WO2000017221A1 - Procede de mesure des interactions proteine-proteine dans des cellules vivantes - Google Patents

Procede de mesure des interactions proteine-proteine dans des cellules vivantes Download PDF

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WO2000017221A1
WO2000017221A1 PCT/US1999/019118 US9919118W WO0017221A1 WO 2000017221 A1 WO2000017221 A1 WO 2000017221A1 US 9919118 W US9919118 W US 9919118W WO 0017221 A1 WO0017221 A1 WO 0017221A1
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protein
cell
interest
nucleic acid
library
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PCT/US1999/019118
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WO2000017221A9 (fr
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Tobias Meyer
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Duke University
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Priority to EP99942401A priority Critical patent/EP1115734A4/fr
Priority to CA002345392A priority patent/CA2345392A1/fr
Priority to AU55789/99A priority patent/AU5578999A/en
Priority to JP2000574129A priority patent/JP2002526756A/ja
Publication of WO2000017221A1 publication Critical patent/WO2000017221A1/fr
Publication of WO2000017221A9 publication Critical patent/WO2000017221A9/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1055—Protein x Protein interaction, e.g. two hybrid selection

Definitions

  • the present invention concerns methods of detecting or determining binding between two different proteins, or oligomer formation by the same protein, in living cells, along with cells and kits useful for carrying out such methods.
  • binding of proteins to one another, or the disruption of binding of one protein to another by a competitive inhibitor is typically measured in vitro.
  • Such binding assays are typically used as a model for in vivo, including intracellular, binding events. While such techniques are well established, the in vitro binding conditions do not control for the vast number of variables introduced when a binding event occurs within a cell. Because of the importance of screening for new binding partners, or inhibitors of known binding partners, to the development of new therapeutic molecules, the development of techniques that measure binding within a cell is extremely important.
  • the "pull down” assay is known, in which the binding of a pair of proteins of interest is determined by forming a co-precipitate with an antibody in vitro and then centrifuging down, or “pulling down” the aggregate so formed. Disadvantages of this technique are that it is carried out in vitro, and that substantial nonspecific binding occurs.
  • the yeast "two hybrid” technique employs a pair of transcription factors that trigger the transcription of a selectable or detectable protein.
  • the technique has been adapted and extended to a number of situations, including examination of enzyme- substrate interactions. R. Sikorski and R. Peters, Science 281, 1822-1823 (18 Sept. 1998).
  • a first hybrid is formed of a first protein of interest and one of the transcription factors;
  • a second hybrid is formed of a second protein of interest and another of the transcription factors. If the two proteins of interest associate, then the two transcription factors associate and transcription of the detectable or selectable protein is initiated.
  • binding occurs in a cell, and it can be readily adapted to the screening of combinatorial libraries by inserting various members of the library in different "two hybrid" cells and expressing the library transcription products therein.
  • Disadvantages of this technique are that it is limited to the use of transcription factors, the binding reaction must be in a narrow range, is typically carried out in yeast, and the binding event must occur in the cell nucleus.
  • a first aspect of the present invention is a method of detecting a protein- protein interaction in a living cell.
  • the method comprises (a) providing a cell that contains a first heterologous conjugate and a second heterologous conjugate, wherein the first heterologous conjugate comprises a first protein of interest conjugated to a detectable group, and wherein the second heterologous conjugate comprises a second protein of interest conjugated to a protein that specifically binds to an internal structure within the cell, and then (b) detecting the presence or absence of binding of the detectable group to the internal structure, the presence of the binding indicating that the first and second proteins of interest specifically bind to one another.
  • the proteins of interest may be the same or different; the proteins of interest may be members of a specific binding pair.
  • the detectable group is a protein, and the first protein of interest and the detectable group together comprise a fusion protem.
  • the second heterologous conjugate is also a fusion protein.
  • the cell may contain and express a nucleic acid encoding either, or both, fusion proteins, or the heterologous constructs may be administered exogenously to the cells.
  • the cell is preferably a eukaryotic cell.
  • Additional aspects of the present invention include nucleic acids encoding fusion proteins as described above, cells containing and expressing such fusion proteins, kits useful for carrying out the methods described above, and nucleic acid libraries useful as screening tools for carrying out the methods described above.
  • the invention is useful for screening compounds for the ability to disrupt or inhibit the binding of known binding pairs and thereby identifying competitive inhibitors thereof.
  • the invention is useful for screening one known protein of interest against a library of other proteins of interest to identify compounds that bind to the known protein of interest.
  • FIG. 1A Schematic representation of the domain organization of the GFP- tagged CaMKII isoforms.
  • the catalytic-domain (C), regulatory-domain (R), variable- domain (V) and oligomerization-domain (A) are shown.
  • Figure IB Autophosphorylation of GFP-tagged CaMKII isoforms. Comparison of the baseline (left), calcium/CaM-dependent (middle) and burst (right) autophosphorylation activity of CaMKIIa, GFP-CaMKIIa and GFP-CaMKIIb. The kinase activity of the in vitro translated constructs are shown. Translated GFP alone was included as a control.
  • Figure lC Relative kinase activity corrected for the amount of expressed CaMKII or GFP-CaMKII protein (measured as the ratio of 32 P incorporation and 35 S- Met incorporation).
  • the dark bars show CaMKII autophosphorylation after incubation with ->2p_ATP in high Ca 2+ /CaM for 30 seconds.
  • the light bars show the calcium independent "burst" autophosphorylation after 30 s in high calcium and 120 seconds in EGTA.
  • Figure ID Confocal image of GFP-tagged CaMKIIa (left) and CaMKIIb (right) expressed in living hippocampal CA1-CA3 neurons.
  • FIG. 2 A Principle of the Pull-out binding assay.
  • the binding interaction between a Protein X and Protein Y can be measured by tagging Protein X with an inducible plasma membrane binding domain (PM-domain) and Protein Y with GFP. If a significant fraction of the two proteins bind to each other, drug addition targets the GFP to the plasma membrane. In contrast, the cytosolic distribution remains unaltered if Protein X and Y do not bind to each other.
  • PM-domain inducible plasma membrane binding domain
  • Figure 2B Property of a minimal phorbol ester binding domain used as an inducible PM-domain in the Pull-Out binding assay.
  • a fusion protein between GFP and the phorbol ester binding domain can be pulled from the cytosol to the plasma membrane by addition of phorbol ester.
  • Left distribution of the fusion protein before phorbol ester addition.
  • Right distribution of the fusion protein after phorbol ester addition.
  • Bottom line scans of the fluorescence intensity across the cell before and after phorbol ester addition.
  • FIG. 2C Demonstration that nearly all CaMKIIa molecules are part of oligomers. Phorbol ester addition to cells with co-expressed PM-CaMKIIa and GFP- CaMKIIa leads to the near complete plasma membrane translocation of GFP- CaMKIIa.
  • Figure 2D Control measurements showing that expressed GFP itself is not affected by phorbol ester addition. Calibration bars are lO ⁇ m.
  • FIG. 3 CaMKIIa forms larger oligomers than CaMKIIb.
  • Figure 3 A Schematic representation of the assay to measure the size of CaMKIIa and CaMKIIb oligomers in living cells.
  • Figure 3B Quantitative comparison of the concentrations of expressed GFP- CaMKIIa and PM-CaMKIIa measured by in vitro translation. The relative concentration of the expressed proteins was compared by 35 S-Met incorporation into in vitro translated proteins. The same RNA was used for the in vitro translation and the RNA transfection of cells.
  • Figure 3D Line scan profiles of three different dilution after phorbol ester addition.
  • FIG. 3E Schematic representations of the quantitative analysis used to measure the relative plasma membrane translocation. I pre and DPM are measured before and after PMA addition, respectively.
  • FIG. 3F Plot of the relative plasma membrane translocation of CaMKIIa and CaMKIIb at decreasing ratios of expressed PM-CaMKII and GFP-CaMKII. Each point is an average of at least 10 experiments. The solid curves are best fits to the two set of data and the dashed lines show the confidence interval. Best fits were obtained assuming an average of 13.5 subunits for CaMKIIa and 4.2 subunits for CaMKIIb.
  • Figure 4. Requirement for more than one CaMKIIb subunits for targeting CaMKIIa/b hetero-oligomers to the actin cytoskeleton.
  • FIG. 4 A Insertion of CaMKIIb into hetero-oligomers of mostly CaMKIIa is a stochastic process. Plot of the relative plasma membrane translocation of GFP- CaMKIIa at decreasing ratios of expressed PM-CaMKIIb. Each point is an average of at least 10 experiments.
  • FIG. 4C Relative cortical localization of GFP-CaMKIIa plotted as a function of increasing dilutions of CaMKIIb.
  • Relative cortical localization is defined as DpM Iav 3 with DPM as the intensity difference between the PM and the cytosol and I av as the average fluorescence intensity of a particular cell. Each point is an average of at least 10 experiments.
  • Figure 4D Measurement of the change in the diffusion coefficient as a function of an increasing dilution of CaMKIIb to GFP-CaMKIIa.
  • the apparent diffusion coefficient of GFP-CaMKIIa increased from 0.2 to 1 mrn ⁇ /s as the ratio of CaMKIIb to CaMKIIa was lowered from 1 :2 to 1 :9.
  • the outermost left and right data points show the diffusion coefficients of GFP-CaMKIIb and CaMKIIa, respectively.
  • Detectable groups or “detectable proteins” used to carry out the present invention include fluorescent proteins, such as green fluorescent protein (GFP) and apoaequorin, including analogs and derivatives thereof.
  • Green fluorescent protein is obtained from the jellyfish A eq uorea victoria and has been expressed in a wide variety of microbial, plant, insect and mammalian cells. A. Crameri et al., Nature Biotech. 14, 315-319 (1996).
  • Any detectable group may be employed, and other suitable detectable groups include other fluorophores or fluorescent indicators, such as a fusion tag with any binding domain such as avidin, streptavidin and ligand binding domains of receptors.
  • Coupling of biotin or other ligands to the fluorophore or indicator of interest may be achieved using a dextran matrix or other linker system.
  • the detectable protein may be one which specifically binds a fluorophore, as in FLASH technology. Fluorescent detectable groups (including both fluorescent proteins and proteins that bind a separate fluorophore molecule thereto) are currently preferred.
  • Internal structure refers to a separate, discreet, identifiable component contained within a cell.
  • structure as applied to the constituent parts of a cell is known (see, e.g., R. Dyson, Cell Biology: A Molecular Approach, pg, 10 (2d ed. 1978)), and the term “internal structure” is intended to exclude external structures such as flagella and pili.
  • Such internal structures are, in general, anatomical structures of the cell in which they are contained. Examples of internal structures include both structure located in the cytosol or cytoplasm outside of the nucleus (also called “cytoplasmic structures"), and structures located within the nucleus (also called “nuclear structures”).
  • the nucleus itself including the nuclear membrane are internal structures. Structures located within the cytoplasm outside of the nucleus are currently preferred. Thus the term “internal structure” is specifically intended to include any non-uniformly distributed cellular component, including proteins, lipids, carbohydrates, nucleic acids, etc., and derivatives thereof.
  • Library refers to a collection of different compounds, typically organic compounds, assembled or gathered together in a form that they can be used together, either simultaneously or serially.
  • the compounds may be small organic compounds or biopolymers, including proteins and peptides.
  • the compounds may be encoded and produced by nucleic acids as intermediates, with the collection of nucleic acids also being referred to as a library.
  • a nucleic acid library it may be a random or partially random library, commonly known as a "combinatorial library” or “combinatorial chemistry library”, or it may be a library obtained from a particular cell or organism, such as a genomic library or a cDNA library.
  • Small organic molecules can be produced by combinatorial chemistry techniques as well.
  • such libraries comprise are organic compounds, including but not limited oligomers, non-oligomers, or combinations thereof.
  • Non-oligomers include a wide variety of organic molecules, such as heterocyclics, aromatics, alicyclics, aliphatics and combinations thereof, comprising steroids, antibiotics, enzyme inhibitors, ligands, hormones, drugs, alkaloids, opioids, benzodiazepenes, terpenes, prophyrins, toxins, catalysts, as well as combinations thereof.
  • Oligomers include peptides (that is, oligopeptides) and proteins, oligonucleotides (the term oligonucleotide also referred to simply as "nucleotide, herein) such as DNA and RNA, oligosaccharides, polylipids, polyesters, polyamides, polyurethanes, polyureas, polyethers, poly (phosphorus derivatives) such as phosphates, phosphonates, phosphoramides, phosphonamides, phosphites, phosphinamides, etc., poly (sulfur derivatives) such as sulfones, sulfonates, sulfites, sulfonamides, sulfenamides, etc., where for the phosphorous and sulfur derivatives the indicated heteroatom for the most part will be bonded to C, H, N, O or S, and combinations thereof.
  • oligonucleotides such as DNA and RNA
  • Nucleic acid refers to both DNA and RNA.
  • Protein as used herein is intended to include protein fragments, or peptides.
  • protein is used synonymously with the phrase “protein or fragment thereof (for the purpose of brevity), particularly with reference to proteins that are “proteins of interest” or members of a specific binding pair.
  • Protein fragments may or may not assume a secondary or tertiary structure. Protein fragments may be of any length, from 2, 3, 5 or 10 peptides in length up to 50, 100, or 200 peptides in length or more, up to the full length of the corresponding protein.
  • Specifically binds and “specific binding” as used herein includes but is not limited to stereospecific binding, electrostatic binding, or hydrophlic binding interactions. Thus, specifically binds and specific binding are exhibited by at least a two or three fold (or two or three times), greater apparent binding affinity between the binding partners as compared to other proteins or binding partners within the cell in which binding is being detected.
  • Specific binding pair refers to a pair of molecules (e.g., a pair of proteins) that specifically bind to one another.
  • a pair of molecules that specifically bind to one another which may be the same or different, are referred to as members of a specific binding pair.
  • a protein that is a member of a specific binding pair may be a protein that has been previously determined to be a member of a specific binding pair or a protein that is a putative member of a specific binding pair.
  • Translocation refers to a change in distribution of a protein or conjugate (including a fusion protein) from one physical distribution within a cell to another, different, physical distribution within a cell. Preferably, translocation is from either a uniform or non-uniform distribution to a non-uniform distribution. Translocation could also be from a non-uniform to a uniform distribution.
  • the present invention provides a method of detecting a protein-protein interaction.
  • the method comprises first providing a cell that contains a first heterologous conjugate and a second heterologous conjugate.
  • the first heterologous conjugate comprises a first protein of interest conjugated to a detectable group.
  • the second heterologous conjugate comprises a second protein of interest
  • the binding of the protein that specifically binds to an internal structure may be immediate, may be induced (as discussed below), or may be a prior binding in the case of a protein that is previously localized to or permanently located at the internal structure of interest.
  • the two conjugates are preferably each present in the cell at a total concentration between about 1 or 10 nM to about 1 or 10 mM.
  • the presence or absence of binding of the detectable group to the internal structure is then detected, the presence of the binding indicating that the first and second proteins of interest specifically bind to one another.
  • Detection may be by any suitable means depending upon the detectable group employed, but preferably the detectable group is a fluorescent group and detection is carried out by optical or visual reading, which may be done manually, by an automated apparatus, or by combinations thereof.
  • the second heterologous conjugate can further comprise a detectable group, which detectable group is preferably different from the detectable group located on the first heterologous conjugate and fluoresces at a different wavelength therefrom.
  • both detectable groups could be a green fluorescent proteins, yet simply different mutants of green fluorescent protein that fluoresce at different wavelengths.
  • heterologous conjugates may be introduced directly in the cell by any suitable means, such as by electroporation or lipofection.
  • a nucleic acid typically a DNA
  • nucleic acid may be stable introduced into the cell (for example, as a plasmid), which nucleic acid includes a suitable promoter segment that controls and causes the expression of a nucleic acid encoding the fusion protein.
  • binding events in the instant invention may be direct or indirect binding events. Indirect binding events are those mediated through an intermediate, or bridging, molecule or conjugate.
  • the bridging molecule may be a covalent conjugate of FK506 and cyclosporin, to cause the indirect binding of FKBP12 and cyclophilin (both conventionally cytosolic proteins) to one another.
  • FKBP12 or the cyclophilin can be modified so that it binds to the plasmazieane, such as by lipidating the protein or forming a fusion protein with the transmembrane domain of a transmembrane protein.
  • Cells used to carry out the present invention are typically eukaryotic cells, which may be yeast, plant, or animal cells. Yeast and animal cells, particularly mammalian cells, are currently preferred.
  • Example plant cells include, but are not limited to, arabidopsis, tobacco, tomato and potato plant cells.
  • Example animal cells include, but are not limited to, human, monkey, chimpanzee, rat, cat, dog, and mouse cells.
  • Any internal structure as defined above can be used to carry out the present invention, as long as the binding of the detectable group to the internal structure provides a different detectable signal from the cell than when the detectable group is not bound to the internal structure.
  • the internal structure is contained in the cell cytoplasm.
  • internal structures include, but are not limited to, plasma membrane, cytoskeleton (including but not limited to actin cytoskeleton, tubulin cytoskeleton, intermediate filaments, focal adhesions, etc.), centromere, nucleus, mitochondria, endoplasmic reticulum, vacuoles, golgi apparatus, and chloroplasts.
  • the internal structure is either the plasma membrane or cortical cytoskeleton.
  • the protein that specifically binds to an internal structure is a translocatable protein.
  • the method further comprises the step of inducing translocation of the second heterologous conjugate prior to the detecting step.
  • Induction of translocation may be carried out by any suitable means, such as by administration of a physical or chemical signal (e.g., administration of a compound such as a phorbol ester).
  • a protein may be selected from the group consisting of cytosolic protein kinases, protein phosphatases, adapter proteins, cytoskeletal proteins, cytoskeleton associated proteins, GTP-binding proteins, plasma transmembrane proteins, plasma membrane associated proteins, ⁇ - arrestin, and visual arrestin (including fragments thereof that specifically bind to an internal structure).
  • the protein is a protein kinase C isoform or a fragment thereof that specifically binds to an internal structure, such as a Cl domain fragment or a C2 domain fragment, where the induction signal is administration of a phorbol ester.
  • induction of translocation may be induced by stimulation of a receptor, such as a glutamate receptor, beta-adrenergic receptor, or PAF receptor, with a receptor agonist to induce a signaling step which in turn induces translocation.
  • a receptor such as a glutamate receptor, beta-adrenergic receptor, or PAF receptor
  • numerous proteins may be modified to make them translocatable by employing bridging molecules, as discussed above.
  • the first and second proteins of interest may together comprise members of a specific binding pair.
  • the invention may further include the step of administering a test compound to the cell prior to the detecting step, wherein the absence of binding of the detectable group to the internal structure indicates that the test compound inhibits the binding of the members of the specific binding pair.
  • a test compound can be used, including peptides, oligonucleotides, expressed proteins, small organic molecules, known drugs and derivatives thereof, natural or non-natural compounds, etc.
  • Administration of the test compound may be by any suitable means, including direct administration such as by electroporation or lipofection if the compound is not otherwise membrane permeable, or (where the test compound is a protein), by introducing a heterologous nucleic acid that encodes and expresses the test compound into the cell. Such methods are useful for screening libraries of compounds for new compounds which disrupt the binding of a known binding pair.
  • the method may be used to quantitatively determine binding affinity by varying the concentration of either construct to measure the binding affinity of the constructs at different concentrations, or (where the members of the specific binding pair are the same) to establish the size of the oligomers formed.
  • the method further comprises the step of: (c) repeating steps (a) and (b) a plurality of times with a library of proteins of interest, wherein one of the first and second proteins of interest is maintained the same and the other of the first and second proteins of interest (the variable protein or the protein being screened) is replaced with a different member of the library, so that the library is screened for proteins that specifically bind to one of the first or second proteins of interest.
  • Administration of the protein of interest that is varied may be by any suitable means, including direct administration such as by electroporation or lipofection if the compound is not otherwise membrane permeable, or (where the test compound is a protein), by introducing a heterologous nucleic acid that encodes and expresses the variable protein of interest into the cell. Such methods are useful for screening libraries of compounds for new candidates for binding to a known protein.
  • the invention provides fusion proteins comprising a protein that specifically binds to an internal structure within a cell and a protein of interest, such as a protein that is a member of a specific binding pair, along with nucleic acids encoding such fusion proteins and cells that contain and express such nucleic acids (the nucleic acid thus including regulatory sequences operative in the cell and operatively associated with the nucleic acid segment that encodes the fusion protein).
  • a protein of interest such as a protein that is a member of a specific binding pair
  • a kit useful for detecting protein-protein interactions within a living cell comprises (a) a cell as described above that contains and expresses a nucleic acid encoding a first fusion protein, the fusion protein comprising a protein that specifically binds to an internal structure within the cell and a first protein of interest; together with (b) a vector for the cell, the vector containing an expression cassette.
  • the expression cassette comprises a promoter operable in the cell and operatively associated with a nucleic acid encoding a detectable protein, and has a splice site positioned adjacent the nucleic acid encoding a detectable protein so that a heterologous nucleic acid encoding a second protein of interest can be inserted therein to produce a nucleic acid segment encoding a second fusion protein.
  • the second fusion protein comprising the detectable protein and the second protein of interest.
  • a kit useful for detecting protein-protein interactions within a living cell comprises (a) a cell as described above that contains and expresses a nucleic acid encoding a first fusion protein, the fusion protein comprising a detectable protein and a first protein of interest; together with (b) a vector for the cell, the vector containing an expression cassette, the expression cassette comprising a promoter operable in the cell and operatively associated with a nucleic acid encoding a protein that specifically binds to an internal structure within the cell, as described above.
  • the expression cassette likewise has a splice site positioned adjacent the nucleic acid encoding the protein that specifically binds to an internal structure within the cell (as described above; preferably a translocatable protein as described above) so that a heterologous nucleic acid encoding a second protein of interest can be inserted therein to produce a nucleic acid segment encoding a second fusion protein.
  • the second fusion protein comprises the protein that specifically binds to an internal structure within the cell and the second protein of interest.
  • kits can be provided in any suitable form, and are typically packed together with suitable instructions. Any vector may be employed, but the vector is typically a plasmid vector.
  • the vector is used in one embodiment to create, from a source nucleic acid library as described above, a product nucleic acid library comprising a plurality of separate nucleic acids, each of the separate nucleic acids encoding a fusion protein, the fusion protein comprising a protein of interest (encoded by the source library) and a detectable protein, wherein the protein of interest encoded by each of the separate nucleic acids is different from the protein of interest encoded by the other nucleic acids of the library.
  • the product library, in the vector can then be used to transform multiple cells so that the library constituents can be screened in the manner described above.
  • the vector is used to create, from a source nucleic acid library as described above, a product nucleic acid library comprising a plurality of separate nucleic acids, each of the separate nucleic acids encoding a fusion protein, the fusion protein comprising a protein of interest (from the source library) and a protein that specifically binds to an internal structure within a cell as described above (preferably a translocatable protein as described above), wherein the protein of interest encoded by each of the separate nucleic acids is different from the protein of interest encoded by the other nucleic acids of the library.
  • the product library, in the vector can then be used to transform or transfect multiple cells so that the library constituents can be screened in the manner described above.
  • Screening of libraries may be carried out in accordance with conventional techniques.
  • the screening will be carried out by transfecting pools (subsets of the members of the library) into different groups of cells to express the protein of interest. This allows one to identify pools that contain binding partners that interact with the protein of interest. Pools with binding partners can then again be divided into subpools until individual members
  • Ca 2+ /calmodulin dependent protein kinase II (CaMKII) is a ubiquitous kinase which is expressed at high concentrations in neurons and at lower concentrations in most other cell types.
  • CaMKII is an essential mediator for long term potentiation and other forms of synaptic plasticity (reviewed in Braun and Schulman, 1995; Soderling, 1993) .
  • CaMKII activity may have an important role in stabilizing the dendritic architecture (Wu and Cline, 1998).
  • a critical neuronal function of the ⁇ -isoform of CaMKII (CaMKIIa) was directly demonstrated by studying mice which were either lacking CaMKIIa or which expressed mutated CaMKIIa.
  • CaMKIIa deficient mice as well as transgenic mice expressing an autonomously active or an autophosphorylation deficient CaMKIIa showed impaired long term potentiation as well as defects in spatial learning and memory (Chapman et al., 1995; Glazewski et al., 1996; Gordon et al., 1996; Mayford et al, 1996; Mayford et al., 1995; Silva et al., 1992; Giese et al., 1998). Since not only CaMKIIa but also CaMKII ⁇ is a prominent isoform in the central nervous system, the question arises whether CaMKIIa and CaMKII ⁇ have different roles in regulating neuronal functions.
  • CaMKII ⁇ and CaMKIIa have a similar overall domain organization and corresponding autophosphorylation consensus sequences and even though the calmodulin binding affinity of CaMKII ⁇ is slightly higher than that of CaMKIIa, the regulation of different CaMKII isoforms by Ca 2+ /CaM and autophosphorylation is overall similar (Miller and Kennedy, 1985; De Koninck and Schulman, 1998; GuptaRoy and Griffith, 1996).
  • GFP green fluorescent protein
  • CaMKIIa and CaMKII ⁇ are the primary docking sites for expressed CaMKII ⁇ .
  • expressed CaMKIIa was uniformly distributed in the soma and processes and was largely absent from spines.
  • CaMKII ⁇ targeted CaMKIIa to dendritic spines and the cell cortex. In vitro binding studies suggested that this targeting results from a direct binding interaction of CaMKII ⁇ with F-actin.
  • a GFP-based protein-protein interaction assay (Pull-Out binding assay) was then developed to explore the binding interactions between CaMKIIa and CaMKII ⁇ isoforms in living cells.
  • CaMKIIb When expressed alone, CaMKIIb was found to form homo- oligomers with an average size that is markedly smaller than the approximately thirteen subunits measured for CaMKIIa homo-oligomers.
  • CaMKIIb isoforms incorporated equally well into either CaMKIIa or CaMKIIb oligomers (and vice versa).
  • PROCEDURES a. Cloning of CaMKII fusion constructs.
  • the cDNA for rat CaMKIIa , ⁇ and ⁇ 1 were generous gifts from Dr. Howard Schulman.
  • the construction of the GFP-CaMKII ⁇ vector was described previously (Shen and Meyer, 1998).
  • the CaMKIIa cDNA was amplified by PCR and cloned into the in vitro transcription vector dSHiro3. DNA sequencing were performed to exclude PCR errors.
  • GFP-CaMKIIb and CaMKIIb were also cloned into the SHiro3 and dSHiro3 vectors using a similar PCR strategy.
  • PM-GFP or Cys-GFP was described previously (Oancea et al., 1998).
  • PM-CaMKII ⁇ and ⁇ were made by replacing the GFP sequence with CaMKIIa and ⁇ coding sequence in the same SHiro3 vector.
  • In vitro translation with SP6 RNA polymerase was performed according to the manufacturer's protocol using a commercial kit (TNT Coupled reticulocyte lysate system, Promega). In vitro transcription reactions were performed using mRNAs as templates. The relative molar concentration of the different translated proteins was calculated by calibration using 35 S-methionine incorporation and by counting the number of methionines in the respective protein. Non-radioactive methionine was used to obtain CaMKIIs and fusion constructs for the autophosphorylation assay. c. Ca 2+ dependent and independent autophosphorylation of CaMKII and GFP fusion proteins.
  • CaMKII autophosphorylation assay were performed as described previously (Hanson et al., 1994). Briefly, CaMKII isoforms and fusion constructs were autophosphorylated at 30°C in 25ml reactions containing 50 mM PIPES (pH 7.0), lOmM MgCl2, 500 mM CaC , 600 nM calmodulin, 50 mg/ml BSA, and 200 mM [g-
  • Poly adenylation was carried out for 30 minutes at 37°C in a 50 ⁇ l reaction mixture containing 40 mM Tris- HC1 (pH 8.0), 10 mM MgCl2, 2.5 mM MnCl2, 250 mM NaCl, 0.25 mg / ⁇ l RNA, 250 mM ATP, 5 units poly(A) polymerase (Life Technologies). The reaction was terminated by addition of 20 mM EDTA. Unincorporated ATP and salts were removed by applying the mRNA to a RNeasy column.
  • the eluent was dried and mRNA was dissolved at 1 ⁇ g / ⁇ l in the electroporation buffer (5 mM KCl, 125 mM NaCl, 20 mM HEPES pH 7.4 and 10 mM glucose).
  • electroporation buffer 5 mM KCl, 125 mM NaCl, 20 mM HEPES pH 7.4 and 10 mM glucose.
  • RBL 2H3 cells were maintained in Dulbecco's Minimum Essential Medium (DMEM) supplemented with 20% fetal bovine serum (Life Technologies,
  • Hippocampal neurons obtained from 2 to 4 days postnatal rats were cultured as described in Ryan and Smith (1995) and used 10 days to three weeks after plating.
  • a self-built small volume electroporation device for adherent cells was used for electroporation (Teruel and Meyer, 1997).
  • modified versions of the device and buffer conditions were used. After transfection, the electroporation buffers were replaced with the same culture medium.
  • FM 4-64 N-(3- triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl) pyridinium,dibromide; Molecular Probes.
  • FM 4-64 (Henkel and Betz, 1995 ) is similar in structure and properties to FM 1-43 but its longer wavelength emission spectra make it more suitable for dual-channel fluorescence microscopy in conjunction with green fluorescent protein (Ziv and Smith, 1996).
  • Cells were loaded with FM 4-64 by replacing the saline in imaging chamber with high potassium solution (lOOmM KCL, 20 mM HEPES, 1.5mM CaCl2, 30mM NaCL, 1.5mMMgCL2, pH 7.4 and 6 ⁇ M of FM 4-64) for 20 seconds and switch back to a saline solution for 5-10 min. After collecting a digital image of the labeled field, the cells were stimulated again by switching to the same high potassium solution. The spatial distribution of the active presynaptic terminals could then be determined from a difference image.
  • the diffusion coefficients were determined using an analysis by which a photobleached area is produced by a focused laser pulse and the fluorescence recovery is fit to sequential 2-dimensional Gaussian distributions. Ratio images of the fluorescence distribution after the bleach pulse to the distribution before the pulse were used for the analysis. This analysis follows at the same time the decrease in the bleach amplitude and the increase in the bleach radius. Assuming mass conservation, the resulting fluorescence distributions were fit by:
  • F n ( x , y ) - (Fo * a 0 2 / a n 2 ) * exp ( - ( (x - x 0 ) 2 + (y - y 0 ) 2 ) / a n 2 ), with x and y as the pixel coordinates, a n as the radius of the bleach diameter in the n-th image, and Fn( ⁇ 5 y) as me l oca l relative fluorescence intensity.
  • a least square fit routine was used to fit at the same time Gaussian profiles to all images in the time series.
  • NIH-3T3, RBL cells, and hippocampal neurons were cultured on glass coverslips and transfected with mRNA encoding GFP-CaMKIIb fusion construct.
  • NIH-3T3 cells and RBL cells were permeabilized for 5 minutes at 4 °C with 0.1% Triton in PBS.
  • Hippocampal neurons were permeabilized for 10 minutes at 4 °C with 0.1% Triton.
  • rhodamine phalloidin (Molecular Probes) was incubated with the cells for 30 min at room temperature at a dilution of 1:300. in PBS.
  • hippocampal neurons were incubated with an monolonal PSD-95 antibody (Cat.# 05-428, Upstate Biotechnology, Lake Placid, NY) overnight at 4 °C at a dilution of 1 :200 and then in secondary Cy3 labeled anti-mouse antibody (Cat.# 1 15- 165-062, lackson ImmunoResearch, West Grove, PA) for 1 hour at room temperature.
  • the cells were washed three times with PBS and coverslips were mounted onto glass slides using buffered glycerol mounting medium.
  • NIH-3T3 cells were plated in 35 mm dishes at a density of 1.0 X 10 ⁇ per dish and incubated overnight at 37 °C in a humid atmosphere containing 5% CO2- Cells were transfected with 1.5 -2.2 ⁇ g of pSRa-CaMKIIb or pSRa-CaMKIIa or cotransfected with pSRa-CaMKIIb and pSRa-CaMKIIa using lipofectin plus
  • Poly-adenylated mRNA was made as described above. In many experiments, mRNA species were mixed and used for electroporation at a final concentration of typically 1 ⁇ g/ ⁇ l total. The relative translation efficiency was determined by a separate in vitro translation reaction using the same mRNA as a template. Images of transfected RBL or NIH 3T3 cells were taken on a Zeiss confocal microscope 8-12 hours after electroporation. 1 ⁇ M of PMA was added and images of single cells were taken under the same configuration. Images were taken before and after PMA addition and were analyzed using NIH-image software. A plasma membrane translocation factor was defined as DP / I pre ( Figure 2E). 2. RESULTS a. Expressed CaMKIIb but not CaMKIIa is enriched in dendritic branches and cell cortex
  • CaMKIIa When GFP-tagged CaMKIIa or b isoforms were expressed in cultured hippocampal neurons, CaMKIIa was largely homogeneous in the soma and main processes ( Figure ID, left) but was only minimally present in the finer branch structures. In contrast, CaMKIIb showed a striking enrichment in dendritic branches as well as at the cell cortex ( Figure ID, right). When expressed in RBL-cells, CaMKIIa was nearly homogeneously distributed in the cytosol and CaMKIIb had a distinct cortical localization (Fig, IE, right).
  • CaMKIIb is an F-actin docking module enriched in dendritic spines
  • actin is highly enriched in dendritic spines and cell cortex (Fisher et al., 1998; Landis and Reese, 1983, Caceres el al, 1983), it is conceivable that the localization of CaMKIIb to dendritic spines is mediated by a direct or indirect binding interaction of CaMKIIb with F-actin.
  • the possible co-localization of CaMKIIb and F- actin in two model cell lines was tested by using rhodamine-phalloidin as a marker for polymerized actin (F-actin).
  • CaMKIIa is targeted to dendritic spines when co-expressed with CaMKIIb
  • RNA transfection method a large number of translation competent RNA molecules are directly introduced into the cytosol of adherent cells by microporation (Teruel and Meyer, 1997; Yokoe and Meyer, 1996). Thus, RNA encoding different proteins can be mixed and expressed at a defined ratio within each transfected cell.
  • GFP-CaMKIIa when GFP-CaMKIIa was expressed together with CaMKIIb (without a GFP-tag) in hippocampal neurons, GFP- CaMKIIa became associated with the same dendritic spine and cortical structures (data not shown). A largely cortical localization of GFP-CaMKIIa was also observed in RBL-cells in the presence of CaMKIIb. In contrast, expression of CaMKIIa (without a GFP-tag) together with a similar amount of GFP-CaMKIIb did not affect the cortical localization of GFP-CaMKIIb (data not shown).
  • CaMKIIb target CaMKIIa to the actin cytoskeleton?
  • CaMKIIa does not undergo cytoskeletal binding interactions of its own but binds to CaMKIIb which then anchors the complex to F-actin.
  • several important questions about the binding interaction and oligomerization of CaMKIIa and CaMKIIb have to be answered: 1. Can CaMKIIb form oligomers on its own and if it does, how large are these oligomers compared to those formed by CaMKIIa?, 2. do co-expressed CaMKIIa and CaMKIIb isoforms assemble into hetero- or homo- oligomers in living cells?, 3.
  • an assay was developed to quantitatively study protein-protein binding interactions in living cells ("Pull-Out” binding assay, Figure 2A).
  • the strategy was to use a protein-domain (PM-domain) that translocates to the plasma membrane in response to the addition of a drug.
  • the potential binding interaction between the investigated Protein X and Protein Y could then be investigated by fusing this PM-domain to Protein X and a GFP-tag to Protein Y (or vice versa). Both fusion proteins could then be expressed in the same cell and their binding interaction investigated. 1. If the initially cytosolic GFP -Protein Y remains cytosolic after drug addition, no significant binding interaction occurs between Proteins X and Y. 2. If the drug addition leads to the GFP- Protein Y translocation to the plasma membrane (along with the non-fluorescent PM- Protein X), Proteins X and Y bind to each other.
  • This translocation process occurs in less than a minute and is mediated by a diffusion-dependent high affinity binding interaction of the fusion protein with plasma membrane localized phorbol ester (Oancea et al., 1998). It should be noted that the also visible nuclear localized GFP fusion protein translocates to the plasma membrane much slower due to its slow diffusion through nuclear pores.
  • CaMKIIb homo-oligomers are significantly smaller than CaMKIIa homo- oligomers
  • RNA transfection method allows one to quantitatively titrate the amount of the two CaMKII fusion proteins in the same cell.
  • concentration of the translated proteins was measured in parallel by in vitro translation of the same RNAs in the presence of 35 S-Met ( Figure 3B).
  • Figure 3C shows the GFP-CaMKIIa plasma membrane translocation at increasing dilutions of co-expressed PM-CaMKIIa.
  • the left images show the distribution before and the right images after phorbol ester addition.
  • the phorbol ester induced targeting to the plasma membrane was still measurable when PM-CaMKIIa was diluted to less than 3% of GFP-CaMKIIa.
  • the sequential reduction in plasma membrane translocation can be seen more clearly in fluorescence line intensity traces ( Figure 3D).
  • a titration curve can be obtained, showing the relative plasma membrane translocation at decreasing dilution ratios of PM-CaMKIIa to GFP- CaMKIIa ( Figure 3F).
  • a 50% reduction in plasma membrane targeting was observed at an approximate dilution ratio of one PM-CaMKIIa per fourteen GFP-CaMKIIa.
  • a small number of CaMKIIb isoforms are sufficient to target CaMKII hetero- oligomers to the actin-cytoskeleton
  • CaMKIIb subunits are required for the targeting of CaMKIIa/b hetero-oligomers to the actin cytoskeleton.
  • the same RNA dilution strategy was used to determine at which ratio of GFP-CaMKIIa to CaMKIIb the cytoskeletal localization still occurs.
  • the distinct cortical actin cytoskeleton localization of CaMKIIb in RBL-cells was used in this assay ( Figure 4C). While CaMKIIb was less potent in targeting GFP- CaMKIIa to the plasma membrane than the PM-CaMKII constructs, 50% translocation to the cortical actin cytoskeleton required a ratio of approximately 6.5 : 1 of GFP-CaMKIIa to CaMKIIb. Since CaMKIIa isoforms contain approximately 13 subunits, this suggests that a small number of CaMKIIb subunits are sufficient to target CaMKIIa/b hetero-oligomers to the actin cytoskeleton.

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Abstract

Un procédé de détection des interactions protéine-protéine dans une cellule vivante consiste (a) à fournir une cellule contenant un premier conjugué hétérologue ainsi qu'un second conjugué hétérologue, dans laquelle le premier conjugué hétérologue comprend une première protéine étudiée conjuguée à un groupe détectable, et le second conjugué hétérologue comprend une seconde protéine étudiée conjuguée à une protéine se liant spécifiquement à une structure interne dans la cellule, et ensuite (b) à détecter la présence ou l'absence de liaison du groupe détectable à la structure interne, la présence de la liaison indiquant que les première et seconde protéines étudiées se lient spécifiquement l'une à l'autre. D'autres aspects de l'invention concernent des acides nucléiques codant des protéines de fusion telles que décrit ci-dessus, des cellules contenant et exprimant ces protéines de fusion, des matériels utiles pour la mise en oeuvre des méthodes décrites ci-dessus, ainsi que des banques d'acides nucléiques utiles en tant qu'outils de criblage pour la mise en oeuvre des méthodes décrites ci-dessus. L'invention est utile pour cribler des composés présentant une capacité à interrompre ou à inhiber la liaison des paires de liaisons connues et ainsi identifier leurs inhibiteurs en concurrence. L'invention est utile pour cribler une protéine connue étudiée par rapport à une banque d'autres protéines étudiées afin d'identifier des composés se liant à la protéine étudiée connue.
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AU55789/99A AU5578999A (en) 1998-09-24 1999-08-24 Method of measuring protein-protein interactions in living cells
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EP1198586A1 (fr) * 1999-06-26 2002-04-24 Odyssey Pharmaceuticals, Inc. Selection in vivo bibliotheque contre bibliotheque d'interactions proteine-proteine optimisees
WO2003029827A2 (fr) * 2001-10-01 2003-04-10 Bioimage A/S Methode amelioree permettant de detecter des interactions entre des constituants cellulaires presents dans des cellules vivantes intactes et d'extraire des informations quantitatives se rapportant auxdites interactions par nouvelle repartition de fluorescence
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WO2004005899A1 (fr) * 2002-07-05 2004-01-15 Kansai Technology Licensing Organization Co., Ltd. Procede et dispositif de detection d'association de proteines par variation de constante de dispersion
EP1533619A2 (fr) 2003-11-20 2005-05-25 F. Hoffmann-La Roche Ag Marqueurs spécifiques du syndrome métabolique.
EP2187213A1 (fr) * 2008-11-12 2010-05-19 Korea Basic Science Institute Procédé de détection d'interaction entre deux macromolécules biologiques ou plus
US7771952B2 (en) 2002-06-26 2010-08-10 Abott Laboratories Modulators and modulation of the interaction between RGM and Neogenin
WO2011083147A1 (fr) 2010-01-08 2011-07-14 Cemm-Forschungsinstitut Für Molekulare Medizin Gmbh Inhibition de wave1 dans l'intervention médicale de maladies inflammatoires et/ou d'infections causées par un pathogène
WO2011131626A1 (fr) 2010-04-19 2011-10-27 Medizinische Universität Innsbruck Tmem195 code pour l'activité alkylglycérol monooxygénase dépendante de la tétrahydrobioptérine
WO2013084950A1 (fr) 2011-12-05 2013-06-13 Ａｍａｌｇａａｍ有限会社 Procédé de détection d'une interaction protéine-protéine
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WO2018048063A1 (fr) * 2016-09-07 2018-03-15 포항공과대학교 산학협력단 Procédé de mesure quantitative de l'interaction entre des protéines membranaires dans des cellules vivantes
US10591486B2 (en) 2014-07-23 2020-03-17 Institut National De La Sante Et De La Recherche Medicale (Inserm) Methods and tools for detecting interactions in eukaryotic cells using microtubule structures and dynamics
US10761085B2 (en) 2014-06-10 2020-09-01 Medical & Biological Laboratories Co., Ltd. Method for determining a protein-protein interaction

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EP1198586A4 (fr) * 1999-06-26 2004-03-24 Odyssey Pharmaceuticals Inc Selection in vivo bibliotheque contre bibliotheque d'interactions proteine-proteine optimisees
EP1198586A1 (fr) * 1999-06-26 2002-04-24 Odyssey Pharmaceuticals, Inc. Selection in vivo bibliotheque contre bibliotheque d'interactions proteine-proteine optimisees
WO2002003072A3 (fr) * 2000-07-04 2002-06-13 Bioimage As Technique permettant d'extraire des informations quantitatives relatives aux interactions entre des composants cellulaires
WO2002003072A2 (fr) * 2000-07-04 2002-01-10 Bioimage A/S Technique permettant d'extraire des informations quantitatives relatives aux interactions entre des composants cellulaires
US7282347B2 (en) 2000-07-04 2007-10-16 Fisher Bioimage Aps Method for extracting quantitative information relating to interactions between cellular components
WO2003029827A2 (fr) * 2001-10-01 2003-04-10 Bioimage A/S Methode amelioree permettant de detecter des interactions entre des constituants cellulaires presents dans des cellules vivantes intactes et d'extraire des informations quantitatives se rapportant auxdites interactions par nouvelle repartition de fluorescence
WO2003029827A3 (fr) * 2001-10-01 2003-11-13 Biolmage As Methode amelioree permettant de detecter des interactions entre des constituants cellulaires presents dans des cellules vivantes intactes et d'extraire des informations quantitatives se rapportant auxdites interactions par nouvelle repartition de fluorescence
WO2003083112A1 (fr) * 2002-04-01 2003-10-09 Japan Science And Technology Agency Population de cellules pourvues de codes d'identification et procedes pour le criblage de population de cellules
US7771952B2 (en) 2002-06-26 2010-08-10 Abott Laboratories Modulators and modulation of the interaction between RGM and Neogenin
WO2004005899A1 (fr) * 2002-07-05 2004-01-15 Kansai Technology Licensing Organization Co., Ltd. Procede et dispositif de detection d'association de proteines par variation de constante de dispersion
EP1533619A2 (fr) 2003-11-20 2005-05-25 F. Hoffmann-La Roche Ag Marqueurs spécifiques du syndrome métabolique.
EP2053409A1 (fr) 2003-11-20 2009-04-29 F. Hoffmann-La Roche Ag Marqueurs spécifiques du syndrome métabolique
US8906864B2 (en) 2005-09-30 2014-12-09 AbbVie Deutschland GmbH & Co. KG Binding domains of proteins of the repulsive guidance molecule (RGM) protein family and functional fragments thereof, and their use
US9605069B2 (en) 2008-02-29 2017-03-28 AbbVie Deutschland GmbH & Co. KG Antibodies against the RGM a protein and uses thereof
US8962803B2 (en) 2008-02-29 2015-02-24 AbbVie Deutschland GmbH & Co. KG Antibodies against the RGM A protein and uses thereof
EP2187213A1 (fr) * 2008-11-12 2010-05-19 Korea Basic Science Institute Procédé de détection d'interaction entre deux macromolécules biologiques ou plus
US9175075B2 (en) 2009-12-08 2015-11-03 AbbVie Deutschland GmbH & Co. KG Methods of treating retinal nerve fiber layer degeneration with monoclonal antibodies against a retinal guidance molecule (RGM) protein
WO2011083147A1 (fr) 2010-01-08 2011-07-14 Cemm-Forschungsinstitut Für Molekulare Medizin Gmbh Inhibition de wave1 dans l'intervention médicale de maladies inflammatoires et/ou d'infections causées par un pathogène
WO2011131626A1 (fr) 2010-04-19 2011-10-27 Medizinische Universität Innsbruck Tmem195 code pour l'activité alkylglycérol monooxygénase dépendante de la tétrahydrobioptérine
US9494595B2 (en) 2011-12-05 2016-11-15 Medical & Biological Laboratories Co., Ltd. Method for detecting protein-protein interaction
WO2013084950A1 (fr) 2011-12-05 2013-06-13 Ａｍａｌｇａａｍ有限会社 Procédé de détection d'une interaction protéine-protéine
US9766250B2 (en) 2011-12-05 2017-09-19 Medical & Biological Laboratories Co., Ltd. Method for detecting protein-protein interaction
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US9102722B2 (en) 2012-01-27 2015-08-11 AbbVie Deutschland GmbH & Co. KG Composition and method for the diagnosis and treatment of diseases associated with neurite degeneration
US10106602B2 (en) 2012-01-27 2018-10-23 AbbVie Deutschland GmbH & Co. KG Isolated monoclonal anti-repulsive guidance molecule A antibodies and uses thereof
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US10591486B2 (en) 2014-07-23 2020-03-17 Institut National De La Sante Et De La Recherche Medicale (Inserm) Methods and tools for detecting interactions in eukaryotic cells using microtubule structures and dynamics
WO2018048063A1 (fr) * 2016-09-07 2018-03-15 포항공과대학교 산학협력단 Procédé de mesure quantitative de l'interaction entre des protéines membranaires dans des cellules vivantes

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