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. 2022 Jan 1;12(1):62.
doi: 10.3390/biom12010062.

Rational Development and Characterization of a Ubiquitin Variant with Selectivity for Ubiquitin C-Terminal Hydrolase L3

Chad S Hewitt  1 , Chittaranjan Das  2 , Daniel P Flaherty  1   3   4
Affiliations

Rational Development and Characterization of a Ubiquitin Variant with Selectivity for Ubiquitin C-Terminal Hydrolase L3

Chad S Hewitt et al. Biomolecules. .

Abstract

There is currently a lack of reliable methods and strategies to probe the deubiquitinating enzyme UCHL3. Current small molecules reported for this purpose display reduced potency and selectivity in cellular assays. To bridge this gap and provide an alternative approach to probe UCHL3, our group has carried out the rational design of ubiquitin-variant activity-based probes with selectivity for UCHL3 over the closely related UCHL1 and other DUBs. The approach successfully produced a triple-mutant ubiquitin variant activity-based probe, UbVQ40V/T66K/V70F-PRG, that was ultimately 20,000-fold more selective for UCHL3 over UCHL1 when assessed by rate of inactivation assays. This same variant was shown to selectively form covalent adducts with UCHL3 in MDA-MB-231 breast cancer cells and no reactivity toward other DUBs expressed. Overall, this study demonstrates the feasibility of the approach and also provides insight into how this approach may be applied to other DUB targets.

Keywords: DUBs; UCHL3; activity-based probes; ubiquitin variants.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Time-dependent Western blot for Ub-ABPs. HA-WT-Ub-PRG and HA-UbVT66K/V70F-PRG, 5 µM, were both dosed into HEK293 lysates (0.5 mg/mL). Samples were tested at the indicated time points. (Top) Western blot for UCHL3 indicating the free-UCHL3 and Ub-UCHL3 complex gel shifted. (Bottom) Western blot for HA (green fluorescence) and UCHL3 (red fluorescence).
Figure 2
Figure 2
Electrostatic potential surface map representations of Ub (green ribbons) with Q40 (sticks) interaction with UCHL3 (A) and UCHL1 (B). Electrostatic potential ranges from negative (red) to positive (blue).
Figure 3
Figure 3
Steady-state binding curves for UbVQ40V/T66K/V70F for determination Kd using BLI for UCHL3 (A, blue) and UCHL1 (B, red). Corresponding association/dissociation curves for each replicate are provided in the Supplementary Materials Figure S2.
Figure 4
Figure 4
UbVQ40V/T66K/V70F displays selective inhibition of UCHL3. The percent activity of each enzyme normalized to 100% activity for the DMSO control as a function of UbV concentration are plotted for UCHL3 (blue) and UCHL1 (red).
Figure 5
Figure 5
Time-dependent Western blot for Ub-ABPs. HA-WT-Ub-PRG and HA-UbVQ40V/T66K/V70F-PRG, 5 µM, were both dosed into MDA-MB-231 cell lysates (total protein concentration of 0.5 mg/mL). Samples were tested at the indicated time points. (A) Anti-HA Western blot indicates several non-specific covalent adducts formed by HA-WT-Ub-PRG while the HA-UbVQ40V/T66K/V70F-PRG displayed significantly reduced non-specific binding. The blots were imaged with both a normal exposure (top) and dark exposure (bottom) to identify lower concentration or faint bands. (B) Anti-UCHL3 and anti-HA colocalization blot that displays the gel-shift of the UCHL3-Ub adducts over time.
See this image and copyright information in PMC

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