Exploring Cooperation and Competition in Molecular Chaperone Networks with Chemical Genetics and Peptide Microarrays.

Abstract

Molecular chaperones are conserved proteins that maintain proteostasis in all organisms. Chaperones bind to unfolded protein substrates and promote outcomes such as folding, localization, aggregation prevention, and degradation. One chaperone, 70-kDa heat shock protein (Hsp70), is ubiquitously expressed and acts as a coordinating hub for a large network of co-chaperones, including J proteins, nucleotide exchange factors (NEFs), and tetratricopeptide-repeat (TPR) domain-containing proteins. The complexity of this network has posed challenges to understanding its roles in cellular health and how these roles may fail in diseases of protein misfolding, such as Alzheimer’s disease (AD). We attempted to gain a more detailed understanding of these networks using techniques that allow us to probe the function of many chaperones in parallel. Using chemical genetics, we investigated the functional roles of individual J proteins in Saccharomyces cerevisiae. We also designed peptide microarrays derived from the sequences of two model chaperone substrates, luciferase and tau. We used the luciferase array to investigate the mechanistic basis of the divergent abilities of two homologous J proteins to refold luciferase. We also mapped the binding of 17 chaperones to tau, a protein that aggregates in a class of neurodegenerative diseases called tauopathies, including AD. We find that chaperones bind a set of “hotspots” on tau, many of which are rich in residues known to be phosphorylated in AD tau or sites of mutations linked to tauopathies. These results suggest that chaperones compete with one another and other tau binding partners to direct tau metabolism in both healthy and diseased systems. Finally, we designed a peptide microarray to test the hypothesis that chaperones might preferentially bind amyloidogenic sequences to prevent the formation of toxic amyloid fibrils. We found that some chaperones, including Hsp70 and some J proteins, have a slight but consistent preference to bind amyloid-forming peptides over non-aggregating ones. In subsequent solution-phase binding assays, we confirm this finding but also conclude that other peptide properties contribute strongly to Hsp70 binding. Taken together, these studies advance our understanding of chaperone networks and suggest novel future avenues of inquiry.