RT Journal Article
SR Electronic
T1 Mapping of a N-terminal α-helix domain required for human PINK1 stabilisation, Serine228 autophosphorylation and activation in cells
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2021.09.06.459138
DO 10.1101/2021.09.06.459138
A1 Poonam Kakade
A1 Hina Ojha
A1 Olawale G. Raimi
A1 Andrew Shaw
A1 Andrew D. Waddell
A1 James R. Ault
A1 Sophie Burel
A1 Kathrin Brockmann
A1 Atul Kumar
A1 Mohd Syed Ahangar
A1 Ewelina M. Krysztofinska
A1 Thomas Macartney
A1 Richard Bayliss
A1 Julia C. Fitzgerald
A1 Miratul M. K. Muqit
YR 2021
UL http://biorxiv.org/content/early/2021/09/06/2021.09.06.459138.abstract
AB Human autosomal recessive mutations in the PINK1 gene are causal for Parkinson’s disease (PD). PINK1 encodes a mitochondrial localised 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 are unknown. We have employed mutagenesis studies of human PINK1 in cells to define the minimal region of PINK1, required for optimal ubiquitin phosphorylation, beginning at residue Ile111. Bioinformatic analysis of the region spanning Ile111 to the kinase domain and inspection of the AlphaFold human PINK1 structure model predicts a conserved N-terminal α-helical domain extension (NTE domain) within this region corroborated by hydrogen/deuterium exchange mass spectrometry (HDX-MS) of recombinant insect PINK1 protein. The AlphaFold structure also predicts the NTE domain forms an intramolecular interaction with the C-terminal extension (CTE). 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 domain mutants do not affect intrinsic catalytic kinase activity but do disrupt PINK1 stabilisation at the mitochondrial Translocase of outer membrane (TOM) 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 stabilisation and activation and show that loss of NTE:CTE interactions is a major mechanism of PINK1-associated mutations linked to PD.Competing Interest StatementMMKM is a SAB member of Mitokinin Inc.