Fig. 1: Design, characterization and inhibition of chimeric USP30.
a, USP30 antagonizes PINK1–parkin-mediated mitophagy. MOMP, mitochondrial outer membrane protein. b, Chemical structures of a covalent and a non-covalent USP30 inhibitor. Inhibition of USP30 to enhance mitochondrial quality control is explored as a therapeutic strategy for Parkinson’s and kidney diseases. ABPP, activity-based protein profiling. c, Crystal structure of human USP30 obtained with a previously engineered construct as the Ub-PA complex (PDB 5OHK). USP subdomains are shown in different colors. d, Architecture of full-length human USP30, the previously used c1 (named c13 in ref. 37) and the USP30 ch3 described here. See Extended Data Fig. 2 for other chimeras. e, AlphaFold2-predicted model of USP30c1 (top left), crystal structures of the catalytic domains of USP14 (PDB 2AYN, top right) and USP35 (PDB 5TXK, bottom left) and AlphaFold2-predicted model of USP30 ch3 (bottom right). Regions used for grafting are shown in corresponding colors. f, Catalytic efficiencies of the indicated USP30 constructs, determined from Ub–RhoG cleavage assays. See Extended Data Fig. 3 for raw data. Mean ± s.e.m. (derived from curve fitting). g, Stability assessment of USP30 constructs in their apo states derived from thermal shift assays (TSAs). Tm, protein melting temperature. Mean (n = 3 independent replicates). h, Changes in protein stability upon binding to the ubiquitin probe Ub-PA. Mean ± s.d. (n = 3 independent replicates). i, Gel-based Ub-PA binding assay. j, Crystal structure of USP30ch3~Ub-PA. Regions within the chimeric USP30 construct derived from different USP DUBs are shown in blue (USP30), orange (USP14) and red (USP35). The Ub-PA probe is shown in yellow. See Table 1 for statistics. k, Structure of NK036, a solubility-enhanced derivative of compound 39. l, Inhibitory potencies of NK036 for the indicated USP30 constructs. IC50 values are given as mean ± s.e.m. (derived from curve fitting, with activity data for each concentration recorded as n = 3 independent replicates and shown as mean ± s.d.). m, Protein stability of the indicated USP30 constructs in the presence of NK036. Mean values are shown (n = 3 independent replicates). Panel a created with BioRender.com.
