The generation and testing of a cell-permeable constitutively active recombinant parkin protein for its neuroprotective properties

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Parkinson's disease (PD) is the second most common neurodegenerative disease in Canada, with ~50% of cases with an early onset linked to mutations in the PARK2 gene, which encodes the parkin protein. Parkin functions as an E3 ubiquitin (Ub) ligase that plays a neuroprotective role in a range of cellular and animal models of PD. Intriguingly, a recent study from my lab has provided the first evidence that parkin exists in an autoinhibited state. Thus, basal E3 activity of parkin is low, requiring activation. Remarkably, changing the W403 in parkin to an alanine abolished this inhibition, causing hyperactivation of parkin. Considering the neuroprotective properties of parkin, developing an active parkin protein presents a novel and exciting therapeutic avenue to explore in protecting neurons against stress in PD patients. Here we propose to partner with iProgen to take advantage of their proprietary iProgen Protein Transduction Domain (iPTD) to generate novel cell-permeable parkin proteins, that we would otherwise be unable to generate. Using this iPTD tag, iProgen will generate a W403A constitutively active parkin protein whose protective properties we will compare against a basally inactive wild-type parkin in cellular models of PD. In the first part of the project, iProgen will engineer, express and purify recombinant parkin proteins. In the second part of the project, we will test the in vitro ubiquitin ligase activity of the parkin proteins, and investigate their biological activity in cellular models of PD. If the present study demonstrates that the derepressed parkin protein can enhance cellular protection relative to the wild-type protein, these results provide impetus for further studies exploring the protective properties of this constitutively active parkin. For iProgen these results enable them to proceed towards the next stage of producing greater quanitites of the parkin protein for use in future trials.