JP2006081413A - Method for screening anti-eukaryotic microbial agent - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for efficiently screening a highly effective anti-eukaryotic microbial agent and to obtain the highly effective anti-eukaryotic microbial agent. <P>SOLUTION: A substance binding to the N-terminal region of a sub-unit composing a 14-3-3 protein and inhibiting the dimer formation of the 14-3-3 protein is detected. Thereby, a substance having ultrahigh probability to be the anti-eukaryotic microbial agent is screened. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a screening method for anti-eukaryotic microbial agents and the like.

  The 14-3-3 protein is a dimeric acidic protein with a relative molecular weight of about 30 KD. This protein is thought to be expressed in almost all eukaryotic tissue activities as a molecular chaperone involved in many biological pathways such as cell cycle, signal transduction, intracellular transfer / targeting, transcription, host defense, and cell death. It has been. Over 100 interacting proteins are listed, most of which are via conserved phosphorylated serine / threonine peptide motifs (eg, Non-Patent Documents 1, 2, 3 and 4).

  Most species have one or more 14-3-3 protein isoforms. In mammals, there are at least 7 isoforms, and in plants, there are at least 15 isoforms (for example, Non-Patent Document 5). Thus, 14-3-3 protein dimer combinations may confer diversity to the 14-3-3 protein dimer molecule. The binding partners of some 14-3-3 proteins are not altered in their binding by dimer formation or monomer formation, but the binding strength of the binding target protein is generally different. Is seen. (For example, nonpatent literature 6).

  The 14-3-3 protein has been reported to exist almost in the form of a dimer (Non-patent Document 7), but recently, MAPKAPK2, protein kinase B / Akt, sphingosine-dependent kinase, etc. It has also been reported that phosphorylation of a serine residue by some of the kinases prevents dimerization of 14-3-3 protein in cells (Non-patent Document 8). Furthermore, dimerized 14-3-3 protein binds to c-Raf1 and DAF-16 with phosphorylation at specific sites, but in the case of monomer, c-Raf1 is independent of phosphorylation. It binds to Raf1 and DAF-16 (for example, Non-Patent Document 9). Furthermore, in plants, it is strictly required to bind to a fusicosin receptor for dimer formation (eg, Non-patent Document 10). Thus, it is important to elucidate the molecular mechanism in dimerization of 14-3-3 protein.

  By the way, in countries such as Africa, Trypanosoma brucei, Trypanosoma cruzi, Leishmania major, Toxoplasma gondii, Plasmodium falciparum and Pneumocystis Infectious diseases caused by many eukaryotic microorganisms such as carini (Pneumocystis carinii) are a serious problem. For example, more than 1 billion and 2 billion cattle are at risk of infection or risk of infection with Trypanosoma brucei, resulting in serious damage to African economic and health problems. Yes.

  T. brucei is infected by biting the testse fly and therefore has two distinct life cycles. One is a procyclic (insect) form (PCF), and the other is a blood stream (animal) form (BSF). This pathogen is the eukaryote most distant from homo sapiens in an evolutionary tree based on ribosome sequences. Many biological features have been revealed for T. brucei, including RNA editing, kinetoplast DNA replication, RNA transcription-splicing, and Glycosylphosphatidyl Inositol protein anchors (For example, nonpatent literature 11).

  However, specific drugs for infections caused by these eukaryotic microorganisms have not been developed yet, and rapid development is required.

Tzivion G, Shen YH, Zhu J. 14-3-3 proteins; bringing new definitions to scaffolding,. Oncogene. 20 (2001) 6331-6338. Tzivion G, Avruch J. 14-3-3 proteins: active cofactors in cellular regulation by serine / threonine phosphorylation. J. Biol. Chem. 277 (2002) 3061-3064. Yaffe MB.How do 14-3-3 proteins work-- Gatekeeper phosphorylation and the molecular anvil hypothesis.FEBS Lett. 513 (2002) 53-57. van Hemert MJ, Steensma HY, van Heusden GP. 14-3-3 proteins: key regulators of cell division, signaling and apoptosis.Bioessays. 23 (2001) 936-946. Rosenquist M, Alsterfjord M, Larsson C, Sommarin M. Data mining the Arabidopsis genome reveals fifteen 14-3-3 genes.Expression is demonstrated for two out of five novel genes.Plant.Physiol. 127 (2001) 142-149. Tzivion G, Luo ZJ, Avruch J. Calyculin A-induced vimentin phosphorylation sequesters 14-3-3 and displaces other 14-3-3 partners in vivo. J. Biol. Chem. 275 (2000) 29772-29778. Jones DH, Ley S, Aitken A. Isoforms of 14-3-3 protein can form homo- and heterodimers in vivo and in vitro: implications for function as adapter proteins.FEBS lett. 368 (1995) 55-58. Powell DW, Rane MJ, Joughin BA, Kalmukova R, Hong JH, Tidor B, Dean WL, Pierce WM, Klein JB, Yaffe MB, McLeish KR.Proteomic identification of 14-3-3zeta as a mitogen-activated protein kinase-activated protein kinase 2 substrate: role in dimer formation and ligand binding. Mol. Cell. Biol. 23 (2003) 5376-5387. Shen YH, Godlewski J, Bronisz A, Zhu J, Comb MJ, Avruch J, Tzivion G. Significance of 14-3-3 Self-Dimerization for Phosphorylation-dependent Target Binding. Mol. Biol. Cell. 14 (2003) 4721- 4733. Jaspert N, Oecking C. Regulatory 14-3-3 proteins bind the atypical motif within the C terminus of the plant plasma membrane H (+)-ATPase via their typical amphipathic groove.216 (2002) 136-139. Molecular Biology of Parasitic Protozoa, Edited by Smith DF and Parsons M. IRL PRESS OXFORD UNIVERSITY PRESS (1996) 6-24, 75-86, 88-107, 134-153, 205-222. Protocols in Molecular Parasitology, Edited by Hide JE Methods in Molecular Biology-21 Humana Press, (1993) 1-13. Rittinger K, Budman J, Xu J, Volinia S, Cantley LC, Smerdon SJ, Gamblin SJ, Yaffe MB.Structural analysis of 14-3-3 phosphopeptide complexes identifies a dual role for the nuclear export signal of 14-3-3 in ligand binding. Mol. Cell. 4 (1999) 153-166. Tzivion G, Luo Z, Avruch J. A dimeric 14-3-3 protein is an essential cofactor for Raf kinase activity.Nature. 394 (1998) 88-92. Obsil T, Ghirlando R, Klein DC, Ganguly S, Dyda F. Crystal structure of the 14-3-3zeta: serotonin N-acetyltransferase complex.a role for scaffolding in enzyme regulation.Cell. 105 (2001) 257-267. Chaudhri M, Scarabel M, Aitken A. Mammalian and yeast 14-3-3 isoforms form distinct patterns of dimers in vivo.Biochem. Biophys. Res. Commun. 300 (2003) 679-685. Liu D, Bienkowska J, Petosa C, Collier RJ, Fu H, Liddington R. Crystal structure of the zeta isoform of the 14-3-3 protein.Nature. 376 (1995) 191-194. Xiao B, Smerdon SJ, Jones DH, Dodson GG, Soneji Y, Aitken A, Gamblin SJ. Structure of a 14-3-3 protein and implications for coordination of multiple signaling pathways. Nature. 376 (1995) 188-191. McEwan NR, Eschenlauer SC, Calza RE, Wallace RJ, Newbold CJ. 19 Protozoal sequences may reveal additional isoforms of the 14-3-3 protein family.Protist. 150 (1999) 257-264.

  Under the circumstances as described above, development of anti-eukaryotic microbial agents for suppressing the growth of eukaryotic microorganisms and treating infectious diseases caused by eukaryotic microorganisms is required. An object of the present invention is to provide a method for efficiently screening an anti-eukaryotic microbial agent having high efficacy and an anti-eukaryotic microbial agent having high effectiveness.

  For the T. brucei with unique properties, the analysis of 14-3-3 protein is necessary for further biological information about 14-3-3 protein and for the development of therapeutic drugs Thought to provide useful information. Therefore, the present inventors cloned two isoforms of 14-3-3 protein from T. brucei, prepared antibodies specific for the isoform, and examined their expression in BSF together with PCF. Furthermore, the present inventors conducted various experiments on dimerization, which is the most conserved feature of the 14-3-3 protein, in addition to binding with a phosphorylated binding motif. As a result, we succeeded in finding a new site that is extremely important in dimer formation of 14-3-3 protein, which is usually difficult to predict simply by comparing and comparing amino acid sequence alignments, and completes the present invention. It came to.

The present invention provides the following screening methods for substances that can be anti-eukaryotic microbial agents, and anti-eukaryotic microbial agents.
[1] It can be an anti-eukaryotic microbial agent that detects a substance that binds to the N-terminal region of the subunit constituting 14-3-3 protein and inhibits 14-3-3 protein dimer formation. Substance screening method.
[2] The anti-true according to [1], wherein the N-terminal region is a region from the leucine site to the N-terminus that is conserved in the first α-helix region from the N-terminal side of the subunit. A screening method for substances that can become nuclear microbial agents.
[3] An amino acid sequence wherein the N-terminal region is selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and 14 in the sequence listing The method for screening an anti-eukaryotic microbial agent according to the above [1] or [2].
[4] The eukaryotic microorganisms are Trypanosoma brucei, Trypanosoma cruzi, Leishmania major, Toxoplasma gondii, Plasmodium falciparum, and Pneumocystis. The screening method for a substance that can be an anti-eukaryotic microbial agent according to any one of [1] to [3] above, which is at least one eukaryotic microorganism selected from the group consisting of carini (Pneumocystis carinii).
[5] An anti-eukaryotic microbial agent that binds to the N-terminal region of a subunit constituting 14-3-3 protein and inhibits dimer formation of 14-3-3 protein.
[Supplementary Note 1] A disease that is caused by a eukaryotic microorganism that administers a substance that binds to the N-terminal region of the subunit constituting 14-3-3 protein and inhibits 14-3-3 protein dimer formation. Method of treatment.

In the present specification, unless otherwise specified, the term “14-3-3 protein” refers to a form in which a dimer is formed, and one constituent unit of the dimer is referred to as “subunit”.
In this specification, a eukaryotic microorganism is a microorganism having a eukaryotic cell, and includes, for example, filamentous fungi, yeast, deformed fungi, basidiomycetes, cellular algae and protozoa.
Unless otherwise specified, each SEQ ID NO refers to a SEQ ID NO in the sequence listing.

  The present invention provides a method capable of efficiently screening a substance that can be an anti-eukaryotic microbial agent that suppresses the growth of eukaryotic microorganisms, etc. According to this screening method, a highly effective anti-eukaryotic microbial agent can be obtained. can get.

  Hereinafter, the present invention will be described in more detail while showing embodiments of the present invention. In carrying out the biochemical or genetic engineering technique in the present invention, for example, Molecular Cloning: A LABORATORY MANUAL, 3rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (2001), New genetic engineering handbook (Muramatsu, M. et al., Yodosha, Experimental medicine separate volume, 3rd edition, 1999), How to proceed with protein experiments (Masato Okada, Kaori Miyazaki, Yodosha, 1st edition, 1998) Protein Experiment notes (Masato Okada, Kaori Miyazaki, Yodosha, 2nd edition, 1999), protein experiment handbook (edited by Tadaomi Takenawa, separate volume of experimental medicine, first edition, August 15, 2003), etc. Refer to the description in the experiment manual.

  The method for screening an anti-eukaryotic microorganism drug of the present invention (hereinafter sometimes referred to as “the drug screening method of the present invention”) binds to the N-terminal region of a subunit constituting 14-3-3 protein, and It detects substances that inhibit 14-3-3 protein dimer formation. . A substance that inhibits dimer formation is a highly probable substance that can be used as an anti-eukaryotic microbial agent, but it is desirable that the substance is finally used after undergoing other tests such as clinical tests. The substance obtained by the screening method of the present invention may be subjected to treatments such as isolation and purification as necessary. In addition, the substance obtained by the screening method of the present invention can be further chemically modified to obtain a final anti-eukaryotic microbial agent.

  The dimer in 14-3-3 protein is formed by two subunits. The dimer may be a homodimer or a heterodimer. In the present invention, a substance that binds to at least one N-terminal region of the subunit and inhibits dimer formation is detected. The binding of the inhibitor is not limited to that in which the binding state is maintained, but once bound to the N-terminal region, chemically modified to leave the N-terminal region, and dimer due to the applied chemical modification Substances that inhibit the formation of are also included.

  As a preferred form of the drug screening method of the present invention, a region from the leucine site to the N-terminus conserved in the first α-helix region from the N-terminal side of 14-3-3 protein as the N-terminal region is used. The form used is mentioned. “Conservation” as used herein means that it is conserved across species in terms of molecular biology or genetics. The 14-3-3 protein is known to have multiple α-helices conserved, and the first α-helix region refers to the region of the α-helix that exists most from the N-terminus. . As a characteristic of 14-3-3 protein, leucine residues are conserved with a very high probability in the first α-helix of 14-3-3 protein.

  As an example, FIG. 1 shows T. brucei derived type I 14-3-3 (SEQ ID NO: 15), type II 14-3-3 (SEQ ID NO: 16) and human 14-3-3τ (SEQ ID NO: 17). The amino acid sequence of the subunit is shown. As shown in FIG. 1, these three 14-3-3 subunits contain 1 to 9 α-helices. In addition, a leucine residue is conserved in the first α helix.

  The present inventors have found that the region from the leucine residue to the N-terminus plays an extremely important role in dimer formation (see Examples below). A substance that binds to this N-terminal region is very likely to inhibit dimer formation.

  Specifically, the N-terminal region used in the drug screening method of the present invention consists of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and 14. Peptides having an amino acid sequence selected from the group are exemplified. These sequences are derived from the 14-3-3 N-terminal region of the following eukaryotic microorganisms. By using the peptides of the respective sequences of SEQ ID NOs: 1 to 14, it is very likely that dimer formation of 14-3-3 protein in at least the original microorganism can be inhibited.

The origin of each sequence is as follows (numbers in parentheses are Accession numbers in GenBank / EMBL / DDBJ or Sanger Institute database).
SEQ ID NO: 1; Leishmania major (LmjF36.3210)
MTETIKWKNVAIQDEVVPRPSDIKLPDDLAEL
SEQ ID NO: 2; Plasmodium falciparum (NP_705504)
MNQYIDNDISVSNKEELIYYIKILNHLGSYDGKNIYEY
Sequence number 3; Trypanosoma cruzi (Tc00.1047053508851.18)
MAEGIKWHNAAIQDELVPKKSPLEFKLPDSAKEL
SEQ ID NO: 4; Trypanosoma brucei I (AB59827)
MTDCIKWYNAVLLDEVVPKKNLSEFKLPDATKDL
Sequence number 5; Trypanosoma cruzi (Tc00.1047053)
MAEGIKWHNAAIQDELVPKKSPLEFKLPDSAKEL
SEQ ID NO: 6; Trypanosoma brucei II (AB066565)
MAGFQIPEKREQL
SEQ ID NO: 7; Candida albicans (AF038154.2)
MPASREDS
Sequence number 8; Leishmania major (LmjF11.0350)
MTNVFKVPEKREEL
SEQ ID NO: 9; Plasmodium falciparum (MAL8P1.69)
MATSEELKQLRCDC
SEQ ID NO: 10; Plasmodium knowlesi (AF065986.1)
MATSEDLKQLRSDC
Sequence number 11; Plasmodium yoeii (AABL01000000)
MATPEELKQLRSDC
SEQ ID NO: 12; Pneumocystis carinii (AF461162.1)
MTSRENL
SEQ ID NO: 13; Trypanosoma cruzi (Tc00.1047053506775.80)
MSSFTVPEKREQL
SEQ ID NO: 14; Toxoplasma gondii (BAA25996)
MAEEIKNLRDEY

  In addition, the drug screening method of the present invention includes Trypanosoma brucei, Trypanosoma cruzi, Leishmania major, Toxoplasma gondii, Plasmodium falciparum and Pneumocystis. -It is suitable for the screening of the substance which inhibits 14-3-3 protein dimer formation in the eukaryotic microorganism selected from the group which consists of Carini (Pneumocystis carinii). These eukaryotic microorganisms are pathogens. In addition, when the dimer formation of 14-3-3 protein is inhibited, the original function of 14-3-3 protein in the cell is lost. The 14-3-3 protein is a substance involved in various metabolic pathways and signal transduction in the cell, and the vital activity of the cell is greatly inhibited by the cessation or inhibition of the function of the 14-3-3 protein. Therefore, by the drug screening method of the present invention, a substance that can be a prophylactic and / or therapeutic drug for various infectious diseases caused by the above pathogens can be discovered.

Infections caused by the above pathogens are as follows.
Trypanosoma brucei; Trypanosoma cruzi, African sleeping sickness; Trypanosoma cruzi; Leishmania major; Chaos disease tropical Leishmania major; Toxoplasma gondii; Congenital and acquired toxoplasmosis P. falciparum Plasmodium falciparum; P. falciparum pneumocystis carinii; Carini pneumonia

  There is no particular limitation on the type of the inhibitor as a substance. The inhibitory substance may be an inorganic compound or an organic compound. The inhibitory substance may be an artificially chemically synthesized substance or a biological substance. Examples of inhibitory substances include substances such as peptides, low molecular weight synthetic compounds, RNA, DNA, and lipids. Inhibitor candidates are supplied from, for example, a compound library prepared by a method such as combinatorial chemistry. The detection probability of a novel inhibitory substance can be improved by performing high-throughput detection using a compound library.

  In the drug screening method of the present invention, a substance that binds to the N-terminal region of the subunit constituting 14-3-3 protein and inhibits 14-3-3 protein dimer formation is screened. As the subunit of 14-3-3 protein, one having an N-terminal region is used. Alternatively, only the N-terminal region may be used. A method for detecting a substance that binds to the N-terminal region is not particularly limited, and a known method that can detect an interaction with a specific protein can be employed. Moreover, you may employ | adopt the method of detecting the coupling | bonding with N terminal area | region, and dimer formation inhibition together.

  Methods for detecting protein interaction include, for example, immunoprecipitation, pull-down assay, yeast two-hybrid method, yeast three-hybrid method, PCA (eg, protein fragment complimentation assay using β-galactamase), protein microassay Examples include analysis, BIAcore analysis, gel shift method, and activity gel method. More specifically, the following method is exemplified as a preferred form used in the drug screening method of the present invention.

Screening method by BIAcore ("BIAcore" is a registered trademark);
The amino acid sequence of the N-terminal variable site important for dimer formation is synthesized by a peptide synthesizer. A biotin group is introduced into the C-terminus of the synthesized peptide. After the obtained biotin-introduced peptide (ligand) is immobilized on a BIAcore streptoavidin-sensor chip, a chemical library (ligand library) candidate compound (analyte) is allowed to flow. When the ligand fixed to the sensor chip and the analyte are combined, a change in the refractive index of the reflected light of a predetermined wavelength is observed and output as a sensorgram. Based on the obtained sensorgram, the presence or absence of binding is determined.

Screening method by radioisotope labeling;
Prepare a peptide labeled with radioisotope (RI). In synthesizing peptides, amino acids labeled with isotopes are used. The isotope-labeled peptide is reacted with a filter or well on which a chemical ligand is spotted. After the reaction, after washing with PBS (-) (Phosphate buffered saline (-)), the amount of bound peptide is quantified as the RI count. Those with high RI counts are collected as binding inhibitor candidates.

Screening method with fluorescently labeled peptides;
Peptides are fluorescently labeled and screened in the same way as radioisotopes. The amount of bound peptide is quantified as fluorescence intensity. Those having high fluorescence intensity are candidates for inhibitors. Examples of the substance used for the fluorescent label include FITC (fluorescein isothiocyanate) RITC (rhodoamine isothiocyanate), Alexa Fluor 488, 633, 680 (manufactured by Invitrogen) and the like.

Detection method of dimer formation;
The following method is illustrated as a detection method of a dimer.
Prepare 14-3-3 protein with a different tag at the C-terminus. 14-3-3 protein with tag is co-expressed in expression systems such as 293 mammalian cells, cos cells, E. coli, wheat germ extract, rabbit reticulocyte extract, and co-immunoprecipitation with one tag antibody ( Co-immunoprecipitation), and the other Tag antibody is detected by Western blotting.

Detection of amino acids essential for dimer formation;
Examples of methods for detecting amino acids essential for dimers include the following methods.
A 14-3-3 protein is prepared by modifying the amino acid sequence of the N-terminal region. For example, a 14-3-3 protein with a different tag at the C-terminus is expressed in a 14-3-3 protein such as Plasmodium, Trypanosoma cruzi, Toxoplasma, Pneumocystis carini. What has co-immunoprecipitated is detected by Western blotting. The dimer formation of 14-3-3 protein is examined for each protein having a different modification site in the N-terminal region, and amino acids essential for dimer formation are detected.

  The present invention also provides anti-eukaryotic microorganism inhibitors. The anti-eukaryotic microorganism inhibitor of the present invention is an inhibitor obtained by the above drug screening method. The product obtained by the screening method of the present invention is preferably used after confirming its safety and the like by a pharmacologically appropriate test. As mentioned above, dimer formation of 14-3-3 protein is often extremely important for 14-3-3 protein to exhibit its original function. That is, an inhibitor of dimer formation is a substance that has a very high possibility of significantly inhibiting the life activity of a eukaryotic microorganism having a 14-3-3 protein. In addition, as described above, there are pathogens that cause serious infections in eukaryotic microorganisms. The anti-eukaryotic microorganism inhibitors of the present invention are likely to be effective preventive and / or therapeutic agents for diseases caused by these pathogens.

Specific examples of the inhibitor candidate include peptides described in (A) and / or (B) below.
(A) Peptide having an amino acid sequence constituting the N-terminal region of the subunit constituting the 14-3-3 protein (B) The amino acid sequence constitutes the N-terminal region of the subunit constituting the 14-3-3 protein Peptide in which at least one mutation among substitution, deletion, insertion and addition is introduced into a peptide having an amino acid sequence The N-terminal region preferably contains a leucine residue conserved in the first α helix . More specifically, specific examples of inhibitors include, for example, a peptide whose amino acid sequence is the sequence described in any of SEQ ID NOs: 1 to 14, or any of substitution, deletion, insertion and addition thereto Peptides into which the above mutation is introduced are also exemplified.

  Furthermore, the present invention provides a method of inhibiting the dimer formation of 14-3-3 protein by modifying the amino acid sequence constituting the N-terminal region of the subunit constituting 14-3-3 protein. Our study revealed that the N-terminal region of the subunit of 14-3-3 protein is extremely important in dimer formation of 14-3-3 protein. By altering the amino acid sequence of the N-terminal region, a subunit of 14-3-3 protein that cannot form a dimer is obtained.

  The amino acid sequence can be modified by a known method. For example, the amino acid sequence can be changed site-specifically by changing the base sequence corresponding to the amino acid sequence to be modified. Amino acids can also be modified by introducing mutations with radiation or drugs. More specifically, for example, a commercially available product such as a Quick change site directed Mutagenesis kit (manufactured by Stratagene) can be used.

[Experiment 1] Cloning of 14-3-3 gene from T. brucei
BSF (Blood Stream Form) type 427 is grown in ddY mice, and according to the method described in Protocols in Molecular Parasitology, Edited by Hide JE Methods in Molecular Biology-21 Humana Press, (1993) 1-13 (Non-patent Document 12). Purified. Using 20 ml of TRIzol Reagent (Invitrogen), total RNA was purified from about 1 × 10 ⁹ cells, and mRNA was purified using MACS mRNA isolation kit (Miltenyi Biotec). CDNA was synthesized from 1 μg of total RNA or 3 μg of mRNA using Cap-finder oligonucleotide oligo-dt (manufactured by Clontech). Based on KMKGDY (SEQ ID NO: 18) and VFYYEI (SEQ ID NO: 19), which are conserved amino acid sequences of 14-3-3 between yeast, plants and mammals; primers; 5'-AARATGAARGGNGAYTAY-3 '(SEQ ID NO: 20) and 5′-DATYTCRTARTARAANAC-3 ′ (SEQ ID NO: 21) was designed.

  PCR was performed under the following conditions: 95 ° C, 1 minute; 95 ° C, 15 seconds, 30 cycles (48 ° C, 30 seconds; and 72 ° C, 15 seconds) plus 95 ° C, 15 seconds, 10 cycles ( 45 ° C., 30 seconds; and 72 ° C., 15 seconds), followed by 72 ° C., 7 minutes. The PCR product was subcloned into pGEM-T Easy (Promega), and the sequence of 11 clones was analyzed using an ABI310 sequencer (Applied Biosystems). Using T. brucei mini exon sequence, 5'-CGCTATTATTAGAACAGTTTCTGTACTATATTTG-3 '(SEQ ID NO: 22) and 5'-GGTGCCAGTGAACTGTTCGCCA-3' (SEQ ID NO: 23) obtained from the PCR product, 5'RACE (Rapid amplification of cDNA ends) was performed. In addition, using PCR primer sequence of T. brucei Cap-finder (manufactured by Clontech) and 5'-CACGGAGGTGGCGAACAGTTCA-3 '(SEQ ID NO: 24) obtained from the PCR product, 3'RACE (Rapid amplification of cDNA) ends).

  The 5 ′ and 3 ′ RACE products were cloned and their sequences were examined to confirm that the full-length coding sequence of 14-3-3 protein was obtained as described above. The obtained full-length coding sequence of 14-3-3 protein was used for screening of a genomic library of a procyclic type 427 strain constructed using λ BluesSTAR BamHI kit (Novagen). The λ BluesSTAR BamHI kit (Novagen) is obtained by completely digesting genomic DNA with BamHI and ligating a fragment group of about 9 kb to λ BluesSTAR.

  Under the conditions of low stringency hybridization, 5 weak and 3 strong signals were obtained and the plaques were purified. For hybridization conditions, the wash condition of the protocol of the Alphos Direct Labeling and Detection Kit was changed from 55 ° C. to 50 ° C., and other points were in accordance with the protocol attached to the kit. PBluesSTAR plasmid clones were obtained by excising from these phage clones in vivo. Synthetic pBluesSTAR plasmid DNA was digested with several restriction enzymes including PstI or SalI, various restriction digestion patterns were prepared, and Southern blot hybridization was performed using the coding sequence of 14-3-3 protein .

  A weakly hybridized digested fragment of PstI and a strongly hybridized digested fragment of SalI with the coding sequence of 14-3-3 protein were isolated and subcloned into pGEM 3Zf (+) (Promega). The sequence was identified using EN :: TN <KAN-2> Insertion kit (EPICENTRE TECHNOLOGIES). A T. brucei cDNA library was constructed using λZipLox and NotI-SalI Arms (GIBCO BRL). Clones were obtained by hybridization with the coding sequences of two different 14-3-3 proteins. The coding sequence of 14-3-3 protein was also verified using these clones.

Construction of GST, MAL-14-3-3 and pcDNA3-6myc 14-3-3 expression vectors
14-3-3 cDNA of type I, type II and human 14-3-3τ (hereinafter human 14-3-3τ is also referred to as “τ-type”) were prepared using LA Taq (TAKARA Bio Amplification).
I, sense strand: CGAATTCATGACGGACTGCATCAAGT (SEQ ID NO: 25)
I, antisense strand: GGTCTCCACCCTTGTCAGTT (SEQ ID NO: 26)
II, sense strand: CGGAATTCATGGCGGGCTTTCAAATAC (SEQ ID NO: 27)
II, antisense strand: GAGCTCACTCCAATTCCTCCATC (SEQ ID NO: 28)
τ, sense strand: GAATTCATGGAGAAGACTGAGCTGATCC (SEQ ID NO: 29)
τ, antisense strand: CTTAGTTTTCAGCCCCTTCTGCCGCAT (SEQ ID NO: 30)

  The PCR product was subcloned into pGEM-T Easy vector and sequenced. Each of the EcoRI digested cDNA fragments was subcloned into pMAL-c2X (New England Biolabs), pGEX-1λT (Amersham Biosciences) and pcDNA3-6myc vector (Kunihiro Tsuchida, University of Tokushima). pcDNA3-6myc vector is a vector in which six myc are linked between the BamHI site and EcoRI site of pcDNA3 vector (manufactured by Invitorogen).

Construction of pcDNA3.1 TOPO I, II and τSTOP (+) or (-) A list of PCR primers for constructing the construct is shown below.
I, sense strand: CACCATGGACGGACTGCATCAAGTGGTACAA (SEQ ID NO: 31)
I, antisense STOP (-): GTTCACGGGTTGCTCGTCAGTCCAGA (SEQ ID NO: 32)
I, antisense STOP (+): GTCAGTTCACGGGTTGCTCGTCAGTCCAGA (SEQ ID NO: 33)
II, sense strand: CACCATGGCGGGCTTTCAAATACCTGA (SEQ ID NO: 34)
II, antisense STOP (-): CTCCAATTCCTCCATCGCAGTTC (SEQ ID NO: 35)
II, antisense STOP (+): CTCACTCCAATTCCTCCATCGCAGTTC (SEQ ID NO: 36)
τ, sense strand: CACCATGGAGAAGACTGAGCTGATCC (SEQ ID NO: 37)
τ, antisense strand STOP (−): GTTTTCAGCCCCTTCTGCCGCAT (SEQ ID NO: 38)
τ, antisense strand STOP (+): TTAGTTTTCAGCCCCTTCTGCCGCAT (SEQ ID NO: 39)

  TOPO cloning reaction was performed according to the protocol attached to pcDNA3.1 Directional TOPO Expression Kit (Invitrogen).

pcDNA3.1 TOPO-MS.Tag vector construction Six oligonucleotides for construction are listed below.
SEQ ID NO: 40: ATCGCCTCTATGGAGCAAAAGCTCATTTCTGAAGAGGACTTGAATGA
SEQ ID NO: 41: ATTTCATTCAAGTCCTCTTCAGAAATGAGCTTTTGCTCCATAGAGGCGAT
SEQ ID NO: 42: AATGGAGCAAAAGCTCATTTCTGAAGAGGACTTGAATGAAGCCTCTGC
SEQ ID NO: 43: TTTGGCAGAGGCTTCATTCAAGTCCTCTTCAGAAATGAGCTTTTGCTCC
Sequence number 44: CAAAGAAACCGCCGCCGCTAAATTTGAACGCCAGCACATCGACAGCTAGC
Sequence number 45: TCGAGCTAGCTGTCGATGTGCTGGCGTTCAAATTTAGCGGCGGCGGTTTC

  Two complementary oligonucleotides were each annealed and ligated. The DNA fragment obtained by ligation was isolated from a 4% low-dissolution agarose gel and subcloned into the EcoRV and XhoI sites of pcDNA3.1 TOPO I, II and τ STOP (−). When the vector obtained is transfected into mammalian cells, it expresses a protein containing the C-terminal tag sequence: IASMEQKLISEEDLNEMEQKLISEEDLNEASAKETAAAKFERQHIDS (2xMyc-Tag & S-Tag, SEQ ID NO: 46).

pcDNA3.1 (+) SM-Tag Vector construction
In order to introduce a tag with S-Tag (Novagen) Myc-Tag attached to the N-terminus of pcDNA3.1 (+) (Invitrogen), four oligonucleotides,
Sequence number 47: GAAGCTTGCCACCATGGGTGTTACCAAAGAAACCGCTGCTGCTAAATTCG
SEQ ID NO: 48: AAAGAAACCGCTGCTGCTAAATTCGAACGCCAGCAC
SEQ ID NO: 49: TTTTGCTCCATGCTGCCCCCGGTTCCGCTGTCGATGTGCTGGCGTTCGA
Sequence number 50: CGAATTCCCCTTTCAAGTCCTCTTCAGAAATGAGCTTTTGCTCCATGCTG
Designed. Ex-Tag was used for annealing and extension reaction of these four oligonucleotides. The product was subcloned into pGEM-T Easy. The obtained plasmid was cleaved with HindIII and EcoRI and subcloned into the corresponding site of pcDNA3.1 (+). The resulting vector, when transfected into mammalian cells, expresses a protein with MGVTKETAAAKFERQHIDSGTGGSMEQKLISEEDLKGEF (S-Tag & Myc-Tag, SEQ ID NO: 51) as the amino terminal tag sequence. pGEM-T Easy 14-3-3, type I, type II and τ type EcoRI site inserts were inserted into the EcoRI site of pcDNA3.1 (+) SM-Tag.

14-3-3 Construction of chimeric expression
Using pcDNA3 6myc-I, pcDNA3 6myc-II and pcDNA3 6myc-τ, a 14-3-3 chimeric expression construct was prepared as follows. Sense and antisense primers for the β-lactamase gene in pcDNA3 6myc plasmid to replace 14-3-3 type I and / or 14-3-3 type II domains with 14-3-3 τ type domains Was used. Two primers of sequence 5′-CCCTTCCGGCTGGCTGGTT-3 ′ (SEQ ID NO: 52) and 5′-CCGAGCGCAGAAGTGGTCCT-3 ′ (SEQ ID NO: 53) can be used to amplify the full length plasmid by PCR and ligation reaction.

  For example, using different combinations of β-lactamase gene sense primer and 14-3-3 cDNA antisense primer, and β-lactamase gene antisense primer and 14-3-3 cDNA sense primer, -3 The vector sequence added with cDNA was amplified. After digestion with DpnI, two fragments derived from cDNAs of different isoforms separated by TAE / agarose gel electrophoresis were ligated. DH5α competent cells were used for transformation. The sequence of each chimera was carefully verified. A list of primers used for the 14-3-3 chimeric expression construct is shown below.

Recombinant protein expression and analysis
The recombinant protein was expressed in DH5α and / or BL21 and purified according to the protocol attached by the manufacturer.

  The proteins were separated by 10-20% or 4-20% gradient SDS-PAGE (Daiichi Kagaku, Japan) and transferred to a PVDF membrane (Millipore) under electrical load. The membrane was covered with either 3% non-fat milk or Block Ace (Dainippon Pharmaceutical). Primary antibody for Western blotting, anti-Myc antibody (9E11) (Oncogene), anti-V5 monoclonal antibody (Invitrogen), horseradish peroxidase (HRP) labeled S protein (Novagen) were purchased. . Using only complete Freund's adjuvant as an accelerator, 250 μg each of purified GST-I and purified GST-II proteins were immunized to Wistar rats to trypanosoma 14-3-3 type I and type II A primary antibody against was obtained. Secondary antibody with minimal cross-reaction to other animal proteins, Peroxidase-AffiniPure F (ab ') 2 Fragment Goat anti-Rat Fc (gamma) (Code 112-036-071), Peroxidase-conjugated AffiniPure F (ab' ) 2 Fragment Donkey AntiRabbt IgG (H + L) (Code 711-036-152) was all purchased from Jackson ImmunoResearch. The purchased primary antibody ((Anti-Myc, V5,14-3-3: K19 (Santa Cruz), 14-3-3τ: C-17) was used according to the protocol attached by the manufacturer. Type II rat serum was diluted 10,000 times with TTBS (50 mM Tris pH 7.5, 150 mM, NaCl, 0.02% Tween 20) containing 20% Block Ace, and all secondary antibodies were diluted 10,000 times with TTBS. The blot was visualized on X-ray film using Super Signal West Pico Chemiluminescent Substrate (PIERCE).

For transfection, 293 and 293T cells were grown using Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% FCS and 20 μg / ml gentamicin (GIBCO BRL), and FuGENE 6 transfection 16 hours before standard transfection using reagents (Roche), 5 × 10 ⁵ cells were seeded in 6-well culture dishes.

Two days after transfection, the cells were washed three times with ice-cold PBS (-) (manufactured by Sigma) and 100 μg of NP-40 lysis buffer (0.5%, NP-40, 150 mM, NaCl, 10 mM, Tris-). HCl, pH 7.5, 5 mM EDTA, 50 mM NaF, 1 mM, NaVO ₄ , protease inhibitor cocktail (manufactured by Roche)). Lysates were washed twice with low speed and high speed centrifugation. Of those obtained after washing, 10 μl was saved for Western blot analysis (Input) and 400 μl of NP-40 lysis buffer was added to each sample. For each sample, 1 μg of anti-Myc antibody 5 μl of rec-protain G beads (manufactured by ZYMED) or S-Tag pull-down using 20 μl of S-protain beads (manufactured by Novagen) The assay was performed. The precipitated sample was washed three times, transferred to an empty Auto-Seq G50 (Amersham Bioscience), rotated at 5000 rpm, and the NP-40 lysis buffer was discharged. After discharging the lysis buffer, 20 μl of 1 × SDS sample buffer containing 2-mercaptoethanol was added, allowed to stand for 5 minutes, and then rotated at 5000 rpm for 1 minute. The sample was boiled and subjected to SDS-PAGE, followed by Western blotting as described above.

Construction of T. brucei T7 polymerase-based expression vector Aldolase splicing acceptor sequence was isolated from pT11-bs using SmaI and HindIII and inserted into pSP73 (Promega). Was named pKI-0. In addition, 2xTetO cassettes were prepared by annealing oligonucleotides having the base sequences shown as SEQ ID NOs: 72 and 73.
5'-TCGAGTCCCTATCAGTGATAGAGATCTCCCTATCAGTGATAGAGC-3 '(SEQ ID NO: 72)
5'-GCTCTATCACTGATAGGGAGATCTCTATCACTGATAGGGAC-3 '(SEQ ID NO: 73)

The annealed oligonucleotide was inserted into pKI-0, and the annealed T7 terminator oligonucleotide (SEQ ID NOs: 74 and 75) was inserted into the BamHI and EcoRV sites.
5'-GATCCCTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGTAT-3 '(SEQ ID NO: 74)
5'-ATACAAAAAACCCCTCAAGACCCGTTTAGAGGCCCCAAGGGGTTATGCTAGG-3 '(SEQ ID NO: 75)

The resulting plasmid is T7 promoter and promoter: 5'-ATACAAAAAACCCCTCAAGAC-3 '
Amplified by PCR using (SEQ ID NO: 76). The PCR product was inserted into the BssHII blunt cleavage site of pBluescript II KS (+) and named pKI-2. pKI-2 was amplified by PCR using M13 forward and reverse primers, and the PCR product was cleaved with BamHI. The fragment containing T7 promoter and 2xTetO obtained by cleavage was inserted into the KpnI blunt cleavage site and BamHI site of pLew100. The 3 ′ untranslated region and 2 × T7 terminator were isolated from pLew111 by digestion with StuI followed by blunting and digestion with NheI. This fragment was inserted into the bluntly cut BamHI and NheI sites incorporating the T7 promoter, 2xTetO and aldolase SAS of pLew100. The resulting plasmid construct was named pKI-6. pKI-7 was prepared by exchanging the XhoI-HindIII fragment of pKI-0 and used in the following experiments.

N-terminal 6myc-I, II and τ expression using PKI-7 29-13 Establishment and analysis of PCF cell clones
pKI-7 was digested with BamHI and blunted with T4 DNA polymerase, and further digested with HindIII. Fragments digested with HindIII and EcoRV were isolated from pcDNA3 6myc-I, II and τ. PKI-7 6myc-I, II and τ were transfected into 29-13 PCF cells by electroporation. 29-13 PCF cells are cells that express TetR and T7 polymerase. One day after transfection, phleomycin (Phleomaycine) selection was performed in MULTIWELL 96 wells (Bacton Dickenson, FALCON). Within 2 weeks, 5-10 clones were isolated for each plate. Protein expression was confirmed by Western blot using anti-Myc, I, II and τ antibodies, and high expression clones were used in the following experiments.

Using 1 × 10 ⁸ cells excluding the Myc-peptide eluate, immunoprecipitation and Western blot were performed in the same manner as described above. The eluate was subjected to 4-20% SDS-PAGE and further silver stained. Western blots were performed using these eluates and antibodies against type I, type II and type τ. As a result, it was confirmed that heterodimization of type I and type II and homodimization of type I and type I also occur in trypanosoma cells.

Results We have already cloned two isoforms of 14-3-3 protein from Trypanosoma brucei strain 427 cDNA and genomic library. They are named 14-3-3 type I and type II and are registered with GenBank / EMBL / DDBJ as accession numbers AB059827 and AB066565. Genomic library screening under low stringency conditions and direct sequence analysis of RT-PCR products using several primers derived from the 14-3-3 sequence help T. brucei Only two isoforms were identified. The sequence became available for viewing at Sanger Institute Blast Server as accession No. TRYPtp 25a10ec07.plk_63. As shown in the server, both sequences were found on chromosome 11.

  However, these isoforms have not been confirmed for biochemical and biological properties to date. The start codon is determined by homologues of other 14-3-3 proteins identified from mammals to yeast, and no sequence that can be a start codon is found upstream of the start codon specified by the present inventors. It was. The deduced amino acid sequence is predicted to form an amphipathic groove for phosphoserine binding; in type I, 77K, 84R, 88R, 150K, 154D, 157R, 158Y, 199L, 203V, 243L, 247L , And 255W, type II, 56K, 63R, 67R, 128K, 132D, 135R, 136Y, 174L, 178V, 221L, 225L, and 233W are all stored (see FIG. 1).

The nuclear exporting signal (Nuclear exporting signal (NES), Non-Patent Document 13) exists in α-helix 9 (see FIG. 1). These data indicate the characteristics of the 14-3-3 protein that were conserved during evolution, i.e.
1) Usually present in the form of a dimer,
2) Depending on the sequence, it binds to a motif containing phosphoserine,
3) including nuclear translocation signals,
It matches.

  As such, the 14-3-3 protein can act as a receptor molecule that enhances protein-protein interactions, presumably through binding to sequence motifs containing diverse partners, phosphoserine, to target protein cells Control localization, enzyme activation or inhibition.

  In order to confirm the expression of 14-3-3 protein in procyclic foam (PCF) and blood stream foam (BSF), rats were immunized with GST-I protein and GST-II protein (FIG. 2a), respectively. The specificity of the antibody was examined by Western blotting using purified maltose binding fusion protein (MAL) of type I, type II and human 14-3-3τ. The result is shown in FIG. Surprisingly, in the absence of booster immunization, GST-I protein or GST-II, despite having a sequence with as high as 45% identity (see FIG. 1) There was no cross-reaction between the two antibodies immunized with the protein.

Therefore, the specificity of these antibodies was examined by Western blot using a PCF cell lysate. Both antibodies revealed that the relative molecular weight of 14-3-3 type I and type II was 30 KD with some background (FIG. 2c). Further, Northern blotting analysis of PCF cells detected a 2.2 kb transcript for type I and a 3.8 kb transcript for type II (FIG. 2d). The amount of type II transcript corresponded to almost three times that of type I transcript (FIG. 2d). In addition, quantitative Western Blot analysis showed that PCF cells had more type I transcript than type II (type I: 107 ng / 1x10 ⁶ cells, type II). : 70ng / 1x10 ⁶ cells). In contrast, in BSF cells, the amounts of transcripts of type I and type II are almost the same (type I: 92 ng / 1 × 10 ⁶ cells, type II: 84 ng / 1 × 10 ⁶ cells). Theoretically, the expression level of 14-3-3 protein in one cell is 1 × 10 ⁶ −2 × 10 ⁶ molecules (FIG. 2e). When combined with Northern and Western blotting results, it was shown that the expression level of 14-3-3 protein is post-transcriptionally regulated, as are many trypanosoma proteins. Immunostaining of PCF cells using anti-type I and type II antibodies confirmed that both isoforms were mainly localized in the cytoplasm, indicating that the predicted nuclear translocation signal This suggests that (NES) functions in vivo.

  The importance of 14-3-3 protein dimerization has only been shown in a few cases. For example, the dimer of 14-3-3 protein in vivo is required to maintain Raf kinase activity (Non-patent Document 14). Furthermore, 14-3-3 protein and serotonin / N- From the crystal structure of the co-crystal with acetyltransferase, the binding of dimeric 14-3-3 protein to the two sites of the enzyme is necessary to induce conformational changes that enhance catalytic activity ( Non-patent document 15) has been reported. This report focuses on the exact mechanism of dimerization, an important feature of the 14-3-3 protein. Therefore, as described in the materials and methods above, the present inventors used an easy-to-use expression vector pcDNA3.1-SM.Tag or pcDNA3.1TOPO-MS.Tag for the detection of dimers in 293 cells. Created. These vectors contain Myc-Tag and S-Tag that bind to anti-Myc antibody and S-protein, respectively.

  The pull-down assay using S-protein beads is less susceptible to steric hindrance than antibodies because the S-protein is as small as 104 amino acids. In addition, since both Myc-Tag and S-Tag can adopt α-helix conformation, there is little structural change in the binding partner. Therefore, the construct pcDNA3.1 TOPO I, II or τ and pcDNA3.1TOPO-MS.Tag I, II or τ were co-transfected and S-protein was used for all possible dimers. A pull-down assay was performed. The result is shown in FIG. As shown in FIG. 3, the I-MS.Tag protein binds to both type I and type II. In addition, II-MS.Tag protein binds only to type I. However, type τ did not bind to either type I or type II.

  In the experiment conducted separately from the above experiment, even when pcDNA3.1-SM.Tag I, II, or τ, and pcDNA3.1 TOPO I, II, or τSTOP (+) were used, the same binding pattern was obtained. confirmed. On the other hand, when the pcDNA3-6mycII construct was used, dimerization was not confirmed between 6Myc-II and type I itself. That is, from these, it was presumed that steric hindrance may have occurred in the 14-3-3 protein due to an antibody that binds to six Nc-terminal myc sequences.

  Then, it examined using MS. And SM.Tag vector. Furthermore, dimerization tests were carried out using pcDNA3.1 TOPO I, II and τ (−) that form the C5 terminal V5 antibody recognition sequence of the protein, and consistent results were obtained. Recent reports also indicate that mammalian and yeast 14-3-3 isoforms also exhibit characteristic dimer patterns (Non-patent Document 16).

  We next tested the amino acid sequence required for dimerization. Since the present inventors have clarified that T. brucei 14-3-3 protein (type I, type II) and τ type do not form a dimer, further, T. brucei 14-3-3 protein and human Chimeric constructs with the derived τ form were made and tested to see if they formed dimers. As shown in FIG. 4, all kinds of constructs were prepared. However, in the test for identifying the sequence required for dimerization, No. Only 3, 4, 5, 9, 10 and 11 constructs were used. The reason for this is that, in the crystal structure analysis of mammalian 14-3-3 isoforms, it has been clarified that the N-terminal amino acid sequence including the 1st to 4th helix part is responsible for dimerization. (Non-patent Documents 17 and 18).

  In detection with anti-I and anti-II antibodies, no bands were observed in the lanes showing S-protein pull-down, SM-Tag.Iα3 + II, and SM-Tag.IIα3 + I (FIG. 5). This data shows that the N-terminal sequence containing helix 1-4 is essential for heterodimer formation in both isoforms of T. brucei 14-3-3 protein.

  Furthermore, we investigated whether other mammalian 14-3-3 isoforms can form dimers with T. brucei 14-3-3 protein. An S-protein pull-down assay was performed using lysates of 293T cells transfected with pcDNA3.1TOPO-MS.Tag I and II, respectively. Detection by Western blotting was performed using the K19 antibody that recognizes all 14-3-3 isoforms. The results are shown in FIG. The results revealed that MS-Tag.I binds to certain mammalian isoforms with low affinity, but MS-Tag.II does not. This suggests the unique property that in both type I and type II, perhaps the N-terminal region plays an important role in the formation of these dimers. This data suggests that the details of the dimerization system may be different from the mechanism in mammalian isoforms because type II does not form dimers with any of the isoforms. is doing. Comparison of the N-terminal amino acid sequence of the T. brucei 14-3-3 protein with that of mammals revealed that one proline residue immediately before α-helix 1 is characteristic.

  From the data that the mammalian 14-3-3 protein hardly binds to the T. brucei 14-3-3 protein, the sequence from the start codon to the proline residue in each isoform It was speculated that it was a factor determining the palatability.

  Therefore, the present inventors deleted the type I and type II N-terminal sequences so that the proline site present in the N-terminal region is the start position of the start codon, and further substitutes this proline for methionine. Thus, a cDNA having a modified base sequence was prepared. The cDNA was transfected into 293T cells, and it was examined by pull-down assay whether dimers were formed. The results are shown in FIG. In FIG. 7, for example, the column indicated by “P → M II-V5 + I-MS” is a type II in which the amino acid residue at the position of proline (P) which is newly N-terminal is substituted with methionine (M). The result of having experimented about the combination of the composite_body | complex which combined V5 antibody, and the composite_body | complex of type I and MS.Tag is shown. Surprisingly, type I 14-3-3 with removal of the N-terminus shown as I-V5 (P → M), type II 14-3 with removal of the N-terminus shown as II-V5 (P → M) None of -3 formed dimers (Figure 7). This data suggested that a sequence composed of 7 amino acids of the N-terminal sequence in type II; 28 amino acids in type I is extremely important in dimer formation of type II.

  Furthermore, the amino acid sequence essential for dimer formation in type II was identified by alanine scanning. As shown in FIG. 8, as a result of determining amino acids essential for dimer formation in type II using an alanine scanning method, among 13 amino acids from leucine (L) to the N-terminal, phenylalanine (F), isoleucine It became clear that when any one of (I), proline (P) and leucine (L) was changed to alanine (A), the dimer formation ability was lost.

  According to the alanine scanning method, it is possible to substitute a predetermined amino acid residue so as not to change the three-dimensional structure of the polypeptide. Usually, if the three-dimensional structure does not change, it is no wonder that the subunits are bound to each other. However, the above results indicate that when a specific amino acid residue in the N-terminal region is substituted, binding between subunits may be inhibited even if the subunits can assume the original three-dimensional structure. It shows that. That is, dimerization can be inhibited if any influence is exerted on the amino acid residue at a specific site in the N-terminal region regardless of whether or not a change in conformation is caused. Therefore, even when some substance is bound to the N-terminal region, the original function of the amino acid residue at the specific site as described above does not work, and the probability that dimerization is inhibited is extremely high.

  Since τ type derived from human does not have a site corresponding to the region from N-terminal to proline (P) in type I and type II, phenylalanine (F), isoleucine (I) from N-terminal to proline (P) A substance that binds proline (P) to the target and inhibits dimerization is considered to be more preferable as an anti-eukaryotic drug.

  Peptides and / or small ligands that bind to this short sequence of type II inhibit dimerization of T. brucei 14-3-3 protein, while inhibiting mammalian 14-3-3 protein It is estimated not to. This leads to a novel idea that the 14-3-3 protein is a unique drug discovery target for T. brucei. We are currently searching for ligands that bind to this N-terminal characteristic sequence and inhibit dimerization. Furthermore, since 14-3-3 proteins are expressed in all eukaryotes, and their N-terminal sequences are diverse in non-higher eukaryotes (Non-patent Document 19), 14-3 -3 protein dimerization can be performed by Trypanosoma brucei, Trypanosoma cruzi, Leishmania major, Toxoplasma gondii, Plasmodium falciparum and Pneumocystis. It will be a potential therapeutic target for all eukaryotic pathogens including carini (Pneumocystis carinii).

  The present invention contributes to the development of preventive drugs and therapeutic drugs for eukaryotic pathogens and the like.

It is a contrast diagram of amino acid sequences of subunits of type I 14-3-3, type II 14-3-3 and human 14-3-3τ derived from T. brucei. It is a figure which shows the electrophoresis image of a MAL complex and a GST complex. It is a figure which shows the result of the western blotting which shows the specificity of an antibody. It is a figure which shows the result of the western blotting which looked at the reactivity in the trypanosoma lysate of the serum immunized with normal serum and type I and type II. It is a figure which shows the result of the Northern blot which investigated the amount of transcripts of type I and type II. It is a figure which shows the expression level of 14-3-3 protein of type I and type II in one cell. It is a figure which shows the result of the pull-down assay using S-protein. It is a figure which shows the design drawing of the chimera construct of each of I type, II type, and (tau) type. It is a figure which shows the result of the pull-down assay using S-protein. It is a figure which shows the result of the pull-down assay using S-protein. It is a figure which shows the result of the pull-down assay using S-protein about 14-3-3 protein of the type I and type II which deleted a part of N terminal region. FIG. 3 is a diagram showing the results of alanine scanning performed to identify amino acids necessary for dimer formation in type II.

SEQ ID NO: 20: PCR primer SEQ ID NO: 21: PCR primer SEQ ID NO: 23: PCR primer SEQ ID NO: 24: PCR primer SEQ ID NO: 25: PCR primer SEQ ID NO: 26: PCR primer SEQ ID NO: 27: PCR primer No. 28: PCR primer SEQ ID NO: 29: PCR primer SEQ ID NO: 30: PCR primer SEQ ID NO: 31: PCR primer SEQ ID NO: 32: PCR primer SEQ ID NO: 33: PCR primer SEQ ID NO: 34: PCR primer SEQ ID NO: 35: PCR primer SEQ ID NO: 36: PCR primer SEQ ID NO: 37: PCR primer SEQ ID NO: 38: PCR primer SEQ ID NO: 39: PCR primer SEQ ID NO: 40: pcDNA3.1 TOPO-MS. -Oligonucleotide for construction of Tag vector SEQ ID NO: 41: pcDNA3.1 TOPO-MS. -Oligonucleotide for construction of Tag vector SEQ ID NO: 42: pcDNA3.1 TOPO-MS. -Oligonucleotide for construction of Tag vector SEQ ID NO: 43: pcDNA3.1 TOPO-MS. -Oligonucleotide for construction of Tag vector SEQ ID NO: 44: pcDNA3.1 TOPO-MS. -Oligonucleotide for construction of Tag vector SEQ ID NO: 45: pcDNA3.1 TOPO-MS. -Oligonucleotide for construction of Tag vector SEQ ID NO: 46: 2xMyc-Tag & S-Tag
SEQ ID NO: 47: pcDNA3.1 (+) SM. -Oligonucleotide for construction of Tag vector SEQ ID NO: 48: pcDNA3.1 (+) SM. -Oligonucleotide for construction of Tag vector SEQ ID NO: 49: pcDNA3.1 (+) SM. -Oligonucleotide for construction of Tag vector SEQ ID NO: 50: pcDNA3.1 (+) SM. -Oligonucleotide for construction of Tag vector SEQ ID NO: 51: S-Tag & Myc-Tag
SEQ ID NO: 53: PCR primer SEQ ID NO: 54: Primer used for 14-3-3 chimeric expression construct SEQ ID NO: 55: Primer used for 14-3-3 chimeric expression construct SEQ ID NO: 56: 14-3-3 chimeric expression construct Primer used for SEQ ID NO: 57: Primer used for 14-3-3 chimeric expression construct SEQ ID NO: 58: Primer used for 14-3-3 chimeric expression construct SEQ ID NO: 59: Used for 14-3-3 chimeric expression construct Primer used SEQ ID NO: 60: Primer used for 14-3-3 chimeric expression construct SEQ ID NO: 61: Primer used for 14-3-3 chimeric expression construct SEQ ID NO: 62: Primer used for 14-3-3 chimeric expression construct SEQ ID NO: 63: Primer used for 14-3-3 chimeric expression construct 4: Primers used for 14-3-3 chimeric expression constructs SEQ ID NO: 65: Primers used for 14-3-3 chimeric expression constructs SEQ ID NO: 66: Primers used for 14-3-3 chimeric expression constructs SEQ ID NO: 67: Primers used for 14-3-3 chimeric expression constructs SEQ ID NO: 68: Primers used for 14-3-3 chimeric expression constructs SEQ ID NO: 69: Primers used for 14-3-3 chimeric expression constructs SEQ ID NO: 70: 14 3-3 Primer used for chimeric expression construct SEQ ID NO: 71: Primer used for 14-3-3 chimeric expression construct SEQ ID NO: 72: oligonucleotide for pKI-0 construction SEQ ID NO: 73: oligonucleotide for pKI-0 construction SEQ ID NO: 74: T7 terminator SEQ ID NO: 75: T7 terminator SEQ ID NO: 76 : PCR primer

Claims (5)

  Screening for a substance that can be an anti-eukaryotic microbial drug that detects a substance that binds to the N-terminal region of the subunit that constitutes the 14-3-3 protein and inhibits the dimer formation of the 14-3-3 protein Method.   2. The anti-eukaryotic microbial agent according to claim 1, wherein the N-terminal region is a region from the leucine site to the N-terminus conserved in the first α-helix region from the N-terminal side of the subunit. A screening method for substances to be obtained.   The N-terminal region has an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and 14 in the sequence listing; The method for screening an anti-eukaryotic microbial agent according to claim 1 or 2.   The eukaryotic microorganisms are Trypanosoma brucei, Trypanosoma cruzi, Leishmania major, Toxoplasma gondii, Plasmodium falciparum and Pneumocystis cystis The method for screening a substance that can be an anti-eukaryotic microbial agent according to any one of claims 1 to 3, which is at least one eukaryotic microorganism selected from the group consisting of carinii).   An anti-eukaryotic microbial agent that binds to the N-terminal region of a subunit constituting 14-3-3 protein and inhibits dimer formation of 14-3-3 protein.