This is a competing revision (NOT-OD-09-058, Notice title: NIH Announces the Availability of Recovery Act Funds for Competitive Revision Applications) for grant 1 R01 GM076536-01, """"""""Cell chips for genome-wide protein and RNA localization in single cells"""""""". Proteins are likely to organize into complexes that assemble and disassemble depending on cellular needs. However, proteins must also avoid catastrophic aggregation, which underlies many important diseases (e.g., Alzheimer's &prion diseases) and even aging itself. This competitive revision pursues a striking discovery we recently made during work on the primary aims of this grant: hundreds of distinct, normally cytosolic protein species form metabolite-induced, reversible, macroscopic punctate foci in quiescent cells of the yeast S. cerevisae. Are these macroscopic assemblies functional or catastrophic? The answers to this question are important for our understanding of quiescent cells and of aggregation-based disease. If these assemblies are functional or contain functional proteins, this represents unexpectedly high levels of organization in quiescent cells, with important ramifications for human cells, which are mostly in the quiescent state. If these assemblies represent widespread aggregation, such structures are likely important factors for aggregation-related disease and aging. In this revision, we propose experiments to distinguish these two hypotheses, with Aim 1 to measure kinetics of formation and dissolution of the punctate foci, relating kinetic properties to functional and sequence properties of the proteins, Aim 2 to rationally design &express proteins with altered aggregation propensities in yeast and monitor the formation of punctate foci. Our goal is to further assess the aggregation hypothesis by computationally designing proteins with a non-native aggregation propensity, expressing these proteins in S. cerevisiae, and monitoring formation of punctate foci. Finally, Aim 3 is to apply microscopy to determine which proteins co-localize to the same punctate foci in quiescent cells, thus testing if proteins from the same pathways co-localize, supporting functional assemblies. The results of these Aims will be concrete evidence, on a proteinby- protein basis, either for forming functional protein assemblies in quiescent cells or for catastrophic aggregation, and will thus accelerate the tempo of research in our original grant, as well as recruiting/retaining scientists consistent with Recovery Act aims.
Proteins must organize into complexes that assemble and disassemble depending on cellular needs, while simultaneously avoiding catastrophic aggregation. Such aggregation underlies many important diseases, including Alzheimer's disease and prion diseases, and even aging itself. This grant proposes cell biology and imaging experiments to understand the mechanistic basis for widespread, reversible protein aggregation observed in quiescent cells. This work will increase our understanding of the mechanisms and circumstances under which proteins aggregate, and thus will be a step towards better understanding of the forces underlying aggregation diseases and aging.
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