Article Figures & Data
Figures
- Figure 1. CMPD-39 is a selective USP30 inhibitor.
(A) Chemical structure of CMPD-39. (B) DUB specificity screen (DUB profiler, Ubiquigent) with 1-100 μM CMPD-39. (C) Activity-based ubiquitin probe assay shows that CMPD-39 engages USP30 in cells at nanomolar concentrations in intact SHSY5Y cells. Samples were incubated with CMPD-39 for 2 h at the indicated concentrations, then incubated with HA-Ub-PA probe for 10 min at 37°C and immunoblotted as shown. Red arrow indicates unbound USP30; blue arrow represents probe bound USP30. (D) Inhibition of USP30 enhances the ubiquitylation of TOMM20 in YFP-PRKN over-expressing hTERT-RPE1 cells in a concentration dependent manner in response to mitophagy induction. Cells were treated for 1 h with DMSO or antimycin A and oligomycin A (AO; 1 μM each) in the absence or presence of CMPD-39 at the indicated concentrations, lysed, and analyzed by Western blotting. Black arrow indicates unmodified TOMM20, ubiquitylated species are indicated by red (mono-ubiquitylated) or blue arrow heads. Quantitation shows percentage mono-ubiquitylated.
Source data are available for this figure.
Source Data for Figure 1[LSA-2021-01287_SdataF1.xlsx]
- Figure 2. CMPD-39 promotes depolarization-dependent ubiquitylation of previously described USP30 substrates.
(A) USP30 inhibitor (CMPD-39) treatment of Parental (PAR) hTERT-RPE1-YFP-PRKN cells phenocopies USP30 deletion (KO1E) by promoting TOMM20 degradation, whereas TOMM20 degradation is unaffected by CMPD-39 in the USP30 KO (KO1E) cells. Cells were treated for 4 h ± AO (1 μM) ± 200 nM CMPD-39. (B) TOMM20 and SYNJ2BP ubiquitylation is enhanced within 1 h by CMPD-39 but is unaffected in the USP30 KO (KO1E) cells. Cells were treated for 1 h ± AO (1 μM) ± 200 nM CMPD-39. (C, D) Graphs show quantification of mono-ubiquitylated TOMM20 and SYNJ2BP in AO-treated samples as a percentage of total for two independent experiments (error bars indicate the range). (E) USP30 inhibitor (CMPD-39) treatment of cells expressing endogenous PRKN (SHSY5Y cells) similarly phenocopies USP30 deletion (KO11) by promoting TOMM20 and SYNJ2BP ubiquitylation, as well as increasing levels of phospho-Ser65 Ubiquitin (pUb). Cells were treated for 4 h ± AO (1 μM) in the absence or presence of 1 μM CMPD-39. (E, F, G) Graphs show quantification of AO treated samples for mono-ubiquitylated TOMM20 as a percentage of total TOMM20 (F) and the pUb signal normalised to AO treated SHSY5Y cells (G) in the 38–76 kD range, for two independent experiments represented by (E) (error bars indicate the range). (B, E): Black arrow indicates unmodified species, and red and blue arrow heads indicate the mono- and multi-ubiquitylated species, respectively.
Source data are available for this figure.
Source Data for Figure 2[LSA-2021-01287_SdataF2.1.xlsx][LSA-2021-01287_SdataF2.2.pdf][LSA-2021-01287_SdataF2.3.pdf]
- Figure 3. Enhancement of basal mitophagy by pharmacological inhibition of USP30.
(A) Representative images of SHSY5Y-mitoQC cells. Cells were treated with DMSO or CMPD-39 (200, 500, or 1,000 nM) for 96 h before imaging. Scale bar 20 μm. (B, C) Quantification of the data from three independent experiments is shown. Median and interquartile range are indicated; 65 cells were quantified per condition. (B) Graph illustrates the number of mitolysosomes. **P < 0.01, One-way ANOVA with Dunnett’s multiple comparisons test. (C) Graph shows the mean mitolysosome area per cell. *P < 0.05, **P < 0.01, one-way ANOVA with Dunnett’s multiple comparisons test. (D) Mitophagy index for patient derived dopaminergic iNeurons derived from two control individuals and two individuals with PRKN loss of function mutations. CMPD-39 was administered for 24 h before measurement. Error bars indicate SEM. Two-way ANOVA with Tukey’s multiple-comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (E) Mitochondrial membrane potential in dopaminergic neurons derived from control and PRKN patients. CMPD-39 was administered for 24 h before measurement. Error bars indicate SEM. Data are normalised to gender matched untreated control. Statistical analysis was conducted on raw non-normalised data using Mixed effect analysis with Tukey’s multiple-comparisons test. *P < 0.05, **P < 0.01.
Source data are available for this figure.
Source Data for Figure 3[LSA-2021-01287_SdataF3.xlsx]
- Figure 4. Enhancement of basal pexophagy by pharmacological inhibition of USP30.
(A) Representative images of U2OS-Keima-SKL cells. Cells were treated with DMSO or CMPD-39 (200, 500 or 1,000 nM) for 96 h before imaging. Scale bar 20 μm. (B, C) Quantification of the data is derived from three independent experiments. Median and interquartile range are indicated; 85 cells were quantified per condition. (B) Graph indicates the number of pexolysosomes in U2OS-Keima-SKL cells. One-way ANOVA with Dunnett’s multiple comparisons test, ****P < 0.0001. (C) Graph indicates the mean pexolysosome area per cell. One-way ANOVA with Dunnett’s multiple-comparisons test, ****P < 0.0001.
Source data are available for this figure.
Source Data for Figure 4[LSA-2021-01287_SdataF4.xlsx]
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