Methods for modulating signal transduction mediated by TGF-beta related proteins

Abstract

Methods are provided for identifying agents that modulate signaling mediated by transforming growth factor beta (TGF-β) and members of the TGF-β family, such as bone morphogenic protein (BMP). Such agents may be identified using screens that evaluate candidate agents for the ability to modulate Smad protein degradation. Agents identified as described herein may be used to augment or inhibit signaling mediated by one or more TGF-β family members in a variety of cell types and for therapeutic purposes.

1 . A method for screening for an agent that modulates TGF-β- and/or BMP-mediated signaling, comprising the steps of: 
 (a) contacting 
 (i) a first polypeptide comprising a HECT E3 ubiquitin ligase WW domain, or a variant thereof in which the ability of the polypeptide to bind to a Smad protein is not substantially diminished relative to the HECT E3 ubiquitin ligase;  
 (ii) a second polypeptide comprising a Smad PY motif, or a variant thereof in which the ability of the polypeptide to bind to an E3 ubiquitin ligase is not substantially diminished relative to a native Smad protein comprising the PY motif; and  
 (iii) a candidate agent; under conditions that permit a detectable level of binding of the first polypeptide to the second polypeptide in the absence of candidate agent;  
 
 (b) determining a level of binding of the first polypeptide to the second polypeptide; and  
 (c) comparing the level of binding to a control level of binding of the first polypeptide to the second polypeptide in the absence of candidate agent, and therefrom determining whether the candidate agent modulates TGF-β- and/or BMP-mediated signaling.  
 
     
     
         2 . A method according to  claim 1 , wherein the HECT E3 ubiquitin ligase WW domain comprises the sequence 
 GPLPXGWEX 3 tttGtXYYhXHNTtTTtWXtPt (SEQ ID NO:2)    wherein each t is an independently selected polar amino acid residue (e.g., S, H, P, D, E, T or Y), h is a hydrophobic residue (e.g., I, V, L or M) and each X is an independently selected amino acid residue.    
     
     
         3 . A method according to  claim 1 , wherein the Smad PY motif comprises the sequence Ser/Thr-Pro-Pro-Pro-Pro/Ala/Gly-Tyr (SEQ ID NO:15), wherein Ser/Thr is an amino acid residue that is serine or threonine and Pro/Ala/Gly is an amino acid residue that is selected from the group consisting of proline, alanine and glycine.  
     
     
         4 . A method according to  claim 3 , wherein the Smad PY motif comprises the sequence TPPPAY (SEQ ID NO:16) or TPPPGY (SEQ ID NO:18).  
     
     
         5 . A method according to  claim 1 , wherein the candidate agent is a small molecule within a combinatorial library.  
     
     
         6 . A method according to  claim 1 , wherein the first polypeptide is immobilized on a solid support and the second polypeptide comprises a tag.  
     
     
         7 . A method according to  claim 1 , wherein the second polypeptide is immobilized on a solid support and the first polypeptide comprises a tag.  
     
     
         8 . A method according to  claim 6  or  claim 7 , wherein the tag is biotin or a radioactive group.  
     
     
         9 . A method according to  claim 1 , wherein the level of binding is determined via a two-antibody sandwich assay.  
     
     
         10 . A method according to  claim 1 , wherein the level of binding is determined via a competitive assay.  
     
     
         11 . A method for screening for an agent that modulates TGF-β- and/or BMP-mediated signaling, comprising the steps of: 
 (a) contacting 
 (i) a candidate agent;  
 (ii) a ubiquitinated HECT E3 ubiquitin ligase; and  
 (iii) a Smad protein or a variant thereof that comprises a PY motif; wherein the contact takes place under conditions and for a time sufficient to permit ubiquitination of the Smad protein or variant thereof by the HECT E3 ubiquitin ligase in the absence of candidate agent;  
 
 (b) determining a level of ubiquitination of the Smad protein or variant thereof; and  
 (c) comparing the level of ubiquitination to a control level of ubiquitination in the absence of candidate agent, and therefrom determining whether the candidate agent modulates TGF-β- and/or BMP-mediated signaling.  
 
     
     
         12 . A method according to  claim 11 , wherein the method comprises a coupled ubiquitination assay.  
     
     
         13 . A method according to  claim 11 , wherein the ubiquitinated HECT E3 ubiquitin ligase is present within a cell extract fraction.  
     
     
         14 . A method according to  claim 11 , wherein the level of ubiquitination is determined by Western blot analysis.  
     
     
         15 . A method according to  claim 11 , wherein the Smad protein or variant thereof comprises a tag.  
     
     
         16 . A method for screening for an agent that modulates BMP-mediated signaling, comprising the steps of: 
 (a) contacting a cell that expresses a BMP receptor with a bone morphogenic protein and a candidate agent; and    (b) detecting a level of a Smad protein in the cell, relative to a level of the Smad protein in a cell that is contacted with the bone morphogenic protein in the absence of the candidate agent, and therefrom determining whether the candidate agent is a modulator of BMP-mediated signaling.    
     
     
         17 . A method according to  claim 16 , wherein the Smad protein is Smad1 or Smad5.  
     
     
         18 . A method according to  claim 16 , wherein the cell is a bone cell.  
     
     
         19 . A method according to  claim 16 , wherein the cell is a neuron or kidney cell.  
     
     
         20 . A method according to  claim 16 , wherein the agent enhances BMP-mediated signaling.  
     
     
         21 . A method for screening for an agent that modulates BMP-mediated signaling, comprising the steps of 
 (a) contacting a cell that expresses a BMP receptor with a bone morphogenic protein and a candidate agent; and    (b) detecting a level of ubiquitination of a Smad protein in the cell, relative to a level of the Smad protein ubiquitination in a cell that is contacted with the bone morphogenic protein but is not contacted with the candidate agent, and therefrom determining whether the candidate agent modulates BMP-mediated signaling.    
     
     
         22 . A method according to  claim 21 , wherein the Smad protein is Smad1 or Smad5.  
     
     
         23 . A method according to  claim 21  wherein the cell is a bone cell.  
     
     
         24 . A method according to  claim 21 , wherein the cell is a neuron or kidney cell.  
     
     
         25 . A method according to  claim 21 , wherein the agent enhances BMP-mediated signaling.  
     
     
         26 . A method for screening for an agent that modulates TGF-β-mediated signaling, comprising the steps of: 
 (a) contacting a cell that expresses a TGF-β receptor with TGF-β and a candidate agent; and  
 (b) detecting a level of a Smad protein in the cell, relative to a level of the Smad protein in a cell that is contacted with the bone morphogenic protein in the absence of the candidate agent, and therefrom determining whether the candidate agent is a modulator of TGF-β-mediated signaling.  
 
     
     
         27 . A method according to  claim 26 , wherein the Smad protein is Smad2 or Smad3.  
     
     
         28 . A method according to  claim 26 , wherein the agent enhances TGF-β-mediated signaling.  
     
     
         29 . A method for screening for an agent that modulates TGF-β-mediated signaling, comprising the steps of: 
 (a) contacting a cell that expresses a TGF-βreceptor with TGF-β and a candidate agent; and  
 (b) detecting a level of ubiquitination of a Smad protein in the cell, relative to a level of the Smad protein ubiquitination in a cell that is contacted with the bone morphogenic protein but is not contacted with the candidate agent, and therefrom determining whether the candidate agent modulates TGF-β-mediated signaling.  
 
     
     
         30 . A method according to  claim 29 , wherein the Smad protein is Smad2 or Smad3.  
     
     
         31 . A method according to  claim 29 , wherein the agent enhances TGF-β-mediated signaling.  
     
     
         32 . A method for screening for an agent that modulates BMP-mediated signaling, comprising the steps of: 
 (a) contacting a cell that expresses a BMP receptor with bone morphogenic protein and a candidate agent; and    (b) detecting a level of a HECT E3 ubiquitin ligase activity in the cell, relative to a level of HECT E3 ubiquitin ligase activity in a cell that is contacted with the bone morphogenic protein in the absence of the candidate agent, and therefrom determining whether the candidate agent modulates BMP-mediated signaling.    
     
     
         33 . A method according to  claim 32 , wherein the cell is a bone cell.  
     
     
         34 . A method according to  claim 32 , wherein the cell is a neuron or kidney cell.  
     
     
         35 . A method according to  claim 32 , wherein the agent enhances BMP-mediated signaling.  
     
     
         36 . A method for screening for an agent that modulates TGF-β-mediated signaling, comprising the steps of: 
 (a) contacting a cell that expresses a TGF-β receptor with TGF-β and a candidate agent; and  
 (b) detecting a level of a HECT E3 ubiquitin ligase activity in the cell, relative to a level of HECT E3 ubiquitin ligase activity in a cell that is contacted with the bone morphogenic protein in the absence of the candidate agent, and therefrom determining whether the candidate agent modulates TGF-β- mediated signaling.  
 
     
     
         37 . A method according to  claim 36 , wherein the agent enhances TGF-β-mediated signaling.  
     
     
         38 . A method for augmenting TGF-β- and/or BMP-mediated signaling in a cell, comprising contacting a cell with an agent that inhibits binding of a HECT E3 ubiquitin ligase WW domain to a Smad PY motif.  
     
     
         39 . A method according to  claim 38 , wherein the Smad PY motif comprises the sequence TPPPAY (SEQ ID NO:16).  
     
     
         40 . A method according to  claim 38 , wherein the Smad PY motif comprises the sequence TPPPGY (SEQ ID NO:18).  
     
     
         41 . A method for augmenting TGF-β and/or BMP-mediated signaling in a cell, comprising contacting a cell with an agent that inhibits ubiquitination of a Smad protein.  
     
     
         42 . A method according to  claim 41 , wherein the Smad protein is Smad1 or Smad5.  
     
     
         43 . A method according to  claim 41 , wherein the Smad protein is Smad2 or Smad3.  
     
     
         44 . A method for stimulating bone formation in a patient, comprising administering to a patient a therapeutically effective amount of an agent that inhibits binding of a HECT E3 ubiquitin ligase WW domain to a Smad PY motif.  
     
     
         45 . A method according to  claim 44 , wherein the Smad PY motif comprises the sequence TPPPAY (SEQ ID NO:16).  
     
     
         46 . A method for stimulating bone formation in a patient, comprising administering to a patient a therapeutically effective amount of an agent that inhibits ubiquitination of a Smad protein.  
     
     
         47 . A method according to  claim 46 , wherein the Smad protein is Smad1 or Smad5.  
     
     
         48 . A method for preventing or treating a condition associated with insufficient TGF-β and/or BMP-mediated cell signaling, comprising administering to a patient a therapeutically effective amount of an agent that inhibits binding of a HECT E3 ubiquitin ligase WW domain to a Smad PY motif.  
     
     
         49 . A method according to  claim 48 , wherein the Smad PY motif comprises the sequence TPPPAY (SEQ ID NO:16).  
     
     
         50 . A method according to  claim 48 , wherein the Smad PY motif comprises the sequence TPPPGY (SEQ ID NO:17).  
     
     
         51 . A method for preventing or treating a condition associated with insufficient TGF-β and/or BMP-mediated cell signaling, comprising administering to a patient a therapeutically effective amount of an agent that inhibits ubiquitination of a Smad protein.  
     
     
         52 . A method according to  claim 51 , wherein the Smad protein is Smad1 or Smad5.  
     
     
         53 . A method according to  claim 51 , wherein the Smad protein is Smad2 or Smad3.  
     
     
         53 . A method according to  claim 48  or  claim 51 , wherein the condition is a cancer or inflammation.