Florey Department of Neuroscience and Mental Health - Theses

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    Spatial and temporal surveillance of the mechanisms controlling proteome foldedness via a FRET-based biosensor
    Raeburn, Candice ( 2018)
    Proteostasis (protein homeostasis) is essential for keeping the proteome functional. This process controls protein synthesis, folding and degradation and involves hundreds of genes, including those encoding chaperones, to form extensive quality control (QC) networks (Kim et al., 2013). Imbalances in proteostasis are implicated in a range of aggregation-based neurodegenerative diseases including Amyotrophic Lateral Sclerosis (ALS), Huntington’s and Alzheimer’s diseases (Morimoto et al., 2014; Vilchez et al., 2014). Currently there is a lack of capacity to quantitatively measure proteostasis imbalance and therefore we are limited in understanding how proteostasis imbalance manifests during disease. A new biosensor system has been developed by our lab to address this shortfall. The biosensor is a genetically encoded unfolded “bait” flanked by two fluorescent proteins to assay foldedness by fluorescence resonance energy transfer (FRET). Proteostasis efficiency is reported by measurement of the efficiency to which the bait interacts with the QC network. In this master’s project, the biosensor was further targeted to organelles to allow for a higher degree of spatiotemporal control. Signalling peptides were used to target the biosensor to specialised microenvironments, and successful targeting was achieved in the Golgi apparatus and nucleus. Investigations into nuclear proteostasis revealed the biosensor behaved predictably to chaperone overexpression (Hsp40 and Hsp70 co-expression) or inhibition (Hsp70 or Hsp90 inhibition). Polyglutamine (PolyQ) expansions of non-pathogenic (Q25) to pathogenic (Q72) lengths reduced the biosensor foldedness and decreased aggregation, which is consistent with an increase in chaperone supply. The biosensor was also adapted to express in the body wall muscles of Caenorhabditis elegans to examine change in proteostasis across age and in an organismal context. The biosensor was successfully expressed in the model organism, with potential sub-microscopic and variant biosensor expression level confounding data analysis. The C. elegans reporter lines were successfully crossed with lines expressing Aβ (1-42) demonstrating the ability of the biosensor to report on disease states. Moving forward, the generation of low-expression, single-copy C. elegans biosensor lines would allow for steady, matched expression and enhanced capacity for comparison between worm lines.