EP1370868A2 - Methode et procede de depistage permettant de detecter des interactions reversibles proteine-proteine - Google Patents

Methode et procede de depistage permettant de detecter des interactions reversibles proteine-proteine

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Publication number
EP1370868A2
EP1370868A2 EP01986933A EP01986933A EP1370868A2 EP 1370868 A2 EP1370868 A2 EP 1370868A2 EP 01986933 A EP01986933 A EP 01986933A EP 01986933 A EP01986933 A EP 01986933A EP 1370868 A2 EP1370868 A2 EP 1370868A2
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European Patent Office
Prior art keywords
protein
ras
rafrbd
gfp
interaction
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EP01986933A
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German (de)
English (en)
Inventor
Oliver c/o Max-Planck-Inst. für molekulare ROCKS
Alfred c/o Max-Planck-Institut für WITTINGHOFER
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Max Planck Gesellschaft zur Foerderung der Wissenschaften eV
Evotec OAI AG
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Max Planck Gesellschaft zur Foerderung der Wissenschaften eV
Evotec OAI AG
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Publication of EP1370868A2 publication Critical patent/EP1370868A2/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B30/00—Methods of screening libraries
    • C40B30/04—Methods of screening libraries by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6842—Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6845—Methods of identifying protein-protein interactions in protein mixtures

Definitions

  • the present invention is in the field of cell biology and describes a method for the detection of protein-protein interactions and an assay for testing active substances which act on protein-protein interactions.
  • the detection of such protein-protein interactions is of great interest for the elucidation of cellular processes.
  • it is of increasing importance to identify substances that influence such interactions. Screening methods in which active substances are tested on a large scale are becoming increasingly important. These must be as effective and inexpensive as possible and should be used at the earliest possible stage in drug discovery.
  • Protein-protein interactions are of central importance for a variety of cell biological processes and are based on different processes such as receptor-ligand interactions, signal transduction cascades, gene expression and a number of other processes, such as cell adhesion phenomena, antigen-antibody interactions and many more .
  • the physiology of a cell is largely determined by protein-protein interactions.
  • this also implies that even the slightest disruption of protein-protein interactions can have far-reaching consequences for the normal function of a cell and can therefore also be the cause of the development of diseases.
  • the biological activity of a target protein to be tested is usually an enzymatic activity or the ability to bind an effector (Silverman et al, 1993).
  • Various methods can be used to detect a protein-protein interaction or the effect of a substance on the extent of a protein-protein interaction, which are suitable for use in vivo and / or in vitro. In general, there is a trend here to switch from radioactive to fluorescence-based methods, which enable faster and more precise evaluation and at the same time do not limit the miniaturization of a test procedure.
  • FRET effect fluorescence resonance energy transfer
  • both proteins are provided with fluorophores which are coordinated with one another with regard to the absorption and emission wavelengths.
  • the emission light of one dye can now be absorbed by the other dye, so that longer-wave light is emitted.
  • the FRET effect is only possible if both proteins are bound to each other so that the fluorophores are in close proximity to one another.
  • the advantage of this method is the general applicability in vivo with different variants of the genetic encoded fluorophore GFP (green fluorescent protein) (Mitra et al, 1996).
  • the disadvantage is the fact that the FRET effect only occurs with optimal spatial orientation of the two fluorophores, which can only be achieved empirically and with great effort for a protein pair.
  • a second method is fluorescence polarization spectroscopy (Nasir & Jolley, 1999). If fluorescent molecules are excited in solution with polarized light, the extent to which the emitted light maintains this polarization depends on the rapidity of the rotational movement of the molecule during the fluorescence lifetime. Since this rotational speed and thus the loss of polarization is inversely proportional to the molecular weight, this effect can be used to distinguish a free protein from a complexed protein (Lynch et al, 1997). An application is only practicable in vivo.
  • FCS fluorescence correlation spectroscopy
  • both proteins must be components of a signal path connected in direct succession, the biological effect of which can be used to detect the interaction.
  • These include the release of low-molecular messenger substances such as calcium ions, cAMP and inositol triphosphate, the changes in concentration of which are measured after the activation of the corresponding signaling pathways through the binding of both partner proteins.
  • a reporter gene assay can be carried out. This takes advantage of the fact that the extent of the binding of both proteins - at least that is the hypothesis - correlates with the extent of the activation of the signaling pathway and thus with the transcription rate of the corresponding gene. If its promoter sequence is fused with the coding sequence of a reporter gene, the expression rate of the reporter protein can be measured as a representative.
  • One such method is, for example, the "two-hybrid method" (Yang et al., 1995), in which the interaction of two proteins in yeast cells is tested.
  • the disadvantage of the common cell-based methods is the fact that the binding of the two partner proteins is not detected directly, but always a downstream effect.
  • Non-specific interactions and the influence of other signal proteins can increase the number of false positive results. It cannot be ruled out that signal components that were only switched within the signal path after the two partner proteins to be examined were blocked. In the reporter gene assays, gene expression also results in a longer period of time before an effect can be detected.
  • Such a translocation assay was developed by Schneider et al. (1999, Nature Biotechn. 17, pl70-175). Two fusion proteins are expressed in one cell. The first contains a potential interaction domain and a core localization signal, the second contains a potential interaction domain and a GFP domain. Both proteins are synthesized in the cytosol, after which the first protein migrates to the nucleus due to its signal sequence. However, if it binds to the second protein through the interaction domains in the cytosol, the second protein is also transferred into the nucleus and can be detected there because of the fluorescence of the GFP domain. This technique enables interactions between protein domains to be determined in vivo.
  • the transport of proteins from the cytosol to the The nucleus occurs via the so-called nuclear pore complex, a cross-membrane multiprotein complex consisting of eight copies of approx. 100 different proteins (in the higher eukaryotes) with a total molecular weight of 125 MDa (Doye & Hurt, 1997; Obno et al, 1998; Gorlich & Kutay, 1999 ).
  • the core transport takes place not only by targeting the proteins with nuclear localization signals, but also by diffusion (Talcott & Moore, 1999).
  • the architecture of the nuclear pore complex allows proteins with a diameter of up to approx.
  • the problem underlying the invention is to provide a method for determining protein-protein interactions. This process is said to be feasible in vivo and to overcome the disadvantages of the prior art processes. In particular, the problem is to provide a method that enables the direct detection of an interaction and therefore does not require indirect detection via an additional signal path. In addition, the method is said to be applicable to proteins, protein domains and peptides of any size and to be suitable for testing a large number of interaction partners or of substances which could influence the protein-protein interaction in the context of assays.
  • the problem is solved according to the invention by a method for the detection of protein-protein interactions in expression systems such as cells, whereby a protein I interacts with a protein II, whereby a transfer of the protein I takes place in a cell compartment in which both protein I and protein II do not occur naturally, and when protein II is detected in the compartment into which protein I has been transferred, an interaction between protein I and protein II is determined.
  • the cell compartment into which the transfer takes place is preferably the nucleus.
  • Other cellular compartments are the endoplasmic reticulum, the nucleolus, the lysosomes, the Golgi apparatus, the dictyosomes, mitochondria, chloroplasts, peroxisomes, vacuoles, endosomes, a periplasmic space or a membrane.
  • Protein I is preferably transferred by a signal sequence, in particular by a nuclear localization sequence.
  • the protein II preferably has an export signal, in particular a core export signal.
  • protein II is furthermore a fusion protein with a fluorescent protein, preferably GFP.
  • Cellular expression systems include, among others, primary cells or immortalized cell lines.
  • a cellular expression system can preferably be derived from mammalian cells, such as human cells Origin, or cells from rodents (mouse, rat), but also from other eukaryotic cells, such as plant cells.
  • the protein-protein interaction is preferably detected by spectroscopic detection, fluorescence detection, colorimetry or radiometry.
  • the invention also relates to an assay using the method according to the invention, substances being added which may interfere with the protein-protein interaction.
  • the method and the assay according to the invention are thus suitable for screening (screening, HTS) proteins or substances on a large scale, which potentially influence protein protein-protein interactions or participate in them.
  • the method according to the invention is suitable for checking putative protein-protein interactions. Due to the high sensitivity of the method according to the invention, it is also possible to investigate dose-dependent effects of chemical substances on protein-protein interactions.
  • the combination of protein II with a nuclear export signal can achieve that a nuclear localization assay can be carried out in vivo with proteins, domains or peptides of any size. It has been found that when such a fusion protein is used as Protein II, the problem of the undesired diffusion of Protein II into and (after the transfer has taken place) from the cell nucleus is avoided. The signals obtained are extraordinarily rich in contrast when using the method and assay according to the invention. This makes it much easier to carry out tests in which active ingredients are tested or differential differences in binding strength are determined quantitatively.
  • the aim of this invention was to produce various constructs of the oncogenic variant G12V of Ras and the Ras-binding domain of Raf kinase (RafRBD) which allow fluorescent labeling or are fused to the genetically encoded fluorophore GFP (green fluorescent protein) and for Localization studies and translocation studies in the cell can be used.
  • the focus should be on the translocation of Ras and RafRBD constructs into the cell nucleus.
  • NLS nuclear localization signal
  • Ras should be enabled to accumulate in the cell nucleus and thus be able to transport RafRBD into the cell nucleus.
  • RafRBD should receive a nuclear export signal (NES).
  • HTS high-throughput screening
  • H-Ras constitutively active H-Ras (G12V mutant) was used, in which the C-terminal CAAX motif, which is responsible for the attachment to the plasma membrane (aa 1-174), and the Ras binding domain of c-Rafl ( RafRBD, aa 51-132) is missing.
  • RafRBD binds to Ras-GTP with affinity in the nanomolar range and is sufficient but necessary for the Ras-Raf interaction (Scheffler et al., 1994; Herrmann et al., 1995).
  • a modified pcDNA3 vector (Invitrogen) was constructed, which made it possible to express a protein of choice, which is fused to a 22 amino acid sequence which is the commonly used nuclear localization signal (NLS) , derived from the simian virus 40 large tumor antigen (SV40 T-ag), and a predicted phosphorylation site for casein kinase II (Serlll / 112), which has been shown to greatly increase the core import of SV40 T-ag .
  • NLS nuclear localization signal
  • Serlll / 112 a predicted phosphorylation site for casein kinase II
  • the RafRBD sequence was inserted into the pEGFP-Cl vector (Clontech).
  • the well-characterized core export signal derived from the HIV Rev protein (Fischer et al., 1995) was fused to the C-terminal end.
  • the expression of the constructs used in NIH3T3 fibroblasts was confirmed by Western blot analyzes (FIGS. 2A and 2B).
  • the constructs for Ras and RafRBD were expressed either alone or together transiently and the intracellular localization of the proteins was examined 12-48 hours after the transfection using a confocal laser microscope. The functionality of both signals, NLS and NES, could be shown here (FIG. 2C).
  • Ras (G12V) -NLS (just like wt-Ras-NLS) but not a control construct that lacks the NLS is completely accumulated in the core.
  • GFP-Raf-RBD-NES was localized exclusively in the cytosol.
  • a construct without NES showed the same distribution of fluorescence as GFP, somewhat more accumulated in the nucleus. Due to these unfavorable properties of GFP, the use of a single NLS in a translocation system is not sufficient to produce a clear, interaction-dependent distinction between the fluorescence intensities between the nucleus and the cytosol.
  • GFP-RafRBD-NES2% Only GFP-RafRBD-NES2%, with a significantly weakened export activity, was completely localized within the core with Ras (G12V) -NLS (FIG. 3B). Individually transfected GFP-RafRBD-NES2% was evenly distributed in the core and cytosol, significantly less enriched within the core than GFP-RafRBD without NES. The other GFP-RafRBD-NES mutants with 8% or higher export activity were completely localized within the cytosol. These experiments clarified that GFP-RafRBD-NES2%, due to its relatively low molecular weight of 38 kDa, is subject to a balanced balance between weak signal-controlled nuclear export and free diffusion through the nuclear pore complex.
  • GFP-RafRBD-NES was increased by adding two more GFP units. Despite its molecular weight of 84 kDa, which should prevent diffusion into the nucleus, GFP3 was still found significantly enriched in the nucleus. This fact indicates that GFP contains a cryptic NLS (Fig. 4A-C). The expression of GFP3-RafRBD-NES2% resulted in the expected accumulation in the cytoplasm. After co-transfection with Ras (G12V) -NLS, colocalization of both proteins in the nucleus was indeed observed.
  • Quantification software can be further improved.
  • GTPase activating proteins The primary defect of oncogenic Ras mutants is their inability to hydrolyze GTP both in the presence and in the absence of GTPase activating proteins (GAPs). Considering that the catalytic effect of GTPase stimulation by GAPs is related to the involvement of an arginine finger of GAP, it was argued that stimulation of the GTPase response of oncogenic Ras in Ras-dependent tumors would be an anti-cancer target (Scheffzek et al., 1997). Recent studies using a GTP analog have supported the idea that small molecules that would induce the GTPase reaction would be preferred lead structures for drug development. This raises the question of whether the GFP-RafRBD translocation assay would be suitable for detecting such compounds.
  • Cdc42-NLS G12V and wt
  • Grb2-NLS were expressed alone, they were localized exclusively in the nucleus, while GFP3-RalGDSRBD-NES2%, GFP3-WASP-CRIB-NES2% and GFP3-Sosl (C- ter) - NES2% accumulated in the cytosol.
  • GFP3-RalGDSRBD-NES2% were predominant, and the other two GFP fusion proteins were completely localized in the nucleus after transfection with their corresponding NLS-containing interaction partners (FIG. 6C).
  • the P49L mutation within the N-terminal SH3 domain of Grb2 is known to prevent interaction with Sosl (Chardin et al., 1993).
  • the method according to the invention can be used for cell-based drug screening search methods for antagonists of protein interactions and, after appropriate modification, may also be applicable for the search for agonists.
  • An enormous advantage of this method is the ability to directly and reversibly demonstrate the interaction, which considerably reduces the amount of false positives in a drug screening search.
  • small molecules could also be identified that block the core transport machinery and, as an alternative, would lead to a weakening of the nuclear GFP fluorescence.
  • Such molecules, which are also of particular interest could be simple by immunodetection of the NLS-containing protein, which would also no longer be located in the nucleus.
  • the simple experimental conditions of the method according to the invention support simple automated quantification and high throughput.
  • this method enables not only quantitative information about active substances, but possibly also mechanistic studies on protein interactions and their regulation, e.g. structure-function studies by using point mutations or analysis of the nucleotide hydrolysis of GTP binding proteins.
  • a further application of the method according to the invention, using the principle of core-cytosol translocation, could be an alternative “mammalian two-hybrid method” for verifying possible interactions or for screening searches of unknown interaction partners of individual proteins, or even a complete genetic expression. Be a library.
  • Fig. 1 Schematic representation of NIFTY: A fluorescence-based protein-protein interaction assay based on a nuclear cytoplasm translocation of reporter fluorescence.
  • Fig. 2 Construction and expression of fusion proteins for the initial studies.
  • A Schematic representation of the constructs produced.
  • B Immunoblots of the initial constructs after transient expression in NIH3T3 cells. For this purpose, cell lysates were separated on 15% SDS polyacrylamide gels, transferred to membranes and stained with anti-GFP or anti-HA antibodies.
  • C Intracellular localization of the initial Ras and RafRBD constructs after expression in NIH3T3 cells. For this purpose, the samples were scanned with a Leica TCS SP2 confocal microscope and a 63x oil immersion objective.
  • Fig. 3 Fine-tuning the NES strength.
  • A Schematic representation of the pool of the GFP-RafRBD-NES constructs produced with weakened export signals. Changes in the amino acids of the NES are marked with red. The percentage NES activities given relate to the export activity of NES-mutated Rev protein in comparison to wild-type Rev (Zhang & Dayton, 1998).
  • B Intracellular localization of GFP-RafRBD-NES2% after single expression (a) and of Ras (G12V) -NLS and GFP-RafRBD-NES2% after co-expression.
  • Fig. 4 Fine-tuning the property of GFP-RafRBD to diffuse through the nuclear pore complex.
  • A Schematic representation of the construct GFP3-RafRBD-NES2%.
  • B Immunoblots of GFP, GFP2, GFP3 and GFP3-RafRBD-NES2% after transient expression in NIH3T3 cells.
  • C Intracellular localization of GFP3, GFP3-RafRBD-NES2% (a) and GFP3-Raf-NES2% (b) after single expression and of Ras (G12V) - NLS after co-expression with either GFP3-RafRBD-NES2% or GFP3-Raf-NES2%. The inverse subcellular is to be compared here Localization of the reporter fluorescence in the second and third image of the lower half of the image with that in FIG. 1.
  • FIG. 5 Correlation of the amount of nuclear accumulation of GFP3-RafRBD-NES2% and Ras * RafRBD affinity or complex concentration.
  • A Co-expression of Ras (G12V) -NLS with different constructs of GFP3-RafRBD-NES2% with the point mutations indicated in each case. Lower half of the figure: Co-expression of wt-Ras-NLS and GFP3-RafRBD-NES2%.
  • B Correlation of Ras * RafRBD binding affinity and the level of nuclear accumulation of reporter fluorescence. The relative nuclear fluorescence is shown against the logarithm of the dissociation constant of the wt-Ras * RafRBD complex (Block et al., 1997).
  • Fig. 6 General application of NIFTY for other interacting proteins.
  • A Schematic representation of the generated constructs.
  • B Immunoblot analysis of the indicated constructs after expression of NIH3T3 cells.
  • C Intracellular localization of individually expressed GFP3-RalGDSRBD-NES2% (a), GFP3-WASP-CRIB-NES2% (b) or GFP-SOS (C-ter) -NES2% (c) or from the indicated constructs Co-expression.
  • the Ras protein is a central link in various signal transduction pathways, the growth and differentiation processes regulate. As a GTP-binding protein, it acts as a regulated molecular switch. Ras switches between two states in which it has either bound GTP (switch position: on), or the GTP is hydrolyzed to GDP (switch position: off). The activated Ras can now recruit the serine threonine kinase Raf (rapid fibrosarcoma), the best characterized effector of Ras, to the plasma membrane, which leads to the activation of this kinase.
  • Raf serine threonine kinase
  • Raf can in turn activate the protein kinase MEK (MAPK / Erk kinase) by phosphorylation, which then in turn activates the protein kinase Erk (extracellular-signal-related kinase).
  • MEK protein kinase MEK
  • Erk extracellular-signal-related kinase
  • MAP kinase module for mitogen activated protein. The activation of this module leads to the phosphorylation of several transcription factors and finally to the expression of different genes (Campbell et al, 1998).
  • Raf interacts with activated Ras via the Ras binding domain (RBD) in the regulatory N-terminus of Raf (Vojtek et al, 1993), which is an independently folding, stable module of 80 amino acids (Scheffler et al, 1994).
  • Ras can bind the isolated RBD in a GTP-dependent manner in the same way as complete Raf.
  • Ras variants with point mutations in the effector region that lead to the blocking of the biological Ras activity are likewise not able to interact with the RBD.
  • the isolated RafRBD is believed to have the same properties as the domain in the intact total protein (Koide et al, 1993; Van Aelst et al, 1993; Moodie et al, 1993).
  • Plasmids pcDNA3-HA-CKII-NLS was obtained by inserting a Hindlll / Kpnl fragment which contains a Kozak sequence, a start codon and a sequence coding for the hemagglutinin epitope (YPYDVPDYA), and an EcoRI / XhoI fragment which is used for the casein kinase II phosphorylation site (SSDDEATADSQHSST) and the nuclear localization sequence (NLS) (PPKKKRKV) of the SV40 T-ag, in the cloning site of the plasmid vector pcDNA3 (Invitrogen).
  • PCR-amplified sequences of H-Ras (aa 1-174), hCdc42 (aa 1-178) and hGrb2 were cloned into the BamHI / EcoRI site of pcDNA3-HA-CKII-NLS.
  • the Ras control construct was prepared by cloning the H-Ras sequence with a stop codon into the plasmid pcDNA3-HA, which lacks the second sequence insertion.
  • the G12V mutations were generated using directed mutagenesis.
  • a c-Rafl fragment coding for the Ras binding domain (aa 51-132) was amplified by PCR and cloned into the XhoI / EcoRI site of plasmid pEGFP-Cl (Clontech) (pGFP-RafRBD).
  • pGFP-RafRBD-NES the sequence coding for the core export signal (NES) of the Rev protein was inserted into the plasmid pGFP-RafRBD by means of a 3 "- overlapping primer.
  • Point mutations within the NES sequences were added
  • the plasmid pEGFP3-Cl-NES2% enables the expression of proteins with an N-terminal fusion of three units GFP and also with a C-terminal fusion of an attenuated NES protein.
  • PEGFP3-C1-NES2% was produced by subsequently adding a Nhel / Agel and an Agel / Agel / fragment of GFP, each containing a Kozak sequence, and one for a strongly attenuated Rev protein NES (into the pEGFP-Cl plasmid. LPPLERLETLD) coding EcoRI / Pstl fragment were inserted.
  • PCR-amplified sequences of hRalGDS-RBD (aa 788-884), hWASP-CRIB (aa 221-257) and the C-terminal region of hSOSl (aa 1132-1333) were used to prepare the GFP fusion constructs (see FIG. 6A). inserted into the XhoI / EcoRI restriction site of pEGFP3-Cl-NES2%.
  • the plasmid pGFP3-RafRBD-NES2% was constructed by subcloning the XhoI / PstI fragment from pGFP-RafRBD2% into the plasmid pEGFP3-Cl-NES2%. Mutations in the RafRBD coding sequence were generated by directed mutagenesis. Finally, all constructs were sequenced from both the 5 "end and the 3" end, which confirmed the correct base sequence.
  • NIH3T3 cells were in DMEM (Dulbecco's modified Eagle medium) supplemented with 10% calf serum with penicillin (1000 IU / ml), streptomycin (1000 ⁇ g / ml) at 37 ° C and a CO 2 content of 7.5% cultivated. The same culture conditions also existed for MDCK and 293 cells. For transient transfection, cells were sown on 21x26 mm coverslips in 6-well tissue culture dishes and cultivated to a confluence of 70%.
  • DMEM Dulbecco's modified Eagle medium
  • penicillin 1000 IU / ml
  • streptomycin 1000 ⁇ g / ml
  • the transfection was carried out with LipofectAMINE PLUS reagent (Invitrogen) according to the manufacturer's instructions using 0.4 ⁇ g pcDNA3 plasmid and 0.7 ⁇ g pEGFP plasmid.
  • the cells were washed in washing buffer (PBS, 0.5 mM CaCl 2 , 0.25 mM MgCl 2 ), fixed in 3.7% formaldehyde for 15 min and in 0.1% Triton for 10 min X-100 permeabilized in Tris / HCl pH 7.5 and 100 mM NaCl.
  • the cells were washed with the primary antibody (rat, anti-HA; Sigma) in a dilution of 1: 100 for 1 h, then washed three times with PBS and with the fluorescence-labeled secondary antibody (Cy-3 or Cy-5, goat, anti-rat; Dianova) in a dilution of 1: 100 for 1 h. After the incubation, three washing steps were carried out with PBS. The coverslips were embedded on slides with Mowiol.
  • NIH3T3 cells were placed on a 10 cm tissue culture dish 48 h after transfection with corresponding plasmid constructs in lysis buffer (20 mM Tris / HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.5% Triton X-100, 0 , 1% SDS, 1% NaDOC and proteinase inhibitors).
  • the cells were detached from the dishes using a rubber spatula. After centrifugation at 4 ° C for 10 min at 22000 g, the supernatant was separated on an SDS-PAGE. The electrophoretically separated proteins were then transferred to a PVDF membrane.
  • the membranes were then incubated for one hour either with rat anti-HA antibody (Sigma) coupled with horseradish peroxidase or with rabbit anti-GFP antibody (Dianova).
  • the membranes incubated with the anti-GFP antibody were washed and then incubated with secondary horseradish peroxidase-coupled anti-rabbit antibodies (Amersham Pharmacia).
  • An ECL chemiluminescent substrate (Pierce) was used to visualize the protein bands.
  • a focus on the equatorial level of the cell nucleus and the determination of the compartment boundaries within which the image analysis should take place was made possible by the Cy5 staining of the protein carrying the nuclear localization signal.
  • the cytoplasmic area was defined by two closely spaced rings near the border to the cell nucleus. The average fluorescence intensity per area was measured for both the GFP and the Cy5 signal and the percentage intensity of the core signal was determined therefrom.
  • the HIV- 1 Rev activation domain is a nuclear export signal that accesses an export pathway used by speeifie cellular RNAs. Cell 82, 475-483 (1995).
  • Urano, T., Emkey, R. & Feig, L.A. Ral-GTPases mediate a distinct downstream signaling pathway from Ras that facilitates cellular transformation. EMBO J. 15, 810-816 (1996).

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Abstract

L'invention a pour objet un procédé et des analyses permettant de détecter des interactions protéine-protéine et de tester des substances actives dans des systèmes d'expression tels que des cellules. L'invention est caractérisée en ce qu'on fait inter-réagir une protéine I avec une protéine II, de manière à transférer la protéine I dans un compartiment cellulaire dans lequel aussi bien la protéine I que la protéine II ne se présentent pas à l'état naturel, et en ce que lorsqu'on détecte la protéine II dans le compartiment dans lequel la protéine I a été transférée, on établit une interaction entre la protéine I et la protéine II.
EP01986933A 2000-12-23 2001-12-21 Methode et procede de depistage permettant de detecter des interactions reversibles proteine-proteine Withdrawn EP1370868A2 (fr)

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DE10064972 2000-12-23
DE10064972 2000-12-23
PCT/EP2001/015265 WO2002052272A2 (fr) 2000-12-23 2001-12-21 Methode et procede de depistage permettant de detecter des interactions reversibles proteine-proteine

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