CN101308142A - Novel function of disheveled protein and beta-catenins interactive function in classical Wnt signal transduction path - Google Patents
Novel function of disheveled protein and beta-catenins interactive function in classical Wnt signal transduction path Download PDFInfo
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Abstract
The invention discloses a screening method of a regulator which modulates the interaction between disheveled protein and beta-catenin. The invention also discloses a matter which can inhibit the interaction between disheveled protein and beta-catenin, namely, a Dvl 1-C polypeptide. The invention reveals the interaction between disheveled protein and beta-catenin in the Wnt signal transduction pathway for the first time, thus affecting the downstream Wnt signal transduction. As abnormal activation of the downstream Wnt signal transduction in the pathway is closely related to the occurrence and development of tumor, so the method provides a new target for screening drugs inhibiting the occurrence and development of tumor.
Description
Technical field
The invention belongs to the cell biology field, be specifically related to interaction protein in the classical Wnt signal transduction path and uses thereof.
Background technology
Classical Wnt signal transduction path is regulated and has been controlled many life processes, and these processes comprise growth, growth, disease, aging and death of biosome or the like; The differentiation that also comprises cellular morphology and function with keep, immunity, stress, cell carcinogenesis and Apoptosis or the like.The Wnt signal transduction pathway has the conservative property of height between different plant species, by the research to fruit bat, Xenopus laevis, mouse isotype biology, established the branch subframe of classical Wnt signal transduction path substantially.In the growth course, specific time and particular environment are induced down, some tissue or cell colony secretion Wnt albumen, and bind receptor FZ (Frizzled) family member and co-receptor low-density lipoprotein LRP5/6 are passed to signal in the cell.When not having the Wnt signal stimulus, white (the β-catenin) participate in of the key molecule beta-catenin of this bars transduction pathway by Axin, colon cancer inhibiting factor (APC), glycogen synthase kinase 3 β (GSK3 β), casein kinase 1 (CK1), and the huge protein complex of β-TrCP albumen formation, under GSK3 β and CK1 effect, be subjected to the phosphorylation mark, and then after the protein mediated ubiquitin modification of β-TrCP, degraded by proteasome.Under the Wnt signal stimulus, the forward of Wnt approach such as Dishevelled (Dvl) in the born of the same parents and Frat is regulated molecule and is broken up the degraded compound that Axin, APC and GSK3 etc. form, and Axin is with film and then degraded by LRP.The pancake of beta-catenin white phosphorus acidifying water is low, and then can accumulation in a large number in tenuigenin.The part beta-catenin enters nucleus in vain, interact with the albumen (as TCF-4) of examining the interior transcription factor that contains HMG-Box-lymphocyte enhancer 1/T cell transcription factor (LEF1/TCF) family, start opening (the GumbinerBM.1998.van Noort M.﹠amp of downstream target gene; Clevers is A.﹠amp H.2002.Wodarz; Nusse R.1998.).
A series of important early stage incidents such as the Wnt signal transduction pathway influences not only that axon in the embryonic development is induced, germinal layer foundation, body segment differentiation, tissue or organ formation participate in somatic differentiation and polarization; And the abnormal activation of Wnt signal pathway is verified closely related with generation kinds of tumors in adult.Discover; about 90% colon cancer all is owing to the sudden change of the activity of Wnt signal transduction pathway causes; the abnormal activation of Wnt signal pathway also takes place relevant with a series of tumour in addition; such as small intestine adenoma, liver cancer, cancer of the stomach, esophageal adenocarcinoma, malignant mela noma, breast cancer (Giles RH; van Es JH, Clevers is H.2003) in all found the sudden change of signaling molecule in Wnt abnormal signal or the transduction pathway.Therefore to the Molecular Study and the understanding of Wnt signal transduction pathway, for explore anti-tumor medicine, to seek new drug target and theoretical foundation significant.
The biological function of classical Wnt signal transduction path mainly is to be achieved by the downstream target gene expression.And a lot of target genes play an important role in cell proliferation such as c-myc, cyclinD1.Studies show that, in a lot of cancer cell, can detect the beta-catenin high expressed of a large amount of accumulation in nucleus and c-myc, cyclinD1 in vain, illustrate that the transcription complex in Wnt signal transduction pathway downstream in these tumour cells is in high transcriptional activity state.Because the active height of transcription complex is directly connected to the Wnt signal effect, to the molecular regulation Study on Mechanism of transcription complex, also just has crucial meaning so.
To sum up, although at present have gained some understanding for the molecular mechanism of classical Wnt signal pathway in this area, however still unclear for the links such as startup of the abnormal activation of this approach.Therefore, this area needs further to be studied some key influence factors in the Wnt signal pathway, in the hope of finding the material of scalable Wnt signal transduction pathway, thus the relevant cell incident of regulation and control Wnt signal transduction.
Summary of the invention
The object of the present invention is to provide a kind of method of regulating disheveled protein and the white interactional correctives of beta-catenin of screening.
Another object of the present invention is to provide a kind of active fragment of disheveled protein, it can be used for preparation and regulates (particularly suppressing) disheveled protein and the white interactional correctives of beta-catenin (particularly inhibitor), or is used for screening adjusting disheveled protein and the white interactional correctives of beta-catenin.
In a first aspect of the present invention, provide a kind of screening regulate disheveled protein (Dishevelled, Dvl) with beta-catenin white (β-catenin, the method for interactional correctives of β-cat) said method comprising the steps of:
(a) the white interactional system of candidate substances and disheveled protein and beta-catenin is contacted; With
(b) detect candidate substances to disheveled protein and beta-catenin white between interactional influence;
Wherein, if described candidate substances can suppress or promote the interaction between white of disheveled protein and beta-catenin, show that then this candidate substances is adjusting disheveled protein and the white interactional correctives of beta-catenin.
In another preference of the present invention, described interaction is that disheveled protein and beta-catenin white hair are given birth to combination.
In another preference of the present invention, described disheveled protein is selected from: disheveled protein 1 (Dvl 1), disheveled protein 2 (Dvl 2) or disheveled protein 3 (Dvl 3).
In another preference of the present invention, step (a) comprising: add candidate substances in disheveled protein and the white interactional system of beta-catenin; With
Step (b) comprising: detect disheveled protein and the beta-catenin situation that interacts in vain, and with control group relatively, wherein said control group be do not add described candidate substances, disheveled protein and the white interactional system of beta-catenin;
(preferably significantly be lower than, if disheveled protein and the white interaction of beta-catenin are lower than statistically in the test group as low 20%; Preferred low 40%) control group just shows that this candidate substances is the white interactional correctives of the inhibition of dishevelled proteins and beta-catenin;
If disheveled protein and the white interaction of beta-catenin are higher than statistically and (preferably significantly are lower than, as high by 20% in the test group; Preferred high by 40%) control group, just show that this candidate substances is to promote disheveled protein and the white interactional correctives of beta-catenin.
In another preference of the present invention, described system is selected from: solution system, cell system or organizational framework.
In another preference of the present invention, also comprise Wnt albumen in the described system.
In another preference of the present invention, described Wnt albumen includes but not limited to: Wnt1 albumen, Wnt3a albumen or Wnt8 albumen.
In another preference of the present invention, described method also comprises: for the correctives that filters out, further test its influence for Wnt signal pathway downstream gene; If it can raise the expression of Wnt signal pathway downstream gene, show that then this correctives is to promote disheveled protein and the beta-catenin interactional promoter between white; If it can reduce the expression of Wnt signal pathway downstream gene, show that then this correctives is the inhibition of dishevelled proteins and the beta-catenin interactional inhibitor between white.
In another preference of the present invention, Wnt signal pathway downstream gene is (but being not limited to) for example: the c-myc gene in the mammalian cell, cyclinD1 gene, Axin2 gene, LEF1 gene, PPARdelta gene; Or vox gene, tbx6 gene in fish (as the zebra fish) cell.
In another preference of the present invention, described Wnt signal pathway downstream gene abnormal activation (as raising) causes the generation of tumour (as colon cancer).
In a second aspect of the present invention, provide a kind of adjusting disheveled protein and white interactional correctives of beta-catenin that obtains by described method.
In another preference of the present invention, described correctives is an inhibitor, by the inhibition of dishevelled proteins and the white interaction of beta-catenin, thereby suppresses Wnt signal pathway downstream gene abnormal activation.
In another preference of the present invention, described correctives is an inhibitor, and described inhibitor is a peptide species, contains 496～695 amino acids sequences among the SEQ ID NO:5.
In another preference of the present invention, this polypeptide also contains the amino acid sequence of nuclear localization signal albumen (NLS).
In another preference of the present invention, the amino acid sequence of described nuclear localization signal albumen is positioned at the C end of described polypeptide.
In another preference of the present invention, described correctives is a kind of nucleic acid molecules or its transcript (mRNA), and described nucleic acid molecules contains the nucleotide sequence shown in the SEQ ID NO:6.
In a third aspect of the present invention, a kind of purposes of disheveled protein is provided, be used for screening and regulate disheveled protein and the white interactional correctives of beta-catenin.
Others of the present invention are because the disclosure of this paper is conspicuous to those skilled in the art.
Description of drawings
Fig. 1 has shown the distribution situation of Dvl3 in normal structure and the colon cancer sample.Being presented at by figure has a large amount of Dvl3 accumulation in the nucleus of colon cancer sample, and Dvl3 has distribution at tenuigenin and nucleus in normal cell, and the amount in the kytoplasm is far above in the nuclear.Wherein, position shown in the arrow is a nucleus in the enlarged drawing of the upper right corner.
Fig. 2 A has shown that β-cat itself can not be by obtaining with anti-Flag antibodies immunoprecipitation, only in transfection under the situation of Dvl3-NLS-Flag endogenous β-cat could in the immunoprecipitation sample, detect.
Fig. 2 B has shown in external Pull Down experiment can co-precipitation arrive His-Dvl3 with anti-myc antibody mediated immunity precipitation His-β-cat-myc.
Fig. 2 C only shown under the condition that Wnt3a stimulates Dvl3 could co-precipitation to endogenous β-cat.
Can co-precipitation when Fig. 3 A has shown with anti-myc antibody mediated immunity precipitation His-β-cat-myc to His-C-flag.
Fig. 3 B has shown at cotransfection under the situation of C-NLS-Flag, can not co-precipitation arrive β-cat-myc with anti-HA antibody mediated immunity deposit D vl1-NLS-HA.
Fig. 3 C has shown that Dvl 1-C-NLS can suppress the Topflash activity that Wnt3a activates effectively.
Fig. 3 D has shown that in the SW480 cell Dvl 1-C-NLS is dose-dependent to the inhibition of Topflash activity, and wherein, "+" expression contains 100ng Dvl-C-NLS, and " ++ " expression contains 200ngDvl-C-NLS.
Fig. 3 E has shown under Wnt3a stimulates, β-cat and TCF-4 can be attached on the target gene c-myc promoter of Wnt signal downstream, in the cell of transfection Dvl 1-C-NLS fragment, the ability that β-cat is attached on the target gene c-myc promoter of downstream reduces significantly, and TCF-4 is unaffected substantially.
Fig. 4 A has shown that the expression of vox and tbx6 descends than the control group embryo significantly among the embryo of mRNA of injection Dvl 1-C-NLS.
After Fig. 4 B had shown the mRNA of injection Dvl-C-NLS, tbx6 and vox expressed embryo's normal and that obviously reduce statistical conditions.The expression that tbx6 among 51% the embryo is arranged after 99 embryos statistics there is obvious downward modulation, the expression of vox is had obvious downward modulation (n=71, embryo's number of 83,89,99 expression statistics respectively) 59% embryo is arranged after 83 embryos' statistics.
Embodiment
The inventor is through extensive and deep research, find first in classical Wnt signal transduction path, disheveled protein (Dvl) can white (β-cat) can interact under the condition that Wnt activates, thereby the abnormal activation of the signal transduction pathway in the accumulation of promotion β-cat and Wnt downstream with beta-catenin.Because the abnormal activation of the signal transduction in Wnt downstream and the generation of tumour development are closely related, therefore according to the interaction mechanism between this newfound Dvl and the β-cat, realize suppressing tumorigenic new drug target thereby can provide by the signal transduction that suppresses the Wnt downstream.Finished the present invention on this basis.
Disheveled protein and active fragment thereof
At present disheveled protein (Dvl) family member who finds has three kinds, i.e. Dvl1, Dvl2, Dvl3, and these three kinds of albumen have higher homology.In research in the past, Dvl albumen is used as the transmission of a cytoplasm protein participating in classical Wnt signal at first, thereby its major function is to accept the Wnt signal to impel β-cat to accumulate in tenuigenin can to enter in a large number and start transcribing of downstream target gene in the nucleus in tenuigenin.Recently, those skilled in the art find not only transduction Wnt signal in tenuigenin of Dvl albumen, and also have important effect in nucleus, but its mechanism of action is not still understood.Yet the inventor has had further announcement through studying for a long period of time to this mechanism of action.
In the present invention, used Dvl albumen can be naturally occurring, can be purified and separates from mammal such as it.Preferably, the amino acid sequence of described naturally occurring Dvl 1 albumen can be substantially the same with the amino acid sequence shown in the SEQ IDNO:2; The amino acid sequence of Dvl 2 albumen can be substantially the same with the amino acid sequence shown in the SEQ ID NO:3; The amino acid sequence of Dvl 3 albumen can be substantially the same with the amino acid sequence shown in the SEQ ID NO:4.In addition, described Dvl albumen also can be the reorganization preparation, such as producing the Dvl albumen of reorganization according to the gene recombination technology of routine.Preferably, the present invention adopts the Dvl albumen of reorganization.In addition, any bioactive version that does not influence Dvl albumen all can be used among the present invention, the for example active fragment of Dvl albumen, derivant, as long as it also has and the interactional function of β-cat, and can not bring other harmful effect, as being the protein fragments that contains Dvl albumen the 496th～695 amino acids.
The present invention also provides a kind of polypeptide of being made up of Dvl 1 albumen the 496th～695 amino acids, inventor general's called after Dvl 1-C polypeptide (fragment), and this polypeptide has the amino acid sequence shown in the SEQ ID NO:5.
Described Dvl 1-C polypeptide can be recombinant polypeptide, natural polypeptides, synthetic polypeptide, preferably synthetic fragment.Described Dvl 1-C polypeptide can be the product of natural purifying, or the product of chemosynthesis, or uses recombinant technique to produce from protokaryon or eucaryon host (for example, bacterium, yeast, higher plant, insect and mammalian cell).In addition, any bioactive version that does not influence Dvl 1-C polypeptide all can be used among the present invention, for example the active fragment of Dvl 1-C polypeptide, derivant or the one or more amino acid of process (are generally 1-50, preferably 1-30, more preferably 1-20,1-10 best) disappearance, insert and/or replace the variant that forms, as long as it also has and the interactional function of β-cat.
The present invention also provides the encoding gene of Dvl 1-C polypeptide.Described encoding gene can be strand or double-stranded.
Preferably, in order to make Dvl 1-C be positioned in the nucleus, Dvl 1-C can be connected with nuclear localization signal (going into nuclear signal); Preferred, can be at the terminal nuclear localization signal that adds of the C of Dvl 1-C.Therefore, the present invention also provides a kind of Dvl 1-C-NLS albumen, its can with β-cat protein-interacting.In cell, described Dvl 1-C-NLS is positioned in the nucleus, by combining with β-cat protein competition, suppresses the interaction of the interior Dvl of nucleus and β-cat.
Described nuclear localization signal is a kind of signal peptide, and it can help albumen to enter nucleus.The present invention can adopt any nuclear localization signal that does not influence Dvl 1-C and β-cat protein-interacting.For example, it is as follows that described nuclear localization signal has amino acid sequence: DPKKKRKV DPKKKRKV DPKKKRKV (SEQ ID NO:7).
In case obtained relevant sequence, just can obtain relevant sequence in large quantity with recombination method or synthetic method.Recombination method normally is cloned into carrier with it, changes cell again over to, separates obtaining relevant sequence then from the host cell after the propagation by conventional method.
Its active fragment of beta-catenin bletilla
Beta-catenin is white, and (β-cat) is a kind of multi-functional intracellular protein, not only participates in cell and intercellular sticking, and can mediate in the born of the same parents of Wnt signal and transduce.The inventor finds that Dvl albumen can interact with β-cat albumen.
In the present invention, used β-cat albumen can be naturally occurring, can be purified and separates from mammal such as it.Preferably, the amino acid sequence of described naturally occurring β-cat albumen can be substantially the same with the amino acid sequence shown in the SEQ IDNO:1.In addition, described β-cat albumen also can be the reorganization preparation, such as producing the β-cat albumen of reorganization according to the gene recombination technology of routine.Preferably, the present invention adopts the β-cat albumen of reorganization.
In addition, any bioactive version that does not influence β-cat albumen all can be used among the present invention, for example β-the bioactive fragment of cat albumen, derivant as long as it also has the function with the Dvl protein-interacting, and can not bring other harmful effect.
Use and screening technique
In research process, the inventor by external in conjunction with experiment, inside and outside, endogenous in conjunction with experiment confirm exist between Dvl and the β-cat and interact, and in eukaryotic and model organism zebra fish, carry out various functional analyses respectively and test, illustrated to exist between Dvl albumen and β-cat albumen and interacted, and described interaction has produced influence for classical Wnt signal transduction path, suppresses the genetic transcription that described interaction can suppress the classical Wnt signal transduction path downstream.
At first, the inventor utilizes immunohistochemistry technique to detect the distribution situation of Dvl3 in colon cancer tissue and the normal structure.Found that Dvl3 has a large amount of accumulation in nucleus in a lot of colon cancer samples, and Dvl has distribution at tenuigenin and nucleus in normal histocyte, and the amount in the kytoplasm is far above in the nuclear.Therefore, as seen, Dvl albumen can accumulation in a large number in nucleus in tumor tissues.
Then, the inventor utilizes the immunoprecipitation technology, has analyzed the interaction situation of Dvl and β-cat albumen in nucleus.Found that, β-cat itself can not be by obtaining with Flag antibodies immunoprecipitation, only in transfection under the situation of Dvl3-NLS-Flag endogenous β-cat could in the immunoprecipitation sample, detect, this prompting Dvl in cell can and β-cat protein interaction takes place.Further external Pull Down experiment showed, with myc antibody mediated immunity precipitation His-β-cat-myc can co-precipitation arrive His-Dvl3, and protein interaction is direct between this explanation Dvl and the β-cat.And Dvl and β-catenin exist protein interaction to be subjected to the adjusting of Wnt signal in vivo.
Further, the inventor has furtherd investigate the function of interaction in classical Wnt signal transduction path between Dvl and the β-cat by making up the Dvl deletion mutant.There is direct interaction in conjunction with finding between His-C-flag and the His-β-cat-myc by external.Further cotransfection contains C-NLS-Flag, Dvl1-NLS-HA and β-catenin-myc plasmid into nuclear signal in HEK293T, and competition finds that in conjunction with experiment C-NLS-Flag can block the interaction between Dvl and the β-catenin.
The C fragment that the inventor also will have nuclear signal (NLS) is each separately transfected in the HEK293T cell, and the activity by the Topflash reporter gene detects their influences to the transcriptional activity of transcription complex.The result confirms that the Dvl-C-NLS fragment is that the downstream of β-cat in nuclear plays a role, and suppresses the transcriptional activity of Wnt3a activation by the interaction of blocking-up Dvl and β-catenin.
The interaction partners Wnt3a that the inventor also utilizes the method (Chip) of nuclear chromatin immunoprecipitation to study between Dvl and the β-cat activates the influence that the front and back transcription complex forms.The result shows that the interaction between Dvl and the β-catenin is essential for the formation of the transcription complex that Wnt3a induces.
The inventor also utilizes model organism zebra fish to detect the function of interaction in biosome between Dvl and the β-catenin.The mRNA that injects Dvl-C-NLS period at zebrafish embryo one to four cell, respectively in two periods of embryonic development: period gathered in the crops the embryo by animal pole cell envelope 40% and embryonic shield, utilizes the technology of in situ hybridization to detect the expression of Wnt signal pathway downstream gene vox and tbx6.Found that the expression of vox and tbx6 descends than the control group embryo significantly among the embryo of the mRNA of injection Dvl-C-NLS.This result shows, the function of the interaction between Dvl and the β-cat in the growth course of zebra fish.
New discovery based on the inventor, Dvl albumen or its active fragment and β-cat albumen or its active fragment Study of Interaction there is many-sided purposes, described purposes includes, but is not limited to: screening suppresses Dvl albumen or its active fragment and β-cat albumen or the interactional material of its active fragment, thereby the abnormal activation that suppresses classical Wnt signal transduction path, with control because the disease that the abnormal activation of classical Wnt signal transduction path causes, as tumour.
Therefore, the invention provides a kind of correctives method of regulating Dvl albumen and β-cat protein-interacting of screening, by interpolation material standed for to be screened in the reaction system of Dvl albumen and β-cat protein-interacting, and the interaction situation of observation Dvl albumen and β-cat albumen is screened.Said method comprising the steps of:
(a) candidate substances is contacted with the system of Dvl albumen with β-cat protein-interacting; With
(b) detect the influence of candidate substances to Dvl albumen and β-cat protein-interacting;
Wherein, if described candidate substances can suppress or promote Dvl albumen and β-cat protein-interacting, show that then this candidate substances is to regulate the correctives of Dvl albumen and β-cat protein-interacting.
Preferred, step (a) comprising: add candidate substances in the system of Dvl albumen and β-cat protein-interacting; And step (b) comprising: detect Dvl albumen and β-cat protein-interacting situation, and with the control group comparison, wherein said control group is not add system described candidate substances, Dvl albumen and β-cat protein-interacting.
As the condition of screening, (preferably significantly be lower than, as hanging down 20% if Dvl albumen and β-cat protein-interacting are lower than statistically in the test group; Preferred low 40%) control group just shows that this candidate substances is to suppress the correctives of Dvl albumen and β-cat protein-interacting; If Dvl albumen and β-cat protein-interacting are higher than statistically and (preferably significantly are lower than, as high by 20% in the test group; Preferred high by 40%) control group, just show that this candidate substances is the correctives that promotes Dvl albumen and β-cat protein-interacting.
As used herein, described " disheveled protein and (or " with ") white interactional system of beta-catenin " refers to a kind of system, wherein contain disheveled protein and beta-catenin is white, and disheveled protein and beta-catenin can interact in vain (as mutually combining).
In the present invention, described system is selected from (but being not limited to): solution system, cell system, subcellular fraction system, organizational framework, organ systems or animal system.
As optimal way of the present invention, also comprise in the described system: the Wnt family protein.For example, described Wnt family protein includes, but is not limited to: Wnt1, Wnt3a, Wnt8.
Regulate disheveled protein and white interactional material of beta-catenin and composition thereof
The correctives that goes out by the said method preliminary screening can constitute a screening storehouse so that people finally can therefrom filter out can be for regulating the useful medicine of Wnt signal pathway.
It is a kind of by the adjusting Dvl albumen of described method screening acquisition and the material of β-cat protein-interacting that the present invention also provides.For example, described material is the material that suppresses Dvl albumen and β-cat protein-interacting, as inhibitor, antagonist, retarding agent; Perhaps, described material is the material that promotes Dvl albumen and β-cat protein-interacting, as promoter, activator.Usually, suppress Dvl albumen and the β-material of cat protein-interacting and can suppress the abnormal activation of Wnt signal pathway, thereby the disease that the abnormal activation that can prevent and treat the Wnt signal pathway causes, therefore, Dvl albumen and the β-inhibitor of cat protein-interacting, antagonist, retarding agent are particularly useful.
In addition, at the Dvl albumen of the present invention's discovery and the interaction property between β-cat albumen, also can design some and stop Dvl albumen and the protein bound material of β-cat by competition combination or wherein a kind of albumen of inhibition.Except as a kind of optimal way of the present invention, provide a kind of and can stop the material of Dvl albumen and β-cat protein-interacting in the born of the same parents, i.e. Dvl 1-C polypeptide in conjunction with β-cat albumen by competitiveness based on Dvl 1 design.Preferred, Dvl 1-C polypeptide is connected with nuclear localization signal (NLS), thereby it is after being imported into cell, can directly be positioned in the nucleus, in nucleus, stop the interaction of Dvl albumen and β-cat albumen.
The correctives of described Dvl albumen and β-cat protein-interacting can be applied to individuality, is used to regulate the activity of Wnt signal pathway in the cell.Usually, these materials can be formulated in nontoxic, inertia with pharmaceutically acceptable aqueous carrier medium in, wherein β H is about 5-8 usually, and preferably β H is about 6-8, although the pH value can change to some extent with being prepared Substance Properties and illness to be treated.The pharmaceutical composition for preparing can carry out administration by conventional route, comprising (but being not limited to): intramuscular, intravenous or subcutaneous administration.
The present invention also provides a kind of composition (as pharmaceutical composition), and it contains (a) the Dvl albumen of safe and effective amount and the correctives of β-cat protein-interacting, and (b) pharmaceutically acceptable carrier or excipient.The preferred a kind of inhibitor of the correctives of described Dvl albumen and β-cat protein-interacting is as Dvl 1-C polypeptide.
At present, known dna sequence, it is routine techniques in this area that this target DNA sequence is incorporated in various known dna molecules (as carrier) and the cell, as long as general personnel can operate easily according to prompting of the present invention.In addition, various forms of sudden changes being incorporated in the various dna moleculars also is technology well known in the art.Persons skilled in the art all know how to select appropriate carriers, promoter, enhancer and host cell.
With the recombinant DNA transformed host cell also is routine techniques well known to those skilled in the art.When the host was prokaryotes such as Escherichia coli, the competent cell that can absorb DNA can be used CaCl in exponential growth after date results
2Method is handled, and used step is well-known in this area.Alternative is to use Mg Cl
2If desired, transforming also the method for available electroporation carries out.When the host is an eucaryote, can select following DNA transfection method for use: coprecipitation of calcium phosphate method, conventional mechanical method such as microinjection, electroporation, liposome packing etc.The transformant that obtains can be cultivated with conventional method, expresses desired polypeptides.
In the present invention, interaction and interactional power can adopt multiple technology well known to those skilled in the art between detection albumen and the albumen, such as GST sedimentation techniques, display technique of bacteriophage, yeast two-hybrid system or co-immunoprecipitation technology.
In a kind of optimal way of the present invention, adopt the immunoprecipitation technology to verify that the specificity between albumen and the albumen interacts.The principle of described co-immunoprecipitation technology is: keeping under the condition of protein-protein interaction, and results and cell lysis, immunoprecipitation destination protein specifically from cell extract is then by method separating immune sediments such as electrophoresis.Co-precipitation with albumen of known features can be adopted the antibody that resists this albumen, by detecting such as the Western trace.In addition, if cell carried out mark with label before cracking, then can observe the albumen of co-precipitation by radioautograph or other immunological technique.
Major advantage of the present invention is:
(1) be disclosed in first in the classical Wnt signal transduction path, Dvl albumen can interact under the condition that Wnt activates with β-cat albumen, thus the abnormal activation of the signal transduction pathway in the accumulation of promotion β-cat and Wnt downstream.
(2), therefore,, thereby can provide the signal transduction that suppresses the Wnt downstream to realize suppressing tumorigenic new drug target according to new discovery of the present invention because the generation of the abnormal activation of the signal transduction in known Wnt approach downstream and tumour development is closely related.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: lab guide (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise number percent and umber calculate by weight.
Unless otherwise defined, the same meaning that employed all specialties and scientific words and one skilled in the art are familiar with in the literary composition.In addition, any method similar or impartial to described content and material all can be applicable among the present invention.The usefulness that preferable implementation method described in the literary composition and material only present a demonstration.
Embodiment 1
Dvl protein navigates in the nucleus in colon cancer tissue
The inventor utilizes conventional immunohistochemistry technique to detect the colon cancer tissue chip, organization chip takes off cured, rehydration, multiple, the dyeing of antigen hot repair through dimethylbenzene, carry out micro-imaging then and detect (chip that is adopted derives from Xinchao Biotech Co., Ltd., Shanghai, and detection method is with reference to its appended operation instructions).
The result contrasts normal histocyte as shown in Figure 1, has to find in the sample about 30% that Dvl3 has a large amount of accumulation in nucleus in 93 routine colon cancer samples; And Dvl3 has distribution at tenuigenin and nucleus in normal cell, and the amount in the kytoplasm is far above in the nuclear.
Embodiment 2
Dvl and β-cat interacts
1.Dvl and the acquisition of β-cat gene
The acquisition of Dvl1 gene:
The design primer is as follows: forward: 5 '-CTAGTCTAGAGGCGGAGACCAAAATCA-3 ' (SEQID NO:9), oppositely: 5 '-CTAGCTCGAGCATGATGTCCACAAAGAA-3 ' (SEQ ID NO:10).Mouse cDNA storehouse with the conventional method preparation is a template, obtains containing the sequence of total length Dvl1 by the PCR reaction.
The acquisition of Dvl3 gene:
The design primer is as follows: forward: 5 '-CTAGTCTAGAGGGCGAGACCAAGAT-3 ' (SEQIDNO:11), and is reverse: 5 '-CTAGCTCGAGCATCACATCCACAAAGA-3 ' (SEQ ID NO:12).People cDNA storehouse with the conventional method preparation is a template, obtains containing the sequence of total length Dvl3 by the PCR reaction.
The acquisition of β-cat gene:
Utilize conventional method respectively at 5 ' and 3 ' tip designs forward of β-cat albumen (sequence is seen SEQ ID NO:1) encoding gene and reverse primer (in primer, adding the XbaI/XhoI restriction enzyme site), the people cDNA storehouse that utilize described primer, prepares with conventional method is a template, obtains containing the sequence of total length β-cat by the PCR reaction.
2.Dvl in cell can and β-cat protein interaction takes place
In order to verify the function of Dvl in nucleus, the inventor adopts conventional method to introduce a nuclear localization signal in the back of Dvl gene, by the transient transfection transfered cell, carries out functional analysis.
In order to confirm this interaction, the inventor has at first made up the carrier pCMV/Dvl3-NLS-Flag that carries Dvl 3-NLS-Flag, method is as follows: the cohesive end that utilizes the XbaI/NotI restriction enzyme site that two ends have behind the primer annealing, (sequence is: 5 '-CTAGACTAGACCTAGCTCGAGGATCCAAAAAAGAAGAGAAAGGTAGATCCAAAAAA GAAGAGAAAGGTAGATCCAAAAAAGAAGAGAAAGGTAG-3 ' (SEQ ID NO:8)) introduce pCMV/Flag (available from Bioscience with the gene order of NLS, carry the Flag label) in, pCMV/NLS-Flag formed; And then utilize the XbaI/XhoI enzyme to cut Dvl3, and enzyme is cut product cloning go in the pCMV/NLS-Flag that same enzyme is cut, obtain recombinant vector pCMV/Dvl3-NLS-Flag.
Then, the pCMV/Dvl3-NLS-Flag recombinant vector of this external source of transient transfection in people's embryonic kidney cell HEK293T cell (ATCC), (Wnt3a obtains by the culture supernatant of results wnt3a/NIH3T3 cell, and wnt3a/NIH3T3 cell and cultural method thereof be referring to Yin Zongsheng, Zhang Hui etc. to add Wnt3a after the transfection, " The Fourth Military Medical University's journal ", Vol.26, No.24, p2212-2215,2006) irritation cell, harvesting is with 1 * lysis buffer (NP401%, glycerine 10%, NaCl 0.135M, Tris-Cl PH8.0, PMSF 0.5M, PPi 1mM, NaF 10mM, Na
3VO
42mM) cell lysis, centrifugal (14, get supernatant 000rpm/10min), add anti-Flag antibody (anti-Flag, available from Sigma) and protein A/G Plusagarose pearl, low temperature rotation 3 hours, centrifugal (5000rpm/1min) collects pearl, by the Western trace detect on the pearl with albumen (said process is that Pull Down tests).The results are shown in Figure 2A.
Shown in Fig. 2 A, β-cat itself can not be by obtaining with anti-Flag antibody (anti-flag) binding immunoassay precipitation, only in transfection under the situation of Dvl3-NLS-Flag endogenous β-cat could in the immunoprecipitation sample, detect.The above results prompting, Dvl in cell can and β-cat protein interaction takes place.
3.Dvl and beta-catenin white between protein interaction be direct
In order to determine whether the protein interaction between Dvl and the β-cat is direct effect, and the inventor has at first made up the recombinant vector that contains His-Dvl3 and His-β-cat-myc.The construction of recombinant vector that contains His-Dvl3 is as follows: cut Dvl3 with the SalI/NotI enzyme, enzyme is cut product cloning go among the pET28c/Hi s (available from Novagen, carrying the His label) that cuts through same enzyme, obtain recombinant vector pET28c/His-Dvl3.The construction of recombinant vector that contains His-β-cat-myc is as follows: connect the conventional myc label that uses at the C of β-cat gene end, form β-cat-myc, design of primers and round pcr by routine is provided with Sal I/Not I restriction enzyme site at the end of β-cat-myc then, be cloned into the pET28c/His carrier cut through same enzyme (available from Novagen, carry the His label) in, recombinant vector pET28c/His-β-cat-myc obtained.
Then, utilize Bacillus coli expression to obtain His-Dvl3 and His-β-cat-myc (β-cat-myc).Recombinant expression plasmid pET28c/His-Dvl3 and pET28c/His-β-cat-myc is transformed into escherichia coli BL21 respectively, inoculates the monoclonal bacterium colony in the LB nutrient solution, 37 ℃ of overnight incubation; Get the above-mentioned bacterium that spends the night and be inoculated at 1: 100 in the fresh LB nutrient culture media, 22 ℃ are cultured to A600 is 0.6～0.8, adds isopropylthiogalactoside (IPTG) respectively and reaches final concentration 1 μ M, cultivates 16h for 22 ℃.Centrifugal collection thalline is dissolved in thalline in the broken bacterium damping fluid that contains the 0.5mg/ml lysozyme, frozen-thawed, three times repeatedly, add DNase I to final concentration be 25mg/ml and MgSO
4To final concentration 20mM, digestion is on ice got supernatant behind the high speed centrifugation (16000rpm/20min) and is obtained brokenly the bacterium extract to not sticking, carries out external Pull Down experiment (experimental technique is with aforementioned).
Shown in Fig. 2 B, in external Pull Down experiment, can co-precipitation arrive His-Dvl3 with anti-myc antibody (available from Sigma) immunoprecipitation His-β-cat-myc, protein interaction is direct between this explanation Dvl and the β-cat.
4.Dvl and beta-catenin interacts between white and needs the stimulation of Wnt3a
In order further to confirm whether β-cat and Dvl exist interaction endogenous, the inventor gathers in the crops Wnt3a respectively to be stimulated or inirritative HEK293T cell (non-transfection), separating nucleus obtains the nuclear consitution supernatant, use IgG and anti-Dvl3 antibody (anti-Dvl3 is available from Santz Cruz) the endogenous Dvl3 of immunoprecipitation respectively.
Shown in Fig. 2 C, only Dvl3 could co-precipitation arrive endogenous β-cat under the condition that Wnt3a stimulates.
To sum up, these test consistent the proof, and Dvl and β-cat exist protein interaction and this interaction to be subjected to the adjusting of Wnt3a signal in vivo.
Embodiment 3
Interaction between Dvl and beta-catenin are white is in the function of classical Wnt signal transduction path
The inventor carries out deletion mutation by the deletion mutation technology of routine to Dvl 1, and verifies the interaction situation of each mutant and β-cat.Found that the 496th～695 amino acids is and the interactional fragment of β-cat (Dvl 1-C is called for short C) on the Dvl 1.For the ease of follow-up clone, design of primers and the round pcr by routine is provided with SalI/NotI (or XbaI/XhoI) restriction enzyme site at the end of Dvl 1-C genetic fragment sequence; Under the another kind of situation, connect conventional Flag (or myc) label that uses at the C of Dvl 1-C genetic fragment end, form Dvl1-C-flag (or Dvl1-C-myc), the end at Dvl 1-C-flag (or myc) is provided with SalI/NotI (or XbaI/XhoI) restriction enzyme site then.
At first, make up the recombinant vector that contains His-C-flag and His-β-cat-myc.The construction of recombinant vector that contains His-C-flag is as follows: the Dvl 1-C-flag that the C end is had the Flag label cuts with the SalI/NotI enzyme, is cloned in the pET28c/His carrier that same enzyme is cut, and obtains recombinant vector pET28c/His-C-flag.The same (that is: pET28c/His-β-cat-myc) of the construction of recombinant vector that contains His-β-cat-myc.
Then, the recombinant vector of aforementioned structure is transformed into e. coli bl21 respectively, in e. coli bl21, express respectively and obtain His-C-flag albumen and the His-β-cat-myc (albumen of His-β-cat-myc), there be direct interaction in conjunction with finding between His-C-flag and the His-β-cat-myc by external, can co-precipitation during as shown in Figure 3A, with anti-myc antibody (anti-myc) immunoprecipitation His-β-cat-myc to His-C-flag.
Further, inventor's cotransfection in HEK293T contains plasmid pCMV/C-NLS-Flag, pCMV/Dvl 1-NLS-HA and the pCMV/ β-cat-myc of C-NLS-Flag, Dvl1-NLS-HA and β-cat-myc.Wherein, the same pCMV/Dvl3-NLS-Flag construction method of pCMV/C-NLS-Flag construction of recombinant plasmid method replaces Dvl3 with Dvl 1-C.The same pCMV/Dvl3-NLS-Flag construction method of pCMV/Dvl1-NLS-HA construction of recombinant plasmid method is similar, adopts pCMV/HA (available from available from Bioscience, carrying the HA label) to replace pCMV/Flag, and replaces Dvl3 with Dvl1.PCMV/ β-cat-myc construction of recombinant plasmid method is as follows: β-cat gene is cut with the XbaI/XhoI enzyme, be cloned in the pCMV/myc carrier of cutting through same enzyme (available from Bioscience, carrying the myc label), obtain recombinant vector pCMV/ β-cat-myc.The recombinant vector of aforementioned preparation is transfected among the HEK293T, expresses C-NLS-Flag, Dvl1-NLS-HA and β-cat-myc respectively.Find that in conjunction with experiment C-NLS-Flag can block the interaction between Dvl and the β-cat by competition.
Shown in Fig. 3 B, at cotransfection under the situation of C-NLS-Flag, with anti-HA antibody (anti-HA is available from Sigma) immunoprecipitation Dvl1-NLS-HA can not co-precipitation to β-cat-myc.Above experimental result explanation, the fragment C on the Dvl 1 is responsible for and the interactional fragment of β-cat.
For the further effect of the described interaction of research in Wnt/ β-cat signal pathway, the inventor has made up the recombinant vector pCMV/C-NLS that has into nuclear signal (NLS) and Dvl 1-C fragment and (C-NLS has been cut with the XbaI/XhoI enzyme, be cloned in the pCMV carrier (available from Bioscience) that same enzyme is cut), this recombinant vector is transfected in the HEK293T cell, and the activity by the Topflash reporter gene detects their influences to the transcriptional activity of transcription complex.LacZ with routine contrasts as blank plasmid, and Wnt3a is the situation that adds Wnt3a in the nutrient culture media of HEK293T cell, and Ctr is not for utilizing the situation that Wnt3a stimulates, other condition is identical.
Shown in Fig. 3 C, Dvl 1-C-NLS can suppress the Topflash activity that Wnt3a activates effectively.
In addition, the inventor also with above-mentioned recombinant vector transfection SW180 cell (available from ATCC, being the colon cancer cell line of APC protein function disappearance), detects their influences to the transcriptional activity of transcription complex by the activity of Topflash reporter gene.The Topflash reporter gene derives from Upstate company; The activity of reporter gene detects and adopts: Luciferase Reporter Gene Assay, and High Sensitivity derives from Roche company, and detection method is referring to the operation instructions that provide simultaneously.
The result is shown in Fig. 3 D, and in the SW480 cell, Dvl 1-C-NLS is dose-dependent (wherein, "+" expression contains 100ng Dvl-C-NLS, and " ++ " expression contains 300ngDvl-C-NLS) to the inhibition of Topflash activity.This result has confirmed that further Dvl 1-C-NLS fragment is that the downstream of β-cat plays a role in nuclear, and the interaction by blocking-up Dvl and β-cat suppresses the transcriptional activity that Wnt3a activates.
Proved that from the above experiment inventor interaction between Dvl and the β-cat is essential for the transcriptional activity that Wnt3a activates.Further the inventor utilizes the method (ChIP) of nuclear chromatin immunoprecipitation to study the influence that the interaction partners Wnt3a activation front and back transcription complex between Dvl and the β-cat forms.The HEK293T cell adds Wnt3a to stimulate 3 hours, then by the formaldehyde fixed cell, the ultrasonic degradation cell, steps such as pre-combination obtain can be used for the supernatant of immunoprecipitation, add IgG antibody (available from sigma), β-cat antibody (available from BD.Biotech) or TCF-4 antibody (available from Upstate) and protein A/GPlus agarose pearl respectively and carry out immunoprecipitation, wash the sample that obtains can be used for pcr amplification with wash-out then, at last by the Agarose electrophoresis detection.
The result is shown in Fig. 3 E, under Wnt3a stimulates, β-cat and TCF-4 can be attached on the target gene c-myc promoter of Wnt signal downstream, in the cell of transfection Dvl 1-C-NLS fragment, the ability that β-cat is attached on the target gene c-myc promoter of downstream reduces significantly, and TCF-4 is unaffected substantially.This formation that shows the transcription complex that the interaction between Dvl and the β-cat is induced for Wnt3a is essential.
The last inventor utilizes model organism zebra fish to detect the function of interaction in biosome between Dvl and the β-cat.
The inventor is at the mRNA that injects Dvl 1-C-NLS period of zebrafish embryo one to four cell.Then, respectively in two periods of embryonic development: animal pole cell envelope 40% and embryonic shield period (shield phage) results embryo, utilize conventional hybridization in situ technique to detect the expression of Wnt signal pathway downstream gene vox and tbx6.And, be used as contrast with mRNA to zebrafish embryo endocellular injection green fluorescent protein (GFP).
The result as shown in Figure 4, the expression of vox and tbx6 is significantly than control group embryo decline (Fig. 4 A) among the embryo of the mRNA of injection Dvl 1-C-NLS, the expression that tbx6 among 51% the embryo is arranged after 99 embryos statistics there is obvious downward modulation, the expression of vox is had obvious downward modulation (Fig. 4 B) 59% embryo is arranged after 83 embryos' statistics.This result has shown the function of interaction in the growth course of zebra fish between Dvl and the β-cat, and it is essential for the formation of the transcription complex of β-cat/TCF-4 on the promoter of Wnt target gene.
The inventor's above-mentioned research has disclosed the molecular mechanism that Dvl regulates the classical Wnt signal pathway in the nucleus, has disclosed the interactional importance between Dvl and the β-cat.And find that fragment Dvl 1-C-NLS can suppress the formation of transcription complex, take place that this screening that is found to be tumour medicine provides good screening model scheme, the target site of effect is provided for the design molecular medicine thereby suppress tumour.
Embodiment 4 screenings suppress interactional material between Dvl and the β-cat
As identical method as described in " 2 " among the embodiment 2, make up the recombinant vector that has pET28c/His-Dvl3 and pET28c/His-β-cat-myc.
Test group: add the e. coli bl21 cell system that material standed for and transfection have pET28c/His-Dvl3 and pET28c/His-β-cat-myc.
Control group: do not add the e. coli bl21 cell system that material standed for and transfection have pET28c/His-Dvl3 and pET28c/His-β-cat-myc.
Adopt Pull Down experiment, detect Dvl 3 albumen and β-cat protein-interacting situation in test group and the control group as embodiment 2.
If compare with the control group that does not add candidate substances, add that the interaction of Dvl 3 albumen and β-cat albumen obviously is suppressed after the candidate substances (can't co-precipitation arriving His-Dvl3) as the system of utilizing anti-myc antibody mediated immunity precipitation cotransfection His-β-cat-myc and His-Dvl3, illustrate that then this candidate substances is to suppress interactional material between Dvl albumen and the β-cat albumen, thereby be the material that a kind of expection can influence the Wnt signal transduction pathway.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Sequence table
＜110〉Shanghai Inst. of Life Science, CAS
＜120〉disheveled protein and the beta-catenin new function of interaction in classical Wnt signal transduction path between white
<130>071528
<160>12
<170>PatentIn?version?3.3
<210>1
<211>781
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<400>1
Met?Ala?Thr?Gln?Ala?Asp?Leu?Met?Glu?Leu?Asp?Met?Ala?Met?Glu?Pro
1 5 10 15
Asp?Arg?Lys?Ala?Ala?Val?Ser?His?Trp?Gln?Gln?Gln?Ser?Tyr?Leu?Asp
20 25 30
Ser?Gly?Ile?His?Ser?Gly?Ala?Thr?Thr?Thr?Ala?Pro?Ser?Leu?Ser?Gly
35 40 45
Lys?Gly?Asn?Pro?Glu?Glu?Glu?Asp?Val?Asp?Thr?Ser?Gln?Val?Leu?Tyr
50 55 60
Glu?Trp?Glu?Gln?Gly?Phe?Ser?Gln?Ser?Phe?Thr?Gln?Glu?Gln?Val?Ala
65 70 75 80
Asp?Ile?Asp?Gly?Gln?Tyr?Ala?Met?Thr?Arg?Ala?Gln?Arg?Val?Arg?Ala
85 90 95
Ala?Met?Phe?Pro?Glu?Thr?Leu?Asp?Glu?Gly?Met?Gln?Ile?Pro?Ser?Thr
100 105 110
Gln?Phe?Asp?Ala?Ala?His?Pro?Thr?Asn?Val?Gln?Arg?Leu?Ala?Glu?Pro
115 120 125
Ser?Gln?Met?Leu?Lys?His?Ala?Val?Val?Asn?Leu?Ile?Asn?Tyr?Gln?Asp
130 135 140
Asp?Ala?Glu?Leu?Ala?Thr?Arg?Ala?Ile?Pro?Glu?Leu?Thr?Lys?Leu?Leu
145 150 155 160
Asn?Asp?Glu?Asp?Gln?Val?Val?Val?Asn?Lys?Ala?Ala?Val?Met?Val?His
165 170 175
Gln?Leu?Ser?Lys?Lys?Glu?Ala?Ser?Arg?His?Ala?Ile?Met?Arg?Ser?Pro
180 185 190
Gln?Met?Val?Ser?Ala?Ile?Val?Arg?Thr?Met?Gln?Asn?Thr?Asn?Asp?Val
195 200 205
Glu?Thr?Ala?Arg?Cys?Thr?Ala?Gly?Thr?Leu?His?Asn?Leu?Ser?His?His
210 215 220
Arg?Glu?Gly?Leu?Leu?Ala?Ile?Phe?Lys?Ser?Gly?Gly?Ile?Pro?Ala?Leu
225 230 235 240
Val?Lys?Met?Leu?Gly?Ser?Pro?Val?Asp?Ser?Val?Leu?Phe?Tyr?Ala?Ile
245 250 255
Thr?Thr?Leu?His?Asn?Leu?Leu?Leu?His?Gln?Glu?Gly?Ala?Lys?Met?Ala
260 265 270
Val?Arg?Leu?Ala?Gly?Gly?Leu?Gln?Lys?Met?Val?Ala?Leu?Leu?Asn?Lys
275 280 285
Thr?Asn?Val?Lys?Phe?Leu?Ala?Ile?Thr?Thr?Asp?Cys?Leu?Gln?Ile?Leu
290 295 300
Ala?Tyr?Gly?Asn?Gln?Glu?Ser?Lys?Leu?Ile?Ile?Leu?Ala?Ser?Gly?Gly
305 310 315 320
Pro?Gln?Ala?Leu?Val?Asn?Ile?Met?Arg?Thr?Tyr?Thr?Tyr?Glu?Lys?Leu
325 330 335
Leu?Trp?Thr?Thr?Ser?Arg?Val?Leu?Lys?Val?Leu?Ser?Val?Cys?Ser?Ser
340 345 350
Asn?Lys?Pro?Ala?Ile?Val?Glu?Ala?Gly?Gly?Met?Gln?Ala?Leu?Gly?Leu
355 360 365
His?Leu?Thr?Asp?Pro?Ser?Gln?Arg?Leu?Val?Gln?Asn?Cys?Leu?Trp?Thr
370 375 380
Leu?Arg?Asn?Leu?Ser?Asp?Ala?Ala?Thr?Lys?Gln?Glu?Gly?Met?Glu?Gly
385 390 395 400
Leu?Leu?Gly?Thr?Leu?Val?Gln?Leu?Leu?Gly?Ser?Asp?Asp?Ile?Asn?Val
405 410 415
Val?Thr?Cys?Ala?Ala?Gly?Ile?Leu?Ser?Asn?Leu?Thr?Cys?Asn?Asn?Tyr
420 425 430
Lys?Asn?Lys?Met?Met?Val?Cys?Gln?Val?Gly?Gly?Ile?Glu?Ala?Leu?Val
435 440 445
Arg?Thr?Val?Leu?Arg?Ala?Gly?Asp?Arg?Glu?Asp?Ile?Thr?Glu?Pro?Ala
450 455 460
Ile?Cys?Ala?Leu?Arg?His?Leu?Thr?Ser?Arg?His?Gln?Glu?Ala?Glu?Met
465 470 475 480
Ala?Gln?Asn?Ala?Val?Arg?Leu?His?Tyr?Gly?Leu?Pro?Val?Val?Val?Lys
485 490 495
Leu?Leu?His?Pro?Pro?Ser?His?Trp?Pro?Leu?Ile?Lys?Ala?Thr?Val?Gly
500 505 510
Leu?Ile?Arg?Asn?Leu?Ala?Leu?Cys?Pro?Ala?Asn?His?Ala?Pro?Leu?Arg
515 520 525
Glu?Gln?Gly?Ala?Ile?Pro?Arg?Leu?Val?Gln?Leu?Leu?Val?Arg?Ala?His
530 535 540
Gln?Asp?Thr?Gln?Arg?Arg?Thr?Ser?Met?Gly?Gly?Thr?Gln?Gln?Gln?Phe
545 550 555 560
Val?Glu?Gly?Val?Arg?Met?Glu?Glu?Ile?Val?Glu?Gly?Cys?Thr?Gly?Ala
565 570 575
Leu?His?Ile?Leu?Ala?Arg?Asp?Val?His?Asn?Arg?Ile?Val?Ile?Arg?Gly
580 585 590
Leu?Asn?Thr?Ile?Pro?Leu?Phe?Val?Gln?Leu?Leu?Tyr?Ser?Pro?Ile?Glu
595 600 605
Asn?Ile?Gln?Arg?Val?Ala?Ala?Gly?Val?Leu?Cys?Glu?Leu?Ala?Gln?Asp
610 615 620
Lys?Glu?Ala?Ala?Glu?Ala?Ile?Glu?Ala?Glu?Gly?Ala?Thr?Ala?Pro?Leu
625 630 635 640
Thr?Glu?Leu?Leu?His?Ser?Arg?Asn?Glu?Gly?Val?Ala?Thr?Tyr?Ala?Ala
645 650 655
Ala?Val?Leu?Phe?Arg?Met?Ser?Glu?Asp?Lys?Pro?Gln?Asp?Tyr?Lys?Lys
660 665 670
Arg?Leu?Ser?Val?Glu?Leu?Thr?Ser?Ser?Leu?Phe?Arg?Thr?Glu?Pro?Met
675 680 685
Ala?Trp?Asn?Glu?Thr?Ala?Asp?Leu?Gly?Leu?Asp?Ile?Gly?Ala?Gln?Gly
690 695 700
Glu?Ala?Leu?Gly?Tyr?Arg?Gln?Asp?Asp?Pro?Ser?Tyr?Arg?Ser?Phe?His
705 710 715 720
Ser?Gly?Gly?Tyr?Gly?Gln?Asp?Ala?Leu?Gly?Met?Asp?Pro?Met?Met?Glu
725 730 735
His?Glu?Met?Gly?Gly?His?His?Pro?Gly?Ala?Asp?Tyr?Pro?Val?Asp?Gly
740 745 750
Leu?Pro?Asp?Leu?Gly?His?Ala?Gln?Asp?Leu?Met?Asp?Gly?Leu?Pro?Pro
755 760 765
Gly?Asp?Ser?Asn?Gln?Leu?Ala?Trp?Phe?Asp?Thr?Asp?Leu
770 775 780
<210>2
<211>690
<212>PRT
＜213〉mouse (Mus musculus)
<400>2
Met?Ala?Glu?Thr?Lys?Ile?Ile?Tyr?His?Met?Asp?Glu?Glu?Glu?Thr?Pro
1 5 10 15
Tyr?Leu?Val?Lys?Leu?Pro?Val?Ala?Pro?Glu?Arg?Val?Thr?Leu?Ala?Asp
20 25 30
Phe?Lys?Asn?Val?Leu?Ser?Asn?Arg?Pro?Val?His?Ala?Tyr?Lys?Phe?Phe
35 40 45
Phe?Lys?Ser?Met?Asp?Gln?Asp?Phe?Gly?Val?Val?Lys?Glu?Glu?Ile?Phe
50 55 60
Asp?Asp?Asn?Ala?Lys?Leu?Pro?Cys?Phe?Asn?Gly?Arg?Val?Val?Ser?Trp
65 70 75 80
Leu?Val?Leu?Ala?Glu?Gly?Ala?His?Ser?Asp?Ala?Gly?Ser?Gln?Gly?Thr
85 90 95
Asp?Ser?His?Thr?Asp?Leu?Pro?Pro?Pro?Leu?Glu?Arg?Thr?Gly?Gly?Ile
100 105 110
Gly?Asp?Ser?Arg?Pro?Pro?Ser?Phe?Gln?Ser?Ser?Arg?Asp?Gly?Met?Asp
115 120 125
Asn?Glu?Thr?Gly?Thr?Glu?Ser?Met?Val?Ser?His?Arg?Arg?Glu?Arg?Ala
130 135 140
Arg?Arg?Arg?Asn?Arg?Asp?Glu?Ala?Ala?Arg?Thr?Asn?Gly?His?Pro?Arg
145 150 155 160
Gly?Asp?Arg?Arg?Arg?Asp?Leu?Gly?Leu?Pro?Pro?Asp?Ser?Ala?Ser?Thr
165 170 175
Val?Leu?Ser?Ser?Glu?Leu?Glu?Ser?Ser?Ser?Phe?Ile?Asp?Ser?Asp?Glu
180 185 190
Glu?Asp?Asn?Thr?Ser?Arg?Leu?Ser?Ser?Ser?Thr?Glu?Gln?Ser?Asn?Ser
195 200 205
Ser?Arg?Leu?Val?Arg?Lys?His?Lys?Cys?Arg?Arg?Arg?Lys?Gln?Arg?Leu
210 215 220
Arg?Gln?Thr?Asp?Arg?Ala?Ser?Ser?Phe?Ser?Ser?Ile?Thr?Asp?Ser?Thr
225 230 235 240
Met?Ser?Leu?Asn?Ile?Ile?Thr?Val?Thr?Leu?Asn?Met?Glu?Arg?His?His
245 250 255
Phe?Leu?Gly?Ile?Ser?Ile?Val?Gly?Gln?Ser?Asn?Asp?Arg?Gly?Asp?Gly
260 265 270
Gly?Ile?Tyr?Ile?Gly?Ser?Ile?Met?Lys?Gly?Gly?Ala?Val?Ala?Ala?Asp
275 280 285
Gly?Arg?Ile?Glu?Pro?Gly?Asp?Met?Leu?Leu?Gln?Val?Asn?Asp?Val?Asn
290 295 300
Phe?Glu?Asn?Met?Ser?Asn?Asp?Asp?Ala?Val?Arg?Val?Leu?Arg?Glu?Ile
305 310 315 320
Val?Ser?Gln?Thr?Gly?Pro?Ile?Ser?Leu?Thr?Val?Ala?Lys?Cys?Trp?Asp
325 330 335
Pro?Thr?Pro?Arg?Ser?Tyr?Phe?Thr?Ile?Pro?Arg?Ala?Asp?Pro?Val?Arg
340 345 350
Pro?Ile?Asp?Pro?Ala?Ala?Trp?Leu?Ser?His?Thr?Ala?Ala?Leu?Thr?Gly
355 360 365
Ala?Leu?Pro?Arg?Tyr?Gly?Thr?Ser?Pro?Cys?Ser?Ser?Ala?Ile?Thr?Arg
370 375 380
Thr?Ser?Ser?Ser?Ser?Leu?Thr?Ser?Ser?Val?Pro?Gly?Ala?Pro?Gln?Leu
385 390 395 400
Glu?Glu?Ala?Pro?Leu?Thr?Val?Lys?Ser?Asp?Met?Ser?Ala?Ile?Val?Arg
405 410 415
Val?Met?Gln?Leu?Pro?Asp?Ser?Gly?Leu?Glu?Ile?Arg?Asp?Arg?Met?Trp
420 425 430
Leu?Lys?Ile?Thr?Ile?Ala?Asn?Ala?Val?Ile?Gly?Ala?Asp?Val?Val?Asp
435 440 445
Trp?Leu?Tyr?Thr?His?Val?Glu?Gly?Phe?Lys?Glu?Arg?Arg?Glu?Ala?Arg
450 455 460
Lys?Tyr?Ala?Ser?Ser?Met?Leu?Lys?His?Gly?Phe?Leu?Arg?His?Thr?Val
465 470 475 480
Asn?Lys?Ile?Thr?Phe?Ser?Glu?Gln?Cys?Tyr?Tyr?Val?Phe?Gly?Asp?Leu
485 490 495
Cys?Ser?Asn?Leu?Ala?Ser?Leu?Asn?Leu?Asn?Ser?Gly?Ser?Ser?Gly?Ala
500 505 510
Ser?Asp?Gln?Asp?Thr?Leu?Ala?Pro?Leu?Pro?His?Pro?Ser?Val?Pro?Trp
515 520 525
Pro?Leu?Gly?Gln?Gly?Tyr?Pro?Tyr?Gln?Tyr?Pro?Gly?Pro?Pro?Pro?Cys
530 535 540
Phe?Pro?Pro?Ala?Tyr?Gln?Asp?Pro?Gly?Phe?Ser?Cys?Gly?Ser?Gly?Ser
545 550 555 560
Ala?Gly?Ser?Gln?Gln?Ser?Glu?Gly?Ser?Lys?Ser?Ser?Gly?Ser?Thr?Arg
565 570 575
Ser?Ser?His?Arg?Thr?Pro?Gly?Arg?Glu?Glu?Arg?Arg?Ala?Thr?Gly?Ala
580 585 590
Gly?Gly?Ser?Gly?Ser?Glu?Ser?Asp?His?Thr?Val?Pro?Ser?Gly?Ser?Gly
595 600 605
Ser?Thr?Gly?Trp?Trp?Glu?Arg?Pro?Val?Ser?Gln?Leu?Ser?Arg?Gly?Ser
610 615 620
Ser?Pro?Arg?Ser?Gln?Ala?Ser?Ala?Val?Ala?Pro?Gly?Leu?Pro?Pro?Leu
625 630 635 640
His?Pro?Leu?Thr?Lys?Ala?Tyr?Ala?Val?Val?Gly?Gly?Pro?Pro?Gly?Gly
645 650 655
Pro?Pro?Val?Arg?Glu?Leu?Ala?Ala?Val?Pro?Pro?Glu?Leu?Thr?Gly?Ser
660 665 670
Arg?Gln?Ser?Phe?Gln?Lys?Ala?Met?Gly?Asn?Pro?Cys?Glu?Phe?Phe?Val
675 680 685
Asp?Ile
690
<210>3
<211>736
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<400>3
Met?Ala?Gly?Ser?Ser?Thr?Gly?Gly?Gly?Gly?Val?Gly?Glu?Thr?Lys?Val
1 5 10 15
Ile?Tyr?His?Leu?Asp?Glu?Glu?Glu?Thr?Pro?Tyr?Leu?Val?Lys?Ile?Pro
20 25 30
Val?Pro?Ala?Glu?Arg?Ile?Thr?Leu?Gly?Asp?Phe?Lys?Ser?Val?Leu?Gln
35 40 45
Arg?Pro?Ala?Gly?Ala?Lys?Tyr?Phe?Phe?Lys?Ser?Met?Asp?Gln?Asp?Phe
50 55 60
Gly?Val?Val?Lys?Glu?Glu?Ile?Ser?Asp?Asp?Asn?Ala?Arg?Leu?Pro?Cys
65 70 75 80
Phe?Asn?Gly?Arg?Val?Val?Ser?Trp?Leu?Val?Ser?Ser?Asp?Asn?Pro?Gln
85 90 95
Pro?Glu?Met?Ala?Pro?Pro?Val?His?Glu?Pro?Arg?Ala?Glu?Leu?Ala?Pro
100 105 110
Pro?Ala?Pro?Pro?Leu?Pro?Pro?Leu?Pro?Pro?Glu?Arg?Thr?Ser?Gly?Ile
115 120 125
Gly?Asp?Ser?Arg?Pro?Pro?Ser?Phe?His?Pro?Asn?Val?Ser?Ser?Ser?His
130 135 140
Glu?Asn?Leu?Glu?Pro?Glu?Thr?Glu?Thr?Glu?Ser?Val?Val?Ser?Leu?Arg
145 150 155 160
Arg?Glu?Arg?Pro?Arg?Arg?Arg?Asp?Ser?Ser?Glu?His?Gly?Ala?Gly?Gly
165 170 175
His?Arg?Thr?Gly?Gly?Pro?Ser?Arg?Leu?Glu?Arg?His?Leu?Ala?Gly?Tyr
180 185 190
Glu?Ser?Ser?Ser?Thr?Leu?Met?Thr?Ser?Glu?Leu?Glu?Ser?Thr?Ser?Leu
195 200 205
Gly?Asp?Ser?Asp?Glu?Glu?Asp?Thr?Met?Ser?Arg?Phe?Ser?Ser?Ser?Thr
210 215 220
Glu?Gln?Ser?Ser?Ala?Ser?Arg?Leu?Leu?Lys?Arg?His?Arg?Arg?Arg?Arg
225 230 235 240
Lys?Gln?Arg?Pro?Pro?Arg?Leu?Glu?Arg?Thr?Ser?Ser?Phe?Ser?Ser?Val
245 250 255
Thr?Asp?Ser?Thr?Met?Ser?Leu?Asn?Ile?Ile?Thr?Val?Thr?Leu?Asn?Met
260 265 270
Glu?Lys?Tyr?Asn?Phe?Leu?Gly?Ile?Ser?Ile?Val?Gly?Gln?Ser?Asn?Glu
275 280 285
Arg?Gly?Asp?Gly?Gly?Ile?Tyr?Ile?Gly?Ser?Ile?Met?Lys?Gly?Gly?Ala
290 295 300
Val?Ala?Ala?Asp?Gly?Arg?Ile?Glu?Pro?Gly?Asp?Met?Leu?Leu?Gln?Val
305 310 315 320
Asn?Asp?Met?Asn?Phe?Glu?Asn?Met?Ser?Asn?Asp?Asp?Ala?Val?Arg?Val
325 330 335
Leu?Arg?Asp?Ile?Val?His?Lys?Pro?Gly?Pro?Ile?Val?Leu?Thr?Val?Ala
340 345 350
Lys?Cys?Trp?Asp?Pro?Ser?Pro?Gln?Ala?Tyr?Phe?Thr?Leu?Pro?Arg?Asn
355 360 365
Glu?Pro?Ile?Gln?Pro?Ile?Asp?Pro?Ala?Ala?Trp?Val?Ser?His?Ser?Ala
370 375 380
Ala?Leu?Thr?Gly?Thr?Phe?Pro?Ala?Tyr?Pro?Gly?Ser?Ser?Ser?Met?Ser
385 390 395 400
Thr?Ile?Thr?Ser?Gly?Ser?Ser?Leu?Pro?Asp?Gly?Cys?Glu?Gly?Arg?Gly
405 410 415
Leu?Ser?Val?His?Thr?Asp?Met?Ala?Ser?Val?Thr?Lys?Ala?Met?Ala?Ala
420 425 430
Pro?Glu?Ser?Gly?Leu?Glu?Val?Arg?Asp?Arg?Met?Trp?Leu?Lys?Ile?Thr
435 440 445
Ile?Pro?Asn?Ala?Phe?Leu?Gly?Ser?Asp?Val?Val?Asp?Trp?Leu?Tyr?His
450 455 460
His?Val?Glu?Gly?Phe?Pro?Glu?Arg?Arg?Glu?Ala?Arg?Lys?Tyr?Ala?Ser
465 470 475 480
Gly?Leu?Leu?Lys?Ala?Gly?Leu?Ile?Arg?His?Thr?Val?Asn?Lys?Ile?Thr
485 490 495
Phe?Ser?Glu?Gln?Cys?Tyr?Tyr?Val?Phe?Gly?Asp?Leu?Ser?Gly?Gly?Cys
500 505 510
Glu?Ser?Tyr?Leu?Val?Asn?Leu?Ser?Leu?Asn?Asp?Asn?Asp?Gly?Ser?Ser
515 520 525
Gly?Ala?Ser?Asp?Gln?Asp?Thr?Leu?Ala?Pro?Leu?Pro?Gly?Ala?Thr?Pro
530 535 540
Trp?Pro?Leu?Leu?Pro?Thr?Phe?Ser?Tyr?Gln?Tyr?Pro?Ala?Pro?His?Pro
545 550 555 560
Tyr?Ser?Pro?Gln?Pro?Pro?Pro?Tyr?His?Glu?Leu?Ser?Ser?Tyr?Thr?Tyr
565 570 575
Gly?Gly?Gly?Ser?Ala?Ser?Ser?Gln?His?Ser?Glu?Gly?Ser?Arg?Ser?Ser
580 585 590
Gly?Ser?Thr?Arg?Ser?Asp?Gly?Gly?Ala?Gly?Arg?Thr?Gly?Arg?Pro?Glu
595 600 605
Glu?Arg?Ala?Pro?Glu?Ser?Lys?Ser?Gly?Ser?Gly?Ser?Glu?Ser?Glu?Pro
610 615 620
Ser?Ser?Arg?Gly?Gly?Ser?Leu?Arg?Arg?Gly?Gly?Glu?Ala?Ser?Gly?Thr
625 630 635 640
Ser?Asp?Gly?Gly?Pro?Pro?Pro?Ser?Arg?Gly?Ser?Thr?Gly?Gly?Ala?Pro
645 650 655
Asn?Leu?Arg?Ala?His?Pro?Gly?Leu?His?Pro?Tyr?Gly?Pro?Pro?Pro?Gly
660 665 670
Met?Ala?Leu?Pro?Tyr?Asn?Pro?Met?Met?Val?Val?Met?Met?Pro?Pro?Pro
675 680 685
Pro?Pro?Pro?Val?Pro?Pro?Ala?Val?Gln?Pro?Pro?Gly?Ala?Pro?Pro?Val
690 695 700
Arg?Asp?Leu?Gly?Ser?Val?Pro?Pro?Glu?Leu?Thr?Ala?Ser?Arg?Gln?Ser
705 710 715 720
Phe?His?Met?Ala?Met?Gly?Asn?Pro?Ser?Glu?Phe?Phe?Val?Asp?Val?Met
725 730 735
<210>4
<211>716
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<400>4
Met?Gly?Glu?Thr?Lys?Ile?Ile?Tyr?His?Leu?Asp?Gly?Gln?Glu?Thr?Pro
1 5 10 15
Tyr?Leu?Val?Lys?Leu?Pro?Leu?Pro?Ala?Glu?Arg?Val?Thr?Leu?Ala?Asp
20 25 30
Phe?Lys?Gly?Val?Leu?Gln?Arg?Pro?Ser?Tyr?Lys?Phe?Phe?Phe?Lys?Ser
35 40 45
Met?Asp?Asp?Asp?Phe?Gly?Val?Val?Lys?Glu?Glu?Ile?Ser?Asp?Asp?Asn
50 55 60
Ala?Lys?Leu?Pro?Cys?Phe?Asn?Gly?Arg?Val?Val?Tyr?Trp?Leu?Val?Ser
65 70 75 80
Ala?Glu?Gly?Ser?His?Pro?Asp?Pro?Ala?Pro?Phe?Cys?Ala?Asp?Asn?Pro
85 90 95
Ser?Glu?Leu?Pro?Pro?Pro?Met?Glu?Arg?Thr?Gly?Gly?Ile?Gly?Asp?Ser
100 105 110
Arg?Pro?Pro?Ser?Phe?His?Pro?His?Ala?Gly?Gly?Gly?Ser?Gln?Glu?Asn
115 120 125
Leu?Asp?Asn?Asp?Thr?Glu?Thr?Asp?Ser?Leu?Val?Ser?Ala?Gln?Arg?Glu
130 135 140
Arg?Pro?Arg?Arg?Arg?Asp?Gly?Pro?Glu?His?Ala?Thr?Arg?Leu?Asn?Gly
145 150 155 160
Thr?Ala?Lys?Gly?Glu?Arg?Arg?Arg?Glu?Pro?Gly?Gly?Tyr?Asp?Ser?Ser
165 170 175
Ser?Thr?Leu?Met?Ser?Ser?Glu?Leu?Glu?Thr?Thr?Ser?Phe?Phe?Asp?Ser
180 185 190
Asp?Glu?Asp?Asp?Ser?Thr?Ser?Arg?Phe?Ser?Ser?Ser?Thr?Glu?Gln?Ser
195 200 205
Ser?Ala?Ser?Arg?Leu?Met?Arg?Arg?His?Lys?Arg?Arg?Arg?Arg?Lys?Gln
210 215 220
Lys?Val?Ser?Arg?Ile?Glu?Arg?Ser?Ser?Ser?Phe?Ser?Ser?Ile?Thr?Asp
225 230 235 240
Ser?Thr?Met?Ser?Leu?Asn?Ile?Ile?Thr?Val?Thr?Leu?Asn?Met?Glu?Lys
245 250 255
Tyr?Asn?Phe?Leu?Gly?Ile?Ser?Ile?Val?Gly?Gln?Ser?Asn?Glu?Arg?Gly
260 265 270
Asp?Gly?Gly?Ile?Tyr?Ile?Gly?Ser?Ile?Met?Lys?Gly?Gly?Ala?Val?Ala
275 280 285
Ala?Asp?Gly?Arg?Ile?Glu?Pro?Gly?Asp?Met?Leu?Leu?Gln?Val?Asn?Glu
290 295 300
Ile?Asn?Phe?Glu?Asn?Met?Ser?Asn?Asp?Asp?Ala?Val?Arg?Val?Leu?Arg
305 310 315 320
Glu?Ile?Val?His?Lys?Pro?Gly?Pro?Ile?Thr?Leu?Thr?Val?Ala?Lys?Cys
325 330 335
Trp?Asp?Pro?Ser?Pro?Arg?Gly?Cys?Phe?Thr?Leu?Pro?Arg?Ser?Glu?Pro
340 345 350
Ile?Arg?Pro?Ile?Asp?Pro?Ala?Ala?Trp?Val?Ser?His?Thr?Ala?Ala?Met
355 360 365
Thr?Gly?Thr?Phe?Pro?Ala?Tyr?Gly?Met?Ser?Pro?Ser?Leu?Ser?Thr?Ile
370 375 380
Thr?Ser?Thr?Ser?Ser?Ser?Ile?Thr?Ser?Ser?Ile?Pro?Asp?Thr?Glu?Arg
385 390 395 400
Leu?Asp?Asp?Phe?His?Leu?Ser?Ile?His?Ser?Asp?Met?Ala?Ala?Ile?Val
405 410 415
Lys?Ala?Met?Ala?Ser?Pro?Glu?Ser?Gly?Leu?Glu?Val?Arg?Asp?Arg?Met
420 425 430
Trp?Leu?Lys?Ile?Thr?Ile?Pro?Asn?Ala?Phe?Ile?Gly?Ser?Asp?Val?Val
435 440 445
Asp?Trp?Leu?Tyr?His?Asn?Val?Glu?Gly?Phe?Thr?Asp?Arg?Arg?Glu?Ala
450 455 460
Arg?Lys?Tyr?Ala?Ser?Asn?Leu?Leu?Lys?Ala?Gly?Phe?Ile?Arg?His?Thr
465 470 475 480
Val?Asn?Lys?Ile?Thr?Phe?Ser?Glu?Gln?Cys?Tyr?Tyr?Ile?Phe?Gly?Asp
485 490 495
Leu?Cys?Gly?Asn?Met?Ala?Asn?Leu?Ser?Leu?His?Asp?His?Asp?Gly?Ser
500 505 510
Ser?Gly?Ala?Ser?Asp?Gln?Asp?Thr?Leu?Ala?Pro?Leu?Pro?His?Pro?Gly
515 520 525
Ala?Ala?Pro?Trp?Pro?Met?Ala?Phe?Pro?Tyr?Gln?Tyr?Pro?Pro?Pro?Pro
530 535 540
His?Pro?Tyr?Asn?Pro?His?Pro?Gly?Phe?Pro?Glu?Leu?Gly?Tyr?Ser?Tyr
545 550 555 560
Gly?Gly?Gly?Ser?Ala?Ser?Ser?Gln?His?Ser?Glu?Gly?Ser?Arg?Ser?Ser
565 570 575
Gly?Ser?Asn?Arg?Ser?Gly?Ser?Asp?Arg?Arg?Lys?Glu?Lys?Asp?Pro?Lys
580 585 590
Ala?Gly?Asp?Ser?Lys?Ser?Gly?Gly?Ser?Gly?Ser?Glu?Ser?Asp?His?Thr
595 600 605
Thr?Arg?Ser?Ser?Leu?Arg?Gly?Pro?Arg?Glu?Arg?Ala?Pro?Ser?Glu?Arg
610 615 620
Ser?Gly?Pro?Ala?Ala?Ser?Glu?His?Ser?His?Arg?Ser?His?His?Ser?Leu
625 630 635 640
Ala?Ser?Ser?Leu?Arg?Ser?His?His?Thr?His?Pro?Ser?Tyr?Gly?Pro?Pro
645 650 655
Gly?Val?Pro?Pro?Leu?Tyr?Gly?Pro?Pro?Met?Leu?Met?Met?Pro?Pro?Pro
660 665 670
Pro?Ala?Ala?Met?Gly?Pro?Pro?Gly?Ala?Pro?Pro?Gly?Arg?Asp?Leu?Ala
675 680 685
Ser?Val?Pro?Pro?Glu?Leu?Thr?Ala?Ser?Arg?Gln?Ser?Phe?Arg?Met?Ala
690 695 700
Met?Gly?Asn?Pro?Ser?Glu?Phe?Phe?Val?Asp?Val?Met
705 710 715
<210>5
<211>195
<212>PRT
＜213〉mouse (Mus musculus)
<400>5
Leu?Cys?Ser?Asn?Leu?Ala?Ser?Leu?Asn?Leu?Asn?Ser?Gly?Ser?Ser?Gly
1 5 10 15
Ala?Ser?Asp?Gln?Asp?Thr?Leu?Ala?Pro?Leu?Pro?His?Pro?Ser?Val?Pro
20 25 30
Trp?Pro?Leu?Gly?Gln?Gly?Tyr?Pro?Tyr?Gln?Tyr?Pro?Gly?Pro?Pro?Pro
35 40 45
Cys?Phe?Pro?Pro?Ala?Tyr?Gln?Asp?Pro?Gly?Phe?Ser?Cys?Gly?Ser?Gly
50 55 60
Ser?Ala?Gly?Ser?Gln?Gln?Ser?Glu?Gly?Ser?Lys?Ser?Ser?Gly?Ser?Thr
65 70 75 80
Arg?Ser?Ser?His?Arg?Thr?Pro?Gly?Arg?Glu?Glu?Arg?Arg?Ala?Thr?Gly
85 90 95
Ala?Gly?Gly?Ser?Gly?Ser?Glu?Ser?Asp?His?Thr?Val?Pro?Ser?Gly?Ser
100 105 110
Gly?Ser?Thr?Gly?Trp?Trp?Glu?Arg?Pro?Val?Ser?Gln?Leu?Ser?Arg?Gly
115 120 125
Ser?Ser?Pro?Arg?Ser?Gln?Ala?Ser?Ala?Val?Ala?Pro?Gly?Leu?Pro?Pro
130 135 140
Leu?His?Pro?Leu?Thr?Lys?Ala?Tyr?Ala?Val?Val?Gly?Gly?Pro?Pro?Gly
145 150 155 160
Gly?Pro?Pro?Val?Arg?Glu?Leu?Ala?Ala?Val?Pro?Pro?Glu?Leu?Thr?Gly
165 170 175
Ser?Arg?Gln?Ser?Phe?Gln?Lys?Ala?Met?Gly?Asn?Pro?Cys?Glu?Phe?Phe
180 185 190
Val?Asp?Ile
195
<210>6
<211>588
<212>DNA
＜213〉mouse (Mus musculus)
<400>6
ctgtgcagta?acctcgcatc?cctgaacctc?aacagtggct?ccagtggagc?ctcagatcag 60
gacacactgg?ccccactgcc?ccacccatca?gtaccctggc?ccttgggtca?aggctacccc 120
taccagtacc?caggaccccc?gccctgcttc?ccacctgctt?accaggaccc?tggcttcagc 180
tgcggcagcg?gcagtgctgg?gagccagcag?agtgaaggga?gcaagagcag?tgggtccaca 240
cggagcagcc?atcggacccc?aggccgagag?gagcgccggg?caactggagc?tgggggtagt 300
ggcagtgaat?cagaccacac?agtaccaagt?gggtctggta?gcaccggctg?gtgggagcgg 360
cctgtcagcc?agcttagccg?tggcagtagc?cctcgaagtc?aggcttcagc?tgttgcccca 420
gggctccccc?cactgcaccc?ccttacaaag?gcctatgcag?tagtgggtgg?gccacctgga 480
gggccacctg?tccgggagct?ggctgctgtc?cctccagaac?ttacaggtag?ccgccagtcc 540
ttccaaaagg?ccatgggaaa?cccctgtgag?ttctttgtgg?acatcatg 588
<210>7
<211>24
<212>PRT
＜213〉artificial sequence
<220>
<221>MISC_FEATURE
＜223〉nuclear localization signal
<400>7
Asp?Pro?Lys?Lys?Lys?Arg?Lys?Val?Asp?Pro?Lys?Lys?Lys?Arg?Lys?Val
1 5 10 15
Asp?Pro?Lys?Lys?Lys?Arg?Lys?Val
20
<210>8
<211>94
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉nuclear localization signal
<400>8
ctagactaga?cctagctcga?ggatccaaaa?aagaagagaa?aggtagatcc?aaaaaagaag 60
agaaaggtag?atccaaaaaa?gaagagaaag?gtag 94
<210>9
<211>27
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>9
ctagtctaga?ggcggagacc?aaaatca 27
<210>10
<211>28
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>10
ctagctcgag?catgatgtcc?acaaagaa 28
<210>11
<211>25
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>11
ctagtctaga?gggcgagacc?aagat 25
<210>12
<211>27
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>12
ctagctcgag?catcacatcc?acaaaga 27
Claims (10)
1. one kind is screened the method for regulating disheveled protein and the white interactional correctives of beta-catenin, it is characterized in that, said method comprising the steps of:
(a) the white interactional system of candidate substances and disheveled protein and beta-catenin is contacted; With
(b) detect candidate substances to disheveled protein and beta-catenin white between interactional influence;
Wherein, if described candidate substances can suppress or promote the interaction between white of disheveled protein and beta-catenin, show that then this candidate substances is adjusting disheveled protein and the white interactional correctives of beta-catenin.
2. the method for claim 1 is characterized in that, step (a) comprising: add candidate substances in disheveled protein and the white interactional system of beta-catenin; With
Step (b) comprising: detect disheveled protein and the beta-catenin situation that interacts in vain, and with control group relatively, wherein said control group be do not add described candidate substances, disheveled protein and the white interactional system of beta-catenin;
If disheveled protein and the white interaction of beta-catenin are lower than control group statistically in the test group, just show that this candidate substances is the white interactional correctives of the inhibition of dishevelled proteins and beta-catenin;
If disheveled protein and the white interaction of beta-catenin are higher than control group statistically in the test group, just show that this candidate substances is to promote disheveled protein and the white interactional correctives of beta-catenin.
3. the method for claim 1 is characterized in that, described system is selected from: solution system, cell system or organizational framework.
4. the method for claim 1 is characterized in that, also comprises Wnt albumen in the described system.
5. the method for claim 1 is characterized in that, described method also comprises: for the correctives that filters out, further test its influence for Wnt signal pathway downstream gene; If it can raise the expression of Wnt signal pathway downstream gene, show that then this correctives is to promote disheveled protein and the beta-catenin interactional promoter between white; If it can reduce the expression of Wnt signal pathway downstream gene, show that then this correctives is the inhibition of dishevelled proteins and the beta-catenin interactional inhibitor between white.
6. one kind is passed through adjusting disheveled protein and the white interactional correctives of beta-catenin that the described method of claim 1 obtains.
7. correctives as claimed in claim 6 is characterized in that it is an inhibitor, and described inhibitor is a peptide species, contains the amino acid sequence shown in the SEQ ID NO:5.
8. polypeptide as claimed in claim 7 is characterized in that this polypeptide also contains the amino acid sequence of nuclear localization signal albumen.
9. correctives as claimed in claim 6 is characterized in that, described correctives is a kind of nucleic acid molecules or its transcript, and described nucleic acid molecules contains the nucleotide sequence shown in the SEQ ID NO:6.
10. the purposes of a disheveled protein is characterized in that, is used for screening and regulates disheveled protein and the white interactional correctives of beta-catenin.
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| CN 200710040600 CN101308142B (en) | 2007-05-14 | 2007-05-14 | Interactive modulator of disheveled protein and beta-catenins interactive function |
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| CN 200710040600 CN101308142B (en) | 2007-05-14 | 2007-05-14 | Interactive modulator of disheveled protein and beta-catenins interactive function |
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| CN101308142A true CN101308142A (en) | 2008-11-19 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105203774A (en) * | 2015-10-20 | 2015-12-30 | 首都医科大学宣武医院 | Applications of DVL (disheveled) as stroke biomarker |
| CN112041329A (en) * | 2019-04-04 | 2020-12-04 | 碧睿制封有限公司 | Composition for preventing hair loss or promoting hair growth |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1253586A (en) * | 1997-03-24 | 2000-05-17 | 昂尼克斯药物公司 | Compositions and methods for diagnosing/treating disease based on beta-catenin/transcription actor interactions |
| US20120178697A9 (en) * | 2003-09-22 | 2012-07-12 | Jie Zheng | Compositions and methods for the inhibition of dishevelled proteins |
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2007
- 2007-05-14 CN CN 200710040600 patent/CN101308142B/en not_active Expired - Fee Related
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105203774A (en) * | 2015-10-20 | 2015-12-30 | 首都医科大学宣武医院 | Applications of DVL (disheveled) as stroke biomarker |
| CN112041329A (en) * | 2019-04-04 | 2020-12-04 | 碧睿制封有限公司 | Composition for preventing hair loss or promoting hair growth |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101308142B (en) | 2012-12-19 |
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