Controlling availability or activity of proteins by use of protease inhibitors or receptor fragments

Abstract

The invention relates to the field of proteins, more specifically to those proteins that are located on the surface of the cell. The invention, among other things, provides an inhibitor or pharmaceutical composition capable of inhibiting down-regulation of a cell surface receptor. The invention provides a method to control or up-regulate hormone activity by using inhibitors or reagents that modify down-regulation of a protein. The invention further provides a method to control or up-regulate protein activity wherein ligand-induced receptor uptake and/or degradation by endocytosis of a receptor is inhibited, preferably by inhibiting the ubiquitin/proteasome system.

1 . A method for controlling or up-regulating the availability or activity of a protein, said method comprising: 
 regulating binding of the ubiquitin/proteasome system at a ubiquitin/proteasome system binding site of the protein.    
     
     
         2 . The method according to  claim 1  wherein the binding site comprises the amino acid sequence motif xEFIxxDx or a sequence essentially corresponding thereto, wherein D is aspartic acid, E is glutamic acid, F is phenylalanine, I is isoleucine and X is any other amino acid.  
     
     
         3 . A method for controlling the availability and/or signal transduction capability of a cell surface receptor, said method comprising providing an inhibitor capable of inhibiting proteolytic cleavage of said cell surface receptor.  
     
     
         4 . The method according to  claim 3  wherein said inhibitor is capable of inhibiting proteolytic cleavage of an intracellular part of the cell surface receptor.  
     
     
         5 . The method according to  claim 3  wherein said inhibitor is capable of inhibiting proteolytic cleavage of an extra-cellular part of the cell surface receptor.  
     
     
         6 . The method according to  claim 3 , wherein said cell surface receptor is selected from the group consisting of a hormone receptor, an amino acid derivative, prostaglandin, peptide, polypeptide, and protein hormone receptors.  
     
     
         7 . The method according to  claim 6  wherein said cell surface receptor is a growth hormone receptor.  
     
     
         8 . The method according to  claim 1  wherein said protein is a transport protein.  
     
     
         9 . The method according to  claim 8  wherein said transport protein is Glut4 insulin regulated glucose transporter.  
     
     
         10 . A peptide or peptide analogue or mimeticum that is derived from, competes with, or binds to an amino acid sequence located at or around a ubiquitin/proteasome system binding site located in a protein.  
     
     
         11 . The peptide or peptide analogue or mimeticum of  claim 10  wherein said binding site comprises the amino acid sequence motif xEFIxxDx or a sequence essentially corresponding thereto, wherein D is aspartic acid, E is glutamic acid, F is phenylalanine, I is isoleucine, and X is any other amino acid.  
     
     
         12 . An inhibitor capable of inhibiting proteolytic cleavage of a cell surface receptor for use in a method according  claim 1 .  
     
     
         13 . The inhibitor of  claim 12  that is capable of inhibiting proteolytic cleavage of the intra-cellular part of said cell surface receptor.  
     
     
         14 . The inhibitor of  claim 13  selected from the group consisting of proteasome inhibitors, MG132, carboxybenzyl-leucyl-leucyl-leucinal, lactacystin, carboxybenzyl-leucyl-leucyl-leucyl vinylsulfone, and β-lacton form of lactacystin.  
     
     
         15 . he inhibitor of  claim 13  comprising a peptide or peptide analogue or mimeticum that is derived from, competes with, or binds to an amino acid sequence located at or around a ubiquitin and/or ubiquitin/proteasome system binding site located in the intra-cellular part of a cell-surface receptor.  
     
     
         16 . The inhibitor of  claim 15  wherein said binding site comprises the amino acid sequence motif xEFIxxDx or a sequence essentially corresponding thereto, wherein D is aspartic acid, E is glutamic acid, F is phenylalanine, I is isoleucine, and X is any other amino acid.  
     
     
         17 . The inhibitor of  claim 15  wherein said binding site comprises the amino acid sequence DDSWVEFIELDI (SEQ ID NO:2) or DSWVEFIELD (SEQ ID NO:3).  
     
     
         18 . The inhibitor of  claim 12  capable of inhibiting proteolytic cleavage of an extra-cellular part of said cell surface receptor.  
     
     
         19 . The inhibitor of  claim 18  wherein said extra-cellular part comprises an approximately 60 kDa fragment of an extra-cellular domain of the growth hormone receptor.  
     
     
         20 . The inhibitor of  claim 18  or  claim 19  comprising a peptide or peptide analogue or mimeticum that is derived from, competes with, or binds to an amino acid sequence located at or around a proteolytic cleavage signal site located in an extra-cellular part of said cell surface receptor.  
     
     
         21 . The inhibitor of  claim 20  wherein said cleavage signal site comprises the amino acid sequence CEEDFYR (SEQ ID NO:7) or a sequence essentially corresponding thereto.  
     
     
         22 . A peptide or peptide analogue or mimeticum that is derived from, competes with, or binds to an amino acid sequence located at or around a ubiquitin and/or ubiquitin/proteasome system binding site located in the intra-cellular part of a cell-surface receptor.  
     
     
         23 . The peptide or peptide analogue or mimeticum of  claim 22  wherein said binding site comprises an amino acid sequence motif xEFIxxDx or a sequence essentially corresponding thereto, wherein D is aspartic acid, E is glutamic acid, F is phenylalanine, I is isoleucine, and X is any other amino acid.  
     
     
         24 . A peptide or peptide analogue or mimeticum that is derived from, competes with, or binds to an amino acid sequence located at or around a proteolytic cleavage signal site located in the extra-cellular part of a receptor.  
     
     
         25 . The peptide or peptide analogue or mimeticum of  claim 24  wherein said cleavage signal site comprises the amino acid sequence CEEDFYR (SEQ ID NO:7) or a sequence essentially corresponding thereto.  
     
     
         26 . A pharmaceutical composition comprising: 
 the peptide or peptide analogue or mimeticum of  claim 10 .    
     
     
         27 . A method of regulating the activity of a hormone in a subject, said method comprising: 
 administering the pharmaceutical composition of  claim 26  to the subject so as to regulate the activity of the hormone.    
     
     
         28 . The method according to  claim 27  further comprising: 
 administering the pharmaceutical composition in conjunction with a hormone.    
     
     
         29 . The method according to  claim 27  wherein the hormone is up-regulated.  
     
     
         30 . A method of controlling a cell surface receptor's availability and/or signal transduction capability in a subject, the method comprising: 
 administering the pharmaceutical composition of  claim 26  to the subject so as to control the availability and/or signal transduction capability of a cell surface receptor.    
     
     
         31 . A method of treating muscle wasting in a subject, the method comprising: 
 administering the pharmaceutical composition of  claim 26  to the subject so as to treat muscle wasting.