Methods to identify the substrates of thiol-disulfide oxidoreductases

Takushi Fujimoto, Kenji Inaba, Hiroshi Kadokura

Research output: Contribution to journalReview articlepeer-review

11 Citations (Scopus)


The formation of a disulfide bond is a critical step in the folding of numerous secretory and membrane proteins and catalyzed in vivo. A variety of mechanisms and protein structures have evolved to catalyze oxidative protein folding. Those enzymes that directly interact with a folding protein to accelerate its oxidative folding are mostly thiol-disulfide oxidoreductases that belong to the thioredoxin superfamily. The enzymes of this class often use a CXXC active-site motif embedded in their thioredoxin-like fold to promote formation, isomerization, and reduction of a disulfide bond in their target proteins. Over the past decade or so, an increasing number of substrates of the thiol-disulfide oxidoreductases that are present in the ER of mammalian cells have been discovered, revealing that the enzymes play unexpectedly diverse physiological functions. However, functions of some of these enzymes still remain unclear due to the lack of information on their substrates. Here, we review the methods used by researchers to identify the substrates of these enzymes and provide data that show the importance of using trichloroacetic acid in sample preparation for the substrate identification, hoping to aid future studies. We particularly focus on successful studies that have uncovered physiological substrates and functions of the enzymes in the periplasm of Gram-negative bacteria and the endoplasmic reticulum of mammalian cells. Similar approaches should be applicable to enzymes in other cellular compartments or in other organisms.

Original languageEnglish
Pages (from-to)30-40
Number of pages11
JournalProtein Science
Issue number1
Publication statusPublished - 2019 Jan


  • disulfide bond
  • disulfide-linked enzyme–substrate complex
  • oxidative protein folding
  • protein disulfide isomerase
  • thiol-based redox regulation
  • thioredoxin superfamily member

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology


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