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

Autophagy. 2022 Sep;18(9):2178-2197. doi: 10.1080/15548627.2021.2022360. Epub 2022 Jan 6.

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.

Keywords: Autoinhibition; USP30; mitophagy; peptide inhibitor; transmembrane.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Apoptosis Regulatory Proteins / metabolism
  • Autophagy*
  • Carbonyl Cyanide m-Chlorophenyl Hydrazone
  • DNA, Mitochondrial
  • Mitophagy* / genetics
  • Proto-Oncogene Proteins c-bcl-2
  • Ubiquitin
  • Ubiquitin-Protein Ligases / metabolism
  • Ubiquitin-Specific Proteases

Substances

  • Apoptosis Regulatory Proteins
  • DNA, Mitochondrial
  • Proto-Oncogene Proteins c-bcl-2
  • Ubiquitin
  • Carbonyl Cyanide m-Chlorophenyl Hydrazone
  • Ubiquitin-Protein Ligases
  • Ubiquitin-Specific Proteases

Grants and funding

This work was supported by the China Postdoctoral Science Foundation [2021M690220]; Natural Science Foundation of China [21778009]; Natural Science Foundation of China [21977010]; Natural Science Foundation of Guangdong Province [2020A1515010522]; Natural Science Foundation of Guangdong Province [2019A1515111184]; National Key Research and Development Program Synthetic Biology Key Special Project of China [2018YFA0902504]; Shenzhen Science and Technology Innovation Committee [JCYJ20170817161908969]; Shenzhen Science and Technology Innovation Committee [JCYJ20180507181527112]; Shenzhen Science and Technology Innovation Committee [JCYJ20180508152213145]; Shenzhen Science and Technology Innovation Committee [JCYJ20170817172023838]; Beijing National Laboratory of Molecular Science open grant [BNLMS20160112]; Shenzhen-Hong Kong Institute of Brain Science-Shenzhen Fundamental Research Institutions grant [2019SHIBS0004]; Natural Science Foundation of Guangdong Province [2019A1515110487]; Shenzhen Science and Technology Innovation Committee [RCJC20200714114433053].