Abstract
Reactive oxygen species (ROS) are key molecules regulating various cellular processes. However, what the cellular targets of ROS are and how their functions are regulated is unclear. This study explored the cellular proteomic changes in response to oxidative stress using H2O2 in dose- and recovery time-dependent ways. We found discernible changes in 76 proteins appearing as 103 spots on 2D-PAGE. Of these, Prxs, DJ-1, UCH-L3 and Rla0 are readily oxidized in response to mild H2O2 stress, and then degraded and active proteins are newly synthesized during recovery. In studies designed to understand the degradation process, multiple cellular modifications of redox-sensitive proteins were identified by peptide sequencing with nanoUPLC-ESI-q-TOF tandem mass spectrometry and the oxidative structural changes of Prx2 explored employing hydrogen/deuterium exchange-mass spectrometry (HDX-MS). We found that hydrogen/deuterium exchange rate increased in C-terminal region of oxidized Prx2, suggesting the exposure of this region to solvent under oxidation. We also found that Lys191 residue in this exposed C-terminal region of oxidized Prx2 is polyubiquitinated and the ubiquitinated Prx2 is readily degraded in proteasome and autophagy. These findings suggest that oxidation-induced ubiquitination and degradation can be a quality control mechanism of oxidized redox-sensitive proteins including Prxs and DJ-1.
Highlights
Reactive oxygen species (ROS) including superoxide anion (O2−), hydrogen peroxide (H2O2), and hydroxyl radical (OH∙), act on lipids, proteins, DNA and their targets and cause oxidative modifications[1]
We identified the proteins differentially appearing in 2D-PAGE, employing peptide sequencing with nanoUPLC-ESI-q-TOF tandem MS (MS/MS)
This study showed that the functions of protein targets of H2O2 could be divided into two groups: one group of proteins closely associated with each other based on protein-protein interactions, is involved in stress response, mitochondria, carbohydrate metabolism, protein synthesis, ubiquitin-proteasome system (UPS), RNA metabolism and cytoskeleton
Summary
Reactive oxygen species (ROS) including superoxide anion (O2−), hydrogen peroxide (H2O2), and hydroxyl radical (OH∙), act on lipids, proteins, DNA and their targets and cause oxidative modifications[1]. To understand the molecular action of H2O2 in target proteins, it is necessary to identify the oxidative modifications in redox-sensitive Cys residues. It was demonstrated in Nm23-H1, a metastasis suppressor protein, that formation of disulfide bond between Cys[4] and Cys[145] induces structural changes, which cause Cys[109] residue to be readily oxidized to various oxidation states in stepwise oxidations[29,30] These oxidative modifications of redox-sensitive proteins are presumed to play significant but poorly understood roles in cellular functions and regulations of proteins. Further studies on the actions of ROS at molecular level, so called ‘ROSics’, are needed for identifying redox-sensitive Cys residues, their oxidative modifications and structural changes caused by oxidation, and understanding how biological activities are regulated by oxidation[31]
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