Insect ferritins: Typical or atypical?

Insects transmit millions of cases of disease each year, and cost millions of dollars in agricultural losses. The control of insect-borne diseases is vital for numerous developing countries, and the management of agricultural insect pests is a very serious business for developed countries. Control methods should target insect-specific traits in order to avoid non-target effects, especially in mammals. Since insect cells have had a billion years of evolutionary divergence from those of vertebrates, they differ in many ways that might be promising for the insect control field—especially, in iron metabolism because current studies have indicated that significant differences exist between insect and mammalian systems. Insect iron metabolism differs from that of vertebrates in the following respects. Insect ferritins have a heavier mass than mammalian ferritins. Unlike their mammalian counterparts, the insect ferritin subunits are often glycosylated and are synthesized with a signal peptide. The crystal structure of insect ferritin also shows a tetrahedral symmetry consisting of 12 heavy chain and 12 light chain subunits in contrast to that of mammalian ferritin that exhibits an octahedral symmetry made of 24 heavy chain and 24 light chain subunits. Insect ferritins associate primarily with the vacuolar system and serve as iron transporters—quite the opposite of the mammalian ferritins, which are mainly cytoplasmic and serve as iron storage proteins. This review will discuss these differences.     

Ferritins,iron uptake and storage from the bacterioferritin viewpoint

Ferritins constitute a broad superfamily of iron storage proteins, widespread in all domains of life, in aerobic or anaerobic organisms. Ferritins isolated from bacteria may be haem-free or contain a haem. In the latter case they are called bacterioferritins. The primary function of ferritins inside cells is to store iron in the ferric form. A secondary function may be detoxification of iron or protection against O(2) and its radical products. Indeed, for bacterioferritins this is likely to be their primary function. Ferritins and bacteroferritins have essentially the same architecture, assembling in a 24mer cluster to form a hollow, roughly spherical construction. In this review, special emphasis is given to the structure of the ferroxidase centres with native iron-containing sites, since oxidation of ferrous iron by molecular oxygen takes place in these sites. Although present in other ferritins, a specific entry route for iron, coupled with the ferroxidase reaction, has been proposed and described in some structural studies. Electrostatic calculations on a few selected proteins indicate further ion channels assumed to be an entry route in the later mineralization processes of core formation.     

Coordinating responses to iron and oxygen stress with DNA and mRNA promoters: The ferritin story

Combinations of DNA antioxidant response element and mRNA iron responsive element regulate ferritin expression in animals in response to oxidant and iron stress, or normal developmental signals. Ferritins are protein nanocages, found in animals, plants, bacteria, and archaea, that convert iron and oxygen to ferric oxy biominerals in the protein central cavity; the mineral traps potentially toxic reactants and concentrates iron for the future synthesis of other iron/heme proteins. Regulatory signals and the nanocage gene products are the same throughout biology, but the genetic mechanisms, DNA versus DNA + mRNA, vary. The number of genes, temporal regulation, tissue distribution in multi-cellular organisms, and gene product size (maxi-ferritins have 24 subunits and mini-ferritins, or Dps proteins, have 12 subunits and are restricted to bacteria and archaea) suggest an overwhelming diversity and variability. However, common themes of regulation and function are described which indicate not only that the three-dimensional protein structure and the functions of the ferritins are conserved, but also that broad features of genetic regulation are conserved relative to organismal and/or community needs. The analysis illustrates the centrality of the ferritins to life with iron and oxygen and models how Nature harnesses potentially dangerous chemistry for biology.     

The role of lysosomes in iron metabolism and recycling

Iron is the most abundant transition metal in the earth's crust. It cycles easily between ferric (oxidized; Fe(III)) and ferrous (reduced; Fe(II)) and readily forms complexes with oxygen, making this metal a central player in respiration and related redox processes. However, 'loose' iron, not within heme or iron-sulfur cluster proteins, can be destructively redox-active, causing damage to almost all cellular components, killing both cells and organisms. This may explain why iron is so carefully handled by aerobic organisms. Iron uptake from the environment is carefully limited and carried out by specialized iron transport mechanisms. One reason that iron uptake is tightly controlled is that most organisms and cells cannot efficiently excrete excess iron. When even small amounts of intracellular free iron occur, most of it is safely stored in a non-redox-active form in ferritins. Within nucleated cells, iron is constantly being recycled from aged iron-rich organelles such as mitochondria and used for construction of new organelles. Much of this recycling occurs within the lysosome, an acidic digestive organelle. Because of this, most lysosomes contain relatively large amounts of redox-active iron and are therefore unusually susceptible to oxidant-mediated destabilization or rupture. In many cell types, iron transit through the lysosomal compartment can be remarkably brisk. However, conditions adversely affecting lysosomal iron handling (or oxidant stress) can contribute to a variety of acute and chronic diseases. These considerations make normal and abnormal lysosomal handling of iron central to the understanding and, perhaps, therapy of a wide range of diseases.     

Effect of the charge distribution along the “ferritin-like” pores of the proteins from the Dps family on the iron incorporation process

DNA-binding proteins from starved cells (Dps) differ in the number and position of charged residues along the “ferritin-like” pores that are used by iron to reach the ferroxidase center and the protein cavity. These differences are shown to affect significantly the electrostatic potential at the pores, which determines the extent of cooperativity in the iron uptake kinetics and thereby the mass distribution of the ferric hydroxide micelles inside the protein cavity. These conclusions are of biotechnological value in the preparation of protein-enclosed nanomaterials and are expected to apply also to ferritins. They were reached after characterization of the Dps from Listeria innocua, Helicobacter pylori, Thermosynechococcus elongatus, Escherichia coli, and Mycobacterium smegmatis. The characterization comprised the calculation of the electrostatic potential at the pores, determination of the iron uptake kinetics in the presence of molecular oxygen or hydrogen peroxide, and analysis of the proteins by means of the sedimentation velocity after iron incorporation.     

Ferritins: furnishing proteins with iron

Ferritins are a superfamily of iron oxidation, storage and mineralization proteins found throughout the animal, plant, and microbial kingdoms. The majority of ferritins consist of 24 subunits that individually fold into 4-α-helix bundles and assemble in a highly symmetric manner to form an approximately spherical protein coat around a central cavity into which an iron-containing mineral can be formed. Channels through the coat at inter-subunit contact points facilitate passage of iron ions to and from the central cavity, and intrasubunit catalytic sites, called ferroxidase centers, drive Fe²⁺ oxidation and O₂ reduction. Though the different members of the superfamily share a common structure, there is often little amino acid sequence identity between them. Even where there is a high degree of sequence identity between two ferritins there can be major differences in how the proteins handle iron. In this review we describe some of the important structural features of ferritins and their mineralized iron cores, consider how iron might be released from ferritins, and examine in detail how three selected ferritins oxidise Fe²⁺ to explore the mechanistic variations that exist amongst ferritins. We suggest that the mechanistic differences reflect differing evolutionary pressures on amino acid sequences, and that these differing pressures are a consequence of different primary functions for different ferritins.     

Cellular regulation of iron assimilation

Cells of plants, most microorganisms, and animals require well-defined amounts of iron for survival, replication, and differentiation. The metal is an important component of such processes as synthesis of DNA, RNA, and chlorophyll; electron transport; oxygen metabolism; and nitrogen fixation. Because of the insolubility of iron in aerobic environments at neutral and alkaline pH values, cells have had to devise specific strategies to assimilate the metal. These include (1) development of systems for reducing ferric ions to the more soluble ferrous ions at the cell surface, (2) employment of small carrier molecules (termed siderophores) that have high affinity for ferric ions and receptor proteins for the ferrated molecules, and (3) use of transferrin and other proteins that can transport ferric ions. Excessive amounts of iron are toxic, however, and intracellular storage capacity is limited and efflux mechanisms generally are lacking. Thus, cells have had to develop methods of preventing over-accumulation of the metal. These include use of (1) oxygen to convert ferrous to ferric ions, (2) small molecules that can bind ferrous ions, termed siderophraxes, and (3) proteins that, when combined with ferrous ions, repress the expression of iron transport genes. Often, one organism can prevent growth of neighbors by restricting their access to iron. In other cases, cells assist each other by sharing iron acquisition systems or by restricting influx of excess iron. Homeostatic control of other essential trace metals also is required for optimal cell function. Nevertheless, since iron thus far has received most attention, it serves as the model of mineral metabolism. Moreover, many of the observations made on control of iron metabolism suggest possible applications in prevention and management of plant and animal infections as well as of neoplastic diseases, arthropathy, and cardiomyopathy. This review will focus on (1) problems at the cellular level of iron acquisition, storage, and exclusion; and (2) the strategies devised by cells of plants, microorganisms, and animals to solve these problems.     

The iron redox and hydrolysis chemistry of the ferritins

Background

Ferritins are ubiquitous and well-characterized iron storage and detoxification proteins. In bacteria and plants, ferritins are homopolymers composed of H-type subunits, while in vertebrates, they typically consist of 24 similar subunits of two types, H and L. The H-subunit is responsible for the rapid oxidation of Fe(II) to Fe(III) at a dinuclear center, whereas the L-subunit appears to help iron clearance from the ferroxidase center of the H-subunit and support iron nucleation and mineralization.

Scope of review

Despite their overall similar structures, ferritins from different origins markedly differ in their iron binding, oxidation, detoxification, and mineralization properties. This chapter provides a brief overview of the structure and function of ferritin, reviews our current knowledge of the process of iron uptake and mineral core formation, and highlights the similarities and differences of the iron oxidation and hydrolysis chemistry in a number of ferritins including those from archaea, bacteria, amphibians, and animals.

General Significance

Prokaryotic ferritins and ferritin-like proteins (Dps) appear to preferentially use H₂O₂ over O₂ as the iron oxidant during ferritin core formation. While the product of iron oxidation at the ferroxidase centers of these and other ferritins is labile and is retained inside the protein cavity, the iron complex in the di-iron cofactor proteins is stable and remains at the catalytic site. Differences in the identity and affinity of the ferroxidase center ligands to iron have been suggested to influence the distinct reaction pathways in ferritins and the di-iron cofactor enzymes.

Major conclusions

The ferritin 3-fold channels are shown to be flexible structures that allow the entry and exit of different ions and molecules through the protein shell. The H- and L-subunits are shown to have complementary roles in iron oxidation and mineralization, and hydrogen peroxide appears to be a by-product of oxygen reduction at the FC of most ferritins. The di-iron(III) complex at the FC of some ferritins acts as a stable cofactor during iron oxidation rather than a catalytic center where Fe(II) is oxidized at the FC followed by its translocation to the protein cavity.     

Iron incorporation into Escherichia coli Dps gives rise to a ferritin-like microcrystalline core

Escherichia coli Dps belongs to a family of bacterial stress-induced proteins to protect DNA from oxidative damage. It shares with Listeria innocua ferritin several structural features, such as the quaternary assemblage and the presence of an unusual ferroxidase center. Indeed, it was recently recognized to be able to oxidize and incorporate iron. Since ferritins are endowed with the unique capacity to direct iron deposition toward formation of a microcrystalline core, the structure of iron deposited in the E. coli Dps cavity was studied. Polarized single crystal absorption microspectrophotometry of iron-loaded Dps shows that iron ions are oriented. The spectral properties in the high spin 3d(5) configuration point to a crystal form with tetrahedral symmetry where the tetrahedron center is occupied by iron ions and the vertices by oxygen. Crystals of iron-loaded Dps also show that, as in mammalian ferritins, iron does not remain bound to the site after oxidation has taken place. The kinetics of the iron reduction/release process induced by dithionite were measured in the crystal and in solution. The reaction appears to have two phases, with t(12) of a few seconds and several minutes at neutral pH values, as in canonical ferritins. This behavior is attributed to a similar composition of the iron core.     

Ferritins in Chordata: Potential evolutionary trajectory marked by discrete selective pressures

Ferritins (FTs) are iron storage proteins that are involved in managing iron‐oxygen balance. In our work, we present a hypothesis on the putative effect of geological changes that have affected the evolution and radiation of ferritin proteins. Based on sequence analysis and phylogeny reconstruction, we hypothesize that two significant factors have been involved in the evolution of ferritin proteins: fluctuations of atmospheric oxygen concentrations, altering redox potential, and changing availability of water rich in bioavailable ferric ions. Fish, ancient amphibians, reptiles, and placental mammals developed the broadest repertoire of singular FTs, attributable to embryonic growth in aquatic environments containing low oxygen levels and abundant forms of soluble iron. In contrast, oviparous land vertebrates, like reptiles and birds, that have developed in high oxygen levels and limited levels of environmental Fe²⁺ exhibit a lower diversity of singular FTs, but display a broad repertoire of subfamilies, particularly notable in early reptiles.     

Ferritins control interaction between iron homeostasis and oxidative stress in Arabidopsis

Ferritin protein nanocages are the main iron store in mammals. They have been predicted to fulfil the same function in plants but direct evidence was lacking. To address this, a loss-of-function approach was developed in Arabidopsis. We present evidence that ferritins do not constitute the major iron pool either in seeds for seedling development or in leaves for proper functioning of the photosynthetic apparatus. Loss of ferritins in vegetative and reproductive organs resulted in sensitivity to excess iron, as shown by reduced growth and strong defects in flower development. Furthermore, the absence of ferritin led to a strong deregulation of expression of several metal transporters genes in the stalk, over-accumulation of iron in reproductive organs, and a decrease in fertility. Finally, we show that, in the absence of ferritin, plants have higher levels of reactive oxygen species, and increased activity of enzymes involved in their detoxification. Seed germination also showed higher sensitivity to pro-oxidant treatments. Arabidopsis ferritins are therefore essential to protect cells against oxidative damage.     

Iron in evolution

Iron chemistry in the environment and in organisms is entwined. The iron surface minerals in solution for the first billion years of the planet were ferrous compounds. This ion became and has remained a major participant in organisms. The evolution of iron was due to its oxidation to insoluble ferric ions by oxygen released from organisms. The evolution of cellular iron chemistry then required uptake from this oxidised state. Use was expanded from the mainly electron transfer properties in the original reductive cell interior to employment in external oxidative chemistry. The environment/organisms evolution is that of one predictable chemical system.     

Microbial ferric iron reductases

Almost all organisms require iron for enzymes involved in essential cellular reactions. Aerobic microbes living at neutral or alkaline pH encounter poor iron availability due to the insolubility of ferric iron. Assimilatory ferric reductases are essential components of the iron assimilatory pathway that generate the more soluble ferrous iron, which is then incorporated into cellular proteins. Dissimilatory ferric reductases are essential terminal reductases of the iron respiratory pathway in iron-reducing bacteria. While our understanding of dissimilatory ferric reductases is still limited, it is clear that these enzymes are distinct from the assimilatory-type ferric reductases. Research over the last 10 years has revealed that most bacterial assimilatory ferric reductases are flavin reductases, which can serve several physiological roles. This article reviews the physiological function and structure of assimilatory and dissimilatory ferric reductases present in the Bacteria, Archaea and Yeast. Ferric reductases do not form a single family, but appear to be distinct enzymes suggesting that several independent strategies for iron reduction may have evolved.     

Ferric citrate transport in Escherichia coli requires outer membrane receptor protein fecA.

Mutants of Escherichia coli K-12 AB2847 and of E. coli K-12 AN92 were isolated which were unable to grow on ferric citrate as the sole iron source. Of 22 mutants, 6 lacked an outer membrane protein, designated FecA protein, which was expressed by growing cells in the presence of 1 mM citrate. Outer membranes showed an enhanced binding of radioactive iron, supplied as a citrate complex, depending on the amount of FecA protein. The FecA protein was the most resistant of the proteins involved in ferric irion iron translocation across the outer membrane (FhuA = TonA, FepA, Cir, or 83K proteins) to the action of pronase P. It is also shown that previously isolated fec mutants (G. C. Woodrow et al., J. Bacteriol. 133:1524-1526, 1978) which are cotransducible with argF all lack the FecA protein. They were termed fecA to distinguish them from the other ferric citrate transport mutants, now designated fecB, which mapped in the same gene region at 7 min but were not cotransducible with ArgF. E. coli W83-24 and Salmonella typhimurium, which are devoid of a citrate-dependent iron transport system, lacked the FecA protein. It is proposed that the FecA protein participates in the transport of ferric citrate.     

A novel mechanism of iron-core formation by Pyrococcus furiosus archaeoferritin, a member of an uncharacterized branch of the ferritin-like superfamily

Storage of iron in a nontoxic and bioavailable form is essential for many forms of life. Three subfamilies of the ferritin-like superfamily, namely, ferritin, bacterioferritin, and Dps (DNA-binding proteins from starved cells), are able to store iron. Although the function of these iron-storage proteins is constitutive to many organisms to sustain life, the genome of some organisms appears not to encode any of these proteins. In an attempt to identify new iron-storage systems, we have found and characterized a new member of the ferritin-like superfamily of proteins, which unlike the multimeric storage system of ferritin, bacterioferritin, and Dps is monomeric in the absence of iron. Monomers catalyze oxidation of Fe(II) and they store the Fe(III) product as they assemble to form structures comparable to those of 24-meric ferritin. We propose that this mechanism is an alternative method of iron storage by the ferritin-like superfamily of proteins in organisms that lack the regular preassociated 24-meric/12-meric ferritins.     

Isolation and characterization of the iron-binding properties of a primitive monolobal transferrin from Ciona intestinalis

Transferrins are bilobal glycoproteins responsible for iron binding, transport, and delivery in many higher organisms. The two homologous lobes of transferrins are thought to have evolved by gene duplication of an ancestral monolobal form. In the present study, a 37.7-kDa primitive monolobal transferrin (nicatransferrin, or nicaTf) from the serum of the model ascidian species Ciona intestinalis was isolated by using an immobilized iron-affinity column and characterized by using mass spectrometry and N-terminal sequencing. The protein binds one equivalent of iron(III) and exhibits an electron paramagnetic resonance spectrum that is anion-dependent. The UV/vis spectrum of nicaTf has a shoulder at 330 nm in both the iron-depleted and the iron-replete forms, but does not display the approximately 460 nm tyrosine-to-iron charge transfer band common to vertebrate serum transferrins under the conditions investigated. This result suggests that iron may adopt a different binding mode in nicaTf compared with the more highly evolved transferrin proteins. This difference in binding mode could have implications for the physiological role of the protein in the ascidian. The genome of C. intestinalis has genes for both a monolobal and a bilobal transferrin, and the sequences of both proteins are discussed in light of the known features of vertebrate serum transferrins as well as other transferrin homologs.     

In vitro interactions of coumarins with iron

Coumarins are a large group of natural substances with diverse pharmacological properties that may predetermine some of them for the prevention and/or treatment of cardiovascular diseases and also other pathologies. Free iron participates in the production of reactive oxygen species (ROS) and plays an important role in the pathogenesis of cardiovascular diseases. Therefore, chelation of iron may attenuate some ROS consequences, but on the other hand, reduction of ferric ions to ferrous ones is unfavourable and leads to intensification of ROS production. In this study, we have examined the interaction of iron with coumarins which has been rarely analyzed.     

昆虫固醇转运蛋白的结构与功能

在昆虫中, 胆固醇不仅是细胞膜的重要成分之一, 也是昆虫蜕皮激素生物合成的前体。由于昆虫体内缺少两种合成胆固醇所必需的关键性酶, 所以昆虫不能自主地从简单的前体化合物从头合成胆固醇, 而必须通过吸收食物中的甾醇转化为胆固醇来满足生长、发育和繁殖的需要。胆固醇在组织和细胞内的运输主要由固醇转运蛋白 (sterol carrier proteins, SCPs) 执行。因此, 对固醇转运蛋白结构与功能的研究对于阐明昆虫中固醇运输具有重要的意义。本文对固醇转运蛋白的基因和蛋白结构、 细胞内表达和定位、 翻译后修饰、 蛋白三维结构、底物特异性和可能的运输途径等方面的研究进展进行了综述, 并对其作为害虫防治分子靶标的可能性进行了初步的讨论。研究发现, 不同物种的SCP蛋白的基因编码形式和蛋白剪切形式不同; 双翅目昆虫埃及伊蚊Aedes aegypti和黑腹果蝇Drosophila melanogaster除了SCP-x基因可编码SCP-x和SCP-2蛋白外, 还有另外的SCP-2和类SCP-2 (SCP-2L)基因编码SCP-2和类SCP-2蛋白; 而鳞翅目昆虫棉贪夜蛾Spodoptera littoralis、 斜纹夜蛾Spodoptera litura和家蚕Bombyx mori中SCP-x 基因的表达和转录方式与脊椎动物的SCP-x 基因类似, 通过转录和翻译后剪切形成SCP-2蛋白。SCP-x和SCP-2蛋白定位于过氧化物酶体。SCP-2蛋白由5个α-螺旋和5个β-折叠组成, 其中α5-螺旋可影响蛋白与底物的结合。SCP-2蛋白以不同的亲和力与固醇、胆固醇衍生物、脂肪酸、脂酰辅酶A和磷脂等化合物结合。超表达斜纹夜蛾SlSCP-x 和SlSCP-2基因可增加细胞对胆固醇的吸收; 而利用RNAi技术抑制幼虫体内SlSCP-x表达, 可导致血淋巴中的胆固醇含量降低, 并导致幼虫生长缓慢, 蜕皮化蛹延迟。     

X-ray structures of ferritins and related proteins

Ferritins are members of a much larger superfamily of proteins, which are characterised by a structural motif consisting of a bundle of four parallel and anti-parallel α helices. The ferritin superfamily itself is widely distributed across all three living kingdoms, in both aerobic and anaerobic organisms, and a considerable number of X-ray structures are available, some at extremely high resolution. We describe first of all the subunit structure of mammalian H and L chain ferritins and then discuss intersubunit interactions in the 24-subunit quaternary structure of these ferritins. Bacteria contain two types of ferritins, FTNs, which like mammalian ferritins do not contain haem, and the haem-containing BFRs. The characteristic carboxylate-bridged di-iron ferroxidase sites of H chain ferritins, FTNs and BFRs are compared, as are the potential entry sites for iron and the ‘nucleation’ site of L chain ferritins. Finally we discuss the three-dimensional structures of the 12-subunit bacterial Dps (DNA-binding protein from starved cells) proteins as well as their intersubunit di-iron ferroxidase site.