Abstract
Iron-regulatory protein 1 (IRP1) belongs to a family of RNA-binding proteins that modulate metazoan iron metabolism. Multiple mechanisms are employed to control the action of IRP1 in dictating changes in the uptake and metabolic fate of iron. Inactivation of IRP1 RNA binding by iron primarily involves insertion of a [4Fe-4S] cluster by the cytosolic iron-sulfur cluster assembly (CIA) system, converting it into cytosolic aconitase (c-acon), but can also involve iron-mediated degradation of IRP1 by the E3 ligase FBXL5 that also targets IRP2. How CIA and FBXL5 collaborate to maintain cellular iron homeostasis through IRP1 and other pathways is poorly understood. Because impaired Fe-S cluster biogenesis associates with human disease, we determined the importance of FBXL5 for regulating IRP1 when CIA is impaired. Suppression of FBXL5 expression coupled with induction of an IRP1 mutant (IRP13C>3S) that cannot insert the Fe-S cluster, or along with knockdown of the CIA factors NUBP2 or FAM96A, reduced cell viability. Iron supplementation reversed this growth defect and was associated with FBXL5-dependent polyubiquitination of IRP1. Phosphorylation of IRP1 at Ser-138 increased when CIA was inhibited and was required for iron rescue. Impaired CIA activity, as noted by reduced c-acon activity, was associated with enhanced FBXL5 expression and a concomitant reduction in IRP1 and IRP2 protein level and RNA-binding activity. Conversely, expression of either IRP induced FBXL5 protein level, demonstrating a negative feedback loop limiting excessive accumulation of iron-response element RNA-binding activity, whose disruption reduces cell growth. We conclude that a regulatory circuit involving FBXL5 and CIA acts through both IRPs to control iron metabolism and promote optimal cell growth.
Highlights
Our studies demonstrate that both Fe-S cluster insertion and protein degradation are critical components of a regulatory network controlling the level of IRP1 RNA-binding activity
Whereas previous studies have shown that IRP1 is subject to these modes of regulation [40, 41], the work described here identified novel interactions between the Fe-S biogenesis and protein degradation pathways that control IRP1 action
Over the continuum of iron levels that cells experience, from deficient to overloaded, is there a hierarchical role for the iron-dependent processes of the Fe-S switch versus protein degradation that control IRP1? Second, do tissue-specific differences in oxygenation selectively influence the control of IRP1 by FBXL5 versus the CIA system? FBXL5 requires oxygen to function
Summary
A synthetic growth defect in human embryonic kidney (HEK) 293 cells is induced by uncoupling IRP1 from the Fe-S switch along with FBXL5 knockdown. IRP13CϾ3S but not IRP1WT substantially inhibited the down-regulation of IRP2 protein by iron (Fig. 7A, compare lanes 3 and 8) These findings suggest that the dysregulation of cellular iron metabolism in response to CIA inhibition is in part a consequence of increased accumulation of the IRP1 apoprotein coupled with reduced ability of FBXL5 to downregulate IRP2. The reduction in IRP2 protein level when CIA factors NUBP2 and FAM96A [10] are knocked down (Fig. 2C) suggests that cytosolic iron is increased, which is in line with increased TfR1 expression (Fig. 5A) and binding of iron-loaded transferrin to cells with impaired CIA activity [10, 11]. To maintain cell viability in the face of inhibition of Fe-S cluster biogenesis
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