A genetically encoded probe for cysteine sulfenic acid protein modification in vivo.
All organisms have defense mechanisms to combat the deleterious effects of oxidative damage produced by reactive oxidative species (ROS). Although it is known that ROS play a major role in oxidative damage, increasing evidence reveals that ROS have wider cellular effects through their role in many signal transduction pathways. Here we have adapted a redox-regulated domain from the Yap1 transcription factor in Saccharomyces cerevisiae to function as a general trap for proteins that form cysteine sulfenic acid (Cys-SOH) in vivo. In response to H2O2, the Yap1 probe forms mixed disulfide bonds with a variety of proteins. The formation of these protein complexes is time dependent and peroxide concentration dependent. Disulfide-bonded complex formation can be attenuated by the addition of dimedone, a compound that specifically reacts with Cys-SOH, indicating the specificity of the probe toward Cys-SOH. An efficient one-step purification procedure was developed for proteins trapped by the Yap1 probe, and the constituents were identified by mass spectrometry. This methodology identified six proteins in Escherichia coli that contain redox-active cysteine residues known to form Cys-SOH as part of their catalytic cycle. The results suggest that the Yap1 probe is useful for identifying Cys-SOH-regulated proteins and can be employed in any genetically tractable organism to monitor transient Cys-SOH formation in vivo.
Takanishi
Christina L
CL
Department of Environmental Toxicology, University of California, Davis, California 95616, USA.
Ma
Li-Hua
LH
Wood
Matthew J
MJ
D000595:Amino Acid Sequence; D003545:Cysteine / Q000031:analogs & derivatives* / Q000737:chemistry; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000737:chemistry; D008954:Models, Biological; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011499:Protein Processing, Post-Translational; D017386:Sequence Homology, Amino Acid; D013434:Sulfenic Acids / Q000737:chemistry*
D005656:Fungal Proteins; D013434:Sulfenic Acids; D006861:Hydrogen Peroxide; C100870:cysteinesulfenic acid; D003545:Cysteine