Fig 2: Characterization of Fubi-based probes.a. Schematic of the chemoproteomics experimental workflow used to identify Fubi proteases. b. Characterization of indicated samples by intact protein mass spectrometry. HA-Fubi-MesNa and derived proteins featured Cys57 mutations to suppress reaction of the thiol with the VS warhead during probe assembly. Analogously mutated and wild-type HA-Fubi-PA probes were prepared for comparison. *, species with a C-terminal carboxylate which was separated during purification. c. Overview of different versions of HA-tagged Fubi probes used in this work. Source data

Fig 3: Molecular basis for Fubi/ubiquitin cross-reactivity.a, Average distance clustering of sequences from the shown human ubiquitin and Ubl modifier proteins; CTD, C-terminal Ubl domain. b, Sequences of ubiquitin and closely related Ubl modifiers. Numbering and secondary structure elements are shown according to Fubi. Numbers of equivalent residues in ubiquitin and other Ubl modifiers are shifted by two after the two-amino-acid-long deletion near the N terminus of Fubi. Black arrowheads indicate residues critical for Fubi recognition by USP DUBs, as validated by mutation. c, Cartoon representations of ubiquitin (left), Fubi (middle) and ISG15 (right) in their USP-engaged conformations. Various residues are shown as sticks and with the following labels: C-terminal tail residues (Leu 71, Arg 72, Leu 73 and Arg 74 for ubiquitin), hydrophobic patch residues (Phe 4, Ile 36 and Ile 44 together with Gln 49, Arg 42 and Val 70 for ubiquitin) and ubiquitin core residues (Ile 13 and Leu 15). Equivalent residues critical for ubiquitin/Ubl modifier recognition are shown in highlighted areas. d, Cartoon representation of USP36 in complex with ubiquitin and Fubi showing the area around the a5 helix that engages the Ile 44 (ubiquitin)/Leu 42 (Fubi) patches. e–g, Zoom-in images for the latter area for USP36 in complex with ubiquitin-PA (e), USP36 in complex with Fubi-PA (f) and mouse USP18 in complex with ISG15-PA15 (g); PDB, Protein Data Base. Hydrogen bonds are indicated as dotted lines. h, Sequence alignment of the a5 helix of the indicated human USP DUBs grouped according to ubiquitin/Ubl modifier cleavage activity as indicated by ‘+’ and ‘–’. The prospective Fubi recognition motif RY and Fubi activity are highlighted in purple. i, Probe reactivity assay for the indicated USP16 proteins analyzed by SDS–PAGE and Coomassie staining. j, Probe reactivity assay for the indicated USP36 proteins as in i. k, Probe reactivity assay for the indicated USP42 proteins as in i.Source data

Fig 4: Chemoproteomic identification of USP16 and USP36 as Fubi probe-cross-reactive DUBs that cleave Fubi-S30 in cells.a, Schematic representation of the roles of DUBs and Fubi protease/deFubiylase enzymes in protein precursor maturation (top) and processing of protein conjugates (bottom). Names of genes are given in italics; Ub, ubiquitin. b, Workflow of Fubi probe semisynthesis for Fubi protease trapping. C-terminally functionalized HA–Fubi species were obtained through expressed protein ligation and native purification. Candidate enzymes were trapped in lysates and enriched on beads, followed by identification by MS; CBD, chitin-binding domain. c, Chemoproteomic identification of HA–FubiC57A-VS-bound proteins from HeLa cell lysates. The volcano plot shows the relative label-free abundance ratio (fold change) of proteins detected in samples with or without cysteine alkylation through IAA pretreatment. USP16 and USP36 as the only hits with annotated peptidase domains are highlighted. Other significantly enriched proteins include enzymes with hyperreactive cysteines. d, Assessment of USP16 and USP36 probe reactivity. Flag-tagged full-length USP16 and USP36 were overexpressed in HEK293 cells as wild-type (WT) or catalytic cysteine mutants. Lysates were incubated with the indicated probes for 1 h at 37 °C, and reactivity was assessed by western blotting. e, Assessment of HA–Fubi-S30–Flag accumulation after depletion of USP16 and USP36. Proteins were depleted in HEK293 cells for 24 h, followed by overexpression of HA–Fubi-S30–Flag for another 24 h; siScr, scrambled control siRNA. f, Quantification of HA–Fubi-S30–Flag intensity in samples described in e; FAU, Fubi-S30. Values shown correspond to the mean intensities of HA bands of four biologically independent experiments; standard deviations are indicated. Statistical significance was analyzed using individual one-sample, two-tailed t-tests comparing to the hypothetical mean of 1 as set for siUSP36 samples; *P = 0.05; **P = 0.01. Additional details on the analysis are given in Statistics and reproducibility. g, Assessment of endogenous Fubi-S30 accumulation after USP36 and USP16 depletion in HEK293 cells after 48 h; USP7 was depleted as a control. h, Quantification of Fubi-S30 intensity in samples described in g. Values shown correspond to the mean of four biologically independent experiments; standard deviations are indicated. Statistical significance was analyzed as in f; **P = 0.01; NS, not significant.Source data

Fig 5: Assessment of molecular determinants of Fubi/Ub specificity.a. Sequence alignment of protein sequences of human Fubi and selected orthologues with secondary structure elements indicated according to the structure of free Fubi. b. Characterization of mutant Fubi-PA probes by intact protein mass spectrometry. c.-e. Ub/Ubl-PA probe reactivity assay. Wild-type Ub-PA, wild-type Fubi-PA, wild-type ISG15-PA (only for USP18) and mutant Fubi-PA probes were incubated with recombinant USP45 (c), USP18 (d) and USP28 (e). Uncropped gels are provided as Source Data. Source data