CN100379752C - Antagonists of BMP and TGF beta signalling pathway - Google Patents

Abstract

This invention provides unique members of the Hect family of ubiquitin ligases that specifically target BMP and TGF beta /activin pathway-specific Smads. The novel ligases have been named Smurf1 and Smurf2. They directly interact with Smads1 and 5 and Smad7, respectively, and regulate the ubiquitination, turnover and activity of Smads and other proteins of these pathways. Smurf1 interferes with biological responses to BMP, but not activin signalling. In amphibian embryos Smurf1 inhibits endogenous BMP signals, resulting in altered pattern formation and cell fate specification in the mesoderm and ectoderm. The present invention provides a unique regulatory link between the ubiquitination pathway and the control of cell fate determination by the TGF beta superfamily during embryonic development. Thus, Smurf1 is a negative regulator of Smad1 signal transduction, by targeting Smad1, Smurf1 blocks BMP signalling. In mammalian cells, Smurf2 suppresses TGF beta signalling, and in Xenopus, blocks formation of dorsal mesoderm and causes anterior truncation of the embryos. Smurf2 forms a stable complex with Smad7, which induces degradation and downregulation of TGF beta /activin signalling.

Description

The antagonist of BMP and TGF signal transduction pathway

Obtain research of the present invention and obtain NIH and partly subsidize, subsidizing number is SR01HD3242902.So United States Government has certain rights and interests to the present invention.

Background of invention

In fact, the adjusting that the committed step of fetal development is subjected to the signal between cell and the cell or secretes the inducement signal of somatomedin mediation in all animal doors.Specifically, transforming growth factor-beta (TGF β) superfamily member is regulated various kinds of cell and growth course, and for example mitotic division, cytodifferentiation, the graphic formation of embryo and organ take place.In the vertebrates embryo, various TGF signals influence germinal layer specialization, the graphic formation of body (body patterning), cell growth and differentiation (1-4).In Amphibians Xenopus laevis (Xenopus) embryo, different TGF β members induce different cell fates, for example activin, Vgl and nodal inducing embryo dorsal mesoderm feature, for example notochord and muscle.Vgl and activin are also induced the entoderm feature.On the contrary, bone morphogenetic protein (BMP) specificity is induced mesoderm, for example blood and mesenchyme, and regulate ectodermic epidermic cell and neurocyte differentiation (referring to summary (5)).

By the Smad family protein signal is directly transduceed to nuclear dna target by the cell surface receptor mixture, thereby make cell produce reaction (referring to summary (4), (6), (7) and (52)) TGF 'beta ' family part.

Smad relates to fruit bat (Drosophila) Mad (mothers againstdecapentaplegic[dpp]) and 3 kinds of relevant nematode gene Sma2, Sma3 and Sma4 encoded protein.For unified name, term Sma and Mad have been fused to Smad.There are 8 members in Smad family.PSmad1,5 and 8 is functional mediation BMP family signal transduction under Smad4 synergy.Smad2 and 3 is signal transducers of TGF β and activin effect.Smad6 and 7 antagonist actions that suppress TGF β/BMP superfamily signal transduction.What is interesting is that Smad7 is positioned in the nuclear, under the effect of TGF signal transduction, accumulate in (73) in the tenuigenin.And the two expression of Smad6 and Smad7 is subjected to the adjusting of TGFb, BMP, somatomedin and cytokine, therefore the Smad signal transduction pathway produced reverse feedback and regulates (53-58).When pSmad1 enters nucleus, form allos polymerization (heteromeric) mixture with Smad4, and activate early stage response gene and transcribe.Bmp receptor also can send signal by the mitogen activated protein kinase.BMP may regulate cell cycle progression, therefore controls the mescenchymal stem cell differentiation.

The signal transduction of BMP and activin/two main paties of TGF β is existing to be introduced in detail.Two different receptor subunits I types and II type transmembrane serine/threonine kinase form activated camplex when combining with part.In these two kinds of mixtures, II type subunit activates I type subunit, the direct phosphorylation of I type subunit also activates the R-Smad albumen that specific acceptor is regulated: bmp receptor is at Smad1 and closely-related Smad5 and 8, and activin and TGF beta receptor are at Smad1 and closely-related Smad2 and 3.These R-Smad and Smad during activation " common mating partner " Smad4 forms the allos polymer composites.This mixture is transported to nuclear, is attached on the promotor of target gene conjugated protein the working in coordination with down of DNA, at last by raising the coactivator activated transcription.The 3rd class inhibition Smad (I-Smad) Smad6 and Smad7 work the inhibitor effect that hinders formation of Smad-Smad mixture or Smad-acceptor interaction.I-Smad is attached to the cytoplasmic structure territory of acceptor or directly in conjunction with Smad1.The sudden change of component on all levels of this approach all with embryo's defective and various related to cancer, the importance (referring to summarize (4), (7)) of outstanding this growth factor family of explanation in growth and lysis.Especially Smad2 is relevant with colorectal carcinoma and lung cancer with the Smad4 defective, and people Smad4 defective is relevant with carcinoma of the pancreas.

Smad does not have inherent enzymic activity.Therefore, very responsive to the cellular responsibility of the Smad signal transduction intracellular Smad protein level of verifying.In fact, the subtle change of the Smad protein content of cell expressing just can change Xenopus laevis embryo's cell fate decision (8-11).So the Smad protein level of regulating in the cell can be used as a kind of means of regulating form generation signal transduction by the TGF beta superfamily.

The protein modified general signal (referring to summary (12) and (13)) that be considered to by proteasome degraded target protein covalently bound with ubiquitin.Selectivity ubiquitination target comprises transcription factor, cyclin, signal transducer and membranin (reference in (12)).The important mechanisms that proteic selectivity ubiquitination of specific objective and degraded can be used as control cell cycle progression, apoptosis, differentiation and fetal development plays a role.Ubiquitination approach function obstruction is relevant with disease and heteroplasia.The ubiquitin ligase enzyme is the integral part of the covalently bound polycomplex of the ubiquitin of catalysis 12.5kD polypeptide and target protein.Ubiquitin is connected as sign by being called the proteic molecule of organoid proteolysis ubiquitination " sign " of 26S proteasome with its target protein.Have at least 3 kinds of enzymes participation ubiquitins and target protein to put together, i.e. E1, E2 and E3.The E1 enzyme activates the ubiquitin molecule, and it is combined with the E2 enzyme, and the E2 enzyme directly makes ubiquitin be connected on the target protein then, perhaps sends it on the E3 ubiquitin ligase enzyme.E3 discerns specific substrates and instructs the substrate ubiquitination.

Dictyostelium (14-16) and Drosophila (17-21) have introduced the ubiquitination system and have regulated several examples of growing.Various different systems utilize ubiquitin and acceptor to put together control endocytosis and signal transduction and the two the control acceptor steady-state level (59-60) of degraded by proteasome and lysosome mediation.In many systems characterized the direct ubiquitination of membrane receptor, but as if the ubiquitin dependency is regulated and not to be related to directly the puting together of ubiquitin and acceptor (61-62) in some cases.Although many cell surface receptors are subjected to the adjusting of ubiquitin dependent pathway, only clear and definite seldom several combine with membranin and its effect is the E3 ubiquitin ligase enzyme that makes the membranin ubiquitination.Nedd4 is a C2-WW-HECT structural domain E3 ubiquitin ligase enzyme, and it can directly combine the conversion (63-66) of regulating described sodium channel by the PPXY primitive with the existence of amiloride responsive type sodium channel C-terminal.In addition, confirm that recently FING finger protein c-cbl has the effect of E3 ubiquitin ligase enzyme, c-cbl is in conjunction with receptor-mediated ubiquitination of EGF and downward modulation receptor complex (67-68).As if in these examples, the membranin ubiquitination relates to the direct interaction of E3 ligase enzyme and target protein.Whether do not know to connect albumen also can play a part to raise the E3 ligase enzyme to the special receptor mixture.Control still unpredictable of the mechanism of ubiquitination in the graphic formation and purpose so far.

After in whole specification sheets, listing in embodiment with the reference of numeric reference.All reference that this paper quotes are attached to herein by reference.

Summary of the invention

The present invention advantageously provides a class and relates to BMP and the beta mediated property of TGF activatory adjusting albumen.Specifically, these albumen are regulated Smad albumen and/or the degraded of promotion TGF beta receptor mixture in the presence of Smad albumen.By the proteic activity of control the present invention, those of skill in the art can raise or the downward modulation cellular activity by for example BMP or TGF β.

Therefore, first aspect the invention provides isolating Smurf albumen, especially people Smurf albumen.In one embodiment, Smurf albumen is Smurf1 albumen.In an alternate embodiment, Smurf albumen is Smurf2 albumen.In specific embodiments, for instance, the aminoacid sequence of people Smurf1 is seen Figure 10 (SEQ ID NO:2), and the aminoacid sequence of people Smurf2 is seen Figure 12 (SEQ ID NO:4).Smurf albumen of the present invention can contain SEQ ID NO:2 and 4 describe sequence at least about 5 continuous amino acids, preferably at least about 10 continuous amino acids.

The present invention also provides specificity in conjunction with the proteic antibody of Smurf.

The present invention also provides code book invention Smurf proteic nucleic acid.In specific embodiments, described nucleic acid has the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3 description.

The present invention also is provided under the high stringent condition and the nucleic acid of the sequence with coding Smurf or the oligonucleotide or the nucleic acid of its complementary sequence specific hybrid.This hybrid nucleic acid comprises the nucleic acid of probe (be them can mark), primer (for example being used for pcr amplification), antisense nucleic acid, ribozyme and formation triple helical.

The present invention also provides the carrier of the nucleic acid that comprises the Smurf that encodes, and this carrier is for example under expression control sequenc control.Host cell that has this carrier and the method for producing Smurf by the described host cell of cultivation under the proteic condition of the described vector expression Smurf of permission also are provided.

The present invention has also considered proteic non-human transgenic animal of expressing human Smurf and the proteic non-human animal of disappearance endogenous Smurf.

The present invention also provides the bone morphogenetic protein that suppresses in the cell or the method for transforming growth factor-beta activated pathway.This method comprises under the condition that allows the vector expression Smurf of cell in permission transduces cell grows.On the other hand, the invention provides the promotion bone morphogenetic protein in the cell or the method for transforming growth factor-beta activated pathway, this method comprises that the endogenous Smurf that suppresses in the cell expresses.

In addition, this discovery allows screening Smurf active regulator.Screening of the present invention comprises that the detection test-compound is to the active regulating effect of host cell Smurf (with respect to the host cell Smurf activity that does not have test-compound).As described in embodiment, a kind of such activity is a Smad albumen ubiquitination.Another kind of activity is the degraded that strengthens the TGF beta receptor.

The accompanying drawing summary

Fig. 1 .Smurf1 coding E3 ubiquitin ligase enzyme.Provide the protein sequence comparison of Xenopus laevis and people Smurf1 and yeast (S.pombe) pub1 among the figure respectively with SMURF1, hSMURF1 and PUB1.Same amino acid is dark-grey shade, the conservative light gray shade that is substituted by.According to primary structure, Smurf1 and pub1 are the Hect family member of E3 ubiquitin ligase enzyme, and they have the several conservative characteristic of this family: lipid/Ca2+ binding domains is positioned at N-terminal (residue 22-37), the catalytic Hect structural domain that has 2 WW protein-interacting structural domains (thick line is represented) and begin to extend to C-terminal from residue 347 in 236-271 and 282-311 position.Use Clustal W analysis and carry out sequence contrast (Mac Vector).

The RNA trace of Fig. 2 A and 2B. embryo and adult mice tissue expression mSmurf1.Analyze the indication stage of equivalent and the PolyA+mRNA of tissue.(2A) embryonic tissue-in this trace, indicated embryo's fate of post-coitum.Adult group is woven with (2B): T, testis; K, kidney; M, skeletal muscle; L, lung; Sp, spleen; Br, brain; H, heart.

The growth of Fig. 3 A and 3B. Xenopus laevis Smurf1 is expressed.

(A) RT-PCR of each stage embryo cDNA shows, Xenopus laevis Smurf1 is source of parents mRNA, and the level of ovum phase, blastula stage and gastrula stage morning is the highest.Zygote Smurf1mRNA level descends during gastrula, but keeps stably express up to the swimming tadpole phase.Each numeral that detects on the road is equivalent to Nieuwkoop and each phase of Faber: 7 and 9, and blastula stage; 11 and 13, gastrula stage; 15 and 20, neurula stage; 25 and 35, the tadpole phase.Measure ornithine decarboxylase (ODC) mRNA of omnipresence expression in the cell, so that make RNA reclaim normalization method.CDNA carries out RT-PCR (non-ThermoScript II) to simulation.

(B) the bulk sample slide glass in situ hybridization of the Smurf1 in the Xenopus laevis fetal development.In ovum and blastula stage, the Smurf1 transcript is positioned animal pole hemisphere (animal pole half) ((bracket) demarcates in the ovum).Express disperse in whole ectoderm, and disappear at the peripheral zone of gastrula; Watch from animal pole (an) and vegetative pole (Veg).There is certain enrichment in transcript at the neural fold of neurula stage 17. Tadpole phase 25 and 35 o'clock, Smurf1 expresses and comprises brain (b), eye (e), otic capsule (o), body segment (s), bursa pharyngea (p) and developmental kidney (k).

Fig. 4 A, 4B, 4C and 4D.Smurf1 cause mammal cell line Smad1 and Smad5 steady-state level selectivity to reduce.With indicated expression vector (μ g measures DNA) transient transfection cell, make about 0.4% total cell lysate of equal portions carry out SDS-PAGE and immunoblotting after 2 days.In order to measure the Smad steady-state level, the suitable Smad antibody shown in using is surveyed trace.By surveying total cell lysate trace, confirmed the expression level of Flag-hSmurf1 with anti-Flag monoclonal antibody.

(A) with constant pCMV5-Smad1 and shown in concentration the pCMV5-Flag-hSmurf1 transfection COS-1 or the 293T cell that increase gradually.For the steady-state level of measuring Smad1 and the expression of hSmurfl, survey the western blotting of two kinds of total cell lysates of clone with α-Smad1 and α-Flag antibody (α-Smad1 and α-Flag trace).

(B) with pCMV5-Smad1, wild-type or activation (Q203D) pCMV5-ALK6-HA and shown in the pCMV5-Flag-hSmurf1 transient transfection 293T cell of ascending-dose gradually.With α-Smad1 antibody (the total cell lysate in the immunoblotting western blotting of α-Smad1), thus detect the steady-state level of Smad1.Use respectively α-Flag (α-Flag) or α-HA (and the total cell lysate of antibody mediated immunity trace of α-HA), thus detect the expression level of hSmurf1 and wild-type or activation ALK6.

(C) with the pCMV5-Flag-hSmurf1 of constant pCMV5-Smad1 or pCMV5-Smad2 and indication concentration transfection 293T cell together.As above detect Smad1 (α-Smad1 trace), Smad2 (α-Smad2 trace) in the cell lysate and the stable state protein level of Flag-hSmurf1 (α-Flag trace).

(D) lacking or existing under the situation of pCMV5-Flag-hSmurf1 pCMV5 transfection 293T cell with Smad1, Smad3, Smad4 or Smad5.Make α-Smad1 antibody of described western blotting and Smad1 and Smad5, α-Smad3 antibody of Smad3 and α-Smad4 antibody (α-Smad trace) incubation that Smad4 detects, thereby detect the Smad steady-state level in total cell lysate.(α-Flag trace) confirmed that identical hSmurf1 expresses as mentioned above.

Fig. 5 A, 5B and 5C.hSmurf1 regulate the Smad1 conversion and ubiquitination: hSmurf1 strengthens the Smad1 conversion.

(A) use hSmurf1 (F/hSmurf1) the transient transfection COS-1 cell of lipofection amine reagent with independent pCMV5-Smad1 or Flag mark.After 2 days, with [ ³⁵S] methionine(Met) makes the transfection body carry out pulse-chase analysis.Fixed time lysing cell in tracing process makes it carry out α-Smad1 immunoprecipitation.Immunoprecipitate separates by SDS-PAGE and shows (left group) by radioactive automatic developing.Also use the quantitative radiolabeled Smad1 of phosphorus imaging, with the result with each time point with respect to 0 time horizon [ ³⁵S] the Smad1 amount mapping (right part of flg) of methionine mark.

(B) ubiquitination of 293T cell Smad1.With ubiquitin (HA-Ub), pCMV5-Smad1 and the wild-type (WT) of the HA mark of indicating combination or the hSmurf1 transient transfection cell of ubiquitin ligase enzyme mutant (CA) Flag mark.After the transfection 2 days, make lysate carry out α-Smad1 immunoprecipitation (α-Smad1 IP), carry out SDS-PAGE then and carry out immunoblotting (α-HA trace) with α-HA monoclonal antibody.Presenting immunoreactive protein band with α-HA indicates with square brackets.Use α-Smad1 polyclonal antibody (α-Smad1 trace) or α-Flag monoclonal antibody (α-Flag trace) to make total cell lysate carry out immunoblotting respectively, confirmed the expression of Smad1 or Flag-hSmurf1.

(C) hSmurf1 reduces the complete ubiquitin ligase enzyme activity that the Smad1 steady-state level needs the Hect structural domain.The wild-type (WT) or ubiquitin ligase enzyme mutant (C710A) the transfection 293T cell that increase gradually with Smad1 and dosage.By the total cell lysate of usefulness suitable antibodies immunoblotting, the Smad1 in the analyzing total cell lysate, hSmurf1 or hSmurf1 (C710A) albumen, as shown in Figure 3.

The interaction of Fig. 6 A, 6B and 6C.Smurf1 and Smad.

(A) Smurf1 and Smad1 interact.The yeast that Xenopus laevis Smurf1 (Xsmurf) and Smad1, Smad2, lamin or independent carrier are united cotransformation together carries out yeast two-hybrid and measures.Have only Smad1 and Smurf1 combination to have tangible beta galactosidase enzyme activity (left figure, the dyeing yeast colony photo that duplication is measured).Make ³⁵The Smurf1 of S mark and be fixed in vitro translated on the anti-Flag affinity gel matrix ³⁵The Flag mark Smad1 of S-Met trace mark ( ³⁵S-F/Smad1), Smad2 ( ³⁵S-F/Smad2) or Smad4 ( ³⁵S-F/Smad4) incubation, thus in vitro translated albumen is carried out coimmunoprecipitation.After the washing, elution of bound albumen also carries out SDS-PAGE and analyzes (right figure).

(B) hSmurf1 optionally with Smad1 and Smad5 two-way interaction.With the pCMV5 expression vector that only contains Smad1 (S1), Smad5 (S5) or Smad2 or with the pCMV5 expression vector transient transfection 293T cell of Flag mark hSmurf1 (F/hSmurf1) that contains Smad1 (S1), Smad5 (S5) or Smad2 and wild-type (WT) or ubiquitin ligase enzyme sudden change (CA).In order to detect the interaction of Smad1 or Smad5 and hSmurf1, survey α-Flag immunoprecipitation with α-Smad1 polyclonal antibody (α-Smad1 trace).In order to detect the interaction of Smad2 and hSmurf1, survey α-Flag immunoprecipitation with α-Smad2 polyclonal antibody (α-Smad2 trace).Smad1, Smad5 in hSmurf1 level in the immunoprecipitation and the total cell lysate and Smad2 level be face two picture groups as follows.

(C) hSmurf1 is to the specificity of Smad1 effect.HSmurf1 dependency ubiquitin ligase enzyme Nedd4 not with the Smad1 effect, and do not reduce its steady-state level.With shown in Smad1 and hSmurf (WT or CA) or Nedd4, (WT) or ligase enzyme mutant (CS) the transient cotransfection 293T cell of Flag mark.Measure the coimmunoprecipitation (left hand view) of Smad1 and hSmurf1 as mentioned above.In order to measure the interaction of Smad1 and Nedd4, (α-Nedd4IP) make cell lysate carry out immunoprecipitation uses α-Smad1 polyclonal antibody (α-Smad1 trace) to carry out immunoblotting, right part of flg then with the Hect-Nedd4 polyclonal antibody.For the Nedd4 expression level in the test sample, (α-Nedd4 trace) surveys corresponding trace once more with the WW2Nedd4 polyclonal antibody.The western blotting of figure below shows the Smad1 level in total cell lysate of these mensuration.

Fig. 7 A and 7B.Smurf1 make the abdomen mesoderm back sideization of expection and make the ectoderm neuralization.

(A) at the peripheral zone injection Xenopus laevis SmurfmRNA (50pg Smurf1 mRNA/ cell) of 2 veutro blastomeres of 4 cell Xenopus laevis blastaeas.The tadpole phase embryo who grows from embryonal vaccination forms dystopy back of the body axle construction (lower-left figure, arrow).Inject the dystopy axle construction (bottom tadpole) under 100pg Smad1 and the 50pg Smurf1mRNA redemption all situations simultaneously.When early stage gastrula forms, from another embryo transfer VMZ sheet of organizing wild-type embryo or injecting Smurf1 mRNA separately or inject Smurf1 and Smad1 simultaneously at the veutro peripheral zone (VMZ) of 4 cell stages.Excision VMZ graft when gastrula forms in early days as shown in the figure, cultivate then up to the contrast embryo and reach tadpole mid-term 28, after preparing total VMZ RNA, measure the expression of red corpuscle specificity α-Zhu Danbai, muscle specific Actin muscle and the general cell mRNA eFl-a (bottom-right graph) that recovery contrasts as RNA by RT-PCR.To reverse transcription or there is not the total RNA of embryo of reverse transcription to contrast the PCR reaction.

(B) to zygote animal pole injection Smurf1 and/or Smad1 mRNA or do not inject.Transplant animal cap (animal cap) in blastula stages 8, be cultured to gastrula mid-term 11, prepare total RNA then, use the expression of RT-PCR analyzing and testing cement gland mark XAG and general neural mark NCAM.Contrast the same.

Fig. 8 A, 8B and 8C.Smurf1 change the response capacity of embryonic cell to Smad1 and Smad2.

(A) Smurf1 obstruction Smad1 induces the abdomen mesoderm.In the zygote animal pole, only be injected into constant (1ng) Smad1 mRNA or also be injected into the Smurf1 of dosage escalation simultaneously, utilize the primer of Xhox3 and Xcad3 homeodomain gene to induce situation by the abdomen mesoderm that RT-PCR measures in the animal cap graft.To respectively the detecting of right side, respectively animal cap is not injected or is injected 100,0,25,50,100 and the Smurf1 mRNA of 200pg dosage from the left side.

(B) Smurf1 strengthens Smad2 inducing dorsal mesoderm.Only inject constant (50pg) Smad2 mRNA or also inject the Smurf1 of ascending-dose simultaneously, by expressing the homeodomain gene goosecoid that significant myoD of muscle and modal back of the body type mesoderm (Spemann organizer (the SpemannOrganizer)) are expressed, thus the monitoring dorsal mesoderm induce situation.Attention is along with Smurf1 dosage increases, by causing the goosecoid expression by detection level.A is the same to be injected animal cap with scheming.

(C) animal cap is to the dose response of Smad2.With synthetic Smad2 mRNA injection animal cap.50pg Smad2 induces MyoD, and injection 100pg or 100pg induces MyoD to reach maximum horizontal during above Smad2.Smad2 induces goosecoid when minimum 250pg dosage.Independent 250pg Smad2 inductive gooscoid level is identical with the goosecoid level of 50pg Smad2 and 100pg Smurf1 combined induction (figure b).

In each figure, two detection roads of right-hand end are respectively wild-type embryo RNA are added reverse transcription (RT+) and do not add the PCR of reverse transcription (RT-).EFl-a contrasts same Fig. 7 among each figure.

Fig. 9. the cDNA sequence of people Smurf1 [SEQ ID NO:1].

Figure 10. the protein sequence of people Smurf1 [SEQ ID NO:2].

Figure 11. the cDNA sequence of people Smurf2 [SEQ ID NO:3].

Figure 12. the protein sequence of people Smurf2 [SEQ ID NO:4].

Smurf2 is HECT E3 ubiquitin ligase enzyme family member.Diagram C2 (going up line), WW (shade) and HECT (two going up rule) structural domain and Cys716Ala sudden change (frame).

The proteic comparison of Figure 13 .Smurf1 and Smurf2.

Diagram Smurf1 and Smurf2.There is shown the amino acid same degree (%) of C2, WW and HECT structural domain.

Figure 14 A and the 14B.Smurf2 expression in mouse tissue.

(A) Smurf2 expresses in whole mice embryonic takes place.The 1kb XhoI/NotI fragment that comprises the 3 ' UTR of mouse Smurf2 is used for surveying mice embryonic RNA trace (Clontech).

(B) expression of Smurf2 in the adult mice tissue.A is the same with scheming, and surveys the RNA trace (Clontch) of a plurality of tissues with mouse Smurf2 fragment.

Figure 15 A, 15B, 15C, 15D, 15E and 15F.Smurf2 and Smad7 interact.

(A and B) Smurf2 expresses the steady-state level that can not reduce Smad.Separately with the Smad7 of Smad1, Smad2, Smad4 or the HA mark of Flag mark or with the Smurf2 transfection 293T cell of Myc mark.The total cell lysate of equal portions is carried out immunoblotting,, perhaps utilize anti-Myc antibody to make it carry out immunoprecipitation, carry out anti-Flag or anti-HA immunoblotting then, with the Smad (Figure 15 B) that detects any co-precipitation to detect the expression (Figure 15 A) of Smurf2 and Smad.The migration of anti-Myc heavy chain (IgH) as a token of.

(C) Smurf2 expresses and can not change the Smad7 conversion.Separately with Smad7-HA or with Flag-Smurf2 transfection COS-1 cell, with [ ³⁵S]-methionine(Met) pulse labelling transfectional cell, in containing the substratum of unmarked methionine(Met), follow the trail of the fixed time then.By in the quantitative anti-HA immunoprecipitation of phosphorus imaging [ ³⁵S] mark Smad7-HA, will be with respect to the control cells (square frame) and the mapping of Smurf2 express cell (circle) level of 0 time content.Data be 2 times the experiment mean value+/-SD.

(D) Smurf2 of bacterial expression and Smad7 are in external interaction.Make bacteriogenic His-Smad7-HA albumen and Ni ²⁺-NTA, GST and GST-Smurf2 incubation.By SDS-PAGE and utilize the immunoblotting of anti-HA antibody to manifest binding substance.Measure GST protein level (base map) by coomassie dyeing.

(E) the PY primitive of Smad7 is the important factor of determination of mediation and Smurf2 effect.Separately with Flag-Smurf2 or with wild-type (WT) or sudden change Y211A (YA) or Δ PY type Smad7-HA transfection 293T cell.Make cell lysate carry out the immunoprecipitation of anti-Flag, by detect the Smad7 albumen of co-precipitation with the immunoblotting of anti-Smad7 antibody.Total cell lysate of immunoblotting equal portions has confirmed the expression (base map) of Smad7.

(F) the WW structural domain of Smurf2 is necessary in conjunction with Smad7.With Smad7-HA and wild-type (WT) or sudden change (Δ WW1, Δ WW2 or Δ WW3) type Flag-Smurf2 transfection 293T cell.Make cell lysate carry out the immunoprecipitation of anti-Flag, by detect the Smad7 of co-precipitation with the immunoblotting of anti-HA antibody.Confirmed the expression (base map) of Smad7 by total cell lysate of immunoblotting equal portions.

Figure 16 .Smad7 raises Smurf2 to TGF beta receptor mixture.

T β RII, T β RI-HA, Smad7-HA and wild-type (WT) or sudden change (C716A) Flag-Smurf2 transfection COS-1 cell with the various combinations of indicating.With [ ¹²⁵I] TGF β affinity labelling cell, and utilize anti-Flga or anti-Smad7 antibody mediated immunity precipitation lysate.The receptor complex that manifests co-precipitation by SDS-PAGE and radioactive automatic developing.Utilize the quantitative co-precipitation T of phosphorus imaging β RI amount (right part of flg).The total cell lysate that manifests equal portions by radioactive automatic developing has confirmed receptor expression.Total cell lysate with anti-Flag and anti-HA antibody mediated immunity trace equal portions has confirmed Smurf2 and Smad7 level respectively.

Figure 17 A, 17B, 17C, 17D and 17E.Smurf2 induce TGF beta receptor and Smad7 degraded.

(A) the Smurf2 expression that lacks Smad7 can not reduce the acceptor steady-state level.With T β RII-HA, the T β RI-HA transfection 293T cell of various combination, indicated the various dose (μ g plasmid DNA) of Flag-Smurf2.Total cell lysate of the suitable antibodies immunoblotting equal portions shown in the employing, thus the protein expression level measured.

(B) exist the Smurf2 of Smad7 to cause that the stable state receptor level descends.With Smad7-HA or T β RII-HA and T β RI-HA (left side) or composition active form I acceptor T β RI-HA (T204D) (right side) and incremental change wild-type (WT) or Flag-Smurf2 (C716A) the transfection 293T cell together that suddenlys change.By shown in anti-HA or anti-Flag immunoblotting measure the steady-state level of acceptor, Smad7 and Smurf2.

(C) Smurf2 improves the transformation efficiency of receptor complex.Independent with TGF beta receptor (T β RII-HA and T β RI-HA) or with Smad7-HA, Flag-Smurf2 or this two transfection COS-1 cell, with [ ³⁵S]-methionine(Met) pulse labelling transfectional cell, in containing the substratum of unmarked methionine(Met), follow the trail of the fixed time then.Make cell lysate carry out the immunoprecipitation of anti-HA,, it is mapped with respect to 0 time content by the amount of phosphorus imaging quantitative mark acceptor and Smad7.

(D) proteasome and lysosome inhibitor hinder the degraded of Smurf2 inductive receptor complex.With TGF beta receptor (T β RII-HA and T β RI-HA), Smad7-HA and Flag-Smurf2 transfection COS-1 cell, with [ ³⁵S]-methionine(Met) pulse labelling transfectional cell, lacking inhibitor or in the presence of 30mM lactacystin or 0.4mM chloroquine, following the trail of the fixed time then.Make cell lysate carry out anti-HA immunoprecipitation, manifest acceptor and Smad7 level by SDS-PAGE and radioactive automatic developing.

(E) Smurf2 induces the Smad7 ubiquitination in the presence of described acceptor.With the ubiquitin of HA mark and shown in Smad7, T β RII, T β RI-Flag and the wild-type (WT) of various combinations or sudden change (C716A) Myc-Smurf2 transfection 293T cell together.Make cell lysate carry out two-way immunoprecipitation (double immunoprecipitation) with anti-Smad7 antibody, then carry out immunoblotting with anti-HA antibody.Immunoblotting has confirmed the protein expression in total cell lysate of equal portions.

Figure 18 A, 18B, 18C and 18D.Smurf2 and Smad7 associate and strengthen Smad7 inhibition activity.

(A) Smad7 (Y211A) is in conjunction with the TGF beta receptor, reduces but Smurf2 is raised to the ability of receptor complex.Lacking or existing Flag-Smurf2 (C716A) transfection COS-1 cell down with TGF beta receptor (T β RII and T β RI-HA) and wild-type (WT) or (Y211A) Smad7/HA that suddenlys change.With [ ¹²⁵I] TGF β affinity labelling cell, with anti-Smad7 or anti-Flag antibody mediated immunity sedimentation cell lysate.Manifest the co-precipitation receptor complex by SDS-PAGE and radioactive automatic developing.The application of radiation autography is measured total acceptor of expressing, and confirms Smad7 and Smurf2 protein expression level by total cell lysate of anti-HA or anti-Flag immunoblotting equal portions.

(B and C) Smad7 (Y211A) activates and does not have wild-type Smad7 effective aspect transcribing suppressing the TGFb dependency.Separately with the 3TP-Lux reporter gene or with the wild-type (WT) of different concns or (Y211A) Smad7-HA transfection HepG 2 cell that suddenlys change.In (B), use the various Smad7 plasmids of 0.3ng/ml.Existing or lacking 1 time incubation cell of TGF β, make uciferase activity to the normalization method of b-galactosidase activity, with the mean value of 3 duplications of model experiment+/-the SD mapping.

(D) pattern of the TGF beta receptor mixture degraded of Smad7 and Smurf2 mediation.Smad7 is directly in conjunction with Smurf2, and associates with TGF beta receptor mixture.Therefore, Smad7 plays the proteic effect of connection of mediation TGF beta receptor mixture degraded.

Detailed Description Of The Invention

The E3 ubiquitin ligase gene man of (being called Smurf albumen) the present invention relates to encode Family, and comprise any animal, especially mammal or amphibian, more especially the people comes The total length in source or natural type gene and any functional activity thereof or antigenicity fragment. At implementation In the scheme, characterization be called the E3 ubiquitin ligase of Smurf1 and Smurf2.

The present invention part is found based on such accident: 2 novel Hect family ubiquitins connect Meeting enzyme member and Smad interacts. This two kinds of ligase called after Smurf1 and Smurf2. Therefore one of them Smurf1 specificity plays the BMP letter for BMP approach specificity Smad Number the transduction antagonist or the effect of negative regulation agent. Smurf1 directly acts on Smad1 and 5, And regulate its ubiquitination, conversion and activity. In the amphibian embryo, Smurf1 presses down Endogenous BMP signal processed causes mesoderm and ectodermic survival rate and cell fate to change Become. So, the invention provides between ubiquitination approach and cell fate control and play connection The uniqueness of effect is regulated albumen, for example during embryonic development. Except Smurf1 albumen [SEQ ID NO:2] and the sudden change variant outside, the present invention also provide the coding Smurf1 nucleic acid [SEQ ID NO:1].

Another novel Hect ubiquitin ligase member of family is Smurf2. Smurf2 is C2-WW-HECT domain E3 ubiquitin ligase, it is relevant with Smurf1. Smurf2 Neither with Smad1,2 or 4 effects, do not change Smad1,2 or 4 steady-state level yet. But Be, Smurf2 really with the Smad7 effect, directly in conjunction with the PPXY primitive among the Smad7. Smurf2 is as antagonist or the negative regulation agent of the transduction of TGF signal beta, at the collaborative work of Smad7 With lower participation degraded TGF beta receptor. Activating the TGF signal beta causes the Smad7 dependence to be raised Smurf2 to TGF beta receptor compound. When lacking activation TGF beta receptor compound, Smurf2 Can not change steady-state level and the conversion of Smad7. Smad7 raises to TGF β Smurf2 and is subjected to Body promotes by proteasome and the two degraded Smad7-TGF beta receptor of lysosomal pathway compound Thing. Studies confirm that of this paper introduction, Smad7 rises and raises Smurf2 to TGF beta receptor compound The effect of connection albumen, promoting its degraded, therefore downward modulation activation TGF beta receptor compound. Regulate that Smad7 is positioned to nuclear and to can be used to control Smad7-Smurf2 multiple with the effect of Smurf2 The inhibition activity of compound.

Except Smurf2 albumen [SEQ ID NO:4] and variant thereof, the present invention also provides The nucleic acid [SEQ ID NO:3] of coding Smurf2.

The discovery of discussing according to this paper as can be known, E3 ubiquitin ligase has at the bottom of the high selectivity The thing specificity. For example Smurf1 is in conjunction with BMP approach specificity Smad. And, Smurf1 The distinct signal transducin of BMP approach, because it is only in conjunction with Smad1 and Smad5, To the affinity of Smad2 (TGF β and activin acceptor approach specificity) very a little less than, and right Smad4 (common Smad signal transduction gametophyte) does not have affinity. Because this substrate specificity The property, Smurf1 can effectively disturb or regulate the biological respinse to BMP, and to the activin approach Not impact, i.e. effect to other TGF signal beta transduction pathway is limited or does not exist.

BMP/TGF signal beta transduction pathway takes place and Growth of Cells control in tissue differentiation, form In work (for example (52)). As the antagonist of the signal transduction pathway of TGF 'beta ' family mediation, Smurf1 incites somebody to action in vivo or external inhibition BMP approach. Adjusting system as degraded TGF beta receptor The organic component of system, Smurf2 will suppress TGF signal beta transduction pathway. Therefore, Smurf To hinder cartilage generation, ostosis, blood differentiation, cartilage formation, nerve channel survival rate, Kidney is grown, heart induce and form generation, hair growth, tooth form, gamete form with And various histoorgan forming processes, and obstruction bone and other dependence BMP approach Regeneration, grow, keep etc.

In one embodiment, foregoing saltant Smurf albumen or Smurf's is little Molecule antagonist can be used for preventing protein (for example Smad or TGF beta receptor) ubiquitination, Thereby the signal transduction pathway that keeps the BMP mediation. In addition, can produce in conjunction with Smurf albumen The Smad fragment of Hect domain, described domain have urging of Smad ubiquitination needs Change actively, perhaps can produce combination in the WW domain of Smad PPXY domain The Smad fragment, thus prevent Smurf1 combination and ubiquitination Smad. These fragments also Can be used for the Screening test of the interactional micromolecular inhibitor of Smurf-Smad, for example suppress knot Close mensuration. Smurf protein variants and Smad fragment can be used to improve because may cause disease The BMP that the Smurf overexpression of state or Smurf increased activity cause and/or TGF β/activation Plain signal transduction defective.

BMP control bone differentiation and growth, and carried out repairing with bone growth and connective tissue Multiple relevant clinical trial and application. Smurf albumen is for finding the New Target of medicine, described medicine Thing can affect the Smurf function, affects thus the cell effect to BMP, thereby has clinical usefulness On the way. So, Smurf albumen can be used for screening can strengthen the BMP approach or by antagonism respectively or Simulation Smurf protein active and suppress various medicines and/or the antibody of BMP approach. For example because of For Smurf1 to being high specials in conjunction with Smad1 and 5, so it can be used for screening and stop Or activate BMP approach and selective impact the cell effect of BMP is not affected TGF β Other member's of superfamily medicine.

Smurf albumen works in intracellular Smad signal transduction level, so Smurf Albumen provides the alternative method that affects BMP and TGF β/activin signal. Yet, because Smurf Albumen is intracellular protein, thus purpose be directly to change any operation of Smurf activity must Must in cell, carry out. Such operation comprises antisense and ribozyme technology and intrabody skill Art.

Can Smurf be passed to cell by the gene therapy scheme, to hinder the excessive signal transduction of the particular growth factor approach that Smad (for example be the Smad of Smurf1 or Smurf2 target) controls.The embodiment of this paper shows, but the Smurf1 expression level in the cell that only raises is with regard to antagonism Smad signal transduction pathway.Therefore, can be used to the clinical disease that excessive correction Smad signal transduction causes by gene therapy overexpression Smurf.Such illness comprises for example bone, tendon or cartilaginous tissue hyperplasia or utilizes Smad1 or other tissue of the acceptor of Smad5 transduction signal (for example bmp receptor) Signal Regulation forms.

Smurf nucleic acid or its partial sequence (for example PCR probe) can be used as the molecular probe of identifying the defective Smurf gene in the human genome, especially when finding that the Smurf transgenation is relevant with specified disease.Smurf albumen can be used as the reagent of the external test of the cell protein that is accredited as the ubiquitination target.Purifying Smurf can regenerate with purifying ubiquitination enzyme (being E1 and E2 component), and can be used for functional (ubiquitination) mensuration, the purpose of this functional assays is to identify the novel target protein (being the cDNA of the translation of purifying protein or unknown identity) of introducing described mensuration.

According to the present invention, can use conventional Protocols in Molecular Biology, microbial technique and recombinant DNA technology in the art technology scope.This class technology has detailed explanation in the literature.Referring to for example Sambrook, Fritsch ﹠amp; Maniatis, Molecular Cloning:A LaboratoryManual, second edition (1989) Cold Spring Harbor Laboratory Press, Cold SpringHarbor, New York (this paper is called " Sambrook etc., 1989 "); DNA Cloning:A PracticalApproach, I and II volume (D.N.Glover writes 1985); OgigonucleotideSynthesis (M.J.Gait writes 1984); Nucleic Acid Hybridization[B.D.Hames﹠amp; S.J.Higgins writes (1985)]; Transcription And Translation[B.D.Hames﹠amp; S.J.Higgins writes (1984)]; Animal Cell Culture[R.I.Freshney writes (1986)]; Immobilized Cells And Enzymes[IRL Press, (1986)]; B.Perbal, APractical Guide To Molecular Cloning (1984); F.M.Ausubel etc. (writing), Current Protocols in Molecular Biology, John Wiley ﹠amp; Sons, Inc. (1994).

If in this article, following term has the following definition that provides.

Term " approximately " or " pact " be meant set-point or scope 20% in, in preferred 10%, more preferably in 5%.

Term used herein " isolating " is meant that the material of indication does not contain the component that exists in the natural surroundings of the described material of common discovery.Specifically, isolating biological substance does not contain cellular component.If be nucleic acid molecule, then isolating nucleic acid comprises PCR product, isolating mRNA, cDNA or restriction fragment.In another embodiment, isolating nucleic acid preferably cuts out from its karyomit(e) that may exist, more preferably no longer link together with non-adjusting, non-coding region, if be present in the karyomit(e), more preferably not be positioned at upstream region of gene that described isolated nucleic acid molecule comprises or other gene in downstream and link together.In an embodiment again, isolating nucleic acid lacks one or more introns.Isolated nucleic acid molecule can be inserted in plasmid, clay, the artificial chromosome etc.Therefore in specific embodiments, recombinant nucleic acid is an isolating nucleic acid.Protein isolate can with other albumen or nucleic acid or the two (protein isolate combines in cell with it, if protein isolate is an embrane-associated protein, then it combines with cytolemma) combination.Isolated cells device, cell or tissue take out from the region of anatomy of the organism of its existence.But isolating material can be to need not be purifying substance.

Term used herein " purifying " is to reduce or eliminate isolating material under the condition that irrelevant substance is a pollutent.For example, purifying protein preferably is not contained in the cell and other albumen of its bonded or nucleic acid substantially; Purification of nucleic acid molecules does not preferably contain protein or other substantially and is present in uncorrelated nucleic acid molecule in the cell with it.Term used herein " does not contain substantially " in the calculating when the described material of analytical test and uses.Substantially the purifying substance that does not contain pollutent preferably is at least 50% purity; More preferably be at least 90% purity, even more preferably be at least 99% purity.Purity can be passed through chromatography, gel electrophoresis, immunoassay, composition analysis, biological assay and other means known in the art and measure.

" gene " in order to refer to the part of dna molecular, it comprises the polypeptid coding sequence that effectively is connected with expression control sequenc in this article.In one embodiment, gene can be genome sequence or portion gene group sequence, because it contains one or more introns.In another embodiment, the term gene is meant cDNA molecule (encoding sequence that does not promptly have intron).

DNA " encoding sequence " is meant when being in the suitable adjustable sequence and controls following time can transcribe and be translated as the double chain DNA sequence of polypeptide in external or intravital cell.The border of encoding sequence is by the terminal translation stop codon decision of the terminal initiator codon and 3 ' (carboxyl) of 5 ' (amino).Encoding sequence can include but not limited to the cDNA of prokaryotic organism sequence, eukaryote mRNA, genomic dna sequence even the synthetic DNA sequence of eukaryote (for example Mammals) DNA.If encoding sequence is used for expressing at eukaryotic cells, then polyadenylic acid signal and transcription termination sequence are usually located at 3 ' end of encoding sequence.

" expression control sequenc " (for example transcribing and translate control sequence) is the adjusting sequence of encoding sequence flank, for example promotor, enhanser, prevent son, terminator etc., they express encoding sequence in host cell.In eukaryotic cell, the polyadenylic acid signal is a control sequence.If be mRNA, ribosome bind site is an expression control sequenc.

" promoter sequence " is the DNA regulatory region that can transcribe downstream (3 ' direction) encoding sequence in conjunction with intracellular RNA polymerase and startup.In order to define the present invention, the border of promoter sequence be from 3 ' end of transcription initiation site upstream (5 ' direction) but extend to and comprise with initial minimum essential base number of transcribing of the detection level that is higher than background or essential parts number.Promoter sequence contains transcription initiation site (usually for example with s1 nuclease mapping definition) and responsible protein binding structural domain (consensus sequence) in conjunction with RNA polymerase.

When the rna polymerase transcribe encoding sequence was mRNA, encoding sequence was in " under the control " of transcribing and translating control sequence in the cell, and trans then RNA montage mRNA (if it contains intron) also is translated as encoding sequence encoded protein matter.

When strand type nucleic acid molecule can be annealed to another nucleic acid molecule (cDAN for example under suitable temp and solution ion strength condition, genomic dna or RNA) time, then described nucleic acid molecule and described another nucleic acid molecule " can be hybridized " (referring to above-mentioned Sambrook etc.)." severity " of temperature and ionic strength conditions decision hybridization.For the preliminary screening homologous nucleic acid, can use low stringent hybridization condition, equaling Tm is 55 ℃, 5 * SSC for example, 0.1%SDS, 0.25% milk and do not contain methane amide; Perhaps 30% methane amide, 5 * SSC, 0.5%SDS.In stringent hybridization condition to equal Tm higher, 40% methane amide for example, 5 * or 6 * SCC.It is the highest that high stringent hybridization condition equals Tm, 50% methane amide for example, 5 * or 6 * SCC.Hybridization requires two kinds of nucleic acid to contain complementary sequence, but according to the hybridization severity, can mispairing between the base.The suitable severity of hybrid nucleic acid depends on the length and the complementary degree of nucleic acid, and its variation is that this area is known.The similarity of two kinds of nucleotide sequences or homology degree are high more, and the Tm value of nucleic acid hybrids that contains these two kinds of sequences is high more.The relative stability of nucleic acid hybridization (equaling higher Tm) descends in the following order: RNA:RNA, DNA:RNA, DNA:DNA.For the crossbred of length greater than 100 Nucleotide, the existing equation that calculates Tm (referring to for example above-mentioned Sambrook etc., 9.50-9.51).For the hybridization of shorter nucleic acid (being oligonucleotide), the mispairing position is more important, and the length of oligonucleotide determines its specificity (referring to above-mentioned Sambrook etc., 11.7-11.8).But the minimum length of hybrid nucleic acid is at least about 10 Nucleotide; Preferably at least about 15 Nucleotide; More preferably length is at least about 20 Nucleotide.

In a specific embodiments, term " standard hybridization conditions " is meant that Tm is 55 ℃, and uses above-mentioned condition.In a preferred embodiment, Tm is 60 ℃; In a preferred embodiment, Tm is 65 ℃.In a specific embodiments, " high severity " hybridization and/or wash conditions are 68 ℃, 0.2 * SSC, and 42 ℃, 50% methane amide, 4 * SSC perhaps are equivalent under the condition of any hybridization level that observes down in these two kinds of conditions obtaining.

" carrier " is the recombinant nucleic acid construction, and for example plasmid, phage genome, viral genome, clay or another DNA sections can connected artificial chromosomes.In a specific embodiments, carrier can make the sections of connection duplicate, for example under the situation of cloning vector.The DNA sections inserts carrier in the specific limited site.DNA sections coding desired polypeptides designs described DNA sections and restriction site and inserts the suitable frame of transcribing and translating to guarantee described sections.

When exogenous DNA or heterology DNA transfered cell, described DNA just " transfection " cell.When transfection DNA has been realized phenotypic alternation, exogenous DNA or heterology DNA just " conversion " cell.Preferably transfering DNA is integrated the chromosomal DNA that (covalently bound) goes into to form cellular genome.

Term " allos " is meant the non-natural element of combination.For example allogeneic dna sequence DNA is meant that non-natural is positioned at the DNA of the chromosomal foci of described cell or described cell.Preferably allogeneic dna sequence DNA comprises the allogenic gene of described cell.The heterogenous expression regulatory element is the element that effectively connects with the gene that is different from its natural effective gene that is connected.In the present invention, Smurf1 or Smurf2 gene are the heterologous genes of plasmid vector DNA, and its inserts in carrier DNA so that clone or express, and it is a heterology to the non-human host cell (for example Chinese hamster ovary celI) of expressing it.

" nucleic acid molecule " is meant phosphoric acid ester aggretion type ribonucleoside (adenosine, guanosine, uridine or cytidine; " RNA molecule ") or dezyribonucleoside (Desoxyadenosine, pancreatic desoxyribonuclease, deoxythymidine or Deoxyribose cytidine; " dna molecular ") or any phosphoric acid ester analogue of its strand type or double-stranded spiral, for example thiophosphatephosphorothioate and thioesters.Can be double-stranded DNA-DNA, DNA-RNA and RNA-RNA spiral.Term nucleic acid molecule, especially DNA or RNA molecule only are meant the firsts and seconds structure of molecule, and unqualified it be any specific tertiary structure form.Therefore, this term comprises double-stranded DNA (for example restriction fragment), plasmid and the karyomit(e) that particularly exists with linearity or circular DNA molecule.When discussing concrete double chain DNA molecule structure, this paper can describe sequence according to the routine that only provides along 5 ' to 3 ' direction sequence of DNA non-transcribed chain (promptly having the chain with described mRNA homologous sequence)." recombinant DNA molecules " is the dna molecular that has carried out the molecular biology operation.

The invention provides antisense nucleic acid (comprising ribozyme), it can be used to suppress Smurf1 and expresses, and especially can be used to strengthen the BMP approach by Smad1 and 4.Therefore, antisense nucleic acid or its fragment that is equivalent to the Smurf1 gene can be used to change the BMP approach.The present invention also provides and suppresses the antisense nucleic acid that Smurf2 expresses, strengthens TGF signal transduction pathway." antisense nucleic acid " is single stranded nucleic acid molecule, when the hybridization of the complementary base in it and RNA or the dna molecular, suppresses the latter's effect.If RNA is the messenger RNA(mRNA) transcript, then antisense nucleic acid is reverse transcription thing (countertranscript) or mRNA interference complementary nucleic acid." antisense " that uses roughly comprises the inhibition of RNA-RNA interaction, RNA-DNA interaction, ribozyme and ribonuclease H mediation at present.Antisense nucleic acid molecule can by the recombination of in cell, expressing coding (for example U.S. Patent number 5,814,500; U.S. Patent number 5,811,234), perhaps they can synthesize and make (for example U.S. Patent number 5,780,607).

Term used herein " oligonucleotide " be meant can with the nucleic acid of mRNA molecule, mRNA, cDNA or other purpose nucleic acid hybridization of genomic dna molecule, cDNA molecule or encoding gene, generally at least 10, preferably at least 15, more preferably at least 20 Nucleotide.For example available ³²P Nucleotide or marker (as vitamin H) Nucleotide labeled oligonucleotide covalently bound with it.In one embodiment, labeled oligonucleotide can be used as the probe that detects the nucleic acid that exists.In another embodiment, oligonucleotide (but mark one of them or two Nucleotide) can be used as PCR primer, Smurf1 or Smurf2 coding nucleic acid that this primer is used to clone total length Smurf1 or Smurf2 or its fragment or is used to detect existence.In an embodiment again, oligonucleotide of the present invention can form triple helical with Smurf1 or Smurf2DNA molecule.In yet another embodiment, be configured in oligonucleotide library on the solid support such as silicon wafer or chip and can be used to detect various purpose polymorphisms.The general preferred preparation oligonucleotide that on the nucleic acid synthesizer, synthesizes.Therefore, preparation oligonucleotide such as available non-natural phosphoric acid ester analogue key such as thioester bond.

The specific examples of the synthetic oligonucleotide of the present invention design comprises the oligonucleotide that contains key between key between thiophosphatephosphorothioate, phosphotriester, methyl orthophosphoric acid, short-chain alkyl or cycloalkyl sugar or short chain heteroatoms or heterocycle sugar.Most preferably has CH ₂-NH-O-CH ₂, CH ₂-N (CH ₃)-O-CH ₂, CH ₂-O-N (CH ₃)-CH ₂, CH ₂-N (CH ₃)-N (CH ₃)-CH ₂And O-N (CH ₃)-CH ₂-CH ₂(wherein phosphodiester is O-PO to main chain ₂-O-CH ₂).U.S. Patent number 5,677,437 have introduced heteroaromatic oligonucleoside key.Nitrogen key or nitrogenous base also can be used for preparing oligonucleotide mimetic (U.S. Patent number 5,792,844 and 5,783,682).U.S. Patent number 5,637,684 have introduced phosphoramidate and sulfo-amino phosphoric acid ester oligomeric compounds.Also designed oligonucleotide (U.S. Patent number 5,034,506) with morpholinyl backbone structure.In other embodiments, peptide nucleic acid(PNA) (PNA) main chain for example, available multiamide main chain replaces the phosphodiester backbone of described oligonucleotide, and described base directly or indirectly is connected (Nielsen etc. with the nitrogen heteroatom of multiamide main chain, Science 254:1497,1991).Other synthetic oligonucleotide can contain the replacement sugar moieties that contains a following groups at 2 ': OH, SH, SCH ₃, F, OCN, O (CH ₂) _nNH ₂Or O (CH ₂) _nCH ₃, wherein n is 1 to about 10; C ₁-C ₁₀Low alkyl group, replacement low alkyl group, alkaryl or aralkyl; Cl; Br; CN; CF ₃OCF ₃O-; S-or N-alkyl; O-, S-or N-alkenyl; SOCH ₃SO ₂CH ₃ONO ₂NO ₂N ₃NH ₂Heterocyclylalkyl; The Heterocyclylalkyl aryl; Aminoalkyl group amino; Many alkylaminos; The silyl that replaces; The fluorescein part; The RNA leavings group; Reporter group; Intercalator; Improve the group of oligonucleotide pharmacokinetic properties; Other substituting group that perhaps improves the group of oligonucleotide pharmacodynamic profiles and have similar characteristics.Oligonucleotide also can contain sugar analogue such as cyclobutyl or replace other carbocyclic ring of pentose furyl glycosyl (pentofuranosyl).Can use the nucleotide unit (unit) that contains adenosine, cytidine, guanosine, thymidine and uridine nucleosides in addition, for example inosine.

" homology " that its all grammatical forms of term used herein and spelling change is meant the albumen relation with " the common source of evolving ", homologous protein (for example Smad (people), Mad (fruit bat) etc. the) (Reeck etc. that comprise superfamily albumen (for example TGF beta superfamily) and different plant species, Cell 50:667,1987).According to the situation of its height sequence similarity reflection, such albumen and encoding gene thereof have sequence homology.

Therefore, the term of all grammatical forms " sequence similarity " is meant the proteic nucleic acid that may have or may not have the common source of evolving or the identity between the aminoacid sequence or corresponding degree (referring to above-mentioned Reeck etc.).But in routine use and the present invention's use, when modifying as " to heavens " with adverbial word, term " homology " is meant sequence similarity, and it may relate to or may not relate to common evolution source.

In specific embodiments, when coupling Nucleotide is at least about 70-75%, most preferably at least about 80-85% on the dna sequence dna in specified length, two kinds of dna sequence dnas " basic homology " or " similar substantially ".Allelic variation body that an example of this sequence is a Smurf gene of the present invention or kind varient.Use the obtainable standard software comparative sequences of sequence library or, can identify basic homologous sequence with for example DNA hybrid experiment under the stringent condition of described concrete system definition.Define suitable hybridization conditions in the art technology scope, referring to for example above-mentioned Maniatis etc.; Above-mentioned DNA Cloning, I ﹠amp; The II volume; Above-mentioned Nucleic AcidHybridization.

Equally, in specific embodiments, when the amino acid more than 70% identical or about similar more than 90% (similar on the function), two seed amino acid sequences are " basic homology " or " similar substantially ".Preferably by using for example GCG (Genetics Computer Group, ProgramManual for the GCG Package, the 7th edition, Madison, Wisconsin) pile up program BLAST and Clustal W analysis (Mac Vector) and carry out the sequence contrast, thereby identify similar or homologous sequence.Sequence comparison algorithm also can find at http://www.nwfsc.noaa.gov/bioinformatics.html.

Term " correspondence " is used to refer to similar sequences or homologous sequence at this paper, and no matter accurate location is identical or different with the molecule of measuring similarity or homology.Nucleic acid or aminoacid sequence contrast can comprise the room.Therefore, term " correspondence " is meant sequence similarity, and does not count amino-acid residue or nucleotide base.

" homologous recombination " is meant that the carrier exogenous DNA array inserts in the karyomit(e).Preferably the lead specific chromosomal foci of homologous recombination of carrier.For specific homologous recombination, carrier will contain and the sufficiently long section of described chromosome sequence homologous, so as to allow carrier complementary in conjunction with and mix in the karyomit(e).The longer homology segment and the sequence similarity of higher degree can improve the efficient of homologous recombination.

DNA " encoding sequence " controls following time when being in the suitable adjustable sequence, transcribes and be translated as the double chain DNA sequence of polypeptide in external or intravital cell.The border of encoding sequence is by the terminal translation stop codon decision of the initiator codon and 3 ' (carboxyl) of 5 ' (amino) end.Encoding sequence can include but not limited to the cDNA of prokaryotic organism sequence, eukaryote mRNA, genomic dna sequence even the synthetic DNA sequence of eukaryote (for example Mammals) DNA.If encoding sequence is used for expressing at eukaryotic cells, then polyadenylic acid signal and transcription termination sequence are usually located at 3 ' end of described encoding sequence.

Transcribe and translate control sequence and be DNA and regulate sequence, for example promotor, enhanser, terminator etc., they express encoding sequence in host cell.In eukaryotic cells, the polyadenylic acid signal is a control sequence.

" promoter sequence " is for can and starting the DNA adjusting section that downstream (3 ' direction) encoding sequence is transcribed in conjunction with the RNA polymerase in the cell.In order to define the present invention, the border of promoter sequence is a 3 ' end of transcription initiation site, upstream (5 ' direction) but extend to and comprise with initial minimum essential base number of transcribing of the detection level that is higher than background or essential parts number.Promoter sequence contains transcription initiation site (usually for example with s1 nuclease mapping definition) and responsible protein binding structural domain (consensus sequence) in conjunction with RNA polymerase.

When the rna polymerase transcribe encoding sequence was mRNA, encoding sequence was in " under the control " of transcribing and translating control sequence in the cell, and trans then RNA montage mRNA also is translated as encoding sequence encoded protein matter.

Can no matter be genomic dna or cDNA from any source, especially from the nucleic acid of the mankind or Xenopus laevis cDNA or genomic library separation coding Smurf family protein (for example Smurf1 or Smurf2).As mentioned above, the method that obtains gene is this area known (referring to for example above-mentioned Sambrook etc., 1989).In specific embodiments, the invention provides the cDNA sequence [SEQ ID NO:1 and SEQ ID NO:3] of people Smurf1 (hSmurf1) and Smurf2 (hSmurf2) gene.

Therefore, any zooblast all can potential nucleic acid source as molecular cloning Smurf gene.Described DNA can obtain from cloned DNA (for example DNA " library ") with standard technique known in the art, and described library comprises the EST library and from the cDNA library (for example Xenopus laevis stage 9 (blastaea) cDNA library-highest level is expressed the cell of Smurf1) of the described proteic tissue preparation of high level expression.Other clone that can express Smurf1 or Smurf2 has frog blastaea and gastrula ectoderm, mesoderm and entoderm; Mouse embryo stem cell; And various mammalian cells, for example NIH3T3, PC12,293T, Hela and COS.Coding Smurf proteic DNA also can by chemosynthesis, cDNA clone or clone from genomic dna or its fragment that needs cell purification obtain (referring to for example above-mentioned Sambrook etc., 1989; Glover, D.M. (writing), 1985, DNA Cloning:A Practical Approach, MRL Press, Ltd., Oxford, U.K. I, II volume).The clone who obtains from genomic dna also contains and regulates section and introne DNA section except containing the coding section; The clone who obtains from cDNA does not contain intron sequences.Regardless of originating, described gene molecule should be cloned into suitable carrier, to breed described gene.

Available various technical evaluation known in the art contains the specific DNA fragments that needs the Smurf gene.But the part of purifying and the Smurf gene that exemplifies below the mark for example, so that the preparation label probe, the DNA that is produced can screen (Benton and Davis, Science 196:180,1977 by the nucleic acid hybridization with label probe; Grunstein and Hogness, Proc.Natl.Acad.Sci.U.S.A.72:3961,1975).Can hybridize with the basic homologous dna fragmentation of described probe (for example another individual allelic variation body).In specific embodiments, high stringent hybridization condition is used for identifying homology Smurf1 or Smurf2 gene.

The invention still further relates to the genes encoding analogue that contains Smurf gene of the present invention (for example Smurf1 or Smurf2) and the cloning vector of derivative, described analogue has similar with derivative or the homology functionally active.The generation and the use of relevant derivative with Smurf2 of Smurf1 and analogue belong to category of the present invention.The Smurf1 or the Smurf2 of clipped form for example can be provided.Such clipped form comprises Smurf1 or the Smurf2 with disappearance.In specific embodiments, described derivative or analogue are the functionally active things, promptly can present one or more and total length wild-type Smurf1 of the present invention or Smurf2 function associated activity.Such function comprises in conjunction with Smad albumen for example Smad1, Smad5 or Smad7, and another proteic ubiquitination in conjunction with Smad or TGF β/activin approach of catalytic activation TGF beta receptor/Smad mixture.In another embodiment, described fragment has binding affinity, but lacks catalytic capability or catalytic capability reduction.

Change nucleic acid coding sequence by replacement, interpolation or the disappearance that produces the functional equivalent molecule and can prepare the Smurf derivative.On the other hand, can prepare NOT-function mutant Smurf albumen, this albumen for example can with the wild-type Smurf protein competition in the BMP approach, but its ubiquitination to Smad is invalid substantially, they can be used for treating aforesaid and the impaired diseases associated of BMP signal transduction pathway.In specific embodiments, said mutation is C710A, sees the embodiment introduction for details.In another embodiment, Smurf2's sports C716A.

If for example genes encoding has the protein product of the proteic following characteristic of Smurf disclosed herein: wait electrically, electrophoresis, amino acid are formed, partly or entirely aminoacid sequence, antibody binding activity or part binding pattern, then can further select according to described genetic characteristics.Therefore, whether can detect described gene by the mensuration based on physics, chemistry, immunity or the functional performance of its expression product exists.For example, can select the dna clone of suitable mRNA to select to cDNA clone or hybridization, the albumen that described clone produces for example has similar or identical electrophoretic migration, isoelectrofocusing or non-equilibrium pH gel electrophoresis characteristic, proteolysis digestion figure or antigenicity with Smurf1 that is measured or Smurf2.

The invention still further relates to the homologue and the sudden change varient of proteic analogue of Smurf and derivative, other species, they have identical or the homology functionally active.The generation and the use of the relevant derivative of Smurf albumen, analogue and sudden change varient belong to category of the present invention.In specific embodiments, described derivative or analogue are the functionally active things, promptly can show one or more and total length wild-type Smurf1 of the present invention or Smurf2 albumen function associated activity.

Change nucleotide sequence by replacement, interpolation or the disappearance that produces the functional equivalent molecule and can prepare the Smurf derivative.The functionally active of the derivative that preferably makes perhaps lacks functionally active with respect to for example Smurf1 or Smurf2 enhancing of former Smurf, for example lacks the catalytic activity of the Hect ubiquitin ligase enzyme structural domain of Smurf PROTEIN C terminal portions.In a specific embodiments, be provided at the sudden change hSmurf1 that there is point mutation in C710A, described point mutation has destroyed the catalytic activity of Hect structural domain, therefore prevents Smad1 and 4 ubiquitinations.In another embodiment, be provided at the sudden change hSmurf2 that there is point mutation in C716A, described sudden change has destroyed the catalytic activity of Hect structural domain, therefore prevents proteasome degradation TGF beta receptor-Smad7 mixture.On the other hand, the soluble fragments of Smurf protein derivatives codified Smurf protein structure domain (for example WW structural domain), but described active fragments is identical or higher to the avidity of native ligand such as Smad1, Smad5 or Smad7.Such soluble derivative may be part (being Smad1,5 or 7) and the protein bound potent inhibitor of Smurf.

In another embodiment, can prepare the derivative or the fragment of Smad1 and 4, they can and prevent that the E3 ligase enzyme is further combined with cell Smad and prevent its ubiquitination in conjunction with Smurf1.In another embodiment, can prepare derivative and the fragment of Smad7, they can reduce the two combine of Smurf2 and Smad7 and TGF beta receptor.Therefore, in one embodiment, the present invention has designed the peptide that comprises the R-Smad connector area with PPXY sequence and the application of nucleotide sequence thereof, the PPXY sequence for the conservative primitive of WW structural domain identification (referring to Rotin, Curr.Topics Microbiol.Immunol., 228:115-133,1998 and (33)), described W/W structural domain for example sees the structural domain of Smurf1 or Smurf2.

Because the degeneracy of nucleotide coding sequence, can be used for implementing the present invention with other dna sequence dna of the essentially identical aminoacid sequence of Smurf genes encoding.Such dna sequence dna includes but not limited to the homologous gene and the nucleotide sequence of allelotrope, other species, and it is all or part of that described nucleotide sequence comprises the Smurf gene that by the different codons changes that replace the same amino acid residue in the described sequence of coding, therefore produces silent mutation.Equally, Smurf derivative of the present invention includes but not limited to such sequence: as the one-level aminoacid sequence, comprise all or part of Smurf Argine Monohydrochloride sequence, described Smurf Argine Monohydrochloride sequence comprises that the residue in the described sequence is replaced, produces the change sequence that conserved amino acid replaces by the functional equivalent amino-acid residue.One or more amino-acid residues in for example described sequence can be acted on aminoacid replacement like identical, the polar phase by another, cause reticent the change.The amino acid whose substituted amino acid of described sequence can be selected from other member of the affiliated classification of described amino acid.For example nonpolar (hydrophobic) amino acid comprises L-Ala, leucine, Isoleucine, Xie Ansuan, proline(Pro), phenylalanine, tryptophane and methionine(Met).The amino acid that contains aromatic ring structure has phenylalanine, tryptophane and tyrosine.Polar neutral amino acid comprises glycine, Serine, Threonine, halfcystine, tyrosine, l-asparagine and glutamine.The amino acid of positively charged (alkalescence) comprises arginine, Methionin and Histidine.The amino acid of electronegative (acidity) comprises aspartic acid and L-glutamic acid.Can not predict that this class changes the influence according to the apparent molecular weight of polyacrylamide gel electrophoresis or isoelectric point determination.Particularly preferred replacement comprises:

-Lys replaces Arg, and vice versa, can keep positive charge;

-Glu replaces Asp, and vice versa, can keep negative charge;

-Ser replaces Thr, can keep free-OH; And

-Gln replaces Asn, can keep free CONH ₂

Also can import aminoacid replacement, with another amino acid of aminoacid replacement with special preferred characteristics.For example a Cys can be imported the potential site of disulfide linkage with another Cys.Import His (be His can play acid or alkali effect and be common amino acid in the biochemical catalysis) as special " catalysis " site.Because the special two dimensional structure of Pro can import Pro, Pro can import b corner (turn) in protein structure.

The gene of available prepared in various methods code book invention Smurf derivative known in the art and analogue.But gene or protein level are prepared operation.Any modification clone's Smurf1 or Smurf2 gene order in for example available many strategies known in the art (Sambrook etc., 1989, the same).The available constraints restriction endonuclease cuts described sequence in appropriate site, further carries out enzyme modification then if necessary, separates and external connection.In the gene that produces coding Smurf derivative or analogue, should notice guaranteeing that modifying factor still in coding needs the identical translation frame of active gene regions gene, is not translated termination signal and is interrupted.

In addition, can in external or body, make the sudden change of Smurf nucleic acid sequence encoding, to produce and/or to destroy translation, initial and/or terminator sequence; Perhaps produce the coding region of various variations and/or form new restriction endonuclease site or destroy already present restriction endonuclease site, to help further external modification.Preferably described sudden change strengthens the functionally active of sudden change Smurf gene product.Any induced-mutation technique known in the art be can use, external site-directed mutagenesis (Hutchinson, C. etc., 1978, J.Biol.Chem.253:6551 included but not limited to; Zoller and Smith, 1984, DNA 3:479-488; Oliphant etc., 1986, Gene 44:177; Hutchinson etc., 1986, Proc.Natl.Acad.Sci.U.S.A.83:710), use TAB joint (Pharmacia) etc.Preferred round pcr is used for site-directed mutagenesis (referring to Higuchi, 1989, " PCR is used for engineering DNA ", be stated from: PCR Technology:Principles andApplications for DNA Amplification, H.Erlich writes, Stockton Press, chapter 6,61-70 page or leaf).

Can will identify that isolating gene inserts in the suitable cloning vector then.Can use numerous carrier host system known in the art.Possible carrier includes but not limited to plasmid or modification virus, but carrier system must mate with employed host cell.The carrier example includes but not limited to coliphage such as λ derivative; Perhaps plasmid such as pBR322 derivative or pUC plasmid derivative thing, for example pGEX carrier, pMal-c, pFLAG, pGBT9 etc.Described dna fragmentation is connected in the cloning vector with complementary sticky end, thereby finish the insertion cloning vector.Yet, if in cloning vector, do not have in order to the complementary restriction site of the described DNA of fragmentation, end that can the described dna molecular of enzyme sex modification.Perhaps can produce any site that needs by making nucleotide sequence (joint) connect into described DNA end; These jointings can comprise the chemical especially synthetic oligonucleotide of coding restriction endonuclease recognition sequence.Recombinant molecule can import host cell by conversion, transfection, infection, electroporation etc., can produce the described gene order of countless copies like this.Clone gene preferably is included on the shuttling expression plasmid vector, and shuttle vectors is in for example amplification and help purifying in the intestinal bacteria of clone cell, so that insert subsequently in the suitable express cell system, and words so if desired.For example can prepare the shuttle vectors that not only can duplicate but also can duplicate by connecting escherichia coli plasmid sequence and yeast 2 μ plasmid sequences in intestinal bacteria in yeast saccharomyces cerevisiae, shuttle vectors is the carrier that can duplicate in more than one organisms.

Express the Smurf polypeptide

The nucleotide sequence of coding Smurf albumen or its antigen fragment, derivative or analogue or functionally active derivative (comprising its chimeric protein) can insert in the suitable expression vector, and suitable expression vector just contains transcribes and translate the carrier that inserts the essential element of albumen coded sequence.Such element is referred to herein as " promotor ".Therefore, the proteic nucleic acid of code book invention Smurf effectively is connected with the promotor of expression vector of the present invention.The two all can clone cDNA and genome sequence and express under such adjusting sequence control.Expression vector preferably also comprises replication orgin.

Must transcribe with translation signals and can be provided on the recombinant expression vector, perhaps can provide by the coding proteic natural gene of Smurf and/or its flanking region.

The mammal cell line system that the virus (for example vaccinia virus, adenovirus etc.) that includes but not limited to potential host carrier system infects; The insect cell system that virus (for example baculovirus) infects; Microorganism for example contains the yeast of yeast vector; The perhaps bacterium that transforms of phage, DNA, plasmid DNA or cosmid DNA.The vector expression element with its intensity with specificity and different.According to used host carrier system, can use numerous suitable any of element of transcribing and translate.Cultivate host cell making under the condition of described cell expressing in suitable cell culture medium, this host cell contains the recombinant vectors that comprises the proteic nucleic acid of code book invention Smurf.Effective host cell of expressing Smurf comprises C ₂C ₁₂, 293T, CHO, COS, HEK, Hela, HepG2, NIH3T3, PC12, P19 and other clone, and kidney, brain and osteocyte.

By behind the recombination and integration encoding sequence, karyomit(e) can be expressed the present invention recombinate Smurf albumen or its function fragment, derivative, chimeric construct thing or analogue.About this, can use any in numerous enhanced system, so that realize high-level stably express gene (referring to above-mentioned Sambrook etc., 1989).

Can use any preceding method construction of expression vector that dna fragmentation is inserted cloning vector, described expression vector contains the gene that suitable transcribing/translate control signal and albumen coded sequence are formed.Such method can comprise reorganization (genetic recombination) in extracorporeal recombinant DNA and synthetic technology and the body.

Any promotor known in the art/enhancer element may command Smurf protein expression, but these regulatory elements must play a role the selected host who is used for expressing.The promotor that can be used for controlling Smurf genetic expression includes but not limited to SV40 early promoter district (Benoist and Chambon, 1981, Nature 290:304-310), the promotor (Yamamoto etc. that comprise of Rous sarcoma virus 3 ' long terminal repeat, 1980, Cell 22:787-797), herpes thymidine kinase promoter (Wagner etc., 1981, Proc.Natl.Acad.Sci.U.SA.78:1441-1445), the adjusting sequence (Brinster etc. of metallothionein gene, 1982, Nature 296:39-42); The prokaryotic expression carrier, for example the beta lactamase promotor (Villa-Kamaroff etc., 1978, Proc.Natl.Acad.Sci.U.S.A.75:3727-3731) or tac promotor (DeBoer etc., 1983, Proc.Natl.Acad.Sci.U.S.A.80:21-25); Referring to " producing useful proteins ", be stated from addition: Scientific American, 1980,242:74-94 with recombinant bacteria; The promoter element of yeast or other fungi, for example Gal4 promotor, ADC (ethanol dehydrogenase) promotor, PGK (phosphoglycerokinase) promotor, alkaline phosphatase promoter; And have tissue specificity and be used for the animal transcripting controling areas of transgenic animal: have active elastoser I gene-controlled area (Swift etc., 1984, Cell 38:639-646 at pancreatic acinar cell; Ornitz etc., 1986, Cold Spring Harbor Symp.Quant.Biol.50:399-409; MacDonald, 1987, Hepatology 7:425-515); In pancreatic beta cell, have active insulin gene control region (Hanahan, 1985, Nature 315; 115-122), in lymphoidocyte, have active immunoglobulin gene control region (Grosschedl etc., 1984, Cell 38:647-658; Adames etc., 1985, Natrue 318:533-538; Alexander etc., 1987, Mol.Cell.Biol.7:1436-1444), in testis, mammary gland, lymphoidocyte and mastocyte, have active mouse mammary tumor virus control region (Leder etc., 1986, Cell 45:485-495), in liver, have active albumin gene control region (Pinkert etc., 1987, Genes and Devel.1:268-276), in liver, have active α a-fetoprotein gene control region (Krumlauf etc., 1985, Mol.Cell.Biol.5:1639-1648; Hammer etc., 1987, Science 235:53-58), in liver, have active alpha1-antitrypsin gene-controlled area (Kelsey etc., 1987, Genes and Devel.1:161-171), in medullary cell, have active beta globin genes control region (Mogram etc., 1985, Nature 315:338-340; Kollias etc., 1986, Cell 46:89-94), in the brain oligodendrocyte, have active MBP gene control region (Readhead etc., 1987, Cell 48:703-712), in skeletal muscle, have active myosin light chain 2 gene-controlled areas (Sani, 1985, Nature 314:283-286) and in hypothalamus, have active gonadotropin releasing hormone gene-controlled area (Mason etc., 1986, Science 234:1372-1378).

Dna sequence dna of the present invention is expressed in available various host/expression vector combination.For example the effectively expressing carrier can be made up of karyomit(e) sections, non-chromosome sections and synthetic DNA sequence.Suitable carrier comprises SV40 derivative and known bacterial plasmid, for example escherichia coli plasmid col E1, pCR1, pBR322, pMal-C2, pET, pGEX (Smith etc., 1988, Gene67:31-40), pMB9 and derivative, plasmid such as RP4; Phage DNA S, many derivatives of phage 1 for example, for example NM989, and other phage DNA, for example M13 and thread single stranded phage DNA; Yeast plasmid, for example 2m plasmid or derivatives thereof; Effective carrier in eukaryotic cell, for example effective carrier in insect or mammalian cell; From the plasmid of combination and the carrier of phage DNA acquisition, for example carried out modifying so that the plasmid of use phage DNA or other expression control sequenc etc.

For example can use non-fusion transfer vector and merge transfer vector in baculovirus expression system, non-fusion transfer vector is (but being not limited to) pVL941 (BamH1 cloning site for example; Summers), pVL1393 (BamH1, SmaI, XbaI, EcoR1, NotI, XmaIII, BglII and PstI cloning site; Invitrogen), pVL1392 (BglII, PstI, NotI, XmaIII, EcoRI, XbaI, SmaI and BamH 1 cloning site; Summers and Invitrogen) and pBlueBacIII (BamH1, BglII, PstI, NcoI and HindIII cloning site may have blue/white reorganization screening; Invitrogen); Merge transfer vector for example (but being not limited to) pAc700 (BamH1 and KpnI cloning site, wherein the BamH1 recognition site is from initiator codon; Summers), (the BamH1 cloning site is at 36 base pairs in polyhedrin initiator codon downstream with pAC702 (identical with pAc700, as to have different frames), pAc360 for pAc701; Invitrogen (195)) and pBlueBacHisA, B, C (3 kinds of different frames have BamH1, BglII, PstI, NcoI and HindIII cloning site, and the N-terminal peptide is used for the ProBond purifying, and blue/white reorganization screening plaque; Invitrogen (220)).

Consider to be used for mammalian expression vector of the present invention and comprise the have inducible promoter carrier of (as Tetrahydrofolate dehydrogenase (DHFR) promotor), for example have any expression vector of DHFR expression vector; Perhaps DHFR/ methotrexate coamplification carrier, for example pED (PstI, SalI, SbaI, SmaI and EcoRI cloning site, the carrier of cloning by expression gene and DHFR; Referring to for example Kaufman, Current Protocols in Molecular Biology, 16.12 (1991)).Perhaps, glutamine synthetase/methionine(Met) sulfoximine coamplification carrier, for example pEE14 (HindIII, XbaI, SmaI, SbaI, EcoRI and BclI cloning site, wherein said vector expression glutamine synthase and clone gene; Celltech).In another embodiment, can use the carrier that under epstein-Barr virus (EBV) control, instructs additive type to express, pREP4 (BamH1, SfiI, XhoI, NotI, NheI, HindIII, NheI, PvuII and KpnI cloning site for example, composing type RSV-LTR promotor, hygromycin selectable marker; Invitrogen), pCEP4 (BamH1, SfiI, XhoI, NotI, NheI, HindIII, NheI, PvuII and KpnI cloning site, composing type hCMV immediate early gene, hygromycin selectable marker; Invitrogen), pMEP4 (XpnI, PvuI, NheI, HindIII, NotI, XhoI, SfiI, BamH1 cloning site, induction type metallothionein(MT) IIa gene promoter, hygromycin selectable marker; Invitrogen), pREP8 (BamH1, XhoI, NotI, HindIII, NheI and KpnI cloning site, RSV-LTR promotor, histidinol selective marker; Invitrogen), pREP9 (KpnI, NheI, HindIII, NotI, XhoI, SfiI and BamHI cloning site, RSV-LTR promotor, G418 selective marker; Invitrogen) and pEBVHis (the N-terminal peptide can be cut by the ProBond resin purification and with enteropeptidase for RSV-LTR promotor, hygromycin selectable marker; Invitrogen).Be used for selectivity mammalian expression vector of the present invention and comprise pRc/CMV (HindIII, BstXI, NotI, SbaI and ApaI cloning site, G418 selective marker; Invitrogen), pRc/RSV (HindIII, SpeI, BstXI, NotI, XbaI cloning site, G418 selective marker; Invitrogen) etc.The vaccinia virus mammalian expression vector is (referring to Kaufman used according to the present invention, 1991, the same) include but not limited to pSC11 (SmaI cloning site, TK-and b-gal select), pMJ601 (SalI, SmaI, AflI, NarI, BspMII, BamHI, ApaI, NheI, SacII, KpnI and HindIII cloning site; TK-and b-gal select) and pTKgptFlS (EcoRI, PstI, SalI, AccI, HindII, SabI, BamHI and Hpa cloning site, TK or XPRT select).

Also can use yeast expression system to express Smurf1 according to the present invention.For example can use non-fusion pYES2 carrier (XbaI, SphI, ShoI, NotI, GstX1, EcoRI, BstXI, BamH1, SacI, Kpn1 and HindIII cloning site according to the present invention; Invitrogen) or merge pYESHisA, B, C (XbaI, SphI, ShoI, NotI, BstXI, EcoRI, BamH1, SacI, KpnI and HindIII cloning site, the N-terminal peptide is cut with the ProBond resin purification and with enteropeptidase; Invitrogen), just enumerate above two classes here.

After identifying the specific reorganization SmurfDNA molecule of separation, can use several means known in the art to make its propagation.After setting up suitable host system and growth conditions, can breed and quantitatively prepare recombinant expression vector.As previously mentioned, spendable expression vector includes but not limited to following carrier or derivatives thereof: human or animal's virus is as vaccinia virus or adenovirus; Insect viruses such as baculovirus; Yeast vector; Phage vector (for example lambda particles phage) and plasmid and cosmid DNA carrier are enumerated several at this.

In addition, can select to regulate that insertion sequence is expressed or modify and the host cell strain of processed gene product with the ad hoc fashion of needs.Different host cells have specifically mechanism of translation and translation post-treatment and modifying protein characteristic.Can select suitable clone or host system to guarantee to modify on demand the foreign protein with expression processing.For example bacterial system is expressed and be can be used to produce non-glycosylated core protein product, and yeast expression can be used to produce glycation product.Eukaryotic cell expression can improve the folding possibility of allos mammalian proteins " natural ".And mammalian cell expression can provide reconstruction or set up the active instrument of Smurf.In addition, different carriers/host expression system known in the art can influence processing reaction (for example proteolytic cleavage) to some extent.

Carrier can import with means known in the art needs host cell, for example transfection, electroporation, microinjection, transduction, cytogamy, DEAE dextran, calcium phosphate precipitation, lipofection (lysosome fusion), applying gene rifle or dna vector transhipment thing (referring to for example Wu etc., 1992, J.Biol.Chem.267:963-967; Wu and Wu, 1988, J.Biol.Chem.263:14621-14624; Hartmut etc., the Canadian Patent of submitting to March 15 nineteen ninety numbers 2,012,311).

Analyze the genetic expression of Smurf functionally active mediation

In one embodiment, the oligonucleotide arrays technology can be used to for example to estimate Smurf1 in conjunction with Smad1 or Smad5 or the Smurf2 genetic expression after in conjunction with Smad7, and in order to identify the genetic expression relevant or different with the genetic expression of following situation: TGF β or BMP treat gene expression of cells or damage and/or callus or have the cellular gene expression that tumour forms the tissue of state cell, and the genetic expression in cell and the growth course, for example mitotic division, cytodifferentiation, embryo's diagram formation and growth and organogenetic genetic expression.For example can relatively there be or lacks Smurf in people and express TGF β down or BMP and treat genetic expression in the cell.(CA) data of gene expression profile and other biological assay are estimated in acquisition to the GeneChip expression analysis for Affymetrix, Santa Clara.Oligonucleotide is expressed array thousands of mRNA transcripts of quantitative check (gene or EST) simultaneously, has simplified extensive genome research.Each transcript can be presented on the right probe array of the multiprobe of distinguishing the closely related member of gene family.The specific oligonucleotides probe of thousands of copies is contained in each probe chamber, thereby allows accurately to detect sensitively low density mRNA crossing pattern.The differential expression data can make people be well understood to cellular pathways.

For example use Hewlett-Packard GeneArray after the hybridization ^TMScanner is caught intensity data by force.Available software is calculated the intensity level of each probe chamber automatically.Probe chamber intensity can be used to calculate the average intensity of each gene, and the average intensity of each gene is directly related with mRNA abundance level.For the gene of any selected subgroup, expression data can be classified and with various graphic representations apace according to any analytical parameters.The probe array of standard normal GeneChip expression at present can be used for the mankind, mouse, yeast and other biology.To expand the GeneChip product line, to comprise expression array and the broadened application field that is used to analyze other biology, for example toxicology and pharmacogenomics.

Transgene carrier

Can will activate diseases associated, for example cancer with treatment with excessive BMP or TGF β in the Smurf transfered cell.Monitor described cell by in cell, importing when Smurf carrier and Smurf express, thereby estimate the Smurf activity.This can carry out in external or body, perhaps is implanted into behind manipulation in vitro in the body, is also referred to as in vitro (ex vivo).On the other hand, as mentioned above, the carrier of available adjustment Smad is sent Smurf or Smurf inhibitor (antisense nucleic acid, ribozyme or intrabody), its objective is for example to prevent when needs BMP or TGF 'beta ' activity (for example osteanagenesis) that Smurf from regulating Smad; Perhaps study the interior regulate process of body of Smurf.

The inhibition that can in all sorts of ways of Smurf activity, the carrier transfer that comprises coding dominant Smurf growth (for example Cys710 sports the Ala mutant) to cell, antisense nucleic acid (comprises ribozyme and forms the oligonucleotide of triple helical; Have a detailed description in above-mentioned document about this) and express anti-Smurf intrabody, for example single-chain Fv antibody (generally referring to Chen, Mol.Med.Today, 3:160-167,1997; Spitz etc., Anticancer Res., 16:3415-3422,1996; Indolfi etc., Nat.Med., 2:634-635,1996; Kijima etc., Pharmacol.Ther., 68:247-267,1995).

As mentioned above, carrier is that nucleic acid of the present invention is transferred to any material in the host cell.Such carrier comprises virus vector, for example slow virus, retrovirus, simplexvirus, adenovirus and adeno associated virus.Therefore, can be with virus vector or by directly importing DNA in the body, in vitro or external importing encoding function or suddenly change Smurf albumen or its segmental gene in polypeptide structure territory.By (for example using virus vector or receptors ligand) with transgene carrier lead specific cells or by with tissue-specific promoter or above the two, can be implemented in the target tissue and express.October nineteen ninety-five, disclosed international patent publications WO 95/28494 introduced the target gene transmission.

Routine be used in the body or in vitro the virus vector of target and methods of treatment be DNA type carrier and retrovirus vector.The method that makes up and use virus vector is [referring to for example, Miller and Rosman, Bio Techniques 7:980-990 (1992)] known in the art.Preferably virus vector is a replication defect type, that is to say they can not be in target cell self-replicating.In general, the genome of the replication-defective virus carrier that uses within the scope of the present invention lacks the viral essential section that duplicates at least in cells infected.These sections can be eliminated (all or part of) or make its no function with any technology well known by persons skilled in the art.These technology comprise fully removes, replaces one or more bases that required area is duplicated in (replaced by other sequence, especially be inserted into nucleic acid and replace), excalation or increase.Described technology can be utilized Genetic Manipulative Technology or utilize mutagenic compound to handle and carry out in external (to separated DNA) or original position.Preferably replication-defective virus keeps the essential genome sequence of its virion dressing shell.

Dna viral vector comprises attenuation or defective type dna virus, such as but not limited to hsv (HSV), papilloma virus, epstein-Barr virus (EBV), adenovirus, adeno associated virus (AAV) etc.The preferred fully or almost completely defective virus of deficiency disease virus gene.Do not have infectious behind the defective virus transfered cell.Use the defective virus carrier to allow to give cell, and do not worry that described carrier infects other cell to the particular limitations zone.So, but specificity guiding particular organization.The example of concrete carrier includes but not limited to defective type 1 type simplexvirus (HSV1) carrier [Kaplitt etc., Molec.Cell.Neurosci., 2:320-330 (1991)], lack the defective herpesvirus carrier [patent disclosure RD371005A] of glycoprotein L gene or other defective herpesvirus carrier [on September 29th, 1994 disclosed International Patent Publication No. WO 94/21807; On April 2nd, 1994 disclosed International Patent Publication No. WO 92/05263]; Attenuation adenovirus carrier, for example carrier [J.Clin.Invest., 90:626-630,1992 introduced such as Stratford-Perricaudet; In addition referring to La Salle etc., Science, 259:988-990,1993]; And defective type adeno-associated virus vector [Samulski etc., J.Virol., 61:3096-3101,1987; Samulski etc., J.Virol., 63:3822-3828,1989; Lebkowski etc., Mol.Cell.Biol., 8:3988-3996,1988].

For giving in the body, for example adenovirus carrier and suitable immunosuppressant therapy combined utilization of virus vector preferably is so that avoid immunization to make virus vector and transfectional cell inactivation.For example can give inhibitive ability of immunity cytokine such as il-1 2 (IL-12), Interferon, rabbit-g (IFN-g) or anti-CD 4 antibodies, with humoral immunization or the cell immune response [referring to for example Wilson, Nature Medicine (1995)] of blocking-up to described virus vector.In addition, preferably use engineering to express the antigenic virus vector of minimal number.

The adenovirus carrier adenovirus is an eukaryotic dna virus, can make improvements effectively nucleic acid of the present invention is passed to various cell types.There are various serotype adenovirus.Preferably use 2 types in these serotypes or the adenovirus (referring to WO 94/26914) of 5 type human adenovirus (Ad2 or Ad5) or animal-origin within the scope of the present invention.Spendable within the scope of the present invention animal-origin adenovirus (for example: SAV) Lai Yuan adenovirus comprises dog, ox, mouse (for example: Mavl, Beard etc., Virology 75 (1990) 81), sheep, pig, bird and ape and monkey.The adenovirus of animal-origin is preferably hepatitis infectiosa canis virus, more preferably CAV2 adenovirus (for example Manhattan or A26/61 strain (ATCC VR-800)).

Replication-defective adenoviral vector of the present invention preferably comprises ITR, dressing shell sequence and purpose nucleic acid.Even more preferably at least the E1 district of adenovirus carrier be nonfunctional area.E1 district disappearance preferably extends to 3329 (PvuII-BglII fragments) or extends to 3446 (HinfII-Sau3A fragments) by Nucleotide 382 from the Nucleotide 455 of Ad5 adenoviral sequence.Other section also can be modified, especially E3 district (WO 95/02697), E2 district (WO 94/28938), E4 district (WO94/28152, WO 94/12649 and WO 95/02697) or any late gene L1-L5.

In a specific embodiments, there is disappearance (Ad 1.0) in adenovirus carrier E1 district.EP185,573 disclose the example of E1 deleted adenovirus, and described patent content is attached to herein by reference.In another embodiment, there is disappearance (Ad 3.0) in adenovirus carrier in E1 and E4 district.WO 95/02697 and WO 96/22378 disclose the example of E1/E4 deleted adenovirus, and described patent content is attached to herein by reference.In an embodiment again, described adenovirus carrier exists in the E1 district that E4 district and nucleotide sequence insert and lacks (referring to FR9413355, its content is attached to herein by reference).

Duplicate deficit type recombinant adenovirus of the present invention can be used any technology well known by persons skilled in the art (Levrero etc., Gene 101:1951991; EP 185573; Graham, EMBO J.3:2917,1984) preparation.The homologous recombination of especially can be by adenovirus and carrying especially between the plasmid of target DNA sequence makes.Homologous recombination realizes after described adenovirus and plasmid co-transfection are gone into suitable clone.The clone of using preferably (i) can be transformed by described element, and (ii) contain can with the portion gene group complementary sequence of replication-defective adenoviral, described adenovirus is preferably integrated danger to avoid recombinating.It is 293 (Graham etc. that spendable clone example has the human embryonic kidney cell, J.Gen.Virol.36:591977), 293 clones contain the left-hand part (12%) that is integrated into its genomic Ad5 adenoviral gene group, and the clone that can have complementary functions with E1 and E4, see that WO 94/26914 and WO 95/02697 application introduce.Recombinant adenovirus can use the known standard molecular biological technique of those of ordinary skills to reclaim purifying.

Adeno associated virus adeno associated virus (AAV) is less dna virus, they stably site-specific integration go in the genome of the cell that their infect.They can infect various cells, and can cell growth, morphology or differentiation produce any influence, as and if they are irrelevant with human pathology.The AAV genome clone, order-checking and characterized have been carried out.It comprises about 4700 bases, and the end that contains 145 bases of having an appointment at two ends oppositely repeats (ITR) section, and this section plays viral replication orgin.The genomic rest part of adeno associated virus is divided into 2 basic sections with dressing shell function: genome left-hand part, this part contain the rep gene that participates in virus replication and viral gene expression; And the genome right hand portion, this part contains the cap gene of the viral capsid proteins of encoding.

Existing about the introduction of using metastatic gene in the external and body of AAV derivative vector (referring to WO 91/18088; WO 93/09239; US 4,797,368, US 5,139,941, EP 488528).These publications have been introduced the various AAV construction of deriving, wherein rep and/or cap genetically deficient and replaced by goal gene, and introduced to use (directly to be transferred in the organism) in these constructions external (being transferred in the culturing cell) or the body and shifted described goal gene.Replication defect type of the present invention reorganization AAV can be prepared as follows: make that to contain purpose nucleotide sequence, its both sides be that the terminal plasmid co-transfections that oppositely repeat the plasmids in (ITR) district and carry AAV dressing shell gene (rep and cap gene) of 2 AAV are gone in the clone that human helper virus (for example adenovirus) infects.The AAV recombinant chou that produces of available standards technology purifying then.

Retrovirus vector in another embodiment, described gene can import in the retrovirus vector, for example sees following document introduction: Anderson etc., U.S. Patent number 5,399,346; Mann etc., 1983, Cell 33:153; Temin etc., U.S. Patent number 4,650,764; Temin etc., U.S. Patent number 4,980,289; Markowitz etc., 1988, J.Virol.62:1120; Temin etc., U.S. Patent number 5,124,263; EP 453242, EP 178220; Bernstein etc., Genet.Eng.7 (1985) 235; McCormick, BioTechnology 3 (1985) 689; May 16 nineteen ninety-five such as Dougherty disclosed International Patent Publication No. WO 95/07358; And Kuo etc., 1993, Blood 82:845.Retrovirus is the integration virus that infects somatoblast.The retrovirus genome comprises 2 LTR, 1 dressing shell sequence and 3 coding regions (gag, pol and env).In recombinant retrovirus carrier, general gap, pol and env gene lack wholly or in part, and are replaced by external source purpose nucleotide sequence.These carriers can make up with dissimilar retroviruss, for example HIV, MoMuLV (" mouse Mo Luoni leukosis virus "), MSV (" mouse Mo Luoni sarcoma virus "), HaSV (" Harvey sarcoma virus "); SNV (" spleen necrosis virus "); RSV (" Rous sarcoma virus ") and Friend virus.WO 95/02697 discloses the defective type retrovirus vector.

In general, in order to make up the recombinant Retroviruses that contains a kind of nucleotide sequence, make up the plasmid that contains LTR, dressing shell sequence and encoding sequence.Use this construction transfection package cell line, this clone can transly provide the retrovirus function of described plasmid defective.Therefore, in general package cell line can be expressed gag, pol and env gene.Prior art by the agency of such package cell line, especially clone PA317 (US 4,861,719); PsiCRIP clone (WO 90/02806) and GP+envAm-12 clone (WO 89/07150).In addition, recombinant retrovirus carrier can contain LTR modification that suppresses transcriptional activity and the expansion dressing shell sequence (Bender etc., J.Virol.61 (1987) 1639) that may comprise part gag gene.Recombinant retrovirus carrier can be used the known standard technique purifying of persons skilled in the art.

Can make up the retrovirus vector that works or only carry out taking turns transfection with infectious particles.In the former case, can improve virus to keep its all genes and expression of heterologous genes except the gene that causes the carinogenicity conversion characteristic.Can prepare the non-infectious virus carrier that has destroyed viral packaging signal, but keep the common structure gene that imports the virus needs that contains heterologous gene and packaging signal behind the Packaging Engineering.Therefore, the virion of generation can not produce extra virus.

Lentiviral vectors in another embodiment, lentiviral vectors can be used as the genetically modified factor of direct transmission and also in some types of organizations the continuous expression transgenosis, described types of organization comprises brain, retina, muscle, liver and blood.Lentiviral vectors can effectively transduce division and Unseparated Cell in the described tissue, and keep the goal gene long-term expression.

Slow virus contains at least 2 auxiliary genes [referring to summary Naldini, L., Curr.Opin.Biotechnol., 9:457-63,1998] that duplicate essential regulatory gene tat and rev and 4 important virulence factors of coding.Therefore remove the nonessential virus sequence of transduction, improve the biological safety of this specific support thus.The HIV-1 carrier of oneself's inactivation is the known carrier that has disappearance in the 3 ' long terminal repeat (LTR) that comprises the TATA frame, they produce the biological safety (Zufferey etc. that produce the possibility of reproducible retrovirus in cell and the target cell and significantly improved the HIV derivative vector because reduced at carrier, J.Virol., 72:9873-80,1998).In addition, lack by suppressing relevant LTR sequence and the potential performance of improving described carrier by permission stricter tissue specificity of structure or modulability carrier before removing with in transcribing interference and body.

Various slow virus package cell lines available and also they be that this area is generally understood.They help to produce the high titre lentiviral vectors that is used for gene therapy.An example is a tsiklomitsin induction type VSV-G pseudotyping slow virus package cell line, and this clone can continue at least to produce virion (Kafri etc., J.Virol., 73:576-584,1999) with the above titre of 10 (6) IU/ml in 3-4 days.The carrier that induction type clone produces can concentrate as required, so that effectively transduce Unseparated Cell in external and the body.

The non-viral carrier on the other hand can be by importing carrier in the lipofection body.Since past 10 years, used liposome to carry out nucleic acid encapsulate capsule and transfection is more and more external.Can design liposome-mediated difficulty that transfection ran into and the dangerous synthesizing cationic lipid that obtains limiting, such lipid can be used to prepare liposome [Felgner etc., Proc.Natl.Acad.Sci.U.S.A., 84 that are used for the gene of transfection coded markings in the body, 7413-7417,1987; Referring to Mackey etc., Proc.Natl.Acad.Sci.U.SA., 85:8027-8031,1988; Ulmer etc., Science, 259:1745-1748,1993].Use cation lipid can promote that the bag of electronegative nucleic acid is encapsulated, and can promote the fusion [Felgner and Ringold, Science, 337:387-388,1989] with electronegative cytolemma.Open WO95/18863 of international monopoly and WO 96/17823 and U.S. Patent number 5,459,127 have been introduced useful especially lipid compounds of transfer nucleic acid and composition.Use lipofection to have certain practical advantage with importing certain organs in the foreign gene body.The molecular targeted specific cells of liposome is represented a useful especially field.Obviously, direct transfection to particular cell types should have special advantage aspect the heterogeneous tissue of cell (as pancreas, liver, kidney and brain).Lipid can with other molecular chemistry coupling so that target gives [referring to Mackey etc., the same].The target peptide for example hormone or neurotransmitter and protein for example antibody or non-peptide molecule can with the liposome chemical coupling.

Other molecule also can be used to promote body interior transfection, for example positively charged ion oligopeptides (for example international patent publications WO 95/21931), the conjugated protein derived peptide of DNA (for example international patent publications WO 96/25508) or the cationic polymers (for example international patent publications WO 95/21931) of nucleic acid.

Described carrier also can import in the body with the naked DNA plasmid.The naked DNA carrier that is used for gene therapy can import the host cell of needs with methods known in the art, the for example transfection of described method, electroporation, microinjection, transduction, cytogamy, DEAE dextran, calcium phosphate precipitation, use particle gun or use dna vector transhipment thing [referring to for example Wu etc., J.Biol.Chem., 267:963-967,1992; Wu and Wu, J.Biol.Chem., 263:14621-14624,1988; Hartmut etc., the Canadian Patent of submitting to March 15 nineteen ninety numbers 2,012,311; Williams etc., Proc.Natl.Acad.Sci.USA, 88:2726-2730,1991].Also can use receptor-mediated DNA transmission method [Curiel etc., Hum.Gene Ther., 3:147-154,1992; Wu and Wu, J.Biol.Chem., 262:4429-4432,1987].U.S. Patent number 5,580,859 disclose in mammalian body without lipofection promotor transmission exogenous DNA array.

Anti-Smurf protein antibodies

The Smurf polypeptide that produces according to reorganization of the present invention or chemosynthesis or its fragment, varient or other derivative or analogue (comprising fusion rotein) can be used as the antibody that immunogen produces identification Smurf polypeptide.Such antibody includes but not limited to polyclonal antibody, monoclonal antibody, chimeric antibody, single-chain antibody, Fab fragment and Fab expression library.This antibody is the Smurf protein-specific; It can discern mutant Smurf or wild-type Smurf.These antibody can be used to change the BMP approach or be used for diagnostic purpose by suppressing Smurf albumen (for example anti-Smurf intrabody).

Various means known in the art can be used for producing the polyclonal antibody of anti-Smurf polypeptide or derivatives thereof or analogue.In order to produce antibody, can be by to including but not limited to the immune various host animals of injection Smurf polypeptide or derivatives thereof (for example fragment or fusion rotein) such as rabbit, mouse, rat, sheep, goat.In one embodiment, Smurf polypeptide or its fragment can be puted together with immunogenic carrier, and described immunogenic carrier is bovine serum albumin (BSA) or keyhole  hemocyanin (KLH) for example.Can use various adjuvant enhancing immunity reactions according to host type, described adjuvant includes but not limited to freund's adjuvant (complete and incomplete Freund's adjuvant), mineral coagulant such as aluminium hydroxide, surfactant such as lysolecithin, poly alcohol, polyanion, peptide, oiliness emulsion, keyhole  hemocyanin, dinitrophenol and potential effective human adjuvant such as BCG (bacille Calmette-Guerin vaccine) and spillikin bacillus.

In order to prepare direct monoclonal antibody, can use any technology that produces antibody molecule by continuous cell line at Smurf polypeptide or its fragment, analogue or derivative.Such technology includes but not limited to that Kohler and Milstein develop (Nature 256:495-497 at first, 1975) hybridoma technology and three knurls (trioma) technology, human B cell hybridoma technology (Kozbor etc., Immunology Today 4:72,1983; Cote etc., Proc.Natl.Acad.Sci.U.S.A.80:2026-2030,1983) and produce human monoclonal antibody's EBV-hybridoma technology (Cole etc., be stated from: Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, Inc., the 77-96 page or leaf, 1985).In another embodiment of the invention, available germ-free animal produces monoclonal antibody (the International Patent Publication No. W WO89/12690 that on November 28th, 1989 announced).In fact, according to the present invention, can use by montage Smurf polypeptid specificity mouse antibodies molecular gene and have technology (Morrison etc., J.Bacteriol.159:870,1984 that suitable bioactive human antibody molecules gene development is used for producing " chimeric antibody "; Neuberger etc., Nature 312:604-608,1984; Takeda etc., Nature 314:452-454,1985); Such antibody belongs to category of the present invention.Preferred described people's antibody or humanization chimeric antibody are used for human disease treatment (as mentioned above), because people's antibody or humanized antibody auto-induction immunne response, especially anaphylaxis are more much lower than the possibility of heteroantibody.

According to the present invention, introduce the technology (U.S. Patent number 5,476,786 and 5,132,405 of Huston that is used to produce single-chain antibody; United States Patent (USP) 4,946,778) can be used for producing Smurf polypeptid specificity single-chain antibody.Another embodiment of the present invention utilization introduction is used to make up the technology (Huse etc. of Fab expression library, Science 246:1275-1281,1989), Smurf polypeptide or derivatives thereof or analogue had the specific mono-clonal Fab fragment of needs to allow to identify quickly and easily.

Use known technology and can prepare the antibody fragment that contains the idiotype antibody molecule.For example, described fragment includes but not limited to: the F that can produce by the described antibody molecule of gastric pepsin digestion (ab ') ₂Fragment; Can be by reduction F (ab ') ₂Fab ' fragment that segmental disulfide linkage produces and the Fab fragment that can pass through to handle the antibody molecule generation with papoid and reductive agent.Adopt technology known in the art (generally referring to Chen, Mol.Med.Today 3:160-167,1997; Spitz etc., Anticancer Res.16:3415-3422,1996; Indolfi etc., Nat.Med.2:634-635,1996; Kijima etc., Pharmacol.Ther.68:247-267,1995), also can suppress the Smurf activity by expressing anti-Smurf intrabody such as single-chain Fv antibody.

When producing antibody, can use technology screening known in the art needs antibody, and described known technology is radioimmunoassay for example, ELISA (enzyme-linked immunosorbent assay), " sandwich " immunoassay, immunoradiometric assay, gel diffusion precipitation reaction, immunodiffusion(ID) is measured, the original position immunoassay (are for example used Radioactive colloidal gold, enzyme labelling thing or radioisotopic tracer), western blotting, precipitin reaction, CA (example gel CA, hemagglutination is measured), complement fixation(CF) is measured, immunofluorescence assay, A protein determination and immunoelectrophoresis mensuration etc.In one embodiment, detect antibodies by detecting a marker that resists.In another embodiment, detect one with anti-combining and resist by detecting two anti-or reagent.In an embodiment again, mark described two is anti-.It is known in the art and within the scope of the present invention that many immunoassay detect the bonded methods.For example in order to select to discern the antibody of Smurf polypeptid specificity epi-position, people can detect it to the hybridoma that is produced in conjunction with the product that contains the Smurf polypeptide fragment of described epi-position.In order to select the specific antibody of concrete animal species Smurf polypeptide, people can according to this animal species cell expressing or separate from the Smurf of this animal species cell polypeptide just in conjunction with selecting.

Above-mentioned antibody can be used for relating to Smurf polypeptide location and active means known in the art, for example utilize any above-mentioned detection technique or detection technique known in the art western blotting, the imaging of Smurf polypeptide original position, detect its level in suitable physiologically sample etc.Described antibody also can be used for part in conjunction with measuring, and for example U.S. Patent number 5,679, and 582 is described.

In a specific embodiments, can prepare the antibody of enhancing or antagonism Smurf polypeptide active.Can adopt the such antibody of above-mentioned detection test, to identify part.

Screening

Can be used as target according to the encode peptide sequence of Smurf gene deutero-nucleotide sequence and its generation of the present invention, to identify the medicine of the disease that effective treatment BMP signal transduction pathway mediates, as previously mentioned (referring to Schmitt etc., J.Orthopedic Res., 17:269,1999), described nucleotide sequence or peptide sequence can be used to strengthen or antagonism Smurf albumen.The medicine target includes but not limited to that (i) is from the isolating nucleic acid of the gene acquisition of coding Smurf and isolated peptides and the polypeptide that (ii) obtains from the Smurf polypeptide.

Specifically, identify that isolating Smurf albumen can be used for developing screening assay, raises or the active molecule of downward modulation Smurf in particular for high flux screening, for example allow to express Smurf albumen, perhaps utilize after the special engineering improvement behind indication transfection or the transformant from the proteic active indicator cells of genetic expression Smurf to be higher than the natural origin fractional dose.Therefore, the present invention has designed the method that Smurf protein-specific part is identified in various screening assay known in the art of using.

Any triage techniques known in the art all can be used for screening proteic agonist of Smurf or antagonist.The present invention has designed in screening small molecules part or ligand analogs and stand-in and the screen body in conjunction with also strengthening or the proteic native ligand of antagonism Smurf.Enhancing or the active molecule of antagonism Smurf in for example available mensuration screening natural product libraries of the present invention.

Provide the useful information of proteic inhibitor of definite Smurf or antagonist to the primary sequence of Smurf1 and Smurf2 with the those skilled in the art that are appreciated that of the similarity of known functional protein matter sequence.For example adopt X ray crystallography, neutron diffraction, nuclear magnetic resonance spectrometry and other structure determination technology to determine that described proteic constitutional features further helps to identify and the screening antagonist.These technology can be used for appropriate design or identify agonist and antagonist.

Another kind method adopts recombinant phage to produce big library.Adopt " phage method " (Scott and Smith, Science 249:386-390,1990; Cwirla etc., Proc.Natl.Acad.Sci., 87:6378-6382,1990; Devlin etc., Science, 49:404-406,1990) can make up very large library (10 ⁶-10 ⁸Individual chemical entities).Second method is mainly used chemical process, wherein Geysen method (Geysen etc., Molecular Immunology 23:709-715,1986; Geysen etc., J.Immunologic Method 102:259-274,1987) and the method (Science251:767-773,1991) of Fodor etc. be example.(14th International Congress ofBiochemistry, Volume #5, Abstract FR:013,1988 such as Furka; Furka, Int.J.PeptideProtein Res.37:487-493,1991), the Houghton (U.S. Patent number 4 that authorize in November, 1986,631,211) and (U.S. Patent number 5 that on April 23rd, 1991 authorized such as Rutter, 010,175) introduced the method that produces peptide mixt, described peptide mixt can be tested as agonist or antagonist.

On the other hand, synthetic library (Needels etc., Proc.Natl.Acad.Sci.USA 90:10700-4,1993; Ohlmeyer etc., Proc.Natl.Acad.Sci.USA 90:10922-10926,1993; Lam etc., International Patent Publication No. W WO 92/00252; Kocis etc., International Patent Publication No. W WO 9428028) etc. can be used to screen the Smurf part according to the present invention.

From synthetic or the big library screening test-compound of natural compounds.Present numerous method is used for synthesizing sugar, peptide and nucleic acid based compound with orientation at random.The synthetic compound library can be from Maybridge Chemical Co. (Trevillet on market, Cornwall, UK), Comgenex (Princeton, NJ), Brandon Associates (Merrimack, NH) and Microsource (New Milford CT) buys.Rare chemical library can (Milwaukee WI) obtains from Aldrich.On the other hand, the natural compounds library of bacterium, fungi, plant and animal form of extract can from for example Pan Laboratories (Bothell, WA) or MycoSearch (NC) obtain, perhaps can easily produce.In addition, the library of natural and synthetic acquisition and compound can easily improve by conventional chemical, physics and biochemical method (Blondelle etc., Tib Tech, 14:60,1996).

In-vitro screening method

In a serial embodiment, vitro test comprises the isolating nucleic acid of Smurf gene with the ability of sequence-specific mode in conjunction with test-compound.This method comprises:

(i) provide to comprise and be equivalent to nucleic acid whole or the proteic particular sequence of part Smurf;

(ii) make described nucleic acid under suitable bonded condition, contact a plurality of test-compounds; And

(iii) identify the compound of the described nucleotide sequence of selective binding.

Selective binding used herein is meant any detectable difference of any incorporating parametric such as binding affinity, binding ability etc.

In a serial embodiment, vitro test comprises proteic isolated peptides of Smurf or polypeptide or its fragment with the ability of sequence-specific mode in conjunction with test-compound.This screening method comprises:

(i) provide and be equivalent to Smurf albumen or its segmental peptide or polypeptide or its fragment;

(ii) make described peptide under suitable bonded condition, contact a plurality of test-compounds; And

(iii) identify the compound of the described peptide of selective binding.

In preferred embodiments, high-throughput screening method is used for detecting a large amount of test-compounds with the ability of sequence-specific mode in conjunction with above disclosed gene or peptide.

Screening method in the body

Express the intact cell of the proteic gene variant of coding Smurf or the screening method that whole animal can be used for identifying drug candidate.Following method is applicable to normal or wild-type Smurf.

In a serial embodiment, set up permanent cell line from the individuality of expressing variation Smurf gene.On the other hand, make cell (including but not limited to Mammals, insect, yeast and bacterial cell) sequencing expression comprise the gene of one or more variations Smurf sequences by importing suitable carrier.Can adopt any suitable mensuration to identify candidate compound, described mensuration includes but not limited to: the mensuration that (i) detects the specific varient of test-compound selective binding Smurf; (ii) detect test-compound change (promptly suppress or strengthen) but the mensuration of the ability of the detection of active of Smurf or function; And (iii) detect the mensuration of ability that a kind of compound changes the transcriptional activity of (promptly suppress or strengthen) Smurf gene promoter (promptly regulating) district derived sequence.

In another serial embodiment, the preparation transgenic animal, wherein (i) stably inserts in the transgenic animal genome people Smurf with one or more sudden changes; And/or (ii) make endogenous Smurf gene inactivation and replace the people Smurf gene of variation.Referring to for example Coffman, Semin.Nephrol.17:404,1997; Esther etc., Lab.Invest.74:953,1996; Murakami etc., Blood Press. supplementary issue, 2:36,1996.A kind of preferred method that produces such transgenic animal is so-called " knocking in (knock-in) " technology, wherein a kind of people's gene can insert to replace the endogenous gene regulatory element to express endogenous gene under the control (referring to Rodriguez etc., Cell, 97:199,1999).This animal available candidate compound treatment is also monitored its BMP and any change of TGF β/activin approach.

In addition, select various crowds to be used for the test replacement therapy, what described crowd was not suitable for having established is used for tissue or bone deterioration (for example osteoporosis) or strengthens the regenerated therapy.And it is invalid and to the selected very effective therapy of crowd to identify in the general population, otherwise should ignore.This is an especially effectively advantage of the present invention, because it has eliminated some randomnesss relevant with clinical trial.

High-throughput screening method

Can identify medicine of the present invention by high throughput assay (including but not limited to cellular type or acellular type mensuration) screening.One skilled in the art will appreciate that dissimilar mensuration can be used to detect different types of drugs.In order to screen tens thousand of compounds in a short time, some automatic testing methods have been developed in recent years.Preferred especially such high-throughput screening method.The utilization of large-scale purification polypeptide provided by the invention has promoted use high-throughput screening method testing drug greatly.

Reporter gene

Smad albumen has been accredited as the mesosome of transforming growth factor-beta (TGF β) the various members' of superfamily intracellular signal transduction, and the TGF beta superfamily member influences cell proliferation, differentiation and the developmental diagram of early stage vertebrates and forms.Behind the receptor activation, Smad is assembled into the allos polymer composites of restricted Smad of approach and common Smad4 composition, and mixture is transferred in the nuclear then, thinks that described mixture plays an important role to genetic transcription in nuclear.The Smad binding member (SBE) that contains sequence C AGACA in the JunB gene promoter is the immediate early gene of TGF β, activin and bone morphogenetic protein (BMP) 2 induced strong (Jonk etc., J.Biol.Chem., 273:21145,1998).2 JunB SBE are with reverse repeated arrangement, and it by trans-activation, and shows that inducibility is handled in conjunction with TGF β in the nuclear extract of cell and contain Smad3-and Smad4-mixture under Smad3 and the common overexpression effect of Smad4.Smad albumen is directly in conjunction with SBE.Poly SBE produces the beta induced type enhanser of effective TGF that activin and BMP are also reacted (referring to for example Kim etc., Nature, 388:304,1997).

Reporter gene is expressed and can be linked together with the expression or the activation of any component in the BMP signal transduction pathway.Can use any reporter gene known in the art.Therefore, can use technical point known in the art, and it effectively is connected with reporter gene, whether change the activity of described promotor to determine Smurf and derivative thereof and varient from nucleotide sequence with SBE sequence.In addition, other known in the BMP signal transduction pathway such as T1 * 2 or Smad7 effectively are connected with reporter gene, described known has and is subjected to Smad1 and 5 promotors of regulating.In these are measured, can test candidate compound and change the BMP of the cell of expressing Smad1, Smad5, Smad7, Smurf1 or Smurf2, or derivatives thereof or varient or the ability of TGF signal transduction.

Various reporter gene assay methods are known, and can be used for measuring the influence of Smurf to BMP or TGF β/activin signal transduction pathway.Reporter gene comprises luciferase, beta-galactosidase enzymes (β-gal or lac-Z), E.C. 2.3.1.28 (CAT), horseradish peroxidase and alkaline phosphatase.In addition, adopt specific antibody can detect almost any proteic expression.For example green fluorescent protein (GFP) detection of expression allows to detect Smad inductive SBE activity.Modified GFP to produce reservation function but the albumen with different fluorescent characteristics.Some United States Patent (USP)s are pointed out, use GFP to show the signal transduction pathway relevant with the reporter gene of inducible promoter.For example referring to U.S. Patent number 5,625,048 (the modification GFP that causes amino acid to change produces visible different colours and strengthened luminous), WO 96/23898 (coding also contains the construction of the modification GFP of enzyme recognition site), WO 97/11094 (strengthened fluorescin), WO 97/266333 (optimize humanization GFP albumen and make the mammalian cell expression higher level), WO 97/42320 (the fluorescence intensity enhanced is modified GFP), WO 98/06737 (with modification GFP green and that blue fluorescent protein is distinguished easily), WO 98/21355 (using blue and white light activated GFP mutant).

The screening reagent box

The component that the above-mentioned screening method of enforcement can be needed is made the kit form that makes things convenient for the user.Such test kit is applicable to that preferably the automatic screening instrument uses.

Identify the Smurf binding partners

Except Smad1, Smad7 and Smad5, in an embodiment again, the invention provides evaluation to the Smurf binding partners, can analyze the sudden change of the disease that causes BMP or TGF β/activin approach mediation then.A kind ofly identify that the method for such binding partners is a yeast two-hybrid system, the yeast that preferably uses hemopoietic stem cell library and reorganization Smurf to transform.On the other hand, Smurf albumen can be used for for example utilizing endogenous to produce albumen in the cell affinity purification cell product of Smurf.Serviceable indicia Smurf albumen probe portion purifying goods are so that for example identify that with western blotting type or other antibody test type system specific binding partner is (referring to the explanation of above-mentioned antibody about this mensuration example; But the certain any antibody protein of mark detects itself and the combining of binding partners then).

Embodiment

Can understand the present invention better with reference to following examples, following examples are not to be used for limiting the present invention.

Embodiment 1

Smurf1 is at the BMP approach and influence the graphic formation TGF of embryo beta superfamily and regulate various bioprocesss, comprises cell growth, differentiation and graphic formation.The imbalance of any TGF signal all may cause disease.In the signal of activation TGF beta receptor is directly extremely examined from the acceptor transduction by Smad albumen.Some contacts between the adjusting of the degraded of ubiquitin-mediation and the form generation signal in the growth course have been established at present.This embodiment illustrates a kind of new E3 ubiquitin ligase enzyme Smurf1, and it optionally interacts with BMP approach specificity Smad, makes its ubiquitination, degraded and loss of activity.In the Amphibians embryo, Smurf1 specific inhibition BMP signal and influence graphic formation.Therefore, the ubiquitination at Smad can play control fetal development and various cell response to the TGF signal.

Method

Yeast two-hybrid screening makes Xenopus laevis Smad1 cDNA (36) be cloned into the pGBT9 carrier, and is used to use yeast two-hybrid method (46) the screening xenopus leavis oocytes cDNA library (Clonetech) of Xenopus laevis Smad1 as bait protein.Separate and use Partial cDNA screening Xenopus laevis stage 9 (blastaea) cDNA library, to obtain total length Smurf1 cDNA[SEQ ID NO:1].In est database, identified the people Smurf1 cDNA (AA292123) of preceding 8 amino acid all parts in addition of encoding, made up people Smurf1 with it.Utilize corresponding Xenopus laevis Smurf1cDNA sequence to rebuild preceding 8 amino acid.At its N-terminal with FLAG mark hSmurfcDNA.Adopt PCR type method to make L-Ala replace halfcystine 710, thereby produce ubiquitin ligase enzyme mutant hSmurf1, confirmed described sudden change by order-checking.

Coimmunoprecipitation is for immune precipitation determination, at its C-terminal with FLAG mark Xenopus laevis Smad1 (36), mouse Smad4 and people Smad2 (47), and ³⁵There is external translation the (rabbit reticulocyte extract down in S-Met; Promega).FLAG mark Smad is attached on the pearl (Kodak) that anti-FLAG antibody puts together, with the co-IP damping fluid (10mM Tris, pH 7.5,03.90mM NaCl, 1mM EDTA, 1%Triton- X 100,10% glycerine, 1mM phenylmethylsulfonyl fluoride) washing, then in same buffer with ³⁵The Smurf1 incubation of S-Met mark.With co-IP damping fluid washing and with behind the gel loading buffer wash-out,, and manifest by radioautograph by the SDS-PAGE protein isolate.

Embryo's method and RT-PCR according to document described (36,48) prepare that the synthetic mRNA of embryo, preparation and injection goes among the embryo, animal cap measures, separates embryo RNA with the veutro peripheral zone, fixedly embryo and in situ hybridization of bulk sample slide glass and growth (develomental) RT-PCR.The Smurf1RT-PCR primer is 5 '-GTCCTGTGACTGGAACCC-3 ' (justice is arranged) [SEQ ID NO:5] and 5 '-GAGGACTGCTAGACAAT-3 ' (antisense) [SEQ ID NO:6], and its 5 ' end lays respectively at 482 and 726 of Smurf1 cDNA.

MSmurf1 carries out the RNA trace to the mSmurf1 of embryo and adult mice tissue expression.Analyze PolyA+mRNA and testis, kidney, skeletal muscle, lung, spleen, brain and the heart tissue sample of 7,11 and 15 days the embryonic tissue of post-coitum of equivalent.To the expression of identical trace detection actin cytoskeleton, it is identical to confirm that the mRNA load detects roads at all.Also mice embryonic has been carried out the in situ hybridization of bulk sample slide glass.

Immunoprecipitation and immunoblotting are used lipofection amine (GibroBRL) and calcium sulfate precipitation method (49) transient transfection COS-1 and 293T cell respectively.Use anti-Flag M2 monoclonal antibody (Sigma), anti-Smad1/5 polyclonal antibody (50) or anti-WW2Nedd4 (51) polyclonal antibody according to described immunoprecipitation and the immunoblotting of carrying out of former document (50).Adopting suitable HRP to put together the anti-and enhanced chemoluminescence (Amersham) of goat anti-mouse or goat antirabbit two detects.

Pulse-chase analysis is transfection COS-1 cell as mentioned above.After the transfection 2 days, with 50 μ Ci[ ³⁵S] methionine(Met)/ml do not have methionine(Met) DMEM in 37 ℃ of labeled cells 10 minutes (pulse).The washed cell layer is 2 times then, the incubation fixed time (tracking) in DMEM+10%FCS.Each time point of following the trail of, using anti-Smad1 polyclonal antibody makes to contain TNTE lysis buffer (the 50mM Tris/HCl of proteolytic enzyme and inhibitors of phosphatases, pH7.4,150mM NaCl, 0.5%Triton X-100 and 1mM EDTA) cell lysate of preparation carries out immunoprecipitation.Manifest by SDS-PAGE separating immune mixture and by radioactive automatic developing.Use the quantitatively metabolic marker Smad1 amount of each time point existence of phosphorus image instrument (Molecular Dynamics).

Ubiquitination is measured with HA mark ubiquitin, unmarked Smad1 and above-mentioned Flag-hSmurf1 or Flag-hSmurf1 (C710A) transfection 293T cell.After the transfection 2 days, lysing cell made it carry out the a-Smad1 immunoprecipitation.Then with TNTE+0.1%triton, SDS-RIPA (TNTE lysis buffer, 0.1% sodium lauryl sulphate and 1% deoxycholate salt) and 500mM LiCl, 50mM Tris/HCl, the sequential washing immunoprecipitate of pH 7.4 and 0.1%triton 2 times.Behind the SDS-PAGE, manifest the HA ubiquitination Smad1 that exists in the immunocomplex with monoclonal anti HA 12CA5 antibody mediated immunity trace.By analyze the protein level of unmarked Smad1, Flag-hSmurf1 and Flag-hSmurf1 (C710A) with the total cell lysate of suitable antibodies immunoblotting equal portions.

The result

Separate with the interactional E3 ubiquitin of BMP signal transducer Smad1 ligase enzyme for centrifugation in and also the factor of potential adjusting Smad1, we adopt Xenopus laevis Smad1 to carry out yeast two-hybrid screening as bait protein.Some kinds of codings and the remarkable proteic overlapping cDNA of homologous of Hect subclass E3 ubiquitin ligase enzyme (22) have been separated.This novel gene and closely-related people's homologue (being respectively Smurf1 and hSmurf1) (for Smad ubiquitination regulatory factor-1), they are the protein of 731 amino acid lengths, have 91% sequence identity and partly contain Hect ubiquitin ligase enzyme structural domain at the C-terminal of described molecule.This structural domain is E3 ubiquitin ligase enzyme (22) E6-AP and Nedd4 (is RSP5p and Publ at the yeast) feature (23-26) of the particular type that contains of Mammals.

Identifier Smurf1 genome people from position Smurf1 genomic clone is positioned at pac clone DJ0808A01, and chromosomal region is 7q21.1-q31.1, and nucleotide position is 2669..53763.Intron/exon montage product is carried out computer forecast obtained to have directly single notion mRNA corresponding to the open reading-frame (ORF) of reality human cloning cDNA.

E3 ubiquitin ligase enzyme works with E1 and E2 enzyme one ubiquitin is combined with the specific protein substrate, its objective is subsequently by the proteasome described albumen (12) of degrading.Ubiquitin is raised and activated to the E1 enzyme, and to be transferred to E2 be ubiquitin-conjugating enzyme to ubiquitin then.E2 makes ubiquitin directly be connected to modify with described albumen again, and perhaps E2 is transferred to E3 ubiquitin ligase enzyme with ubiquitin, and E3 ubiquitin ligase enzyme makes substrate selective ground form the ubiquitination mixture.Raise particular target albumen to the effect of puting together organoid by E3, the E3 activity is given the ubiquitination process and is had selectivity.The different albumen of structure function has the E3 activity.For example E3 can be the polyprotein mixture, and it helps to discern substrate, but may have or may not have direct ligase enzyme activity.E3 enzyme example comprises N-terminal rule (end rule) E3 and Skp1/Cullin/F-box (SCF) mixture (27-29).If the E3 of Hect family ubiquitin ligase enzyme (Smurf1 be wherein a member), single albumen not only has substrate specificity but also have catalytic activity (22,30).

Smurf1 also has some other constitutional featuress, and these features are features of RSP5, Nedd4 and Pub1 ubiquitin ligase enzyme.Such feature comprises N-terminal phosphatide and calcium in conjunction with C2 structural domain and 2 WW structural domains, and the WW structural domain helps protein-protein interaction (31-33) by PPXY primitive on the binding partners albumen.Generally speaking, Smurf1 and Pub1 relation the closest (Fig. 1), Pub1 is yeast saccharomyces cerevisiae (Sacromycespombe) ubiquitin ligase enzyme, it regulates mitotic division (23) by pilot protein enzyme body degraded cdc25.

The RNA trace of the expression mSmurf1 of mSmurf1 in embryo and adult tissue shows that the transcript of about 6kb is the principal mode (Fig. 2 A) of the Smurf1 of embryo's expression.In adult tissue, observe main transcript and be 3.0 and 6.0kb (Fig. 2 B).On identical trace, detect actin cytoskeleton and express, and it is identical to confirm that the mRNA load detects the road at each.From mice embryonic at least the 7 day, very just there was mSmurf1 in Zao growth period.At adult mice, mSmurf1 expresses in the great majority tissue that is detected, but the transcript abundance has nothing in common with each other.We do not observe detectable mSmurf1 transcript at skeletal muscle, and kidney and spleen are expressed low-level big transcript.Little transcript content in testis is very abundant, expresses very weak or basic not expression at kidney, lung, spleen, brain and heart.Prepare two kinds of traces by detecting road application of sample equivalent polyA+RNA, but in order to explain any variation of RNA load and transfer, we have surveyed described trace with the tubulin probe, found between each road significantly not different at each.Therefore, in trace, observe variation and reflected the relative expression of mSmurf1 in embryo and tissue.

The bulk sample slide glass in situ hybridization prompting that mice embryonic is carried out, Smurf1 expresses in the appendage bud of neural system, eye, cheek bow, axial mesoderm (notochord), body segment and post-coitum 10-14 days.

Xenopus laevis Smurf1 navigates to the oozooid utmost point and embryonic ectoderm in the Xenopus laevis fetal development, finds all to express Smurf1 mRNA from ovum to the tadpole phase of swimming, and observes high expression level in early days in the growth of ovum, blastaea and gastrula stage.Fall sharply near gastrula Smurf1 in latter stage level, express to late period with low-level zygote again and swim the tadpole phase (Fig. 3 A).Bulk sample slide glass in situ hybridization (Fig. 3 B) shows, Disease in Infants Smurf1 transcript is positioned the animal pole hemisphere of ovum and spilting of an egg blastaea (cleavingblastulae), and this is by carrying out the rna blot analysis (not shown) from the RNA of blastaea embryo's in mid-term animal pole hemisphere and vegetative pole hemisphere and be confirmed separating.When gastrula, Smurf1 is expressed among the embryo more extensive, and RT-PCR observes its transcriptional level decline simultaneously.Yet when neural plate was closed, the Smurf1 expression and localization was in the neural system of growing, and during the phase, Smurf1 expresses at central nervous system, eye, bursa pharyngea and body segment camber to tadpole.The embryo's expression pattern of Smurf1 and blastaea and gastrula stage ectoderm and express overlap (34-36) with Smad1 and BMP-4 in neural system, eye, body segment and the bursa pharyngea of tadpole phase.

HSmurf1 expresses the stable state protein level selectivity decline Smurf1 cause mammalian cell Smad1 and Smad5 and contains and infer E3 ligase enzyme or Hect structural domain, so it is the candidate albumen that acts on Smad1.Whether regulate Smad1 albumen steady-state level in order to study Smurf1, separately with Smad1 or with 2 kinds of mammal cell line 293T of incremental change Flag-hSmurf1 transfection and COS-1.Estimate Smad1 albumen steady-state level with the immunoblotting of full cell lysate then.Detect Smad1 (Fig. 4 A) when lacking hSmurf1 at an easy rate.Yet,, also cause Smad1 protein level dose-dependently significantly to descend even hSmurf1 expresses and can not be easy to detectedly when low-level at western blotting.When hSmurf1 expresses highest level, do not detect Smad1 albumen.In addition, make the composition of Smad1 phosphorylation (37,38) activate the hSmurf1 dependency decline (Fig. 4 B) that I type bmp receptor ALK6 coexpression can not change the Smad1 level.Therefore, hSmurf1 expresses and causes that Smad1 steady-state level dose-dependently descends.This effect can be independent of I type bmp receptor and activate Smad1.

Whether in order to study the Smurf1 activity is exclusive to Smad1, and we have studied its effect to Smad2, and Smad2 is receptor modulating Smad, and it works in TGF β and activin signal transduction pathway.With to the Smad1 of lowest dose level hSmurf1 sensitivity different be, hSmurf1 has only affects to Smad2 albumen steady-state level, only just there be (Fig. 4 C) in this humble influence when the F1ag-hSmurf1 of highest level expresses.Also tested other Smad:Flag-hSmurf1 to Smad3 or the almost not effect of Smad4 protein level, albumen significantly descends but it makes Smad5, Smad5 albumen and Smad1 closely related (Fig. 4 D).Generally speaking, these data acknowledgements hSmurf1 preferably regulates Smad1 and Smad5 steady-state level, and Smad1 and Smad5 are two kinds of receptor modulating Smad that play a role in the BMP signal transduction.

HSmurf1 regulates Smad1 degraded and ubiquitination Smurf1 and comprises and infer the E3 ligase enzyme or the such prediction of Hect structural domain support: Smurf1 plays E3 ubiquitin ligase enzyme.Whether regulate Smad degraded in order to study Smurf1, various researchs concentrate on Smad1 (with the homology of Smad5 be 91%).The apply pulse chase experiment is analyzed Smad1 and is transformed demonstration, when lacking hSmurf, and about 6 hours of the transformation period of Smad1.Yet when having hSmurf1, Smad1 transforms and significantly strengthens (transformation period is lower than 2 hours) (Fig. 5 A).Therefore, hSmurf1 strengthens the Smad1 conversion rate.

For the Smad1 that determines the hSmurf1 mediation shifts to new management mechanisms, estimated the Smad1 ubiquitination in the intact cell.In order to help to detect ubiquitin, there is or lacking transfection 293T cell under the Flag-hSmurf1 together with HA mark ubiquitin and Smad1.When lacking hSmurf1, Smad1 does not almost have detectable ubiquitination.Yet during with the hSmurf1 cotransfection, we observe and ubiquitin occurs and put together Smad1 ladder (Fig. 5 B).In order to confirm that the Smad1 ubiquitination needs the catalytic activity of hSmurf1 Hect structural domain, in hSmurf1, make up point mutation (hSmurf1 (C710A)).This residue is critical to Hect structural domain catalytic activity, thinks that this sudden change is at forming the cysteine residues (22) of thiolic acid ester bond with ubiquitin.HSmurf1 is opposite with wild-type, and hSmurf1 (C710A) expresses can not produce ubiquitination Smad1.And compare with wild-type hSmurf1, hSmurf1 (C710A) does not influence Smad1 stable state protein level, although effective expression mutain (Fig. 5 C).In a word, these Notes of Key Datas, hSmurf1 induces the Smad1 degraded of ubiquitin mediation to change the Smad1 steady-state level by its Hect structural domain.

Smurf1 selectively acting has in vivo studied Smurf1 and the effect of Smad albumen in Smad1 and Smad5, with the ultimate principle of the selectivity target of estimating Smurf1 degraded Smad1 and Smad5.At first the ability of test Xenopus laevis Smurf1 and Smad1, Smad4 or the effect of non-specific reference protein lamin is measured in the using yeast double cross.The yeast of Smurf1 and Smad1 cotransfection has significant beta galactosidase enzyme activity; Smurf1 and Smad4 or lamin cotransfection yeast do not show beta galactosidase enzyme activity (Fig. 6 A, left side).According to ³⁵The ability of external combination of S-mark Smurf1 and immunoprecipitation Smad shows, Smurf1 selective binding Smad1, and debond Smad2 or Smad4 (Fig. 6 A, right side).

In order to test ³⁵The ability of external combination of the Smurf1 of S-mark and immunoprecipitation Smad has been studied Smurf1 and Smad interactional specificity in the intact cell, the association of hSmurf1 and various Smad in the 293T cell.Do not detect the interaction of wild-type hSmurf1 and Smad1.But may be in intact cell Smurf1 and Smad1 interact and be actually instantaneity be difficult to confirm ubiquitin ligase enzyme and the association of its substrate because proved with other system.By detecting the interaction of Smad1 and ubiquitin ligase enzyme mutant Flag-HSmurf1 (C710A), can detect described proteic association (Fig. 6 B, C).And the influence (not display data) of the coexpression of active I type bmp receptor ALK2 is not formed in this interaction, and this and hSmurf1 regulate the Smad1 conversion and be independent of the of the same mind of BMP signal transduction.HSmurf1 (C710A) is also effectively in conjunction with Smad5, but analyzes it and the association announcement of Smad2, if there are interactional words, also is very faint (Fig. 6 A and B).

The R-Smad connector area contains the PPXY sequence, and the PPXY sequence is the conservative primitive as seen in the WW structural domain identification of Smurf1.Smad1 is different with wild-type, the Smad1 mutant that wherein lacks the PY primitive only with the faint association of Smurf1, and the Degradation (data does not show) of opposing Smurf1 mediation.These results show, hSmurf1 and Smad1 and Smad5 specificity are associated, this interaction by Smad1 PY primitive and Smurf1WW structural domain in conjunction with mediation.

By comparing the relation of hSmurf1 and a kind of structurally associated ubiquitin ligase enzyme Nedd4, further tested hSmurf1 as the specificity of ubiquitin ligase enzyme at Smad.Although effectively co-precipitation of Smad1 and hSmurf (C710A), it does not act on corresponding N edd4 mutant.Consistent is that the Nedd4 of overexpression does not influence Smad1 albumen steady-state level (Fig. 6 C, figure below) therewith.In a word, these data declarations, Smad1 that people Smurf1 albumen and the two selectivity of Xenopus laevis Smurf1 albumen and BMP regulate and Smad5 associate, and this effect is that Smurf1 is exclusive, rather than the general feature of Hect class ubiquitin ligase enzyme.

Endogenous BMP signal among the Smurf1 antagonism Xenopus laevis embryo, make the abdomen mesoderm back sideization and ectoderm neuralization in the Xenopus laevis blastaea, the Smurf1 expression and localization is in the animal pole ectoderm and the peripheral zone of overlapping, and promptly a cell band is positioned at and forms mesoblastic blastaea equator.This expression pattern and Smurf1 act on BMP approach specificity Smad, the ability integration of its ubiquitination and degraded are considered, illustrate that Smurf1 influences ectoderm and the graphic formation of mesoderm by Smad1 or Smad5 antagonism BMP signal.

Introduced the natural inhibitor of TGF 'beta ' activity at present in ligand level.These factors comprise chordin, Progynon statin and noggin, and they are by playing a role in the extracellular in conjunction with specific ligand (comprising BMP and activin).When the Xenopus laevis mesoderm was induced with graphic formation, the BMP signal of peripheral zone veutro portion made the tissue development specialization, for example blood and mesenchyme, and this is the feature of tadpole phase lateral region of abdomen.Yet, if the BMP signal transduction is by ligand antagonists such as chordin, Progynon statin or noggin (secretion of back Spemann organizer) or the artificial inhibitor of quilt such as dominant negative BMP part or receptor blocking, then Yu Qi abdomen mesoderm is divided into back tissue such as muscle and notochord (a kind of process that is called " back sideization ") (5,39).

Smurf1 has the potentiality of regulating peripheral zone BMP signal, has therefore tested dystopy Smurf1 and has expressed the interference of veutro being organized graphic formation.Smurf1mRNA is gone into 4 cell blastula embryos in veutro peripheral zone (VMZ) microinjection, form second axle construction of dystopy in the hope of exciting at 52% tadpole phase embryo (n=25) lateral region of abdomen.These Smurf1 inductive second axle construction are back side features that the BMP inhibition causes.Its formation can obtain saving by Smad1 and Smurf1 coexpression together, confirms that Smurf1 effect is limited to interference BMP/Smad1 approach (Fig. 7 A).In addition, the overexpression of Smurf1 in back edge band (DMZ) cell that forms head and back of the body axle construction is without any influence (data does not show).Back one observed result is not consistent at the discovery of the Smad2 in the culturing cell about Smurf1 with front of the present invention.

Further with VMZ graft characterized the back side of Smurf1 turn usefulness into.In the case, Smurf1 expresses and causes that blood breaks up the muscle differentiation that weakens and follow and weakens, and turns into consistent (Fig. 7 A, right figure) with the Smurf1 back side.The same with axle formation mensuration, Smad1 and Smurf1 coexpression reverse these back ofs the body and turn usefulness into, have confirmed its BMP approach specificity.

In ectoderm, endogenous BMP expresses the specialization epidermis, but when BMP signal weakening or elimination, ectoderm is divided into cement gland or nervous tissue (40,41) respectively.Smurf1mRNA is positioned the ectoderm of Xenopus laevis blastaea and early stage gastrula, illustrates that Smurf1 can regulate ectodermic graphic formation by regulating the BMP signal transduction.So, tested Smurf1 and had a situation in the ectoderm tissue.Find that the overexpression of Smurf1 in animal cap causes neural and the cement gland differentiation, this is the feature that the BMP signal transduction weakens.These effects can be reversed (Fig. 7 B) by coexpression Smad1 in animal cap.Comprehensive these results confirm, Smurf1 ectoderm capable of blocking and mesoblastic BMP signal, and prompting Smurf1 weakens the BMP signal transduction of these tissues and influences the graphic formation of embryo.

It is active but strengthening Smad2 activity Smurf1 in culturing cell excites BMP approach specificity Smad ubiquitination and degraded that Smurf1 suppresses the Smad1 of embryonic cell, and in the Xenopus laevis embryo Smurf1 antagonism endogenous BMP signal.In the Xenopus laevis animal cap, overexpression Smad simulation is by the effect of the TGF β factor of specificity R-Smad transduction signal.Therefore, the Smad1 specificity is induced veutro/back mesoderm, and as the BMP part, and Smad2 induces dorsal mesoderm (Spemann organizer), as activin, Vgl and nodal.So whether we have tested Smurf1 can directly antagonism Smad1 or Smad2 induce mesoblastic activity by the Smurf1 of each Smad of overexpression in animal cap and different amounts.We find, show according to expressing veutro/back mesoderm marker Xhox3 and Xcad1, only express Smad1 (1ng mRNA) and induce the abdomen mesoderm.But Smurf1 and Smad1 coexpression all hinder at whole Smurf1 dosage of being tested induces these markers (Fig. 8 A), but confirms Smurf1 antagonism Smad1 activity.

In order to determine whether Smurf1 can disturb Smad2, use Smad2 dosage (50pgmRNA) (Fig. 7 B, C) enough to induce myoD (the vertebrates muscle mark of back/lateral mesoderm) still to be not enough to induce goosecoid (the frog mark of Spemann organizer dorsal mesoderm).When Smurf1 and Smad2 coexpression, all do not suppress myoD at any Smurf1 dosage of being tested and induce (Fig. 8 B).This is not consistent at other discovery of Smad2 with Smurf1.What is interesting is that the dosage along with Smurf1 under the situation of the Smad2 (50pg) that has this limited volume increases, and observes inductive goosecoid genetic expression.Thisly induce that Smad2 is expressed is dependent, because independent Smurf1 does not induce goosecoid.Cap cell is similar to the reaction (Fig. 8 C) that independent Smad2 dosage is increased to the response class of the Smurf1 of incremental change in the presence of restriction Smad2.Therefore, 50pg Smad2 and 100pg Smurf1 combined induction goosecoid expression levels equal the raise level (Fig. 8 C) of 5 times (250pg) of independent Smad2 dosage.Therefore, Smurf1 suppresses the Smad2 activity, but strengthens the susceptibility of animal cap to Smad2.These experiment confirms, Smurf1 also strengthens the reaction of these cells to the activin approach except hindering the reaction of animal pole cell to the BMP approach.Although the present invention does not rely on any concrete mechanism, the inventor proposes, and the animal pole cell response of Smurf1 mediation sexually revises the endogenous BMP signal transduction that causes because of the specific aim ubiquitination by Smurf1 substrate Smad1 and Smad5 and weakens.Thereby Smurf1 plays a role in growth course, to change the response capacity of cell to multiple TGF beta ligands by the specific Smad approach of selectivity inactivation.

Discuss

The inventor's discovery makes that people are clear have been understood the selectivity ubiquitination and regulating effect, approach and the mechanism of growing in the graphic formation.The embodiment that is provided confirms that the transduction of TGF signal is controlled by the ubiquitination of Smad signal transducers, and this activity influence is grown.Say exactly, confirm that it is Smad1 and Smad5 that a kind of Hect E3 of family ubiquitin ligase enzyme Smurf1 selectivity directly acts on BMP approach specificity Smad, causes its ubiquitination and degraded.Yet Smurf1 does not act on or influences TGF β/activin approach specificity Smad2, does not also influence the common mating partner Smad4 in the Smad signal transduction mixture.In addition, Hect ubiquitin ligase enzyme is not the general feature of E3 ligase enzyme at Smad, because Nedd4 does not act on Smad.What is interesting is that described result shows that the Smad of Smurf1 mediation transforms the influence that is not subjected to the BMP signal transduction, prompting activated form Smad1 or Smad5 do not need the substrate as Smurf1.Therefore, Smurf1 does not work in activation Smad downstream and changes the BMP signal, but controls the response capacity of cell to BMP by regulating cell Smad albumen steady-state level.

The mSmurf1 gene is being grown early stage just expression, and we predict this moment, and mSmurf1 regulates the reaction of cell to the BMP signal.Smurf1 is present in adult tissue, and indication mSmurf1 regulates BMP signal in late period.Possible these signals play a role in organizing the continuation differentiation of stagnating or may be relevant with growth.Neurula stage is very consistent with the expression pattern that observes in the frog embryos in similar stage with mice develop camber expression Smurf1 after this.

The phenotypic effect of Smurf1 in the Xenopus laevis embryo points out that the selectivity ubiquitination is the important conditioning agent of inducement signal at embryo development procedure.Smurf1 makes the mesoderm back sideization and ectoderm neuralization by disturbing the BMP signal of controlling mesoderm and the graphic formation of ectoderm in normal development.Think at present and regulate the graphic formation of BMP dependency by secretor type BMP is conjugated protein as chordin, noggin and Progynon statin, secretor type BMP is conjugated protein by directly suppressing extracellular BMP signal in conjunction with the BMP part.Yet Smurf1 provides a kind of new mechanism of regulating the BMP signal, and Smurf1 works in the signal transduction level, and infers that Smurf1 influences cell naturally, because Smurf1 is a kind of intracellular protein.In addition, with the secretor type BMP with restricted ligand specificity conjugated protein different be that Smurf1 produces the BMP approach and extensively suppresses active, because many I receptors (ALK, ALK2, ALK3 and ALK6) are by Smad1 and Smad5 transduction signal (42).Therefore, in embryo development procedure, Smurf1 and the graphic formation of extracellular BMP inhibitor synergy specialization.Whether Smurf1 is subjected to the adjusting of certain mode to remain an open question.

What attract people's attention especially is that Smurf1 mRNA is positioned xenopus oocyte and blastaea animal pole.This district forms the ectoderm tissue, and for example epidermis, cement gland and neural system are organized the BMP signal transduction level control of destiny by the cell perception.When the animal pole ectoderm is not accepted endogenous BMP signal, Neural Differentiation spontaneous generation, but when the Smad1 of BMP ligand level that puts on cell or cell inner expression amount increased gradually, the cell fate spectrum was epidermis from nerve by carrying out property of cement gland specialization.In addition, Xenopus laevis embryo's expection abdomen mesoderm BMP signal transduction weaken cause this tissue again specialization be dorsal mesoderm.Because the adjustable ganglion cell of Smurf1 is to the reactivity of BMP signal, it can influence ectoderm and mesoderm destiny characteristic by control Smad1 or Smad5 level.High level Smurf1 can block the response capacity of cell to BMP fully, but moderate or low-level Smurf1 can regulate the BMP strength of signal by control Smad1 protein level, thereby changes the character of cell response.Smurf1 can be by the active cell fate decision of controlling under the BMP effect of the Smad1 that sets up morphogenetic gradient in the cell as a result.In various animals, the source of parents mRNA that is positioned in the ovum plays cell fate determiner (43).In Xenopus laevis, coding Vg1 (a TGF β member) and the mRNA of Brat/VgT are positioned the ovum vegetative pole, at this their specialization entoderm and mesoderm (44,45).The extremely middle Smurf1 of localization of the presentation of results that this embodiment provides, oozooid may play ectoderm cell destiny determiner.

Smurf1 interesting result of overexpression in the Xenopus laevis animal cap changes the response capacity of cell to Smad2, and Smad2 is activin/TGF beta pathway specificity Smad.Smurf1 strengthens the susceptibility of cell to the Smad2 of fixing horizontal ectopic expression, and therefore along with the Smurf1 level raises, carrying out property of inductive mesoderm becomes the Spemann organizer one modal back of the body type mesoderm of more characterization.This response class is similar to the influence that takes place when Smad2 level independent in the animal cap raises.Therefore, Smurf1 can change the response capacity of cell to different TGF signals simultaneously: it suppresses the reaction to the Smad1 approach, and stimulates the reaction to the Smad2 approach.Other biological function that Smurf1 regulates fetal development, cell growth, tissue stagnation and TGF beta superfamily the effect that has nothing in common with each other of different TGF signal transduction pathway produces material impact.

Embodiment 2:The characterized of people Smurf2 and clone

Use Xenopus laevis Smurf1 Sequence Identification and human cloning Smurf2 in the est database.Obtain 2 overlapping EST clones, make up the full length sequence of hSmurf2 with it corresponding to hSmurf2.Smurf2 and Smurf1 are closely related, are about 75% with the homology of hSmurf1 aminoacid sequence.Smurf2 contains a N-terminal C2 structural domain, is thereafter 3 WW structural domains and a HECT ubiquitin ligase enzyme structural domain (Figure 12).Smurf2 and Smurf1 are closely related, but C2 structural domain downstream has an extra WW structural domain (Figure 13).Find Smurf2 at whole growth early expression, and be present in most of adult tissues the level in spleen and skeletal muscle lower (Figure 14 A and Figure 14 B).RT-PCR analyzes further and show that Smurf2 expresses in comprising the various clones of P19, HepG2 and 293T.

Separation of human Smurf2 and mouse Smurf2 have identified that from expressed sequence mark (EST) database several and Smurf1 have the overlapping people clone of similarity, adopt 2 overlapping EST clones to make up full-length Smurf2 by PCR.For rna blot analysis, in est database, having identified coding comprise terminator codon open reading-frame (ORF) 225 amino acid and also with the amino acid identity of people Smurf2 be 96% mouse part Smurf2 cDNA clone (I.M.A.G.E. clones ID 638876).

Make up the Mammals expression constructs of plasmid,, make it go into to have the pCMV5 (69) of N-terminal Flag or Myc mark by the frame subclone by polymerase chain reaction (PCR) amplification open reading-frame (ORF) for Smurf2.About Smurf2WW structural domain disappearance, DWW1 disappearance amino acid/11 63-185, DWW2 disappearance amino acid 257-279, and DWW3 disappearance amino acid 303-325.In order to produce the Smurf2 ubiquitin ligase enzyme mutant of catalytic activity forfeiture, replace halfcystine 716 with L-Ala.In order to produce the Smad7PY mutant, replace tyrosine 211 (Y211A) with L-Ala, perhaps lack the PPPPY sequence (Δ PY) between the amino-acid residue 206-212.For T β RI-Flag, the Flag mark imports the C-terminal of acceptor.All constructions confirm by the PCR preparation and through order-checking.Utilize conventional restriction site to prepare bacterial expression vector pET15-Smad7-HA and pGEX4T-1-Smurf2.

Immunoprecipitation, immunoblotting and affinity labelling are about the research of mammalian cell, respectively with calcium phosphate precipitation or deae dextran method transient transfection 293T and COS-1 cell.Use anti-HA monoclonal antibody (12CA5, Boehringer), anti-HA rabbit polyclonal antibody (SantaCruz), anti-Myc monoclonal antibody (9E10 ascites, Developmental Studies HybridomaBank), anti-Flag M2 monoclonal antibody (Sigma) or anti-Smad7 rabbit polyclonal antibody carry out immunoprecipitation and immunoblotting.About anti-Smad7 antibody, with the bacteriogenic GST-Smad7 immunize rabbit of coded amino acid 202-260.After antibody is adsorbed onto G albumen or A-Sepharose, and usefulness TNTE 0.1% (50mM Tris, pH 7.4,150mM NaCl, 1mM EDTA, 0.1%Triton X-100) washing precipitate 5 times, separate by SDS-PAGE, be transferred on the nitrocellulose, with the suitable antibodies immunoblotting.Adopt sheep anti mouse that suitable horseradish peroxidase (HRP) puts together or anti-rabbit two anti-and strengthen chemoluminescence (Amersham) and detect.Make bacteriogenic His-Smad7-HA and Ni ²⁺-NTA pearl (Qiagen) or GST or GST-Smurf2-with TNTE (0.5%Triton X-100) washing 3 times, pass through immunoblotting assay throw out with anti-HA antibody in conjunction with gsh pearl (Amersham) incubation.For affinity labelling, make the COS-1 cell and the 250pM[of transfection ¹²⁵I] TGF-bl is in 4 ℃ of incubations 1 hour, makes acceptor and part crosslinked according to document described (70) with DSS.Quantitatively measure by phosphorus imaging (Molecular Dynamics) in conjunction with the T β RI of Smurf2 or Smad7.

Pulse-chase analysis and ubiquitination are measured with specifying construction transfection COS-1 cell, use 50mCi/ml[after the transfection in 2 days ³⁵S] methionine(Met) (Trans[ ³⁵S]-marker; Fig. 2 B) or 150mCi/ml[ ³⁵S] methionine(Met) (Fig. 5 C and D) in the Dulbecco of no methionine(Met) modified form Eagle substratum (DMEM) in 37 ℃ of described cells of mark 15 minutes.The washed cell layer is 2 times then, exist or lack 30mM lactacystin (derive from E.J.Corey, HarvardUniversity) or under the 0.4mM chloroquine in the DMEM that contains 10% foetal calf serum the incubation fixed time.At each time point,, show through the SDS-PAGE separation and by radioautograph with anti-HA monoclonal antibody immunity sedimentation cell lysate.Use the protein of the quantitative metabolic marker of phosphorus imaging.Measure about ubiquitination, with HA mark ubiquitin and combination acceptor Smad7 and Smurf2 transfection 293T cell.Make cell lysate carry out anti-Smad7 immunoprecipitation, immunoprecipitate was boiled in 1%SDS 5 minutes,, behind anti-Smad7 antibody redeposition, carry out anti-HA immunoblotting with the 0.1%TNTE dilution.

Using immunofluorescence (deconvolution) microscopy of deconvoluting carries out Subcellular Localization and is tiled in MvlLu cell on the Permanox cell sheet (Nunc) of gelatin bag quilt with the appointment construction by the transfection of calcium phosphate precipitation method.According to document described (69) pair cell fix, change thoroughly processing and with anti-and two anti-reactions.The deconvolute Olympus 1X70 inverted microscope (Applied Precision) of microscopy software of application configuration fluorescence part and Deltavision obtains image.

Responsive transcription is measured with specified CMV-β gal, 3TP-Lux report construction and Smad7-HA construction and is utilized calcium phosphate DNA precipitator method transient transfection HepG2 cell.Keep total DNA constant by adding the pCMV5 empty carrier.Next day, with or spend the night without 100pM TGF β incubation cell.Utilize luciferase detection system (Promega) to detect uciferase activity with Berthold Lumat LB 96V luminometer, and make it betagalactosidase activity normalization method.

The result

Smurf2 does not regulate the Smad steady-state level and is lacking or existing 293T cell expressing Smad under the Smurf2.Be that different with the Smurf1 of the proteolysis that causes ubiquitin mediation at Smad1, Smurf2 expresses and do not change Smad1,2,4 or 7 steady-state level (Figure 15 A) unexpectedly.For determine Smurf2 whether can with any association among these Smad, carry out anti-Smurf2 immunoprecipitation with cell lysate, and detect association Smad by immunoblotting.In coexpression Smad1,2 or 4 cell, do not find any Smad under such condition with Smurf2 co-precipitation (Figure 15 B).On the contrary, in the cell of expressing Smad7, detect the strong interaction between Smurf2 and the Smad7.Can not change the Smad7 conversion in order to confirm that Smurf2 expresses, we have also carried out pulse-chase analysis to Smad7.Lacking or existing the transformation period of the Smad7 that expresses under the Smurf2 similar, about 4 hours (Figure 15 C).For characterized Smad7-Smurf2 associates,, make itself and bacteriogenic Smad7 incubation from bacteria purification GST-Smurf2 fusion rotein.Under such conditions in vitro, Smad7 illustrates the direct and Smad7 association (Figure 15 D) of Smurf2 effectively in conjunction with Smurf2.We also analyze the factor of determination on its interactional Smad7 of mediation and the Smurf2.Smad7 has PPXY sequence (PY primitive) at its connector area, and the PPXY sequence can mediate and the interaction (71) of WW structural domain as seen in the WW structural domain of Smurf2.The Smad7PY primitive also can by make tyrosine residues change into L-Ala (Smad7 (Y211A)) or by the disappearance whole primitive (Smad7 (Δ PY)) change.Smurf2 shows in conjunction with the 293T cell analysis of any Smad7 mutant, weakens with the interaction of Smurf2, but do not eliminate (Figure 15 E) fully.Illustrate that interaction produces material impact to the PY primitive to Smad7-Smurf2, but be unique factor of determination that Smad7 is also played a role to its association in other district.We have also prepared wherein the Smurf2 mutant of each structural domain disappearance in 3 WW structural domains.Smad7 is suitable with wild-type Smurf2 with the effect of the Smurf2 mutant that lacks first WW structural domain, and any one all eliminates any detectable effect (Figure 15 F) with Smad7 yet lack WW2 or WW3.Therefore, mediation combines WW2 and two structural domains of WW3 that need Smurf2 simultaneously with Smad7.In a word, these presentation of results, Smurf2 by WW2 and WW3 structural domain directly in conjunction with Smad7, but it can not produce the proteolysis of ubiquitin mediation to Smad7 under the situation that lacks the transduction of TGF signal.

Smad7 makes Smurf2 raise that Smad7 combines the allos polymer composites (72,56) of TGF β II type (T β RII) and I type (T β RI) acceptor by interacting with activation I receptor subunit in the mixture with the TGF beta receptor.Therefore the composing type of Smad7 and Smurf2 association generation absorbing possibility: Smad7 may work to raise Smurf2 to TGF beta receptor mixture.In order to test this kind possibility, we express the FTG beta receptor in the COS-1 cell under the situation of existence and shortage Smad7 and Smurf2.Then by with [ ¹²⁵I]-the crosslinked and labeled receptor of TGF β.Manifest affinity labelling receptor complex with the Smurf2 co-precipitation through autography.Lack or when having Smad7, find almost not have TGF beta receptor mixture and wild-type Smurf2 co-precipitation (Figure 16).Make up Smurf2 catalysis mutant with Smurf2, wherein halfcystine 716 is converted into alanine residue (C716A).This sudden change thinks that at the cysteine residues in the HECT ubiquitin ligase enzyme structural domain this cysteine residues and ubiquitin form the thiolic acid ester bond.When only expressing Smurf2 (C716A), we detect the slight interaction (Figure 16) with the TGF beta receptor.Yet when having Smad7, Smurf2 (C716A) significantly strengthens with the effect of described acceptor.Thereby Smad7 mediation Smurf2 and the interaction of TGF beta receptor.

We have also detected the Smad7 with described receptors bind.Smad7 combines allos polymerization TGF beta receptor mixture (72,56) by acting on activation I receptor subunit.When having wild-type Smurf2, the TGF beta receptor mixture amount that we observe with the Smad7 co-precipitation significantly reduces (Figure 16, the 3rd road).This reduces relevant with the I receptor total amount that these transfection bodies exist.Because Smad7 is by the bind receptor mixture with the effect of activation I receptor, so these presentation of results Smurf2 reduces the level of Smad7 bind receptor mixture.Identical of viewsly with this be when the mutant coexpression of Smad7 and Smurf2 catalytic inactivation, not observe Smad7 bind receptor level descend (Figure 16, the 5th road).These presentation of results, the downward modulation of mediation of Smurf2 catalytic activity and Smad7 bonded TGF beta receptor.

The Subcellular Localization Smad7 of Smad7 control Smurf2 is positioned in the nuclear, outputs in the tenuigenin when the transduction of TGF signal activates, and Smad7 combines TGF beta receptor (73) by acting on acceptor I subunit in tenuigenin.Whether raise Smurf2 to the described acceptor of intact cell in order to study Smad7, whether we have measured Smad7 can regulate the Smurf2 location.For this reason, we express T β RII, T β RI and Smurf2 (C716A) lacking or exist under the situation of Smad7, distribute by the suitable proteic ubcellular of IFM.

Smad7 is positioned in the nuclear, but is mainly seen in the tenuigenin when having signal transduction, in tenuigenin Smad7 also with the TGF beta receptor of transient expression with point-like pattern co.We and other scholar had just observed this distribution (74-76) of TGF beta receptor in the past.Similar is, Smurf2 is mainly seen in the nucleus, but this being positioned at when having the transduction of TGF signal do not change.Yet when the Smad7 coexpression, the described ubcellular of Smurf2 distributes and obviously changes, and Smurf2 albumen is mainly seen in outside the nucleus and with the TGF beta receptor extensively locatees altogether.These results suggest, Smad7 expresses and causes Smurf2 to export from nucleus, and Smurf2 is raised to TGF beta receptor mixture.

Smurf2 induces TGF beta receptor and Smad7 degraded Smad7 association acceptor in that have wild-type Smurf2 but not significantly reduce the such possibility of prompting: Smurf2 during mutant Smurf2 may the degraded of catalysis Smad7 bind receptor mixture.Transform in order to analyze TGF beta receptor mixture Smurf2 dependency, we are at 293T cell coexpression T β RII and T β RI.Under such condition, T β RII and T β RI effectively are assembled into functional allos polymer composites, make us can study the conversion of whole receptoire.We have at first studied the acceptor steady-state level (Figure 17 A) in the cell of expressing Smurf2, observe when II type or I receptor single expression or when expressing together Smurf2 minimum to the steady-state level influence of II type or I receptor.Yet when having wild-type Smad7, Smurf2 expresses to increase and causes the steady-state level of I receptor significantly to descend (Figure 17 B).On the contrary, not effect of Smurf2 (C716A).When expressing wild-type Smurf2, II receptor level also descends, and the decline degree that observes than I receptor is low.This species diversity may not act on independent T β RII by activation I receptor bind receptor mixture because of such fact: Smad7.Whether we have also tested Smurf2 at forming activated form I receptor T β RI (T204D).This receptor mediates the transduction of TGF signal under the situation that does not have the II receptor, and in conjunction with Smad7 (72).Similar to receptor complex, wild-type Smurf2 rather than Smurf2 (C716A) cause activation I receptor steady-state level decline (Figure 17 B).In order to confirm that the acceptor steady-state level changes the change that has reflected that acceptor transforms, our apply pulse follow-up analysis has been analyzed the transformation period (Figure 17 C) of T β RII and T β RI.Analysis to the II receptor discloses, new about 1 hour of the proteic transformation period of synthetic II receptor.Opposite T β RI is much stable, about 4-6 of its transformation period hour.In addition, as Smad7 or Smurf2 during respectively with described expression of receptor, the transformation period of I receptor is constant.But as Smurf2 and Smad7 during with TGF beta receptor mixture coexpression, the transformation period of I receptor was reduced to about 1 hour.Therefore, Smad7 and Smurf2 strengthen the conversion of I receptor.

The proteolysis membrane receptor of ubiquitin mediation can be by proteasome and the two common mediation (60) of lysosome.Whether depend on proteasome and lysosome effect in order to test the degraded of Smurf2 dependency enhancing TGF beta receptor, acceptor when we have detected existence and have lacked lactacystin and chloroquine transforms, and lactacystin and chloroquine suppress respectively by proteasome and lysosome protein degradation.Pulse-chase analysis to acceptor shows that each inhibitor itself plays stabilization (Figure 17 D) to a subgroup in total TGF beta receptor storehouse.These presentation of results, the two acceptor that observes when having Smad7 and Smurf2 of proteasome and lysosome transforms and strengthens the generation effect.

In the process of analyzing the TGF beta receptor, we have also detected the Smad7 protein level.When not having TGF beta receptor mixture, Smad7 steady-state level and conversion are not subjected to the influence (seeing Figure 15) of Smurf2.Yet when having TGF beta receptor mixture, Smurf2 expresses and reduces Smad7 steady-state level and transformation period (seeing Figure 17 B and 17C respectively).And this Smad7 reduces the catalytic activity that depends on Smurf2 HECT structural domain, does not change the Smad7 level because Smurf2 (C716A) mutant is expressed.Lactacystin and chloroquine are also stablized Smad7 and are transformed, and point outs equally with receptor complex, and Smad7 degrades by proteasome and two approach of lysosome.Therefore, under the situation that has the transduction of TGF signal, Smurf2 induces the Smad7 degraded, and its mechanism may be by working at complete acceptor-Smad7 mixture.

In order to study the ubiquitination of described acceptor and Smad7, we have expressed HA epi-position marking type ubiquitin, have detected the ubiquitin conjugate of T β RII, T β RI or Smad7 by the immunoblotting behind the immunoprecipitation.In order to guarantee specificity, in SDS, boil the immunoprecipitation sample, carry out immunoblotting with the suitable antibodies post precipitation again.The analysis of Smad7 ubiquitination is shown, lack or exist Smurf2 to have only that seldom ubiquitin and Smad7 albumen are puted together (Figure 17 E).Yet when Smurf2 and Smad7 and TGF beta receptor coexpression, we observe high-molecular weight Smad7 ubiquitin conjugate, and can not detect described conjugate when using Smurf2 catalysis mutant.Opposite with Smad7, we fail to detect the obvious Smurf2 dependency ubiquitination of I receptor.Because acceptor transform to strengthen when having Smurf2 and Smad7, put together acceptor may illustrate that they acceptor may take place degrade fast when ubiquitination so can not detect ubiquitin.On the other hand, the Smad7 ubiquitination may be as the signal that makes complete acceptor-Smad7 mixture targeting proteins enzyme body.Comprehensive these results show that the Smad7 dependency is raised Smurf2 to TGF beta receptor, cause proteasome and lysosome mediation property degraded TGF beta receptor mixture and Smad7.

Smurf2 associates and strengthens our the research explanation of Smad7 inhibition activity, and Smurf2 may raise to TGF beta receptor mixture by Smad7, thereby causes described acceptor degraded.The Smad7 that the degraded of prompting ubiquitin mediation may influence the TGF beta pathway suppresses active.In order to study this point, we have at first studied with the very weak Smad7 of Smurf2 effect (Y211A) (seeing Figure 15 D) whether have the ability of raising Smurf2 to TGF beta receptor that changes.For this reason, under the situation of existence or shortage Smurf2 (C716A), make wild-type Smad7 or Smad7 (Y211A) and TGF beta receptor coexpression,, detect acceptor interaction by the affinity labelling acceptor of analysis and Smad7 or Smurf2 co-precipitation.The effect of TGF beta receptor mixture and Smad (Y211A) and the effect that observes with wild-type Smad7 suitable (Figure 18 A) confirm that this sudden change of PY primitive does not influence the interaction of Smad7 and TGF beta receptor.Yet when having sudden change Smad7, Smurf2 (C716A) significantly reduces with the associating ability of TGF beta receptor, and the reduction of the functioning efficiency between above-mentioned Smurf2 that observes and the Smad7 (Y211A) is related.Next, whether we have studied Smad7 (Y211A) and have and change the ability that suppresses the transduction of HepG2 cell TGF signal, described HepG2 cell expressing endogenous Smurf2.For this reason, we utilize the 3TP-lux report construction of abundant characterized to detect the transduction of TGF signal.Wild-type Smad7 expresses strong the reduction TGF-b dependency of this report thing is induced (Figure 18 B).On the contrary, the inhibition of Smad7 (Y211A) mutant is active significantly to be reduced, although it has the ability with the useful effect of TGF beta receptor mixture.Illustrate that Smurf2 combines the inhibition activity of enhancing Smad7 to TGF signal transduction pathway with Smad7.

Studies confirm that in the past prevents that in conjunction with the Smad7 of TGF beta receptor mixture Smad2 is in conjunction with described acceptor.Because Smad7 (Y211A) useful effect is in the TGF beta receptor, we seek to determine whether described mutain still keeps the inhibition activity of the TGF beta pathway that higher level is expressed.In order to detect this point, we have compared Smad7 and Smad7 (Y211A) and have blocked relative effectiveness aspect the transduction of TGF signal by changing the Smad7 expression amount.Wild-type Smad7 is in the lowest dosage levels strongly inhibited TGF signal transduction that is detected, and the inhibition efficient of Smad7 (Y211A) much lower (Figure 18 C).Yet at the maximum dose level of being tested, Smad7 (Y211A) can suppress the TGF beta response.These presentation of results, Smad7 (Y211A) may prevent that in conjunction with described acceptor Smad2 or Smad3 from having kept certain inhibition activity in conjunction with the ability of described acceptor by it.Comprehensive these data declarations, Smurf2 and Smad7 synergy promote it to suppress active.

Discuss

This embodiment has confirmed to identify a kind of new ubiquitin ligase enzyme Smurf2, and it and Smad7 synergy make the degraded of TGF beta receptor.Smurf2 is very faint in conjunction with the TGF beta receptor, and can not influence the conversion of TGF beta receptor.Yet when having Smad7, Smurf2 and allos polymerization TGF beta receptor form effective mixture, cause the degraded of TGF beta receptor.Because Smad7 directly associates in conjunction with Smurf2 and with TGF beta receptor mixture, so these results show that Smad7 plays the proteic effect (Figure 18 D) that is connected of mediation Smurf2 and the effect of TGF beta receptor mixture.Consistent therewith is to have destroyed the interactional Smad7PY primitive sudden change of Smad7-Smurf2 and also disturbed Smad7 to raise the ability of Smurf2 to the mixture of described acceptor.The ability that this Smad7 mutant also makes it hinder the transduction of TGF signal is impaired, illustrates that Smurf2 has certain effect in mediation Smad7 restraining effect.Because Smad7 combines activation TGF beta receptor with the R-Smad competition, so this synergy particularly important (53-58) when Smad7 transient expression or low expression level.Therefore, Smurf2 provides the mechanism of permanent deactivation Smad7 bind receptor mixture by making the acceptor degraded.We also observe, in Smurf2 dependency degraded TGF beta receptor, and the dependent Smad7 degraded of transduceing of TGF signal.

* * *

The invention is not restricted to the specific embodiments scope that this paper introduces.In fact, except the specific embodiments that this paper introduces,, various improvement of the present invention be will be apparent to those skilled in the art according to above-mentioned introduction and accompanying drawing.Such improvement belongs to the category of appended claims.

Should be understood that further all base sizes or amino acid size and all molecular weight or molecular weight values are approximations, provide such approximation just in order to explanation the present invention.

Other data that all patents, patent application, public publication and this paper quote all by reference integral body be attached to herein.

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Claims (42)

1. isolating Smad ubiquitination regulatory factor Smurf polypeptide, it comprises with aminoacid sequence shown in the SEQ ID NO:2 the aminoacid sequence that surpasses 90% similarity.

2. the Smurf polypeptide of claim 1, it comprises the aminoacid sequence shown in the SEQ ID NO:2.

3. isolating Smurf polypeptide, it comprises the aminoacid sequence shown in the SEQ ID NO:4.

4. the Smurf polypeptide of claim 3, it comprises the sudden change corresponding to C716A.

5. the aminoacid sequence shown in isolating nucleic acid, its coding SEQ ID NO:2.

6. isolating nucleic acid, it comprises the nucleotide sequence that has 80% similarity with nucleotide sequence shown in the SEQ ID NO:1 at least, and described nucleic acid encoding has the albumen of Smurf polypeptide.

7. the nucleic acid of claim 6, it comprises the nucleotide sequence that has 85% similarity with nucleotide sequence shown in the SEQ ID NO:1 at least.

8. the nucleic acid of claim 7, it comprises the nucleotide sequence shown in the SEQ ID NO:1.

9. the aminoacid sequence shown in isolating nucleic acid, its coding SEQ ID NO:4.

10. the nucleic acid of claim 9, it comprises the nucleotide sequence shown in the SEQ ID NO:3.

11. the nucleic acid of claim 9, it comprises the sudden change corresponding to C716A.

12. a carrier, it comprises the nucleic acid of coding SEQ ID NO:2.

13. a host cell, it comprises the carrier of claim 12.

14. a carrier, it comprises the nucleic acid of coding SEQ ID NO:4.

15. a host cell, it comprises the carrier of claim 14.

16. a method that produces aminoacid sequence shown in the SEQ ID NO:2, this method comprise the host cell of aminoacid sequence shown in the culture expression SEQ ID NO:2.

17. a method that produces aminoacid sequence shown in the SEQ ID NO:4, this method comprise the host cell of aminoacid sequence shown in the culture expression SEQ ID NO:4.

18. the method for the bone morphogenetic protein activated pathway in the vitro inhibition cell, this method comprise the isolating nucleic acid of expressing coding SEQ ID NO:2.

19. the method for the bone morphogenetic protein activated pathway in the external promotion cell, this method comprise the endogenous expression that suppresses aminoacid sequence shown in the SEQ ID NO:2.

20. the bone morphogenetic protein in the vitro inhibition cell or the method for transforming growth factor-beta activated pathway, this method comprise the isolating nucleic acid of expressing coding SEQ ID NO:4.

21. the method for claim 20, this method is carried out on from the cell as the transgenic animal of research tool.

22. the bone morphogenetic protein in the external promotion cell or the method for transforming growth factor-beta activated pathway, this method comprise the endogenous expression that suppresses aminoacid sequence shown in the SEQ ID NO:4.

23. the method for claim 22, this method is carried out on from the cell as the transgenic animal of research tool.

24. the method for an in-vitro screening Smurf active regulator, Smurf activity when this method comprises detection and lacks test-compound is compared when having this test-compound the active regulating effect of Smurf, and the Smurf activity that is wherein detected is 90% similarity of surpassing to be arranged and have the activity of the active Smurf of Smurf with aminoacid sequence shown in the SEQ ID NO:2.

25. the method for claim 24, wherein said Smurf activity are the ubiquitinations of host cell Smad polypeptide.

26. the method for claim 24, wherein said Smurf activity are the interactions of the PPXY structural domain of Smurf WW structural domain and Smad polypeptide.

27. the method for claim 26 is wherein screened described test-compound and is suppressed described interactional ability.

28. the method for claim 24, wherein said Smurf activity are the ubiquitinations of transform growth factor-beta receptor.

29. the method for claim 24, the Smurf activity that is wherein detected is the activity that comprises the Smurf of aminoacid sequence shown in the SEQ IDNO:2.

30. the method for an in-vitro screening Smurf active regulator, Smurf activity when this method comprises detection and lacks test-compound is compared when having this test-compound the active regulating effect of Smurf, and the Smurf activity that is wherein detected is the activity that comprises the Smurf of aminoacid sequence shown in the SEQ ID NO:4.

31. the method for claim 30, wherein said Smurf activity are the ubiquitinations of Smad polypeptide.

32. the method for claim 30, wherein said Smurf activity are the interactions of the PPXY structural domain of Smurf WW structural domain and Smad polypeptide.

33. the method for claim 32, wherein said test-compound is according to described interactional inhibition ability is screened.

34. the method for claim 30, wherein said Smurf activity are the ubiquitinations of transform growth factor-beta receptor.

35. the method for an in-vitro screening Smurf active regulator, Smurf activity when this method comprises detection and lacks test-compound is compared when having this test-compound the active regulating effect of Smurf, wherein said Smurf activity is the ubiquitination of Smad polypeptide, and wherein said Smurf activity is the activity of polypeptide with aminoacid sequence of SEQ ID NO:2 or SEQ ID NO:4.

36. the method for claim 35, wherein said Smurf activity are the ubiquitination of Smad polypeptide in host cell, and wherein said Smurf activity is the activity of polypeptide with aminoacid sequence of SEQ IDNO:2 or SEQ ID NO:4.

37. the method for an in-vitro screening Smurf active regulator, Smurf activity when this method comprises detection and lacks test-compound is compared when having this test-compound the active regulating effect of Smurf, wherein said Smurf activity is the ubiquitination of transform growth factor-beta receptor, and wherein said Smurf activity is the activity of polypeptide with aminoacid sequence of SEQ ID NO:2 or SEQ ID NO:4.

38. the method for an in-vitro screening Smurf active regulator, Smurf activity when this method comprises detection and lacks test-compound is compared when having this test-compound the active regulating effect of Smurf, wherein said Smurf activity is the interaction of the PPXY structural domain of Smurf WW structural domain and Smad polypeptide, and wherein said Smurf activity is the activity of polypeptide with aminoacid sequence of SEQ ID NO:2 or SEQ ID NO:4.

39. the method for claim 38, wherein said test-compound is according to described interactional inhibition ability is screened.

40. claim 24,30,31,37 or 38 method, wherein said screening assay is carried out in host cell.

41. an antibody, it is in conjunction with the aminoacid sequence shown in the SEQ ID NO:2.

42. an antibody, it is in conjunction with the aminoacid sequence shown in the SEQ ID NO:4.

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