Mutations in PINK1 are the second most frequent cause of autosomal recessive early-onset Parkinson’s disease. PINK1 is activated upon mitochondrial damage to phosphorylate Ubiquitin and Parkin to stimulate Parkin E3 ligase activity, and this is critical for removal of damaged mitochondria by autophagy (mitophagy).
In 2011, Helen Woodroof, who was a PhD student in the Muqit lab, developed the first biochemical assay of PINK1 kinase activity using an insect orthologue and demonstrated that mutations found within the kinase domain disrupted catalytic activity. In 2017, in collaboration with Daan van Aalten, post-doctoral scientists, Atul Kumar and Jevgenia Tamjar, in the Muqit Lab, solved the first high resolution x-ray crystal structure of insect PINK1 that revealed the molecular mechanism of Parkinson’s-causing mutations located within the kinase domain.
An outstanding question has been the mechanism of Parkinson’s mutations that lie outside the kinase domain of PINK1. This has now been solved, in new work published yesterday in Open Biology, by a collaborative team of researchers from the Muqit Lab; the Astbury Centre for Structural Biology at Leeds University, and the Hertie Institute for Brain Research at the University of Tubingen in Germany.
Post-docs Poonam Kakade and Hina Ojha together with PhD students Andrew Shaw and Andrew Waddell employed mutagenesis studies of human PINK1 in cells to map and characterize the key regions of the N-terminus and C-terminus of PINK1 required for activation. A structural model of human PINK1 predicted by AlphaFold revealed an intramolecular interface formed by an N-terminal a-helix extension (NTE) and C-terminal a-helix extension (CTE) of PINK1. This was then corroborated by post-doc Olawale Raimi in collaboration with James Ault and Richard Bayliss in Leeds using HDX mass spectrometry analysis of recombinant insect PINK1.
To investigate the functional importance of the interface, Hina, Andrew S, and Andrew W studied the impact of Parkinson’s causing mutations located within this interface in human cell lines and found that this disrupted PINK1 stabilisation and activation in response to mitochondrial damage. Furthermore, Hina discovered that the NTE:CTE interaction is critical for PINK1 stabilisation at the TOM complex and was abolished by disease-associated point mutations within the interface.
Finally in collaboration with brain researchers Kathrin Brockmann and Julia Fitzgerald at the University of Tubingen, a German Parkinson’s patient was identified with homozygous mutation of PINK1 Glutamine126 to Proline residue (Q126P). Andrew W and postdoc Sophie Burel were able to demonstrate that PINK1 stabilization was disrupted in primary skin fibroblasts from the patient compared to a human control, validating the clinical relevance of the molecular findings
The research was funded by the Wellcome Trust, Michael J Fox Foundation, Medical Research Council, Biotechnology and Biological Sciences Research Council (BBSRC), EMBO, CRUK, and the German Federal Ministry of Education and Research.
To read a copy of the paper published in Open Biology.
Image - Left: Current lab members Hina Ojha and Olawale Raimi who co-led the work. Right: AlphaFold model of human PINK1.