Poster Presentation The 43rd Lorne Conference on Protein Structure and Function 2018

New tools to study protein aggregation and the molecular chaperone action of heat shock proteins in cells. (#172)

Heath Ecroyd 1 2 , Rebecca San Gil 1 2 , Dezerae Cox 1 2 , Daniel Whiten 1 2 , Luke McAlary 1 2 , Justin Yerbury 1 2 , Mark Wilson 1 2
  1. Illawarra Health and Medical Research Institute, University of Wollongong, Wollongong, NSW, Australia
  2. School of Biology, University of Wollongong, Wollongong, NSW, Australia

Protein homeostasis, or proteostasis, is the process of maintaining the conformational and functional integrity of the proteome. Proteinaceous cytoplasmic inclusions are an indicator of dysfunctions in normal cellular proteostasis and a hallmark of many neurodegenerative diseases. Proteostasis is preserved in the face of stress by a complex network of cellular machinery, which includes the heat shock proteins (Hsps). The heat shock proteins (Hsps) are molecular chaperones that prevent the aggregation of client proteins by facilitating their refolding, or trafficking them for degradation. We have developed new tools to study (i) the intracellular aggregation of proteins into inclusions, and (ii) the molecular chaperone activities of Hsps in cells. Our simple and rapid new flow cytometry-based method enumerates, characterises and, if desired, can physically recover protein inclusions from cells (1). This technique can analyse and resolve a broad variety of inclusions differing in both size and protein composition, making it applicable to essentially any model of intracellular protein aggregation. Since the chaperone activities of Hsps are dependent on dynamic protein-protein interactions, including their oligomerisation into large multi-subunit complexes, we have avoided tagging them with a fluorescent reporter. Instead, we have exploited bicistronic constructs for the concurrent expression of the non-tagged heat shock protein and fluorescent reporter from a single mRNA in cells (2). Here we report on work in which we have combined these new tools in order to elucidate the impact on Hsps on the intracellular aggregation of disease-related and model aggregation-prone proteins.

  1. Whiten, D.R., San Gil, R., McAlary, L., Yerbury, J. J., Ecroyd, H. and Wilson, M.R. (2016) Rapid flow cytometric measurement of protein inclusions and nuclear trafficking. Sci Reports. 6, 31138.
  2. San Gil, R., Berg, T. and Ecroyd, H. (2017) Using bicistronic constructs to evaluate the chaperone activities of heat shock proteins in cells. Sci Reports. 7, 2387