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Cited by 42Parkinson's Disease Mechanisms and TreatmentsAutophagy in Disease and TherapyNuclear Receptors and Signaling

Mapping of a N-terminal α-helix domain required for human PINK1 stabilization, Serine228 autophosphorylation and activation in cells

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Kakade, Poonam, Ojha, Hina, Raimi, Olawale G., Shaw, Andrew, Waddell, Andrew D., Ault, James R., Burel, Sophie, Brockmann, Kathrin +7 more·2022·Open Biology
DOI 10.1098/rsob.210264PMC PMC8767193

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Abstract

Autosomal recessive mutations in the PINK1 gene are causal for Parkinson's disease (PD). PINK1 encodes a mitochondrial localized protein kinase that is a master-regulator of mitochondrial quality control pathways. Structural studies to date have elaborated the mechanism of how mutations located within the kinase domain disrupt PINK1 function; however, the molecular mechanism of PINK1 mutations located upstream and downstream of the kinase domain is unknown. We have employed mutagenesis studies to define the minimal region of human PINK1 required for optimal ubiquitin phosphorylation, beginning at residue Ile111. Inspection of the AlphaFold human PINK1 structure model predicts a conserved N-terminal α-helical extension (NTE) domain forming an intramolecular interaction with the C-terminal extension (CTE), which we corroborate using hydrogen/deuterium exchange mass spectrometry of recombinant insect PINK1 protein. Cell-based analysis of human PINK1 reveals that PD-associated mutations (e.g. Q126P), located within the NTE : CTE interface, markedly inhibit stabilization of PINK1; autophosphorylation at Serine228 (Ser228) and Ubiquitin Serine65 (Ser65) phosphorylation. Furthermore, we provide evidence that NTE and CTE domain mutants disrupt PINK1 stabilization at the mitochondrial Translocase of outer membrane complex. The clinical relevance of our findings is supported by the demonstration of defective stabilization and activation of endogenous PINK1 in human fibroblasts of a patient with early-onset PD due to homozygous PINK1 Q126P mutations. Overall, we define a functional role of the NTE : CTE interface towards PINK1 stabilization and activation and show that loss of NTE : CTE interactions is a major mechanism of PINK1-associated mutations linked to PD.

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  • Mitochondrial depolarization stabilizes PINK1 by arresting its import at the TOM complex, preventing turnover and allowing accumulation of full-length, dimerized PINK1 on the outer mitochondrial membrane.

    “Current models suggest that mitochondrial depolarization stabilizes PINK1 by arresting its import at the TOM complex, thereby preventing turnover and allowing accumulation of full-length, dimerized PINK1 on the outer mitochondrial membrane [59–62].”

    From miRNA family miR-29 inhibits PINK1-PRKN signaling via ATG9A (2026) · cites this paper as [61]

    The Introduction says that mitochondrial depolarization stabilizes full-length PINK1 at mitochondria, and the Discussion links stabilization to reduced proteolysis and association with the TOM complex (page numbers unavailable). The Results also report reduced cleavage after depolarization. Our copy does not establish that PINK1 accumulates specifically as a dimer on the outer mitochondrial membrane or that its import is arrested at the TOM complex. (LLM, conf=0.85)

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References 34 · Cited by 42

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  1. [1]Valente EM et al. 2004 Hereditary early-onset Parkinson's disease caused by mutations in PINK1. Science 304, 1158- 1160. (10.1126/science.1096284) 15087508 · DOI · Paper page
  2. [2]Woodroof HI et al. 2011 Discovery of catalytically active orthologues of the Parkinson's disease kinase PINK1: analysis of substrate specificity and impact of mutations. Open Biol. 1, 110012. (10.1098/rsob.110012) 22645651 PMC3352081 · DOI
  3. [3]Yamano K, Youle RJ. 2013 PINK1 is degraded through the N-end rule pathway. Autophagy 9, 1758- 1769. (10.4161/auto.24633) 24121706 PMC4028335 · DOI
  4. [4]Kondapalli C et al. 2012 PINK1 is activated by mitochondrial membrane potential depolarization and stimulates Parkin E3 ligase activity by phosphorylating Serine 65. Open Biol. 2, 120080. (10.1098/rsob.120080) 22724072 PMC3376738 · DOI · Paper page
  5. [5]Harper JW, Ordureau A, Heo JM. 2018 Building and decoding ubiquitin chains for mitophagy. Nat. Rev. Mol. Cell Biol. 19, 93- 108. (10.1038/nrm.2017.129) 29358684 · DOI
  6. [6]Moehlman AT, Youle RJ. 2020 Mitochondrial quality control and restraining innate immunity. Annu. Rev. Cell Dev. Biol. 36, 265- 289. (10.1146/annurev-cellbio-021820-101354) 33021820 · DOI
  7. [7]Singh PK, Muqit MMK. 2020 Parkinson's: a disease of aberrant vesicle trafficking. Annu. Rev. Cell Dev. Biol. 36, 237- 264. (10.1146/annurev-cellbio-100818-125512) 32749865 · DOI
  8. [8]Lai YC et al. 2015 Phosphoproteomic screening identifies Rab GTPases as novel downstream targets of PINK1. EMBO J. 34, 2840- 2861. (10.15252/embj.201591593) 26471730 PMC4654935 · DOI
  9. [9]Kumar A et al. 2017 Structure of PINK1 and mechanisms of Parkinson's disease-associated mutations. Elife 6, e29985. (10.7554/eLife.29985) 28980524 PMC5679756 · DOI
  10. [10]Schubert AF, Gladkova C, Pardon E, Wagstaff JL, Freund SMV, Steyaert J, Maslen SL, Komander D. 2017 Structure of PINK1 in complex with its substrate ubiquitin. Nature 552, 51- 56. (10.1038/nature24645) 29160309 PMC6020998 · DOI
  11. [11]Lazarou M, Jin SM, Kane LA, Youle RJ. 2012 Role of PINK1 binding to the TOM complex and alternate intracellular membranes in recruitment and activation of the E3 ligase Parkin. Dev. Cell 22, 320- 333. (10.1016/j.devcel.2011.12.014) 22280891 PMC3288275 · DOI · Paper page
  12. [12]Okatsu K, Uno M, Koyano F, Go E, Kimura M, Oka T, Tanaka K, Matsuda N. 2013 A dimeric PINK1-containing complex on depolarized mitochondria stimulates Parkin recruitment. J. Biol. Chem. 288, 36 372- 36 384. (10.1074/jbc.M113.509653) PMC3868751 24189060 · DOI
  13. [14]Fell MJ et al. 2015 MLi-2, a potent, selective, and centrally active compound for exploring the therapeutic potential and safety of LRRK2 kinase inhibition. J. Pharmacol. Exp. Ther. 355, 397- 409. (10.1124/jpet.115.227587) 26407721 · DOI
  14. [15]Prestel J et al. 2008 Clinical and molecular characterisation of a Parkinson family with a novel PINK1 mutation. J. Neurol. 255, 643- 648. (10.1007/s00415-008-0763-4) 18286320 · DOI
  15. [16]McWilliams TG et al. 2018 Phosphorylation of Parkin at serine 65 is essential for its activation in vivo. Open Biol. 8, 180108. (10.1098/rsob.180108) 30404819 PMC6282074 · DOI
  16. [17]Hjerpe R, Aillet F, Lopitz-Otsoa F, Lang V, England P, Rodriguez MS. 2009 Efficient protection and isolation of ubiquitylated proteins using tandem ubiquitin-binding entities. EMBO Rep. 10, 1250- 1258. (10.1038/embor.2009.192) 19798103 PMC2775171 · DOI
  17. [18]Koyano F et al. 2014 Ubiquitin is phosphorylated by PINK1 to activate Parkin. Nature 510, 162- 166. (10.1038/nature13392) 24784582 · DOI · Paper page
  18. [19]Cornwell O, Radford SE, Ashcroft AE, Ault JR. 2018 Comparing hydrogen deuterium exchange and fast photochemical oxidation of proteins: a structural characterisation of wild-type and DeltaN6 beta2-Microglobulin. J. Am. Soc. Mass Spectrom. 29, 2413- 2426. (10.1007/s13361-018-2067-y) 30267362 PMC6276068 · DOI
  19. [20]Cryar A, Groves K, Quaglia M. 2017 Online hydrogen-deuterium exchange traveling wave ion mobility mass spectrometry (HDX-IM-MS): a systematic evaluation. J. Am. Soc. Mass Spectrom. 28, 1192- 1202. (10.1007/s13361-017-1633-z) 28374315 PMC5438439 · DOI
  20. [21]Narendra DP, Jin SM, Tanaka A, Suen D-F, Gautier CA, Shen J, Cookson MR, Youle RJ. 2010 PINK1 is selectively stabilized on impaired mitochondria to activate Parkin. PLoS Biol. 8, e1000298. (10.1371/journal.pbio.1000298) 20126261 PMC2811155 · DOI · Paper page
  21. [23]Mukhopadhyay A, Yang CS, Weiner H. 2006 Binding of mitochondrial leader sequences to Tom20 assessed using a bacterial two-hybrid system shows that hydrophobic interactions are essential and that some mutated leaders that do not bind Tom20 can still be imported. Protein Sci. 15, 2739- 2748. (10.1110/ps.062462006) 17088320 PMC2242433 · DOI
  22. [24]Muqit MM et al. 2006 Altered cleavage and localization of PINK1 to aggresomes in the presence of proteasomal stress. J. Neurochem. 98, 156- 169. (10.1111/j.1471-4159.2006.03845.x) 16805805 · DOI
  23. [25]Okatsu K, Kimura M, Oka T, Tanaka K, Matsuda N. 2015 Unconventional PINK1 localization to the outer membrane of depolarized mitochondria drives Parkin recruitment. J. Cell Sci. 128, 964- 978. (10.1242/jcs.161000) 25609704 PMC4342580 · DOI
  24. [26]Sekine S, Wang C, Sideris DP, Bunker E, Zhang Z, Youle RJ. 2019 Reciprocal roles of Tom7 and OMA1 during mitochondrial import and activation of PINK1. Mol. Cell 73, 1028- 1043; e5. (10.1016/j.molcel.2019.01.002) 30733118 · DOI
  25. [27]Lin HN, Sung TY, Ho SY, Hsu WL. 2010 Improving protein secondary structure prediction based on short subsequences with local structure similarity. BMC Genomics 11(Suppl. 4), S4. (10.1186/1471-2164-11-S4-S4) PMC3005913 21143813 · DOI
  26. [28]Ben El Haj R, Regragui W, Tazi-Ahnini R, Skalli A, Bouslam N, Benomar A, Yahyaoui M, Bouhouche A. 2016 A novel homozygous p.L539F mutation identified in PINK1 gene in a moroccan patient with Parkinsonism. Biomed. Res. Int. 2016, 3460234. (10.1155/2016/3460234) 27413743 PMC4931068 · DOI
  27. [29]Ha BH, Boggon TJ. 2018 The crystal structure of pseudokinase PEAK1 (Sugen kinase 269) reveals an unusual catalytic cleft and a novel mode of kinase fold dimerization. J. Biol. Chem. 293, 1642- 1650. (10.1074/jbc.RA117.000751) 29212708 PMC5798295 · DOI
  28. [30]Lecointre C et al. 2018 Dimerization of the Pragmin pseudo-kinase regulates protein tyrosine phosphorylation. Structure 26, 545- 554; e4. (10.1016/j.str.2018.01.017) 29503074 · DOI
  29. [31]Masson GR et al. 2019 Recommendations for performing, interpreting and reporting hydrogen deuterium exchange mass spectrometry (HDX-MS) experiments. Nat. Methods 16, 595- 602. (10.1038/s41592-019-0459-y) 31249422 PMC6614034 · DOI
  30. [33]Soya N, Truong L, Croteau N, Lukacs GL, Trempe J. 2018 PINK1 autophosphorylation is required for ubiquitin recognition. EMBO Rep. 19, e44981. (10.15252/embr.201744981) 29475881 PMC5891426 · DOI
  31. [34]Sekine S, Youle RJ. 2018 PINK1 import regulation; a fine system to convey mitochondrial stress to the cytosol. BMC Biol. 16, 2. (10.1186/s12915-017-0470-7) 29325568 PMC5795276 · DOI
  32. [35]Rasool S et al. 2021 Structure of PINK1 reveals autophosphorylation dimer and provides insights into binding to the TOM complex. BioRxiv. (10.1101/2021.08.05.455304) · DOI
  33. [36]Deutsch EW et al. 2020 The ProteomeXchange consortium in 2020: enabling ‘big data’ approaches in proteomics. Nucleic Acids Res. 48, D1145- D1152. 31686107 PMC7145525

Authors
Kakade, P., Ojha, H., Raimi, O. G., Shaw, A., Waddell, A. D., Ault, J. R., Burel, S., Brockmann, K., Kumar, A., Ahangar, M. S., Krysztofinska, E. M., Macartney, T., Bayliss, R., Fitzgerald, J. C., & Muqit, M. M. K.
Affiliations
  • University of Dundee (GB)
  • University of Leeds (GB)
  • German Center for Neurodegenerative Diseases (DE)
  • Hertie Institute for Clinical Brain Research (DE)
Corresponding author
Muqit, Miratul M. K.
ORCID
Venue
Open Biology
Publisher
The Royal Society
Published
2022-01
Volume
12
Issue
1
Pages
210264-210264
ISSN
2046-2441 (ISSN-L)
DOI
10.1098/rsob.210264
PMC
PMC8767193
OpenAlex
W4206131125
Cited by
42
References
34
Citations by year
2026: 7 · 2025: 10 · 2024: 8 · 2023: 11 · 2022: 6
FWCI
4.61
Type
journal article
Language
en
Open access
Licence
Locations
  • Open Biology (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • White Rose Research Online (University of Leeds, The University of Sheffield, University of York) (acceptedVersion)
  • Discovery Research Portal (University of Dundee) (submittedVersion, cc-by-nc)
  • Discovery Research Portal (University of Dundee) (submittedVersion, cc-by)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • DZNE Pub (submittedVersion, other-oa)
Funders
  • Wellcome Trust (210753/Z/18/Z)
  • BBSRC Studentship
  • Michael J. Fox Foundation for Parkinson's Research
  • European Molecular Biology Organization
  • Medical Research Council (MR/P00704X/1 COEN award)
  • German Federal Ministry of Eduction & Research
  • Cancer Research UK (C24461/A23303)
Topics
  • Health Sciences › Medicine › Neurology › Parkinson's Disease Mechanisms and Treatments
  • Health Sciences › Medicine › Epidemiology › Autophagy in Disease and Therapy
  • Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Nuclear Receptors and Signaling
Keywords
PINK1, Parkinson's disease, C-terminal extension, protein stabilization, ubiquitin phosphorylation, mitochondrial quality control, TOM complex, early-onset Parkinson's disease, protein-protein interaction, hydrogen-deuterium exchange
MeSH terms
Protein Conformation, alpha-Helical, PTEN-Induced Putative Kinase, Enzyme Activation, Humans, Phosphorylation, Protein Kinases, Ubiquitin, Ubiquitin-Protein Ligases

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