Fig 1: Optimizing the ABPP-HT workflow. (A). Number of DUBS identified by timsTOF MS with increasing amounts of HA-Ub-PA-labelled MCF-7 lysate protein, after immunoprecipitation and elution with 0.15% TFA. (B). Western blot densitometry quantification (full blot in Figure S2D) of USP7 in the immunoprecipitation loading flow-through, with increasing amounts of HA-Ub-PA-labelled MCF-7 lysate protein quantity immunoprecipitated and eluted with 0.15% TFA. (C). Number of DUBs identified by LC-MS/MS with different IP elutions: 0.15% TFA, 6 M urea, HEPES, * = On column digestion. (D). Log2 intensities of DUBS identified with different elution methods by a QE orbitrap MS. 0.15% TFA, 6 M urea, HEPES, * = On column digestion. (E). Log2 intensities of DUBs identified with different elution methods by a timsTOF MS. 0.15% TFA, 6 M urea, 5% SDS. F. USP30 immunoblots showing mouse brain lysate displacement of a covalent (3-b) and non-covalent (39) USP30 inhibitor with increasing HA-Ub-PA (at 37°C) incubation times. (G). The densitometric quantification of Figure 3F from the intensity of the HA-Ub-PA-labelled band, normalized to the intensity of both USP30 bands together. (H). timsTOF DUB intensities of MCF-7 labeled with HA-Ub-PA for 10 min normalized to 45 min at 37°C (SEM, n = 3). I. Optimization workflow for high-throughput DUB inhibitor screening using ABPP LC-MS/MS.
Fig 2: ABPP‐HT reveals DUB inhibitor selectivity and specificity compatible with higher throughput. (A). The activity of a panel of DUBs from MCF‐7 identified from timsTOF MS, in response to USP7 specific inhibitors FT671 (n 3 (for 0.2 μM n 2)), FT827, HBX108 and P22077. (B). The activity of a panel of DUBs from mouse brain lysate identified from timsTOF MS, in response to USP30 specific inhibitors 3‐b and 39. (C). The activity of a panel of DUBs in MCF‐7 lysates identified by timsTOF LC‐MS/MS, in response to the cysteine modifier NEM, and broad spectrum DUB inhibitor PR619 (PR619 n 2). D‐I. From left to right concentration dependences of USP7 from inhibitors FT671, FT827, HBX41108, and P22077 in MCF‐7 lysates, and USP30 inhibitors for 3‐b and 39 in mouse brain. (J). IC50 values extracted from D‐I, fit to equation: Y 100/(1 + X/IC50). * normalized raw intensities, not LFQ intensities.
Fig 3: USP30 promotes c-Myc deubiquitination. A Interaction between USP30 and c-Myc in HSC4 cells. c-Myc expression in B HSC4 and C SCC4 cells with or without USP30 knockdown and overexpression. c-Myc levels in HSC4 cells with USP30 knockdown with or without D protease inhibitor MG132 (10 μM) or E CHX treatment. F Effect of USP30 overexpression on c-Myc ubiquitination in SCC4 cells transfected with WT-USP30, C77S-mutant USP30, or vector. G Effect of USP30 knockdown on c-Myc ubiquitination in HSC4 cells
Fig 4: USP30 overexpression increased SCC4 cell viability and glutamine consumption, and inhibits apoptosis. SCC4 cells were transfected with WT-USP30, C77S-mutant USP30, or vector, and (A) USP30 expression, (B) cell viability, (C, D) apoptosis, (E) glutamine consumption, and (F) expression of c-Myc, GLS1, and SLC1A5 were measured. *P < 0.05, ***P < 0.001
Fig 5: USP30 upregulation is associated with poor prognosis. A mRNA and B protein levels of USP30 in stage 1 or 2 OSCC tissues (I/II), stage 3 OSCC tissues (III), and adjacent nontumor tissues (N). C USP30 protein levels in human OSCC tissue microarrays detected by IHC (scale bars: 100 µm). D Overall survival rate analysis. E USP30 expression in human OSCC cell lines and oral epithelial cell HOEC. **P < 0.01, ***P < 0.001
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