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

  • Nat Commun. 2017 Sep 7;8(1):474. doi: 10.1038/s41467-017-00203-5.
Moore Z Chen  1 ,  Nagaraj S Moily  1 ,  Jessica L Bridgford  1 ,  Rebecca J Wood  1 ,  Mona Radwan  1 ,  Trevor A Smith  2 ,  Zhegang Song  3 ,  Ben Zhong Tang  3 ,  Leann Tilley  1 ,  Xiaohong Xu  4 ,  Gavin E Reid  1  2 ,  Mahmoud A Pouladi  4  5 ,  Yuning Hong  6  7  8 ,  Danny M Hatters  9
Affiliations
  • 1. Department of Biochemistry and Molecular Biology and Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Parkville, VIC, 3010, Australia.
  • 2. School of Chemistry, The University of Melbourne, Parkville, VIC, 3010, Australia.
  • 3. Department of Chemistry, The Hong Kong University of Science & Technology, Clear Water Bay, Kowloon, Hong Kong, China.
  • 4. Translational Laboratory in Genetic Medicine, Agency for Science, Technology and Research, Singapore (A*STAR), 8A Biomedical Grove, Immunos, Level 5, Singapore, 138648, Singapore.
  • 5. Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, 117597, Singapore.
  • 6. Department of Biochemistry and Molecular Biology and Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Parkville, VIC, 3010, Australia. [email protected].
  • 7. School of Chemistry, The University of Melbourne, Parkville, VIC, 3010, Australia. [email protected].
  • 8. Department of Chemistry and Physics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, VIC, 3083, Australia. [email protected].
  • 9. Department of Biochemistry and Molecular Biology and Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Parkville, VIC, 3010, Australia. [email protected].
PMID: 28883394 DOI: 10.1038/s41467-017-00203-5
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 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.

Products