Systemic investigation of redox regulated proteome

Plants and animals developed different strategies to adapt to oxygen availability. One is to regulate gene expression by the hypoxia-inducible transcription factor (HIF). Second is via the Arg/Cys branch of the N-end rule pathway, which directs proteins for proteasomal degradation. To date, Regulator Of G Protein Signaling 4 (RGS4) and RGS16, members of the RGS family of GTPase-activating proteins, and interleukin (IL)-32 were found to be physiological substrates regulated by oxygen. In our study we used systematic proteomic approaches to search for additional oxygen regulated proteins by analyzing proteome stability in response to hypoxia and oxidative stress conditions. Next, using mutagenesis approach, we mapped the degradation signal (degron) of the proteins whose stability changes in response to hypoxia. CRISPR/Cas9 approach was used to characterize E3 ligases and other regulators that are responsible for the proteolysis of our substrate candidates. Overall, our approach identified novel substrates, allowing better understanding of redox regulated proteome that help to shed light on pathways important to cell and animal physiology in the adaptation response to oxygen fluctuations.