PPIX-binding Proteins Reveal Porphyrin Synthesis and Ferroptosis Link

Abstract

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 PPIX's 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 ferroptotic death that is pharmacologically distinct from canonical ferroptosis driven by erastin inhibition 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.