Frataxin acts as an iron chaperone protein to modulate mitochondrial aconitase activity

Numerous degenerative disorders are associated with elevated levels of prooxidants and declines in mitochondrial aconitase activity. Deficiency in the mitochondrial iron-binding protein frataxin results in diminished activity of various mitochondrial iron-sulfur proteins including aconitase. We found that aconitase can undergo reversible citrate-dependent modulation in activity in response to pro-oxidants. Frataxin interacted with aconitase in a citrate-dependent fashion, reduced the level of oxidant-induced inactivation, and converted inactive [3Fe-4S]1+ enzyme to the active [4Fe-4S]2+ form of the protein. Thus, frataxin is an iron chaperone protein that protects the aconitase [4Fe-4S]2+ cluster from disassembly and promotes enzyme reactivation.     

Proapoptotic BAX and BAK modulate the unfolded protein response by a direct interaction with IRE1alpha

Accumulation of misfolded protein in the endoplasmic reticulum (ER) triggers an adaptive stress response-termed the unfolded protein response (UPR)-mediated by the ER transmembrane protein kinase and endoribonuclease inositol-requiring enzyme-1alpha (IRE1alpha). We investigated UPR signaling events in mice in the absence of the proapoptotic BCL-2 family members BAX and BAK [double knockout (DKO)]. DKO mice responded abnormally to tunicamycin-induced ER stress in the liver, with extensive tissue damage and decreased expression of the IRE1 substrate X-box-binding protein 1 and its target genes. ER-stressed DKO cells showed deficient IRE1alpha signaling. BAX and BAK formed a protein complex with the cytosolic domain of IRE1alpha that was essential for IRE1alpha activation. Thus, BAX and BAK function at the ER membrane to activate IRE1alpha signaling and to provide a physical link between members of the core apoptotic pathway and the UPR.     

MMS19 assembles iron-sulfur proteins required for DNA metabolism and genomic integrity

Instability of the nuclear genome is a hallmark of cancer and aging. MMS19 protein has been linked to maintenance of genomic integrity, but the molecular basis of this connection is unknown. Here, we identify MMS19 as a member of the cytosolic iron-sulfur protein assembly (CIA) machinery. MMS19 functions as part of the CIA targeting complex that specifically interacts with and facilitates iron-sulfur cluster insertion into apoproteins involved in methionine biosynthesis, DNA replication, DNA repair, and telomere maintenance. MMS19 thus serves as an adapter between early-acting CIA components and a subset of cellular iron-sulfur proteins. The function of MMS19 in the maturation of crucial components of DNA metabolism may explain the sensitivity of MMS19 mutants to DNA damage and the presence of extended telomeres.     

Oxidation-reduction and the molecular mechanism of a regulatory RNA-protein interaction

Iron-responsive elements (IREs) are RNA motifs that have been identified within the 5' untranslated region of ferritin messenger RNA and the 3' untranslated region of transferrin receptor mRNA. A single IRE mediates iron-dependent control of ferritin translation, whereas multiple IREs are found in the region of the transferrin receptor mRNA responsible for iron-dependent control of mRNA stability. A cytosolic protein binds in vitro to the IREs of both mRNAs. The IRE-binding protein (IRE-BP) is shown to require free sulfhydryl groups for its specific interaction with the IRE. Treatment of lysates with reducing agents increases the binding activity, whereas agents that block sulfhydryls inhibit binding. Iron starvation, leading to decreased ferritin translation, results in increased binding activity, which is explained by an increase in the fraction of the IRE-BP that is in a fully reduced state.     

Binding of a cytosolic protein to the iron-responsive element of human ferritin messenger RNA

The human ferritin H chain messenger RNA contains a specific iron-responsive element (IRE) in its 5' untranslated region, which mediates regulation by iron of ferritin translation. An RNA gel retardation assay was used to demonstrate the affinity of a specific cytosolic binding protein for the IRE. A single-base deletion in the IRE eliminated both the interaction of the cytoplasmic protein with the IRE and translational regulation. Thus, the regulatory potential of the IRE correlates with its capacity to specifically interact with proteins. Titration curves of binding activity after treatment of cells with an iron chelator suggest that the factor acts as a repressor of ferritin translation.     

MMS19 links cytoplasmic iron-sulfur cluster assembly to DNA metabolism

The function of many DNA metabolism proteins depends on their ability to coordinate an iron-sulfur (Fe-S) cluster. Biogenesis of Fe-S proteins is a multistep process that takes place in mitochondria and the cytoplasm, but how it is linked to nuclear Fe-S proteins is not known. Here, we demonstrate that MMS19 forms a complex with the cytoplasmic Fe-S assembly (CIA) proteins CIAO1, IOP1, and MIP18. Cytoplasmic MMS19 also binds to multiple nuclear Fe-S proteins involved in DNA metabolism. In the absence of MMS19, a failure to transfer Fe-S clusters to target proteins is associated with Fe-S protein instability and preimplantation death of mice in which Mms19 has been knocked out. We propose that MMS19 functions as a platform to facilitate Fe-S cluster transfer to proteins critical for DNA replication and repair.     

Structure of the hydrophilic domain of respiratory complex I from Thermus thermophilus

Respiratory complex I plays a central role in cellular energy production in bacteria and mitochondria. Its dysfunction is implicated in many human neurodegenerative diseases, as well as in aging. The crystal structure of the hydrophilic domain (peripheral arm) of complex I from Thermus thermophilus has been solved at 3.3 angstrom resolution. This subcomplex consists of eight subunits and contains all the redox centers of the enzyme, including nine iron-sulfur clusters. The primary electron acceptor, flavin-mononucleotide, is within electron transfer distance of cluster N3, leading to the main redox pathway, and of the distal cluster N1a, a possible antioxidant. The structure reveals new aspects of the mechanism and evolution of the enzyme. The terminal cluster N2 is coordinated, uniquely, by two consecutive cysteines. The novel subunit Nqo15 has a similar fold to the mitochondrial iron chaperone frataxin, and it may be involved in iron-sulfur cluster regeneration in the complex.     

Redox signaling in chloroplasts: cleavage of disulfides by an iron-sulfur cluster

Light generates reducing equivalents in chloroplasts that are used not only for carbon reduction, but also for the regulation of the activity of chloroplast enzymes by reduction of regulatory disulfides via the ferredoxin:thioredoxin reductase (FTR) system. FTR, the key electron/thiol transducer enzyme in this pathway, is unique in that it can reduce disulfides by an iron-sulfur cluster, a property that is explained by the tight contact of its active-site disulfide and the iron-sulfur center. The thin, flat FTR molecule makes the two-electron reduction possible by forming on one side a mixed disulfide with thioredoxin and by providing on the opposite side access to ferredoxin for delivering electrons.     

Coupling of stress in the ER to activation of JNK protein kinases by transmembrane protein kinase IRE1

Malfolded proteins in the endoplasmic reticulum (ER) induce cellular stress and activate c-Jun amino-terminal kinases (JNKs or SAPKs). Mammalian homologs of yeast IRE1, which activate chaperone genes in response to ER stress, also activated JNK, and IRE1alpha-/- fibroblasts were impaired in JNK activation by ER stress. The cytoplasmic part of IRE1 bound TRAF2, an adaptor protein that couples plasma membrane receptors to JNK activation. Dominant-negative TRAF2 inhibited activation of JNK by IRE1. Activation of JNK by endogenous signals initiated in the ER proceeds by a pathway similar to that initiated by cell surface receptors in response to extracellular signals.     

Solvent tuning of electrochemical potentials in the active sites of HiPIP versus ferredoxin

A persistent puzzle in the field of biological electron transfer is the conserved iron-sulfur cluster motif in both high potential iron-sulfur protein (HiPIP) and ferredoxin (Fd) active sites. Despite this structural similarity, HiPIPs react oxidatively at physiological potentials, whereas Fds are reduced. Sulfur K-edge x-ray absorption spectroscopy uncovers the substantial influence of hydration on this variation in reactivity. Fe-S covalency is much lower in natively hydrated Fd active sites than in HiPIPs but increases upon water removal; similarly, HiPIP covalency decreases when unfolding exposes an otherwise hydrophobically shielded active site to water. Studies on model compounds and accompanying density functional theory calculations support a correlation of Fe-S covalency with ease of oxidation and therefore suggest that hydration accounts for most of the difference between Fd and HiPIP reduction potentials.     

The actin-binding protein profilin binds to PIP2 and inhibits its hydrolysis by phospholipase C

Profilin is generally thought to regulate actin polymerization, but the observation that acidic phospholipids dissociate the complex of profilin and actin raised the possibility that profilin might also regulate lipid metabolism. Profilin isolated from platelets binds with high affinity to small clusters of phosphatidylinositol 4,5-bisphosphate (PIP2) molecules in micelles and also in bilayers with other phospholipids. The molar ratio of the complex of profilin with PIP2 is 1:7 in micelles of pure PIP2 and 1:5 in bilayers composed largely of other phospholipids. Profilin competes efficiently with platelet cytosolic phosphoinositide-specific phospholipase C for interaction with the PIP2 substrate and thereby inhibits PIP2 hydrolysis by this enzyme. The cellular concentrations and binding characteristics of these molecules are consistent with profilin being a negative regulator of the phosphoinositide signaling pathway in addition to its established function as an inhibitor of actin polymerization.     

Nitrite inhibition of Clostridium botulinum: electron spin resonance detection of iron-nitric oxide complexes

Vegetative cells of Clostridium botulinum were shown to contain iron-sulfur proteins that react with added nitrite to form iron-nitric oxide complexes, with resultant destruction of the iron-sulfur cluster. Inactivation of iron-sulfur enzymes (especially ferredoxin) by binding of nitric oxide would almost certainly inhibit growth, and thus is probably the mechanism of botulinal inhibition by nitrite in foods.     

柑橘果实发育过程中有机酸含量及相关代谢酶活性的变化

 测定了柠檬 (高酸 ,CitruslimonL .Burm)、锦橙 (中酸C .sinesisL .Osbeck)、冰糖橙 (低酸 ,C .sinesisL .Os beck)和奉节脐橙 (C .sinesisL .Osbeck)及其低酸芽变株系、晚熟芽变株系果实在发育过程中柠檬酸的含量及其相关代谢酶活性。结果表明 ,除柠檬外 ,汁胞柠檬酸含量均在花后 10 0～ 130d达到高峰 ,以后逐渐下降 ;柠檬酸合成酶活性变化与各类型柑橘果实中柠檬酸含量差异没有明显联系 ;细胞溶液中的乌头酸酶后期升高对柑橘果实中酸的降解有明显影响 ;磷酸烯醇式丙酮酸羧化酶活性和磷酸烯醇式丙酮酸羧化酶 /NAD 异柠檬酸脱氢酶值较高 ,酸积累较高。     

Aconitase couples metabolic regulation to mitochondrial DNA maintenance

Mitochondrial DNA (mtDNA) is essential for cells to maintain respiratory competency and is inherited as a protein-DNA complex called the nucleoid. We have identified 22 mtDNA-associated proteins in yeast, among which is mitochondrial aconitase (Aco1p). We show that this Krebs-cycle enzyme is essential for mtDNA maintenance independent of its catalytic activity. Regulation of ACO1 expression by the HAP and retrograde metabolic signaling pathways directly affects mtDNA maintenance. When constitutively expressed, Aco1p can replace the mtDNA packaging function of the high-mobility-group protein Abf2p. Thus, Aco1p may integrate metabolic signals and mtDNA maintenance.     

Organic Acid Concentrations and the Relative Enzymatic Changes During the Development of the Citrus Fruits

Seasonal changes in enzyme activities and citrate concentration during the development of citrus fruits were investigated. The result showed that the organic acid concentrations reached a peak in the 100 - 130days after anthesis and gradually declined during later stages of fruit maturation in most varieties of citrus,but declined only slightly thereafter in lemon [Citrus lin on (L.) Burm]; there is no relation between the activity of citrate synthetase(CS) and the different acid concentration in different citrus fruits; the increase of the activity of the cytosolic aconitase in the late period of the development of citrus fruits accelerated the degradation of citric acid in citrus fruits; the higher the activity of phosphoenolpyruvate carboxylase(PEPC) and the ratio of NAD-dependent isocitrate hydrogenase(PEPC/NAD-IDH- ), the more the concentrations of organic acids in citrus fruit.     

Protein carbon-13 spin systems by a single two-dimensional nuclear magnetic resonance experiment

By applying a two-dimensional double-quantum carbon-13 nuclear magnetic resonance experiment to a protein uniformly enriched to 26 percent carbon-13, networks of directly bonded carbon atoms were identified by virtue of their one-bond spin-spin couplings and were classified by amino acid type according to their particular single- and double-quantum chemical shift patterns. Spin systems of 75 of the 98 amino acid residues in a protein, oxidized Anabaena 7120 ferredoxin (molecular weight 11,000), were identified by this approach, which represents a key step in an improved methodology for assigning protein nuclear magnetic resonance spectra. Missing spin systems corresponded primarily to residues located adjacent to the paramagnetic iron-sulfur cluster.     

放线菌A5295激发子的稳定性及活性物质分析

为明确对致病疫霉无拮抗作用但能显著诱导马铃薯抗晚疫病的放线菌A5295发酵液的稳定性及其有效激发物质,以不同pH、温度和蛋白酶K处理A5295菌株发酵原液(激发子),用硫酸铵、乙醇和丙酮分别沉淀发酵液中的有效物质,通过马铃薯块茎切片测试其诱导抗病效果,并以考马斯亮蓝法和蒽酮法测定乙醇粗提物中蛋白质和糖的含量。结果显示,该激发子耐高温、耐强酸和强碱,对蛋白酶K不敏感,不同处理下的诱抗效果均接近发酵原液(69%)、甚至高达93%;乙醇粗提物的诱抗效果达到73%,明显优于硫酸铵和丙酮粗提物;该粗提物中蛋白和糖的含量分别为1.32%和53.4%,且发现经Sevag法去除蛋白后的诱抗效果略有增加(80%),能耐受121℃高温高压和强酸强碱。表明A5295的有效物质为糖类或以糖类为主,对环境稳定。