CN1970074A - Application of beta-protein inhibitor in preparation of medicament for treating genetic correlated disease - Google Patents

Abstract

The invention discloses a beta-inhibiting protein, nucleonic sequence and application of composition with nucleonic sequence to treat relative disease with histone acetylated reduction level, which also relates to the application of delta opitum receptor stimulating agent or k opitum receptor exciting agent to treat relative disease with histone acetylated reduction level. The invention provides a drug sieving method from candidate material to treat relative disease with histone acetylated reduction level.

Description

The application of beta-protein inhibitor in the medicine of preparation treatment genetic correlated disease

Technical field

The present invention relates to biomedicine field, be specifically related to people's beta-protein inhibitor is used for reducing with the acetylation of histone level medicine of relevant disease in preparation application.

Background technology

Nearest discovers, human numerous disease is all disorderly relevant with the epigenetic adjusting, and the epigenetic treatment will become the effective means that solves these diseases.And, there have been at present many such preparations carrying out preclinical phase research (.Nature 2004 such as Egger; 429:457-463).Mechanisms of Histone Acetylation Modification effect in epigenetic is regulated is great, is described as the early stage switch that eukaryotic gene transcribes (.Science 2003 such as Grewal; 301:798-802; .Science such as Fry 2002; 295:1847-1848).

Recent discovers, it is the important cause of disease of cell carcinogenesis and many nervous system disease that the acetylation of histone level reduces, and the preparation that can promote the acetylation of histone level to raise has been considered to treat new formulation (the .Curr Opin Genet Develop 1999 such as Archer of these diseases; 9:171-174; .Nature such as Egger 2004; 429:457-463).There are two kinds of enzymes regulating and control the acetylation level of histone in the body, are respectively acetylation of histone enzyme and histon deacetylase (HDAC).The mode of regulating the acetylation of histone level in the body has two kinds, and a kind of is by influencing the activity of acetylation of histone enzyme or histon deacetylase (HDAC), and another kind is by influencing contacting of the two and histone.It should be noted that the preparation that influences acetylation of histone that is used for clinical trial at present all is the inhibitor of histon deacetylase (HDAC), though these preparations have clear and definite antitumaous effect, they have almost suppressed the activity of whole histon deacetylase (HDAC)s in the body.Unquestionable, such preparation makes a big impact to normal groups of cells albumen acetylation level, does patent medicine and can produce very big side effect later.In fact, the non-specific of these preparation effects is the key that limits its patent medicine.

Therefore, press for a kind of ideal acetylation of histone promoter of searching.This new formulation can not influence the activity of interior whole acetylation of histone enzyme of body or histon deacetylase (HDAC); but can promote the acetylation of histone level specifically in the zone; especially influence the acetylation of histone level of disease related gene region, thereby reach the ideal effect of curing the disease.

Summary of the invention

For addressing the above problem, the present inventor has carried out deep research, found that beta-protein inhibitor 1 can address the above problem.

The construction that first aspect present invention relates to beta-protein inhibitor 1, its nucleotide sequence, contain this nucleotide sequence is used for the treatment of with the acetylation of histone level in preparation and reduces application in the relevant disease.In preferable embodiment, the described disease relevant with the reduction of acetylation of histone level is cancer, nervous system disease or epigenetic disease.

Second aspect present invention relates to DOR receptor stimulating agent or KOR receptor stimulating agent and is used for the treatment of with the acetylation of histone level in preparation and reduces application in the relevant disease.In preferable embodiment, the described disease relevant with the reduction of acetylation of histone level is cancer, nervous system disease or epigenetic disease.In another preferable embodiment, described DOR receptor stimulating agent is selected from [D-Pen2, D-Pen5] enkephalin (DPDPE), (D-Ala2, D-Leu5) enkephalin (DADLE) or deltorphin-I.

A further aspect of the invention relates to a kind of being used for the treatment of and the relevant disease medicament compositions of acetylation of histone level reduction; described compositions comprises at least a material that is selected from the nucleotide sequence of beta-protein inhibitor 1, beta-protein inhibitor 1, the construction that contains described nucleotide sequence, delta opiate receptor agonist or kappa opioid receptor agonist of effective dose as active component, and pharmaceutically acceptable carrier.

The present invention also provides a kind of method that filters out the medicine of the treatment disease relevant with the reduction of acetylation of histone level from candidate substances on the other hand, and this method comprises:

A) make described candidate substances and the cells contacting of expressing beta-protein inhibitor 1;

B) identify the endonuclear expression of beta-protein inhibitor 1, and this expression is compared with interior beta-protein inhibitor 1 expression of the nucleus of the cell that does not contact described candidate substances at described cell;

C) select the material that makes beta-protein inhibitor 1 expression raising in the nucleus reduces relevant disease as described treatment and acetylation of histone level medicine.

Through research, the present inventor finds that (β-arrestin1) can promote the level of acetylation of histone in the body to beta-protein inhibitor 1 well, but can not influence the activity of whole acetylation of histone enzyme or histon deacetylase (HDAC).The mode that it influences acetylation of histone is that the enrichment of the p27 gene of anticancer propagation (for example can) realizes in specific chromatin zone by influencing some acetylation of histone enzyme; therefore more much smaller to the influence of acetylation of histone level than Antibiotic FR 901228, and certain gene specific is arranged.As seen, beta-protein inhibitor 1 is a kind of targeting acetylation of histone booster of novelty, and is extremely promising aspect cancer and other epigenetic treatment of diseases.Other purpose of the present invention and advantage can be known by hereinafter detailed description and specific embodiment.

Description of drawings

Fig. 1 has shown that karyon β arr1 has promoted the histone H 4 acetylation.Figure A has shown from the HeLa cell separation and has gone out histone; β arrs+ /+, β arrs-/-, or β arrs-/-MEF cell transient expression shown in plasmid (figure B); Carry out the Western trace with different acetylation (Ac) HAB equity duplicate samples (6 microgram).Also measure the amount of histone H 4 in addition, to confirm to have in each swimming lane the histone of equivalent.Western result is carried out quantitatively being standardized into histone H 4 albumen, shown H4, H4K12 and the H4K16 acetylation meansigma methods of three independent trialss among the figure. ^*P＜0.05, ^*P＜0.01 (with respect to matched group).

Fig. 2 has shown the acetylation of nuclear gathering promotion p27 and the c-fos promoter place histone H 4 of β arr1.Antibody with anti-acetylation H4 (H4Ac), H3 (H3Ac), β arr1 and people or mice IgG (as negative control) carries out the ChIp experiment.Analyze through qPCR, have p27, c-fos and c-jun promoter sequence in the regenerant of the DNA of input and antibodies chromatin section.The data normalization that obtains is become corresponding input contrast.Adopt the primer sets of the zones of different of identical promoters, obtained similar result.Shown in data are meansigma methodss of three independent experiments of every group of primer.Figure A: HeLa of plasmid shown in the expression (left side) and β arrs-/-MEF (right side) cell.NSD represents not detect signal.Figure B: the HeLa cell of single DOR of expression of time shown in 1 μ M DP, cultivating.Figure C: with or handle to express the HeLa cell 5 minutes of DOR or DOR and β arr1 siRNA without 100nM Na1, then with 1 μ M DP cultivation 60 minutes.Figure D: chromatin-protein binding test.(WCE) is centrifugal to full cell pyrolysis liquid, and precipitation (Crude Pel) is handled with micrococcal nuclease (Mnase), recentrifuge.Gained supernatant (Mnase Sup) carries out super centrifugal, obtains precipitation (Ultra Pel) and supernatant (Ultra Sup).With shown in antibody sample carried out Western analyze.Chromobindins H4 and p300 and cytosol albumen tubulin and β arr1 Q394L are in contrast.Figure E, the nuclear of the wild type of transient expression and saltant β arr1 distributes and to the effect of H4 acetylation and genetic transcription in the HeLa cell.Under Laser Scanning Confocal Microscope, observe β arr1-HA and change nuclear.Record H4 acetylation in the p27 promoter region with the ChIP test.Analyze transcribing of p27 with RT-qPCR.Data are taken from three independent trialss, ^*P＜0.05, ^*P＜0.01 (corresponding to contrast).

Fig. 3 has shown that β arr1 is to HAT, HDAC and the active influence of CREB.Figure A: measure the β arr1 of purification, the 3 ' HA-β arr1 and the proteic potential HAT activity of 5 ' HA-β arr1 of immunoprecipitation.Histone acetyltransferase PCAF (0.1 μ g) is as positive control.Figure B, C: extract HAT and the rough thing of HDAC from the full cell homogenates thing of HeLa cell, measure external acetylation of histone (B) and deacetylation (C) under the not commensurability GST-β arr1 and the 3 ' HA-and 5 ' of immunoprecipitation-HA β arr1 albumen existence.Use 600 μ M NiCl respectively ₂With 100nM TSA in contrast.Figure D: exist or not down at 100nMTSA and 25 μ M sirtinol (STN), analysis β arrs-/-MEF in β arr1 to the acetylizad influence of histone H 4 in p27 and the c-fos promoter region.H4 acetylation level in p27 and the c-fos promoter is carried out quantitatively, according to the input value standardization of correspondence.Figure E: report plasmid (left side) and CREB-luciferase report plasmid (right side) separately or the cell in β arr1 plasmid transfection 6 orifice plates shown in commensurability with GAL4-CREB, tables of data is shown as with respect to the multiple of inducing that contrasts.Make (FK) as positive control with 10 μ M Forskolins.Data are from three independent experiments. ^*P＜0.01 (with respect to the contrast of correspondence).

Fig. 4 has shown that β arr1 raises p27 and c-fos promoter region with p300.Figure A and B: carry out the ChIP experiment with anti-p300, CBP, Tip60 and people or mice IgG (as negative control).Analyze through qPCR, have p27, c-fos and c-jun promoter sequence in the regenerant of the DNA of input and antibodies chromatin section.The data normalization that obtains is become corresponding input value.Shown in data are meansigma methodss of three independent experiments of every group of primer. ^*P＜0.05, ^*P＜0.01 (corresponding to contrast).Figure A: HeLa of plasmid shown in the expression (left side) and β arrs-/-MEF (right side) cell.Figure B: the HeLa cell (last figure) of the expression DOR of time shown in 1 μ M DP, cultivating.With or handle to express the HeLa cell 5 minutes of DOR or DOR and β arr1 siRNA without 100nM Nal, then with 60 minutes (middle figure and figure below) of 1 μ M DP cultivation.Figure C: make HeLa karyon extract and p300 or β arr antibody mediated immunity precipitation, the antibody with anti-p300 or β arr carries out the Western analysis to immune complex then.5% total karyon extract in contrast.

Fig. 5 has shown the adjusting of genetic transcription in the people neuroblast oncocyte of β arr1-mediation.Figure A: with the time shown in the SK cell of 1 μ MDP, 1 μ M DADLE (DA) or 1 μ M δ-deltorphin delta-I (DEL) cultivation endogenous expression DOR and β arr1.In right figure, cell is used in advance or was handled 5 minutes with 100nM Na1, cultivates 20 minutes with 1 μ M DP then.Also quantitative with the β arr1 content that Western analyzes in the karyon extract.Figure B, with or handle 5 minutes with 100nM Na1 after, make the SK cell of transfection or untransfected β arr1 siRNA in 1 μ M DP, cultivate shown in time (left side) or 60 minutes (the neutralization right side).With the H4Ac level at ChIP and RT-qPCR analysis p27 and c-jun promoter place, and the transcriptional level of p27 and c-jun. ^*P＜0.05, ^*P＜0.01 (corresponding to contrast).Figure C: cultivate the SK cell fixed time with 1 μ M DA or 1 μ M DEL, analyze the H4Ac level (left side) at p27 and c-jun promoter place then with ChIP and RT-qPCR, and the transcriptional level of p27 and c-jun (right side). ^*P＜0.05, ^*P＜0.01 (with respect to contrast in 0 minute in the correspondence group).Figure D:SK cell is through β ga1, β arr1, and β arr1Q394L, NSsiRNA, β arr1 siRNA, p27 siRNA or given combination transfection were cultivated 60 minutes under 1 μ M DP and the DA or were cultivated 30 minutes under 1 μ M DEL being with or without.Measuring [3H] thymidine and mix, is the data from three independent experiments shown in the figure. ^*P＜0.01 (with respect to the contrast of correspondence).

The specific embodiment

Beta-protein inhibitor 1 (gene number: be the important adjusting albumen of GPCRs NM-004041), distribution is all arranged in Cytoplasm and nucleus.The function of the beta-protein inhibitor 1 now all is to betide Cytoplasm or cytoplasma membrane, and it does not still have report in endonuclear function.The present inventor discovers that activating delta opiate receptor (DOR) causes beta-protein inhibitor 1 to shift in nucleus; cause beta-protein inhibitor 1 in p27 and the enrichment of c-fos gene promoter area; cause these promoter region histone H 4 acetylations to increase, finally promoted these gene transcription.Discover that further beta-protein inhibitor 1 has mediated the recruitment of acetylation of histone enzyme p300 at the target gene promoter region, and this recruitment to be that receptor activation causes acetylation of histone and specific gene to be transcribed increasing necessary.These results have not only showed the epigenetic mechanism that the GPCR signal transmits to nuclear from film, and have disclosed beta-protein inhibitor 1 as function in the nuclear of kytoplasm and internuclear transmission GPCR signal courier material.

The present inventor at first uses the level of beta-protein inhibitor 1 in transgenic and the interferential method affect nucleus of RNA, finds that beta-protein inhibitor 1 significantly improves the level of whole acetylation of histone in the cell.Further discover; the adjusting of 1 pair of acetylation of histone of beta-protein inhibitor has gene specific; for example it can significantly promote to concern with cell proliferation the acetylation of extremely close gene p27 and c-fos promoter region histone H 4; but do not influence c-jun; cyclin A, and the acetylation of cyclin D1 promoter region histone H 4.Employing GST-β Profilin 1 and immunoprecipitation obtains in eukaryotic cell lines HA-β Profilin 1 external acetylation of histone that carries out and dna methylase inhibitor the analysis showed that; β Profilin 1 does not influence the overall activity of acetylation of histone enzyme and histon deacetylase (HDAC) neither acetylation of histone enzyme and histon deacetylase (HDAC) yet.Use protein--chromatin is found in conjunction with experiment and the experiment of chromatin co-immunoprecipitation: 1, beta-protein inhibitor 1 energy and chromatin combination; 2, as DOR, the agonist of KOR etc. stimulates down at g protein coupled receptor, and beta-protein inhibitor 1 can advance to examine Fig.1 and the district's combination of specific staining matter.Further co-immunoprecipitation experiment and mass spectral analysis are found: beta-protein inhibitor 1 can bonding histone acetylase P300.Comprehensive these presentation of results: no matter which kind of factor causes that beta-protein inhibitor 1 increases, and can both promote the level of the acetylation of histone in the special zone of chromatin in nucleus.

Therefore, first aspect present invention relates to beta-protein inhibitor 1, its nucleotide sequence, the construction that contains this nucleotide sequence is used for the treatment of with the acetylation of histone level in preparation and reduces application in the relevant disease.

Not only comprise the full length sequence of beta-protein inhibitor 1 in the implication of term used herein " beta-protein inhibitor 1 ", but also comprised variant form, fragment, the derivant of same or similar biological activity of having of described protein polypeptide or function.These variant forms comprise (but being not limited to): with respect to described amino acid sequence of polypeptide several (preferably 1-10 is individual, is more preferred from 1-5, and the best is 1-3) amino acid whose disappearances, insertion and/or replacement are arranged.Above-mentioned variant form also comprises the analog of above-mentioned albumen or polypeptide.The difference of these analog and native protein can be the difference on difference on the aminoacid sequence and/or the modified forms that does not influence sequence.Term of the present invention " beta-protein inhibitor 1 " also comprises identical with it (homology) or substantially the same (homology) and the polypeptide of same or similar biological activity or function is arranged, for example, have at least 60%, better 70%, also want good more than 80%, 90% even 95% homology or the polypeptide of homogeny.Equally, the variant form with same or similar biological activity or function, fragment that can the encoding said proteins polypeptide, the nucleotide sequence of derivant also contained in term " nucleotide sequence of beta-protein inhibitor 1 ".The construction that comprises described nucleotide sequence also can be used for the present invention.

Term used herein " acetylation of histone level reduce relevant disease " expression can from treated benefit reduce relevant any symptom with the acetylation of histone level, for example comprise cancer, nervous system disease or epigenetic disease." cancer " is meant in the mammal there not to be the physiological symptom that controlled cell is grown to typical characteristic.Can comprise from the cancer example that the present invention benefits, but be not limited to squamous cell carcinoma, pulmonary carcinoma, the intestines and stomach cancer, pancreas cancer, cervical cancer, ovarian cancer, hepatocarcinoma, bladder cancer, hepatoma, mastocarcinoma, colon cancer, colorectal carcinoma, carcinoma of endometrium, salivary-gland carcinoma, renal carcinoma, renal carcinoma, carcinoma of prostate, carcinoma vulvae, thyroid carcinoma etc.

The present inventor has also carried out the experiment of gene chip, RT-PCR, immunoblotting and chromatin co-immunoprecipitation; found that: cause the factor that beta-protein inhibitor 1 increases in the nucleus; can regulate some important gene relevant with cytopathy such as p27 by influencing this epigenetic modification of acetylation of histone, c-fos transcribing and expressing.Prompting beta-protein inhibitor 1, can cause that the g protein coupled receptor that increases in beta-protein inhibitor 1 nuclear is as DOR, KOR (opiate receptor is a kind of of G protein-coupled receptor, and the opiate receptor of finding has three kinds of hypotype: δ, μ, κ at present) etc. might be as the important molecule or the target spot of epigenetic disease treatment.

The present inventor also at the verification experimental verification of people's neuroma cell line SK-N-SH this probability.The stimulation (processing of DOR agonist) of finding to express beta-protein inhibitor 1 or promoting beta-protein inhibitor 1 to advance nuclear all suppresses the neuromatous propagation of SK-N-SH Fig.7.Prompting beta-protein inhibitor 1, and can cause that the g protein coupled receptor that increases in beta-protein inhibitor 1 nuclear is as DOR, the good prospect aspect the epigenetic disease treatment such as KOR.

Therefore, the present invention also relates to delta opiate receptor agonist or kappa opioid receptor agonist on the other hand and is used for the treatment of with the acetylation of histone level in preparation and reduces application in the relevant disease.

Based on above-mentioned application; third aspect present invention relates to a kind of being used for the treatment of and the relevant disease medicament compositions of acetylation of histone level reduction; it is characterized in that; described compositions comprises at least a material that is selected from the nucleotide sequence of beta-protein inhibitor 1, beta-protein inhibitor 1, the construction that contains described nucleotide sequence, delta opiate receptor agonist or kappa opioid receptor agonist of effective dose as active component, and pharmaceutically acceptable carrier.Term used herein " effective dose or treatment effective dose " refers to the therapeutic agent treatment, alleviates or prevent the amount of target disease or situation, or shows the amount of detectable treatment or preventive effect.Depend on the nature and extent of the build of this object and health status, disease and the therapeutic agent selecting to give and/or the combination of therapeutic agent for the accurate effective dose of a certain object.Therefore, specifying accurately in advance, effective dose is useless.Yet, for certain given symptom, can determine this effective dose with normal experiment, the clinicist can judge.Term " pharmaceutically acceptable carrier " refers to be used for the treatment of the carrier or the excipient of agent (for example polypeptide, gene or other therapeutic agent) administration, its should with albumen of the present invention, nucleotide sequence, construction or cytocompatibility, can not reduce the effect of pharmaceutical composition with its blend under normal conditions significantly.The object lesson that can be used as some materials of pharmaceutically acceptable carrier or excipient or its component is a saccharide, as lactose, dextrose plus saccharose; Starch is as corn starch and potato starch; Cellulose and derivant thereof are as sodium carboxymethyl cellulose, ethyl cellulose and methylcellulose; The tragakanta powder; Fructus Hordei Germinatus; Gelatin; Talcum; Kollag is as stearic acid and magnesium stearate; Calcium sulfate; Vegetable oil is as Oleum Arachidis hypogaeae semen, Oleum Gossypii semen, Oleum sesami, olive oil, Semen Maydis oil and cupu oil; Polyhydric alcohol is as propylene glycol, glycerol, Sorbitol, mannitol and Polyethylene Glycol; Alginic acid; Emulsifying agent is as Tween; Wetting agent is as sodium lauryl sulfate; Coloring agent; Flavoring agent; Tablet agent, stabilizing agent; Antioxidant; Antiseptic; Apirogen water; Deng oozing saline solution; With phosphate buffer etc.In addition, (Mack Pub.Co. can find discussing fully about pharmaceutically acceptable excipient in N.J.1991) at Remington ' s Pharmaceutical Sciences.

Pharmaceutical composition of the present invention can be made various dosage forms as required, pharmaceutical composition can be made injectable agent, for example liquid solution or suspension usually; Also can be made into before injection, be fit to allocate in solution or the suspension, the solid form of liquid-carrier.Liposome is also included within the definition of pharmaceutically acceptable carrier.Pharmaceutical composition of the present invention can by the doctor according to patient's kind, age, body weight and roughly factor such as disease condition, administering mode determine the useful dosage of patient is used.In order to improve the medication effect, compositions of the present invention also can be used jointly with other drug.

The present invention also provides a kind of method that filters out the medicine of the treatment disease relevant with the reduction of acetylation of histone level from candidate substances on the other hand, and this method comprises:

A) make described candidate substances and the cells contacting of expressing beta-protein inhibitor 1;

B) identify the endonuclear expression of beta-protein inhibitor 1, and this expression is compared with interior beta-protein inhibitor 1 expression of the nucleus of the described cell that does not contact described candidate substances at described cell;

C) select the material that makes beta-protein inhibitor 1 expression raising in the nucleus reduces relevant disease as described treatment and acetylation of histone level medicine.

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.Unless description is arranged in addition, enforcement of the present invention will be adopted molecular biology, microbiology, recombinant DNA and immunologic routine techniques, and these all are known to those skilled in the art.These technology have complete description in following document: for example, and Sambrook " molecular cloning experiment guide " the 2nd edition (1989); " dna clone " I and II volume (D.N.Glover edits 1985); " oligonucleotide is synthetic " (M.J.Gait edits, 1984); " nucleic acid hybridization " (B.D.Hames and S.J.Higgins edit .1984); " protein purification: principle and put into practice " the 2nd edition (Springer-Verlag, N.Y.), and " experiment immunization is learned handbook I-IV volume (D.C.Weir and C.C.Blackwell edit 1986).Perhaps, can carry out according to the description that the reagent manufacturer is provided.

Embodiment

1 materials and methods

1.1 antibody and reagent

Beta-protein inhibitor 1 mouse-anti, p27 and c-Jun antibody available from BD Biosciences (San Jose, CA).The beta-protein inhibitor multi-clone rabbit is anti-, and (Duke University Medical Center Durham) gives for Robert J.Lefkowitz.P300 CT, CBP, histone H 4, acetylated histones H4 and H3 antibody available from Upstate Biotechnology (Lake Placid, NY).At the mouse-anti 12CA5 of HA-tag available from Roche Molecular Biochemicals (Indianapolis, IN).Tip60 antibody available from Santa Cruz Biotechnology (Santa Cruz, CA).CREB antibody available from Cell Signaling Technology (Beverly, MA).Texas Red coupling mountain sheep anti-mouse igg and FITC coupling goat anti-rabbit igg available from Jackson Immunoresearch (West Grove, PA).IRDye ^TMAnti-mice IgG of the link coupled protein affinity purification of 800CW and anti-rabbit igg available from Rockland Inc. (Gilbertsville, PA).Following antibody all available from Sigma (Saint Louis, MO): the HA rabbit is anti-, the actin rabbit is anti-, Flag, tubulin mouse-anti and SP-1 rabbit are anti-.Two luciferase reporter gene system test kit available from Promega (Madison, WI).Following reagent available from Sigma (Saint Louis, MO): different third (nor-) epinephrine (isoproterenol), [D-Pen2, D-Pen5] enkephalin (DPDPE), (D-Ala 2, and D-Leu 5) enkephalin (DADLE), U69593, forskolin, naltrindole.Δ deltorphin delta-I available from BACHEM California Inc (Torrance, CA).

1.2 cell culture and transfection

The HEK-293 cell, SK-N-SH cell and HeLa cell available from American Type Culture Collection (Rockville, MD).(Gibco-BRL, Gaithersburg MD) cultivate with the MEM culture medium of adding 10% heat inactivation hyclone.Cell culture is at 37 ℃, 5%CO ₂In the incubator.After cultivating 20 hours, the calcium phosphate transfection method transfection of HEK-293 cell, method sees that (Kang, J. wait (2003) .Toxicol Sci 74,279-286.).To the HeLa cell with liposome (Invitrogen, Carlsbad, CA) transfection, method is seen Invitrogen company standard rule of operation.Wild type and β arr1/2 are two to knock out (β arrs-/-) rat embryo fibroblast cell (MEFs) is by Dr.Robert J.Lefkowitz (Duke University Medical Center, Durham) present, cultivate with DMEMmedium (Gibco-BRL), liposome 2000 (Invitrogen, Carlsbad, CA) transfection.

1.3 plasmid construction

Beta-protein inhibitor 1 all is contained in pcDNA 3.0 carriers with the deletion mutant of beta-protein inhibitor 1, and HA-tag is positioned at 3 ' end.The p300 plasmid available from Upstate Biotechnology (Lake Placid, NY).

1.4 laser co-focusing fluorescence microscope

Behind the cell transfecting 44 hours, the MEM culture fluid that changes serum-free was in 37 ℃ of preincubates 2 hours.Add agonist subsequently, 37 ℃ of preincubates 5 minutes, the PBS washing.Use 4% paraformaldehyde room temperature to fix 15 minutes, room temperature was sealed 1 hour in 1%BSA/PBS then.After sealing finished, cell and 12CA5 (1 μ g/ml) incubated at room 1.5 hours in 1%BSA/PBS was washed with PBS, two anti-incubated at room of adding Texas-Red labelling 1 hour, the PBS washing adds 50% glycerol at last coverslip is placed on the microscope slide, and 4 ℃ keep in Dark Place after the mounting.The excitation wavelength of GFP is 488nm, measures wavelength 515-540nm, and the excitation wavelength of Texas-Red is 543nm, measures wavelength 590nm.TCS NT laser confocal microscope writing scan image (Leica Microsystems, Bensheim, Germany).When carrying out the living cells Real Time Observation, behind the cell transfecting 44 hours, the MEM culture fluid that changes serum-free was in 37 ℃ of preincubates 2 hours.In the temperature control cover, use the laser confocal microscope Real Time Observation.Image before at first scanning stimulates, the variation and the real time scan record of the same aspect cell of observation behind the adding agonist.Then, a certain fixed area of choosing homocellular nuclear, endochylema and after birth in the image of scanning is carried out the fluorescence intensity analysis.The fluorescence intensity analysis software is provided by Leica company.

1.5 nuclear preparation

The preparation nuclear extract, according to the method for having reported (Neves, S.R., Ram, P.T., and Iyengar, R. (2002) .Science 296 1636-1639.), slightly changes, and is summarized as follows:

Behind hungry 12 hours of the cell non-serum, hatch different time points altogether with 1 μ M DPDPE; PBS (pH7.4) with 4 ℃ of pre-coolings washes one time, and reuse 400 μ l buffer (containing: 10mM HEPES, pH7.9,10mM KCl, 0.1mM EDTA, 0.1mM EGTA, 1mM DTT, 0.5mM PMSF) are resuspended; After hatching 10 minutes on ice, if HEK293 cell or SK cell then add 3 μ l1%NP-40, if the HeLa cell then adds 3 μ l10%NP-40, mixing was placed 5 minutes on ice gently; Centrifugal 1 minute of 13000g.The gained supernatant is the Cytoplasm extract, and the precipitation part is a nucleus; Nucleus is washed 3 times with hypotonic buffer liquid, adds high osmotic buffer (containing 20mMHEPES, pH7.9,25% glycerol, 400mM NaCl, 1mM EDTA, 1mM EGTA, 1mM DTT and 100 μ g/ml PMSF) then in 4 ℃ of cracking 1 hour; Centrifugal 1 minute of 13000g.The supernatant that obtains is nuclear extract.Behind Bradford method mensuration protein concentration, separate with SDS-PAGE glue.SP-1 is as confidential reference items, show add the nucleoprotein amount unanimity of different time points sample; In addition, also as carrying the proteic indicator protein of nuclear test process center, be presented at that nucleoprotein is outer under the extractive process condition rushes down.Tubulin is also as confidential reference items, show add the cytoplasmic protein amount unanimity of different time points sample; In addition, as the indicator protein of carrying cytoplasmic protein in the nuclear test process, being presented at the nuclear that is proposed under the extractive process condition does not have cytoplasmic protein to pollute yet.

1.6Western immunoblotting

Nuclear extract or cell pyrolysis liquid, ultrasonic 5 minutes, degeneration is 10 minutes in 95 ℃ of water-baths, get about 50 μ g protein samples electrophoretic separation in the 10%SDS polyacrylamide gel, the electrophoresis taking-up gel that finishes, protein electrophorese is transferred on the nitrocellulose filter, film is placed with TBST buffer (150mM NaCl, 50mM Tris-HCl pH7.4,0.2%Tween-20) room temperature sealing 1 hour in Pei Zhi 10% skim milk, afterwards with special one anti-and two anti-hatching (antibody is with the dilution of TBST buffer, washes film three times with the TBST buffer after anti-and the two anti-reactions, each 10 minutes), and detect with the ECL test kit.

1.7 reporter gene analysis

Adopt Promega company's test kit and undertaken by the experimental technique that is provided.Cultivate the HEK293 cell with 12 well culture plates, transfection CREB-luc (0.1 μ g/ hole) and RL-tk-luc (0.2 μ g/ hole, in contrast), corotation or corotation β arr1 (0.1,0.2,0.4,0.8 μ g/ hole) not, and with the amount of pcDNA3-β ga1 polishing plasmid.The MEM culture fluid of serum-free is changed in transfection after 44 hours, continue to hatch 2 hours at 37 ℃.The agonist (Forsklin:10 μ M) that adds the MEM preparation of serum-free then continues 37 ℃ and hatched 6 hours.Behind 1 * PBS washed cell, the passive lysis buffer 100 μ l room temperature cracking in the two luciferase reporter gene test kits that provide with Promega company 20 minutes.Get 5 μ l cell pyrolysis liquids with the reaction of the substrate (test kit provides) of volume, use photometer to detect.Remaining sample detects expressing quantity with the Western immunoblotting.

1.8 immuno-precipitation

Cell is washed one time with the PBS (pH7.4) of 4 ℃ of pre-coolings, the reuse lysis buffer (comprises 50mM Tris-HCl, pH7.5,1mM EDTA, 150mM NaCl, 20mM NaF, 1%Triton-X100,10% glycerol, the 1mM Benzylsulfonyl chloride, 1x protease inhibitor cocktail (Roche Molecular Biochemicals) was in 4 ℃ of cracking 2 hours.Centrifugal 15 minutes of 13000g.Get supernatant and p300 antibody, CREB antibody, β Profilin s antibody or HA antibody were hatched 4 hours 4 ℃ of rotations.Add Protein G (Amersham Biosciences), rotation was hatched 1 hour again.After centrifugal, pearl adds the SDS sample-loading buffer, 50 ℃ of water-baths 20 minutes with lysis buffer washing 3 times.Albumen separates with SDS-PAGE glue.The step and the titre of recommending according to the antibody description detect with Western marking method.

1.9RT-PCR

Total RNA of extracting HeLa, SK, D2P cell and mice Hippocampus, brain forehead page or leaf cortex.Method with quantitative RT-PCR detects p27 in the cell, c-fos, c-jun, the expression of cyclinA and cyclinD1 mRNA.Use primer following listed: in HeLa and SK cell, HPRT-has justice, 5 '-CCT GCT GGA TTA CAT CAA AGCACT G-3 ' (SEQ ID NO:1), antisense, 5 '-TCC AAC ACT TCG TGG GGT CCT-3 ' (SEQ IDNO:2); P27-has justice, 5 '-GTT AAC CCG GGA CTT GGA GA-3 ' (SEQ ID NO:3), antisense, 5 '-CAC AGA ACC GGC ATT TGG GGA ACC-3 ' (SEQ ID NO:4); C-fos-has justice, 5 '-CTCCAG TGC CAA CTT CAT TC-3 ' (SEQ ID NO:5), antisense, 5 '-CAG CCA TCT TAT TCCTTT CC-3 ' (SEQ ID NO:6); C-jun-has justice, 5 '-GCA TGA GGA ACC GCA TCG CTG CCTCCA AGT-3 ' (SEQ ID NO:7), antisense, 5 '-GCG ACC AAG TCC TTC CCA CTC GTGCAC ACT-3 ' (SEQ ID NO:8); Cyclin A-has justice, 5 '-TTA GGG AAA TGG AGG TTA-3 ' (SEQ ID NO:9), antisense, 5 '-TAG TTC ACA GCC AAA TGC-3 ' (SEQ ID NO:10); CyclinDl-has justice, 5 '-GAA CAG AAG TGC GAG GAG GAG-3 ' (SEQ ID NO:11), antisense, 5 '-AGG CGG TAG TAG GAC AGG AAG-3 ' (SEQ ID NO:12).β arrs-/-MEFs and murine brain in, HPRT-has justice, 5 '-CCT GCT GGA TTA CAT TAA AGC ACT G-3 ' (SEQ ID NO:13), antisense, 5 '-TTC AAC ACT TCG AGA GGT CCT-3 ' (SEQ ID NO:14); TBP-has justice, 5 '-TGCACA GGA GCC AAG AGT GAA-3 ' (SEQ ID NO:15), antisense, 5 '-CAC ATC ACA GCTCCC CAC CA-3 ' (SEQ ID NO:16), p27-has justice, 5 '-CAG CTT GCC CGA GTT CTA C-3 ' (SEQ ID NO:17), antisense, 5 '-CCG GGC TTC TTG GGC GTC TGC T-3 ' (SEQ ID NO:18); C-fos-has justice, 5 '-CGA AGG GAA CGG AAT AAG-3 ' (SEQ ID NO:19), antisense, 5 '-CTCTGG GAA GCC AAG GTC-3 ' (SEQ ID NO:20); C-jun-has justice, 5 '-CTT CTA CGA CGATGC CCT CA-3 ' (SEQ ID NO:21), antisense, 5 '-GAA GCG TGT TCT GGC TAT GC-3 ' (SEQ ID NO:22); Cyclin A-has justice, 5 '-CCG CCC GAC GTG GAT GAG-3 ' (SEQ ID NO:23), antisense, 5 '-CTG CGG TCG ATG GGG GAT ACT-3 ' (SEQ ID NO:24); Cyclin D1-has justice, 5 '-TCC CGC TGG CCA TGA ACT ACC-3 ' (SEQ ID NO:25), antisense, 5 '-GGC GCAGGC TTG ACT CCA GAA-3 ' (SEQ ID NO:26).

1.10 chromatin immunoprecipitation (ChIP)

The standard method that the chromatin immunoprecipitation provides by Upstate Biology company.Method with quantitative PCR detects p27 in the cell, c-fos, c-jun, cyclinA and cyclinD1 gene promoter area β arr1, p300, CBP, Tip60, histone H 4, the enrichment degree of acetylation group egg H3 and H4.Use primer following listed: in HeLa and SK cell, p27 promoter-have justice, 5 '-GCT CCC GCC GCC GCA ACC AAT-3 ' (SEQ IDNO:27); Antisense, 5 '-CGA ACC CAG CCG CTC TCC AAA CC-3 ' (SEQ ID NO:28); C-fos promoter-justice is arranged, 5 '-ATT AGG ACA CGC GCC AAG GC-3 ' (SEQ ID NO:29), antisense, 5 '-ACG GTC ACT GCT CGT TCG CT-3 ' (SEQ ID NO:30); C-jun promoter-justice is arranged, 5 '-GGGCGG GCC GGG AAA AAC-3 ' (SEQ ID NO:31), antisense, 5 '-CAT TGG CTC GCG TCGCTC TCA GG-3 ' (SEQ ID NO:32); Cyclin A promoter-justice is arranged, 5 '-GTG TTG TTC CGGACA CAT AGA AAG-3 ' (SEQ ID NO:33), antisense, 5 '-CAG TGC ATT GCT TCA GACTCC AC-3 ' (SEQ ID NO:34); Cyclin D1 promoter-justice is arranged, 5 '-TGC CCC TGT AGT CCGGTT TTC ATA-3 ' (SEQ ID NO:35), antisense, 5 '-GCG CCC CAG TCA CCC CTT CTC-3 ' (SEQ ID NO:36).β arrs-/-MEFs and murine brain in, p27 promoter-justice is arranged, 5 '-TCC GAG GCCAGC CAG AGC AGG TTT-3 ' (SEQ ID NO:37), antisense, 5 '-AGC GGG GAC AGG GGAGGG GGA GAA-3 ' (SEQ ID NO:38); C-fos promoter-justice is arranged, 5 '-CGT CAA TCC CTC CCTCCT-3 ' (SEQ ID NO:39), antisense, 5 '-GGC GCT CTG TCG TCA ACT-3 ' (SEQ ID NO:40); C-jun promoter-justice is arranged, 5 '-CTA CCG GCC AGC AAC TTT C-3 ' (SEQ ID NO:41), antisense, 5 '-CCG CCC GGC CTC CAA CAA-3 ' (SEQ ID NO:42); Cyclin A promoter-have justice, 5 '-CAG GGG AAG TGG GAT GGA TAG-3 ' (SEQ ID NO:43); Antisense, 5 '-CAG CGC TGGATA CCC CTT GAG-3 ' (SEQ ID NO:44); Cyclin D1 promoter-have justice, 5 '-CCC CCA GCGAGG AGG AAT AGA TGA-3 ' (SEQ ID NO:45); Antisense, 5 '-GTA ACA CCC GAA GATGCC AGA CGA-3 ' (SEQ ID NO:46).

1.11 external acetylation of histone and deacetylation analysis

External acetylation of histone enzymatic activity, external acetylation of histone and deacetylation analysis adopt the test kit of UpstateBiology to carry out.Acetylation of histone enzyme PCAF (0.1 μ g), nickel ion (600 μ M) and Antibiotic FR 901228 TSA (100nM) are used as external acetylation of histone enzymatic activity respectively, positive control [the Broday that external acetylation of histone and deacetylation are analyzed, L, Deng (2000) .Cancer Res 60,238-241; Kang, J. waits (2003) .Toxicol Sci 74,279-286; Koyama, Y. waits (2000) .Blood 96,1490-1495].

1.12 albumen-chromatin is in conjunction with experiment

[Donovan, S. wait (1997) .Proc NatlAcad Sci U S A 94,5611-5616 to albumen-chromatin with reference to the method for delivering in the past in conjunction with experiment; Liang, C. waits (1997) Genes Dev 11,3375-338618,19].With extracting buffer (EB; 100mM KCl, 50mM HEPES-KOH, pH7.5,2.5mM MgCl ₂, 50mM NaF, 5mM Na ₄P ₂O ₇, 0.1mM NaVO ₃, 0.5%Triton X-100 is with before adding protease inhibitor cocktail (RocheMolecular Biochemicals)) and cracking about 2 * 10 ⁷Cell.Put half volume 30% sucrose solution, the centrifugal 10min of 10,000 * g in lysate (WCE) below.Precipitation (Crude Pel) is used CaCl ₂(to 1mM) and 5 unit micrococcal nucleases (MNase, Takara Biotechnology) digestion 20 seconds.Stop digestion with EGTA (to 1mM), 10,000 * g, 4 ℃ are centrifugal 5 minutes. centrifugal hour of supernatant (MNase Sup) 500,000 * g, results precipitation (Ultra Pel) and supernatant (Ultra Sup).The all samples that obtains with 8% or the 15%SDS-PAGE gel separation, is detected β arr1, p300, the existence of histone H 4 and tubulin.

1.13[ ³H] thymidine mixes

With β arrs or β arr1 siRNA plasmid transfection SK cell 42 or 90 hours; Or the SK cell is with β arr1 siRNA or p27 siRNA plasmid transfection 90 hours, and hungry 2 hours, reuse 1 μ M of DPDPE or DADLE handled 1 hour, or 1 μ M of DEL handles 30min.The different disposal cell is containing 1 μ Ci/ml of [3H] thymidine (24.0Ci/mmol; Amersham) cultivated 6 hours in the fresh training liquid, detect [3H] thymidine with Beckman multipurpose scintillationS6500 numeration instrument and mix situation.

1.14 mice intracerebroventricular injection

Behind urethra anesthesia three week C57 mices in age (Chinese Academy of Sciences's Shanghai Experimental Animal Center), tricorn (anterior fontanelle-0.25mm; Sidepiece, 1mm; The degree of depth, 2.25mm) injection 2pmol DPDPE (DP, 1.5 μ l/ mices).Some group was being injected DPDPE preceding 15 minutes, intracerebroventricular injection normal saline or 20fmol naltridole (1.5 μ l/mice). and different time points is got mice Hippocampus and cerebral cortex behind the injection DPDPE, carries out RT-PCR and ChIP and analyzes.The regulation that NIH is healthy about laboratory animal and use is followed in zoopery.

1.15 data statistics

Experimental data is represented with meansigma methods ± standard error, carries out curve fitting with the SigmaPlot4.0 mapping software, and experimental group differences significance adopts Student t check, or adopts ANOVA and do many group differences relatively with Bonferroni post-hoc test.

2 results

2.1 activating, DOR cause beta-protein inhibitor 1 to change nuclear

The present inventor is transient expression DOR receptor and β arrs-GFP fusion rotein in the HEK293 cell at first, adopts laser confocal microscope to observe the variation that β arrs distributes under agonist stimulates in cell.When not having agonist to exist, β arr1-GFP is evenly distributed in cytoplasm and nucleus.And β arr2-GFP only is distributed in cytoplasm.After the specific agonist DPDPE of DOR (1 μ M) handled 5 minutes, β arr1-GFP and the β arr2-GFP fluorescence intensity in endochylema descended, and the fluorescence intensity on cell membrane increases.Different with β arr2-GFP is to stimulate back β arr1-GFP also obviously to increase in endonuclear fluorescence intensity at opiate agonist.Arr2 is similar with wild-type beta, and β arr1Q394L mainly is distributed in endochylema, and nuclear translocation does not take place behind receptor activation yet.Be to confirm that β arr1 changes nuclear phenomenon, the present inventor again on the HEK293 living cells of expressing DOR and β arr1-GFP the heavily distribution to the β arr1-GFP of agonist induction carried out real-time monitored.The result shows that before agonist was handled, β arr1-GFP was evenly distributed in cell cytosol and the nuclear.Added behind the agonist 1 minute, β arr1-GFP fluorescence intensity just obviously increases simultaneously intracytoplasmic fluorescence intensity and reduces in cell membrane and the nuclear, and cell membrane and nuclear fluorescence intensity reach maximum and fluorescence intensity in the cytoplasm reaches minimum during by 3 minutes.These results suggest under agonist stimulates β arr1-GFP in the cell membrane transposition also to the nucleus transposition.For getting rid of GFP may influence to viewed β arr1 transposition, the present inventor at coexpression the HEK293 cell and only having expressed in the HeLa cell of DOR of DOR and β arr1-HA stimulate with 1 μ M DPDPE, adopt the method for western immunoblotting to detect the variation of β arr1-HA and endogenous β arr1 content in the nuclear extract then.The result shows it is that β arr1-HA or the content of β arr1 in nuclear all are significantly increased after agonist is handled.These results show that activating DOR has induced β arr1 to nuclear transposition really.Discover that further the activation of KOR is the same with the activation of DOR, can cause that β arr1 is to the nucleus transposition.But activate MOR or β 2AR, can not cause that β arr1 is to the nucleus transposition.

Promote the p27 and the c-fos of β arr1-mediation to transcribe 2.2DOR activate

In conjunction with the PRELIMINARY RESULTS of gene chip, the present inventor at first uses quantitative RT-PCR and Western trace method has been studied β arr1 to p27, c-fos, c-jun, cyclinA and cyclin D1 these and cell proliferation related influence because of expressing.The result shows, compares with contrast HeLa cell, crosses the obvious rising that expression β arr1 (endogenous approximately β arr1 8 times) causes p27mRNA and protein level, and the horizontal expression β arr2 or the β arr1Q394L that mainly are distributed in endochylema then do not have this effect equally.Corresponding with it, the application of β arr1 siRNA and β arr2 siRNA has reduced the expression (reducing by 80% approximately) of endogenous β arr1 in the cell or β arr2 equally significantly, but has only the β arr1 siRNA can significantly to reduce p27 and the mRNA level of c-fos and the protein level of p27 in the cell.Because c-Fos albumen instability, the present inventor does not detect c-Fos albumen.In contrast, β arr1 and its siRNA do not influence c-jun in the cell, cyclin A, and the expression of cyclin D1.β arr1 to this influence of genetic transcription β arrs-/-also be confirmed in MEF (β arr1 and β arr2 the are two to knock out) cell [Kohout, T.A. wait (2001) .Proc Nat1 Acad Sci U S A 98,1601-1606].These results prove that β arr1 has regulated p27 and c-fos gene transcription really, and this adjusting is relevant with the distribution of β arr1 in nuclear.Therefore, the present inventor has studied and can cause that β arr1 changes the DOR that examines and activates the influence that said gene is transcribed.DPDPE handles the obvious rising that causes p27mRNA and protein level, to then not effect of c-jun.This effect of DPDPE can be blocked by the antagonist of DOR or β arr1 siRNA, but be not subjected to Gi/Go (pertussis toxin, PT), PI3K (wortmannin), p38 (SB203580), JNK (SP600125), or the influence of the inhibitor of ERK (PD98059), demonstration DOR activation can be examined direct regulator gene by the commentaries on classics of inducing β arr1 and transcribe.

2.3 the rising of concentration has promoted the acetylation of p27 and c-fos promoter region histone H 4 in the β arr1 nuclear

The present inventor has studied the influence of β arr1 to acetylation of histone.The result of Figure 1A shows in the HeLa cell, expresses β arr1 and its siRNA and significantly promotes or inhibition of histone H4 acetylation, does not influence the histone H 3 acetylation.Find that further in the histone H 4 aminoterminal can be acetylation four sites [Grunstein, M. (1997) .Nature 389,349-352] of modifying, β arr1 mainly influenced the acetylation of Lys-12 and Lys-16.The same with β arr1 to the influence of genetic transcription, β arr1 to this regulating action of acetylation of histone equally can β arrs-/-be confirmed in the MEF cell (Figure 1B).Estimate as the present inventor, β arr2, β arr1 Q394L and β arr2 siRNA do not influence acetylation of histone (Fig. 1).These results show that the β arr1 in the nuclear can regulate the acetylation of histone H 4, thereby influence comprises the transcribing of some specific genes of p27 and c-fos.

Whether acetylation of histone changes has regulating action to depend on that especially whether whether this variation occurs near the specific gene, occur in the promoter region of this gene to genetic transcription.Chromatin immunoprecipitation experiment (ChIP) shows; no matter at HeLa; still β arrs-/-the MEF cell in, β arr1 significantly promotes the acetylation of p27 and c-fos promoter region histone H 4, β arr1 siRNA then significantly suppresses the acetylation (Fig. 2 A) of these regional histone H 4s among the HeLa.With top the same, β arr2 and β arr1Q394L do not have this effect (Fig. 2 A), and the enrichment of prompting β arr1 in nuclear is that histone H 4 acetylation variation is needed.β arr1 does not influence c-jun; cyclin A; and the acetylation of cyclin D1 promoter region histone H 4; do not influence acetylation (Fig. 2 A that all measure the gene promoter area histone H 3 yet; cyclin A; the data not shown of cyclin D1), acetylizad adjusting has gene specific to histone H 4 to show β arr1.Accordingly; DPDPE handles β arr1-and receptor activation relies on ground promotion p27 (Fig. 2 B; 2C) and the acetylation of c-fos (data not shown) promoter region histone H 4, illustrate that the inductive β arr1 of receptor activation changes the acetylation that karyogene has promoted some promoter region histone H 4 specifically.The data show β arr1 that obtains from different β arr1 mutants is to the acetylizad promotion of p27 promoter region histone H 4 and it the adjusting positive correlation (Fig. 2 E) to these genetic transcriptions.

Regulate the acetylizad mechanism of histone H 4 in order to study β arr1, whether the present inventor has at first studied β arr1 and has combined with chromatin.Seven component proteins-chromatin shows (Fig. 2 D) in conjunction with experiment, similar with chromobindins histone H 4 and p300, outer commentaries on classics and endogenous β arr1 are distributed in chromatin deposited components (swimming lane 3) just, micrococcal nuclease (MNase) digestion back supernatant (swimming lane 4), and in the precipitation (swimming lane 7) behind the MNase digestion supernatant ultracentrifugation, show that β arr1 can combine with chromatin.The ChIP experiment confirms that further β arr1 is enriched in p27 and c-fos promoter region specifically in the HeLa cell, and not at c-jun (Fig. 2 A), cyclin A or cyclin D1 (data not shown) promoter region.As estimating, β arr1 antibody β arrs-/-be settled out any DNA sequence (Fig. 2 A, the data not shown of cyclin A and cyclin D1) among the MEFs, proved the specificity of this antibody.The same to the acetylizad adjusting of histone H 4 with the DOR activation, DOR activates and has also promoted the enrichment (Fig. 2 B, the data not shown of c-fos) of β arr1 at p27 and c-fos promoter region greatly.These results show that β arr1 may be relevant to the acetylizad promotion of these regional histone H 4s with it in the enrichment of p27 and c-fos promoter region.

2.4 β arr1 recruits p300 to p27 and c-fos promoter region

The level of acetylation of histone is subjected to acetylation of histone enzyme (HAT) and histon deacetylase (HDAC) (HDAC) control in the body.The present inventor adopts the external acetylation of histone that carries out of HA-β arr1 and the dna methylase inhibitor of GST-β arr1, immunoprecipitation the analysis showed that; β arr1 is neither acetylation of histone enzyme and histon deacetylase (HDAC), the external activity (Fig. 3 A-3C) that does not also influence acetylation of histone enzyme and histon deacetylase (HDAC).The more important thing is that I, II, III histone deacetylases inhibitor trichostatin A (TSA) and sirtinol (STN) do not influence the acetylation (Fig. 3 D) of β arr1 to p27 and c-fos promoter region histone.These presentation of results β arr1 may advance acetylation of histone by recruiting the acetylation of histone enzymatic in the specific gene zone.

P300, CBP and Tip60 are very important acetylation of histone enzymes.There is report to find that relevant with c-fos promoter region acetylation of histone with CBP [Chan, H.M. wait (2001) .J Cell Sci 114,2363-2373 to p300; Usenko, T. waits (2003) .Oncogene 22,7661-7666.], Tip60 then may combine [Salwinski, L, Miller, (2004) .Nucleic Acids Res 32, D449-451] with β arr1.Therefore, the present inventor has studied β arr1 to the influence of these enzymes in p27 and the enrichment of c-fos promoter region.Studies show that, express β arr1 siRNA or β arr1 reduces respectively or promote p300 in p27 and the enrichment of c-fos promoter region, but do not influence CBP and Tip60 and distinguish enrichment condition (Fig. 4 A) at these, do not influence p300 yet, CBP and Tip60 be at c-jun (Fig. 4 A), the enrichment of cyclin A and cyclin D1 (data not shown) promoter region.Stimulate the result of promotion p27 and c-fos promoter region β arr1 enrichment and acetylation of histone consistent with DPDPE, DPDPE stimulates and has also promoted the enrichment (Fig. 2 B, 4B, c-fos β data not shown) of p300 at p27 and c-fos promoter region.Carry out co-immunoprecipitation with HeLa nucleus extract and find between β arr1 in the nuclear and p300 interaction is arranged.These results show that β arr1 gene specific ground promotes that the mechanism of acetylation of histone may be that it has recruited these gene regions to p300.

2.5p300 in the acetylation of histone of β arr1-mediation and the effect in the genetic transcription

No matter HeLa still be β arrs-/-the MEF cell in, p300 crosses to express and significantly promotes p27 and the acetylation of c-fos promoter region histone H 4, and this facilitation of p300 can be promoted by β arr1 or β arr1 siRNA or stop.The more important thing is that p300 dominant negative mutant (p300 DN) has significantly reduced β arr1 to p27 and c-fos promoter region histone H 4.These data show p300 plays a significant role in the acetylation of histone of β arr1-mediation.Corresponding with it, to cross and express p300 DN and also significantly reduced the transcriptional activation of β arr1 p27 and c-fos, the gene specific acetylation of histone of prompting β arr1-mediation is relevant with the transcriptional activation that its mediates.

Have report to show that transcription factor cAMP response element conjugated protein (CREB) can be recruited CBP and p300, and regulate p27 and c-fos transcribe that [Mayr, B. wait (2001) .Nat Rev Mol Cell Biol 2,599-609; Garcia, C. waits (2004) J Neurosci Res 78,338-346].Nearest research also shows acetylation of histone play an important role [Johannessen, M. wait (2004) .Cell Signal16,1211-1227] in the gene (as c-fos) that CREB-relies on is transcribed.The experiment of present inventor's co-immunoprecipitation shows between endogenous β arr1 and CREB that interaction is arranged.The expression that further studies show that CREB siRNA has significantly reduced the enrichment of β arr1 at p27 and c-fos promoter region, and conversely, the expression of β arr1 and β arr1 siRNA does not influence the enrichment condition of these zone C REB.β arr1 crosses the transcriptional activity that expression does not influence CREB in addition; these results show that β arr1 transcribes by the transcriptional activity regulator gene of direct activating ELK 1 B; under the GPCR signal stimulus; interaction between β arr1 and CREB may participate in the enrichment of β arr1 in the specific gene zone, the variation of these regional acetylation of histone and these gene transcription.

2.6 activate the acetylation of histone that neuronal cell surface DOR promotes that β arr1-relies on, p27 transcribes and breeds inhibition

DOR is expressed in central nervous system's an important neurotransmitter receptor.In order to verify under more approaching physiological condition; DOR activates whether cause equally that histone H 4 acetylation and p27 transcribe; the present inventor expresses the human brain neuroblastoma SK cell [Yu of DOR with 1 μ M DPDPE stimulation of endogenous; V.C.; Deng (1986) .JBiol Chem 261,1065-1070].Fig. 5 A and 5B show that DPDPE processing time dependency ground promotes β arr1 to change nuclear, and histone H 4 acetylation of p27 promoter region and p27 transcribe.These effects of DPDPE can be blocked by naltridole and β arr1 siRNA.Similar with it, intracerebroventricular injection DPDPE also DOR-promotes mice hippocampus and the histone H 4 acetylation of cerebral cortex p27 promoter region and p27 to transcribe specifically, shows that vivo activation DOR causes that equally histone H 4 acetylation and p27 transcribe.[Kiyokawa, H. wait (1996) .Cell 85,721-732 because p27 mainly participates in cell inhibitory effect; Nakayama, K. waits (1996) .Cell 85,707-720], the present inventor has studied β arr1 and DOR activates whether can rely on the propagation that ground suppresses the SK cell by p27-.Fig. 5 D shows that the expression p27-dependency ground of β arr1 or β arr1 siRNA significantly suppresses or promoted the propagation of SK cell.Corresponding, DOR activation β arr1-and p27-dependency ground have suppressed the propagation of SK cell.Simultaneously; other agonist of DOR such as DADLE (with the DPDPE structural similarity) or deltorphin-I (different with the DPDPE structure) can cause that also β arr1 changes nuclear (Fig. 5 A), histone H 4 acetylation, p27 transcribes (Fig. 5 C), and the propagation of SK cell suppresses (Fig. 5 D).The epigenetic mechanism of these Notes of Key Datas β arr1-mediation activates to regulate in the physiological activity process at neurocyte DOR and plays a significant role.

3. discuss

β arrs is the moderator and the joint albumen (scaffold protein) of signal in the well-known kytoplasm.Film has played all in receptor desensitization and signal transduction that very important [Claing, A. wait (2002) .Prog Neurobiol 66,61-79 on the β arrs of receptor activation mediation; Luttrell, L.M. waits (2002) .J Cell Sci 115,455-465].The β arr1 function of all reports all occurs on Cytoplasm or the cell membrane, never has related for the function of β arr1 in nuclear.The present invention finds that DOR activates and causes β arr1 to change nuclear, and the p27 and the c-fos that promote β arr1-to rely on transcribe, and has disclosed β arr1 and not only can mediate the transduction of signal in kytoplasm, and might be delivered to the signal that comes from receptor the nucleus.β arr1 crosses and expresses and β arr1 that receptor activation causes changes nuclear and promoted the acetylation of histone of p27 and c-fos promoter region and p27 and c-fos to transcribe, and shows that by changing that the epigenetic modification regulator gene transcribes be the function of β arr1 in examining.Put it briefly, studies have shown that originally chromatin is the direct target of GPCR-Mediated Signal Transduction, disclosed the epigenetic mechanism of GPCR signal from film to nuclear.It simultaneously also is first report about function in the β arr1 nuclear.

Nearest studies show that, during eukaryotic gene is transcribed, and epigenetic modification variation effect great [Grewal, S.I. wait (2003) .Science 301,798-802].Wherein, the variation of the acetylation modification of nucleosome histone is that [Cheung, W.L. wait (2000) .Curr Opin Cell Biol 12,326-333 to the active important decision maker of chromatin Structure and genetic transcription; Grunstein, M. (1997) .Nature 389,349-352].The accumulation of β arr1 had promoted the acetylation of p27 and c-fos promoter region histone H 4 and p27 and c-fos to transcribe in the present inventor found to examine, and prompting promotes acetylation of histone to represent β arr as crucial new function of courier in the nuclear.Discover that further the acetylation of histone and the genetic transcription of β arr-mediation are subjected to outer signals, activate the accent sky as DOR and KOR, the modification of prompting histone may be the outer signals of cellular integration GPCR-mediation and the epigenetic mechanism that produces transcription response.

Acetylation of histone enzyme (HAT) and histon deacetylase (HDAC) (HDAC) are the pherons of control acetylation of histone height in the cell.Present inventor's result shows that HDAC does not participate in the adjusting of β arr1 to acetylation of histone, and β arr1 does not also have or influence the activity of HAT.Therefore the present inventor infers that β arr1 regulates acetylation of histone by influencing HAT albumen raising of gene region.The expression of present inventor's experiment confirm β arr1 and β arr1 siRNA significantly improves respectively or has reduced the accumulation of p300 at p27 and c-fos promoter region, and p300 DN suppresses the acetylation of histone and the genetic transcription of β arr-mediation.In conjunction with having interaction between p300 and the β arr1, behind present inventor's the data show receptor activation, in the specific gene zone, β arr as p300, promotes these regional acetylation of histone by raising HATs.

There are some researches show that before genetic transcription was initial, the transcription factor of promoter region needed harmony ground to recruit large quantities of albumen that comprise the chromatin reconstitution enzyme.Specific chromatin reconstitution enzyme is by guaranteeing that with transcription factor interaction chromatin reconstitution can take place on the correct gene of suitable cell of suitable time.The present inventor studies show that, transcription factor CREB intermediary the accumulation of β arr1 at p27 and c-fos promoter region, but,, do not have the existence of β arr1 here although CREB also is present in the c-jun promoter region.More and more evidences shows outside the transcription factor, the nucleoprotein structure that specific promoter region is pre-existing in play a significant role equally in this zone chromatin reconstitution [Urnov, F.D. and (2001) .Oncogene 20,2991-3006].The data show of present inventor's mass spectral analysis remains at multiple nucleoprotein in the β arr1 complex except that p300 and CREB.Therefore, p27 and c-fos promoter region may also exist recruits other essential factor of β arr1, and this is the β arr1 regulator gene mechanism of transcribing specifically just.

One of previous critical function that studies show that β arrs goes to strengthen the effect of GPCR signal or/and specificity [Hall, R.A., (2002) .Circ Res 91,672-680] as scaffolding protein exactly.As, after GPCR activated, β arrs in turn in conjunction with the kinases that works among the JNK3 cascade, caused the activation of JNK3 and the retardance in Cytoplasm [McDonald, P.H. wait (2000) .Science 290,1574-1577] in Cytoplasm.The previous research of present inventor finds that also β arr2 can be targeted to activated GPCR receptor to Mdm2, thereby suppresses self ubiquitinization of Mdm2 and the p53 degraded of Mdm2 mediation.Similar with it, β arr1 may also be as support molecule and signaling molecule in nuclear, by with transcription factor and other nucleoprotein effect recruitment HAT to the specific gene zone, thereby regulator gene is transcribed.

The present inventor discovers; β arr1 crosses expression and DOR activates the acetylation of histone that promotes β arr1 commentaries on classics nuclear to promote p27 and cfos promoter region; improved the expression of p27; but do not significantly improve transcribing of c-fos; prompting is for the normal transcription of c-fos, and endogenous β arr1 and/or basic acetylation of histone are enough in the cell.β arr1siRNA reduces c-fos promoter region acetylation of histone simultaneously and c-fos transcribes the above-mentioned viewpoint of support.In the gene that detects, β arr1 crosses and expresses and DOR activates the β arr1 that causes and changes to examine and do not influence c-jun, and cyclin A and cyclin D1 transcribe acetylation of histone with these gene regions.In p27 and c-fos promoter region, the present inventor infers that GPCR activates histone modification and the genetic transcription that can induce to β arr dependence a series of specific genes zone in conjunction with accumulation specific effect in the β arr1 nuclear.

Barrs is the important moderator of GPCR signal, and two kinds of hypotypes of β arr1 and β arr2 are arranged.Although the two enjoys～80% homology, and the present inventor discovers, after agonist stimulates, has only β arr1 transfer in nuclear.These two kinds of β arrs that studies show that in the past can shuttle back and forth between Cytoplasm and nucleus.But different with β arr1 is, the C end of β arr2 has one and strong goes out nuclear signal [20,39].The point mutation β arr1 Q394L of β arr1 have with β arr2 same go out nuclear signal, the present inventor finds that stimulant can not induce β arr1 Q394L to go out nuclear, shows that the C end of β arrs has important effect aspect the concentration in regulating different subtype β arrs nuclear.These results point out in the nuclear of regulating the GPCR mediation in the signal simultaneously, β arr1 hypotype performance more important role.This research is also observed, and DOR and KOR stimulate can cause that β arr1 changes nuclear, and β ₂AR or MOR stimulate does not but have this function, shows that some specific GPCR signals may more prefer to the epigenetic path of this β arr1 mediation.

The GPCR signal can be transmitted to nuclear by film by transduction cascade paths different in the Cytoplasm.GPCR activates the typical born of the same parents' inner gateway cause and comprises: the proteic activation of trimer G that links to each other with receptor, with the adjusting of variation, unlike signal molecule such as the MAPKs and the PKA of the hydrolysis of the bonded GTP of G α subunit, cAMP generation, finally cause target gene transcribe variation [Shaywitz, A.J., Deng (1999) .Annu Rev Biochem 68,821-861; Neves, S.R. waits (2002) .Science 296,1636-1639.].DOR activates born of the same parents' inner gateway that may cause and comprises: [Eisinger, D.A. wait (2004) .J Pharmacol ExpTher 309,776-785 for Gi albumen, ERK1/2, JNK, P38 and the road activation of PI3K grade UNICOM; Persson, A.I. waits (2003) .Mol Cell Neurosci 23,360-372; Shahabi, N.A. waits (2003) .Cell Immunol 221,122-127; Zhang, Z. waits (1999) .J Neurochem 73,1502-1509].The present inventor finds that the gene specific acetylation of histone of agonist induction and genetic transcription can be blocked by the antagonist of DOR or β arr1 siRNA, but be not subjected to Gi/Go, the influence of PI3K, p38, JNK or ERK inhibitor.Need the commentaries on classics nuclear of β arr1 to reach in conjunction with regional acetylation of histone of the specific gene of p300 mediation and genetic transcription in the zone accumulation of specific chromatin; it is to change nuclear by β arr1 by film to the transduction of examining that these results suggest DOR activates the outer signals that causes, rather than mediate by above-mentioned known intracellular signal path.

Research in the past thinks that endocytosis is the negative feedback regulatory mechanism of membrane receptor signal.Recent studies show that accept to stimulate after, particular group become second nature the endocytosis albumen that between Cytoplasm and nucleus, shuttles back and forth also participate in directly outer signals by film to the transmission of examining [Benmerah, A. (2004) .Curr Biol 14, R314-316].As, under environmental stimuli, endocytosis protein A PPL1 changes nuclear and transmits signal [Miaczynska, M. wait (2004) .Cell 116,445-456.] by interacting with the NuRD/MeCP1 complex.Present inventor's result shows, thereby DOR activation causing endocytosis albumen β arr1 changes acetylation and these gene transcription that nuclear, specific gene region beta arr1 and HAT protein enrichment have promoted specific gene zone histone.These data displays β arr as the scaffolding protein of tool signal effect in a direct courier and the nuclear GPCR signal is delivered to nuclear by film, and the new function of transcribing by the epigenetic modification regulator gene.The experiment that obtains from people's neuroma cell and Mus Hippocampus further confirms, activates endogenous DOR and causes that equally β arr1 changes the acetylation and the p27 gene transcription of nuclear, p27 gene region histone.

The epigenetic network equalize is significant to people's normal development, destroys to cause that then [Egger, G. wait (2004) .Nature 429 to multiple pathological changes such as comprising cancer, the unstable syndrome of chromosome and dysmnesia, 457-463; Sutherland, J.E. waits (2003) .Ann N Y Acad Sci 983,151-160].Increasing research thinks that it is cell produces whole transcription response to environmental stimulus important path [Levenson, J.M. wait (2005) .Nat RevNeurosci 6,108-118] that epigenetic is regulated.Present inventor's the DOR activation-inducing β arr1 that studies show that changes nuclear, cause histone modification to change, gene activation and cell inhibitory effect disclose the GPCR signal and can regulate by the epigenetic modification pair cell, in this course, β arr plays an important role once more.Further investigation will help to disclose as signal support albumen between cell membrane and nucleus, and the mechanism that β arr regulator gene is transcribed also helps to disclose the molecular mechanism of epigenetic signal path of the dependence β arr of receptor-inducible.

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 read of the present invention above-mentioned tell about content after, those skilled in the art can make various changes or modifications 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

Fudan University

＜120〉application of beta-protein inhibitor in the medicine of preparation treatment genetic correlated disease

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Claims (9)

1. beta-protein inhibitor 1, its nucleotide sequence, the construction that contains this nucleotide sequence are used for the treatment of with the acetylation of histone level in preparation and reduce application in the relevant disease.

2. application as claimed in claim 1 is characterized in that, the described disease relevant with the reduction of acetylation of histone level is cancer, nervous system disease or epigenetic disease.

3. delta opiate receptor agonist or k opioid receptor agonist are used for the treatment of with the acetylation of histone level in preparation and reduce application in the relevant disease.

4. application as claimed in claim 3 is characterized in that, the described disease relevant with the reduction of acetylation of histone level is cancer, nervous system disease or epigenetic disease.

5. application as claimed in claim 3 is characterized in that, described delta opiate receptor agonist is selected from [D-Pen2, D-Pen5] enkephalin, (D-Ala2, D-Leu5) enkephalin or deltorphin-I.

6. one kind is used for the treatment of and the relevant disease medicament compositions of acetylation of histone level reduction; it is characterized in that; described compositions comprises at least a material that is selected from the nucleotide sequence of beta-protein inhibitor 1, beta-protein inhibitor 1, the construction that contains described nucleotide sequence, delta opiate receptor agonist or k opioid receptor agonist of effective dose as active component, and pharmaceutically acceptable carrier.

7. pharmaceutical composition as claimed in claim 6 is characterized in that, the described disease relevant with the reduction of acetylation of histone level is cancer, nervous system disease or epigenetic disease.

8. pharmaceutical composition as claimed in claim 6 is characterized in that, described delta opiate receptor agonist is selected from [D-Pen2, D-Pen5] enkephalin, (D-Ala2, D-Leu5) enkephalin or deltorphin-I.

9. method that from candidate substances, filters out the medicine of the treatment disease relevant with acetylation of histone level reduction, this method comprises:

A) make described candidate substances and the cells contacting of expressing beta-protein inhibitor 1;

B) identify the endonuclear expression of beta-protein inhibitor 1, and this expression is compared with interior beta-protein inhibitor 1 expression of the nucleus of the cell that does not contact described candidate substances at described cell;

C) select the material that makes beta-protein inhibitor 1 expression raising in the nucleus reduces relevant disease as described treatment and acetylation of histone level medicine.

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