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[Preprint]. 2024 Apr 8:2024.04.08.588544.
doi: 10.1101/2024.04.08.588544.

Chemical tools to define and manipulate interferon-inducible Ubl protease USP18

Griffin J Davis  1 , Anthony O Omole  1 , Yejin Jung  1 , Wioletta Rut  2 , Ronald Holewinski  3 , Kiall F Suazo  3 , Hong-Rae Kim  1   4 , Mo Yang  1 , Thorkell Andresson  3 , Marcin Drag  2 , Euna Yoo  1
Affiliations

Chemical tools to define and manipulate interferon-inducible Ubl protease USP18

Griffin J Davis et al. bioRxiv. .

Abstract

Ubiquitin-specific protease 18 (USP18) is a multifunctional cysteine protease primarily responsible for deconjugating interferon-inducible ubiquitin-like (Ubl) modifier ISG15 from protein substrates. Here, we report the design and synthesis of activity-based probes (ABPs) capable of selectively detecting USP18 activity over other ISG15 cross-reactive deubiquitinases (DUBs) by incorporating unnatural amino acids into the C-terminal tail of ISG15. Combining with a ubiquitin-based DUB ABP, the selective USP18 ABP is employed in a chemoproteomic screening platform to identify and assess inhibitors of DUBs including USP18. We further demonstrate that USP18 ABPs can be utilized to profile differential activities of USP18 in lung cancer cell lines, providing a strategy that will help define the activity-related landscape of USP18 in different disease states and unravel important (de)ISGylation-dependent biological processes.

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Figures

Figure 1.
Figure 1.
Fluorogenic substrates for USP18. (A) Catalytic triad of USP18. The isopeptide bond between the C-terminal Gly residue of ISG15 and the Lys residue of the substrate protein is hydrolyzed by the USP18 protease activity. (B) Structure of USP18 complexed with ISG15 (PDB: 5CHV). Active site residues of USP18 that interact with conserved C-terminal LRLRGG of ISG15 are highlighted in comparison to other ISG15 reactive DUBs. (C) Structure of ISG15-based ACC-labeled substrate. The C-terminal LRGG motif is highlighted. (D) Schematic overview of HyCoSuL screening. (E) Substrate profiles presented as heatmaps indicating the percentage of relative cleavage measured by a fluorescent signal produced upon USP18 binding with two sub-libraries, Ac-Mix-P3-Gly-Gly-ACC and Ac-P4-Mix-Gly-Gly-ACC (where Mix represents an equimolar mixture of natural amino acids). (F) Structures (left panel) and rate of hydrolysis (right panel) of selected tetrapeptide fluorogenic substrates. mUSP18 (10 μM), USP2 (2 μM), and USP5 (1 μM) were incubated with 100 μM of Ac-LXGG-ACC substrates for 1 h at RT and initial release of fluorescent ACC (V0, RFU/s) by enzyme was measured at Ex: 360 nm / Em: 460 nm. Agb: 2-amino-4-guanidino-butyric acid, hArg: homoarginine, Phe(guan): guanidino-phenylalanine.
Figure 2.
Figure 2.
Preparation of mISG15CTD-based probes. (A) Synthesis of C-terminal domain of mouse ISG15 and ISG15 variants. Leu80~Gly154 sequence was assembled using SPPS and the N-terminal end was modified by either an acetyl group, fluorescent cyanine dye, or biotin affinity handle. After the cleavage from the solid support, the C-terminal end was functionalized with propargylamine (PA) replacing Gly155. Arg153 was varied with unnatural amino acids such as Agb (2-amino-4-guanidino-butyric acid), hArg (homoarginine), Phe(guan)(guanidine-phenylalanine). (B) LC-MS analysis of synthetic Ac-mISG15CTD-PA and Cy5-mISG15CTD-PA probes.
Figure 3.
Figure 3.
Reactivity and selectivity of mISG15CTD-based probes. (A-B) Recombinant mouse and human USP18 (2 μM) or USP5 (1 μM) were incubated with Ac-mISG15CTD-PA probes (10 μM) for 3 h at RT. Protein samples were analyzed by SDS-PAGE and SYPRO Ruby staining. (C) WT or C64/65A mutant of USP18-FLAG overexpressing HEK293T cell lysates were incubated with Cy5-mISG15CTD-PA probes at indicated concentrations for 3 h at RT. Protein samples were analyzed by SDS-PAGE and in-gel fluorescence scanning for Cy5 signal. Red marks indicate the appearance of new protein bands only in USP18WT overexpressing cell lysates after labeling by probes corresponding to the expected molecular weight of USP18–probe conjugate. Expression of USP18 was confirmed by western blotting. (D) Volcano (left) and protein rank (right) plots of quantitative proteomic analysis of streptavidin beads pulldowns after labeling of USP18-FLAG overexpressing HEK293T cell lysates by Biotin-mISG15CTD[R153Agb]-PA probe (5 μM, 3 h, RT) showing significantly enriched proteins (log2 ratio > 1, p-value ≤ 0.05). USP18 is marked and other DUBs are colored based on subfamilies.
Figure 4.
Figure 4.
USP18 inhibitor assay by competitive activity-based protein profiling. (A) Schematic workflow for quantitative activity-based protein profiling. Lysates were treated with DMSO or compound, followed by treatment with an activity-based DUB probe. Labeled proteins were enriched using biotin-streptavidin pull-down, digested, and analyzed by LC-MS/MS. (B) Chemical structure of WP1130. (C) Recombinant human USP18 (2 μM) was pretreated with WP1130 for 30 min and incubated with Ac-mISG15CTD[WT]-PA probe (10 μM) for 2 h at RT. Protein samples were analyzed by SDS-PAGE and SYPRO Ruby staining. (D) USP18WT-FLAG overexpressing HEK293T cell lysates were preincubated with WP1130 for 1 h followed by labeling with 1 μM of Cy5-Ubl-PA for 2 h at RT. Protein samples were analyzed by SDS-PAGE and in-gel fluorescence scanning for Cy5 signal. (E-G) USP18WT-FLAG overexpressing HEK293T cell lysates were preincubated with WP1130 for 2 h followed by labeling with 1 μM of Biotin-Ub-PA or 5 μM of Biotin-mISG15CTD[R153Agb]-PA or a cocktail of probes (1 μM of Biotin-Ub-PA + 5 μM of Biotin-mISG15CTD[R153Agb]-PA) for 3 h at RT. Protein samples were analyzed by SDS-PAGE and immunoblotting (E). Volcano plots of quantitative proteomic analysis of streptavidin beads pulldowns after pretreating cell lysates with either DMSO (F) or 100 μM of WP1130 (G) and labeling by a cocktail of probes. Significantly enriched proteins are shown (log2 ratio > 1, p-value ≤ 0.05). USP18 is marked and other DUBs are colored based on subfamilies.
Figure 5.
Figure 5.
USP18 expression and activity profiles in lung cancer cell lines. (A) Constitutive and induced USP18 expression levels upon IFN-β stimulation (48 h) across lung cancer cell lines. (B) Each cell lysates were incubated with 1 μM of Cy5-mISG15CTD[R153Agb]-PA probe for 3 h at RT. Left, protein samples were analyzed by SDS-PAGE and in-gel fluorescence scanning for Cy5 signal. Red marks indicate the appearance of USP18–probe conjugates. Expression of USP18 was confirmed by western blotting. Right, normalized intensity of fluorescent protein band corresponding to the USP18sf–probe conjugate.
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