Identification of a Conserved and Functional Iron-responsive Element in the 5′-Untranslated Region of Mammalian Mitochondrial Aconitase

Self Cite

115

Hereditary hyperferritinemia-cataract syndrome (HHCS) is a novel genetic disorder characterized by elevated serum ferritin and early onset cataract formation. The excessive ferritin production in HHCS patients arises from aberrant regulation of L-ferritin translation caused by mutations within the ironresponsive element (IRE) of the L-ferritin transcript. IREs serve as binding sites for iron regulatory proteins (IRPs), iron-sensing proteins that regulate ferritin translation. Previous observations suggested that each unique HHCS mutation conferred a characteristic degree of hyperferritinemia and cataract severity in affected individuals. Here we have measured the in vitro affinity of the IRPs for the mutant IREs and correlated decreases in binding affinity with clinical severity. Thermodynamic analysis of these IREs has also revealed that although some HHCS mutations lead to changes in the stability and secondary structure of the IRE, others appear to disrupt IRP-IRE recognition with minimal effect on IRE stability. HHCS is a noteworthy example of a human genetic disorder that arises from mutations within a protein-binding site of an mRNA cis-acting element. Analysis of the effects of these mutations on the energetics of the RNA-protein interaction explains the phenotypic variabilities of the disease state. Several reports have described a new autosomal dominant disorder called hereditary hyperferritinemia-cataract syndrome (HHCS)1 (1-12). This condition is characterized by a combination of elevated levels of serum ferritin and congenital nuclear cataract. Human ferritin is composed of two different types of subunit (H-ferritin and L-ferritin), although the relative proportion of each subunit in the ferritin copolymer varies among tissues (13). Through the use of monoclonal antibodies specific for either H-or L-ferritin, the excess serum ferritin in HHCS patients has been shown to be predominantly L-ferritin (3, 7). A close relationship has also been established between monocyte L-ferritin content and serum ferritin concentration (7), suggesting that the excess production of L-ferritin in cells is directly responsible for the hyperferritinemia. Although serum ferritin is thought to reflect body iron stores, individuals with HHCS do not appear to suffer from iron overload (1, 2).The expression of both H-ferritin and L-ferritin is normally regulated according to intracellular iron concentration by means of iron-responsive elements (IREs) that are found within the 5Ј-untranslated regions of their transcripts (14, 15). These regulatory elements interact with the iron regulatory proteins IRP1 and IRP2, which are both capable of sensing cellular iron status. Under conditions of intracellular iron depletion, both IRP1 and IRP2 bind to IREs with high affinity. The binding of an IRP to the ferritin IRE prevents the binding of the 43 S translation initiation complex to the transcript (16, 17) and thus blocks translation of the ferritin protein. When intracellular iron concentrations rise, IRP1 acquires a [4Fe-4S] cluster ...

Section: Characteristics For Ires Containing Hhcs Mutationsmentioning

confidence: 80%

Clinical Severity and Thermodynamic Effects of Iron-responsive Element Mutations in Hereditary Hyperferritinemia-Cataract Syndrome

Allerson¹,

Cazzola²,

Rouault³

1999

Self Cite

115

“…IRPs also regulate enzymes involved in cellular energy homeostasis such as ACO2 and succinate dehydrogenase (SDH) (32,39,40). We found that EPI or NE enhanced binding of IRP to the IREs present in the TfR1 3Ј-UTR to increase its transcript stability (Fig.…”

Section: Discussionmentioning

confidence: 93%

Catecholamine Stress Hormones Regulate Cellular Iron Homeostasis by a Posttranscriptional Mechanism Mediated by Iron Regulatory Protein

Tapryal

Mukhopadhyay

2015

Background:The role of catecholamines in cellular iron homeostasis is not understood. Results: Epinephrine and norepinephrine up-regulate transferrin receptor-1, inhibit ferritin expression, and increase mitochondrial iron content in hepatic and skeletal muscle cells. Conclusion: Catecholamine stress hormones are novel regulators of cellular iron homeostasis. Significance: Catecholamines may act to increase cellular iron content to meet an increased demand during stress.

“…Given that 1) aconitases catalyze the interconversion of citrate to isocitrate and thus require the presence of substrate to function, 2) aconitase function in the citric acid cycle is more ancient than a direct role of the protein in iron metabolism, 3) eukaryotic cells need to regulate intermediary metabolism both in the mitochondria and in the cytosol, utilizing citrate and isocitrate for different purposes in each compartment, 4) IRP1 control of the citric acid enzyme mitochondrial aconitase is present in mammalian cells (54,55), and 5) the IRE of Fer1HCH is contained in an alternatively spliced exon, enabling a bypass of IRP-1A-dependent regulation of ferritin (29,30), we speculate that the initial IRE evolved in succinate dehydrogenase subunit B. After generation by gene duplication of two cytosolic aconitases, one of them acquired the ability to bind to the succinate dehydrogenase subunit B transcripts, which would down-regulate the citric acid cycle and secure higher citrate availability for the cytosol.…”

Section: Discussionmentioning

confidence: 99%

Of Two Cytosolic Aconitases Expressed in Drosophila, Only One Functions as an Iron-regulatory Protein

Lind

Missirlis

Melefors

et al. 2006

Self Cite

In mammalian cells, iron homeostasis is largely regulated by post-transcriptional control of gene expression through the binding of iron-regulatory proteins (IRP1 and IRP2) to iron-responsive elements (IREs) contained in the untranslated regions of target mRNAs. IRP2 is the dominant iron sensor in mammalian cells under normoxia, but IRP1 is the more ancient protein in evolutionary terms and has an additional function as a cytosolic aconitase. The Caenorhabditis elegans genome does not contain an IRP2 homolog or identifiable IREs; its IRP1 homolog has aconitase activity but does not bind to mammalian IREs. The Drosophila genome offers an evolutionary intermediate containing two IRP1-like proteins (IRP-1A and IRP-1B) and target genes with IREs. Here, we used purified recombinant IRP-1A and IRP-1B from Drosophila melanogaster and showed that only IRP-1A can bind to IREs, although both proteins possess aconitase activity. These results were also corroborated in whole-fly homogenates from transgenic flies that overexpress IRP-1A and IRP-1B in their fat bodies. Ubiquitous and muscle-specific overexpression of IRP-1A, but not of IRP-1B, resulted in pre-adult lethality, underscoring the importance of the biochemical difference between the two proteins. Domain-swap experiments showed that multiple amino acid substitutions scattered throughout the IRP1 domains are synergistically required for conferring IRE binding activity. Our data suggest that as a first step during the evolution of the IRP/IRE system, the ancient cytosolic aconitase was duplicated in insects with one variant acquiring IRE-specific binding.Iron is required for aerobic metabolism and is actively sensed, sequestered, and regulated by organisms, including hosts and pathogens, that often compete for metal acquisition (1). In multicellular organisms, signals exist for systemic control of iron metabolism (2). One of the central regulators of cellular iron metabolism is the IRP/IRE system (3). In brief, absence of iron is sensed by IRP1 and IRP2, which then bind to ironresponsive elements (IREs) 4 in the 5Ј-untranslated regions of mRNAs encoding several storage or consumer iron proteins, inhibiting their translation. IRP1 and IRP2 also bind to IREs in the 3Ј-untranslated regions of mRNAs encoding proteins that function in cellular iron import, stabilizing the transcripts and enhancing iron sequestration. There is much interest in defining how IRP1 and IRP2 sense iron levels. IRP1 interconverts between an iron-sulfur protein with aconitase activity and the apoprotein that binds to the IRE (4). IRP2 has an additional 73-amino acid domain inserted in the protein and has no aconitase activity (5, 6). Extensive analysis of knock-out mice has led to the conclusion that IRP2 dominates mammalian cellular iron metabolism (7). IRP1 also contributes to the regulation of cellular iron metabolism, albeit to a lesser extent, because its iron-sulfur cluster is efficiently repaired and the protein functions mostly as an aconitase (8 -10). It is also well established that b...