Targeted polypeptide degradation
This invention pertains to compositions, methods, cells and organisms useful for selectively localizing polypeptides to the proteasome for degradation. Therapeutic methods and pharmaceutical compositions for treating disorders associated with the expression and/or activity of a polypeptide by targeting these polypeptides for degradation, as well as methods for targeting therapeutic polypeptides for degradation and/or activating therapeutic polypeptides by degradation are provided. The invention provides methods for identifying compounds that mediate proteasome localization and/or polypeptide degradation. The invention also provides research tools for the study of protein function.
1. An in vitro method for targeted polypeptide degradation in a ubiquitin-independent manner comprising:
a) providing a proteasome having a first member of a heterodimer pair covalently linked thereto;
b) providing a target polypeptide having a second member of a heterodimer pair covalently linked thereto; and
c) providing a heterodimerizer that binds the first member of the heterodimer pair and causes heterodimerization of the first member of the heterodimer pair and the second member of the heterodimer pair, such that the proteasome degrades the target polypeptide.
2. The method of claim 1 , wherein said heterodimerizer is selected from the group consisting of FK-506, FK-506-cyclosporin A, an aptamer, coumermycin, bismethotrexate, dexamethasone-methotrexate, an RNA-protein binder, and a rapamycin derivative.
3. The method of claim 1 , wherein said heterodimerizer comprises a first module that binds a proteasome and a second module that binds a target polypeptide.
4. The method of claim 3 , wherein said first module is chemically crosslinked to said second module.
5. The method of claim 3 , wherein said second module is an epitope tag or an antibody.
6. The method of claim 5 , wherein said epitope tag is selected from the group consisting of hemagglutinin, c-myc and TAP.
7. An in vitro method for targeted polypeptide degradation in a ubiquitin-independent manner comprising:
a) providing a proteasome comprising an Fpr1 tag;
b) providing a target polypeptide comprising Tor; and
c) providing rapamycin, such that binding of rapamycin to Tor forms a complex, said complex binding the proteasome comprising the Fpr1 tag, and such that the proteasome degrades the target polypeptide.