Dissecting the cellular response to unfolded proteins in the cytosol and nucleus of mammalian cells [electronic resource]

Proteins are involved in every aspect of life owing to their highly diverse enzymatic and structural properties. Despite their importance in the regulation of life, proteins are vulnerable in living cells. Since this trait can easily lead to cellular defects, the stability of the protein folding process must be monitored. Failure to clear aberrant proteins results in the loss as well as gain of protein function, which may disrupt various cellular functions and lead to diseases in humans such as Alzheimer's, Parkinson's, and Huntington's diseases. Furthermore, cancer, one of the biggest causes of death, and aging, an inevitable defect for every human being, have been reported to be related to unfolded proteins. Thus, the maintenance of protein folding homeostasis is indispensable to all organisms, and the cell develops and contributes a significant amount of energy to ensuring that nascent proteins become their native state upon translation, to preventing the accumulation of aberrant proteins, and to maintaining the proper folding state of proteins. In chapter one I have briefly summarized the current understandings about the stress response related to unfolded proteins. However, we do not fully comprehend the cellular response to unfolded proteins. One reason why the response has not been well elucidated is because of lack of specific perturbants that induced the specific stresses in an acute manner. To overcome the barrier in elucidating the mechanisms inderlying the cellular response to unfolded proteins, we implemented a unique chemical biology approach developed by our group, known as destabilizing domains (DDs). Because the folding state of DDs can be conditionally controlled by a cell-permeable small molecule, we were able to create specific amounts of unfolded proteins inside cells in a simple and sudden manner by removing the ligand. Chapter two reports that acute unfolded protein stress from unfolded DDs elicit a coordinated transcriptional response from mammalian cells. The response is distinct from heat stress and the conventional unfolded protein response, and cells that trigger this response are more resistant than unstressed cells when challenged with other stressors. In addition, by changing the location of DDs, we demonstrated for the first time that the nucleus and cytoplasm have compartment specific responding elements to the stress from unfolded proteins. In the third chapter, we describe a series of experiments designed to elucidate another aspect of unfolded proteins, aggregation, by developing new method based on DD. We report a chemically controllable fluorescent aggregating protein that allows us to monitor the entire life of aggregates in living cells. This technology allows us to rapidly produce many small aggregates in seconds while monitoring the movement and coalescence of the aggregates in cells. We found that Hsc70 captures the aggregates extremely fast and prevents aggregation. This method is applicable for various experimental systems including living organisms, and further collaborations are currently taking place. The fourth and final chapter focus on our development of the new and third DD system to regulate a specific protein by a cell-permeable small molecule. The system based on the ligand binding domain of the estrogen receptor that can be regulated by one of the two synthetic ligands, CMP8 or 4-hydroxytamoxifen. It is orthogonal to other FKBP- and DHFR-based DD systems and will enable us to simultaneously and independently regulate three proteins in biological studies. We believe that this new technology to control protein activities will contribute to the advancement of science.

Keywords: Chemical and Systems Biology.
Advisor: Wandless Thomas primary advisor. Thesis advisor
Frydman Judith advisor. Thesis advisor
Meyer Tobias advisor. Thesis advisor
Snyder Michael