AbstractParkinson’s disease (PD) is the second most common neurodegenerative disorder, affecting approximately 1% of the population over the age of 65. Around 5% of these cases can be linked to mutations in known genes, one of which is the PINK1 gene, first linked to PD a decade ago. Since then, over 30 mutations in PINK have been described. The PINK1 gene encodes an unusual serine/threonine protein kinase; uniquely among protein kinases, PINK1 is anchored to the mitochondria and furthermore possesses three unusual insertions of unknown function in the N-lobe of its kinase domain. Recently, two important PINK1 substrates have been identified - the ubiquitin E3 ligase Parkin and ubiquitin itself. Phosphorylation of both of these substrates has further been shown to be necessary for the activation of Parkin E3 ligase activity. At the start of this project, little was known about the catalytic properties of PINK1 or the effects of the identified PD- linked mutations due to the lack of a robust in vitro assay for PINK1 activity. In addition, the mechanisms by which PINK1 phosphorylation activated Parkin were not understood. The work described in this thesis was carried out with the aim of overcoming these hurdles by using a PINK1 homologue from a more tractable species.
In Chapter III of this thesis, I describe the identification of a PINK1 homologue from the insect species Tribolium castaneum (TcPINK1) and its validation as a bona fide model for human PINK1. The activity assay established with TcPINK1 is used to show the PINK1 C-terminal domain is required for kinase activity and that TcPINK1 strongly prefers peptide substrates with a +1 proline. Interestingly, this requirement is absent when intact protein substrates are used. TcPINK1 is then used as a model to analyse the effects of PD- linked disease mutations, revealing that all the tested point and truncating mutations lead to a decrease in kinase activity. In most cases activity is completely abolished. In Chapter IV, progress towards the crystal structure of TcPINK1 is described. Several TcPINK1
￼constructs were expressed and purified to homogeneity and used for crystallisation trials. Crystals were obtained under multiple conditions and some progress was made towards improving their diffraction resolution, although this project did not reach a point at which data could be collected. Lastly, in Chapter V, preliminary data describing the molecular mechanism by which PINK1 phosphorylation of the Parkin Ubl domain at residue Ser65 leads to Parkin activation is presented. An AlphaScreen assay was used to produce data suggesting that PINK1 phosphorylation of the Parkin Ubl domain relieves autoinhibitory binding of the Ubl domain to Parkin.
Overall, this work provides the PINK1 field with a useful tool in the form of a robust and reproducible activity for PINK1 activity using TcPINK1, which has already been utilised several times to facilitate discoveries in both this lab and others. Significant progress has been made towards determination of the crystal structure of TcPINK1, which will provide insight into the molecular mechanisms of PD-linked mutations and may shed light onto PINK1 regulation and function. Finally, preliminary data suggests that PINK1- phosphorylation of the Parkin Ubl domain may activate Parkin via alterations in autoinhibitory intramolecular interactions.
|Date of Award||2014|
|Supervisor||Miratul Muqit (Supervisor), Daan van Aalten (Supervisor) & Dario Alessi (Supervisor)|