Phosphorylation and ubiquitylation are reversible signalling events and there is significant interest in in their interplay in the regulation of normal biological processes and disruption in human diseases. Previous research in our lab has defined a Parkinson’s linked enzyme, PINK1 as the first protein kinase to target ubiquitin via phosphorylation at Serine65. PINK1 is unique amongst all kinases, since it possesses an N-terminal mitochondrial targeting domain that localizes it to the mitochondria. Under basal conditions PINK1 is inactive however upon exposure to mitochondrial uncouplers that induce mitochondrial depolarization, PINK1 becomes stabilized and activated. Upon activation PINK1 phosphorylates a Parkinson’s disease-linked ubiquitin ligase, Parkin and ubiquitin. Phosphorylated ubiquitin binds to Parkin with high affinity and triggers activation of its catalytic activity leading to ubiquitylation of multiple protein substrates at the outer mitochondrial membrane .
The mechanism by which PINK1 is activated how it targets ubiquitin remains mysterious. The primary aim of the project will be to identify novel PINK1 binding partners using state-of-the-art proteomic technologies from a variety of cellular systems including relevant primary neuronal cell systems. The student will also train in genome-editing technologies to harness the powerful CRISPR/Cas9 methodology to knockout candidate interactors to determine their effect on PINK1 complexes and activation. A major challenge in studying mitochondrial protein biology is that many mitochondrial proteins are essential limiting the ability to generate null cell types. The Sapkota laboratory has recently developed a novel technology to target proteins for degradation called affinity-directed protein missile (AdPROM) system . The von-Hippel-Lindau protein that normally functions to degrade HIF1a via the 26S proteasome has been engineered to recognize GFP tagged proteins in cells and efficiently degrade them. Homozygous knock-in cells in which GFP is targeted to essential mitochondrial proteins by CRISPR will be generated to enable knockdown by AdPROM. These studies will lead to greater understanding on how PINK1 is regulated that will be of relevance for Parkinson’s disease mechanisms.
- Kazlauskaite, A., Martinez-Torres, R.J., Wilkie, S., Kimar, A., Peltier, J., Gonzalez, A., Johnson, C., Zhang, J., Hope, A.G., Peggie, M., Trost, M., van Aalten, D.M.F., Alessi, D.R., Prescott, A.R., Knebel, A., Walden, H., Muqit, M.M. (2015) Binding to Serine 65-phosphorylated ubiquitin primes Parkin for optimal PINK1-dependent phosphorylation and activation. EMBO Reports 16: 939-54.
- Fulcher LJ, Macartney T, Bozatzi P, Hornberger A, Rojas-Fernandez A, Sapkota GP (2016) An affinity-directed protein missile system for targeted proteolysis. Open Biol 6: 160255.