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

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.

LUBAC assembles a ubiquitin signaling platform at mitochondria for signal amplification and transport of NF‐κB to the nucleus

“…In this pathway, depolarization of the inner mitochondrial membrane leads to an import arrest and stabilization of the mitochondrial kinase PINK1 at the outer mitochondrial membrane. PINK1 is then activated by autophosphorylation and phosphorylates ubiquitin and the ubiquitin‐like domain of the E3 ubiquitin ligase Parkin at serine 65, thereby activating Parkin (Gan et al , 2022; Kakade et al , 2022; Rasool et al , 2022; Sauve et al , 2022). Parkin‐mediated ubiquitination of outer membrane proteins recruits the autophagic machinery to eliminate damaged mitochondria.…”

Systematic Functional Analysis of PINK1 and PRKN Coding Variants

“…This effect was not apparent upon transient expression of this mutant, confirming that overexpression of PINK1 is prone to mask such cryptic phenotypes. Interestingly, both Q115L and D525N are located just outside two α-helical regions in PINK1 that were recently shown to interact with each other and to be important for PINK1 activation in cells [ 43 ]. Several other changes in these regions presented additionally with reduced auto- and substrate phosphorylation, suggesting that intramolecular interaction between an N- and C-terminus of PINK1 are critical for its stabilization at the TOM complex [ 43 ].…”

MTSviewer: a database to visualize mitochondrial targeting sequences, cleavage sites, and mutations on protein structures

“…While some of these PINK1 N-MTS variants are still cleaved by PARL in healthy mitochondria and accumulate following mitochondrial damage (Sekine et al 2019), their import rates and effects on MPP processing remain unstudied. Experiments which swap the PINK1 N-MTS with those from other mitochondrial proteins have shown that PINK1 can still be imported into mitochondria with chimeric N-MTS’s, though PINK1 accumulation is prevented (Kakade et al 2022). While many of these N-MTS PINK1 chimeras can still be imported, their specific rates of import have also yet to be measured.…”