Modulators of leukocyte activation, compositions and methods of use

Abstract

The present invention provides compositions and methods for modulating leukocyte activation. Nucleic acids encoding proteins and proteins so encoded which are capable of modulating leukocyte activation are provided. Compositions and methods for the treatment of disorders related to leukocyte dysfunction or dysregulation are also provided. Prophylactics and methods for the prevention of such disorders are also provided. Also provided are compositions and methods for diagnostic and prognostic determination of such disorders. Further provided are assays for the identification of bioactive agents capable of modulating leukocyte activation.

1 - 17 . (canceled)  
     
     
         18 . A method for inhibiting immunoglobulin production in a B cell, comprising introducing into a B cell an antagonist of a USP- 25  protein, wherein said USP-25 protein comprises an amino acid sequence having at least about 95% identity to the amino acid sequence in SEQ ID NO:2.  
     
     
         19 . A method for inhibiting immunoglobulin production in a B cell, comprising introducing into a B cell an antisense USP-25 nucleic acid.  
     
     
         20 . A method for inhibiting immunoglobulin production in a B cell, comprising introducing into a B cell a double stranded nucleic acid capable of eliciting USP-25 mRNA interference.  
     
     
         21 . A method for inhibiting immunoglobulin production in a B cell, comprising introducing into a B cell an intrabody that specifically binds to USP-25 and reduces or eliminates USP-25 bioactivity.  
     
     
         22 . A method for modulating lymphocyte activation in a patient having a lymphocyte activation disorder, comprising administering to a patient having a lymphocyte activation disorder a medicament comprising an antagonist of USP-25 protein activity.  
     
     
         23 . A method for modulating lymphocyte activation in a patient having a lymphocyte activation disorder, comprising administering to a patient having a lymphocyte activation disorder a medicament comprising an agonist of USP-25 protein activity.  
     
     
         24 . A method for inhibiting B cell and T cell activation in a patient having an autoimmune disease, comprising administering to a patient having an autoimmune disease a medicament comprising an antagonist of USP-25 protein activity.  
     
     
         25 . A method for prolonging the survival of a graft in a mammalian host, comprising administering to a mammalian host receiving a graft a medicament comprising an antagonist of USP-25 protein activity.  
     
     
         26 . A method for inhibiting CD40L expression, comprising contacting a T lymphocyte with an antagonist of USP-25 activity.  
     
     
         27 . A method for inhibiting IL-2 production, comprising contacting a T-lymphocyte with an antagonist of USP-25 activity.  
     
     
         28 . A method of screening for an immunosuppressant, comprising: 
 a) combining a candidate bioactive agent, a USP-25 protein and a USP-25 substrate protein; and    b) determining the level of ubiquitination of said USP-25 substrate protein in the presence and absence of said candidate bioactive agent;    wherein said USP-25 protein comprises an amino acid sequence having at least about 95% identity to the amino acid sequence set forth in SEQ ID NO: 2, wherein said USP-25 protein has ubiquitin-specific peptidase activity directed at said USP-25 substrate protein, and wherein an increase in the level of ubiquitin-conjugated or ubiquitin-like protein-conjugated substrate protein in the presence of said candidate bioactive agent indicates that said candidate bioactive agent is an immunosupressant.    
     
     
         29 . The method according to  claim 30 , wherein said USP-25 substrate protein is selected from the group consisting of UBC9, IkB, SYK and calcineurin.  
     
     
         30 . A method for screening for a bioactive agent capable of modulating USP-25 protein activity, comprising: 
 a) combining a USP-25 protein, a USP-25 target protein conjugated to ubiquitin or ubiquitin like protein, and a candidate bioactive agent; and    b) determining the level of ubiquitin-conjugated or ubiquitin-like protein-conjugated target protein in the presence and absence of said candidate bioactive agent;    wherein said USP-25 protein comprises an amino acid sequence having at least about 95% identity to the amino acid sequence set forth in SEQ ID NO: 2, wherein said USP-25 protein has ubiquitin-specific peptidase activity directed at said USP-25 target protein, and wherein a difference in the level of ubiquitin-conjugated or ubiquitin-like protein-conjugated target protein in the presence of said candidate bioactive agent indicates that said candidate bioactive agent is capable of modulating USP-25 protein activity.    
     
     
         31 . A method of screening for a bioactive agent capable of modulating lymphocyte activation, comprising: 
 i) contacting a candidate bioactive agent to a lymphocyte comprising a recombinant nucleic acid encoding a USP-25 protein;    ii) inducing activation of said lymphocyte; and    iii) determining the activation of said lymphocyte in the presence and absence of said candidate bioactive agent;    wherein said USP-25 protein comprises an amino acid sequence having at least about 95% identity to the amino acid sequence in SEQ ID NO:2, and wherein a difference in the activation of said lymphocyte in the presence and absence of said candidate bioactive agent indicates that said candidate bioactive agent is capable of modulating lymphocyte activation.    
     
     
         32 . The method according to  claim 33 , wherein said USP-25 protein comprises the amino acid sequence set forth in  FIG. 2  (SEQ ID NO:2).  
     
     
         33 . The method according to  claim 33 , wherein said determining the activation of said lymphocyte comprises determining the activity of the immunoglobulin heavy chain gene promoter or the nuclear factor in activated T cells (NFAT) gene promoter.  
     
     
         34 . A method for screening for a bioactive agent capable of modulating lymphocyte activation, comprising: 
 i) contacting a candidate bioactive agent to a lymphocyte comprising a recombinant nucleic acid encoding a USP-25 protein;    ii) inducing activation of said lymphocyte; and    iii) determining the activation of said lymphocyte in the presence and absence of said candidate bioactive agent;    wherein said USP-25 protein comprises an amino acid sequence having at least about 95% identity to the amino acid sequence set forth in  FIG. 4  (SEQ ID NO:4), and wherein a difference in the activation of said lymphocyte in the presence and absence of said candidate bioactive agent indicates that said candidate bioactive agent is capable of modulating lymphocyte activation.    
     
     
         35 . The method according to  claim 36 , wherein said USP-25 protein comprises the amino acid sequence set forth in  FIG. 4  (SEQ ID NO:4).  
     
     
         36 . The method according to  claim 36 , wherein said determining the activation of said lymphocyte comprises determining the activity of the immunoglobulin heavy chain gene promoter or the nuclear factor in activated T cells (NFAT) gene promoter.  
     
     
         37 . The method according to  claim 35  or  38 , wherein said determining the activation of said lymphocyte further comprises determining the expression of CD69.