Alex D. Sheftel

Mitochondrial iron trafficking and the integration of iron metabolism between the mitochondrion and cytosol

The mitochondrion is well known for its key role in energy transduction. However, it is less well appreciated that it is also a focal point of iron metabolism. Iron is needed not only for heme and iron sulfur cluster (ISC)-containing proteins involved in electron transport and oxidative phosphorylation, but also for a wide variety of cytoplasmic and nuclear functions, including DNA synthesis. The mitochondrial pathways involved in the generation of both heme and ISCs have been characterized to some extent. However, little is known concerning the regulation of iron uptake by the mitochondrion and how this is coordinated with iron metabolism in the cytosol and other organelles (e.g., lysosomes). In this article, we discuss the burgeoning field of mitochondrial iron metabolism and trafficking that has recently been stimulated by the discovery of proteins involved in mitochondrial iron storage (mitochondrial ferritin) and transport (mitoferrin-1 and -2). In addition, recent work examining mitochondrial diseases (e.g., Friedreichs ataxia) has established that communication exists between iron metabolism in the mitochondrion and the cytosol. This finding has revealed the ability of the mitochondrion to modulate whole-cell iron-processing to satisfy its own requirements for the crucial processes of heme and ISC synthesis. Knowledge of mitochondrial iron-processing pathways and the interaction between organelles and the cytosol could revolutionize the investigation of iron metabolism.

Human Ind1, an Iron-Sulfur Cluster Assembly Factor for Respiratory Complex I

ABSTRACT Respiratory complex I (NADH:ubiquinone oxidoreductase) is a large mitochondrial inner membrane enzyme consisting of 45 subunits and 8 iron-sulfur (Fe/S) clusters. While complex I dysfunction is the most common reason for mitochondrial diseases, the assembly of complex I and its Fe/S cofactors remains elusive. Here, we identify the human mitochondrial P-loop NTPase, designated huInd1, that is critically required for the assembly of complex I. huInd1 can bind an Fe/S cluster via a conserved CXXC motif in a labile fashion. Knockdown of huInd1 in HeLa cells by RNA interference technology led to strong decreases in complex I protein and activity levels, remodeling of respiratory supercomplexes, and alteration of mitochondrial morphology. In addition, huInd1 depletion resulted in massive decreases in several subunits (NDUFS1, NDUFV1, NDUFS3, and NDUFA13) of the peripheral arm of complex I, with the concomitant appearance of a 450-kDa subcomplex representing part of the membrane arm. By a novel radiolabeling technique, the amount of iron associated with complex I was also shown to reflect the dependence of this enzyme on huInd1 for assembly. Together, these data identify huInd1 as a new assembly factor for human respiratory complex I with a possible role in the delivery of one or more Fe/S clusters to complex I subunits.

Iron–sulfur proteins in health and disease

Iron-sulfur (Fe/S) proteins are a class of ubiquitous components that assist in vital and diverse biochemical tasks in virtually every living cell. These tasks include respiration, iron homeostasis and gene expression. The past decade has led to the discovery of novel Fe/S proteins and insights into how their Fe/S cofactors are formed and incorporated into apoproteins. This review summarizes our current knowledge of mammalian Fe/S proteins, diseases related to deficiencies in these proteins and on disorders stemming from their defective biogenesis. Understanding both the physiological functions of Fe/S proteins and how Fe/S clusters are formed will undoubtedly enhance our ability to identify and treat known disorders of Fe/S cluster biogenesis and to recognize hitherto undescribed Fe/S cluster-related diseases.

Humans possess two mitochondrial ferredoxins, Fdx1 and Fdx2, with distinct roles in steroidogenesis, heme, and Fe/S cluster biosynthesis

Mammalian adrenodoxin (ferredoxin 1; Fdx1) is essential for the synthesis of various steroid hormones in adrenal glands. As a member of the [2Fe-2S] cluster-containing ferredoxin family, Fdx1 reduces mitochondrial cytochrome P450 enzymes, which then catalyze; e.g., the conversion of cholesterol to pregnenolone, aldosterone, and cortisol. The high protein sequence similarity between Fdx1 and its yeast adrenodoxin homologue (Yah1) suggested that Fdx1, like Yah1, may be involved in the biosynthesis of heme A and Fe/S clusters, two versatile and essential protein cofactors. Our study, employing RNAi technology to deplete human Fdx1, did not confirm this expectation. Instead, we identified a Fdx1-related mitochondrial protein, designated ferredoxin 2 (Fdx2) and found it to be essential for heme A and Fe/S protein biosynthesis. Unlike Fdx1, Fdx2 was unable to efficiently reduce mitochondrial cytochromes P450 and convert steroids, indicating that the two ferredoxin isoforms are highly specific for their substrates in distinct biochemical pathways. Moreover, Fdx2 deficiency had a severe impact, via impaired Fe/S protein biogenesis, on cellular iron homeostasis, leading to increased cellular iron uptake and iron accumulation in mitochondria. We conclude that mammals depend on two distinct mitochondrial ferredoxins for the specific production of either steroid hormones or heme A and Fe/S proteins.

The human mitochondrial ISCA1, ISCA2, and IBA57 proteins are required for [4Fe-4S] protein maturation

The human mitochondrial proteins ISCA1, ISCA2, and IBA57 are essential for the generation of mitochondrial [4Fe-4S] proteins in a late step of Fe/S protein biogenesis. This process is important for mitochondrial physiology, as documented by drastic enlargement of the organelles and the loss of cristae membranes in the absence of these proteins.

Human CIA2A-FAM96A and CIA2B-FAM96B Integrate Iron Homeostasis and Maturation of Different Subsets of Cytosolic-Nuclear Iron-Sulfur Proteins

Numerous cytosolic and nuclear proteins involved in metabolism, DNA maintenance, protein translation, or iron homeostasis depend on iron-sulfur (Fe/S) cofactors, yet their assembly is poorly defined. Here, we identify and characterize human CIA2A (FAM96A), CIA2B (FAM96B), and CIA1 (CIAO1) as components of the cytosolic Fe/S protein assembly (CIA) machinery. CIA1 associates with either CIA2A or CIA2B and the CIA-targeting factor MMS19. The CIA2B-CIA1-MMS19 complex binds to and facilitates assembly of most cytosolic-nuclear Fe/S proteins. In contrast, CIA2A specifically matures iron regulatory protein 1 (IRP1), which is critical for cellular iron homeostasis. Surprisingly, a second layer of iron regulation involves the stabilization of IRP2 by CIA2A binding or upon depletion of CIA2B or MMS19, even though IRP2 lacks an Fe/S cluster. In summary, CIA2B-CIA1-MMS19 and CIA2A-CIA1 assist different branches of Fe/S protein assembly and intimately link this process to cellular iron regulation via IRP1 Fe/S cluster maturation and IRP2 stabilization.

Non-heme Induction of Heme Oxygenase-1 Does Not Alter Cellular Iron Metabolism

The catabolism of heme is carried out by members of the heme oxygenase (HO) family. The products of heme catabolism by HO-1 are ferrous iron, biliverdin (subsequently converted to bilirubin), and carbon monoxide. In addition to its function in the recycling of hemoglobin iron, this microsomal enzyme has been shown to protect cells in various stress models. Implicit in the reports of HO-1 cytoprotection to date are its effects on the cellular handling of heme/iron. However, the limited amount of uncommitted heme in non-erythroid cells brings to question the source of substrate for this enzyme in non-hemolytic circumstances. In the present study, HO-1 was induced by either sodium arsenite (reactive oxygen species producer) or hemin or overexpressed in the murine macrophage-like cell line, RAW 264.7. Both of the inducers elicited an increase in active HO-1; however, only hemin exposure caused an increase in the synthesis rate of the iron storage protein, ferritin. This effect of hemin was the direct result of the liberation of iron from heme by HO. Cells stably overexpressing HO-1, although protected from oxidative stress, did not display elevated basal ferritin synthesis. However, these cells did exhibit an increase in ferritin synthesis, compared with untransfected controls, in response to hemin treatment, suggesting that heme levels, and not HO-1, limit cellular heme catabolism. Our results suggest that the protection of cells from oxidative insult afforded by HO-1 is not due to the catabolism of significant amounts of cellular heme as thought previously.

The power plant of the cell is also a smithy: the emerging role of mitochondria in cellular iron homeostasis.

Iron is required for a barrage of essential biochemical functions in virtually every species of life. Perturbation of the availability or utilization of iron in these functions or disruption of other components along iron-requiring pathways can not only lead to cellular/organismal insufficiency of respective biochemical end-products but also result in a broad derangement of iron homeostasis. This is largely because of the elaborate regulatory mechanisms that connect cellular iron utilization with uptake and distribution. Such mechanisms are necessitated by the ‘double-edged’ nature of the metal, whose very property as a useful biological catalyst also makes it able to generate highly toxic compounds. Since the majority of iron is dispatched onto a functional course by mitochondria-localized pathways, these organelles are in an ideal position within the cellular iron anabolic pathways to be a central site for regulation of iron homeostasis. The goal of this article is to provide an overview of how mitochondria acquire and use iron and examine the ramifications of disturbances in these processes on overall cellular iron homeostasis.

Mitochondrial iron metabolism and sideroblastic anemia.

Sideroblastic anemias are a heterogeneous group of disorders, characterized by mitochondrial iron overload in developing red blood cells. The unifying characteristic of all sideroblastic anemias is the ring sideroblast, which is a pathological erythroid precursor containing excessive deposits of non-heme iron in mitochondria with perinuclear distribution creating a ring appearance. Sideroblastic anemias may be hereditary or acquired. Hereditary sideroblastic anemias are caused by defects in genes present on the X chromosome (mutations in the ALAS2, ABCB7, or GRLX5 gene), genes on autosomal chromosomes, or mitochondrial genes. Acquired sideroblastic anemias are either primary (refractory anemia with ring sideroblasts, RARS, representing one subtype of the myelodysplastic syndrome) or secondary due to some drugs, toxins, copper deficiency, or chronic neoplastic disease. The pathogenesis of mitochondrial iron loading in developing erythroblasts is diverse. Ring sideroblasts can develop as a result of a heme synthesis defect in erythroblasts (ALAS2 mutations), a defect in iron-sulfur cluster assembly, iron-sulfur protein precursor release from mitochondria (ABCB7 mutations), or by a defect in intracellular iron metabolism in erythroid cells (e.g. RARS).

Iron regulatory protein‐independent regulation of ferritin synthesis by nitrogen monoxide

The discovery of iron‐responsive elements (IREs), along with the identification of iron regulatory proteins (IRP1, IRP2), has provided a molecular basis for our current understanding of the remarkable post‐transcriptional regulation of intracellular iron homeostasis. In iron‐depleted conditions, IRPs bind to IREs present in the 5′‐UTR of ferritin mRNA and the 3′‐UTR of transferrin receptor (TfR) mRNA. Such binding blocks the translation of ferritin, the iron storage protein, and stabilizes TfR mRNA, whereas the opposite scenario develops when iron in the intracellular transit pool is plentiful. Nitrogen monoxide (commonly designated nitric oxide; NO), a gaseous molecule involved in numerous functions, is known to affect cellular iron metabolism via the IRP/IRE system. We previously demonstrated that the oxidized form of NO, NO+, causes IRP2 degradation that is associated with an increase in ferritin synthesis [Kim, S & Ponka, P (2002) Proc Natl Acad Sci USA99, 12214–12219]. Here we report that sodium nitroprusside (SNP), an NO+ donor, causes a dramatic and rapid increase in ferritin synthesis that initially occurs without changes in the RNA‐binding activities of IRPs. Moreover, we demonstrate that the translational efficiency of ferritin mRNA is significantly higher in cells treated with SNP compared with those incubated with ferric ammonium citrate, an iron donor. Importantly, we also provide definitive evidence that the iron moiety of SNP is not responsible for such changes. These results indicate that SNP‐mediated increase in ferritin synthesis is, in part, due to an IRP‐independent and NO+‐dependent post‐transcriptional, regulatory mechanism.