Identification of redox-active cell-surface proteins by mechanism-based kinetic trapping

Sci STKE. 2007 Dec 18;2007(417):pl8. doi: 10.1126/stke.4172007pl8.

Abstract

A number of thiol-dependent oxidoreductases are released from cells and act on the cell surface. Correspondingly, several cell-surface processes appear to depend on catalyzed thiol-disulfide exchange, including integrin activation and the fusion of viral particles with the host membrane. Tumor cells frequently increase the abundance of secreted and cell-surface forms of particular oxidoreductases, and evidence suggests that oxidoreductases released from tumor cells promote growth and contribute to the remodeling of the cellular microenvironment. Few cell-surface or membrane proteins that are targeted by extracellular redox enzymes have been identified. One major reason for this slow progress is the highly transient nature of thiol-disulfide exchange, making its detection by conventional techniques difficult or impossible. Here we describe the application of an activity-based proteomics approach, also known as "mechanism-based kinetic trapping," to identify individual cell-surface target proteins that engage in disulfide exchange with thiol-dependent oxidoreductases. Although we have applied this approach to thioredoxin-1, it should also be applicable to other members of the thioredoxin superfamily whose activity is based on the CXXC active-site motif.

MeSH terms

  • Bacterial Proteins / genetics
  • Bacterial Proteins / physiology
  • Base Sequence
  • Cell Culture Techniques / methods
  • Cell Membrane / physiology*
  • Escherichia coli / genetics
  • Escherichia coli / ultrastructure
  • Kinetics
  • Membrane Proteins / genetics
  • Membrane Proteins / physiology*
  • Molecular Sequence Data
  • Oxidation-Reduction
  • Polymorphism, Single Nucleotide
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Thioredoxins / genetics

Substances

  • Bacterial Proteins
  • Membrane Proteins
  • Recombinant Proteins
  • Thioredoxins