Csn5 polypeptides and uses thereof for screening therapeutic agents

Abstract

The present invention relates to mutated CSN5 polypeptides and their use in a method of screening modulators of CSN5 activity that could be used as therapeutic agents.

1 . A polypeptide comprising
 an amino acid sequence ranging from amino acid at position 53 to amino acid at position 252 in SEQ ID NO: 1 wherein the amino acid at position 106 in SEQ ID NO: 1 is substituted by another amino acid,   an amino acid sequence ranging from amino acid at position 98 to amino acid at position 297 in SEQ ID NO: 2 wherein the amino acid at position 151 in SEQ ID NO: 2 is substituted by another amino acid,   an amino acid sequence ranging from amino acid at position 58 to amino acid at position 257 in SEQ ID NO: 3 wherein the amino acid at position 111 in SEQ ID NO: 3 is substituted by another amino acid,   an amino acid sequence ranging from amino acid at position 57 to amino acid at position 256 in SEQ ID NO: 4 wherein the amino acid at position 110 in SEQ ID NO: 4 is substituted by another amino acid,   an amino acid sequence ranging from amino acid at position 55 to amino acid at position 254 in SEQ ID NO: 5 wherein the amino acid at position 108 in SEQ ID NO: 5 is substituted by another amino acid,   an amino acid sequence ranging from amino acid at position 56 to amino acid at position 255 in SEQ ID NO: 6 wherein the amino acid at position 109 in SEQ ID NO: 6 is substituted by another amino acid,   an amino acid sequence ranging from amino acid at position 61 to amino acid at position 260 in SEQ ID NO: 7 wherein the amino acid at position 114 in SEQ ID NO: 7 is substituted by another amino acid,   an amino acid sequence ranging from amino acid at position 51 to amino acid at position 250 in SEQ ID NO: 8 wherein the amino acid at position 104 in SEQ ID NO: 8 is substituted by another amino acid,   an amino acid sequence ranging from amino acid at position 52 to amino acid at position 251 in SEQ ID NO: 9 wherein the amino acid at position 105 in SEQ ID NO: 9 is substituted by another amino acid,   an amino acid sequence ranging from amino acid at position 50 to amino acid at position 249 in SEQ ID NO: 10 wherein the amino acid at position 103 in SEQ ID NO: 10 is substituted by another amino acid,   an amino acid sequence ranging from amino acid at position 50 to amino acid at position 249 in SEQ ID NO: 11 wherein the amino acid at position 103 in SEQ ID NO: 11 is substituted by another amino acid,   an amino acid sequence ranging from amino acid at position 57 to amino acid at position 256 in SEQ ID NO: 12 wherein the amino acid at position 110 in SEQ ID NO: 12 is substituted by another amino acid,   an amino acid sequence ranging from amino acid at position 57 to amino acid at position 256 in SEQ ID NO: 13 wherein the amino acid at position 110 in SEQ ID NO: 13 is substituted by another amino acid,   an amino acid sequence ranging from amino acid at position 48 to amino acid at position 247 in SEQ ID NO: 14 wherein the amino acid at position 101 in SEQ ID NO: 14 is substituted by another amino acid,   an amino acid sequence ranging from amino acid at position 56 to amino acid at position 255 in SEQ ID NO: 15 wherein the amino acid at position 109 in SEQ ID NO: 15 is substituted by another amino acid,   or an amino acid sequence ranging from amino acid at position 75 to amino acid at position 273 in SEQ ID NO: 16 wherein the amino acid at position 127 in SEQ ID NO: 16 is substituted by another amino acid, and function-conservative variants thereof.   
     
     
         2 . The polypeptide according to  claim 1  comprising
 an amino acid sequence SEQ ID NO: 1 wherein the amino acid at position 106 in SEQ ID NO: 1 is substituted by another amino acid, 
 an amino acid sequence SEQ ID NO: 2 wherein the amino acid at position 151 in SEQ ID NO: 2 is substituted by another amino acid, 
 an amino acid sequence SEQ ID NO: 3 wherein the amino acid at position 111 in SEQ ID NO: 3 is substituted by another amino acid, 
 an amino acid sequence SEQ ID NO: 4 wherein the amino acid at position 110 in SEQ ID NO: 4 is substituted by another amino acid, 
 an amino acid sequence SEQ ID NO: 5 wherein the amino acid at position 108 in SEQ ID NO: 5 is substituted by another amino acid, 
 an amino acid sequence SEQ ID NO: 6 wherein the amino acid at position 109 in SEQ ID NO: 6 is substituted by another amino acid, 
 an amino acid sequence SEQ ID NO: 7 wherein the amino acid at position 114 in SEQ ID NO: 7 is substituted by another amino acid, 
 an amino acid sequence SEQ ID NO: 8 wherein the amino acid at position 104 in SEQ ID NO: 8 is substituted by another amino acid, 
 an amino acid sequence SEQ ID NO: 9 wherein the amino acid at position 105 in SEQ ID NO: 9 is substituted by another amino acid, 
 an amino acid sequence SEQ ID NO: 10 wherein the amino acid at position 103 in SEQ ID NO: 10 is substituted by another amino acid, 
 an amino acid sequence SEQ ID NO: 11 wherein the amino acid at position 103 in SEQ ID NO: 11 is substituted by another amino acid, 
 an amino acid sequence SEQ ID NO: 12 wherein the amino acid at position 110 in SEQ ID NO: 12 is substituted by another amino acid, 
 an amino acid sequence SEQ ID NO: 13 wherein the amino acid at position 110 in SEQ ID NO: 13 is substituted by another amino acid, 
 an amino acid sequence SEQ ID NO: 14 wherein the amino acid at position 101 in SEQ ID NO: 14 is substituted by another amino acid, 
 an amino acid sequence SEQ ID NO: 15 wherein the amino acid at position 109 in SEQ ID NO: 15 is substituted by another amino acid, 
 or an amino acid sequence SEQ ID NO: 16 wherein the amino acid at position 127 in SEQ ID NO: 16 is substituted by another amino acid, and function-conservative variants thereof. 
 
     
     
         3 . The polypeptide according to  claim 1 , wherein the amino acid substituted by another amino acid is substituted by any amino acid excepting proline. 
     
     
         4 . A kit of parts comprising at least one CSN5 polypeptide according to  claim 1  or a fragment thereof and at least one CSN6 polypeptide or a fragment thereof. 
     
     
         5 . A nucleic acid encoding for a polypeptide according to  claim 1 . 
     
     
         6 . A vector comprising the nucleic acid according to  claim 5 . 
     
     
         7 . A host cell, which has been transformed by the nucleic acid according to  claim 5 . 
     
     
         8 . A method of screening therapeutic agents comprising the steps of:
 i) providing a CSN5 polypeptide according to  claim 1  or a fragment thereof,   ii) providing at least one CSN5 isopeptidase substrate,   iii) providing a candidate agent,   iv) measuring the binding of the substrate using appropriate biophysical techniques and measuring the activity of the CSN5 polypeptide,   v) and positively selecting candidate agents that modulates CSN5 activity.   
     
     
         9 . A method of screening therapeutic agents comprising the steps of:
 i) providing a CSN5 polypeptide selected from the group consisting of SEQ ID NO: 1-16, or a CSN5 polypeptide according to  claim 1  or a fragment thereof,   ii) providing a CSN6 polypeptide or a fragment thereof,   iii) providing at least one CSN5 isopeptidase substrate,   iv) providing a candidate agent,   v) measuring the binding of the substrate using appropriate biophysical techniques and measuring the activity of the CSN5 polypeptide,   vi) and positively selecting candidate agents that modulates CSN5 activity.   
     
     
         10 . A method of screening therapeutic agents comprising the steps of:
 i) providing a CSN5 polypeptide according to  claim 1  or a fragment thereof,   ii) providing a candidate agent,   iv) measuring the binding of the candidate agent to the CSN5 polypeptide using appropriate biophysical techniques,   v) and positively selecting candidate agents that bind to the CSN5 polypeptide.