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Title: Molecular characterization of the Parkinson's associated protein DJ-1
Author: Hassanjani, Mahdieh
ISNI:       0000 0004 6350 7001
Awarding Body: University of Leicester
Current Institution: University of Leicester
Date of Award: 2015
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Mutations in DJ-1 (PARK7), a conserved protein of 189 amino acids, cause autosomal recessive cases of Parkinson’s disease (PD). DJ-1 appears to play a central role in protecting cells from oxidative stress, which likely has relevance for its role in PD pathogenesis. Biochemical and crystallographic approaches indicate that DJ-1 dimerizes, which is likely fundamental for its stability and normal function. A main focus of this thesis was identifying and characterizing DJ-1 dimerization modifiers through an unbiased screen of a kinase and phosphatase inhibitor library, taking advantage of bimolecular fluorescence complementation (BiFC) as a readout for DJ-1 dimerisation in living cells. To address this aim, we generated HEK 293T cell clones stably over-expressing DJ-1 BiFC constructs and optimised high throughput Cell^RScan^ R screening. This approach identified two kinase inhibitors (Bosutinib and KW2449) which decrease DJ-1 dimerisation in an oxidative stress-dependent manner. Furthermore, to indicate whether or not the observed effects of the kinase inhibitors on DJ-1 dimerization were due to a direct alteration of DJ-1 phosphorylation status or were indirect effects, we studied DJ-1 phosphorylation at all potential sites on its dimerization and stability by generating phosphomimic and phosphoblocking mutants. This work indicates that phosphorylation of key residues of DJ-1 likely dramatically reduces its stablilty. Additionally, by using the BiFC approach we found a direct interaction in living cells of DJ-1 with microtubule-associated protein tau, which is known to be hyper phosphorylated and aggregated in neurodegenerative disorders like Alzheimer’s disease and Parkinson’s disease. These analyses suggest that alterations in DJ-1 dimerization, stability and phosphorylation status in normal conditions and in response to oxidative stress may shed more light on DJ-1 function and its role associated with PD pathogenesis.
Supervisor: Giorgini, Flaviano Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available