EP1192174A2 - Antagonists of bmp and tgf beta signalling pathways - Google Patents
Antagonists of bmp and tgf beta signalling pathwaysInfo
- Publication number
- EP1192174A2 EP1192174A2 EP00941398A EP00941398A EP1192174A2 EP 1192174 A2 EP1192174 A2 EP 1192174A2 EP 00941398 A EP00941398 A EP 00941398A EP 00941398 A EP00941398 A EP 00941398A EP 1192174 A2 EP1192174 A2 EP 1192174A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- smurfl
- smurf
- protein
- smad7
- expression
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
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Classifications
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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- A—HUMAN NECESSITIES
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A—HUMAN NECESSITIES
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- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
Definitions
- TGF ⁇ transforming growth factor ⁇
- TGF ⁇ transforming growth factor ⁇
- a variety of TGF ⁇ signals affect germ layer specification, body patterning, cell growth and differentiation (1- 4).
- distinct TGF ⁇ members induce different cell fates, e.g., activin, Vgl and nodal induce mesoderm characteristic of the dorsal part of the embryo, such as notochord and muscle.
- BMPs Bone Mo ⁇ hogenetic Proteins
- Cells respond to ligands in the TGF ⁇ family by transducing signals directly from cell surface receptor complexes to nuclear DNA targets via the Smad family of proteins (see (4), (6), (7), and (52) for a review).
- Smads are related to Drosophila Mad (mothers against decapentaplegic [dpp]) and proteins encoded by three related nematode genes Sma 2, Sma 3, and Sma 4.
- the terms Sma and Mad have been fused as Smad to unify the nomenclature.
- Phosphorylated Smads 1 , 5 and 8 are functional mediators of BMP family signaling in partnership with Smad 4.
- Smads 2 and 3 are signal transducers for actions of TGF ⁇ and activins.
- Smad 6 and Smad 7 function as antagonists to inhibit TGF ⁇ /BMP superfamily signaling.
- Smad7 is localized in the nucleus and accumulates in the cytoplasm in response to TGF ⁇ signalling (73).
- Smad ⁇ and Smad7 are regulated by TGFbs, BMPs, growth factors and cytokines thereby providing for negative feedback regulation of the Smad signalling pathway (53-58).
- Phosphorylated Smad 1 forms a heteromeric complex with Smad 4 when entering the nucleus and activates transcription of early response genes.
- the BMP receptors may also signal via the mitogen activated protein kinase. It is likely that BMPs regulate cell cycle progression and thus govern differentiation of mesenchymal stem cells.
- BMP and activin/TGF ⁇ Two distinct receptor subunits, the Type I and II transmembrane serine/threonine kinases, form activated complexes upon ligand binding.
- the Type II subunit activates the Type I subunit, which directly phosphorylates and activates particular receptor-regulated R-Smad proteins: BMP receptors target Smadl and closely-related Smads 5 and 8, while activin and TGF ⁇ receptors target Smadl and closely-related Smads 2 and 3.
- BMP receptors target Smadl and closely-related Smads 5 and 8
- activin and TGF ⁇ receptors target Smadl and closely-related Smads 2 and 3.
- Upon activation these R-Smads form a heteromeric complex with Smad4, the "common partner" Smad.
- I-Smads A third class of inhibitory Smads (I-Smads), Smad ⁇ and Smad7, function as inhibitors that block Smad-Smad complex formation or Smad-receptor interactions.
- I-Smads bind to the cytoplasmic domain of receptors or directly to Smadl . Mutations in components at all levels in this pathway are associated with embryonic defects and various cancers, underscoring the importance of this growth factor family in developmental and disease processes. (See, (4), (7) for reviews.) In particular, defects of Smad2 and Smad4 are associated with colon and lung cancer and defects in human Smad4 are associated with pancreatic cancer.
- Smads do not have intrinsic enzymatic activity.
- the nature of the cellular response to Smad signaling is extraordinarly sensitive to the level of Smad protein in the cell.
- alternative cell fate determinations in Xenopus embryos can be achieved by relatively small changes in the amount of Smad protein expressed in the cell (8-11). Therefore, regulating the level of Smad protein in the cell can be used as one means of modulating morphogenetic signaling by the TGF ⁇ superfamily.
- Protein modification by covalent attachment of ubiquitin is recognized as a general signal to target proteins for degradation via the proteasome (see (12), and (13) for a review).
- Targets of selective ubiquitination include transcription factors, cell cycle regulators, signal transduction proteins, and membrane proteins (references in (12)).
- Selective ubiquitination and degradation of specific target proteins can function as an important mechanism to control cell cycle progression, programmed cell death, differentiation and embryonic development. Dysfunction of the ubiquitination pathway is associated with disease and abnormal development.
- Ubiquitin ligases are part of a multimeric complex that catalyzes the covalent attachment of ubiquitin, a 12.5 kD polypeptide, to target proteins.
- Attachment of ubiquitin to its target serves as a molecular "flag" that marks the ubiquitinated protein for proteolytic degradation via an organelle known as the 26S proteosome.
- the El enzyme activates a ubiquitin molecule and conjugates it to the E2 enzyme which then either directly attaches ubiquitin onto a target protein, or passes it to the E3 ubiquitin ligase.
- the E3 recognizes a particular substrate and directs it ubiquitination.
- ubiquitination system A few examples of developmental regulation by the ubiquitination system have been described in Dictyostelium (14-16) and Drosophila (17-21). Conjugation of ubiquitin to receptors is used in diverse systems to control endocytosis and signalling, as well as receptor steady state levels by both proteasome- and lysosome-mediated degradation (59-60). Direct ubiquitination of membrane receptors has been characterized in a number of systems, although in some cases ubiquitin-dependent regulation does not appear to involve direct conjugation of ubiquitin to the receptor (61-62). Although many cell surface receptors are regulated by ubiquitin-dependent pathways, few E3 ubiquitin ligases that bind to membrane proteins and target them for ubiquitination have been defined.
- Nedd4 a C2-WW-HECT domain E3 ubiquitin ligase, can regulate the turnover of the amiloride-sensitive sodium channel by binding directly to a PPXY motif present in the carboxy-terminus of the channel (63-66).
- the RING finger protein, c-cbl has recently been shown to function as an E3 ubiquitin ligase that binds to the EGF receptor to mediate ubiquitination and downregulation of the receptor complex (67-68).
- ubiquitination of the membrane proteins appears to involve direct interactions between the E3 ligase and the target protein. Whether adaptor proteins might also function to recruit E3 ligases to specific receptor complexes is unknown.
- the mechanism and targets of ubiquitination in the control of patterning have heretofore remained elusive. References that are cited throughout the specification by number are listed at the end of the Example. All references cited herein are inco ⁇ orated by reference.
- the present invention advantageously provides a class of regulatory proteins that are involved in BMP and TGF ⁇ -mediated activation.
- these proteins regulate Smad proteins and/or promote degradation of TGF ⁇ receptor complexes in the presence of Smad proteins.
- the skilled artisan can up or down regulate cellular activation, e.g., via BMP or TGF ⁇ .
- the invention provides an isolated Smurf protein, and particularly a human Smurf protein.
- it is a Smurfl protein.
- it is a SmurfZ protein.
- a human Smurfl has the amino acid sequence depicted in Figure 10 (SEQ ID NO:2)
- a human Smurf2 has the amino acid sequence depicted in Figure 12 (SEQ ID NO:4).
- Smurf proteins of the invention may contain at least about 5 and preferably at least about 10 contiguous amino acids from the sequences depicted in SEQ ID NO:2 and 4.
- the invention further provides an antibody that specifically binds to Smurf protein.
- the invention further provides a nucleic acid encoding the Smurf proteins of the invention.
- the nucleic acid has a nucleotide sequence as depicted in SEQ ID NO:l or SEQ ID NO:3.
- the invention further provides an oligonucleotide or nucleic acid that specifically hybridizes under highly stringent conditions to a nucleic acid having a sequence encoding Smurf, or the complementary sequence thereof.
- hybridizable nucleic acids include probes (i.e., they may be labeled), primers (e.g., for PCR amplification), anti-sense nucleic acids, ribozymes, and triple-helix forming nucleic acids.
- the invention further provides a vector comprising the nucleic acid encoding Smurf, e.g., under control of an expression control sequence.
- host cells harboring such a vector, and methods for producing Smurf by culturing such host cells under conditions that permit expression of Smurf protein from the vector.
- a transgenic non-human animal that expresses a human Smurf protein and non-human animals in which endogenous Smurf protein is deleted.
- the invention further provides a method for inhibiting a bone mo ⁇ hogenic protein or transforming growth factor-beta activation pathway in a cell.
- This method comprises permitting the cell to grow under conditions that permit expression of Smurf from a vector of introduced into the cell.
- the invention provides a method for promoting a bone mo ⁇ hogenic protein or transforming growth factor-beta activation pathway in a cell, which method comprises suppressing expression of endogenous Smurf in the cell.
- the present discoveries permit screening for a modulator of Smurf activity.
- Screens of the invention comprise detecting modulation of Smurf activity in the host cell in the presence of a test compound relative to Smurf activity of the host cell in the absence of the test compound. As shown in the examples, one such activity is ubiquitination of Smad proteins. Another activity is the enhancement of TGF ⁇ receptor degradation.
- Smurfl encodes an E3 ubiquitin ligase. Protein sequence of Xenopus and human Smurfl compared to yeast (S. pombe) publ, given as SMURFl, hSMURFl and PUB1, respectively in the figure. Identical amino acids are shaded dark gray and conservative substitutions are shaded light gray.
- Smurfl and publ are members of the Hect family of E3 ubiquitin ligases and display several conserved features of the family: A Iipid/Ca2+ binding domain is located at the N-terminus (residues 22-37), two WW protein interaction domains at 236 - 271 and 282 -311 (indicated by thick lines) , and a catalytic Hect domain beginning at residue 347 and extending to the C-terminus. Alignment was by Clustal W analysis (Mac Vector).
- FIGS 2A and 2B Northern blots ofmSmurfl expression in embryonic and adult mouse tissues . Equal amounts of Poly A+ mRNA from indicated stages and tissues were analyzed.
- (2B) Adult tissues shown are T, testes; K, kidney; M, skeletal muscle; L, lung; Sp, spleen; Br, brain; H, heart.
- FIGS 3A and 3B Developmental expression of Xenopus Smurfl.
- A RT-PCR on staged embryonic cDNA revealed that Xenopus Smurfl is a maternal mRNA, present at highest levels in egg, blastula and early gastrula stages.
- Zygotic Smurfl mRNA levels decline at gastrulation but maintain steady expression into swimming tadpole stages. Numbers above each lane correspond to Nieuwkoop and Faber stages: 7 and 9, blastula; 1 1 and 13, gastrula; 15 and 20, neurula; 25 and 35, tadpole.
- ODC Ornithine decarboxylase
- Smurfl transcripts are localized to the animal pole half (bracket in egg). Expression is diffuse throughout the ectoderm and involuting marginal zone of the gastrula; views from , animal pole (an) and vegetal pole (Veg). There is some enrichment of transcripts in the neural folds at neurula stage 17.
- Smurfl expression includes the brain (b), eye (e), otic vesicle (o), somites (s), pharyngeal pouches (p) and developing kidney (k).
- FIGS. 4A, 4B, 4C, and 4D Smurfl leads to a selective decrease in steady-state level of Smadl and SmadS in mammalian cell lines.
- Cells were transiently transfected with the indicated expression vectors (DNA quantities in ⁇ g) and two days later an aliquot representing approximately 0.4% of total cell lysates were subjected to SDS-PAGE and immunoblotting.
- the blots were probed with the appropriate Smad antibody as shown.
- Flag-hSmurfl expression level was confirmed by probing blots of total cell lysates with the anti-Flag monoclonal antibody.
- COS-1 or 293T-cells were transfected with a constant amount of pCMV5-Smadl and increasing concentrations of pCMV5-Flag-hSmurfl as indicated.
- Smadl steady- state level and the expression of hSmurfl Western blots of total cell lysates from both cell lines were probed with ⁇ -Smadl and ⁇ -Flag antibodies ( ⁇ -Smadl and ⁇ -Flag blot).
- B 293T cells were transiently transfected with pCMV5-Smadl, wild type or activated (Q203D) pCMV5-ALK6-HA, and increasing amounts of pCMV5-Flag-hSmurfl as indicated.
- Smadl steady-state levels were examined by immunoblotting total cell lysates in Western blots with ⁇ -Smadl antibody ( ⁇ -Smadl).
- hSmurfl and wild type or activated ALK6 expression levels were determined by immunoblotting the total cell lysates with ⁇ -Flag ( ⁇ -Flag) or ⁇ -HA ( ⁇ -HA) antibody, respectively.
- (C) 293T cells were transfected with a constant amount of pCMV5-Smadl or pCMV5-Smad2 along with the indicated concentrations of pCMV5-Flag-hSmurfl .
- Smadl ( ⁇ -Smadl blot), Smad2 ( ⁇ -Smad2 blot) and Flag-hSmurfl ( ⁇ -Flag blot) steady-state protein levels were determined in cell lysates, as above.
- (D) 293T cells were transfected with Smadl, Smad3, Smad4 or Smad5 in pCMV5 in the absence or presence of pCMV5-Flag-hSmurfl .
- the steady-state level of the Smads in total cell lysates was determined by incubating the Western blots with ⁇ -Smadl antibody for Smadl and Smad5, ⁇ -Smad3 antibody for Smad3, and ⁇ -Smad4 antibody for Smad4 detection ( ⁇ -Smads blot). Equivalent hSmurfl expression was confirmed as described above ( ⁇ -Flag blot).
- COS-1 cells were transiently transfected with pCMV5-Smadl alone or with Flag-tagged hSmurfl (F/hSmurfl) using lipofectAMINE. Two days later, transfectants were subjected to pulse-chase analysis using [ 35 S]methionine. At the indicated time during the chase, cells were lysed and subjected to an ⁇ -Smadl immunoprecipitation. The immunoprecipitates were resolved by SDS-PAGE and visualized by autoradiography (left panel). Radiolabelled Smadl was also quantitated by phosphoimaging, and the results are plotted as the amount of [ 35 S] methionine-labelled Smadl present at each time point relative to the level at time 0 (right panel).
- Smadl or Flag-hSmurfl was confirmed by subjecting total cell lysates to immunoblotting with an ⁇ -Smadl polyclonal antibody ( ⁇ -Smadl blot) or an ⁇ -Flag monoclonal antibody ( ⁇ -Flag blot), respectively.
- Yeast two-hybrid assays were done on yeast co-transformed with combinations of Xenopus Smurfl (Xsmurf) together with Smadl, Smad2, lamin or vector alone. Only the combination of Smadl and Smurfl exhibited significant ⁇ -galactosidase activity (left panel, photograph of stained yeast colonies assayed in duplicate).
- 293T cells were transiently transfected with the pCMV5 expression vectors containing Smadl (SI), Smad5 (S5) or Smad2 either alone, with wild type (WT) or the ubiquitin ligase mutant (CA) Flag-tagged hSmurfl (F/hSmurfl).
- Smadl or Smad5 interaction with hSmurfl blots of ⁇ -Flag immunoprecipitates were probed with ⁇ -Smadl polyclonal antibody ( ⁇ -Smadl blot).
- ⁇ -Smad2 blot To examine Smad2 interaction with hSmurfl , blots of ⁇ -Flag immunoprecipitates were probed with ⁇ -Smad2 polyclonal antibody ( ⁇ -Smad2 blot). The level of hSmurfl in the immunoprecipitates and Smadl, Smad5 and Smad2 in the total cell lysates, are shown in the lower two panels.
- Nedd4 a ubiquitin ligase related to hSmurfl , does not interact with and does not reduce the steady-state level of Smadl .
- 293T cells were transiently co-transfected with Smadl and either Flag-tagged hSmurfl (WT or CA) or Nedd4, (WT) or ligase mutant (CS) as indicated.
- Smadl coimmunprecipitation with hSmurfl (left panels) was determined as described above.
- VMZ ventral marginal zone
- FIGS 8A, 8B, and 8C Smurfl alters embryonic cell competence to respond to Smadl and Smad2.
- Smurfl blocks ventral mesoderm induction by Smadl.
- a constant amount (1 ng) of Smadl mRNA was injected alone, or together with an increasing amount of Smurfl . into fertilized egg animal poles and ventral mesoderm induction in animal cap explants was assayed by RT-PCR with primers for the Xhox3 and Xcad3 homeodomain genes.
- animal caps were not injected or injected with Smurfl mRNA at doses of 100, 0, 25, 50, 100, and 200 pg.
- Smurfl enhances dorsal mesoderm induction by Smad2.
- a constant amount (50 pg) of Smad2 mRNA was injected alone, or together with an increasing amount of Smurfl, and dorsal mesoderm induction was monitored by expression of myoD, which marks muscle, and goosecoid, a homeodomain gene expressed in the most dorsal type of mesoderm, the Spemann Organizer. Note that goosecoid expression was triggered from undetectable levels, as the dose of Smurfl was increased. Animal caps were injected as in panel a.
- Figure 10 Protein sequence of human Smurfl [SEQ ID NO:2].
- Figure 11 cDNA sequence of human Smurf2 [SEQ ID NO: 3].
- FIG. 12 Protein sequence of human Smurfl [SEQ ID NO: 4].
- SmurfZ is member of the HECT E3 ubiquitin-ligase family.
- the C2 (overline), WW (shaded) and HECT (double overline) domains and the Cys716Ala mutation (boxed) are shown.
- FIG. 13 Comparison of Smurfl and Smurfl proteins . Schematic representation of Smurfl and SmurfZ. The degree of amino acid identity (%) between the C2, WW and HECT domains is shown.
- FIGS 14A and 14B Smurfl is expressed in mouse tissues.
- FIGS. 15A, 15B, 15C, 15D, 15E and 15F Smurfl interacts with Smadl.
- a and B Expression of SmurfZ does not decrease steady-state levels of the Smads.
- 293T cells were transfected with Flag-tagged Smadl, Smad2, Smad4 or HA- tagged Smad7 either alone or together with the Myc -tagged Smurf2.
- Aliquots of total cell lysates were immunoblotted to detect expression of SmurfZ and the Smads (Fig. 15 A) or were subjected to immunoprecipitation with anti-Myc antibody followed by anti-Flag or anti-HA immunoblotting to detect any coprecipitating Smads (Fig. 15B).
- the migration of the anti-Myc heavy chain (IgH) is marked.
- the PY motif in Smad7 is an important determinant for mediating interaction with SmurfZ.
- 293T cells were transfected with Flag-SmurfZ either alone or together with wild type (WT) or mutant Y211A (YA) or ⁇ PYversions of Smad7-HA.
- Cell lysates were subjected to anti-Flag immunoprecipitation and coprecipitating Smad7 proteins were detected by immunoblotting with anti-Smad7 antibody. Smad7 expression was confirmed by immunoblotting aliquots of total cell lysates (bottom panel).
- Cells were affinity-labelled with [ 12 T]TGF ⁇ and lysates immunoprecipitated with anti-Flag or anti-Smad7 antibodies. Coprecipitating receptor complexes were visualized by SDS- PAGE and autoradiography. The amount of coprecipitating T ⁇ RI was quantified by phosphorimaging (right panels). Receptor expression was confirmed by visualizing aliquots of total cell lysates by autoradiography. Smurf2 and Smad7 levels were confirmed by immunoblotting aliquots of total cell lysates with anti-Flag and anti-HA antibodies, respectively.
- FIGS 17A, 17B, 17C, 17D and 17E Figures 17A, 17B, 17C, 17D and 17E. Smurfl induces degradation of TGF ⁇ receptors and Smadl.
- C Smurfl increases the turnover rate of the receptor complex.
- COS-1 cells transfected with TGF ⁇ receptors (T ⁇ RII-HA and T ⁇ RI-HA) alone or together with Smad7-HA, Flag-SmurfZ or both were pulse-labelled with [ 35 S] -methionine and then chased for the indicated times in media containing unlabelled methionine.
- Cell lysates were subjected to anti-HA immunoprecipitation and the amount of labelled receptors and Smad7 was quantified by phosphorimaging and is plotted relative to the amount present at time 0.
- 293T cells were transfected with HA-tagged ubiquitin together with various combinations of Smad7, T ⁇ RII, T ⁇ RI-Flag, and wild type (WT) or mutant (C716A) Myc- SmurfZ as indicated.
- Cell lysates were subjected to a double immunoprecipitation with anti-Smad7 antibodies followed by immunoblotting with anti-HA antibodies. Protein expression in aliquots of total cell lysates was confirmed by immunblotting.
- FIGS. 18A, 18B, 18C and 18D Association of Smurfl with Smadl enhances the Smad? inhibitory activity.
- COS-1 cells were transfected with TGF ⁇ receptors (T ⁇ RII and T ⁇ RI-HA) and either wild type (WT) or mutant ( Y211 A) Smad7/HA in the absence or presence of Flag-SmurfZ(C716A).
- Cells were affinity-labelled with [ 125 I]TGF ⁇ and lysates immunoprecipitated with anti-Smad7 or anti-Flag antibodies. Coprecipitating receptor complexes were visualized by SDS-PAGE and autoradiography. Total receptor expression was determined by autoradiography and Smad7 and SmurfZ protein levels were confirmed by anti-HA or anti-Flag immunoblotting of aliquots of total cell lysates.
- Smad7(Y211A) is not as effective as wild type Smad? in inhibiting TGFb-dependent activation of transcription.
- HepG2 cells were transfected with the 3TP-Lux reporter alone or together with varying concentrations of wild type (WT) or mutant (Y21 1A) Smad7-HA.
- WT wild type
- Y21 1A mutant Smad7-HA.
- 0.3 ng/ml of each Smad7 plasmid was used.
- Cells were incubated in the presence or absence of TGF ⁇ 1, and luciferase activity was normalized to b-galactosidase activity and is plotted as the mean +/- SD of triplicates from representative experiments.
- the present invention is directed to a family of genes encoding E3 ubiquitin ligases, called Smurf proteins, and includes full length, or naturally occurring forms, and any functionally active or antigenic fragments thereof, from any animal, particularly mammal or amphibian, and more particularly from a human source.
- E3 ubiquitin ligases called Smurfl and SmurfZ are characterized.
- This invention is based, in part, on the su ⁇ rising discovery that two novel members of the Hect family of ubiquitin ligases interact with Smads. These ligases have been named Smurfl and Smurf2. One of these, Smurfl, specifically targets BMP pathway-specific Smads, thereby acting as an antagonist or negative regulator of BMP signaling. Smurfl directly interacts with Smadsl and 5 and regulates their ubiquitination, turnover, and activity. In amphibian embryos, Smurfl inhibits endogenous BMP signals, resulting in altered pattern formation and cell fate in the mesoderm and ectoderm.
- the invention provides a unique regulatory protein link between the ubiquitination pathway and the control of cell fate, e.g., during embryonic development.
- the invention further provides a nucleic acid encoding Smurfl [SEQ ID NO: 1], in addition to the Smurfl protein, [SEQ ID NO: 2], and mutant variants thereof.
- Smurfl SEQ ID NO: 1
- SEQ ID NO: 2 SEQ ID NO: 2
- SmurfZ a C2-WW-HECT domain E3 ubiquitin ligase is related to Smurfl .
- SmurfZ does not interact with Smadl, 2 or 4, nor does SmurfZ alter steady state levels of Smad 1, 2 or 4.
- SmurfZ does interact with Smad 7, binding directly to a PPXY motif in Smad7.
- Smurf is involved with TGF ⁇ receptor degradation acting in partnership with Smad7 as an antagonist or negative regulator of TGF ⁇ signaling. Activation of TGF ⁇ signalling results in Smad7-dependent recruitment of SmurfZ to the TGF ⁇ receptor complex. In the absence of activated TGF ⁇ receptor complex, SmurfZ does not alter the steady state level and turnover of Smad7.
- Recruitment of Smurf2 to the TGF ⁇ receptor by Smad7 promotes the degradation of the Smad7- TGF ⁇ receptor complex by both proteasomal and lysosomal pathways.
- Smad7 functions as an adaptor protein that recruits SmurfZ to the TGF ⁇ receptor complex to promote its degradation and thereby down-regulate activated TGF ⁇ receptor complexes. Regulation of Smad 7 localization to the nucleus and interaction with SmurfZ may be used to control the inhibitory activity of the Smad 7-Smurf2 complex.
- the invention further provides a nucleic acid encoding SmurfZ [SEQ ID NO: 3], in addition to the Smurf 2 protein [SEQ ID NO: 4] and variants thereof.
- E3 ubiquitin ligases display very selective substrate specificity, as is evident in the findings discussed herein.
- Smurfl binds BMP pathway-specific Smads.
- Smurfl is a unique signaling protein of the BMP pathway because it binds only to Smadl and Smad5 and has little affinity for Smad 2 (specific for TGF ⁇ and activin receptor pathways), and no affinity for Smad4 (common Smad signaling partner).
- Smurfl can effectively interfere or regulate biological responses to BMPs without consequence to activin pathways, i.e., effects on other TGF ⁇ signaling pathways are limited, or non-existent.
- BMPs/TGF ⁇ signalling pathway functions in tissue differentiation, mo ⁇ hogenesis, and cell growth control (e.g., (52)).
- Smurfl As an antagonist to the signal transduction pathway mediated by the TGF ⁇ family, Smurfl will inhibit the BMP pathway in vivo or in vitro.
- Smurf As an integral component of regulatory system for degradation of TGF ⁇ receptors, Smurf will inhibit the TGF ⁇ signalling pathway.
- the Smurfs will block chondrogenesis, osteogenesis, blood differentiation, cartilage formation, neural tube patterning, retinal development, heart induction and mo ⁇ hogenesis, hair growth, tooth formation, gamete formation and a wide variety of tissue and organ formation processes, and hinder the regeneration, growth, maintenance, etc., of bone and other tissues that are dependent on the BMP pathway.
- mutant forms of a Smurf protein or small molecule antagonists of Smurf can be used to prevent ubiquitination of proteins, e.g., Smads or TGF ⁇ receptor, and therefore to preserve the signal transduction pathway mediated by BMPs.
- fragments of Smads can be generated that bind to either the Hect domain of a Smurf protein, which domain has the catalytic activity needed for ubiquitination of Smads, or to the WW domains that interact with the PPXY domains of Smads, thus precluding Smurfl from binding and ubiquitinating Smads.
- fragments can also be used in screening assays for small molecule inhibitors of Smurf-Smad interaction, e.g., in an inhibition binding assay.
- Variants of a Smurf protein and fragments of Smads can be used to improve defective BMP and/or TGF ⁇ /activin signaling as a result of overexpressed or increased Smurf activity, which may contribute to a disease state.
- Smurf proteins represent a novel target for the discovery of drugs that can influence its function, thereby affecting cellular responses to BMPs, and thus having clinical applications. Therefore, a Smurf protein can be used for screening for various drugs and/or antibodies that can either enhance the BMP pathway, or inhibit it by antagonizing or mimicking the activity of the protein, respectively. For example, since Smurfl is highly specific for binding Smadsl and 5, it can be used to screen for drugs that block or activate the BMP pathway, and selectively affect cellular responses to BMPs without consequence to other members of the TGF ⁇ superfamily.
- Smurf proteins operate within cells, at the level of Smad signal transduction, and therefore provide an alternative means to affect BMP and TGF ⁇ /activin signals.
- Smurf proteins are intracellular proteins, any manipulations aimed at directly altering Smurf activity must operate intracellularly. Such manipulations include antisense and ribozyme technology, and intracellular antibody technology.
- Smurf may be delivered to cells in gene therapy regimens to block excessive signaling by particular growth factor pathways controlled by Smads, e.g., Smads that are targets for Smurfl or Smurf2.
- Smads e.g., Smads that are targets for Smurfl or Smurf2.
- Smurfs by gene therapy may be used to correct clinical conditions that result from excessive Smad signaling.
- These may include, for example, hype ⁇ lasia of bone, tendon or cartilage tissues, or formation of other tissues, that are regulated by signals from receptors (such as BMP receptors) that utilize Smadl or Smad5 for signal transduction.
- Smurf nucleic acids or partial sequences thereof would be useful as molecular probes for identification of defective Smurf genes in the human genome, particularly where a mutation of a Smurf gene is found in association with a particular disease.
- Smurf proteins may be used as reagents for in vitro assays to identify proteins in cells that are targets for ubiquitination.
- Purified Smurfs may be reconstituted with purified ubiquitination enzymes (i.e. El and E2 components) and utilized in functional (ubiquitination) assays that are aimed at identifying novel target proteins introduced into the assays (as purified proteins or translated cDNAs of unknown identity).
- purified ubiquitination enzymes i.e. El and E2 components
- functional (ubiquitination) assays that are aimed at identifying novel target proteins introduced into the assays (as purified proteins or translated cDNAs of unknown identity).
- an isolated nucleic acid includes a PCR product, an isolated mRNA, a cDNA, or a restriction fragment.
- an isolated nucleic acid is preferably excised from the chromosome in which it may be found, and more preferably is no longer joined to non-regulatory, non-coding regions, or to other genes, located upstream or downstream of the gene contained by the isolated nucleic acid molecule when found in the chromosome.
- the isolated nucleic acid lacks one or more introns.
- Isolated nucleic acid molecules can be inserted into plasmids, cosmids, artificial chromosomes, and the like.
- a recombinant nucleic acid is an isolated nucleic acid.
- An isolated protein may be associated with other proteins or nucleic acids, or both, with which it associates in the cell, or with cellular membranes if it is a membrane-associated protein.
- An isolated organelle, cell, or tissue is removed from the anatomical site in which it is found in an organism.
- An isolated material may be, but need not be, purified.
- purified refers to material that has been isolated under conditions that reduce or eliminate unrelated materials, i.e., contaminants.
- a purified protein is preferably substantially free of other proteins or nucleic acids with which it is associated in a cell; a purified nucleic acid molecule is preferably substantially free of proteins or other unrelated nucleic acid molecules with which it can be found within a cell.
- substantially free is used operationally, in the context of analytical testing of the material.
- purified material substantially free of contaminants is at least 50% pure; more preferably, at least 90% pure, and more preferably still at least 99% pure.
- a “gene” is used herein to refer to a portion of a DNA molecule that includes a polypeptide coding sequence operatively associated with expression control
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CA2531226A1 (en) * | 2003-07-01 | 2005-01-13 | Vib Vzw | Ubiquitinated tnf receptor 2 and its uses |
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JP2009529699A (en) | 2006-03-01 | 2009-08-20 | ソフトマックス,インコーポレイテッド | System and method for generating separated signals |
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US20100183514A1 (en) | 2007-05-29 | 2010-07-22 | President And Fellows Of Harvard College | Molecules involved in regulation of osteoblast activity and osteoclast activity, and methods of use thereof |
WO2011088163A1 (en) | 2010-01-14 | 2011-07-21 | President And Fellows Of Harvard College | Methods for modulating skeletal remodeling and patterning by modulating shn2 activity, shn3 activity, or shn2 and shn3 activity in combination |
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US9403810B2 (en) * | 2014-05-14 | 2016-08-02 | Novartis Ag | Carboxamide derivatives |
KR102360028B1 (en) * | 2014-12-26 | 2022-02-08 | 삼성전자주식회사 | Composition for reducing senescence of a cell or a subject comprising Smurf2 inhibitor and use thereof |
CN110196326A (en) * | 2019-06-05 | 2019-09-03 | 武汉合研生物医药科技有限公司 | A kind of TGF β R1(T204D) enzymatic activity rapid detection method and its application |
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