Sheila A. Anderson

Iron-responsive degradation of iron-regulatory protein 1 does not require the Fe-S cluster.

The generally accepted role of iron‐regulatory protein 1 (IRP1) in orchestrating the fate of iron‐regulated mRNAs depends on the interconversion of its cytosolic aconitase and RNA‐binding forms through assembly/disassembly of its Fe–S cluster, without altering protein abundance. Here, we show that IRP1 protein abundance can be iron‐regulated. Modulation of IRP1 abundance by iron did not require assembly of the Fe–S cluster, since a mutant with all cluster‐ligating cysteines mutated to serine underwent iron‐induced protein degradation. Phosphorylation of IRP1 at S138 favored the RNA‐binding form and promoted iron‐dependent degradation. However, phosphorylation at S138 was not required for degradation. Further, degradation of an S138 phosphomimetic mutant was not blocked by mutation of cluster‐ligating cysteines. These findings were confirmed in mouse models with genetic defects in cytosolic Fe–S cluster assembly/disassembly. IRP1 RNA‐binding activity was primarily regulated by IRP1 degradation in these animals. Our results reveal a mechanism for regulating IRP1 action relevant to the control of iron homeostasis during cell proliferation, inflammation, and in response to diseases altering cytosolic Fe–S cluster assembly or disassembly.

Suppression of hepatic hepcidin expression in response to acute iron deprivation is associated with an increase of matriptase-2 protein

Recent studies demonstrate a pivotal role for bone morphogenic protein-6 (BMP6) and matriptase-2, a protein encoded by the TMPRSS6 gene, in the induction and suppression of hepatic hepcidin expression, respectively. We examined their expression profiles in the liver and showed a predominant localization of BMP6 mRNA in nonparenchymal cells and exclusive expression of TMPRSS6 mRNA in hepatocytes. In rats fed an iron-deficient (ID) diet for 24 hours, the rapid decrease of transferrin saturation from 71% to 24% (control vs ID diet) was associated with a 100-fold decrease in hepcidin mRNA compared with the corresponding controls. These results indicated a close correlation of low transferrin saturation with decreased hepcidin mRNA. The lower phosphorylated Smad1/5/8 detected in the ID rat livers suggests that the suppressed hepcidin expression results from the inhibition of BMP signaling. Quantitative real-time reverse transcription polymerase chain reaction analysis revealed no significant change in either BMP6 or TMPRSS6 mRNA in the liver. However, an increase in matriptase-2 protein in the liver from ID rats was detected, suggesting that suppression of hepcidin expression in response to acute iron deprivation is mediated by an increase in matriptase-2 protein levels.

F-box and Leucine-rich Repeat Protein 5 (FBXL5) Is Required for Maintenance of Cellular and Systemic Iron Homeostasis

Background: FBXL5 is an iron-responsive E3 ubiquitin ligase. Results: FBXL5-null mice die during embryogenesis, whereas Fbxl5 heterozygotes perform better than wild type littermates when fed a low iron diet due to enhanced iron absorption. Conclusion: FBXL5 plays an essential role in the in vivo maintenance of cellular and systemic iron homeostasis. Significance: FBXL5 is an essential physiological iron sensor. Maintenance of cellular iron homeostasis requires post-transcriptional regulation of iron metabolism genes by iron regulatory protein 2 (IRP2). The hemerythrin-like domain of F-box and leucine-rich repeat protein 5 (FBXL5), an E3 ubiquitin ligase subunit, senses iron and oxygen availability and facilitates IRP2 degradation in iron replete cells. Disruption of the ubiquitously expressed murine Fbxl5 gene results in a failure to sense increased cellular iron availability, accompanied by constitutive IRP2 accumulation and misexpression of IRP2 target genes. FBXL5-null mice die during embryogenesis, although viability is restored by simultaneous deletion of the IRP2, but not IRP1, gene. Mice containing a single functional Fbxl5 allele behave like their wild type littermates when fed an iron-sufficient diet. However, unlike wild type mice that manifest decreased hematocrit and hemoglobin levels when fed a low-iron diet, Fbxl5 heterozygotes maintain normal hematologic values due to increased iron absorption. The responsiveness of IRP2 to low iron is specifically enhanced in the duodena of the heterozygotes and is accompanied by increased expression of the divalent metal transporter-1. These results confirm the role of FBXL5 in the in vivo maintenance of cellular and systemic iron homeostasis and reveal a privileged role for the intestine in their regulation by virtue of its unique FBXL5 iron sensitivity.

Evidence that phosphorylation of iron regulatory protein 1 at Serine 138 destabilizes the [4Fe-4S] cluster in cytosolic aconitase by enhancing 4Fe-3Fe cycling.

Iron-sulfur cluster-dependent interconversion of iron regulatory protein 1 (IRP1) between its RNA binding and cytosolic aconitase (c-acon) forms controls vertebrate iron homeostasis. Cluster removal from c-acon is thought to include oxidative demetallation as a required step, but little else is understood about the process of conversion to IRP1. In comparison with c-aconWT, Ser138 phosphomimetic mutants of c-acon contain an unstable [4Fe-4S] cluster and were used as tools to further define the pathway(s) of iron-sulfur cluster disassembly. Under anaerobic conditions cluster insertion into purified IRP1S138E and cluster loss on treatment with NO regulated aconitase and RNA binding activity over a similar range as observed for IRP1WT. However, activation of RNA binding of c-aconS138E was an order of magnitude more sensitive to NO than for c-aconWT. Consistent with this, an altered set point between RNA-binding and aconitase forms was observed for IRP1S138E when expressed in HEK cells. Active c-aconS138E could only accumulate under hypoxic conditions, suggesting enhanced cluster disassembly in normoxia. Cluster disassembly mechanisms were further probed by determining the impact of iron chelation on acon activity. Unexpectedly EDTA rapidly inhibited c-aconS138E activity without affecting c-aconWT. Additional chelator experiments suggested that cluster loss can be initiated in c-aconS138E through a spontaneous nonoxidative demetallation process. Taken together, our results support a model wherein Ser138 phosphorylation sensitizes IRP1/c-acon to decreased iron availability by allowing the [4Fe-4S]2+ cluster to cycle with [3Fe-4S]0 in the absence of cluster perturbants, indicating that regulation can be initiated merely by changes in iron availability.

Low Intracellular Iron Increases the Stability of Matriptase-2

Background: Matriptase-2 (MT2) is essential for iron homeostasis. The mechanism for its regulation is controversial. Results: The cytoplasmic domain of MT2 is necessary for its stabilization by iron depletion. MT2 expression is not regulated at either the transcriptional mRNA or translational level by iron. Conclusion: Depletion of cellular iron stabilizes MT2. Significance: Low iron levels in hepatocytes stabilize MT2 to suppress hepcidin expression. Matriptase-2 (MT2) is a type II transmembrane serine protease that is predominantly expressed in hepatocytes. It suppresses the expression of hepatic hepcidin, an iron regulatory hormone, by cleaving membrane hemojuvelin into an inactive form. Hemojuvelin is a bone morphogenetic protein (BMP) co-receptor. Here, we report that MT2 is up-regulated under iron deprivation. In HepG2 cells stably expressing the coding sequence of the MT2 gene, TMPRSS6, incubation with apo-transferrin or the membrane-impermeable iron chelator, deferoxamine mesylate salt, was able to increase MT2 levels. This increase did not result from the inhibition of MT2 shedding from the cells. Rather, studies using a membrane-permeable iron chelator, salicylaldehyde isonicotinoyl hydrazone, revealed that depletion of cellular iron was able to decrease the degradation of MT2 independently of internalization. We found that lack of the putative endocytosis motif in its cytoplasmic domain largely abolished the sensitivity of MT2 to iron depletion. Neither acute nor chronic iron deficiency was able to alter the association of Tmprss6 mRNA with polyribosomes in the liver of rats indicating a lack of translational regulation by low iron levels. Studies in mice showed that Tmprss6 mRNA was not regulated by iron nor the BMP-mediated signaling with no evident correlation with either Bmp6 mRNA or Id1 mRNA, a target of BMP signaling. These results suggest that regulation of MT2 occurs at the level of protein degradation rather than by changes in the rate of internalization and translational or transcriptional mechanisms and that the cytoplasmic domain of MT2 is necessary for its regulation.

Iron regulatory protein-1 protects against mitoferrin-1-deficient porphyria.

Background: Heme and [Fe-S] cluster assembly are tightly regulated processes that require mitochondrial iron. Results: Loss of mitochondrial iron activates the [Fe-S]-dependent RNA-binding activity of IRP1 that inhibits protoporphyrin biosynthesis. Conclusion: IRP1 forms a critical feedback mechanism, preventing protoporphyrin accumulation under limiting mitochondrial iron conditions. Significance: This study provides evidence linking heme biogenesis to that of [Fe-S] clusters synthesis. Mitochondrial iron is essential for the biosynthesis of heme and iron-sulfur ([Fe-S]) clusters in mammalian cells. In developing erythrocytes, iron is imported into the mitochondria by MFRN1 (mitoferrin-1, SLC25A37). Although loss of MFRN1 in zebrafish and mice leads to profound anemia, mutant animals showed no overt signs of porphyria, suggesting that mitochondrial iron deficiency does not result in an accumulation of protoporphyrins. Here, we developed a gene trap model to provide in vitro and in vivo evidence that iron regulatory protein-1 (IRP1) inhibits protoporphyrin accumulation. Mfrn1+/gt;Irp1−/− erythroid cells exhibit a significant increase in protoporphyrin levels. IRP1 attenuates protoporphyrin biosynthesis by binding to the 5′-iron response element (IRE) of alas2 mRNA, inhibiting its translation. Ectopic expression of alas2 harboring a mutant IRE, preventing IRP1 binding, in Mfrn1gt/gt cells mimics Irp1 deficiency. Together, our data support a model whereby impaired mitochondrial [Fe-S] cluster biogenesis in Mfrn1gt/gt cells results in elevated IRP1 RNA-binding that attenuates ALAS2 mRNA translation and protoporphyrin accumulation.