Substrate for Rpn11 enzymatic activity
View Patent ↗The present application provides peptides that serve as substrates for proteasome enzymatic activity, e.g., the enzymatic activity of Rpn11, a metalloprotease of the 19S regulatory particle. The present application also provides methods and compositions employing the peptide substrates.
1. An isolated peptide comprising an amino acid sequence at least 80% identical to SEQ ID NO:1 and at least one ubiquitin moiety that is at least 90% identical to the first 76-amino acid sequence of SEQ ID NO:2, wherein a deubiquitinating protease deubiquitinates the peptide.
2. The peptide of claim 1 , wherein the protease is a deubiquitinating metalloprotease.
3. The peptide of claim 1 , wherein the protease is a deubiquitinating non-metalloprotease.
4. The peptide of claim 1 , wherein the protease is Rpn11.
5. The peptide of claim 1 , further comprising at least 3 additional amino acids at the C-terminus of the peptide.
6. The peptide of claim 1 , wherein the amino acid sequence has the amino acid sequence of SEQ ID NO:1 wherein at least one non-Ala amino acid is replaced by Ala.
7. The peptide of claim 1 , wherein the amino acid sequence has the amino acid sequence of SEQ ID NO:4 wherein at least one non-Ala amino acid is replaced by Ala.
8. The peptide of claim 1 , further comprising one or more ubiquitin moieties at the N-terminus of the peptide.
9. The peptide of claim 8 , wherein the ubiquitin moiety consists of two or more repeats of an amino acid sequence that is at least 90% identical to the first 76-amino acid sequence of SEQ ID NO:2.
10. The peptide of claim 1 , wherein the ubiquitin moiety comprises the first 76-amino acid sequence of SEQ ID NO:2 modified such that the 75th amino acid is an Ala.
11. The peptide of claim 1 , wherein the ubiquitin moiety comprises the first 76-amino acid sequence of SEQ ID NO:2 modified such that the 76th amino acid is an Ala.
12. The peptide of claim 1 , further comprising a detectable agent linked to the peptide.
13. The peptide of claim 12 , wherein the detectable agent is a fluorescent label.
14. The peptide of claim 13 , wherein the fluorescent label is a fluorescent peptide linked to the C-terminus of the peptide by a peptide bond.
15. The peptide of claim 12 , wherein the detectable agent is a radioactive label.
16. The peptide of claim 1 further comprising an N-terminal target portion or C-terminal target portion that specifically binds to a selection agent.
17. The peptide of claim 16 , wherein the selection agent is an antibody.
18. The peptide of claim 16 , wherein the selection agent is a divalent metal ion.
19. The peptide of claim 1 , wherein the peptide comprises SEQ ID NO: 1 and at least one ubiquitin moiety that is at least 90% identical to the first 76-amino acid sequence of SEQ ID NO:2.
20. The peptide of claim 19 , wherein the peptide comprises SEQ ID NO: 1 and at least one ubiquitin moiety that is identical to the first 76-amino acid sequence of SEQ ID NO:2.
21. The peptide of claim 1 , wherein the peptide comprises at least one of the amino acid sequences as set forth in SEQ ID NOs: 3-15 and at least one ubiquitin moiety that is at least 90% identical to the first 76-amino acid sequence of SEQ ID NO:2.
22. The peptide of claim 21 , wherein the peptide comprises at least one of the amino acid sequences as set forth in SEQ ID NOs: 3-15 and at least one ubiquitin moiety that is identical to the first 76-amino acid sequence of SEQ ID NO:2.
23. The peptide of claim 10 , wherein the peptide comprises SEQ ID NO: 1.
24. The peptide of claim 11 , wherein the peptide comprises SEQ ID NO: 1.
25. The peptide of claim 10 , wherein the peptide comprises at least one of the amino acid sequences as set forth in SEQ ID NOs: 3-15.
26. The peptide of claim 11 , wherein the peptide comprises at least one of the amino acid sequences as set forth in SEQ ID NOs: 3-15.
27. The peptide of claim 1 , wherein the deubiquitinating protease comprises a JAMM motif.
28. The peptide of claim 27 , wherein the JAMM motif comprises the amino acid sequence of SEQ ID NO: 17 or SEQ ID NO:18.