Electronic Thesis and Dissertation Repository

Degree

Master of Science

Program

Biochemistry

Supervisor

Dr. Patrick O'Donoghue

Abstract

Parkin is a Parkinson’s disease-linked E3 ubiquitin (Ub) ligase that promotes mitophagy by ubiquitination of mitochondrial outer membrane proteins. Phosphorylation of Ub at Ser65 by the PTEN-induced putative kinase 1 activates parkin. The role of other Ub phosphorylation sites and the associated kinases remain unknown. We optimized genetic code expansion to produce pure site-specfically phosphorylated Ub (pUb) variants (pUbS7, pUbS12, pUbS20, pUbS65) and investigated their activity in a key neurodegenerative pathway. Purification of pUbS7 revealed a +3 frameshifted protein (Ub ∆7) that was successfully purified away from the pUb. Parkin was significantly activated when pUbS12 or pUbS65 was the sole parkin substrate. pUbS20 showed dose-dependent inhibition of stimulated parkin. Producing pure phosphoprotein was essential, as we showed parkin activity is sensitive to the stoichiometry and location of phosphorylation on its substrate Ub. Further mechanistic investigations of Ub phosphorylation will identify new pathways and potential drug targets in neurodegenerative signalling networks.

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