Building a Toolbox of Sensors and Approaches to Monitor the Proteostasis Network Core B

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Core B – Frydman/Finkbeiner - Sensors and Tools for Proteostasis Analyses Summary: This Core will develop tools to define the proteostasis network (PN) during aging and in the context of disease-associated aggregation-prone proteins and will provide technical support to Projects 1–4. The resources will also be used to test and validate compounds from Core D. Core B will have two sites in close geographical proximity. The Frydman group at Stanford will build molecular sensors and assays to measure proteostasis changes during aging, starting with reporters developed for yeast chaperone machinery and the ubiquitin-proteasome system (UPS). The Finkbeiner group at Gladstone/UCSF will build on mammalian expression and analysis tools to assess protein aggregation and autophagy and help PLs address specific questions for which their multiplexed longitudinal single-cell imaging platform is critical. By working hand-in- hand and testing the ability of the sensors to report on different proteostasis events in experiments used by all Projects, we will produce a set of assays that provide a comprehensive view of the PN of any cell. Tools will be available to the research community. A primary task of Core B will be to develop molecular sensors that robustly and quantitatively report on each branch of the PN and that change in aging and in the context of disease-associated misfolded proteins. Imaging sensors will also allow us to examine how proteostasis mechanisms are communicated from one cell to another. Answers to these questions will provide key insights into mechanisms of proteostasis dysfunction and protein aggregation during aging and will open new avenues for maintaining health and treating disease. We Propose to: (1) Develop and validate a panel of proteostasis sensors and reporters and (2) Assist project leaders (PLs) in the analysis of the PN sensors: towards a comprehensive exam of the PN status of any cell. Our ultimate goal is to build a comprehensive set of sensors compatible with 384-well plates that could be used as an integrated panel to interrogate all aspects of the PN. By synthesizing the collective body of knowledge of the proteostasis network into a set of fluorescence-based sensors and reporters, Core B will produce an invaluable toolbox for the whole proteostasis field that will facilitate progress, not only in the labs associated with this PPG but also in other labs interested in linking proteostasis dysfunction with aging and disease.