Darius J.R. Lane

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.

Voltage-dependent anion-selective channel (VDAC) in the plasma membrane

Voltage‐dependent anion channels (VDACs) have originally been characterized as mitochondrial porins. Starting in the late 1980s, however, evidence began to accumulate that VDACs can also be expressed in plasma membranes. In this review, we briefly revisit the historical milestones in the discovery of plasma membrane‐bound VDAC, and we critically analyze the evidence for VDAC plasma membrane localization obtained from various purification strategies and recently from plasma membrane proteomics studies. We discuss the possible biological function and relevance of VDAC in the plasma membrane and finally discuss a hypothetical model of how VDAC may be targeted to the plasma membrane.

Cellular iron uptake, trafficking and metabolism: Key molecules and mechanisms and their roles in disease.

Iron is a crucial transition metal for virtually all life. Two major destinations of iron within mammalian cells are the cytosolic iron-storage protein, ferritin, and mitochondria. In mitochondria, iron is utilized in critical anabolic pathways, including: iron-storage in mitochondrial ferritin, heme synthesis, and iron-sulfur cluster (ISC) biogenesis. Although the pathways involved in ISC synthesis in the mitochondria and cytosol have begun to be characterized, many crucial details remain unknown. In this review, we discuss major aspects of the journey of iron from its initial cellular uptake, its modes of trafficking within cells, to an overview of its downstream utilization in the cytoplasm and within mitochondria. The understanding of mitochondrial iron processing and its communication with other organelles/subcellular locations, such as the cytosol, has been elucidated by the analysis of certain diseases e.g., Friedreichs ataxia. Increased knowledge of the molecules and their mechanisms of action in iron processing pathways (e.g., ISC biogenesis) will shape the investigation of iron metabolism in human health and disease.

Mammalian Iron Homeostasis in Health and Disease: Uptake, Storage, Transport, and Molecular Mechanisms of Action

Iron is a crucial factor for life. However, it also has the potential to cause the formation of noxious free radicals. These double-edged sword characteristics demand a tight regulation of cellular iron metabolism. In this review, we discuss the various pathways of cellular iron uptake, cellular iron storage, and transport. Recent advances in understanding the reduction and uptake of non-transferrin-bound iron are discussed. We also discuss the recent progress in the understanding of transcriptional and translational regulation by iron. Furthermore, we discuss recent advances in the understanding of the regulation of cellular and systemic iron homeostasis and several key diseases resulting from iron deficiency and overload. We also discuss the knockout mice available for studying iron metabolism and the related human conditions.

The active role of vitamin C in mammalian iron metabolism: much more than just enhanced iron absorption!

Ascorbate is a cofactor in numerous metabolic reactions. Humans cannot synthesize ascorbate owing to inactivation of the gene encoding the enzyme l-gulono-γ-lactone oxidase, which is essential for ascorbate synthesis. Accumulating evidence strongly suggests that in addition to the known ability of dietary ascorbate to enhance nonheme iron absorption in the gut, ascorbate within mammalian systems can regulate cellular iron uptake and metabolism. Ascorbate modulates iron metabolism by stimulating ferritin synthesis, inhibiting lysosomal ferritin degradation, and decreasing cellular iron efflux. Furthermore, ascorbate cycling across the plasma membrane is responsible for ascorbate-stimulated iron uptake from low-molecular-weight iron-citrate complexes, which are prominent in the plasma of individuals with iron-overload disorders. Importantly, this iron-uptake pathway is of particular relevance to astrocyte brain iron metabolism and tissue iron loading in disorders such as hereditary hemochromatosis and β-thalassemia. Recent evidence also indicates that ascorbate is a novel modulator of the classical transferrin-iron uptake pathway, which provides almost all iron for cellular demands and erythropoiesis under physiological conditions. Ascorbate acts to stimulate transferrin-dependent iron uptake by an intracellular reductive mechanism, strongly suggesting that it may act to stimulate iron mobilization from the endosome. The ability of ascorbate to regulate transferrin iron uptake could help explain the metabolic defect that contributes to ascorbate-deficiency-induced anemia.

Two routes of iron accumulation in astrocytes: ascorbate-dependent ferrous iron uptake via the divalent metal transporter (DMT1) plus an independent route for ferric iron

Astrocytes are central to iron and ascorbate homoeostasis within the brain. Although NTBI (non-transferrin-bound iron) may be a major form of iron imported by astrocytes in vivo, the mechanisms responsible remain unclear. The present study examines NTBI uptake by cultured astrocytes and the involvement of ascorbate and DMT1 (divalent metal transporter 1). We demonstrate that iron accumulation by ascorbate-deficient astrocytes is insensitive to both membrane-impermeant Fe(II) chelators and to the addition of the ferroxidase caeruloplasmin. However, when astrocytes are ascorbate-replete, as occurs in vivo, their rate of iron accumulation is doubled. The acquisition of this additional iron depends on effluxed ascorbate and can be blocked by the DMT1 inhibitor ferristatin/NSC306711. Furthermore, the calcein-accessible component of intracellular labile iron, which appears during iron uptake, appears to consist of only Fe(III) in ascorbate-deficient astrocytes, whereas that of ascorbate-replete astrocytes comprises both valencies. Our data suggest that an Fe(III)-uptake pathway predominates when astrocytes are ascorbate-deficient, but that in ascorbate-replete astrocytes, at least half of the accumulated iron is initially reduced by effluxed ascorbate and then imported by DMT1. These results suggest that ascorbate is intimately involved in iron accumulation by astrocytes, and is thus an important contributor to iron homoeostasis in the mammalian brain.

Molecular functions of the iron-regulated metastasis suppressor, NDRG1, and its potential as a molecular target for cancer therapy.

N-myc down-regulated gene 1 (NDRG1) is a known metastasis suppressor in multiple cancers, being also involved in embryogenesis and development, cell growth and differentiation, lipid biosynthesis and myelination, stress responses and immunity. In addition to its primary role as a metastasis suppressor, NDRG1 can also influence other stages of carcinogenesis, namely angiogenesis and primary tumour growth. NDRG1 is regulated by multiple effectors in normal and neoplastic cells, including N-myc, histone acetylation, hypoxia, cellular iron levels and intracellular calcium. Further, studies have found that NDRG1 is up-regulated in neoplastic cells after treatment with novel iron chelators, which are a promising therapy for effective cancer management. Although the pathways by which NDRG1 exerts its functions in cancers have been documented, the relationship between the molecular structure of this protein and its functions remains unclear. In fact, recent studies suggest that, in certain cancers, NDRG1 is post-translationally modified, possibly by the activity of endogenous trypsins, leading to a subsequent alteration in its metastasis suppressor activity. This review describes the role of this important metastasis suppressor and discusses interesting unresolved issues regarding this protein.

Expression of the glaucoma gene myocilin (MYOC) in the human optic nerve head

Glaucoma is a leading cause of blindness in the world, and several glaucoma genes have been recently identified. The glaucoma gene myocilin (MYOC) (also known as GLC1A and TIGR) is responsible for juvenile open‐angle glaucoma and a subset of adult‐onset primary open‐angle glaucoma. Previous studies have shown that MYOC is expressed in the trabecular meshwork, an ocular tissue involved in the development of ocular hypertension, which is often associated with the development of glaucoma. Because all forms of glaucoma involve pathogenic changes to the optic nerve head (ONH), including cupping, excavation, and collapse of the connective support tissue, we sought to determine whether MYOC mRNA and protein are expressed in cells of the human ONH. Reverse transcriptase PCR, in situ hybridization, immunofluorescent microscopy, and immunoblotting of polyacrylamide gels were used to demonstrate that myocilin is expressed in ONH tissue and cultured ONH cells. A secondary purpose was to determine whether MYOC sequence variations are associated with a clinically significant fraction of normal tension glaucoma (NTG), a form of glaucoma in which intraocular pressure is not appreciably elevated. Nonconservative MYOC sequence variations were observed in only 6 of 307 patients with NTG and 4 of 193 control individuals (P=1.0, Fishers exact test). Thus, although MYOC is expressed in a pattern that is consistent with its involvement in NTG, variations in the MYOC coding sequence are not commonly or significantly associated with this disease.

Mitochondrial Mayhem: The Mitochondrion as a Modulator of Iron Metabolism and Its Role in Disease

The mitochondrion plays vital roles in various aspects of cellular metabolism, ranging from energy transduction and apoptosis to the synthesis of important molecules such as heme. Mitochondria are also centrally involved in iron metabolism, as exemplified by disruptions in mitochondrial proteins that lead to perturbations in whole-cell iron processing. Recent investigations have identified a host of mitochondrial proteins (e.g., mitochondrial ferritin; mitoferrins 1 and 2; ABCBs 6, 7, and 10; and frataxin) that may play roles in the homeostasis of mitochondrial iron. These mitochondrial proteins appear to participate in one or more processes of iron storage, iron uptake, and heme and iron-sulfur cluster synthesis. In this review, we present and critically discuss the evidence suggesting that the mitochondrion may contribute to the regulation of whole-cell iron metabolism. Further, human diseases that arise from a dysregulation of these mitochondrial molecules reveal the ability of the mitochondrion to communicate with cytosolic iron metabolism to coordinate whole-cell iron processing and to fulfill the high demands of this organelle for iron. This review highlights new advances in understanding iron metabolism in terms of novel molecular players and diseases associated with its dysregulation.

Non-transferrin iron reduction and uptake are regulated by transmembrane ascorbate cycling in K562 cells

K562 erythroleukemia cells import non-transferrin-bound iron (NTBI) by an incompletely understood process that requires initial iron reduction. The mechanism of NTBI ferrireduction remains unknown but probably involves transplasma membrane electron transport. We here provide evidence for a novel mechanism of NTBI reduction and uptake by K562 cells that utilizes transplasma membrane ascorbate cycling. Incubation of cells with dehydroascorbic acid, but not ascorbate, resulted in (i) accumulation of intracellular ascorbate that was blocked by the glucose transporter inhibitor, cytochalasin B, and (ii) subsequent release of micromolar concentrations of ascorbate into the external medium via a route that was sensitive to the anion channel inhibitor, 4,4′-diisothiocyanatostilbene-2,2′-disulfonate. Ascorbate-deficient control cells demonstrated low levels of ferric citrate reduction. However, incubation of the cells with dehydroascorbic acid resulted in a dose-dependent stimulation of both iron reduction and uptake from radiolabeled [55Fe]ferric citrate. This stimulation was abrogated by ascorbate oxidase treatment, suggesting dependence on direct chemical reduction by ascorbate. These results support a novel model of NTBI reduction and uptake by K562 cells in which uptake is preceded by reduction of iron by extracellular ascorbate, the latter of which is subsequently regenerated by transplasma membrane ascorbate cycling.