A genetically encoded probe for the identification of proteins that form sulfenic acid.

摘要

The damage caused by reactive oxygen species (ROS) has been linked to a wide variety of human pathologies, such as Alzheimer's disease, atherosclerosis, cancer, and aging. ROS, such as hydrogen peroxide (H2O2), are also involved in regulation of cellular signal transduction pathways through thiol modifications on protein cysteine residues. The principle product produced from the reaction of protein cysteine with H2O2 is cysteine sulfenic acid (Cys-SOH). The reactivity of Cys-SOH toward other thiols as well as its ability to reversibly participate in disulfide bond exchange allows for the modification of signal transduction pathways. Cys-SOH is relatively unstable and has no unique UVvisible or fluorescent characteristics. A lack of tools for the detection of Cys-SOH has made the identification of proteins that form Cys-SOH difficult. By utilizing the region of the Yap1 transcription factor known to form a disulfide bond with the Cys-SOH on the thiol peroxidase Orp1, I have designed a genetically encoded probe for the general detection and identification of proteins that form Cys-SOH in vivo. This dissertation demonstrates the successful application of the Yap1-cCRD probe for the identification of proteins that form Cys-SOH in both Escherichia coli and Saccharomyces cerevisiae in response to H2O2. I have also identified proteins that form Cys-SOH in response to other peroxides, such as tert-butyl hydroperoxide and cumene hydroperoxide in an investigation of protein peroxide specificity. Additional insight into peroxide specificity was also gained in a study of the mechanisms of Cys-SOH formation on Orp1. The data presented here will hopefully aid in further understanding of the physiological roles of proteins as well as providing insight into dysfunction linked to human pathologies.