2. Academic Validation
  3. Activation of endogenous PRKN by structural derepression is linked to increased turnover of the E3 ubiquitin ligase

Activation of endogenous PRKN by structural derepression is linked to increased turnover of the E3 ubiquitin ligase

  • Autophagy. 2025 Jul 18:1-21. doi: 10.1080/15548627.2025.2531025.
Fabienne C Fiesel 1 2,  Bernardo A Bustillos 1,  Jens O Watzlawik 1,  Carol X Q Chen 3 4,  Martin H Berryer 3 4,  Jiazhen Zhang 5,  Paige K Boneski 1,  Caleb S Hayes 1,  Jenny M Bredenberg 1,  Eric Deneault 3 6,  Zhipeng You 3 4,  Narges Abdien 3 4,  Nathalia Aprahamian 3 4,  Taylor M Goldsmith 3 4,  Zahra Baninameh 1,  Liam T Cocker 1,  Haonan Zhang 1,  Matthew S Goldberg 7,  Edward A Fon 3 8,  Jean-François Trempe 8 9 10 11,  Satpal Virdee 5,  Thomas M Durcan 3 4,  Wolfdieter Springer 1 2
Affiliations

Affiliations

  • 1 Department of Neuroscience, Mayo Clinic, Jacksonville, FL, USA.
  • 2 Neuroscience PhD Program, Mayo Graduate School of Biomedical Sciences, Mayo Clinic, Jacksonville, FL, USA.
  • 3 McGill Parkinson Program, Neurodegenerative Diseases Group, Department of Neurology and Neurosurgery, Montreal Neurological Institute-Hospital, McGill University, Montreal, Québec, Canada.
  • 4 The Neuro's Early Drug Discovery Unit, McGill University, Montreal, Canada.
  • 5 MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee, UK.
  • 6 Centre for Oncology, Radiopharmaceuticals and Research (CORR), Biologic and Radiopharmaceutical Drugs Directorate (BRDD), Health Products and Food Branch (HPFB), Ottawa, Canada.
  • 7 Center for Neurodegeneration and Experimental Therapeutics, Department of Neurology, University of Alabama at Birmingham, Birmingham, AL, USA.
  • 8 Structural Genomics Consortium, McGill University, Montréal, Canada.
  • 9 Department of Pharmacology & Therapeutics, McGill University, Canada.
  • 10 Centre de Recherche en Biologie Structurale, McGill University, Montréal, Montréal, Canada.
  • 11 Brain Repair and Integrative Neuroscience (BRaIN) Program, Research Institute of the McGill University Health Centre, Montreal, Canada.
PMID: 40624741 DOI: 10.1080/15548627.2025.2531025
Abstract

Loss-of-function mutations in the PINK1 and PRKN genes are the most common cause of early-onset Parkinson disease (PD). The encoded enzymatic pair selectively identifies, labels, and targets damaged mitochondria for degradation via the macroautophagy/autophagy-lysosome system (Mitophagy). This pathway is cytoprotective and efforts to activate Mitophagy are pursued as therapeutic avenues to combat PD and Other neurodegenerative disorders. When mitochondria are damaged, the ubiquitin kinase PINK1 accumulates and recruits PRKN from the cytosol to activate the E3 ubiquitin Ligase from its auto-inhibited conformation. We have previously designed several mutations that effectively derepress the structure of PRKN and activate its enzymatic functions in vitro. However, it remained unclear how these PRKN-activating mutations would perform endogenously in cultured neurons or in vivo in the brain. Here, we gene-edited neural progenitor cells and induced pluripotent stem cells to express PRKN-activating mutations in dopaminergic cultures. All tested PRKN-activating mutations indeed enhanced the enzymatic activity of PRKN in the absence of exogenous stress, but their hyperactivity was linked to their own PINK1-dependent degradation. Strikingly, in vivo in a mouse model expressing an equivalent activating mutation, we find the same relationship between PRKN enzymatic activity and protein stability. We conclude that PRKN degradation is the consequence of its structural derepression and enzymatic activation, thus resulting only in a temporary gain of activity. Our findings imply that pharmacological activation of endogenous PRKN will lead to increased turnover and suggest that additional considerations might be necessary to achieve sustained E3 ubiquitin Ligase activity for disease treatment.Abbreviations: BSA: bovine serum album, CCCP: carbonyl cyanide 3-chlorophenylhydrazone; ECL: electrochemiluminescence; EGF: epidermal growth factor; ELISA: enzyme-linked immunosorbent assay; FGF: fibroblast growth factor; iPSC: induced pluripotent stem cell; KI: knock-in; KO: knockout; MAP2: microtubule associated protein 2; MFN2: mitofusin 2; MSD: Meso Scale Discovery; mt-Keima: mitochondrial targeted Keima; NPC: neural progenitor cell; PD: Parkinson disease; PDH: pyruvate dehydrogenase; p-S65-PRKN: Serine 65 phosphorylated PRKN; p-S65-Ub: Serine 65 phosphorylated ubiquitin; REP: repressor element of PRKN; TH: tyrosine hydroxylase; TX: Triton X-100, Ub: ubiquitin; UBL: ubiquitin-like; WT: wild-type.

Keywords

Autophagy; PINK1; Parkin; Parkinson’s disease; mitophagy.

Figures
Products