JP2009065909A - Method for screening material for inhibiting nuclear exportation of protein - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new technical means for simply and reliably identifying a new material for inhibiting nuclear exportation. <P>SOLUTION: The method for screening the material for inhibiting the nuclear exportation of the protein in a eukaryotic cell includes (1) expressing a fusant of a reporter protein and a nuclear export signal (NES) peptide in the eukaryotic cell, (2) introducing a candidate material into the eukaryotic cell, and (3) identifying the candidate material as a target material when the signal of the reporter protein positions in the nuclear of the eukaryotic cell. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a method for screening a protein nuclear export inhibitor and a eukaryotic cell used in this method. The present invention also relates to a novel screening method for nuclear export signal (NES) peptides.

  Eukaryotic cells roughly have a nucleus and a cytoplasm, and the nucleus and the cytoplasm are separated by a nuclear membrane. In the nucleus, gene transcription is performed from cellular DNA, and the transcribed RNA is transported out of the nucleus through the nuclear pore (Nuclear Pore Complex, NPC) and translated into protein on the ribosome. The translated protein has various functions in the cell, and is transported on the cell membrane, into the cytoplasm, and into the nucleus in accordance with each function.

  There are two main methods for transporting proteins into the nucleus. Passive transport and active transport. Passive transport is a phenomenon in which relatively low molecular weight proteins pass through NPCs by diffusion, and active transport is a transport form mainly seen in relatively high molecular weight proteins, and is involved in transport from the cytoplasm to the nucleus in cells. It is an energy-dependent process performed by various proteins. For this active nuclear translocation, a sequence called NLS (Nuclear Localization Signal) is important, and nuclear translocation is performed by binding to a protein called importin of the host cell.

  There are two main methods for transporting proteins out of the nucleus (passive transport and active transport). The former is a phenomenon that passes by diffusion as in the case of nuclear transfer. In the latter, a protein having the sequence of nuclear export NES (Nuclear Export Signal) directly binds to the host cell nuclear export factor Exportin / CRM1, and is actively exported. Compared to nuclear translocation, little is known about the factors and pathways involved in nuclear translocation. The only pathway recognized so far for active nuclear export of proteins is the CRM1-dependent pathway. Leptomycin B is the only known drug that specifically inhibits CRM1-dependent nuclear export, and few others have been reported.

Leptomycin B (LMB) was discovered from actinomycetes by searching for antifungal antibiotics using fungal morphological abnormalities as an index (Non-patent Document 1). Fission yeast causes growth arrest and morphological elongation at a low concentration of 20 ng / mL (Non-patent Document 2). It was also known to induce G1 arrest in the cell cycle in animal cells and to exhibit antitumor activity (Non-Patent Documents 3 and 4).
On the other hand, it has been discovered that LMB has an activity of inhibiting the export of proteins to the nucleus, in which LMB inhibits the binding of NES sequence and CRM1, completely independent of such effects on the cell cycle (Non-patent Document 5). . This has led to the use of LMB as a nuclear export inhibitor, but it effectively inhibits nuclear export at the level of cultured cells (in vitro), but LMB has a highly hydrophobic structure. The problem is that the effect is greatly diminished in vivo. To date, CRM1-dependent nuclear export inhibitors have been mainly used only at the laboratory level. In addition, LMB has no nuclear export inhibitors that have been clinically applied due to lipid solubility problems.
Hamamoto, T. et al. J. Antibiotics 36, 639- (1983) Hamamoto T. et al. J Antibiotics 36, 646- (1983) Komiyama K et al. J. Antibiotics 38, 427- (1985) (Yoshida M et al. Exp. Cell Res. 187, 150- (1990)) Kudo N et al., Proc Natl Acad Sci US A. 96, 9112- (1999)

  Protein nuclear export plays an important role in maintaining homeostasis, proliferation of cancer cells, and growth of several pathogenic viruses that infect living organisms (cells), and the development of specific inhibitors is clinically However, drugs with the action of inhibiting nuclear export have never been applied as pharmaceuticals. Therefore, if a useful inhibitor candidate is found, it is considered possible to develop various drugs with this point as an action point. In particular, influenza virus NS2 protein, human immunodeficiency virus (HIV-1) Rev protein, etc. are known as proteins with NES, and these proteins play an important role in virus growth. Development of a novel antiviral agent whose action is transport inhibition is also expected.

  Note that a novel inhibitor for a protein having an NES sequence can be searched for by observing the localization of the protein. In other words, proteins with NES sequences are actively transported from the nucleus to the cytoplasm. Therefore, when cultured in the presence of LMB, migration to the nucleus is inhibited, and accumulation in the nucleus (localization) Change) is observed. Therefore, when a candidate substance is added to a medium, if protein nuclear export is inhibited as in the case of LMB addition, the candidate substance can be identified as a nuclear export inhibitor. As a method of knowing the localization and behavior of proteins in cells, an antibody is reacted with cells, a fluorescently labeled secondary antibody is bound, and the localization of the proteins is observed indirectly by observing the fluorescence localization. The indirect fluorescent antibody method is widely used. This method requires labor and time, and there are problems such as differences in the observed localization due to cell fixation methods and cell membrane permeabilization with surfactants, and difficulty in processing a large number of specimens. .

  Accordingly, an object of the present invention is to provide a new technical means for easily and reliably identifying a novel nuclear export inhibitor.

  To date, only CRM1 has been found as the major nuclear exporter that binds to protein NES. However, if a new NES sequence is found in addition to the known CRM1-binding NES sequence, a new nuclear export factor that binds to the new NES sequence can be identified, and a specific inhibitor for that factor can be developed. Expected.

The present invention is a method for screening a substance that inhibits the nuclear export of a protein in a eukaryotic cell as a means for solving the above problems,
(1) A fusion of a reporter protein and a nuclear export signal (NES) peptide is expressed in a eukaryotic cell,
(2) introducing a candidate substance into a eukaryotic cell;
(3) When a reporter protein signal is located in the nucleus of a eukaryotic cell, the candidate substance is identified as the target substance.
A screening method for a protein nuclear export inhibitor is provided.

  In this screening method, the NES peptide is a mutant NES peptide in which one or more amino acid residues are deleted, replaced with another amino acid residue, or one or more amino acid residues are added. It is one.

  Furthermore, the present invention provides a eukaryotic cell that expresses a fusion of a reporter protein and an NES peptide in the cell. This eukaryotic cell has a preferred embodiment in which DNA encoding the fusion is incorporated into chromosomal DNA. Furthermore, in this eukaryotic cell, the NES peptide is a mutant NES peptide in which one or more amino acid residues are deleted, replaced with another amino acid residue, or one or more amino acid residues are added. One embodiment is adopted. In addition, as a specific example of such a eukaryotic cell, a canine kidney-derived Madin-Darby Canine Kidney (MDCK) cell in which the fusion is incorporated into chromosomal DNA is provided.

  According to the screening method of the present invention, the nuclear transport inhibitory activity of a candidate substance is evaluated using the intracellular localization of a fusion of a reporter protein and an NES peptide (hereinafter sometimes referred to as Rep-NES) as an index. . The localization of Rep-NES can be observed by visually observing the reporter protein signal. For example, when green fluorescent protein (GFP) is used as the reporter protein, the localization of GFP-NES in the cell is performed by applying appropriate excitation light (according to excitation light normally used for GFP). Since it emits bright green fluorescence, visual observation is easy.

  In addition, the cells can be observed without being fixed, and a change in localization (effect of the inhibitor) can be determined in a short time (15 to 30 minutes). Furthermore, since a large number of cells can be targeted identically, a high-throughput assay is possible.

  MDCK cells incorporating Rep-NES into chromosomal DNA are stable expression cell lines in which Rep-NES is stably maintained even after cell division, and thus can be easily maintained by passage. Moreover, MDCK cells can be maintained in Eagle's medium (MEM) that is used for normal eukaryotic cell culture, and have good adherence among adherent cells and are easy to handle. Cell doubling time grows well at around 18-24 hours.

  Furthermore, by using mutant NES, it is possible to cope with screening of substances having various action points.

  The reporter protein can be appropriately selected from, for example, known green fluorescent protein (GFP), blue fluorescent protein (CFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), and mutants thereof. . In addition, a photoprotein (for example, firefly luciferase, click beetle luciferase, Renilla luciferase, or a mutant thereof) can also be used. These reporter proteins have known amino acid sequences and cDNAs encoding the same (for example, the GenBank database).

As the nuclear export signal (NES) peptide, for example, an NES sequence portion (LALKLAGLDI) such as human PKIα protein shown in the Examples can be used. NES sequence portions such as influenza virus NS2 protein and human immunodeficiency virus (HIV-1) Rev protein can also be used. Furthermore, 298 proteins are registered in the GenBank database as “Nuclear Export Signal”. The basic structure of the amino acid sequence of the NES peptide, which is the center of NES activity, consists of 8-9 amino acids, specifically LX _(1-2) LX ₂ LXL. However, L is leucine and X is any amino acid. In some cases, methionine M, valine V, and isoleucine I may be substituted for L. The number of any amino acid X may be different. Furthermore, in the case of what has already been reported as a NES sequence, it may refer to the entire sequence several amino acids longer than this. In the present invention, an NES peptide can be selected from these known NES proteins and used. A mutant NES peptide can be obtained by a method such as creating a mutant peptide by a mutagenesis PCR method using a DNA encoding a known NES peptide as a target.

The reporter protein and NES peptide fusion (Rep-NES), for example, links the DNA encoding the reporter protein and the DNA encoding the NES peptide, and expresses this fusion DNA in the nucleus of a eukaryotic cell. That is, the above DNA is ligated and inserted into a eukaryotic cell expression vector, and this vector is introduced into the eukaryotic cell by a known method. Examples of expression vectors that can be used include vectors such as pKA1, pCDM8, pSVK3, pSVL, pBK-CMV, pBK-RSV, EBV vector, pRS, and pYE82. In order to introduce the expression vector into eukaryotic cells, known methods such as electroporation, calcium phosphate method, cationic liposome method, DEAE dextran method and the like can be used.
In the above example, when an appropriate antibiotic resistance gene for selection of a stable expression cell line in eukaryotic cells is not encoded in the expression vector, the neomycin resistant pCMV-neo, together with the vector in which the DNA is ligated and inserted, A vector such as hygromycin resistant pCMV-Hyg is introduced (co-transfected) together.

  As eukaryotic cells, cells of animals, plants, fungi and protists can be used, but mammalian cells are preferable, and canine kidney-derived Madin-Darby Canine Kidney (MDCK) shown in the Examples is particularly preferable. First, because it is an adherent cell, it is easy to observe its morphology with a microscope. Second, since the cell itself is the larger of mammalian cells, the nucleus and cytoplasm can be observed with a conventional optical microscope. It can be clearly discriminated, it can be grown on MEM medium and serum widely used for culturing mammalian cells, and fourth, it is adherent and proliferative among mammalian cells. It is relatively strong and easy to handle during screening.

  Candidate substances in the screening method of the present invention are, for example, organic or inorganic compounds (particularly low molecular weight compounds), proteins, peptides and the like. These substances may be known or unknown in function or structure, may be pure purified preparations, or may be a mixture having an unknown concentration or content. For example, in the case of a compound derived from a natural product, a bacterial culture supernatant (bacterial metabolite), an actinomycete culture supernatant (actinolytic metabolite), a plant extract, etc. Conceivable. Further, the “combinatorial chemical library” is an effective means as a test substance group for efficiently specifying a target substance. The preparation and screening of combinatorial chemical libraries is well known in the art (see, eg, US Pat. Nos. 6,004,617; 5,985,365). Furthermore, commercially available libraries (for example, libraries made by ComGenex, USA, Asinex, USA, Tripos, Inc., Russia ChemStar, Ltd, 3D Pharmaceuticals, Martek Biosciences, etc.) Can also be used. In addition, so-called “high-throughput screening” can be performed by applying various types of candidate substances to a population of Rep-NES expressing cells.

  The genetic engineering and molecular biological techniques in the above methods are described in literature (eg Sambrook and Maniatis, in Molecular Cloning-A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1989; Ausubel, FM et al. The current protocol in Molecular Biology, John Wiley & Sons, New York, NY, 1995).

  EXAMPLES Hereinafter, although an Example is shown and this invention is demonstrated further more concretely, this invention is not limited by the following examples.

A plasmid was prepared by fusing the NES sequence of PKI to the Clontech GFP expression plasmid. Specifically, the human PKIα (GenBank / BC022265) NES sequence (LALKLAGLDI: SEQ ID No. 1) is added to pEGFP-C1, 5'-aatttagccttgaaattagcaggtcttgatatcg-3 '(SEQ ID No. 2) and 5'-gatccgatatcaagacctgctaatttcaaggcta- The 3 ′ (SEQ ID No. 3) primer was prepared by in vitro phosphorylation and annealing and ligation to the BamHI site of pEGFP-C1. This plasmid may hereinafter be called pEGFP-NES. Canine kidney-derived Madin-Darby Canine Kidney (MDCK) cells were seeded in 24-well cell plates and cultured overnight at 37 ° C. 2 μg of plasmid pEGFP-NES, 6 μl of transfection reagent Lipofectamine 2000 (Invtrogen) and 100 μl of Opti-MEM (Invitrogen) were added, and the solution was added to MDCK cells (transfection). After 4 hours, the medium was changed and replaced with a culture medium of MEM 5% serum. After 24 hours in a 37 ° C CO ₂ incubator, the cells are detached from the dish by trypsinization, re-spread to 90 mm dish, and stable in MEM 5% serum medium containing 800 ug / mL G418 (Nacalai Tesque). An expression cell line was selected, colonies were isolated, and cells with high fluorescence intensity in the cytoplasm were selected by fluorescence microscopy. Since pEGFP-C1 encodes a G418 resistance gene, cells in which the plasmid is stably integrated into the chromosome can survive, but the other cells die.

  The established MDCK-GFP-NES cells (GFPNES # 5 cell line) were maintained by adding 5% FBS to the MEM medium.

  The nuclear export inhibitor sensitivity test was performed as follows. 24 hours after seeding the GFPNES # 5 cell line in a culture dish, leptomycin B (LMB), a known nuclear export inhibitor, was added at different concentrations, and the change in localization was observed over time using a fluorescence microscope. Observation was performed and images were taken (FIG. 1).

  As shown in Fig. 1, in the absence of LMB (LMB (-)), GFP-NES is transported into the cytoplasm by the action of the NES sequence, so that the cytoplasm is relatively strongly fluorescent. Is observed. In contrast, the addition of LMB at 300 pg / mL, 1, 3, and 10 ng / mL at different concentrations inhibits the function of the NES sequence, thereby inhibiting the transfer of GFP-NES from the nucleus to the cytoplasm. And accumulation in the nucleus is observed from 1 ng / mL. Added at the concentration (10 ng / mL) used in previous reports (Watanabe K et al., Virus Res 77, 31- (2001), Sato H et al., Virology 352, 121- (2006), etc.) After 25 minutes, localization (accumulation) in the nucleus was observed. This accumulation in the nucleus can be observed stably for more than 24 hours.

  Screening for nuclear export inhibitors can be performed as follows. Inoculate a 96-well plate with the MDCK-GFP-NES cell line, and observe whether the localization of GFP fluorescence changes by adding a candidate substance (eg, bacterial culture supernatant previously filtered and sterilized by filtration) to this medium. Can be done. To screen a large number of specimens, for example, when screening metabolite from 10,000 kinds of bacteria, 1000 kinds of mixed liquids of 10 kinds of bacterial culture supernatants are prepared in advance. First, add 1000 kinds of liquid mixture to the cell culture medium, select the liquid mixture candidate that causes the change in localization of GFP fluorescence (primary screening), and then select the liquid mixture that became the candidate one cell at a time. Add to the culture and select bacteria with metabolites that cause local changes in GFP fluorescence (secondary screening).

It is the microscope image which observed the green fluorescence signal at the time of processing MDCK-GFP-NES cell line with LMB of various concentration.

Claims (6)

A method for screening a substance that inhibits nuclear export of a protein in a eukaryotic cell,
(1) A fusion of a reporter protein and a nuclear export signal (NES) peptide is expressed in a eukaryotic cell,
(3) When a reporter protein signal is located in the nucleus of a eukaryotic cell, the candidate substance is identified as the target substance.
A screening method for a protein nuclear export inhibitor. The screening method according to claim 1, wherein the NES peptide is a mutant NES peptide in which one or more amino acid residues are deleted, substituted with another amino acid residue, or one or more amino acid residues are added. A eukaryotic cell that expresses a fusion of a reporter protein and an NES peptide in the cell. The eukaryotic cell according to claim 3, wherein the DNA encoding the fusion is incorporated into chromosomal DNA. The eukaryotic cell according to claim 3 or 4, wherein the NES peptide is a mutant NES peptide in which one or more amino acid residues are deleted, substituted with another amino acid residue, or one or more amino acid residues are added. The eukaryotic cell according to claim 3, 4 or 5, which is a canine kidney-derived Madin-Darby Canine Kidney (MDCK) cell.