Biophysical methods to study the ubiquitination of amyloidogenic proteins and proteasomal responses to protein aggregates
The accumulation of misfolded proteins is a universal mechanism in neurodegenerative diseases such as Parkinson’s disease (PD), Alzheimer’s disease (AD) and prion disease. In mammalian cells, the majority of misfolded proteins are degraded by the ubiquitin-proteasome system (UPS), in which selective substrates are unfolded and cleaved into small peptides when passing through proteasome. The underlying pathogenesis of neurodegenerative diseases is at least partially contributed by the dysfunction of UPS. In this thesis, the kinetics of ubiquitination conjugation, which is the first step to induce selective degradation in the UPS was studied using fluorescence-based methods. From the change of FRET efficiency, the rates of specific combinations of ubiquitination enzymes E1-E2-E3 can be determined. This method does not require labelling of enzymes to study the ubiquitination activity. Next, this ubiquitination activity was investigated upon incubation with α-synuclein and tau proteins. Biochemical and biophysical analysis revealed the ubiquitination sites on the amyloid proteins and its effects on the formation of protein aggregates in vitro. To obtain further insights into how UPS maintains the quality control of the cellular proteome in response to a proteotoxic stress induced by protein aggregates, two cell models were generated to visualise the proteasome and ubiquitin (Ub) in live cells. Proteasomes were observed to form transient foci with distinct characteristics under the proteolytic stress, some foci co-localised with aggregates. Single molecule tracking experiments indicated these proteasome-containing bodies act as active proteolytic centres to provide cells with an extra defence function against stress. In addition, a photoconvertible Ub construct enables the direct observation of cellular degradation rates by UPS under various stresses. Initial results showed that proteotoxic stress inhibits the UPS function. Overall, my studies on UPS and neurodegenerative proteins provide a new assay to study ubiquitination kinetics and characterise how UPS responses to proteolytic stress. These data may also have implications for the mechanism of the formation of pathogenic protein deposits and its links with downregulated UPS function due to aging and degradation-resistant aggregates.
Fluorescence Microscopy, proteasome, protein aggregation, ubiquitination
Networked Digital Library of Theses and Dissertations