Fig. 6: BIO-2007817 rescues parkin Ubl mutations in organello and in cells.

a Schematic of in organello ubiquitination assay of parkin. PINK1 expression is induced in cultured cells by CCCP and parkin activity measured by ubiquitination of mitofusin-2 (Mfn2). b Immunoblots of in organello assays showing the rescue of ubiquitination activity of the non-phosphorylatable parkin mutants, S65A and R0RBR, by BIO-2007817. Quantification of the percentage of ubiquitinated Mfn2 is shown as chart bar. c Immunoblots of in organello assays showing the rescue of ubiquitination activity of Parkinson’s disease parkin mutants, R42P and V56E, by BIO-2007817, but not by the diastereomer, BIO-2007818. Quantification of the percentage of ubiquitinated Mfn2 is shown as chart bar. d Quantification of average percentage of mitophagy detected by fluorescence‐activated cell sorting (FACS) of mitochondrially targeted mitoKeima U2OS cells expressing transient WT or mutant GFP‐parkin. Cells were treated with DMSO (white), CCCP (black), or pre-treated with BIO-2007817 followed by CCCP treatment (gray). Percentages were normalized to WT Parkin pre-treated with DMSO followed by CCCP treatment. A partial rescue by BIO-2007817 was observed in cells expressing parkin with defective Ubl. Error bars indicate s.e.m. For statistical analysis, a one‐way ANOVA with Tukey’s post‐test was performed. The number of independent, biological replicates varied between 5 and 23. ****P < 0.0001; ***P = 0.0004; ns, not significant. e Model for THPP activation of parkin with a defective Ubl ___domain.