A biosensor-based framework to measure latent proteostasis capacity

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.

Widespread remodelling of proteome solubility in response to different protein homeostasis stresses

“…To investigate whether the proteins that changed solubility upon Httex1 aggregation are relevant to protein homeostasis stress more generally, we expanded our analysis to examine proteome solubility changes associated with 5 other triggers of protein homeostasis stress that have previously reported roles leading to protein misfolding and aggregation. These stresses included three specific inhibitors of key protein homeostasis hubs (Hsp70, Hsp90 and the proteasome) whereby defects are reported in models of Huntington's disease, protein aggregation, degradation of misfolded proteins and-or other markers of protein homeostasis stress (9,22,23,(38)(39)(40)(41)(42)(43) and two exogenous stress states that reflect pathology observed in neurodegenerative disease settings and protein aggregation in cell models (namely, oxidative stress and ER stress) (44-50). The Hsp70 chaperone system was targeted by the small molecule inhibitor Ver-155008, which binds to the ATPase domain of Hsp70 family proteins (K d of 0.3 µM and IC 50 of 0.5 to 2.6 µM) (51, 52).…”

“…The Hsp70 chaperone system was targeted by the small molecule inhibitor Ver-155008, which binds to the ATPase domain of Hsp70 family proteins (K d of 0.3 µM and IC 50 of 0.5 to 2.6 µM) (51, 52). We have previously demonstrated Ver-155008 impairs protein homeostasis in cell culture models and can increase the aggregation propensity of an ectopically expressed metastable bait protein (38). Hsp90 was targeted with the ATP binding competitor novobiocin, which binds to the C-terminal nucleotide binding pocket to inhibit activity (40,42,43,53).…”

“…Unlike other drugs that target the N-terminal domain of Hsp90, novobiocin does not induce a heat shock response (54). We showed previously that novobiocin can unbalance the protein homeostasis system and induce the aggregation of a metastable bait protein (38).…”

Quantification of Cellular Proteostasis in Live Cells by Fluorogenic Assay Using the AgHalo Sensor

“…To this end, fusion of fluorescent protein tags to these sensors or labeling with fluorescent probes has been utilized to visualize the sensors’ aggregation by observing fluorescent granules in live cells. These sensors are exemplified by destabilized client proteins that are fused to green fluorescent protein (GFP) (Gupta et al., ; Winkler et al., ), thermally labile proteins incorporating fluorescent unnatural amino acids (Hsieh et al., ), thermally labile proteins that are fused to a tetracysteine motif and labeled with FlAsH dye (Ignatova & Gierasch, ), destabilized retroaldolase enzyme that is labeled with a fluorophore (Liu, Zhang, Chen, Tan, & Kelly, ), destabilized barnase enzyme (Wood et al., ), GFP‐fused heat‐shock proteins (Pereira et al., ), and destabilized GFP (Waldo, Standish, Berendzen, & Terwilliger, ; Wigley, Stidham, Smith, Hunt, & Thomas, ). The limitation of these sensors is that they exhibit fluorescence before and after aggregation (i.e., they are non‐fluorogenic), so their aggregation can only be judged based on formation of fluorescent granules.…”

Heat Shock Protein Reports on Proteome Stress

“…Within a compromised proteostasis, metastable protein sensors aggregate and produce measurable signal for detection. Examples are provided by firefly luciferase, a de novo designed retroaldolase, Halo‐Tag, and barnase . Thus, most present proteome stress sensors use aggregation of a specific metastable protein to flag proteome stress.…”