfer flavoprotein alpha subunit, medium chain fatty acyl acyl CoA dehydrogenase, Long chain acyl CoA synthetase, TBP-related factors (TAFs), glyceraldehyde-3-phosphate dehydrogenase, importin α, DNA2 helicase, ribosomal protein L7, von Willebrand factor (vWF), N-ethylmaleimide sensitive factor (NSF) ), S-adenosylhomocysteine hydrolase, serine protease, asymmetric acetylcholinesterase collagen-like tail subunit (ColQ), serine carboxypeptider 1 (SCEP1), histone cluster 1, Δ3, Δ2-enoyl-CoA isomerase, acrosin-binding protein (ACRPB), semaphorin 5A, aurora-A-kinase interaction protein 1, p53, phosphoinositide 3-kinase (PI3K), phosphoinositide 3-kinase (PI3K) p85 subunit, β-catenin, Zap-70, T cell receptor (TCR), Fyn tyrosine kinase, growth factor binding protein binding protein 2 (Grb2), Sos protein, DNA repair kinase Ku70 / Ku80 Complex (Ku70 / 80), Crk-like protein (Crkl), phospholipase C-γ (PLCγ), protein kinase Cθ (PKCθ), estrogen receptor binding protein (ERBP), ubiquitin ligase HIP, cyclin (Cdc13), hypoxia-inducible factor 1α (HIF-1α), mitochondrial-related protein (Drp1), histone H2B, H2AX complex (histone), IκBα, IκB p100, TNF receptor-related factor 6 (TRAF-6) ) And NF-kB p-105, the method according to any one of (1) to (12) above.

(14) The method according to any one of (1) to (12) above, wherein the target protein (A) is ubiquitin.

(15) The method according to any one of (1) to (14) above, which is a method for screening an inhibitor of degradation of the target protein (A) by the proteasome.

  According to the present invention, it is possible to provide a method for quantitatively detecting polyubiquitination and / or deubiquitination of a target protein in a cell.

Moreover, according to this invention, the difference in polyubiquitination for every cell can be compared.
Further, according to the present invention, a large number of samples can be easily measured with a high throughput and in a short time.
Furthermore, according to the present invention, polyubiquitination and / or deubiquitination in living cells can be detected in real time.
Furthermore, according to the present invention, polyubiquitination and / or deubiquitination can be detected even when the expression level of the fusion protein is smaller than when only the fluorescence intensity is measured.

The present invention is “a method for detecting polyubiquitination and / or deubiquitination of a target protein in a cell, comprising the following steps:
1) A transformed cell that expresses both a fusion protein (X) of a target protein (A) and a tag (T ₁ ) and a fusion protein (Y) of a ubiquitin (B) and a tag (T ₂ ), Preparing a transformed cell in which the fusion protein (X) is labeled with a fluorescent dye (D ₁ ) and the fusion protein (Y) is labeled with a fluorescent dye (D ₂ );
2) Incubating the transformed cell under conditions where polyubiquitination and / or deubiquitination of the target protein is performed,
3) The transformed cells are irradiated with laser light, and 4) the fluorescence lifetime of the fluorescence emitted by the transformed cells is measured. Is.
Hereinafter, the present invention will be described in detail.

<Target protein (A)>
The target protein (A) is not particularly limited, but is preferably a protein or ubiquitin that is degraded by a ubiquitin-proteasome system (which refers to a system that degrades a protein labeled with ubiquitin with the proteasome).

  The protein degraded by the ubiquitin-proteasome system is not particularly limited, but is preferably one selected from the group consisting of the following proteins: carbamoyl phosphate synthase I (CPSI), glutathione S-transferase ( GST), fructose-1,6-bisphosphate aldolase, electron transfer flavoprotein alpha subunit, medium chain fatty acyl CoA dehydrogenase, long chain acyl CoA synthetase, TBP-related factors (TAFs), glyceraldehyde-3-phosphate dehydrogenase , Importin α, DNA2 helicase, ribosomal protein L7, von Willebrand factor (vWF), N-ethylmaleimide sensitive factor (NSF), S-adenosylhomocysteine hydride Ase, serine protease, asymmetric acetylcholinesterase collagen-like tail subunit (ColQ), serine carboxypeptidase 1 (SCEP1), histone cluster 1, Δ3, Δ2-enoyl-CoA isomerase, acrosin-binding protein (ACRPB), semaphorin 5A , Aurora-A-kinase interacting protein 1, p53, phosphoinositide 3-kinase (PI3K), phosphoinositide 3-kinase (PI3K) p85 subunit, β-catenin, Zap-70, T cell receptor (TCR), Fyn tyrosine Kinase, growth factor binding protein binding protein 2 (Grb2), Sos protein, DNA repair kinase Ku70 / Ku80 complex (Ku70 / 80), Crk-like protein (Crkl), e Spholipase C-γ (PLCγ), protein kinase Cθ (PKCθ), estrogen receptor binding protein (ERBP), ubiquitin ligase CHIP, cyclin (Cdc13), hypoxia-inducible factor 1α (HIF-1α), mitochondrial related protein (Drp1), Histone H2B, H2AX complex (histone), IκBα, IκB p100, TNF receptor-related factor 6 (TRAF-6) and NF-kB p-105.

<Ubiquitin (B)>
Ubiquitin (B) is not particularly limited, but preferably has a function as ubiquitin in transformed cells. For example, when preparing transformed cells using human-derived cells, it is preferable to use human ubiquitin.

<Polyubiquitination / Deubiquitination>
Polyubiquitination refers to a reaction of adding ubiquitin to a target protein. This reaction system involves a complex enzyme reaction system of ubiquitin activating enzyme (El), ubiquitin-binding enzyme (E2), and ubiquitin ligase (E3). By this complex enzyme reaction system, an isopeptide bond is formed with a specific lysine residue of the target protein, and the same bond is repeated between ubiquitin molecules to form a plurality of (poly) ubiquitin chains.
Deubiquitination refers to a reaction that removes a (poly) ubiquitin chain from a polyubiquitinated target protein. This reaction system involves deubiquitinating enzymes (DUBs).

<Tag (T ₁ ) / Tag (T ₂ )>
A tag (referred to as “protein tag” or “peptide tag”) is a protein or peptide that serves as a marker of a target protein (A) or ubiquitin (B). In the present invention, the tagged target protein or ubiquitin is expressed as a fusion protein. In the present invention, a tag attached to a target protein is referred to as a tag (T ₁ ), and a tag attached to ubiquitin is referred to as a tag (T ₂ ), which may be distinguished. The tag (T ₁ ) and the tag (T ₂ ) are preferably different. The tag (T ₁ ) is preferably attached to its N-terminal and / or C-terminal depending on the function, structure, etc. of the target protein. The tag (T ₂ ) may be either N-terminal or C-terminal of ubiquitin, It is preferable to attach to the terminal.
In the present invention, the tag is preferably a tag relating to a fluorescent label, and more preferably an affinity tag or a fluorescent protein.

<Tags related to fluorescent labels>
A tag relating to a fluorescent label refers to a tag for binding a fluorescent dye directly or via a ligand or a tag which itself is a fluorescent label. An example of a tag for binding a fluorescent label is an affinity tag, and a tag that is itself a fluorescent label includes a fluorescent protein tag.
In the present invention, when the tag (T ₁ ) and / or the tag (T ₂ ) is an affinity tag, they may be referred to as an affinity tag (AT ₁ ) and / or an affinity tag (AT ₂ ), respectively.

《Affinity tag》
An affinity tag is a tag that utilizes specific affinity (binding, affinity) with other molecules. In addition, what is a fluorescent protein is mentioned later.
Specific examples of the affinity tag include His tag, HQ tag, HN tag, HAT tag, GST (glutathione-S-transferase) tag, MBP (maltose binding protein) tag, FLAG tag, HA tag, c- Examples include Myc tag, TC (tetracysteine) tag, SNAP tag, CLIP tag, Halo tag, BCCP (biotinylated peptide) tag and the like.
The fluorescent dye is preferably bound directly or indirectly to these affinity tags via bonds such as covalent bonds and hydrogen bonds.
The affinity tag (AT ₁ ) fused with the target protein (A) and the affinity tag (AT ₂ ) fused with ubiquitin (B) are different tags and have specific affinity with different chemical substances. It is preferable. The fluorescent dye (D ₁ ) and fluorescent dye (D ₂ ) that fluorescently label each tag are preferably different fluorescent dyes. The fluorescent dye is preferably bound to the affinity tag by direct interaction or via a moiety having affinity with the affinity tag such as an antibody.

《Fluorescent protein tag》
The following fluorescent protein is used as a tag. At this time, the fusion protein (X) or the fusion protein (Y) is obtained by binding a fluorescent protein to the N-terminus or C-terminus of the target protein (A) directly or via a linker peptide, or ubiquitin (B), respectively. It is preferable that the fluorescent protein is bound to the N-terminus or C-terminus directly or via a linker peptide.
In the present invention, when the tag (T ₁ ) and / or the tag (T ₂ ) is a protein tag containing a fluorescent protein (P ₁ ) and / or a protein tag containing a fluorescent protein (P ₂ ), respectively, Sometimes referred to as a fluorescent protein tag (PT ₁ ) and / or a fluorescent protein tag (PT ₂ ).
The fluorescent protein tag (PT ₁ ) and the fluorescent protein tag (PT ₂ ) are preferably tags with different fluorescent proteins.

"Fluorescent dye _(D 1) / fluorescent dye _(D 2)"
The fluorescent dye is not particularly limited, but an organic synthetic fluorescent dye and / or fluorescent protein is preferable.
In the present invention, the fluorescent dye, organic synthetic fluorescent dye, and fluorescent protein that label the target protein are referred to as fluorescent dye (D ₁ ), organic synthetic fluorescent dye (C ₁ ), and fluorescent protein (P ₁ ), respectively. The fluorescent dye, organic synthetic fluorescent dye, and fluorescent protein that label ubiquitin may be referred to as a fluorescent dye (D ₂ ), an organic synthetic fluorescent dye (C ₂ ), and a fluorescent protein (P ₂ ), respectively. The fluorescent dye (D ₁ ) and the fluorescent dye (D ₂ ) are preferably different from each other, and fluorescence energy transfer (FRET) between the fluorescent dye (D ₁ ) and the fluorescent dye (D ₂ ). Preferably occurs (is a FRET pair).

(Organic synthetic fluorescent dye)
The organic synthetic fluorescent dye is not particularly limited. For example, coumarin-based dyes such as hydroxycoumarin, 6-methylcoumarin, 4-methoxycoumarin, 7-methoxycoumarin, and 7-aminocoumarin; rhodamine B, rhodamine 6G, rhodamine 123, Sulforhodamine 101, Texas Red (sulforhodamine 101 acid chloride), 5-TAMRA (5-carboxytetramethylrhodamine), 6-TAMRA, TMR (tetramethylrhodamine), TRITC (tetramethylrhodamine-5 (6) -isothiocyanate NATO), 5-ROX (5-carboxy-X-rhodamine), rhodamine dyes such as 6-ROX (6-carboxy-X-rhodamine); fluorescein, 5-FAM (5-carboxyfluorescein), 6-FAM ( 6-cal Boxyfluorescein), 5-HEX (5-carboxy-2 ′, 4,4 ′, 5 ′, 7,7′-hexachlorofluorescein), 6-HEX (6-carboxy-2 ′, 4,4 ′, 5 ', 7,7'-hexachlorofluorescein), 5-TET (5-carboxy-2', 4,7,7'-tetrachlorofluorescein), 6-TET (6-carboxy-2 ', 4,7,7 Fluorescein dyes such as' -tetrachlorofluorescein), FITC (fluorescein isothiocyanate), FlAsH (Fluorescein Arsenical Hairpin); cyanine dyes such as Cy3 and Cy5; resorufin dyes such as resorufin, ReAsH (Resorufin Arsenical Hairpin); Alexa Fluor ^(R) 350, Alexa Fluor 405, Alexa Fluor 430, Alexa F Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 680, Alexa Fluor 680, Alexa Fluor 680, Alexa Fluor 680, Alexa Fluor 680 Dye series (manufactured by Life Technologies); DyLight 350, DyLight 405, DyLight 488, DyLight 549, DyLight 594, DyLight 633, DyLight 649, DyLight 680, DyLight Light 850D, DyLight Light 850D -Oyster dye series such as Oyster-500, Oyster-550P, Oyster-556, Oyster-645, Oyster-650P, Oyster-656 (manufactured by Denovo Biolabels); .

  Organic synthetic fluorescent dyes can be attached via a ligand that has affinity for the tag, a ligand that has affinity for the modified tag, an antibody that has affinity for the tag, or an antibody that has affinity for the target protein or ubiquitin. It is preferable to bind to the fusion protein (Y) or the fusion protein (X).

  A combination of organic synthetic fluorescent dyes preferable as a FRET pair is not particularly limited as long as the fluorescence spectrum emitted from the donor overlaps with the excitation spectrum of the acceptor. For example, Alexa Fluor 430 and Cy3, FITC and Alexa Fluor 532, Cy3 and TAMRA (the left side is the donor and the right side is the acceptor).

  In the case of the FRET pair, one of the organic synthetic fluorescent dye for labeling the target protein and the organic synthetic fluorescent dye for labeling ubiquitin may be a donor and the other an acceptor. It is preferable that the synthetic fluorescent dye serves as a donor and the organic synthetic fluorescent dye that labels ubiquitin serves as an acceptor. When the target protein is ubiquitin, one of the organic synthetic fluorescent dye that labels the target protein and the organic synthetic fluorescent dye that labels ubiquitin may be a donor and the other may be an acceptor.

(Fluorescent protein)
The fluorescent protein is not particularly limited, but Sirius, EBFP, SBP2, EBP2, Azurite, mKalama1, TagBFP, mBlueberry, mTurquoise, ECFP, Cerulean, TagCFP, AmCyan, GTP, MiCy, GTP, MiCyP , AG (Azami-Green), mAG1, ZsGreen, EmGFP (Emerald), EGFP, GP2, T-Sapphire, HyPer, and other blue fluorescent proteins; TagYFP, mAmetrine, EYFP, YFP, Venus, CitriYP, PhineP, PhineP turboYFP, ZsYellow, mBana, etc. Yellow fluorescent protein; orange fluorescent protein such as mKO1, KO (Kusabila Orange), mOrange, mOrange2, mKO2, etc .; , TurboFP602, mRP1, JRed, KillerRed, mCherry, KeimaRed, HcRed, mRasberry, mKate2, TagFP635, mPlum, eggFP650, Neptune, mNeptune, egFPT, etc. Thailand Chromatography; and the like.

  A combination of fluorescent proteins preferable as a FRET pair is not particularly limited as long as the fluorescence spectrum emitted from the donor overlaps with the excitation spectrum of the acceptor. For example, CFP and YFP, AG and KO, EGFP and Venus, etc. (The left side is a donor, and the right side is an acceptor).

  In the case of the FRET pair, one of the fluorescent protein that labels the target protein and the fluorescent protein that labels ubiquitin may be a donor and the other may be an acceptor, but the fluorescent protein that labels the target protein is a donor. The fluorescent protein that labels ubiquitin is preferably an acceptor. When the target protein is ubiquitin, one of the fluorescent protein that labels the target protein and the fluorescent protein that labels ubiquitin may be a donor and the other may be an acceptor.

  Fluorescent proteins can be passed through ligands with affinity for the tag, via ligands with affinity for the modified tag, via antibodies with affinity for the tag, or via antibodies with affinity for the target protein or ubiquitin. It is preferable to bind to the fusion protein (Y) or the fusion protein (X), or to be expressed in the cell as a fusion protein with the target protein or ubiquitin as a tag itself.

  The fluorescent labeling of the target protein (A) and ubiquitin (B) may be both labeled with a fluorescent protein, or both may be labeled with an organic synthetic fluorescent dye, or one may be labeled with a fluorescent protein, May be labeled with an organic synthetic fluorescent dye.

<Transformed cells>
A transformed cell refers to a cell transformed to express the fusion protein (Y) and the fusion protein (X) in the cell. Preferably, the cell which integrated the gene which codes a fusion protein (Y), and the gene which codes a fusion protein (X) is said. More preferably, it refers to a cell double-transfected with a vector encoding the fusion protein (Y) and a vector encoding the fusion protein (X).

The cell may be a cell derived from an animal such as a human or a mouse, an insect cell, or a yeast.
Specific examples of human-derived cells include adherent cells such as HEK293 and HeLa; suspension cells such as 293-F, 293-FT, and Jurkat; In addition, primary cells and stem cells established from various tissues can be used.
Examples of cells derived from mammals other than humans include cells derived from mouse-derived MC systems, Chinese hamster-derived CHO, and the like.
Examples of insect cultured cells include Sf strains established from Spodoptera frugiperda such as Sf-9 and Sf-21.

<Incubation>
Incubation can appropriately determine conditions under which polyubiquitination can be induced, depending on the cell type. For example, in the case of human-derived 293-F free cells, it is preferable to culture while shaking at 37 ° C. in a 5 to 10% carbon dioxide atmosphere. In addition, adherent cells such as HeLa are preferably cultured in a static state at 37 ° C. in a 5% carbon dioxide atmosphere.

<Laser light>
The wavelength of the laser light can be determined as appropriate depending on the excitation wavelength of the fluorescent dye to be used, but is preferably a visible light region wavelength from the viewpoint of relatively little stimulation to cells.

<Fluorescence lifetime / fluorescence intensity>
The fluorescence lifetime is the time required for the fluorescence intensity to drop to 1 / e. The fluorescence lifetime depends on the molecular structure and properties, and the state of the molecule.
In the method of the present invention, when measuring the fluorescence lifetime, the fluorescence intensity may be measured simultaneously.

<Flow cytometer>
In the method of the present invention, it is preferable to use a flow cytometer from the viewpoint of increasing the throughput. As the flow cytometer, one capable of simultaneously measuring the fluorescence lifetime and the fluorescence intensity is preferable, and specifically, for example, Flicyme ^(R) 300 (manufactured by Mitsui Engineering & Shipbuilding) can be mentioned. When a flow cytometer is used, measurement can be performed for each cell, and more information can be obtained.
In addition, when using a flow cytometer, it is preferable to use a floating cell, but the cell can also be used in a floating state by treating the adherent cell with trypsin or the like.

<Fluorescence energy transfer (FRET)>
FRET is a phenomenon that occurs when two types of fluorescent molecule pairs (donor and acceptor) approach a distance of 10 nm or less, and a part of the excitation energy received by the donor from the laser is transferred to the acceptor without interposing fluorescence. It refers to the phenomenon of passing. The occurrence of FRET causes a decrease in donor fluorescence intensity and an increase in acceptor fluorescence intensity.

In the present invention, it is sufficient that fluorescence energy transfer (FRET) occurs between the fluorescent dye (D ₁ ) and the fluorescent dye (D ₂ ), which may be a donor and which may be an acceptor. The fluorescent dye that labels the protein is preferably a donor, and the fluorescent dye that labels ubiquitin is preferably an acceptor.

[Screening method]
The present invention also provides a method for screening for inhibitors of proteasome degradation of a target protein.
This is because the target protein is a protein that is degraded in the ubiquitin-proteasome system, and thus a substance that inhibits polyubiquitination can also be an inhibitor of the degradation of the protein.

[Detection of polyubiquitination of IκBα]
<Features of NF-κB>
NF-κB is one of transcription factors that play a central role in immune responses, and is involved in many physiological phenomena such as acute and chronic inflammatory reactions, cell proliferation, and apoptosis. NF-κB is activated by stimuli such as stress, cytokines, and ultraviolet rays. Poor control of NF-κB activity causes inflammatory diseases such as Crohn's disease and rheumatoid arthritis, as well as cancer and septic shock, and in many cases, NF-κB is constantly activated in malignant tumors. . Furthermore, NF-κB is also involved in the growth of cytomegalovirus (CMV) and human immunodeficiency virus (HIV).

  NF-κB forms a homo- or hetero-dimer in the cytoplasm, and exists in an inactive state by binding to its inhibitor IκB. NF-κB belongs to the Rel family and is classified into five types according to its structure: RelA (p65), RelB, c-Rel, NF-κB1 (p105 / p50), and NF-κB2 (p100 / p52). Of these, p50 and p52 are produced as precursors of p105 and p100, and are produced by limited degradation by the proteasome. These have an amino acid sequence called Rel homology domain (RHD) as a region common to each other, and a transition signal (NLS) into the nucleus also exists in this region.

<Features of IκB>
IκB is a suppressor of nuclear factor κB1 (NF-κB). There are seven families of IκB (IκBα / β / γ / ε, Bcl3, p105, p100). These molecules have a repetitive sequence of amino acids called ankyrin repeats, thereby associating with NF-κB RHD and masking NLS present in NF-κB to block NF-κB translocation into the nucleus. is doing.

<Activation of NF-κB>
In normal cells, NF-κB exists in the cytoplasm as an inactive form associated with inhibitory proteins such as IκBα and p100. However, when an activation signal is received, the inhibitory protein is degraded and NF-κB moves into the nucleus. Activated. At present, the activation pathway of NF-κB is known to include the following three pathways (Tsuchiya, Yoshihiro, five others, “Nuclear IKKbeta is an adaptor protein for IkappaBalpha ubiquitination and degradation in UV-induced NF-kappaB activation ", Molecular Cell, August 27, 2010, Vol. 39, No. 4, pp. 570-582).

  The first pathway (classical pathway) of NF-κB activation is a pathway in which inflammatory cytokines such as tumor necrosis factor α (TNFα) activate IKKβ to induce degradation of IκBα. Activated IKKβ phosphorylates two serine residues present at the N-terminus of IκBα, and the phosphorylated IκBα undergoes polyubiquitination by ubiquitin ligase β-TrCP and is degraded by the proteasome. Finally, NF-κB composed of RelA and p50 moves into the nucleus and induces gene expression. Normally, activated NF-κB induces the expression of inhibitory proteins such as IκBα and the expression of activation-suppressing proteins such as A20, so that NF-κB is quickly inactivated.

The second pathway of NF-κB activation is a pathway in which lymphocyte control-related factors such as CD40 activate IKKα. Activated IKKα phosphorylates p100, and the phosphorylated p100 is converted to p52 by limited degradation via polyubiquitination by β-TrCP, and finally NF-κB composed of RelB and p52 Is activated.
The point that β-TrCP directly associates with an inhibitory protein phosphorylated by IKKα or IKKβ to induce polyubiquitination is a common point between the first pathway and the second pathway.

  On the other hand, the third pathway of NF-κB activation is a pathway in which IκB is inducibly degraded without phosphorylation. In this pathway, IKKβ does not function as a kinase, but functions as an adapter protein that mediates the association between β-TrCP and IκB. When cells are irradiated with ultraviolet rays, IκBα moves into the nucleus and associates with IKKβ. Β-TrCP is associated with IKKβ. On this complex, IκBα undergoes polyubiquitination and is finally degraded by the proteasome. Casein kinase 2 and p38 MAP kinase also promote the degradation of IκBα via IKKβ.

  In this example, irradiation with a visible light laser is performed to detect polyubiquitination of IκBα, so that polyubiquitination of the inhibitory protein IκBα in the first pathway can be detected.

[Example 1] Detection of polyubiquitination of fusion protein in transformed cells <Method>
<< Expression construct of fusion protein of IκBα and GST tag >>
(Entry clone construction)
Using the pENTR / D-TOPO ^(R) cloning kit (Life Technologies), the DNA fragment having the nucleotide sequence shown in SEQ ID NO: 1 was incorporated into the pENTR / D-TOPO ^(R) entry vector by Directional TOPO cloning. Obtain a recombinant vector. Furthermore, this recombinant vector is transfected into Escherichia coli (OneShot ^(R) OmniMax ^™ 2T1 cells, Life Technologies), cultured in a kanamycin-containing medium, and the recombinant vector is isolated and purified to obtain an entry clone.

(Construction of expression clone)
The constructed entry clone, Gateway ^(R) expression vector for mammalian cells (pDEST ^™ 27, manufactured by Life Technologies; N-terminal GST tag, neomycin resistant), and Gateway® LR Clonase ^™ II enzyme mix (produced by Life Technologies ⁾ ) Are mixed and incubated, and an LR recombination reaction is performed to obtain a recombinant vector. Furthermore, the recombinant vector E. coli ^(OneShot (R) TOP10 cells, Life Technologies, Inc.) and transfected into, and cultured in medium containing kanamycin, and separating and purifying the recombinant vector, expressing clones (hereinafter, Example 1 is referred to as “expression clone 1”).

<< Expression construct of fusion protein of ubiquitin and TC tag >>
(Entry clone construction)
Using a pENTR / D-TOPO ^(R) cloning kit (manufactured by Life Technologies), a DNA fragment having the nucleotide sequence shown in SEQ ID NO: 3 is incorporated into the pENTR / D-TOPO ^(R) entry vector to obtain a recombinant vector . Furthermore, this vector is transfected into Escherichia coli (OneShot ^(R) OmniMax ^™ 2T1 cells, manufactured by Life Technologies), cultured in a kanamycin-containing medium, and the recombinant vector is separated and purified to obtain an entry clone.

(Construction of expression clone)
The constructed entry clone, expression vector (pcDNA ^TM 6.2 / nTC-Tag-DEST, manufactured by Life Technologies; N-terminal TC tag, blasticidine resistant), and LR clonase ^TM II enzyme mix (manufactured by Life Technologies) The mixture is incubated, and an LR recombination reaction is performed to obtain a recombinant vector. Furthermore, it was transfected into Escherichia coli (OneShot ^(R) TOP10 cells, manufactured by Life Technologies), cultured in a kanamycin-containing medium, the recombinant vector was isolated and purified, and an expression clone (hereinafter referred to as “expression clone in Example 1”). 2 ”).

<Transfection>
Expression clone 1 and expression clone 2 are double-transfected into 293-F suspension cells (FreeStyle ^™ 293-F cells; manufactured by Life Technologies) by a conventional method. Cultivation in a medium containing neomycin sulfate and blasticidine S hydrochloride at 37 ° C. for 48 hours in an atmosphere of 8% carbon dioxide with shaking, fusion of GST tag and IκBα, TC tag and ubiquitin Express the protein.

<Fluorescent labeling>
Binding to GST tag, Alexa ^{Fluor (R)} 488 dye-labeled anti-glutathione -S- transferase antibody (excitation maximum 495 nm, maximum fluorescence wavelength 520 nm; manufactured by Life Technologies) and binds to TC tag, ReAsH-EDT Fluorescent labeling is performed using a _two- labeling reagent (maximum excitation wavelength 593 nm, maximum fluorescence wavelength 608 nm; manufactured by Life Technologies).

<Fluorescence lifetime measurement>
Using a flow cytometer Flicyme ^(R) 300 (manufactured by Mitsui Engineering & Shipbuilding), 100,000 pieces of data are measured for each transformed cell. A band-pass filter having a center wavelength of 494 nm and a half-value width of 41 nm is used for the donor, and a long-pass filter having a cut-on wavelength of 600 nm is used for the acceptor.

<result>
The occurrence of FRET is detected. Moreover, the polyubiquitination in a cell can be measured quantitatively, and also the difference in polyubiquitination for each cell can be compared. And shortening of the fluorescence lifetime of a donor area | region is recognized.

[Comparative Example 1] Measurement with non-transformed cells <Method>
Use 293-F suspension cells without transformation. Measurement and others are performed in the same manner as in the first embodiment.
<result>
The occurrence of FRET is not detected.

[Comparative Example 2] Detection of polyubiquitination of fusion protein in transformed cells by fluorescence intensity <Method>
The fluorescence intensity is measured using Flicyme-300, but the fluorescence lifetime is not measured, and the measurement is performed in the same manner as in Example 1 except that the fluorescence intensity ratio is used as an index of FRET.
<result>
The occurrence of FRET is detected. However, the background is high and intracellular polyubiquitination cannot be quantitatively measured.

  Screening of a substance that inhibits or promotes polyubiquitination of a target protein can be performed with high throughput, and can be used as a cell assay technique for drug discovery and development.

Claims (15)

A method for detecting polyubiquitination and / or deubiquitination of a target protein in a cell, comprising the following steps:
1) A transformed cell that expresses both a fusion protein (X) of a target protein (A) and a tag (T ₁ ) and a fusion protein (Y) of a ubiquitin (B) and a tag (T ₂ ), Preparing a transformed cell in which the fusion protein (X) is labeled with a fluorescent dye (D ₁ ) and the fusion protein (Y) is labeled with a fluorescent dye (D ₂ );
2) Incubating the transformed cells under conditions that allow polyubiquitination and / or deubiquitination of the target protein,
3) Irradiating the transformed cells with laser light,
4) The fluorescence lifetime of the fluorescence emitted from the transformed cells is measured.   The method according to claim 1, wherein in the step of measuring the fluorescence lifetime, the fluorescence intensity is simultaneously measured.   The method according to claim 1 or 2, wherein the step of irradiating the laser beam and the step of measuring the fluorescence lifetime are performed using a flow cytometer. The method according to claim 1, wherein fluorescence energy transfer (FRET) occurs between the fluorescent dye (D ₁ ) and the fluorescent dye (D ₂ ). The method according to claim 4, wherein the fluorescent dye (D ₁ ) is a donor and the fluorescent dye (D ₂ ) is an acceptor. The tag (T ₁ ) is an affinity tag (AT ₁ ), and the fluorescent dye (D ₁ ) is bound to the affinity tag (AT ₁ ). Method. The tag (T ₂ ) is an affinity tag (AT ₂ ), and the fluorescent dye (D ₂ ) is bound to the affinity tag (AT ₂ ). Method. The tag (T ₁ ) and the tag (T ₂ ) are an affinity tag (AT ₁ ) and an affinity tag (AT ₂ ), respectively, and the fluorescent dye (D ₁ ) and the fluorescent dye (D ₂ ) Are bound to the affinity tag (AT ₁ ) and the affinity tag substance (AT ₂ ), respectively. The fluorescent dye (D ₁ ) is an organic synthetic fluorescent dye (C ₁ ) or fluorescent protein (P ₁ ), and / or the fluorescent dye (D ₂ ) is an organic synthetic dye (C ₂ ) or fluorescent protein (P _2). The method according to any one of claims 1 to 8, wherein The fluorescent dye (D ₁ ) is a fluorescent protein (P ₁ ), and the tag (T ₁ ) is a fluorescent protein tag (PT ₁ ) containing the fluorescent protein (P ₁ ). The method in any one of. The fluorescent a dye _{(D 2)} is fluorescent protein _{(P 2),} and said tag _{(T 2)} is a fluorescent protein tag containing fluorescent protein _{(P 2) (PT 2)} , according to claim 1 to 5 The method in any one of. The fluorescent dye (D ₁ ) and the fluorescent dye (D ₂ ) are a fluorescent protein (P ₁ ) and a fluorescent protein (P ₂ ), respectively, and the tag (T ₁ ) and the tag (T ₂ ) but each is a fluorescent protein tag containing fluorescent protein _{(P 1)} (PT ₁₎ and a fluorescent protein tag containing fluorescent protein _{(P 2) (PT 2)} , according to any of claims 1 to 5 the method of.   The target protein is carbamoyl phosphate synthase I (CPSI), glutathione S-transferase (GST), fructose-1,6-bisphosphate aldolase, electron transfer flavoprotein alpha subunit, medium chain fatty acyl CoA dehydrogenase, long chain acyl CoA synthetase, TBP-related factors (TAFs), glyceraldehyde-3-phosphate dehydrogenase, importin α, DNA2 helicase, ribosomal protein L7, von Willebrand factor (vWF), N-ethylmaleimide sensitive factor (NSF), S -Adenosyl homocysteine hydrolase, serine protease, asymmetric acetylcholinesterase collagen-like tail subunit (ColQ), serine carboxypeptidase 1 (S EP1), histone cluster 1, Δ3, Δ2-enoyl-CoA isomerase, acrosin binding protein (ACRPB), semaphorin 5A, aurora-A-kinase interacting protein 1, p53, phosphoinositide 3-kinase (PI3K), phosphoinositide 3- Kinase (PI3K) p85 subunit, β-catenin, Zap-70, T cell receptor (TCR), Fyn tyrosine kinase, growth factor binding protein binding protein 2 (Grb2), Sos protein, DNA repair kinase Ku70 / Ku80 complex (Ku70 / 80), Crk-like protein (Crkl), phospholipase C-γ (PLCγ), protein kinase Cθ (PKCθ), estrogen receptor binding protein (ERBP), ubiquitin ligase CHIP Cyclin (Cdc13), hypoxia inducible factor 1α (HIF-1α), mitochondrial associated protein (Drp1), histone H2B, H2AX complex (histone), IκBα, IκB p100, TNF receptor associated factor 6 (TRAF-6) and The method according to claim 1, which is one selected from the group consisting of NF-kB p-105.   The method according to any one of claims 1 to 12, wherein the target protein (A) is ubiquitin.   The method according to any one of claims 1 to 14, which is a method for screening an inhibitor of degradation of the target protein by proteasome.