School of Chemistry - Research Publications

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Author: Reid, Gavin × Author: Hatters, Daniel × End date: 2019 × Type: Journal Article × Start date: 2017 ×

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    A thiol probe for measuring unfolded protein load and proteostasis in cells
    Chen, MZ ; Moily, NS ; Bridgford, JL ; Wood, RJ ; Radwan, M ; Smith, TA ; Song, Z ; Tang, BZ ; Tilley, L ; Xu, X ; Reid, GE ; Pouladi, MA ; Hong, Y ; Hatters, DM (NATURE PUBLISHING GROUP, 2017-09-07)
    When proteostasis becomes unbalanced, unfolded proteins can accumulate and aggregate. Here we report that the dye, tetraphenylethene maleimide (TPE-MI) can be used to measure cellular unfolded protein load. TPE-MI fluorescence is activated upon labelling free cysteine thiols, normally buried in the core of globular proteins that are exposed upon unfolding. Crucially TPE-MI does not become fluorescent when conjugated to soluble glutathione. We find that TPE-MI fluorescence is enhanced upon reaction with cellular proteomes under conditions promoting accumulation of unfolded proteins. TPE-MI reactivity can be used to track which proteins expose more cysteine residues under stress through proteomic analysis. We show that TPE-MI can report imbalances in proteostasis in induced pluripotent stem cell models of Huntington disease, as well as cells transfected with mutant Huntington exon 1 before the formation of visible aggregates. TPE-MI also detects protein damage following dihydroartemisinin treatment of the malaria parasites Plasmodium falciparum. TPE-MI therefore holds promise as a tool to probe proteostasis mechanisms in disease.Proteostasis is maintained through a number of molecular mechanisms, some of which function to protect the folded state of proteins. Here the authors demonstrate the use of TPE-MI in a fluorigenic dye assay for the quantitation of unfolded proteins that can be used to assess proteostasis on a cellular or proteome scale.
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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.