Development of Ubiquitin Variants with Selectivity for Ubiquitin C-Terminal Hydrolase Deubiquitinases

Abstract

Ubiquitin (Ub) is a highly conserved protein that is covalently attached to substrate proteins as a post-translational modification to regulate signaling pathways such as proteasomal degradation and cell cycle/transcriptional regulation in the eukaryotic cellular environment. Ub signaling is regulated by the homeostasis of substrate protein ubiquitination/deubiquitination by E3 ligases and deubiquitinating enzymes (DUBs) in healthy eukaryotic systems. One such DUB, ubiquitin C-terminal hydrolase L1 (UCHL1), is endogenously expressed in the central nervous system under normal physiological conditions, but overexpression and/or mutation has been linked to various cancers and neurodegenerative diseases. The lack of UCHL1 probing strategies suggests development of a selective Ub variant (UbV) for probing UCHL1's role in these disease states would be beneficial. We describe a computational design approach to investigate UbVs that lend selectivity, both binding and inhibition, to UCHL1 over the close structural homologue UCHL3 and members of other DUB families. A number of UbVs, mainly those containing Thr9 mutations, displayed appreciable binding and inhibition selectivity for UCHL1 over UCHL3, compared to wild-type Ub in in vitro assays. By appending reactive electrophiles to the C-terminus of the UbVs, we created the first activity-based probe (ABP) with demonstrated reaction selectivity for UCH family DUBs over other families in cell lysates. Further kinetic analysis of covalent inhibition by the UbV-ABP with UCHL1 and UCHL3 offers insight into the future design of UCHL1 selective UbV-ABP. These studies serve as a proof of concept of the viability of the in silico design of ubiquitin variants for UCH family DUBs as a step toward the development of macromolecular UCHL1 inhibitors.

Optimization and Anti-Cancer Properties of Fluoromethylketones as Covalent Inhibitors for Ubiquitin C-Terminal Hydrolase L1

“…His-UCHL1 was preincubated with an excess of 1 (2 mM) or DMSO overnight at room temperature to ensure full covalent modification of the Cys90 was achieved. The dissociation constant ( K d ) of Ub towards UCHL1 pre-treated with DMSO treated control was determined and proved to be similar to previously published affinities ( Figure 6 B) [ 69 ]. However, the presence of 1 conjugated to the Cys90 completely abrogated the ability of UCHL1 to interact with Ub ( Figure 6 C).…”

“…This assay was carried out according to our previously published protocol using an Octet RED384 biolayer interferometer (ForteBio, Fremont, CA, USA) [ 69 ]. A solution containing 5 µM His-UCHL1 was incubated with 2 mM VAEFMK 1 or DMSO overnight in reaction buffer (50 mM Tris pH 7.6, 0.5 mM EDTA, 5 mM DTT) at room temperature before buffer exchanging into water using Zeba spin desalting columns (Thermo Scientific, catalog no.…”

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

“…UbV site-directed mutagenesis and validation in the pRSET-A monoubiquitin plasmid ordered through GenScript. Plasmids were transformed and expressed as previously described by our group [ 20 ].…”

“…The recombinant UCHL3 and UCHL1 proteins were recombinantly expressed and purified according to our previously published protocols [ 20 , 29 , 30 ] and summarized below. A pET-15b plasmid construct was used for the expression of both hexa-histidine (His 6 )-tagged UCHL1 and His 6 -UCHL3 in bacterial culture.…”

“…One such alternative strategy is the development of selective Ub-variants (UbVs) for DUBs of interest. Previous work has utilized phage display libraries [ 16 , 17 , 18 ] or computational [ 19 ] approaches, including a report from our group for the design of UCH selective activity-based probes (ABPs) [ 20 ], to accomplish this goal. Moreover, the Ovaa group combined both phage-display and computational approached to generate selective UbV activity-based probes for USP family DUBs [ 21 , 22 ].…”