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WO2003010507A2 - Systeme permettant de surveiller un processus intracellulaire et criblage de medicaments in vivo - Google Patents

Systeme permettant de surveiller un processus intracellulaire et criblage de medicaments in vivo Download PDF

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Publication number
WO2003010507A2
WO2003010507A2 PCT/IB2002/004257 IB0204257W WO03010507A2 WO 2003010507 A2 WO2003010507 A2 WO 2003010507A2 IB 0204257 W IB0204257 W IB 0204257W WO 03010507 A2 WO03010507 A2 WO 03010507A2
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Prior art keywords
protein
cell
signal
trafficking
reporter
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WO2003010507A3 (fr
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Inhwan Hwang
Dae Heon Kim
Yong Jik Lee
Jing Bo Jin
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Ahram Biosystems Inc
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Ahram Biosystems Inc
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Priority to AU2002339228A priority Critical patent/AU2002339228A1/en
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Publication of WO2003010507A3 publication Critical patent/WO2003010507A3/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters
    • 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
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5076—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving cell organelles, e.g. Golgi complex, endoplasmic reticulum
    • 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
    • G01N33/6875—Nucleoproteins

Definitions

  • the present invention generally relates to methods for detecting intracellular movement (trafficking) of a selected protein inside cells.
  • the invention further relates to methods for detecting compounds (screens) that modulate at least one of expression and trafficking of the protein in vivo.
  • the invention has a wide spectrum of applications including use in the detection of drugs that impact protein trafficking to a pre-selected subcellular organelle.
  • the assay method to evaluate the effects of drug candidates on a specific target can be categorized into three classes.
  • the first is a protein- or a molecule-based method in which the effect of the drug candidate on the activity of an enzyme or a receptor critical in the induction mechanism of a disease is directly observed to screen the drug candidates.
  • the second is an organism-based method in which the effect of the drug candidate on the morphological or biochemical characteristics of an organism is observed to screen the drug candidates.
  • the last is a cell-based method in which the effect of the drug candidate on the morphology of the cell or subcellular organelles, and expression, trafficking, and metabolism of proteins in the cell is observed to screen the drug candidates.
  • the protein-based method has the advantage that the assay can be performed for a specific target protein.
  • the assay is conducted in vitro, it is not possible to examine various complex cellular factors. Therefore, it requires many additional time-consuming experiments before examining the drug candidate to a living organism.
  • the organism- or cell-based method can detect the composite changes of the organism or the cell induced by the drug candidates. But it also requires additional time-consuming experiments to confirm that the drug candidate specifically affects the target gene or protein.
  • fluorescent proteins such as green fluorescent protein (GFP), red fluorescent protein (REF) (Moris et al., 1974), and their derivatives (Evans, W098/21355) that can be expressed in the cell have been developed, and various researches have been reported using expression of these fluorescent proteins.
  • Examples of using the fluorescent proteins include observation of the gene expression and subcellular protein localization (Chalfie and Prasher, US5491084), visualization of the subcellular organelles (Kost et al., 1998), visualization of the protein trafficking in the secretory pathway (Kaether and Gerdes, 1995), and visualization of the protein expression pattern in a plant cell (Hu and Cheng, 1995) and in Drosophila embryo (Davis and Viestra, 1998).
  • the fluorescent proteins have been also used to develop new drug screening methods. Examples include a method using mutated organisms transformed to express a modified GFP (Ward and Chalfie, W095/21191), a method using a cell transformed to express a chimeric protein comprising a fluorescent protein and a transcription factor related to the activity of a cell surface receptor which regulates signal transduction (Harpold et al., US5401629), and a method using a cell transformed to express a chimeric protein comprising a fluorescent protein and the active site of the protein kinase (Thastrup et al., W096/23898).
  • the chlorophyll-binding proteins are transported to chloroplast, the nuclear localization signal domain (NLS domain) of SV40 is targeted to the nucleus (Goldfarb et al., 1986), the peroxisome targeting motif SKL is targeted to peroxisome (Davis and Viestra, 1998), and FI- H + -ATPase is transported to mitochondria (Niwa et al., 1999).
  • NLS domain nuclear localization signal domain
  • SKL is targeted to peroxisome
  • FI- H + -ATPase is transported to mitochondria (Niwa et al., 1999).
  • most of proteins in the cell are transported to specific subcellular organelles related to their functions. Therefore, it is possible to visualize and observe intracellular trafficking of a specific signal protein (Harpold et al., US5401629; Kost et al., 1998).
  • Phospholipid-specific intracellular processes can also be observed by expressing phospholipid-binding proteins linked to a fluorescent protein.
  • PLC-Delta PH Stauffer et al., 1998)
  • AtPH AtPH
  • FAPP1 Lowler et al., 2000
  • a chimeric protein having the PH domain and GFP was shown to be translocated to the plasma membrane when expressed in a cell (Kost et al., 1998).
  • the invention generally relates to methods for detecting intracellular movement (trafficking) of at least one selected protein inside cells. Additional invention methods involve screening compounds for capacity to modulate at least one of expression and trafficking of the protein in vivo.
  • the invention has many important uses including detecting compounds that modulate protein trafficking to at least one preselected subcellular organelle.
  • the invention provides useful methods for detecting trafficking of at least one selected (reporter) protein inside living cells. Particular reporters of interest include at least one "signal" sequence and at least one detectable tag. Preferred signal sequences help shuttle the protein to at least one other intracellular location.
  • More particular reporters include at least one detectable tag, usually a fluorescent (or potentially fluorescent) sequence, that is operably and covalently attached to the signal sequence in- frame, thereby labeling the reporter protein.
  • Particular signal sequences of interest include or consist of amino acid residues that are known to help shuttle the reporter protein to at least one subcellular organelle.
  • such tagged reporter proteins can be used to screen compounds for capacity to modulate (increase or decrease) trafficking to one or more of the organelles.
  • the signal sequence of a reporter include or consist of recognition site(s) that facilitate movement of the reporter protein to a sub-cellular organelle.
  • recognition site(s) that facilitate movement of the reporter protein to a sub-cellular organelle.
  • An example of such proteins are characterized herein as Group I signal proteins (see Figure 1).
  • the proteins include or consist of amino acid sequences that are recognized by and typically contacted by endoplasmic reticulum (ER). Preferred examples of such proteins are featured herein as Group II signal proteins (see Figure 4).
  • ER endoplasmic reticulum
  • These reporter protein groups, as well as others discussed below, can be used as important tools to detect compounds that modulate such trafficking in vivo. Such proteins can also be used to confirm trafficking activity in cases in which such function is known or suspected.
  • the present invention provides important advantages.
  • the invention provides, for the first time, recognition that operably linked fusions of a signal protein and a detectable tag can be used to screen compounds for capacity to modulate intracellular trafficking in vivo. That is, the invention is one of general application in which a wide spectrum of suitable fusion proteins can be made and used to detect compounds for capacity to increase, decrease or preserve existing intracellular protein trafficking to one or more pre-selected subcellular organelles.
  • any of the Group I or II protems can be fused to at least one detectable tag to create useful reporter protein fusions.
  • Such protems can be used, for instance, to detect compounds for ability to modulate trafficking to at least one of the nucleus, chloroplast, mitochondria, and endoplasmic reticulum.
  • reporter proteins in accord with the invention will include signaling features that provide for trafficking of the reporter to a single pre-selected subcellular organelle. Also envisioned however are reporters with multiple signal proteins (eg., two or three) that can provide trafficking to more than one of pre-selected organelle.
  • each reporter protein includes at least one fluorescent sequence preferably bound in-frame, and either directly or indirectly, to at least one signal protein.
  • the fluorescent sequence is intended to provide a highly sensitive and reproducible tag that can be used to detect minute amounts of trafficked protein inside cells.
  • Such advantages enhance the sensitivity, selectivity and reproducibility of the invention particularly in screens that are designed to detect compounds with potential trafficking activity.
  • the invention is fully compatible with existing screening platforms including what is often referred to as "high” or “ultra-high” throughput strategies.
  • the invention can provide an important and valuable in vivo confirmation of such strategies where trafficking activity of a subject compound is known or suspected.
  • such a method includes introducing and expressing at least one type of, preferably less than about five, more preferably one type of reporter protein inside cells and detecting the cellular distribution of that reporter to detect trafficking and localization of the desired protein within the cells.
  • the general detection method can be used as a screening platform to detect and optionally quantify trafficking activity of one or more compounds to be tested.
  • the cell expressing the reporter protein can be contacted with at least one desired compound to be tested, preferably less than about fifty of such compounds, more preferably less than about ten to fifteen of such compounds, even more preferably one compound to be tested.
  • the compound to be tested is added to the cells under conditions conducive to providing a protein trafficking effect. That is, the compound can be added to cultured cells before, during or after introduction of the reporter protein. In this example of the invention, cellular distribution of the reporter protein is visualized in the presence of the compound to detect any modulation of protein trafficking inside the cells.
  • Typical practice of the invention involves comparison with a suitable control eg., as when water or buffer is added to the cells in lieu of the tested compound.
  • the invention further encompasses diverse and complex intracellular processes and a detailed method for selectively screening compounds (eg., chemicals) that affect a specific intracellular process based on the above detection method.
  • compounds eg., chemicals
  • the present invention also provides a systematic method for transforming a cell with the recombinant plasmid and expressing efficiently the reporter protein in the transformed cell. Furthermore, the present invention provides a detailed method for monitoring details of the intracellular distribution of the reporter protein in a living cell by observing the fluorescence image either continuously or step by step during the expression and trafficking processes. In addition to providing the efficient systematic method for measuring the spatial distribution specific to the expression and trafficking processes of a selected protem in the transformed cell, it is another objective of the present invention to provide a method for selectively screening drug candidates that affect a specific intracellular process in the transformed cell.
  • the present invention provides recombinant genes for diverse signal proteins that direct targeting to specific intracellular organelles such as nucleus, mitochondria, chloroplast, peroxisome, plasma membrane, endoplasmic reticulum, Golgi apparatus, storage vacuole, lytic vacuole, prevacuolar compartment, etc.
  • the present invention aims to provide an optimized overall procedure from the construction of the recombinant plasmids to the preparation of the transformed cells.
  • the invention also establishes a method for determining the detailed effects of chemicals by examining the effects of several known chemicals on the intracellular processes.
  • the present invention provides (1) the method for selectively screening chemicals that affect the intracellular trafficking and localization of a selected protem, (2) the method for screening chemicals that inhibit or enhance the transcription or translation process by monitoring the effects of the chemicals on the expression of the reporter protein, and (3) the method for screening cytotoxic chemicals by observing the effect of chemicals that cause deformation, damage, or disruption of subcellular cellular organelles.
  • FIG 1 shows schematic diagrams of the reporter protems targeting to the subcellular organelles enclosed by membrane, which are classified as Group I proteins in the present invention.
  • Figure 2 shows fluorescence images showing the expression of the Group I reporter proteins in the cell, wherein
  • AtOEP7:GFP is localized to the envelope of chloroplast, wherein the red fluorescent signal is the auto-fluorescence signal of chloroplast and the yellow fluorescence signal is an merged image of the auto-fluorescence signal of chloroplast and the green fluorescence signal of the reporter protein;
  • (b) is a photograph where the red auto-fluorescence of chloroplast in (a) is eliminated by using a filter;
  • FIG. 3 shows a photograph of a Western blot analysis of the expressed AtOEP7:GFP, wherein T, S, and M indicate the total protein extract, the soluble fraction, and the membrane fraction, respectively.
  • FIG 4 shows schematic diagrams of the reporter protems targeting to the subcellular organelles by endosomal trafficking, which are classified as Group II protems in the present invention.
  • Figure 5 shows fluorescence images showing the expression of the Group II reporter proteins in the cell, wherein
  • (b) shows the fluorescence image of sialtransferase (ST) translocated in the Golgi apparatus, wherein the red fluorescence signal is the auto-fluorescence signal of chloroplast;
  • Figure 6 shows schematic diagrams of the reporter proteins binding specifically to phospholipids, which are classified as the Group III proteins in the present invention.
  • Figure 7 shows fluorescent images showing the expression of the Group III reporter proteins, wherein
  • Figure 8 shows photographs representing the inhibitory effect of wortmannin on the intracellular trafficking of a Group I reporter protein RbcS:GFP.
  • Wortmannin is known to be an inhibitor of PI(3)P and PI(4)P.
  • Figure 10 shows photographs showing the change induced by a specific inhibitor upon co-expression of two reporter proteins, wherein
  • FIG. 11 shows photographs that visualize the effects of brefeldin A disrupting the subcellular organelles, wherein (a) shows that BiP:RFP is distributed along the structure of the endoplasmic reticulum in the control protoplast;
  • Figure 12 shows photographs representing the inhibitory effect of chemicals on Group III reporter proteins, wherein
  • the invention relates to a method detecting intracellular movement (trafficking) of a selected reporter protein inside cells.
  • the invention provides methods for screening compounds for capacity to modulate at least one of expression and trafficking of the protein in vivo.
  • Particular use of the invention involves screening compounds for capacity to modulate protein trafficking to a subcellular organelle.
  • the present invention provides a method for detecting specific characteristics related to trafficking and localization of a selected protein in a cell.
  • the method comprises at least one of:
  • the present invention provides a method for screening chemicals that affect specific characteristics related to trafficking and localization of a selected protein in a cell.
  • the method comprises at least one of:
  • step (f) determining the effect of the chemical by comparing the fluorescence image obtained in step (e) with that of a control transformed cell which is not treated with the chemical.
  • the present invention provides recombinant genes encoding the reporter protems that are used in the above methods to visualize the trafficking of the reporter protems and their distributions in subcellular organelles.
  • the recombinant gene comprises a gene encoding a signal protein which includes a trafficking signal targeting to a specific subcellular organelle and a gene encoding a fluorescent protein linked thereto. Details of the compositions of the present invention are described below.
  • the signal protein is selected from the proteins that have trafficking signals targeting to nucleus, mitochondria, chloroplast, peroxisome, plasma membrane, endoplasmic reticulum, Golgi apparatus, storage vacuole, lytic vacuole, and prevacuolar compartment, and also those proteins targeting to 3 classes of phospolipids.
  • Examples of the signal protems include NLS (nuclear localization sequence), AtOEP7, Cab (chlorophyll a/b binding protein), SKL (peroxisome targeting motif), RbcS (rubisco small subunit), RA (rubisco activase), Fl-H ⁇ -ATPase, ETVATPase, BiP (chaperone binding protein), ST (Sialyltransferase).
  • Chi chitinase), recombinant clone 526, clone 491, clone 500, AtVTIla, SPO (sporamin), EBD, AtPH, FAPP, PH, etc.
  • the present invention provides methods for preparing transformed cells that can express reporter proteins, each comprising one of the signal proteins described above and a fluorescent protein label linked thereto.
  • the present invention also provides a systematic method for selectively detecting the intracellular processes using the transformed cells and a systematic method for selectively screening drug candidates based on this detection method.
  • the methods of the present invention include a step of selecting a specific protein that has a property of translocating to a specific subcellular location, a step of synthesizing the whole gene of the selected signal protein or a portion thereof encoding the trafficking signal of the selected protein, a step of synthesizing a gene encoding a fluorescent protein that can be linked to the signal protem to fluorescently visualize the subcellular localization, and a step of constructing a recombinant gene comprising a gene encoding the signal protem and a gene encoding the fluorescent protein linked thereto.
  • the function of the signal protem can change depending on the way that the fluorescent protein is linked to the signal protein.
  • the present invention therefore provides compositions of the reporter proteins whose signal protems can correctly direct the trafficking, and also construction methods thereof.
  • the present invention also provides a procedure for constructing recombinant plasmids that can be used to express the recombinant genes in a cell.
  • the recombinant plasmid can be constructed by ligating a recombinant gene into a vector containing a promoter, a terminator, and other necessary factors.
  • methods for transforming a cell by introducing the recombinant plasmid include, but are not limited to, chemical-mediated methods using PEG (polyethylene glycole), potassium phosphate, or DEAE-dextran, cationic lipid-mediated lipofection, microinjection, electroporation, and electrofusion.
  • one type of the recombinant plasmid could be introduced, or else two or more types of the recombinant plasmids can be introduced to express two or more reporter protems simultaneously.
  • the conditions need to be optimized to efficiently express the reporter protein comprising the signal protein and the fluorescent protein in the transformed cell.
  • the persons skilled in the art can select appropriate conditions depending on the signal protem and the fluorescent protein used. Detailed structures of the recombinant plasmids are explained in the examples of the present invention to present the amino acid sequences of the reporter proteins or corresponding nucleic acid sequences.
  • the signal proteins were classified into three classes based on the protem trafficking mechanisms, in order to show that the methods of the present invention for constructing the reporter proteins can be commonly used for various mechanisms of protein trafficking to different subcellular organelles.
  • Signal proteins targeting to nucleus, chloroplast, mitochondria, etc. correspond to the case that a specific portion of the signal protein directly acts as a recognition signal to direct the intracellular trafficking.
  • These signal proteins are classified as Group I for convenience in the specification of the present invention (see Figure 1).
  • Signal protems targeting to endoplasmic reticulum, Golgi apparatus, lytic vacuole, storage vacuole, plasma membrane, etc. correspond to the case of the endosomal trafficking in which a specific portion of the signal protein acts as a signal to be captured by endoplasmic reticulum so that the signal protein is translocated as enclosed in endoplasmic reticulum.
  • These signal proteins are classified as Group II for convenience in the specification of the present invention (see Figure 4). In the present invention, detailed methods are provided for visualizing the trafficking processes and the cellular distributions of these classes of the signal proteins.
  • signal proteins related to intracellular signal transduction via specific binding to phospolipids are selected in the present invention, and methods are provided for observing the subcellular organelles that contain specific phospholipid. These signal proteins are classified as Group III for convenience in the specifications of this invention (see Figure 6).
  • reporter proteins having specific trafficking features It will often be useful to practice the invention with reporter proteins having specific trafficking features. Thus for example in embodiments in which it is desirable to detect trafficking and localization of reporters with Group I or Group II signal proteins (or both Group I and Group II signal proteins), it will not be as useful to detect trafficking of report proteins having specific Group III signal protems.
  • the invention can be employed to detect trafficking and localization of a selected reporter protein in which the reporter includes a Group I or Group II signal protein.
  • a reporter does not include a Group III signal protein as defined herein.
  • the reporter protein of interest does not include the endosome binding domain (EBD) of human early endosome antigen 1 (a Group III signal protein) as defined by Kim D.H et al. (2001) in Plant Cell 13: 287 ie., amino acids 1257 to 1411 of EBD.
  • the reporter protem does not include one or more of the following signal proteins as defined in Figure 6: AfPH; FAAPl; and PH.
  • the reporter protein for use with the present invention does not include a recognition site(s) that binds specifically to subcellular organelles that contain specific phospholipid as defined herein for the Group III family of signal protems.
  • the present invention thus provides a method for detecting trafficking and localization of a selected reporter protein inside a cell in which the protein does not include one or more of the EBD signal protem, AtPH; FAAPl; or PH as defined in Figure 6.
  • the method includes introducing and expressing the reporter protein inside cells and detecting the cellular distribution of that reporter to detect trafficking and localization of the desired protein within the cells.
  • Such a method can also be used in accord with the invention as a screening platform to detect and optionally quantify trafficking activity of one or more compounds to be tested.
  • the cell expressing the reporter protein (without one or more of the EBD, AtPH; FAAPl; or PH signal proteins as defined in Figure 6) can be contacted with at least one desired compound to be tested.
  • the compound to be tested can be added to cultured cells before, during or after introduction of the reporter protein as discussed previously.
  • Such a specific invention method is useful in a variety of settings eg., where detection of Group I, Group II, or both Group I and II trafficking is desired.
  • reporter proteins that do not include Group II signal protems it will often be useful to practice the invention methods with reporter proteins that do not include Group II signal protems. For instance, it will be useful to detect trafficking with reporters that do not include one or more of the following Group II signal proteins: ST; BiP; Sporamin; and H + -ATPase as defined by Kim D.H et al. (2001) in Plant Cell 13: 287. In other embodiments, it will be more useful to detect trafficking with reporters in which the signal protein does not include a recognition site(s) that is involved with endosomal trafficking such that a specific portion of the signal protein acts as a signal to be captured by endoplasmic reticulum.
  • the invention provides a method for detecting trafficking and localization of a selected reporter protem inside a cell in which the protein does not include one or more of the ST; BiP; Sporamin; and H -ATPase signal proteins as defined eg., in Figure 4.
  • the method includes introducing and expressing the reporter protein inside cells and detecting the cellular distribution of that reporter to detect trafficking and localization of the desired protein within the cells.
  • the method can also be used in accord with the invention as a screening platform to detect and optionally quantify trafficking activity of one or more compounds to be tested.
  • the cell expressing the reporter protein (without one or more of the ST: BiP; Sporamin; and H + -ATPase signal protems as defined in Figure 4) can be contacted with at least one desired compound to be tested.
  • the compound to be tested can be added to cultured cells before, during or after introduction of the reporter protein as discussed previously.
  • Such a specific invention method has a variety of important uses such as in settings in which detection of Group I, Group III, or both Group I and III trafficking is desired.
  • the present invention also provides a method for visualizing the localization of two or more proteins simultaneously by using two or more fluorescent proteins with different colors.
  • green fluorescent protein (GFP, Davis and Viestra, 1998) and red fluorescent protein (RFP) are used for constructing the reporter protems to visualize their cellular localization.
  • RFP red fluorescent protein
  • the reporter proteins of the present invention can be constructed by using fluorescent proteins other than GFP and RFP.
  • the expression and trafficking processes of the reporter protein can be visualized in details for each stage of the processes by continuously monitoring the images of the fluorescence emitted by the reporter protem expressed in the transformed cell, using a fluorescence microscope at a specific wavelength.
  • a selective drug screening system for identifying chemicals inhibiting or enhancing the intracellular trafficking of the selected protein is established using this detection method. More particularly, it is demonstrated that chemicals affecting the intracellular trafficking can be identified by treating the transformed cell with a chemical before, after, or at the same time as the expression of the reporter protein, and then identifying the effect of the chemical by comparing the cellular distribution of the reporter protein in the transformed cell treated with the chemical with that in the control transformed cell which is not treated with the chemical. The same method can be used to screen chemicals inhibiting or enhancing the transcription or the translation of proteins because the level of the protein expression can be examined from decrease or increase in the intensity of the fluorescence signal from the reporter protein. This is also demonstrated in the examples of the present invention.
  • Morphological changes induced by a chemical such as modification, damage, or destruction of the subcellular organelles can be detected by observing the distribution or pattern of the fluorescence signal from the reporter protein. Therefore, it is also possible to screen cytotoxic chemicals that cause alteration of the subcellular organelles. This is also demonstrated in the examples of the present invention.
  • Example 1 Construction of recombinant plasmids for expression of Group I proteins targeting to the organelles across the membrane.
  • D88374 was amplified by polymerase chain reaction (PCR) from a ⁇ ZAPII cDNA library using two specific primers (5'-CTTTAATCAATGGCAATG and 5'-
  • Rubisco complex was PCR amplified from a ⁇ ZAPII cDNA library using two specific primers (5'-CCTCAGTCACACAAAGAG and 5'-
  • the resulting PCR product was subcloned into pBluescript and subsequently ligated in-frame to the 5' end of the coding regions of GFP and RFP to construct recombinant plasmids for RbcS:GFP and RbcS:RFP, respectively.
  • the coding region of the chloroplast a/b binding protein was PCR amplified from a ⁇ ZAPII cDNA library using two specific primers (5'-
  • TAGAGAGAAACGATGGCG and 5'-GGATCCCGTTTGGGAGTGGAACTCC used to construct a recombinant plasmid for Cab:GFP .
  • RA rubisco activase
  • GGATCCATCTGTCTCCATCGGTTTG GGATCCATCTGTCTCCATCGGTTTG
  • AtOEP7 a homolog of OEP14 of pea was PCR amplified from a Arabidopsis genomic
  • a recombinant plasmid for the nuclear localization signal (NLS), NLS:GFP was constructed as described previously (Pih et al., 2000).
  • a recombinant plasmid for the nuclear localization signal (NLS), NLS:GFP was constructed as described previously (Pih et al., 2000).
  • NLS:RFP was constructed by replacing the GFP coding region with the RFP coding region in the recombinant gene for NLS:GFP.
  • the recombinant plasmid for the peroxisomal reporter GFP:SKL was constructed by PCR with 326GFP (Davis and Viestra, 1998) as a template using two specific primers (5'-CCGTATGTTACATCACC and 5'- TTATAGCTTTGATTTGTATAGTTCATCCAT).
  • the protoplast suspension was filtered through a 100 ⁇ m mesh and protoplasts were collected by centrifugation at 46xg for 5 min.
  • the pelleted protoplasts were resuspended in 5 to 10 ml of the W5 solution (154 mM NaCl, 125 mM CaCl 2 , 5 mM KC1, 5 mM glucose, 1.5 mM Mes-KOH, pH 5.6), overlaid on top of 20 ml of 21% sucrose, and centrifuged for 10 min. at 78xg.
  • the intact protoplasts at the interface were transferred to a new tube containing 20 ml of the W5 solution.
  • the protoplasts were pelleted again by centrifugation at 55xg for 5 min and resuspended in 20 ml of the W5 solution.
  • the protoplasts were incubated on ice for 30 minutes.
  • Plasmid DNA (about 20-50 ⁇ g at a concentration of 2 ⁇ g/ ⁇ l) was added to 300 ⁇ l of the protoplast suspension, and subsequently 325 ⁇ l of the PEG solution (400 mM Mannitol, 100 mM Ca(N0 3 ) 2 , 40% PEG 4000) was added. The mixture was gently mixed and incubated for 30 min at room temperature. After incubation, the mixture was diluted with 10 ml of the W5 solution. Protoplasts were recovered by centrifugation at 50xg for 5 min and resuspended in 3 ml of the W5 solution and incubated at 22°C in the dark.
  • Example 3 Expression of Group I reporter proteins and observation of their expression and localization.
  • the recombinant plasmids constructed in Example 1 were used to transform the protoplasts according to the method described in Example 2.
  • the expression of the reporter proteins after the transformation was monitored as a function of time by capturing images using a fluorescence microscope (Axioplan fluorescence microscope, Zeiss, Germany) equipped with a cooled charge-coupled device (CCD) camera.
  • a fluorescence microscope Alxioplan fluorescence microscope, Zeiss, Germany
  • CCD charge-coupled device
  • the filter sets used were XF116 (exciter: 474AF20, dichroic: 500DRLP, emitter: 510AF23), XF33/E (exciter: 535DF35, dichroic: 570DRLP; emitter, 605DF50), and XF137 (exciter, 540AF30; dichroic, 570DRLP, emitter: 585ALP) (Omega, Inc, Brattleboro, VT) for GFP, RFP, and auto-fluorescence of chlorophyll, respectively. Data were then processed using Adobe (Mountain View, CA) Photoshop software, and the images were rendered in pseudo-color.
  • the green fluorescence of the reporter protein AtOEP7:GFP was observed at the outer envelop membrane of the chloroplast.
  • the red fluorescence in Figure 2a is the auto-fluorescence of chloroplasts.
  • Figure 2b shows the image obtained by eliminating this auto-fluorescence by using a filter. This result indicates that the chimeric protein comprising the signal protein with the chloroplast envelope targeting signal and the fluorescent protein label was correctly targeted to the chloroplast envelope membrane.
  • AtOEP7 GFP by Western blot analysis.
  • the recombinant plasmid for AtOEP7:GFP was constructed according to the method in Example 1. This recombinant plasmid was used to transform protoplasts according to the method in Example 2, and the transformed protoplasts were incubated for 24 hrs at 22°C.
  • the total protein extract was prepared as follows. Five ml of cell lysate was centrifuged, suspended in 5 ml of the extraction solution (10 mM EDTA, 50 mM HEPES-KOH, 0.33 M sorbitol, 0.5 g/1 BSA, 5 mM sodium ascorbate) at 4°C, and homogenized every three seconds for 20 min.
  • the total protein extract was fractionated by ultra-centrifugation at 100,000xg to separate the soluble and membrane fractions. Both fractions were then electrophoresed on a 7.5% SDS/PAGE gel and transferred onto the PVDF membrane. The blot was probed with a polyclonal anti-GFP antibody.
  • the result shows that the expressed signal protein was transported to the chloroplast envelope membrane and not present in the cytosol.
  • This result indicates that localization of proteins, which is conventionally determined by Western blot analysis, can be identified by the method provided by the present invention.
  • Example 5 Construction of recombinant plasmids for expression of Group II protems that are transported to subcellular organelles by endosomal trafficking.
  • H + - ATPase (Arabidopsis AHA2) was amplified with two specific primers (5'-GAGATGTCGAGTCTCGAA and 5'-
  • the coding sequence of the chaperone binding protein (BiP) (access number D82817) was amplified from an Arabidodsis cDNA library using two specific primers, BIP5 (5'-TACGCAAAAGTTTCCGAT-3') and BIP3 (5'-
  • the sialtransferase (ST) cDNA was amplified from a ⁇ ZAPII cDNA library using two specific primers (5'-ATGATTCATACCAACTTGAAG and 5'- GGATCCACAACGAATGTTCCGGAA). GFP or RFP was ligated in-frame to the carboxyl terminus of ST to construct ST:GFP or ST:RFP.
  • ST:GFP or ST:RFP was constructed from construct a recombinant plasmid for the chimeric protem Chi-n:RFP:Chi-c.
  • DNA fragment including the RFP coding sequence without the termination codon was inserted into the Sma I and Eco RV sites of the chitinase cDNA of pea (access number M13968).
  • plasmid for 500:GFP was constructed by inserting the GFP coding region without the termination codon into the EcoRI site of clone 500 (Kim et al., 2001).
  • plasmid for 526:GFP was constructed by inserting the coding region of GFP into the EcoRI site of clone 526.
  • recombinant plasmids for 491:GFP and 491:RFP were constructed by inserting clone 491 into the 5' end of the coding regions of GFP and RFP without the termination codon, respectively.
  • a recombinant plasmid for 500:GFP:KKXX was generated as follows: The
  • GFP coding region without the termination codon was inserted into the EcoRI site of clone 500 (Jiang and Rogers, 1998) and KKXX was then added to the C-terminus of 500:GFP by PCR amplification using two specific primers (5'- GGATCCTCTAGAGGATCGATCCGG and 5'- TTAGATGAGTTTCTTTTTCTCAAAGAAAGTTTTCAAAAGGAATCCCCCTCC).
  • AtVTIla a homolog of Arabidopsis t-SNARE which is transported from the trans-Golgi network to the storage prevacuole (Zheng et al., 1999), a recombinant plasmid for RFP: AtVTIla was constructed by ligating the coding region of AtVTIla to the C-terminus of the RFP coding region.
  • a recombinant plasmid for AtVTIl :GFP was constructed by ligating the coding region of GFP to the C-terminus of the coding region of AtVTIla.
  • a recombinant plasmid for SPO:GFP was constructed by ligating GFP to the carboxyl terminus of the sporamin B gene.
  • Example 6 Observation of the expression and localization of Group II reporter proteins.
  • Recombinant plasmids for H + -ATPase:GFP, ST:GFP, BiP:GFP, 526:GFP, Chi- n:RFP:Chi-c, and 500:GFP:KKXX were constructed as described in Example 5 and used to transform the protoplasts by the method of Example 2. Expression of the reporter proteins was monitored as a function of time using a fluorescence microscope as explained in Example 3. A part of the results is given in the following.
  • Chi-n:RFP in which the carboxyl region of chitinase was not ligated, was not targeted to the storage vacuole, but it was present as speckles in the endoplasmic reticulum ( Figure 5(g)). Fluorescence of SPO:GFP was distributed uniformly throughout the lytic vacuole ( Figure 5(h)).
  • Example 7 Construction of recombinant plasmids to express Group III proteins that are specific to phospholipids.
  • the C-terminal coding region (amino acid residue 1257 to 1411) of human early endosome antigen 1 (EEA1) was PCR amplified with two primers 5'-GAATTCGTGGCAATCTAGTCAACGG-3' and 5'-CTAATGTTAGTGTAATATTAC-3', and ligated to the C-terminus of the GFP coding sequence without the termination codon.
  • This recombinant DNA was inserted to a pUC vector under the control of the 35S promoter to construct a recombinant plasmid. The same cloning procedure was applied in the examples hereafter.
  • a recombinant plasmid for the chimeric protein of Arabidopsis Pleckstrin homology (PH) domain, GFP:AtPH was constructed by PCR amplification using two primers 5'-CCCGGGAAATGGAGAGTATGTGGCGA-3' and 5'-
  • a recombinant plasmid for the chimeric protein of FAPP including the PH domain, GFP:FAPP was constructed by PCR amplification using two primers 5'-CTCGAGATGGAGGGGGTTCTGTACAAG-3' and 5'- TCACGCTTTGGAGCTCCCAAGGGC-3'.
  • a recombinant plasmid for PH:GFP was constructed by the method of Kost B et al. (1998).
  • Example 8 Observation of the expression and localization of Group III reporter proteins.
  • Recombinant plasmids for GFP:EBD, GFP:AtPH, GFP:FAPP, and GFP:PH were constructed as described in Example 7 and used to transform the protoplasts by the method of Example 2. Expression of the reporter proteins was monitored as a function of time using a fluorescence microscope as explained in Example 3. A part of the results is given in the following.
  • Example 9 The effect of wortmannin on the intracellular trafficking of RbcS:GFP.
  • Plasmid construction, transformation, and expression of the chimeric protem A recombinant plasmid for RbcS:GFP was constructed as described in Example 1. Isolation of the recombinant plasmid, preparation of protoplasts, and transformation of protoplasts were performed as in Example 2. The protoplast suspension was treated with wortmannin at a concentration of 5 ⁇ g/ml. Then the protoplasts were transformed and incubated in the dark. Expression of the chimeric protem was observed as in Example 3.
  • Wortmannin is known as a specific inhibitor of phosphatidyl 3 -phosphate (PI(3)P) and phosphatidylmositol 4-phos ⁇ hate (PI(4)P) (Ui et al., 1995). Localization of the green fluorescence in the wortmannin-treated protoplast was compared with that of the control protoplast that was not treated with wortmannin. As shown in Figure 8, in contrast to the control protoplast in which the green fluorescence of RbcS:GFP was targeted to the chloroplast as expected ( Figure 8(a)), the green fluorescence was not translocated to the chloroplast in the presence of wortmannin, but observed as either speckles or aggregates ( Figure 8(b) and 8(c)). This result indicates that wortmannin inhibits trafficking of the chloroplast-targeting protem from the cytosol to the chloroplast.
  • PI(3)P phosphatidyl 3 -phosphate
  • PI(4)P phosphatidylmositol 4-
  • Example 10 The effect of bafilomycin Al (BafAl), known as an inhibitor of the vacuolar type H -ATPase, on retrograde trafficking of 500:GFP:KKXX.
  • Example 11 The effect of a specific inhibitor on the localization of two reporter proteins.
  • BFA Brefeldin A
  • Arfs ADP-ribosylation factors
  • Example 12 The effect of brefeldin A (BFA) on the biogenesis and structure conservation of the endoplasmic reticulum.
  • a recombinant plasmid for BiP:RFP was constructed as described in Example 5.
  • Example 2 Isolation of the recombinant plasmid, preparation of protoplasts, and transformation of protoplasts were performed as in Example 2.
  • the protoplast suspension was treated with brefeldin A at a concentration of 5 ⁇ g/ml. Then the protoplasts were transformed and incubated in the dark. Expression of the chimeric protein was observed as in Example 3.
  • Examplel3 Inhibition of trafficking of proteins that are specific to phospholipids.
  • Plasmid construction, transformation, and expression of the chimeric protein Plasmid construction, transformation, and expression of the chimeric protein.
  • GFP:EBDC1 58S were constructed as described in Example 7. These recombinant plasmids were used to transform the protoplasts according to the method of Example 2.
  • the transformed protoplasts were treated with wortmannin at a concentration of 1.0 ⁇ g/ml or with 2-(4-morpholinyl)-8-phenyl-4H-l-benzopyran-4-on, a specific inhibitor of phosphatidylmositol 3 -kinase, at a concentration of 10 ⁇ g/ml (LY294002, Vlahos et al., 1994) and incubated at 22°C. Fluorescence images were monitored at various time points.
  • Example 14 Change in the expression level induced by cycloheximide, an inhibitor of the protein expression.
  • Example 3 Plasmid construction, transformation, and expression of the chimeric protein.
  • a recombinant palsmid for RA:GFP was constructed as described in Example 1. Isolation of the recombinant plasmid, preparation of protoplasts, and transformation of protoplasts were performed as in Example 2. The protoplast suspension was treated with cycloheximide a concentration of 5 ⁇ g/ml. Then the protoplasts were transformed and incubated at 22°C in the dark. Expression of the chimeric protein was observed as in Example 3.
  • 13(c) and 13(d) shows the auto-fluorescence of the chloroplast, which are measured to relatively compare the growth and metabolism of the cells.
  • a new Dynamin-like protein, ADL6 is involved in trafficking from the traras-Golgi network to the central vacuole in Arabidopsis. Plant Cell (in press)
  • Brefeldin A inhibited activity of the sec7 domain of p200, a mammalian guanine nucleotide- exchange protein for ADP-ribosylation factors. J. Biol. Chem. 274, 17417- 17423.
  • the plant vesicle-associated SNARE AtVTIla likely mediates vesicle transport from the trans-Golgi network to the prevacuolar compartment. Mol. Biol. Cell 10, 2251- 2264.

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Abstract

L'invention concerne un procédé permettant de détecter les caractéristiques associées au trafic intracellulaire et à la localisation sous-cellulaire d'une protéine reporter sélectionnée. L'invention concerne également un large éventail d'applications importantes notamment l'utilisation de ces caractéristiques dans des cribles pour détecter des composés qui modulent le trafic protéique in vivo.
PCT/IB2002/004257 2001-07-16 2002-07-16 Systeme permettant de surveiller un processus intracellulaire et criblage de medicaments in vivo Ceased WO2003010507A2 (fr)

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JP2022513056A (ja) * 2018-11-15 2022-02-07 バイオアプリケーションズ インコーポレイテッド 植物体でウイルス様粒子を発現する組み換えベクター及びこれを利用したサーコウイルス様粒子を含むワクチン組成物の製造方法
CN114317593A (zh) * 2021-12-31 2022-04-12 北京林业大学 一种超分辨显微技术观察植物原生质体膜蛋白动态的方法

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JP2022513056A (ja) * 2018-11-15 2022-02-07 バイオアプリケーションズ インコーポレイテッド 植物体でウイルス様粒子を発現する組み換えベクター及びこれを利用したサーコウイルス様粒子を含むワクチン組成物の製造方法
JP7212968B2 (ja) 2018-11-15 2023-01-26 バイオアプリケーションズ インコーポレイテッド 植物体でウイルス様粒子を発現する組み換えベクター及びこれを利用したサーコウイルス様粒子を含むワクチン組成物の製造方法
CN114317593A (zh) * 2021-12-31 2022-04-12 北京林业大学 一种超分辨显微技术观察植物原生质体膜蛋白动态的方法

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