FBXL5:一种维持细胞内铁稳态的泛素连接酶

细胞内铁稳态的维持主要通过铁调节蛋白(ironregulatory protein,IRP)与几种铁代谢基因如转铁蛋白受体和铁蛋白mRNA上铁应答元件结合来实现。铁不足可增加IRP2活性和含量,而铁过载则诱导了IRP2的泛素化和蛋白降解。F-盒蛋白FBXL5是一种铁和氧依赖的E3泛素连接酶,在铁和氧存在的情况下催化IRP2的泛素化,而缺铁或缺氧则造成FBXL5自身被泛素化修饰和随后的蛋白酶体降解。FBXL5铁调节功能的发现使人们对细胞内铁稳态的理解更为清晰。     

The FBXL5-IRP2 axis is integral to control of iron metabolism in vivo

Iron-dependent degradation of iron-regulatory protein 2 (IRP2) is a key event for maintenance of an appropriate intracellular concentration of iron. Although FBXL5 (F box and leucine-rich repeat protein 5) is thought to mediate this degradation, the role of FBXL5 in the control of iron homeostasis in?vivo has been poorly understood. We have now found that mice deficient in FBXL5 died in utero, associated with excessive iron accumulation. This embryonic mortality was prevented by additional ablation of IRP2, suggesting that impaired IRP2 degradation is primarily responsible for the death of Fbxl5(-)(/-) mice. We also found that liver-specific deletion of Fbxl5 resulted in deregulation of both hepatic and systemic iron homeostasis, leading to the development of steatohepatitis. The liver-specific mutant mice died with acute liver failure when fed a high-iron diet. Thus, our results uncover a major role for FBXL5 in ensuring an appropriate supply of iron to cells.     

Iron regulatory protein 2 as iron sensor. Iron-dependent oxidative modification of cysteine

Iron regulatory protein 2 coordinates cellular regulation of iron metabolism by binding to iron responsive elements in mRNA. The protein is synthesized constitutively but is rapidly degraded when iron stores are replete. This iron-dependent degradation requires the presence of a 73-residue degradation domain, but its functions have not yet been established. We now show that the domain can act as an iron sensor, mediating its own covalent modification. The domain forms an iron-binding site with three cysteine residues located in the middle of the domain. It then reacts with molecular oxygen to generate a reactive oxidizing species at the iron-binding site. One cysteine residue is oxidized to dehydrocysteine and other products. This covalent modification may thus mark the protein molecule for degradation by the proteasome system.     

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.     

铁代谢与蛋白质泛素化．降解调控研究进展

铁元素为几乎所有的生命体所必需,维持铁代谢稳态对机体的正常功能至关重要。铁代谢紊乱与人类多种疾病的发生和发展有关。已知铁代谢稳态受到一系列参与铁代谢环节的关键蛋白质,如IRP2等的精确调节。这些重要蛋白质的稳定性、生理活性的动态变化及其协调作用是细胞维持铁代谢平衡的分子基础。除了转录和转录后水平的调控,泛素化等翻译后修饰方式和蛋白质降解是细胞精确调控参与铁代谢的蛋白质的水平及功能普遍而有效的方式之一;同时,细胞的铁代谢状态也影响细胞内参与泛素化等翻译后修饰途径的酶类的活性和稳定性,从而在铁代谢和蛋白质修饰．降解途径之间形成反馈机制,实时和动态地完成对细胞内铁代谢水平的精确调控。就相关领域的最新进展作简要综述。     

Leishmania donovani inhibits ferroportin translation by modulating FBXL5‐IRP2 axis for its growth within host macrophages

Hepcidin mediated ferroportin (Fpn) degradation in macrophages is a well adopted strategy to limit iron availability towards invading pathogens. Leishmania donovani (LD), a protozoan parasite, resides within macrophage and competes with host for availing iron. Using in vitro and in vivo model of infection, we reveal that LD decreases Fpn abundance in host macrophages by hepcidin independent mechanism. Unaffected level of Fpn‐FLAG in LD infected J774 macrophage confirms that Fpn down‐regulation is not due its degradation. While increased Fpn mRNA but decreased protein expression in macrophages suggests blocking of Fpn translation by LD infection that is confirmed by ³⁵S‐methionine labelling assay. We further reveal that LD blocks Fpn translation by induced binding of iron regulatory proteins (IRPs) to the iron responsive element present in its 5′UTR. Supershift analysis provides evidence of involvement of IRP2 particularly during in vivo infection. Accordingly, a significant increase in IRP2 protein expression with simultaneous decrease in its stability regulator F‐box and leucine‐rich repeat Protein 5 (FBXL5) is detected in splenocytes of LD‐infected mice. Increased intracellular growth due to compromised expressions of Fpn and FBXL5 by specific siRNAs reveals that LD uses a novel strategy of manipulating IRP2‐FBXL5 axis to inhibit host Fpn expression.     

Redox control of iron regulatory protein 2 stability

Iron regulatory protein 2 (IRP2) is a critical switch for cellular and systemic iron homeostasis. In iron-deficient or hypoxic cells, IRP2 binds to mRNAs containing iron responsive elements (IREs) and regulates their expression. Iron promotes proteasomal degradation of IRP2 via the F-box protein FBXL5. Here, we explored the effects of oxygen and cellular redox status on IRP2 stability. We show that iron-dependent decay of tetracycline-inducible IRP2 proceeds efficiently under mild hypoxic conditions (3% oxygen) but is compromised in severe hypoxia (0.1% oxygen). A treatment of cells with exogenous H(2)O(2) protects IRP2 against iron and increases its IRE-binding activity. IRP2 is also stabilized during menadione-induced oxidative stress. These data demonstrate that the degradation of IRP2 in iron-replete cells is not only oxygen-dependent but also sensitive to redox perturbations.     

A Cellular Stress Model for the Sequestration of Redox-Active Glial Iron in the Aging and Degenerating Nervous System

Abstract: The mechanisms responsible for the accumulation of redox-active brain iron in normal senescence and in Parkinson's disease remain poorly understood. The aminothiol compound cysteamine (CSH) induces the appearance of autofluorescent, iron-rich cytoplasmic granules in cultured astroglia that are identical to glial inclusions that progressively accumulate in the aging periventricular brain. Both in situ and in culture, these glial inclusions appear to arise in the context of a generalized cellular stress (heat shock) response. Several laboratories have previously concluded that porphyrins and heme ferrous iron are responsible, respectively, for red-orange autofluorescence and nonenzymatic peroxidase activity in the glial inclusions. In the present study we found that, contrary to hypothesis, CSH suppresses the incorporation of the heme precursors δ-amino[¹⁴C]levulinic acid and [¹⁴C]glycine into astroglial porphyrin and heme in primary culture. Similar results were obtained when the cells were preloaded with radiolabeled heme precursors for 24 h before CSH treatment, suggesting that the latter directly inhibits porphyrin-heme biosynthesis rather than limiting precursor uptake by these cells. We also demonstrated that CSH exposure results in the sequestration of iron-59 by astroglial mitochondria (granule precursors). The results of this study suggest that stress-related trapping of nonheme iron by astroglial mitochondria may be an important mechanism underlying the pathological accumulation of redox-active iron in the basal ganglia of subjects with Parkinson's disease. CSH-treated astrocytes provide a useful model to investigate the role of stress-related dysregulation of neuroglial iron metabolism in the aging and degenerating nervous system.     

A new role for heme, facilitating release of iron from the bacterioferritin iron biomineral

Bacterioferritin (BFR) from Escherichia coli is a member of the ferritin family of iron storage proteins and has the capacity to store very large amounts of iron as an Fe(3+) mineral inside its central cavity. The ability of organisms to tap into their cellular stores in times of iron deprivation requires that iron must be released from ferritin mineral stores. Currently, relatively little is known about the mechanisms by which this occurs, particularly in prokaryotic ferritins. Here we show that the bis-Met-coordinated heme groups of E. coli BFR, which are not found in other members of the ferritin family, play an important role in iron release from the BFR iron biomineral: kinetic iron release experiments revealed that the transfer of electrons into the internal cavity is the rate-limiting step of the release reaction and that the rate and extent of iron release were significantly increased in the presence of heme. Despite previous reports that a high affinity Fe(2+) chelator is required for iron release, we show that a large proportion of BFR core iron is released in the absence of such a chelator and further that chelators are not passive participants in iron release reactions. Finally, we show that the catalytic ferroxidase center, which is central to the mechanism of mineralization, is not involved in iron release; thus, core mineralization and release processes utilize distinct pathways.     

Iron regulatory protein 2 turnover through a nonproteasomal pathway

Iron regulatory protein 2 (IRP2) controls the synthesis of many proteins involved in iron metabolism, and the level of IRP2 itself is regulated by varying the rate of its degradation. The proteasome is known to mediate degradation, with specificity conferred by an iron-sensing E3 ligase. Most studies on the degradation of IRP2 have employed cells overexpressing IRP2 and also rendered iron deficient to further increase IRP2 levels. We utilized a sensitive, quantitative assay for IRP2, which allowed study of endogenous IRP2 degradation in HEK293A cells under more physiologic conditions. We found that under these conditions, the proteasome plays only a minor role in the degradation of IRP2, with almost all the IRP2 being degraded by a nonproteasomal pathway. This new pathway is calcium-dependent but is not mediated by calpain. Elevating the cellular level of IRP2 by inducing iron deficiency or by transfection causes the proteasomal pathway to account for the major fraction of IRP2 degradation. We conclude that under physiological, iron-sufficient conditions, the steady-state level of IRP2 in HEK293A cells is regulated by the nonproteasomal pathway.     

Is serum gamma glutamyltransferase a marker of oxidative stress?

The primary role of cellular gamma glutamyltransferase (GGT) is to metabolize extracellular reduced glutathione (GSH), allowing for precursor amino acids to be assimilated and reutilized for intracellular GSH synthesis. Paradoxically, recent experimental studies indicate that cellular GGT may also be involved in the generation of reactive oxygen species in the presence of iron or other transition metals. Although the relationship between cellular GGT and serum GGT is not known and serum GGT activity has been commonly used as a marker for excessive alcohol consumption or liver diseases, our series of epidemiological studies consistently suggest that serum GGT within its normal range might be an early and sensitive enzyme related to oxidative stress. For example, serum and dietary antioxidant vitamins had inverse, dose-response relations to serum GGT level within its normal range, whereas dietary heme iron was positively related to serum GGT level. More importantly, serum GGT level within its normal range positively predicted F2-isoprostanes, an oxidative damage product of arachidonic acid, and fibrinogen and C-reactive protein, markers of inflammation, which were measured 5 or 15 years later, in dose-response manners. These findings suggest that strong associations of serum GGT with many cardiovascular risk factors and/or events might be explained by a mechanism related to oxidative stress. Even though studies on serum and/or cellular GGT is at a beginning stage, our epidemiological findings suggest that serum GGT might be useful in studying oxidative stress-related issues in both epidemiological and clinical settings.     

Iron and heme utilization in Porphyromonas gingivalis

Porphyromonas gingivalis is a Gram-negative anaerobic bacterium associated with the initiation and progression of adult periodontal disease. Iron is utilized by this pathogen in the form of heme and has been shown to play an essential role in its growth and virulence. Recently, considerable attention has been given to the characterization of various secreted and surface-associated proteins of P. gingivalis and their contribution to virulence. In particular, the properties of proteins involved in the uptake of iron and heme have been extensively studied. Unlike other Gram-negative bacteria, P. gingivalis does not produce siderophores. Instead it employs specific outer membrane receptors, proteases (particularly gingipains), and lipoproteins to acquire iron/heme. In this review, we will focus on the diverse mechanisms of iron and heme acquisition in P. gingivalis. Specific proteins involved in iron and heme capture will be described. In addition, we will discuss new genes for iron/heme utilization identified by nucleotide sequencing of the P. gingivalis W83 genome. Putative iron- and heme-responsive gene regulation in P. gingivalis will be discussed. We will also examine the significance of heme/hemoglobin acquisition for the virulence of this pathogen.     

Identification of a heme-sensing domain in iron regulatory protein 2

Iron regulatory protein 2 coordinates the cellular regulation of iron metabolism by binding to iron-responsive elements in mRNA. The protein is synthesized constitutively but is rapidly degraded when iron stores are replete. The mechanisms that prevent degradation during iron deficiency or promote degradation during iron sufficiency are not delineated. Iron regulatory protein 2 contains a domain not present in the closely related iron regulatory protein 1, and we found that this domain binds heme with high affinity. A cysteine within the domain is axially liganded to the heme, as occurs in cytochrome P450. The protein-bound heme reacts with molecular oxygen to mediate the oxidation of cysteine, including beta-elimination of the sulfur to yield alanine. This covalent modification may thus mark the protein molecule for degradation by the proteasome system, providing another mechanism by which heme can regulate the level of iron regulatory protein 2.