Fig 2: Enhanced p-Ser65-Ub levels upon pharmacological inhibition of USP30 in Parkin heterozygote fibroblasts.(A) The Parkin+/+, Parkin+/R275W and ParkinR275W/R275Q fibroblasts were treated with 3 µM USP30Inh-1 for 6 days before inducing mitophagy with 10 µM FCCP for 2 h. Lysates were immunoblotted for total ubiquitin, p-Ser65-Ub and Mfn2. *indicates a non-specific band coming from the used p-Ser65-Ub antibody. (B) Same conditions as at (A) but TUBE enrichment was used to enhance the levels of poly-ubiquitinated proteins before conducting immunoblotting. The Parkin+/+, Parkin+/R275W and ParkinR275W/R275Q fibroblasts were treated with the indicated concentrations of USP30Inh-1 for (C) 6 h or (D) 6 days before inducing mitophagy with 10 µM FCCP for 2 h. The immunoreactivity of p-Ser65-Ub was quantified from three independent experiments for each condition. The expression levels were normalised to DMSO Parkin+/+ treated cells (+/+ = 100% and ParkinR275W/R275Q = 0%). (E) The Parkin+/+ fibroblasts were treated with the indicated concentrations of USP30Inh-1 for 1 day or 6 days. On the assay day, cells were incubated with 50 nM TMRM/200 nM MTG before imaging on the Opera Phenix. The intensity of TMRM from three independent experiments was quantified and no change was observed. (F) The Parkin+/+ fibroblasts were treated with the indicated concentrations of USP30Inh-1 for 6 h or 6 days. Cells were counted after staining for nuclei and average from three independent experiment was calculated. Reduced numbers of nuclei and therefore cells was observed only at 10 µM USP30Inh-1 after 6 days incubation.

Fig 3: USP30 inhibition increases p-Ser65-Ub in dopaminergic neurones and astrocytes.(A) Representative immunostaining of p-Ser65-Ub in a co-culture of dopaminergic neurones and astrocytes. Cells were co-cultured for 5 days before incubating with 3 µM USP30Inh-1 for 4 more days. Mitophagy was induced by adding 10 µM FCCP for 2 h. Cells were immunostained with TH, GFAP and p-Ser65-Ub. (B) Immunoreactivity levels of p-Ser65-Ub in the dopaminergic TH-positive neurones was quantified from three independent experiments. (C) Immunoreactivity levels of p-Ser65-Ub in the astrocytes-GFAP positive cells was quantified from three independent experiments. (D) The dopaminergic neurones/astrocytes co-cultures were treated with the indicated USP30Inh-1 concentrations for 4 days before 50 nM TMRM/200 nM Mitotracker green (MTG) were added and imaged with the Opera Phenix. Intensity of TMRM from three independent experiments was quantified. Data are pooled from three independent experiments. Error bars show means ± SD. * P < 0.05, ** P < 0.01, **** P < 0.0001. Data were analysed with One-Way ANOVA with Dunnett's test.

Fig 4: 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.

Fig 5: Pharmacological inhibition of USP30 enhances mitoKeima signal in SHSY5Y cells.(A) SHSY5Y cells were treated with the indicated USP30Inh-1 concentration for 1 h, 1 day and 3 days. On the assay day, fresh media containing the tested concentration as well as 50 nM TMRM was added and live images were acquired by the Opera Phenix. Fold change in TMRM intensity from three independent experiments was quantified. (B) SHSY5Y mitoKeima cells were treated with the indicated USP30Inh-1 concentration for 3 days. On the assay day, media was replaced with fresh medium containing fresh compound before inducing mitophagy with 1 µM A/O or 10 µM CCCP and live images were acquired with the Opera Phenix for a further 10 h. The mitophagy index was calculated as the fold change of the ratio of the total lysosomal red mitochondria area divided by the total cytoplasmic green mitochondria area from three independent experiments. Data are pooled from three independent experiments. Error bars show means ± SD. * P < 0.05, ** P < 0.01, *** P < 0.00, **** P < 0.0001. Data were analysed with Two-way ANOVA with Sidak's test.