Mechanisms of small heat shock protein sequestrases
Final Report Abstract
Small heat shock proteins (sHsps) are crucial components of protein quality control (PQC) systems and bind misfolded proteins to form sHsp/substrate complexes. The sizes of these complexes can be very large, including microscopically visible inclusions, defining a sHsp sequestrase activity. Using the specific phenotypes of S. cerevisiae sequestrase mutants we screened for the ability of sHsps from diverse species to restore substrate sequestration. We show that the sequestrase activity of sHsps is evolutionarily conserved and is found in E. coli, C. elegans, and human sHsps. Sequestrase-positive C. elegans sHsps harbour extended disordered N-terminal extensions with aromatic residues that are crucial for substrate interaction. To dissect sHsp sequestrase activity we tried to obtain high-resolution structures of the yeast Hsp42 sequestrase by cryoEM and of sHsp/substrate complexes by cryoET. This could not be achieved due to domain flexibilities, oligomer dynamics (Hsp42), and sample heterogeneity (sHsp/substrate complexes). We therefore performed a detailed structure-function analysis of Hsp42. We developed an AlphaFold3 model of Hsp42 octamers based on size determination of the oligomers by various biochemical methods. Hsp42 appears as a disk-like structure, which is formed by the conserved a-crystallin domain. DSSO-crosslinking and identification of crosslinked peptides by mass spectrometry largely confirms this architecture. The N-terminal and disordered PrLD and IDD domains are largely surface-exposed as shown by limited proteolysis. We confirm that the PrLD is essential for Hsp42 sequestrase activity and show that the IDD controls sHsp/substrate complex size as it is crucial for the formation of large complexes. Furthermore, the IDD plays an important function in the cooperation with downstreamacting Hsp70/Hsp100 disaggregases. Recovery of bound substrates by Hsp70/Hsp100 is strongly reduced upon IDD deletion. This finding indicates that both phases of sHsp function, the sequestration of misfolded proteins and the release of bound substrates by protein disaggregases, are precisely controlled in Hsp42. We additionally dissected the cooperation of sHsps with protein disaggregases using Pseudomonas aeruginosa as model. P. aeruginosa harbours two sHsps, IbpA and Hsp20, and two disaggregation systems, Hsp70/ClpB and ClpG. The genes encoding for Hsp20 and ClpG are co-organized and have been implicated in protecting bacteria against severe heat stress. We tested whether the expansion of sHsps and disaggregase repertoires are functionally linked. We show that both sHsps boost Hsp70/ClpB disaggregation activity, as observed before for multiple sHsps from diverse species. Notably, IbpA strongly inhibited ClpG-mediated disaggregation, while Hsp20 could enhance ClpG activity in a concentration-dependent manner. The specific cooperation between Hsp20 and ClpG is in line with their gene organization and broadens sHsp function in PQC systems.
Publications
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The cytoprotective sequestration activity of small heat shock proteins is evolutionarily conserved. Journal of Cell Biology, 221(10).
Shrivastava, Aseem; Sandhof, Carl Alexander; Reinle, Kevin; Jawed, Areeb; Ruger-Herreros, Carmen; Schwarz, Dominic; Creamer, Declan; Nussbaum-Krammer, Carmen; Mogk, Axel & Bukau, Bernd
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The Diverse Functions of Small Heat Shock Proteins in the Proteostasis Network. Journal of Molecular Biology, 434(1), 167157.
Reinle, Kevin; Mogk, Axel & Bukau, Bernd
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Balanced activities of Hsp70 and the ubiquitin proteasome system underlie cellular protein homeostasis. Frontiers in Molecular Biosciences, 9.
Jawed, Areeb; Ho, Chi-Ting; Grousl, Tomas; Shrivastava, Aseem; Ruppert, Thomas; Bukau, Bernd & Mogk, Axel
