Organized protein aggregation: a regulatory strategy to adapt to stress conditions

Failure of proteins to adopt an appropriately folded conformation is a prevalent cause of human disease and hinders the biotechnological production of valuable proteins. Misfolded proteins usually either refold to their functional states or are degraded by quality control mechanisms. When misfolded proteins evade quality control, they form aggregates that have been viewed as a hallmark of proteostasis. For instance, the organized sequestration of misfolded proteins to defined inclusion sites is a regulated process that depends on dedicated molecular chaperones, termed sequestrases. Appropriate sequestration of protein in aggregates is a defence strategy that prevents the potential dysfunction and toxicity associated with protein misfolding diseases. Although aggregation is a well-studied phenomenon and many key players are known, it remains unclear how changes in cellular states including stress, quiescence, or nutritional alterations cause protein aggregation, and the degree of stress specificity in the chaperone response to aggregate formation is unknown. Sequestrase chaperones help cells to cope with accumulating misfolded protein by sequestering proteins away from their normal productive pathways to protect against cytotoxic effects and facilitate targeted degradation. More recently, chaperone-mediated sequestration has also been identified as a regulatory response that compartmentalises key signalling molecules. For example, sequestration can act to separate signalling molecules away from their substrates and inhibitors under stress conditions. This current proposal builds upon our recent studies where we show that chaperone-mediated sequestration provides a non-canonical mechanism to fine-tune protein kinase A (PKA) signalling in yeast. We have identified a PKA inactivation mechanism based on sequestration of a specific PKA isoform. Sequestration is controlled by the Hsp42 sequestrase and is reversible such that PKA activity is restored following stress recovery. These studies reveal a previously unknown mechanism that regulates a conserved cellular signalling pathway and provides a new rationale for protein aggregation upon stress. This proposal addresses fundamental questions concerning sequestration as a protein quality control (PQC) mechanism in eukaryotic cells including: 1) How do sequestrase chaperones mediate the spatial sequestration of proteins as a stress defence strategy? The overall aim is to provide a mechanistic understanding of protein sequestration and the stress specificity of sequestrase chaperones. 2) How and why do cells sequester misfolded proteins into aggregates? As a part of this project, we aim to define rules and principles governing the formation and resolution of aggregates, which can then be exploited to design strategies to mitigate aggregate toxicity. 3) How does sequestration act as a reversible response to control enzyme activity and key signalling pathways during stress exposure and recovery. This research will provide a greater understanding of how protein aggregates form in cells and the rules determining this will address the BBSRC strategic objective: Understanding the rules of life. Beneficiaries will include researchers in the immediate 'protein homeostasis' and 'chaperone biology' communities, as well as scientists broadly interested in protein misfolding and stress. The use of yeast biochemical and genetic techniques has in the past served as a paradigm for the study of protein misfolding across all eukaryotes and much of the current knowledge in these fields has stemmed from such work. Importantly, the proposed work will expand skills, training and experience for a RA1A and Technician. This project will provide a conduit for researcher progression to positions that positively impact on academic and industrial sectors, addressing the BBSRC's strategic objective: World-class people and careers.