PPIX-binding Proteins Reveal Porphyrin Synthesis and Ferroptosis Link

All aerobic organisms require the cofactor heme to survive, but its synthesis requires formation of a potentially toxic intermediate protoporphyrin IX (PPIX). Little is known about the extent of PPIXs cellular interactions. Here, we report the development of PPB, a biotin-conjugated, PPIX-probe that captures proteins capable of interacting with PPIX. Quantitative proteomics with PPB identified common proteins among a diverse panel of mammalian cell lineages. Pathway and quantitative difference analysis revealed PPB-bound proteins related to iron and heme metabolism and suggested that these processes might be altered by heme/porphyrin synthesis. We show that increased heme/porphyrin synthesis in cells promotes ferroptosis that is pharmacologically distinct from canonical ferroptosis driven by erastin, an inhibitor of the cystine/glutamate antiporter. Proteomic data derived from PPB revealed an interactor, PRDX3, a mitochondrial peroxidase, that modulated heme/porphyrin biosynthesis driven ferroptosis. Consistent with a role in porphyrin-induced ferroptotic death targeted gene knockdown of PRDX3, but not peroxidases, PRDX1 or 2, enhanced porphyrin-induced ferroptotic death. The relationship between increased heme/porphyrin synthesis and ferroptosis was also found in a ferrochelatase-deficient T-lymphoblastoid leukemia cell line, suggesting potential strategy for treating certain cancers. We demonstrate that when the PPB probe is coupled with unbiased proteomics a previously unreported relationship between heme/porphyrin synthesis, and ferroptosis was discovered.