Biochemistry and Pharmacology - Research Publications

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Start date: 2010 × End date: 2019 × Author: Hatters, Daniel × Author: Haynes, Angelique × Author: Reid, Gavin ×

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    A biosensor-based framework to measure latent proteostasis capacity
    Wood, RJ ; Ormsby, AR ; Radwan, M ; Cox, D ; Sharma, A ; Voepel, T ; Ebbinghaus, S ; Oliveberg, M ; Reid, GE ; Dickson, A ; Hatters, DM (NATURE PUBLISHING GROUP, 2018-01-18)
    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.
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    Huntingtin Inclusions Trigger Cellular Quiescence, Deactivate Apoptosis, and Lead to Delayed Necrosis
    Ramdzan, YM ; Trubetskov, MM ; Ormsby, AR ; Newcombe, EA ; Sui, X ; Tobin, MJ ; Bongiovanni, MN ; Gras, SL ; Dewson, G ; Miller, JML ; Finkbeiner, S ; Moily, NS ; Niclis, J ; Parish, CL ; Purcell, AW ; Baker, MJ ; Wilce, JA ; Waris, S ; Stojanovski, D ; Bocking, T ; Ang, C-S ; Ascher, DB ; Reid, GE ; Hatters, DM (CELL PRESS, 2017-05-02)
    Competing models exist in the literature for the relationship between mutant Huntingtin exon 1 (Httex1) inclusion formation and toxicity. In one, inclusions are adaptive by sequestering the proteotoxicity of soluble Httex1. In the other, inclusions compromise cellular activity as a result of proteome co-aggregation. Using a biosensor of Httex1 conformation in mammalian cell models, we discovered a mechanism that reconciles these competing models. Newly formed inclusions were composed of disordered Httex1 and ribonucleoproteins. As inclusions matured, Httex1 reconfigured into amyloid, and other glutamine-rich and prion domain-containing proteins were recruited. Soluble Httex1 caused a hyperpolarized mitochondrial membrane potential, increased reactive oxygen species, and promoted apoptosis. Inclusion formation triggered a collapsed mitochondrial potential, cellular quiescence, and deactivated apoptosis. We propose a revised model where sequestration of soluble Httex1 inclusions can remove the trigger for apoptosis but also co-aggregate other proteins, which curtails cellular metabolism and leads to a slow death by necrosis.