AbstractParkinson’s disease (PD) is the second most common neurodegenerative disorder affecting around 1% of the population over the age of 65 worldwide. While the majority of cases are sporadic, groundbreaking genetic research over the last two decades has revealed a complicated network of genes underlying susceptibility and pathology of PD. Mutations in Parkin are the most common cause of early onset Parkinson’s disease. Parkin encodes a RING-In Between RING-RING E3 ligase that normally resides in the cytoplasm and is
regulated by autoinhibitory intramolecular interactions. PINK1 is a mitochondrial Ser/Thr kinase that is unique in possessing a N-terminal mitochondrial targeting sequence and three loop insertions within its kinase domain. At the outset of my PhD, PINK1 was discovered to phosphorylate Parkin at Serine 65 (Ser65), however, little was known about how this phosphorylation affected Parkin function and E3 ligase activity. The work described in this thesis aims to address in detail the molecular interactions between these proteins.
In Chapter 3, I describe the development of novel tools and assays to study the PINK1- Parkin interaction. I demonstrate that PINK1 robustly phosphorylates Parkin at Ser65 in vitro as well as in cells. I further show that Parkin is activated via PINK1 mediated phosphorylation of Ser65. I validate the mitochondrial GTPase Miro1 as a direct substrate of Parkin and employ this novel substrate-based assay to demonstrate a multitude of effects conferred by Parkin disease mutations on its phosphorylation and E3 ligase activity.
In Chapter 4, I describe work that revealed a novel PINK1 substrate – ubiquitin. I demonstrate that Ubiquitin gets phosphorylated at a homologous Ser65 residue and this is critical for PINK1-mediated Parkin activation. I provide further evidence that both phosphorylation events are necessary for optimal Parkin E3 ligase activity.
Following up on this work, in Chapter 5, I investigate the mechanism by which phosphorylated ubiquitin contributes to Parkin activation. I demonstrate that binding of
phosphorylated ubiquitin to Parkin significantly enhances its phosphorylation by PINK1 and thereby its activation. I further identify Histidine 302 and Lysine 151 as Parkin residues critically involved in binding phosphorylated ubiquitin and confirm their importance for Parkin phosphorylation and activation in vitro as well as in cells.
Overall, the work described in this thesis elaborates the mechanism by which Parkin is regulated by PINK1-dependent phosphorylation. In parallel this work provides the
PINK1/Parkin field with a set of valuable tools to evaluate this mechanism. My work not only reveals PINK1-dependent Parkin activation via direct phosphorylation of the Parkin
Ser65 residue, but goes on to identify a new key player in this pathway – Ser65 phosphorylated ubiquitin. Finally my studies provide novel and original insights into the interplay of these three proteins towards driving Parkin activation.
|Date of Award||2015|
|Sponsors||Parkinson’s UK & J. Macdonald Menzies Charitable Trust|
|Supervisor||Miratul Muqit (Supervisor) & Dario Alessi (Supervisor)|