A thiol probe for measuring unfolded protein load and proteostasis in cells Academic Article uri icon

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.

authors

  • Chen, Moore Z
  • Moily, Nagaraj S
  • Bridgford, Jessica L
  • Wood, Rebecca J
  • Radwan, Mona
  • Smith, Trevor A
  • Song, Zhegang
  • Tang, Ben Zhong
  • Tilley, Leann
  • Xu, Xiaohong
  • Reid, Gavin E
  • Pouladi, Mahmoud A
  • Hong, Yuning
  • Hatters, Danny M

publication date

  • 2017