Chaperone-Facilitated Aggregation of Thermo-Sensitive Proteins Shields Them from Degradation during Heat Stress

Abstract

Graphical Abstract Highlights d Misfolded proteins collapse into protein aggregate centers (PACs) upon heat shock d PAC formation requires the Hsp40 chaperone Mas5 d PACs serve as seed for the nucleation of stress granules at severe temperatures d The fate of PACs is refolding, as they protect misfolded proteins from degradation SUMMARY Cells have developed protein quality-control strategies to manage the accumulation of misfolded substrates during heat stress. Using a soluble reporter of misfolding in fission yeast, Rho1.C17R-GFP, we demonstrate that upon mild heat shock, the reporter collapses in protein aggregate centers (PACs). They contain and/or require several chaperones, such as Hsp104, Hsp16, and the Hsp40/70 couple Mas5/ Ssa2. Stress granules do not assemble at mild temperatures and, therefore, are not required for PAC formation; on the contrary, PACs may serve as nucleation centers for the assembly of stress granules. In contrast to the general belief, the dominant fate of these PACs is not degradation, and the aggregated reporter can be disassembled by chaperones and recovers native structure and activity. Using mass spectrometry, we show that thermo-unstable endogenous proteins form PACs as well. In conclusion, formation of PACs during heat shock is a chaperone-mediated adaptation strategy. Image Studio Lite Li-Cor Biosciences https://www.licor.com/bio/ image-studio-lite/ e1

Acute Heat Stress Leads to Reversible Aggregation of Nuclear Proteins into Nucleolar Rings in Fission Yeast

“…98.1% (98.1% ± 2%, n = 185) of wild-type cells and 95.8% (95.8% ± 2.3%, n = 241) of hsp104D cells assembled NuRs upon HS; however, during the recovery, the absence of Hsp104 resulted in a strong delay in NuRs dissolution ( Figure 4D). hsp104D cells also delayed cytoplasmic SG dissolution ( Figure 4D), in agreement with a recent report (Cabrera et al, 2020). Consistently, the absence of Hsp104 resulted in a delay in cell growth recovery after the HS and in a significant decline in cell survival ( Figure 4D).…”

“…Although increased protein synthesis and turnover, as well as protection by molecular chaperones, predominate during mild HS, protein aggregation that stops translation and ceases cell growth has a prominent role during extreme conditions (Cabrera et al, 2020;M€ uhlhofer et al, 2019;Wallace et al, 2015). In S. pombe under mild HS (37 C), Hsp70/40 chaperones facilitate the aggregation of non-terminally unfolded proteins in cytosolic PACs, which might serve as nucleation sites for SGs formed at extreme temperatures (Cabrera et al, 2020). Here, we show that during acute HS, several nuclear proteins aggregate and immobilize in a reversible manner at the nucleolar region ( Figure S5).…”

“…The disaggregation of NuRs, as occur with other cytosolic and nuclear aggregates during the recovery from HS, depends on the activity of Hsf1 and Hsp104 disaggregase (Figure 4). This leads to the recovery of the native structure and activity of aggregated proteins to promote cell fitness and rapid restoration of cell growth after the HS (Cabrera et al, 2020;Cherkasov et al, 2013;Glover and Lindquist, 1998;Parsell et al, 1994). Therefore, NuRs represent a reversible state of protein aggregation, which functions as a resilience mechanism for heat adaptation and contributes to the general inhibition of nuclear activities upon acute HS.…”

“…In S. pombe, Hsp104 is required to disassemble cytoplasmic protein aggregate centers (PACs) formed after mild and severe HS (Cabrera et al, 2020). To test whether Hsp104 activity is required for NuR dissolution, we monitored Nup85 as a NuR marker during acute HS in the hsp104D mutant.…”

Evolutionarily conserved chaperone-mediated proteasomal degradation of a disease-linked aspartoacylase variant

“…This is consistent with a previous study showing that proteins initially form small amorphous stress foci, and then depend on Hsp104 to remodel these smaller aggregates into larger inclusions (Spokoini et al, 2012). In addition, a recent study in fission yeast found that chaperone-mediated sequestration of misfolded proteins into protein aggregate centres (PACs) protected proteins from degradation and allowed for subsequent refolding (Cabrera et al, 2020). Hence, without Hsp104, ASPA C152W fails to undergo refolding-linked disaggregation and is routed by parallel pathways to degradation or remains in the smaller inclusions.…”