Abstract
The pool of quality control proteins (QC) that maintains protein-folding homeostasis (proteostasis) is dynamic but can become depleted in human disease. A challenge has been in quantitatively defining the depth of the QC pool. With a new biosensor, flow cytometry-based methods and mathematical modeling we measure the QC capacity to act as holdases and suppress biosensor aggregation. The biosensor system comprises a series of barnase kernels with differing folding stability that engage primarily with HSP70 and HSP90 family proteins. Conditions of proteostasis stimulation and stress alter QC holdase activity and aggregation rates. The method reveals the HSP70 chaperone cycle to be rate limited by HSP70 holdase activity under normal conditions, but this is overcome by increasing levels of the BAG1 nucleotide exchange factor to HSPA1A or activation of the heat shock gene cluster by HSF1 overexpression. This scheme opens new paths for biosensors of disease and proteostasis systems.
Highlights
The pool of quality control proteins (QC) that maintains protein-folding homeostasis is dynamic but can become depleted in human disease
Others have examined the in-cell folding rates of a test protein through rapid temperature jumps to follow rates for reestablishment of equilibrium[7]. These approaches lack a quantitative capacity to understanding proteostasis; namely through the inability to define the effectiveness of QC systems to engage with the bait proteins
We describe a biosensor system based on a series of metastable bait proteins that report on foldedness and aggregation state by fluorescence resonance energy transfer (FRET)
Summary
The pool of quality control proteins (QC) that maintains protein-folding homeostasis (proteostasis) is dynamic but can become depleted in human disease. The method reveals the HSP70 chaperone cycle to be rate limited by HSP70 holdase activity under normal conditions, but this is overcome by increasing levels of the BAG1 nucleotide exchange factor to HSPA1A or activation of the heat shock gene cluster by HSF1 overexpression This scheme opens new paths for biosensors of disease and proteostasis systems. Others have examined the in-cell folding rates of a test protein through rapid temperature jumps to follow rates for reestablishment of equilibrium[7] These approaches lack a quantitative capacity to understanding proteostasis; namely through the inability to define the effectiveness of QC systems to engage with the bait proteins. We describe a new mathematical framework that can extract quantitative information from the holdase activity of QC as well as ability to suppress aggregation These approaches provide insight to the depth of the pool of QC resources that regulate proteostasis
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