Fig. 5: Cellular evaluation of compound-resistant USP30 mutations.
a, Mitochondrial ubiquitination analysis. HeLa cells expressing YFP–parkin were treated with USP30 inhibitors for 19 h where indicated. Mitophagy was induced with carbonyl cyanide m-chlorophenyl hydrazone (CCCP) for 1 h. Ubiquitinated proteins were enriched through pulldowns with OtUBD, and samples were analyzed by western blot. Cmpd, compound; IB, immunoblot. b, Target engagement assay with endogenous USP30. HEK293 cells were treated with the indicated compounds. Lysates were then incubated with ubiquitin probe where indicated and analyzed by western blot for USP30. The asterisk denotes an unspecific band. c, Cellular assessment of the USP30 inhibition mechanism. C-terminally Flag-tagged USP30 (wild type or with the compound-resistant mutation F453Y) was overexpressed in HEK293 cells. Cells were analyzed as described in b, with a western blot for the Flag tag. d, Catalytic activities of additional USP30 mutants, assessed by Ub–RhoG cleavage as described for Fig. 4e. e, Protein stability of the indicated USP30 proteins by NK036 as described for Fig. 4f. f, Cellular probe competition assay as described in c with mutations characterized in d and e. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
