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.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Artemisinins / pharmacology
  • Cells / metabolism*
  • Cysteine / chemistry
  • Fluorescent Dyes / chemistry
  • HEK293 Cells
  • HeLa Cells
  • Humans
  • Huntingtin Protein / metabolism
  • Malaria / parasitology
  • Maleimides / chemistry
  • Mice
  • Molecular Probes / chemistry*
  • Mutant Proteins / metabolism
  • Oligopeptides / pharmacology
  • Parasites / drug effects
  • Parasites / metabolism
  • Protein Folding* / drug effects
  • Proteome / metabolism
  • Proteostasis* / drug effects
  • Solubility
  • Sulfhydryl Compounds / metabolism*
  • Tunicamycin / pharmacology


  • Artemisinins
  • Fluorescent Dyes
  • Huntingtin Protein
  • Maleimides
  • Molecular Probes
  • Mutant Proteins
  • Oligopeptides
  • Proteome
  • Sulfhydryl Compounds
  • Tunicamycin
  • maleimide
  • artenimol
  • Cysteine
  • epoxomicin