ENO1 suppresses cancer cell ferroptosis by degrading the mRNA of iron regulatory protein 1

α-Enolase 1 (ENO1) is a critical glycolytic enzyme whose aberrant expression drives the pathogenesis of various cancers. ENO1 has been indicated to have additional roles beyond its conventional metabolic activity, but the underlying mechanisms and biological consequences remain elusive. Here, we show that ENO1 suppresses iron regulatory protein 1 (IRP1) expression to regulate iron homeostasis and survival of hepatocellular carcinoma (HCC) cells. Mechanistically, we unprecedentedly uncover that ENO1, as an RNA-binding protein, recruits CNOT6 to accelerate the mRNA decay of IRP1 in cancer cells, leading to inhibition of mtioferin-1 (Mfrn1) expression and subsequent repression of mitochondrial iron-induced ferroptosis. Moreover, through in vitro and in vivo experiments and clinical sample analysis, we identified IRP1 and Mfrn1 as tumor suppressors by inducing ferroptosis in HCC cells. Taken together, this study establishes a novel role for the ENO1/IRP1/Mfrn1 pathway in the pathogenesis of HCC and reveals a previously unknown connection between the ENO1/IRP1/Mfrn1 pathway and ferroptosis, suggesting a potential innovative cancer therapy.

FeS-cluster coordination of vertebrate thioredoxins regulates suppression of hypoxia-induced factor 2α through iron regulatory protein 1

Thioredoxins (Trxs) provide electrons to essential cellular processes such as DNA synthesis. Here, we characterize human and murine Trx1 as new iron-sulfur proteins. The [2Fe-2S] cluster is complexed using cysteinyl side chains 32 and 73 in a dimeric holocomplex. Formation of the holo-dimer depends on small structural changes of the loop connecting helices three and four and is stabilized by the formation of a direct electrostatic interaction between Lys72 and Asp60 of two monomers. The not strictly conserved Cys73 in vertebrates co-evolved with the regulation of cellular iron homeostasis through the iron-regulatory proteins (IRP). Active apo-Trx1 is required for the reduction of cysteinyl residues in IRP1 and its binding to the iron-responsive elements in the mRNA encoding hypoxiainducible factor (HIF) 2α. Depletion of Trx1 increased the mRNA levels of HIF2α, an important target of IRP1. Hence, translation of the HIF2α mRNA requires either sufficient iron-supply or the lack of reducing power of the Trx system under iron-limiting conditions. Only then, HIF2α protein may accumulate under hypoxic conditions to transcriptionally regulate processes like erythropoiesis.Significance StatementThioredoxins are, in general, cofactor-less key proteins in redox regulation and provide electrons to many essential cellular processes such as DNA synthesis. 55 years after its discovery, we show that mammalian thioredoxin 1 coordinates an iron-sulfur cluster using one of its active site cysteinyl residues and a non-conserved additional cysteinyl residue located outside the active site. This particular residue co-evolved with the vertebratespecific iron regulatory system. Our study demonstrates that this system is regulated by thioredoxin 1 at the level of the iron-regulatory protein 1, thus linking redox and iron homeostases.

Glycogen branching enzyme controls cellular iron homeostasis via Iron Regulatory Protein 1 and mitoNEET

Iron Regulatory Protein 1 (IRP1) is a bifunctional cytosolic iron sensor. When iron levels are normal, IRP1 harbours an iron-sulphur cluster (holo-IRP1), an enzyme with aconitase activity. When iron levels fall, IRP1 loses the cluster (apo-IRP1) and binds to iron-responsive elements (IREs) in messenger RNAs (mRNAs) encoding proteins involved in cellular iron uptake, distribution, and storage. Here we show that mutations in the Drosophila 1,4-Alpha-Glucan Branching Enzyme (AGBE) gene cause porphyria. AGBE was hitherto only linked to glycogen metabolism and a fatal human disorder known as glycogen storage disease type IV. AGBE binds specifically to holo-IRP1 and to mitoNEET, a protein capable of repairing IRP1 iron-sulphur clusters. This interaction ensures nuclear translocation of holo-IRP1 and downregulation of iron-dependent processes, demonstrating that holo-IRP1 functions not just as an aconitase, but throttles target gene expression in anticipation of declining iron requirements.