Identification of redox-active cell-surface proteins by mechanism-based kinetic trapping.
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.
Schwertassek
Ulla
U
Redox Regulation Research Group (A160), German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany.
Weingarten
Lars
L
Dick
Tobias P
TP
D001426:Bacterial Proteins / Q000235:genetics / Q000502:physiology; D001483:Base Sequence; D018929:Cell Culture Techniques / Q000379:methods; D002462:Cell Membrane / Q000502:physiology*; D004926:Escherichia coli / Q000235:genetics / Q000648:ultrastructure; D007700:Kinetics; D008565:Membrane Proteins / Q000235:genetics / Q000502:physiology*; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D020641:Polymorphism, Single Nucleotide; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013879:Thioredoxins / Q000235:genetics
D001426:Bacterial Proteins; D008565:Membrane Proteins; D011994:Recombinant Proteins; D013879:Thioredoxins