Biochemistry Commons^™

Modulating Parkin E3 Ubiquitin Ligase Activity Using Phospho-Ubiquitin Variants, Susanna George

Electronic Thesis and Dissertation Repository

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 (pUb^S7, pUb^S12, pUb^S20, pUb^S65) and investigated their activity in a key neurodegenerative pathway. Purification of pUb^S7 revealed a +3 frameshifted protein (Ub ∆7) that was successfully purified away from the pUb. Parkin was …