Compositions and methods for using engineered deubiquitinases for probing ubiquitin-dependent cellular processes
View Patent ↗The present disclosure provides, inter alia, a recombinant engineered deubiquitinase (DUB) and methods for treating or ameliorating an inherited ion channelopathy, such as long QT syndrome, Brugada syndrome, or cystic fibrosis, in a subject. Further provided are methods for screening mutations causing such inherited ion channelopathies for a trafficking-deficient mutation that is treatable by the recombinant engineered DUB disclosed herein.
1. A recombinant engineered deubiquitinase (DUB) comprising:
a) a catalytic unit comprising the catalytic domain of a deubiquitinase;
b) a protein binder comprising an antibody, or antigen binding fragment thereof, that specifically binds a target substrate protein for deubiquitination by the engineered DUB; and
c) a variable linker between the catalytic unit and the protein binder.
2. The recombinant engineered DUB of claim 1 , wherein the antibody is a nanobody, scFv, (scFv)2, Fab, Fab′, F(ab′)2, Fv, diabody, or a DARPin.
3. The engineered DUB of claim 1 , wherein the antibody is a nanobody.
4. The recombinant engineered DUB of claim 1 , wherein the protein binder specifically binds an ion channel.
5. The recombinant engineered DUB of claim 4 , wherein the ion channel is KCNQ1, HERG, or CFTR.
6. The engineered DUB of claim 1 , wherein the catalytic unit comprising the catalytic domain of a deubiquitinase from the OTU family.
7. The recombinant engineered DUB of claim 1 , wherein the catalytic unit comprises the catalytic domain of Cezanne.
8. The recombinant engineered DUB of claim 1 , wherein the antibody is a single domain antibody (dAb).
9. The recombinant engineered DUB of claim 1 , wherein the catalytic unit is selective for a particular ubiquitin linkage type.
10. The recombinant engineered DUB of claim 1 , wherein the catalytic unit comprises the catalytic domain of a deubiquitinase, wherein the deubiquitinase is from the ubiquitin specific proteases (USP) family, the ovarian tumor proteases (OTU) family, the ubiquitin C-terminal hydrolases (UCH) family, the Josephin domain (Josephin) family, the motif interacting with ubiquitin-containing novel DUB (MINDY) family, or the JAB1/MPN/Mov34 metalloenzyme domain (JAMM) family.
11. The recombinant engineered DUB of claim 10 , wherein the catalytic unit comprises the catalytic domain of a deubiquitinase from the USP family.
12. The recombinant engineered DUB of claim 11 , wherein the catalytic unit comprises the catalytic domain of USP21.
13. The recombinant engineered DUB of claim 10 , wherein the catalytic unit comprises the catalytic domain of a deubiquitinase from the OTU family.
14. The recombinant engineered DUB of claim 13 , wherein the catalytic unit comprises the catalytic domain of OTUD1.
15. The recombinant engineered DUB of claim 13 , wherein the catalytic unit comprises the catalytic domain of OTUD4.
16. The recombinant engineered DUB of claim 13 , wherein the catalytic unit comprises the catalytic domain of Cezanne.
17. The recombinant engineered DUB of claim 13 , wherein the catalytic unit comprises the catalytic domain of TRABID.
18. The recombinant engineered DUB of claim 13 , wherein the catalytic unit comprises the catalytic domain of OTULIN.
19. The recombinant engineered DUB of claim 1 , wherein the catalytic unit comprises the catalytic domain of USP21 and is capable of non-selectively eliminating all ubiquitin linkage types.
20. The recombinant engineered DUB of claim 1 , wherein the catalytic unit comprises the catalytic domain of OTUD1 and is capable of selectively eliminating K63 ubiquitin linkages.
21. The recombinant engineered DUB of claim 1 , wherein the catalytic unit comprises the catalytic domain of OTUD4 and is capable of selectively eliminating K48 ubiquitin linkages.
22. The recombinant engineered DUB of claim 1 , wherein the catalytic unit comprises the catalytic domain of Cezanne and is capable of selectively eliminating K11 ubiquitin linkages.
23. The recombinant engineered DUB of claim 1 , wherein the catalytic unit comprises the catalytic domain of TRABID and is capable of selectively eliminating K29 ubiquitin linkages, K33 ubiquitin linkages, or K29 and K33 ubiquitin linkages.
24. The recombinant engineered DUB of claim 1 , wherein the catalytic unit comprises the catalytic domain of OTULIN and is capable of selectively eliminating Met1 ubiquitin linkages.
25. The recombinant engineered DUB of claim 1 , wherein the catalytic unit is non-selective for a particular ubiquitin linkage type.