Identification of an autoinhibitory, mitophagy-inducing peptide derived from the transmembrane domain of USP30.

ABSTRACT

The mitochondrial-anchored deubiquitinating enzyme USP30 (ubiquitin specific peptidase 30) antagonizes PRKN/parkin-mediated mitophagy, making it a potential target for treating Parkinson disease. However, few inhibitors targeting USP30 have been reported. Here, we report a novel peptide (Q14) derived from the transmembrane (TM) domain of USP30 that can target mitochondrial-anchored USP30 directly and increase mitophagy through two intriguing and distinct mechanisms a novel autoinhibition mechanism in USP30 and accelerated autophagosome formation via the LC3-interacting region (LIR) of the Q14 peptide. We identified the potential binding sites between the Q14 peptide and USP30 and postulated that an allosteric autoinhibition mechanism regulates USP30 activity. Furthermore, the LIR motif in the Q14 peptide offers additional binding with LC3 and accelerated autophagosome formation. The two mechanisms synergistically enhance mitophagy. Our work provides novel insight and direction to the design of inhibitors for USP30 or other deubiquitinating enzymes (DUBs).Abbreviations 3-MA 3-methyladenine; ATTEC autophagosome-tethering compound; BafA1 bafilomycin A1; BNIP3 BCL2 interacting protein 3; BNIP3L/NIX BCL2 interacting protein 3 like; CCCP carbonyl cyanide m-chlorophenyl hydrazone; DMSO dimethyl sulfoxide; FP fluorescence polarization; FUNDC1 FUN14 domain containing 1; HCQ hydroxychloroquine; LIR LC3-interacting region; MST microscale thermophoresis; mtDNA mitochondrial DNA; mtPA-GFP mitochondria-targeted photoactive fluorescence protein; OMM outer mitochondrial membrane; PINK1 PTEN induced kinase 1; PRKN/parkin parkin RBR E3 ubiquitin protein ligase; Rap rapamycin; SA streptavidin; TM transmembrane; Ub ubiquitin; Ub-AMC Ub-7-amido-4-methylcoumarin; UPS ubiquitin-protease system; USP ubiquitin specific peptidase; USP30 ubiquitin specific peptidase 30.