Poster Presentation The 44th Lorne Conference on Protein Structure and Function 2019

Investigating the role of proteostasis pathways in regulating the intracellular protein aggregation of firefly luciferase (#212)

Shannon McMahon 1 2 , Harm Kampinga 1 , Heath Ecroyd 2
  1. University Medical Center Groningen, Groningen, The Netherlands
  2. University of Wollongong, Wollongong, NSW, Australia

A hallmark of many neurodegenerative diseases, including Alzheimer’s disease and motor neurone disease, is the chronic expression of aggregation-prone proteins, which oligomerise and deposit into inclusions. Cellular protein homeostasis (proteostasis) relies upon the interconnected network of pathways that together maintain the stability and functionality of the proteome [1]. The heat shock proteins (Hsps) are a family of evolutionarily conserved molecular chaperones which have a range of protective functions in response to stress [2]. By binding to aggregation-prone proteins, Hsps can either refold or traffic misfolded proteins for proteolytic degradation. To date, the majority of cell-based Hsp screens conducted have investigated the effect on aggregation-prone proteins associated with disease. However, as these proteins are expressed endogenously in cells, their misfolding in conditions of proteostasis deficiency may be associated with loss-of-function effects. This project utilises a double mutant form of firefly luciferase (FlucDM), the aggregation of which mimics a disease-like phenotype. Since FlucDM has no biological role in cells, any aggregation resulting from a loss of function will not impact the analysis [3]. Using quantitative flow cytometry, candidates from the small Hsp, Hsp40 and Hsp70 families were screened to identify modulators of FlucDM aggregation in cells. Of the chaperones screened, every member of the Hsp40 family significantly reduced FlucDM aggregation. However, when the interaction of Hsp40 with Hsp70 was inhibited, this ability to reduce aggregation was lost, implying that Hsp70 activity is essential for the suppression of aggregation. Finally, when the amyloid-specific binding domain of Hsp40 was mutated, its ability to inhibit FlucDM aggregation remained, suggesting that Hsp40 may have other regions responsible for the recognition of different misfolded substrates. Uncovering the mechanisms by which Hsps prevent the accumulation of aggregation-prone proteins into inclusions is essential, as these may reveal novel therapeutic targets to treat such pathologies in the future.

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  3. Gupta, R., Kasturi, P., Bracher, A., Loew, C., Zheng, M., Villella, A., Garza, D., Hartl, F.U. & Raychaudhuri, S. 2011. Nature Methods, 8, 879-884.