Mutations in PINK1 can cause Parkinson’s disease in certain familial PD cases. The exact cellular function of PINK1 and the impact of disease-relevant mutations remain unknown. PINK1 is known to function as a kinase but its cellular substrates remain to be identified.
In this project, Dr. Culvenor has isolated PINK1 protein and will utilize biochemical assays to identify physiologically relevant PINK1 substrates and determine whether the interaction between PINK1 and these identified substrates is affected by PD-relevant mutations. These studies will increase our understanding of the role of PINK1 in PD and identify specific therapeutic targets for the development of disease-modifying approaches.
Kinases are important therapeutic targets for disease modification. Genetic linkage of kinase proteins indicates phosphorylation defects can cause Parkinson’s. Finding the target sites of action for PINK1 will provide new insight into disease mechanism and opportunity for novel therapy development. Improved kinase assays and target identification for PINK1 will enable selection and production of activators and inhibitors for this Parkinson’s associated neuroprotective enzyme.
Final Outcome
Dr. Culvenor and colleagues successfully developed the procedure for expression and purification of sufficient active recombinant PINK1 protein to examine phosphorylation of various candidate substrates and to interrogate rodent brain protein extracts for potential physiological substrates. Expression and stability of purified recombinant PINK1 has proved challenging. To overcome some of these problems they are now generating modified enzyme with mutations in the N-lobe of the kinase domain of PINK1 to improve stability of recombinant PINK1 expressed in both Sf9 cells and mammalian cells.
The expression and purification of the “kinase-dead” PINK1 protein is under way. When available, it will be used as the control to confirm that phosphorylation of the substrates presented in this report was solely attributed to the intrinsic kinase activity of recombinant PINK1 protein and the purified recombinant PINK1 protein preparation is free from any contaminating kinase activity. This study has generated significant leads for further investigation towards the identification of PINK1 substrates.
Results of this work were published in the Journal of Neurochemistry. Additionally, The MJFF grant contributed to a strengthened collaboration and preliminary data toward successful follow-on funding from the Australian National Health and Medical Research Council.