Expression and structural and functional properties of human ferritin L-chain from Escherichia coli

Levi, Sonia; Salfeld, Jochen; Franceschinelli, F; Cozzi, Anna; M, Dorner; Arosio, Paolo

doi:10.1021/bi00438a040

Cited by 122 publications

(91 citation statements)

References 24 publications

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“…Recombinant human H-chain ferritin was prepared as previously described (26). The Sigma holoHoSF and HuHF were rendered iron free by anaerobic ultrafiltration with dithionite and bipyridyl (27).…”

Section: Methodsmentioning

confidence: 99%

Is Hydrogen Peroxide Produced during Iron(II) Oxidation in Mammalian Apoferritins?

et al. 2001

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The ferritins are a class of iron storage and detoxification proteins that play a central role in the biological management of iron. These proteins have a catalytic site, "the ferroxidase site", located on the H-type subunit that facilitates the oxidation of Fe(II) to Fe(III) by O 2 . Measurements during the past 10 years on a number of vertebrate ferritins have provided evidence that H 2 O 2 is produced at this diiron ferroxidase site. Recently reported experiments using three different analytical methods with horse spleen ferritin (HoSF) have failed to detect H 2 O 2 production in this protein [Lindsay, S., Brosnahan, D., and Watt, G. D. (2001) Biochemistry 40, 3340-3347]. These findings contrast with earlier results reporting H 2 O 2 production in

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Section: Methodsmentioning

confidence: 99%

Is Hydrogen Peroxide Produced during Iron(II) Oxidation in Mammalian Apoferritins?

et al. 2001

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“…After 2 h, the samples were centrifuged at 14,000 ϫ g for 15 min. Iron incorporation was initially monitored by measuring absorbance of the supernatants at 310 nm (14,17). Iron incorporation into ferritin was more precisely determined by densitometric analysis of Prussian blue staining of supernatants run on nondenaturing gel electrophoresis.…”

Section: Cloning and Expression Of Ferritin Polypeptides-cdnasmentioning

confidence: 99%

“…Enhanced Precipitation of MT-FTL Homopolymers-Iron loading of apoferritin homopolymers was examined by an often used and well described procedure (14,16,17). In brief, WTand MT-FTL apoferritin homopolymers were incubated aerobically with increasing amounts of iron (ferrous ammonium sulfate) up to 4500 iron atoms per 24-mer.…”

Section: Recombinant Mt-ftl Polypeptides Assemble Into 24-mermentioning

confidence: 99%

Iron-mediated Aggregation and a Localized Structural Change Characterize Ferritin from a Mutant Light Chain Polypeptide That Causes Neurodegeneration

Baraibar

Barbeito

Muhoberac

et al. 2008

Journal of Biological Chemistry

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Nucleotide insertions in the ferritin light chain (FTL) polypeptide gene cause hereditary ferritinopathy, a neurodegenerative disease characterized by abnormal accumulation of ferritin and iron in the central nervous system. Here we describe for the first time the protein structure and iron storage function of the FTL mutant p.Phe167SerfsX26 (MT-FTL), which has a C terminus altered in sequence and extended in length. MT-FTL polypeptides assembled spontaneously into soluble, spherical 24-mers that were ultrastructurally indistinguishable from those of the wild type. Far-UV CD showed a decrease in ␣-helical content, and 8-anilino-1-naphthalenesulfonate fluorescence revealed the appearance of hydrophobic binding sites. Near-UV CD and proteolysis studies suggested little or no structural alteration outside of the C-terminal region. In contrast to wild type, MT-FTL homopolymers precipitated at much lower iron loading, had a diminished capacity to incorporate iron, and were less thermostable. However, precipitation was significantly reversed by addition of iron chelators both in vitro and in vivo. Our results reveal substantial protein conformational changes localized at the 4-fold pore of MT-FTL homopolymers and imply that the C terminus of the MT-FTL polypeptide plays an important role in ferritin solubility, stability, and iron management. We propose that the protrusion of some portion of the C terminus above the spherical shell allows it to cross-link with other mutant polypeptides through iron bridging, leading to enhanced mutant precipitation by iron. Our data suggest that hereditary ferritinopathy pathogenesis is likely to result from a combination of reduction in iron storage function and enhanced toxicity associated with iron-induced ferritin aggregates.Iron is an essential element needed for vital processes such as neuronal development, myelination, synthesis, and catabolism of neurotransmitters and electron transport, as well as heme and iron-sulfur cluster synthesis (1). Iron that is not utilized immediately in the cell is stored in ferritin. However, when iron is improperly regulated, it is potentially toxic leading to cell death. Mammalian ferritin is a large, iron-storage heteropolymer composed of two conformationally equivalent subunit types, light (FTL) 2 and heavy (FTH1) polypeptides, which are expressed in most kinds of cells (2-5). A single ferritin protein is composed of 24 self-assembled polypeptide subunits related by 4-, 3-, and 2-fold symmetry axes with one polypeptide per asymmetric unit. Each polypeptide subunit consists of a bundle of four parallel ␣-helices (A-D), a long extended loop (connecting helices B and C), and a C terminus with a short ␣-helix (E), which is involved in important stabilizing interactions around the 4-fold symmetry axes (2, 6, 7). Although both types of polypeptide subunits share a high degree of conformational similarity, they have diverse functional roles. The FTH1 subunit has a potent ferroxidase activity that catalyzes the oxidation of ferrous iron, whereas the F...

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“…The H chain carries ferroxidase active sites that oxidize ferrous to ferric iron, a prerequisite for mineralization in the core (reviewed by Chasteen 1998). The L chain, present only in vertebrates, does not have a ferroxidase site but is able to form a complex and bind iron at neutral but not acidic pH (Levi et al 1989). Therefore changes in the ratio of H/L subunits, e.g., in response to iron nutrition or in particular cell types, can affect the iron storage capability of ferritin (reviewed by Koorts and Viljoen 2007).…”

mentioning

confidence: 99%

FER1 and FER2 Encoding Two Ferritin Complexes in Chlamydomonas reinhardtii Chloroplasts Are Regulated by Iron

Long¹,

Sommer²,

Allen

et al. 2008

Genetics

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Two unlinked genes FER1 and FER2 encoding ferritin subunits were identified in the Chlamydomonas genome. An improved FER2 gene model, built on the basis of manual sequencing and incorporation of unplaced reads, indicated 49% identity between the ferritin subunits. Both FER1 and FER2 transcripts are increased in abundance as iron nutrition is decreased but the pattern for each gene is distinct. Using subunitspecific antibodies, we monitored expression at the protein level. In response to low iron, ferritin1 subunits and the ferritin1 complex are increased in parallel to the increase in FER1 mRNA. Nevertheless, the iron content of the ferritin1 complex is decreased. This suggests that increased expression results in increased capacity for iron binding in the chloroplast of iron-limited cells, which supports a role for ferritin1 as an iron buffer. On the other hand, ferritin2 abundance is decreased in iron-deprived cells, indicative of the operation of iron-nutrition-responsive regulation at the translational or post-translational level for FER2. Both ferritin subunits are plastid localized but ferritin1 is quantitatively recovered in soluble extracts of cells while ferritin2 is found in the particulate fraction. Partial purification of the ferritin1 complex indicates that the two ferritins are associated in distinct complexes and do not coassemble. The ratio of ferritin1 to ferritin2 is 70:1 in iron-replete cells, suggestive of a more dominant role of ferritin1 in iron homeostasis. The Volvox genome contains orthologs of each FER gene, indicating that the duplication of FER genes and potential diversification of function occurred prior to the divergence of species in the Volvocales.A LTHOUGH iron is abundant on earth, its bioavailability is limited in the aerobic world because of the insolubility of ferric salts, and iron can be a limiting nutrient for most forms of life. A third of the agricultural land and the same fraction of the ocean are considered iron deficient (reviewed by Boyd et al. 2007). Therefore, iron nutrition is a key component of global productivity. Organisms have evolved multiple and varied pathways for assimilating iron in its various chemical forms and at a range of concentrations in the nutrient environment (Staiger 2002; reviewed in Curie and Briat 2003;Hentze et al. 2004). Even though iron can be toxic to cells as a consequence of its propensity for participating in redox chemistry, cells do not generally excrete iron because it is a limiting nutrient (Liochev and Fridovich 1999). Instead, cells tend to store intracellular iron in a less reactive and hence nontoxic form. Because of the importance of iron for life, these pathways of uptake and storage are subject to layers of homeostatic regulation.We have developed Chlamydomonas as a model organism for understanding trace metal nutrition in plants, especially in the context of chloroplast function and photosynthesis (Merchant et al. 2006). As a microorganism, Chlamydomonas lends itself to nutritional studies because of the ease wit...

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Expression and structural and functional properties of human ferritin L-chain from Escherichia coli

Cited by 122 publications

References 24 publications

Is Hydrogen Peroxide Produced during Iron(II) Oxidation in Mammalian Apoferritins?

Is Hydrogen Peroxide Produced during Iron(II) Oxidation in Mammalian Apoferritins?

Iron-mediated Aggregation and a Localized Structural Change Characterize Ferritin from a Mutant Light Chain Polypeptide That Causes Neurodegeneration

FER1 and FER2 Encoding Two Ferritin Complexes in Chlamydomonas reinhardtii Chloroplasts Are Regulated by Iron

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