Mechanisms of small heat shock protein sequestrases

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The imbalance of protein homeostasis caused by stress conditions or cellular ageing results in the accumulation of misfolded protein species, which is linked to cell toxicity. A sophisticated proteostasis network removes misfolded proteins by their refolding, degradation or sequestration into intracellular inclusions. Sequestration is a protein quality control strategy that has been recognized only recently, and the cellular factors involved, underlying mechanisms and physiological functions of sequestration are just beginning to unfold.Protein sequestration is best characterized in Saccharomyces cerevisiae where it is executed by the Hsp42 and Btn2 sequestrases. Hsp42 is member of the small heat shock protein (sHsp) family and shares common features with Btn2, including the presence of large disordered regions, the ability to self-assemble and to direct bound substrates to Hsp70/Hsp100-dependent refolding pathways. In previous work we investigated the basic mechanism of Hsp42 sequestrase activity, which involves a prion-like domain that binds substrates and a charged, disordered domain that regulates sequestrase activity. Both Hsp42 and Btn2 promote cell survival under conditions of limited Hsp70 availability, presumably because misfolded proteins become secluded into inclusions which frees Hsp70 capacity. This unique phenotype of sequestrase mutants enabled us to set up a screen for C. elegans sequestrases that restore growth and protein sequestration in yeast mutants with lacking sequestrase and limiting Hsp70 activities. Our preliminary experiments identified selected C. elegans sHsps as sequestrases, which contribute to life span of the animals. Other sHsps do not show such activity, indicating functional specialization among sHsps in metazoa. Here, we plan to determine the mechanism of sHsp sequestrases, their physiological relevance in a metazoan and how sequestrases are triaging bound substrates to refolding pathways. Our major research aims are: (1) explore whether sequestration represents a conserved activity of sHsps by testing sHsps from different kingdoms of life in our yeast sequestrase mutant screen; (2) define the biochemical and structural features that underlie sHsp sequestrase activity; (3) determine the physiological role of sequestrase positive sHsps in C. elegans; (4) dissect how sequestrase activity is regulated by environmental cues using Hsp42 as model; (5) determine the structures of sequestrase/substrate complexes by cryo electron tomography and how they are modulated by Hsp70/Hsp100 disaggregases. With these experiments we expect to reveal key mechanisms driving the protein sequestration function of sHsps, which constitutes a prime line of defense of cells against proteotoxic stress.