铜、铁离子代谢调节隐球菌致病力及宿主免疫的研究进展

金属离子参与真菌和哺乳动物众多重要的生长代谢过程。隐球菌利用金属离子参与毒力因子的形成;宿主利用金属离子进行自身代谢,增强机体免疫,抵御隐球菌感染。因此深入研究隐球菌金属离子代谢,以及探索宿主利用金属离子抵御隐球菌感染的调控机制是至关重要的,可为真菌感染的治疗提供理论依据。     

Thematic series: metals in biology

Metals are important in biochemistry, and the concentrations of many are highly regulated. This prologue introduces the thematic series "Metals in Biology," which includes minireviews on three metals: iron, copper, and selenium. Deficiencies and excesses of all three of these metals cause problems in human health. The three minireviews deal with regulation of iron homeostasis, the roles of copper metabolism in cell regulation and disease, and the functions of selenoproteins.     

Thematic minireview series: Metals in Biology 2012

Metals are present in about one-half of the protein structures available and also have critical roles in nucleic acid biochemistry. This prologue introduces the fourth of the Thematic Minireview Series on Metals in Biology, which deals with several topics involving iron, manganese, copper, and other metals. The six minireviews discuss metal transport and intracellular homeostasis, including chaperones and siderophores, maturation of the diiron active sites in hydrogenases, the balance between manganese and iron, and copper homeostasis relevant to pathogens.     

Cellular and mitochondrial iron homeostasis in vertebrates

Iron plays an essential role in cellular metabolism and biological processes. However, due to its intrinsic redox activity, free iron is a potentially toxic molecule in cellular biochemistry. Thus, organisms have developed sophisticated ways to import, sequester, and utilize iron. The transferrin cycle is a well-studied iron uptake pathway that is important for most vertebrate cells. Circulating iron can also be imported into cells by mechanisms that are independent of transferrin. Once imported into erythroid cells, iron is predominantly consumed by the mitochondria for the biosynthesis of heme and iron sulfur clusters. This review focuses on canonical transferrin-mediated and the newly discovered, non-transferrin mediated iron uptake pathways, as well as, mitochondrial iron homeostasis in higher eukaryotes. This article is part of a Special Issue entitled: Cell Biology of Metals.     

Metabolic remodeling in iron-deficient fungi

Eukaryotic cells contain dozens, perhaps hundreds, of iron-dependent proteins, which perform critical functions in nearly every major cellular process. Nutritional iron is frequently available to cells in only limited amounts; thus, unicellular and higher eukaryotes have evolved mechanisms to cope with iron scarcity. These mechanisms have been studied at the molecular level in the model eukaryotes Saccharomyces cerevisiae and Schizosaccharomyces pombe, as well as in some pathogenic fungi. Each of these fungal species exhibits metabolic adaptations to iron deficiency that serve to reduce the cell's reliance on iron. However, the regulatory mechanisms that accomplish these adaptations differ greatly between fungal species. This article is part of a Special Issue entitled: Cell Biology of Metals.     

IscA mediates iron delivery for assembly of iron-sulfur clusters in IscU under the limited accessible free iron conditions

Increasing evidence suggests that IscS, a cysteine desulfurase, provides sulfur for assembly of transient iron-sulfur clusters in IscU. IscU appears to act as a scaffold and eventually transfers the assembled clusters to target proteins. However, the iron donor for the iron-sulfur cluster assembly largely remains elusive. Here we find that Escherichia coli IscU fails to assemble iron-sulfur clusters when the accessible "free" iron in solution is limited by an iron chelator sodium citrate. Remarkably, IscA, an iron-sulfur cluster assembly protein with an iron association constant of 3.0 x 10(19) m(-1), is able to overcome the iron limitation due to sodium citrate and deliver iron for the IscS-mediated iron-sulfur cluster assembly in IscU. Substitution of the invariant cysteine residues Cys-99 or Cys-101 in IscA with serine completely abolishes the iron binding activity of the protein. The IscA mutants that fail to bind iron are unable to mediate iron delivery for the iron-sulfur cluster assembly in IscU under the limited accessible "free" iron conditions. The results suggest that IscA is capable of recruiting intracellular iron and providing iron for the iron-sulfur cluster assembly in IscU in cells in which the accessible "free" iron content is probably restricted.     

Mammalian iron metabolism and its control by iron regulatory proteins

Cellular iron homeostasis is maintained by iron regulatory proteins 1 and 2 (IRP1 and IRP2). IRPs bind to iron-responsive elements (IREs) located in the untranslated regions of mRNAs encoding protein involved in iron uptake, storage, utilization and export. Over the past decade, significant progress has been made in understanding how IRPs are regulated by iron-dependent and iron-independent mechanisms and the pathological consequences of IRP2 deficiency in mice. The identification of novel IREs involved in diverse cellular pathways has revealed that the IRP-IRE network extends to processes other than iron homeostasis. A mechanistic understanding of IRP regulation will likely yield important insights into the basis of disorders of iron metabolism. This article is part of a Special Issue entitled: Cell Biology of Metals.     

Murine mutants in the study of systemic iron metabolism and its disorders: an update on recent advances

Many past and recent advances in the field of iron metabolism have relied upon the use of mouse models of disease. These models have arisen spontaneously in breeder colonies or have been engineered for global or conditional ablation or overexpression of select genes. Full phenotypic characterization of these models typically involves maintenance on iron-loaded or -deficient diets, treatment with oxidative or hemolytic agents, breeding to other mutant lines or other stresses. In this review, we focus on systemic iron biology and the contributions that mouse model-based studies have made to the field. We have divided the field into three broad areas of research: dietary iron absorption, regulation of hepcidin expression and cellular iron metabolism. For each area, we begin with an overview of the current understanding of key molecular and cellular determinants then discuss recent advances. Finally, we conclude with brief comments on prospects for future study. This article is part of a Special Issue entitled: Cell Biology of Metals.     

Distinct iron binding property of two putative iron donors for the iron-sulfur cluster assembly: IscA and the bacterial frataxin ortholog CyaY under physiological and oxidative stress conditions

Frataxin, a small mitochondrial protein linked to the neurodegenerative disease Friedreich ataxia, has recently been proposed as an iron donor for the iron-sulfur cluster assembly. An analogous function has also been attributed to IscA, a key member of the iron-sulfur cluster assembly machinery found in bacteria, yeast, and humans. Here we have compared the iron binding property of IscA and the frataxin ortholog CyaY from Escherichia coli under physiological and oxidative stress conditions. In the presence of the thioredoxin reductase system, which emulates the intracellular redox potential, CyaY fails to bind any iron even at a 10-fold excess of iron in the incubation solution. Under the same physiologically relevant conditions, IscA efficiently recruits iron and transfers the iron for the iron-sulfur cluster assembly in a proposed scaffold IscU. In the presence of hydrogen peroxide, however, IscA completely loses its iron binding activity, whereas CyaY becomes a competent iron-binding protein and attenuates the iron-mediated production of hydroxyl free radicals. Hydrogen peroxide appears to oxidize the iron binding thiol groups in IscA, thus blocking the iron binding in the protein. Once the oxidized thiol groups in IscA are re-reduced with the thioredoxin reductase system, the iron binding activity of IscA is fully restored. On the other hand, hydrogen peroxide has no effect on the iron binding carboxyl groups in CyaY, allowing the protein to bind iron under oxidative stress conditions. The results suggest that IscA is capable of recruiting intracellular iron for the iron-sulfur cluster assembly under normal physiological conditions, whereas CyaY may serve as an iron chaperon to sequester redox active free iron and alleviate cellular oxidative damage under oxidative stress conditions.     

Protein degradation and iron homeostasis

Regulation of both systemic and cellular iron homeostasis requires the capacity to sense iron levels and appropriately modify the expression of iron metabolism genes. These responses are coordinated through the efforts of several key regulatory factors including F-box and Leucine-rich Repeat Protein 5 (FBXL5), Iron Regulatory Proteins (IRPs), Hypoxia Inducible Factor (HIF), and ferroportin. Notably, the stability of each of these proteins is regulated in response to iron. Recent discoveries have greatly advanced our understanding of the molecular mechanisms governing iron-sensing and protein degradation within these pathways. It has become clear that iron's privileged roles in both enzyme catalysis and protein structure contribute to its regulation of protein stability. Moreover, these multiple pathways intersect with one another in larger regulatory networks to maintain iron homeostasis. This article is part of a Special Issue entitled: Cell Biology of Metals.     

Yeast frataxin sequentially chaperones and stores iron by coupling protein assembly with iron oxidation

We have investigated the mechanism of frataxin, a conserved mitochondrial protein involved in iron metabolism and neurodegenerative disease. Previous studies revealed that the yeast frataxin homologue (mYfh1p) is activated by Fe(II) in the presence of O2 and assembles stepwise into a 48-subunit multimer (alpha48) that sequesters >2000 atoms of iron in 2-4-nm cores structurally similar to ferritin iron cores. Here we show that mYfh1p assembly is driven by two sequential iron oxidation reactions: A ferroxidase reaction catalyzed by mYfh1p induces the first assembly step (alpha --> alpha3), followed by a slower autoxidation reaction that promotes the assembly of higher order oligomers yielding alpha48. Depending on the ionic environment, stepwise assembly is associated with accumulation of 50-75 Fe(II)/subunit. Initially, this Fe(II) is loosely bound to mYfh1p and can be readily mobilized by chelators or made available to the mitochondrial enzyme ferrochelatase to synthesize heme. Transfer of mYfh1p-bound Fe(II) to ferrochelatase occurs in the presence of citrate, a physiologic ferrous iron chelator, suggesting that the transfer involves an intermolecular interaction. If mYfh1p-bound Fe(II) is not transferred to a ligand, iron oxidation, and mineralization proceed to completion, Fe(III) becomes progressively less accessible, and a stable iron-protein complex is formed. Iron oxidation-driven stepwise assembly is a novel mechanism by which yeast frataxin can function as an iron chaperone or an iron store.     

Genetic regulation of fluxes: iron homeostasis of Escherichia coli

Iron is an essential trace-element for most organisms. However, because high concentration of free intracellular iron is cytotoxic, cells have developed complex regulatory networks that keep free intracellular iron concentration at optimal range, allowing the incorporation of the metal into iron-using enzymes and minimizing damage to the cell. We built a mathematical model of the network that controls iron uptake and usage in the bacterium Escherichia coli to explore the dynamics of iron flow. We simulate the effect of sudden decrease or increase in the extracellular iron level on intracellular iron distribution. Based on the results of simulations we discuss the possible roles of the small RNA RyhB and the Fe–S cluster assembly systems in the optimal redistribution of iron flows. We suggest that Fe–S cluster assembly is crucial to prevent the accumulation of toxic levels of free intracellular iron when the environment suddenly becomes iron rich.     

Assembly of the iron-binding protein frataxin in Saccharomyces cerevisiae responds to dynamic changes in mitochondrial iron influx and stress level

Defects in frataxin result in Friedreich ataxia, a genetic disease characterized by early onset of neurodegeneration, cardiomyopathy, and diabetes. Frataxin is a conserved mitochondrial protein that controls iron needed for iron-sulfur cluster assembly and heme synthesis and also detoxifies excess iron. Studies in vitro have shown that either monomeric or oligomeric frataxin delivers iron to other proteins, whereas ferritin-like frataxin particles convert redox-active iron to an inert mineral. We have investigated how these different forms of frataxin are regulated in vivo. In Saccharomyces cerevisiae, only monomeric yeast frataxin (Yfh1) was detected in unstressed cells when mitochondrial iron uptake was maintained at a steady, low nanomolar level. Increments in mitochondrial iron uptake induced stepwise assembly of Yfh1 species ranging from trimer to > or = 24-mer, independent of interactions between Yfh1 and its major iron-binding partners, Isu1/Nfs1 or aconitase. The rate-limiting step in Yfh1 assembly was a structural transition that preceded conversion of monomer to trimer. This step was induced, independently or synergistically, by mitochondrial iron increments, overexpression of wild type Yfh1 monomer, mutations that stabilize Yfh1 trimer, or heat stress. Faster assembly kinetics correlated with reduced oxidative damage and higher levels of aconitase activity, respiratory capacity, and cell survival. However, deregulation of Yfh1 assembly resulted in Yfh1 aggregation, aconitase sequestration, and mitochondrial DNA depletion. The data suggest that Yfh1 assembly responds to dynamic changes in mitochondrial iron uptake or stress exposure in a highly controlled fashion and that this may enable frataxin to simultaneously promote respiratory function and stress tolerance.     

Expression, Purification, and Characterization of an Iron Chaperon Protein CyaY from Acidithiobacillus ferrooxidans

CyaY is the bacterial homolog of frataxin, proposed to be involved in the assembly of iron-sulfur clusters. While, the physiological iron donor for the iron-sulfur clusters assembly remains controversial. In this study, the gene of CyaY from Acidithiobacillus ferrooxidans was cloned and expressed in Escherichia coli, the protein was purified by one-step affinity chromatography to homogeneity. The CyaY protein can bind ferric iron and serve as an iron donor for the biogenesis of iron-sulfur clusters on the scaffold protein IscU in the presence of IscS and L-cysteine in vitro.     

The ubiquity of iron

The importance of iron in living systems can be traced to the many complexes within which it is found, to its chemical mobility in undergoing oxidation-reduction reactions, and to the abundance of iron in Earth's crust. Iron is the most abundant element, by mass, in the Earth, constituting about 80% of the inner and outer cores of Earth. The molten outer core is about 8000 km in diameter, and the solid inner core is about 2400 km in diameter. Iron is the fourth most abundant element in Earth's crust. It is the chemically functional component of mononuclear iron complexes, dinuclear iron complexes, [2Fe-2S] and [4Fe-4S] clusters, [Fe-Ni-S] clusters, iron protophorphyrin IX, and many other complexes in protein biochemistry. Metals such as nickel, cobalt, copper, and manganese are present in the crust and could in principle function chemically in place of iron, but they are scarce in Earth's crust. Iron is plentiful because of its nuclear stability in stellar nuclear fusion reactions. It seems likely that other solid planets, formed by the same processes as Earth, would also foster the evolution of life and that iron would be similarly important to life on those planets as it is on Earth.     

Thioredoxin reductase system mediates iron binding in IscA and iron delivery for the iron-sulfur cluster assembly in IscU

IscA is a key member of the iron-sulfur cluster assembly machinery found in bacteria and eukaryotes. Previously, IscA was characterized as an alternative iron-sulfur cluster assembly scaffold, as purified IscA can host transient iron-sulfur clusters. However, recent studies indicated that IscA is an iron-binding protein that can provide iron for the iron-sulfur cluster assembly in a proposed scaffold IscU (Ding H., Clark, R. J., and Ding, B. (2004) J. Biol. Chem. 279, 37499-37504). To further elucidate the roles of IscA in the biogenesis of iron-sulfur clusters, we reevaluate the iron binding activity of IscA under physiologically relevant conditions. The results indicate that in the presence of the thioredoxin reductase system, Escherichia coli IscA binds iron with an iron association constant of 2.0 x 10(19) M(-1) in vitro. Whereas all three components (thioredoxin 1, thioredoxin reductase and NADPH) in the thioredoxin reductase system are essential for mediating the iron binding in IscA, only catalytic amounts of thioredoxin 1 and thioredoxin reductase are required. In contrast, IscU fails to bind iron in the presence of the thioredoxin reductase system, suggesting that the iron binding in IscA is specific. Nevertheless, the thioredoxin reductase system can promote the iron-sulfur cluster assembly in IscU in the presence of the iron-loaded IscA, cysteine desulfurase (IscS), and L-cysteine, demonstrating a physiologically relevant system for the biogenesis of iron-sulfur clusters. The results provide additional evidence for the hypothesis that IscA is capable of recruiting intracellular "free" iron and delivering the iron for the iron-sulfur cluster assembly in IscU.     

Complementary roles of SufA and IscA in the biogenesis of iron-sulfur clusters in Escherichia coli

Biogenesis of iron-sulfur clusters requires a concerted delivery of iron and sulfur to target proteins. It is now clear that sulfur in iron-sulfur clusters is derived from L-cysteine via cysteine desulfurases. However, the specific iron donor for the iron-sulfur cluster assembly still remains elusive. Previous studies showed that IscA, a member of the iron-sulfur cluster assembly machinery in Escherichia coli, is a novel iron-binding protein, and that the iron-bound IscA can provide iron for the iron-sulfur cluster assembly in a proposed scaffold IscU in vitro. However, genetic studies have indicated that IscA is not essential for the cell growth of E. coli. In the present paper, we report that SufA, an IscA paralogue in E. coli, may represent the redundant activity of IscA. Although deletion of IscA or SufA has only a mild effect on cell growth, deletion of both IscA and SufA in E. coli results in a severe growth phenotype in minimal medium under aerobic growth conditions. Cell growth is restored when either IscA or SufA is re-introduced into the iscA-/sufA- double mutant, demonstrating further that either IscA or SufA is sufficient for their functions in vivo. Purified SufA, like IscA, is an iron-binding protein that can provide iron for the iron-sulfur cluster assembly in IscU in the presence of a thioredoxin reductase system which emulates the intracellular redox potential. Site-directed mutagenesis studies show that the SufA/IscA variants that lose the specific iron-binding activity fail to restore the cell growth of the iscA-/sufA- double mutant. The results suggest that SufA and IscA may constitute the redundant cellular activities to recruit intracellular iron and deliver iron for the iron-sulfur cluster assembly in E. coli.