Cysteine reactivity and thiol-disulfide interchange pathways in AhpF and AhpC of the bacterial alkyl hydroperoxide reductase system

AhpC and AhpF from Salmonella typhimurium undergo a series of electron transfers to catalyze the pyridine nucleotide-dependent reduction of hydroperoxide substrates. AhpC, the peroxide-reducing (peroxiredoxin) component of this alkyl hydroperoxidase system, is an important scavenger of endogenous hydrogen peroxide in bacteria and acts through a reactive, peroxidatic cysteine, Cys46, and a second cysteine, Cys165, that forms an active site disulfide bond. AhpF, a separate disulfide reductase protein, regenerates AhpC every catalytic cycle via electrons from NADH which are transferred to AhpC through a tightly bound flavin and two disulfide centers, Cys345-Cys348 and Cys129-Cys132, through putative large domain movements. In order to assess cysteine reactivity and interdomain interactions in both proteins, a comprehensive set of single and double cysteine mutants (replacing cysteine with serine) of both proteins were prepared. Based on 5,5-dithiobis(2-nitrobenzoic acid) (DTNB) and AhpC reactivity with multiple mutants of AhpF, the thiolate of Cys129 in the N-terminal domain of AhpF initiates attack on Cys165 of the intersubunit disulfide bond within AhpC for electron transfer between proteins. Cys348 of AhpF has also been identified as the nucleophile attacking the Cys129 sulfur of the N-terminal disulfide bond to initiate electron transfer between these two redox centers. These findings support the modular architecture of AhpF and its need for domain rotations for function, and emphasize the importance of Cys165 in the reductive reactivation of AhpC. In addition, two new constructs have been generated, an AhpF-AhpC complex and a "twisted" form of AhpF, in which redox centers are locked together by stable disulfide bonds which mimic catalytic intermediates.     

Hydrogen peroxide and peroxiredoxins in redox regulation of intracellular signaling

A growing body of evidence indicates that hydrogen peroxide is generated in response to ligand-receptor interaction and is involved in redox-regulation of signaling pathways in various cell types. The mechanism of redox-regulation is based on post-translational modification of key regulatory proteins that contain essential cysteinyl residues at their catalytic sites. Hydrogen peroxide acts as a chemical mediator that affects signaling through reversible oxidation of thiol groups of essential cysteins, which results in changes in protein catalytic activity. The accumulation and propagation of H₂O₂ signal in a cell is regulated by antioxidant proteins peroxiredoxins. The catalytic cycle of peroxiredoxins enables them to keep a low resting level of H₂O₂ in a cell, while the hyperoxidation-reduction cycle allows for H₂O₂ bursts during signal transduction. H₂O₂, being a small, diffusible, highly reactive molecule generated and destroyed in enzymatic reactions and capable of regulating the phosphorylation-dephosphorylation events in a cell, meets all major criteria of the second messenger in signal transduction but the criterion of specificity. How a toxic molecule that is potentially dangerous to all biomolecules in a cell can specifically relay information through signaling cascades is discussed in this review.     

Cyclophilin a binds to peroxiredoxins and activates its peroxidase activity

Six distinct peroxiredoxin (Prx) proteins (Prx I-VI) from distinct genes have been identified in mammalian tissues. Prxs are members of a group of peroxidases that have conserved reactive cysteine residue(s) in the active site(s). An immediate physiological electron donor for the peroxidase catalysis for five Prx proteins (Prx I-V) has been identified as thioredoxin (Trx), but that for Prx VI (1-Cys Prx) is still unclear. To identify an immediate electron donor and a binding protein for Prx VI, we performed a Prx VI protein overlay assay. A 20-kDa binding protein was identified by the Prx VI protein overlay assay with flow-through fractions from a High-Q column with rat lung crude extracts. Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) and MS-Fit, we identified the 20-kDa Prx VI-binding protein as a cyclophilin A (CyP-A). The binding of recombinant human CyP-A (hCyP-A) to Prx VI was confirmed by using the hCyP-A protein overlay assay and Western immunoblot analysis with hCyP-A-specific antibodies. hCyP-A enhanced the antioxidant activity of Prx VI, as well as the other known mammalian Prx isotypes. hCyP-A supported antioxidant activity of Prx II and Prx VI both against thiol (dithiothreitol)-containing metal-catalyzed oxidation (MCO) systems and ascorbate-containing MCO systems. Prx II was reduced by hCyP-A without help from any other reductant, and the reduction was cyclosporin A-independent. These results strongly suggest that CyP-A not only binds to Prx proteins but also supports its peroxidase activity as an immediate electron donor. In addition, Cys(115) and Cys(161) of hCyP-A were found to be involved in the activation and the reduction of Prx.     

Mutant forms of tufA and tufB independently suppress nonsense mutations

The level of nonsense suppression in Salmonella typhimurium carrying error-enhancing mutations in either or both of the genes coding for the elongation factor EF-Tu has been measured. Suppression of both UGA and UAG is observed. There is no significant suppression of any of six UAA sites tested. Nonsense suppression does not require that both genes for EF-Tu be mutant. Strains carrying one mutant and one wild-type tuf gene suppress nonsense mutations. The level of suppression increases approximately additively when both tuf genes are mutant. It is suggested that these mutant forms of EF-Tu act independently of each other to suppress nonsense mutations. Suppression is not observed at all UGA and UAG sites, but instead shows a strong site specificity.     

Mutations in the thiol-disulfide oxidoreductases BdbC and BdbD can suppress cytochrome c deficiency of CcdA-defective Bacillus subtilis cells

Cytochromes of the c type in the gram-positive bacterium Bacillus subtilis are all membrane anchored, with their heme domains exposed on the outer side of the cytoplasmic membrane. They are distinguished from other cytochromes by having heme covalently attached by two thioether bonds. The cysteinyls in the heme-binding site (CXXCH) in apocytochrome c must be reduced in order for the covalent attachment of the heme to occur. It has been proposed that CcdA, a membrane protein, transfers reducing equivalents from thioredoxin in the cytoplasm to proteins on the outer side of the cytoplasmic membrane. Strains deficient in the CcdA protein are defective in cytochrome c and spore synthesis. We have discovered that mutations in the bdbC and bdbD genes can suppress the defects caused by lack of CcdA. BdbC and BdbD are thiol-disulfide oxidoreductases. Our experimental findings indicate that these B. subtilis proteins functionally correspond to the well-characterized Escherichia coli DsbB and DsbA proteins, which catalyze the formation of disulfide bonds in proteins in the periplasmic space.     

Analysis of the link between enzymatic activity and oligomeric state in AhpC, a bacterial peroxiredoxin

Peroxiredoxins (Prxs) make up a ubiquitous class (proposed EC 1.11.1.15) of cysteine-dependent peroxidases with roles in oxidant protection and signal transduction. An intriguing biophysical property of typical 2-Cys Prxs is the redox-dependent modulation of their oligomeric state between decamers and dimers at physiological concentrations. The functional consequences of this linkage are unknown, but on the basis of structural considerations, we hypothesized that decamer-building (dimer-dimer) interactions serve to stabilize a loop that forms the peroxidatic active site. Here, we address this important issue by studying mutations of Thr77 at the decamer-building interface of AhpC from Salmonella typhimurium. Ultracentrifugation studies revealed that two of the substitutions (T77I and T77D) successfully disrupted the decamer, while the third (T77V) actually enhanced decamer stability. Crystal structures of the decameric forms of all three mutant proteins provide a rationale for their properties. A new assay allowed the first ever measurement of the true k(cat) and K(m) values of wild-type AhpC with H(2)O(2), placing the catalytic efficiency at 4 x 10(7) M(-)(1) s(-)(1). T77V had slightly higher activity than wild-type enzyme, and both T77I and T77D exhibited ca. 100-fold lower catalytic efficiency, indicating that the decameric structure is quite important for, but not essential to, activity. The interplay between decamer formation and active site loop dynamics is emphasized by a decreased susceptibility of T77I and T77D to peroxide-mediated inactivation, and by an increase in the crystallographic B-factors in the active site loop, rather than at the site of the mutation, in the T77D variant.     

Human embryonic stem cell-derived NK cells acquire functional receptors and cytolytic activity

Human embryonic stem cells (hESCs) provide a unique resource to analyze early stages of human hematopoiesis. However, little is known about the ability to use hESCs to evaluate lymphocyte development. In the present study, we use a two-step culture method to demonstrate efficient generation of functional NK cells from hESCs. The CD56(+)CD45(+) hESC-derived lymphocytes express inhibitory and activating receptors typical of mature NK cells, including killer cell Ig-like receptors, natural cytotoxicity receptors, and CD16. Limiting dilution analysis suggests that these cells can be produced from hESC-derived hemopoietic progenitors at a clonal frequency similar to CD34(+) cells isolated from cord blood. The hESC-derived NK cells acquire the ability to lyse human tumor cells by both direct cell-mediated cytotoxicity and Ab-dependent cellular cytotoxicity. Additionally, activated hESC-derived NK cells up-regulate cytokine production. hESC-derived lymphoid progenitors provide a novel means to characterize specific cellular and molecular mechanisms that lead to development of specific human lymphocyte populations. These cells may also provide a source for innovative cellular immune therapies.     

Magnetic fields suppress Pseudomonas aeruginosa biofilms and enhance ciprofloxacin activity

Due to the refractory nature of pathogenic microbial biofilms, innovative biofilm eradication strategies are constantly being sought. Thus, this study addresses a novel approach to eradicate Pseudomonas aeruginosa biofilms. Magnetic nanoparticles (MNP), ciprofloxacin (Cipro), and magnetic fields were systematically evaluated in vitro for their relative anti-biofilm contributions. Twenty-four-hour biofilms exposed to aerosolized MNPs, Cipro, or a combination of both, were assessed in the presence or absence of magnetic fields (Static one-sided, Static switched, Oscillating, Static + oscillating) using changes in bacterial metabolism, biofilm biomass, and biofilm imaging. The biofilms exposed to magnetic fields alone exhibited significant metabolic and biomass reductions (p < 0.05). When biofilms were treated with a MNP/Cipro combination, the most significant metabolic and biomass reductions were observed when exposed to static switched magnetic fields (p < 0.05). The exposure of P. aeruginosa biofilms to a static switched magnetic field alone, or co-administration with MNP/Cipro/MNP + Cipro appears to be a promising approach to eradicate biofilms of this bacterium.     

Light modulation of enzyme activity: activation of the light effect mediators by reduction and modulation of enzyme activity by thiol-disulfide exchange

Light and dark modulation experiments with pea (Pisum sativum L.) chloroplast stromal fractions pretreated with dithiothreitol (to reduce protein disulfide bonds) or with 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) (to block sulfhydryl groups) suggest that light modulation involves thiol-disulfide exchange on the modulatable stromal enzyme protein. Light-dependent reduction of DTNB involves a photosynthetic electron transport chain component located on the reducing side of photosystem I prior to ferredoxin; DTNB may be acting as a light effect mediator substitute. The thylakoid-bound light effect mediator system, then, in its light-activated reduced form probably catalyzes thiol-disulfide exchange reactions on stromal enzymes.     

Impact of SIN-1-derived peroxynitrite flux on endothelial cell redox homeostasis and bioenergetics: protective role of diphenyl diselenide via induction of peroxiredoxins

Abstract

Increased production of reactive nitrogen (RNS) and oxygen (ROS) species and its detrimental effect to mitochondria are associated with endothelial dysfunction. This study was designed to determine the effect of a peroxynitrite flux, promoted by 1,3-morpholinosydnonimine (SIN-1), in mitochondrial function and some redox homeostasis parameters in bovine aortic endothelial cells (BAEC). Moreover, the effect of diphenyl diselenide (PhSe)₂, a simple organic selenium compound, in preventing peroxynitrite-mediated cytotoxicity was also investigated. Our results showed that overnight exposure to SIN-1 (250 μM) caused a profound impairment of oxygen consumption, energy generation and reserve capacity in mitochondria of BAEC. Mitochondrial dysfunction resulted in an additional intracellular production of peroxynitrite, amplifying the phenomenon and leading to changes in redox homeostasis. Moreover, we observed an extensive decline in mitochondrial membrane potential (ΔΨ_m) induced by peroxynitrite and this event was associated with apoptotic-type cell death. Alternatively, the pretreatment of BAEC with (PhSe)₂, hindered peroxynitrite-mediated cell damage by preserving mitochondrial and endothelial function and consequently preventing apoptosis. The protective effect of (PhSe)₂ was related to its ability to improve the intracellular redox state by increasing the expression of different isoforms of peroxiredoxins (Prx–1–3), efficient enzymes in peroxynitrite detoxification.     

Determination of microbial respiratory and redox activity in activated sludge

The tetrazolium salt 5-cyano-2,3-ditolyltetrazolium chloride (CTC) was used for the determination of metabolically active bacteria in active sludge. The method was adapted and optimized to the conditions of activated sludge. The colorless and nonfluorescent tetrazolium salt is readily reduced to a water-insoluble fluorescent formazan product via the microbial electron transport system and indicates mainly dehydrogenase activity. After more than 2 h incubation, no further formation of new formazan crystals was observed, although the existing crystals in active cells continued to grow at the optimal CTC-concentration of 4 mM. The dehydrogenase activity determined by direct epifluorescence microscopic enumeration did not correlate with cumulative measured activity as determined by formazan extraction. The addition of nutrients did not lead to an increase of CTC-active cells. Sample storage conditions such as low temperature or aeration resulted in a significant decrease in dehydrogenase activity within 30 min. The rapid and sensitive method is well suited for the detection and enumeration of metabolically active microorganisms in activated sludge. Extracellular redox activity was measured with the tetrazolium salt 3′-{1-[phenylamino-) carbonyl]-3,4-tetrazolium}-bis(4-methoxy-6-nitro)benzene-sulfonic acid hydrate (XTT), which remains soluble in its reduced state, after extraction of extracellular polymeric substances (EPS) with a cation exchange resin. Received 12 August 1996/ Accepted in revised form 29 May 1997     

Warming and drying suppress microbial activity and carbon cycling in boreal forest soils

Climate warming is expected to have particularly strong effects on tundra and boreal ecosystems, yet relatively few studies have examined soil responses to temperature change in these systems. We used closed‐top greenhouses to examine the response of soil respiration, nutrient availability, microbial abundance, and active fungal communities to soil warming in an Alaskan boreal forest dominated by mature black spruce. This treatment raised soil temperature by 0.5 °C and also resulted in a 22% decline in soil water content. We hypothesized that microbial abundance and activity would increase with the greenhouse treatment. Instead, we found that bacterial and fungal abundance declined by over 50%, and there was a trend toward lower activity of the chitin‐degrading enzyme N‐acetyl‐glucosaminidase. Soil respiration also declined by up to 50%, but only late in the growing season. These changes were accompanied by significant shifts in the community structure of active fungi, with decreased relative abundance of a dominant Thelephoroid fungus and increased relative abundance of Ascomycetes and Zygomycetes in response to warming. In line with our hypothesis, we found that warming marginally increased soil ammonium and nitrate availability as well as the overall diversity of active fungi. Our results indicate that rising temperatures in northern‐latitude ecosystems may not always cause a positive feedback to the soil carbon cycle, particularly in boreal forests with drier soils. Models of carbon cycle‐climate feedbacks could increase their predictive power by incorporating heterogeneity in soil properties and microbial communities across the boreal zone.     

海洋浮游藻类细胞质膜氧化还原活性与细胞外CA活性的关系（简报）

Exofacial ferricyanide reduction at the plasma membrane of intact cells, and the link between plasma membrane redox activity, inorganic carbon status of the cells and extracellular carbonic anhydrase (CAext) activity were assayed using 10 marine phytoplankton species. In species Chaetocceros compressus, Cocolithus pelagicus and Gephyrocapsa ocetanica with no extracellular CA activity under carbon-limited or carbon-replete conditions, barely detectable ferricyanide reduction was observed. Species Skeletonema costatum, Melosira sp., Thalassiosira rotula, Thalassiosira weisflogi and Pleurochrysis carterae in which extracellular CA activity was only detected under carbon-limited conditions showed high rates of exofacial ferricyanide reduction. Western blotting and immunolocalization showed the presence of enzyme protein under carbon-limited and replete conditions at the cell surface, even though the CA activity could only detected when inorganic carbon was limiting, which suggests that the development of extracellular CA in response to carbon limitation is an activation of a preexisting protein rather than de novo synthesis. The results suggest that inorganic carbon limitation in the light increases plasma membrane redox activity and promotes proton extrusion, which result in the protonation and activation of the extracellular CA.     

Respiratory activity,energy and redox status in sulphur-deficient bean plants

Sulphur (S) is an essential nutrient that due to its chemistry plays important roles in many metabolic processes. S-deficient bean plants (Phaseolus vulgaris L. cv. Złota saxa) showed decreased sulphate concentrations and sulphur to nitrogen ratios in the leaves and roots, less chloroplastic pigments and lower dry matter production. Phenotypic effects of S deficiency appeared as depressed shoot growth, paling and curling of the youngest leaves, chlorotic and/or necrotic spots on the leaf surface. Our results show that S deficiency changes mitochondrial function, cellular energy status and redox homeostasis. ATP production in bean leaf and root mitochondria was lower as the result of decreased activity of Complex I. Increased activities of internal NADH dehydrogenases (ND_in) may at least partially compensate for Complex I impairment. External NADH dehydrogenases (ND_ex) activities, as well as protein level and capacity of alternative oxidase (AOX), did not change in S-deficient bean plants. Total ATP concentration severely decreased in leaf and root tissues. Pyridine nucleotide level was changed in S-deficient bean plants, NAD(H) pool became more reduced in leaf and root tissues whereas NADP(H) pool was more oxidized in the leaves. Our findings indicate that flexible function of plant mitochondrial respiratory chain could be an important target during adaptations to S deficiency.     

Resemblance and dissemblance of Arabidopsis type II peroxiredoxins: similar sequences for divergent gene expression,protein localization,and activity

The Arabidopsis type II peroxiredoxin (PRXII) family is composed of six different genes, five of which are expressed. On the basis of the nucleotide and protein sequences, we were able to define three subgroups among the PRXII family. The first subgroup is composed of AtPRXII-B, -C, and -D, which are highly similar and localized in the cytosol. AtPRXII-B is ubiquitously expressed. More striking is the specific expression of AtPRXII-C and AtPRXII-D localized in pollen. The second subgroup comprises the mitochondrial AtPRXII-F, the corresponding gene of which is expressed constitutively. We show that AtPRXII-E, belonging to the last subgroup, is expressed mostly in reproductive tissues and that its product is addressed to the plastid. By in vitro enzymatic experiments, we demonstrate that glutaredoxin is the electron donor of recombinant AtPRXII-B for peroxidase reaction, but the donors of AtPRXII-E and AtPRXII-F have still to be identified.     

Mutant LRRK2 enhances glutamatergic synapse activity and evokes excitotoxic dendrite degeneration

Mutations in leucine-rich repeat kinase 2 (LRRK2), which are associated with autosomal dominant Parkinson's disease, elicit progressive dendrite degeneration in neurons. We hypothesized that synaptic dysregulation contributes to mutant LRRK2-induced dendritic injury. We performed in vitro whole-cell voltage clamp studies of glutamatergic receptor agonist responses and glutamatergic synaptic activity in cultured rat cortical neurons expressing full-length wild-type and mutant forms of LRRK2. Expression of the pathogenic G2019S or R1441C LRRK2 mutants resulted in larger whole-cell current responses to direct application of AMPA and NMDA receptor agonists. In addition, mutant LRRK2-expressing neurons exhibited an increased frequency of spontaneous miniature excitatory postsynaptic currents (mEPSCs) in conjunction with increased excitatory synapse density as assessed by immunofluorescence for PSD95 and VGLUT1. Mutant LRRK2-expressing neurons showed enhanced vulnerability to acute synaptic glutamate stress. Furthermore, treatment with the NMDA receptor antagonist memantine significantly protected against subsequent losses in dendrite length and branching complexity. These data demonstrate an early association between mutant LRRK2 and increased excitatory synapse activity, implicating an excitotoxic contribution to mutant LRRK2 induced dendrite degeneration.     

BRCA1 and BRCA2 bind Stat5a and suppress its transcriptional activity

Germline mutations in the breast cancer susceptibility genes, BRCA1 and BRCA2, are thought to account for a large portion of familial breast cancer. The increased risk of breast cancer in women carrying such mutations suggests that these proteins play a critical role in the growth regulation of mammary epithelial cells. Another protein, Stat5a, is known to be essential for growth and terminal differentiation of breast epithelial cells. Here we show that Stat5a forms a complex with both BRCA1 and BRCA2 in breast epithelial cells upon stimulation with prolactin. In addition, we show that the activity of Stat5a on the beta-casein promoter is modulated by both BRCA1 and BRCA2. This interaction may be important during the expansion and terminal differentiation of breast epithelial cells, as happens during pregnancy and lactation.     

Tumor suppressor PALB2 maintains redox and mitochondrial homeostasis in the brain and cooperates with ATG7/autophagy to suppress neurodegeneration

The PALB2 tumor suppressor plays key roles in DNA repair and has been implicated in redox homeostasis. Autophagy maintains mitochondrial quality, mitigates oxidative stress and suppresses neurodegeneration. Here we show that Palb2 deletion in the mouse brain leads to mild motor deficits and that co-deletion of Palb2 with the essential autophagy gene Atg7 accelerates and exacerbates neurodegeneration induced by ATG7 loss. Palb2 deletion leads to elevated DNA damage, oxidative stress and mitochondrial markers, especially in Purkinje cells, and co-deletion of Palb2 and Atg7 results in accelerated Purkinje cell loss. Further analyses suggest that the accelerated Purkinje cell loss and severe neurodegeneration in the double deletion mice are due to excessive oxidative stress and mitochondrial dysfunction, rather than DNA damage, and partially dependent on p53 activity. Our studies uncover a role of PALB2 in mitochondrial homeostasis and a cooperation between PALB2 and ATG7/autophagy in maintaining redox and mitochondrial homeostasis essential for neuronal survival.     

Characterization of redox state and reductase activity of protein disulfide isomerase under different redox environments using a sensitive fluorescent assay

In this study, dieosin glutathione disulfide (Di-E-GSSG) was synthesized by the reaction of eosin isothiocyanate with GSSG. Di-E-GSSG had low fluorescence which increased approximately 70-fold on reduction of its disulfide bond. The substrate was used to monitor the disulfide reductase activity of PDI. Di-E-GSSG is the most sensitive pseudo substrate for PDI reductase activity reported to date. This probe was further used as an analytical reagent to develop an end point assay for measuring the redox state of PDI. The reduction of Di-E-GSSG by reduced enzyme was studied in the absence of reducing agents and the redox state of PDI was monitored as a function of the stoichiometric changes in the amount of eosin-glutathione (EGSH) generated by the active-site dithiols of PDI. The redox state of PDI was also studied under variable [GSH]/[GSSG] ratios. The results indicate that PDI is in approximately 1/2-reduced state where the [GSH]/[GSSG] ratio is between 1:1 and 3:1, conditions similar to the lumen of endoplasmic reticulum or in the extracellular environment. On the other hand, [GSH]/[GSSG] ratios of > or =8:1, such as in cytosol, all active-site thiols would be reduced. The study was extended to utilize Di-E-GSSG to investigate the effect of variable redox ratios on the platelet surface PDI reductase activity.