KR20170060418A - Protein crystal of CEP55-TEX14 complex and its use - Google Patents

Abstract

This disclosure discloses three-dimensional crystal structures of CEP55-EABR and TEX complexes and their uses. The complex crystals according to the present invention provide detailed information on the precise three-dimensional structure of the CEP55-TEX14 binding site and use it to identify peptide derivatives or low molecular compounds capable of binding to the CEP55 protein to inhibit cell division, Can be used to develop anticancer drugs in the form of suppression.

Description

CEP55-TEX14 protein crystal and its use {Protein crystal of CEP55-TEX14 complex and its use}

This is a field related to protein complex crystals involved in cell division.

The three-dimensional structure of anticancer target proteins can be very useful in developing new anticancer drugs. In order for somatic cells to divide into two daughter cells, the cell membrane must be cleaved at the end of cytoplasmic cleavage, known as ALIX (ALG-2 interacting protein X, also called programmed cell death 6 interacting protein) for CEP55 (Centrosomal Protein 55) ) Bonding is essential.

However, in germ cells, TEX14 (Testis Expressed Gene 14) binds to CEP55 instead of ALIX, which leads to a 0.5 to 3-μm intercellular bridge without complete cell division at the end of cell division.

United States Patent Application Publication No. 2013-0225503 discloses an anticancer composition using a TEX14 peptide and a method for inhibiting cell proliferation.

However, if a TEX14 peptide derivative or a low molecular weight compound, which is more tightly bound to CEP55 than TEX14, is discovered, it is possible to develop a new anticancer agent that inhibits the cytoplasmic division of cancer cells.

Therefore, the development of these new types of anticancer agents requires a detailed analysis of the three-dimensional structure of CEP55-TEX14 conjugates.

The present disclosure provides a three-dimensional structure of a complex of CEP55-TEX14 and its use.

In one embodiment, the present disclosure provides a three-dimensional crystal structure of a CEP55-EABR and TEX14 peptide complex.

In one embodiment, the crystal of the complex has a space group of P22 ₁ 2 ₁ and a unit cell dimension of a = 53.9 A, b = 102.6 A, c = 132.5 A, and the CEP (Centrosomal Protein 55 ) Protein is represented by the amino acid sequence of residues 160 to 217 of SEQ ID NO: 1, and TEX14 (Testis-Expressed Gene 14) is represented by the amino acid sequence of residues 792 to 804 of SEQ ID NO:

According to one embodiment, the crystal according to the present invention shows X-ray diffraction data of a structure coordinate determination defined by the data described in Table 3 at a resolution of 2.3 Å.

According to one embodiment, the amino acid residues that interact with CEP55 of TEX in the complex according to the present invention are residues Ala793, Gly795, Pro796, Pro797, Tyr801, Pro803 and Pro804 or Gly795, Pro796, Pro797, Tyr801, Pro803 and Pro804, The residues interacting with the TEX of CEP55 are Trp184, Tyr187, Lys180, Glu183 and Arg191.

According to one embodiment, the Gly795, Pro796, and Pro797 residues of TEX are contacted with the Trp184, Tyr187, Lys180, and Gln183 residues of the CEP55, and the Pro796, Tyr801, and Pro804 residues of the TEX are contacted with the Tyr187 residue of the CEP55.

In another aspect, the present invention is also directed to compositions comprising protein complex crystals and salts according to the present invention.

In another aspect, the present invention provides a method for preparing a protein complex crystal according to the present invention.

In one embodiment, the method comprises the steps of: a) providing a purified CEP55 protein and a TEX 14 peptide, wherein the CEP55 protein is in a 20 mM Tris-HCL (pH 7.4) solution, and the TEX14 peptide is 150 mM Tris- 0) solution; b) mixing the purified proteins and peptides in a molar ratio of 1: 2; And c) crystallizing the mixed proteins and peptides in 0.1 M imidazole (pH 8.0), 1 M ammonium phosphate solution.

In another aspect, the present invention also provides a method of designing or screening a substance that inhibits or enhances binding between complexes using the complex crystals according to the present invention.

In another aspect, the invention also provides a method of screening for a TEX14 derivative that is an anticancer agent candidate, which binds to the CEP55-EABR region.

In one embodiment, the method comprises providing a CEP55-EABR region represented by the amino acid residues 160 to 217 of SEQ ID NO: 1, or a CEP55 fragment comprising the CEP55 full region or region comprising the region and a TEX14 peptide represented by AxGPPx ₃ YxPP a step of, in the AxGPPx ₃ YxPP a is Ala, G is Gly, P is Pro, Y is Tyr, X is any amino acid, the CEP55-EABR area, CEP55 full-length or a fragment and the TEX14 peptide comprising the same Contacting the test substance with a test substance, wherein the test substance is a TEX14 derivative capable of competitively binding to the CEP55-EABR region with the TEX14 peptide; Measuring binding of said TEX14 peptide to said CEP55-EABR in the presence of said test substance; And selecting the test substance as an anticancer candidate substance when the binding of the TEX14 peptide to the CEP55-EABR is decreased in the test group that is contacted with the test substance as compared with the control group not in contact with the test substance as a result of the measurement.

In one embodiment, the TEX peptide is represented by an amino acid sequence of residues 793 to 804 of SEQ ID NO: 2, an amino acid sequence of residues 792 to 804 of SEQ ID NO: 2, or residues 791 to 804 of SEQ ID NO:

In one embodiment, the CEP55-EABR region is represented by an amino acid sequence represented by 160 to 217 residues of SEQ ID NO: 1 from human, and the full length of CEP55 is represented by an amino acid sequence of SEQ ID NO: 1.

In one embodiment TEX14 derivative is a polypeptide capable of binding to the EABR area, AxGPPx ₃ YxPP in the polypeptide of the amino acid sequence is any one of Ala, Gly, Pro, Pro, Tyr, Pro and Pro, the TEX14 derivative Wherein the modification is a chemical modification of a substitution or amino acid side chain of an amino acid residue, wherein the substitution comprises non-conservative substitution or conservative substitution.

The complex crystals according to the present invention provide detailed information on the precise three-dimensional structure of the CEP55-TEX14 binding site and use it to identify peptide derivatives or low molecular compounds capable of binding to the CEP55 protein to inhibit cell division, Can be used to develop anticancer drugs in the form of suppression.

Figure 1 depicts a CEP55-EABR crystal structure and competition assay coupled to a TEX14 peptide according to one embodiment of the invention. (A) Represents the domain structure of CEP55 and TEX14. ANK represents ankyrin repeats. CC represents a coiled-coil structure. (B) the CEP55-binding sequence of TEX14, ALIX, TSGlOl, and the corresponding chimeric peptide. The area marked in yellow means a strictly conserved residue. The bold and gray areas highlight important features. (C) Surface plasmon resonance (SPR) analysis of CEP55-EABR with TEX14 or ALIX peptides. RU means the response unit. (D) an amount of _{792 -807} TEX14 CEP55 EABR-pool by the presence of a competitor peptide to GST-ALIX _{795 -811} Instruction-down (pull-down) shows an analysis. (E) Competitive binding of ALIX to TEX14-bound CEP55-EABR is shown by SPR analysis. (F) Superposition of TEX14 (orange) and ALIX (green) peptides bound to CEP55-EABR. (G) TEX14 _{792 -804} 2F _o -F _c around peptide I draw the map (1.0 σ).
Figure 2 illustrates the interaction with CEP55-EABR and TEX14 (or ALIX) according to one embodiment of the present disclosure. (A) Close-up of the selection area of the CEP55-EABR surface with the TEX14 peptide. (B) SPR analysis of wild type and mutant structures affecting CEP55-EABR bound to TEX14 fragment. (C) NMR 1H-15N TROSY spectra obtained from ¹⁵ N-labeled CEP55-EAB in complex with TEX14 or ALIX peptide in free (black) and molar ratios of 1: 0.25 (red) and 1: S4 A and B).
Figure 3 depicts the crystal structure of CEP55-EABR bound to a chimeric peptide, according to one embodiment of the disclosure. (A) GST pull-down analysis of the interaction between CEP55-EABR and chimeric peptides. The separated proteins on each gel were visualized by Coomassie blue staining. Each structure is exhibited over a suitable gel image, the arrow a CEP55 ₁₆₀ coupled to GST resin containing GST-TEX14 ^N -ALIX ^C or GST-ALIX ^N -TEX14 ^C _- shows a _217. M represents a protein marker. (B) SPR sensorgram of CEP55-EABR chimera (GST-TEX14 ^N -ALIX ^C and GST-ALIX ^N- TEX14 ^C ) interaction. All sensorgrams are GST-TEX14 ^N -ALIX ^C or GST-ALIX ^N- TEX14 ^C _Lt; RTI ID ₌ 0.0 > CEP55 _{< /} RTI &gt; _160-217 protein. (C) It overlaps TEX14 ^N- ALIX ^C (cyan) and ALIX ^N -TEX14 ^C (yellow). (D and E), respectively TEX14 ^N -ALIX peptide ^C (1.0 σ) and ALIX ^N -TEX14 ^C around the peptide _{(1.0 σ) 2F o - F} c I draw the map.
Figure 4 shows the shape of the point mutation of TEX14 for midbody localization and polynucleation. (A) RT-PCR analysis of expression of ALIX, TSG101, MKLP1, CEP55, and TEX14 in both somatic and mouse tests. Quantitative RT-PCR analysis was additionally performed to determine the relative expression levels of ALIX, TSGlOl, MKLPl, CEP55, and TEX14 in mouse tests (Fig. S4C). DAZL was used as a marker for male reproductive cells and GAPDH was used as an internal control. (B) centralized localization of wild-type TEX14 and mutants. GFP-TEX14 WT and its mutants were transiently transfected into HeLa cells. All mediators captured in HeLa cells were stained with GFP (green), alpha -tubulin (red), and DAPI (blue). (Inset) dashed-line box (scale bar, 10 μm; 1 μm (inset).]. (C) In the experiment shown in B, GFP intensity was quantified in the center sieve. A total of 150 entities captured in the cells were counted in three repeated experiments. Relative GRP intensities were normalized to GFP mean intensities in TEX14 WT transfected cells. The error bar indicates the SEM. ND indicates not determined. (D) Percentage of polynucleated cells containing 2n, 4n, and 8n DNA in wild type and mutant TEX14-flag stable cell lines (n = 3; number of cells per condition> 200). The error bar indicates the SEM. (E) Representative microscopic images of TEX12 WT-flagstabilized cells in the absence of doxycycline (-dox) and presence of 1 μg / mL doxycycline (+ dox). Both centrally captured stable cells were stained with GFP (green), alpha -tubulin (red), and DAPI (blue). (Inset) dashed-line box (scale bar, 10 μm; 1 μm (inset).].
Figure 5 shows the ALIX substituted centrally with TEX14. (A) Representative microscopic images of endogenous ALIX substitutions in wild-type TEX14 and in the center of mutant stable cells. Stable cells were stained with flag (green), ALIX (red), α-tubulin (cyan), and DAPI (blue). The dashed line represents the rim of the medulla fetus cell. (Inset) dashed-line box (scale bar, 10 μm; 1 μm (inset).]. (B) In the experiment described in A, the ALIX intensity was quantified in the center sieve. 150 or more centrioles of captured cells were counted in each stable cell line and the experiment was repeated three times. Relative ALIX intensity was normalized to mean intensity in empty HeLa FRT / TO cells. The error bar indicates the SEM. *** P <0.001.
6 is a SPR analysis of the CEP55-TEX14 and CEP55-ALIX interactions and the overall model according to one embodiment of the present disclosure. (A) SPR analysis of dissociation rates of CEP55-EABR-TEX14 and CEP55-EABR-ALIX interactions. , It is focused by measuring the dissociation curve of the combination at the fixed GST-TEX14 _{792 -807} or _{GST-ALIX 795 -811 CEP55-EABR} ( the indicated amount) to calculate the dissociation rate. (B) Models for the construction (or ganization) of the CEP55-TEX14 and CEP55-ALIX complexes in the central body. FIG. 1 is a schematic diagram of a multi-layered system for delivering intercellular bridges of germ cells disclosed in the present invention. The cell schematic diagrams at each central stage are shown and the domain architecture of CEP55, TEX14, ALIX, and MKLP1 is shown.

The structural features, interacting residues, and interaction mechanism of the complex were identified through the determination of the tertiary structure of the CEP55-TEX14 complex crystals. The germ cells have a function to inactivate the cell cleavage by binding to TEX14 (testis-expressed gene 14). Here we demonstrate that TEX14 is a component of the ESCRT device in the CEP55 (centrosomal protein 55 kDa) -EABR [ESCRT and ALIX-binding region] through the AxGPPx ₃ YxPP motif and competes with the ALIX containing the AxGPPx3Y motif To prevent recruiting of ALIX, thereby revealing that TEX14 protects intercellular bridges of germ cells by predominantly binding to CEP55-EABR in the presence of ALIX.

Accordingly, the present invention relates to a CEP55-TEX14 protein complex crystal.

CEP55 according to the present invention is a protein involved in attachment of carinat core-microtubule in intercellular bridge formation and somatic cell division in the process of germ cell production. It is indicated by NP_001120654 (SEQ ID NO: 1). In particular, the present invention encompasses the CEP55-EABR region. In one embodiment, the CEP55-EABR region is represented by amino acid residues 160 to 217 of the sequence of SEQ ID NO: 1. The protein according to the present invention is a wild-type protein, or a mutant, or a protein crystal having a tertiary structure which has a substitution in the above sequence but has the same tertiary structure as the wild-type protein.

TEX14 according to the present invention is a protein necessary for the formation of intercellular bridges of germ cells in the process of sperm production. Can be represented by the amino acid sequence of AAH40526 (SEQ ID NO: 2). The complex crystals according to the present invention include polypeptides having an amino acid sequence of 792 to 804 residues of the above sequence.

The crystals herein may take a variety of forms and may be provided, for example, in the form of a composition, wherein in one embodiment the complex crystals are provided in the form of a composition comprising a salt.

In one embodiment, the crystals herein have a tertiary structure as set forth in Table 3. In the crystal of the present invention, the complex crystal has a space group of P22 ₁ 2 ₁ , and a unit cell dimension is a = 53.9 Å, b = 102.6 Å, and c = 132.5 Å.

In another embodiment, the determination of the present disclosure can be described by the coordinates of Table 3, where the data in Table 3 provides a data set of relative positions that determine the shape in three dimensions. An entirely different set of positional data with different origins and axes can also determine similar three-dimensional shapes. Similarly, the overall tertiary structure defined by the data set of Table 3 will not change significantly, even if the data set is adjusted to increase or decrease the distance between various atoms or to add or remove solvent molecules. Thus, the reference to Table 3 includes a Coordinate set of the same generic CEP55-TEX14 complex or a data set to which a slight modification has been made. A structure similar to that defined by the data set of Table 3, although not identical, may be a computer program such as the Molecular Similarity application of QUANTA (Molecular Simulations Inc, USA).

Furthermore, the tertiary structure defined by the data set of Table 3 includes those in which addition, substitution or deletion occurs in one or more of the CEP55 or TEX14 protein sequences, without modifying the tertiary structure of the general CEP55-TEX14 complex. Methods for altering the primary structure (sequence) of a protein without altering its tertiary structure and / or activity are known in the art. Those skilled in the art will also be able to readily identify sites that do not alter the folding / structure, activity, etc. of the protein by viewing the provided tertiary structure, and such variations do not lead to a significant change in tertiary structure or activity, These are also included in the tertiary structure defined by Table 3.

In another embodiment, the present invention is a CEP55-TEX14 protein complex showing X-ray diffraction data of structure coordinates for structure determination in Table 3 at a resolution of 2.3 A.

In the complex according to the present invention, CEP55-EABR and TEX14 polypeptides interact via hydrophobic binding.

In one embodiment, the residue interacting with the CEP55 in the TEX is selected from the group consisting of Ala793, Gly795, Pro796, Pro797, Tyr801, Pro803 and Pro804 residues; Or a CEP55-TEX14 protein complex crystal wherein the residues interacting with the TEX in CEP55 are Trp184, Tyr187, Lys180, Glu183 and Arg191, wherein Gly795, Pro796, Pro797, Tyr801, Pro803 and Pro804.

In another embodiment, the Gly795, Pro796, and Pro797 residues of TEX are contacted with the Trp184, Tyr187, Lys180, and Gln183 residues of the CEP55, and the Pro796, Tyr801, and Pro804 residues of the TEX are contacted with the Tyr187 residue of the CEP55.

The crystals herein may take a variety of forms, for example in the form of a composition, and in one embodiment the complex crystals according to the invention are provided in the form of a composition comprising a salt.

In another aspect, the present invention is directed to a method of making a protein complex crystal according to the present invention, including: In one embodiment, a method according to the present invention comprises the steps of: a) providing a purified CEP55-EABR region, a purified CEP55 protein or a fragment comprising said region and a TEX14 peptide, wherein the CEP55-EABR region, the purified CEP55 protein, The TEX14 peptide is contained in a solution of 20 mM Tris-HCl (pH 7.4), the TEX14 peptide is contained in a solution of 150 mM Tris-HCl (pH 8.0), and b) the CEP55-EABR region, And a peptide at a molar ratio of 1: 2; And c) crystallizing the mixed proteins and peptides in 0.1 M imidazole (pH 8.0), 1 M ammonium phosphate dibasic solution.

In a CEP55-EABR region, a purified CEP55 protein or a fragment comprising such a region as used in the method according to the present invention, the CEP55-EABR region may be represented by a human 160 to 217 amino acid sequence of SEQ ID NO: 1, The total length may be represented by the amino acid sequence of SEQ ID NO: 1, and the fragment includes the CEP55-EABR region and is apparent to those skilled in the art to mean shorter than the total length.

The TEX14 peptide used in the method according to the present invention may be represented by any one of the amino acid residues 791 to 804, 792 to 804, or 793 to 804 residues of SEQ ID NO: 2.

The polypeptide or protein expression used in the method according to the present invention can be carried out using conventional methods known in the art. For example, a nucleotide sequence encoding a protein may be inserted into a commonly used vector such as pQE30, pGEM-T, pSC101, ColE1, pBR322, pUC8 / 9, pHC79, pUC19 Or pET, and then transformed into a suitable host cell, gram-negative bacterium or the like, and the transformed cells are cultured to express a desired protein in a large amount.

The expressed protein can be purified according to various purification methods known in the art. For example, the protein may be purified by applying a crude extract obtained by disrupting the cultured transformed cells, but not limited thereto, to various chromatographies (prepared for separation by size, charge, hydrophobicity, or affinity) have.

The method is particularly effective for crystallizing CEP55-EABR and TEX14 peptides. In the method according to the invention, the purified protein is treated under conditions which allow the complex to stabilize for crystallization. Solutions for obtaining such crystals are carried out under alkaline conditions, especially weakly alkaline conditions, and various buffers known in the art can be used. In one embodiment of the invention, tris is used. The solution also includes imidazole and ammonium phosphate. In one embodiment, the solution comprises 0.1 mM imidazole and 1 mM ammonium phosphate. The process according to the present invention may be carried out at about 4 ° C to about 30 ° C, and in one embodiment at 22 ° C.

Throughout the various biochemical experiments described herein in the tertiary structure and in the Examples herein, the residues that bind to CEP55 in the TEX peptide are residues Ala793, Gly795, Pro796, Pro797, Tyr801, Pro803 and Pro804 and the residue of CEP55 that binds to the TEX Were Trp184, Tyr187, Lys180, Glu183 and Arg191. Specifically, the Gly795, Pro796, and Pro797 residues of TEX are contacted with Trp184, Tyr187, Lys180, and Gln183 residues of CEP55, and the Pro796, Tyr801, and Pro804 residues of the TEX interact with the Tyr187 residue of the CEP55.

All or part of the crystals of the crystallized complex according to the present invention and the crystallization data and information can be used for various purposes. Prior to this, this information did not exist.

The three-dimensional structure of anticancer target proteins can be very useful in developing new anticancer drugs. Therefore, by using the information of the three-dimensional structure of the complex of the anticancer target protein CEP55-EABR and TEX14 peptide identified herein, it is possible to identify a peptide derivative or a low molecular compound capable of binding to the CEP55 EABR region to inhibit cell division And may be useful for the development of anticancer agents that inhibit cytoplasmic division.

In this regard, the present invention provides in one aspect a method of screening or designing a substance that inhibits and / or increases binding thereof using a complex crystal according to the present disclosure. In this method, a tertiary structure based on the complex binding site obtained in the present invention can be used, and at least one candidate substance can be evaluated for the effect of overlapping the binding site of the tertiary structure, Suitable substances can be selected as drugs.

In other embodiments, the methods herein may use rational drug design using the tertiary structure of the present disclosure. The candidate compound can be bound to all or a portion of the binding region, and the binding potential of the candidate compound can be measured and compared with the binding force of existing known substances to select candidates.

In another embodiment, the methods of the invention relate to methods of designing compounds that enhance binding of the complex. The method provides data for the tertiary structure disclosed herein to a computer and designs such materials using software. The method includes synthesizing the designed compounds using known methods and then evaluating them in Invitro and / or Invivo.

Protein crystallization conditions according to the present invention can also be used to make complex crystals with lead compounds, and the structure of such complexes is essential for effective anti-cancer agent redesign.

This material means low molecular compounds, antibodies, peptides, proteins interacting on a complex contact, and methods for designing or screening such materials are well known in the art.

The present invention also relates to a method of screening a substance that increases the interaction of CEP55 and TEX14, comprising the steps of:

The method according to the present invention comprises providing a CEP55-EABR (residues 160-217 of SEQ ID NO: 1) and a TEX14 peptide (AxGPPxxxYxPP) or a peptide (DLAxGPPxxxYxPP); Contacting the mixture of the protein and the peptide with the test substance; And comparing the interaction of the CEP55-EABR and TEX14 peptides in a control not contacted with the mixture contacted with the test substance.

In another aspect, the invention also relates to a method of screening a TEX14 derivative as an anticancer candidate agent, which binds to a CEP55-EABR region, comprising the steps of:

In one embodiment, the method comprises providing a CEP55-EABR region, a CEP55 full-length protein comprising a CEP55-EABR region or a CEP55 protein fragment comprising a CEP55-EABR region, and a TEX14 peptide comprising an amino acid sequence represented by AxGPPx ₃ YxPP Wherein A is Ala, G is Gly, P is Pro, Y is Tyr, and X is any amino acid in the above AxGPPx ₃ YxPP; Contacting the CEP55 fragment comprising the CEP55-EABR region, the CEP55 full-length or CEP55-EABR region comprising the CEP55-EABR region and the TEX14 peptide with the test material, wherein the test material comprises the TEX14 A TEX14 derivative capable of competitive binding to a peptide; Measuring the binding of the TEX14 peptide to the CEP55-EABR region in the presence of the test substance; And selecting the test substance as an anticancer candidate substance when the binding of the TEX14 peptide to the CEP55-EABR region is decreased in the test group which is contacted with the test substance as compared with the control group not in contact with the test substance as a result of the measurement do.

In another embodiment, the method according to the present invention comprises: a TEX14 peptide represented by the CEP55-EABR region represented by the amino acid residues 160 to 217 of SEQ ID NO: 1 and the 793 to 804 amino acid residues of SEQ ID NO: 2; the 792 to 804 amino acids And a TEX14 peptide represented by the amino acid residues 791 to 804 of SEQ ID NO: 2, wherein the TEX14 peptide is selected from the group consisting of: And contacting the CEP55-EABR region and the TEX14 peptide with a test substance, wherein the test substance is a TEX14 derivative that competitively binds to the CEP55-EABR region with the TEX14 peptide, wherein the CEP55- Lt; RTI ID = 0.0 &gt; TEX14 &lt; / RTI &gt; peptide to EABR.

The CEP55-EABR region which can be used in the method according to the present invention is represented by the amino acid sequence represented by 160 to 217 residues of SEQ ID NO: 1 from human and the CEP55 total length is represented by the amino acid sequence of SEQ ID NO:

The TEX14 peptide which can be used in the method according to the present invention is a wild-type sequence derived from a human, which comprises an amino acid sequence of residues 793 to 804 of SEQ ID NO: 2, an amino acid sequence of residues 792 to 804 of SEQ ID NO: 2 or residues 791 to 804 of SEQ ID NO: &Lt; / RTI &gt;

In the method according to the present invention, when the binding of the TEX14 peptide to the CEP55-EABR is decreased in the test group that is contacted with the test group as compared with the control group not in contact with the TEX14 peptide derivative as a test substance, the test substance is selected as an anticancer candidate substance .

In the method according to the present invention, the peptide derivative is a polypeptide comprising the amino acid sequence of AxGPPx ₃ YxPP, which has a different amino acid sequence and / or structure from the TEX14 peptide and has been modified in such a way as to increase binding in the EABR region.

In one embodiment, the TEX14 derivative according to the present invention is selected from the group consisting of Ala, Gly, Pro, Pro, Tyr, Pro and Pro in the order indicated in the polypeptide of the AxGPPx ₃ YxPP amino acid sequence, Wherein said modification comprises substitution of amino acid residues and / or chemical modification of amino acid side chains.

TEX14 peptide or peptide derivatives according to the present invention include natural or unnatural amino acids and include amino acid mimetics and amino acid mimetics that act in a manner similar to amino acids found in nature. Naturally occurring amino acids include the known 20 amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine) and pyrrolic acid and cellocysteine. Amino acid analogs include those having the same basic chemical structure as the amino acids found in nature, that is, the structure in which the alpha carbon is bonded to hydrogen, a carboxyl group, an amino group and an R group, in which the R group is modified or a peptide skeleton (backbone) For example, 2-amino adipic acid, hydroxy lysine homoserine, norleucine, sulfoxide, methylsulfonium.

Peptide derivatives according to the present invention include peptidomimetics (typically synthetic peptides), which include the known 20 amino acids and amino acid residues other than pyrrolicin or selenocysteine, and peptide analogs such as peptoids and semipeptides .

In one embodiment, the peptide derivative is selected from the group consisting of N-terminal modifications, C-terminal modifications, peptide bond modifications such as CH ₂ -NH, CH ₂ -S, CH ₂ -S═O, CH ₂ -CH ₂ , . Methods for preparing peptide mimetic compounds are well known in the art and can be found in, for example, Quantitative Drug Design, CA Ramsden Gd., Choplin Pergamon Press (1992).

In one embodiment, the peptide derivative means that at least one of the amino acid residues constituting the derivative is modified.

Means that the TEX peptide derivative is modified in a manner that has at least the same or higher binding force as compared to the binding force of the wild-type TEX14 peptide to CEP55, in order to obtain a peptide derivative having characteristics meeting the purpose according to the present invention .

In one embodiment, it involves physical, i.e., chemical or biochemical, modification of one or more residues that make up the peptide.

In other embodiments, the modification may be, for example, a conservative or non-conservative substitution, deletion or addition of one or more amino acid residues constituting the peptide, or a chemical or biochemical modification of the side chain (R) group constituting the amino acid residue . The amino acid in which the R group has been modified may be classified as an amino acid analogue.

In one embodiment, the TEX14 peptide derivative according to the present invention is a modification of one or more residues selected from the group consisting of Gly795, Pro796, Pro797, Tyr801, Pro803 and Pro804 among residues of TEX peptide shown in SEQ ID NO: 2.

In another embodiment, the TEX14 peptide derivative according to the present invention is one wherein at least one of the residues selected from the group consisting of Gly795, Pro796, Pro797, Tyr801, Pro803 and Pro804 among the residues of the TEX peptide shown in SEQ ID NO: 2 is different conservative or non- It is completely replaced by an amino acid residue.

Substitutions according to the present disclosure include conservative substitutions or non-conservative substitutions with other amino acids that can increase the binding of TEX14 peptide derivatives to CEP55. A conservative substitution is one in which an amino acid has similar characteristics in terms of side chain size, charge, hydrophobicity, hydrophilicity, and / or aromatic character, such that residues in the following group may be conservative substitutions: small aliphatic, nonpolar, Residues group: Ala, Ser, Thr, Pro, Gly; Polar, negatively charged residues and their amides and esters: Asp, Asn, Glu, Gln, cysteic acid and homocysteic acid; Polarity, positive charge residue group: His, Arg, Lys; Ornithine (Orn); And large branched, aliphatic, nonpolar residue groups: Met, Leu, Ile, Val, Cys, Norleucine (Nle), homocysteine; Large side chain aromatic moieties: Phe, Tyr, Trp, acetyl phenylalanine.

The chemical modification of the R group according to the present invention is modified in such a way as to increase the binding force of the CEP55 of the peptide containing the residue, for example, modified in such a manner as to bring about changes in hydrophobicity, hydrophilicity, degree of charge, and / And those skilled in the art will be able to make modifications to suitable functional groups in view of the tertiary structure information disclosed herein.

For example, in introducing an amino acid substitution, the hydropathic index of the amino acid can be considered. Each amino acid is assigned a hydrophobic index according to its hydrophobicity and charge: isoruicin (+4.5); Valine (+4.2); Leucine (+3.8); Phenylalanine (+2.8); Cysteine / cysteine (+2.5); Methionine (+1.9); Alanine (+1.8); Glycine (-0.4); Threonine (-0.7); Serine (-0.8); Tryptophan (-0.9); Tyrosine (-1.3); Proline (-1.6); Histidine (-3.2); Glutamate (-3.5); Glutamine (-3.5); Aspartate (-3.5); Asparagine (-3.5); Lysine (-3.9); And arginine (-4.5). Such hydrophobic indexes are particularly important in imparting interactions or binding forces between proteins.

Similar hydrophilicity values are also important for determining protein interactions or binding forces. Thus, for example, as disclosed in U.S. Patent No. 4,554,101, arginine (+3.0); Lysine (+3.0); Aspartate (+ 3.0 ± 1); Glutamate (+ 3.0 ± 1); Serine (+0.3); Asparagine (+0.2); Glutamine (+0.2); Glycine (0); Threonine (-0.4); Proline (-0.5 ± 1); Alanine (-0.5); Histidine (-0.5); Cysteine (-1.0); Methionine (-1.3); Valine (-1.5); Leucine (-1.8); Isoru Isin (-1.8); Tyrosine (-2.3); Phenylalanine (-2.5); Amino acid substitution can be performed taking into account the hydrophilicity of tryptophan (-3.4).

Proteins used herein may be referred to above and may be prepared using methods known in the art. Particularly, a method for producing a protein is to use a gene recombination technique. For example, a plasmid containing the corresponding gene encoding the protein may be transferred to prokaryotic or eukaryotic cells such as insect cells and mammalian cells for overexpression, followed by purification.

Alternatively, a DNA or RNA sequence encoding the screening protein may be expressed in an appropriate host cell to produce a cell lysate thereof, or the mRNA of the screening protein may be translated in vitro, followed by purification of the screening protein by a protein separation method known in the art can do. Usually, the cell lysate or the translation product in vitro is centrifuged to remove cell debris and the like, and then precipitation, dialysis, various column chromatography and the like are applied. Ion exchange chromatography, gel-permeation chromatography, HPLC, reversed phase-HPLC, SDS-PAGE for affinity column, and affinity column are examples of column chromatography. Affinity columns can be made, for example, using anti-screening protein antibodies.

Peptides can also be artificially synthesized by commercial companies.

The contact of the purified protein can be directly confirmed in the absence or presence of the test substance by directly using them in vitro. In this case, the protein may be labeled using various known markers such as biotin, fluorescent material, acetylation, radioisotope or the like using a known protein labeling kit or a commercially available labeled substance Lt; RTI ID = 0.0 &gt; detectors &lt; / RTI &gt; Protein-protein interactions can be measured using a variety of methods known in the art. For example, the yeast two-hybrid method, the confocal microscope method, which confirms the binding / interaction of intracellular proteins. (SPR) and spectroscopy methods, and further references to comparative and detailed experimental methods for such methods are provided in Berggard et al., (2007) "Methods for protein-protein interactions &quot;, PROTEOMICS Vol 7: pp 2833-2842.

The amount of the protein used, the type and concentration of the cells used, the amount and type of the test substance, and the like are dependent on the specific experimental method used and the kind of the test substance. Those skilled in the art will be able to select an appropriate amount.

The TEX14 derivative to be screened in the method of the present invention has a higher binding capacity to CEP55 than wild-type TEX14. Such TEX14 derivatives can be screened by competitive binding methods of CEP55 in the presence of wild-type TEX14 or by measuring their binding capacity to CEP55 with wild-type TEX14.

These TEX 14 derivatives increased about 105% more, about 110%, about 115%, about 120%, about 125%, about 130%, about 135% more than the wild type control, About 140% increase, about 145% decrease, about 150% increase.

In the present invention, the interaction refers to the protein / protein / protein / protein interaction, which includes both covalent and noncovalent interactions.

Hereinafter, embodiments are provided to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited to the following examples.

Example

Experimental methods and results

Protein purification and crystallization / data collection, structure determination and refinement

CEP55 _{160 - 217} then cloned into the BamHI and XhoI pGST2 vector by using the restriction enzymes was transformed to CEP55 (160-217, pGST2) plasmid in BL21 (DE3) E. coli. The transformed Escherichia coli was inoculated into 1 L of LB broth (containing 100 μg / ml ampicillin) and cultured at 37 ° C until the absorbance A600 reached 0.8. 0.5 mM IPTG (Isopropyl-β-D-Thiogalactopyranoside) was added to the culture solution and cultured at 25 ° C. for 18 hours. After centrifuging the culture at 5,000 rpm for 10 minutes, discard the supernatant and dissolve the precipitated pellet in 20 mM Tris-HCl pH 7.4, 100 mM NaCl buffer (1 mM PMSF), and then use a Microfluidizer (French press) After crushing, the mixture was centrifuged at 12,000 rpm for 1 hour to obtain a supernatant. The separated supernatant was passed through a GST-column and eluted with 20 mM Tris-HCl pH 8.0, 15 mM GSH buffer. GST-column was treated with TEV protease to remove GST tag. (4 ° C overnight). The sample obtained from TEV protease treatment was concentrated to 4 ml and injected into a gel filtration column (Superdex 200 pg 16/60). The CEP55 was incubated at about 92-102 ml (flow rate: 1 ml / min) The protein and GST protein were eluted together. (Running buffer: 20 mM Tris-HCl pH 7.4, 100 mM NaCl). GST was passed through a Hi-trap Q HP column. The buffer was 20 mM Tris-HCl pH 7.4, 100 mM NaCl and 20 mM Tris-HCl pH 7.4, 1M NaCl as the elution buffer. Was passed through a Q-column CEP55 ₁₆₀ the GST is removed _- Collect ₂₁₇ protein 7mg / ml in mixed ice is TEX14 peptide after concentration in (20mM Tris-HCl pH 7.4, 100 mM NaCl) so that the molar ratio of twice the CEP55 After incubation for 1 hour, crystallization screening was carried out to obtain crystals. Crystallization conditions were 0.1 M imidazole (pH 8.0), 1 M ammonium phosphate. Lt; RTI ID = 0.0 &gt; 22 C &lt; / RTI &gt; using sitting-drop vapor diffusion. X-ray diffraction data were obtained from beamline 7A of the Pohang Acceleration Research Center (PAL) in Korea. The crystals were directly frozen using 30% (v / v) glycerol and cooled instantaneously with 110 K of liquid nitrogen . The original data were processed using HKL2000 and the diffraction data were collected with a maximum resolution of 2.3 A with a space group of P22 ₁ 2 ₁ . Table 2 summarizes the statistics of the collected data. CEP55 _{160 -217} -TEX14 _792-804 composite crystal is a = 53.9 Å, b = 102.6 Å, and c = with a unit cell parameter of 132.5 Å, belongs to the space group P22 ₁ 2 ₁ (Table 2). CEP55 _{160 -217} -TEX14 ^N -ALIX ^C composite and CEP55 _{160 -217} -ALIX ^N crystallization and data collection of -TEX14 ^C composite has been engaged in the similar steps used for CEP55 _160-217 -TEX14 _792-804 complexes. CEP55 _160-217- TEX14 ^N- ALIX ^C composite crystals were _{prepared by} mixing 20% (wt / vol) polyacrylic acid 5100, 0.2 M magnesium chloride, 0.1 M 4- (2-hydroxyethyl) -1 -piperazine ethanesulfonic acid (HEPES) sikyeotgo generated using _{(pH 7.5), CEP55 160 -217} -ALIX N -TEX14 C complex crystal was used as a 0.8M ammonium sulfate, 0.1M sodium citrate (pH 5.0). CEP55 _{160 -217} -TEX14 ^N -ALIX ^C composite and CEP55 _{160 -217} determines the -ALIX ^N -TEX14 ^C composite was frozen rapidly with liquid nitrogen and then put in a solution of glycerol 25-30% (v / v).

Structure determination and purification

The crystal structure of CEP55 _{160 -217 792 -804} -TEX14 complex, CEP55 _{160 -217} -TEX14 ^N -ALIX ^C composite and CEP55 _{160 -217} -ALIX ^N -TEX14 ^C composite _{human CEP55 160 -217 [Protein Data Bank} (PDB) ID code 3E1R] using the molecular replacement method, and the crystallographic calculation was performed using CCP4. A cross-rotational search was performed after a translational search using the Phaser program (McCoy AJ, et al., Acta Crystallogr D Biol Crystallogr 61 (4): 458-464.). Manual model building was then performed using the Coot program (Emsley P, et al., Acta Crystallogr D Biol Crystallogr 60 (12): 2126-2132) and the REFMAC5 program (Murshudov GN, et al., Acta Crystallogr D Biol Crystallogr 53 (3): 240-255.) Was used for restrained refinement. Several model building, simulated annealing, position fly refinement, and individual B-factor refinement were performed. Table 2 shows refinement statistics. CEP55 _{160 -217} crystallographic asymmetric unit of the -TEX14 _{792 -804} complex comprises four homodimer of four homodimer and TEX14 CEP55 peptide of the peptide, in the A, B, D, E, G, H, J , and K chain corresponds to _{160 -217} CEP55, C, F, I, and the L chain corresponds to TEX14 peptide. The refined model contains 188 water molecules, with 98.8% residues in the most-allowed region of the Ramachandran plot. 160-167 residues of residues 212-217 A, B, and J chain in the chain of CEP55 _{160 -217,} 210-217, and residues of the D and E chain, G residues 211-217, residues 209-217 of the H chain of the chain, And no electron density was observed in the 209-217 residues of the K chain. CEP55 _{160 -217} -TEX14 _{792 -804} complex, CEP55 _{160 -217} -TEX14 ^N -ALIX ^C composite and CEP55 _{160 -217} -ALIX ^N atom coordinates and crystal structure of the complex is -TEX14 ^C Protein Data Bank (PDB ID codes each 3WUT , 3 WUU, and 3 WUV).

Competition  Analysis method

To the Competition analysis, CEP55 ₁₆₀ of 350㎍ _{- 217} were cultured at 4 ℃ for 1 hour with the peptide TEX14 (20, 40, 160, or 250 ㎍). GST-ALIX _{795 - 811} a glutathione-combines the sepharose beads (GE Healthcare), was then incubated followed by the addition of _{160 -217} and CEP55 TEX14 peptide mixture for 1 hour. The beads were washed using 20 mM Tris (pH 7.4), 100 mM NaCl buffer. The bound proteins were eluted with 20 mM Tris-HCl pH 8.0, 15 mM GSH buffer, and then separated by SDS-PAGE and stained with Coomassie Brilliant Blue.

GST  Pull-Down Assay.

GST-TEX14 ^N -ALIX ^C and GST-ALIX ^N- TEX14 ^C fusion proteins were conjugated to glutathion-Sepharose beads (GE Healthcare). The CEP55 _{160 -217} protein (also labeled with TEV) GST, the 100 mM NaCl with the ^GST-N TEX14 -ALIX ^C (also labeled with No TEV), or GST-ALIX ^N -TEX14 ^C (also labeled with No TEV) In 20 mM Tris.HCl (pH 7.4) for 1 h at 4 &lt; 0 &gt; C with gentle shaking. After washing, the bound proteins were eluted with elution buffer (30 mM glutathione, 150 mM Tris.HCl, pH 8.0), separated by SDS / PAGE and stained with Coomassie Brilliant Blue.

NMR Spectroscopy.

In the minimal medium supplemented with ¹⁵ NH ₄ Cl as the only nitrogen source, Escherichia an coli BL21 (DE3) cells at 20 ℃ grow for 18 h 0.5 mM isopropyl-β-D-1- thio going to induce the expression in Lactococcus pyrano side (IPTG), a uniformly labeled ¹⁵ N- human CEP55 _{160 - 217} (pGST2, 160217 residue). The protein was purified using GST-affinity and size-exclusion chromatography. Using a 600 μl sample at 298 K (200 μM protein, 50 mM Tris.HCl, pH 7.2, 10% D ₂ O) in a ruker Avance 600 spectrometer equipped with a cryoprobe, homogenous ¹⁵ N-labeled CEP ₁₆₀₂₁₇ NMR titration experiments were carried out. ¹⁵ N- labeled CEP _{160 - 217} alone or TEX14 (or ALIX) peptide (50 and 100 μM) and ^{1 H- 15 N TROSY (transverse relaxation} optimized spectroscopy) to perform spectral TopSpin with (versions 1.3 and 3.0; Bruker) Respectively. CEP55 _160-217 alone, CEP55 _{160 -217} -TEX14 complex, and CEP55 ₁₆₀ for sequential alignment of _-217 -ALIX complex could never collect the NMR spectrum, which is 20 mM Tris · HCl (pH 7.2 ), 10% D 2 uniformly ¹⁵ N, ¹³ C- labeled CEP55 _{160 -217} constructs (1 mM) in the O is due to a tendency to flocculation during the measurement.

Reverse transcription  And quantitative real-time PCR

All experimental animals (C57BL / 6) were examined and tested under the approval of Seoul National University 's Animal Experimental Ethics Committee. In the present invention, maturation mice (7 weeks) and 7-d- and 17-d-postpartum mice were used for the PCR test. Total RNA was extracted from 7, 17, and 42 days old mice using TRIzol reagent (Invitrogen). Reverse transcription was performed using a random hexamer. Real-time PCR was performed using the gene-specific primers listed in Table 1 below. Relative gene expression levels were calculated using the relative Ct method and normalized to the expression of GAPDH .

[Table 1]

surface Plasmon  Resonance Plasmon  Resonance SPR )

Surface plasmon resonance (SPR) measures bond strengths by kinetic measurements of on-rate and off-rate constants. The binding of the wild-type and mutant CEP55 _160-217 construct to the TEX14 peptide was analyzed by SPR (ProteOn XPR36 system, Bio-Rad) at 25 ° C at a flow rate of 100 μl / min. To ₈₁₁ were fixed to the surface via a covalent bond between the N- terminus of the GST _- GST-TEX14 _792-807 (wild-type, G795A, P796A, P797A, Y801A , P803A, P804A and mutations) and GST-ALIX ₇₉₅ for the SPR experiment . The carboxymethylated dextran surface of the GLM sensor chip (Bio-Rad) was treated with 1: 1 N-hydroxysuccinimide (NHS) / 1-ethyl-3- (3- dimethylaminopropyl) carbodiimide EDC) at a flow rate of 30 μL / min for 5 minutes. Each protein sample (10 μM) in 10 mM sodium acetate (pH 4.5) was passed through the activated surface for 5 minutes at a flow rate of 30 μL / min and then eluted with ethanolamine (1 M, pH 8.5) at a flow rate of 30 μL / . All binding experiments were performed in 20 mM Tris (pH 7.5), 100 mM NaCl. CEP55 _{160 - 217.} Binding of the wild-type protein and is TEX14, CEP55 _{160 - 217} protein (wild-type, W184A, Y187A, R191A, and E192A) to each of the coupling 300 and 400s and that dissociation is a GST-binding TEX14 _792-807 for a time and passed through a flow cell (flow cell) association rate constant, Kon ^- was measured (in M ^-1 s ¹⁾ and dissociation rate constants koff (unit s-1). Wild type CEP55 _{160 -217} and ALIX, TEX14 mutation, and key combination of chimeric peptides CEP55 _{160 - 217} respectively, for the 300 and association and dissociation time of _{400s GST-ALIX 795 -811, GST} -TEX14 792 -807 mutations (G795A, P796A, P797A, Y801A, P803A, and P804A) or a GST-chimeric peptide (TEX14 ^N- ALIX ^C and ALIX ^N- TEX14 ^C ). All proteins were regenerated by continuously injecting 20 mM ris (pH 7.5), 500 mM NaCl at a flow rate of 30 μL / min for 10-30 seconds between successive injections.

Cell biology experiment Construct  And the generation of stabilizing cell lines

The GFP-TEX14 construct was cloned into the Eco RI / Xma I site of the pEGFP-C2 vector (Clontech). Positional mutations of GFP-TEX14 (G795A, P796A, P797A, Y801A, P803A, and P804A) were generated using the QuikChange Mutagenesis Kit (Stratagene) and this mutation was confirmed by DNA sequencing. The wild-type and mutant TEX14 (Y801A, P804A, Y801A and / P804A) encoding gene from the cut Hind III / Sma I pre clones GFPTEX14 WT and mutant constructs were inserted into a root pcDNA5 / TO / FRT / 3xflag vector. In order to establish a stable cell line expressing TEX14 WT and mutants, a 9: 1 ratio of pcDNA5 / TO / FRT / 3xflag containing TEX14 and POG44 according to the manual (Roche) was used to generate HeLa FRT / TO cells with FuGENE Lt; / RTI &gt; Hygromycin was chosen to collect survival colonies. Established stable cell lines were maintained in DMEM supplemented with 10% FBS in a 5% CO ₂ incubator at 37 ° C. To induce TEX14 expression, the cells were treated with doxycycline (1 [mu] g / mL).

Example  1. TEX 14th CEP55 - EABR  Identification of crystal structure of complex

SPR experiments showed that CEP55-EABR binds to 13 residues of TEX14 and the dissociation constant (Kd) of this bond is ~300 nM (see Figure 1 A-C). These results indicate that the peptide has a higher binding capacity than ALIX and TSG101 (1-3 uM) (Lee HH, et al, Science 322 (5901): 576-580.). (Fig. 1 D, E). As shown in the results of competitive experiments using an increasing amount of TEX14 peptide, TEX14 and ALIX peptides bind to the same site of CEP55-EABR. These results indicate that TEX14, ALIX and TSG101 peptides bind to the same site of CEP55-EABR.

The diffraction data were collected at a maximum resolution of 2.3 A with a space group of P22 ₁ 2 ₁ . Table 2 summarizes the statistics of the collected data. As a result of analyzing the crystal structure of the CEP55-EABR and TEX14 complexes with a resolution of 2.3 Å, a coiled coil structure parallel to the whole length was formed as shown in FIGS. 1 G and F, and seven heptad repeats . Furthermore, TEX peptide binds to the same site as ALIX, and the stoichiometry of CEP55-EABR versus TEX14 was 2: 1.

[Table 2]

Table 3 is the structure coordinate X-ray diffraction data of the composite crystal.

[Table 3]

Example  2. CEP55 - EABR - TEX14  Important of coupling of complex Residue  Identification

In CEP55-EABR, the interfacial residue in contact with TEX14 was replaced with Ala, and their interaction was identified by SPR. The N-terminus of TEX14 is folded between the GPP motif and Tyr801 while surrounding the Tyr 187 residue of CEP55-EABR. The Y187A substitution mutation resulted in a decrease in binding to the TEX14 peptide (Kd 149.1 [mu] M) (Fig. 2A, B). The GPP sequence of the TEX14 peptide makes substantial contact with Trp184 and Tyr187 of CEP55-EABR, and the aliphatic side chains of Lys180 and Gln183 (Figure 2A). The CEP55-EABR Trp184 mutation almost disappeared with TEX14 (Fig. 2B).

Pro796 and Pro797 of TEX14 also showed significant contact with CEP55-EABR (Fig. 2A) and showed a significant decrease in binding when substituting these residues with Ala (Kd 116.1 and 40.4 μM, respectively) (Fig. 2B). Furthermore, the G795A mutation was also shown to reduce the binding force to Fact or ~ 10 (Figure 2B). TEX14 Tyr801 (corresponding to Tyr806 of ALIX) also showed strong hydrogen bonding (2.8 A) with OE1 atoms of Glu192 '(prime represents residues of surrounding subunits) and contributed to binding (Fig. 2A).

When the Tyr801 and Glu192 'residues were substituted with Ala, the binding capacity decreased (Kd 184.3 and 119.2 μM, respectively) (Fig. 2B). Substitution of Arg191 for CEP55 with Ala has shown that mid-body localization is severely affected and also has an effect on binding to TEX14 (Kd 222.3 [mu] M) (Fig. 2B). In CEP55-EABR and TEX14, the double mutation appeared to have a more serious impact on the binding force than either single substitution (Fig. 2B).

Tyr187 of CEP55-EABR was found to have significant contact with Pro796, Tyr801, and Pro804 of TEX14 (Fig. 2A), and double substitutions involving the Y187A mutation in CEP55-EABR showed severe adverse effects on interaction CEP55-EABR-TEX14 Y187A / P796A, Y187A / Y801A, and Y187A / P804A) (Fig. 2B). These results indicate that binding of TEX14 peptide around Tyr187 of CEP55-EABR is important for CEP55-EABR and TEX14 interaction.

Example  3. Of TEX14  Identification of C terminal structure (Conformation)

The overall structure of the TEX14 peptide is similar to the ALIX peptide except at the C-terminus (Lee HH, et al, Science 322 (5901): 576-580. When the CEP55-EABR-TEX14 complex is overlapped with the CEP55-EABR-ALIX complex, it can be seen that the C-terminal ring of TEX14 is oriented in the opposite direction to the ALIX (FIG. 1F).

A 15N-1H HSQC (heteronuclear single-quantum coherence) spectroscopy of CEP55-EABR was performed with increasing concentrations of each peptide to see if this structure was retained in solution. The perturbation peaks showed that overall interactions were very slow in terms of chemical exchange (data not shown). A single peak of CEP55 Trp184 showed strong contact with Val794, Gly795, and Pro796 (Gln799, Gly800 and Pro801 of ALIX) of TEX14, showing a separate migration in CEP55-EABR-TEX14 and CEP55-EABR-ALIX This indicates that the environment of Trp184 and Trp184 'is unbalanced when bound to TEX14 or ALIX peptide (Fig. 2C).

The chemical shifts of Gly197 and Gly197 'residues near the C-terminus of TEX14 or ALIX peptide (913A apart) were also different (FIG. 2C). These results indicate that the C-terminal tail of TEX14 and ALIX peptides bind differently to CEP55-EABR (Fig. 1F).

Example  4. Different structures TEX14  or ALIX , Peptides  Identifying the role of Pro at the C-terminus

The cause of the C-terminal of TEX14 (or ALIX) peptide with different structure was identified. In TEX14, one residue Ile802 was added between Tyr801 and Pro803 compared to ALIX (Fig. 1F). The tyrosine residue tends to cause the proline residue in the Tyr-Pro sequence to assume the cis structure (16). The Pro807 residue of the ALIX and TEX14 peptides appeared to take a trans structure (Figures 1F and 2A). To further characterize the role of the proline residue (Pro803 of TEX14; Pro807 of ALIX) on the orientation of the C-terminus, the position of the proline residue was changed. To this end, two chimeric peptides (TEX14 ^N- ALIX ^C and ALIX ^N- TEX14 ^C ): N-terminal TEX14 (residues 792-801) are linked to the C-terminal ALIX (residues 807-811) 795-806) is linked to C-terminal TEX14 (residues 802-807) (Fig. 1B). GST pulldown analysis and SPR experiments showed that both TEX14 ^N- ALIX ^C and ALIX ^N- TEX14 ^C peptides had low binding (relative to wild-type TEX14 or ALIX peptide to CEP55-EABR (Kd 7.3 and 6.7 μM, respectively) 3 A and B). The actual crystal structure CEP55-EABR in the formation of the ^N TEX14 -ALIX ^C or ^N ALIX -TEX14 ^C-peptide and the complex was found to have changed direction of the C- terminal. These results indicate that the Pro residue plays a role in determining the orientation of the C-terminus (Fig. 3 CE).

Example  5. Of TEX14 CEP55 - To EABR Predominant  Identification of binding mechanism

In somatic cells, TEX14 is not expressed, resulting in binding to ALIX (or TSG101) CEP55-EABR (1). In contrast, the binding of TEX14 to CEP55-EABR requires elaborate regulation, since TEX 14, ALIX and TSG101 are expressed in germ cells as shown in the real-time RT quantitation PCR of FIG. 4A.

Thus, we have identified the mechanism by which TEX14 in germ cells is responsible for binding to CEP55 in the presence of competitor ALIX or TSG101. For this, we hypothesized that the binding force of TEX14 to CEP55 (Kd 323 nM) is stronger than that of ALIX (or TSG101) against CEP55 (Kd 1-3 μM), so it is differentiated or temporarily recoated in midbodies.

However, affinity itself alone can not completely prevent the recruitment of CEP55-EABR from ALIX (or TSG101), which is recruited from the germ cells to the midbodies in order of MKLP1 → TEX14 → CEP55, whereas in somatic cells, MKLP1 → CEP55 → ALIX TSG101) (Iwamori T, et al. (2011) PLoS One 6 (2): e17066.). In the model according to the present invention, TEX14 in germ cells is recycled mid-body without the aid of CEP55-EABR before ALIX or TSG101, resulting in a local concentration of TEX14 792-804 peptide in the midbodies higher than ALIX and TSG101 peptides, In the end, TEX14 792-804 will take over CEP55-EABR. In this case, the CEP55-EABR-TEX14 region involved in the recruitment of TEX14 was analyzed. For this, we investigated whether the mutations that interfere with CEP55-EABR-TEX14 binding affect local concentrations in the midbodies. Expression of GFP-TEX14 was analyzed in HeLa cells and its intracellular location was examined. As a result, wild-type and mutant GFP-TEX14 were recruited to the midbodies without the aid of CEP55-EABR (Fig. 4B and C). These results are in agreement with the fact that they bind to MKLP1 and are recruited in midbodies before the binding of CEP55 to germ cells ((Iwamori T, et al., PLoS One 6 (2): e17066.) These results suggest that the overexpression of TEX14 In order to exclude the possibility of inducing TEX14 expression, a stabilized cell line expressing TEX14 was prepared, in which the basal expression of wild-type TEX14 was reduced, but the leakage of TEX14 was also found to be low even in the absence of doxycycline (FIG. (Fig. 5A). Further, the effect of wild-type and mutant TEX14-stabilized cell lines on the division of normal cells was investigated. For this, 2n, 4n, and 8n DNA (Fig. 4D). As a result, the cells expressing wild-type TEX14 showed accumulation of polynuclear intercellular bridges (P <0.0001), indicating that TEX14 prevents ESCRT from being recruited into the midbodies. In addition, TEX14 mutations (Y801A, P804A, and Y801A / P804A) (Fig. 4D). These results indicate that TEX14 can inhibit cell division due to the interaction between CEP55-EABR and TEX14 (Fig. .

Example  6. In Invivo TEX14 ALIX Mid-body  Analysis of impact on replacement

ALIX displacement experiments were performed using wild-type and mutant TEX14-stabilized cell lines to investigate whether TEX14 prevented ALIX from being recruited through competitive binding to CEP55-EABR (FIGS. 5A and B). Specifically, the presence of TEX14 or its mutants in the midbodies investigated whether the endogenous ALIX could go to the midbodies, which is based on the hypothesis that TEX14 expression can prevent the endogenous ALIX from going to the midbodies. As shown in Figures 5A and B, when wild-type TEX14 is expressed, ALIX has been shown to be completely replaced, indicating that TEX14 interferes with the interaction between ALIX and CEP55-EABR. To investigate the effect of the TEX14 mutation, the relative fluorescence intensity between the wild type and the mutant was analyzed (Fig. 5B). The intensity of endogenous ALIX in the non-transfected cells was significantly higher (P &lt;0.001; FIG. 5B) when wild-type TEX14 was expressed compared to the intensity. However, when the TEX14 mutations (Y801A, P803A, and Y801A / P803A) were expressed, the relative intensity of ALIX was significantly increased (P <0.001) compared to wild-type TEX14 expression (FIG. These results indicate that when the mutation of the AxGPPx ₃ YxPP motif of TEX14 is induced, the binding between TEX14 and CEP55-EABR is weakened and as a result the ALIX replacement effect is reduced.

Example  7. TEX14 Of peptide  Increased retention time Of TEX14 CEP55 - EABR of Predominant  Bond identification

ESCRT devices are necessary for normal cell division, but intercellular bridges in germ cells may damage cell membranes because they must not be cleaved during spermatogenesis. Thus, even small numbers of ALIX molecules can be problematic when bound to CEP55-EABR. The high affinity of TEX14 peptide for CEP55-EABR and the locally high concentration of TEX14 were found to contribute to the dominant binding of TEX14. However, if the binding kinetic of the TEX14 peptide to CEP55-EABR is not beneficial for predominant binding of the cell, the cell bridge is still not safe. If the time for TEX14 to stay in CEP55-EABR is short, ALIX will bind to CEP55-EABR. To identify these problems, dissociation rates of TEX14 and ALIX peptides were measured using SPR (Fig. 6A). It was shown to be the dissociation constant of 0.012 ± 0.001 s ^-1 TEX14 ALIX peptide which ^peptide is to 15 times slower in comparison with (0.19 ± 0.02 s ¹⁾ (Fig. 6A). These results indicate that the residence time of the TEX14 peptide is longer to ~80 s, which is more advantageous for the predominant binding of the TEX14 peptide to CEP55-EABR.

Through the SPR experiment CEP55 _{160 -217} -TEX14 peptide dissociation constant (Kd) has been able to check that it has a high affinity compared to ~300 nM, CEP55 _160-217 -ALIX peptide dissociation constant (Kd) is ALIX peptide to ~3 μM , And the dissociation constant (Kd) of the GST-TEX14 _{792 -807} mutants was lowered by experiments. In addition, the dissociation rate constant of the TEX14 peptide is 0.012 ± 0.001 s ^-1 and the dissociation rate constant of the ALIX peptide is 0.19 ± 0.02 s ^-1 . The TEX 14 peptide is much slower than the Alix peptide, indicating that the TEX 14 peptide binds to CEP 55 Which is dominantly advantageous. TEX14's multi-layered safeguard system has shown that ALIX prevents ALIX from binding to the EABR of CEP55, ultimately protecting the intercellular bridges of germ cells from ESCRT machinery, a potential risk factor acting as a cell membrane scissors. Therefore, TEX14 is an effective means of preventing the division of abnormal cells such as cancer cells by identifying the cell division mechanism of germ cells inhibiting cell division.

Thus, according to the results of the present application, CEP55 can act as an effective target protein that can artificially control cell division as a regulator of cell division through competitive interplay of TEX14 and ALIX. In particular, in view of the increased expression of CEP55 in tumor cells, CEP55 can be a therapeutic target for a variety of diseases. The TEX14 peptide derivative containing the AxGPPX ₃ YxPP motif may be a therapeutic target for inhibiting the division and proliferation of cancer cells by binding to CEP55-EABR to form a stable intercellular bridge.

Example  8. Assay model of germ cell division inhibition and its use

The results presented herein demonstrate that multiple safety systems of TEX14 inhibit the binding of ALIX (and TSG101) to CEP55-EABR and protect germ cell intercellular bridges from ESCRT devices, thereby preventing possible membrane damage. Therefore, it would be possible to find a method of preventing the division of abnormal cells such as cancer cells by identifying the mechanism of cleaving the cells in germ cells having endogenous ability to inactivate the cell cleavage through TEX14 (Fig. 6B).

Taking into consideration the role of CEP55 as a modulator of cell cleavage through competitive interactions with TEX14 and ALIX (or TSG101), CEP55 can be a target for effectively controlling cell division. In fact, CEP55 is one of the 70 genes associated with chromosomal instability and its expression has been shown to be increased in many cancer cells (18). Thus, CEP55 can be a therapeutic target for various diseases. Thus, using TEX14 peptide or derivatives thereof containing AxGPPx ₃ YxPP motif, it is possible to inhibit cancer cell proliferation through binding to CEP55-EABR and stable intercellular bridge formation There will be. These proposals are supported by previous research showing that it is possible to express TEX14 and convert the somatic midbodies into germ cell intercellular bridges (Greenbaum MP, et al. (2007) Dev Biol 305 (2): 389-396; Iwamori T, et al (2010) Mol Cell Biol 30 (9): 2280-2292.).

While the present invention has been described in connection with what is presently considered to be the preferred embodiments, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, .

All technical terms used in the present invention are used in the sense that they are generally understood by those of ordinary skill in the relevant field of the present invention unless otherwise defined. The contents of all publications referred to herein are incorporated herein by reference.

<110> SEOUL NATIONAL UNIVERSITY R & D FOUNDATION <120> Protein crystal of CEP55-TEX14 complex and its use <130> DP201510006P <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 464 <212> PRT <213> Homo sapiens <220> <221> DOMAIN &Lt; 222 > (160) <223> EABR <400> 1 Met Ser Ser Arg Ser Thr Lys Asp Leu Ile Lys Ser Lys Trp Gly Ser   1 5 10 15 Lys Pro Ser Asn Ser Lys Ser Glu Thr Thr Leu Glu Lys Leu Lys Gly              20 25 30 Glu Ile Ala His Leu Lys Thr Ser Val Asp Glu Ile Thr Ser Gly Lys          35 40 45 Gly Lys Leu Thr Asp Lys Glu Arg His Arg Leu Leu Glu Lys Ile Arg      50 55 60 Val Leu Glu Ala Glu Lys Glu Lys Asn Ala Tyr Gln Leu Thr Glu Lys  65 70 75 80 Asp Lys Glu Ile Gln Arg Leu Arg Asp Gln Leu Lys Ala Arg Tyr Ser                  85 90 95 Thr Ala Leu Leu Glu Gln Leu Glu Glu Thr Thr Arg Glu Gly Glu             100 105 110 Arg Arg Glu Gln Val Leu Lys Ala Leu Ser Glu Glu Lys Asp Val Leu         115 120 125 Lys Gln Gln Leu Ser Ala Ala Thr Ser Arg Ile Ala Glu Leu Glu Ser     130 135 140 Lys Thr Asn Thr Leu Arg Leu Ser Gln Thr Val Ala Pro Asn Cys Phe 145 150 155 160 Asn Ser Ser Ile Asn Asn Ile His Glu Met Glu Ile Gln Leu Lys Asp                 165 170 175 Ala Leu Glu Lys Asn Gln Gln Trp Leu Val Tyr Asp Gln Gln Arg Glu             180 185 190 Val Tyr Val Lys Gly Leu Leu Ala Lys Ile Phe Glu Leu Glu Lys Lys         195 200 205 Thr Glu Thr Ala Ala His Ser Leu Pro Gln Gln Thr Lys Lys Pro Glu     210 215 220 Ser Glu Gly Tyr Leu Gln Glu Glu Lys Gln Lys Cys Tyr Asn Asp Leu 225 230 235 240 Leu Ala Ser Ala Lys Lys Asp Leu Glu Val Glu Arg Gln Thr Ile Thr                 245 250 255 Gln Leu Ser Phe Glu Leu Ser Glu Phe Arg Arg Lys Tyr Glu Glu Thr             260 265 270 Gln Lys Glu Val His Asn Leu Asn Gln Leu Leu Tyr Ser Gln Arg Arg         275 280 285 Ala Asp Val Gln His Leu Glu Asp Asp Arg His Lys Thr Glu Lys Ile     290 295 300 Gln Lys Leu Arg Glu Glu Asn Asp Ile Ala Arg Gly Lys Leu Glu Glu 305 310 315 320 Glu Lys Lys Arg Ser Glu Glu Leu Ser Ser Gln Val Gln Phe Leu Tyr                 325 330 335 Thr Ser Leu Leu Lys Gln Gln Glu Glu Gln Thr Arg Val Ala Leu Leu             340 345 350 Glu Gln Gln Met Gln Ala Cys Thr Leu Asp Phe Glu Asn Glu Lys Leu         355 360 365 Asp Arg Gln His Val Gln His Gln Leu His Val Ile Leu Lys Glu Leu     370 375 380 Arg Lys Ala Arg Asn Gln Ile Thr Gln Leu Glu Ser Leu Lys Gln Leu 385 390 395 400 His Glu Phe Ala Ile Thr Glu Pro Leu Val Thr Phe Gln Gly Glu Thr                 405 410 415 Glu Asn Arg Glu Lys Val Ala Ala Ser Pro Lys Ser Pro Thr Ala Ala             420 425 430 Leu Asn Glu Ser Leu Val Glu Cys Pro Lys Cys Asn Ile Gln Tyr Pro         435 440 445 Ala Thr Glu His Arg Asp Leu Leu Val His Val Glu Tyr Cys Ser Lys     450 455 460 <210> 2 <211> 1497 <212> PRT <213> Homo sapiens <400> 2 Met Ser Arg Ala Val Arg Leu Pro Val Pro Cys Pro Val Gln Leu Gly   1 5 10 15 Thr Leu Arg Asn Asp Ser Leu Glu Ala Gln Leu His Glu Tyr Val Lys              20 25 30 Gln Gly Asn Tyr Val Lys Val Lys Lys Ile Leu Lys Lys Gly Ile Tyr          35 40 45 Val Asp Ala Val Asn Ser Leu Gly Gln Thr Ala Leu Phe Val Ala      50 55 60 Leu Leu Gly Leu Arg Lys Phe Val Asp Val Leu Val Asp Tyr Gly Ser  65 70 75 80 Asp Pro Asn His Arg Cys Phe Asp Gly Ser Thr Pro Val His Ala Ala                  85 90 95 Ala Phe Ser Gly Asn Gln Trp Ile Leu Ser Lys Leu Leu Asp Ala Gly             100 105 110 Gly Asp Leu Arg Leu His Asp Glu Arg Gly Gln Asn Pro Lys Thr Trp         115 120 125 Ala Leu Thr Ala Gly Lys Glu Arg Ser Thr Gln Ile Val Glu Phe Met     130 135 140 Gln Arg Cys Ala Ser His Met Gln Ala Ile Ile Gln Gly Phe Ser Tyr 145 150 155 160 Asp Leu Leu Lys Lys Ile Asp Ser Pro Gln Arg Leu Val Tyr Ser Pro                 165 170 175 Ser Trp Cys Gly Gly Leu Val Gln Gly Asn Pro Asn Gly Ser Pro Asn             180 185 190 Arg Leu Leu Lys Ala Gly Val Ile Ser Ala Gln Asn Ile Tyr Ser Phe         195 200 205 Gly Phe Gly Lys Ala Met Pro Trp Phe Gln Phe Tyr Leu Thr Gly Ala     210 215 220 Thr Gln Met Ala Tyr Leu Gly Ser Leu Pro Val Ile Gly Glu Lys Glu 225 230 235 240 Val Ile Gln Ala Asp Asp Glu Pro Thr Phe Ser Phe Ser Ser Gly Pro                 245 250 255 Tyr Met Val Met Thr Asn Leu Val Trp Asn Gly Ser Arg Val Thr Val             260 265 270 Lys Glu Leu Asn Leu Pro Thr His Pro His Cys Ser Arg Leu Arg Leu         275 280 285 Ala Asp Leu Leu Ile Ala Glu Gln Glu His Ser Ser Lys Leu Arg His     290 295 300 Pro Tyr Leu Leu Gln Leu Met Ala Val Cys Leu Ser Gln Asp Leu Glu 305 310 315 320 Lys Thr Arg Leu Val Tyr Glu Arg Ile Thr Ile Gly Thr Leu Phe Ser                 325 330 335 Val Leu His Glu Arg Arg Ser Gln Phe Pro Val Leu His Met Glu Val             340 345 350 Ile Val His Leu Leu Leu Gln Ile Ser Asp Ala Leu Arg Tyr Leu His         355 360 365 Phe Gln Gly Phe Ile His Arg Ser Leu Ser Ser Tyr Ala Val His Ile     370 375 380 Ile Ser Pro Gly Glu Ala Arg Leu Thr Asn Leu Glu Tyr Met Leu Glu 385 390 395 400 Ser Glu Asp Arg Gly Val Gln Arg Asp Leu Thr Arg Val Pro Leu Pro                 405 410 415 Thr Gln Leu Tyr Asn Trp Ala Ala Pro Glu Val Ile Leu Gln Lys Ala             420 425 430 Ala Thr Val Lys Ser Asp Ile Tyr Ser Phe Ser Met Ile Met Gln Glu         435 440 445 Ile Leu Thr Asp Asp Ile Pro Trp Lys Gly Leu Asp Gly Ser Val Val     450 455 460 Lys Lys Ala Val Val Ser Gly Asn Tyr Leu Glu Ala Asp Val Arg Leu 465 470 475 480 Pro Lys Pro Tyr Tyr Asp Ile Val Lys Ser Gly Ile His Val Lys Gln                 485 490 495 Lys Asp Arg Thr Met Asn Leu Gln Asp Ile Arg Tyr Ile Leu Lys Asn             500 505 510 Asp Leu Lys Asp Phe Thr Gly Ala Gln Arg Thr Gln Pro Thr Glu Ser         515 520 525 Pro Arg Val Gln Arg Tyr Gly Leu His Pro Asp Val Asn Val Tyr Leu     530 535 540 Gly Leu Thr Ser Glu His Pro Arg Glu Thr Pro Asp Met Glu Ile Ile 545 550 555 560 Glu Leu Lys Glu Met Gly Ser Gln Pro His Ser Pro Arg Val His Ser                 565 570 575 Leu Phe Thr Glu Gly Thr Leu Asp Pro Gln Ala Pro Asp Pro Cys Leu             580 585 590 Met Ala Arg Glu Thr Gln Asn Gln Asp Ala Pro Cys Pro Ala Pro Phe         595 600 605 Met Ala Glu Glu Ala Ser Ser Pro Thre Gly Gln Pro Ser Leu Cys     610 615 620 Ser Phe Glu Ile Asn Glu Ile Tyr Ser Gly Cys Leu Ile Leu Glu Asp 625 630 635 640 Asp Ile Glu Glu Pro Pro Gly Ala Ser Ser Leu Glu Ala Asp Gly                 645 650 655 Pro Asn Gln Val Asp Glu Leu Lys Ser Met Glu Glu Glu Leu Asp Lys             660 665 670 Met Glu Arg Glu Ala Cys Cys Phe Gly Ser Glu Asp Glu Ser Ser Ser         675 680 685 Lys Ala Glu Thr Glu Tyr Ser Phe Asp Asp Trp Asp Trp Gln Asn Gly     690 695 700 Ser Leu Ser Ser Leu Ser Leu Pro Glu Ser Thr Arg Glu Ala Lys Ser 705 710 715 720 Asn Leu Asn Asn Met Ser Thr Thr Glu Glu Tyr Leu Ile Ser Lys Cys                 725 730 735 Val Leu Asp Leu Lys Ile Met Gln Thr Ile Met His Glu Asn Asp Asp             740 745 750 Arg Leu Arg Asn Ile Glu Gln Ile Leu Asp Glu Val Glu Met Lys Gln         755 760 765 Lys Glu Gln Glu Glu Arg Met Ser Leu Trp Ala Thr Ser Arg Glu Phe     770 775 780 Thr Asn Ala Tyr Lys Leu Pro Leu Ala Val Gly Pro Pro Ser Leu Asn 785 790 795 800 Tyr Ile Pro Pro Val Leu Gln Leu Ser Gly Gly Gln Lys Pro Asp Thr                 805 810 815 Ser Gly Asn Tyr Pro Thr Leu Pro Arg Phe Pro Arg Met Leu Pro Thr             820 825 830 Leu Cys Asp Pro Gly Lys Gln Asn Thr Asp Glu Gln Phe Gln Cys Thr         835 840 845 Gln Gly Ala Lys Asp Ser Leu Glu Thr Ser Arg Ile Gln Asn Thr Ser     850 855 860 Ser Gln Gly Arg Pro Arg Glu Ser Thr Ala Gln Ala Lys Ala Thr Gln 865 870 875 880 Phe Asn Ser Ala Leu Phe Thr Leu Ser Ser His Arg Gln Gly Pro Ser                 885 890 895 Ala Ser Pro Ser Cys His Trp Asp Ser Thr Arg Met Ser Val Glu Pro             900 905 910 Val Ser Ser Glu Ile Tyr Asn Ala Glu Ser Arg Asn Lys Asp Asp Gly         915 920 925 Lys Val His Leu Lys Trp Lys Met Glu Val Lys Glu Met Ala Lys Lys     930 935 940 Ala Ala Thr Gly Gln Leu Thr Val Pro Pro Trp His Pro Gln Ser Ser 945 950 955 960 Leu Thr Leu Glu Ser Glu Ala Glu Asn Glu Pro Asp Ala Leu Leu Gln                 965 970 975 Pro Pro Ile Arg Ser Pro Glu Asn Thr Asp Trp Gln Arg Val Ile Glu             980 985 990 Tyr His Arg Glu Asn Asp Glu Pro Arg Gly Asn Gly Lys Phe Asp Lys         995 1000 1005 Thr Gly Asn Asn Asp Cys Asp Ser Asp Gln His Gly Arg Gln Pro Arg    1010 1015 1020 Leu Gly Ser Phe Thr Ser Ile Arg His Pro Ser Pro Arg Gln Lys Glu 1025 1030 1035 1040 Gln Pro Glu His Ser Glu Ala Phe Gln Ala Ser Ser Asp Thr Leu Val                1045 1050 1055 Ala Val Glu Lys Ser Tyr Ser His Gln Ser Met Gln Ser Thr Cys Ser            1060 1065 1070 Pro Glu Ser Ser Glu Asp Ile Thr Asp Glu Phe Leu Thr Pro Asp Gly        1075 1080 1085 Glu Tyr Phe Tyr Ser Ser Thr Ala Gln Glu Asn Leu Ala Leu Glu Thr    1090 1095 1100 Ser Ser Pro Ile Glu Glu Asp Phe Glu Gly Ile Gln Gly Ala Phe Ala 1105 1110 1115 1120 Gln Pro Gln Val Ser Gly Glu Glu Lys Phe Gln Met Arg Lys Ile Leu                1125 1130 1135 Gly Lys Asn Ala Glu Ile Leu Pro Arg Ser Ser Gln Phe Gln Pro Val Arg            1140 1145 1150 Ser Thr Glu Asp Glu Glu Glu Glu Thr Ser Lys Glu Ser Pro Lys Glu        1155 1160 1165 Leu Lys Glu Lys Asp Ile Ser Leu Thr Asp Ile Gln Asp Leu Ser Ser    1170 1175 1180 Ile Ser Tyr Glu Pro Asp Ser Ser Phe Lys Glu Ala Ser Cys Lys Thr 1185 1190 1195 1200 Pro Lys Ile Asn His Ala Pro Thr Ser Val Ser Thr Pro Leu Ser Pro                1205 1210 1215 Gly Ser Val Ser Ser Ala Ala Ser Gln Tyr Lys Asp Cys Leu Glu Ser            1220 1225 1230 Ile Thr Phe Gln Val Lys Thr Glu Phe Ala Ser Cys Trp Asn Ser Gln        1235 1240 1245 Glu Phe Ile Gln Thr Leu Ser Asp Asp Phe Ile Ser Val Arg Glu Arg    1250 1255 1260 Ala Lys Lys Leu Asp Ser Leu Leu Thr Ser Ser Glu Thr Pro Pro Ser 1265 1270 1275 1280 Arg Leu Thr Gly Leu Lys Arg Leu Ser Ser Phe Ile Gly Ala Gly Ser                1285 1290 1295 Pro Ser Leu Val Lys Ala Cys Asp Ser Ser Pro Pro His Ala Thr Gln            1300 1305 1310 Arg Arg Ser Leu Pro Lys Val Glu Ala Phe Ser Gln His His Ile Asp        1315 1320 1325 Glu Leu Pro Pro Ser Glu Glu Leu Leu Asp Asp Ile Glu Leu Leu    1330 1335 1340 Lys Gln Gln Gln Gly Ser Ser Thr Val Leu His Glu Asn Thr Ala Ser 1345 1350 1355 1360 Asp Gly Gly Gly Thr Ala Asn Asp Gln Arg His Leu Glu Glu Gln Glu                1365 1370 1375 Thr Asp Ser Lys Lys Glu Asp Ser Ser Met Pro Leu Ser Lys Glu Thr            1380 1385 1390 Glu Asp Leu Gly Glu Asp Thr Glu Arg Ala His Ser Thr Leu Asp Glu        1395 1400 1405 Asp Leu Glu Arg Trp Leu Gln Pro Pro Glu Glu Ser Val Glu Leu Gln    1410 1415 1420 Asp Leu Pro Lys Gly Ser Glu Arg Glu Thr Asn Ile Lys Asp Gln Lys 1425 1430 1435 1440 Val Gly Glu Glu Lys Arg Lys Arg Glu Asp Ser Ile Thr Pro Glu Arg                1445 1450 1455 Arg Lys Ser Glu Gly Val Leu Gly Thr Ser Glu Glu Asp Glu Leu Lys            1460 1465 1470 Ser Cys Phe Trp Lys Arg Leu Gly Trp Ser Glu Ser Ser Arg Ile Ile        1475 1480 1485 Val Leu Asp Gln Ser Asp Leu Ser Asp    1490 1495

Claims (11)

The space group is P22 ₁ 2 ₁ and the unit cell dimension is a CEP55-TEX14 protein complex crystal having a = 53.9 Å, b = 102.6 Å and c = 132.5 Å. The CEP (Centrosomal Protein 55) Is expressed by the amino acid sequence of residues 160 to 217 of SEQ ID NO: 1, and TEX14 (Testis-Expressed Gene 14) is represented by the amino acid sequence of residues 792 to 804 of SEQ ID NO: 2.
The method according to claim 1,
Wherein the crystal shows X-ray diffraction data of structure coordinate determinations defined by the data set forth in Table 3 at a resolution of 2.3 A; a CEP55-TEX14 protein complex crystal.
3. The method according to claim 1 or 2,
Residues interacting with the CEP55 of the TEX in the complex include residues Ala793, Gly795, Pro796, Pro797, Tyr801, Pro803 and Pro804; Or Gly795, Pro796, Pro797, Tyr801, Pro803 and Pro804,
Wherein the residues interacting with the TEX of the CEP55 are Trp184, Tyr187, Lys180, Glu183 and Arg191.
The method of claim 3,
Wherein the Gly795, Pro796, and Pro797 residues of the TEX are in contact with the Trp184, Tyr187, Lys180 and Gln183 residues of the CEP55 and the Pro796, Tyr801, and Pro804 residues of the TEX are in contact with the Tyr187 residue of the CEP55. Protein complex.
A composition comprising a protein complex crystal according to any one of claims 1 to 4 and a salt.
A process for producing a composite crystal according to any one of claims 1 to 4, comprising:
a) providing an EABR region of purified CEP55 or a purified CEP55 protein comprising said region, or a CEP55 fragment comprising said region, and a TEX14 peptide, wherein said CEP55 EABR region or CEP55 protein or CEP55 fragment comprises 20 mM Tris -HCL (pH 7.4) solution, the TEX14 peptide was contained in a solution of 150 mM Tris-HCl (pH 8.0)
b) mixing the EABR region of the purified CEP55 or the CEP55 protein or CEP55 fragment, and the TEX14 peptide in a molar ratio of 1: 2; And
c) crystallizing the mixture of step b) in 0.1 M imidazole (pH 8.0), 1 M ammonium phosphate solution.
A method of designing or screening a substance that inhibits or enhances binding between said complexes using the complex crystals of any one of claims 1 to 4.
A method for screening a TEX14 derivative which is an anticancer agent candidate, which binds to a CEP55-EABR region comprising the steps of:
A step of providing a CEP55-EABR area, TEX14 peptide comprising the amino acid sequence shown in CEP55 protein fragments, and AxGPPx ₃ YxPP containing CEP55 full-length protein or CEP55-EABR region including the CEP55-EABR region, said AxGPPx ₃ In YxPP, A is Ala, G is Gly, P is Pro, Y is Tyr, X is any amino acid,
Contacting the CEP55 fragment comprising the CEP55-EABR region, the CEP55 full-length or CEP55-EABR region comprising the CEP55-EABR region and the TEX14 peptide with the test material, wherein the test material comprises the TEX14 As a TEX14 derivative capable of competitive binding with peptides,
Measuring the binding of the TEX14 peptide to the CEP55-EABR region in the presence of the test substance; And
Selecting the test substance as an anticancer candidate substance when binding of the TEX14 peptide to the CEP55-EABR region is decreased in the test group that is contacted with the test substance as compared with the control group not in contact with the test substance as a result of the measurement Way.
9. The method of claim 8,
Wherein the CEP55-EABR region is derived from human by an amino acid sequence of 160 to 217 residues of SEQ ID NO: 1 and the CEP55 total length is represented by an amino acid sequence of SEQ ID NO: 1.
9. The method of claim 8,
Wherein said TEX14 peptide is represented by an amino acid sequence of residues 793 to 804 of SEQ ID NO: 2, an amino acid sequence of residues 792 to 804 of SEQ ID NO: 2, or residues 791 to 804 of SEQ ID NO: 2.
9. The method of claim 8,
Wherein the TEX14 derivative is modified in such a manner that any one of Ala, Gly, Pro, Pro, Tyr, Pro and Pro in the polypeptide of the amino acid sequence of AxGPPx ₃ YxPP is increased in the EABR region of the TEX14 derivative, Wherein said modification is a chemical modification of a substitution or amino acid side chain of an amino acid residue.

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