Cytoplasmic redox imbalance in the thioredoxin system activates Hsf1 and results in hyperaccumulation of the sequestrase Hsp42 with misfolded proteins.

Cells employ multiple systems to maintain homeostasis when experiencing environmental stress. For example, the folding of nascent polypeptides is exquisitely sensitive to proteotoxic stressors including heat, pH, and oxidative stress, and is safeguarded by a network of protein chaperones that concentrate potentially toxic misfolded proteins into transient assemblies to promote folding or degradation. The redox environment itself is buffered by both cytosolic and organellar thioredoxin and glutathione pathways. How these systems are linked is poorly understood. Here, we determine that specific disruption of the cytosolic thioredoxin system resulted in constitutive activation of the heat shock response in Saccharomyces cerevisiae and accumulation of the sequestrase Hsp42 into an exaggerated and persistent juxtanuclear quality control (JUNQ) compartment. Terminally misfolded proteins also accumulated in this compartment in thioredoxin reductase (TRR1)-deficient cells, despite apparently normal formation and dissolution of transient cytoplasmic quality control (CytoQ) bodies during heat shock. Notably, cells lacking TRR1 and HSP42 exhibited severe synthetic slow growth exacerbated by oxidative stress, signifying a critical role for Hsp42 under redox-challenged conditions. Finally, we demonstrated that Hsp42 localization patterns in trr1∆ cells mimic those observed in chronically aging and glucose-starved cells, linking nutrient depletion and redox imbalance with management of misfolded proteins via a process of long-term sequestration.

Cytoplasmic redox imbalance in the thioredoxin system activates Hsf1 and results in hyperaccumulation of the sequestrase Hsp42 with misfolded proteins.

Cells employ multiple systems to maintain homeostasis when experiencing environmental stress. For example, the folding of nascent polypeptides is exquisitely sensitive to proteotoxic stressors including heat, pH and oxidative stress, and is safeguarded by a network of protein chaperones that concentrate potentially toxic misfolded proteins into transient assemblies to promote folding or degradation. The redox environment itself is buffered by both cytosolic and organellar thioredoxin and glutathione pathways. How these systems are linked is poorly understood. Here, we determine that specific disruption of the cytosolic thioredoxin system resulted in constitutive activation of the heat shock response in Saccharomyces cerevisiae and accumulation of the sequestrase Hsp42 into an exaggerated and persistent juxtanuclear quality control (JUNQ) compartment. Terminally misfolded proteins also accumulated in this compartment in thioredoxin reductase (TRR1)-deficient cells, despite apparently normal formation and dissolution of transient cytoplasmic quality control (CytoQ) bodies during heat shock. Notably, cells lacking TRR1 and HSP42 exhibited severe synthetic slow growth exacerbated by oxidative stress, signifying a critical role for Hsp42 under redox-challenged conditions. Finally, we demonstrated that Hsp42 localization patterns in trr1∆ cells mimic those observed in chronically aging and glucose-starved cells, linking nutrient depletion and redox imbalance with management of misfolded proteins via a mechanism of long-term sequestration.

Regulation of the Hsf1-dependent transcriptome via conserved bipartite contacts with Hsp70 promotes survival in yeast.

Protein homeostasis and cellular fitness in the presence of proteotoxic stress is promoted by heat shock factor 1 (Hsf1), which controls basal and stress-induced expression of molecular chaperones and other targets. The major heat shock proteins and molecular chaperones Hsp70 and Hsp90, in turn, participate in a negative feedback loop that ensures appropriate coordination of the heat shock response with environmental conditions. Features of this regulatory circuit in the budding yeast Saccharomyces cerevisiae have been recently defined, most notably regarding direct interaction between Hsf1 and the constitutively expressed Hsp70 protein Ssa1. Here, we sought to further examine the Ssa1/Hsf1 regulation. We found that Ssa1 interacts independently with both the previously defined CE2 site in the Hsf1 C-terminal transcriptional activation domain and with an additional site that we identified within the N-terminal activation domain. Consistent with both sites bearing a recognition signature for Hsp70, we demonstrate that Ssa1 contacts Hsf1 via its substrate-binding domain and that abolishing either regulatory site results in loss of Ssa1 interaction. Removing Hsp70 regulation of Hsf1 globally dysregulated Hsf1 transcriptional activity, with synergistic effects on both gene expression and cellular fitness when both sites are disrupted together. Finally, we report that Hsp70 interacts with both transcriptional activation domains of Hsf1 in the related yeast Lachancea kluyveri Our findings indicate that Hsf1 transcriptional activity is tightly regulated to ensure cellular fitness and that a general and conserved Hsp70-HSF1 feedback loop regulates cellular proteostasis in yeast.

Unraveling protein misfolding diseases using model systems.

Experimental model systems have long been used to probe the causes, consequences and mechanisms of pathology leading to human disease. Ideally, such information can be exploited to inform the development of therapeutic strategies or treatments to combat disease progression. In the case of protein misfolding diseases, a wide range of model systems have been developed to investigate different aspects of disorders including Huntington's disease, Parkinson's disease, Alzheimer's disease as well as amyotrophic lateral sclerosis. Utility of these systems broadly correlates with evolutionary complexity: small animal models such as rodents and the fruit fly are appropriate for pharmacological modeling and cognitive/behavioral assessment, the roundworm Caenorhabditis elegans allows analysis of tissue-specific disease features, and unicellular organisms such as the yeast Saccharomyces cerevisiae and the bacterium Escherichia coli are ideal for molecular studies. In this chapter, we highlight key advances in our understanding of protein misfolding/unfolding disease provided by model systems.