Identification of Novel Mediators of Ferroptosis Using Proteomic Methods

Abstract

Over the past two decades there have been a variety of programmed cell death (PCD) pathways to emerge. Among these emerging PCD pathways ferroptosis is has been of especial interest as its iron-dependent reliance on the generation of reactive oxygen species (ROS) links this PCD pathway to some of the most pervasive pathologies including cancer and neurodegeneration. In order to broaden our understanding of ferroptosis we applied a number of proteomic based methods in effort to identify novel ferroptotic mediators. Before the application of proteomic methods, we developed complementary pharmacological and genetic ferroptosis models. With these models we identified ferroptosis-induced changes in protein abundance. Using these data, we generated CRISPR-Cas9-mediated knockouts of protein disulfide isomerase A1 (PDIA1) and cytosolic acetyl-CoA acetyltransferase (ACAT2) that were found to exhibit altered susceptibility to the induction of ferroptosis. With data on protein abundance changes we then profiled ferroptosis-induced changes in abundance corrected cysteine reactivity. Many proteins displayed significant changes in cysteine reactivity that will require further investigation in order to determine if they are drivers or a downstream consequence of ferroptosis. Finally, we used a cell surface biotinylation reagent together with proteomics in effort to identify potential cell surface markers of ferroptosis. This lead to the identification of multiple known cell membrane proteins with ferroptosis altered cell surface labeling. Future studies will seek to confirm the observed alterations with complementary methods. Together these studies illustrate the dynamic responses of the proteome to the induction of ferroptosis.