Understanding how cells sense and respond to stressful environmental changes is a major goal in cell biology. The two representative features of the cellular stress response that are conserved in all eukaryotes are 1) upregulation of molecular chaperones and 2) aggregation of RNA and protein into stress granules. These clusters had long been interpreted to be aggregates of misfolded proteins. However, we recently demonstrated (Riback et al., 2017) that poly(A)-binding protein (Pab1), a universally conserved stress-granule marker, forms clusters by phase separating without misfolding, and that disrupting phase separation impairs cellular growth during stress, indicating that stress-triggered aggregation of Pab1 is a part of the adaptive stress response. We refer to such fitness-promoting stress-triggered aggregates as adaptive protein assemblies. The realization that stress triggers formation of two distinct class of aggregates, adaptive protein assemblies and misfolded protein aggregates, motivated us to investigate whether and how molecular chaperones can distinguish these different substrates. Pab1 phase-separates into a hydrogel under physiologically stressful conditions. To investigate how Pab1 hydrogels are disaggregated by molecular chaperones?specifically the stress-induced Hsp104 disaggregation system?we developed a fluorescence anisotropy assay that permits both quantitative and kinetic measurement of Pab1 dispersal. This is the first in vitro system for studying disaggregation of adaptive protein assemblies. The yeast Hsp104 consists of three molecular chaperones, Hsp104/70/40. We will ask how Pab1 disaggregation differs from disaggregation of misfolded aggregates. Our preliminary data show that disaggregation of Pab1 hydrogel is much faster than disaggregation of the model substrate firefly luciferase. We will also ask what molecular features of Pab1 are recognized by molecular chaperones. Hsp70 preferentially binds unfolded proteins with an extended hydrophobic sequence, and hydrophobic residues in the proline-rich low-complexity domain of Pab1 modulate phase-separation of Pab1. We thus hypothesize that features which alter phase-separation of Pab1 will also affect disaggregation. We will test this hypothesis using our recently reported Pab1 mutants with altered phase-separation behavior. Finally, we have a strong prediction based on both literature and preliminary data that phosphorylation of Hsp70/Ssa1 at T36 will increase the efficiency of the Hsp104 disaggregation system toward Pab1 hydrogels. We will test this prediction using the fluorescence anisotropy assay. Additionally, we employ an evolutionary analysis approach to identify other regulatory phosphorylation sites; we will test our findings biochemically using the fluorescence anisotropy assay. The outcome of this research will advance our understanding of how the Hsp104 disaggregation system disperses different stress-induced structures.
(Public Health Relevance Statement) Compromised cellular capacity to dismantle environmental stress-induced protein aggregates is linked to a number of age-associated neurodegenerative diseases such as Alzheimer and Parkinson?s. This proposed research will study how molecular chaperones dismantle a relatively recently described class of environmental stress-induced aggregates by focusing on the disaggregation of stress-induced poly(A)-binding protein (Pab1 in yeast; PABPC1 in human) hydrogels.
