A wide range of important human diseases are characterized by protein misfolding. In addition to disease, protein misfolding is also caused by a range of cellular stresses. In the cell, protein chaperones maintain proteostasis by ensuring correct folding of newly synthesized proteins and the refolding of stress-denatured proteins. It follows that a full understanding of protein misfolding diseases depends on understanding both how chaperones function and how they are regulated in response to stress. High temperature stress is one of the stresses which causes protein misfolding. The two goals of this project are to understand 1) how cells sense high temperature stress, and 2) how the Arabidopsis protein chaperone BOBBER1 (BOB1) ensures normal cellular function under both non-stressful and stressful condition. The mechanisms which cells use to sense heat stress and misfolded proteins are only partially understood. Forward genetic approaches have been of limited use in understanding these mechanisms because of genetic redundancy. We plan on identifying genes involved in high temperature sensing and signaling, genes we call thermostat genes, using the RootScope, a custom automated microscopy system. This high-throughput system allows us to quantitatively monitor cellular responses to heat stress with high spatial and temporal resolution. It enables us to identify subtle changes in the kinetics of heat shock responses caused by mutations in redundant genes. The RootScope will allow us to identify and characterize genes involved in responding to protein misfolding which cannot be identified using conventional phenotyping approaches. BOB1 encodes a protein chaperone with functions in both development and temperature responses. BOB1 contains a conserved NudC protein domain. NudC genes are essential genes in animals, fungi, and plants suggesting that they have conserved, important, and unique functions. NudC genes are found in humans so understanding BOB1 function will contribute to our understanding of protein quality control in disease. We have taken advantage of the developmental defects in bob1 mutants to identify genetic interactions between a BOB1 partial loss of function allele and multiple genes encoding proteasome subunits. We plan on characterizing these interactions in order to understand how protein folding and degradation mechanisms are linked. We have also identified two additional genes which interact genetically with BOB1 which we will clone and characterize. Finally, we will continue an ongoing BOB1 genetic modifier screen to identify and characterize additional genes and cellular pathways which rely on BOB1 function. The identification and characterization of both heat shock response thermostat and BOB1 interacting genes will contribute to a mechanistic understanding of how eukaryotic cells sense and respond to disruptions to proteostasis caused by elevated temperatures and other stresses.
This project takes advantage of a newly developed automated microscopy system to quantitatively investigate how cells sense and respond to high temperature stresses. Additionally, the cellular functions of BOBBER1, a molecular chaperone which helps other proteins fold and function correctly will be investigated using a genetic screen. Both of these approaches will generate insights into how cells respond to misfolded proteins, a characteristic feature of many human diseases.