Methods of Protein Destabilization and Uses Thereof

Abstract

This invention is directed towards methods of destabilizing proteins in living cells, and their use for the development of novel assays. In one embodiment, the invention comprises the use of non-cleavable multimerized ubiquitin fusion proteins to destabilize a target protein, such as a reporter moiety. In one aspect of this method the constructs also comprises a linker that operatively couples the reporter moiety to the multimerized ubiquitin fusion protein. In this embodiment, enzymatic modification of the linker results in a modulation of the coupling of the reporter protein to the multimerized ubiquitin domains resulting in a change in the stability of the reporter moiety. The level of the reporter moiety in the cell can then be used as a measure of the enzymatic activity in the cell. In another embodiment the invention provides for a generalized way of coordinately regulating the cellular concentration of a plurality of target proteins. In one aspect of this method, the target proteins are operatively coupled to a ubiquitin fusion protein via a linker containing a protease cleavage site. Cleavage of the linker by a protease results in uncoupling of the target protein from the multimerized ubiquitin construct, and results in an increase in the stability and concentration of the target protein.

1 - 37 . (canceled) 
     
     
         38 . A method of destabilizing a target protein in a cell, comprising;
 operatively coupling a target protein to a linear multimerized destabilization domain, wherein said linear multimerized destabilization domain is non-cleavable by a α-NH-ubiquitin protein endoproteases, and comprises at least two copies of a destabilization domain.   
     
     
         39 . The method of  claim 38 , wherein said destabilization domain comprises a ubiquitin homolog. 
     
     
         40 . The method of  claim 39 , wherein said ubiquitin homolog comprises a mutation that prevents cleavage by α-NH-ubiquitin protein endoproteases. 
     
     
         41 . The method of  claim 39 , wherein said ubiquitin homolog comprises a mutation at glycine 76. 
     
     
         42 . The method of  claim 38 , wherein said protein of interest is fused in frame to said multimerized destabilization domain. 
     
     
         43 . The method of  claim 38 , wherein said protein of interest is non-covalently coupled to said multimerized ubiquitin fusion protein. 
     
     
         44 . The method of  claim 38 , wherein said cell is a mammalian cell. 
     
     
         45 . The method of  claim 38 , wherein said cell is a yeast cell. 
     
     
         46 . The method of  claim 38 , wherein said cell is an insect cell. 
     
     
         47 . The method of  claim 38 , wherein said cell is a plant cell. 
     
     
         48 . The method of  claim 38 , wherein said target protein is coupled to said multimerized destabilization domain by a linker. 
     
     
         49 . The method of  claim 48 , wherein said linker is between 1 and 10 amino acid residues. 
     
     
         50 . A recombinant DNA molecule, comprising a nucleic acid sequence encoding for;
 a) a linear multimerized destabilization domain, wherein said linear multimerized destabilization domain is non-cleavable by a α-NH-ubiquitin protein endoproteases, and comprises at least two copies of a destabilization domain,   b) a target protein, and   c) a linker moiety that operatively couples said multimerized destabilization domain to said reporter moiety, wherein said linker is non-cleavable by a α-NH-ubiquitin protein endoproteases.   
     
     
         51 . The method of  claim 50 , wherein said linker moiety comprises an enzyme modification site for an activity, and modification of said linker moiety by said activity modulates the coupling of said multimerized destabilization domain to said reporter moiety, thereby modulating the stability of said reporter moiety. 
     
     
         52 . The method of  claim 50 , wherein said destabilization domain comprises a ubiquitin homolog. 
     
     
         53 . The method of  claim 52 , wherein said ubiquitin homolog comprises a mutation that prevents cleavage by α-NH-ubiquitin protein endoproteases. 
     
     
         54 . The method of  claim 52 , wherein said ubiquitin homolog comprises a mutation at glycine 76. 
     
     
         55 . A recombinant protein molecule, comprising an amino acid sequence encoding for;
 a) a linear multimerized destabilization domain, wherein said multimerized destabilization domain is non-cleavable by a α-NH-ubiquitin protein endoproteases, and comprises at least two copies of said destabilization domain,   b) a target protein, and   c) a linker moiety that operatively couples said multimerized destabilization domain to said reporter moiety, wherein said linker is non-cleavable by a α-NH-ubiquitin protein endoproteases.   
     
     
         56 . The method of  claim 55 , wherein said linker moiety comprises a recognition motif for an activity, and modification of said linker moiety by said activity modulates the coupling of said multimerized destabilization domain to said reporter moiety, thereby modulating the stability of said reporter moiety. 
     
     
         57 . The method of  claim 55 , wherein said destabilization domain comprises a ubiquitin homolog. 
     
     
         58 . The method of  claim 57 , wherein said ubiquitin homolog comprises a mutation that prevents cleavage by α-NH-ubiquitin protein endoproteases. 
     
     
         59 . The method of  claim 57 , wherein said ubiquitin homolog comprises a mutation at glycine 76. 
     
     
         60 . A host cell, comprising a nucleic acid sequence encoding for;
 a) a linear multimerized destabilization domain, wherein said multimerized destabilization domain is non-cleavable by a α-NH-ubiquitin protein endoproteases, and comprises at least two copies of said destabilization domain,   b) a target protein, and   c) a linker moiety that operatively couples said multimerized destabilization domain to said reporter moiety, wherein said linker is non-cleavable by a α-NH-ubiquitin protein endoproteases.   
     
     
         61 . The method of  claim 60 , wherein said destabilization domain comprises a ubiquitin homolog. 
     
     
         62 . The method of  claim 61 , wherein said ubiquitin homolog comprises a mutation that prevents cleavage by α-NH-ubiquitin protein endoproteases. 
     
     
         63 . The method of  claim 61 , wherein said ubiquitin homolog comprises a mutation at glycine 76. 
     
     
         64 . A transgenic animal, comprising a nucleic acid sequence encoding for;
 a linear multimerized destabilization domain, wherein said multimerized destabilization domain is non-cleavable by a α-NH-ubiquitin protein endoproteases, and comprises at least two copies of said destabilization domain,   b) a target protein, and   c) a linker moiety that operatively couples said multimerized destabilization domain to said reporter moiety, wherein said linker is non-cleavable by a α-NH-ubiquitin protein endoproteases.   
     
     
         65 . The method of  claim 64 , wherein said destabilization domain comprises a ubiquitin homolog. 
     
     
         66 . The method of  claim 65 , wherein said ubiquitin homolog comprises a mutation that prevents cleavage by α-NH-ubiquitin protein endoproteases. 
     
     
         67 . The method of  claim 65 , wherein said ubiquitin homolog comprises a mutation at glycine 76. 
     
     
         68 . A transgenic plant, comprising a nucleic acid sequence encoding for;
 a) a linear multimerized destabilization domain, wherein said multimerized destabilization domain is non-cleavable by a α-NH-ubiquitin protein endoproteases, and comprises at least two copies of said destabilization domain,   b) a target protein, and   c) a linker moiety that operatively couples said multimerized destabilization domain to said reporter moiety, wherein said linker is non-cleavable by a α-NH-ubiquitin protein endoproteases.   
     
     
         69 . The method of  claim 68 , wherein said destabilization domain comprises a ubiquitin homolog. 
     
     
         70 . The method of  claim 69 , wherein said ubiquitin homolog comprises a mutation that prevents cleavage by α-NH-ubiquitin protein endoproteases. 
     
     
         71 . The method of  claim 69 , wherein said ubiquitin homolog comprises a mutation at glycine 76. 
     
     
         72 . A method for identifying a modulator of an activity, comprising;
 a) contacting a cell with a test chemical, wherein said cell comprises,
 i) at least one destabilization domain, wherein said destabilization domain is non-cleavable by α-NH-ubiquitin protein endoproteases, 
 ii) a reporter moiety, and iii) a linker moiety that operatively couples said destabilization domain to said reporter moiety, wherein said linker moiety comprises a recognition motif for said activity and modification of said linker moiety by said activity modulates the coupling of said destabilization domain to said reporter moiety thereby modulating the stability of said reporter moiety, and wherein said linker moiety is non-cleavable by said α-NH-ubiquitin protein endoproteases, 
   b) detecting said reporter moiety, or a product of said reporter moiety in the presence of said test chemical, and   c) comparing said reporter moiety activity from step b) to the reporter moiety activity in a control cell in the absence of said test chemical.   
     
     
         73 . The method of  claim 72 , further comprising the step of contacting said cell with an activator of said activity prior to the addition said test chemical. 
     
     
         74 . The method of  claim 72 , further comprising the step of detecting the viability of said cell. 
     
     
         75 . The method of  claim 72 , wherein said activity is selected from the group consisting of a protease activity, a protein kinase activity and a phosphoprotein phosphatase activity. 
     
     
         76 . The method of  claim 72 , wherein said reporter moiety is selected from the group consisting of a naturally fluorescent protein homolog, a β-lactamase homolog, a β-galactosidase homolog, an alkaline phosphatase homolog, a CAT homolog, and a luciferase homolog. 
     
     
         77 - 79 . (canceled)