A thiol probe for measuring unfolded protein load and proteostasis in cells

MZ Chen; NS Moily; JL Bridgford; RJ Wood; M Radwan; TA Smith; Z Song; BZ Tang; L Tilley; X Xu; GE Reid; MA Pouladi; Y Hong; DM Hatters

Journal article

A thiol probe for measuring unfolded protein load and proteostasis in cells

MZ Chen, NS Moily, JL Bridgford, RJ Wood, M Radwan, TA Smith, Z Song, BZ Tang, L Tilley, X Xu, GE Reid, MA Pouladi, Y Hong, DM Hatters

Nature Communications | NATURE PUBLISHING GROUP | Published : 2017

DOI: 10.1038/s41467-017-00203-5

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Abstract

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 proteo..

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University of Melbourne Researchers

Trevor Smith Author

Leann Tilley Author

Gavin Reid Author

Danny M Hatters Author

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Awarded by University of Melbourne

Funding Acknowledgements

We thank Peng Zeng and Sijie Chen from The University of Melbourne for technical support and helpful discussion. We also thank Michael Parker from St Vincents Institute of Medical Research, VIC, Australia for helpful discussion and feedback. We thank Juliet Gerrard from the University of Auckland, NZ for provision of the human peroxiredoxin expression construct. This work was supported by grants to D.M.H (National Health and Medical Research Council grants APP1049458, APP1049459 and APP1102059 and Australian Research Council (FT120100039 and DP170103093)) Y.H.(Australian Research Council DE170100058, Bruce Stone Fellowship from La Trobe University, Melbourne Neuroscience Institute Interdisciplinary Seed Funding (University of Melbourne), and McKenzie Fellowship from University of Melbourne) and grants to M.A.P. (Ministry of Education Singapore Tier 1 grant R-172-000-297-112, and Agency for Science, Technology and Research Strategic Positioning Fund for Genetic Orphan Diseases grant SPF2012/005).