In a previous post, I described a ubiquitin-rhodamine110 (UbRho110) assay, and showed that a ZnF-UBD ligand antagonizes USP5 catalytic activity. UbRho110 is not a native substrate, and validation with native polyubiquitin species in an orthogonal assay is required. To address this, I developed and optimized an internally quenched fluorophore pair (IQF) assay. You can see details on Zenodo.
The IQF assay uses di-ubiquitin substrates, one of which carries a fluorophore that is silenced by a nearby quencher. After the di-ubiquitin substrate is cleaved by a deubiquitinase (DUB) such as USP5, the free fluorophore can be detected through a fluorescence signal (Figure 1).
Figure 1. Summary of IQF assay. Image taken from LifeSensors.
I tested two di-ubiquitin IQF substrates: Ub2K63 and Ub2K48. A ZnF-UBD ligand, UBTR020994a (KD= 8 ± 2 µM [n=3]) equipotently antagonizes USP5 DUB activity for both diubiquitin substrates in a dose-dependent manner (Figure 2).
Figure 2. a) Chemical structure of UBTR020994a (KD= 8 ± 2 µM [n=3]) b) Dose-response of UBTR020994a and Ub2K63 (n=3) IC50 ̴ 63 µM c) Dose-response of UBTR020994a and Ub2K48 (n=3) IC50=45 ± 17 µM
In addition to the wild-type (WT) USP5, I tested a R221A and C335S mutant. The R221A is a ZnF-UBD mutant, where R221 co-ordinates ubiquitin binding at the ZnF-UBD. C335S is a mutant of the catalytic cysteine and prevents the cleavage of the isopeptide bond in ubiquitin chains. The R221A mutant has little to no activity with Ub2K48 substrate-comparable to inhibition of the WT-USP5 with a ZnF-UBD antagonist, UBTR020994a. IQF Ub2K63 has approximately 50% activity for R221A mutant, suggesting R221 may be more important for recruitment and placement of Ub2K48 substrate than Ub2K63 substrate. C335S is inactive in the assay.
Figure 3. USP5 DUB Activity for wild-type (WT) USP5, a ZnF-UBD mutant (R221A), a catalytic mutant (C335S) ± 2 mM UBTR020994a: a) Ub2K63 (N=2) b) Ub2K48 (N=3)
In the future, I will be repeating experiments with the USP5 mutants and dose-response curves for UBTR020994a for both IQF substrates to confirm the results. I also hope to test other lysine-linked di-ubiquitin substrates, such as K11, using this IQF assay.