Zhang, Jiatong
(2025)
Probing USP4 Specificity through Engineered Substrate Trapping and a Fluorescence Polarization Assay Platform Suitable for Measuring DUB Activity and Inhibition.
PhD thesis, University of Nottingham.
Abstract
Deubiquitinases (DUBs) play a critical role in the regulation of various cellular processes, such as protein homeostasis and signaling, rendering them attractive drug targets. However, the generation of reagents for measuring DUB activity typically involve several steps and is not straight forward. To dissect their activity and substrate recognition. Here, we report the development and characterization of a novel fluorescent polarization assay using an isopeptide bond substrate mimetic (IsoMim) that can be made recombinantly in high yields. The IsoMim assay was able to discern the differential activity of ubiquitin specific protease family members (USP4, USP15, USP11 and USP2), the ubiquitin C-terminal hydrolase UCHL3 and the Machado-Joseph Domain deubiquitinase JOSD2. A competition assay format of the assay was developed that discerned differences between the close paralogues USP15, USP4 and USP11 in interacting with monoubiquitin, the isopeptide mimetic ubiquitin-GGG and the C-terminal truncation variant ubiquitin (1-74). Moreover, dose-response curves and associated pIC50 values using the broad-spectrum inhibitor PR-619 confirmed differential inhibition in the low µM range for four tested DUBs, demonstrating assay reliability. The successful discrimination of DUB activity and inhibition, and the high obtainable yields of the substrate make the IsoMim assay method applicable for high-throughput screening (HTS). This was ascertained in a ‘pseudo HTS screen’ for USP4 inhibitors in which PR-619 was successfully identified as a ‘pseudo hit’. USP4 was then used to dissect the molecular basis of substrate recognition and specificity, we determined high-resolution crystal structures of USP4 catalytic core (D1D2) bond to both linear diubiquitin and an engineered Ub-GG-Ub mimetic, revealing an induced-fit mechanism in which blocking loops (BL1 and BL2), switching loop (SL) and finger domain undergo concerted rearrangements to accommodate diubiquitin. A potential secondary S2 binding site on the finger domain was identified in the Ub-GG-Ub complex. Mutagenesis of S2-localized USP4 residue His805 to glutamate (H805E) and subsequent cleavage assays showed minimal impact on activity against linear tetraUb and K63-linked polyUb. We also examined a critical gate keeper residue mutation localized in BL2 S1’ site, V879D in blocking loop 2 (BL2), selectively impacted USP4’s ability to cleave linear diubiquitin, as confirmed by both gel-based assays and FP analysis. To further probe the S1’ site and assess substrate preferences, we developed a series of monoubiquitin based FP probes with distinct C-terminal extensions, Ub-GGGC-FM, Ub-MGGC-FM, and Ub-MQIC-FM. These tools enabled quantitative profiling of USP4 specificity and revealed altered binding behaviors upon mutation of conserved S1’ site residues His369 and Asn901.
Altogether, the developed assay provides a valuable tool for probing DUB activity and the identification and characterization of DUB inhibitors and has the potential to accelerate drug discovery efforts in this area. Our study uncovers key structural and functional features that govern USP4’s substrate recognition, defines roles of auxiliary binding sites and loop regions, and introduces versatile FP probes suitable for mechanistic and screening applications.
Actions (Archive Staff Only)
 |
Edit View |
Tools
Tools
![[thumbnail of Zhang Jiatong 20161350 correction 2.pdf]](https://eprints.nottingham.ac.uk/style/images/fileicons/application_pdf.png "Zhang Jiatong 20161350 correction 2.pdf")